Biotechnology for Agro-Industrial Residues
Utilisation
Poonam Singh nee’ Nigam · Ashok Pandey
Editors
Biotechnology
for Agro-Industrial
Residues Utilisation
Utilisation of Agro-Residues
123
Editors
Poonam Singh nee’ Nigam
University of Ulster
Faculty of Life & Health
Sciences
Coleraine
Northern Ireland
United Kingdom BT52 1SA
P.Singh@ulster.ac.uk
ISBN 978-1-4020-9941-0
Ashok Pandey
National Institute for
Interdisciplinary
Science & Technology
CSIR, Industrial Estate PO
Trivandrum-695019
India
ashokpandey56@yahoo.co.in
e-ISBN 978-1-4020-9942-7
DOI 10.1007/978-1-4020-9942-7
Library of Congress Control Number: 2009920465
c Springer Science+Business Media B.V. 2009
No part of this work may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming, recording
or otherwise, without written permission from the Publisher, with the exception
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Printed on acid-free paper
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DEDICATED BY POONAM TO her dearest mother ROOPA NIGAM
And in loving memory of her late father
MAHESH CHANDRA NIGAM, who left too early.
Preface
Industries from two large sectors, i.e. agriculture and food for most of its history
have been environmentally benign. Industrial activity has always resulted in some
kind of pollution, be it solid waste, wastewater or gaseous pollution. Even when
technology began to have an impact, reliance on natural and ecological processes
remained crucial. Crop residues were incorporated into the soil or fed to livestock
and the manure returned to the land in amounts that could be absorbed and utilized.
Since farms have become highly mechanized and reliant on synthetic fertilizers and
pesticides, the crop residues, which were once recycled, are now largely wastes
whose disposal presents a continuing problem for the farmer.
The agro-industrial residues consist of many and varied wastes from agriculture
and food industry, which in total account for over 250 million tonnes of waste per
year in the UK alone. The prospects and application of biotechnical principles facilitates these problems to be seen in a new approach, as resources, which in many
cases have tremendous potential. As a result an extensive range of valuable and
usable products can be recovered from what was previously considered waste. This
encompasses a huge area of microbial-biotechnology with many possibilities that
have been researched; and such findings have shown the massive potential when
they are practically and economically applied.
Although several agricultural residues can be disposed of safely (due to biodegradable nature) in the environment, the vast quantities in which they are generated as
a result of diverse agricultural and industrial practices, necessitates the requirement
to look for some avenues where these could be utilized for some application. Since
these are rich in organic nature, they represent one of the most energy-rich resources
on the planet. Accumulation of this kind of biomass in large quantities every year
results not only in the deterioration of the environment, but also in the loss of potentially valuable material which can be processed to yield a number of valuable
added products, such as food, fuel, feed and a variety of chemicals. These residues
include renewable lignocellulosic materials such as the stalks, stems, straws, hulls
and cobs which all vary slightly in composition. Cellulose and hemicellulose, the
major constituents of these materials, can be referred to as valuable resources for
a number of reasons, largely due to the fact that they can be bio-converted for the
production of several valuable products.
vii
viii
Preface
Thus, today, for better or for worse, we live in a society with a throw away attitude which often chooses in many cases to ignore the potential that is all around
it. Particularly in the case of agriculture, there can be considerable damage to the
environment which is already being continually put under increasing stress by waste
disposal. Furthermore it is often quite expensive to dispose of these wastes this is
not to mention the economic loss of not exploiting them properly in the first place.
Biotechnology can offer many viable alternatives to the disposal of agricultural
waste with the production of many much needed products such as fuels, feeds, and
pharmaceutical products.
Therefore, this book has been presented with the up-to-date information available
on a biotechnology approach for the utilisation of agro-industrial residues. The book
contains twenty four chapters by the experts working in the field of Biotechnology
for Agro-Industrial Residues Processing. Each of the chapters includes information
on materials and suitable technology for their utilization and bioconversion methods
to obtain products of economic importance. The chapters have been categorised in
appropriate sections: (1) General; (2) Production of industrial products using agroindustrial residues as substrates; (3) Biotechnological potential of agro-industrial
residues for bioprocesses; (4) Enzymes degrading agro-industrial residues and their
production; and (5) Bioconversion of agro-industrial residues.
It is hoped that the book will provide a useful information resource for academics,
researchers, and industries.
Northern Ireland, UK
Trivandrum, Kerala, India
Poonam Singh nee’ Nigam
Ashok Pandey
Acknowledgements
Editors sincerely thank all contributing authors of the chapters included in this book
for their cooperation in submitting and revising their manuscripts on due dates as per
guidelines of the publisher Springer. Thanks for the approval of our book-proposal
on this particular topic are due to Peter Butler, publishing director; Dugald MacGlashan, senior publishing editor; and Sara Huisman, publishing assistant, Springer
Science & Business Media B.V. Efforts of Max Haring and Agnieszka Brodawka,
are acknowledged for realising the production of this book. Finally, the support
extended by our families could make this project possible.
Northern Ireland, UK
Trivandrum, Kerala, India
Poonam Singh nee’ Nigam
Ashok Pandey
ix
Contents
Part I General
1 Agro-Industrial Residue Utilization for Industrial Biotechnology
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Erick J. Vandamme
3
2 Pre-treatment of Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . 13
Poonam Singh nee’ Nigam, Nutan Gupta and Ashish Anthwal
Part II Production of Industrial Products Using Agro-Industrial Residues
as Substrates
3 Production of Organic Acids from Agro-Industrial Residues . . . . . . . . 37
Poonam Singh nee’ Nigam
4 Biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Soham Chattopadhyay, Asmita Mukerji and Ramkrishna Sen
5 Production of Protein-Enriched Feed Using Agro-Industrial
Residues as Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
J. Obeta Ugwuanyi, Brian McNeil and Linda M. Harvey
6 Aroma Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Syed G. Dastager
7 Production of Bioactive Secondary Metabolites . . . . . . . . . . . . . . . . . . . . 129
Poonam Singh nee’ Nigam
8 Microbial Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Sumathy Babitha
xi
xii
Contents
9 Production of Mushrooms Using Agro-Industrial Residues as
Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Antonios N. Philippoussis
10 Solid-State Fermentation Technology for Bioconversion of Biomass
and Agricultural Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Poonam Singh nee’ Nigam and Ashok Pandey
Part III Biotechnological Potential of Agro-Industrial Residues for
Bioprocesses
11 Biotechnological Potentials of Cassava Bagasse . . . . . . . . . . . . . . . . . . . . 225
Rojan P. John
12 Sugarcane Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Binod Parameswaran
13 Edible Oil Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Swetha Sivaramakrishnan and Dhanya Gangadharan
14 Biotechnological Potential of Fruit Processing Industry Residues . . . . 273
Diomi Mamma, Evangelos Topakas, Christina Vafiadi and Paul
Christakopoulos
15 Wine Industry Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Bo Jin and Joan M. Kelly
16 Biotechnological Potential of Brewing Industry By-Products . . . . . . . . 313
Solange I. Mussatto
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and
Bran Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Hongzhang Chen, Ye Yang and Jianxing Zhang
18 Palm Oil Industry Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Mynepalli K.C. Sridhar and Olugbenga O. AdeOluwa
Part IV Enzymes Degrading Agro-Industrial Residues and Their Production
19 Amylolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Dhanya Gangadharan and Swetha Sivaramakrishnan
Contents
xiii
20 Cellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Reeta Rani Singhania
21 Pectinolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Nicemol Jacob
22 Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
K.N. Niladevi
Part V Bioconversion of Agro-Industrial Residues
23 Anaerobic Treatment of Solid Agro-Industrial Residues . . . . . . . . . . . . 417
Michael Ward and Poonam Singh nee’ Nigam
24 Vermicomposting of Agro-Industrial Processing Waste . . . . . . . . . . . . . 431
V.K. Garg and Renuka Gupta
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Contributors
Olugbenga O. AdeOluwa Department of Agronomy, Faculty of Agriculture,
University of Ibadan, Ibadan, Nigeria, adeoluwaoo@yahoo.com
Ashish Anthwal Department of Earth and Environmental Sciences, Atmospheric
Environment Laboratory, Sejong University, Gwangjin-Gu, Seoul 143-747,
Republic of Korea, ashishaanthwal25@rediffmail.com
Sumathy Babitha Skin Bioactive Material Laboratory, Inha University, Yonghyunong, Nam-gu, Incheon 402-751, Republic of Korea, babisp2003@yahoo.com
Soham Chattopadhyay Department of Biotechnology, Indian Institute of
Technology, Kharagpur, West Bengal 721302, India, sohamcrj@gmail.com
Hongzhang Chen State Key Laboratory of Biochemical Engineering, Institute of
Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China,
hzchen@home.ipe.ac.cn
Paul Christakopoulos Biotechnology Laboratory, School of Chemical
Engineering, National Technical University of Athens, Zografou 157 80, Athens,
Greece, hristako@orfeas.chemeng.ntua.gr
Syed G. Dastager National Institute of Interdisciplinary Science and Technology
(Formerly RRL), CSIR, Industrial Estate, Thiruvananthapuram-695019, Kerala,
India, syedmicro@gmail.com
Dhanya Gangadharan Biotechnology Division, National Institute for
Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum-695 019,
Kerala, India, dhanya 28@yahoo.com
V.K. Garg Department of Environmental Science and Engineering, Guru
Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India,
vinodkgarg@yahoo.com
Nutan Gupta School of Biomedical Sciences, Faculty of Life and Health
Sciences, University of Ulster, Coleraine, BT521SA, Northern Ireland, UK,
nutangupta100@rediffmail.com
xv
xvi
Contributors
Renuka Gupta Department of Environmental Science and Engineering, Guru
Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India,
renug77@gmail.com
Linda M. Harvey Strathclyde Fermentation Centre, Strathclyde Institute for
Pharmacy & Biomedical Science, University of Strathclyde, Glasgow, Scotland,
UK, lm.harvey@strath.ac.uk
Nicemol Jacob Biotechnology Division, National Institute for Interdisciplinary Science and, Technology (CSIR), Trivandrum, 695019, Kerala, India,
nicemariacyril@yahoo.co.in
Bo Jin School of Earth and Environmental Sciences, School of Chemical
Engineering, The University of Adelaide, Adelaide, SA 5005, Australia,
bo.jin@adelaide.edu.au
Rojan P. John Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum 695 019, Kerala, India,
rojanpj@yahoo.co.in
Joan M. Kelly School of Molecular and Biomedical Sciences, The University of
Adelaide, Adelaide, SA 5005, Australia, joan.kelly@adelaide.edu.au
Diomi Mamma Biotechnology Laboratory, School of Chemical Engineering,
National Technical University of Athens, Zografou 157 80, Athens Greece,
dmamma@chemeng.ntua.gr
Brian McNeil Strathclyde Fermentation Centre, Strathclyde Institute for
Pharmacy & Biomedical Science, University of Strathclyde, Glasgow, Scotland,
UK, B.mcneil@strath.ac.uk
Asmita Mukerji Department of Biotechnology, Anandapur, East Kolkata
Township, Kolkata 700 107, India, asmita.mukerji@gmail.com
Solange I. Mussatto Institute for Biotechnology and Bioengineering, Centre
of Biological Engineering, University of Minho, Braga 4710-057, Portugal,
solange@deb.uminho.pt
Poonam Singh nee’ Nigam Faculty of Life and Health Sciences, School of
Biomedical Sciences, University of Ulster, Coleraine BT521SA, Northern Ireland,
UK, P.Singh@ulster.ac.uk
K.N Niladevi Biotechnology Division, National Institute for Interdisciplinary
Science and, Technology (CSIR), Trivandrum 695019, Kerala, India,
nilanandini@yahoo.co.in
Ashok Pandey National Institue for Interdisciplinary Science and Technology, CSIR, Industrial Estate PO, Trivandrum-695 019, Kerala, India,
ashokpandey56@yahoo.co.in
Contributors
xvii
Binod Parameswaran Bioenergy Research Centre, Korea Institute of
Energy Research (KIER), Yusong, Daejon 305-343, Republic of Korea,
binodkannur@yahoo.com
Antonios N. Philippoussis National Agricultural Research Foundation, I.A.A.C.,
Laboratory of Edible and Medicinal Fungi, 13561 Ag, Anargyri, Athens, Greece,
iamc@ath.forthnet.gr
Ramkrishna Sen Department of Biotechnology, Indian Institute of Technology
Kharagpur, West Bengal 721302, India, rksen@yahoo.com
Reeta Rani Singhania Biotechnology division, National Institute for Interdisciplinary Science and Technology, Trivandrum 695 019, Kerala, India,
reetasinghania 123@rediffmail.com
Swetha Sivaramakrishnan Biotechnology Division, National Institute for
Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum 695 019,
Kerala, India, swsj79@yahoo.co.in
Mynepalli K.C. Sridhar Division of Environmental Health Sciences, Faculty of
Public Health, University of Ibadan, Ibadan, Nigeria, mkcsridhar@yahoo.com
Evangelos Topakas Biotechnology Laboratory, School of Chemical Engineering,
National Technical University of Athens, Zografou 157 80, Athens, Greece,
vtopakas@central.ntua.gr
J. Obeta Ugwuanyi Department of Microbiology, University of Nigeria, Nsukka,
Enugu, Nigeria, jerryugwuanyi@yahoo.com
Christina Vafiadi Biotechnology Laboratory, School of Chemical Engineering,
National Technical University of Athens, Zografou 157 80, Athens, Greece,
cvafiadi@central.ntua.gr
Erick J. Vandamme Laboratory of Industrial Microbiology and Biocatalysis,
Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, B-9000 GENT, Belgium, Erick.Vandamme@UGent.be
Michael Ward Centre for Vision Science, Queens University of Belfast,
Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland, UK, michaelward@
hotmail.co.uk
Ye Yang State Key Laboratory of Biochemical Engineering, Institute of
Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China,
yeyang@home.ipe.ac.cn
Jianxing Zhang State Key Laboratory of Biochemical Engineering, Institute of
Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China,
zhangjx04@gmail.com
Part I
General
Chapter 1
Agro-Industrial Residue Utilization
for Industrial Biotechnology Products
Erick J. Vandamme
Contents
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Fermentation and Biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 Currently Used Renewable Agrosubstrates as Industrial Fermentation Substrates . . . . . . 5
1.3.1 Carbohydrates as Carbon Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3.2 Plant Oils as Carbon Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.3 Nitrogen Sources, Used in Industrial Fermentation Processes . . . . . . . . . . . . . . . . . 8
1.3.4 Nutrient Selection Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Towards Agro-Industrial Residue Utilization Technology in Industrial Biotechnology . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Keywords Renewable-resources · Microbial-nutrition · Industrial fermentation
substrates · Bio-chemicals versus petrochemicals · Submerged and solid state
fermentation
1.1 Introduction
As worldwide demand for petroleum, our main fossil-resource to produce energy,
chemicals and materials is steadily increasing, particularly to satisfy the fast growing economies of countries such as China and India, petroleum prices are expected
to continue to rise further. The effect can be seen today, with petroleum prices over
130 $/barrel at the time of writing (May 2008). Whereas this fossil resource will
certainly not become exhausted from one day to another, it is clear that its price will
follow a long-term upward trend. Its scarcity and high price will not only afflict the
chemical industries and energy sectors drastically all around the world, but it will
impact on society as a whole (Soetaert and Vandamme 2006).
E.J. Vandamme (B)
Laboratory of Industrial Microbiology and Biocatalysis, Department Biochemical and Microbial
Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000
GENT, Belgium
e-mail: Erick.Vandamme@UGent.be
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 1,
C Springer Science+Business Media B.V. 2009
3
4
E.J. Vandamme
Consequently, concerns have arisen about our future energy and chemicals supply. In the first place, this has caused an ongoing search for renewable energy
sources that will in principle never run out, such as hydraulic energy, solar energy,
wind energy, tidal energy, geothermal energy and also energy from renewable raw
materials such as biomass. Biomass can be defined as “all organic material of vegetal
or animal origin, which is produced in natural or managed ecosystems (agriculture,
aquaculture, forestry), all or not industrially transformed”. Bioenergy, the renewable
energy released from biomass, is indeed expected to contribute significantly in the
mid to long term. According to the International Energy Agency (IEA), bioenergy
offers the potential to meet 50% of our world energy needs in the 21st century. The
same hold true for the synthesis of fine and bulk chemicals, materials and polymers,
now also mainly based on fossil resources, petroleum, gas and coal. The chemical
industry will be confronted with the switch to utilize biomass sooner than anticipated.
In contrast to these fossil resources, bulk agricultural raw materials such as
wheat, rice or corn have till a few years ago been continuously low (and even
declining) in price because of increasing agricultural yields, a tendency that has
recently drastically changed, with the competition between biomass for food use
versus biomass for chemicals or biofuels use, becoming a societal issue. However,
climate changes, droughts, high oil prices and the switch to non-vegetarian diets in
fast developing economies such as China are actually the main underlying causes of
the increasing food prices. New developments such as plant genetic engineering –
specifically of industrial or energy crops (Van Beilen 2008) – and the production of
bioenergy and chemicals from agricultural waste and agro-industrial residues can
relieve these trends (Morris 2006, Zhang 2008). Agricultural crops such as corn,
wheat, rice and other cereals, sugar cane and beet, potato, tapioca, etc. are now
already processed in the starch and sugar refineries into relatively pure carbohydrate
feedstocks (starch, sugars,. . .), primary substrates for the food industries, but also
for most industrial fermentation processes and for some chemical processes (Dahod
1999, Kamm and Kamm 2004). Especially fermentation processes can convert those
agro-feedstocks into a wide variety of valuable chemical products, including biofuels such as bioethanol, and organic solvents such as butanol (Demain 2000, 2007,
Kunz 2008, Soetaert and Vandamme 2005, 2008, Wall et al. 2008).
Oilseeds such as soybeans, rapeseed (canola) and oilpalm seeds (but also waste
vegetal oils and animal fats) are equally processed into oils that are subsequently
converted into food ingredients and oleo-chemicals, but recently increasingly into
biodiesel (Canakci and Sanli 2008, Vasudevan and Briggs 2008).
While these technologies are rather mature, agro-industrial residues or waste
streams such as straw, bran, beet pulp, corn cobs, corn stover, oil cakes, waste
wood,. . .all rich in lignocellulosic materials, are now either poorly valorized or left
to decay on the land. (Sarath et al. 2008, Zhang 2008). These residues are now
already efficiently converted into biogas and used for heat, steam or electricity generation. These waste materials attract now increasingly attention as an abundantly
available and cheap renewable feedstock for chemicals, materials and biofuels production. Improved physical, chemical and biotechnological treatments must now
quickly be developed to upgrade and valorize these agro-industrial side streams.
1 Agro-Industrial Residue Utilization for Industrial Biotechnology Products
5
Estimates from the USA Department of Energy have shown that up to 500 million
tonnes of such raw materials can be made available into the USA each year, at prices
ranging between 20 – 50 $/ton.
This volume will focus on the biotechnological potential of using and upgrading
renewable resource-“leftovers”, especially the agro-industrial processing residues
(now left unused when biomass is processed largely into food, but also into chemicals or fiber/materials). Emphasis will be put on fermentation and biocatalysis
principles and processes as very suitable technologies for upgrading these agroindustrial residues in a sustainable way.
1.2 Fermentation and Biocatalysis
It is only now being fully realized by the chemical industry that microorganisms
(bacteria, yeast and fungi, micro-algae) are an inexhaustible source of a wide range
of useful chemical compounds: indeed, an ever increasing number of fine and
bulk chemicals, solvents, food additives, enzymes, agrochemicals and biopharmaceuticals is now being produced based on microbial biotechnology via industrial
fermentation or biocatalysis processes (Demain 2007, Vandamme 2007). Often,
there is no alternative route for their synthesis but fermentation. Also bioconversion reactions, based on the use of (immobilised) microbial biocatalysts (cells or
enzymes), yield useful interesting regio- and enantioselective molecules under mild
reaction conditions, often starting from racemic precursors (Vandamme et al. 2005,
2006). Furthermore, all these microbial processes have a positive environmental
impact (Table 1.1). These microbial products generally display desired chirality,
are biodegradable and practically all are produced, starting from renewable (agro)substrates, till now mainly starch and sugars. Indeed, these nutrient substrates, which
are the “workhorse” ingredients in industrial fermentation processes worldwide, are
mainly derived from agricultural crops, being processed in the established sugar and
starch refineries. Agricultural practice as well as this industrial processing leads to
agro-industrial residues, which should be considered now also as nutrient substrates,
rather than as a waste!
Table 1.1 Sustainability-related properties of fermentation and bioconversion derived chemicals
- Produced from renewable agrosubstrates and agro-industrial residues
- Mild reaction conditions→ “green chemistry”
- Biodegradable
- Desired chirality
1.3 Currently Used Renewable Agrosubstrates as Industrial
Fermentation Substrates
Worldwide, the feedstock for fermentation processes is provided directly or indirectly by agriculture: indeed cereal grains, plant tubers, plant oils, crop residues
and agro-industrial products, side or waste streams are main sources of microbial
6
E.J. Vandamme
Table 1.2 Currently used typical carbon sources in industrial fermentation processes
Carbohydrates:
Corn flour
Starch (from various plant sources)
Dextrins
Glucose syrups
Dextrose
Maltose
Whey
Lactose
Oils and Alcohols:
Soybean oil, methyloleate
Corn oil, cottonseed oil, peanut oil,. . .
Palm oil, . . .
Lardoil, fish oil
Cane molasses
Beet molasses
Sucrose
Sulfite waste liquor
Wood hydrolysate
Organic acids
Agro-industrial waste
Glycerol
Polyols
Hydrocarbons
Methanol
Ethanol
nutrients. With respect to the carbon and nitrogen source, most are plant derived,
but certain microbial nutrients are of animal origin (i.e. peptones, lactose, whey,
. . .) or are derived from yeast (Dahod 1999) (Table 1.2). There is a general trend to
replace these animal derived nutrients for plant derived ones, due to the threat and
transfer of prion diseases.
1.3.1 Carbohydrates as Carbon Substrate
Although carbohydrates in general serve many other important functions, especially
bulk carbohydrates serve as a nutrient source of carbon for the large scale cultivation of microorganisms (Table 1.3). Cheap carbohydrates such as beet and cane
molasses, sucrose, starch or its hydrolysates and glucose syrups are almost universally used as renewable carbon sources in large scale fermentation processes. The
worldwide total usage of carbohydrate-nature feedstock for industrial fermentation
processes has been estimated at 4.107 tons per year. Molasses are produced both
from cane or beet; the product is actually the mother liquor separated from the crystallized sucrose. The total fermentable sugar is in the range of 50–55% by weight
and it is used extensively (often as a mix) in the fermentation of bulk products such
as yeast, ethanol, monosodium glutamate, citric acid, industrial enzymes, and many
others. It is also a source of nitrogen, minerals, vitamins and growth factors. Its
varying composition is often a drawback, such that standardisation, pre-treatment
and addition of further nutrients are needed, depending on the fermentation process
envisaged.
Starch generally cannot be used in its native form as far as most fermentation
applications are concerned, since it undergoes gelatinisation during sterilisation of
the fermentation broth, resulting in high viscosity. Liquefaction with an ␣-amylase is
needed to decrease this viscosity. Such liquefied starch can be used as carbon source,
if the microorganisms involved produce the needed glucoamylases i.e. many bacilli
1 Agro-Industrial Residue Utilization for Industrial Biotechnology Products
7
Table 1.3 Important functions of carbohydrates
Common:
– Diet of living organisms, including microorganisms
– Energy source
– Storage compounds
– Biological “construction” material
– Substrate for chemical derivatisation
Special:
– “Messenger” molecules: receptors, recognition sites,
lectin interactions, immunostimulants, . . .
– Unusual sugars
– Chiral intermediates
– Biopolymers, bioplastics
Bulk:
Sucrose: > 100 × 106 tons/year
Glucose: > 10 × 106 tons/year
Carbohydrate fermentation feedstock: 4 × 107 tons/year
and fungi. Maltodextrins result from enzymatic or acid hydrolysis of starch and can
be used as such i.e. for the production of antibiotics (to avoid glucose catabolite
repression) i.e. penicillin, cephalosporin, streptomycin,
Glucose syrups are obtained by the action of amylases on starch liquefacts, a process called saccharification. These syrups (85–90% glucose), also known as starch
hydrolysates, are most frequently used in fermentation applications: i.e. for production of citric acid, gluconic acid, itaconic acid, L-amino acids (monosodiumglutamate, L-lysine, L-threonine,. . .), xanthan, curdlan, scleroglucan, erythritol, several
antibiotics, . . .. For the production of lactic acid and several other chemicals, pure
glucose (dextrose) is often preferred to facilitate the product recovery.
Maltose syrups, obtained by -amylase action on starch liquefacts, are suitable
in fermentations, where a glucose repression effect is active, as is the case in several
antibiotic fermentations.
Sulfite waste liquor, a side product of the paperpulp manufacturing process, is
rich in pentose-sugars and can be utilized by Candida yeasts and several other microorganisms as a carbon source.
Currently, well defined – rather pure – carbon sources are preferred in industrial
fermentations, due to constraints imposed by the microorganisms involved, but also
with a simple downstream processing of the end product in mind.
1.3.2 Plant Oils as Carbon Substrate
Another interesting substrate for fermentation processes are the plant lipids and
oils (Table 1.2), commonly used in fermentations of bulk antibiotics such as the
-lactam group (penicillins and cephalosporins), the tetracyclines, the macrolides
and the antifungal polyenes.
8
E.J. Vandamme
Although carbohydrate substrates are relatively easily handled, as compared to
plant oils, molasses and starch sources may need costly pre-treatment or hydrolysis. However oils contain about 2,5 times the energy content of glucose per weight
basis: 8880 kcal/kg oil versus 3722 kcal/kg glucose. On a volume basis, oils display
another advantage: it takes 1.24 litres of soybean oil to provide 10 kcal of energy
into a fermentor, while it takes about 5 litres of sugar (50% ww) solution to reach
that value (Stowell et al. 1987). Oils also display anti-foam properties and can act
as a precursor in certain biosurfactant and antibiotic fermentations i.e. the polyene
antifungal. On the other hand, utilization of oils necessitates handling two-phase
system fermentations, demands a higher oxygen input and relies on microbial strains
displaying lipase activity.
1.3.3 Nitrogen Sources, Used in Industrial
Fermentation Processes
Crude pertinacious plant, animal and yeast derived products are commonly used
as complex nitrogen sources in fermentation processes: in addition to nitrogen and
carbon, they also supply vitamins, growth factors and minerals for microbial growth.
Some examples are given in Table 1.4.
Yeast derived products are generally produced from baker’s and brewer’s yeast,
grown themselves on molasses, malt extract or occasionally on other agro-waste
substrates. Yeast extracts as well as yeast autolysates and hydrolysates are in use;
all of them should be tested as to their suitability for a given microbial strain, used
in a particular fermentation process.
Peptones are obtained by partial enzymatic hydrolysis of proteins of animal,
dairy or plant origin (meat, gelatin, casein, whey protein, soy protein,. . .). The
recent emergence of prion diseases among breeded animals has created a greater
demand for protein hydrolysates derived from other sources such plants, fish and
other marine sources.
Corn steep liquor is a fermented by-product of the cornwet-milling process; it is
rich in minerals as well as in amino acids, vitamins and growth factors and is in use
Table 1.4 Typical nitrogen sources, used in fermentation processes
Plant derived
Yeast derived
Corn steep liquor (CSL)
Corn gluten meal
Cottonseed flour
Peanut meal, linseed meal, rice meal, . . .
wheat flour,. . ..
Soybean meal
Yeast extract
Yeast autolysate
Yeast hydrolysate
Distillers dried solubles
Others
−
NH+
4 , NO3 , N2
Urea
Animal Derived
Peptones (meat, fish,. . .)
Lard water
Milk proteins (casein,. . .)
1 Agro-Industrial Residue Utilization for Industrial Biotechnology Products
9
as a nutrient source in many industrial fermentation processes for i.e. penicillin G,
amino acids, enzymes, biopesticides.
Again, all these crude nitrogen sources are directly or indirectly derived from
agricultural products or their industrial processing.
1.3.4 Nutrient Selection Criteria
The ultimate choice of nutrient source type for a given fermentation process is a
complex decision, based on imperatives given by the microbial strain involved, or
the nature of the end product (Table 1.5) and on technical and economic considerations (Table 1.6). For a single antibiotic process, as many as ten quite different
carbon substrates have been used for commercial production, depending on prevailing economics and on geographical location of the production plant.
Table 1.5 Selection of starch, maltose or glucose based feedstock for fermentation processes
Fermentation product
Starch maltose glucose
Reason of preferred use (+)
Polyols: Erythritol
+
Organic acids: Gluconic:
Itaconic
+
Higher yield and reduced purification
steps, as compared to sucrose or
molasses
Molecular structure
Amino acids: Lysine
Polysaccharides:
Xanthan Cyclodextrins
Enzymes:
Carbohydrases:
Proteases
Antibiotics:
Macrolides:
Tetracyclines:
Penicillin G:
Vitamins: B12
+
+
+
+
+
+
+(fedbatch)
+
+(fedbatch)
+
+
+
+(fedbatch)
+
Molecular structure
Yield and reduced purification steps
Molecular structure
Molecular structure
Catabolite repression
Purity of product
Molecular structure
Catabolite repression
Purity of product
Table 1.6 Economic and technical considerations in the selection of fermentation nutrient sources
Availability
Cost per unit of nutrient
Transportation cost
Price stability
Pre-treatment costs
Stabilization costs
Storage costs
Safety factors
Consistency of nutritional quality
Flexibility in application
Rheological properties
Surface tension factors
Product recovery impact
Process yield
Product concentration and type
Overall productivity
10
E.J. Vandamme
Some important factors in comparing the benefits and/or disadvantages of using
crude or refined carbohydrates or oils as carbon source in industrial fermentations
have been compiled by Stowell et al. (1987). The key point here is that microorganisms can convert these abundantly available and renewable nutrient sources into
a vast range of very complex biochemical’s with often unsuspected application potential (Demain 2000). Submerged fermentation has been the mainstay industrial
biotechnology production process in use, but as increasingly crude (solid) agroindustrial residues will become available, solid state fermentation processes will
experience a remarkable revival in the near future (Robinson et al. 2001).
1.4 Towards Agro-Industrial Residue Utilization
Technology in Industrial Biotechnology
When switching to agro-industrial residues or even agro-waste streams, the bottleneck remains to release the fermentable sugars, left in the lignocellulosic matrix, the
main component of these residues (Zhang 2008, Sarath et al. 2008, Vasudevan and
Briggs 2008, Canakci and Sanli 2008).
Special pre-treatments of these agro-industrial side streams is a prerequisite:
mechanical (thermo) physical, chemical and enzymatic pre-treatments will be
primordial in most cases, before microbial fermentation technology or enzymatic
upgrading (biocatalysis) can start. An exception here is the use of solid state fermentation technology, where crude lignocellulosics are directly provided as a substrate for microbial productions (Robinson et al. 2001). The switch to agro-industrial
residues will also put even more emphasis on pre-treatment (upstream) – and on
downstream-processing costs in the overall economics of such “second generation”
fermentation processes!
These physical, (thermo) chemical, mechanical and enzymatic pre-treatments are
covered by experts in detail in the first chapters of this volume, as well as the principles of solid state versus submerged fermentation.
Subsequently, the potential of a wide range of agro-industrial residues to serve as
nutrient source for industrial biotechnology processes is covered. Also the production potential of a wide range of fine and bulk chemicals, fuels and materials based
on these agro-industrial residues is discussed. If these processes materialize in the
near future, it will relief drastically current societal tension whether to use biomass
and crops for food or for platform chemicals and biofuels (Morris 2006).
References
Canakci M and Sanli H (2008) Biodiesel production from various feed stocks and their effects on
the fuel properties. J Ind Microbiol Biotechnol 35: 431–441
Dahod SK (1999) Raw materials selection and medium development for industrial fermentation
processes, pp. 213–220 In “Manual of Industrial Microbiology and Biotechnology” (2nd ed);
(Demain, A.L. and Davies, J.E., eds.) ASM Press, Washington DC
1 Agro-Industrial Residue Utilization for Industrial Biotechnology Products
11
Demain AL (2000) Small bugs, big business: the economic power of the microbe. Biotechnol Adv
18: 499–514
Demain AL (2007) The business of biotechnology. Ind Biotechnol 3: 269–283
Kamm B and Kamm M (2004) Principles of biorefineries. Appl Microbiol Biotechnol 64: 137–145
Kunz M (2008) Bio-ethanol: Experiences from running plants, optimization and prospects. Biocat
Biotransf 26: 128–132
Morris D (2006) The next economy: From dead to living carbon. J Sci Food Agric 86: 1743–1746
Robinson T, Singh D, and Nigam P (2001) Solid state fermentation: A promising microbial technology for secondary metabolite production. Appl Microbiol Biotechnol 55: 284–289
Sarath G, Mitchel RB, Satler SE, Funnell D, Pedersen JF, Graybosch RA, and Vogel KP (2008)
Opportunities and roadblocks in utilizing orages and small grains for liquid fuels. J Ind Microbiol Biotechnol 35: 343–354
Soetaert W and Vandamme EJ (2005) Biofuel production from agricultural crops. In: Biofuels
for fuel cells: Renewable energy from biomass fermentation, pp. 37–50 In “Series Integrated
Environmental Technology” (Lens, P., Westerman, P., Haberbauer, M., and Moreno, A., eds.)
IWA Publ., UK.
Soetaert W and Vandamme EJ (2006) The impact of industrial biotechnology. Biotechnol J 1(7–8):
756–769
Soetaert W and Vandamme EJ (2009) Biofuels, 242 pp. In “Renewable Resources series” (Stevens,
C., Series ed.) J Wiley & Sons Ltd. ISBN 978-0-470-02674-8
Stowell JD, Beardsmore AS, Keevil CW, and Woodward JR (1987) Carbon Substrates in Biotechnology IRL-Press, Oxford-Washington DC
Van Beilen JB (2008) Transgenic plant factories for the production of biopolymers and platform
chemicals. Biofuels Bioprod Bioref 2: 215–228
Vandamme EJ (2007) Microbial gems: Microorganisms without frontiers. SIM-News 57(3): 81–91
Vandamme EJ, Cerdobbel A, and Soetaert W (2005) Biocatalysis on the rise: Part 1 Principles.
Chem Today 23(6): 47–51
Vandamme EJ, Cerdobbel A, and Soetaer W (2006) Biocatalysis on the rise: Part 2 Applications.
Chem Today 24(1): 57–61
Vasudevan PT and Briggs M (2008) Biodiesel production: Current state of art and challenges. J Ind
Biotechnol 35: 421–430
Wall JD, Harwood CS, and Demain AL (2008) “Bioenergy”. ASM-Press, Washington DC
Zhang YHP (2008) Reviving the carbohydrate economy via multi-product lignocellulose biorefineries. J Ind Microbiol Biotechnol 35: 367–375
Chapter 2
Pre-treatment of Agro-Industrial Residues
Poonam Singh nee’ Nigam, Nutan Gupta and Ashish Anthwal
Contents
2.1 Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1
Types of Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Composition of Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 Annual Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4 Uses of Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5 Pre-treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6 Physical Pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.1
Steam Explosion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.2
Hydrothermal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.3
Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7 Chemical Pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.1
Hydrogen Peroxide (H2 O2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.2
Organosolvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.3
Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.4
Peroxyformic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8 Biological and Enzymatic Pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8.1
White-Rot Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9 Combined Pre-treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9.1
Gamma Irradiation and Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9.2
Sodium Hydroxide and Solid State Fermentation . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
14
15
16
17
19
20
20
21
21
21
22
22
23
23
23
26
28
28
29
29
Abstract Problem of management of agro-industrial residues complicate the farming economies. Agro-industrial residues are the most abundant and renewable resources on earth. Accumulation of this biomass in large quantities every year results
not only in the deterioration of the environment, but also in the loss of potentially
valuable material which can be processed to yield a number of valuable added products, such as food, fuel, feed and a variety of chemicals. The agro-industrial residues
P. Singh nee’ Nigam (B)
Faculty of Life and Health Sciences, School of Biomedical Sciences, University of Ulster,
Coleraine, BT521SA, Northern Ireland, UK
e-mail: P.Singh@ulster.ac.uk
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 2,
C Springer Science+Business Media B.V. 2009
13
14
P. Singh nee’ Nigam et al.
have alternative uses or markets. Pre-treatment is an important tool for breakdown of
the structure of these residues mainly formed of cellulose, hemicellulose and lignin.
Cellulose is present in large quantities in agro-industrial residues. As hemicellulose
and cellulose are present in the cell wall they undergo lignification hence there is
an increasing need to have an effective and economic method to separate cellulose
and hemicellulose from cell wall. Various pre-treatment methods such as physical,
chemical, biological (enzymatic) and combined are available. Physical and chemical treatments breakdown the materials present in the agro-industrial residues. As
glucose is readily used by the microorganisms and is present in cellulose, biological pre-treatment by microrganisms is also a good method. Enzymes like phytase,
laccase, LiP, MnP are produced by these microrganisms and help in delignification,
bleaching, and manufacture of animal feed etc.
Keywords Agro-industrial · Pre-treatment · Physical · Enzymatic · Chemical ·
Microorganisms
2.1 Agro-Industrial Residues
Agro-industrial residues are directly burnt as fuel in developing world that includes
crop residues, forest litter, grass and animal garbage. Crop residues are more widely
burnt than animal waste and forest litter.
Agro-industrial residues are derived from the processing of a particular crop or
animal product usually by an agricultural firm. Included in this category are materials like molasses, bagasse, oilseed cakes and maize milling by-products and
brewer’s wastes. Crop residues encompass all agricultural wastes such as straw,
stem, stalk, leaves, husk, shell, peel, lint, seed/stones, pulp, stubble, etc. which come
from cereals (rice, wheat, maize or corn, sorghum, barley, millet), cotton, groundnut,
jute, legumes (tomato, bean, soya) coffee, cacao, olive, tea, fruits (banana, mango,
coco, cashew) and palm oil.
2.1.1 Types of Agro-Industrial Residues
Agro-industrial residues are of a wide variety of types, and the most appropriate
energy conversion technologies and handling protocols vary from type to type. The
most significant division is between those residues that are predominantly dry (such
as straw) and those that are wet (such as animal slurry).
2.1.1.1 Dry Residues
These include those parts of arable crops not to be used for the primary purpose of
producing food, feed or fibre.
2 Pre-treatment of Agro-Industrial Residues
15
a. Field and Seed Crop
Field and seed crop residues are the materials remaining above the ground after
harvesting, including straw or stubble from barley, beans, oats, rice, rye, and wheat,
stalks, or stovers from corn, cotton, sorghum, soybeans, and alfalfa.
b. Fruit and Nut crop
Fruit and nut crop residues include orchard prunings and brushes. The types of
fruit and nut crops include almonds, apples, apricots, avocados, cherries, dates, figs,
grapefruit, grapes, lemons, limes, olives, oranges, peaches, pears, plums, prunes,
and walnuts.
c. Vegetable Crop
Vegetable crop residues consist mostly of vines and leaves that remain on the ground
after harvesting. The types of vegetable crops include such plants as artichokes,
asparagus, cucumbers, lettuce, melon, potatoes, squash, and tomatoes.
d. Nursery Crop
Nursery crop residues include the prunings and trimmings taken from the plants during their growth and in the preparation for market. There are more than 30 different
species of nursery crops (e.g. flowers and indoor plants, etc.) that are grown.
2.1.1.2 Wet Residues
These are residues and wastes that have high water content as collected. These include:
a. Animal Slurry
b. Farmyard manure
c. Grass silage
Silage is forage biomass harvested and fermented for use as winter fodder for cattle
and sheep. Grass silage is harvested in the summer and stored anaerobically in a
silage clamp under plastic sheeting.
2.2 Composition of Residues
Agro-industrial residues consist of lignocellulose that is compact, partly crystalline
structure consisting of linear and crystalline polysaccharides cellulose, branched
non cellulosic and non-crystalline heteropolysaccharides (hemicelluloses), and
branched (non crystalline) lignin (Glasser et al. 2000).
Cellulose is made up of a linear polymer chain, which in turn consists of a series
of hydroglucose units in glucan chains (Fig. 2.1). The hydroglucose units are held
together by -1-4 glycosidic linkages, producing a crystalline structure that can be
16
P. Singh nee’ Nigam et al.
OH
OH
O
OH
O
OH
OH
OH
O
O
O
OH
O
OH
OH
O
OH
O
OH
OH
Fig. 2.1 Structure of cellulose
broken down more readily to monomeric sugars. Another major component of the
lignocellulose structure is hemicelloluse, which is made up of various polysaccharides, namely, xylose, galactose, mannose and arabinose. The function of hemicellulose has been proposed as a bonding agent between lignin and cellulose. Mannose
has been used as a fermentable substrate since many years, with more specific yeast
being able to utilize arabinose and xylose. Hemicellulose is composed of linear
and branched heteropolymers of L-arabinose, D-galactose, D-glucose, D-mannose,
and D-xylose. Methyl or acetyl groups are attached to the carbon chain to various
degrees. Hemicellulose and cellulose, constitute 13–39% and 36–61% of the total
dry matter, respectively.
Lignin found in nature is made of three monomers which are biosynthesized in
plants through shikimic acid pathway. It is made by an oxidative coupling of three
major C6 –C3 phenypropanoid units, namely sinapyl alcohol, coniferyl alcohol and
p-coumaryl alcohol. These are arranged in a random, irregular three dimensional
network that provide strength and structure and is consequently very resistant to
enzymatic degradation (Fig. 2.2).
CH2OH
CH2OH
CH2OH
OMe = OCH3
methoxyl group
OMe
OMe MeO
OH
OH
OH
p-coumaryl
alcohol
coniferyl
alcohol
sinapyl
alcohol
Fig. 2.2 Monomers of lignin
p-Coumaryl alcohol is a minor component of grass and forage type lignins.
Coniferyl alcohol is the predominant lignin monomer found in softwoods. Both
coniferyl and sinapyl alcohols are the building blocks of hardwood lignin.
2.3 Annual Yield
Agro-industrial residues are an ideal energy source if the two components can be
successfully separated or treated. Over 300 million tons of lignocellulose are produced annually worldwide. In UK there are nearly 2 million ha of wheat and 1 million ha of barley. Over half a million ha of oilseed rape which is generally ploughed
2 Pre-treatment of Agro-Industrial Residues
17
back, partly as it is very friable and does not lend itself conveniently to collection.
Smaller areas of oats (100,000 ha), rye (9,000 ha) and triticale (13,000 ha), all of
which can yield straw. In the UK, as a result of insufficient summer warmth to fully
ripen the grain, most maize production (around 100,000 ha) is grown as a forage
crop and used for high quality silage, with only about 2,500 ha for grain, in the far
south of England.
This renewable biomass has the potential to be used for the production of fuels,
chemicals, animal feed etc. Sometimes these agro-industrial residues are seen as
waste and pose disposal problems for the associated industries. This can be solved
through its utilization, turning a valueless waste into a valuable substrate for fermentation processes. The main components of agro-industrial residues are shown in
Table 2.1.
Table 2.1 Main components of agro-industrial residues
Agro-industrial
residues
Lignin
(wt %)
Cellulose
(wt %)
Hemicellulose
(wt %)
References
Corn cobs
Sugarcane
baggase
Wheat straw
Rice straw
Corn stalks
Barley straw
Rye straw
Oat straw
Flax
Soya stalks
Sunflower
stalks
Vine shoots
Cotton stalks
Sunflower
seed hulls
Thistle
6.1±15.9
10±20
33.7±41.2
40±41.3
31.9±36.6
27±37.5
Ropars et al. 1992
Schaffeld 1994a
8.9±17.3
9.9±24
7±18.4
13.8±14.5
19.0
17.5
22.3
19.8
13.44
32.9±50
36.2±47
35±39.6
33.8±37.5
37.6
39.4
34.9
34.5
42.10
24±35.5
19±24.5
16.8±35
21.9±24.7
30.5
27.1
23.6
24.8
29.66
Bjerre et al. 1996.
Patel and Bhatt 1992
Barrier et al. 1985.
Fan et al. 1987
Fan et al. 1987
Fan et al. 1987
Fan et al. 1987
Fan et al. 1987
Jiménez et al. (1990)
20.27
21.45
29.40
41.14
58.48
24.10
26.00
14.38
28.60
Jiménez et al. (2007)
Jiménez et al. (2007)
Dekker and Wallis 1983
22.1
31.1
12.2
JimeÂnez and Loâpez 1993
2.4 Uses of Agro-Industrial Residues
Agro-industrial residues can be used in many ways because they are cheap, abundant
and their use will provide us with environmental and economic benefits:
a. Barley straw is used for animal bedding and feed
b. In UK around 40% of wheat straw is chopped and returned to the soil, 30% used
on the farm (for animal bedding and feed), and 30% is sold.
c. Chopped straw can reduce phosphate and potassium needed for the following
crop, and can help conserve soil moisture and structure.
18
P. Singh nee’ Nigam et al.
d. The ash from burning or gasifying straw can be used to return minerals to the soil
however cannot contribute organic matter or help soil structure.
e. Corn Stover is used as biomass which is low carbon sustainable fuel that can
deliver a significant reduction in net carbon emissions when compared with fossil
fuels.
f. Rice straw can be used as pulp for paper becoming an ideal solution for the
California and Oregon rice burning conundrum.
g. Agro-industrial residues produce ethanol, bioethanol a product of high potential
value containing minor quantities of soluble sugars, pectin, proteins, minerals
and vitamins. Bioethanol produced from renewable biomass has received considerable attention in current years. Using ethanol as a gasoline fuel additive as
well as transportation fuel helps to alleviate global warming and environmental
pollution (Fig. 2.3).
h. They also have potential to produce biogas under anaerobic fermentation
conditions.
i. For soil nutrient recycling and improvement purposes and may therefore be displacing significant quantities of synthetic fertilizers or other products.
j. In USA and Canada, the straws of wheat, barley, oats and rye, and the husks of
rice have been utilised in mixture with wood fibers in the production of pulp,
particleboards and fibreboards (Hesch 1978, Loken et al. 1991, Knowles 1992).
Fig. 2.3 Ethanol production
from ligocellulosic material
i.e agroindustrial residues.
Adapted from Olsson and
Hagerdal (1996)
2 Pre-treatment of Agro-Industrial Residues
19
k. In Asia, husks of rice have been used to produce cement-boards (Govindarao 1980).
l. China and Japan also have made attempts to utilize Indian cane fibers in combination with wood fibers and foamy plastics to produce various kinds of woodboards (Wang and Joe 1983)
m. Production of charcoal and briquettes (Hulscher et al. 1992, TDRI 1983).
2.5 Pre-treatments
As glucose is readily fermented by most microorganisms, yielding a variety of
products, it is very much in demand by the fermentation industries. Glucose as
cellulose is present in large quantities in agro-industrial residues. Because hemicellulose and cellulose present in the cell wall undergo lignification, an effective
and economic method must be used to separate cellulose and hemicellulose from
cell wall. To make monomeric sugar utilization from these residues a viable option, various physical, chemical and biological pre-treatments have been explored
Table 2.2.
Table 2.2 Pre-treatment of Agro-industrial residues
Pre-treatment
Examples
Effect of Pre-treatment
References
Physical
Milling
Fine, highly decrystalized
structure
Increased pore
size/hemicellulosehydrolysis
Hemicellulose hydrolysis,
alteration in properties of
cellulose and lignin.
Depolymerization
Li et al. 2007
Steam Explosion,
Steaming treatment
Hydrothermal
Irradiation
Kokta et al. 1992
Sun and
Tomkinson 2002
Aoyama et al. 1995
NaOH, NH3 , H2 02
Peroxyformic acid,
Organosolvents
Peroxymonosulphate
Lignin/ hemicellulose
degradation
Singh et al. 1988
Activates deliginification
Stewart 2000
Biological
White-rot fungi
(Bjerkendra adusta,
Phanerochaete
chysoporium,
Ceriporiopsis
subvermispora)
Specific bacteria
Lignin degradation
Diana et al (2002)
Enzymatic
Lignin Peroxidases (LiP, Selective
MnP, laccase)
lignin/hemicellulose
degradation
Chemical
Aoyama 1996
20
P. Singh nee’ Nigam et al.
2.6 Physical Pre-treatment
Mechanical and thermal methods exist to treat agro-industrial residues, but these
methods tend to require a high energy input which can increase the processing cost
considerably. Product separation for fermentation purposes can also make physical
pre-treatments expensive.
2.6.1 Steam Explosion
Cellulose and hemicellulose in agro-industrial wastes are natural organic resources
which can be harvested for energy production. To get products like methane and alcohol, biological treatments cannot be applied directly because these agroindustrial residues have a covering of lignin in their cell walls hence one efficient
physical method is steam explosion, in which agro-industrial residue is pressurised
with steam for a period of time followed by a rapid decompression, producing an
explosive reaction that acts on lignocellulose structure. This process is carried out
◦
at a high pressure and high temperature (180 to 240 C) and breaks up the lignocellulose structure by blowing apart the three dimensional lignin components, as well
as causing the decomposition of some hemicellulose into uronic and acetic acids,
which catalyzes the depolymerization of hemicelluloses and lignin.
Once the lignin and cellulose components have been separated, efficient isolation
of these components is required, which is a major constraint in this technology.
However it forms a necessary part of delignification or pre-treatment as lignin needs
to be fully removed depending on the subsequent product. For instance, if cellulose
is to be used for high quality paper or chemical production, then the amount of lignin
present in the final product will affect its purity. Separating the major components
(fractionation) can be carried out by solvent extraction.
Pre-treatment of agro-industrial residues such as wheat and rice straw by steam
explosion has been successfully used for ethanol production (Ballerini et al. 1994),
bioconversion of olive oil cake. Similarly other agro-industrial residues such as
sugar cane bagasse, cassava bagasse have been pretreated by steam for producing
multiple other industrial products.
Using steam explosion as a pre-treatment method for agro-industrial residues,
various advantages like allowing more susceptibility to cellulose degrading enzymes. As this method is environmental friendly there are no costs for recycling
as in case of chemical treatment. As compared to mechanical methods like milling
there is low energy input which can further be minimized using high efficiency
equipments.
But if this method is to be used on a large industrial scale then high running cost
and high energy requirement must be kept in view as large amounts of energy are
needed.
Energy efficient and economically viable approaches like optimizing the thickness of heat-insulating material of the explosion apparatus and also the pressure and
holding times should be used.
2 Pre-treatment of Agro-Industrial Residues
21
2.6.2 Hydrothermal Processing
Hydrothermal processing of agro-industrial residues causes a variety of effects
including extractive removal, hemicellulose hydrolysis and alteration of the properties of both cellulose and lignin.
Water treatments provide an interesting alternative for the chemical utilization of
lignocellulose owing to the following reasons:
i. No chemicals different from water are necessary, the whole process being
environment-friendly
ii. Hemicelluloses can be converted into hemicellulosic sugars at good yields with
low byproduct generation (Lamptey et al. 1985), leading to solutions of sugar
oligomers and/or sugars that can be utilized for a variety of practical purposes (Modler 1994, Saska and Ozer 1995, Aoyama et al. 1995, Aoyama 1996,
LoÂpez-Alegret 1996)
iii. In comparison with acid pre-hydrolysis, no problems derived from equipment
corrosion are expected owing to the mild pH of the reaction media
iv. Stages of sludge handling and acid recycling are avoided, resulting in a simplified process structure
v. The physico-chemical alteration provoked by treatments on lignin and cellulose
facilitates the further separation and processing of these fractions
vi. Economic estimates (Schaffeld 1994b, Kubikova et al. 1996) showed advantages for water treatments over alternative technologies.
The studies on the processing of Lignocellulosic materials by water or steam
have been referred to in literature as autohydrolysis (Lora and Wayman 1978,
Conner 1984, Carrasco 1989, Tortosa et al. 1995), hydrothermolysis (Bonn et al.
1983, HoÈrmeyer et al. 1988, Kubikova et al. 1996), aqueous liquefaction or extraction (Heitz et al. 1986, Saska and Ozer 1995), aquasolv (Kubikova et al. 1996), water
prehydrolysis (Conner 1984), hydrothermal pre-treatment or treatment (Overend
and Chornet 1987, Schaffeld 1994b, Kubikova et al. 1996). All these studies are
based on the same kind of reactions and are referred to as “hydrothermal treatments”
in this work.
2.6.3 Irradiation
Irradiation produces delignification, depolymerization and destruction of the crystalline structure of cellulose (Lowton 1952). Pritchard et al. 1962 have reported that
the solubility and digestibility of wheat straw increased by gamma irradiation.
2.7 Chemical Pre-treatment
Chemical treatment is generally used to remove lignin content of agro-industrial
residues. Chemical pre-treatments by alkali or acid hydrolysis, are common in paper
and pulp industries to recover cellulose for paper production. These treatments tend
22
P. Singh nee’ Nigam et al.
to be expensive hence are not used for bioconversion purposes. Caustic welling is
common chemical method that has the effect of increasing the surface area of the
agro-industrial residue due to the swelling and disruption of lignin.
The need for corrosion-resistant apparatus, an effective washing strategy and the
capability for the safe disposal of used chemicals are the disadvantages of chemical
pre-treatments for lignin removal.
2.7.1 Hydrogen Peroxide (H2 O2 )
Hydrogen peroxide is used as a pre-treatment for agro-industrial residues at opera◦
tional temperatures of ≥ 100 C in alkaline solution i.e. hydroperoxide anion reacts
with the lignin present in pulps acting as a nucleophile as well as an oxidant and as at
this high temperature decomposition of H2 O2 takes place certain chelants are added
to suppress the decomposition. In paper and pulp industries it is used for bleaching
and delignification purposes (to improve the brightness of pulp as it reacts with
coloured carbonyl-containing structures in the lignin structure). Decomposition of
H2 O2 in alkaline condition is rapid and as a consequence more reactive radicals such
as hydroxyl radicals (HO) and superoxide anions (O2– ) are produced which are responsible for lignin degradation. However, the H2 O2 treatment process is expensive.
It becomes particularly unstable in the presence of certain transition metals e.g. Mn,
Fe and Cu, at high temperatures, necessitating the addition of chelants to reduce the
rate of decomposition.
◦
Delignification by this process on a large scale can therefore be costly. At 25 C
in alkaline solution of 1% H2 O2 about half of hemicelluloses and lignin content of
wheat straw and corn stover is solubilized yielding a cellulose rich insoluble residue
that can be enzymatically converted to glucose (Gould 1984).
This treatment of agro-industrial residues like wheat straw increases the susceptibility of plant structural carbohydrates to fibre digesting microorganisms present
in the digestive tract of ruminants. Also H2 O2 treatment results in both partial
delignification of the cell wall and at least partial decrystalization of cellulose
microfibrils.
Current research has focussed on reducing energy requirements of this process
and working temperatures has been reduced by over 50%, with 82% and 88% lignin
◦
dissolution occurring at temperatures of 40 and 70 C, respectively in 2% H2 O2 .
2.7.2 Organosolvents
The pre-treatment of lignocellulosic with organosolvents involves the use of an
aqueous solvent such as ethanol, butanol, phenol, etc., in the presence of a catalyst.
This hydrolyzes lignin bonds as well as lignin-carbohydrate bonds, but many of
the carbohydrate bonds in the hemicellulose components are also broken. Lignin
is dissolved as a result of the action of the solvent and cellulose remains in
solid form.
2 Pre-treatment of Agro-Industrial Residues
23
The use of organsolvents for lignin removal is an attractive process because solvents can be recovered and recycled. It also has the advantage of being able to
separate lignin from the solid cellulose with the hemicellulose hydrolysate found in
liquid form.
2.7.3 Ozone
Ozone has restricted use as a pre-treatment for lignocellulosic substrates because
it not only attacks the lignin molecule but also degrades the cellulose component.
Lignin attacks as a scavenger during this pre-treatment because lignin consumes
most of ozone during the degradation of the carbohydrate content of agro-industrial
residues, but this pre-treatment has a positive side that it lowers the amount of ozone
available for cellulose degradation hence providing a substantial hydroxyl radical
reactivity. Cellulose degradation has been attributed partly to a direct reaction of
ozone with the glucosidic linkage and partly to a free radical mediated oxidation of
hydroxyl groups in glucose (Johansson et al. 2000). Hydroxyl radicals are formed
during ozonation and although lignin is attacked more rapidly, the cellulose is also
targeted when the protective layer of lignin has been removed.
Double bonds react readily with ozone hence pulp bleaching is initially fairly
selective, it also has the effect of disrupting the association between carbohydrates
polymers and lignin, yielding a residue that is more susceptible to attack cellulases.
Due to this unspecific nature, ozonation is more widely used as a pre-treatment for
pulping industry.
2.7.4 Peroxyformic Acid
Peroxyformic acid is generated in situ by mixing formic acid with hydrogen perox◦
ide. The agro-industrial material is added to it and cooked at 80 C for three hours.
Formic acid has the ability to act as a solvent for lignin and breaks down hemicellulose chains, hence peroxyformic acid causes oxidative depolymerization of lignin
increasing its solubility. The formation of peroxyformic acid causes the production
of electrophilic HO+ ions, which react with lignin. The next stage involves an increase in the reaction temperature for a period of time and it is in this stage that
most delignification occurs. At these temperatures, the cellulose component may
be detrimentally affected. The third and final stage of the treatment is designed to
degrade any remaining lignin. This pre-treatment is known as the Milox process,
derived from “mileu pure oxidative pulping”.
2.8 Biological and Enzymatic Pre-treatment
As said earlier in the chapter lignin is associated with cellulose and hemicellulose
in the cell wall thus acting as a barrier preventing the availability of carbohydrates
for further transformation processes. Therefore, pre-treatment becomes a necessity
for utilization of agro-industrial residues to obtain a good degree of fermentable
24
P. Singh nee’ Nigam et al.
Table 2.3 Biological treatment of agro-industrial residues by microorganisms
Lignocellulose
substrate
Fungi
Product
References
Production of
bioethanol
Patel et al. 2007
Wood chips
Aspergillus niger
A.awamori
T. reesei
P. chrysosporium,
P. sajor-caju
Bjerkendra adusta
Rice straw
Cyathus stercoreus
Wheat straw
Pleurotus ostreatus
Trametes versicolor
Trametes versicolor
Wheat straw/Rice
straw
Kraft pulp
Lignocellulosic
hydrolysates
Trametes versicolor
Delignification for pulp Dorado et al. 2001
refining
Improving nutrional
Orth et al. 1993
quality
Animal feed
Eichlerova et al. 2000
Zafar et al. 1996
Delignification and
Dumonceaux et al. 2001
bleaching
Peroxidase and laccase Valmaseda et al. 1991
for delignification
prior to ethanol
fermentation
sugars. Pre-treatment is required for alteration in the cellular structure of cellulose
containing agro-industrial residues to make more accessible to the enzymes that
convert the carbohydrate polymers into fermentable sugars (Mosier et al. 2005) and
to cellulase producing microorganisms. White-rot fungi and certain bacteria have
been commonly used for biological pre-treatments of lignocellulosics (Table 2.3).
Although lignin removal through lignin degradation is possible using biological
methods, it is however, unselective as lignin is degraded only to obtain the more
readily metabolized cellulose and/or hemicellulose. Solid state fermentation allows
the enhancement of enzyme productivity through immobilization on agricultural
residue and the reduction in cost for growth substrates of fungus because agricultural
or industrial waste material can be used as cheap substrates.
Ligninolytic peroxidases gained attention by their industrial applications in pulp
and paper industries such as biochemical pulping and decolorization of bleach
plant effluent. The use of natural solid substrates, especially lignocellulosic agroindustrial residues, as growth substrates of fungi was done for laccase production.
LiP and MnP, were used because of anticipated effects on cost reduction, waste
reuse, and enhanced enzyme production. The high oxidative potential of many enzymes have a positive effect on many unusable and unwanted wastes. Enzymes
obtained from agricultural sources are produced by both brown-rot and white-rot
fungi. The studies about the enzyme production in brown rot fungi have been done
on chemically defined liquid medium under conditions to produce that particular
enzyme. But in solid state fermentation and in natural environment all wood rot
fungi grow under different conditions and hence the growth of fungi in solid state
fermentation (fungus immobilized) and in liquid submerged fermentation is different and therefore the enzymes produced are different. The biochemical mechanisms
required for degradation of lignin have been studied on lignolytic systems of
2 Pre-treatment of Agro-Industrial Residues
25
white-rot fungi while the polysaccharide degradation is efficiently done by brownrot fungi and other ascomycetous fungi.
High amounts of laccase was produced by C. gallica UAMH8260 on cereal bran
liquid medium (Pickard et al. 1999b). Growth of Trametes versicolor FPRL-28A
and its laccase production on wood chips, cereal grain, wheat husk, and wheat bran
was good (Ullah et al. 2000). Wheat bran was also successfully used as a solid-state
medium for the laccase production by Pleurotus pulmonarius CCB-19 (De Souza
et al. 2002) and Fomes sclerodermeus BAFC 2752 (Papinutti et al. 2003). Wheat
straw is a better substrate of P. ostreatus than wheat extract for the laccase production (Morais et al. 2001b). Stimulation effect of wheat straw on the laccase production was observed in the cultures of Lentinula edodes 610 (Hatvani and Mecs 2002).
Barley bran is a good substrate for laccase production by T. versicolor CBS100.29
than were grape seed and grape stalks (Lorenzo et al. 2002). Olive mill wastewaterbased medium, containing large amounts of recalcitrant aromatic compounds, was
used for the production of laccase and MnP in submerged and solid-state cultures of
Panus tigrinus (Fenice et al. 2003).
White-rot and brown-rot fungi produce several enzymes when grown on
Eucalyptus grandis using solid state fermentation technique. All fungi produced
hydrolytic activities but brown-rot fungi produced higher levels of cellulose and
xylanase than white rot fungi whereas phenol peroxidases were produced only by
white-rot fungi (Machuca and Ferraz 2001).
Phytase is either absent or present at a very low level in the gastrointestinal
tract of monogastric animals (Selle and Ravindran 2007). Dietary phytate is not
digested in the intestine and consequently accumulates in faecal materials. Phytase
is produced by bacteria, fungi and yeasts. Among them, strains of Aspergillus niger
produce large amounts of extracellular phytase (Chelius and Wodzinski 1994) and
show more acid tolerance than bacteria and yeasts (Kim et al. 1998). In view of its
industrial importance the ultimate objective is to produce this enzyme at cost effective level and establish conditions for its industrial application. Phytase production
under submerged fermentation conditions using pretreated agriculture residues is
useful to remove excess inorganic phosphate which otherwise inhibit phytase production. Phytase production by SSF of agriculture residue using A. niger NCIM
563 which was highly active at pH 5.0 (Mandviwala and Khire 2000) and process
for preparation of acidic phytase using dextrin glucose medium under submerged
fermentation condition (Soni and Khire 2005, 2007).
Maximum increase of 20.3 times in phytase activity was observed in case of
wheat bran as compared to de-oiled rice bran, coconut cake, peanut cake high and
low oil. Phytase production under submerged fermentation conditions by A. niger
NCIM 563, indicates that pre-treatment of agriculture residues with distilled water
was useful There was substantial increase in phytase activity when this excess phosphate was removed by pre-treatment. Similarly there was increase in productivity
and reduction in fermentation time when agriculture residue was used instead of
dextrin in submerged fermentation hence, being more economic as the cost of any
agriculture residue is much cheaper than dextrin (Bhavsar et al. 2008). More details
about the enzymatic treatment is discussed in chapter 22.
26
P. Singh nee’ Nigam et al.
2.8.1 White-Rot Fungi
Due to their ability to degrade lignin as well as polysaccharides found in cellulose
and hemicellulose, white-rot fungi have the potential not only to act as a biological
pre-treatment but also to degrade all the major components of lignocellulose to yield
a valuable product. Although lignin can be degraded by these microorganisms, the
enzymes responsible are produced only when other widely available substrates are
unavailable. The purpose of lignin degradation by white-rot fungi is to allow better
access to the cellulose and hemicellulose components.
Three main enzymes are thought to be involved in ligninolytic biodegradation,
namely lignin peroxidise (LiP), manganese peroxidise (MnP) and laccase (Fig. 2.4).
LiP has the ability to take electron from the lignin molecule to create a cation radical,
which then initiates an oxidative reaction that results in the oxygenation and depolymerising of the lignin. MnP oxidizes Mn (II) to Mn (III), which has the ability to
diffuse into the lignin structure and initiate the oxidation process. Laccase is phenol
oxidase that differs from peroxidises in that it does not require hydrogen peroxide
to directly attack lignocellulose.
White-rot fungi such as Phanerochaete chrysosporium, Trametes versicolor,
Trametes hirsuta and Bjerkandera adusta has the ability to degrade lignin and can be
used as an effective biological pre-treatment (Table 2.4). This is a cheap and effective method of delignification. As fungi grow on these agro-industrial residues, they
utilize the polysaccharides after lignin degradation in order to grow and reproduce.
This, in turn, has the effect of increasing the nutritional value of the agro-industrial
substrates that are genrally low. After fermentation, this may be used as an animal
feed or soil fertilizer. This process is mostly carried out under solid state fermentation conditions.
Recent work has concentrated on trying to make biological treatments more selective through the use of genetically manipulated fungi with cellulose promoting
enzymes inactivated. This allows lignin degradation without affecting the cellulose
component of the complex. Disadvantages of using mutant fungi include their high
dependence on an external carbon source and an increase in hemicellulose degradation. This is true of Sporotrichum pulverulentum mutants.
Some processes have focussed on directly converting lignocellulosic residues
to single-cell protein (SCP). In a method known as the Institut Armand-Frappier
process, a Chaetomium cellulolyticum mutant and Pleurotus sajor-caju as well as
strains of Aspergillus and Penicillium spp., are used. This cocultivation of fungi
has the ability to utilize cellulose and hemicellulose, after lignin degradation, for
SCP production. In this case, there is no need for any other pre-treatment method,
as together these fungi are capable of separating lignocellulose into its individual
components. The cellulose obtained may be also used for paper production or as
SCP for animal or human feed.
Biological pre-treatments require a long time period in comparison to other tried
and tested physical and chemical methods. A period of two to five weeks may be
required for sufficient delignification. The direct application of ligninolytic enzymes
has also been investigated in order to reduce the length of the treatment period, but
2 Pre-treatment of Agro-Industrial Residues
27
Fig. 2.4 A scheme showing lignin biodegradation showing enzymatic reactions. (Source : International Micobiology (2005) 8: 195-204 updated from Gutièrrez and Martı̀nez, 1996)
28
P. Singh nee’ Nigam et al.
Table 2.4 lignin degrading Enzyme production using agricultural residues
Fungus
Enzyme
Agricultural residues
Reference
B. adusta
C. gallica
F. sclerodermeus
L. edodes
MnP
Laccase
MnP, laccase
Laccase
Rice bran
Wheat bran
Wheat bran
Wheat straw
P. ostreatus
P. ostreatus
P. ostreatus,
P. sajor-caju
P. pulmonarius
P. tigrinus
T. hirsute
Laccase
MnP
LiP (MnP),
laccase
Laccase
MnP, laccase
Laccase
Wheat straw
Wood sawdust
Banana waste
Wang et al. (2001)
Pickard et al. (1999b)
Papinutti et al. (2003)
Hatvani and
Mecs (2002)
Morais et al. (2001b)
Giardina et al. (2000)
Reddy et al. (2003)
T. versicolor, Funlia
trogii
Laccase
T. versicolor
T. versicolor
T. versicolor
T. versicolor
Laccase
Laccase
Laccase
Laccase
Wheat bran
Olive mill wastewater
Potato peeling with barley
bran
Molasses wastewater
(vinasse) with cotton
stalk
Barley straw
Grape stalk, grape seed
Wood shaving
Barley bran
T. versicolor
T. versicolor
Laccase
Laccase
Wheat Straw
Wheat bran, wheat husk
De Souza et al. (2002)
Fenice et al. (2003)
Rosales et al. (2002)
Kahraman and
Gurdal (2002)
Couto et al. (2002d)
Lorenzo et al. (2002)
2002d (2002d)
Lorenzo et al. (2002),
Couto et al. (2002d)
Couto et al. (2002d)
Ullah et al. (2000)
the direct use of enzymes for delignification is expensive and suffers from poor
enzymes on the lignocellulose material.
2.9 Combined Pre-treatments
These treatments are a combination of physical, chemical, biological and enzymatic
treatments used in order to get the best of all treatments hence minimizing the disadvantages of individual treatments.
2.9.1 Gamma Irradiation and Sodium Hydroxide
The physical and chemical pre-treatment when used individually breakdown the
material of the agro-industrial residues whereas a combined treatment acts on the
cell wall structure. Agricultural residues when pretreated alone with sodium hydroxide increase the organic matter digestibility from 5 to 54% and gamma irradiation alone increase the organic matter digestibility from 8 to 46% but a combination of the two increases the organic matter digestibility by many folds (p<0.05)
(Al-Masri 1999). As most of the agro-industrial residues contain high concentration
of cell wall constituents therefore the increase in the apparent digestibility, as a
result of alkali or irradiation pre-treatments, could be attributed to the decrease in
2 Pre-treatment of Agro-Industrial Residues
29
cell wall constituents of agro-industrial residues. Individual treatments with NaOH
or irradiation decrease the cellulose and hemicellulose content. Other studies have
shown that alkali treatment alone decreases the lignin and hemicellulose percentage
with swelling in the cellulose content.
Treatment of roughages with alkali at a higher level might upset the rumen fermentation because the high sodium intakes of unreacted alkali prevent the potential
digestibility of the feed from being realized.
Irradiation can produce delignification, depolymerization and destruction of the
crystalline structure of cellulose (Lowton 1952). Combined treatment of irradiation
and sodium hydroxide had better effects in increasing the values of organic matter
digestibility hence having a greater effect of the combined treatment on the cellwall constituents (Al-Masri 1994). Treatment by alkali when given after irradiation
of the agricultural residues, was more effective in reducing the values of cellulose
and hemicellulose, which could be the cause of the degradation of cellulose and
hemicellulose into soluble materials.
NaOH and irradiation on the enzymatic hydrolysis of treated rice straw increased
as the irradiation dose increased (Xin and Kumakura 1993).The irradiation may have
broken the structure of the lignocellulose so that the NaOH solution could enter
easily into the lignocellulose complex, thus the rate of reaction was increased.
2.9.2 Sodium Hydroxide and Solid State Fermentation
In this combined treatment of solid state fermentation when the agro-industrial
residue is pre-treated with sodium hydroxide, factors like pH, steam sterilization and
urea are important in formulating the substrate for the growth of the microorganisms
hence cellulose production which leads to commercial application in the fruit and
food industries.
Abbreviations
LiP:
MnP:
SCP:
SSF:
Lignin peroxidases
Manganese peroxidases
Single cell protein
Solid State Fermentation
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Part II
Production of Industrial Products Using
Agro-Industrial Residues as Substrates
Chapter 3
Production of Organic Acids
from Agro-Industrial Residues
Poonam Singh nee’ Nigam
Contents
3.1
3.2
3.3
Use of Agro-Wastes for Organic Acid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Commercial Importance of Organic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Organic Acid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1 The Production-System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2 The Production-Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3 The Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4 Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.1 Citric Acid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.2 Industrial Applications of Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.3 Production of Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.4 Process-Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.5 Commercial Strains for the Synthesis of Citric Acid . . . . . . . . . . . . . . . . . . . . . .
3.5 Preparation of Production Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.1 Pre-culture for Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.2 Medium and Culture Conditions for SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.3 Bioreactors for Citric Acid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6 Optimisation of Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.1 Influence of Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2 Influence of Micronutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3 Influence of Various Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7 Mechanism of Citric Acid Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.1 Pathways of Citric Acid-Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.2 Biochemistry of Citric Acid-Overproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8 Extraction of Citric Acid from Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9 Productivity of Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.10 Prospects of R & D in Citric Acid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11 Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11.1 Industrial Applications of Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11.2 Lactic Acid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11.3 Parameters Influencing Lactic Acid-Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11.4 Downstream Processing of Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.12 Prospects of R & D in Lactic Acid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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P. Singh nee’ Nigam (B)
Faculty of Life and Health Sciences, School of Biomedical Sciences, University of Ulster,
Coleraine BT52 1SA, Northern Ireland, UK
e-mail: P. Singh@ulster.ac.uk
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 3,
C Springer Science+Business Media B.V. 2009
37
38
P. Singh nee’ Nigam
Keywords Organic acids · Citric acid · Lactic acid · Food-acidulant · Beverage ·
Buffering · Chelation
3.1 Use of Agro-Wastes for Organic Acid Production
Most of the chapters in this book include information on the variety of agricultural wastes, agro-industrial by-products and residues produced globally and the
authors of some of these chapters also have presented data on the annual yield of
these renewable resources. Hence, not to repeat this information again here, this
chapter deals with the utilization of these bio-resources for organic acid production.
Couto (2008) has recently published an article on the exploitation of large scale
biological wastes for the production of value-added products. The agricultural raw
materials have been used for the bioconversion of their appropriate components for
the production of various bio-products and in bio-refining (Koutinas et al. 2007b).
The surplus agricultural materials have been used for the production of important products of industrial value such as lactic acid (Koller et al. 2005; Koutinas
et al. 2007a,b,c; Reddy and Yang 2005; Venus and Richter 2006). Novel approaches
have been tried for the utilisation of waste corn and corn fiber (Johnson 2006;
Kalman et al. 2006; Murthy et al. 2006). Other agricultural residues and wheat straw
have been studied for the production of industrial products (Chang and Ma 2007;
Canilha et al. 2006; Thomsen 2005; Soccol and Vandenberghe 2003). Citric acid
production has been studied using solid substrates in solid state fermentation by
Prado et al. (2004) and Xie and West (2006) used corn distillers’ grain for citric
acid production. Oat cereal (Koutinas et al. 2007a) and wheat bran (John et al. 2007)
could be utilised as substrates for Lactic acid production.
3.2 Commercial Importance of Organic Acids
Organic acids have established their important application not only in the food and
beverage industry, but also in a variety of industries. These acids possess three
main characteristics, which make them suitable in various formulations as one of
the main ingredients. Their much sought after characteristics include their room
temperature solubility, hygroscopic, buffering and chelating nature. Organic acids
are used mainly as food acidulants (Taing and Taing 2007; Wang et al. 2008). The
main organic acids in industrial use are citric, acetic, tartaric, malic, gluconic and
lactic acids. The most utilised organic acid is citric acid (Milson et al. 1985; Moeller
et al. 2007). The compound of citric acid, iron citrate is used in pharmaceutical
industry as a source of iron and citric acid to preserve cosmetic preparations, ointments, stored blood, and tablets. Organic acids are obtained as the end-products or
as the intermediate component of a particular biochemical cycle.
Citric acid is used as a suitable and cheaper replacement of polyphosphates in
the detergent industry. The higher cost of polyphosphates formerly restricted its
use, however, detergents containing polyphosphates have been prohibited in some
countries and therefore their use has been completely replaced by citric acid. Citric
3 Production of Organic Acids from Agro-Industrial Residues
39
Table 3.1 Global production of organic, acids of industrial importance (adapted from Pandey
et al. 2001)
Type of acid
Process of synthesis
Total global production∗
Acetic Acid
Citric Acid
Gluconic Acid
100% by microbial Fermentaion
100% by microbial Fermentation
[i] 67% by Fermentation
[ii] 33% by Chemical-synthesis
[i] 50% by Fermentation
[ii] 50% by Chemical-synthesis
[i] 70% by Chemical-synthesis
[ii] 30% by Fermentation
100% by Chemical-synthesis
100% by fermentation
120,000
840,000
50,000
Lactic Acid
Malic Acid
Propionic Acid
Tartaric Acid
35,000
30,000
50,000
30,000
(∗ measured in metric tonnes)
acid is used in chemical industry as an antifoaming agent, as a softener, and in the
treatment of textiles. Pure metals are first produced as metal citrates in metal industry. Organic acids are synthesised commercially either chemically or biotechnologically. The important data has been presented in Table 3.1 on worldwide production
of industrially important organic acids.
Though organic acids have wide applications in the food industry as additives and
as chemical feed-stocks, but the 75% of food industry usage requires two main acids,
citric and acetic acid. Citric acid has established itself as the most widely produced
organic acid due to its widest range of applications. The biosynthesis of organic
acids has been widely studied (Soccol et al. 2004) using agricultural residues in solid
state fermentation (SSF). Citric acid has been produced through SSF technology
(Shankaranand and Lonsane 1993; Hang et al. 1987; Hang and Woodams 1984,
1986) for many years, while other organic acids such as lactic, fumaric and oxalic
acid have been reported to be produced in SSF only in the last few years.
Fermentation processes play an important role in the production of organic acids.
All acids produced as a result of the tricarboxylic acid cycle can be produced in high
yields in microbiological processes (Kapritchkoff et al. 2006). Therefore, there is
increasing demand for their production. Some of the factors involved in the bulkproduction of organic acids are discussed under Section 3.3.
3.3 Organic Acid Biosynthesis
3.3.1 The Production-System
The agricultural residues and by-products are the ideal substrates as the raw
material. For economical production the substrates used as raw materials and fermentation medium used for the synthesis process should be cheaper and easily available globally and in large quantities. Therefore, specific medium and fermentation
parameters should be simple and easy to optimise. The control of several factors
including, bioreactors, oxygen, temperature, humidity and aeration have been optimised by various researchers (Moeller et al. 2007; Murthy et al. 2006).
40
P. Singh nee’ Nigam
3.3.2 The Production-Strains
For the production of a bio-product such as organic acid, strain selection is very
important. The organism must have relatively stable characteristics and the ability
to grow rapidly and vigorously (Prescott and Dunn 1959). The microorganism of
choice should also be non-pathogenic and suitable for the studies related to the
optimisation of parameters (Wang et al. 2008; Richter and Nottelmann 2004). The
most important economic characteristic to note in the selection of an organism is its
ability to produce high yields of the desired product (Zhang et al. 2007).
3.3.3 The Product
The fermentation end-product should be present in a heterogeneous mixture as a result of the metabolism of cultured microorganism. It should be accepted worldwide
as GRAS, approved by the Joint FAO/WHO Expert committee on Food additives.
At the same time the production of undesirable side-products, such as other acids,
should be efficiently suppressed. An economical, easy to follow and efficient largescale strategy for product-recovery, and purification must be developed.
There is great competition between microbiological and chemical processes for
the production of various organic acids. However, the production of citric acid is the
exception, which is now synthesised 100% by fermentation. Table 3.2 summarises
the production of organic acids in SSF.
3.4 Citric Acid
3.4.1 Citric Acid Biosynthesis
Citric acid was a produced by Mucor and Penicillium sp. as fungal metabolite in
media limited in phosphate. The presence of citric acid was detected as a by-product
of calcium oxalate produced by a culture of Penicillium glaucum. A great number of
problems had to overcome, before an effective fermentation process could be used
commercially (Lockwood and Schweiger 1967). Other investigation showed the isolation of two varieties of fungi belonging to genus Citromyces (namely Penicllium).
Initially for the production of citric acid microorganisms were cultivated in surface
culture. At present over 99% of the world’s output of citric acid is produced using
the process of microbial fermentation. Ikramul et al. (2007) have used Sugar industry by-product molasses for citric acid production. The optimisation of citric acid
production was recently studied by Moeller et al. (2007); and Maria et al. (2007).
Citric acid is a natural constituent and common metabolite of plants and animals. It is the most versatile and widely used organic acid. Citric acid is an important commercial product with a global production reaching 840,000 tons per
year. Currently citric acid is produced by fermentation-technology, using the filamentous fungus Aspergillus niger mainly through surface (solid or liquid) and
3 Production of Organic Acids from Agro-Industrial Residues
41
Table 3.2 Use of agro-industrial residues and by-products for the production of organic acids
(adapted from Pandey et al. (2001))
Agri-industrial waste
Production-strain
Fermentation product
Oat cereal
Molasses
Sweet Potato
Wheat bran
Starch
Pineapple Waste
Pineapple Waste
Sugar Cane Bagasse
Carrot-Processing Waste
Gallo Seeds-Cover Powder
Okara, Soy-Residues
Carob-Pods
Corn-Cobs
Sweet Potato
Cassava
Cassava Bagasse
Myroballan seeds
Sugarcane Press Mud
Kiwifruit peel
Cassava
Cassava
Sweet Sorghum
Sugar Cane Press-Mud
Sugar Cane Press-Mud
Coffee Husk
Sugar Cane Press-Mud
Starch Containing root Kumara
Carrot-Processing Waste
Amberlite (Inert Solid Support)
Polyurethane (Inert Solid Support)
Rhizopus oryzae
Aspergillus niger GCMC-7
Aspergillus niger
Mixed lactobacilli
Immobilised Rhizopus oryzae
Aspergillus Foetidus
Aspergillus niger
Rhizopus oryzae
Aspergillus niger
Aspergillus nige, A. oryzae
Aspergillus niger
Aspergillus niger
Aspergillus niger
Rhizopus Sp.
Aspergillus niger
Aspergillus niger
Rhizopus oryzae
Aspergillus niger
Aspergillus niger NRRL 567
Aspergillus niger
Streptococcus thermophilus
Lactobacillus helveticus
Rhizopus oryzae
Lactobacillus casei
Aspergillus niger
Lactobacillus Paracasei
Aspergillus niger
Rhizopus Sp.
Aspergillus niger
Aspergillus niger
Lactic acid
Citric acid
Citric Acid
Lactic Acid
Lactic acid
Citric acid
Citric Acid
Lactic Acid
Citric Acid
Gallic Acid
Citric Acid
Citric Acid
Citric Acid
Oxalic Acid
Citric Acid
Citric Acid
Gallic Acid
Citric Acid
Citric Acid
Lactic Acid
Furmaric Acid
Lactic Acid
Lactic Acid
Lactic Acid
Citric Acid
Lactic Acid
Citric Acid
Lactic Acid
Citric Acid
Citric Acid
submerged fermentation of starch or sucrose-based media, (Jianlong 2000; Vandenberghe et al. 2000). The food industry utilises about 70% of the total production
of citric acid (Rohr et al. 1983), the pharmaceutical industry consumes 12% and the
rest 18% has market for other applications.
3.4.2 Industrial Applications of Citric Acid
Citric acid has a long list of applications in industrial sectors such as the food, beverage, and pharmaceutical industries. It is mainly used for preservation, antioxidation,
chelation and acidulation. It is used as flavour enhancer, plasticizer and synergistic
as well as sequestering agent. Its rising demand is subsequently causing an increase
in global production.
Citric acid is mainly used in the food industry because of its pleasant acidic taste
and its high solubility in water. It is worldwide accepted as “GRAS” (generally
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Table 3.3 Industrial Applications of citric acid (adapted from Pandey et al. (2001))
Properties and functions of citric acid
Commercial sector
Tartness; complementary fruits and berries flavours;
effective antimicrobial preservative; pH adjuster,
providing uniform acidity.
Tartness; Producer of dark colour in hard sugar-candies;
Acidulant; restricting sucrose inversion
pH adjustment, antioxidant as a metallic-ion chelator,
buffering agent.
Emulsifier in ice creams and processed cheese; acidifying
agent in many cheese products; antioxidant.
Synergist for other antioxidants, as sequestrant.
Soft drinks; canned fruit-juices;
bottled beverages
Lowers pH to inactivate oxidative enzymes. Protects
ascorbic acid by inactivating trace metals.
Sequestrant of metal ions, neutralizant, buffer agent
Frozen fruit
confectionery
Cosmetics
Dairy (Ice-cream & Cheese)
Fats and oils
commercial applications
Provides tartness, pH adjustment.
Fruit-preserves; marmalade;
confiture
Removes metal oxides from surface of ferrous and
nonferrous metals, for preperational and operational
cleaning of iron and copper oxides.
As effervescent in powders and tablets in combination with
bicarbonates. Provides rapid dissolution of active
ingredients. Acidulant in mild astringent formulation.
Anticoagulant.
electroplating, copper plating, metal cleaning, leather
tanning, printing inks, bottle washing compounds, floor
cement, textiles, photographic reagents, concrete, plaster,
refractories and moulds, adhesives, paper, polymers,
tobacco, waste treatment
Purification of Metal oxides
Pharmaceuticals
Industrial; miscellaneous in world
market
recognised as safe), approved by the joint FAO/WHO Expert Committee on Food
Additives. Table 3.3 summarises the main industrial and commercial uses of citric acid.
3.4.3 Production of Citric Acid
Citric acid has always been a subject of interest and therefore of research. Solid state
fermentation has been regarded as an alternative method to produce citric acid from
agro-industrial residues. It is also produced from starch or sucrose-based media using liquid and surface fermentation (Milson et al. 1985; Peller and Perlman 1979).
Bioproduction can be regarded as a combination of fermentation and processing
techniques. The two methods should be considered as a single production unit because of the competition afforded by chemical synthesis.
The choice of the production method is dictated, amongst other things, by the
effect of energy metabolism on the product formation. Production of citric acid
is a good example of production of a metabolite and its downstream processing
method. Citric acid is a tricarboxylic acid and is an intermediate in the Kreb’s cycle.
It was discovered that Aspergillus niger could accumulate citric acid in a media rich
3 Production of Organic Acids from Agro-Industrial Residues
43
in carbohydrate but deficient in phosphate and trace elements like Fe+2 , Zn+2 and
Mn+2 . The regulation of glycolysis is most important for citric acid production. The
fermentation is carried out aerobically in large fermenters and a key requirement for
high citric acid yields is that the medium be iron-deficient. This is because citric
acid is overproduced by the fungus as a chelator to scavenge on.
3.4.4 Process-Technology
The process depends on the type of substrate used for organic acid production,
therefore different workers have used different methods (Pandey et al. 2001; Soccol
et al. 2004). Wheat bran agricultural residue has been used as a fermentation raw
material in the Koji-process (Yamada 1977). Koji process was first developed in
Japan and is used for commercial production that accounts for 20% of all the citric
acid produced in the country annually. The pH of the bran is adjusted to 4.5 before sterilization, and is then steam-sterilised. During steaming, wheat bran gathers
moisture so the final water-saturation becomes 70–80%. The sterilised bran at pH
4.5 is converted into a mash. The bran-mash is then cooled to 30–36◦ C prior to inoculation. The mash is mixed well and inoculated with “Koji”, which is a preparation
of fermented grains covered with fungal growth.
A variety of different cereal grains such as soybean, wheat and rice can be used.
Koji is used to inoculate the wheat bran mash. A special strain of Aspergillus niger
is used for this fermentation process because wheat bran contains a high concentration of undesirable trace elements such as like Fe+2 , Zn2+ and Mn2+ . This special
strain is capable of producing citric acid unaffected in the presence of these ions.
Koji provides as a source of important enzymes such as amylase, protease and lipases, which are used in the hydrolysis process required for cereal grains containing
considerable percentage of carbohydrate-starch. Amylase helps to hydrolyse starch
in the wheat bran to glucose, which is further fermented to citrate by the fungus.
The inoculated wheat bran is then spread into trays at a depth of 3 to 5 cm or is
arranged in long rows on the surface of fermenting units. Solid state cultivation is
carried out for a week at a temperature of 30–36◦ C. The pH in the mash drops to
1.8–2.0. After the fermentation is over, the fermented wheat bran is harvested and
placed in percolators. The fermented mass is extracted with water for citric acid.
The aqueous extract is subjected to modern downstream processing, for purification
and concentration of the organic acid.
3.4.5 Commercial Strains for the Synthesis of Citric Acid
Some fungal strains are capable of producing citric acid as a metabolite in their
primary metabolism. Citric acid is produced by several moulds such as Penicillium luteum, P. citrinum, Aspergillus niger, A. wentii, A. clavatus, Mucor piriformis,
Citromyces pfefferianus, Paecilomyces divaricatum, Trichoderma viride etc. Citric
acid can also be produced by yeasts, such as Yarrowia lipolytica and Candida guilliermondii. However, for commercial production only mutants of Aspergillus niger
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P. Singh nee’ Nigam
should be used. The reason being that compared to Penicillium strains, the aspergilli
are capable of producing higher yield of citric acid per unit time. Moreover, the
production of undesirable side products such as oxalic acid, isocitric acid and gluconic acid, could be easily and efficiently suppressed in the mutants of Aspergillus
niger.
Production strains are optimised based on the carbon sources used. A strain,
which produces citric acid in good yields using one substrate, may not be able to
produce the same product yield with another carbon source. If the raw material
used is starch, the employed fungus should be able to form amylases for the hydrolysis of starch. Using non-amylase producing strains with starch as the starting
substrate, amylase enzymes need to be supplemented in the fermentation medium
(Wang et al. 2008). Since starch forms the largest part of food waste, external enzymes, especially glucoamylase (EC 3.2.1.3) which could hydrolyse starch to produce glucose would be necessary for the bioconversion of food waste into organic
acids (Pandey et al. 2000). Industrial strains that produce commercial citric acid are
not freely available. A few can be obtained from international culture collections.
These are Aspergillus niger NRRL 2270, NRRL 599, ATCC 11414 and ATCC 9142;
and Y. Iipolytica ATCC 20346, ATCC 20390, NRRL Y-7576 AND NRRL Y-1095.
Improvement of strains for citric acid production is focused on obtaining strains
with reduced levels of activity of isocitrate dehydrogenase and aconitase.
3.5 Preparation of Production Medium
3.5.1 Pre-culture for Fermentation
The fungal-preculture used as inoculum for citric acid production is in the form
of a spore-suspension. Spores are produced in glass bottles on solid substrates at
optimum temperatures. The incubation period for the formation of spores is 8–14
days. Besides the total number of spores, the viability of spore crops is critical.
Aspergillus niger spores are produced in Czapeck Dox Broth solidified with agar
in bottles or flasks. Medium is incubated at 28◦ C for minimum of 8 days. Spores
are removed in the form of a suspension using distilled water with 0.1% Tween-80.
This spore-suspension can then be stored at 4◦ C for a maximum of two weeks. A
spore-suspension used to inoculate the solid culture medium should contain at least
108 spores per ml suspension.
3.5.2 Medium and Culture Conditions for SSF
SSF can be carried out using several raw materials (Shankaranand and Lonsane
1993, 1994) and agricultural wastes (Tongwen and Weihua 2002). A variety of
substrates can be used for citric acid production especially in SSF. Various agroindustrial by-products and residues such as sugarcane press mud, coffee-husk,
3 Production of Organic Acids from Agro-Industrial Residues
45
wheat-bran, cassava fibrous residue, rice bran and de-oiled rice bran have been
evaluated as the potential substrates for citric acid production in SSF. The list of
other potential substrates includes molasses distributed on spaghnum moss with
calcium carbonate, saw dust impregnated with molasses, beet pulp impregnated
with pine apple juice, sweet potato residue and wheat bran, sugar-free sugarcane
bagasse with molasses, sugarcane bagasse impregnated with concentrated liquor of
pine apple juice, rice-bran, apple-pomace, grape-pomace, kiwi-fruit peel, sugarcane
bagasse impregnated with cellulose hydrolysate, cassava waste, citrus waste, mandarin wastes, polyurethane foam, starch cake with rice-bran as well as cassava and
coffee wastes.
Generally, the substrates are moistened to about 70% depending on the substrate
absorption capacity. The initial pH is normally adjusted to a lower value before
sterilisation. After sterilisation the material is inoculated with spore-suspension and
spread on trays in layers of 5 cm maximum thickness. The temperature of incubation varies according to the microorganisms used. Growth can be accelerated by
adding (x-amylases), although Aspergillus niger can hydrolyse starch with its own
amylase. The solid culture process takes 90 hours, at the end of fermentation period
the fermented mass is extracted with hot water to isolate citric acid from the solid
medium.
One of the important advantages of the SSF process is that the presence of trace
elements may not affect the citric acid production as it does in submerged liquidfermentation. Consequently, substrate pre-treatment is not required. Citric acid production is directly influenced by the nitrogen source. Physiologically, ammonium
salts are preferred such as urea, ammonium chloride and ammonium sulphate.
Nitrogen consumption leads to a pH decrease, which is very favourable for citric
acid fermentation. Urea has a tampon effect that assures pH control. A high nitrogen
concentration increases fungal growth and the consumption of substrate, but this
result in a decrease yield.
The presence of phosphate in the fermentation medium has a great influence on
the yield of citric acid. Potassium dihydrogen phosphate has been found to be the
most suitable and effective phosphorus source. Low levels of phosphorus favour
citric acid production. The higher concentrations of phosphorus in the fermentation
medium results in the formation of certain sugar-acids, a decrease in the fixation
of carbon dioxide and the stimulation of culture-growth. Phosphates act at the level
of enzyme activity. Different strains require distinct nitrogen and phosphorus concentrations in the medium. In fact, nitrogen and phosphorus limitation is the crucial
factor in citric acid production as there is interaction between them.
The specificity of solid culture is largely due to a lower diffusion rate of nutrients and metabolites occurring at lower water activity in SSF. Consequently, the
strains with large requirements of nitrogen and phosphorus are not the ideal organisms for SSF. The production process employing solid culture medium is in use
solely in small production-plants and a maximum of 500 tons per year are produced in these plants. The other production processes used are surface-fermentation,
which are performed using solid and liquid media. Several factors affect the choice
of fermentation processes or production-type. Important ones are: availability of
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investment capital, energy availability, cost of labour and training, and the availability of techniques for the measurement and regulation of the process.
3.5.3 Bioreactors for Citric Acid Production
Various types of fermenters can be used for the production of citric acid in SSF
(Prado et al. 2004; Hang, 1988). The most common fermenters for this purpose
are glass-incubators, glass-columns, trays, Erlenmeyer conical flasks, rotating drum
bioreactors, single-layer and multi-layer packed-bed bioreactors and packed-bed
column bioreactors (Hang and Woodams 1985; Raimbault and Alazard 1980). Production is usually better in flasks while the yields could be lower in tray and rotatingdrum bioreactors. A multi-layered packed-bed bioreactor is capable of considerably
improved mass-transfer compared to a single-layer packed-bed bioreactor operated
under similar conditions. Packed-bed bioreactors show superior production of citric
acid than the production in flask culture under similar conditions. Higher yields of
citric acid could be obtained using cassava bagasse as a SSF-substrate in a packedbed column bioreactor than in flasks. Aeration, heat and mass-transfer effects can be
controlled and improved in bioreactors. Different mechanisms of heat removal such
as conductive, convective and evaporative from packed-bed bioreactors in SSF may
also affect citric acid production. The conductive heat transfer could be the least efficient mechanism compared to convective and evaporative mechanisms. Evaporative
heat-transfer is the best mechanism of heat removal from packed-bed bioreactors
is SSF.
3.6 Optimisation of Factors
One of the most important factors is the employment of a suitable microorganism
in fermentation. The strain of microorganism should have the ability to accumulate in the presence of high concentrations of various trace elements (Sikander and
Ikramul 2005). The ability of microorganisms to produce amylolytic enzymes is of
great importance if the SSF-substrates used are starchy in nature. The selection of
strain is done for the utilisation and fermentation of specific substrates in SSF. The
rate of citric acid production may become slower if the sufficient enzymes are not
produced by the culture for the fermentation of substrates (Maria et al. 2007). In
such cases the saccharification of the substrate can be performed separately, or by
adding commercial preparations of enzymes to the fermentation medium at the time
of culture-inoculation the rate of saccharification can be enhanced.
3.6.1 Influence of Environmental Factors
The pH of a culture may change in response to microbial-metabolic activities. The
most obvious reason is the secretion of organic acids such as citric acid, acetic or lactic acids. Production and accumulation of organic acids in the fermentation medium
3 Production of Organic Acids from Agro-Industrial Residues
47
will cause the pH of the medium to decrease. Changes in pH kinetics depend mainly
also on the microorganisms employed in SSF. The pH can drop very quickly to less
than 3.0 with the organisms such as Aspergillus sp., Penicillium sp., and Rhizopus
sp. The pH is more stable between 4.0 and 5.0 for other groups of fungi such as
Trichoderma, Sporotrichum, and Pleurotus sp. The nature of the substrate may also
influence the pH kinetics in the fermentation medium. Generally, a pH below 2.0 is
required for optimum production of citric acid. A low initial pH has the advantage
of preventing bacterial contamination and also helps in inhibiting the formation of
oxalic acid. A lower pH is the optimum for the growth of the fungal cultures and at
the same time for accumulation of citric acid (Kolicheski 1995).
The synthesis of citric acid by Aspergillus niger using different agro-industrial
residues of variable composition requires a number of problems to be encountered
due to high sensitivity of the culture to a number of medium-ingredients. Citric acid
synthesis is directly influenced by the nitrogen source and its consumption leads to a
pH decrease, which is very important point in citric acid fermentation. The presence
of phosphate and trace elements also has a significant influence. Another important
factor is the presence of lower alcohols, which has been found to enhance the citric
acid synthesis. Appropriate alcohols being methanol and ethanol and their optimal
amount depend on the strain being used and the composition of the fermentation
medium.
Relationship between citric acid production and respiration rate of the cultivated
microorganism has been studied in detail by Prado et al. (2004). Glass column fermenters have been used in order to study the influence of aeration on citric acid
production. On-line respirometry monitoring is used to evaluate the performance of
SSF-process. A nutritive solution composed of urea, potassium dihydrogen phosphate, methanol and FeSO4 . 7H2 O has been found to increase the production of
citric acid to 347.7 g per kg dry substrate. The yield of citric acid was achieved 81%
based on starch-consumption.
Other factors can also influence the citric acid production in SSF. The presence and concentration of lower alcohols affect the yield. A list of parameters
affecting the yield of citric acid includes: weight of the substrate in the bioreactor, the availability of surface-area of the SSF-substrate for microbial-colonisation,
pre-culture condition such as the type, age and size of inoculum, initial moisture content of the prepared SSF-medium, various fermentation-parameters such
as incubation-temperature, rate of aeration and mixing, concentration and nature
of the carbohydrates in SSF-substrate used and the pH of the SSF-medium. It has
been generally found that the addition of methanol may increase production. Glycolytic rate can influence the production of citric acid and oxalic acid in SSF. The
overproduction of citric acid is found to be related to an increased glucose-flux
through glycolysis. At a lower glucose-flux oxalic acid is found to accumulate in
the medium. The specificity of solid state culture is largely due to a lower diffusion rate of nutrients and metabolites, which occurs in lower water activity conditions. Consequently, strains with large requirements of nitrogen and phosphorus
are not favoured, due to the restriction of accessibility to the nutrients in SSFmedium.
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3.6.2 Influence of Micronutrients
Trace-element nutrition is the main factor influencing the yield of citric acid. A
number of divalent metals such as zinc, manganese, iron, copper and magnesium
have been found to affect the citric acid yield in fermentation by Aspergillus niger
(Shankaranand and Lonsane 1994). Different additives and metallic micro minerals
have been reported for the enhanced production of citric acid by Aspergillus niger
cultivated on different carbohydrate materials (Sikander and Ikramul 2005). It is
crucial to take into account the interdependence of medium-constituents probably in
SSF. Zinc favours the production of citric acid when added with potassium hydrogen
phosphate. The presence of manganese ions and iron and zinc in high concentrations
could cause the reduction of citric acid yields only in phosphate-free medium. Some
microorganism such as Aspergillus niger show a difference in response to metal ions
and minerals in different fermentation systems, therefore the response of the strain
may be different in SSF compared to submerged fermentation. SSF-systems are
able to overcome the adverse effects of high concentrations of metals and minerals
in the medium. As a consequence of this, the addition of chelating agents such as
potassium ferrocyanide to the solid medium proves to be of no use.
Copper is found to complement the ability of iron at optimum level, to enhance
the biosynthesis of citric acid. Manganese deficiency results in the repression of
the anaerobic and TCA cycle enzymes with the exception of citrate synthetase.
This leads to overflow of citric acid as an end-product of glycolysis. A low level
of manganese (ppm) is capable of reducing the yield of citric acid by 10%. Citric
acid accumulation decreases with the addition of iron, which also has some effect
on mycelial growth and the colonisation of the fungus. Presence of different copper
concentrations in the pellet formation medium could be very important in order to
enhance a suitable structure, related to the cellular physiology and for citric acid
production. Magnesium is required for growth as well as for citric acid production.
3.6.3 Influence of Various Supplements
Some compounds that can inhibit metabolism, such as calcium fluoride, sodium
fluoride and potassium fluoride have been found to accelerate the citric acid production, but potassium ferrocyanide decreases the yield. There are many compounds,
which could act in many ways to favour the production of citric acid. These favouring compounds are capable of impairing the action of metal-ions and other toxic
compounds, which influence the growth during the initial phase. Some of the favouring compounds are: 4-methyl-umbelliferone, 3-hydroxi-2-naphtoic, benzoic acid,
2-naphtoic acid, iron cyanide, quaternary ammonium compounds, amine oximes,
starch, EDTA and vermiculite etc.
Addition of lower alcohols enhances citric acid production from crude carbohydrates. Appropriate alcohols could be methanol, ethanol, isopropanol or methyl
acetate. The optimal amount of methanol/ethanol depends upon the strain and the
composition of the fermentation medium. The addition of ethanol may result in
3 Production of Organic Acids from Agro-Industrial Residues
49
two-fold increase in citrate synthetase activity and a 75% decrease in aconitase activity. Whereas the activities of other TCA cycle enzymes increase slightly. Alcohols
have been shown to principally act on membrane permeability in microorganisms
by affecting phospholipid composition on the cytoplasmatic membrane. The role of
membrane permeability may be argued in the citric acid accumulation. The alcohols
stimulate citric acid production by affecting the growth and sporulation through by
not only acting on the cell-permeability but also on the spatial organisation of the
membrane, or changes in lipid composition of the cell wall.
3.7 Mechanism of Citric Acid Synthesis
Recently advances in citric acid fermentation including biochemical aspects, membrane transport system and the modelling of the process are well documented by
Maria et al. (2007). Citric acid (2-hydroxy propane-1,2,3, tricarboxylic acid) is a
primary metabolite, which is formed in the tricarboxylic acid cycle. Glucose is
the starting carbon-source in metabolism, which is released from the enzymatichydrolysis of various substrates used as raw material in the citric acid fermentation.
A part of glucose coming from the raw materials used in the fermentation is consumed in the trophophase for the production of fungal-mycelium and is converted
through respiration into CO2 . The rest of the glucose, in the idiophase, is converted
into organic acids. There is a minimal loss through respiration during this phase.
The theoretical yield is 123 g citric acid-1 -hydrate or 112 g anhydrous citric acid per
100 g sugar. These yields are theoretical, and are not obtained in practice because of
loss during the trophophase.
3.7.1 Pathways of Citric Acid-Biosynthesis
Biochemical aspects, membrane transport and modelling of citric acid fermentation
have been recently published by Maria et al. (2007). Glucose used for the citric
acid synthesis is metabolised in two pathways; 80% is broken down by reactions of
the Embden-Meyerhof-Parnas (EMP) pathway and the rest 20% goes through the
reactions of the Pentose-phosphate cycle. The relationship between these two pathways is 2:1 during the growth phase. The enzymes of the EMP-pathway are present
throughout the fermentation process in the strains of citric acid production. The
activity of the pathway is regulated in a positive manner with phosphofructokinase
but pyruvate kinase regulates the activity in a negative manner. The acetate residue
is channelled into the tricarboxylic acid cycle after pyruvate is decarboxylated with
the formation of acetyl-CoA. All enzymes of the Krebs cycle are expressed during
the idiophase except ␣-ketoglutarate dehydrogenase. During the production of citric
acid, the citrate synthase activity, a condensing enzyme, is increased by a factor of
10. Those enzyme activities, which catabolise citric acid, are aconitase and isocitrate
dehydrogenase, both activities are sharply reduced in the trophophase. A mitochondrial enzyme, one of the three isocitrate dehydrogenase isozymes, is specific for
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NADP, which is inhibited by glycerol that accumulates during the spore germination process. In addition, citric acid production is inhibited by high intracellular
concentrations of ammonium ion.
Citrate synthase can not be solely responsible for maintaining the activity of
the tricarboxylic acid cycle, because the cycle would cease if citric acid were
removed. The tricarboxylic acid cycle intermediates, replenished by distinct sequences (anaplerotic sequences), must exist in the production phase. The first
anaplerotic enzymes present in Aspergillus is a pyruvatecarboxylase. Pyruvate and
carbon dioxide are converted into oxalacetate, inorganic phosphate, and ADP with
the consumption of ATP. The reaction is dependent on Mg2+ and K+ ions. Acetyl
CoA is not required for this reaction, although this is required in the metabolic
reactions of other microorganisms. Therefore, pyruvate carboxylase is the essential
enzyme for citric acid biosynthesis. The second anaplerotic sequence involves a
phosphoenol pyruvate (PEP) carboxykinase enzyme. This enzyme converts PEP and
CO2 into oxalacetate and ATP in the presence of ADP. This system needs Mg2+ or
Mn2+ and K+ or NH+
4.
3.7.2 Biochemistry of Citric Acid-Overproduction
The metabolic pathways involved in the overproduction of citric acid by Aspergillus
niger have been studied (Moresi and Parente 2000). Essential for overproduction
are a high flux of metabolites through the glycolysis (5–10 mmol/min per mg of
protein), a block of reactions of the tricarboxylic acid (TCA) cycle that degrade
citrate, and an anaplerotic sequence, to replenish the oxaloacetate (OAA) used for
the synthesis of citric acid.
Although the regulation of enzyme activity is critical in controlling the metabolic
flux toward citric acid overproduction, the most important steps in controlling the
rate of the pathway are glucose transport and hexokinase activity (HK). Ikramul
et al. (2002) have studied that the improvement of strains of micro-organisms for
the production of citric acid has been and can be traditionally achieved by mutagenesis and screening. But it has been postulated that the overexpression of HK
and the glucose-transport systems would result in the maximum possible increase in
citric acid synthesis. This strategy appears to be viable. The overexpression of about
20–30-fold of pyruvate kinase (PK) and phosphofructokinase (PFKI) has been
achieved in Aspergillus niger. The metabolic changes necessary for the overproduction of citric acid by Aspergillus niger and Yarrowia lipolytica are induced by
high sugar concentrations (Moeller et al. 2007) and manganese deficiency, although
other factors are also important mainly the concentrations of phosphate, nitrogen
and trace metals, a low pH, and high dissolved oxygen concentration.
3.8 Extraction of Citric Acid from Fermentation
Citric acid from the fermented solid material is leached out using water. The extract
obtained contains citric acid, which is recovered following downstream processing
by conventional methods. Recovery from the extracts is generally accomplished
3 Production of Organic Acids from Agro-Industrial Residues
51
in three processes, precipitation, extraction and adsorption and absorption using
ion-exchange resins. The Food and Drug Administration of the United States have
described citric acid extraction. Citric acid extracted by this method has been recommended suitable for use in food and drugs. Precipitation is the classical method and
it is performed by the addition of calcium oxidehydrate (milk of lime) to form the
slightly soluble tri-calcium citrate tetrahydrate. The precipitated tri-calcium citrate
is removed by filtration and washed several times with water. The precipitate is then
treated with sulphuric acid to form calcium sulphate, which is removed by filtration.
Mother liquor contains citric acid is treated with active carbon and passed through
cation and anion exchangers. Several anion-exchange resins are commercially available. Finally the liquor is concentrated in vacuum crystallisers at 20–25◦ C, forming
citric acid monohydrate. Anhydrous citric acid is prepared following the process of
crystallisation at higher temperature than 25◦ C.
3.9 Productivity of Citric Acid
Few examples of citric acid production using various agricultural residues and industrial by-products have been presented in Table 3.4
Different agro-industrial residues have been investigated (as presented above) for
citric acid production (Kolicheski et al. 1997). In order to achieve economic development, focus has shifted to the industrial application of cassava for value addition.
Industrial processing of cassava tubers is mainly done to isolate flour and starch.
Processing for flour generates solid-residues including brown peel, inner peel, unusable roots, crude bran and bagasse. Cassava bagasse is a fibrous residue from
the extraction process, which is generated during the separation stage. This bagasse
contains about 40–70% starch that physically could not be extracted. It has a large
absorption capacity and may hold up to 70% moisture. This waste can be used for
citric acid production along with two other potential substrates, sugarcane bagasse
and coffee-husk.
Citric acid production from cassava bagasse can be carried out using a culture of Aspergillus niger NRRL 2001 in solid state fermentation. SSF is performed at 30◦ C for 120 h. These three substrates are ground to a particle size of
0.8–2.0 mm and dried at 55-60◦ C for 12 h. The fermentation-nutrient medium containing ZnSO4 7H∼O (0.2 g/l) and FeCl3 . H2 O (0.014 g/l) are sterilised at 121◦ C
for 15 min. After cooling methanol (4%) is added aseptically to the medium. Sugar
cane bagasse and coffee-husk are supplemented with 40% of glucose (corresponding to the starch content of cassava bagasse). Solid substrates are inoculated with
the spore-suspension, mixed with the nutrient medium. The inoculum size must
contain 107 spores per g of dry substrates. The initial moisture contents of the three
substrates vary due to their structure and composition. Solid culture medium should
contain 90, 65 and 62% of moisture contents for sugar cane bagasse, coffee-husk and
cassava bagasse, respectively. Fermentation of these solid substrates can be carried
out in vertical column fermenters, further details and the kinetics of this process are
discussed by Pandey et al. (2001).
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Table 3.4 Biosynthesis of citric acid from agro-industrial substrates (adapted from Pandey et al.
2001)
Agro-industrial material
Culture used for Biosynthesis
Recovery
Food wastes
Wheat bran
Apple-pomace
Cassava residue
Apple-pomace
Rice-bran
Apple pomace
De-oiled rice bran
Sugar cane press-mud+
Wheat bran (4: 1)
Apple pomace
Apple pomace
Grape pomace
Grape pomace
Grape pomace
Grape pomace
Grape pomace
Kiwifruit peel
Sugar cane
Orange waste
Beet molasses
Sugar cane Bagasse
Coffee husk
Carrot waste
Okara (soy residue)
Pineapple waste
Pineapple waste
Glucose (Sugar can bagasse)
Kumara (starch containing)
Cassava bagasse
A. niger UV60
A. niger CFTRI30
A. niger NRRL2001
A. niger CFTRI30
A. nigerNRRL2270
A. niger CFTRI30
A. niger NRRL2270
A. niger CFTRI30
A. niger CFTRI30
45.5 g/l
85 g/kg
766 g/kg
234 g/kg
816 g/kg
127 g/kg
771 g/kg
92 g/kg
116 g/kg
A. niger NRRL 328
A. niger NRRL 567
A. niger NRRL 2001
A. niger NRRL 2270
A. niger NRRL 599
A. niger NRRL 328
A.. niger NRRL 567
A. niger NRRL 567
A. niger
A. niger
A. niger ATCC 942
A. niger CFTRI 30
A.. niger CFTRI 30
A. niger NRRL 2270
A. niger
A. niger ATCC 1015
A. niger ACM 4942
A. niger CBS 733.88
A. niger
A. niger LPB– 21
789 g/kga
883 g/kga
413 g/kga
511 g/kg a
498 g/kg a
523 g/kg a
600 g/kg a
100 g/kg a
29 g/kg
46 g/kg
35 g/l
174 g/kgb
150 g/kgb
29 g/kga
96 g/kga
132 g/kgb
194 g/kgb
21.24 g/l
103 g/kgb
347 g/kgb
a – based on sugar consumed; b - based on dry matter
3.10 Prospects of R & D in Citric Acid Biosynthesis
The production of citric acid has not received much attention in the form of modern methods of molecular biology, presumably because it is considered a wellestablished area. Mutagenesis and selection have carried out the improvement of
strains of Aspergillus niger. The two principal methods of selecting populations,
namely, “the single-spore technique” and the “passage method” have been used for
selecting citric acid producing strains. The single-spore technique has the disadvantage in that mineral acid and organic acids (gluconic and oxalic acids) simulate the
presence of citric acid; this method has been improved by incorporating a specific
stain para-di-methylamino benzaldehyde instead of using the indicator for citric
acid. The most employed technique to improve citric acid producing strains has
been by inducing mutations in parental strains using mutagenesis. Among physical
mutagens, ␥-radiation and UV-radiation have often used. Hyperproducer strains are
3 Production of Organic Acids from Agro-Industrial Residues
53
obtained by combining UV treatment with some chemical mutagens such as aziridine, N-nitrosoN-methylurea, or ethyl methane-sulphonate.
Another approach for strain improvement has been the para-sexual cycle. Diploid
strains display higher citric acid yields compared to their parent haploids, but they
tend to be less stable. Protoplast fusion appears to be a promising tool to extend
the range of genetic manipulation of Aspergillus niger with respect to citric acid
production. Protoplast fusion techniques have produced fusant-strains that have acid
production capacities exceeding those of the parent strains in solid state fermentation. The other important aspects of strain improvement could be the resistance to
detrimental constituents of fermentation raw-materials, capability of utilizing a variety of raw materials e.g. starch, cellulose, pectin-containing and other waste materials. However, there is no single effective technique to achieve the hyper-producing
mutants and much remains to be done in this area.
Metabolic engineering is now possible, in the light of increased knowledge about
the regulation of acidogenesis. The biochemistry of the citric acid biosynthesis
has been studied in detail and knowledge of pathways is necessary to regulate the
biosynthesis.
3.11 Lactic Acid
Food industry wastes have been used for lactic acid synthesis using integrated glucoamylase production (Wang et al. 2008). Development of an oat-based biorefinery
for the production of lactic acid by Rhizopus oryzae has been recently reported by
Koutinas et al. (2007a). Lactic acid (C3 H6 O3 : 2-hydroxypropionic acid) is produced
by fermentation (50%) and by chemical-synthesis (50%). Lactic acid is common in
nature; it is present in plants and microorganisms. Lactic acid has two enantiomers,
L-(+) and D-(−)-lactic acid. The L-(+) isomer is used by human metabolism and
is preferred for food use because the D-(−) isomer is slightly toxic. Nevertheless,
synthetic racemic (DL) lactic acid is the primary commercial form. Lactic acid is
widely used in the food industry as taste-enhancing additive. Ferrous salts and the
various L(+)-lactic acid salts are used in the pharmaceutical industry for their therapeutic qualities.
3.11.1 Industrial Applications of Lactic Acid
The free acid is used as an acidulant and preservative in several food products such
as cheese, meat, beer and jellies. Ammonium lactate is used as a source of nonprotein nitrogen in feeds; sodium and calcium stearoyl lactylates are used as emulsifiers and dough conditioners. Only the L(+) form of the lactic acid is metabolised
in animal and human cells, because these cells can synthesize only L(+)-lactate
dehydrogenase enzyme for the metabolism. Therefore, consumption of large quantity of D(−)-lactic acid will result in its accumulation in the blood. As a result of
D(−)lactic acid accumulation hyper-acidity of urine and decalcification may occur.
54
P. Singh nee’ Nigam
In the pharmaceutical industry lactic acid is used for the adjustment of pH of
pharmaceutical preparations and topical wart preparations. Other applications are as
a blood coagulant and dietary calcium source. Ethyl lactate is used in the preparation
of anti-inflammatory drugs.
Lactic acid also is a good solvent and provides acidity in foods and beverages.
Ethyl and butyl lactates are used as flavour ingredients. Lactic acid has some important applications in food industry such as for the production of fermented foods,
in the pickling-process of gherkins, dill, olives, sauerkrauts, carrot and some leafy
vegetables, and in the processing of oriental-foods. Lactic acid finds its application
for the production of dairy products such as yoghurt, buttermilk, acidophilus milk,
cottage cheese, creamy-cheese and fermented cheese etc.
Lactic acid production has been studied recently with increased interest because
of its application in the synthesis of biodegradable, biocompatible plastics and coatings (Koutinas et al. 2007a). L(+)-lactic acid can be polymerised to form polylactic
acid (PLA). This polymer can be used in the manufacture of new biodegradable
plastics. The plastics prepared in such way are increasingly used in surgery for selfdissolving suture thread. Biodegradable plastics could play an increasing role in the
industry by replacing or minimizing the use of non-degradable ordinary plastics to
solve environmental pollution problems. These biodegradable plastics synthesized
from lactic acid could be considered as a substitute for plastics manufactured from
the petroleum products. Lactic acid has been used in the manufacture of cellophane,
resins and some herbicides and pesticides. Another important application of lactic
acid is in textile and tanning industries.
3.11.2 Lactic Acid Production
Lactic acid using solid state fermentation can be carried out using fungal as well
as bacterial cultures. Strains of Rhizopus sp. (Koutinas et al. 2007a; Tay and
Yang 2002) have been common among the fungal cultures and that of Lactobacillus
sp. among the bacterial cultures (John et al. 2007). The continuous fermentation
systems using total microbial cell retention have been used as an empiric steady
state model for lactic acid production (Richter and Nottelmann 2004).
Different crops such as cassava and sweet sorghum and the various crop-residues
such as sugar cane bagasse, sugar cane press-mud, carrot-processing waste and
starch (Tay and Yang 2002) can be used as substrates. A strain of Rhizopus oryzae
has the potential of producing lactic acid in solid state fermentation. This strain has
been used to evaluate the lactic acid production in different fermentation systems.
An inert solid support, sugar cane bagasse impregnated with a nutrient solution has
been used for solid state fermentation. Both production level and productivity of lactic acid have been found higher in SSF with a yield of 137.0 g/l and the productivity
of 1.43 g/l per hour.
Food wastes produced in large amounts in food and catering industry have been
studied for lactic acid production by Kim et al. (2003) and Wang et al. (2005).
One bacterial strain Lactobacillus paracasei has been found to produce lactic acid
3 Production of Organic Acids from Agro-Industrial Residues
55
in SSF. Lactate concentration and yield of 90 g/kg and 91–95% has been achieved
in SSF using bacterial culture. The time required to complete SSF for lactic acid
production is usually 120–200 h. Sugar cane press-mud has been used as substrate
in SSF employing three bacterial cultures. A strain of Lactobacillus casei has been
found to produce a higher concentration of lactic acid in comparison to Lactobacillus helveticus and Streptococcus thermophilus.
Production of L(+)-lactic acid by Rhizopus oryzae can be carried out in SSF
using a solid medium. This solid medium is prepared by impregnating sugar cane
bagasse with a nutrient solution containing glucose and calcium carbonate. The optimal concentration of glucose is 18%, producing 13.7% lactic acid. In such process
the productivity can be achieved up to 1.43 g/l per h and the fermentation yield is in
the range of 77%. These data are significant for L(+)- lactic acid biosynthesis.
A process can be designed for example to carry out SSF for the maximum production of lactic acid. Sugar cane bagasse, a lignocellulosic waste can be used as
the support for solid culture. Crushed, moulded, carefully washed and 0.8–2.0 mm
size particles are used after sterilisation. Standard fermentation medium is prepared
using 200 g/l glucose. The fermentation medium is sterilised at 110◦ C for 30 min
and the pH is adjusted to 7.5 using a 4% solution of NaOH. A solid-fermentation
medium of 1200 g-wet weight, with moisture content of 70%, consists of 100-g
sugar cane bagasse, 100-g calcium carbonate, and 800-ml fermentation medium.
The fermentation medium contains glucose, ammonium sulphate, potassium dihydrogen phosphate, zinc and magnesium sulphates. Inoculum size of 200 ml is used
and is included on 1200g wet weight. The inoculum of fungal culture is prepared
by growing a sporangiospore suspension using 107 spores per g glucose in a liquid
fermentation medium for about 14–15 h under shaking condition. Inoculum mixed
solid fermentation medium can be incubated in various fermenters such as Erlenmeyer flasks or glass column reactors taking about 0.45 g/cm3 . If the fungal culture
used is Aspergillus oryzae, the SSF should be run for at least 96-98 h at 35◦ C and
the aeration rate is fixed at 1.2 l/h in each glass column reactors.
3.11.3 Parameters Influencing Lactic Acid-Synthesis
Effects of cultivation parameters on morphology of Rhizopus arrhizus and the lactic
acid production in a bubble column reactor has been studied by Zhang et al. 2007).
In solid state fermentation, aeration of the moistened medium is important. It controls the humidity of the solid support and simultaneously the heat-release of the
fermentation metabolism. Aeration also provides the oxygen required for the fungal
colonisation in solid medium. Therefore, the optimisation of aeration rate is very
important in SSF and every SSF-process is different due to the use of different
substrate and microorganism. In one example of lactic acid production as described
above, aeration rates of 0 to 100 ml/min per column reactor were tried; the maximal
L(+)- lactic acid yield was obtained with the aeration rate of 20 ml/min. A decrease
of 35% was caused in lactic acid yield at the aeration rate of 100 ml/min. A higher
56
P. Singh nee’ Nigam
aeration may cause a significant reduction in the lactic acid yield, which is directly
related to an increase in the aerobic respiration rate of the fungus. Similarly, at very
low or with a poor aeration a significant amount of glucose may not be consumed in
the fermentation that may result in decreased biosynthesis of lactic acid.
A second important factor influencing the SSF is the inoculation rate of fungal
spores. The preculture for a SSF is mostly prepared by growing the fungal spores
in liquid fermentation medium, which is used to inoculate the solid fermentation
medium. Various sizes of spore-inoculum using 105 to 108 spores in a suspension
form have been used to produce a preculture of Aspergillus oryzae. The optimal
seed-inoculum has been found to be 106 spores per g glucose used. This inoculum
size produced 120 g/l lactic acid with complete glucose uptake and 75% yield of
lactic acid. Inoculation rate and the lactic acid production are parallel. A similar
variation in lactic acid yield has been observed as any variation occurred in inoculation rate.
Lactic acid synthesis has been found to be affected by the glucose concentration
in the fermentation medium. SSF using 12 to 24% glucose has been performed to
optimize sugar concentration for an enhanced yield of lactic acid. The optimum
glucose concentration, as the initial intake in the medium, was found to be the 18%
producing 137 g/l lactic acid with a 76% yield. A higher concentration of glucose
as 24% led to a decrease in lactic acid biosynthesis. Because of a partial consumption of glucose in the fermentation medium, a glucose concentration of about 16%
produced 117 g/l of lactic acid with a fermentation yield of 74%. If the glucose
concentration used was lower as 12%, lactic acid production was reduced to 75 g/l,
with a lower fermentation yield of 63%.
During the course of fermentation various parameters affect the yield and the
fermentation efficiency. Simultaneously with lactic acid biosynthesis, variation in
pH and the moisture content have been recorded. The pH normally falls by more
than two units from an initial value of 6.96 to 4.80. A slight increase in relative
humidity is observed throughout the fermentation; it changes 70–76% after 96 h of
fermentation. In SSF of lactic acid, fumaric acid has also been found to be produced
at the concentration of 20 g/l after 96 h.
The type of fermenter used is another factor affecting the product-yield. Rotating fibrous bed bio-reactor have been found useful for lactic acid production by
immobilised Rhizopus oryzae (Tay and Yang 2002). Bubble column reactor has
been used for lactic acid synthesis through the cultivation of Rhizopus arrhizus
(Zhang et al. 2007). A comparative study of SSF in column-bioreactor and culture
in Erlenmeyer flasks showed 77 and 74% fermentation yields, respectively. Fumaric
acid production (20 g/l) has been found important in SSF, while this acid has been
noticed in very small quantity of 2.1 g/l in liquid fermentation of lactic acid. The
culture conditions cause the synthesis of fumaric acid. A closer investigation of the
biochemical processes generating fumaric acid is necessary for the understanding
of the metabolic pathways involved. Lactate-dehydrogenase activity is related to the
presence of its substrate, pyruvic acid as well as to the co-factors that are indispensable in the reaction it catalyses. The absence of reduced co-factors such as NADH2
or the degree of cytochrome oxidation may limit the reaction.
3 Production of Organic Acids from Agro-Industrial Residues
57
The decrease in pH during the SSF of lactic acid was noticed from 6.96 to 4.8.
Membrane processes are involved in proton-translocation between the cell medium
and the outside medium. The nature of the proton-pumping activities concerned
should be characterized in relation to L(+)-lactic acid production. This type of approach has been used in numerous studies of lactic acid bacteria.
3.11.4 Downstream Processing of Lactic Acid
The solid-fermented mass obtained from the fermenters is used for the extraction
of lactic acid. A solution of 1M H2 SO4 is mixed with the fermented material. The
whole content is then placed in a press cell with 0.5–1.5 mm orifices over its whole
surface. A hydraulic pressure of 500 to 2,100 kg/cm3 is used to extract concentrated
liquid from the fermented material. The extract obtained consists of L(+)-lactic acid
unfermented sugar, salts and other metabolites formed such as fumaric acid. The
insoluble fraction contains the support used in SSF, calcium phosphate, and mycelial
biomass. Recovery of lactate is complicated by the high solubility of its salts.
The traditional process involves precipitation of calcium lactate and regeneration
of lactic acid by addition of sulphuric acid followed by further purification steps,
which are ion exchange and decolourisation. The extract is treated with hydrated
lime for four purposes; it kills lactic acid bacteria, coagulates proteins, degrades
residual sugars and solubilises calcium lactate precipitated due to the reaction of
lactic acid with calcium hydroxide and calcium carbonate present in fermenter. The
solution is then filtered and the filtrate containing soluble calcium lactate is treated
with sulphuric acid to precipitate calcium sulphate liberating lactic acid into the
solution. The lactic acid solution is filtered to remove calcium sulphate precipitates
and then bleached. The bleaching is performed by treatment with activated carbon.
The bleached lactic acid solution is then concentrated to 35–40% acidity in evaporator. The process of bleaching and evaporation is repeated 2–3 times to obtain
lactic acid of different grades, such as technical-grade and food-grade lactic acid.
Alternative processes are the extraction by liquid membranes, electro dialysis and
ion exchange.
3.12 Prospects of R & D in Lactic Acid Biosynthesis
The concept of biorefinery using agricultural substrate such as oat is certainly
has good prospects (Koutinas et al. 2007a). A physiological and biochemical approach is required for a better understanding of the contribution of the enzymes
of the metabolic pathway or pathways leading to the synthesis of L(+)-lactic acid.
An NAD+ -dependent lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC
1.1.1.27) catalysing the reduction of pyruvate into lactate is certainly one of the
fundamental enzymes involved. A shuttle between pyruvic acid and oxaloacetic acid
involving a carboxylation reaction often forms the furmaric acid. Better understanding of formation of both ethanol and fumaric acid is required.
58
P. Singh nee’ Nigam
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Chapter 4
Biofuels
Soham Chattopadhyay, Asmita Mukerji and Ramkrishna Sen
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Bio-Fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 Biogas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1
Biomethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2
The Phases of Anaerobic Methane Production . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3
The Strategy of Co-digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.4
Influence of Process Parameters and Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4 Biohydrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.1
Hydrogen from Agro-Industrial Residue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.2
Production of Biohydrogen in Fermenter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5 Coupling of Biohydrogen and Biomethane Production . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6 Bio Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.1
Fermentative Production of Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.2
Bioethanol from Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.3
Major Raw Materials Used in Bioethanol Production . . . . . . . . . . . . . . . . . . . . . .
4.7 Biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.1
Chemical Catalyzed Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.2
Enzymatic Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.3
Biodiesel from Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8 Future Prospectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
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74
Abstract It is the cost and abundant availability of raw materials that determine
the economic feasibility of biofuel production. Considering these constrains, agroindustrial residues may offer cheaper options as raw materials for biofuel production.
This chapter thus aims at presenting the current status and future directions of
biofuel production using both conventional substrates and agro-industrial residues
R. Sen (B)
Department of Biotechnology, Indian Institute of Technology, Kharagpur,
West Bengal 721302, India
e-mail: rksen@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 4,
C Springer Science+Business Media B.V. 2009
61
62
S. Chattopadhyay et al.
as raw materials and critically analyzing the prospect of agro-industrial residue
based production of biofuels. Utmost care has been taken to address all the critical
economic and environmental issues related to the production of each of the gaseous
and liquid biofuels namely biomethane, biohydrogen, bioethanol and biodiesel in
the light of available published literature information. While the important process parameters involved in anaerobic digestion and co-digestion of agro-industrial
residues in various judicious combinations have been discussed for biomethane
production, roles of different reactor configurations, designs and various types
of cultivation processes including photo and dark fermentation employing agroindustrial waste as substrates for biohydrogen production have been analyzed.
Similarly, a comparative study of bioethanol production using lignocellulosic and
non-lignocellulosic wastes has been presented and discussed. Though the oil cake
as an agro-industrial waste holds some promise, biodiesel production using agroindustrial residues has not been reported in literature.
Keywords Biofuels · Agro-industrial residues · Biomethane · Biohydrogen ·
Bioethanol
4.1 Introduction
Agro-industrial wastes are those end-products of primary production that have not
been reused, recycled or salvaged. Recycling, reprocessing and eventual utilization
of agro-industrial residues can be accomplished for the benefit of man and his
amenities rather than their discharge to the environment which may cause detrimental effects (Hamza 1989). Extensive research has been done on the utilization
of agro-industrial residues and a good number of publications are available on
this (Fernández et al. 2005; Khardenavis et al. 2007; Sellami et al. 2007; Gañán
et al. 2008). Table 4.1 shows chronological use of agro-industrial residues as raw
materials for various industrial applications.
4.2 Bio-Fuels
Bio-fuels can be broadly classified into two major types, gaseous and liquid biofuels. Purification of the conventional biogas into methane-enriched biofuel led to the
development biomethane. Biohydrogen is a relatively new type of gaseous biofuel,
which is produced by anaerobic fermentation of agro-industrial wastes by the synergistic action of a consortium of methanogenic, acidogenic and hydrogenic bacteria.
On the other hand, liquid biofuels have recently been classified into bioethanol and
biodiesel. While bioethanol has recently gained rejuvenated importance on the wake
of present energy crisis worldwide, biodiesel occupied the centre stage as a potential
4 Biofuels
63
Table 4.1 Various uses of agro-industrial residues
Agro-industrial residues
Uses
Reference
Pressmud (waste of sugar
industry), Cow dung, Neem and
Mahua cakes (soil-expelled seed
material), and Neem leaf
Sugarbeet pulp, Potato pulp,
Brewery grain
Grape skin pulp extract, Starch
waste, Olive oil waste effluents
and Molasses
Wheat bran
Sugarcane Bagasse
Improving the nodulation
and dry matter
production of soybean
Jauhri 1989
Methane production
Kang and Wetland 1993
Extracellular
polysaccharide (pullulan)
production
Production of ferulic acid.
Production of
protein-enriched cattle
feed and enzymes
Production of organic acids,
flavour and aroma
compounds, and
mushrooms
Production of Aroma
compounds
Cultivation of edible
Rhizopus strains.
Israilides et al. 1994
Cassava Bagasse
Coffee husk
Cassava bagasse, Apple pomace,
Soyabean, Amaranth grain and
Soyabean oil
Technical oleic acid (TOA), waste
frying oil (WFO) and waste-free
fatty acids from soybean oil
(WFFA)
Wheat bran (WB), Rice bran (RB),
Rice straw (RS), Sawdust (SD),
Coconut pith (CP)
Biomass (corn cobs and stalks,
sugarcane waste, wheat or rice
straw)
Olive-cake with Poultry manure
and Sesame shells
Cotton stalk, Rice straw, Bagasse,
and Banana plant waste
Sugar cane, starchy materials and
lignocellulosic biomass
Faulds et al. 1997
Pandey et al. 2000a
Pandey et al. 2000b
Soares et al. 2000
Christen et al. 2000
Production of poly(3hydroxyalkaonates)
(PHA)
Fernández et al. 2005
Production of
endoxylanase.
Poorna and Prema 2006
Ethanol production
Lin and Tanaka 2006
Composting and soil
amendment
Extraction of
Lignocellulosic fibers
Bioethanol production
Sellami et al. 2007
Habibi et al. 2008
Sánchez and Cardona 2008
substitute for petroleum diesel in the last two decades. Various types of the gaseous
and liquid biofuels are discussed below.
4.3 Biogas
The conventional biogas, which is produced in biogas plants employing anaerobic digestion of organic wastes including manures by mixed microbial cultures,
64
S. Chattopadhyay et al.
is composed primarily of methane (typically 55%–70% by volume) and carbon
dioxide (typically 30%–45%) and may also include smaller amounts of hydrogen
sulfide (typically 50–2000 ppm), water vapor (saturated), oxygen, and various trace
hydrocarbons (Amigun et al. 2008; Kashyap et al. 2003; Siso 1996). Due to its lower
methane content (and therefore lower heating value) compared to natural gas, biogas
use is generally limited to engine-generator sets and boilers (Krich et al. 2005).
4.3.1 Biomethane
Biomethane is upgraded or sweetened biogas after the removal of the bulk of the carbon dioxide, water, hydrogen sulfide and other impurities from raw biogas. From a
functional point of view, biomethane is extremely similar to natural gas (which contains 90% methane) except that it comes from renewable sources. (Krich et al. 2005).
Biogas can also be purified and upgraded and used as vehicle fuel. Over a million
vehicles are now using biogas and fleet operators have reported savings of 40–50%
in vehicle maintenance costs (Parawira 2004). Table 4.2 compares wastes generated
by various agro-industries on the basis of their annual production rate, yield, power
generation capacity and the amount of fossil fuel they can replace.
Anaerobic digestion has proved to be the most feasible strategy for biogas production from agro-industrial wastes. The potential substitution of fossil fuel with
Table 4.2 Various agro-industries and their annual production rate, yield, power generation
capacity and the amount of fossil fuel they can replace
Industry
Olive oil
Wine
Meat
Cereals
Sugar
Coffee
Annual
production
rate of
primary
product
Annual
production
rate of
secondary
product
377.75 Kt
of olive
oil
348.43 Kt
of must
Power
generation
capacity
Yield of
(million
Methane
m3 CH4 /unit kWh)
Amount of
Fossil fuel
replaced
(tons)
References
−
−
−
2.266 Mt of
olive oil
wastewater
135.50 Kt of
wine-grape
residues
315 Kt of
4.185 Mt of
meat
slaughter
annually
house
waste
water
3,733,700 336,060 Bran
Residue
−
−
−
−
−
−
450
454
127333
123,620
56,053
230
650
14
190
3873
53198
20,000
199,200
Adapted from
Fountoulakis
et al. 2008
Adapted from
Kivaisi and
Rubindamayugi
1996
4 Biofuels
65
biogas represents an annual reduction in the atmosphere of 1.05 million m3 of CO2 .
(Kivaisi and Rubindamayugi 1996).
4.3.2 The Phases of Anaerobic Methane Production
The anaerobic digestion process encompasses mainly four interdependent, complex
sequential and parallel biological reactions. The phases are hydrolysis/liquefaction,
acidogenesis, acetogenesis and methanogenesis. Figure 4.1 represents how complex
polymers like carbohydrates, proteins and fats are hydrolyzed into simple sugars,
amino acids, fatty acids and alcohols, respectively which are in turn converted into
CH4 and CO2 on acetogenesis and methanogenesis.
Complex polymer
or organic matter
Carbohydrate
Protein
Fat
Hydrolysis
Cellulase, amylase
Lipase
Protease
Simple
Sugar
Amino acids &
Small peptides
Long chain
Fatty acids
& alcohol
Acidogenesis
Lactate, ethanol,
propionate, butyrate
Acetate
Fig. 4.1 Methane and carbon
di-oxide production from
complex polymers or organic
matter
Acetogenesis
H2 and CO2
Reductive
methanogenesis
Acetidastic
methanogenesis
Methane and CO2
66
S. Chattopadhyay et al.
4.3.3 The Strategy of Co-digestion
Co-digestion of different types of organic by-products has been increasingly applied
in order to improve plant profitability through easier handling of mixed wastes;
common access facilities and the known effect of economy scale are some of the
advantages of co-digestion. LHL (Laying Hen Litter), CW (Cheese Whey), SW
(Slaughter house Wastewater), cattle manure, swine manure or piggery effluent have
been utilized as biogas yield ameliorating agents (Azbar et al. 2008). Co-digestion
of OMW (Olive Mill Wastewater) and WGR (Wine Grape Residues) with SW yields
much improved results, with a 30–57% increase in methane yields as compared to
individual digestion of the substrates (Table 4.3).
Biogas releases by OME increases 90% on co-digestion with LHL, but only
22% increases with CW (Azbar et al. 2008). Methane yields during thermophilic
digestion are 14–35% higher than mesophilic digestion (Table 4.4). Thermophilic
digestion, intrinsically, has higher degrading capability and methanogenic activity
in biogas production. Results of comprehensive studies suggest that thermophilic
anaerobic digestion may be attractive for treating high-temperature industrial effluents and specific types of slurries (Parawira 2004).
Table 4.3 Increase of methane yield and energy yield with co-digestion (adapted from
Fountoulakis et al. 2008)
Agro-industrial
wastes
Methane Yield
(L CH4 /kg COD)
OMW
WGR
SW + OMW
SW + WGR
OMW + WGR
108
147
170
191
163
% Increase in
comparison to
individual digestion.
−
−
57.4
29.93
50.9 (w.r.t. OMW)
10.88 (w.r.t. WGR)
Energy yield (MJ/kg
COD)
3.89
5.3
6.13
6.87
5.87
Table 4.4 Percentage increase in Methane yield in thermophilic condition (adapted from
Fountoulakis et al. 2008)
Materials
Temperature
Methane yield
(l g−1 COD added)
Percentage
increase
SW+OMW
35
55
35
55
35
55
0.184
0.282
0.188
0.219
0.214
0.301
34.5
SW+WGR
OMW+WGR
14.1
28.9
4 Biofuels
67
4.3.4 Influence of Process Parameters and Steps
4.3.4.1 Effect of pH
A pH range of 6.4–7.6 ensures normal functioning of a digester, beyond which inhibitory effects may be due to the toxic effects of the hydrogen ions, which are
closely related to the accumulation of VFAs (Volatile Fatty Acids) (Anderson and
Yang 1992).
4.3.4.2 Effect of VFA: Alkalinity Ratio
It is essential that the reactor contents provide enough buffering capacity to neutralize any possible VFA accumulation in the reactor and maintain pH 6.7 to 7.4, for
stable operation. The VFA: alkalinity ratio < 0 : 4 indicates digester is stable; a
range from 0:4 to 0:8 shows some instability, whereas ≥ 0 : 8 shows marked instability (Switzenbaum and Jewell 1980). The buffering capacity is an added advantage
of using proteins during methane generation and eliminating the requirement of an
external buffer (Mshandete et al. 2004).
4.3.4.3 Effect of C: N Ratio
The C: N ratio in the range of 25–30 has been suggested as optimum for anaerobic
digestion, there has been a contradiction with the ranges of 16–19 (Nyns 1986) and
16.8–18 (Kivaisi and Mtila 1998) having been proposed as ideal when lignin are
taken into account.
4.3.4.4 Effect of VFA’s
The major VFA’s usually produced are acetic acid (A), propionic acid (P) and butyric
acid. It has been seen that a P/A ratio exceeding 1.4 and acetate and butyrate buildup above 200 Mm and that of propionate above 100 Mm leads to inhibition which
culminates into digester failure (Hill et al. 1987; Ahring et al. 1995).
4.3.4.5 Pre-treatment of Lignocellulosics
The pre-treatment of wastes on alkaline hydrolysis before co-digestion with activated sludge demonstrates a steep rise in methane yield from 25L to 222L CH4
(STP)/kg VSinitial . Consequently, the total solids and volatile solids were reduced by
67% and 84%, respectively. Acidogenic bacteria can ferment the pre-treated lignocellulose even though no delignification or cellulose hydrolysis occurs during the
pre-treatment (Neves et al. 2006).
4.3.4.6 Effect of Temperature
Anaerobic digestion reactors are normally operated within the mesophilic and thermophilic ranges. Methanogenesis is also possible under psychrophilic (< 20◦ C)
conditions but occurs at lower rates. Bacterial activity and growth decrease by one
half for every 10◦ C decrease in temperature below 35◦ C (Kashyap et al. 2003).
68
S. Chattopadhyay et al.
4.3.4.7 Accumulation of Inhibitory Compounds
It has been observed that biogas production decreases significantly with time during OMW digestion. The primary reason, appears to be OMW’s high content of
polyphenols inhibit the anaerobic process (Beccari et al. 1998). It was found that the
individual chemical structure of compounds also greatly influences and determines
the rate and mechanisms of methanogenic degradation (Fountoulakis et al. 2008).
4.4 Biohydrogen
Hydrogen is a very high energy (122 kJ/g) yielding fuel in comparison to methane
or ethanol, produces water instead of greenhouse gases when combusted. Photoautotrophically growing bacteria and (micro)-algae, utilize light as primary energy
source to split water into hydrogen and oxygen by the enzyme hydrogenase. The
basic reactions are (de Vrije and Claassen 2003):
C6 H12 O6 + 2H2 O → 4H2 + 2CO2 + 2C2 H4 O2 ⌬Go′ = −206 kJ
C2 H4 O2 + 2H2 O → 2CO2 + 4H2 ⌬Go′ = 104 kJ
Figure 4.3 shows how biomass containing carbohydrates is converted into organic acids and hydrogen by the process of thermophilic heterotrophic fermentation.
The organic acids are subsequently converted into hydrogen by photoheterotrophic
fermentation process. During growth of thermophilic bacteria, hydrogen production is directly linked to central metabolic pathways unlike the case during photoheterotrophic growth (de Vrije and Claassen 2003).
4.4.1 Hydrogen from Agro-Industrial Residue
Several forms of organic waste streams, ranging from solid wastes like rice straw,
black strap molasses (Nath et al. 2005) to waste water from a sugar factory and
a rice winery have been successfully used for hydrogen production. Most experiments have shown considerable hydrogen production with the limited number of
thermophilic strains used. The utilization of potato steam peels by the two phase approach has been examined. The organic acids, already present in the initial substrate
and additionally produced in the first fermentation step, are the substrates of choice
for the photo-heterotrophic fermentation (de Vrije and Claassen 2003). Assessment
of the total production of hydrogen and acetate from glucose and from equivalent
Table 4.5 Hydrogen and Acid Production from different carbon sources
Carbon source
Glucose
Potato steam peel
hydrolysate
Hydrogen
production (mM)
Maximum Hydrogen
productivity (mmol/L.h)
130
218
10.7
11.7
4 Biofuels
69
amount of sugars in potato steam peel hydrolysate (prepared by the action of amylase and glucoamylase) revealed that higher hydrogen production occurred from the
peels as reflected by the data in Table 4.5. (adapted from Claassen et al. 2004).
4.4.2 Production of Biohydrogen in Fermenter
Among three different reactors, namely, Continuously Stirred Tank Reactor (CSTR),
an Up flow Fixed Bed Reactor (UFBR) and an Upflow Anaerobic Sludge Blanket
(UASB) reactor, the CSTR gives the best performance with a yield of 0.30 L H2 /g
carbohydrate and production rate of 4.50 mmol H2 /L reactor, h. Its superior performance has been attributed to the mechanical stirring that promoted both H2 and
CO2 removal from the fermentation broth, therefore reducing feed-back inhibition
phenomena (Camilli and Pedroni 2005).
4.5 Coupling of Biohydrogen and Biomethane Production
The effluent from a hydrogen producing reactor containing high concentrations of
fatty acids may be subjected to a subsequent anaerobic digestion step with the conversion of the remaining organic content to biogas, mainly methane and carbon
dioxide (Antonopoulou et al. 2008). Methane yields from the mesophilic reactor
receiving effluent from the hydrogenogenic reactor emulate the yields of the original feed.
4.6 Bio Ethanol
4.6.1 Fermentative Production of Bioethanol
Bioethanol is a biofuel used as a petrol substitute, produced by simple fermentation
processes involving cheaper and renewable agricultural carbohydrate feedstock and
yeasts as biocatalysts. A variety of common sugar feedstocks including sugarcane
stalks, sugar beet tubers and sweet sorghum are used. The fermentation process is
mediated by two enzymes invertase and zymase, produced by the yeast cells. The
overall process steps are as follows (Zanichelli et al. 2007):
Hydrolysis
Biomass
Yeast
Complex Sugar
Simple Sugar
(Invertase)
(Acid or enzymatic)
(Cellulose or
Hemicellulose)
Yeast
Distillation
99.8% pure Ethanol
Fig. 4.2 The flow chart of basic ethanol production process
Zymase
Crude Ethanol &
Carbon dioxide
70
S. Chattopadhyay et al.
zymase
invertase
(C6 H10 O5 )n + nH2 O −−−−→ nC6 H12 O6 −−−→ 2nC6 H12 OH + 2nCO2
Figure 4.2 describes an overview of bioethanol production from cellulosic or
hemi cellulosic biomass. The first step is a pre-treatment step for the conversion
of cellulosic and hemicellulosic biomass into complex sugars by acid catalysts or
enzyme. The bioconversion of the complex sugars into bioethanol is mediated by
invertase and zymase as discussed above. But the ethanol produced from this fermentation process contains significant amount of water in it. To remove water, fractional distillation process is used, wherein the ethanol-water mixture is vaporized.
Bioethanol gets separated from water due to lower boiling point (78.3◦ C). After
distillation step the final product is enriched with 95% to 99.8% ethanol.
The discovery of continuous mode during 1960s permits recycling of yeast; increase the speed of the process and reducing the cost. The Yield % of theoretical
max in continuous process is around 95% as compared to less than 90% for batch
or fed-batch (Sánchez and Cardona 2008).
4.6.2 Bioethanol from Agro-Industrial Residues
Ethanol produced from renewable and cheap agricultural products reduces the green
house gas emissions like COX , NOX and SOX and eliminate smog from the environment. Agricultural residues and wastes have several advantages as they do not
require any additional lands because they are collected into piles at large agricultural and forestry facilities. Some of the agro-industrial residues and waste materials abundantly available are mentioned in Table 4.6, which are used as potential
substance for ethanol production in various countries.
Table 4.6 Agro-industrial residues and plant waste materials used for bioethanol production in
various countries
Agro-industrial
residues and plant
wastes
Thippi
Switchgrass
Corn steep liquor
(CSL)
Ami-ami solution,
Brewer’s yeast
autolysate and Fish
soluble waste
Waste Potato
Rice straw, oat straw,
wheat straw
Country
Bioethanol yield (% of
theoretical maximum)
Reference
India (Tamilnadu)
USA
USA and Brazil
93.18
72
−
Patle and Lal 2008
Asli et al. 2008
Ruanglek et al. 2006
Thailand
88
Ruanglek et al. 2006
Finland
87
Not specified
−
Liimatainen
et al. 2004
Kim and Dale 2004
4 Biofuels
71
4.6.3 Major Raw Materials Used in Bioethanol Production
4.6.3.1 Lignocellulosic Materials
Removal of lignin from lignocellulosic raw materials is the most critical step.
Among various methods physicochemical and biological methods are mainly used
for pre-treatment. Saturated steam at 160◦ C to 290◦ C and at high pressure (0.69
to 4.65 MPa) is used to convert hemicelluloses into soluble oligomers (Hamelinck
et al. 2005; Ballesteros et al. 2004). Lignin is not solubilized but redistributed.
Ammonia soaking of corn stover at room temperature can remove as much as 74%
of the lignin (Asli et al. 2008). The fungus Phanerochaete chrysosporium can also
be used for degrading lignin (Sánchez and Cardona 2008).
4.6.3.2 Non-lignocellulosic Materials
Thippi
Thippi that is an agro-industrial waste composed of starch, pectin, fiber and protein.
After pre-treatment at 121◦ C for 20 minutes, acid or enzymatic treatment is done.
Acid treatment is done with 0.75% H2 SO4 at 55◦ C for 3 h. Enzymatic treatment
is performed with various enzymes like amylase, pectinase and cellulase at 55◦ C
for 3 h at pH 5. Fermentation is carried out to use the reducing sugars obtained
from above process to produce ethanol (Patle and Lal 2008). Because of higher
yield % of maximum theoretical value (>90%) as compared to other available
non-lignocellulosic materials, thippi can be used as preferred substrate with great
potential for bioethanol production.
Switchgrass
Switchgrass (Panicum virgatum) is a perennial grass grown in warm season and
resistant to harsh conditions, pests, and diseases. It is capable of producing high
biomass yields at low fertilizer application rates. Untreated switch grass contained
42% cellulose, 31% hemi-cellulose, 6% acid detergent lignin (ADL), 22% klason
lignin and 0.7% ash. The yield % of theoretical max is 72% with simultaneous
saccharification and fermentation (Asli et al. 2008).
4.7 Biodiesel
Finite resources and gradually increasing demand for diesel all over the world lead
researchers to find some alternative sources. Emission of toxic green house gases
from the combustion of petroleum diesel is also a major contributing factor for this.
Right from the experiment of Rudolf Diesel using pea nut oil in his self-designed
engine (World Exhibition in Paris in 1900), numerous studies have followed to
establish the potential of triglycerides as alternative sources of diesel. But using
triglycerides directly into a diesel engine leads to some operational difficulties due
to its high viscosity and poor low temperature properties like pour point and cloud
point (Fukuda et al. 2001). This problem could be fixed by developing vegetable oil
72
S. Chattopadhyay et al.
derivatives that resemble properties of petrodiesel. Transesterification is the most
widely used process in which triglycerides react with an alcohol (mainly methanol)
in presence of chemical (acid or alkali) or biological (enzyme) catalysts to produce
mono alkyl esters, popularly known as biodiesel. Some alkali catalyzed batch processes have been commercialized.
4.7.1 Chemical Catalyzed Method
In this method, acid (H2 SO4 ) or alkali (NaOH or KOH) is used for transesterification
of triglycerides. Acid catalyzed reaction has some disadvantages which include very
high temperature and pressure and incomplete conversion. On the other hand, alkali
catalyzed process, though requires high temperature (about 80◦ C) is very quick with
higher conversion rates (Chongkhon et al. 2007; Vicente et al. 2004). Alkali catalyzed methods have been commercialized. This process has certain disadvantages
and transesterification with biocatalyst have been tried. Figure 4.3 shows biodiesel
production using alkali as catalyst.
Alkali + Methanol
Oil
Methanol
Transesterification
Glycerol
Methanol evaporation
Separation of
reaction mixture
Purification
Upper
layer
Waste water
Washing
Methyl ester
Fig. 4.3 The flowchart of Alkali catalyzed Transesterification
4.7.2 Enzymatic Transesterification
Mostly lipases from different sources are used for this process. To make the process cost effective immobilization of lipase has been done. This process can be
performed under normal temperature and pressure, but takes more time than alkali
catalyzed reaction. Percentage conversion is quite high and glycerol obtained in
this method is of good quality and can be directly used to produce some valuable
products (Fukuda et al. 2001). The enzyme catalyzed transesterification process is
summarized in Fig. 4.4. Methanol is preferred to other alcohols for its abundant
supply and diesel like quality of the transesterified product. By process optimization
over 90% conversions can be obtained. (Shimada et al. 2002).
4 Biofuels
73
4.7.3 Biodiesel from Agro-Industrial Residues
Biodiesel is generally produced from vegetable oils or animal fats. Various oils like
palm oil, soybean oil, sunflower oil, rice bran oil, rapeseed oil etc. are used. The
choice of vegetable oil used depends on its abundant availability in the country
where biodiesel is produced. To our knowledge, there are no reports available on
the use of agro-industrial residues for biodiesel production. However, bioethanol
produced from agro-industrial residues can in turn be used for the transesterification
of vegetable oils to produce mono ethyl esters of fatty acids as biodiesel. Some
residues can be successfully utilized as carbon sources for single cell oil production.
It reduces the fermentation costs (Peng and Chen 2007). Whey concentrate and
tomato waste hydrolysate, which contains more than 1 g/l total organic nitrogen, in
turn produce 14.3 % and 39.6 % lipid respectively can be used for gamma-linolenic
acid production. The amount of gamma-linolenic acid produced from these wastes
is 14.1% and 11.5%, which makes whey concentrate and tomato waste hydrolysate,
two good raw materials for biodiesel production (Fakas et al. 2008).
4.8 Future Prospectives
Rapidly spiraling crude oil prices and cost ineffectiveness of most of the biofuel
technologies, mainly due to expensive raw materials and manufacturing processes,
have fueled extensive worldwide search and utilization of agro-industrial residues
for the cost competitive production of alternative biofuels. The major gaseous biofuels, namely, biomethane and biohydrogen and the major liquid biofuels, namely,
bioethanol and biodiesel have evolved as potential alternative to the dwindling fossil
fuel resources. Bioethanol and biodiesel are gaining importance as alternative fuels to petrol and diesel respectively. Bioethanol, which is conventionally produced
from cane molasses by yeast fermentation, can also be produced from various agroindustrial residues and plant wastes. Efficient process optimization and integration
by combining production and recovery processes may lead to economic production of bioethanol. Switchgrass that grows mainly in the USA in drastic climatic
conditions and contains high percentage of cellulose and hemicelluloses generated
some excitement in the field of bioethanol production. Biodiesel on the other hand
Oil
Immobilized
Lipase
Separation of
reaction mixture
Methanol
Glycerol
Fig. 4.4 The flowchart of Enzymatic Transesterification
Methyl
Esters
74
S. Chattopadhyay et al.
is generally produced from vegetable oils. Agro-industrial residues are still not used
as the substrate for biodiesel, though the residual oil present in oil cake, a waste
product of oil extraction units, hold some hope. But in future, suitable residues with
high lipid content may be used as potential raw materials for biodiesel production.
Biotechnological techniques to produce biofuels from agro-industrial wastes and
residues are potentially effective in reducing the emission of toxic pollutants and
greenhouse gases, saving our environment and partly solving the worldwide fuel
crisis. By focusing the transformative power of biotech on challenges in biofuel production, while considering sustainability in all its dimensions, one can reasonably
hope to enable the ’second industrial revolution’ that our society now requires.
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Chapter 5
Production of Protein-Enriched Feed Using
Agro-Industrial Residues as Substrates
J. Obeta Ugwuanyi, Brian McNeil and Linda M. Harvey
Contents
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Technologies Applied to Waste Reprocessing for Protein Enrichment . . . . . . . . . . . . . . . .
5.2.1 Solid Substrate Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2 Silage Making (Ensiling) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.3 Waste Enrichment in Liquid and Slurry Processes . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Agro-Food Industry Wastes and Residues and Their Reuse Potentials . . . . . . . . . . . . . . . .
5.3.1 Lignocellulosic Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.2 Slaughter House Wastes and Manure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3 Fish and Fisheries Industries Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.4 Microbial Biomass (Single Cell Protein) from Animal Wastes . . . . . . . . . . . . . . . .
5.3.5 Wastes from Cassava and Other Roots and Tuber Crops . . . . . . . . . . . . . . . . . . . . .
5.3.6 Protein Enrichment of Fruit and Vegetable Wastes . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.7 Other Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.8 Feed Grade Enzymes from Agro-Food Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4 Conclusion and Safety Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Agricultural and food industry residues, refuse and wastes constitute a
significant proportion (estimated to amount to over 30%) of world wide agricultural
productivity. These wastes, include lignocellulosic materials, fruit and vegetable
wastes, sugar industry wastes as well as animal and fisheries operations refuse and
wastes. They represent valuable biomass and potential solutions to problems of animal nutrition and world wide supply of protein and calories if appropriate technologies can be deployed for their valorization by protein enrichment. Technologies
available for protein enrichment of these wastes include solid substrate fermentation, ensiling and high solid or slurry processes. Technologies to be deployed for
the reprocessing of these wastes will need to take account of the peculiarities of
individual wastes and the environment in which they are generated, reprocessed and
J.O. Ugwuanyi (B)
Department of Microbiology, University of Nigeria, Nsukka, Enugu, Nigeria
e-mail: jerryugwuanyi@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 5,
C Springer Science+Business Media B.V. 2009
77
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used. In particular such technologies need to deliver products that are safe not just
for animal feed use but also from the perspective of human feeding. The use of
organisms that are generally recognized as safe (GRAS) for the protein enrichment
and reprocessing of waste will enhance user confidence
Keywords Protein-enrichment · Solid state fermentation · Silage-making · Liquid
process · Slurry-process
5.1 Introduction
Agricultural and food industry residues, refuse and wastes constitute a significant proportion of world wide agricultural productivity. It has been variously estimated that up to 30% of global agricultural produce are left behind in the farms
as residues and refuse. This is in addition to the proportion of photosynthetically
produced energy that ends up as unused but potentially usable biomass (Robinson
and Nigam 2003; Graminha et al. 2007). Large volumes of wastes, both solids and
liquids, are also generated from the food processing industries. Together, these represent potential solutions to problems of animal nutrition if appropriate technologies
can be deployed for their valorization. The problems posed by the continuing accumulation of these wastes have been compounded by the increasing concentration of
food processing activities in large industries, particularly in the developed countries.
Even in the less developed nations, the need to transport large quantities of food to
the cities has led to increasing movement of processed food. In addition, emphasis on intensive agriculture to meet growing demand, as well as the concentration
of food processing facilities in small land areas have resulted in the production of
agro-food wastes in much higher concentrations than the available land space can
take for disposal. The non-utilization of these vast resources constitutes significant
loss of value.
Growing global demand for environmentally sustainable methods of production,
pollution prevention and also economic motives have changed the way wastes and
refuse are looked at. In other words, wastes have come to be seen more as resources
in the wrong location and form than as a problem to be safely disposed of. Aside
from their capacity to cause pollution, most food processing wastes are potentially
of good enough quality to be recycled as raw materials for other applications or/and
may be reprocessed to higher value products with relative ease. Even without any
biotechnological improvement or upgrading, a wide variety of these wastes are already in use, albeit in small quantities in animal nutrition and other biotechnological
processes (Martin 1998).
Developing and deploying appropriate technologies for the reprocessing and
reuse of these abundant energy rich resources in human or animal feeding will go a
long way in reducing the pressure on agricultural productivity and increase resource
utilization efficiency. In particular, it will help to fight protein-energy malnutrition in
those areas of the world where humans and animals compete for the same sources
of protein and calories. Besides reducing pressure on productivity and improving
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
79
global food security, efficient utilization of agricultural wastes will help to improve
environmental health particularly with respect to those wastes whose accumulation
constitute significant health hazards, and whose treatment and disposal incur considerable cost penalties.
Ultimately, some level of waste will arise from human productive activity, even
with the growing application of clean technology. However, the idea behind waste
minimization, which is the emerging face of waste management, requires that much
of what is considered as waste and refuse, particularly those of considerable calorific
value be seen first as valuable raw material for the production of value added product
than as waste for disposal. This approach is likely to lead to ever decreasing volumes
of waste meant for ultimate disposal. As transport and logistic costs will always
put constraints on the movement of the vast quantities of agro-food wastes away
from their point of production, reprocessing options need necessarily be designed
to handle the wastes at the point of production. Technologies based on small and
medium scale farm and factory based digesters and reactors should go a long way
in making the processes attractive and economically viable. This work will attempt
to review the current trend in biotechnological reprocessing by protein enrichment
of agricultural wastes for feed use.
5.2 Technologies Applied to Waste Reprocessing
for Protein Enrichment
5.2.1 Solid Substrate Fermentation
The term solid substrate fermentation (SSF) has been variously defined as the cultivation of microorganisms on solid, moist substrates in the absence of free aqueous
phase (Pandey 2003), or cultivation of microorganisms in the presence of a liquid
phase at maximal solid substrate concentrations (Mitchell et al. 2000), or on inert
carriers supporting moist substrate (Ooijkaas et al. 2000). SSF has been historically
used for the cultivation of microorganisms as it approximates the natural growth
condition of most microorganisms (particularly of filamentous fungi). In Asia and
Latin America, SSF has a traditional presence in the production of food and condiments such as tempeh, shoyu, and miso (and the fermented oil seeds common in
Africa). On the contrary SSF seems to be employed much less frequently in Europe,
except perhaps for its role in cheese and bread making.
SSF has continued to thrive on account of its labour convenience and generally
low technological level. The majority of studies on the application of SSF have concentrated on its use for production of different enzymes for mostly environmental
and industrial application (Couto and Sanroman 2006). But of particular interest,
and one which appears to have immediate commercial potential is the application of
SSF for the re-processing of bulk agricultural wastes for animal feed use (Laufenberg et al. 2003; Pandey et al. 2000a,b). SSF simulates the living conditions of
filamentous fungi in their natural habitat. It is therefore convenient to grow them in
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such environments. In addition, in reaction environments, such as all SSF processes,
where the ability to penetrate into the tissue or mass of solid substrates is important
these organisms are more suited for cultivation (Santos et al. 2004) than unicellular
ones, even though these have also been successfully employed in SSF (Virupakshi
et al. 2005; Prakasham et al. 2006).
In theory SSF is versatile enough to be applied in a wide variety of biotechnological processes. In practice however, it is currently most suited for processes that
generate low value bulk products such as applications for environmental control,
including the production of compost and animal feed from solid wastes, bioremediation and biodegradation of hazardous compounds, detoxification of industrial
wastes; nutritional enrichment of crops or crop-residues by biotransformation, biopulping, production of fermented foods, industrial enzymes, pigments, biopesticides,
organic acids and flavor compounds. Cultivation of microbial consortia can also
be more easily achieved in SSF than in submerged processes. Over the last few
years, several topical reviews have been written on the available SSF technologies
for the reprocessing and reuse of different agricultural and food industry wastes
particularly lignocellulosic and vegetable wastes (Nigam and Singh 1994; 1996;
Singh et al. 1996; Pandey and Soccol 2000; Pandey et al. 2000a,b; Pandey 2003;
Raghavarao et al. 2003; Tengerdy and Szakacs 2003; Das and Singh 2004; Singh
2004; Couto and Sanroman 2006; Graminha et al. 2007).
5.2.2 Silage Making (Ensiling)
Silage making is the (lactic) fermentation/storage of (forage) for use in animal (ruminant) feeding. Ensiling is a multistage process which ultimately results in low
pH (< 4.0) products that have extended resistance to spoilage. During ensiling,
some bacteria are able to break down some cellulose and hemicellulose to their
components sugars which are subsequently metabolized to low molecular weight
acids, mostly lactic acid. This can also be encouraged by the use of appropriate mix
of enzymes and microbial (lactic acid bacteria) silage inoculants (Okine et al. 2005;
Colombatto et al. 2004. The lactic acid bacteria are also believed to produce bacteriocins that discourage the growth of and spoilage by unwanted populations. Efficient fermentation ensures a palatable and digestible feed. Production of good
quality silage requires that anaerobiosis be achieved quickly to enable the lactic
acid bacteria to develop and predominate, and in the process further bring down
the pH of the mass. This discourages spoilage of the silage by putrefactive aerobic populations and ensures the retention of the most nutrients in the final product
(Arvidsson et al. 2008).
Silage making starts with the impounding of the biomass and is initiated by
aerobic populations. During this stage the aerobic organisms scavenge oxygen and
bring about anaerobiosis. This phase is undesirable, because the aerobic bacteria
consume soluble carbohydrates that should otherwise be available for the beneficial
lactic acid bacteria. It also leads to the production of moisture, and heat generation,
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
81
which if not properly managed are capable of destroying the process. Proteinaceaous
materials may also be rapidly broken down during this phase and this can lead to
loss of nutrients and the accumulation of ammonia (Slottner and Bertilsson 2006).
To encourage rapid acidification during ensiling, fermentable sugars and lactic
acid bacteria inoculants are often added to the silage (Okine et al. 2005; Yang
et al. 2006). This is common during ensiling of protein rich feeds such as manure, slaughter house and fish wastes as well as many agricultural residues such as
wheat straw, tomato or apple pomace and citrus waste (Chaudhry et al. 1998; Shaw
et al. 1998; Scerra et al. 2001; Vidotti et al. 2003; Bampidis and Robinson 2006;
Yang et al. 2006; Volanis et al. 2006; Santana-Delgado et al. 2008; Vázquez et al.
2008).
In the anaerobic stage of ensiling, a mixed population of lactic acid bacteria predominates and metabolizes fermentable sugars, producing lactic acid and reducing
the pH of the mass to acidic levels. As the pH drops, minor acetic fermentation (if
present) ends. This process continues until most of the available sugars have been
consumed, and the pH has dropped to a level low enough to discourage bacterial
activity. The duration of this stage varies with the nature of the biomass being ensiled, particularly, the initial concentration of fermentable sugars and the population
of lactic acid bacteria. Ensiling by itself hardly leads to protein enrichment of the
biomass except if mineral nitrogen is included. However, its capacity to achieve conservation of waste protein for use in animal feeding makes it important in schemes
for the reuse of agricultural refuse.
5.2.3 Waste Enrichment in Liquid and Slurry Processes
A number of agricultural wastes are produced with considerable moisture content
giving them a slurry consistency. In general the evaporation of such wastes to a
solid consistency prior to biotechnological improvement (by SSF) would increase
the cost of reprocessing and make them economically unattractive. Reprocessing
of such residues as citrus pulp, farm house slurries, potato process slurries, cane
process wastes has to be implemented as they are generated to be economically
viable (Stamford and Decamargo 1992; De Gregorio et al. 2002). Production of
protein enriched food (Teniola and Odunfa 2001; Nguyen et al. 2007a,b) and feed
(Ugwuanyi 2008a; Ugwuanyi et al. 2006; 2008) in slurry state fermentation have
been reported. The process has advantages in its capacity to operate at self heating
elevated temperatures, achieve simultaneous protein enrichment and pasteurization
of reprocessed waste, convert mineral nitrogen sources to protein rich microbial
biomass and produce high protein content waste for use in animal feeding, and
in employing high protein accumulating thermophiles to drive the process. Slurry
reactions may be very rapid (Ugwuanyi et al. 2008) and can be adapted for use in
the treatment of vast range of waste biomass, particularly those generated at elevated
temperatures. As slurry reactions are a midpoint between SSF and SmF they may
be expected to enjoy borderline advantages.
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5.3 Agro-Food Industry Wastes and Residues
and Their Reuse Potentials
The nature and quantity of waste generated from agricultural and food manufacturing practices vary with the predominant crop types and processing technology.
Similarly, the need to reuse available waste varies with the pressure on calorie
and the environment. In economies that produce mostly grains, large quantities
of straw are produced, while economies that produce mainly root and tuber crops
contend with high starch containing wastes. Although it is known that reuse of
these wastes can significantly improve the economics of crop production, their effective reuse has often been constrained by the available technology. In general,
the major wastes from agricultural and food industry operations include a variety of lignocellulosic materials, such as rice straw, wheat straw, maize stalk and
cobs, barley straw, cane bagasse, cassava bagasse, vegetable process wastes including starch wastes, sugar industry wastes as well as farm animal refuse and
waste from slaughter house and fisheries operations. Food processing industries
also generate large quantities of rejects, trimmings and other substandard food
materials that do not make it into the production chain. Fruit processing industries produce vast quantities of waste such as pomace and pulp that present disposal problems related to bulk and nutritional insufficiency making them unsuited
for large scale use animal feeding and expensive to dispose. From the fermentation industries, vast amounts of spent media and microbial biomass are also
generated.
5.3.1 Lignocellulosic Wastes
Agricultural refuse in this class are composed principally of cellulose, hemicellulose and lignin, and include cereal and vegetable wastes such as straw, bagasse,
stover, cobs, cotton husk, groundnut husk, fibrous remnants of forage grass among
others. They are arguably the most abundant agricultural wastes (Tengerdy and
Szakacs 2003). In general, wastes in this class are composed of nearly 50% cellulose on a dry weight basis while hemicellulose and lignin account for the balance
on a nearly equal basis (Bisaria 1998; Pandey et al. 2000a,b). On a global scale
the quantity of cellulosic wastes available varies with the predominant agricultural
and industrial crop produced in a given society. Although lignocellulosic wastes
have found significant application as sources of (heat and electric) energy, it is believed that considerable value addition may be achieved by using these wastes for
animal nutrition (Pandey et al. 2000a,b). Unfortunately their use in animal feeding
is constrained by very low content of protein, vitamin, oil and other nutrient and
limited digestibility and palatability to ruminants. However, they may be applied
for animal nutrition following protein enrichment by using a variety of micro and
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
83
macro fungi and bacteria. The predominant efforts in this direction have emphasized the use of SSF. In addition to the protein enrichment of wastes for use in
ruminant nutrition, they have also been employed as principal raw materials, as
a carbon source, for the fermentative production of feed related products such as
enzymes and organic acids, single cell oil and flavour compounds, etc. The use
of these wastes as principal carbon and energy sources for the production of microbial biomass protein such as single cell protein and mushrooms has also received considerable attention and a number of topical reviews exist in the literature
(See Table 5.1).
The choice of microbial type to grow on lignocellulosic wastes depends to a
large extent on the desired end product, and on whether or not a pre-treatment
step is included or needed in the cultivation process. Pre-treatment processes that
have been applied include steam explosion, acid, alkali, peroxide treatment, gamma
irradiation, and combination of two or more of these processes as well as the usually
more expensive but environmentally friendlier enzymatic treatment using a variety
of cellulases hemicellulases and ligninases, (See Chapters 20 and 22 of this volume). Table 5.1 shows some processes and organisms that have been employed at
both laboratory and pilot scale to achieve protein enrichment of some agricultural
produce of mostly lignocellulosic nature and other wastes for food use and animal (ruminant) nutrition. In many instances, protein enrichment of agro-food waste
may be accompanied by the economic extraction of valuable biochemicals such as
food/feed grade enzymes and organic acid. In most cases the production of protein
enriched lignocellulosic waste has been associated with the reduction in the content
of lignocellulose (associated with loss of biomass via microbial respiration as carbon dioxide). Of particular importance in the protein enrichment of lignocellulosic
waste for feed use is the improvement in palatability/acceptability/digestibility of
such treated wastes for ruminants (Misra et al. 2007). This is due to the enzymatic
disintegration of the lignocellulosic structure of plant cell wall. In addition, the low
technology and reduced reactor volume employed in the SSF process (Nigam and
Singh 1996) means that the process may be easily adapted for use in less affluent
farm communities.
Although agricultural and food industry waste can be cheap raw materials for the
production of enriched feed, the deployment of this technology may be constrained
by logistic costs, particularly in the less developed economies. As the feed so produced is bulky and of low economic value, a key “selling point” of the process
is the reduction of the pollution potential of the reprocessed waste. The implication of this for cost of the final product will be central to the economics of the
reprocessing technology. Whatever options are adopted, a direct local need, such
as the utilization of the produced feed for animal nutrition in the vicinity of the
producing facility will go a long way in increasing the competitiveness of the process and product. The challenge is to develop on-site applicable and scalable technologies for the enrichment of agro-food waste in the vicinity of waste generating
facilities.
Principal substrate
Microorganism used
84
Table 5.1 Microorganism grown on lignocellulose and other wastes in solid substrate fermentation and the products
Product/process/
Reference
J.O. Ugwuanyi et al.
Animal feed and food; protein
Cassava wastes (peels; slurry;
Saccharomyces cerevisae; Lactobacillus Ubalua 2007; Oboh and Elusiyan 2007;
enriched biomass, SCP; edible
bagasse; waste water); cassava
spp; Rhizopus oryzae; Rhizopus spp;
Oboh 2006; Obadina et al. 2006;
mushroom; cyanogenic glycoside tubers Cassava starch; wastewater Aspergillus niger; Aspergillus spp.;
Fagbemi and Ijah 2006; Srirotha
detoxification; Protein enriched
Cephalosporium eichhorniae;
et al. 2000; Soccol 1996;
flour Glutamic acid; citric acid;
Pleurotus spp; Lentinus spp,
Balagopalan 1996; Daubresse
volatile compounds
Brevibacterium divaricatum;
et al. 1987; Brook et al. 1969; Zvauya
Geotricum fragrans
and Muzondo 1994; Nguyen
et al. 1992; Noomhorm et al. 1992
Jyothi et al. 2005; Damasceno
et al. 2003
Protein enrichment; anti-nutrient
Coffee pulp; coffee husk; other
Streptomyces; Pleurotus spp
Orozco et al. 2008; Salmones et al. 2005;
removal; protein rich biomass
coffee wastes
Brand et al. 2001; Fan et al. 2000
Single cell oil; protein enriched
Wheat bran/straw/corn
Microsphaeropsis sp; Streptomyces
Peng and Chen 2008; Certik et al. 2006;
straw/feed; single cell protein;
stover/buckwheat/ millet/sugar
cyaneus; various Basidiomycete
Chaudhary and Sharma 2005;
mushroom; gamma linoleic acid;
beet pulp/citrus waste/water
fungi; Coprinus-fimetarius
Varnayte and Raudonene 2004; Basu
citric acid; vitamins; essential
hyacinth; Mustard straw; bean
Micromycetes; Phanerochaete
et al. 2002; Berrocal et al. 2000;
amino acids Medicinal fungus;
straw; agave bagasse
chrysosporium; Pleurotus ostreatus;
Woomer et al. 2000; Dorado
feed
Agro-residues; Perennial grass
Thamnidium elegans; cellulolytic
et al. 1999; Zadrazil 1997; Singh
bacteria; Neurospora sitophila;
et al. 1995; Tripathi and Yadav 1992;
Rhodotorula gracilis; Trametes spp
Jacob 1991; Vladimirova et al. 1995;
Ganoderma spp, Coriolus
Iconomou et al. 1998; Mukherjee and
versicolorTrichoderma spp Lentinus
Nandi 2004 Tripathi et al. 2008; Misra
edodes; Cellulomonas biazoteain
et al. 2007; Ortiz-Tovar et al. 2007,
Philippoussis et al. 2007;
Gaitan-Hernandez et al. 2006;
Rajoka 2005
Protein rich fungi and feed; single Apple pomace; apple waste; apple Rhizopus oligosporus; Candida utilis
Rodriguez-Ramirez et al. 2007;
cell protein
pulp; grape waste; carob pod;
and Pleurotus ostreatus;
Albuquerque et al. 2006; Villas-Boas
pineapple waste
Kloeckera-apiculata; Penicillium
et al. 2003; Nigam and Singh 1996;
funiculosum Myrothecium verrucaria
Nigam 1998; Rahmat et al. 1995;
Aspergillus niger; Saccharomyces spp
Bhalla and Joshi 1994; Kuzmanova
et al. 1991; Correia et al. 2007
Product/process/
Principal substrate
Protein enriched feed
Cactus pear; cactus waste fibre
Protein rich biomass/feed; Protein
rich mushrooms
Microorganism used
Saccharomyces cerevisae; Aspergillus
niger
Rice polishing/rice
Candida utilis; Aspergillus niger;
bran/straw/chaff; sago fibre; saw
Trichoderma viride; Pleurotus
dust; paddy straw;
sajor-caju; Pleurotus ostreatus
Lignocellulosic waste
Trichoderma reesei, Saccharomyces
cerevisiaes; A. oryzae
Protein rich food/feed
Protein enriched silage
Fruit aroma
Protein rich feed
Viticulture waste
Corn straw
Various agro waste
Cane bagasse and residues; other
cane wastes in solid and slurry
Pleurotus spp
Silage population; silage and ssf
Ceratocystis f mbriata
Trichoderma reesei and Trichoderma
viride; Aspergillus niger; white rot
fungi; Pleurotus spp
Protein enriched waste feed
Protein rich waste/feed/single cell
protein
Protein enrichment
Saw dust
Mango waste; date industry waste
Pleurotus spp
Pleurotus spp
Sugar beet pulp
Trichoderma reesei; Trichoderma
aureoviride
Various yeasts
Sclerotium rolfsii, Trichoderma
harzianum, Trichoderma
longiobrachiatum, Trichoderma
koninggi and Aspergillus niger
Candida utilis, Pichia stipitis,
Kluyveromyces marxianus,
Saccharomyces cerevisiae;
Indigenous microbes
Protein enrichment; scp production Cashew waste
Protein enrichment; Cellulose
Palm kernel cake
degration
Protein enriched waste; hydrolytic
enzymes; single cell protein
Cabbage waste; Chinese cabbage
Reference
Araujo et al. 2005; Oliveira et al. 2001
Rajoka et al. 2004; Bonatti et al. 2004;
Ravinder et al. 2003; Vadiveloo 2003;
Yang et al. 2003; Anupama 2001;
Banik and Nandi 2004; Youssef and
Aziz 1999; Patrabansh and
Madan 1997
Sanchez et al. 2002; Zhang et al. 2008
Yang et al. 2001;
Bramorski et al. 1998
Valino et al. 2002; 2003;
Gutierrez-Correa et al. 1999; Zadrazil
and Puniya 1995; Ortega et al. 1992;
Echevarria et al. 1991
Lal and Panda 1995
Jwanny et al. 1995
Israilides et al. 1994; Illanes et al. 1992
De Holanda et al. 1998
Iluyemi et al. 2006
Choi and Park 2003; Krishna and
Chandrasekaran 1995
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
Table 5.1 (Continued)
85
86
J.O. Ugwuanyi et al.
5.3.2 Slaughter House Wastes and Manure
Large scale production of animal products including meat and poultry, and the processing and packaging of these in large scale facilities has resulted in the generation
of large volumes of wastes including blood, feather, hoofs, horns, poultry intestines
among others. The world wide production of chicken intestine runs into several
million tons (Shaw et al. 1998). Often these wastes are treated on site for disposal,
resulting in considerable loss of otherwise useful biomass. The high protein content
of these wastes makes them attractive for use in animal nutrition. However, reprocessing them for use in animal nutrition can be a considerable challenge due to their
ease of spoilage. Consequently, techniques applied to these wastes are essentially
preservative. Fermentative ensiling has been studied extensively as an economical
process to reprocess these wastes for use as ingredients in animal feeds as alternative to the conventional, but more expensive fish and soy meal. As animal offals
are poor in carbohydrates, preservation is usually effected following addition of
fermentable sugars (Shaw et al. 1998). Ensiling of animal wastes also has the advantage of causing reduction in the level of pathogenic organisms present in the
wastes.
Reuse of poultry litter and manure for animal feeding has also attracted considerable attention due to the inefficiency of feed utilization by poultry. Pure and mixed
culture lactic acidification and silage type reactions have been variously applied to
improve the protein content and preservation quality and improve the smell of poultry manure for use in the production of poultry layers, beef cattle and pigs without
impairing performance (Lallo et al. 1997; El Jalil et al. 2001). The process is energy
efficient and has enabled the use of much higher concentration of animal waste in
feed formulation than could be achieved with chemically or physically modified
litter and offal (Kherrati et al., 1998).
Lactic acidification of animal wastes is particularly interesting in the tropics
where the process can be very rapid and the storage life of this form of waste can
be very short, with putrefaction starting only a few hours following the collection
of offal and litter. Lactic acidification also leads to rapid elimination of spoilage and
pathogenic organisms Shaw et al. 1998. A slight decline in protein content of the
waste (if it happens) can be compensated for by the fact of reuse of the waste, rather
than incurring cost penalties in its disposal.
Long term preservation by lactic preservation of slaughter house sludge for use in
animal feeding has been demonstrated in processes that also achieve rapid inactivation of potential pathogens and spoilage organisms (Skrede and Nes 1988; Urbaniak
and Sakson 1999; De Villiers and Pretorius 2001). As regulatory control of sludge
disposal tightens, the process could play pivotal roles in waste minimization during
animal production. A slightly different approach to the protein enrichment of (poultry) manure was reviewed by El Boushy (1991), which involves the use of house fly
pupae as the protein enriching principle. Although this technology appears promising for the protein enrichment/extraction from poultry manure and other protein
rich wastes, the necessity for sterilization at elevated temperature and its attendant
energy cost may be a disincentive for the use of this technology, besides its aesthetic
drawbacks.
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
87
5.3.3 Fish and Fisheries Industries Wastes
In the fish and fisheries industries including shrimp and crustacean processing, large
amounts of wastes including rejects, discards and by-products, are produced worldwide. It is estimated that up to 30% of the total landings in the fisheries industries are considered as underutilized, by-catch and unconventional or unexploited
(Venugopal and Shahidi 1995; Evers and Carroll 1996, 1998). It has also been
estimated that over 32 million tones of fish wastes accumulate annually from the
processing of fish (Kristinsson and Rasco 2000). Although a proportion of these
get reprocessed into fish meal, oil and cake, several tons end up as waste requiring disposal. (Arvanitoyannis and Kassaveti 2008; Arvanitoyannis and Ladas 2008)
provide a current review on the environmental position of fishery and meat wastes
and management processes that are being considered for handling these, particularly
within the context of the European Union where several directives and legislations
aim to control their disposal. A prominent approach to the valorization of these
wastes involves their biotechnological reprocessing (particularly by ensiling) for
use in animal nutrition.
The ensiling of fish and shrimp wastes for use in fishery, as well as other animal
nutrition, is receiving considerable attention and several studies have reported on
the optimization of processes for the reuse of these wastes. In addition to using
the ensiled feed for fish culture the processes lead to effective management of the
waste. De Arruda et al. (2007) have shown that the use of ensiled fish waste in fish
feeding can significantly improve the economics of fish production, considering that
feeding accounts for up to 60% of cost. Dong et al. (2005) and Ngoan et al. (2000)
used ensiled shrimp waste to replace soy meal in the feed of duck and pigs with
comparable efficiency. Coello et al. (2002) optimized fish waste ensiling for the
production of L-lysine using Corynebacterium glutamicum. The lactic preservation
of these wastes by ensiling alone or with a variety of straw, forage and molasses has
been shown to increase storage life of the product and increase acceptability, intake
and digestibility by poultry, cattle, fish and other animals with the possibility of the
silage serving a probiotic function (Faid et al. 1997; Hammoumi et al. 1998; Gerona
et al. 2007; Goncalves and Viegas 2007). Importantly, rapid lactic preservation has
enabled the reuse of an abundant waste, the disposal of which would otherwise
attract considerable cost penalties.
5.3.4 Microbial Biomass (Single Cell Protein)
from Animal Wastes
A slightly different approach to the valorization of animal and fishery waste is the
hydrolysis and conversion wastes to single cell protein. Horn et al. (2005) used
hydrolysate of cord viscera which constitutes about 17% of the fish biomass to grow
Lactobacillus spp. and demonstrated that the medium so formulated was as effective
as commercial peptone based media in the cultivation of the organism. This underscores the potential for the use of fishery waste of this kind for the cultivation of even
fastidious organisms for the production of microbial biomass. Kuhn et al., (2008)
88
J.O. Ugwuanyi et al.
fed microbial biomass produced from fish effluent to shrimps and demonstrated that
the process improved the economics of shrimp production. In addition, the process
led to the effective treatment of the resulting effluent. Single cell protein production
for feed use has been achieved by cultivation of organisms on ram horn hydrolysate
(Kurbanoglu and Algur 2002; Kurbanoglu 2003), poultry process waste Najafpour
et al. 1994 and acid hydrolysed shrimp waste (Ferrer et al. 1996). Composted fish
waste has been used for the production of Scytalidium aciabphilum biomass in submerged fermentation with good protein yield for animal feeding. Amar et al. (2006)
also employed bacterial digestion of fish waste to produce feed for the production
of Indian white prawn and in the process achieved both treatment and reuse the
fish waste. Schneider et al. (2006) produced protein enriched bacterial biomass for
animal feed use from a suspended growth process using aquaculture waste and in
the process achieved treatment of a particularly recalcitrant waste stream. Viera
et al. (2005) used microalgae to treat fish pond waste water effluent, and demonstrated that the protein rich algal biomass could be used as feed for the production
of abalone.
5.3.5 Wastes from Cassava and Other Roots and Tuber Crops
Root and tuber crops including cassava, potato, cocoyams and yams are the principal sources of calories in many countries. The processing of these crops for human
nutrition often results in the generation and disposal of several tones of carbohydrate
rich wastes. Cassava, which is acknowledged to be a most important source of calories for large populations in the tropics, ranks as the world’s sixth most important
food crop (Soccol 1996). Besides its significant place in tropical and global food
security, cassava has recently become recognized as an industrial crop in many countries where it is playing significant roles in animal nutrition and supply of industrial
starch (Obadina et al. 2006).
5.3.5.1 Protein Enrichment of Cassava Wastes
Estimates vary considerably, but in the processing of cassava into food and starch,
waste biomass may account for up to 30% of total produce (Antai and Mbongo 1994).
Cassava wastes are very toxic due to the disproportionate partitioning of cyanogenic
glycoside into the waste. As a result, without treatment, the waste can only be used
in limited quantities in animal nutrition, while the capacity of cassava waste to cause
pollution limits the disposal of the waste to land. Apart from the problem of toxicity,
use of the waste in animal nutrition is also constrained by its limited protein content. Yet cassava waste remains a valuable resource which if widely used in animal
nutrition can reduce pressure on food crops. In order to achieve the reuse of cassava
wastes, a number of processing methods have been reported which result in detoxification and improvement in the protein content of the waste thereby converting this
strong environmental pollutant to a value added product.
A number of processes have been implemented using cassava waste alone or
in combination with other waste types, including poultry droppings to achieve
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
89
reprocessing and protein enrichment of wastes. Organisms that have been employed
in the protein enrichment and detoxification of cassava process wastes for use in
ruminant nutrition include Aspergillus spp, Trichoderma spp. as well as a variety
of bacteria, yeasts and ruminal microflora (Noomhorm et al. 1992; Oboh 2006;
Adeyemi et al. 2007. See also Table 5.1). The carbohydrate content of cassava
waste has also been variously exploited for the production of various food additives
and ingredients, including citric acid and lactic acid (John et al. 2006; Pandey and
Soccol, 2000; Ghofar et al. 2005). Although room exists for improvement of the
protein content of the product, it is interesting that by the application of relatively
inexpensive solid substrate processes cassava waste could be converted into useful
products rather than being disposed of by expensive waste treatment strategies.
5.3.5.2 Protein Enrichment of Cassava
Various approaches have been studied in small laboratory and pilot scales for
the protein enrichment of cassava for food use ((Brook et al. 1969; Daubresse
et al. 1987; Oboh and Akindahunsi 2003). The processes led to slight drop in the
carbohydrate content of the food. However, it was considered that the drop in carbohydrate was compensated for by the increase in the protein content of the resulting
foods. Cassava starch has found considerable application in the production of food
and feed grade single cell protein using various microorganisms (Ejiofor et al. 1996;
Srirotha et al. 2000).
5.3.5.3 Protein Enrichment of Cocoyam Waste
Cocoyams (Colocasia and Xanthosoma spp) are widely cultivated for food in West
Africa, Asia and the Oceania, with Nigeria, China and Ghana leading in world
production (Onwueme and Charles 1994). In 1999, world wide production of cocoyams topped 6.5 million tones with Africa producing over half of the total
(Onwueme 1999). In producing countries, cocoyams account for a significant proportion of the total energy intake, and this varies from about 7% in Ghana to
about 18% in parts of the Oceania (Horton 1988). The processing of cocoyams
to food and starch is associated with the generation of vast quantities of waste
and residue that account for a significant proportion of the entire cocoyam produce
(Ugwuanyi 2008b). The preservation and reuse of these vast wastes in animal nutrition will enhance food security in areas where cocoyams are abundant. Duru and
Uma (2003a,b) have demonstrated the potential of using SSF to achieve over 50%
increase in the protein content of cocoyam process waste using Aspergillus oryzae.
The protein enriched waste could be used for the feeding of both ruminants and
monogastric animals.
5.3.5.4 Protein Enrichment of Potato and Sweet Potato Wastes
Gelinas and Barrette (2007) employed Candida utilis to improve the protein content
of waste potato starch from a chip manufacturing facility. Up to 11% protein was
accumulated in a process that yielded 8% yeast protein in a submerged fermentation.
90
J.O. Ugwuanyi et al.
In a process that mixed sweet potato and sugar cane, Rodriguez et al. (2005)
achieved improvement in the protein content of waste digest. Other processes in
which potato process pulp and waste water, and sweet potato wastes have been
reprocessed for protein enrichment including production of food and feed grade
SCP have been reported (Yang 1993; Yang et al. 1993; Abu et al. 2000; Okine
et al. 2005). The processes have been operated as SSF reactions, silage and as
submerged fermentations.
5.3.6 Protein Enrichment of Fruit and Vegetable Wastes
5.3.6.1 Fruit Industry Wastes
Growing international production and marketing of fruits has led to increasing accumulation of fruit wastes such as citrus pulp, seeds and peels, grape pommace among
others (Volanis et al. 2006; Bampidis and Robinson 2006). Disposal of these wastes
can be a major cost component of fruit production since they may account for up to
50% by weight of fruits (Scerra et al. 2000; Graminha et al. 2007). These materials
are very high in cellulosic materials (cellulose and hemicellulose), but low in lignin,
making them potentially good feed sources for ruminants and promising substrates
for the production of microbial protein. In countries with inadequate supplies of conventional ruminant feeds the use of fruit industry waste can impact quite positively
on the supply of feed for animal nutrition while reducing environmental pollution.
Unfortunately, fruit wastes have only minimal protein content which limits their
value in animal nutrition. So, exploitation of these wastes in animal nutrition will
depend on the deployment of processes for their protein enrichment by biotechnological means (Hang and Woodams 1986; Hang et al. 1987; Shojaosadati and
Babaripour 2002; Volanis et al. 2006).
Fermentative processes in both SSF and slurries employing both filamentous
and unicellular microorganisms have been employed for the protein enrichment of
a variety of fruit industry wastes for animal feed use. (Shojaosadati et al. 1999;
Scerra et al. 2000; De Gregorio et al. 2002; Correia et al. 2007; Plessas et al. 2008;
Vendruscolo et al. 2008). Up to 500% protein enrichment of apple pomace using
a combination of Candida utilis and Pleurotus ostreatus has been reported (VillasBoas et al. 2003) and these wastes have also been employed to produce food and
feed grade SCP. Protein enrichment and detoxification of coffee pulp for animal
feed use has been reported (Orozco et al. 2008). Sunita and Rao (2003) used mango
processing waste to produce blue green algal biomass for the production of Tilapia.
5.3.6.2 Vegetable Waste
Vegetable waste including trimmings, pressing fluids and rejects account for significant proportions of vegetable produce world wide. These wastes have high content
of fermentable sugars and are very perishable. As a result, they have been treated for
protein enrichment by a number of processes including ensiling and solid substrate
5 Production of Protein-Enriched Feed Using Agro-Industrial Residues as Substrates
91
fermentation. Vegetable wastes that have been reprocessed using food grade yeasts
include Chinese cabbage juice, waste brine generated from kimchi production, deproteinized leaf juices, corn silage juice, date waste, tea process waste (Chanda and
Chakrabatri 1996; Nancib et al. 1997; Choi and Park 1999; Choi et al. 2002; Hang
et al. 2003; Murugesan et al. 2005). Stabnikova et al. (2005) produced specialty
selenium enriched Saccharomyces cerevisae biomass by growing the organism in
extracts of cabbage, watermelon, a mixture of residual biomass of green salads and
tropical fruits.
5.3.7 Other Wastes
5.3.7.1 Olive Mill and Other Lipid Wastes
Production of olive oil, an important produce in some Mediterranean countries, particularly Spain, Italy, Greece and Tunisia, results in the production and disposal
of large volumes of strongly polluting and toxic olive mill wastewater (OMW)
(Israilides et al. 1997; Christodoulou et al. 2008). The considerable biomass content of OMW has necessitated efforts being made to develop biotechnological processes for the valorization of the waste. Fermentation and composting of OMW
mixed with a variety of agricultural wastes as bulking agents have been practiced as
means of detoxifying the waste (Haddadin et al. 1999; Garrido Hoyos et al. 2002;
Mantzavinos and Kalogerakis 2005; Laconi et al. 2007; Hachicha et al. 2008;
Cayuela et al. 2008). The use of OMW for production of microbial biomass
as part of the detoxification process, and use of the detoxified waste/produced
biomass as source of vitamins and mineral in animal nutrition has also been studied
(Gharsallah 1993; DeFelice et al. 1997; Haddadin et al. 1999; Sampedro et al. 2005,
2007; Aloui et al. 2007; Christodoulou et al. 2008). Recently, several workers have
studied the possibility of using OMW in media for the production of edible mushrooms with promising results (Zervakis et al. 1996; Tsioulpas et al. 2002; Roig
et al. 2006; Soler-Rivas et al. 2006; Kalmis et al. 2008. See also Table 5.1).
5.3.8 Feed Grade Enzymes from Agro-Food Wastes
A variety of agricultural products contain anti-nutrients that limit their value as feedstuffs. Reduced nutrient utilization due to these anti-nutrients can contribute to environmental pollution due to excessive excretion of unabsorbed nutrients, especially
nitrogen (N) and phosphorus (P), and increase production costs due to inefficient
feed utilization (Woyengo et al. 2008). The use of enzymes to increase digestibility
and nutritional value of feed has increased recently, and various depolymerizing
enzymes have found their way into animal feed as supplement and additives (Selle
et al. 2003; Roopesh et al. 2006; Nortey et al. 2007; Sands and Kay 2007). The
use of solid substrate processes for the production of various feed grade enzymes
including xylanases, cellulases, pectinases, chitinases, phytases, and ligninases is
attractive, because in addition to its process engineering advantages, the enzyme
92
J.O. Ugwuanyi et al.
may be produced and used (in situ) in the feed, in situations where the substrate
is also the feed undergoing (protein and enzyme) enrichment leading to enhanced
digestibility (Karunanandaa and Varga 1996; Kang et al. 2004; Carmona et al. 2005;
Couto and Sanroman 2005; 2006; Mazutti et al. 2006; Roopesh et al. 2006; Mamma
et al. 2008). The application of organisms with GRAS status for this process should
be valuable and promising in the reuse of lignocellulosics for animal feeding. The
use of exogeneous enzymes to improve the nutrient availability of feed has found
most application in silage making, where a number of depolymerising enzymes
are commercially available as silage additives (Schimidt et al. 2001; Colombatto
et al. 2004).
A number of studies and reviews on SSF production, and use of depolymerising
enzymes in animal nutrition, have been published recently (Juanpere et al. 2004;
Titi and Tabbaa 2004; Eun et al. 2006; Cao et al. 2007; Graminha et al. 2007).
5.4 Conclusion and Safety Considerations
Global perception of wastes in general and agricultural wastes in particular is changing rapidly in response to need for environmental conservation sustainable agricultural productivity and global food security. Consequently, wastes are more currently
seen as resources in the wrong form and location that needs to be reprocessed and
reused than as wastes to be disposed of. This has increased the need for appropriate
technologies for the reprocessing of such wastes. Protein enrichment of wastes for
use in animal nutrition offers opportunities for the reuse of abundant agricultural
wastes and refuse. Biotechnological processes such as solid substrate fermentation
and ensiling offer great opportunities for the reuse of abundant agricultural wastes
in animal nutrition. The use organisms with GRAS status to effect the protein enrichment, enzyme production and biomass production and detoxification reactions
will help improve confidence in the final products derived from these processes, and
help drive development of the application of biotechnology for the valorization of
agricultural waste.
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Chapter 6
Aroma Compounds
Syed G. Dastager
Contents
6.1 Aroma Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.1 Types of Aroma Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Production of Aroma Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1 Production of Aroma Compounds from Plant Cell Cultures . . . . . . . . . . . . . . . . . .
6.2.2 Aroma Production from Microbial Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3 Using Agro Wastes as Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4 Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5 Application of Aroma Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
106
106
106
111
112
117
120
120
121
Abstract The increasing demand for natural products in the food industry has
encouraged remarkable efforts towards the development of biotechnological processes for the production of aroma compounds. This chapter deals with major
achievements reported in this field, with a special emphasis on the potential lying in plant cell, microbial cultures and enzyme technology for the production of a
wide range of flavours. The use of solid-state fermentation as a means to improve
economical feasibility of these processes and application of aroma compounds. In
order to understand the flavour of (traditional) foods a multitude of scientific investigations were carried out and a number of appropriate analytical tools for flavour
research were developed in the past few decades.
Keywords Aroma-production · SSF · Aroma-compounds · Microorganisms ·
Aroma-application · Terpenes · Alcohols · Vanillin · Methyl ketones · Diacetyles ·
Pyrizines
S.G. Dastager (B)
National Institute of Interdisciplinary Science and Technology (Formerly
RRL), CSIR, Industrial Estate, Thiruvananthapuram-695019, Kerala, India
e-mail: syedmicro@gmail.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 6,
C Springer Science+Business Media B.V. 2009
105
106
S.G. Dastager
6.1 Aroma Compounds
An aroma compound, also known as odorant, aroma, fragrance or flavor, is a chemical compound that has a smell or odor. Aroma compounds can be found in food,
wine, spices, perfumes, fragrance oils, and essential oils. The world of aroma is
very attractive especially because it concerns the taste of what we eat (Aguedo
et al. 2004). From a scientific and technological point of view, this field is also
highly exciting since it brings together several different branches of science. Aroma
is usually the result of the presence, within complex matrices, of many volatile
components of various chemical and physicochemical properties. The processing
of mixtures of raw food materials can have various sensorial impacts depending on
the properties of each compound. Processing modifies the equilibrium between the
different components and, as a result, the original aroma will be perceived as being
weaker and as artificial or chemical. The work of the aroma formulator consists
in constructing a flavour recalling a true and original aroma in a processed food
product with a specific texture and composition. Aroma compounds can be extracted
from fruits or vegetables but, as they are required in the product in concentrations
comparable to those in the source material, this utilizes high amounts of materials
and is generally not economically realistic. Most of them can also be synthesized
in a chemical way resulting in chemical compounds that are not well perceived
by consumers whose demand, especially in Western Europe, is in favour of natural
products. As an alternative, biotechnology proposes to use enzymes or whole cells to
produce aroma compounds. Flavors and fragrances constitute a world-wide market
of US$ 7 billion a year, with a share of 25% of the food additives market (Armstrong
and Yamazaki 1986). The consumer’s preference for natural food additives is more
important than ever.
6.1.1 Types of Aroma Compounds
There are various families of aroma compounds and the differences used to classify
these families can be based not only on chemical structures, physicochemical properties or sensorial properties of the compounds but also, and in fact more commonly,
on the chemical family of the substrate. On this latter basis, lipid-derived aroma
compounds constitute one of the most important families, which include volatile
fatty acids or esters, lactones, aldehydes, alcohols, ketones and some groups such as
carotenoid-derived aroma compounds (Table 6.1). Although there are many investigations into the natural generation of these compounds in food products, only a few
aroma components are produced by biotechnological routes.
6.2 Production of Aroma Compounds
Aroma production constitutes an important sector in the chemical industry. The
aromas are compounds utilized in the manufacture of cosmetics, perfumes, cleaning products and food processing. Traditionally, aromas have been extracted from
Alcohols
Aldehydes
Esters
Fatty acids
Ketones
Lactones
Aromatic
compounds
1,2-butanediol
Acetaldehyde
methyl acetate
Acetate
acetophenone
␦-decalactone
vanillin
2,3-diethyl-5-methyl
pyrazine
2-butanol
Decanal
ethyl acetate
Butyrate
acetone
␥-decalactone
benzaldehyde
2-ethyl-3,5dimethlypyrazine
2,3-butanediol
Heptanal
ethyl butyrate
Caproate
2,3-butanedione
␥ -butyrolactone
-phenethyl
alcohol
2-methoxy-3isopropylpyrazine
Ethanol
(Z)-4-heptenal
ethyl hexanoate
Decanoate
2,3-pentandione
␦ -dodecalactone
trimethylbenzene
2-methoxy-3isopropylpyrazine
2-ethylbutanol
Hexanal
ethyl isobutanoate Isobutyrate
2-butanone
␦-octalactone
2-ethylhexanol
2-hexanal
ethyl octonate
2-methylbutyric acid
3-hydroxy2-butanone
(z)-6-dodecan␦-lactone
2-heptanol
Isohexanal
ethyl butanoate
3-methylbutyric acid
Hexanol
2-methylbutanal
isobutyl butanoate Octanoate
2-hexanone
Isobutanol
3-methylbutanal
2-methyl-1-butyl
acetate
phenylacetate
3-methyl2-butanone
2-methylbutanol
2-methylpropanal
3-methyl-1-butyl
acetate
propionate
4-methyl2-pentanone
Valerate
3-methylbutanol
Nonanal
3-octyl acetate
2-methylpropanol
(E,E)-2,4nonadienal
pentyl acetate
Pyrazines
6 Aroma Compounds
Table 6.1 Classification of food aroma compounds based on their chemical structure
2-heptanone
2-nonanone
2-octanone
107
108
Table 6.1 (Continued)
Alcohols
Aldehydes
Esters
Fatty acids
Ketones
2-nonanol
(Z)-2-nonenal
phenethyl acetate
(Z)-1,5-octadien-3-ol
(E)-2-nonenal
ethyl butyrate
2-pentanone
2-octanol
Octanal
propyl butyrate
3-pentanone
1-octen-3-ol
Butanal
2-hidroxyethyl
2-tridecanone
1-pentanol
Pentanal
propionate
2-undecanone
Phenylethanol
Propanal
2-methyl-2-ethyl3hydroxyhexyl
propionate
2-phenylethanol
Propenal
ethyl 2methylbutanoate
1-nonanol
thiophen-2aldehyde
ethyl 3methylbutanoate
Lactones
Aromatic
compounds
Pyrazines
1-octen-3-one
S.G. Dastager
6 Aroma Compounds
109
plants, but in general these procedures are low yield processes. Aromas can also be
produced by chemical synthesis, however there is a clear consumer preference for
products of natural origin. For these reasons, there is an increasing scientific interest in searching for aroma production alternatives, different from processes based
on extractive or chemical synthesis. Therefore, several biotechnological approaches
have been considered as real options for aroma production (Berger 1995).
The use of biotechnology for the production of natural aroma compounds by fermentation or bioconversion using micro-organisms is an economic alternative to the
difficult and expensive extraction from raw materials like plants (Harlander 1994;
Janssens et al. 1992). A fungus with aromatic properties and often referred to as a
yeast, Geotrichum candidum, has been used for commercial cheese ripening (Jollivet et al. 1994). Some strains may produce fatty acids esters, often related to specific fruit aroma (Koizumi et al. 1982; Latrasse et al. 1987). G. candidum is highly
lipolytic with a whole range of substrate specificity (Jacobsen et al. 1990; Sidebottom et al. 1991). Its proteolytic activity may also form aroma compounds and
has been partly characterized by Gueguen and Lenoir (Gueguen and Lenoir 1975,
1976). A vast array of compounds may be responsible for the aroma of the food
products, such as alcohols, aldehydes, esters, dicarbonyls, short to medium-chain
free fatty acids, methyl ketones, lactones, phenolic compounds and sulphur compounds (Gatfield 1988; Urbach 1997). Since early times, aroma compounds ranging
from single to complex substances have been extracted from plant sources. Eventually, after elucidation of their structure, synthetic aroma was produced by chemical
synthesis.
Nowadays, aroma represent over a quarter of the world market for food additives and most of the aroma compounds are produced via chemical synthesis or by
extraction from natural materials. However, recent market surveys have shown that
consumers prefer foodstuff that can be labelled as natural. Although aroma may be
produced by chemical transformation of natural substances, the resulting products
cannot legally be labelled as natural. Furthermore, chemical synthesis often results
in environmentally unfriendly production processes and lacks substrate selectivity,
which may cause the formation of undesirable racemic mixtures, thus reducing process efficiency and increasing downstream costs. On the other hand, the production
of natural aroma by direct extraction from plants is also subject to various problems.
These raw materials often contain low concentrations of the desired compounds,
making the extraction expensive. Moreover, their use depends on factors difficult to
control such as weather conditions and plant diseases. The disadvantages of both
methods and the increasing interest in natural products have directed many investigations towards the search for other strategies to produce natural aroma.
An alternative route for flavour synthesis is based on microbial biosynthesis or
bioconversion (Aguedo et al. 2004; Janssens et al. 1992; Krings and Berger 1998;
Vandamme and Soetaert 2002). The most popular approaches involve the use of
microbial cultures or enzyme preparations, although plant cell cultures have also
been reported as suitable production systems (Fig. 6.1). Microorganisms can synthesize aroma as secondary metabolites during fermentation on nutrients such as
sugars and amino acids. This capability may be used in two different ways:
110
S.G. Dastager
MICROBIAL
CULTURES
BIOTECHNOLOGICAL
PROCESSES
ENZYME-CATALYSED
REACTIONS
Yeasts, fungi, bacteria
Secondary metabolism
Precursors
PLANT CELL
CULTURES
Lipases, proteases, glucosidases...
(free/immobilised)
Reaction medium
(aqueous, solvent-free, organic, SC-CO2*)
Precursors
Food/Beverage
fermentation
Callus, suspension, root...
Precursors
Flavours
Specifically
designed processes
NATURAL
FLAVOURS
Fig. 6.1 Biotechnological production of Aroma compounds
r
r
In situ flavour generation, as an integral part of food or beverage production
processes (i.e. cheese, yogurt, beer, wine) which determines the organoleptic
characteristics of the final product
Microbial cultures specifically designed to obtain aroma compounds that can be
isolated and used later as additives in food manufacture. This strategy allows the
obtained aroma to be labelled as natural.
In both cases, precursors or intermediates can be added to the culture medium in
order to promote the biosynthesis of specific aroma. Also, the information obtained
through the investigation of microbial metabolism in food fermentation processes
could be utilized to develop suitable production systems for particular aroma additives. On the other hand, enzyme technology offers a very promising option for natural flavour biosynthesis. A number of enzymes (i.e. lipases, proteases, glucosidases)
catalyse the production of aroma-related compounds from precursor molecules
(Adinarayana et al. 2004; Asther et al. 2002; Kamini et al. 1998; Macris et al.
1987; Miranda et al. 1999). The use of enzyme-catalysed reactions has the notable
advantage of providing higher stereo selectivity than chemical routes. Besides, the
products thus obtained may possess the legal status of natural substances. Although
a considerable amount of current research focuses on the production of aroma
compounds, at the moment only a few are obtained by biotechnological routes.
The challenge is to put a naturally rich source of substrate in contact with highly
active enzymes. In adequate conditions, this can result in the production of aroma
6 Aroma Compounds
111
compounds in mass fractions of the order of several g/kg, instead of mg/kg encountered in raw materials. The resulting aroma compounds are called natural since they
are produced from agro-products through natural biological activities. The ratio of
isomers or isotopes is thus comparable to what can be found in extracted products
and not to what results from chemical synthesis. However, although the productivity of some of these processes is good, the resulting products are usually more
expensive than those from chemical synthesis (Benjamin and Pandey 1997; Besson
et al. 1997; Beuchat 1982).
6.2.1 Production of Aroma Compounds
from Plant Cell Cultures
Plant cell cultures appear as a viable method to produce a wide range of aromas
characteristic of their plant origin (Table 6.2; Dornenburg and Knorr 1996; Kim
et al. 2001; Suvarnalatha et al. 1994; Townsley 1972). This approach is based on
the unique biochemical and genetic capacity, and the totipotency of plant cells
(Scragg 1997). Every cell of a plant culture contains the genetic information necessary to produce numerous chemical components that constitute natural aromas.
Feeding intermediates of the biosynthetic pathway can enhance the production of
aroma metabolites by precursor biotransformation. Some authors (Mulabagal and
Tsay 2004; Rao and Ravishankar 2002) summarised the advantages of plant cell
culture technology over conventional agricultural production.
As for specific efforts related to aroma production by plant cell cultures, several
researchers have investigated the synthesis of vanillin, a much sought-after flavour
compound (Rao and Ravishankar 2000). Plant cell cultures of Vanilla planifolia
have been initiated from various plant cells and tissues (Davidonis and Knorr 1991),
and the convenience of using elicitors to induce vanillic acid synthesis assessed
Table 6.2 Aroma compounds from plant cell cultures
Plant species
Aroma compounds
Agastache rugosa
2,3-butanedione,
(E,Z)-2,6-nonadienal and
(E,Z)-2,6-nonadien-1-ol
Apple aroma
Cinnamic acid
Caryophyllen
Basmati flavour
Cocoa flavour
Flavanol
Garlic
Monoterpenes
Onion
Triterpenoid
Vanillin
Malus silvestris
Nicotiana tabacum
Lindera strychnifolia
Oryza sativa
Theobromo cacao
Polygonum hydropiper
Allium sativum
Perilla frutescens
Allium cepa
Glycyrrhiza glabra glandulifera
Vanilla planifolia
Literature
(Kim et al. 2001)
(Drawert et al. 1984)
(Suvarnalatha et al. 1994)
(Townsley 1972)
(Nakao et al. 1999)
(Ohsumi et al. 1993)
(Nabeta et al. 1983)
(Prince et al. 1997)
(Ayabe et al. 1990)
(Dornenburg and Knorr 1996)
112
S.G. Dastager
(Funk and Brodelius 1990) and also feeding of the precursor ferulic acid resulted
in increase in vanillin accumulation (Romagnoli and Knorr 1988). Furthermore, the
production of vanillin from ferulic acid with vanilla aerial roots on charcoal as a
product reservoir has been described (Westcott et al. 1994). Capsicum frutescens
root cultures have also been used for the bioconversion of ferulic acid to vanillin
(Suresh et al. 2003). Some other works involve the production of monoterpenes (i.e.
limonene, linalool, etc.) in callus tissues and cell suspensions of Perilla frutescens
(Nabeta et al. 1983; Sahai 1994), and basmati rice volatile flavour components in
callus cultures of Oryza sativa (Suvarnalatha et al. 1994). In some cases, the flavour
profiles obtained in plant cell cultures differ from those encountered in the parent
plants. Such was the case in suspension cultures of Agastache rugosa Kuntze (Korean mint), which had a marked cucumber/wine-like aroma, and produced some
interesting flavour-related alcohols (i.e. 2-phenylethanol) (Kim et al. 2001). This
alteration of the original flavour profiles can be deliberately induced by the addition
of precursors, as demonstrated in root cultures of Allium cepa L. (onion) (Prince
et al. 1997).
6.2.2 Aroma Production from Microbial Cultures
Microorganisms have historically played an integral role in the elaboration of the
aroma components of many different foods. Products such as wine, vinegar, beer,
fermented vegetables, milk, soya and meat have been preserved, modified and
flavoured by means of microbial strains. As previously indicated, microbial cultures
can be used to produce aroma compounds (Chandrasekaran 1997), either specifically for application as food additives or in situ as a part of food fermentation
processes. Detailed information on the production of some commonly used food
aroma compounds by microorganisms is presented below.
6.2.2.1 Diacetyl
Diacetyl is mainly related to butter flavour, and therefore extensively used in the
imitation of butter and other dairy flavours, as well as whenever butter notes are
desirable in food or beverages. This compound is produced by lactic acid bacteria and other microorganisms in several foods (e.g.Lactococcus lactis, Lactobacillus sp., Streptococcus thermophilus, Leuconostoc mesenteroides) (EscamillaHurtado et al. 2005). The studies done by Ibragimova et al. (1980) showed that
milk cultures of Streptococcus lactis, S. cremoris and S. diacetilactis produced high
amounts of 2,3-butanedione and acetaldehyde in 24 h at 30◦ C. Cultures with the
best aroma contained 2–5 parts acetaldehyde to 1 part 2,3-butanedione. A number of researchers have investigated the behaviour and/or metabolism of food processing microorganisms, and the enzymes involved in the production of diacetyl
and related compounds. The formation of diacetyl by lactic acid bacteria through
acetoin dehydrogenase-catalysed dehydrogenation of acetoin has been investigated
in the dough products (Bratovanova 2001). Bassit et al. (1995) studied the effect of
temperature on diacetyl and acetoin production by a particular strain of Lactococcus
6 Aroma Compounds
113
lactis, with special reference to lactic dehydrogenase, acetolactate synthase, NADH
oxidase and diacetyl reductase (Escamilla-Hurtado et al. 2000), the main enzymes
involved in pyruvate metabolism. Medina de Figueroa et al. (1998) investigated the
effect of citrate in the repression of diacetyl/acetoin reductase, resulting in the accumulation of diacetyl and acetoin in batch cultures of Lactobacillus rhamnosus. Genetic manipulation of the gene encoding enzymes involved in diacetyl metabolism,
such as diacetyl-acetoin reductase from Lactococcus lactis, has been attempted to
increase the diacetyl production capacity of lactic acid bacteria (Aungpraphapornchai et al. 1999).
Carroll et al. (1999) cloned and expressed in E. coli acetolactate synthase, a
key enzyme for the production of the diacetyl precursor acetolactate, with the final
objective of increasing diacetyl production in lactococcal strains. In some cases,
diacetyl can contribute to off-flavours (i.e. beer production) and strategies should
be designed to avoid their formation. Kronlof and Linko (1992) proposed the use
of genetically modified brewer’s yeast encoding a-acetolactate decarboxylase in immobilised yeast bioreactors for the main fermentation of beer, promoting the direct
conversion of a-acetolactate to acetoin without the formation of diacetyl. Sandine
et al. (1965) assayed the addition of a crude diacetyl reductase from Aerobacter
aerogenes as a means to remove diacetyl and 2,3-pentadione from beer by conversion to flavourless acetoin.
6.2.2.2 Lactones
Lactones are cyclic esters of primarily g- and d-hydroxy acids, and they are ubiquitously found in food, contributing to taste and flavour nuances such as fruity,
coconut-like, buttery, creamy, sweet or nutty. The possibility of producing a lactone using a biotechnological route was discovered in the 1960s by the group of
Okui et al. (1963a, b) during the investigation of hydroxyacid catabolism by several
organisms. Dimick et al. (1969) stated in their review that raw milk does not contain
free lactones, which only appear after heating. The milky, buttery and coconut-like
flavour notes provided by these compounds are generally considered as desirable in
dairy products. However, the presence of lactones may contribute to the stale flavour
of heated milk, although to a lesser extent than ketones. The compound 6-pentyl-2pyrone provides a coconut aroma, highly desired by flavourists. It was found by
Collin and Halim (1972) to be the major volatile constituent in cultures of the fungus Trichoderma viride. Other fungi such as Tyromyces sambuceus and Cladosporium suaveolens efficiently generate the coconut-flavoured lactones g-decalactone
and d-dodecalactone from ricinoleic acid and linoleic acid, respectively (Allegrone
et al. 1991; Kapfer et al. 1989). Yeasts such as Candida tropicalis or Yarrowia
lipolytica degraded ricinoleic acid to C16, C14 and C12 acids and, interestingly,
accumulated d-decalactone, which exhibits fruity and oily notes important in the
formulation of peach, apricot or strawberry aromas. However, the yields of this
biotransformation are commonly poor, and they rarely reach concentrations over
4 to 5 g/L in the fermentation broth (Gatfield 1999). Wache et al. (2001) investigated the enzymes involved in g-decalactone production by Yarrowia lipolytica, and
encountered the reasons for low yields.
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S.G. Dastager
6.2.2.3 Esters
Esters are commonly used flavouring agents, very appreciated for the fruity aromas
they provide. They are employed in fruit-flavoured products (i.e. beverages, candies, jellies, and jams), baked goods, wines, and dairy products (i.e. cultured butter,
sour cream, yogurt, and cheese). Acetate esters, such as ethyl acetate, hexyl acetate,
isoamyl acetate and 2-phenylethyl acetate are recognized as important flavour compounds in wine and other grape-derived alcoholic beverages (Geusz and Anderson
1992). Rojas et al. (2001) studied several so-called non-Saccharomyces wine yeasts
as the producers of acetate ester. Among them, the yeasts Hanseniaspora guilliermondii and Pichia anomala were found to be potent 2-phenylethyl acetate and
isoamyl acetate producers, respectively. In cheese production, ethyl or methyl esters of short-chain fatty acids generally bring about fruity flavours, while thioesters
derived from thiols are associated with cabbage or sulphur aromas (Castanares et al.
1992; Liu et al. 2004). The capacity of lactic acid bacteria to synthesize both ethyl
esters and thioesters has been reported. The role of a unique esterase from Lactococcus lactis in the formation of these aroma compounds has been investigated, and
ascertained as at least partially responsible for the esterification reactions leading to
the production of aroma ester compounds. This was undertaken by using an esterase
negative mutant of L. lactis (Nardi et al. 2002).
6.2.2.4 Pyrazines
Pyrazines are heterocyclic, nitrogen-containing compounds which possess a nutty
and roasted flavour. They are normally formed during conventional cooking/roasting
of food through the Maillard reaction (Seitz 1994). Nowadays, the use of cooking
processes that do not favour pyrazine formation (i.e. microwave cooking) has caused
the need to supply natural pyrazines with a roasty flavour as food additives. A few
microorganisms are also able to synthesize pyrazines. For instance, bacteria such as
Corynebacterium glutamicum produce important quantities of tetramethylpyrazine
from amino acids (Demain et al. 1967).
6.2.2.5 Terpenes
Terpenes are widespread in nature, mainly in plants as constituents of essential oils.
They are composed of isoprene units, and can be cyclic, open-chained, saturated,
unsaturated, oxidized, etc. The biotransformation of these compounds is potentially
of considerable interest for application in the food flavour industry. Among the terpenes, linalool, nerol, geraniol and citronellol are the most flavour-active due to
their low sensory threshold. Most of the terpenes obtained in microbial cultures
are produced by fungi that belong to the ascomycetes and basidiomycetes species.
Schindeler and Bruns (1980) have demonstrated that terpene yields in Ceratocystis
variospora cultures could be improved when toxic end products were removed
using ion exchange resins. The fungus Ceratocystis moniliformis produces several aroma products such as ethyl acetate, propyl acetate, isobutyl acetate, isoamyl
acetate, citronellol and geraniol. In order to avoid the inhibitory effects detected in
6 Aroma Compounds
115
these cultures, it is necessary to decrease product concentrations in the bioreactor.
Bluemke and Schrader (2001) developed an integrated bioprocess to enhance the
production of natural flavours by C. moniliformis. The total yield of aroma compounds produced in the integrated bioprocess, with in situ product removal using
pervaporation, is higher than in conventional batch cultivation. In addition, permeates obtained from pervaporation consist of highly enriched mixtures of flavours and
fragrances.
On the other hand, microbial transformation of terpenes has received considerable attention. Many microorganisms are able to break down terpenes or to
carry out specific conversions, creating products with an added value. Dhavlikar
and Albroscheit (1973) demonstrated that the inexpensive sesquiterpene valencene
can be converted by some bacteria to the important aroma compound nootkatone.
Recently, significant research effort has focused on the enzymes related to terpene
biosynthesis. The nucleic acid sequence of a monoterpene synthase from sweet
basil, a key enzyme for the production of geraniol, has been determined in order to
allow the production of recombinant geraniol synthase (Pichersky et al. 2005). Also,
a geraniol synthase from the evergreen camphor tree Cinnamomum tenuipilum was
cloned and expressed in E. coli (Yang et al. 2005). Functional genomics has also
been applied to identify the genes for monoterpene synthases from Vitis vinifera
grapes in order to characterize the enzymes by expression in E. coli and subsequent
analysis (Martin and Bohlmann 2004).
6.2.2.6 Alcohols
In alcoholic fermentations, apart from ethanol, yeast produces long-chain and complex alcohols. These compounds and their derived esters have interesting organoleptic properties. Some authors have proposed strategies for promoting this kind of
flavour compounds during alcoholic beverage production. Mallouchos et al. (2002)
utilized Saccharomyces cerevisiae immobilised on delignified cellulosic material
and gluten pellets. The former produced higher amounts of esters, whereas the latter
gave higher amounts of alcohols. Kana et al. (1992) evaluated yeast immobilisation
on g-alumina and kissiris, and found in the former case an increase in the concentration of amyl alcohols, total volatiles, and ethyl acetate, which led to a fine
aroma. One of the most relevant aroma-related alcohols is 2-phenylethanol, which
possesses a rose-like smell. It is still predominantly synthesized by petrochemical
routes from toluene, benzene, styrene, or methylphenylacetate (Nomura et al. 2001),
while the natural 2-phenylethanol is mainly extracted from rose petals through a
high-cost process (Fabre et al. 1998; Zheng and Shetty 2000).
Different yeast strains such as Hansenula anomala, Kluyveromyces marxianus or
Saccharomyces cerevisiae have shown a high potential for industrial production of
aroma compounds, such as 2-phenylethanol, which is derived from 2-phenylalanine
by bioconversion (Fabre et al. 1998; Stark et al. 2002). Stark et al. (2002, 2003)
reported that the presence of ethanol and 2-phenylethanol in the medium resulted in
a synergistic inhibition, which reduced the tolerance of Saccharomyces cerevisiae
to 2-phenylethanol and thus its final concentration. As a result, the feed rate had
116
S.G. Dastager
to be reduced in fed-batch cultures to avoid ethanol production. Thus, a maximal 2phenylethanol concentration of 2.35 g/L could be attained in batch cultures, whereas
3.8 g/L were obtained in a fed-batch culture with the limitation of ethanol production
(Stark et al. 2002; Topakas et al. 2004).
To enhance the productivity of the bioconversion of 2-phenylalanine by Saccharomyces cerevisiae, a novel in situ product recovery strategy was proposed by Serp
et al. (2003). An organic solvent (dibutyl sebacate) was entrapped within a polyethylene matrix, in order to form a highly absorbent, chemically and mechanically stable
composite resin. The use of this technique increased twofold the volumetric productivity of 2-phenylethanol and significantly facilitated downstream processing. Fabre
et al. (1997) screened yeast strains for 2-phenylethanol production. Amongst the
different 2-phenylethanol producers, Kluyveromyces marxianus was outstanding,
which makes this strain a promising candidate to be applied in an industrial process.
Moreover, K. marxianus has several advantages such as (Wittman et al. 2002)
r
r
r
It shows optimal production characteristics (Fabre et al. 1997).
2-phenylethanol production depends on the medium and temperature used
(Etschmann et al. 2004).
K. marxianus is Crabtree-negative, which is an advantage for scale production
processes, because the production of toxic by-products (i.e. ethanol) under aerobic conditions can be avoided (Etschmann et al. 2002).
6.2.2.7 Vanillin
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a universally appreciated flavour
chemical that occurs in Vanilla planifolia beans. It is widely used in foods, beverages, perfumes, pharmaceuticals, and in various medical industries (Priefert et al.
2001). Chemically synthesized vanillin accounts nowadays for more than 99 %
of the total market share (Walton et al. 2000), but there is an increasing demand
for natural vanillin. Direct extraction from vanilla beans is expensive and limited
by plant supply, which makes this compound a promising target for biotechnological flavour production. Vanillin is an intermediate in the microbial degradation of several substrates, such as ferulic acid, phenolic stilbenes, lignin, eugenol
and isoeugenol (Funk and Brodelius 1992). The conversion of natural eugenol and
isoeugenol from essential oils into vanillin has been investigated using microbial and
enzymatic biotransformations (Overhage et al. 1999; Rao and Ravishankar 1999).
Strains including Pseudomonas putida, Aspergillus niger, Corynebacterium glutamicum,Corynebacterium sp., Arthrobacter globiformis and Serratia marcescens
(Priefert et al. 2001; Shimoni et al. 2000, 2003) can also convert eugenol or
isoeugenol to vanillin (Washisu et al. 1993).
A two-step bioconversion process using filamentous fungi was developed by
Lesage-Meessen et al. (1996, 2002) to transform ferulic acid into vanillin. First,
Aspergillus niger transformed ferulic acid to vanillic acid, and then vanillic acid
was reduced to vanillin by Pycnoporus cinnabarinus. Bonnin et al. (2000) showed
that the yield of vanillin may be significantly increased by adding cellobiose to
P. cinnabarinus culture medium, due to the decrease in oxidative decarboxylation
6 Aroma Compounds
117
of vanillic acid. The importance of ferulic acid as precursor of vanillin has brought
about a number of efforts in the investigation of its production.
Feruloyl esterase has been identified as the key enzyme in the biosynthesis of
ferulic acid, and some researchers have studied the production of this enzyme in
microbial cultures of several fungi grown on different pretreated cereal brans, such
as wheat, maize, rice bran and sugar cane bagasse (Mathew and Abraham 2005).
The metabolism of ferulic acid in some microorganisms has also been investigated
(Falconnier et al. 1994; Narbad and Gasson 1998). Benzaldehyde It is the second
most important molecule after vanillin for its use in cherry and other natural fruit
flavours. The world consumption of benzaldehyde amounts to approximately 7000
tonnes per year (Clark 1995). Natural benzaldehyde is generally extracted from fruit
kernels such as apricots, leading to the undesirable formation of the toxic hydrocyanic acid.
Nowadays, the fermentation of natural substrates is an alternative route to the
production of benzaldehyde without harmful by-products (Lomascolo et al. 1999).
However, benzaldehyde is toxic towards microbial metabolism and its accumulation
in the culture medium may strongly inhibit cell growth (Lomascolo et al. 2001). For
this reason, only a few microorganisms have been reported as benzaldehyde producers. Amongst them, the bacterium Pseudomonas putida (Wilcocks et al. 1992;
Wilcocks et al. 1992) and the white rot fungi Trametes suaveolens (Lomascolo
et al. 2001), Polyporus tuberaster (Kawabe and Morita 1994), Bjerkandera adusta
(Lapadatescu et al. 1999) and Phanerochaete chrysosporium (Jensen et al. 1994) are
mentioned as biocatalysts in the biosynthesis of benzaldehyde from phenylalanine.
Park and Jung (2002) proposed the use of calcium alginate-encapsulated whole-cell
enzymes from P. putida for the production of benzaldehyde from benzoylformate.
This allowed the accumulation of benzaldehyde in the capsule core, minimising
its subsequent transformation to benzyl alcohol by the action of alcohol dehydrogenase, and thus providing continuous production of benzaldehyde until reactant
exhaustion.
6.2.2.8 Methyl Ketones
The methyl ketones, 2-heptanone, 2-nonanone, and 2-undecanone, are the largest
contributors to stale flavour in UHT milk (Badings et al. 1981). Moio et al. (1994)
similarly report that 2-heptanone and 2-nonanone are the most powerful odorants in
UHT milk. These methyl ketones are aromas employed in a wide range of flavouring
applications, especially those related to blue cheese and fruit flavours (Hagedorn
and Kaphammer 1994). There is not much information on microbial production of
these compounds, although Janssens et al. (1992) mention in their review the methyl
ketone-producing ability of Agaricus bisporus, Aspergillus niger, Penicillium roqueforti and Trichoderma viride TS.
6.3 Using Agro Wastes as Substrates
The tropical agro-industrial residues such as coffee pulp and coffee husk, cassava
bagasse, sugar cane bagasse are generated in large amounts during the processing
and their disposal rather causes serious environmental problems. In recent years,
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S.G. Dastager
there has been constant increase in the efforts to utilize these residues as substrates
(carbon source) in bioprocessing (Asther et al. 2002; Pandey et al. 1999). Microorganisms play an important role in the generation of natural compounds, particularly
in the field of food aromas (Janssens et al. 1992; Jiang 1995). Solid state fermentation (SSF) has been used for the production of aroma compounds by cultivating
yeasts and fungi. Numerous microorganisms are capable of synthesizing potentially
valuable aroma compounds and enzymes used in flavour manufacturing. However,
yields are often disappointingly low, which hampers extensive industrial application.
In the last decades there has been an increasing trend towards the utilization
of the solid-state fermentation (SSF) technique to produce several bulk chemicals
and enzymes (Adinarayana et al. 2004; Cordova et al. 1998; Gombert et al. 1999;
Muniswaran et al. 1994). SSF has been known from ancient times (approximately
2600 BC), and typical examples of this technique are traditional fermentations such
as Japanese koji, Indonesian tempeh and French blue cheese. In recent years, SSF
has received more and more interest from researchers, since several studies on enzymes (Domı́nguez et al. 2003; Pandey et al. 1999), flavours (Beuchat 1982; Feron
et al. 1996), colourants (Johns and Stuart 1991) and other substances of interest to
the food industry have shown that SSF may lead to higher yields or better product characteristics than submerged fermentation (SmF). In addition, costs are much
lower due to the efficient utilization and value-addition of wastes (Robinson and
Nigam 2003). The main drawback of this type of cultivation concerns the scalingup of the process, largely due to heat transfer and culture homogeneity problems
(Di Luccio et al. 2004; Mitchell et al. 2000). However, research attention has been
directed towards the development of designs such as mixed solid-state bioreactor
(Nagel et al. 2001), rotating drum bioreactor (Stuart et al. 1999) and immersion
bioreactor (Rivela et al. 2000), which overcome these difficulties. SSF could be potentially useful for the production of flavour compounds (Berger 1995; Soccol and
Vandenberghe 2003). Feron et al. (1996) reviewed the prospects of microbial production of food flavours and the recommended SSF processes for their manufacture.
Several researchers have studied SSF production of aroma compounds by several microorganisms (Table 6.3; Pastore et al. 1994; Besson et al. 1997; Bramorski
et al. 1998a; Medeiros et al. 2001), such as Neurospora sp, Zygosaccharomyces
rouxii and Aspergillus sp., using pre-gelatinized rice, miso and cellulose fibres,
respectively. Bramorski et al. (1998b) compared fruity aroma production by Ceratocystis fimbriat in solid-state cultures using several agro-industrial wastes (cassava bagasse, apple pomace, amaranth and soybean), and found that the medium
with cassava bagasse, apple pomace or soybean produced a strong fruity aroma.
Soares et al. (2000) also reported the production of strong pineapple aroma when
SSF was carried out using coffee husk as a substrate by this strain. Compounds
such as acetaldehyde, ethanol, ethyl acetate (the major compound produced), ethyl
isobutyrate, isobutyl acetate, isoamyl acetate and ethyl-3-hexanoate were identified
in the headspace of the cultures. The addition of leucine increased ethyl acetate and
isoamyl acetate production, and then a strong odour of banana was detected.
Bramorski et al. (1998a) and Christen et al. (2000) described the production of
volatile compounds such as acetaldehyde and 3-methylbutanol by the edible fun-
6 Aroma Compounds
119
Table 6.3 Agro-industrial wastes used for the aroma production by solid state fermentation
Microorganisms
in SSF
Pediococcus
pentosaceus
Lactobacillus
acidophilus
Kluyveromyces
marxianus
Ceratocystis
fimbriat
Neurospora sp.
Zygosaccharomyces
rouxii
Ceratocystis
fimbriat
Bacillus subtilis
Aspergillus oryzae
Rhizopus oryzae
Substrates
Aroma compounds
Literature
Semisolid maize
Butter flavour
(Escamilla-Hurtado
et al. 2005)
Cassava bagasse and
giant palm bran
Cassava bagasse,
apple pomace,
amaranth and
soybean
Pre-gelatinized rice
Fruity aroma
Fruity aroma
(Medeiros
et al. 2001)
Fruity aroma
(Bramorski
et al. 1998a)
Miso
HEMF
(Pastore et al. 1994)
Coffee husk
Pineapple aroma
Soybeans
Rice koji
Tropical
agro-industrial
substrates
Pyrazine
Volatile compounds
Volatile compounds
(Sugawara
et al. 1994)
(Soares et al. 2000)
(Besson et al. 1997)
(Ito et al. 1990)
(Christen et al. 2000)
gus Rhizopus oryzae during SSF on tropical agro-industrial substrates. The production of 6-pentyl-a-pyrone (6-PP), an unsaturated lactone with a strong coconut-like
aroma, was studied using liquid and solid substrates by De Araujo et al. (2002).
Sugarcane bagasse was adequate for growth and aroma production; it has been
demonstrated that, by solid-state fermentation process, it is possible to produce
6-PP at higher concentration than that reported in literature for submerged process. Kluyveromyces marxianus produced fruity aroma compounds in SSF using
cassava bagasse or giant palm bran (Opuntia ficu indica) as a substrate (Medeiros
et al. 2000). SSF was found to be very suitable for the production of pyrazines.
Besson et al. (1997) and Larroche et al. (1999) studied the biosynthesis of 2,5dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TMP) using SSF cultures of
Bacillus subtilis on soybeans.
Production of dairy flavour compounds, such as butyric acid, lactic acid and diacetyl in mixed cultures of Lactobacillus acidophilus and Pediococcus pentosaceus
growing on a semisolid maize-based culture, has been reported (Escamilla-Hurtado
et al. 2005). Soccol et al. (1994) studied the synthesis of lactic acid by Rhizopus
oryzae in SSF with sugarcane bagasse as a support. They obtained a slightly higher
productivity than in submerged cultivation. Moreover, lactic acid production by
lactic acid bacteria Lactobacillus paracasei and Lactobacillus amylophilus GV6
under SSF conditions using sweet sorghum and wheat bran as both support and
120
S.G. Dastager
substrate, respectively, have been investigated (Naveena et al. 2005a, b; Richter
and Träger 1994). It is known that several methyl ketones such as 2-undecanone,
2-nonanone and 2-heptanone are produced at commercial scale by SSF from Aspergillus niger using coconut fat as substrate with a yield of 40 % (Allegrone
et al. 1991). Several methods have been developed in order to enable vanillin and
furanone or pyranone derivatives of natural origin to be produced from agricultural
wastes.
6.4 Recovery
Aroma compounds are typically organic compounds that are extremely volatile.
Consequently, during thermal processing such as, concentration or pasteurization,
change and/or loss of aroma compounds are likely to occur. In many cases these
changes are unwanted. In the pasteurization step, for instance, chemical changes
can occur due to thermal degradation. The major problem lies through in concentration step by conventional process such as multiple-effect evaporation (Suvarnalatha
et al. 1994). It is a problem that may deteriorate the quality and acceptance of the
final product. A possible way of minimizing the changes is to use separation techniques for recovery of the aromas. Techniques suitable for this task, both commercially available and developing, are distillation, partial condensation, gas injection,
adsorption, super critical fluid extraction and pervaporation (Jørgensen et al. 2004;
Yanniotis et al. 2007; Aroujalian and Raisi 2007).
6.5 Application of Aroma Compounds
Aroma compounds have been of high importance for folk medicine, classical
medicine, food, perfumery and cosmetics since ancient times. The renaissance of the
use of natural products in the last years also led to an increasing interest in aroma
components. Especially because of their high biological activity and low toxicity
aroma compounds are often used in pharmaceutical products. Although there is a
discussion about their usage between researchers in the field of traditional and classical medicine, the number of scientific papers including analytical and biological
data on aroma components is at present higher than ever. Additionally, the flavouring
and conservation of food stuff by odorous volatiles as well as the search for pleasant
smelling raw materials for perfume and cosmetic products in the nature is not only
supported by the food, but also by the perfume and cosmetic industry with great
commercial significance. And it is also used as defoaming agents for ophthalmic
solutions with high concentrations of surfactants Natural aroma compounds are used
to improve the shelf life and safety of minimally processed fruits (Anese et al. 1997;
Lanciotti et al. 2004).
6 Aroma Compounds
121
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Chapter 7
Production of Bioactive Secondary Metabolites
Poonam Singh nee’ Nigam
Contents
7.1 Reasons to Use Agro Residues as Starting Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
7.2 What are Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
7.2.1 Properties of Bioactive Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
7.3 Biotechnology Used for Production of Secondary
Metabolites from Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
7.3.1 Reason for Selecting Solid State Technology
for Bioactive Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
7.4 Biosynthesis of Secondary-Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
7.4.1 Utilization of Agro-Industrial Residues as Substrate . . . . . . . . . . . . . . . . . . . . . . . . . 135
7.4.2 Process Operation for Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
7.5 Process Control in Synthesis of Desired Metabolite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
7.5.1 Preparation of Agro Residues for SM-Production . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
7.5.2 Control of SM Production by Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
7.5.3 Control of SM Production by Agitation of Substrates . . . . . . . . . . . . . . . . . . . . . . . . 138
7.5.4 Control of SM Production by Aeration of Substrates . . . . . . . . . . . . . . . . . . . . . . . . 139
7.5.5 Control of SM Production by Moisture Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.6 Recovery of Secondary Metabolites in Downstream Processing . . . . . . . . . . . . . . . . . . . . . 140
7.7 Scaling-Up of Process For Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.7.1 Control of Temperature in Process Scale-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.7.2 Control of Factors Related to Substrates in Scale-Up . . . . . . . . . . . . . . . . . . . . . . . . 142
7.8 Prospects of Agro Residues for Secondary Metabolites Production . . . . . . . . . . . . . . . . . . 143
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Abstract This chapter includes information based on published literature on utilization of agro industrial residues for the production of bioactive compounds. Various approaches using microbial fermentation technology have been explored for
the production of bioactive compounds which as secondary metabolites could be
produced by selected microorganisms. Certain factors have been found to affect the
P. Singh nee’ Nigam (B)
Faculty of Life and Health Sciences, School of Biomedical Sciences, University of Ulster,
Coleraine BT52 1SA, Northern Ireland, UK
e-mail: P.Singh@ulster.ac.uk
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 7,
C Springer Science+Business Media B.V. 2009
129
130
P. Singh nee’ Nigam
productivity of these compounds, hence the yield of secondary metabolites may be
manipulated by controlling these factors in fermentation system.
Keywords Secondary metabolite · Stationary phase · Idiophase · Biologicallyactive compounds · Antibiotics · Mycotoxins · Ergot-alkaloids
7.1 Reasons to Use Agro Residues as Starting Material
Despite the obvious problems that agricultural waste can create, the vast quantities
of agricultural and agro-industrial residues that are generated as a result of diverse
agricultural and industrial practices represent one of the most energy-rich resources
on the planet. Accumulation of this biomass in large quantities every year results
not only in the deterioration of the environment, but also in the loss of potentially
valuable material which can be processed to yield a number of value added products,
such as food, fuel, feed and a variety of chemicals. The agro-industrial residues are
generated globally and a major portion is left unutilized and left over as wastes in
surrounding environment. Such wastes produced annually can be used as a natural
bioresource for the production of bioactive compounds such as secondary metabolites from various selected microorganisms.
Secondary metabolites are excreted by microbial cultures at the end of primary
growth and during the stationary phase of growth. Secondary metabolites represent some of the most economically important industrial products and are of huge
interest. The best known and most extensively studied secondary metabolites are
the antibiotics, steroids and alkaloids. A variety of agricultural residues (Table 7.1)
such as wheat straw, rice hulls, spent cereal grains, various brans such as wheat
and rice bran, and corncobs, are available globally which can be considered the
cheaper and often free of cost substrates for the commercial production of secondary
metabolites.
7.2 What are Bioactive Compounds
Bioactive compounds are mostly secondary metabolites produced by microorganisms in an active culture cultivation process. Secondary metabolites usually accumulate during the later stage of microbial growth in process of fermentation,
known as the “Idiophase”. This later stage of microbial growth follows the active
growth phase called “Trophophase”. Compounds produced in the idiophase have
no direct relationship to the synthesis of cell material and normal growth of the
microoganisms. Secondary metabolites are formed in a fermentation medium after
the microbial growth is completed. Filamentous fungi synthesize many secondary
metabolites and are rich in genes encoding proteins involved in their biosynthesis.
Genes from the same pathway are often clustered and co-expressed in particular
conditions (Khaldi et al. 2008). Most common secondary metabolites are antibiotics
7 Production of Bioactive Secondary Metabolites
131
Table 7.1 Use of solid-state fermentation for the production of secondary metabolites and their
applications (Adapted from Pandey et al. 2001)
Substrate used
Wheat, oat, rice,
maize, peanuts
Impregnated loam
based compost
Coconut waste
Barley
Wheat straw with
cotton seed cake
and sunflower cake
Wheat straw with
cotton seed cake
and sunflower cake
Wheat bran
Rice, rice bran, rice
husk
Sugarcane bagasse
Wheat bran, corn cob,
cassava flour,
sugarcane baggase
Okara, wheat bran
Wheat bran
Wheat, oat, rice,
maize, peanuts
Corn cob
Sugarcane bagasse
Soyabean residue
Okara
Sweet potato residue
Microorganism
employed
Secondary metabolite
produced
Application of
metabolite
Aspergillus oryzae,
A. panasitus
B. subtillis
Aflatoxin
Mycotoxin
Antifungal volatiles
Antifungal compounds
Bacterial endotoxins
Cephalosporin
Insecticide
Antibiotic
Cephalosporin C
Antibiotic
S. clavulingerus
Clavulanic acid
-Lactamase inhibitor,
antibacterial
Tolypocladium
infautum
Metarhizum
anisopliae
Claviceps purpurea,
C. fusiformis
Gibberella fujikuroi,
Fusarium
moniliforme
Bacillus subtilis
P. brevicompactum
oryzae A. Panasitus
Cyclosporin A
Destrucxins A and B
Immuno suppressive
drug
Cyclodesipeptides
Ergot alkaloids
Disease treatment
Gibberellic acid
Plant growth harmone
Iturin
Mycophenolic
Ochratoxin
Antibiotic
–
Mycotoxin
S. rimosus
Penicillium
chrysogenum
B. Subtillis
Oxytetracycline
Penicillin
Antibiotic
Antibiotic
Surfactin
Antibiotic
S. viridifaciens
Tetracycline,
chlortetracycline
Ustiloxins
Antibiotic
Bacillus thuringenisis
Cephalosporium
armonium
Streptomyces
clavuligerus
Rice panicles
Ustilaginoidea virens
Corn
Fusarium
monoliforme
Zearalenone
Antimitotic cyclic
peptides
Growth promoter
and others include mycotoxins, ergot-alkaloids, the widely used immunosuppresant
cyclosporin, and fumigillin, an inhibitor of angiogenesis and a suppresser of tumour
growth.
7.2.1 Properties of Bioactive Secondary Metabolites
The desired product is released in fermentation medium as secondary metabolite of
a particular microorganism grown for the purpose. These metabolites are usually not
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derived from the primary growth substrate, but a product formed from the primary
growth substrate acts as a substrate for the production of a secondary metabolite.
Secondary metabolites have the following characteristics:
a. Secondary metabolites of choice can be produced only by few selected
microorganisms
b. These compounds are not essential for the organism’s own growth and
reproduction
c. Growth conditions, especially the composition of medium within a fermentation
system, control the formation of secondary metabolites.
d. These compounds are produced as a group of closely related structures.
e. Secondary metabolic compounds can be overproduced.
There are several hypotheses about the role of secondary metabolites. Besides the
five phases of the cell’s own metabolism i.e. intermediary metabolism, regulation,
transport, differentiation and morphogenesis, secondary metabolism is the activity
centre for the evolution of further biochemical development. This development can
proceed without damaging primary metabolite production. Genetic changes leading
to the modification of secondary metabolites would not be expected to have any
major effect on normal cell function. If a genetic change leads to the formation of a
compound that may be beneficial, and then this genetic change would be fixed in the
cell’s genome, and becomes an essential one. Now this secondary metabolite would
be converted into a primary metabolite.
7.3 Biotechnology Used for Production of Secondary
Metabolites from Agro-Industrial Residues
The most commonly used technology is microbial-biotechnology practiced in industry, where secondary metabolites are mostly produced in a microbial fermentation process. This fermentation is performed in liquid state growing culture under
submerged (SmF) conditions. This is mainly because the processes associated with
scale up are much simplified and easy to manipulate for control of factors associated
with whole production process. Liquid state fermentation allows greater control of
parameters, such as pH, heat, nutrient conditions etc.
But for using agro-industrial residues for the production of bioactive compounds,
another type of technology of microbial culture cultivation process would be ideal.
In this technology process is performed under solid state condition rather in liquid
state. This biotechnology is solid state (or substrate) fermentation (SSF) and characterized by a fermentation process on a solid support, which has a low moisture
content (lower limit ≈ 12%), and occurs in a non-septic and natural state. Such
technology describes the microbial transformation of biological materials in their
natural state. The process is carried out in absence or near absence of free flowing
water in the system, and it mainly utilizes fungal species.
7 Production of Bioactive Secondary Metabolites
133
Fungal species are ideal for this type of cultivation as these are capable of growth
at lower water activity while bacteria require the presence of free water in fermentation system. SSF presents a low-cost system, it utilizes naturally occurring
substances such as agricultural residues and forestry as substrates. Fermenters are
easily to construct often only incorporating a tray and microorganism can be natural in some processes. The low water volume in SSF has a large impact on the
economy of the process mainly due to smaller fermenter-size, reduced downstream
processing, obviated or reduced stirring and lower sterilization costs. SSF produces
a high product concentration with a relatively low energy requirement. Due to all
these advantages over submerged fermentations, SSF has been exploited for the
production of primary metabolites as well as secondary metabolite production.
7.3.1 Reason for Selecting Solid State Technology
for Bioactive Secondary Metabolites
The use of SSF technology for the production of secondary metabolites should not
be discounted. The mycelial morphology associated with the microorganisms that
are predominately used for secondary metabolite production is well suited to growth
on a solid support. This can also have a detrimental effect on product formation in
liquid media, as highly viscous liquid media is required for successful metabolite
production and can interfere with oxygen transfer. The filamentous morphology of
these microorganisms and the secretion of these metabolites into the growth media
can increase viscosity further. Therefore, SSF, technology can be exploited as an
alternative, allowing better oxygen circulation (Hesseltine 1977).
Solid-state fermentation (SSF) is an important area of biotechnology, since the
last decade has witnessed an unprecedented increase in interest in this technology
(Nigam and Singh 1996a,b). This culture-technique is increasingly being used in
the development of various bioprocesses in pharmaceutical, industrial and environmental sectors. Solid State (substrate) Fermentation (SSF) can be used successfully
for the production of secondary metabolites (Robinson et al. 2001). These products
(Nigam and Singh 2000) associate with the stationary phase of microbial growth
and can be produced on an industrial scale for use in agriculture and the treatment of
disease. Many of these secondary metabolites are still produced by submerged liquid fermentation (Nigam and Singh 1999), although production by this method has
been shown less efficient in comparison to SSF. As large-scale production increases
further, so does the cost and the growing energy demands. SSF has been shown
to produce a more stable product, requiring less energy, in smaller fermenters with
easier downstream processing measures. SSF technology has several advantages
over submerged fermentation; primarily it represents a low-cost and easy to operate
user-friendly system (Nigam and Singh 1994).
Concerning the production of secondary metabolites, SSF have ability to produce higher yields and productivities in certain cases. If the quality of the products
could be guaranteed and the process-variables such as temperature, and pH could
be controlled, SSF production of secondary metabolites would be very attractive
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(Robinson et al. 2003). If these problems could be overcome, SSF technology could
reduce the production-cost. It would enable third world countries cheaper access
to secondary metabolites. SSF has found important applications particularly in the
production of value-added products, such as biologically active secondary metabolites. Some of the important secondary metabolites produced in SSF are antibiotics,
alkaloids, and plant growth factors (Balakrishnan and Pandey 1996). SSF systems,
which during the previous two decades were termed as “low-technology” systems, appear to be a promising one for the production of value-added “low-volume
and high-cost” products such as bio-pharmaceuticals (Pandey et al. 2000a,b). The
recent evidence indicates that bacteria and fungi, growing under SSF conditions,
are more than capable of supplying the growing global demand for secondary
metabolites.
Though there are certain advantages of SSF-production process over conventional SmF systems but many practical advantages have been attributed to the production of biologically-active secondary metabolites through SSF route. Due to the
lack of free water smaller fermenters are required for SSF and therefore less effort is
required for downstream processing of secondary metabolites. Wild type strains of
bacteria and fungi tend to perform better in SSF conditions than genetically modified
microorganisms reducing energy and cost requirements further.
Different strategies and processes have been developed utilizing SSF technology
for the production of biopharmaceuticals. Potential applications of SSF systems
have been realized to produce high value bioactive secondary metabolites. Various secondary metabolites such as mycotoxins, bacterial endotoxins, plant growth
factors, antibiotics, immuno-suppressive drugs and alkaloids etc. are among the important group of bioactive compound which can be produced by SSF technology
(Table 7.1).
7.4 Biosynthesis of Secondary-Metabolites
Secondary metabolites comprise a diverse range of compounds synthesised by
various fungal cultures (Nigam and Singh 2000) and some bacteria such as Streptomyces. Fungal secondary metabolites are an important source of bioactive compounds for agro chemistry and pharmacology. Over the past decade, many studies
have been undertaken to characterize the biosynthetic pathways of fungal secondary
metabolites. This effort has led to the discovery of new compounds, gene clusters,
and key enzymes, and has been greatly supported by the recent releases of fungal
genome sequences (Collemare et al. 2008). These secondary metabolites are of
great commercial importance. Some are beneficial to life such as antibiotics and
growth-promoters and some metabolites are mycotoxins, a threat to human and
animal life. Various fermentation systems (Nigam and Singh 1999) such as surfaceliquid, submerged, batch or fed-batch processes have been used for the production
of different secondary metabolites. The use of certain liquid fermentation processes
is established industrial practice, there are following reasons for this practice: the
relative ease of scaling up liquid culture process; the greater homogeneity of liquid systems and the use of soluble starch; the superior monitoring for the precise
7 Production of Bioactive Secondary Metabolites
135
regulation of process-parameters which particularly control the biosynthesis of secondary metabolites. As described above due to many other advantages solid state
fermentation have been considered for certain secondary metabolite production.
7.4.1 Utilization of Agro-Industrial Residues as Substrate
Following points are worth consideration for the application and suitability of solid
agricultural residues in the biosynthesis of secondary metabolites:
1. In several productions, the product formation has been found superior using solid
insoluble substrates.
2. The most commonly used microorganisms in the production of secondary metabolites are fungi and Actinomycetes; and the mycelial morphology of such organisms is ideal for their invasive growth on solid and insoluble substrates.
3. The fungal morphology is responsible for considerable difficulties in largescale-submerged processes. These include highly viscous, non-Newtonian broths
and foam production. This results in very high power requirements for mixing and oxygen transfer. The presence of chemical antifoam in fermentation
broths reduces oxygen transfer efficiency and can lead to problems in the product
recovery.
4. In some processes, the final product is required in form of solid consistency, such
as antibiotics present in animal feed.
5. The capital cost of overall production process using solid substrates is claimed
to be significantly less.
6. The yields of certain secondary metabolites as aflatoxin B1 and ochratoxin
A obtained from liquid culture were found to be very poor. This led to the
use of solid substrates and subsequently, a higher yield of 100 g. Similarly the
production of the cyclic pentapeptide mycotoxin, malformin C was performed
using Aspergillus niger in solid culture and a higher yield of 369 mg/kg was
obtained compared to the yield of 15–200 mg/kg from liquid fermentations
(Kobbe et al. 1977).
The production of extremely toxic mycotoxins by fungi has attracted attention,
due to their importance in human and animal food chain. The aflatoxins have considerable economic impact, the poultry industry in U.S. lost US$100 million per year
from aflotoxin poisoning in 1970s. Solid state cultivation has been used to produce
sufficient quantities of these compounds for toxicity studies and these cultivations
have been performed to study the conditions that promote toxin formation on cereal
grains (Greenhalgh et al. 1983). The production of gibberellic acid in SSF has been
adopted to eliminate the need of cell-removal in downstream-processing after submerged culture process, which contributes a significant part in the production cost.
Another concept is the growth of antibiotic producing microorganisms on animalfeed for two purposes, firstly to enrich the protein-content in nutrient-poor feed and
secondly to produce antibiotics, such as cephalosporins, tylosin and monensin.
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7.4.2 Process Operation for Secondary Metabolites
The biotechnology based process for the production of secondary metabolites can be
performed in batch, fed-batch, continuous or plug-flow bioreactor operation. These
modes of operation are well suited to solid state process, but a well-mixed fed-batch
bioreactor is difficult to operate on a large scale. A plug-flow mode process operating in a continuous system could be more straightforward on industrial-scale
compared to a submerged fermentation process. The productivity in a continuous fermentation is higher in such process compared to batch system. Mostly the
secondary metabolites are produced in batch reactor system. The productivity of
compounds is low in batch process because the time required to achieve the phase
for secondary metabolite synthesis is longer, occurring after the primary metabolite production stage and after culture-growth has happened. While a continuous
process runs for a longer time with a continuous yield of secondary metabolites
once the phase of synthesis has started. Genes responsible for biosynthesis of fungal
secondary metabolites are usually tightly clustered in the genome and co-regulated
with metabolite production (Patron et al. 2007).
The substrate addition in the fermentation-medium also affects the processoperation. The addition of soluble starch or glucose in the initial wheat-bran medium
of Gibberella fugikuroi affected the synthesis and a reduced yield of gibberellic
acid was obtained. A fed-batch process with intermittent feeding of soluble starch
instead of including starch in the beginning increased the yield of gibberellic acid by
18% compared to the lower yield obtained in batch process. As catabolite regulation
by glucose is common to the synthesis of many secondary metabolites, solid-state
fed-batch operation is likely to be superior to batch systems for most. For example,
a 47% of increase in product yield has been obtained using feeds of cornstarch
intermittently. A study of the hydrolytic enzymes secreted by G. fugikuroi during
batch and fed-batch solid-substrate fermentation demonstrated that the quantity and
rate of production of enzymes were higher in batch cultures. This was suggested that
the glucose levels in the medium would be increased over fed-batch cultures leading
to catabolite regulation of gibberellic production by glucose and which resulted in
lower yields of the product in batch process.
7.5 Process Control in Synthesis of Desired Metabolite
The production-times for many secondary metabolites have been found similar
time-periods required in many submerged state and solid state fermentation using
solid substrates. SSF processes performed for the synthesis of secondary metabolites
show similar process-kinetic patterns such as microbial-growth, substrate-utilisation
and bioconversion, and product-synthesis, similar to the characteristics of a submerged fermentation. Similar patterns have been observed for the production of
aflatoxin B1, ochratoxin A (Lindenfelser and Ciegler 1975), trichothecene mycotoxins (Greenhalgh et al. 1983), polyketide pigments (Lin and Lizuka 1982) and
7 Production of Bioactive Secondary Metabolites
137
gibberellic acid (Kumar and Lonsane 1987a,b,c). The kinetics of spore formation
by Penicillium roqueforti is similar to those of secondary metabolite production.
Process using agro-residues as starting substrates is controlled by a number of process – regulating factors such as: the initial moisture content of the substrate; rate of
aeration; mixing of the fermenting solid medium; substrate-type, composition and
structure of the substrate, and the constitution of fermenting medium; temperature;
and the choice of microorganism.
7.5.1 Preparation of Agro Residues for SM-Production
A variety of solid substrates for secondary metabolites production have been tried in
solid state fermentation (Table 7.1). These substrates derived from various sources
vary in their nature, structure, and composition. Substrates coming from different
origins have ability to provide a range of easily to poorly metabolizable nutritional
sources and therefore, various substrates have been utilized as single carbon source
or in combination with others, and also some substrates have been used as inert
solid-supports for fungal colonization required for secondary metabolites production. Ultimate choice of a substrate for a particular metabolite is made after extensive trials with various types of substrates. Spent cereal grains such as wheat and
rice have predominated as substrates for secondary metabolites. Aflatoxin has been
produced using corn (Silman et al. 1979), rice (Shotwell et al. 1966), peanuts, corn
meal and crushed wheat (Chang et al. 1963). Many brans such as wheat and rice
brans are used singly or in combination with grains.
Mostly the production yields of secondary metabolites can be improved with
a right choice of substrate or mixture of substrates with appropriate nutrients.
A single-selected substrate performs in a different way changing the overall fermentation efficiency of the process; if the substrate is used in its different forms;
for example pieces, fibres, particles or flour of a same substrate are metabolized in
a different way. Though smaller particle size of a solid substrate has larger surface
area for microbial action, but at the same time small particles have tendency of the
increasing packing density. Densely packed fermentation system results in higher
heat output per unit area of fermenter or output per unit space of fermenting solid
medium. A column and other large size bioreactors would have a problem of poor
aeration if used with smaller particle size substrates. In a process of gibberellic acid
production using Gibberella fuzikuroi, higher yields have been achieved with larger
particle size of wheat bran such as 0.3 to 0.4 cm (Kumar and Lonsane 1987a,b,c)
compared to smaller particles of wheat bran.
7.5.2 Control of SM Production by Temperature
Normally the incubation temperature in a cultivation process is the optimum
growth temperature of the particular microorganism used for secondary metabolite
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production, and this optimal temperature for any secondary metabolite biosynthesis
is similar in solid substrate fermentation to that for liquid fermentation. Each process
has its range of temperature over which secondary metabolite production occurs,
since temperature can not be precisely controlled at all times in all layers of solid
substrates and within same system the temperature can vary by few degrees due to
metabolic heat generation. Excess temperature due to poor heat transfer in fermenting system may adversely affect the yields of secondary metabolite. Therefore, the
temperature regulation is achieved with the mixing of fermenting solid substrates by
rotation or agitation and aeration. In a production process of aflatoxin production,
the effect of temperature was studied over the range of 27◦ C to 40◦ C using flasks
and small column fermenters (Silman et al. 1979). Aflatoxin formation was achieved
over this range, but it ceased above 40◦ C; the production of toxin could be restored
if the temperature was lowered and controlled within the range of 27◦ C to 40◦ C.
Hence, it is possible to control the production of secondary metabolites by simply
controlling the temperature of fermenting agro-industrial substrates in fermentation
system.
7.5.3 Control of SM Production by Agitation of Substrates
The mixing of contents such as solid substrate, nutrient medium and seed-culture is
very important to start any process in effective way, but also the mixing is required
during fermentation to aid the aeration and to facilitate the heat-transfer in some
processes. The extent of mixing or the rate of agitation in system depends primarily
on the type and design of the bioreactor used in that particular process of secondary
metabolite production. Mixing of fermenting contents in a production process where
solid substrates are being used, has generally been found to increase the productivity of the secondary metabolites, whereas lower yields were obtained in a similar
process run without agitation or mixing. The only disadvantages noticed of mixing in fermentation are shear damage to the growing microorganism and the extra
power requirements to run the agitator. In some processes agitation has increased
the products-yield considerably. A possible physiological explanation for the increase or improvement in yields through agitation is that the mixing of fermenting
solid insoluble substrates suppresses the process of sporulation. Sporulation occurs
simultaneously in system with the production of secondary metabolites and it may
compete for common intermediates and substrates. Suppression of this competition
has been suggested as the only reason for the increased yield of the desired product.
In some processes a clear advantage of mixing has been noticed such as the rotation at the speed of 16 rpm was found necessary for the production of high yields
of ochratoxin A by A. ochraceus (Lindenfelser and Ciegler 1975). This culture was
grown on wheat in a small-scale rotary drum bioreactor. This was confirmed with the
low yields obtained if there was no rotation of the drum-bioreactor or the rotation
was just brief and intermittent. Therefore, it was concluded that the superior performance of the rotary-bioreactor was due to the mixing. Similarly, in a utilization
process of pearled barley cultivating a culture of A. clavatus NRRL 5890 (Demain
7 Production of Bioactive Secondary Metabolites
139
et al. 1977), the production of crude toxins was increased by 50% in agitated culture
over static culture and the crude toxin was enriched in cyochalasin E. In contrast,
the cephalosporin production was adversely affected by agitation of solid fermenting
mass (Jermini and Demain 1989).
The production of most mycotoxins has been found to be improved in shaken
cultures compared to stationary fermentations. Some of such improved-yield fermentations are: aflatoxin production by A. flavu NRRL 2999 using rice as substrate
(Shotwell et al. 1966), ochratoxin A production by A. ochraceus, cytochalasin E
and tremorgens production from culture of A. clavatus, cyclochorotine and simatoxin from Penicillium islandicum (Ghose et al. 1978), and cyclopiozonic acid by
A. flavu (Luk et al. 1977). The yield of cyclopiozonic acid was obtained almost ten
times (10×) in agitated fermentation of white wheat compared to the lower yield
(1×) in static culture process (Luk et al. 1977).
7.5.4 Control of SM Production by Aeration of Substrates
Since the production of secondary metabolites has started utilizing agro industrial
residues as the starting materials in fermentation process, the effect of aeration on
the synthesis of various secondary metabolites has been investigated. The most
comprehensive study has been performed on the production of aflatoxin B1 in a
corn storage bin with a capacity of 1266 bushels (Silman et al. 1979). The effect of
aeration was studied by passing humidified air of 80–85% relative humidity through
the bed of corn at flow rates between 0.001 to 0.04 l/kg corn per min and a recirculation rate of 1.5 l/kg per min. It was noticed that the rate of aflatoxin production
and yields were directly proportional to the aeration rate. However, the direction of
air-flow through the corn-bed had no apparent effect on aflatoxin yield or on the rate
of production.
But the aeration may not be necessarily required in some cases, this needs to be
confirmed before the running of process. In one case of secondary-metabolite production aeration proved to be the unnecessary where the production of ochratoxin
A performed in a system using a rotating drum bioreactor was adversely affected by
aeration (Lindenfelser and Ciegler 1975).
7.5.5 Control of SM Production by Moisture Content
The optimal moisture content of solid fermenting substrates varies for various
metabolites production. The control of the water content present in solid substrate
fermenting – medium or the maintenance of the initial moisture content in the system is very important factor. The moisture content of solid substrates greatly affects process of any secondary metabolite production. The optimal initial moisture
contents have been found to be different according to the reactor-type used in the
process. The initial moisture content may vary for the same fermentation process i.e.
using same substrate and same microorganism for same metabolite production but
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during the scaling up of the process it may vary using different designs of bioreactor,
for example from flasks to tray type.
Production of another metabolite such as toxin, using substrate grains or corn
is greatly favoured at low initial moisture contents. The most favourable moisture
content for optimal toxin production has been found to be between 20–40%. The
optimal range is characteristically broad, however outside this range the yield of the
product and the rate of toxin-formation are severely affected. Aflatoxin B1 production from corn by Aspergillus flavu is negligible at initial moisture content below
17% though the fungal growth takes place (Silman et al. 1979); at moisture contents between 18–20% toxin production occurs at a reduced rate and lower yield
is obtained. There is a rapid fungal growth and aflatoxin production at the moisture contents of 20–30% with the optimal moisture content being 22.4% at 33◦ C in
flask culture. Similarly, ochratoxin A production has been found affected due to the
variation in initial moisture content using laboratory-scale rotary drum bioreactors.
In these culturing processes, the most significant parameter identified has been the
effect of initial moisture content that determines the toxin-yields. The optimal moisture content of rice for the production of cephalosporin C was found to be 49–51%
using Acremonium chrysogenum. In such cultivation-systems, yields were severely
affected (inhibited) below the optimal value, whereas at higher levels of moisture
content the bacterial contamination became an unavoidable problem.
7.6 Recovery of Secondary Metabolites
in Downstream Processing
The solid fermented mass obtained after the completion of the process is extracted
using various solvents to recover the product. The extraction is usually performed
using the aqueous or other solvents mixing in proper ratio with the solid fermented
mass. The extracts obtained from the fermentation of solid substrates usually contain higher concentration of secondary metabolites compared to the concentrations
present in the submerged culture medium (Kumar and Lonsane 1988). One advantage of using agro industrial residues as solid substrates in fermentation is that there
is no need of cell removal prior to the extraction of metabolites. The fermented liquid medium from a submerged fermentation is subjected to downstream processing
for the removal of microbial-cells or mycelial-biomass to obtain a clear supernatant
for the extraction of secondary metabolites. The cost of cell-removal from the submerged culture broths prior to the extraction process is estimated to be between 48%
to 76% of the total production cost of the final product (Datar 1986).
There could be certain problems associated with the recovery of metabolites from
system. The recovery of secondary metabolites from the solid fermented mass offers
less flexibility in the choice of the initial unit operation than submerged fermentation. The metabolites diffuse throughout the solid mass during the culturing, which
requires longer extraction-times for complete recovery of metabolites. The extraction of larger amount of solids may increase the concentration of impurities in the
extract. The cost of purification depends on the quality of extract. The presence and
7 Production of Bioactive Secondary Metabolites
141
concentration of inert compounds in the extract increase the cost of purification and
therefore the cost of recovery is increased. Particularly those secondary metabolites
which are used in bulk in the pharmaceutical and health industry and whose purity is
governed by stringent regulations need to go through specific purification strategy.
Now in antibiotic industry, the problem of culture-biomass in submerged process, is
solved to some extent by the application of whole-broth processing. The extraction
process uses the whole fermented broth including the cell-biomass. In such extraction the process is carried out using solvent extraction in multi-stage, centrifugal
decanters.
The important variables in the extraction of an important secondary metabolite
gibberellic acid from the fermented mouldy-bran (Kumar and Lonsane 1987) are
the type of solvent to be used for an efficient extraction, concentration of solvent,
the ratio of solvent to the solid, and pH. An 87% recovery of gibberellic acid in the
extract of 0.9 mg/l was obtained using 2%, v/v ethanol as solvent in a multi-stage,
counter-current extraction system. A considerable loss of solvent is common, with
up to 68% of the initial solvent added to the bran, remaining absorbed into the
solid on separation. In a particular production process the product is left on solid
residue for use. Production of antibiotic monensin on various materials is carried
out by Streptomyces cinnamonensis for use in poultry feeds to control coccidiosis.
Tin such application no extraction cost would be involved.
7.7 Scaling-Up of Process For Secondary Metabolites
The elucidation of important scale-up criteria for SSF is very important to guarantee the successful large-scale operation of the processes. The scale-up of the each
process results in mixed success for various processes employing different microorganisms and different solid substrates for a range of metabolites production. Since a
particular individual process has its own characteristics due to the nature of organism and contents of the system such as type of agro residues used as substrate, each
process behaves differently in scaling-up process. On the other hand the synthesis of
secondary metabolites is very sensitive to the environmental factors and therefore
the control of such factors becomes difficult in a similar process on large-scale,
which is naturally expected.
Any process can be operated successfully regulating the various parameters on
small-scale but the yield of the secondary-metabolites has been found adversely affected during large-scale cultivation. The synthesis of toxin has been found severely
affected in a large-scale system, though the growth of employed microorganisms
were always very obvious. In another process for the synthesis of aflatoxin B1 , the
scaling-up using 75 g to 1266 bushels of corn have been successful producing good
yields of aflatoxin even at larger scale (Silman et al. 1979).
Many of the important scale-up problems are generic to all solid state fermentation processes. The criteria of scale-up of systems utilising solid substrates are
different to those established for submerged processes, such as volume-ratio, mixing
time, k1 a, and foam-control etc.
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7.7.1 Control of Temperature in Process Scale-Up
The precise control of incubation temperature in any bioreactor is a difficult problem, and this problem becomes a difficult engineering challenge with increase in
scale of process for a large-scale production. Unlike submerged fermentation, where
the dominant mechanism of heat-transfer is convection, the heat-transfer in a fermentation system using solid substrates occurs predominantly via two mechanisms
i.e. convection and conduction. The convective medium in solid fermenting biomass
is air and therefore the rate of heat-exchange is much smaller than in liquid submerged cultivations.
The microbial-activity on agro residue substrates for the production of secondarymetabolites is similar and not greatly less than in liquid cultures; consequently the
heat generation is also considerable. The increase in temperature due to heat generation during the production of amyloglucosidase has been found up to 56–57◦ C.
The rise in temperature affects the rate of microbial-growth and the rate of moisture
loss in that process and as a result the production yield of the secondary metabolite
is also affected. Excessive heat production may result in the progressive drying of
the solid substrate with time in a longer process which requires 10 or more days for
the optimum synthesis of secondary metabolites. This is a serious problem, which
may affect the yield of the products.
This is natural to face difficulty in maintaining the temperature control on large
scale such as if the fermentation is carried in large 1266-bushel storage bins. In such
fermenters the temperature up to 47◦ C have been measured, despite the continual
circulation of cool air to regulate the temperature-rise. Even similar difficulties may
be experienced on a small-scale process for the production of secondary metabolites. Using a small-scale rotary-drum bioreactor, the temperature increase may be
normally noticed to be 1–2◦ C per day (Lindenfelser and Ciegler 1975).
7.7.2 Control of Factors Related to Substrates in Scale-Up
The yields of secondary metabolites are affected in scale-up due to few more factors other than temperature-rise, which are directly related to solid substrates. The
importance of the moisture content of the fermenting solid substrates is very significant in determining the yield of metabolites. However, the control of the moisture
content of the solid substrate can be difficult. There are certain reasons for this
difficulty such as the combined effects of heat production and moisture release due
to microbial-respiration. The excessive wetness may result in bacterial and yeast
contamination and also in rotary bioreactors wetness promotes the clumping of
the material. Therefore a purposely-built reactor-design is essential to prevent such
problems. During the typical process using wheat grains as substrate for ochratoxin
A production cycle of 10–12 days, the moisture content has been found to rise from
30–40% and as a result of this the volume of substrate used (wheat) increased by
60–70%. Such problems lead to the reduced yield of the secondary metabolite.
7 Production of Bioactive Secondary Metabolites
143
Another factor in scale-up of SSF is the amount of solid substrates used in
bioreactors. The yields of secondary metabolites are superior in SSF to those obtained in submerged process based on weight of product obtained per gram substrate and per litre, respectively. Frequently considerable amounts of carbon remain unutilized in fermentation. The yields of secondary metabolites calculated
on the basis of the weight of substrate consumed, needs to be ascertained, this is
important to adequately compare the productivity in two types of processes. This
measure of the productivity of secondary metabolites is important for a commercial
operation.
7.8 Prospects of Agro Residues for Secondary
Metabolites Production
The use of agro industrial residues for the production of commercially valuable
metabolites is at present under-utilized, with a strong preference towards conventional and familiar liquid fermentations. This lack of adoption into industry seems
strange, since research in this area clearly shows that SSF produces higher yields
in a shorter time period. It is easy to see why a liquid state fermentation using
simple sugars as substrate is still prevalent for the production of secondary metabolites; it is a familiar technique, scale-up from lab to industrial fermenter level is
much simplified in comparison to SSF with parameters being easier to monitor and
control. There are also problems associated with secondary metabolite production
in liquid fermentation, such as shear forces, increasing viscosity due to metabolite
secretion, and reduction in metabolite stability. There is an increasing demand on
science in regards to antibiotic production, with a growing global demand and cases
of antibiotic resistance. Therefore, the use of residual agro industrial wastes should
be considered by industry, especially when large quantities of secondary metabolites
are required in a shorter fermentation period, with minimal expenditure on media
and downstream processing.
There is clear evidence that high concentrations of secondary metabolites can
be achieved using a variety of cheaper and freely available agro industrial residual substrates, employing best performing microorganisms in suitable properly designed and purposely-built bioreactors for large-scale production. To achieve the
high standard of this technology, a number of studies have been performed to
elucidate the optimal conditions for the synthesis of various secondary metabolites. Some areas of further interest are the use of fed-batch and continuous plugflow modes of operation; the study of broader range of secondary-metabolites; and
analysis of bioreactors to identify criteria for successful scale-up and therefore to
permit effective process control. This last criterion is particularly important for
the lengthy nature of these culture-systems. The residual substrate fermentation
process for the biosynthesis of secondary metabolites is more likely to be seriously considered for the industrial-scale production of some important secondary
metabolites.
144
P. Singh nee’ Nigam
References
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state fermentation. J Sci Ind Res 55: 365–372
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biological activity. Science 142: 1191–1192
Collemare J, Billard A, Bohnert HU, Lebrun MH (2008) Biosynthesis of secondary metabolites
in the rice blast fungus Magnaporthe grisea: the role of hybrid PKS-NRPS in pathogenicity.
Mycol Res Feb 112(Pt 2): 207–215
Datar R (1986) Economics of primary separation steps in relation to fermentation and genetic
engineering. Process Biochem 21: 19–26
Demain AL, Hunt AN, Malik V, Kobbe B, Hawkins H, Matsuo K, Wogan GN (1977) Improved
procedure for production of cytochalasin E and tremorgenic toxins by Aspergillus clavatus.
Appl Environ. Microbiol 31: 138–140
Ghose AC, Manmade A, Townsend JM, Bosquet A, Howes JF, Demain AL (1978) Production
of cyclochlorotine and a new metabolite, simatoxin by Penicillium islandicum. Appl Environ
Microbiol 35: 1074–1078
Greenhalgh R, Neish GA, Miller D (1983) Deoxynivalenol, acetyl deoxynivalenol, and zearalenone
formation by Canadian isolates of Fusarium graminarium on solid substrates. Appl Environ
Microbiol 46: 625–629
Hesseltine CW (1977) Solid state fermentation-Part I. Process Biochem 12(6): 24–27
Jermini MFG, Demain AL (1989) Solid state fermentation for cephalosporin production by Streptomyces clauvligerus and Cephalosporin acremonium. Experienta 45: 1061–1065
Khaldi N, Collemare J, Lebrun MH, Wolfe KH (2008, Jan 24) Evidence for horizontal transfer of
a secondary metabolite gene cluster between fungi. Genome Biol 9(1): R18
Kobbe B, Cushman M, Wogan GN, Demain AL (1977) Production and antibacterial activity of
malformin C, a toxic metabolite of Aspergillus niger. Appl Environ Microbiol 33: 996–997
Kumar PKR, Lonsane BK (1987a) Gibberellic acid by SSF: consistent and improved yields.
Biotechnol Bioeng 30: 267–271
Kumar PKR, Lonsane BK (1987b) Extraction of gibberellic acid from dry mouldy bran produced
under solid-state fermentation. Process Biochem 22: 139–143
Kumar PKR, Lonsane BK (1987c) Potential of fed-batch culture in solid-state fermentation for the
production of gibberellic acid. Biotechnol Lett 9: 179–182
Kumar PKR, Lonsane BK (1988)]kumar1988 Kumar PKR, Lonsane BK (1988) Batch and fedbatch solid-state fermentations: kinetics of cell growth, hydrolytic enzymes production, and
gibberellic acid production. Process Biochem 23(2): 43–47
Lin CF, Lizuka H (1982) Production of pigment by a mutant of Monascus kaoliang sp. nov. Appl
Environ Microbiol 43: 671–676
Lindenfelser LA, Ciegler A (1975) Solid state fermenter for ochratoxin A production. Appl Microbiol 29: 323–327
Luk KC, Kobbe B, Townsend JM (1977) Production of cyclopiazonic acid by Aspergillus flavus
Appl Environ Microbiol 33: 211–212
Nigam P, Singh D (2000) Secondary Metabolites. In Encyclopedea of Food Microbiology (RK
Robinson et al. eds.) Academic Publisher, London
Nigam P, Singh D (1999) Characteristics and techniques of fermentation systems in Biotechnology: Food Fermentation, Vol. II, (VK Joshi, A Pandey eds) Educational Publishers, N Delhi,
pp. 427–466
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Nigam P, Singh D (1996b) Processing of agricultural wastes in solid state fermentation for cellulolytic enzyme production. J Sci Ind Res 55: 457–467
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biotechnology. J Basic Microbiol 34: 405–423
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Pandey A, Soccol CR, Nigam P, Soccol VT, Vandenberghe LPS, Mohan R (2000a) Biotechnological potential of agro-industrial residues: II cassava bagasse. Bioresource Technol 74(1): 81–87
Pandey A, Soccol CR, Nigam P, Soccol VT (2000b) Biotechnological potential of agro-industrial
residues: I sugarcane bagasse. Bioresource Technol 74(1): 69–80
Pandey A, Soccol CR, Rodriguez-Leon JA and Nigam P (2001) Production of organic acids by
solid state fermentation. In Solid state fermentation in Biotechnology-Fundamentals and Applications, Asitech Publishers N. Delhi, pp. 132–158
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Robinson T, Nigam P, Singh D (2004) Secondary metabolites. In Handbook of Fungal Biotechnology, (DK Arora et al. eds.) Marcel Dekker Inc, NY pp. 267–274 ISBN 08247-4018-1
Robinson T, Singh D, Nigam P (2001) Solid-state fermentation: A promising microbial technology
for secondary metabolite production. Appl Microbio Biotechnol 55: 284–289
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large-scale solid-substrate fermentation process. Biotechno Bioeng 21: 1799–1808
Chapter 8
Microbial Pigments
Sumathy Babitha
Contents
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
8.10
8.11
8.12
8.13
Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Food Technology and Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Market Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microbial Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pigment Producing Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biodiversity of Filamentous Fungi: A Promising Source of Colorants . . . . . . . . . . . . . .
Microbial Pigments – Production and Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Role of Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Monascus – A Potent Source of Food Colorant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Monascus Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Monascus Pigment Production by Solid-State Fermentation . . . . . . . . . . . . . . . . . . . . . . .
Agro-Industrial Residues as Substrates for Monascus Pigment Production . . . . . . . . . . .
Other Applications of Monascus Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Recent increasing concern regarding the use of edible coloring agents has
banned various synthetic coloring agents, which have a potential of carcinogenicity
and terratogenicity. This circumstance has inevitably increased the demands for safe
and naturally occurring natural (edible) coloring agents, one of which is pigment
from the fungus Monascus purpureus. It has long been known that the microorganisms of the genus Monascus produce red pigments, which can be used for coloring
the foods. Monascus pigments are a group of fungal secondary metabolites, called
azaphilones, which have similar molecular structures as well as similar chemical
properties. The pigments can easily react with the amino group containing the compounds in the medium to form water-soluble pigments. Due to the high cost of the
currently used technology for the microbial pigment production on an industrial
scale, there is a need for developing low cost process for the production of the
S. Babitha (B)
Skin Bioactive Material Laboratory, Inha University, Yong-hyunong, Nam-gu, Incheon 402-751,
Republic of Korea
e-mail: babisp2003@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 8,
C Springer Science+Business Media B.V. 2009
147
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pigments that could replace the synthetic ones. Utilizing cheaply available agroindustrial residues as substrate through solid-state fermentation can attain such an
objective.
Keywords Pigments · Monascus · Azhaphilones · Polyketide · Solid-state
fermentation
8.1 Pigments
The word pigment has a Latin origin and initially denoted a color (in the sense of
colored matter), but it was later extended to indicate the colored objects such as
makeup. In the beginning of the middle ages, the word was also used to describe the
diverse plant and vegetable extracts, especially those used for the food coloring. The
word pigment is still used in this sense in the biological terminology: the colored
matter present in the animals or the plants, occurring in the granules inside the cells
or cell membranes as the deposits on the tissues, or suspended in the body fluids
(Ullmann 1985).
The modern meaning associated to the word pigment has its origin in the twentieth century, meaning a substance constituted of small particles which is practically
insoluble in the applied medium, and is used due to its colorant, protective or magnetic properties (Ullmann 1985). This definition applies well to the pigments of
the mineral origin, such as titanium dioxide or carbon black, but for the soluble
dyestuffs, usually the organic compounds, the expressions dye, colorant or simply
color (as in the food colors) is more adequate. The terms pigment and color are
usually applied for the food coloring matters, sometimes indistinctly (Timberlake
and Henry 1986).
8.2 Food Technology and Color
Numerous sociological, technical and economic factors have influenced the food
industry over the years in past and the food market has changed rapidly with a much
larger proportion of the food being ‘processed’ before the sale and ready prepared
to meet the needs of new consumers such as the working mothers, single parent
families and the increasing number of older people in the world. The challenge to
the food industry is to provide visually appealing foods that taste good and meet the
consumer’s demands on the quality and price.
The color production industry aims to meet the food and drink manufactures
needs by providing a full range of the colors to suit all the applications, within
the legislative constraints. There is, however, a constant ongoing development to
improve the stability and handling properties of the colors using the formulation
technology, new processing methods and to a much lesser extent (mainly restricted
by the legislative controls), the development of totally new pigments.
8 Microbial Pigments
149
8.3 Market Trends
The market of the natural products on an international level is in a phase of extraordinary expansion. Among the causes, the boom in the markets, projected beyond
US$ 4 milliards in 2000 alone for US, and the emergence of new categories of
the natural substances which are rapidly and fundamentally changing the idea of
health and diseases (Downham and Collins 2000). There are no reliable published
statistics on the size of the color market; however, on a global scale a reasonable
estimate would be $940 m which can be segmented as in Fig. 8.1.
In terms of individual sector size, it is estimated as below:
r
r
r
r
synthetic colors – $400 m;
natural colors – $250 m
nature identical colors – $189 m;
Caramel colors – $100 m.
Consumer pressure, sociological changes, and technological advances leading
to more advances in the food processing industry have increased the overall color
market. The most significant growth has been in the naturally derived colors owing
to the improvements in the stability as well as the food industries aim to meet the
increasing consumer perception that ‘natural is best’. Currently, the cost of the natural colors in most cases is higher than the synthetic colors of similar shades but this
hurdle can be overcome by the mass production of the natural colors which would
bring the cost down, thereby increasing the demand also.
Fig. 8.1 Percentage market
share of food colors
The global food colour market
Synthetic
11%
42%
20%
Natural
Nature identical
Caramel
27%
8.4 Microbial Pigments
An alternative route for the production of the natural food colorants is through the
application of biotechnological tools employing the microorganisms. When the microbial cells are used to produce the color the term refers to ‘Microbial pigments’.
Inspite of the availability of the variety of the pigments from the fruits and vegetables, there is an ever growing interest in the microbial pigments due to several reasons such as their natural character and safety to use; production being independent
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S. Babitha
of the seasons and geographical conditions, controllable and predictable yield and
are not subject to vagaries of natures, for example, the production of cochineal
(Francis 1987). Some provide nutrients like vitamins, and have medicinal properties
also. The microbial pigment such as ß-carotene gradually shifts its color towards
orange-red which is more attractive and this unique property is absent in the plant
derived pigments (Pandey and Babitha 2005). Some of the microbial pigments can
be produced from industrial residues (starch and juice industry), hence reducing
water and environmental pollution (Babitha et al. 2004)
8.5 Pigment Producing Microorganisms
A large number of different species of the bacteria, molds, yeasts and algae can
produce pigments but only a few are considered suitable for this purpose. It must
satisfy several criteria such as the capability to use a wide range of carbon and
nitrogen sources, should have tolerance to pH, temperature, mineral concentration
and possess moderate growth conditions, reasonable color yield, should be nontoxic and non-pathogenic and must be easily separable from the cell mass. Table 8.1
gives a list of microbial sources and color shades of pigments produced by them.
Microalgae and several classes of fungi are known to produce a wide range
of excreted water-soluble pigments, but the low productivity of algal cultures is
a significant bottleneck for their commercialization (Hejazi and Wijffels 2004). Pigments of the basidiomycetous fungi have been used in the past for dying the wool
and silk but such fungi are difficult to grow under industrial large-scale conditions.
Hence, attention was drawn on the ascomycetous fungi. The use of such fungi to
color foodstuffs is not a novel practice; the use of Monascus pigments in the food
has been carried out traditionally in the Orient for hundreds of years (Teng and
Feldheim 2001; Babitha et al. 2004). Considering the apparent heat and pH stability
of the Monascus derivatives during the food processing, taken together with the
socio-climatic independence of such a readily available raw material, fungi seem to
be well-worth for further exploration as an alternative source of natural colorants.
8.6 Biodiversity of Filamentous Fungi: A Promising
Source of Colorants
Like plants, the filamentous fungi synthesize the natural products because they
have an ecological function and are of value to the producer (Firn and Jones 2003,
Babitha et al. 2004). Depending on the type of the compound, they serve different functions—varying from a protective action against the lethal photo-oxidations
(carotenoids) to protection against the environmental stress (melanins) and acting as cofactors in the enzyme catalysis (flavins). Besides providing the functional diversity to the host, these pigments exhibit a unique structural and chemical
diversity with an extraordinary range of the colors. Several characteristic
8 Microbial Pigments
151
Table 8.1 Some microbial sources and color shades of pigments produced by them
Microorganism
Pigment color shade
Bacteria
Janthinobacteriumlividum
Achromobacter
Bacillus sp
Brevibacterium sp
Corynebacterium michigannise
Pseudomonas sp
Rhodococcus maris
Streptomyces sp
Serratia sp
Bluish purple
Creamy
Brown
Orange, Yellow
Greyish to creamish
Yellow
Bluish red
Yellow, red, blue
Red
Fungi
Aspergillus sp
Blakeslea trispora
Monascus purpureus
Helminthosporium catenarium
H. gramineum
H. cynodontis
H. avenae
Penicilliumcyclopium
P. nalgeovnsis
Orange, red
Cream
Yellow, orange, red
Red
Red
Bronze
Bronze
Orange
Yellow
Yeast
Rdodotorula sp
Yarrowialipolytica
Cryptococcus sp
Phaffi rhodozyma
Red
Brown
Red
Red
Algae
Dunaliella salina
Red
non-carotenoid pigments are produced by the filamentous fungi, including quinones
such as anthraquinones and naphthaquinones (Baker and Tatum 1998, Medenstev and Akimenko 1998), dihydroxy naphthalene melanin (a complex aggregate
of polyketides) (Butler and Day 1998), and flavin compounds such as riboflavin.
Anthraquinone (octaketide) pigments such as catenarin, chrysophanol, cynodontin, helminthosporin, tritisporin and erythroglaucin are produced by Eurotium spp.,
Fusarium spp., Curvularia lunata and Drechslera spp. (Duran et al. 2002). Yellow
pigments epurpurins A to C were isolated from Emericella purpurea (Mapari
et al. 2005) and azaphilone derivatives (hexaketides), falconensins A–H and
falconensones A1 and B2, have been produced both by Emericella falconensis and
Emericella fructiculosa (Ogasawara et al. 1997). Moreover, known for centuries,
Monascus spp. produce azaphilone pigments like monascorubrin, rubropunctatin
(Juzlova et al. 1996) and, more recently, monascusones from a yellow Monascus
mutant have been identified (Jongrungruangchok et al. 2004). Another new natural
food colorant of fungal origin has been patented by Sardaryan (2002), a red colorant
which is an extracellular metabolite of the anthraquinone class produced by a variety
of Penicillium oxalicum. It is also said to confer the anticancer effects when used in
food supplements.
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8.7 Microbial Pigments – Production and Market
The development of the products with an attractive appearance has always been an
important goal in the food industry. Microbial pigments are advantageous, in terms
of production, when compared to similar pigments extracted from the vegetables
or animals. The development of superior vegetable or animal organisms is slower
than that of microorganisms and algae; therefore, the production of the pigments by
bioprocesses involving microorganisms, whose growth velocity is relatively high, is
expected to give such a productivity for the processes that rends them industrially
competitive. Furthermore, the isolation and development of new strains may provide
new, different pigments. Currently, the pigments produced by the microorganisms
and used commercially are riboflavin (vitamin B2, a yellow pigment permitted in
most countries), by Eremothecium ashbyii and Ashbya gossypi; the pigments from
Monascus, by M. purpureus and M. ruber; carotenoids (yellow pigments produced
by several microorganisms, but to this moment produced commercially only from
micro algae) such as -carotene (by Dunaliella salina and D. bardawil) and astaxanthin (by Haematococcus pluvialis); and ficobiliproteins such as phycocianin (a blue
pigment used in food and cosmetics), produced by Spirulina sp.; the pigments with
potential use in the future could be indigoids, anthraquinones and naphtoquinones.
The market for the microbial pigments produced by bioprocesses is hard to estimate, due either for the lack of the statistics of the regional, low-technology products
such as annatto extracts, or the fact that the production is pulverized over many small
companies worldwide (Carvalho et al. 2003, Babitha et al. 2004). At one side, there
is a growing preference for the natural additives in food and cosmetics; at the other
side, in some cases, natural substances may be several times more expensive than
synthetic analogs. A unique example is the -carotene produced by the micro-algae,
which has an approximate cost of U$1000/kg against U$ 500/kg by synthetic means;
although more costly, -carotene produced by the microbial means competes in a
market segments where it is important that all the pigments be “natural”; besides,
the microbial pigment is a mixture of cis- and trans- isomers, with therapeutical
effects against the cancer that synthetic -carotene, predominantly cis-, has not
(Ruijter 1998).
The world market for the pigments of the natural sources (excluding natureidentical and caramels) was estimated in 1987, as U$ 35 million; in 2000, this
market was around U$ 250 million (Downham and Collins 2000). Based on this
growth tendency (600% in thirteen years, against 200% for the whole color market
in the same period), currently the market for the natural pigments (which excludes
nature identical and caramels) is probably on the order of U$ 350 to 600 million
(Babitha et al. 2004). The biggest markets for food pigments are Europe and United
States. The utilization distribution is not proportional to the food consumption (or
to the population), because pigments are used in the processed foods: there is a
potential demand for other countries, in which an improvement on the economic
profile possibly will cause an improvement on the consumption of processed foods.
In the specific case of Monascus pigments, the consumption of these pigments in
Japan rose from 100 ton in 1981 to 600 ton in 1992, and was estimated as U$12
8 Microbial Pigments
153
million, according to a study published in the same year (Lee et al. 1995, Hajjaj
et al. 1997).
8.8 Role of Biotechnology
A lot of attention is now paid to the biotechnological synthesis of the colors through
the microorganisms. Plant cell and tissue culture, microbial fermentation and gene
manipulation have been investigated with respect to the production of pigment.
However, extensive safety testing of such products is required before they are given
clearance as safe food additives. Single cell algae and fungi appear better options
for new biotechnologically derived colorants. The biotechnological production of
the natural colors has two fundamental approaches; first is to find new sources of
colors and then enhance their color production capacity. The other approach is to
obtain enhanced and consistent yields from the already recognized good sources
of the colorants either through the strain improvement or through optimizing the
process parameters to maximize the yield. There is also the obstacle of research
and development investment and manufacturing facilities. The appropriate use of
the fermentation physiology together with the metabolic engineering (Nielsen and
Olsson 2002) could allow the efficient mass production of the colorants from the
fungi. With the advances in the gene technology, attempts have been made to create
cell factories for the production of food colorants through the heterologous expression of biosynthetic pathways from either already known or novel pigment producers (Kim et al. 2003, Lakrod et al. 2003).
8.9 Monascus – A Potent Source of Food Colorant
Exactly when Monascus was discovered is very difficult to investigate, but Chinese
have been using it for one thousand years (Su 1970). In the book, Tsu Shuei Ji,
of Hsu Jian, Tang Dynasty, he quoted the poem of Wang Tsan of Han Dynasty, Chi
Shih, describing the foods he was served when he stayed in Gua Chou. It was “White
Steamed Rice blended with Red Aspergillus in one to one ratio, it was soft, rich
and gliding and spread everywhere after stuffing into mouth”. This was the earliest
record of Monascus in China. In 1884, the French botanist Philippe van Tieghem
isolated a purple mold on the potato and linseed cakes and named it Monascus
ruber. This ascomycete was so named as it has only one polyspored ascus. Then
Went in 1895 isolated one from the red mold rice obtained from the market in
Java, Indonesia. This fungus was named Monascus purpureus (Fig. 8.2a). Then
several others species were isolated around the world. Monascus is encountered
often in the oriental foods, especially in Southern China, Japan and Southeastern
Asia. Currently, more than 50 patents have been issued in Japan, United States,
France and Germany, concerning the use of Monascus pigments for the food.
Historically, Monascus has been wildly used in preparing the traditional Chinese
medicine and food. Anka (Monascus-grown rice) (Fig. 8.2b) is widely used in Japan
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Fig. 8.2 (a) Monascus purpureus (b) Anka
for coloring the protein foodstuff. It has been widely used in China since ancient
time up to present for manufacturing the roasted red pork, Chinese-style red sausage,
and the like. Monascus also produces the enzyme that can decompose the starch,
produce alcohol and decompose the protein. It the oriental countries, it also used
to produce the red rice wine and red fermented bean curd and the like. At present,
several industries produce the red, grinded rice as a natural food supplement capable
of lowering the blood cholesterol, and as the dry product or purified extracts as
the food colorants. In Europe, it is sold as a natural product. Its use in the rice
wine manufacture is due to its high content of alpha-amylase, which promotes the
conversion of the starch into glucose (the attractive red color of the rice wine is
caused by Monascus pigments).
The scientific investigations have confirmed the pharmacological effects of
Monascus fermentate. Monascus extract is marketed in Japan as a dietetic product.
The preservative effect of Monascus fermentate has also been confirmed by the scientific investigation. The observation of the bacteriostatic effects has lead to the consideration to use Monascus fermentate, at least partially, as a substitute for nitrite in
the meat preservation (Fink-Gremmels et al. 1991, Fabre et al. 1993). Sodium nitrite
is added to most packaged meat products for imparting the red color to meat products that would otherwise appear to be a putrid gray color is extremely carcinogenic.
When combined with saliva and digestive enzymes, sodium nitrite creates cancercausing compounds known as nitrosamines. The nitrosamines are highly toxic to
biological systems (they are actually used to give lab rats cancer in laboratory tests).
In the humans, the consumption of sodium nitrite has been strongly correlated with
the brain tumors, leukemia, and cancers of the digestive tract (Pszczola 1998). A scientific proof of the flavor-enhancing properties of Monascus fermentate is difficult
to obtain. However, in a tasting panel, tasters called Monascus containing noodles
“more salty” than the normal noodles, although there was actually no difference in
the salt content. It can be speculated that the relishing effect of Monascus could
8 Microbial Pigments
155
be caused by the flavor-enhancing oligopeptides produced by a partial hydrolysis of
rice proteins by Monascus enzymes (Jacobson and Wasileski 1994). Lin et al. (2004)
have reported a method that used Monascus to ferment the alcohol slowly to produce
a beer-like and alcohol-free fermented beverage.
8.10 Monascus Pigments
The pigments are secondary metabolites of Monascus fermentation; they belong
chemically to the group of Azaphilones, which are typical fungus metabolites. Depending on whether the yellow or red pigments predominate or are absent, the color
of M. purpureus varies from orange-yellow to scarlet to purple-red. The color can
be influenced by the culture conditions, in particular by the pH value (Fabre et al.
1993) and by the phosphorus and nitrogen source in the substrate (Wong et al. 1981).
Monascus produces at least six major related pigments which could be categorized
as (1) orange pigments: rubropunctatin (C21 H22 05 ) and monascorubin (C23 H26 05 );
(2) yellow pigments: monascin (C21 H26 05 ) and ankaflavin (C23 H30 05 ); and (3)
red pigments: rubropunctamine (C21 H23 N04 ) and monoscorubramine (C23 H27 N04 )
(Sweeny et al. 1981) (Fig. 8.3). The same colour exists in two molecular structures
differing in the length of the aliphatic chain. These pigments are produced mainly
in the cell- bound state.
Several authors have described the presence of the pigments different from the
six classical azaphilones in the cultures of Monascus. Blanc et al. (1994) reported
the presence of N-glutarylmonascorubramine and N-glutarylrubropunctamine by
NMR in M. purpureus and M ruber grown in defined medium containing glutamate.
R
R
O
R
O
O
O
O
O
O
O
O
R = C5H11 – monascin
R = C7H15 – ankaflavin
O
O
O
R = C5H11 – rubropunctatin
R = C7H15 – monascorubrin
(yellow)
O
R = C5H11 – rubropunctamine
R = C7H15 – monascorubramine
(orange)
(red)
O
C7H15
NH
O
C5H11
O
O
O
HO
HO
O
O
O
Yellow II
(yellow)
O
HO
R = C5H11 – xantomonascin A
R = C7H15 – xantomonascin B
(yellow)
Fig. 8.3 Structure of Monascus pigments
CHO
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S. Babitha
These two compounds probably derive from the incorporation of a full glutamic
acid molecule instead of the amino group occurring before the cyclization of ring c
in the R1 and R2 normal compounds. The addition of a high glucose concentration
to the culture induced the formation of glucosyl derivatives of the pigments, such as
N-glucosylrubropunctamine and N-glucosylmonascorubramine (Hajjaj et al. 1997),
confirming the influence of the media composition in the pigmentation of the cultures. Two yellow pigments, xanthomonascin and yellow-III, were isolated and characterized by Sato et al. (1992) and Yongsmith et al. (1993); these two pigments and
the yellow ankalactone molecule described by Nozaki et al. (1991) are azaphilonederived molecules.
From the previous works (Blanc et al. 1994, Hajjaj et al. 1997), a scheme of the
hypothetical routes for the biosynthesis of these various pigments in the filamentous
fungi is depicted in Fig. 8.4. The condensation of 1 mol of acetate with 5 mol of
malonate leads to the formation of a hexaketide chromophore by the polyketide
synthase. Then a medium-chain fatty acid such as octanoic acid, likely produced
by the fatty acid biosynthetic pathway, is bound to the chromophore structure by
a transesterification reaction to generate the orange pigment monascorubrin (or
rubropunctatin upon trans-esterification with hexanoic acid). The reduction of the
orange pigment gives rise to the yellow pigment ankaflavin from monascorubrin
(or monascin from rubropunctatin), whereas red pigments (monascorubramine and
rubropunctamine) are produced by the amination of orange pigments with NH3 units
(Lin et al. 1992). All these pigments remain essentially intracellular because of their
high hydrophobicity. They are eventually excreted in the medium after reacting with
an NH2 unit of amino acids (Kyoko et al. 1997, Lin et al. 1992).
The biosynthetic reactions leading to each product are completely open, and it
is unclear if they are formed from a common early intermediate by a branched
pathway or not. The structure of these compounds indicates that at least one, and
possibly two, polyketide synthases must be involved in their biosynthesis. Octanoic
or hexanoic acid have been suggested to be precursors of the azaphilone compounds
(Hajjaj et al. 2000). Possibly, these fatty acids are formed by a separate polyketide
synthase as in aflatoxin biosynthesis (Brown et al. 1996).
The orange pigments, monascorubrin and rubropunctatin, are synthesized on
the cytosol from acetyl coenzyme A. These pigments have a structure responsible
by their high affinity to compounds containing primary amino groups (thus called
aminophiles). Reactions with amino acids lead to formations of hydro soluble red
pigments, monascorubramine and rubropunctamine. The mechanism of yellow pigment formations is not yet clear; some authors consider that these are product of
the alteration of orange pigments, as others believe it to be pigments with their own
metabolic pathway (Lin and Demain 1991, Juzlova et al. 1996).
Since the pigments generally show low water solubility, attempts have been made
to make water-soluble pigments. The principle is the substitution of the replaceable
oxygen in monascorubrine or rubropunctatine by nitrogen of the amino group of various compounds such as aminoacids, peptides and proteins, changing the color from
orange to purple. Monascus pigments can be reduced, oxidized and react with other
products, especially amino acids, to form various derivative products sometimes
8 Microbial Pigments
157
Fig. 8.4 Scheme of the hypothetic metabolic routes leading to the final structure of the watersoluble red pigment N-glutarylmonascorubramine in Monascus. Source: Hajjaj et al., 1997
called the complexed pigments. Glutamyl-monascorubrine and glutamylrubropunctatine were isolated from the broth of a submerged culture. Because of their affinity
to amino groups, Monascus pigments are frequently associated to proteins (Wong
and Koehler 1983), amino acids, nucleic acids to form water-soluble pigments or
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S. Babitha
to the cell wall, forming a complex that may be of difficult extraction. Other authors consider that there may be a fixation of the pigments to lipids of the fungal
biomass, so that the extraction would involve cell breaking and dissolution in an
organic solvent (St. Martin 1990). Also due to this affinity for amino groups, it
is possible to convert orange lipossoluble pigments in red hydro soluble ones by
reaction with amino acids and analog compounds in vitro (St. Martin 1990). In that
case, nitrogen from the amino group (from the amino acid or analog) takes the place
of the oxygen of the ring on rubropunctatin or monascorubrin, yielding analogs of
rubropunctamine or monascorubramine, but presenting a radical linked to the N in
substitution to the H of the natural red pigments.
Amongst the possible amino acids to be used in order to induce the formation
of hydro soluble pigments, in vivo is glutamic acid, in the form of monosodium
glutamate (MSG) (Lin 1992). In a study using glucose as a carbon source in a
liquid medium containing MSG, it was observed the formation of N-glucosil derivatives of the red pigments, corresponding to as much as 10% total pigments (Hajjaj
et al. 1997).
Amongst the pigments produced by Monascus, the red ones are the most important, since they may be possible substitutes of the synthetic colors such as erythrosine (FD & C red No. 3) (Johns and Stuart 1991); these are stable pigments on
the range of pH from 2 to 10, with good stability to temperature and that may be
autoclaved (Lin 1992). Some studies show that Monascus pigments may be used
as substitutes for the traditional food additives, such as nitrites and cochineal, in
sausages and other meat products (Fabre et al. 1993). There are other studies on the
sensorial response, allergenicity and toxicity, although eastern countries like Japan
make extensive industrial use of these pigments since several decades – as examples,
yellow hydro soluble pigments for candies, (Watanabe et al. 1997), or red pigments
in red rice wine.
8.11 Monascus Pigment Production by Solid-State
Fermentation
Solid-state fermentation (SSF), however, gives a higher yield and productivity of the
pigment than the liquid fermentation. Lin (1973) has demonstrated that the pigment
production in submerged culture was only 1/10 of that in solid-state culture. In order
to reduce the costs of the industrial-scale fermentations, the use of agro-industrial
byproducts as the sources for the microbial biosynthesis has been the subject of
much research in recent years, which highlights the biotechnological potential of
SSF (Pandey et al. 2000, Carvalho et al. 2007). One another advantage, which SSF
offers is the application of fermented solids directly as a colorant without isolating
the product. Fungi are the most adequate microorganisms for the SSF as solid substrate presents a more adequate habitat for the fungus (Pandey 1994). The solid-state
fermentation of rice by Monascus has a long tradition in the East Asian countries,
which dates back at least to the first century.
8 Microbial Pigments
159
A comparison was made between the liquid and solid media of similar composition, the solid media obtained from the liquid by addition of a gelling agent,
followed by the extrusion in rice-sized particles. The solid media thus prepared supported the production of up to three times more pigment than the corresponding liquid media, but the cultivations over rice were still superior (Johns and Stuart 1991).
The mycelia of Monascus species penetrate into the surface of the solid nutritional
medium and grow during the period of fermentation. The pigments produced are
absorbed so that the color of the medium turns purple. In addition, the mycelia also
penetrate inside the grain particles. This phenomenon facilitates the production of
pigments.
8.12 Agro-Industrial Residues as Substrates
for Monascus Pigment Production
The selection of substrate for the SSF process depends upon several factors, mainly
related with cost and availability. In the SSF process, the solid substrate not only
supplies the nutrients to the microbial culture growing in it, but also serves as an
anchorage for the cells (Pandey et al. 2000). Traditionally, Monascus has been cultivated on the rice (forming ang-kak or red rice), although several other media, have
been tested for the pigment production. Rice is the natural substrate which gives
the best production, compared to other typical cereals, tubers and leguminous plants
(Carvalho et al. 2003). However, some of the other substrates used also presented
good biopigment production, especially corn, wheat and cassava. Cassava bagasse
gave a low pigment yield, but being an agro-industrial residue whose low price
might compensate for its low yield. Very recently jackfruit seed powder has been
identified as a potent substrate for pigment production (Babitha et al. 2007).
8.13 Other Applications of Monascus Pigments
Tsuyoshi et al. (2004) has invented a decoloring ink for ink jet printing containing
a Monascus pigment. Inclusion of Monascus pigment enables the printed characters and/or images to be preserved as long as they are kept in the dark, but they
are quickly decolored on exposure to the visible light and/or ultraviolet light when
they are no longer needed. Another landmark application of Monascus pigment has
been patented by Yamamoto et al. (2006) which relates to a histostain composition
for endoscope containing one or more members selected from colors derived from
Monascus. The stain composition is a staining agent which sharpens the shapes of
digestive tract lumen surfaces and the like with a light in the visible wavelength
range, having a function of being excited by a light of specific wavelength to emit
fluorescence, and being biologically safe and suitable for endoscopy. They unexpectedly found that colors derived from Monascus are characterized by being excellent in staining property under a visible light, having fluorescence whose wavelength
being different from its excitation wavelength, being useful not only as a staining
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agent in usual endoscopy but also as a fluorescent dye for interstitial staining in
confocal endoscopy, to give a vivid stained image useful in detection of a small
affected region, and staining only the cytoplasm without staining cell nuclei, thus
indicating that these colors have reduced cellular mutagenicity. Very recently, inhibitory activity of Monascus pigments against diet related lipase has been reported
(Kim et al. 2007). The lipase produced by pancreatic acinar cells is considered to
be essential for the digestion of dietary fats in the intestinal lumen. Therefore, if
reactions using the pancreatic lipase in the human body are inhibited, fat absorption
and obesity can be controlled.
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Chapter 9
Production of Mushrooms
Using Agro-Industrial Residues as Substrates
Antonios N. Philippoussis
Contents
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2 Residue-Based Substrates and their Solid-State Fermentation by Mushroom Fungi . . . . .
9.2.1 Types, Availability and Chemical Composition of Raw Materials . . . . . . . . . . . . . .
9.2.2 Nutritional and Environmental Aspects of Mushroom Growing . . . . . . . . . . . . . . .
9.2.3 Output and Stages of Mushroom Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3 Bioconversion of Solid Residue-Substrates Through Mushroom Cultivation . . . . . . . . . .
9.3.1 Commercial Mushroom Production Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.2 Efficiency of Residue Conversion to Pleurotus sp. and L. edodes
Fruiting Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Mushroom cultivation as a prominent biotechnological process for the
valorization of agro-industrial residues generated as a result of agro-forestry and
agro-industrial production. A huge amount of lignocellulosic agricultural crop
residues and agro-industrial by-products are annually generated, rich in organic
compounds that are worthy of being recovered and transformed. A number of these
residues have been employed as feedstocks in solid state fermentation (SSF) processes using higher basidiomycetus fungi for the production of mushroom food,
animal feed, enzymes and medicinal compounds. Likewise, the above-mentioned
microorganisms have been successfully employed in processes related with the
bioremediation of hazardous compounds and waste detoxification. Mushroom cultivation presents a worldwide expanded and economically important biotechnological
industry that uses efficient solid-state-fermentation process of food protein recovery
from lignocellulosic materials. Several aspects of mushroom physiology along with
A.N. Philippoussis (B)
National Agricultural Research Foundation, I.A.A.C., Laboratory of Edible and Medicinal Fungi,
13561 Ag, Anargyri, Athens, Greece
e-mail: iamc@ath.forthnet.gr
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 9,
C Springer Science+Business Media B.V. 2009
163
164
A.N. Philippoussis
impacts of different environmental and nutritional conditions on mycelium growth
and fruiting bodies production are highlined. Moreover, cultivation technologies of
Agaricus bisporus, Pleurotus spp and Lentinula edodes, comprising spawn (inoculum) production, substrate preparation and mushroom growing process i.e. inoculation, substrate colonization by the cultivated fungus, fruiting, harvesting and
processing of the fruiting bodies, are outlined. Finally, the efficiency of residues
conversion into fruiting bodies are outlined in two medicinal mushroom genera,
Pleurotus and Lentinula, widely cultivated for their nutritional value and extensively researched for their biodegradation capabilities. Experimental data concerning residue-substrates used, as well as biological efficiencies obtained during their
cultivation were considered and discussed.
Keywords Fungi · Mushroom cultivation · Biotechnology · Agricultural residues ·
By-products · Solid state fermentation · Fruiting bodies · Yield · Biological
efficiency · Agaricus spp. · Pleurotus spp. · Lentinula edodes
9.1 Introduction
On the surface of our planet, around 200 billion tons per year of organic matter are
produced through the photosynthetic process (Zhang 2008). However, the majority
of this organic matter is not directly edible by humans and animals and, in many
cases, becomes a source of environmental problem. Moreover, today’s society, in
which there is a great demand for appropriate nutritional standards, is characterized by rising costs and often decreasing availability of raw materials together
with much concern about environmental pollution (Laufenberg et al. 2003). Consequently, there is a considerable emphasis on recovery, recycling and upgrading of
wastes. This is particularly valid for the agro-food industry, which furnishes large
volumes of solid wastes, residues and by-products, produced either in the primary
agro-forestry sector or by secondary processing industries, posing serious and continuously increasing environmental pollution problems (Boucqué and Fiems 1988,
Koopmans and Koppejan 1997, Lal 2005). It is worth mentioning that only crop
residues production is estimated to be about 4 billion tons per year, 75% originating
from cereals (Lal 2008).
Nevertheless, residues such us cereals straw, corn cobs, cotton stalks, various
grasses and reed stems, maize and sorghum stover, vine prunings, sugarcane and
tequila bagasse, coconut and banana residues, corn husks, coffee pulp and coffee
husk, cottonseed and sunflower seed hulls, peanut shells, rice husks, sunflower
seed hulls, waste paper, wood sawdust and chips, are some examples of residues
and by-products that can be recovered and upgraded to higher value and useful products by chemical or biological processes (Wang 1999, Fan et al. 2000a,
Pandey et al. 2000b, c, Webb et al. 2004). In fact, the chemical properties of such
lignocellulosic agricultural residues make them a substrate of enormous biotechnological value. They can be converted by solid state fermentation (SSF) into
various different value-added products including mushrooms, animal feed enriched
with microbial biomass, compost to be used as biofertilizer or biopesticide, en-
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
165
zymes, organic acids, ethanol, flavours, biologically active secondary metabolites
and also for bioremediation of hazardous compounds, biological detoxification of
agro-industrial residues, biopulping etc. (Pandey et al. 2000a, Bennet et al. 2002,
Sánchez et al. 2002, Tengerdy and Szakacs 2003, Howard et al. 2003, Kim and
Dale 2004, Nigam et al. 2004, Zervakis et al. 2005, Manpreet et al. 2005,
Krishna 2005).
Among applications of SSF, mushroom cultivation has proved its economic
strength and ecological importance for efficient utilization, value-addition and biotransformation of agro-industrial residues (Chang 1999, 2001, 2006, Chiu et al. 2000,
Zervakis and Philippoussis 2000). Current literature shows that lignocellulose degrading mushroom species are used in various SSF applications such as bioremediation and biodegradation of hazardous compounds (Pérez et al. 2007), biological detoxification of toxic agro-industrial residues (Pandey et al. 2000d, Fan
et al. 2000b, Soccol and Vandenberghe 2003), biotransformation of agro-industrial
residues to mushroom food and animal feed (Moore and Chiu 2001, Alborés
et al. 2006, Okano et al. 2006), compost and product developments such as biologically active metabolites, enzymes, and food flavour compounds (Ooi and Liu 2000,
Cohen et al. 2002, Silva et al. 2007, Nikitina et al. 2007). Moreover, recent research
work indicates medicinal attributes in several species, such as antiviral, antibacterial, antiparasitic, antitumor, antihypertension, antiatherosclerosis, hepatoprotective, antidiabetic, anti-inflammatory, and immune modulating effects (Wasser and
Weis 1999, Wasser 2002, Daba and Ezeronye 2003, Paterson 2006).
Commercial mushroom production, carried out in a large or small scale, is an efficient and relatively short biological process of food protein recovery from negativevalue lignocellulosic materials, utilizing the degrading capabilities of mushroom
fungi (Martı́nez-Carrera et al. 2000, Chiu and Moore 2001). Among mushroom
fungi, L. edodes and Pleurotus species reveal high efficiency in degradation of a
wide range of lignocellulosic residues, such as wheat straw, cotton wastes, coffee
pulp, corn cobs, sunflower seed hulls wood chips and sawdust, peanut shells, vine
prunings and others into mushroom protein, (Ragunathan et al. 1996, Campbell
and Racjan 1999, Stamets 2000, Poppe, 2000, Philippoussis et al., 2000, 2001a, b),
the productivity of the conversion being expressed by biological efficiency (Chang
et al. 1981). Their mycelium can produce significant quantities of a plethora of
enzymes, which can degrade lignocellulosic residues and use them as nutrients
for their growth and fructification (Bushwell et al. 1996, Elisashvili et al. 2008).
However, the nature and the nutrient composition of the substrate affect mycelium
growth, mushroom quality and crop yield of this value-added biotransformation process (Kües and Liu 2000, Philippoussis et al. 2001c, 2003, Baldrian and Valášková
2008).
The focus of this work is to highlight significant aspects of utilization of low- or
negative-value agro-industrial residues in mushroom biotechnology, emphasizing
on their biotransformation to fruiting bodies that are nutritious human foodstuff
regarded also as functional food. Aspects to be reviewed in this article include:
an overview of availability, sources and types as well as chemical composition of
solid lignocellulosic agro-residues suitable for mushroom cultivation, some back-
166
A.N. Philippoussis
ground on mushroom degrading abilities and of their nutritional and environmental
demands, an outline of commercial production technologies of A. bisporus, Pleurotus
spp. and L. edodes mushrooms, and finally a consideration and discussion of experimental data regarding productivity (biological efficiency) on various agro-industrial
residues during cultivation ofPleurotus spp. and L. edodes.
9.2 Residue-Based Substrates and their Solid-State
Fermentation by Mushroom Fungi
As a result of agro-forestry and agro-industrial production, a huge amount of livestock waste, agricultural crop residues and agro-industrial by-products are annually
generated, the major part being lignocellulosic biomass (Kuhad et al. 1997). Although agro-industrial residues contain beneficial materials, their apparent value is
smaller than the cost of collection, transportation and processing for beneficial use.
However, if residues are utilized, such as to enhance food production, they are not
considered as wastes but new resources. A number of agro-industrial residues have
been employed as feedstocks in SSF processes, using high basidiomycetus fungi for
the production of valuable metabolites (Rajarathnam et al. 1998, Howard et al. 2003,
Hölker et al. 2004). However, mushroom production is one of the areas with great
potential for exploitation of forest and agricultural residues (Moore and Chiu 2001,
Chang 2006, Gregori et al. 2007, Silva et al. 2007).
9.2.1 Types, Availability and Chemical Composition
of Raw Materials
Reddy and Yang (2005) and very recently, Zhang (2008), reviewing the global world
information about lignocellulose availability, estimated the production of lignocellulosic biomass to be more than 200 × 109 tons per year. Especially, the amount
of crop residues produced annually in the world from 27 food crops is estimated at
about 4 × 109 tons, from which 3 billion tons account per annum for lignocellulosic residues of cereals (Lal 2005, 2008). Cereals, accounting 75% of global world
production (FAO 2004), furnish these outstanding amounts of waste products as
wheat residues, rice straw and hulls, barley residue, maize stalks and leaves, millet
and sorghum stalks. Sugar cane provides the next sizeable residue with two major
crop wastes, leaves and stalk, and bagasse, which is the crop processing residue.
The cotton crop also provides significant residue in the form of stalks and husks,
while no negligible are the residues furnished by minor crops as sunflower, oil palm,
coconut, banana, vines, groundnut and coffee. In fact, this generation of residues is a
result of the limited portions of the crops that are actually used. To give the order of
magnitude, 95% of the total biomass produced in palm and coconut oil plantations
is discarded as a waste material; the respective values for sisal plant and sugar cane
biomass are 98% and 83% (Chang 1998). Moreover, in the flax industry only 2% of
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
167
the produced biomass is effectively used, less than 9% in the palm oil industry and
only 8% in the brewing industry (López et al. 2004).
Biomass availability is a primary factor for bio-based industrial production. Indeed, the available resource potential (the amount of residues used for various purposes) is smaller than the one generated. The quantities of crop residues that can
be available for bioprocesses are estimated using total grain production, residue to
product ratio (RPR), moisture content, and taking into consideration the amount of
residue left on the field to maintain soil quality (i.e. maintain organic matter and
prevent erosion), grazing and other agricultural activities (Koopmans and Koppejan 1997, Giljum et al. 2005). Concerning cereal straw, the RPRs for rice, barley,
wheat and corn are 1.4, 1.2, 1.3 and 1 respectively (Mulkey et al. 2008). Assuming
that one quarter of the residues can be harvested and that roughly one third of the
harvested straw is used in animal husbandry, 0.22 tons straw per ton cereal grain
and 0.25 ton residues per ton maize are available biomass for other uses, as energy,
enzyme production or mushroom growing.
As agro-industrial residues accumulate in fields and factories, availability issue tends to become a regional and local matter. Geographical distribution of crop
residues is skewed by large crop productions in India and China, where increased
quantities of crop residues and agro-industrial by-products are generated because
of expanding agricultural production. Furthermore, Asia along with Europe, North
America and Australia are world leader mushroom producing regions (Chang 2006)
and consequently the major residue demanding for this bio-based industrial activity. Among countries in the Asian and Pacific Region, China produces the largest
quantities of agricultural and forest residues, mainly by-products of rice, corn and
wheat (Zhang 2008). China’s quantities, estimated to reach about 1 billion tons/year
(Qu et al. 2006), are followed by India’s yielding at least 200 million tons/year
of agricultural residues according to Das and Singh (2004), while according to
Mande (2005) India’s total amount of agro-industrial residues reaches 600 million tons. This quantity comprises 480 tons of crop residues (rice, wheat, millet,
sorghum, pulses, oilseed crops, maize stalks and cobs, cotton stalks, sugarcane trash
etc.) and 120 tons of processing–based residues (mainly groundnut shells, rice husk,
sugarcane bagasse, cotton waste, coconut shell and coir pith). Rice and sugar are
Asia’s rest southeast countries dominant crops.
Moghtaderi et al. (2006) report that Australian agro-industrial biomass reaches
100 million tons/year, including bagasse, cane trash, wood residues, energy crops
etc. As far as Africa is concerned, wheat and barley predominate in the north, millet
and sorghum are the main crops in sub-Saharan Africa, while farther south maize is
the dominant crop. Kim and Dale (2004), estimated Africa’s annual lignocellulosic
biomass from rice straw, wheat straw and sugar cane bagasse to be about 40 million
tons, indicating that the fraction of most crop residues collectable is less than 30%
because of low yields. In the same work, Central and South America’s lignocellulosic residues were estimated to be about 140 million tons from rice and wheat
straw, corn stover and sugar cane bagasse, not taking into account coffee, banana
and other agricultural residues. Concerning North America, according to USDA-US
DOE report (2005), USA is able to produce 1.3 billion tons of dry residues per year,
168
A.N. Philippoussis
including agricultural (933 million tons) and forest resources (368 million tons).
Main lignocellulosic by-products in considerable quantity are corn stover, the most
abundant agricultural residue in USA, wheat, rice, barley straw, sorghum stalks,
coconut husks, sugarcane bagasse, pineapple and banana leaves. Canada, the second
largest supplier of wood lignocellulosic biomass, supplies more than 200 million m3
of lignocellulose annually through commercial operations (Mabee et al. 2005). Finally, Europe is not only a great wheat straw producer, but also outstanding quantities of lignocellulosic residues from barley, maize, sunflower, rapeseed, cotton, olive
trees and vines, summarized as 120 million tons/ year (Nikolaou et al. 2003).
Regarding the types of wastes, according to Mande (2005), agricultural residues
can be divided into two groups: crop-based residues (generated in the field) and
processing-based residues (generated during wood and industrial processing). Cropbased residues, which are plant materials left behind in the field or farm after removal of the main crop produce, are consisted of different sizes, shapes, forms, and
densities like straw, stalks sticks, leaves, haulms, fibrous materials, roots, branches,
and twigs. Crop-based residues are produced from various sources such as field
and seed crops (including straw or stubble from barley, beans, oats, rice, rye, and
wheat, stalks or stovers from corn, cotton, sorghum, grasses and reeds, soybeans
and alfalfa), fruit, nut, vegetable or energy crops (brushes and orchard prunings, e.g.
vine shoots or leaves that remain on the ground after harvesting), and livestock manure. Processing-based agro-industrial residues are by-products of the post-harvest
processes of crops such as cleaning, threshing, linting, sieving, and crushing. They
are in the form of husk, dust, stalks etc. Food processing wastes that come from
plant materials are culls, rinds, seeds, pits, pulp, press cakes, marc, malts, hops
and a variety of other by-products from mass food production processes. Some
examples of these materials are coffee processing by-products, sugarcane bagasse,
hulls and husks, wheat middlings, corncobs, seed meals etc. Moreover, this category
comprises wood residues produced either from the primary processing or from secondary manufacturers (producing bark, chips, sawdust, coarse residues, and planer
shavings). During the sawing of a log at a typical sawmill, approximately 50% of
the initial log volume is converted into wood products and 50% is converted into
wood residues (Alderman 1998).
In general, solid agro-industrial residues are heterogeneous water insoluble materials having a common feature, their basic macromolecular structure being cellulose, hemicellulose and lignin and to a lesser extend pectin, starch and other
polysaccharides (Thomsen 2005). Cellulose, the most abundant renewable organic
resource comprising about 45% of dry wood weight, is a linear homopolymer of
glucose units linked with  − 1,4-glucosidic bonds (Baldrian and Valášková 2008).
Hemicelluloses, heteropolysaccharides containing two to four different types of
sugars, are divided in three major groups: xylans, mannans and galactans. They
consist of short-branched chains of hexoses, e.g. mannose units in mannans and
pentoses such as xylose units in xylans (Kuhad et al. 1997). After cellulose, lignin
is the second most abundant renewable biopolymer in nature. Lignin, representing
between 26 to 29% of lignocellulose, is strongly bounded to cellulose and hemicellulose, imparting rigidity and protecting the easily degradable cellulose from
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
169
the hydrolase attack (Raimbault 1998). Lignin is an aromatic polyphenol macromolecule, 3-dimensional and amorphous (Pérez et al. 2002). As the proportions of
these three structural components characterize residue biomass, their percentages in
mushroom substrate ingredients, along with nitrogen content and carbon to nitrogen
ratios, are shown in Table 9.1. Additionally, crop residues contain, on a dry weight
basis, approximately 0.5–1.5% N, 0.15–0.2% P, 1% K, 1% Ca, 0.5% Mg, 0.2% S,
30 mg Kg–1 Mn, 100 mg Kg–1 Fe, 30 mg Kg–1 Zn, 5 mg Kg–1 Cu, 20 mg Kg–1 B and
about 1 mg Kg–1 Mo (Mills and Jones 1996). However, these values differ with crop,
plant part, season, soil moisture as well as other factors that affect plant growth.
Substrates used in mushroom cultivation include both field-based residues and
processing based-residues (Table 9.1). However, as the nutrient composition of the
substrate is one of the factors limiting colonization as well as quantitative and
qualitative yield of cultivated mushrooms (Philippoussis et al. 2000, 2002), supplements containing sugars and starch (easily available carbohydrates) and fats
(slower degraded and time-lasting nutrient sources) are added to the basal ingredient. Supplements are used to increase nutritional content, speed-up growth and
increase mushroom yield, especially in the cultivation of the white-rot mushroom
fungi L. edodes (Royse et al. 1990, Royse 1996) and Pleurotus spp. (Naraian
et al. 2008). The various organic supplements used in mushroom cultivation comprise molasses, brewer’s grain, grasses and waste paper, cotton and coffee wastes
etc. However, soybeans and cereal grains or their milling by-products are the most
commonly used supplements, as they are generated in considerable amounts and
contain increased levels of protein, fats and easily metabolized carbohydrates: soybeans (carbohydrates 21.5%, N 6.3%), wheat bran (carbohydrates 49.8%, N 2.4%),
rice bran (carbohydrates 37.0%, N 2.0%) and millet (carbohydrates 57.3%, N 1.9%),
(Przybylowicz and Donoghue 1990).
9.2.2 Nutritional and Environmental Aspects
of Mushroom Growing
From about 14000 mushroom-forming fungal species, at least 2000 are edible, of
which 80 species are grown experimentally and around 20 are cultivated commercially (Chang 1999, Silva et al. 2007). The most cultivated worldwide species are
A. bisporus, P. ostreatus and L. edodes, followed by Auricularia auricula, Flammulina velutipes and Volvariella volvacea. Other mushroom species produced successfully on various substrates include Agrocybe aegerita, Ganoderma spp., Grifola
frondosa, Hericium erinaceus, Hypsizygus marmoreus, Lepista nuda, Coprinus comatus, Pholiota nameko and Stropharia spp. (Stamets 2000, Royse 2004). Although
the mentioned mushroom species have the ability to degrade lignocellulosic residues
in their original or composted form (Rajarathnam et al. 1998), they exhibit differences regarding production of enzymes necessary to degrade lignocellulosic substrates and thus different abilities to grow and fruit on residue-substrates (Bushwell
et al. 1996, Chen et al. 2003, Baldrian and Valášková 2008).
Table 9.1 Chemical properties (based on dry matter) of agro-industrial residues used as substrate ingredients in mushroom cultivation
a
Hemicellulose Lignin (%)
Cellulose/
lignin
Ash (%)
N (%)
C/N
Referencesb
36.4–40.0
25.0–29.0
13.0–21.0
2.1–2.3
3.6–7.0
0.6–0.9
55.8–77.3
1, 15, 17, 20
25.0–40.0
34.4–42.6
22.8–38.4
34.0–60.8
31.5–39.5
13.0–38.0
28.4–30.6
17.7–28.5
17.0–21.0
21.2–29.0
6.4–17.6
17.1–19.7
6.4–18.0
20–22.9
5.6–15.0
2.4–3.9
1.7–2.0
3.6–5.9
2.0–2.8
2.2–5.3
4.2–6.2
4.3–4.9
8.3–17.8
NA
5.6–8.0
1.3–2.5
0.3–0.5
0.5–1.1
NA
0.4–0.8
28.0–42.0
150.0–170.0
51.4–57.8
NA
48.8–59.6
1, 8, 20, 22
12, 33
8, 14, 17, 29
7, 10, 31
1, 20, 21, 22, 30, 31
16.0–18.0
21.0–36.0
23.0–29.1
28.0–45.0
52.0–90.0
26.4–30.4
12.0–22.7
15.1–17.1
35.0–43.0
5.0–20.0
27.5–28.1
41.0–48.0
13.0–26.0
11.0–17.0
4.0–12.0
0.6–0.8
0.6–1.3
0.89–0.94
2.5–2.7
5.0–11.2
4.6–5.0
2.7–10.2
4.5–6.3/1.0–6.0
4.4–4.8
2.6–8.4
4.1–4.5
0.4–1.1
1.4–1.9/0.9–1.0
0.4–1.1
0.3–1.4
11.6–12.2
77.6–124.2
53.5–59.4
64.2–71.6
40.0–59.0
5, 9, 16
1, 24, 25, 29
2, 26, 27
6, 8, 11, 21, 22, 31
8, 21, 22, 23
24.5–37.5
54.3–70.0
28.0–43.0
26.6–40.0
31.3–42.7
37.7–49.5
20.6–24.9
12.4–25.0
17.5–20.6
19.0–30.0
24.0–25.2
10.7–25.0
29.6–35.1
11.3–29.7
21.5–22.5
19.0–23.3
23.2–28.7
26.1–29.5
0.7–1.2
3.0–6.0
1.3–1.9
1.4–2.2
1.1–1.8
1.4–1.7
8.2–8.7
NAa
16.7–21.4
1.5–5.0
3.0–3.3
0.4–0.5
0.8–0.9
NA
0.3–0.4
0.2–0.8
0.6–0.9
0.1–0.1
50.6–58.6
NA
100.0–136.0
120.0–190.0
60.0–72.4
310.0–520.0
3, 19, 28
8, 15, 34
9, 13, 31
8, 18, 24, 26, 31
4, 28
20, 32, 34
42.9–45.1
22.0–33.0
24.0–26.0
1.7–2.0
0.2–0.3
0.1–0.2
150.0–450.0
6, 20, 21, 32
NA: Data not available
References: [1] USDA-US DOE 2005: http://www.eere.energy.gov/biomass/progs, [2] Brand et al. 2000, [3] Çöpür et al. 2007, [4] Curvetto et al. 2005, [5]
Demeke 2007, [6] Gabriel 2004, [7] Gañan et al. 2006, [8] Howard et al. 2003, [9] Anonymous 2006: http://nkk.naro.affrc.go.jp/eng/topics/reseach/2006/9.pdf,
[10] Jiménez and González 1991, [11] Laufenberg et al. 2003, [12] Lin 2005, [13] Liou et al. 1997, [14] Mata and Savoie 2004, [15] Mosier et al. 2005, [16]
Mussato et al. 2008, [17] Obodai et al. 2003, [18] Ortega et al. 1992, [19] Özçelik and Pekşen 2007, [20] Palonen 2004, [21] Philippoussis et al. 2001a,
[22] Poppe 2004, [23] Quian 2004, [24] Ragunathan et al. 1996, [25] Reddy and Yang 2005, [26] Salmones et al. 1999, [27] Salmones et al. 2005, [28]
Saura-Calixto et al. 1983, [29] Shashirekha and Rajarathnam 2007, [30] Singh 2000, [31] Thomsen 2005, [32] Tisdale et al. 2006, [33] Ververis et al. 2004,
[34] Ward et al. 2000.
b
A.N. Philippoussis
Field-based residues
Corn (maize)
stover/husk
Grass residues
Reed stems/ residues
Rice straw
Vine shoots
Wheat straw
Processing-based
residues
Brewers grains
Coconut husk/coir
Coffee pulp/husk
Corncob
Cotton wastes (gin trash
/hull)
Hazelnut husk
Paper (waste)
Rice husk
Sugarcane bagasse
Sunflower seed hull
Wood chips/ sawdust
(softwood)
Wood chips/ sawdust
(hardwood)
Cellulose
170
Residue-substrates
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
171
In plant residues, cellulose and hemicellulose are the main sources of carbohydrates, often incrusted with lignin, which forms a physical seal around these two
components. Lignocellulose is physically hard, dense and recalcitrant, the degradation of which is a complex process requiring a battery of hydrolytic or oxidative
enzymes. Taking into consideration that the substrates are insoluble, degradation occurs extracellularly, by two types of extracellular enzymatic systems: the hydrolytic
system, which produces hydrolases and is responsible for cellulose and hemicellulose degradation; and a unique oxidative lignolytic system, which depolymerizes
lignin (Pérez et al. 2002, Baldrian 2005). The hydrolytic breakdown of cellulose
by fungi is catalyzed by extracellular cellobiohydrolases, endoglucanases and glucosidases, which hydrolyze the long chains of cellulose, liberating cellobiose
and finally glucose, while the major hemicellulose-degrading enzymes are endoxylanases and endomannanases (Tengerdy and Szakacs, 2003). Most of these enzymes
have been detected in both wood-degrading mushroom fungi (WDF), like P. ostreatus and L. edodes (Elisashvili et al. 2008) and litter-decomposing mushroom
fungi (LDF), such as A. bisporus or V. volvacea (Steffen et al. 2007). Due to its
complicated structure, lignin is more difficult to break down than cellulose or hemicellulose. The main extracellular enzymes participating in lignin degradation are
lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Hatakka 1994),
with MnP, prooving to be the most common lignin-modifying peroxidase produced
by almost all wood-degrading basidiomycetes (Steffen et al. 2007). In addition,
litter-decomposing basidiomycetes can degrade lignin e.g. A. bisporus produces at
least two lignolytic enzymes, laccase and MnP, however, the overall lignin degradation rate by these fungi is lower compared to that of white-rot fungi (Lankinen
et al. 2005, Steffen et al. 2007). Besides the lignocellulosic enzyme complex, lignocellulolytic fungi also produce other enzymes, such as pectinases, proteases, lipases
and phytases on lignocellulosic substrates (Tengerdy and Szakacs, 2003).
Basidiomycetous fungi comprise diverse ecological groups, i.e. WDF (white rots,
brown rots) and LDF, which may insure their nutrition in different ways. White-rot
fungi are able of a simultaneous degradation of all wood components (cellulose,
hemicellulose and lignin), while brown-rot fungi, a relatively small group of Basidiomycetes, degrade only cellulose and hemicellulose. Given that the majority of
cultivated higher basidiomycetes is WDF, while few of them are LDF, emphasis
is given here to the nutritional behaviour and degradation potentials of these two
groups, represented by the most cultivated species A. bisporus, Pleurotus spp. and
L. edodes.
White rot mushroom-forming fungi, comprising cultivated species like Pleurotus spp., L. edodes, Ganoderma spp. etc., are the most efficient degraders, due to
their capability to synthesize relevant hydrolytic (cellulases and hemicellulases)
and unique oxidative (lignolytic) extracellular enzymes. Their general strategy is
to decompose the lignin in wood, so that they can gain access to the cellulose and
hemicelluloses embedded in the lignin matrix (Hatakka 1994). However, laccase
expression in fungi is influenced by culture conditions, such as nature and concentration of carbon and nitrogen sources, media composition, pH, temperature,
presence of inducers and lignocellulosic materials, etc. (Revankar et al. 2007).
172
A.N. Philippoussis
A wide variety of lignin degradation efficiency and selectivity abilities, enzyme
patterns and substrates enhancing lignin degradation are reported from white-rot
fungi (Hatakka 2001, Baldrian and Valášková 2008). An interesting category of
white-rot fungi are selective degraders that degrade lignin rather than cellulose, like
Pleurotus spp., which are used in a wide range of biotechnological applications
(Cohen et al. 2002). Lignin degradation by these fungi is thought to occur during secondary metabolism and typically under nitrogen starvation (Hammel 1997).
Non-composted, chopped and water-soaked straw is sufficient for the cultivation
of Pleurotus spp., while L. edodes is cultivated on logs or in bags on moisturized
sawdust supplemented with cereal bran (Philippoussis et al. 2000, 2004). Although
not necessarily optimal, since they are low in readily accessible nutrients, these
commercially used substrates satisfy the needs of the fungi for growth and fruiting,
and most importantly, help to withstand microbial competitors (Kües and Liu 2000).
In basidiomycetous LDF, comprising cultivated mushroom species like Agaricus
spp., Agrocybe spp., Coprinus spp., Stropharia spp. and V. volvacea, degradation
involves a succession of biodegradative activities that precede attack by lignocellulose degraders. However, the ability to break down lignin and cellulose enables
some of the LDF to function as typical “white-rot fungi” in soil (Hofrichter 2002).
Well known mushroom forming LDF are A. bisporus and V. volvacea, both grown
commercially on composted lignocellulose. As A. bisporus contains lignolytic enzymes, degrades both cellulose and lignin, the former more rapidly (Cai et al. 1999,
Lankinen et al. 2005). Compost prepared from straw, horse or chicken manure, calcium sulphate (gypsum), water and some nutritional supplements is a cheap cultural
substrate for A. bisporus and some other saprophytic basidiomycetes. Manure in the
compost serves as N source, straw as C source. It must be pointed out that after the
initial medium preparation stage, little control can be exerted over the composition
of the solid substrate medium. In composted substrates this is particularly crucial
since the nutrient composition of the initial medium ingredients has to allow both
a successful composting process and good fungal colonization and fruiting (Wood
and Smith 1987).
Mushrooms have a two-phase life cycle, the mycelium (vegetative or colonization phase) and the fruiting body (reproductive phase that bears the spores). The
mycelium grows through the substrate, biodegrades its components and supports
the formation of fruiting bodies. Mushroom growers call the switch from mycelial
extension to the production of mushroom primordia “pinning”, the successive development of primordia into mushrooms “fruiting”. While growth of mycelium lasts for
several days, weeks or months, production of fruiting bodies is short lived, and the
phenomenon is called ‘fructification’. However, both vegetative and reproductive
phases are very much influenced by the physiological condition and nutritional state
of the mycelium (Wood and Smith 1987).
Since the carbon sources utilized by basidiomycetes are usually of a lignocellulosic character, fungi during vegetative growth produce a wide range of enzymes
to degrade the lignocellulosic substrates. Data obtained in various studies demonstrate that the type and composition of lignocellulosic substrate appear to determine
the type and amount of enzyme produced by basidiomycetous fungi during vegetative growth (Baldrian 2005, Baldrian and Valášková 2008, Elisashvili et al. 2008).
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
173
Moreover, cellulose/lignin ratios of wheat straw and cotton waste substrates were
positively correlated to mycelial growth rates and mushroom yields of P. ostreatus
and P. pulmonarius and with the yield of V. volvacea (Philippoussis et al. 2001a).
According to Kües and Liu (2000), considerable changes in enzyme activities occur
during fruiting, indicating a connection to the regulation of fruiting body development. For example, in A. bisporus and L. edodes, laccase activities are highest just
before fruiting body initiation and decline rapidly with primordia formation. Cellulase activities are highest when fruiting body develops (Ohga et al. 2001). Regarding
the influence of nitrogen availability, recent studies revealed a positive correlation
between the C/N ratio and P. eryngii mushroom yield (Philippoussis et al. 2000).
They also demonstrated that mycelium growth rates of Pleurotus spp. and L. edodes
were positively correlated to C/N ratio (Philippoussis et al. 2001a, 2003). Similar
conclusion was drawn by Silva et al. (2005), indicating that L. edodes extension rate
is related to bioavailability of nitrogen and is enhanced by supplementation with
cereal bran. Moreover, both nature and concentration of nitrogen sources are factors
regulating enzyme production by wood rotting basidiomycetes, e.g. in L. edodes
cultivation on wheat straw, nitrogen supplementation represses MnP and enhances
laccase activity (Kachlishvili et al. 2005). According to Kües and Liu (2000), for
fruiting body induction it is of importance to keep a balance between C and N
sources, e.g. in A. bisporus compost, the optimal C/N ratio for fruiting has been
determined to lie between 80:1 and 10:1. In addition, substrate supplementation with
protein-rich materials proved to enhance yield of Agaricus, Pleurotus and Lentinula
strains (Rodriguez-Estrada and Royse 2007, Naraian et al. 2008).
Apart from nutrition, mycelial growth and fruiting of basidiomycetous fungi are
also regulated by temperature, gaseous environment, water activity and in certain
cases by light. During substrate colonization, the effect of environmental parameters plays an essential role on mycelium growth, and hence confers significantly to
the success of the entire cultivation process. In addition, the duration of the substrate colonization phase is of direct economic importance, since media that are
non-thoroughly impregnated with the hyphae, are sensitive to fungal and bacterial
infections resulting in reduced yields (Philippoussis et al. 2001a, Diamantopoulou
et al. 2006). Production of the vegetative mycelium usually occurs over a wide
range of temperatures. Zervakis et al. (2001) examined the influence of temperature
on mycelium linear growth of P. ostreatus, P. eryngii, P. pulmonarius, A. aegerita,
L. edodes, V. volvacea and A. auricula-judae. Their temperature optima were found
to be 35◦ C for V. volvacea strains, while P. eryngii grew faster at 25◦ C, P. ostreatus
and P. pulmonarius at 30◦ C. Moreover, A. aegerita grew faster at 25◦ C or 30◦ C
and A. auricula-judae at 20◦ C or 25◦ C depending on the nutrient medium used, and
L. edodes at 20◦ C or 30◦ C depending on the strain examined. It is generally believed
that basidiomycetes tolerate relatively high levels of salts for growth, but fruiting
body development can be more sensitive. Likewise, mycelial growth is less affected
by pH but fruiting body development of several species occurs best at neutral or
slightly acidic pH values around 6–7 (Wood and Smith 1987) or, in L. edodes,
at a pH 4.0 (Ohga 1999). On lignocellulosic substrates, Pleurotus and Lentinula
species are growing with a linear rate (Philippoussis et al. 2001a, Diamantopoulou
and Philippoussis 2001), which is influenced by substrate salinity and porosity
174
A.N. Philippoussis
(Philippoussis et al. 2002). Measurements of electric conductivity through the entire colonization process of three residue-substrates by L. edodes strains revealed an
increase of salinity values until mycelium colonized 60 to 75% of the substrate, and
then it slightly declined or remained constant until the end of incubation, presenting
the highest and lowest values in the wheat straw and oak sawdust media respectively. In addition, a negative correlation was established between final salt content
of the substrates and mycelium extension rates. Furthermore, monitoring of CO2
concentrations in pilot-scale cultivation of L. edodes on synthetic blocks, revealed
higher respiration rates on oak sawdust and corncobs than on wheat straw, which
are further correlated with substrate colonization rates (Philippoussis et al. 2003).
Following colonization of the substrate, fruiting is induced by environmental
and/or cultural manipulation. The optimal environmental parameters for mycelial
growth and the subsequent fruiting are usually very distinct. Depending on the
species and the degree of investment in environmental control technology, temperature is normally manipulated by heating or cooling systems to maintain the optima
for vegetative growth or fruiting. However, fruiting body development is often induced after drastically altering the environmental parameters, usually favoured by
reducing the temperature by at least five ◦ C compared to mycelium growth. In fact,
fruiting is typically induced, after vegetative growth, e.g. in A. bisporus to 16–18◦ C
(Kües and Liu 2000), in P. ostreatus to 15◦ C (Zadrazil et al. 2004), and in L. edodes
to 10–16◦ C for the cold temperature strains and 16–21◦ C for the warm temperature
strains (Chen et al. 2000). Other parameters of fruiting body initiation and maturation include CO2 concentration, humidity, salinity and pH. High humidity (90–95%)
is favorable for pinning and fruiting but the moisture content of the substrate might
be even more critical. The optimal water content for wooden substrates is 35–60%
and for other substrates 60–80%. The lower values reflect the oxygen demand of
the fungi in the substratum, balanced against their requirement for water (Kües and
Liu 2000). Carbon dioxide (CO2 ) level is also critical for efficient mycelial growth,
fruit body initiation and fruit body development. Higher CO2 concentrations (e.g.
1% v/v in air) may stimulate mycelial growth and inhibit fruiting. Increased aeration
is used to reduce CO2 levels, which otherwise produces increased elongation of stipe
growth and abnormality of cap development (Wood and Smith 1987). Light has been
implicated in the fruiting of several mushroom genera e.g. Lentinula and especially
Pleurotus species have an obligate requirement for light for fruiting induction. Brief
exposure of the culture to daylight or suitable artificial light is sufficient. Usually,
light positively influences hyphal aggregation and fruiting body maturation (Kües
and Liu 2000). However, light is not needed for the fruiting of A. bisporus (Wood
and Smith 1987).
9.2.3 Output and Stages of Mushroom Cultivation
Mushroom industry presents a worldwide expanded and economically important
biotechnological application, which can be divided into three main categories: cultivated edible mushrooms, medicinal mushroom products and wild mushrooms, with
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
175
an annual global market value in excess of $45 billion (Chang 2006). The global
annual mushroom output (including production and wild mushroom collection)
surpass nowadays 10 million metric tons, with China being the top world producer (about 8.000.000 tons), followed by Europe and USA (Desrumaux 2007,
Huang 2007). Commercial mushroom production is an efficient solid state fermentation process of food protein recovery from lignocellulosic materials carried out
on a large or small scale (Martı́nez-Carrera et al. 2000, Chiu et al. 2000). Taking
into account the value and volume of the product, the number of people involved
in the industry, or the geographical area over which the industry is practiced, mushroom cultivation is the greatest application of exploitation of filamentous fungi using SSF and the biggest (non-yeast) biotechnology industry in the world (Moore
and Chiu 2001). The economic strength of mushroom cultivation derives from the
successful use as feedstocks of a variety of low- or negative-value residues from
agriculture, forestry or industry. These wastes are processed using relatively cheap
microbial technology to produce human foodstuff, which could also be regarded as a
functional food or as a source of drugs and pharmaceuticals (Wood and Smith 1987).
Moreover, the effective exploitation of resources from agricultural solid wastes and
by-products, rich in organic compounds that are worthy of being recovered and
transformed, is a sound environmental protection strategy (Zervakis and Philippoussis 2000).
There are three major stages involved in mushroom cultivation: (1) inoculum
(spawn) production, (2) substrate preparation, and (3) mushroom growing i.e. inoculation of the substrate with propagules of the fungus, growth of the fungal mycelium
to colonise the substrate, followed by fruiting, harvesting and processing of the fruiting bodies (Wang 1999, Martı́nez-Carrera et al. 2000).
Inoculum (spawn) production. In order to achieve reliable and vigorous fungal
growth and fruiting bodies production of good quality, inoculum fungal cultures
are necessary. Inoculum is produced by inoculation of sterilized cereal grains (usually wheat, rye or millet) from high quality stock mycelial cultures (Stamets 2000,
Mata and Savoie 2005b). Essential prerequisite is the selection and breeding work
to acquire suitable biological material for commercial cultivation, which ensures
good yield and quality. The various mushroom inocula are often the only microbiologically pure part of the whole technology (Wood 1989). Spawn-making is a
rather complex task, not feasible for the common mushroom grower, and is produced by specialist companies (spawn-makers) using large scale bulk autoclaving,
clean air and other microbiological sterile techniques for vegetative mycelia cultures
onto cereal grains, wood chips and plugs or other materials. The colonized cereal
grain/mycelium mixture is called spawn and is grown under axenic conditions in
autoclavable polyethylene bags, ensuring gas exchange, or rarely in jars. Finally,
after quality control to assure biological purity and vigor, spawn is distributed from
the manufacturer to individual mushroom farms in the same aseptic containers used
for spawn production (Wood and Smith 1987, Royse 2002).
Substrate preparation. Fermentation process involves cultivation on specific
substrates imitating the natural way of life of mushroom fungi (Tengerdy and
Szakacs 2003). Regarding the litter-decomposer A. bisporus, this has come to mean
176
A.N. Philippoussis
cultivation of a mushroom crop on composted plant litter (Moore and Chiu 2001).
On the other hand, the white-rot mushroom fungi Pleurotus spp and L. edodes are
cultivated on non-composted lignocellulosic substrates, using methodologies that
exploit their ability to produce enzymes capable of degrading all wood components
(Chen et al. 2000, Zadrazil et al. 2004). The first stage of mushroom production has
to do with assembly and treatment of the substrate to prepare a growth medium. The
substrates used for mushroom production, varying according to cultivated species,
are prepared from waste agricultural or forest product materials using ingredients
such as manures, cereal straws or other crop residues, sawdusts etc. (Wood and
Smith 1987). In certain cases the substrate is be directly inoculated and require very
little pre-treatment, e.g. L. edodes production using logs. In other cases, the substrate is microbiologically or physically pretreated. Microbiological pre-treatment
normally comprises some form of controlled bulk composting process (Wood 1989).
Physical pre-treatment could include steam treatment or sterilization by autoclaving.
Substrates for fungal growth can be prepared as sterile materials, to produce an
axenic growth medium, e.g. bottle cultures of F. velutipes, or be non-sterile, e.g.
compost substrates to produce A. bisporus. One of the aims of substrate preparation
is to introduce sufficient water into the substrate to ensure that the water activity of
the final medium is optimal for fungal growth. The scale of substrate preparation
varies according to the type of species to be cultivated and the size of the production
unit. For A. bisporus production, many tons of straw are processed per day to produce compost. Thus, large-scale bulk handling machinery is used for this process.
After the initial preparation stage, little control can be exerted over the composition
of the solid substrate medium. In composted substrates, this is particularly crucial
since the nutrient composition of the initial medium ingredients has to allow both a
successful composting process and good fungal colonization and fruiting. Although
nutrient balances and status, e.g. for carbon, nitrogen, pH and other components,
can be measured on the initial ingredients, little can be done to regulate the quantity or feed rate of these once the production processes are under way (Wood and
Smith 1987).
Mushroom growing. This stage deals with the two phases of mushrooms life
cycle i.e. the mycelium (vegetative phase) and the fruiting body formation (reproductive phase). Following inoculation, the mycelium, grows through the substrate,
biodegrades its ingredients and supports the formation of fruiting bodies. Mycelial
growth and fruiting during this stage are regulated by temperature, gaseous environment, nutrient status, water activity and in certain cases by light e.g. Pleurotus
spp. has an obligate requirement for light for fruiting induction, Agaricus spp. have
no light requirement (Wood 1989, Zadrazil et al. 2004). The level of environment
and cultural control used is determined by the type of production technology. In
controlled environment growing system, temperature is manipulated by heating or
cooling systems to maintain the optima for vegetative growth or fruiting. Carbon
dioxide (CO2 ) level and humidity are also controlled. Basidiomata production on
the culture medium surface occurs as a series of cycles (flushes). Depending on the
fate of the harvested product as fresh or preserved material, the fruit bodies are harvested either by hand or mechanically and processed accordingly. After harvesting,
mushrooms are normally cooled down to retard fruiting body metabolism, packed
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
177
and sent to the fresh market, or processed further through freezing, canning, drying
etc., depending on marketing strategies (Martı́nez-Carrera et al. 2000).
9.3 Bioconversion of Solid Residue-Substrates
Through Mushroom Cultivation
9.3.1 Commercial Mushroom Production Processes
In the suite, the principles of production of the litter decomposingAgaricus spp.
and. of two wood-degrading mushroom species (Pleurotus spp. and L. edodes) are
presented.
Agaricus bisporus cultivation. It is the most commonly cultivated mushroom
worldwide, mainly cultivated in Europe, North America, China and Australia
(Chang 1999). A. bisporus, belonging to the Agaricaceae family, is a litterdecomposing basidiomycete that in nature usually grows on grasslands and forests
(Kirk et al. 2001). Western countries, focused on Agaricus for consumption, led to
progress in cultivation technology including farm design, quality control in compost
production, microprocessor control and records for growing, mechanical harvesting
and processing. The Netherlands, practising high-technology cultivation systems,
has the highest mushroom yield per unit area worldwide (Chiu et al. 2000). The
substrate used for A. bisporus cultivation is a complex culture medium made from
straw- and manure-based compost. Its preparation is a two-stage process in which
the first stage includes composting of the raw material consisting of straw, horse
or poultry manure and gypsum (Sánchez 2004). During composting that lasts about
3 weeks, the lignocellulose waste is modified by various bacteria and fungi to a
better-digested form suitable for A. bisporus. In the second week stage, the compost
is pasteurized before inoculation with A. bisporus spawn. The final mushroom compost is a selective growth medium for this organism. Natural drop in temperature
and lack of free ammonia are signs that the composting process has been completed
(Moore and Chiu 2001).
Cultivation begins with inoculation (spawning; the process that introduces the
mushroom mycelium into the compost) and growth of the mushroom mycelia into
the compost under high humidity and temperature 25◦ C. At complete colonization,
after 2–3 weeks, a casing layer containing peat moss and limestone is spread on
the top of the compost. ‘Casing’ is needed only by Agaricus, the procedure is not
necessary when cultivating other species such as Pleurotus spp. and L. edodes. After
allowing 7 to 9 days for the Agaricus mycelium to grow into the casing layer, a
machine with rotating tines is run across the mushroom bed to mix the casing layer
thoroughly. The above process is called ‘ruffling’ and it serves in breaking up the
mycelial strands and encourages the mushroom mycelia to grow and colonize the
surface of the casing layer. The mushroom mycelium grows into the casing layer in
similar conditions to those of compost colonization, and when it reaches the upper
surface of the casing layer the fruiting process starts comprising environmental manipulation. The growing room is ventilated to decrease the concentration of carbon
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A.N. Philippoussis
dioxide (usually to < 0.1%) and to help reduce the temperature to 16–18◦ C. The
temperature, humidity and CO2 level are then adjusted to trigger fructification and
to favor the development of mushrooms. The first pin initials begin to appear about
2 weeks after casing. One layer of compost produces 2–4 crops called flushes. In
general, the production of A. bisporus is time-consuming due to the long composting
stage (Sánchez 2004).
In modern mushroom growing process, a specialist compost producer may complete the outdoor stages of composting and the ready-to-use compost (spawn run or
not) can be delivered in bulk to a mushroom farm. Therefore, the mushroom production industry comprises spawn makers, phase I, phase II and phase III compost suppliers. Phase III compost is completely colonized by the mushroom mycelia, which
if placed in a suitable environment in a mushroom farm will produce the fruit body
crop readily. For a commercial mushroom farmer, the use of phase I compost gives
the most flexibility to optimize farm conditions for cultivation of any mushroom
strain. Purchase of phase II compost enables a farmer to choose which mushroom
strain to spawn. The use of phase III compost, though it is obviously more costly, it
guarantees the production of a crop in a short time and requires the least investment
in facilities (Moore and Chiu 2001). Moreover, there are different growing systems,
while the process can be separated into specialized stages. In the shelf-bed growing
system, shelving is usually made of metal and arranged to give four to six layers
of 1.4 m wide fixed shelves in a cropping room with centre and peripheral access
gangways. Special machinery for compost filling, emptying, spawning, casing and
other cultivation operations is necessary. In the bag growing system, growing bags of
about 25 kg are usually supplied to the farm already spawned and may be arranged
on the floor of the cropping house or on tiered shelving. Each arrangement makes
its own demands on techniques and equipment.
Pleurotus spp. cultivation. Pleurotus species (like P. ostreatus, P. sajor-caju,
P. pulmonarius, P. eryngii, P. cornucopiae, P. tuber-regium, P. citrinopileatus and
P. flabellatu ) are commercially very important edible mushrooms, found all over
the world. These mushrooms present several advantages related with rapid mycelial
growth, high ability for saprophytic colonization, simple, inexpensive cultivation
techniques and several kinds of species available for cultivation under different
climatic conditions. The production of Pleurotus mushrooms is a sharp contrast
with the technology used for Agaricus production. Both pasteurized and sterilized
substrate of a wide range of residues can be used (Fig. 9.1) and no casing is required.
The primary ingredients used for Pleurotus spp. production is chopped wheat straw
(Triticum aestivum L.) or cottonseed hulls (Gossypium hirsutum L.) or mixtures of
them. For production on wheat straw, the material is chopped from 2 to 6 cm, water
is added and pH of the material is adjusted with limestone to about 7.5 or higher to
provide selectivity against Trichoderma green mold (Royse 2004). The substrate is
then pasteurized in tunnels with aerated steam at 60–70◦ C for 12 hr by passing the
air-steam mixture through the substrate. After pasteurization is complete (a proximate two-day process), filtered air is passed through the substrate for cooling to
25◦ C. Then grain spawn (inoculum) is added at about 3–5% of the fresh weight and
the substrate, packed into 15–20 kg plastic bags or blocks, is placed in a dark room at
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
Fig. 9.1 Biological efficiency (BE; % ratio of fruiting body fresh weight over the dry weight of substrate) of P. ostreatus and P. pulmonarius cultivated on
residue-based substrates. References: [1] Coutiño et al. 2004, [2] Curvetto et al. 2002, [3] Croan 2003, [4] Darjania et al. 1997, [5] Das and Mukherjee 2007,
[6] Gezer et al. 2007, [7] Hernández et al. 2003, [8] Kalmiş and Sargin 2004, [9] Lara et al. 2002, [10] Mandeel et al. 2005, [11] Marino et al. 2003, [12]
Martı́nez-Carrera et al. 1985, [13] Martı́nez-Carrera et al. 2000, [14] Moda et al. 2005, [15] Obodai et al. 2003, [16] Pant et al. 2006, [17] Philippoussis
et al. 2000, [18] Philippoussis et al. 2001a, [19] Ragunathan et al. 1996, [20] Royse and Schisler 1987, [21] Salmones et al. 2005, [22] Sánchez et al. 2002,
[23] Serra and Kirby 1999, [24] Shah et al. 2004, [25] Soto et al. 1987, [26] Upadhyay et al. 2002, [27] Velázquez-Cedeño et al. 2002, [28] Vetayasuporn 2006,
[29] Yildiz et al. 2002, [30] Zhang et al. 2002, [31] Zervakis and Balis 1992
179
180
A.N. Philippoussis
25–30◦ C with 80 per cent humidity. Depending on the strain, complete colonization
of the substrate is achieved in 2–3 weeks. In Asia, small containers are used, e.g. in
Japan, bottle production of Pleurotus mushrooms is common. Substrate is filled into
bottles, sterilized and inoculated with Pleurotus spawn. Upon completion of spawn
run, bottle lids are removed and mushroom emerge from the surface of the substrate
(Wood and Smith 1987, Royse 2003a, 2004).
Fructification of P. ostreatus is triggered by lowering the air temperature to
12–15◦ C (cold-shock treatment) although no such treatment is required for other
Pleurotus spp. The fruiting is light-dependent, requiring a 8–12 hour light cycle
(solar or fluorescent lamp light) and adequate ventilation is given to keep CO2
levels lower than 500 ppm. Insufficient ventilation generally leads to mass primordial development with little differentiation into fruit bodies (Zadrazil et al. 1996).
Three to four weeks after spawning depending on strain, amount of supplement used
and temperature of spawn run, mushrooms begin to form around the edges of bag
perforations and they are harvested from the substrate (Royse 2004). Throughout
cropping, mushroom houses are kept at 12–17◦ C, the substrate usually being a few
degrees higher. Under ideal growing conditions, 1 kg of well-colonized substrate
will yield about 1 kg of marketable mushrooms (after two flushes) the completely
growing cycle being completed in about 70 days. Due to the absence of a velum
covering the gills of Pleurotus fruitbodies, spore discharge begins at a very early
stage. Very large spore deposits within mushroom houses can cause allergy problems (Wood and Smith 1987).
Lentinula edodes cultivation. L. edodes (Berk.) Pegler is the second most popular
edible mushroom in the world because of its flavour, taste, nutritional and medicinal
properties. (Smith et al. 2002). This fungus can grow on synthetic logs as well as
natural logs. The most traditional but laborious cultivation is carried out in wood
logs, mainly oak. The wood logs are holed and the mycelia plugs are inserted in
these holes. After inoculation the logs are stored several months for mycelium colonization and finally for the formation of fruit bodies. This method is still used
because of its high quality mushroom product (Royse 2001, Silva et al. 2007),
although leads to a severe threat to natural forests (Chiu et al. 2000). The last
decades, new methods for L. edodes cultivation on residue-based substrates have
been developed using milled wood residues (e.g. oak, hornbean, sweetgum, poplar,
alder, ironwood, beech, willow, pine, maple and birch sawdust) supplemented with
nitrogen sources (e.g. rice bran). The main advantages of using synthetic medium
over natural logs are time and efficiency (Royse 2004). Some formulations used
consist of 80% sawdust and 20% bran; 80% sawdust, 10% bran and 10% wheat or
millet; and 84% sawdust, 5% rice bran, 5% wheat bran, 3% soybean and 3% lime
(Kalberer 1987).
Other agricultural wastes that can be used as substrates (alone or in combination with other supplements) in L. edodes cultivation are cereal straw, corn
cobs, sugarcane bagasse, tea waste, sunflower seed hulls, peanut shells, vineyard prunings, cotton straw and seed hulls etc. (Fig. 9.2; Curvetto et al. 2002;
Philippoussis et al. 2003; Rossi et al. 2003, Gaitán-Hernández and Mata 2004,
Mata and Savoie 2005a, b, Fan and Soccol 2005, Özçelik and Pekşen 2007, Royse
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
Fig. 9.2 Biological efficiency (BE; % ratio of fruiting body fresh weight over the dry weight of substrate) of L. edodes mushroom cultivated on residue-based
substrates. References: [1] Curvetto et al. 2005, [2] Diehle and Royse 1986, [3] Donoghue and Denison 1995, [4] Donoghue and Denison 1996, [5] Fan and
Soccol 2005, [6] Gaitán-Hernández and Mata 2004, [7] Gaitán-Hernández et al. 2006, [8] Hiromoto 1991, [9] Kalberer 2000, [10], Kawai et al. 1996, [11]
Kirchhoff and Lelley 1991, [12] Levanon et al. 1993, [13] López et al. 2004, [14] Martı́nez-Carrera et al. 2000, [15] Özçelik and Pekşen 2007, [16] Philippoussis
et al. 2003, [17] Pire et al. 2001, [18] Rossi et al. 2003, [19], Royse and Bahler 1986, [20] Royse and Sanchez 2007, [21] Royse and Sanchez-Vazquez 2001,
[22] Royse and Sanchez-Vazquez 2001, [23] Royse et al. 1990, [24] Royse 2002, [25] Salmones et al. 1999, [26] Worrall and Yang 1992
181
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A.N. Philippoussis
and Sanchez 2007). The principle of the cultivation method comprises mixing and
compacting ingredients into plastic bags, followed by sterilization, inoculation with
fungal mycelia, incubation in dark rooms with controlled temperature and humidity
for 30 to 80 days and finally fruiting induced by temperature reduction. Regardless
of the main ingredients used, starch-based supplements such as wheat bran, rice
bran, millet, rye, corn, etc. are added to the mixture in a 10 to 40% ratio to the main
ingredient. These supplements serve as nutrients to provide an optimum growing
medium (Royse et al. 1990, Royse 1996, 2003b). Substrate’s ingredients are mixed,
watered to gain a moisture content around 60% and filled into polypropylene bags
1–3 kg/ bag. The filled bags are stacked on racks, loaded into an industrial-sized
autoclave, sterilized for 2 hours at 121◦ C, cooled and inoculated with spawn. After a 20 to 25 days spawn run, the bags are removed and the substrate blocks are
exposed to an environment conducive for browning of the exterior log surfaces.
As the browning process reaches completion (4 weeks), primordia begin to form
about 2 mm under the surface of the bag-log indicating that it is ready to produce
mushrooms (Royse 2004). Primordia maturation is stimulated by soaking the substrate in water (12◦ C) for 3 to 4 hours (or 3 to 4 min if vacuum soaking is used;
Royse 2002). Soaking allows water rapidly to displace carbon dioxide contained
in air spaces, providing enough moisture for one flush of mushrooms. Approximately 9 to 11 days after soaking, mushrooms are ready to harvest (Royse 2001).
This method decreases the production time and increases productivity. While in the
traditional cultivation the logs need 8 months to 1 year of cultivation to produce
10–15 kg/100 kg of substrate, the cultivation on agro-forestry residues can furnish
a yield of 60–80 kg/100 kg of substrate in 80 days harvest period (Israilides and
Philippoussis 2003, Royse 2004, Silva et al. 2007).
9.3.2 Efficiency of Residue Conversion to Pleurotus sp.
and L. edodes Fruiting Bodies
Two particular basidiomycetus mushroom genera that have received considerable
attention for their nutritional value, medicinal properties and biodegradation abilities are Pleurotus and Lentinula (Elisashvili et al. 2008). These widely cultivated
edible mushrooms are efficient colonizers and bioconverters of lignocellulosic agroindustrial residues into palatable human food with medicinal properties
(Zervakis and Philippoussis 2000, Philippoussis et al. 2004, Zadrazil et al. 2004,
Silva et al. 2007, Gregori et al. 2007). The efficacy of this value-added bioconversion process and the productivity of the mushroom crop are assessed by the
biological efficiency (Chang et al. 1981). Biological efficiency (BE) expresses the
bioconversion of dry substrate to fresh fruiting bodies and indicates the fructification
ability of the fungus utilizing the substrate (Fan et al. 2000a). BE is calculated as
the percentage ratio of the fresh weight of harvested mushrooms over the weight
of dry substrate at inoculation (Chang and Chiu 1992, Philippoussis et al. 2001b,
Diamantopoulou et al. 2006). Yet, in a scarce number of reports, biological effi-
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
183
ciency has been defined in terms of dry fruit bodies yield over the dry weight of
the substrate used (Bisaria et al. 1987, Wang et al. 2001). Nevertheless, it should
be made clear that apart from the type of substrate and stain used, yield response is
determined by the duration of the cropping period and cultivation practice applied
e.g. high spawn levels enhance mushroom yields (Obodai et al. 2003). Nevertheless,
for considering the Pleurotus cultivation profitable, BE value must be over 50%
(Patra and Pani 1995).
The genus Pleurotus comprises some the most popular edible mushrooms due
to their favourable organoleptic and medicinal properties, fast mycelial growth and
undemanding cultivation conditions. These mushrooms are commercially grown on
pasteurized straw-based substrates or hardwood sawdust, fermented or not, with
added supplements. However, as these fast-growing mushrooms display a complete
lignocellulolytic enzyme system (Bushwell et al. 1996, Elisashvili et al. 2007), they
can use a wide spectrum of agricultural and industrial wastes that contain lignin
and cellulose for growth and fruiting (Poppe 2000). A significant number of agroindustrial lignocellulosic materials are used as substrates for the production of Pleurotus spp., like corn cobs, various grasses and leaves, reed stems, maize and sorghum
stover, rice and wheat straw, vine shoots, cardboard and paper, wood sawdust and
chips, coffee pulp, cottonseed hulls, peanut shells, sunflower seed hulls, sugarcane
and tequila bagasse etc. Average experimental BE values varying from 14.5–126.0%
are presented in Fig. 9.1. Further evaluation of the overall BE values obtained on
these residue-substrates for P. ostreatus and P. pulmonarius strains indicated that
among all residues cardboard, coffee pulp, paper wastes and softwood residues, presented the highest (≥ 100%) biological efficiencies (Martı́nez-Carrera et al. 2000,
Croan 2003, Mandeel et al. 2005). BEs between 75% and 100% were recorded on
cotton wastes and wheat straw (Upadhyay et al. 2002, Philippoussis et al. 2001a).
Regarding straw pre-treatment, data demonstrated an approximate 20% reduction
of overall BE when P. ostreatus is cultivated on non fermented wheat straw, as
compared to fermented substrate (mean values 70.5% and 85.5% respectively).
On pretreated wheat straw, supplementation with cotton seed cake and soybean
cake proved to enhance productivity of P. ostreatus (Upadhyay et al. 2002, Shah
et al. 2004). Finally, satisfactory productivity (BEs 50–75%) is demonstrated by
most of agro-industrial residues, namely corncobs, various grasses and reed stems,
vine shoots, cottonseed hulls and sugarcane bagasse. From the considered data it
can be assumed that P. pulmonarius furnished significantly better yields than P.
ostreatus on cardboard and soft-wood residues (respective BEs: 126.0 and 124.3%).
Moreover, yield of P. pulmonarius is favored on coffee and cotton wastes as well as
on wheat straw (Pant et al. 2006, Zervakis and Balis 1992, Philippoussis et al. 2001a,
Velázquez-Cedeño et al. 2002).
Our previous studies, concerning evaluation of a wide range of residues available
in the Mediterranean region as P. ostreatus, P. eryngii and P. pulmonarius cultivation
substrates, demonstrated significantly higher colonization rates of these mushrooms
on wheat straw and cotton waste (Philippoussis et al. 2000). Moreover, faster colonization was achieved on non-composted than on composted wheat straw and cotton
waste substrates. Cellulose/lignin ratios in substrates were positively correlated to
184
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mycelial growth rates and mushroom yields of P. ostreatus and P. pulmonarius.
In addition, there was a positive correlation between the C/N ratio and P. eryngii mushroom yield (Philippoussis et al. 2001a). Additional data furnished by the
‘race-tube’method experiments provided an estimate of the potential of wheat straw,
cotton gin-trash, peanut shells, poplar sawdust and corn cobs to serve as alternative
mushroom cultivation substrates (Zervakis et al. 2001). Furthermore, in recent studies conducted to evaluate different grass and reed stalks as cultivation substrates
of Pleurotus species (Philippoussis et al. 2007, Diamantopoulou et al. 2007), bean
plant residues (BRP) and a mixture of reed-grass maces (TCP) supported fast colonization rates for both genera strains, while BRP enhanced laccase and endoglucanase activities (Diamantopoulou et al. 2007). Moreover, fructification assay by the
‘tube fruiting technique’ provided a quick estimate of the potential of these wastes
to support basidiomata formation. Basidiomata produced by both fungi, on all tested
residues, with TCP supporting remarkably better fruiting results compared to wheat
straw (Philippoussis et al. 2007).
Lentinula edodes (Berk.) Pegler is one of the best-known species among cultivated mushrooms, grown on natural or artificial logs, composed of either sawdust
or of locally available agricultural wastes. L. edodes produces hydrolytic and oxidative enzymes responsible for lignocellulose degradation (Ohga and Royse 2001,
Mata and Savoie 2005c). The production of enzymes is specifically related to and
dependent on substrate composition and environmental factors such as temperature and moisture (Bushwell et al. 1996, Silva et al. 2005, Elisashvili et al. 2008).
Since L. edodes is an efficient wood degrader, it can be grown on a variety of
agro-industrial residues such as oak, ash, poplar, alder, eucalypt, beech, pine, maple
and birch sawdust, cereal straws (mainly barley and wheat), corn cobs, sugarcane
bagasse, sunflower seed hulls, peanut shells, cotton straw and seed hulls, vine
shoots, coffee husk and pulp etc. Figure 9.2 presents comparatively the biological efficiencies obtained on these substrates during productivity evaluation experiments. Data indicate that the nature of the substrate affects remarkably L. edodes
basidiomata yield. The highest average biological efficiencies were achieved with
sunflower seed hulls (BE: 107.5%; Curvetto et al. 2005), followed by sugarcane
bagasse (BE: 87.4%; Salmones et al. 1999). More or less similar interesting results (BE ≈ 80%) appeared to be obtained with hard-wood residues (beech) and
barley straw (Kirchhoff and Lelley 1991, Kawai et al. 1996, Gaitán-Hernández
et al. 2006). Among other substrates, progressively lower BEs, in the range of
65–50% and in descending order, were furnished by cereal grains, wheat straw,
vine shoots, hard-wood residues of various trees and hazelnut husk (Hiromoto 1991,
Philippoussis et al. 2003, Gaitán-Hernández et al. 2006, Özçelik and Pekşen 2007).
Oak sawdust (comprising all types of oak), pine sawdust and coffee residues (all
types) exhibited BEs in the range of 40–50% (Donoghue and Denison 1996, Royse
and Sanchez-Vazquez 2001, Royse and Sanchez 2007), while the overall lower
BE values (around 20%) were detected on apple pomace and corncobs (Worrall
and Yang 1992, Philippoussis et al. 2003). Nevertheless, reliable estimations of the
residues impact on L. edodes yield cannot be withdrawn from the presented average BE values, which only as indicative could be regarded. The main problem is
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
185
that data from different substrate formulas, co-substrates and supplements, media
treatments, strains used, cultivation periods, experimental conditions etc. are compared. It is well known that apart from substrate nature and composition, strain and
length of incubation (to mention only a few factors), are important parameters for L.
edodes production on artificial substrates (Royse and Bahler 1986, Zadrazil 1993,
Kalberer 1995, Sabota 1996, Chen et al. 2000, Philippoussis et al. 2002, 2003).
Comparing Figs. 9.1 and 9.2, BEs obtained on residue-substrates used for both
L. edodes and Pleurotus spp. cultivation, the overall assumption is that wood
chips and wheat straw, followed by sugarcane bagasse and vine prunings can support good basidiomata yield of L. edodes, as well as of P. ostreatus and P. pulmonarius strains. Coffee pulp and corncobs supported significantly higher yields
for Pleurotus strains, while sunflower seed hulls favoured higher productivity for
L. edodes.
Our previous studies, evaluating six commercial and wild L. edodes strains as
regards their efficacy of mycelium growth on wheat straw, cotton wastes, oak-wood
sawdust and corncobs, have demonstrated that oak-wood sawdust and wheat straw
supported faster growth than corncobs and cotton wastes (Philippoussis et al. 2001c).
In addition, a strain-dependent behaviour was detected since three strains performed much better on oak-wood sawdust and wheat straw, while one commercial
strain performed satisfactorily on the other two substrates. In general, significantly
lower linear growth rates were recorded for corncobs and cotton wastes (Philippoussis et al. 2003). Results were verified by the fruiting technique conducted in
glass tubes, that furnishes a remarkable reduction in the time necessary for the
first fructification (≥ 2 months), conducing to a quick evaluation of the production potential of tested substrates. This, along with the use of growth rate measurement for valorization of the substrate incubation efficacy, renders the ‘glasstube’ method a dependable technique for screening-selecting purposes. Additionally, measurements of mycelium respiration during the incubation phase in bag-log
cultivation demonstrated very low respiration rates on cotton wastes, irrespective of
the strain used (Philippoussis et al. 2002), while further experiments demonstrated
the suitability of wheat straw and to a lesser extend supplemented corncobs for
the cultivation of L. edodes (Philippoussis et al. 2003, 2004). Nevertheless, cotton
wastes generated in large quantities in many countries, have proved to be a very
good substrate for the cultivation of Pleurotus species (Philippoussis et al. 2001a,
Cohen et al. 2002).
9.4 Closing Remarks
Current mushroom industry is based on both application of techniques for the production of mushroom fruiting bodies and the application of modern biotechnological
techniques to produce medicinally beneficial compounds and nutraceutical products
(Chang 2006, 2007). The medicinal properties of bioactive substances, like polysaccharides with antitumor and immunostimulating properties occurring in higher
186
A.N. Philippoussis
basidiomycetes have become a subject of numerous recent reviews (Mizuno 1999,
Wasser and Weis 1999, Kidd 2000, Ooi and Liu 2000, Wasser 2002, Daba and
Ezeronye 2003, Paterson 2006). Among them, the commercial polysaccharide of
L. edodes, lentinan, has been researched extensively as it offers the most clinical
evidence for antitumor activity (Wasser 2002, Nikitina et al. 2007). Recently, some
mushroom polysaccharides have shown to exert a direct cytotoxic effect on cancer
cells in vitro (Jiang et al. 2004, Wong et al. 2007, Israilides et al. 2008).
Moreover, solid-state fermentations other than fruiting body production are suggested for upgrading and valorizing lignocellulosic residues using basidiomycetous cultures, either through protein enhancement and transformation of residues
into animal feed (Zadrazil et al. 1996, Zadrazil 2000), or for enzyme production
(Revankar et al. 2007, Elisashvili et al. 2008). In the first case, agro-industrial
residues such as rice straw, coffee pulp, sugarcane bagasse, banana leaves etc.
have been fermented by white-rot basidiomycetes to improve the digestibility of the
residues for use as ruminate feed supplement (Vega et al. 2005, Alborés et al. 2006,
Okano et al. 2006). In the second case, lignocellulose degrading mushroom species
like Pleurotus sp, Lentinula edodes, Trametes versicolor, Flammulina velutipes are
used for the production of enzymes of industrial importance, such as cellulases,
xylanases and laccases, using as substrates agro-industrial residues, from which
wheat straw and bagasse are the most commonly used substrates (Krishna 2005,
Silva et al. 2007). Most results, however, come from laboratory, or semi-pilot-scale
experiments (Pandey et al. 2000a, Cohen et al. 2002). Additionally, lignocellulolytic
mushroom fungi likePleurotus ostreatus and Trametes versicolor have been investigated for bioremediation and biodegradation of toxic and hazardous compounds
like caffeinated residues (Fan et al. 2000a, b) as well as toxic chemicals such as
pesticides, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls
(PCBs), chlorinated ethenes (CIUs) etc., in polluted soils or contaminated ground
water (Pointing 2001, Pérez et al. 2007, Rigas et al. 2007).
In terms of food production process, the aim of mushroom growing should be
to follow the holistic concept of production, according to Laufenberg et al. (2003).
This approach tries to connect differing goals, such as highest product quality and
safety, highest production efficiency and integration of environmental aspects into
product development and food production. An outstanding example of integrated
crop management practice of mushroom cultivation is the use of spent substrate,
that is the residual growth medium after cropping (Rinker 2002) (1) as animal
feed, since the mushroom mycelium boosts its protein content (Zhang et al. 1995),
(2) as soil conditioner and fertilizer as it is still rich in nutrients and with polymeric components that enhance soil structure (Castro et al. 2008), (3) as a source
of enzymes (Ko et al. 2005), (4) for the biological control of plant pathogens
(Philippoussis et al. 2004, Davis et al. 2005) and even (5) used for bioremediation
purposes as to digest pollutants on land-fill waste sites because it contains populations of microorganisms able to digest the natural phenolic components of lignin
(Eggen 1999, Fermor et al. 2000). In this concept, solid state fermentation processes
are not only the methods of mushroom production for food and nutraceutical purposes but also examples of an organic system integrated with waste treatment that
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates
187
contributes to sustainability and benefits the human population, health and environment.
Acknowledgments This work was partially financial supported by the General Secretariat of Research and Technology through the 05 PAV 105 project. The author wish to express his gratitude
to Diamantopoulou Panagiota MSc and to Kontogiorgi Ioanna, members of Laboratory of Edible
and Medicinal Fungi of NAGREF, for their great assistance in the preparation of the manuscript.
In addition, Dr. Serapheim Papanikolaou is thanked for his comments and improvements made to
the document.
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Chapter 10
Solid-State Fermentation Technology
for Bioconversion of Biomass
and Agricultural Residues
Poonam Singh nee’ Nigam and Ashok Pandey
Contents
10.1 Agro-Residue Bioconversion in SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.1
Nature of Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2 A Bio-Technology Solid State Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3 Advantages of SSF Over Conventional Liquid Fermentation . . . . . . . . . . . . . . . . . . . . . .
10.4 Performance Control of SSF Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.4.1
Performance Control by Particle Size of Agro Residues . . . . . . . . . . . . . . . . .
10.4.2
Performance Control by Medium Preparation of Agro-Residues . . . . . . . . . .
10.4.3
Performance Control by Moisture Content of Agro Residues . . . . . . . . . . . . .
10.5 Microorganisms Used for Agro-Residues Bioconversion . . . . . . . . . . . . . . . . . . . . . . . . .
10.6 Designing and Types of SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6.1
Fermenter Design for SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6.2
Types of SSF Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6.3
SSF Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.7 Scale-Up Stages of SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.7.1
Flask Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.7.2
Laboratory Fermenter Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.7.3
Pilot Fermenter Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.7.4
Production Fermenter Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.8 Factors Affecting SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.8.1
Significance of Aeration and Mixing in SSF . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.8.2
Significance of Control of Temperature and pH in SSF . . . . . . . . . . . . . . . . . .
10.9 Processes and Products of SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Solid-state fermentations (SSF) have attracted a renewed interest and
attention from researchers due to recent developments in the field of microbialbiotechnology. Hence, for the practical, economical and environmentally-friendly
P. Singh nee’ Nigam (B)
Faculty of Life and Health Sciences∗ University of Ulster, Coleraine BT52 1SA,
Northern Ireland, UK
e-mail: P.Singh@ulster.ac.uk
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 10,
C Springer Science+Business Media B.V. 2009
197
198
P. Singh nee’ Nigam and A. Pandey
bioconversion of agro-industrial wastes, solid state or substrate fermentation has
been researched globally and proved to be the ideal technology for this purpose.
In this chapter some important aspects of solid-state cultivation system have been
discussed, including the variety of substrates and microorganisms used in SSF for
the production of various end products; and the performance control of system by
regulation of important factors.
Keywords Solid substrates · Agricultural residues · Solid state fermentation · Water
activity · Moisture · Bioreactor
10.1 Agro-Residue Bioconversion in SSF
Commonly used substrates in SSF are natural agricultural products, as well as
agro-industrial waste residues and by-products serve as a source of carbon in SSF
(Table 10.1). Lignocellulosic materials of agriculture origin compose more than
60% of plant biomass produced annually through the process of photosynthesis.
This vast resource is the potential and renewable source of biofuels, biofertilizers, animal feed and chemical feedstocks. Lignocellulose may be a substrate for
the production of value-added products (Nigam and Singh 1996a, b; Nigam 1988,
1989a, b) (Table 10.2), such as biofuels, biochemicals, biopesticides, biopromoters,
or may even be a product itself after biotransformation (e.g. compost, biopulp).
In all applications the primary requirement is the hydrolysis of lignocellulose
into fermentable sugars by lignocellulolytic enzymes, or appropriate modification of
Table 10.1 Diverse range of agro-residues utilization in SSF technology
Substrates for SSF
Microorganisms used in SSF
Reference
Starchy raw materials
Bannana waste
Barley Husk
Corn cob
Citrus peel
Sugarcane by-products
Cassava
Sugarbeet pulp
Cassava
Wheat straw
Wheat straw
Sugarbeet pulp
Sugarcane bagasse
Saccharum munjaResidues Wheat straw
Cassava
Straw
Sweet potato
Fodder beets
Aspergillus spp
A. niger
Bjkendra adusta
A. niger
A. niger
A. terreus
Rhizopus oryzae
Trichoderma viride
T. resei & yeast
T. reesei & Endomycopsis fi uleger
T. reesei, Chaeotominum
T. reesei and Fusarium oxysporum
Polyporus spp
Pleurotus spp.
Coprinus spp.
Sporotrichum pulverulentum
Candida utilis
Pichia bartonii
Saccharomyces cerevisiae
Czajkowska and Ilnicka 1988
Baldensperger et al. 1985
Robinson and Nigam 2008
Singh et al. 1989
Rodriquez et al. 1985
Gonzalez-Blanco et al. 1990
Daubresse et al. 1987
Durand 1998
Opoku and Adoga 1980
Laukevics et al. 1984
Abdullah et al. 1985
Nigam and Vogel 1988, 1990
Nigam 1990
Gujral et al. 1987
Yadav 1989, 1988
Smith et al. 1986
Han 1987
Yang 1988
Gibbon et al. 1984
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Table 10.2 Agro-industrial residues used for addcd-value products
Substrate
Microorganisms
Product
Reference
Oat cereal
Rhizopus oryzae
Lactic acid; various
value added
products
Citric acid
Citric acid
Rennet
Rennet
Koutinas et al. (2007)
Prado et al. (2004)
Xie and West (2006)
Thakur et al. 1990
Karanth (1988)
Cheese aroma
Cheese flavour
Aroma
Cephalosporin
Gervais (1988)
Revah and Lebeault (1988)
Yamauchi et al. (1989)
Jermini and Demain (1989)
Aspergillus niger
Aspergillus niger
Mucor meihei
Rhizopus oligosporus,
Mucor meihei
Agar
Trichoderma viride
Agro-residues
Fungal cultures
Polished rice
Neurospora spp
Barley
Strptomyces
Cephcdosportum
aermonium
Sweet potato
Aspergillus
Rice grains
Streptomyces
Bagasse
Penicillium
chrysogenum
Cassava
Aspergillus niger
Corn
A. flavu
Soya
Various moulds
Oat straw
Pofyporovs spp
Birch lignin
Phanerochaete
chrysosporhun
Maple Wood
Polyporus anceps
Bagasse
2 Polyporous spp
Aspen Wood
Phubia tremelloasa
Wheat bran
Fusarium monoliforme,
Gibberrela fujikuroi
Wheat bran
Fusarium monoliforme,
Gibberrela fujikuroi
Wheat straw
Poms tignium
Soya bean Cassava R. oligsoporus
Koji-type SSF
Filamentous fungi
Soya bean
Filamentous fungi
Agro-residues
Distillers grain
Wheat bran
Wheat bran
Tetracyclines
Cephalosporin
Penicillin
Yang and Ling (1984)
Wang et al. (1984)
Barrios-Gonzalez et al.
(1988a, b)
Aflatoxins
Barrios-G et al. (1990)
Mycotoxin
Hesseltine (1972)
Mycotoxin
Bhumiratna et al. 1980
Lignin degradation Bone and Munoz (1984)
Lignin conversion Mudagett and Paradis (1985)
Lignin conversion
Lignin conversion
Delignification
Gibberellic acid
Gibberellic acid
Matteau and Bone (1980)
Nigam (1990)
Reid (1989a, b)
Kumar and Lonsane
(1987a, b, c)
Prema et al. (1988)
Hydroenperoxi de
Tempeh and Koji
Fungal spores
Fungal spores
Maltseva et al. (1989)
Hesseltine (1972)
Vezina and Singh (1975)
Lotong and Suwarnarit (1983)
the structure of lignocellulose. Economical and effective lignocellulolytic enzyme
complexes, containing cellulases, hemicellulases, pectinases and ligninases may be
prepared by SSF (Table 10.3). Lignocellulose is also the raw material of the paper
industry. To fully utilize the potential of lignocellulose, it has to be converted by
chemical and/or biological processes. Solid substrate fermentation (SSF) plays an
important role, and has a great perspective for the bioconversion of plant biomass.
Lignocellulose may be a good feedstock for the production of biofuels, enzymes
and other biochemical products by SSF. Crop residues (straw, corn by-products,
bagasse, etc.) are particularly suitable for this purpose, since they are available in
large quantities in processing facilities (Pandey et al. 2001).
Lignocellulose in wood may be transformed into good quality paper products
with the help of SSF biopulping and biobleaching. Agricultural residues may be
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Table 10.3 Agro-residues used in SSF for enzyme production
Substrates
Microorganisms
Enzymes
Reference
Bagasse,
sawdust,corn
cobs
Corn cobs
Wheat bran
Wheat bran
Sugarbeet pulp
Wheat bran
A. niger
cellulose, beta
glucosidase
Madamwar et al. (1989)
A. niger
A. niger
A. niger
A. phoenicis
A. flavu
cellulose
glucoamylase
glucoamylase
beta glucosidase
protease
Wheat bran
Wheat bran
Sugarbeet pulp
A. carbonarius
A. niveus
T. viride and A. niger
Wheat bran and
rice straw
Trichoderma spp.
A. ustus, Botritis
spp.,
S. pulverulentum
Pencillium spp.
Geotrichwn
Candidum, Mucor
meihei & 2
Rhizopus spp.
P. capsulatum
P. charlesii,
Talaromyces
flavus
Tubercularia
vulgaris
T. vulgaris
Polyporous spp.
pectinase
catalase
cellulase and
amylase
Cellulose, betaglucosidase,
Xylanse
Singh et al. (1989)
Pandey (1990)
Ramakrishnana et al. (1982)
Deschamps and Huet (1984)
Malathi and
Chakrabarty (1991)
Karanth (1988)
Karanth (1988)
Desgrenges and
Durand (1990)
Shamala and
Sreekantiah (1986)
Wheat bran
Sugarbeet pulp
Citrus pulp-pellets
Citrus pulp
Bagasse
Lignocellulo sis
Wheat bran
Wheat bran
Straw
Lentinula edodus
Bacillus
licheniformis
Bacillus subtilis
Neurospora crasse
lipase
Munoz et al. (1991)
enzymes
Pectic enzymes
Considine et al. (1988)
Siessere and Said (1989)
pectic enzymes
Cellulase &
ligninase
enzymes
alpha amylase
Vieira et al. (1991)
Nigam et al. (1987b)
protease
Carboymethyl
cellulase, beta
glucosidase
Mishra and Leatham (1990)
Ramesh and Lonsane
(1987a, b, 1990)
Jermini and Demain (1989)
Macris et al. (1987)
converted into animal feed enriched with microbial biomass, enzymes, biopromoters, and made more digestible by SSF. Lignocellulosic waste may be composted to
targeted biofertilizer, biopesticide and biopromoter products. Post-harvest residue
may be decomposed on site by filamentous fungi and recycled to the soil with improved biofertilizer and bioprotective properties.
10.1.1 Nature of Substrates
The major organic material available in nature are polymeric in nature e.g. polysaccharides (cellulose, hemicellulose, pectins, and starch etc.), lignin and protein,
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201
Table 10.4 Agro-residues used for microbial growth studies
Substrate
Microorganism
Reference
Cassava
A. niger
Barley Husk and Barley straw
Citrus peel
Cassva
Soya bean
Sugarbeet pulp
Sugarrbeet pulp
Bjkendra adusta
A. niger
Rhizosporus oligosporus
R. oligosporus
T. viride, A. niger
T. viride, Sporotrichum
Pulverulentum and
Thermoascus auranticus
Penicillium roqueforti
Corinus fimetariu
Raimbault and Alazard, 1980,
Casteada et al., 1990, Oriol et
al. 1988
Robinson and Nigam 2008
Rodriguez et al. 1985,
Mitchell et al. (1988, 1990)
Rathbun and Shuler 1983
Desgrenges and Durand 1990
Grajek 1988
Buckwheat seeds
Wheat straw
Desfarges et al. 1987
Singh et al. 1990
which can be metabolized by different microorganisms as a source of energy. These
substrates that are insoluble in water, absorb water onto their matrix, which provides
required moisture in SSF system for the growth and metabolic activities of microorganisms. Bacterial and yeast cultures grow on the surface of substrate fibrils and
particles while fungal mycelia penetrate into the particles of substrate for nutrition.
The solid phase in SSF provides a rich and complex source of nutrients that may
be sufficient or sometimes insufficient and incomplete with respect to the overall nutritional requirements of that particular microorganism that is cultivated on that substrate. The constituents in the agricultural solids are approximately analysed in terms
of total carbohydrates, proteins, lipids, various elements and ash content. The solid
substrates generally contain some small carbon compounds whereas the bulk of total
dry weight is a complex polymer. The polymeric forms require enzymatic hydrolysis for their mineralisation as carbon-energy sources in microbialmetabolism. In
comparison with liquid-state fermentation, which generally use less complex carbon
energy sources, solid insoluble substrates provide mixed ingredients of high molecular weight carbon compounds. Such complex carbon compounds may contribute
inhibition, induction, or repression mechanism in microbial metabolism during solid
state cultivation (Table 10.4).
10.2 A Bio-Technology Solid State Fermentation
Solid substrate systems have been defined in several ways:
1. Solid substrate fermentation (SSF) is the microbial transformation of biological
materials in their natural state, in contrast with liquid or submerged fermentation
that is carried out in dilute solutions or slurries (Pandey et al. 2001, 2004).
2. Solid substrate fermentation is generally defined as the growth of microorganisms on solid substrates or sometimes referred to as solid-state fermentation
since the process taking place is in the absence or near-absence of free water
in the system (Nigam and Singh 1994). The substrate however, must contain
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enough moisture, which exists in absorbed form within the solid substrate matrix
and simulates the fermentation reaction occurring in nature. These moist solidsubstrates are insoluble in water and polymeric in nature, are a source of carbon
and energy, vitamins, minerals, nutrients and also provide their absorbed water
for microbial growth as well as anchorage.
3. Solid-state or solid-substrate fermentation means that the substrate is moistened,
often with a thin layer of water on the surface of the particles, but there is not
enough water present to make fluid mixture. Weight ratios of water to substrate
in SSF are usually between 1:1 and 1:10.
4. SSF can be defined as a system with solid matrix particles, a liquid phase bound
to them and a gaseous phase entrapped within the particles. The physical properties of this system such as the water potential and water holding capacity, (can
be used as an index of aeration) and bulk density (which predicates the volume
of pore space) help to define the conditions of solid-state fermentation.
10.3 Advantages of SSF Over Conventional
Liquid Fermentation
Traditional SSF came about for two primary reasons:
1. The desire for more tasty food, as with Oriental fermented foods and mouldripened cheese; and
2. The need to dispose of agricultural and farm waste materials (as in composting).
A closer examination of SSF processes in recent years in several research centres throughout the world has led to the realisation of its numerous economical
and practical advantages (Lonsane et al. 1985; Steinkraus 1984). The attraction
of SSF comes from its simplicity and its closeness to the natural way of life for
many microorganisms. Since large amount of water are not added to the biological systems, fermenter volumes remain small, necessary manipulations become
less expensive and the cost of water removal at the end of fermentation in minimised. This type of fermentation is especially suitable for growing mixed cultures of microorganisms where symbiosis stimulates better growth and productivity
(Bushell and Slater 1981). Solid-state fermentations are clearly distinguished from
submerged cultures by the fact that microbial colonisaton occur at or near the surfaces of solid substrate, or in few cases the soluble substrate supported on the solid
insoluble-matrix in the environment of low-moisture contents. In contrast to liquid
fermentation, the substrates traditionally fermented in the solid-state are renewable
agricultural products, such as wheat, rice, millet, barley, corn and soybeans. The
non-traditional substrates, which can be used in industrial process development,
include an abundant availability of agricultural, forest and food-processing wastes.
From an engineering point of view, SSF offers many attractive features in comparison to conventional stirred tank reactors or aerated liquid medium fermentations
because no free water is present, this leads to many benefits.
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Solid-state fermentations can be used to provide low-shear environments for the
cultivation of shear-sensitive mycelial organisms. Solid state cultivations can be and
have been used for mass production of spores, which can than be used for the transformation of organic compounds such as steroids, antibiotics, fatty acids, and carbohydrates. Fungal spores have applications in the production of food-flavours and
insecticides. The advantage of solid state fermentation includes simplicity, yields
and the homogeneity of spore preparations. The expected advantages of SSF over
submerged fermentations are:
a. Smaller fermenter volume, relative to the yield of the product, as there is no
excess water taking space in the fermenter,
b. Lower sterilisation energy costs, as less volume of water needs to be heated,
c. Seed tanks are not necessary in all cases, as the spore inocula can be successfully
used to inoculate the solid medium.
d. Easier aeration, as air can circulate easily and freely between the substrate particles, and also because the liquid film covering the substrate has a large surface
area compared to its volume. Aeration is facilitated by spaces between substrate
particles and particle mixing.
e. Reduced or eliminated capital and operating costs for stirring, since occasional
stirring is sufficient.
f. Lower costs of product recovery and drying; in many cases the product is concentrated in the substrate and can be used directly e.g. Oriental foods and cheeses,
or the products can be directly incorporated into animal feeds.
g. If the product is to be extracted from the substrate e.g. enzymes and other metabolites, then much less solvent is needed. The fermented solids may be extracted
immediately by direct addition of solvents or maintained in frozen storage before
extraction.
h. Reduced or eliminated capital and operating costs for effluent treatment due to
lower water content in the system.
The other benefits are:
1. The media are relatively simple; a natural, as opposed to a synthetic, medium is
used;
2. A more natural environment for microorganisms, e.g. agricultural wastes degrading organisms: many of these fungi grow and perform better under SSF than
submerged conditions;
3. A less favourable environment for many bacteria, which require a high moisture level to survive, lowering the risk of contamination, therefore many SSF
processes need no sterilisation;
4. SSF is adaptable to either continuous or batch process and the complexity of
equipment is no greater than that required for submerged reactors.
Above described advantages are so attractive for the biological processing of
agricultural by products that most of the work has used SSF process. These
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advantages can outweigh the disadvantages of SSF, which are the slowness of fermentation and the difficulty of controlling the process precisely.
10.4 Performance Control of SSF Process
The difference in process control between SSF and SmF is mainly due to the use of
solid substrates with a very low moisture content in system. The disadvantages of
large-scale solid cultures are due to the problems of process-control, process scaleup and the major problem of heat build-up. Despite these drawbacks, large-scale
SSF processes have been developed successfully in Japan for the manufacture of a
variety of products, including fermented foods and food-products, enzymes, and organic acids. The drawbacks have been overcome by carrying these fermentations in
stationary and rotary tray processes, where the temperature and humidity-controlled
air is circulated through the stacked beds of fermenting solid substrate particles.
These tray methods of cultivation have been used for centuries in the manufacture
of traditional food products and the cultures experience the shear-sensitivity in some
of these processes. These are main reasons of less frequent use of rotary drum-type
fermenters.
Little information is available in the West on the details of modern control systems in large-scale solid-state cultivations. The control of temperature and humidity
within practical limits is exercised through water temperatures, which is used to
humidify the circulating air. The humidified air is circulated at flow-rates to meet
the requirements of heat and mass transfer. The gas environment has been found to
significantly affect the rate and extent of culture colonisation and product formation in SSF. In the commercial production of amylase using rice substrate in SSF,
oxygen pressures above atmospheric have been found to significantly stimulate the
enzyme productivity, suggesting oxygen limitation at normal atmospheric pressure.
The DNA measurements revealed that this only caused a little effect on biomass
formation, but the carbon dioxide pressures above 0.01 atm severely affected the
process through the inhibition in amylase productivity.
In a protein production process by Aspergillus species using alfalfa residues,
cellulase and pectinase activities have been found stimulated by oxygen and carbon dioxide pressures above atmospheric levels, and with no effect on biomass
formation. These studies have been conducted in controlled gas environments at
constant partial pressures, which is maintained by admitting pure oxygen on demand
at pressures below a set point and purging carbon dioxide in 30% KOH at pressures
above a set point in a closed aeration system. In another type of SSF performed for
the degradation of natural birch lignin employing Phanerochaete chrysosporium,
high oxygen pressures have been found to be stimulating, whereas the high carbon
dioxide pressures have been found inhibiting the process. The stimulatory effect of
oxygen on breakdown of lignins has been confirmed in laboratory studies by using
labeled synthetic lignins and natural wood lignins.
Given the present state of the art, the most promising approach in solid state
fermentation processes development happens to be the measurements and control of
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205
various parameters and process variables, similarly as in any liquid fermentation. In
SSF processes, various methods are selected to analyse the temperature, pH, humidity, oxygen and carbon dioxide concentrations in gas phases, biochemical analysis
of fermented and unfermented solids and their extracts. The manufacturing productivities of some industrial scale submerged liquid fermentations have increased significantly over years, e.g. antibiotic production. This development has been possible
due to applied and basic research in microbial-biochemistry, microbial-physiology,
and genetics. To some extent the contribution also goes to engineering research
based on concepts of stoichiometry, kinetics, thermodynamics, and heat and mass
transfer in control of the microbial fermentation process and its environment.
Direct economic comparisons of solid-state and liquid-state fermentations are not
possible, it is apparent that the large-scale solid-state fermentations (known as Koji
in Orient) have been developed in Japan on an economic basis. Potential economic
advantages of such processes to employ suitable microbe-substrate system include:
1. reduced thermal processing requirements, since many processes are not aseptic;
2. reduced energy requirements for agitation, since surface-to-volume ratios for gas
transfer are high and many processes do not require agitation due to their shearsensitivity;
3. high extracellular product concentrations, that can be efficiently recovered by
superficial-extraction or leaching methods.
10.4.1 Performance Control by Particle Size of Agro Residues
SSF processes performance can be varied and controlled by changing physical and
chemical factors. It has been reported that substrates with finer particles showed improved degradation due to an increase in surface area for enzymatic action (Moloney
et al. 1984). The greater growth of fungal cultures has been found stimulated
by smaller particle size substrates. Higher enzyme productivity in SSF has been
achieved with substrates, which contained particles of mixed sizes from 180 m to
1.4 mm.
Particles and kernels of grain must be of suitable size, but not be too small in
order to avoid particle agglomeration. The particle size must be in a limited size
range to be maintained at relatively low moisture content to prevent contamination.
The smaller particle size provides a larger surface area which facilitates heat transfer
and gas exchange. Smaller particle sizes also distribute equivalent moisture concentrations in thinner films on external surfaces exposed to the gas environments, given
the same void volume fraction (porosity) and pore size distribution. Internal pores
maintain the same surface-to-volume ratios with respect to solid surfaces, based on
geometric considerations of spherical particles. This results in higher surface nutrient concentrations and the diffusion of nutrients takes place via shorter pathways at
the surfaces as well as in the pores of those substrates which have same tortuosity.
Too small a particle size may result in closer packing densities of the substrates
and the void space between particles becomes considerable reduced. The reduced
space between particles tends to reduce the available area for heat transfer and
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gas-exchange with the surrounding environment. If such condition arises, densely
packed particles in a cultivation system have to be sufficiently agitated to provide a
better separation of particles for the exchanges of gases and heat transfer. There may
be a lower limit in particle size at which the heat transfer or gas exchange becomes
rate limiting and there may be an upper limit at which the nutrient transfer becomes
limiting. Conclusively under any condition, the particle size of the substrate to be
used is one of the major variables in the SSF-process development. Various methods are available to obtain particle sizes such as milling, grinding, chopping and
sieving to obtain substrates of particular particle-sizes. In the case of lignocellulosic
substrates, smaller particle size substrate is usually obtained through ball-milling.
10.4.2 Performance Control by Medium Preparation
of Agro-Residues
Some SSF systems do not require any nutritional supplements as do most of
the traditional food fermentations. Medium supplementation is necessary in nontraditional SSF fermentations, as it induces enzyme-synthesis, provides balanced
growth conditions for mycelial-colonisation and biomass formation, as well as prolonging the production of secondary metabolites. SSF employing brown-rot fungi,
require an additional carbon source for the induction of enzymes for the celluloseutilisation. Certain fungi including Lentinus lapidus, Poria monticola, and Lezites
trabea can be cultivated on lignin-containing natural wood substrates from aspen,
pine and spruce, when the SSF medium is supplemented with glucose or cellobiose
in smaller quantities of 0.5%, w/v, and an even smaller amount of peptone, asparagine and yeast extract. In unsupplemented media, growth of these fungi was
very slow as negligible. A co-metabolite, such as glucose or cellulose, stimulates the
lignin-degrading system in white-rot fungi such as Phaenerochaete chrysosporium
and Coriolus versicolor when these organisms are cultivated on spruce lignin. Other
supplementations of cellobiose, mannose, xylose, glycerol or succinate have been
found less effective.
Studies for the nutritional requirements for a developmental microbe-substrate
system to be used on a large-scale SSF, can be done in preliminary experiments
in small-scale liquid or SSF on laboratory scale. There is a procedure for evaluating the effects of nutritional supplements on culture-growth and product formation,
in which microbial-cultures and the solid substrate are contained in separate compartments divided by a membrane with a molecular-weight-cut-off. The membrane
permits the passage of enzymes and small molecular weight compounds but restricts
microbial and substrate solids. One of the major difficulties in the development of
solid state fermentations has been the problem in separating microbial biomass from
the solid substrate particles after the mycelial growth has covered the substrate surfaces. In solid culture cultivation the microorganism and substrate are intimately
associated making the analytical methods of limited value in stoichiometric analysis of SSF. The analysis of biomass yield and growth rate by the measurement of
10 Solid-State Fermentation Technology
207
glucosamine, protein, RNA, DNA, oxygen consumption, and carbon dioxide or heat
evolution, can not be accurately used in samples of SSF.
Solid cultures for the production of secondary metabolites may have another
problem in that the nutrient, whose deficiency triggers the pathway leading to formation of secondary metabolite, may be available in excess when the microbial growth
becomes limited by other nutrient. Therefore, the selection of a solid substrate and
required-supplements is more critical for a SSF process for antibiotic production
that for a SSF designed for enzyme and organic acid biosynthesis.
10.4.3 Performance Control by Moisture Content
of Agro Residues
Solid-state or solid-substrate fermentation means that the substrate is moistened,
often with a thin layer of water on the surface of the particles, although there is not
enough water present to make a fluid mixture. Weight ratios of water to substrate
in SSF are usually between 1:1 and 1:10 (Reid 1989a, b). Since biological activity
ceases below a moisture content of about 12%, this establishes the lower limit at
which SSF can take place. The upper limit is a function of absorbency and hence,
moisture content varies with the substrate material type.
Solid substrates may be viewed as gas-liquid-solid mixtures. The aqueous phase
in such mixtures is intimately associated with solid surfaces in various states of
sorption. The aqueous phase in a cultivation system is in contact with the gas phase
continuous with the external gas environment. Different types of solid substrates
can absorb different amounts of water. Depending on the moisture content of the
solid; some of the water is tightly bound to solid surfaces, some amount of water is
less tightly bound and remaining water may exist in a free state inside the capillary
regions of the solid substrates. The gas-liquid interface provides a boundary for
gaseous exchange between carbon dioxide and oxygen as well as for heat exchanges.
Water in biological materials exists in three states. The moisture isotherm measurements determines that the solids sorb or desorb water vapour in equilibrium
with relative humidities in a gas phase (water activities), which can be maintained
by saturated salt solutions at a constant temperature. Water is tightly bound to solid
surfaces at the surface in a monolayer region. In case of agricultural residues, monolayer binding is generally 5 to 10 g per 100 g of dry solids. Beyond the surface
monolayer in a multilayer region, water is less tightly bound in additional layers
at progressively decreasing energy levels. Then beyond the multilayer region, free
water exists in a region of capillary condensation. In terms of relationships between
water activity and moisture content, the distinction between the multilayer and capillary regions is ambiguous. The electric measurements of an agricultural residue
containing high starch content has been used to determine the dividing line between
multilayer and capillary regions. The dividing line was defined by a moisture content
of about 25 to 30% by weight at a water activity of 80 to 85%, which is the lower
limit for microbial growth except for some halophilic or osmophilic microbes.
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The sorption isotherm may vary from one type of product to another, the hysteresis is seen in sorption and desorption isotherms. Water may exist in free state
at moisture levels of interest in solid state fermentation, which is in contrast with
general perception about SSF that the free water does not exist in such systems.
Moisture is a critical factor in SSF of aflatoxin production on rice; the yields of
aflatoxins have been found decreasing rapidly at moistures above 40%. The rice
particles become sticky at moistures above 30 to 35%. Moisture content plays an
important role on the growth of lactic acid bacteria on feedlot wastes liquids mixed
with cracked corn; growth and acid production was limited at moisture level less
than 35%, whereas the higher level above 42% in SSF-mixtures caused the contents
to become gummy and aggregate. One of the secrets of a successful SSF-process
is to keep the fermenting substrate moist enough for fungal-growth and colonisation and to avoid higher moisture level not to promote the unwanted bacterial
growth. Therefore, the optimum moisture content for a particular type of SSF for
its microbe-substrate system should be determined for a particular end-product and
cultivation conditions of that SSF.
The level of moisture content affects the process productivity significantly in
any SSF system, when available in lower or higher quantities than the optimum
value (Lonsane et al. 1985). Hence, it should be in limited and required amounts in
system. The presence of an optimum moisture content in SSF medium has been emphasised also for the cultivation of bacterial cultures (Ramesh and Lonsane 1990).
The process productivities are affected by water content because the physiochemical
properties of the solids depend and vary with moisture available to them. Therefore,
the major key factors determining the outcome of the SSF-process are the moisture
content and the relative humidity levels (Lonsane et al. 1985).
Heat removal during fermentation is mostly achieved by evaporative cooling.
This leads to an uneven distribution of water in system due to large quantities of
water evaporation. Workers have practised various ways to maintain the moisture
content of the solids (Lonsane et al. 1985; Ahmed et al. 1987).
10.4.3.1 Control of Water Activity Factor in SSF
Water activity of the substrate has been proposed as the condition of growth and
viability of the microbes and hence, the importance of aw in SSF has widely been
studied (Nishio et al. 1979; Raimbault and Alazard 1980; Kim et al. 1985). Water
activity is defined as the relative humidity of the gaseous atmosphere in equilibrium
with the substrate and the water activity factor, aw of the substrate quantitatively
expresses the water requirement for microbial activity (Smith et al. 1985).
aw = −Vm /55.5 where,
V = number of ions formed,
m = Molar concentration of solute
= Molar osmotic coefficient, and
55.5 = molar concentration of a solution of pure water.
Pure water has an aw = 1.00 and it will decrease with the presence of solutes.
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209
The types of the microorganisms that can grow in SSF systems are determined
by the water activity factor, aw . Bacteria mainly grow at higher aw values while
filamentous fungi and some yeasts can grow at lower aw values (0.6–0.7). The microorganisms capable of carrying out their metabolic activities at lower aw values
are suitable for SSF processes. High aw favours sporulation in the course of growth
in SSF, but low aw favours spore germination and mycelial growth.
Numerous experiments have demonstrated the influence of aw on microbialmetabolism, such as, on growth rate and sporogenesis of filamentous fungi (Gervais
et al. 1988), on enzyme biosynthesis by fungi (Grajek and Gervais 1987), and on
cheese aroma production (Gervais et al. 1988).
The aw of the medium is a fundamental parameter for mass transfer of the water
and solutes across the cell membrane (Gervais and Sarrette 1990). The control of
this parameter could be used to modify the metabolic production or excretion of a
microorganism (Gervais 1989, 1990). A theoretical calculation based on the Ross
equation showed that aw decreased towards the end of the SSF-culture (Oriol et al.
1988). A kinetic model which relates the rate constant of the death of the microbial
cells to aw and temperature has been proposed by Moser (1988), using the equation
k = k␣ aw exp −EA aw / RT
Constants k␣ and EA are calculated from the experimental value of aw . Regulation of the aw can be controlled by the relative humidity of the air. Gervais and
Bazelin (1986) reported a SSF process allowing the control of aw and Gervais (1989)
developed a new sensor for the continuous aw measurement in SSF.
10.5 Microorganisms Used for Agro-Residues Bioconversion
Selection of a suitable microorganism is one of the most important criteria in SSF.
The vast majority of wild type microorganisms are incapable of producing commercially acceptable yields of the desired products. The unique characteristics of
solid-state cultivations are their ability to provide a selective environment at lower
concentrations of moisture ideal for mycelial organisms. The mycelial organisms are
capable of producing a range of extracellular enzymes required for the hydrolysis
of complex, polymeric solid substrates. Such microorganisms are able to colonise
at high nutrient concentrations near solid surfaces. The mycelial organisms include
a large number of filamentous fungi and a few bacteria of actinomycetes. The importance of microorganisms can be seen from the fact that a culture of Aspergillus
niger can produce as many as 19 types of enzymes, while enzyme alpha amylase
can be produced by some 28 different types of cultures (Fogarty and Kelly 1979;
Pandey 1992). SSF processes can be placed in two main classes based on the type
of microorganism involved:
1. Natural (Indigenous) SSF: Ensiling and composting are SSF processes, that
utilise natural microflora. In nature, SSF is often carried out by mixed cultures
in which several microorganisms show symbiotic cooperation.
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2. Pure culture SSF: Known purified microorganisms are used in such processes
either singly or in mixed culture. SSF using a pure culture is known since antiquity e.g. the Koji process with Aspergillus oryzae. A pure culture is necessary in
industrial SSF process for improved rate of substrate utilisation and controlled
product formation. A typical example of pure mixed culture SSF is the bioconversion of agricultural residues to fungal biomass (protein) using two pure
cultures of Chaetomium cellulolyticum and Candida utilis.
Several microorganisms have been employed in a wide range of SSF processes
for various objectives. The cultivation of filamentous fungi on solid substrates has
been widely used for different purposes at laboratory scale e.g. for Koji fermentation, for lignocellulose fermentation (Matteau and Bone 1980), for fungal spores
(Lotong and Suwarnarit 1983), and for mycotoxin production (Hesseltine 1972;
Bhumiratna et al. 1980). For various purposes, among the filamentous fungi three
classes, viz. Phycomycetes (Mucor and Rhizopus), Ascomycetes (Aspergillus and
Penicillium) and Basidiomycetes (Nigam and Prabhu 1985), have been most
widely used.
SSF has been most commonly used employing Aspergillus niger for protein enrichment (Rodriquez et al. 1985; Baldensperger et al. 1985; Czajkowska and Ilnicka 1988) as well as for enzymes production, such as, cellulase (Singh et al. 1989;
Madamwar et al. 1989), amylase, glucoamylase (Ramakrishna et al. 1982; Pandey
1990), beta glucosidase, and protease (Malathi and Chakrabarty 1991). Production
of alcohols, ketones and aldehyde in rice fermentation was achieved by the use of
A. oryzae (Ito et al. 1990). For protein enrichment and kinetic studies related to
SSF process Rhizopus oligosporus has been employed (Rathbun and Shuler 1983;
Mitchell et al. 1988, 1990).
Fungal rennet has been produced by R. oligosporus and Mucor meihei (Karanth
1988). For enzyme production and protein enrichment cultures of Trichoderma
spp. have been employed in pure, single and mixed SSF (Daubresses et al. 1987;
Grajek 1988). Lipase enzyme production has been reported (Munoz et al. 1991)
using six species of Penicillium, two species of Rhizopus, Geotrichum candidum
and Mucor meihei, whereas the maximum lipase activity was obtained with P. candidum, P. camembertii and M. meihei. For the production of several other enzymes
e.g. hydrolases and pectic enzymes (Siesser and Said 1989) several other species of
Penicillium have been employed in SSF.
Production of the antibiotic penicillin was achieved in a non-sterile SSF process on sugar cane bagasse impregnated with culture medium using Penicillium
chrysogenum. Protein enrichment of lignocellulosic substrates for animal feed production (Nigam 1990; Nigam and Vogel 1990a, b), lignin degradation (Bone and
Munoz 1984), and cellulase and ligninase enzyme production (Nigam et al. 1987a,
b) have been obtained by white-rot cultures in SSF.
Production of gibberellic acid has been reported using Fusarium monoliforme
and Gibberella fugikuroi (Kumar and Lonsane 1987a, b). Bacterial alpha amylase
production is reported using Bacillus licheniformis in SSF (Ramesh and Lonsane
1987a, b, 1990). Several yeasts have been used for protein enrichment and ethanol
10 Solid-State Fermentation Technology
211
fermentation in SSF. For protein enrichment of straw (Han 1987) Candida utilis
was used whereas Saccharomyces cerevisiae has most commonly been employed
for ethanol production (Gibbons et al. 1984; Kargi et al. 1985).
10.6 Designing and Types of SSF
10.6.1 Fermenter Design for SSF
Several miscellaneous types of fermenters have been used in batch or continuous mode in SSF processes (Hardin 2004). Process parameters are very important factors and they have to be considered in a bioreactor design for any SSF.
Design considerations in types of SS-fermenters used by various researchers are
described by Aidoo et al. (1982). The engineering aspects, with major types of fermenters describing their advantages and drawbacks has been reviewed by Fernandez
et al. (2004). Solid state cultivations are not as well characterised on a fundamental
scientific or engineering basis, as are the liquid fermentation systems that are used
in the West for the industrial production of microbial-metabolites. Solid-state fermentations are, however, widely used in the Orient and therefore, the old traditional
methods of cultivation systems which have been used in food-processing for more
than 2,000 years, have now been modernised and well characterised for their extended application to non-traditional products. Mitchell et al. (2004) have described
in detail the modelling aspects of SSF.
The physical state of the substrate and the products to be produced in the system
characterise the design-type of solid state cultivation process:
a. Low-moisture solids are fermented
1. without any agitation for the production of Tempeh and Natto;
2. by occasional stirring for the production of Miso and Soy sauce;
3. with continuous stirring for the production of Aflatoxin.
b. Suspended solids are fermented in packed bed columns
1. through which the liquid is circulated, as for the production of rice-wine;
2. which contain stationary or agitated liquid media, for the production of Kaffir
beer.
10.6.2 Types of SSF Systems
There are two types based on process design:
Type one- Fermentation in static reactor
e.g. Tray fermentations (Lonsane et al. 1985; Viesturs et al. 1987)
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Type two- Fermentation with occasional or continuous agitation
e.g. Production of aflatoxin, ochtratoxin and enzymes (Lindenfelser and Ciegler
1975; Hesseltine 1977; Silman 1980).
Type two has 4 variations according to the need of process:
1.
2.
3.
4.
Occasional agitation, without forced aeration
Slow continuous agitation, without forced aeration
Occasional agitation with forced aeration
Continuous agitation with forced aeration.
10.6.3 SSF Bioreactors
Three basic groups of reactor exist for SSF, and these may be distinguished by the
type of mixing and aeration used. In laboratory scale, SSF occurs mainly in flasks
whereas following reactors are used for large-scale product-formation.
10.6.3.1 Tray Bioreactors
Tray bioreactors tend to be very simple in design, with no forced aeration or mixing
of the solid substrate. Such reactors are restrictive in the amount of substrate that
can be fermented, as only thin layers can be used, so as to avoid overheating and
maintain aerobic conditions. Tray undersides are perforated to allow aeration of
the solid substrate, each arranged above each other. In such reactors, temperature
and relative humidity are the only controllable external parameters (Durand 1998).
Wooden trays were initially used for soy sauce production in Koji fermentations by
Aspergillus oryzae. The use of tray fermenters in large-scale production is limited
as they require a large operational area and tend to be labour intensive. The lack
of adaptability of this type of fermenter makes it an unattractive design for any
large-scale production.
10.6.3.2 Drum Bioreactors
Drum bioreactors are designed to allow adequate aeration and mixing of the solid,
whilst limiting the damage to the inoculum or product. As previously mentioned,
mixing and aeration of the medium has been explored in two ways: by rotating
the entire vessel or through the use of various agitation devices. Rotation or the
use of agitation can be carried out on a continuous or periodic basis. In contrast
to tray reactors, growth of the inoculum in drum bioreactors is considered to be
better and more uniform. Increased sheer forces through mixing, can however, have
a detrimental affect on the ultimate product yield.
Although the mass heat transfer, aeration and mixing of the substrate is increased,
damage to inoculum and heat build up through sheer forces may affect the final
product yield. Application of drum reactors for large-scale fermentations also poses
handling difficulties.
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213
10.6.3.3 Packed Bed Bioreactors
Columns are usually constructed from glass or plastic with the solid substrate supported on a perforated base through which forced aeration is applied. They have
been successfully used for the production of enzymes, organic acids and secondary
metabolites. Forced aeration is generally applied at the bottom of the column, with
the humidity of the air kept high to avoid desiccation of the substrate. Disadvantages
associated with packed bed column bioreactors for SSF include difficulties in retrieving the product, non-uniform growth, poor heat removal and scale-up problems.
10.7 Scale-Up Stages of SSF
Scale-up of SSF has been defined in many ways. There are mainly four stages:
10.7.1 Flask Level
This is smallest scale using 50–1000 g substrate working capacity, and used for the
selection of the organism, optimisation of the process and experimental variables
in a short time and at low cost. The vessels used are conical flasks and beakers
(Mitchell et al. 1986; Nigam et al. 1987a, b), Roux bottles (Gervais et al. 1988;
Nigam 1990), jars (Hang et al. 1986), and glass tubes (Raimbault and Alazard 1980).
10.7.2 Laboratory Fermenter Level
This is next to flask scale using a 5–20 kg substrate working capacity. It is used
for a selection of procedures such as, inoculum development, medium sterilisation,
aeration, agitation and downstream processing. Standardisation of various parameters, selection of control strategies and instruments, evaluation of economics of
the process and its commercial feasibility are also examined at this level. The fermenters used are glass incubators (Deschamps and Huet 1984; Oriol et al. 1988;
Smith et al. 1986), column fermenters (Oriol et al. 1988); polypropylene bags
(Yadav 1988), and miscellaneous types of fermenters (Raimbault and Alazard 1980;
Viesturs et al. 1981).
10.7.3 Pilot Fermenter Level
This scale is a stage before the commercial scale using 50–5000 kg of substrate.
This level is necessary for the confirmation of laboratory data and selection of
optimised procedures. It facilitates market trials of the product, physicochemical characterisation and determination of viability of the process. Most large
scale SSFs employ tray type fermenters as in the oldest soy sauce Koji process
(Daubresse et al. 1987), rotating drum type (Lindenfelser and Ciegler 1975; Han
and Anderson 1975; Hesseltine 1977), horizontal paddle fermenters and mixed layer
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P. Singh nee’ Nigam and A. Pandey
pilot plant fermenters (Laukevics et al. 1984). Durand and Chereau (1988) reported
the use of a pilot reactor having a one ton working capacity.
10.7.4 Production Fermenter Level
The commercial scale fermenter utilises 25–1000 tonnes of substrate and is performed for streamlining of the developed process. Yokotsuka (1985) described deep
trough methods and mechanical continuous equipment for Koji production generating 50–100 tonnes of Koji per day.
10.8 Factors Affecting SSF
Each microbe-substrate system is unique and the process variables must be considered in terms of the physical properties and chemical composition of its substrate, growth characteristics and physiological properties of the microorganisms
to be cultivated in SSF. The nature of the product, if the process involves the
synthesis of primary or secondary metabolite may be based on the synthesis of
extracellular enzymes in growth-associated metabolism. The process variables affecting a solid state cultivation include, pretreatment of substrates, particle-size of
substrates, medium-ingredients, supplementation of growth medium, sterilisation of
SSF-medium, moisture-content, inoculum-density, temperature, pH, agitation and
aeration. These variables should be considered in process-development of a SSF to
be carried out for different purposes. Some of these variables have been discussed
in some sections as above, the rest are discussed below.
10.8.1 Significance of Aeration and Mixing in SSF
In any SSF-process an adequate supply of oxygen is required to maintain the
aerobic conditions and for the transfer of excess carbon dioxide produced during
metabolism. This requirement can be achieved through the process of aeration and
mixing of the fermenting solids. In certain cases, the mixture can not be agitated
vigorously or in some cases, at all, if the microorganism used in SSF is shear sensitive. The shear sensitivity is attributed to disruption of mycelial-substrate contact;
this is particularly concerned to those organisms which possess mycelial-bound enzymes required for the hydrolysis of solid substrate-polymers. Most Koji processes
in Japan performed for the commercial production of enzymes do not involve great
agitation. The fermenting substrate is gently turned periodically just to bring the
bottom of Koji to the top. These processes have been developed in highly controlled
environments, using automated systems for inoculum mixing, and turning of the
fermenting substrate.
Most of the traditional food-fermentation in Japan use the rotary-tray method
for SSF with the circulation of humidified air to create the conditions suitable for
gas-exchange and heat-transfer. In the SSF for the production of certain secondary
10 Solid-State Fermentation Technology
215
metabolites such as aflatoxin and ochratoxin, and in some processes for the enzyme
production, mixing and particle separation are achieved by agitation on shakers or in
rotating vessels with circulating conditioned air. Maximum rotation rates generally
decrease with the size of the fermentation-vessel. Therefore, solid-state fermentations are ideal for the cultivation of those microorganisms that are extremely sensitive to the shear rates of the impeller speeds required for stringent oxygen demand
rates in liquid fermentaton. Such microorganisms colonise the solid substrates by
microbe-substrate attachment and there is no pellet formation in solid-state cultivation, which is added advantage to SSF.
Aeration plays an important role in solid state fermentations as compared to liquid fermentation where it only helps in gas transfer. Aeration facilitates in heat,
gas and moisture transfer between the fermenting solid particles and the gas environment of the system. The temperature of the gas phase serves by supplying or
removing heat, in maintaining the relative humidity in equilibrium with the liquid
phase. In liquid fermentations the substrates are dissolved in at low substrate concentrations in large volumes of fluid, but in solid cultures with respect to moisture
transfer, the loss or gain of moisture during SSF is extremely sensitive to the water
activity of the gas-phase. Therefore, small changes in the relative humidity of the
gas phase in equilibrium with the solids may cause the large changes of moisture
content in the solid state, depending on the sorption-desorption characteristics of the
solid substrate.
There are two main functions of the gas phase in SSF, the primary function is
to supply oxygen and remove the carbon dioxide from the system. The secondary
function of aeration is in heat and moisture transfer that is more important, when
the rates of oxygen and carbon dioxide are not limiting. The gas phase can facilitate in the control of solid cultures, due to the fact that direct measurements can
not be performed to estimate dissolved oxygen or carbon dioxide concentrations in
low-moisture solids during the course of the fermentation on either a continuous or
sampling basis. The methods of aeration may cause the conditions of gas transfer being relatively stagnant. This condition may be responsible for the oxygen limitation
at small penetration depths or may lead to inhibitory carbon dioxide concentrations
in normal atmospheric environments. The gas phase in the SSF during the course
of microbial metabolism, can be analysed for oxygen, and carbon dioxide pressures
using analysers which function on thermal-conductivity, paramagnetism, or infrared
absorption. The technique of gas chromatography can also be used for gas-analysis
of the gas phase of a SSF.
10.8.2 Significance of Control of Temperature and pH in SSF
Two significant variables affecting any SSF are the incubation temperature and the
pH of SSF-medium. Both variables are specific for each SSF process depending on
the microoganisms to be cultivated and the product to be formed. Unlike submerged
fermentation, these factors are difficult to control in SSF. These variables can not
be directly measured in the liquid phase, as these are associated with the solids at
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P. Singh nee’ Nigam and A. Pandey
lower moisture content without any free liquid in the fermenting medium. The other
difficult situation arises when the growth temperature of cultivated microorganism
is different than the optimal temperature for the product formation. Such systems
require a possible need for temperature profiling or shift in the later stages of fermentation. The thermal gradients may be induced within SSF-mixture due to the
rate of heat generation in SSF-system at high levels of biological activity. This
gradient may limit the heat transfer and may lead to sub-optimal conditions for
microbial-biomass and product formation.
The local pH levels at solid surfaces near which the biological activity occurs,
may be considerable different than the bulk pH of the liquid phase. This difference
in pH levels happens due to surface charge effects and ionic equilibria modified
by solute transport effects. There is no suitable method to measure the precise pH
of fermenting solid residues in SSF. A general method used for measuring pH of
solid agricultural residues involves mixing one part of fermented solids (dry weight)
and three parts of freshly boiled and cooled water, and measuring the pH of the
resultant liquid after five minutes using a glass electrode. This procedure can be
used to monitor pH changes during fermentation on intervals using minimum one
gram of the SSF-mixture.
It is easier to measure temperature of the fermenting SSF-mixture, in comparison to pH measurement. Temperature can be measured using thermistor or thermocouple probes at various depths of the SSF-mixture below the medium-surface. In
various SSF-processes for the production of enzymes, mycelial-biomass or organic
acids, total heat generation of up to 600 kcal per kilogram of fermenting solids has
been observed. A study of composting of animal wastes and agricultural residue has
revealed that such heat generations may lead to rapid temperature rise of the fermenting mass in the system limited by heat transfer. The study also revealed that the
biological activity was considerably higher near the surface of the compost pile than
in the depth of pile that was at lower oxygen pressure. This phenomenon happens
due to a decrease in interior oxygen concentrations inside the SSF-mixture pile of
compost. Thus the heat generation in such fermentations is coupled to conditions
for heat as well as mass transfer.
10.9 Processes and Products of SSF
Various processes and products from bioconversion of agro-residues of industrial,
pharmaceutical, and environmental importance have been discussed in detail in further chapters 11–24 under sections II, III.
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Part III
Biotechnological Potential of
Agro-Industrial Residues for Bioprocesses
Chapter 11
Biotechnological Potentials of Cassava Bagasse
Rojan P. John
Contents
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2 Cassava- a Global View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3 Cassava Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.1 General Process in Starch Extraction and Generation of Bagasse . . . . . . . . . . . .
11.3.2 General Properties of Cassava Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.3 General Cultivation Systems Using Cassava Bagasse . . . . . . . . . . . . . . . . . . . . .
11.3.4 Cassava Bagasse Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.5 Value Addition of Cassava Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Crop residues such as cassava bagasse are annually renewable sources
of energy. Though they are rich in carbohydrate, their utilization for any direct
application is very less due to the low content of protein and poor digestibility.
However, the utilization of such agro-industrial residues provides alternative substrate for bioprocesses and will solve the problem of environmental pollution to
an extent. Several processes have been developed to utilize cassava bagasse, the
fibrous residue of the tropical tuber for the production of value added products such
as organic acids, ethanol, aroma, mushroom etc. The chapter focuses on the wide
spectrum applications of cassava bagasse in bioprocess technology.
Keywords Agro-industrial residues · Bioprocess technology · Cassava bagasse ·
Value addition
R.P. John (B)
Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR,
Trivandrum–695 019, Kerala, India
e-mail: rojanpj@yahoo.co.in
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 11,
C Springer Science+Business Media B.V. 2009
225
226
R.P. John
11.1 Introduction
The concept of utilizing excess biomass or waste from agricultural and agroindustrial residues to produce energy, feeds or foods, and other useful products is
not necessarily new. Our whole world thinks in the same path to overcome the pollution problems in environmentally sound methods like composting, reuse, recycling,
bioconversion etc. Researchers from all over the world adopted many technologies
to reduce the pollution and cost of value added products by the use of agricultural
wastes. Several processes have been developed to utilize renewable raw materials
for the production of value added products such as enzyme, organic acids, ethanol,
amino acids, aroma, single cell protein, mushroom etc. Currently, a great deal of
attention is being paid on biotechnological potential of agro industrial residues such
as cassava bagasse, sugarcane bagasse, sugar beet pulp, coffee husk and pulp, apple
pomace, oilcakes, wheat/rice bran etc. for their use as raw material in bioprocesses
(John et al. 2007a). Approximately 3.5 billion tons of agricultural residues are produced per annum in the world. Though they are rich in carbohydrate, their utilization
is very less due to the low content of protein and poor digestibility (Elkholy and
Eltantawy 2000). Recently, fermentation of biomass such as cassava bagasse, wheat
bran etc. has gained considerable attention because of the forthcoming scarcity of
fossil fuels, and because it is necessary to increase the world food and feed supplies. This chapter focuses on the potentials of cassava bagasse, starchy waste generated from the starch extraction, to generate value added products in eco-friendly
manners.
11.2 Cassava- a Global View
Cassava (Manihot esculenta Cranz), tropical root crop, is the third most important source of calories in the tropics, after rice and corn. According to FAO, more
than 600 million people depend on the cassava in Africa, Asia and Latin America
(Tonukari 2004). It gives the highest yield of starch per hectare of any crop ranking
it as 4th crop in worldwide production after rice, wheat and maize. Total cassava
production is estimated as approximately 166 million tons and is projected to reach
approximately 266 million tons by 2020 based on the current production rate. Most
of the contribution in the production is from the developing countries and from these
the sub-Saharan Africa (Soccol 1996). It is known as tapioca in Asian countries, as
aipin, castelinha, and macaxeira in Brazil, as yuca in Spanish-speaking countries
of Latin America, and as manioc in French-speaking countries in Africa (Pandey
et al. 2000). Cassava is considered to have originated in Venezuela during 2700 BC
(Pandey et al. 2000). In a significant research published during May 1999, biologists
from the Washington University in St. Louis discovered that cassava originated from
the southern border of the Amazon River basin in Brazil. It was introduced in Africa
during the 16th century and from there into Asia during the 18th century. It is a
bushy plant producing tubers and is made up of an aerial part and an underground
part. The aerial part can be as high as 2–4 m with a trunk and branches on it. The
11 Biotechnological Potentials of Cassava Bagasse
227
underground part consists of two types of roots: the ones responsible for the plant
nutrition, and the others with axial disposition surrounding the trunk. These are
called tubers and are the edible parts of the plant. Each plant may have 5–20 tubers,
and each tuber may attain a length of 20–80 cm and a diameter of 5–10 cm. The
fresh weight of each tuber may vary between a few hundred grams and 5 kg.
11.3 Cassava Bagasse
Cassava (Manihot esculenta Crantz) belongs to Euphorbiaceae is a short-lived
perennial, 1 to 5 meters tall. The origin of cassava is in South America, presumably
eastern Brazil. From stem cuttings, the plant produces 5 to 10 very fleshy adventitious roots up to 15 centimeters in diameter. Cassava (Manihot esculenta Crantz),
tropical root crop, is the third most important source of calories in the tropics, after
rice and corn.
According to FAO, more than 600 million people depend on the cassava in
Africa, Asia and Latin America (Elkholy and Eltantawy 2000). Total cassava production is estimated as approximately 166 million tons and is projected to reach
approximately 266 million tons by 2020 based on the current production rate. Most
of the contribution in the production is from the developing countries and from these
the sub-Saharan Africa (Tonukari 2004). It is known as tapioca in Asian countries, as
aipin, castelinha, and macaxeira in Brazil, as yuca in Spanish-speaking countries of
Latin America, and as manioc in French-speaking countries in Africa (Soccol 1996;
Pandey et al. 2000). Cassava is considered to have originated in Venezuela during
2700 B.C. (Soccol 1996).
In a significant research published during May 1999, biologists from the Washington University in St. Louis discovered that cassava originated from the southern
border of the Amazon River basin in Brazil. It was introduced in Africa during the
16th century and from there into Asia during the 18th century. It is a bushy plant
producing tubers and is made up of an aerial part and an underground part. The aerial
part can be as high as 2–4 m with a trunk and branches on it. The underground part
consists of two types of roots: the ones responsible for the plant nutrition, and the
others with axial disposition surrounding the trunk. These are called tubers and are
the edible parts of the plant. Each plant may have 5–20 tubers, and each tuber may
attain a length of 20–80 cm and a diameter of 5–10 cm. The fresh weight of each
tuber may vary between a few hundred grams and 5 kg.
Industrial processing of cassava tubers is mainly done to isolate flour and starch,
which generates more liquid and solid residues (processing for flour generates solid
residues while for starch generates more liquid residues). Solid residues include
brown peel, inner peel, unusable roots, bagasse and flour refuse, among which
bagasse is the main residue (Fig. 11.1). Processing about 250–300 t of fresh tubers
results about 280 t of wet cassava bagasse. Cassava bagasse is made up fibrous root
material and contains starch that physically process could not be extracted. Poor
processing conditions may result even higher concentrations of starch in cassava
bagasse.
228
R.P. John
(a)
(b)
Fig. 11.1 Cassava bagasse (a) residue after starch extraction and (b) its powder
11.3.1 General Process in Starch Extraction
and Generation of Bagasse
Generally, factories only purchase sufficient cassava tubers to fulfill a daily crushing
capacity and collected roots are deposited onto a cement floor for short-term storage. Soil and sand are removed as the roots pass through a cylindrical root sieve,
rotating at a low rpm. Soil, sand, pieces of broken peels and impurities pass through
the sieve. Roots after sieving are transported into a water chamber where they are
washed and moved by a paddle blade rotating at low rpm. Water used for washing is
re-circulated from later processing stages. Washed roots are chopped with a cutting
blade. The small root pieces are gravimetrically fed to the raspers. Fresh rasped root
slurry from the rasper is pumped through a series of extractors, from coarse to fine.
The extractors are continuous centrifugal perforated baskets. Pulp from the coarse
extractor is repeatedly re-extracted using the same screen aperture until minimal
starch content of the pulp is achieved. Moisture content of the pulp is then reduced
by a screw press and the pulp finally discharged out of the process. Whole processes
are generalized in Fig. 11.2. The moisture content of pressed pulp is about 60 to
70% and starch content about 40 to 70% (dry basis). High starch content of the pulp
reflects both low efficiency of the rasping stage and composition of the cell membrane and cell walls. The compositions of polysaccharides are in turn influenced by
variety and environmental factors.
11.3.2 General Properties of Cassava Bagasse
Bagasse has a large absorption capacity and may contain about 75% moisture. Cassava tuber contains cyanide content but cassava bagasse does not show any cyanide
content. However, its poor nitrogen content (2.3%) makes it unattractive as an animal feed. Because of the poor nutrient content other than carbohydrates this waste is
11 Biotechnological Potentials of Cassava Bagasse
Fig. 11.2 Schematic of
cassava starch extraction
229
Cassava tuber
Washing and/or peeling
Water
Cutting and rasping
Coarse extraction
Water
Separation
Cassava
bagasse
Fine extraction
Water
Separation
Cassava
bagasse
Starch
mainly used as the land fill and creates environmental pollution. Application of cassava bagasse as substrate would provide an alternative substrate, and solve pollution
problem. Such agro-industrial residues constitute promising alternative substrates
for bioprocesses. On the other hand, because of its low ash content (1.44%), it could
offer numerous advantages in comparison to other crop residues such as rice straw
and wheat straw, which have 7.5 and 11%, respectively, for usage in bioconversion
process using microbial cultures. Other components such as calcium, potassium,
lipids, phosphorous can be also be found in low concentrations (<1%). Table 11.1
shows the major physico-chemical composition of cassava bagasse reported by different studies.
Table 11.1 Physico-chemical composition of cassava bagasse (g/100 g dry weight)
Composition
Soccol 1994
Cereda 1994
Stertz 1997
Vandenberghe 1998
Moisture
Protein
Lipids
Fibers
Ash
Carbohydrates
5.02
1.57
1.06
50.55
1.10
40.50
9.52
0.32
0.83
14.88
0.66
63.85
10.70
1.60
0.53
22.20
1.50
63.40
11.20
1.61
0.54
21.10
1.44
63.00
Source: Pandey et al. 2000.
230
R.P. John
Extraction of starch from the tuber is not fully success as a large part of it trapped
in the fibrous residue. The only alternatives that can help reduce environmental impact of pulpy waste and add value to the pulp is to recover the starch, either as
starch or sugar. In order to accomplish this, physical or biotechnological methods
need to be developed. Improved starch recovery from cassava roots and waste can be
achieved by using multi-enzyme preparations. Cassava starch entangled with fibers
was more efficiently saccharified after treatment with fungal cellulases.
11.3.3 General Cultivation Systems Using Cassava Bagasse
The microbial cultivation on cassava bagasse can broadly be classified into three
groups and are processes based on liquid fermentation using hydrolysate prepared
by enzymatic or acid hydrolysis, simultaneous saccharification of cassava bagasse
and fermentation and processes based on solid-state fermentation (SSF). Due to
high water retention capacity makes it an ideal substrate for SSF processes like cultivation of mushrooms and other fungi. Some of the work carried out using cassava
bagasse hydrolysate and inert support in SSF processes (Rojan et al. 2005; John
et al. 2006a, 2007c). Submerged fermentation (SmF) processes have rarely been
utilized due to obvious reasons of cost effectiveness. But simultaneous saccharification and fermentation can be effectively utilized for the production of organic acid
like lactic acid using bacterial cultures.
11.3.4 Cassava Bagasse Hydrolysis
Cassava bagasse can be hydrolysed using acid or enzymes. Even though acid hydrolysis may be fast or cost effective, it needs the neutralization of medium and thus
increase in salt concentration may occur. Efforts were taken by many researchers for
the hydrolysis of cassava bagasse by commercial starch degrading enzymes (Pandey
et al. 2000). Woiciechowski et al. 2002 studied the hydrolysis of cassava bagasse
starch by acid and enzymatic hydrolysis. They reported both methods were quite
efficient when considering one or other parameter like the percentage of hydrolysis,
time and cost of the chemicals and energy consumption. Although acid hydrolysis
is time saving and cost effective, there will be a neutralizing step after acid hydrolysis and which will create the unnecessary increase of salts in the medium and it
will affect the growth and production of lactic acid. So the enzymatic hydrolysis is
better as it yields a high percentage of reducing sugars from cassava bagasse. But
the enzymatic hydrolysis of 150 kg cassava bagasse required US$ 2470 as power
for long time saccharification and cost of enzymes. Simultaneous saccharification
reduces the cost of energy consumption for the liquefaction and saccharification
and thus it is cost effective and time saving process for bioconversion of cassava
bagasse in production of organic acid like lactic acid.
11 Biotechnological Potentials of Cassava Bagasse
231
11.3.5 Value Addition of Cassava Bagasse
The molds such as Rhizopus stolonifer, Neurospora sitophila and lactic acid bacteria, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Enterococcus
faecium, Weissella cibaria, Lactobacillus plantarum, L. manihotivorans etc. were
identified as the natural microflora in cassava. There is a restriction of the growth
of a wide variety microorganism in cassava due to its cyanogenic content. As there
is no cyanogenic content in cassava bagasse, it was used as a suitable substrate for
many microorganisms to produce the value added products and Table 11.2 shows the
microorganism which grow on the bagasse or bagasse hydrolysate. Fibrous starchy
cassava waste could utilize to produce many value added products by biotechnological, chemical and physical processes.
11.3.5.1 Xanthan Gum
Xanthan gum is a microbial desiccation-resistant polymer prepared commercially
by aerobic submerged fermentation from Xanthomonas campestris. Woiciechowski
et al. (2004), reported use of cassava bagasse hydrolysate for the production of xanthan gum using a bacterial culture of X. campestris. Cassava bagasse hydrolysate
with an initial concentration of approx 20 g of glucose/L proved to be the best
Table 11.2 Microorganisms used for the production of various products from cassava bagasse
Organisms
Process
Product
Reference
Aspergillus niger LPB
21
A. niger NRRL 2001
A. niger CFTRI 30
Candida lipolytica
C. fimbriat
Kluyveromyces
marxianus
Pleurotus sajor-caju
Rhizopus arrahizus
R. ciricians
R. delemer
R. formosa
R. oligosporus
R. oryzae
R. oryzae
Lactobacillus casei
L. delbrueckii
L. delbrueckii
L. casei and
L. delbrueckii
Xanthomonas
campestris
SSF
Citric acid
Kolicheski et al. (1997)
SSF
SSF
SmF
SSF
SSF
Citric acid
Citric acid
Citric acid
Aroma compounds
Aroma compounds
Vandenberghe et al. (2000)
Shankaranand and Lonsane (1994)
Vandenberghe et al. (1998)
Bramorski et al. (1998a)
Medeiros (1998)
SSF
SmF
SmF
SmF
SmF
SmF
SmF
SSF
SSF
SsF
SSF
SsF
Mushroom
Fumaric acid
Fumaric acid
Fumaric acid
Fumaric acid
Fumaric acid
Fumaric acid
Aroma compounds
Lactic acid
Lactic acid
Lactic acid
Lactic acid
Barbosa et al. (1995)
Carta et al. (1999)
Carta et al. (1999)
Carta et al. (1999)
Carta et al. (1998, 1999)
Carta et al. (1999)
Carta et al. (1999)
Bramorski et al. (1998b)
Rojan et al. 2005
Anuradha et al. 1999
John et al. 2006a
John et al. 2006b
SmF
Xanthan gum
Woiciechowski et al. 2004
SSF: Solid-state Fermentation, SmF: Submerged Fermentation, SsF: Simultaneous Saccharification and Fermentation.
232
R.P. John
substrate concentration for xanthan gum production. Maximum xanthan gum (about
14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and
fermented for 72 h, and it remained almost the same until the end of fermentation (96 h).
11.3.5.2 Aroma Compounds
Bramorski et al. 1998a compared fruity aroma production by Ceratocystis fimbriat
in solid cultures from several agro-industrial wastes: cassava bagasse, apple pomace,
amaranthus and soya bean. Cassava bagasse was used in combination with soya
bean or apple pomace. Media containing cassava bagasse with apple pomace or soya
bean produced a strong fruity aroma. The components present in the headspace of
fermenter were acid, alcohols, aldehyde, ketones and esters. Bramorski et al. 1998b
also studied the production of volatile compounds by the edible fungus Rhizopus
oryzae during solid-state cultivation on tropical agro industrial substrates. When
R. oryzae was grown on a medium containing cassava bagasse plus soybean meal
(5:5, w/w), CO2 production rate was at its highest (200 mL/L), whereas the highest volatile metabolite production was with amaranth grain as the sole substrate
(282.8 mL/L). In the headspace, ethanol was the most abundant compound (more
than 80%). A strain of the yeast Kluyveromyces marxianus was used for the production of a fruity aroma in SSF and showed the feasibility of cassava bagasse as a
substrate (Medeiros 1998).
11.3.5.3 Biopigments
Now, people prefer natural colourants to synthetic colours. Some filamentous fungi,
like Monascus purpureous can be used to produce the GRAS level pigments. Carvalho et al. (2005) showed the possibility of producing pigments by Monascus sp.
with cassava bagasse. Good results were obtained with cassava bagasse. In this case,
growth and pigment extraction were higher comparing to that commonly obtained
with rice. An optimized medium composition was found, showing great perspectives
for this process.
11.3.5.4 Mushroom Culturing
Mushrooms are the rich source of nutrients and they can be produced by the utilization of many agro-residues. Barbosa et al. (1995) also compared cassava bagasse
and sugarcane bagasse for mushroom production. They used Pleurotus sajor-caju
and best results were obtained when cassava bagasse was used in a mixture with
sugarcane bagasse (8:2, dry weight basis).
11 Biotechnological Potentials of Cassava Bagasse
233
11.3.5.5 Citric Acid
Citric acid is a weak organic acid and is used as a natural preservative and is also
used to add sour taste to foods and drinks. Citrus fruits are rich source of citric
acid and were used in the industrial production of citric acid. In 1917, the American
food chemist James Currie discovered that certain strains of the mold Aspergillus
niger could be efficient citric acid producers and their microbial production replaced
the position of citrus fruits for the industrial production of citric acid. Kolicheski
et al. 1997 studied citric acid production on three cellulosic supports in SSF. Out
of the six strains of Aspergillus niger one, LPB 21, was selected for cultivation
on cassava bagasse, sugarcane bagasse and vegetable sponge. Cassava bagasse was
found to be a good substrate, giving 13.64 g citric acid per 100 g dry substrate. This
corresponded to 41.78% yield. Under improved fermentation conditions, the citric
acid production increased to 27 g/100 g dry substrate, which corresponded to 70%
yield (based on sugars consumed). Shankaranand and Lonsane (1994) presented a
comparative profile of citric acid production from various agro industrial residues,
such as cassava bagasse, wheat bran, rice bran, sugarcane pressmud, coffee husk,
etc., using an indigenous strain of A. niger CFTRI 30. Cassava bagasse gave the
highest yield of citric acid based on the total starch or sugars present initially in the
medium (Shankaranand and Lonsane 1994). Vandenberghe 2000 used three substrates, sugarcane bagasse, coffee husk and cassava bagasse for citric acid production with A. niger NRRL 2001. Cassava bagasse best supported the mould’s growth,
giving the highest yield of citric acid among the tested substrates. The citric acid
production reached a maximum (88 g/kg dry matter).
11.3.5.6 Fumaric Acid
Fumarate is an intermediate in the citric acid cycle formed by the oxidation of succinate by the enzyme succinate dehydrogenation. Fumaric acid is a food acidulent
and used in beverages. Carta et al. (1998, 1999) studied the prospects of production of fumaric acid from cassava bagasse. Submerged fermentation was carried out
using enzymatic hydrolysate of cassava bagassse nourished with different nitrogen
sources by Rhizopus strains. The strain Rhizopus formosa MUCL 28422 was found
to be the best fumaric acid producer, yielding 21.28 g/L in a medium containing
cassava bagasse hydrolysate as the sole carbon source. Moresi et al. (1992) studied
the production of fumaric acid by Rhizopus arrhizus from hydrolysates of corn,
cassava and potato starch.
11.3.5.7 Bioethanol
Ethanol can be used as a fuel, mainly as a biofuel alternative to gasoline, and is
widely used in cars in countries like Brazil. Because it is easy to manufacture and
process, and can be made from very common crops, such as sugar cane and maize,
it is an increasingly common alternative to gasoline in some parts of the world.
Agu et al. (1997) studied the combined effect of heat treatment and acid hydrol-
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R.P. John
ysis (various concentrations) on cassava grate waste (CGW) biomass for ethanol
production was investigated. Sixty percentage process efficiency was achieved
with 0.3 M H2 SO4 in hydrolysing the cellulose and lignin materials present in the
CGW biomass. From three litres of the CGW biomass hydrolysate obtained from
hydrolysis with 0.3 M H2 SO4 , ethanol yield was 3.5% (v/v) after yeast fermentation. However, although the process resulted in gainful utilization of CGW biomass,
additional costs would be required to effectively dispose new by-products generated
from CGW biomass processing. Improved alcohol production from cassava starch
residues was achieved by saccharification with multi-enzymatic preparation consisting of cellulase, D-xylanase, ␣-D-glucosidase, ␣-amylase, amyloglucosidase,
and pectinase. They got 29–36 mL alcohol/100g of sun-dried cassava starch residue
(Shamala and Sreekantiah 1986).
11.3.5.8 Natural Insecticide
The long residual action and toxicity of the chemical insecticides have brought about
serious environmental problems such as the emergence and spread of insecticide
resistance in many species of vectors, mammalian toxicity, and accumulation of pesticide residues in the food chain. Entomo-pathogenic Bacillus thuringiensis (Bt) and
Bacillus sphaericus (Bs) are two safe biological control agents. In the conventional
Bt production process, the cost of raw materials varied between 30 and 40% of the
total cost depending on the plant production capacity. Therefore, local production of
this insecticide in developing countries should depend on the use of production media made of cheap, locally available sources including agro-industrial by-products.
The cassava waste was proved as the potent carbon source for the production of
natural insecticide Bt (Ejiofore 1991).
11.3.5.9 Biotransformation of Cassava Bagasse for Feed or Food
Using Rhizopus sp., an edible fungus, the high content starch of cassava bagasse
biotransformed to food or feed by the successful improvement in the protein content from 1.67% to 12%. The optimized conditions were incubation temperature
28–32◦ C, inoculum size 105 spores/g dry cassava bagasse, initial moisture 70%
and pH 5.7–6.4. Tray fermenter with the substrate thickness of 6–8 cm was proved
best bioreactor during the scale up studies using different bioreactors (Soccol
et al. 1995a, b, c). The bio-transformed bagasse obtained showed an excellent sanitary condition and is perfectly inside the standards of the sanitary legislation, considering that cassava bagasse was bio-transformed without any thermal process of
sterilization, being only dehydrated at 60◦ C for about 24 h after fermentation. The
results showed no growth of undesirable bacteria such as Staphylococcus aureus,
Bacillus cereus, Salmonella and faecal coliforms. But there was the presence of
some moulds and yeasts in the bio-transformed one and which was very low from
the not bio-transformed one. Great possibilities were found for the biotransformation products obtained from cassava bagasse by Rhizopus showing some facilities
11 Biotechnological Potentials of Cassava Bagasse
235
and economical advantages such as very short time of fermentation (Soccol and
Vandenberghe 2003).
11.3.5.10 Lactic Acid
Lactic acid (2-hydroxypropionic acid), is an important organic acid having a prime
position due to its versatile applications in food, pharmaceutical, textile, leather
and chemical industries. The demand for lactic acid has been increasing considerably, owing to the promising applications of its polymer, the polylactic acid
an environment-friendly alternative to plastics derived from petrochemicals. There
have been numerous investigations on the development of biotechnological processes for lactic acid production, with the ultimate objective to enable the process to
be more effective and economical. Cassava bagasse starch was used as the carbon
source for the lactic acid fermentation by lactobacilli in submerged, solid-state, and
simultaneous saccharification and fermentation (Rojan et al. 2005; John et al. 2006a,
b, 2007b, c, d, 2008). Cassava bagasse was saccharified with amylase enzyme and
hydrolysate was used for the submerged and solid-state fermentation. It was proven
that cassava bagasse under simultaneous saccharification and fermentation is far better than the submerged or solid-state fermentations with respects to its productivity
and yield.
11.4 Conclusion
Biological conversion has an important role in waste utilization, and it is likely
that various food processing wastes may contain useful substrates, which can be
used for value added product production. The biotechnological production of these
products offers several advantages compared to chemical synthesis, like low cost
of substrates, high product specificity, low production temperature and low energy
consumption. Fermentation technologies are widely used in many environmentalfriendly and economic industrial sectors. Environmental pollution is no longer accepted as inevitable in technological societies. Over the past century, there has been
a tremendous increase in awareness of the effects of pollution, and public pressure
has influenced both industry and government. There is increasing demand to replace
traditional chemical processes with biotechnological processes involving microorganisms, which not only provide an economically viable alternative but are also
more environmentally friendly. The use of nutrient-rich renewable resources such
as cassava bagasse opens an avenue in a dual working manner for value addition
through an eco-friendly green technology.
Acknowledgments Rojan P. John thanks Council of Scientific and Industrial Research, New Delhi,
India for the Senior Research Fellowship.
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R.P. John
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Fungos do Genero Rhizopus em Fermentacão no Estado Solido. Tese Mestrado, Universidade
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alternative carbon source for citric acid production by Candida lipolytica. Paper presented in
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synthesis of citric acid by Aspergillus niger. Bioresour Technol 74: 175–178
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Appl Biochem Biotechnol 118: 305–312
Chapter 12
Sugarcane Bagasse
Binod Parameswaran
Contents
12.1
12.2
12.3
12.4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Processing of Sugarcane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Composition of Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biotechnological Potential of Sugarcane Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.1
Sugarcane Bagasse as Animal Feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2
Sugarcane Bagasse for the Production of Industrially Important Enzymes . .
12.5 Pre-treatment Methods for Sugarcane Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Advances in industrial biotechnology offer potential opportunities for
economic utilization of agro-industrial residues. Sugarcane bagasse is the major
by-product of the sugar cane industry. It contains about 50% cellulose, 25% hemicellulose and 25% lignin. Due to its abundant availability, it can serve as an ideal
substrate for microbial processes for the production of value-added products such as
protein enriched animal feed, enzymes, amino acids, organic acids and compounds
of pharmaceutical importance etc. Since untreated bagasse is degraded very slowly
by micro-organisms, a pre-treatment step may be useful for improved substrate
utilization. This chapter reviews the developments on processes and products developed for the value-addition of sugarcane bagasse through the biotechnological
means and it also discuss about various pre-treatment methods for efficient utilization of this substrate for the production of fermentable sugars.
Keywords Sugarcane bagasse · Industrial enzymes · Value-added products ·
Bioethanol · Bioplastics
B. Parameswaran (B)
Bioenergy Research Centre, Korea Institute of Energy Research (KIER), Yusong, Daejon 305-343,
Republic of Korea
e-mail: binodkannur@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 12,
C Springer Science+Business Media B.V. 2009
239
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B. Parameswaran
12.1 Introduction
Sugarcane is the common name of a species of herb belonging to the grass family.
The official classification of sugarcane is Saccharum officinaru , and it belongs to
the family Gramineae. It is common in tropical and subtropical countries throughout the world. It can grow from eight to twenty feet tall, and is generally about 2
inches thick. Several different horticultural varieties are known, and they differ by
their stem color and length. About 200 countries cultivate this crop and Brazil is
the world’s largest sugar cane producer, responsible for around 25 percent of total
world production, followed by India, Pakistan, China and Thailand. India is second
largest producer of sugar in the world. In India there are about 571 sugar mills which
produce a total quantity of 19.2 million tones (MT). Uses of sugar cane include the
production of sugar, Falernum, molasses, rum, soda, cachaça (the national spirit of
Brazil) and ethanol for fuel.
12.2 Processing of Sugarcane
Sugar processing begins when the cane plant arrives at the sugar mill. Rotating
knives, shredders, and crushers extract the juice from the cane. Heating the juice
evaporates off excess water and condenses the juice into thick syrup. Sugar granules
act as seed crystals when they are added to the syrup, making the dissolved sugar in
the syrup crystalize. When as much sugar as possible has crystallized in the syrup,
the mix is spun in a centrifuge, which separates the remaining syrup (now called
molasses) from the raw sugar crystals. The fibrous residue of cane stalk left over
after the crushing and extraction of juice from the sugar cane is called bagasse.
12.3 Composition of Bagasse
Bagasse consists of approximately 50% cellulose and 25% each of hemicellulose
and lignin. Chemically, bagasse contains about 50% ␣-cellulose, 30% pentosans and
2.4% ash. Because of its low ash content, bagasse offers numerous advantages for
usage in bioconversion processes using microbial cultures. Also, in comparison to
other agricultural residues, bagasse can be considered as a rich solar energy reservoir
due to its high yields (about 80 tonnes per hectare in comparison to about 1, 2 and
20 tonnes per hectare for wheat, other grasses and trees, respectively) and annual
regeneration capacity (Pandey et al. 2000).
12.4 Biotechnological Potential of Sugarcane Bagasse
Sugar cane bagasse is a lignocellulosic material providing an abundant and renewable energy source. It is one of the largest cellulosic agro-industrial by-products.
Several processes and products have been reported that utilize sugarcane bagasse
12 Sugarcane Bagasse
241
raw material. These include electricity generation, pulp and paper production, and
products based on fermentation. The various products, which have been obtained
from the processes involving bagasse include chemicals and metabolites such as alcohol and alkaloids, mushrooms, protein enriched animal feed (single cell protein),
and enzymes etc. One of the significant applications of the bagasse is the production
of protein-enriched cattle feed and enzymes. Bagasse could also been used for the
production of industrially important enzymes and biofuel.
12.4.1 Sugarcane Bagasse as Animal Feed
Bagasse has most commonly been used for the production of protein enriched
animal feed. It can be used as a substrate for solid state fermentation (SSF) for
animal feed production. Zadrazil and Puniya (1995) differentiated bagasse into four
fractions of particle size (>1 mm, 1–3 mm, 3–5 mm and 5–10 mm) with a view to
enhancing its nutritive value as animal feed. They found a varying degree of degradation by white-rot fungi and also variation in in vitro rumen digestions. It was
concluded that the mechanical separation of a substrate into different particle size
could be useful if it was utilized as a substrate to be fermented by filamentous fungi
to produce animal feed. Puniya et al. (1996) subjected bagasse to SSF using a strain
of Pleurotus sajor-caju in a closed system, with the aim of optimising the gaseous
atmosphere and developing a cost-effective and simple technology for animal feed
production. A patent was obtained on the application of bagasse, softened with alkali treatment, for feedstuff, fertilizer, and sweetener by cultivating Enterococcus
faecium in SSF (Iritani et al. 1995).
12.4.2 Sugarcane Bagasse for the Production of Industrially
Important Enzymes
Sugarcane bagasse has been used for the production of various industrially important enzymes. Many microorganisms, including filamentous fungi, yeasts and
bacteria, have been cultivated on this material in fermentation processes.
12.4.2.1 Cellulase
Amongst the various enzymes produced in SSF of bagasse, cellulases have most
extensively been studied. It is well established that the hydrolysis of the lignocellulosic residues using enzyme largely depends upon the cost of the production
of cellulases. Application of bagasse in SSF for this purpose appears attractive.
Sharma et al. (1991, 1995) reported production of cellulases from different fungal
strains. Roussos et al. (1992) used a mixture of bagasse and wheat bran (4:1) for the
production of cellulases. They suggested hydraulic pressing as a good technique to
leach out the enzymes from the fermented matter. Modi et al. (1994) reported higher
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B. Parameswaran
yields of cellulase from a strain of Streptomyces sp. HM29 when grown on bagasse
in comparison to rice straw, rye straw and corncobs. Yields were comparable with
those obtained from rice bran but lower than those from wheat straw, wheat bran
and newspaper.
Often, cultivation of two different strains as mixed culture and pre-treatment of
bagasse showed desirable impact on fermentation. Gupte and Madamwar (1997)
reported that production of cellulolytic enzymes under SSF by co-culturing of two
fungal strains showed improved hydrolytic and -glucosidase activities as compared
to the occasions when they were used separately. Alkali pre-treatment improved
the enzyme production (Gupte and Madamwar 1994). Similarly, Gutierrez-Correa
and Tengerdy (1997) also reported higher cellulase productivity in co-culturing
of a basidiomycete strain with another filamentous fungi. A mutual synergism
was observed between the parent strain (of Trichoderma reesei LM-UC4) and the
Aspergillus phoenicis QM 329, resulting in enhanced combined biomass production and corresponding increase in cellulase, endo-glucanase and -glucosidase
activities. When co-culturing was carried out using a mutant strain of T. reesei
LM-UC4E1, such synergism was absent, suggesting that in the hypermutation the
ability for co-operative interaction with other microbes was lost. Treatment of
bagasse with ammonia (80%, w/w moisture content) resulted higher enzyme productivity (Duenas et al. 1995).
When Trichoderma reesei LM-UC4 and its hypercellulolytic mutant LM-UC4E1
were co-cultured with Aspergillus niger ATCC 10864 in solid substrate fermentation on alkali-treated sugar cane bagasse for cellulolytic enzyme production,
the mutant strain was more responsive to mixed culturing than the parent strain
(Gutierrez-Correa et al. 1999). Bagasse was supplemented with either soymeal or
with ammonium sulfate and urea, and fermented at 80% moisture content and
30 degrees C. Mixed culturing produced better results with the inorganic supplement. Their study shows that mixed culturing is beneficial for the economic production of cellulases on nutritionally poor agricultural residues, without the need for
supplementation with expensive organic supplements.
12.4.2.2 Xylanase
Xylanase has been another enzyme produced in SSF of bagasse. Xylanases are
typically important enzymes for the degradation of plant materials (hemicellulose,
which comprises mainly of xylan). Xylans are formed mainly by heteropolysaccharide of a chain of -1,4 xylanopyranose units highly substituted by acetyl, arabinosyl and glucopiranosyl residues. Most of the commercially available xylanases
are being produced from fungi which are active at neutral or acidic pH and their
optimum temperature for activity is below 45◦ C. Thermophilic xylanases, which
are active at alkaline conditions, have great potential for industrial applications.
Jain (1995) used a thermophilic fungus for the production of extra-cellular xylanase
on various agro-residues, including bagasse. Fungus grew well on untreated bagasse
and enzyme titres were lower when fungus was grown on treated (alkali or acid
chlorite treatment) bagasse. Acetyl esterase was produced concurrently, maximal
12 Sugarcane Bagasse
243
activity being with bagasse in comparison to other substrates. Gutierrez-Correa and
Tengerdy (1998) also performed xylanase production in SSF using bagasse. They
co-cultured T. reesei and A. niger or A. phoenicis and achieved high xylanase titres
(2,600–2,800 IU/g dry wt). Sugar cane bagasse was chemically treated to generate
different bagasse samples with varying quantities of lignin and hemicellulose, keeping the cellulose fraction intact. These bagasse samples were evaluated for the production of cellulase and xylanases enzymes by Penicillium janthinellum NCIM 1171
and Trichoderma viride NCIM 1051 in the production medium (Adsul et al. 2004).
12.4.2.3 Amylase
Studies by Rajagopalan and Krishnan (2008) showed that sugar cane bagasse hydrolysate (SBH) can be used for ␣-amylase production. Utilization of sugar cane
bagasse has not been possible for ␣-amylase production by Bacillus sp. and there is
no previous report for the production of ␣-amylase from Bacillus sp. in submerged
or solid state fermentation. This is due to the fact that hydrolysis of sugar cane
bagasse forms simple sugars primarily glucose, xylose and arabinose that repress
␣-amylase synthesis through catabolic repression. A new isolate of Bacillus subtilis
KCC103 showed absence of repression by glucose during ␣-amylase synthesis. The
level of ␣-amylase produced in sugar cane bagasse hydrolysate medium was equivalent to that in starch medium, therefore replacement of starch by SBH in production
medium is highly feasible to produce ␣-amylase at low cost.
12.4.2.4 Inulinase
Optimization of inulinase production by Kluyveromyces marxianus NRRL Y-7571
using sugarcane bagasse as substrate was studied by Marcio et al. (2006). The best
fermentation conditions found after optimization was 36◦ C and 20% of corn steep
liquor, which yielded about 390 Ug−1 . Maximum productivity was 3.34 Ug−1 h−1 ,
the highest reported in literature to date. Sugarcane bagasse seems to present a great
nutritional potential for growth of K. marxianus NRRL Y-7571 and production of
inulinase.
12.4.2.5 Lipase
The use of solid state fermentation for the production of thermostable lipases is
an interesting alternative to the valorization of bagasse and olive oil cake. Lipase
production could be optimized by adding the appropriate precursors found in olive
oil cake. Olive oil cake and sugar cane bagasse were used for lipase production
using thermostable fungal cultures of Rhizomucor pusillus and Rhizopus rhizopodiformis by Cordova et al. (1998). The maximum production of lipase by Rhizomucor pusillus and Rhizopus rhizopodiformis in solid state fermentation using SCB,
was 4.99 Ug−1 DM equivalent to 1.73 Uml−1 and 2.67 Ug−1 DM equivalent to
0.97 Uml−1 , respectively. However, the mixture of olive oil cake and sugarcane
bagasse, 50% each, increased the lipase activity as high as 79.6 Ug−1 DM equivalent
244
B. Parameswaran
to 43.04 Uml−1 and 20.24 Ug−1 DM equivalent to 10.83 Uml−1 obtained by Rhizopus rhizopodiformis and Rhizomucor pusillus, respectively.
12.4.2.6 Other Value-Added Products
Nampoothiri and Pandey (1996) reported production of L-glutamic acid using sugarcane bagasse in which bagasse was impregnated with a medium containing glucose, urea, mineral salts and vitamins. Maximum yields (80-mg glutamic acid/g
dry bagasse) were obtained when bagasse of mixed particle size was fermented
with 85–90% moisture and 10% glucose. Impregnated bagasse was also used by
Hernandez et al. (1993) to grow a fungus culture for the production of ergot alkaloids. They used a total of sixteen different combinations of liquid media and concluded that there existed the possibilities of achieving tailor-made spectra of ergot
alkaloids by changing the liquid nutrient media composition used for impregnation.
Barrios-Gonzalez et al. (1993) studied the effect of particle size, packing density
and agitation on penicillin production in SSF using bagasse as inert substrate. The
use of a large particle size (14-mm) bagasse increased penicillin production by 37%.
Christen et al. (1994, 1997) reported production of fruity aroma on bagasse when it
was fermented with a nutritive medium containing glucose (200 g/L). Twenty-four
compounds were separated and twenty of them were identified from the headspace
analysis of the fermenter by GC. Aroma production was dependent on the growth
and the maximum aroma intensity was detected at about time of the maximum
respirometric activity.
SSF carried out on inert support materials, which differs from the process of
microbial growth on or in solid particles floating in a liquid medium has been
regarded as one of the future development of SSF systems (Aidoo et al. 1982,
Pandey 1991, 1992). The use of solid inert material impregnated with suitable liquid
media would provide homogenous aerobic conditions throughout the bioreactor and
the purity of the product would also be relatively high. Soccol et al. (1994) evaluated
potential of bagasse, impregnated with a liquid medium containing glucose and calcium carbonate, to be used as inert substrate, for lactic acid production from a strain
of Rhizopus oryzae NRRL 395. Keeping glucose level at 120 and 180 gL−1 for liquid
and solid state fermentation, an yield of 93.8 and 137.0 gL−1 of L(+)-lactic acid was
obtained, respectively. The productivity was 1.38 and 1.43 gL−1 h−1 in liquid and
solid fermentation, respectively.
Lactobacillus delbrueckii was able to grow in a solid support of sugarcane
bagasse and it effectively utilized the sugar available in the medium used for moistening the substrate (Rojan et al. 2006). Cassava hydrolysate prepared from the
bagasse was the sole carbon source used with minimum supplementation of ammonium salt and yeast extract. Under the optimised conditions, the strain produced
up to 249.1 mg lactic acid g−1 DM with a conversion efficiency of more than 99%
of the total sugar available to lactic acid. This process offers a cost effective, eco
friendly technology to scale up lactic acid production.
Citric acid was another organic acid, which was produced in SSF using bagasse
as inert carrier. Manonmani and Sreekantiah (1987) conducted citric acid production
12 Sugarcane Bagasse
245
using enzymatic hydrolyzate of alkali treated bagasse by SSF. The studies by
Kianoush and Alaleh (2008) showed that among all pre-treatment methods (acid,
alkali and urea) which had an important role in increasing citric acid productivity
from sugar cane bagasse, urea pre-treatment was the most influential support for
the acid production. Tosmani et al. (1997) compared gibberllic acid production in
submerged fermentation with SSF when latter showed excellent fungal growth.
Pectinases were produced in SSF using bagasse, impregnated with high glucose
concentration solution (Solis-Pereyra et al. 1996). The fermentation was carried out
in packed bed column fermenter for SSF. In a similar study, Huerta et al. (1994)
concluded that SSF carried out on inert substrates (they referred it as adsorbed
substrate fermentation technique) not only allowed the design of culture medium
to produce important metabolites, but also the study of fungal metabolism in artificially controlled SSF processes. Acuna-Argulles et al. (1994) studied the effect of
water activity on pectinases production using bagasse impregnated with a medium
containing pectin and sucrose. Ethylene glycol, sorbitol and glycerol were used as
water activity depressors. Results indicated that although polygalacturonase production decreased at low aw values, this activity was present at aw values as low as 0.90.
The specific activity increased up to 4.5-fold by reducing aw from 0.98 to 0.9.
Chiu and Chan (1992) described production of pigments using bagasse in roller
bottle cultures of Monascus purpurea. Fungus was cultivated in wet bagasse containing PGY medium with corn oil in SSF when it produced red and yellow pigments.
12.4.2.7 Bioethanol
Sugarcane bagasse is a potential lignocellulosic feedstock for ethanol production,
since it is cheap and readily available as an industrial waste product. Because of its
high carbohydrate content and relatively low lignin content, bagasse is particularly
appropriate substrate for bioconversion to ethanol. After glucan, xylan is the most
abundant carbohydrate in bagasse. Xylose can account for almost one-third of the
total sugar content in bagasse hydrolysates. Therefore, micro-organisms able to ferment both glucose and xylose are required for an efficient conversion of bagasse
to ethanol. The geometrical ratio and size of sugar cane bagasse fibres strongly influence the profile of lignocellulosic enzyme activity. The hemicellulose fraction of
sugar cane bagasse contains up to 35% of the total carbohydrate that can be readily
hydrolyzed to monomeric sugars by dilute sulfuric acid. However, the concentration of reducing sugar in hydrolysate is relatively low due to high liquid/solid ratio
during the acid hydrolysis (Cheng et al. 2008). Hydrolysis of bagasse by using dil.
sulfuric acid or hydrochloric acid at elevated temperature and pressure was studied
by Lavarack et al. (2002). In this technique, the elevated temperature softens the
lignin protective layer around the hemicellulose fibres and allows the aid to hydrolyse the hemicellulose to form polysaccharides and monosaccharides of mainly
xylose and arabinose. The potential of a genetically engineered xylose-utilising
Saccharomyces cerevisiae strain for fermenting sugarcane bagasse enzymatic hydrolysates was demonstrated by Martin et al. (2002b).
246
B. Parameswaran
A process have been developed for the continuous production of xylitol from
hemicellulosic hydrolysate utilizing Candida guilliermondii cells immobilized onto
natural sugar cane bagasse fibres. Xylitol is an alternate high added-value sweetner
with anti-carcinogenic properties of great concern for both the food industry and the
biomedical sector (Diego et al. 2008).
12.4.2.8 Bioplastics
Studies by Jian and Heiko (2008) showed that sugarcane bagasse can be pretreated
in dilute acid solution under moderately severe conditions, releasing sugars and
other hydrolysates including volatile organic acids, furfurals and acid soluble lignin
and utilization of these hydrolysates by an aerobic bacterium, Ralstonia eutropha
for biosynthesis of value-added bioplastics, polyhydroxyalkanoates (PHAs). PHA
biopolyesters were synthesized and accumulated to 57 wt% of cell mass under
appropriate C/N ratios. Poly(3-hydroxybutyrate) was the predominant biopolyester
formed on the hydrolysates, but the cells could also synthesize co-polyesters that
exhibit high ductility. Table 12.1 shows microorganisms cultivated on sugarcane
bagasse and the products.
12.5 Pre-treatment Methods for Sugarcane Bagasse
Different pre-treatment methods for preparing bagasse enzymatic hydrolysates have
been investigated with focus on obtaining high sugar yields. The pre-treatment
methods include steam explosion (Martin et al. 2002a), liquid hot water pretreatment (Laser et al. 2002) and pre-treatments with peracetic acid (Teixeira et al.
1999) or with ammonia water (Kurakake et al. 2001). Martin et al. (2007) reported
wet oxidation as a pre-treatment method for enhancing the enzymatic convertibility of sugarcane bagasse. Wet oxidation (WO) is the process of treating material
with water and air or oxygen at temperatures above 120◦ C (McGinnis et al. 1983).
Two types of reactions occur during WO, a low-temperature hydrolytic reaction and
a high-temperature oxidative reaction. The advantages of WO as a pre-treatment
method for lignocellulose have been presented in the 1980s (McGinnis et al. 1983)
and have been confirmed in recent years at Risø National Laboratory (Varga et al.
2003). In a recent work, the enzymatic convertibility and the fermentability of
bagasse pretreated by WO at different pH values were investigated (Martin et al.
2006). The studies by Martin et al. (2007) revealed that wet oxidation is an appropriate method for fractionating sugarcane bagasse and for enhancing its enzymatic hydrolysis. Alkaline WO at 195◦ C during 15 min gave the best results, yielding a solid
material with nearly 70% cellulose content, a solubilisation of approximately 93%
of hemicelluloses and 50% of lignin, and an enzymatic convertibility of cellulose
of around 75%. Although acidic WO at 195◦ C for 15 min gave good fractionation
of bagasse, a significant part of the polysaccharides was lost due to degradation and
formation of by-products, mainly carboxylic acids, and the enzymatic convertibility
of the pretreated material was poor. From the study they found the tendency of WO
12 Sugarcane Bagasse
247
Table 12.1 Micro-organisms cultivated on sugarcane bagasse and the products
Product
Organism
Reference
Ethanol
Pachysolen tannophilus
Escherichia coli KO11,
Pichia stipitis
Pichia stipitis
Recombinant bacteria
Yeast
Yeast
Candida guilliermondii
Candida guilliermondii
Candida guilliermondii
Candida guilliermondii
Yeast
Candida guilliermondii
Candida guilliermondii
Polyporus sp.
Aspergillus niger
Cellulomonas flavi ena and
Xanthomonas sp
white-rot fungus
Candida tropicalis
Trichoderma reesei
Neurospora sitophila
Ceratocystis fimbriat
Piromyces sp.
Aspergillus niger
Trichoderma reesei
white-rot fungi
Aspergillus niger
Aspergillus ellipticus and
Aspergillus fumigatus
Trichoderma harzianum
Cellulomonas flavi ena:
Penicillium janthinellum
Piromyces sp.
Melanocarpus albomyces
Cellulomonas flavi ena
Flammulina velutipes and
Trametes versicolor
Gibberella fujikuroi
Monascus purpurea
Brevibacterium sp.
Claviceps purpurea
Rhizopus oryzae
Lactobacillus delbrueckii
Aspergillus niger
Cheng et al. 2008
Caroline et al. 2000
Roberto et al. 1991
Vanzyl et al. 1991
Katzen and Fowler 1994
Gong et al. 1993
Navarro et al. 1982
Diego et al. 2008
Carvalho et al. 2005
Gurgel et al. 1995
Roberto et al. 1995
Dominguez et al. 1996
Felipe et al. 1996
Alves et al. 1998
Nigam et al. 1987
Zayed and Mostafa 1992
Rodriguez-Vazquez and
Diazcervantes (1994)
Elsayed et al. 1994
Pessoa et al. 1996
Aiello et al. 1996
MooYoung et al. 1993
Christen et al. 1994
Teunissen et al. 1992
Ray et al. 1993
Aiello et al. 1996
Breccia et al. 1997
Ray et al. 1993
Gupte and
Madamwar 1994, 1997
Roussos et al. 1992
Mayorga-Reyes et al. 2002
Milagres et al. 1993
Teunissen et al. 1992
Jain 1995
Perezavalos et al. 1996
Pal et al. 1995
Xylitol
SCP/protein enriched feed
Mycoprotein
Aroma
Cellulases
Xylanases
Laccase
Gibberllic acid
Pigments
Glutamic acid
Ergot alkaloids
Lactic acid
Citric acid
Pectinases
Bioplastics
Aspergillus niger
Aspergillus niger
Ralstonia eutropha
Tosmani et al. 1997
Chiu and Chan 1992
Nampoothiri and Pandey 1996
Hernandez et al. 1993
Soccol et al. 1994
Rojan et al. 2006
Manonmani and
Sreekantiah 1987
Huerta et al. 1994
Acuna-Arguelles et al. 1994
Jian and Heiko 2008
248
B. Parameswaran
to catalyze the transfer of hemicellulose from the solid phase to the liquid phase
without a major hydrolysis of the solubilized hemicellulose molecules. It was also
reported that more xylose was formed by WO and more glucose by steam explosion (Martin et al. 2008). According to McGinnis et al. (1983) monosaccharide are
oxidized to carboxylic acids by WO, while oligosaccharides are more resistant to
oxidation due to the stability of the glycosidic linkages. Therefore sugar oligomers
will be present in the wet oxidation filtrate.
12.6 Conclusions
It can be concluded that bioconversion of bagasse could be economically advantageous for the production of enzymes, animal feed, bioethanol and bioplastics. Since
untreated bagasse is degraded very slowly by micro-organisms, a pre-treatment step
may be useful for improved substrate utilization. Evidently, additional research on
the pre-treatment of bagasse is required to improve components yield and cellulose
digestibility to the extent which would make its use economically viable. Similarly, although many efforts have been made on sugarcane bagasse hydrolysis using
pre-treatment methods as well as enzymatic saccharification, its effective conversion into bioethanol is an area which needs further inputs in terms of research and
development.
Abbreviations
SSF: Solid State Fermentation
PHA: polyhydroxyalkanoates
WO: Wet Oxidation
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Chapter 13
Edible Oil Cakes
Swetha Sivaramakrishnan and Dhanya Gangadharan
Contents
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2 Chemical and Nutrient Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3 Applications of Edible Oil Cakes for Bioprocesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.1 Production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.2 Production of Media Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.3 Production of Secondary Microbial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.4 Production of Mushrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.5 Biological Detoxification of Oil Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.6 Bioconversion of Oil Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3.7 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254
255
259
259
260
263
264
264
266
266
267
267
Abstract The very sustainability of the growing bioprocess industry depends on the
progressive reduction of expensive nutrient inputs into fermentation media. The use
of cheap agricultural and food-processing by-products such as oil cakes, as feedstock is highly favored so as to improve the commercial feasibility of bioprocess
technology. Due to stringent nutritional requirements of these edible oil cakes as
animal feed, there is considerable interest in using them as substrates in the fermentation industry. This chapter will provide an impetus to further research in this area
enabling better utilization of edible oil cakes as sources of protein and carbohydrates
for economic viability of the bioprocess industry.
Keywords Edible oil cakes · Agro-residues · Fermentation · Enzyme production ·
Mushroom production
S. Sivaramakrishnan (B)
Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST),
CSIR, Trivandrum 695 019, Kerala, India
e-mail: swsj79@yahoo.co.in
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 13,
C Springer Science+Business Media B.V. 2009
253
254
S. Sivaramakrishnan and D. Gangadharan
13.1 Introduction
Fermentation medium plays a major role in the commercialization of a bioprocess
in terms of cost, high product yield and efficient recovery. This necessitates the
formulation of a medium developed from cheap agro-industrial by-products which
has to provide nutritional conditions necessary for growth and synthesis of the
product. Various crop-residues such as bran, straw, bagasse, molasses and oil cakes
are being evaluated in terms of efficiency and economics in bioprocesses for the
production of chemicals and value added products such as enzymes, amino acids,
organic acids, surfactants, pigments, flavors, bioactive products etc. For the past
two decades, significant attention is being given to the utilization of oil cakes in
bioprocess and related industries as feedstock because of its high nutritional value,
year-round availability and competitive pricing.
Oil cakes/meals are solid residues obtained after the extraction of oil from the
plant part such as seed, by expelling or solvent extraction. Those cakes resulting
from edible oil-bearing seeds which are being used to meet a part of the nutritional
requirements of either animal-feed or of human consumption are called as edible
oil cakes and those which cannot be used as feed stuff due to the presence of
toxic compounds and other impurities are differentiated as non-edible (Mitra and
Misra 1967). Most of the non edible oil cakes such as from neem, castor, mahua,
karanja are used as manures. The world oil cake market is dominated by eight major
edible oil cakes such as soybean cake, rapeseed cake, cottonseed cake, groundnut
cake, sunflower cake, groundnut cake, copra cake and linseed cake. Among these
soybean cake occupies 54% of the total production volume of the eight cakes followed by rapeseed cake (10%) and cottonseed cake (10%). According to Food and
Agricultural Policy Research Institute (FAPRI) the world oil cake production and
consumption is expected to have a dynamic growth of 2.3% per annum over the
forecast period 2006–2015. The annual production of major oil cakes in the world
for the year 2006–2007 according to Food and Agricultural Organisation (FAO) is
given in the Table 13.1.
Table 13.1 World production of major oil cakes for the year 2006–2007
Oil cake
Production in
2006/07 (Megatons)
Plant source
Plant part
Soybean oil cake
Rapeseed/mustard/canola oil cake
Cotton seed oil cake
Groundnut/peanut oil cake
Sunflower oil cake
Palm kernel oil cake
Copra cake (coconut oil cake)
Linseed/flax oil cake
Safflower oil cake
Sesame/gingelly oil cake
236
47.0
44.5
33.8
29.7
10.0
4.9
1.1
0.3
1.2
Glycine max
Brassica napus
Gossypium
Arachis hypogaea
Helianthus annuus
Elaeis guineensis
Cocos nucifera
Linus usitatissimum
Carthamus tinctorius
Sesamum indicum
seed
seed
seed
seed
seed
kernel
endocarp
seed
seed
seed
13 Edible Oil Cakes
255
13.2 Chemical and Nutrient Composition
The utility of any feed stock depends on its chemical and nutrient composition.
As these oil cakes are the preferred protein sources for livestock rations they have
been a subject of extensive investigation since the oil-production industry started a
century ago. They have been thoroughly examined for use as feedstuff for almost
all economically important species of animals. Even though the feed-conversion is
high, feeding requirements are critical for acceptable production in case of animal
feed (Schumacher 1993). The presence of anti-nutrients is a major constraint to
the practical use of several oil cakes as animal feed, making them less demanding.
Moreover pre-processing of meals for the removal of toxicity will further increase
the operational cost. But these limitations are not encountered in the fermentation
industry creating renewed interest in their efficient utilization and value addition.
The composition of an oil cake depends on the processing and mode of oil extraction from the oil-bearing source. All oilseeds require pre-processing such as
dehulling before oil extraction. Hulls are fibrous outer covering enclosing the seed
which needs to be removed for efficient oil extraction. The extent of dehulling varies
with different oilseeds. The extraction of oil from oilseeds is either by mechanical
pressing or by solvent extraction. The expeller pressed oil cake contains more residual oil than the desolventized cake. The chemical compositions of major oil cakes
(solvent extracted) in the world are given in the Table 13.2. Most of these oil cakes
Table 13.2 Chemical composition of major oil cakes
Oil cake
Soybean oil
cake
Rapeseed
oil cake
Cottonseed
oil cake
Groundnut
oil cake
Sunflower
oil cake
Palm kernel
oil cake
Sesame oil
cake
Linseed oil
cake
Safflower
oil cake
Copra cake
Olive oil
cake
DM
%
CP
%
Carbohydrate Crude
%
fiber %
Ash
%
Fat
%
90.3
51.8
23.6
17.8
7.3
0.9
90.75
42.8
32.2
12.1
7
4.1
91.53
41.5
27.0
14.67
6.46
5.75
90
45.6
14.1
8.3
5.02
2.47
93
35.6
23.0
28.41
7.36
1.68
93
17.5
45.5
11.9
4.8
7.4
93.9
48.2
21.0
6.4
12.6
2.3
88.9
33.2
36.0
8.1
5.4
2.8
93.1
44
20.1
12.1
7.2
5.9
89.9
72.8
20.9
4.77
42.4
10.1
11.5
49.14
5.5
2.36
8.0
8.72
DM- dry matter, CP- crude protein
Reference
(Castro
et al. 2007)
(Bell 1984)
(Briggs and
Heller 1942)
(Batal et al. 2005)
(Villamide and
San Juan 1998)
(Carvalho
et al. 2006)
(Yamauci
et al. 2006)
(Loosli
et al. 1960)
(Lyon et al. 1979)
(Thampan 1975)
(Vlyssides
et al. 2004)
256
S. Sivaramakrishnan and D. Gangadharan
are rich in protein content, the highest for soybean cake and the lowest for olive
oil cake.
Soybean cake is a rich source of protein and energy with lower fiber content than
most other oil cakes. They are widely used as feed ingredients both for animal and
human food products owing to their high digestibility and palatability. Methionine
was found to be the major limiting amino acid in soybean cake while threonine,
valine and lysine were found to be marginal in a study conducted on chick performance (Smith 1986). Carbohydrates in soybeans are largely present as oligosaccharides (15%) such as sucrose, raffinose and stachyose. Low levels of anti-nutrients
such as saponins, lectins and phytates were reported. The comparative amino acid
compositions of important oilseed meals are presented in Table 13.3. Being highly
produced and priced meal in oil-cake market, soybean cake is followed by rapeseed
cake in production. The cultivated rapeseed types are represented by two species
such as Brassica napus and B. campestris. Rapeseed oil cake is comparable well
with that of soybean cake in amino acid balance and is richer in sulphur amino acids
such as methionine and cysteine. Carbohydrates in rapeseed cake are mainly pectins
(14.5%), cellulose, fuco-amyloid, arabinan and arabinogalactan. Its use in poultry
feeding is limited by the presence of anti-nutrients such as glucosinates, sinapine,
tannins, erucic acid and phytates. The cultivars of rapeseed (Brassica campestris)
which have low content of both erucic acid and glucosinates widely cultivated in
Canada are known as canola. Mustard is an oil seed crop as well as a condiment
belonging to the same genus of rapeseed. The genetic types Brassica juncea and
Brassica nigra are widely cultivated in India, the former for oil and the latter is used
only as a condiment and not pressed for oil. The chemical and amino acid composition of mustard oil cake compares closely with that of rapeseed cake, but contains
more glucosinates than rapeseed, although of different kinds (Achaya 1990).
Cotton seed oil cake comprises 45% of the seed and has high protein content. But
its utilization as a feed ingredient for non ruminants faces severe constraints due to
the presence of toxic metabolite gossypol, high fiber content and low lysine, cysteine and methionine levels. Cotton seed genetically devoid of gossypol-containing
glands has been developed and oil cake obtained from this variety is reported to have
immense potential as protein diet. Groundnut oil cake, a high protein content solid
residue is rich in arginine levels, but low in essential amino acids such as lysine.
Aflatoxins, toxic fungal metabolites from Aspergillus flavu group are frequent contaminants of these cakes and their presence has severe implications on animal performance. Sunflower oil cake is similar to that of cotton seed cake in composition
and is rich in sulphur amino acids, but remarkably low in lysine. It is mainly being
used for swine and poultry. Chlorogenic acid, a polyphenolic compound reported to
inhibit hydrolytic enzymes has an adverse effect on animal performance. No antinutritional factors such as tripsin inhibitors are known in sunflower oil cake and its
mineral content is satisfactory. Copra cake or coconut oil cake has a protein content
of about 20% with low concentrations of essential amino acids notably tryptophan,
lysine, methionine and histidine. Palm kernel cake is dry and gritty with high fiber
content. This reduces its suitability for monogastric animals. Like sunflower cake,
it is deficient in lysine and rich in sulphur containing amino acids. It has a better
AA %
SBOC
Gly
Ala
Pro
Val
Leu
Ile
Met
Phe
Tyr
Trp
Ser
Thr
Cys
Lys
His
Arg
Asp
Glu
Reference
2.05
2.15
2.05
2.12
3.81
2.08
0.69
2.44
1.81
0.77
2.57
1.98
0.69
2.99
1.26
3.64
–
–
(Castro
et al. 2007)
RSOC
a
2.58
1.69
1.31
2.09
2.85
1.63
0.73
1.64
0.48
–
1.98
1.92
0.5
2.45
1.21
2.5
2.1
2.14
(Bell 1984)
CSOCb
4.9
4.5
4.3
5.1
6.6
3.4
1.8
5.7
3.4
GOCa
–
1.76
1.96
1.87
2.89
1.54
0.52
2.27
1.64
–
0.45
4.7
1.71
3.6
1.17
1.2
0.64
4.4
1.54
2.8
1.1
11.5
5.04
10.1
4.96
21.7
8.36
(O’Mara
(Batal
et al. 1997) et al. 2005)
SFOCa
PKOCa
SOCa
LSOCa
SfOCa
COCa
OOCc
1.88
1.36
1.21
1.52
2.07
1.2
0.67
1.42
0.71
–
1.29
1.21
0.53
1.01
0.73
2.43
2.88
5.99
(Villamide
and San
Juan 1998)
0.82
–
–
0.93
1.11
0.62
0.3
0.73
0.38
0.17
0.55
0.2
0.2
0.59
0.29
2.18
–
–
(Sundu
et al. 2006)
1.04
0.96
0.79
0.98
1.44
0.77
0.6
0.94
0.67
0.28
0.99
0.76
0.38
0.58
0.52
2.58
1.75
4.15
(Ravindran
and
Blair 1992)
1.74
1.04
0.81
1.74
2.02
1.68
0.54
1.46
1.09
0.51
1.92
1.22
0.61
1.16
0.69
2.94
1.92
4.1
(Barbour
and
Sim 1991)
0.77
0.6
0.62
0.72
0.92
0.61
0.14
0.53
0.35
0.14
0.63
0.45
0.21
0.46
0.35
1.28
1.42
2.77
(Ravindran
and
Blair 1992)
0.89
0.81
0.71
0.89
1.21
0.6
0.37
0.81
0.46
–
0.96
0.66
0.24
0.4
0.41
1.96
1.62
3.64
(Creswell
and
Brooks 1971)
6.64
8.56
4.8
10.7
11.4
4.64
1.41
5.69
1.62
–
3.98
4.05
1.04
6.53
3.16
7.74
2.67
5.54
(Martı́n
et al. 2003)
13 Edible Oil Cakes
Table 13.3 Comparative amino acid composition of important oil cakes
a
a
- % of DM, b - g/ 100 g of total amino acids, c -g/100 g of total nitrogen, Soybean oil cake (SBOC), Rapeseed oil cake (RSOC), Cotton seed oil cake (CSOC),
Groundnut oil cake (GOC), Sunflower oil cake (SFOC), Palm kernel oil cake (PKOC), Sesame oil cake (SOC), Linseed oil cake (LSOC), Safflower oil cake
(SfOC), Coconut oil cake (COC), Olive oil cake (OOC)
257
258
S. Sivaramakrishnan and D. Gangadharan
amino acid index relative to coconut oil cake. The digestibility of the cake is low for
poultry and swine due to high fiber content (Ravindran and Blair 1992).
The nutrient composition of sesame oil cake is comparable to soybean cake with
an average protein content of 40% and fiber content of 8%. It’s an excellent source
of methioinine, cysteine and tryptophan but deficient in lysine. Even though sesame
cake is a rich source of minerals, its availability is lower due to the presence of
oxalates and phytates in the hull fraction of the seed. Removal of hull increases
mineral availabity and reduces fiber content. However complete dehulling is not
always possible due to small size of sesame seeds. Linseed cake comprises of
60–55% of flax seed with a protein content of 32%. But poor protein quality due
to lysine deficiency, presence of antipyridoxine factor linatin, cyanogenic glucoside
linamarin and indigestible mucilage adversely limits its nutritive value as animal
feed. The crude protein content of safflower oil cake ranges from 20–60%, the composition being dependent on dehulling. Partially dehulled seed cakes comprise of
40% crude protein and 15% crude fiber. Two phenolic glucosides 2-hydroxyarctiin
and matairesinol impart a bitter flavour to the cake (Ravindran and Blair 1992).
Crude olive oil cake has a very low protein content of 5–10%, high crude fiber
content (35–50%), phenolic content of 4.3% with high moisture content ranging
35–30%. The fibers comprise of hemicellulose (1.5%), cellulose (1.72%) and lignin
(2.75%) (Vlyssides et al. 2004).
India is known for the production of a wide variety of oil cakes from its native oil
seeds and other oleaginous plant sources. About 86 different types of minor oil cakes
are reported to be produced in India apart from the major oil cakes described earlier.
Production of various non conventional oleaginous sources and oil cakes are given in
Table 13.4. Castor cake, a minor oil cake is produced mainly by Brazil followed by
India and China. It contains 35% crude protein and 25% fiber. It contains three antinutritional compounds such as ricin (toxic protein), ricinine (alkaloid) and castor
bean allergen, a potent allergen. It is mainly used as a fertilizer. Kapok cake (Ceiba
pentandra) is reported to have low feeding value due to its high fiber content and
presence of tannins (Ravindran and Blair 1992). Among the minor oil cakes, water
melon oil cake and musk melon oil cake are rich in proteins, with negligible fiber
content and no anti-nutrients (Achaya 1990).
Table 13.4 Annual production of various non-conventional oil bearing materials and oil cakes in
India during 1983–1984 (Achaya 1990)
Oil bearing material
Plant source
Production of cake (Tonnes)
Rice bran
Neem seed
Karanja seed
Kusum seed
Mahua seed
Mango kernel
Tobacco seed
Spent coffee
Oryza sativa
Azadirachta indica
Pongamia pinnata
Schleichera oleosa
Madhuca indica
Mangifera indica
Nicotiana tabacum
Coffea arabica
1,212,522
62,600
7,895
2,866
55,480
2,507
1,246
224
13 Edible Oil Cakes
259
Table 13.5 Composition of non-conventional oil cakes from native Indian oil-seeds (Achaya 1990)
Oil cake
CP %
Crude fiber %
Ash %
Rice-bran
14
10
Tapioca seed
Maize germ
Tobacco seed
Rubber seed kernel
Jute seed
Spent coffee
Tea seed kernel
Neem kernel
50
18
30
25
22
16
8
21
6.5
10
17
5
19
22
30
28
3.5
3
10
9
7
3.7
2.5
9.5
Karanja kernel
34
6
3.5
Kusum kernel
Mahua kernel
22
16
10
8
5.3
6.3
Teak kernel
Tamarind kernel
Mango kernel
Water melon kernel
(dehulled)
Musk melon kernel
(dehulled)
60
19
6
51
4.5
1.1
4.5
4
66
3
4
12
3.4
3.6
9
9
Unusual constituents
High crude fibre and high sand &
silica (8%)
Cyanogenic glucosides
None
None
Cyanogenic glucosides
High crude fiber
High crude fiber
Saponins (10%)
High crude fiber, high ash, tannins
(1.5%)
Furanoflavones like karanjin,
pongamol & mucilage (13.5%).
None
Toxic saponins (6.8%), tannins
(1.2%)
High ash
–
Tannins
–
–
Presence of anti-nutritional factors, high fiber content and poor protein quality
precludes the utilization of these minor cakes as animal feed. The compositions of
various non-conventional oil cakes produced in India are given in the Table 13.5.
13.3 Applications of Edible Oil Cakes for Bioprocesses
The major challenge of the bioprocess industry lies in developing cost-and ecoefficient processes using renewable raw materials to deliver high value-added products at costs, acceptable to the general public. Oil seeds are second only to grain
crops in the supply of plant proteins for human and animal consumption. The global
oilseed production reached 417 million tonnes in 2006–2007 and the oil cake production was brought to 106 million tonnes (FAO). In view of increasing the utility
of renewable feedstock in bioprocesses, the exploration of potential usefulness of
oil-cakes is desirable.
13.3.1 Production of Enzymes
Enzymes are biological catalysts, the catalysts for cell metabolism. They are widely
occurred throughout the biological system, and complex network of reactions
brought about by enzymes are the basis for the continuity of living world that
260
S. Sivaramakrishnan and D. Gangadharan
evolved over millennia. They offer efficient biocatalytic conversion potentials to
technologies ranging from food industry to personal care industry. They are one of
the most important products obtained from microbial sources for human requirements. Enzymes are produced industrially either by submerged (SmF) or solid state
fermentation (SSF). Both bacteria and fungi are employed for the industrial production of enzymes. The production of various enzymes utilizing oil cakes either as
the major substrate or media supplement is enumerated in Table 13.6. The critical
analysis of literature shows that enzyme production by fungi is favored to bacteria
employing oil cakes under solid state fermentation. This is attributed to the morphology and physiology of these molds which enable them to penetrate and colonize solid substrates (Sivaramakrishnan et al. 2007). Aspergillus, Penicillium and
Rhizopus are the widely reported fungal species for enzyme production. Among
bacteria Bacillus sp has been most commonly used for enzyme production utilizing oil cakes. Oil cakes are used either as a single substrate or mixed with various
other substrates in different combinations for solid state fermentation. In the case
of submerged fermentation, oil cakes are supplemented either as carbon or nitrogen
source for enzyme production. The use of minor oil cakes such as babassu oil cake
and almond oil cake for enzyme production point out to the constant search in the
bioprocess industry to evaluate and valorize indigenous sustainable agro-resources.
13.3.2 Production of Media Supplements
The high cost of synthetic media has a negative impact on the development of studies
in the field of microbiology and fermentation and molecular biology in developing
countries. Efforts are being made to develop organic nitrogen supplements from oil
cakes equivalent to commercially available yeast extract, beef extract and peptone.
Phillipchuk and Jackson, 1979 compared the effect of acidic and enzymic digests
of rapeseed cake with commercial media such as Sabouraud dextrose broth and
mycological media in supporting the growth of Candida utilis. Acidic and trypsin
digests of rapeseed cake supplemented with dextrose gave growth responses equivalent to that on commercial media with the exception of the peptic digest of rapeseed
cake. Gupta et al. 2005 prepared growth medium from oil cakes such as linseed,
mustard and neem to study the growth and morphology of Catenaria anguillulae,
a nematicidal fungus. The oil cake media were prepared by boiling and filtering
5g of oil cake and 15g of agar to yield 0.5% of oil cake. These three media were
compared with commercially available media such as beef extract agar, Emerson
agar and YPSS agar. Optimal growth and morphology of the fungus was recorded
on linseed oil cake agar. Defatted cotton seed protein was used for the production
of protease by Entomophthora coronata in 6L fermenter (Jönsson 1968). Enzymic
digests and water soluble extracts of various oil cakes such as soybean cake, cottonseed cake were used as nitrogen sources in the commercial production of various
bioactive molecules. Soybean cakes are processed to yield an array of products such
as soy flour, soybean protein concentrate, soy protein isolate. Various commercial
media has been developed for micro-organisms utilizing mainly soybean cake such
as trypticase soy agar, soy peptone etc.
Utilization of oil cake
Enzyme
Micro-organism
Method
Magnitude
Reference
Soybean cake
Soybean cake
Soybean cake (1% w/w)
supplemented to spent brewing
grain
Soybean cake (3% w/v)
Soybean cake as nitrogen source
(1.5%w/v)
Soybean cake (4% w/v)
Canola cake
Groundnut oil cake + Wheat bran
(1:1 w/w)
Copra cake
Copra cake
Lipase
Protease
Alpha amylase
Penicillim simplicissimum
Penicillium sp.
Aspergillus oryzae NRRL 6270
SSF
SSF
SSF
Static flask
Static flask
Static flask
(Di Luccio et al. 2004)
(Germano et al. 2003)
(Francis et al. 2003)
Carboxy peptidase
Alkaline protease
Aspergilus saitoi
Bacillus sp. I-312
SmF
SmF
6L Fermenter
Shake flask
(Ichishima et al. 1973)
(Joo and Chang 2003)
Lipase
Phytase
Alpha amylase
SmF
SSF
SSF
Shake flask
Static flask
Static flask
Glucoamylase
Alpha amylase
Penicillium camembertii Thom PG
Aspergillus f cuum NRRL 3135
Bacillus amyloliquefaciens ATCC
23842
Aspergillus niger NCIM 1245
Aspergillus oryzae IFO 30103
SSF
SSF
Static flask
Static flask
Copra cake
Coconut oil cake
Phytase
Lipase
Mucor racemosus ATCC 46129
Candida rugosa
SSF
SSF
Static flask
Static flask
Coconut oil cake + Sesame oil
cake (1:1 w/w)
Sesame oil cake + Wheat bran
(1:3 w/w)
Sesame oil cake
Sesame oil cake
Phytase
Rhizopus oryzae NRRL 1891
SSF
Static flask
Lipase
Aspergillus niger MTCC 2594
SSF
Static flask
(Tan et al. 2004)
(Ebune et al. 1995)
(Gangadharan
et al. 2005)
(Pandey et al. 1995)
(Ramachandran
et al. 2004)
(Boger et al. 2003)
(Benjamin and
Pandey 1997)
(Ramachandran
et al. 2005)
(Mala et al. 2007)
Phytase
l-Glutaminase
SSF
SSF
Shake flask
Static flask
(Roopesh et al. 2006)
(Kashyap et al. 2002)
Sesame oil cake
Sunflower oil cake + Sugar beet
oil cake +Wheat bran (3:1:1)
Lipase
Alpha amylase
Mucor racemosus NRRL 1994
Zygosaccharomyces rouxii NRRL-Y
2547
Aspergillus niger MTCC 2594
Penicillium chrysogenum
SSF
SSF
Static flask
Static flask
(Kamini et al. 1998)
(Ertan et al. 2006)
13 Edible Oil Cakes
Table 13.6 Enzyme production employing oil cakes as nutrient source
261
262
Table 13.6 (continued)
Enzyme
Micro-organism
Method
Magnitude
Reference
Palm kernel oil cake
Palm kernel oil cake + Wheat bran
(2:1)
Palm kernel oil cake
Tannase
Xylanase
Aspergillus niger ATCC 16620
Aspergillus niger ATCC 6275
SSF
SSF
Static flask
Static flask
(Sabu et al. 2005)
(Prasertsan et al. 1997)
Alpha amylase
Bacillus licheniformis CUMC305
SmF
Shake flask
Mustard oil cake (2% w/v)
Lipase
Rhizopus rhizopodiformis
SSF
Olive oil cake + Sugarcane
bagasse (1:1 w/w)
Babassu oil cake
Almond oil cake
Lipase
Penicillium simplicissimum
SSF
Lipase
Lipase
Penicillium restrictum
Rhizopus oligosporus GCBR-3
SSF
SSF
Packed bed
column
reactor
Fixed bed
reactor
Static flask
Static flask
(Krishnan and
Chandra 1982)
(Cordova et al. 1998)
(Cavalcanti et al. 2005)
(Gombert et al. 1999)
(Haq et al. 2002)
S. Sivaramakrishnan and D. Gangadharan
Utilization of oil cake
13 Edible Oil Cakes
263
13.3.3 Production of Secondary Microbial Metabolites
Secondary metabolites of microbial origin such as antibiotics have significant role
in the development of humanity. The fermentation industry received its greatest
impetus for expansion with the advent of antibiotics as chemotherapeutic agents.
The requirement of antibiotics in huge numbers to combat bacterial diseases led
to the extensive research on producing them economically at the highest concentration with minimum energy input. Arun and Dharmalingam (1999) have reported
the production of daunorubicin, an antitumor antibiotic produced by Streptomyces
peucetius using 5% sesame oil cake as carbon source thus reducing the production cost by 96%. Soy peptone, water-soluble enzymatic digest of soybean cake
is reported to enhance the production of pactamycin, an antitumor antibiotic produced by Streptomyces pactum by two fold (Bhuyan 1962). Rifamycin, an antibiotic
used against Mycobacterium tuberculosis and M. leprae inhibits RNA synthesis by
binding to the -subunit of RNA polymerase. Significant improvement (4 fold) of
rifamycin production is acheived by optimization of media which comprised of 1%
peanut cake and 1% soybean cake as nitrogen source (Krishna et al. 1998).
Cotton seed cake (2%) was used as a nitrogen source for the production of
an antimicrobial agent called lomofungin produced by Streptomyces lomondensis
(Johnson and Dietz 1969). Bacitracin A, the predominating antibiotic belonging to
the group polypeptidic antibiotics is produced by Bacillus licheniformis. Different
processes for industrial production of bacitracin utilized soybean cake and cottonseed cake individually as nitrogen sources under submerged conditions. Matelová
and Břečka (1967) studied the influence of peanut cake (6%) as nitrogen source
and oxygen transfer in bacitracin biosynthesis to effect maximal production. Utilization of soybean cake extract (water soluble) as nitrogen source was reported to
trigger significant production of synnematin B (identical to cephalosporin N) by
Emericellopsis terricola var. glabra (Nara and Johnson 1959). Sarada and Sridhar (1998) reported significant enhancement in the production of cephamycin C
produced by Streptomyces clavuligerus LC 21 when sunflower oil cake (12.5%) was
supplemented to the medium. Cephamycin C belongs to the class of microbially
synthesized -lactam antibiotics, highly resistant to -lactamases. Melingimycin
is a potent broad-spectrum insecticide produced by Streptomyces nanchangensis.
It belongs to the family of milbemycins, which have a 16-membered macrocyclic
lactone. Soybean cake (1%) is supplemented as nitrogen source for the optimal production of this biocontrol agent (Zhuang and Chen 2006).
Biosurfactants are surface active compounds having a wide range of industrial
applications such as enhanced oil recovery, lubricants, bioremediation of pollutants,
food processing etc. The structures of these complex molecules include lipopeptides, glycolipids, polysaccharide protein complexes, fatty acids and phospholipids.
Optimal production of biosurfactant (glycolipid) by Bacillus megaterium was obtained in 3L laboratory scale fermenter when peanut oil cake (2%) was used as
carbon source (Thavasi et al. 2008). Carotenoids are important natural pigments
with a range of applications as colorants, feed supplements and neutraceuticals.
Lycopene is a red coloured intermediate of the ß-carotene biosynthetic pathway
264
S. Sivaramakrishnan and D. Gangadharan
and is an important dietary carotenoid. It is reported to inhibit the harmful effect
of ferric nitrilotriacetate on DNA in rats and prevents liver necrosis. López-Nieto
et al. 2004 reported the development of a semi-industrial process (800 L fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+)
and minus (−) strains. This process describes the critical requirement of soybean
cake (44g/L) as nitrogen source for optimal lycopene production. Mustard oil cake
(6%) in the presence of Mg2+ ions is reported to improve lactic acid production
ability of agar-gel immobilized Lactobacillus casei after 48 hours, when further
addition of the substrate (whey lactose) failed to maintain the process efficiency
(Tuli et al. 1985).
13.3.4 Production of Mushrooms
Mushrooms, the fruiting bodies of various Basidiomycetes are used as food and
as flavoring agents in soups and sauces. Pleurotus sajor-caju is a commercially
important mushroom grown on paddy straw. Supplementation of oil-cakes from
mustard, niger, sunflower, cottonseed and sobean as nitrogen source to the rice
straw substrate increased the mushroom yield by 50–100%. This also increased the
digestibility of rice straw by mushrooms resulting in a corresponding increase in the
amino acid and sugar content and significant decrease in cellulo-hemicellulosics.
Oil cake supplementation enhances the secretion of cellulases, hemicellulases and
laccases thereby decreasing the cellulose, hemicellulose and lignin content of rice
straw significantly (Bano et al. 1993). About 40% rice straw is left behind as spent
substrate with each harvest of the mushroom crop. In view of utilizing the large
quantities of spent rice straw, Shashirekha et al. 2002 investigated the evaluation of
the same supplemented with oil cakes for the production of Pleurotus sajor-kaju.
A twelve fold enhancement in mushroom yield was reported with cottonseed cake
(0.15% N levels) illustrating the ability of the cake to supplement necessary nutrients exhausted in the spent rice straw substrate. This research work discusses the
immense potential of oil cakes to support two mushroom harvests utilizing the same
substrate without limiting the production yield.
13.3.5 Biological Detoxification of Oil Cakes
Oil-cakes are mainly used as livestock feed worldwide and there is an increasing
trend, towards its utilization as feed for poultry, non-ruminants and aquaculture.
The presence of certain anti-nutrients in several oil cakes limit its usage as animal
feed. Table 13.7 enumerates the spectrum of anti-nutrients present in various oil
cakes and its effect on animals fed. Several methods are devised to inactivate or
remove toxic components. Physical treatments such as dehulling, heat treatments
(cooking, autoclaving, toasting) are largely used for detoxifying legume seeds. Toxic
substances are withdrawn when cooked in liquid medium. Autoclaving aims at de-
13 Edible Oil Cakes
265
Table 13.7 Principal antinutritional factors in oil cakes and its effect on fed-animals (Ravindran
and Blair 1992)
Oil cake
Antinutrient
Effect on fed-animals
Soybean
Rapeseed
raffinose , stachyose, phytate
glucosinates, sinapine, erucic acid,
tannins, phytates
Cottonseed
Gossypol
Groundnut
aflatoxins as contaminants
Sunflower
Palm kernel cake
Sesame
Linseed
Safflower
chlorogenic acid
high fiber content
oxalates, phytates
linatin, cyanogenic glucosides
phenolic glucosides
Castor
ricin, ricinine, castor bean allergen
Kapok
Tannins
Indigestibility to fish
Glucosinates are goitrogenic & toxic
to monogastrics and erucic acid
cause heart lesions in animals, toxic
to aquaculture
Inhibits hydrolytic enzymes and
reduces palatability
Potent carcinogens and highly toxic to
animals
Inhibit hydrolytic enzymes in poultry
Low palatability in monogastrics
Lowers mineral availability in animals
Toxic to poultry
Bitter flavor thereby lowering
palatability
Ricin, highly toxic protein, ricinine, an
alkaloid, reducing palatability
Lowers palatability and digestion
naturing biologically active proteins such as ricin, protease inhibitors, goitrogenic
factors etc. Chemical treatments include ammoniation, sodium carbonate treatment
(glucosinates), addition of ferrous sulphate (gossypol), supplementation of choline
or methionine (chlorogenic acid) etc (Delort-Laval 1993). These treatments are
highly discouraged as it drastically reduces the suitability of oil cake as animal feed.
Enzymatic and fermentative treatments to enhance the nutritional value of oil
cakes are being explored. The negative effect of phytates on mineral availability
and protein digestibility can be reduced by the addition of microbial phytase to
animal feeds. Fermentative treatment involves the use of micro-organisms for removing anti-nutrients and adding essential nutrients and amino acids. Removal of
unavailable carbohydrates, phytates and tannins is well within the capabilities of
this approach (Ravindran and Blair 1992).
Fermentation of sesame oil cake with Lactobacillus acidophilus completely removed phytates and reduced the tannin content to 50% increasing the suitability of
the oil cake as feed to rohu fingerlings (Labeo rohita) (Mukhopadhyay 2001). Phosphate excretion studies was investigated in chicks fed with soybean cake fermented
with Aspergillus usamii. Results indicated that fermentation improved phosphorus
bioavailability indicating the complete degradation of phytate and thereby reducing
phosphate excretion (Hirabatashi et al. 1998).
Detoxification of gossypol in fermented cottonseed cake using Diploidia (class:
Fungi imperfectii) was evaluated in growing pigs. Liver free and bound gossypol
levels were found to be significantly lower for fermented cottonseed cake fed
pigs, with no mortality when compared with control (50% mortality) (Kornegay
et al. 1972).
266
S. Sivaramakrishnan and D. Gangadharan
Mustard oil cake contains glucosinolates which gets subjected to endogenous
degradation by myrosinase contained in the seeds resulting in the production of toxic
breakdown metabolites such as nitriles, thiocynates and isothiocyanates. Solid state
fermentation of mustard oil cake by Aspergillus sp. NR 4201 resulted in complete
degradation of glucosinolates without any toxic breakdown product rendering the
oil cake for utilization as animal feed (Rakariyatham and Sakorn 2002).
In view of the value addition of palm kernel cake fermented with Rhizopus
stolonifer NAU 07 under solid state fermentation for the production of fructosyl
transferase, chemical and nutrient composition of spent substrate such as crude
protein, crude fiber, ash and lipid contents was studied. The protein content was
observed to increase by 33.3% while ash content decreased by 44.5% thereby improving the nutritional qualities of the oil cake (Lateef et al. 2008).
13.3.6 Bioconversion of Oil Cakes
Hem et al. (2008) reported the development of a rural self sufficient technology in
Republic of Guinea to utilize palm kernel oil cake, the only available agricultural
by-product to generate aquaculture feed for Tilapia (Oreochromis niloticus). Palm
kernel cake was moistened with water (1:2) and kept for fermentation in rectangular
iron tanks. The insect Hermetia illucens (black soldier fly), attracted by the odour
of fermented matter lays its eggs on the fermented matter generating black soldier
(BS) larvae. An inverse correlation was obtained between conversion rate (PKOC
kg/ larval biomass kg) on Y-axis and duration of culture time (days) on X-axis,
indicating efficient bioconversion from the oil-cake to larval biomass. These larvae
grown in the fermented medium are harvested after four weeks to use as feed stuff
along with rice bran (3:7) for the Tilapia fish.
13.3.7 Other Applications
Oil cakes have an array of roles ranging from being a food product to soil amendment. In Indonesia, peanut oil cake is used as a popular fermented food called
Ontjom (lontjom) (Beuchat 1974) and on the contrary it is also used as a promising
soil amendment promoting plant growth in India (Bhattacharya and Goswami 1987).
Olive oil cake has proved to be a good source of combustible gases such as methane.
Combustion efficiency of oil cake is widely investigated for its use as an energy
source (Abu-Qudais and Okasha 1996). Investigations on anaerobic treatment of
cotton seed cake revealed significant methane generation potential indicating it to
be a good source for biogas production (Isci and Demirer 2007). Catalytic steampyrolysis of cottonseed cake generated bio-oils, a high quality liquid fuel comparable with gasoline fraction of petroleum (Pütün et al. 2006).
Combinations of neem oil cake, mustard oil cake and castor oil cake were evaluated for their efficiency against plant parasitic nematodes and soil-inhabiting parasitic fungi infesting crops such as mungbean and chickpea. Application of these
oil cakes as soil amendment reduced the population of plant-parasitic nematodes,
13 Edible Oil Cakes
267
Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus brassicae,
Helicotylenchus indicus and the frequency of the pathogenic fungi Macrophomina phaseolina, Rhizoctonia solani, Phyllosticta phaseolina, Fusarium oxysporum
f. ciceri significantly, but increased the frequency of saprophytic fungi. Plant growth
parameters were observed to be improved by several fold and residual effects of oil
cakes were also noted in the subsequent crop, in the next growing season (Tiyagi
and Alam 1995).
13.4 Conclusion
Oil cakes being rich in proteins, carbohydrates and minerals find various applications in the bioprocess industry such as production of enzymes, secondary metabolites, biomass etc. It has been mainly recognized as an important nitrogen source in
many commercial fermentation studies. Available information indicated the need to
evaluate and refine the methods depicting the digestibility of these substrates and
availability of nutrients provided for micro-organisms. Research must be focused to
develop simple, low-cost and self-sustainable technologies with efficient utilization
of indigenous substrates promoting the social development of rural sectors.
Abbreviations
DM:
CM:
AA:
SBOC:
RSOC:
CSOC:
GOC:
SFOC:
PKOC:
SOC:
LSOC:
SfOC:
COC:
OOC:
FAO:
SSF:
SmF:
dry matter
crude matter
amino acid
soybean oil cake
rapeseed oil cake
cotton seed oil cake
groundnut oil cake
sunflower oil cake
palm kernel oil cake
sesame oil cake
linseed oil cake
Safflower oil cake
coconut oil cake
olive oil cake
Food and Agricultural Organisation
solid state fermentation
submerged fermentation
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Chapter 14
Biotechnological Potential of Fruit
Processing Industry Residues
Diomi Mamma, Evangelos Topakas, Christina Vafiadi
and Paul Christakopoulos
Contents
14.1 World Availability of Citrus, Apples and Grapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2 Composition of Fruit by-Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2.1
Citrus Residue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2.2
Apple Pomace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2.3
Grape Pomace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3 Biotechnological Applications of Fruit Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.1
Microbial Transformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.2
Enzymatic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
274
275
275
276
277
277
277
282
286
Abstract Fruit juices and derived products such as nectars and drinks have experienced growing popularity within the last years. Orange waste, apple pomace
and grape pomace are the solid by-products derived from processing of oranges,
apples and grapes, respectively. Due to increasing production, their disposal represents a growing problem since the plant material is usually prone to microbial
spoilage, thus limiting further exploitation. On the other hand, costs of drying,
storage and shipment of by-products are economically limiting factors. Therefore,
agro-industrial by-products are often utilized as feed or as fertilizer. The application
of agro-industrial by-products in bioprocesses offers a wide range of alternative substrates, thus helping to solve pollution problems related to their disposal. Attempts
have been made to use orange waste, apple pomace and grape pomace to generate
several value-added products through microbial transformations or enzymatic modifications, such as enzymes, bioethanol, organic acids, heteropolysaccharides, aroma
compounds, protein enriched feeds, prebiotic oligosaccharides and biologically active molecules.
P. Christakopoulos (B)
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of
Athens, 9 Iroon Polytechniou Str., Zografou Campus, 157 80, Zografou, Greece
e-mail: hristako@orfeas.chemeng.ntua.gr
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 14,
C Springer Science+Business Media B.V. 2009
273
274
D. Mamma et al.
Keywords Fruit processing industry residues · Biotechnological applications ·
Microbial transformations · Enzymatic modifications
14.1 World Availability of Citrus, Apples and Grapes
According to Food and Agriculture Organization (FAO) estimates the world citrus production for the year 2005 was 94.8 million MT (FAOSTAT-FAO Statistical
Database, 2005). The genus Citrus includes several important fruits (Bampidis and
Robinson 2006), with the most important on a worldwide basis being sweet orange
(C. sinensis: 61.1% of world citrus production), tangerine (C. reticulata: 19.9%),
lemon and lime (C. limon and C. aurantifolia: 12.1%) and grapefruit (C. paradisi:
5.0%). Minor citrus genuses that comprise the bulk of the remaining 2.0% include
sour orange (C. quarantium), shaddock (C. grandis) and citron (C. medica). Table 14.1 presents the major producing countries. About 20.6% of world production
of citrus is in the Mediterranean countries of Spain, Italy, Greece, Egypt, Turkey and
Morocco, with Brazil (20%), China (16%) and the USA (11%) being major individual citrus producing countries (Table 14.1). Approximately, 27 million MT of the
total citrus production for the year 2005 have been processed to yield juice, essential oils and other by-products (FAOSTAT-FAO Statistical Database, 2005). Citrus
by-products are the principal solid by-product of the citrus processing industry and
constitute about 50% of fresh fruit weight (Garzón and Hours 1992). According to
FAO the world citrus production for the year 2006 increased approximately 17%
(FAOSTAT-FAO Statistical Database, 2005).
World apples (Malus sp., Rosaceae) production accounts for approximately
54.2 million MT (FAOSTAT-FAO Statistical Database, 2005). Table 14.2 presents
the major producing countries. About 46% of world production of apples is in
China, followed by USA (8%) and Turkey (5%) (Table 14.2). Fruits that are not
suitable for consumption in natura are processed, generating large amounts of
residues.
Approximately 14 million MT of apples will be processed mainly for the production of juice, jelly and pulp. Apple pomace, the solid residue from juice production,
represents around 30% of the original fruit and is generated during fruit pressing
(Vendruscolo et al. 2008). US Department of Agriculture estimated an increase in
apples production for the year 2006 of about 5%. Grape, one of the world’s largest
fruit crop, with a reported annual production higher than 58 million MT (FAOSTATFAO Statistical Database, 2005), is cultivated mainly as Vitis vinifera. The economical importance of grapes and products obtained therefrom, such as wine, grape juice,
jams and raisins, is therefore obvious. The most important grape producers are Italy
(16%), France (12%), Spain (10%), and the USA (11%) (Table 14.2). About 80%
of the produced grape quantity is used in wine-making. Wine-making affords grape
pomace as a by-product in an estimated amount of 13% by weight of the grapes
(Pinelo et al. 2006).
14 Biotechnological Potential of Fruit Processing Industry Residues
275
Table 14.1 World citrus production (in thousand tons) (FAOSTAT-FAO Statistical Database, 2005)
Total
Northern Hemisphere
Algeria
542.7
China
15227.9
Cuba
216.0
Cyprus
178.7
Egypt
2706.3
Greece
861.0
India
4662.0
Indonesia
1311.7
Iran
3037.0
Israel
639.9
Italy
3320.9
Japan
1341.0
Korea Rep
594.0
Lebanon
339.0
Mexico
6910.0
Morocco
1320.9
Pakistan
504.5
Spain
6181.3
Tunisia
307.9
Turkey
2316.8
USA
10498.5
Southern Hemisphere
Argentina
2670.0
Australia
716.1
Brazil
18902.5
Chile
312.0
Colombia
330.0
Paraguay
289.6
Peru
754.0
South Africa
1543.0
Oranges
390.0
4462.0
200.0
69.5
1759.3
763.2
3100.0
1311.7
1900.0
184.2
2105.1
88.0
Tangerines
Lemon and limes
Grapefruits
111.0
8695.0
40.0
50.4
612.6
59.7
20.9
331.4
31.5
1420.0
1903.0
7.0
37.9
3.0
6.6
142.0
1100.0
68.0
597.4
264.9
6.8
83.0
1890.0
25.0
360.0
5.0
200.0
4300.0
827.0
122.8
611.6
1249.0
594.0
42.0
360.0
463.9
2835.4
174.7
1040.0
8419.1
2500.4
33.2
500.0
367.3
809.5
28.0
670.0
789.4
36.0
72.0
106.8
922.7
770.0
571.0
16565.0
430.0
1300.0
35.0
1000.0
170.0
170.0
15.1
67.5
16.4
220.0
180.0
43.3
47.0
250.0
205.7
315.0
1113.0
1270.0
172.0
14.2 Composition of Fruit by-Products
14.2.1 Citrus Residue
Citrus fruits are principally consumed by humans as fresh fruit or processed juice,
either fresh chilled or concentrated. After juice is extracted from the fruit, a residue
comprised of peel (flavedo and albedo), pulp (juice sac residue), rag (membranes
and cores) and seeds remained, which represents 50% of fresh citrus fruit weight
(Garzón and Hours 1992). The processing of citrus results in approximately 13.5
million MT solid by-products (FAOSTAT-FAO Statistical Database, 2005).
The composition of citrus fruit is affected by factors such as growing conditions, maturity, rootstock, variety and climate. Citrus fruits contain nitrogen, lipids,
sugars, acids, insoluble carbohydrates, enzymes, flavonoids, bitter principles, peel
oil, volatile constituents, pigments, vitamins and minerals. The content of citrus
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D. Mamma et al.
Table 14.2 World apples and grapes production (in thousand MT) (FAOSTAT-FAO Statistical
Database, 2005)
Country
Apples
Country
Grapes
Northern Hemisphere
China
Egypt
France
Germany
Hungary
India
Iran, Islamic Rep of
Italy
Japan
Korea
Poland
Russian Federation
Spain
Turkey
Ukraine
USA
25006.5
550.0
2123.0
1600.0
720.0
1470.0
2400.0
2194.9
870.0
669.0
2050.0
2050.0
797.7
2550.0
700.0
4254.3
Northern Hemisphere
China
Egypt
France
Germany
Greece
Hungary
India
Iran, Islamic Rep of
Italy
Moldova, Republic of
Portugal
Romania
Spain
Turkey
USA
Southern Hemisphere
5698.0
1300.0
6787.0
1122.0
1200.0
815.0
1200.0
2800.0
9256.8
600.0
1000.0
1027.6
5879.8
3650.0
6414.6
Southern Hemisphere
Argentina
Brazil
Chile
South Africa
1262.4
843.9
1350.0
778.6
Argentina
Australia
Brazil
Chile
South Africa
2365.0
1834.0
1208.7
2250.0
1700.0
by-products is influenced by factors that include the source of the fruit and type
of processing (Bampidis and Robinson 2006). Residues of citrus juice production
are a source of dried pulp and molasses, fiber-pectin, cold-pressed oils, essences,
D-limonene, juice pulps and pulp wash, ethanol, seed oil, pectin, limonoids and
flavonoids. Fiber-pectins may easily be recovered from lime peels and are characterized by high fiber contents. The main flavonoids found in citrus species are
hesperidin, narirutin, naringin and eriocitrin. Citrus seeds and peels were found to
possess high antioxidant activity (Schieber et al. 2001).
14.2.2 Apple Pomace
Apples that are not suitable for consumption in natura are processed, generating
large amounts of residues named apple pomace. The apple pomace is a heterogeneous mixture consisting of peel, core, seed, calyx, stem and soft tissue. It has high
water content and is mainly composed of insoluble carbohydrates such as cellulose,
hemicellulose and lignin. Simple sugars, such as glucose, fructose and sucrose, as
well as minerals, proteins, vitamins and polyphenols are part of apple pomace composition. The composition varies according to the apple variety used and the type
of processing applied for juice extraction, especially regarding how many times the
fruits are pressed (Vendruscolo et al. 2008).
14 Biotechnological Potential of Fruit Processing Industry Residues
277
Production of pectin is considered the most reasonable way of utilizing apple
pomace both from an economical and from an ecological point of view. In comparison to citrus pectins, apple pectins are characterized by superior gelling properties.
Apple pomace has been shown to be a good source of polyphenols which are predominantly localized in the peels and are extracted into the juice to a minor extent.
Major compounds isolated and identified include catechins, hydroxycinnamates,
phloretin glycosides, quercetin glycosides and procyanidins. Since some phenolic
constituents have been demonstrated to exhibit strong antioxidant activity in vitro,
commercial exploitation of apple pomace for the recovery of these compounds
seems promising (Schieber et al. 2001).
14.2.3 Grape Pomace
The major part of grapes production is used for wine-making and the major solid
by-product generated is grape pomace. Grape pomace consists of three different
components, seeds, stalks and skins. Taking into account that about 80% is used
in winemaking, approximately 10 million tons of grape pomace arise within a few
weeks during harvest. The chemical composition of grape pomace is rather complex: alcohols, acids, aldehydes, esters, pectins, polyphenols, mineral substances,
sugars etc. are the most represented classes of compounds (Ruberto et al. 2008).
One of the main environmental problems related to the management of the winery
and distillery residues is the generation of large amounts during a short period of
the year (August–October) (FAOSTAT-FAO Statistical Database, 2005), as well as
some polluting characteristics of these residues, such as low pH and a high content
of phytotoxic and antibacterial phenolic substances, which resist biological degradation. A great range of products such as ethanol, tartrates, citric acid, grape seed
oil, hydrocolloids, and dietary fiber are recovered from grape pomace (Schieber
et al. 2001). Anthocyanins, catechins, flavonol glycosides, phenolic acids and alcohols and stilbenes are the principal phenolic constituents of grape pomace (Pinelo
et al. 2006).
14.3 Biotechnological Applications of Fruit Wastes
14.3.1 Microbial Transformations
Bacteria, yeast, and fungi have been cultivated under both submerged (SmF) and
solid state fermentation (SSF) on orange waste, apple pomace and grape pomace
for different purposes. A synopsis of the major strategies for fruit waste utilization
for the microbial production of value-added products is following.
14.3.1.1 Enzymes
The most important area of citrus wastes and apple pomace utilization is the production of enzymes, especially pectinolytic ones. Pectinolytic enzymes or pectinases
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D. Mamma et al.
are a heterogeneous group of related enzymes that hydrolyze the pectic substances.
Pectinolytic enzymes are of significant importance in the current biotechnological
era with their all embracing applications in fruit juice extraction and its clarification,
scouring of cotton, degumming of plant fibers, waste water treatment, vegetable oil
extraction, tea and coffee fermentations, bleaching of paper, in poultry feed additives
and in the alcoholic beverages and food industries (Jayani et al. 2005).
Both SmF and SSF conditions were evaluated by several researchers for the production of the above mentioned enzymes using citrus wastes or apple pomace as
carbon sources. Bacteria, yeasts, and fungi under both SmF and SSF conditions were
able to produce pectinolytic enzymes using citrus wastes (Garzón and Hours 1992;
Fonseca and Said 1994; Ismail 1996; Kapoor et al. 2000; Martins et al. 2002; De
Gregorio et al. 2002; Dhillon et al. 2004; Mamma et al. 2008) or apple pomace
(Hang and Woodams 1994a; Berovic and Ostroversnik 1997; Pericin et al. 1999;
Zheng and Shetty 2000; Joshi et al. 2006) as carbon sources.
Apart from pectinolytic enzymes, several other enzymatic activities have been
produced on apple pomace namely β-fructofuranosidase (Hang and Woodams 1995),
xylanase (Villas-Bôas et al. 2002; Seyis and Aksoz 2005), β-glucosidase (Hang
and Woodams 1994b), manganese-dependent peroxidase and cellulase (Villas-Bôas
et al. 2002). Citrus waste on the other hand has been used for the production
of α-amylase, neutral and alkaline proteases (Mahmood et al. 1998), xylanase
(Ismail 1996; Seyis and Aksoz 2005; Mamma et al. 2008) and cellulase (Ismail 1996;
Mamma et al. 2008).
In literature there are a few reports on grape pomace utilization for the production
of enzymes, namely pectinase, cellulase and xylanase (Botella et al. 2005, 2007;
Dı́az et al. 2007) by different Aspergillus species.
14.3.1.2 Bioethanol
With the inevitable depletion of the world’s petroleum supply and due to increased
prices for oil, there has been an increasing worldwide interest in alternative, nonpetroleum-based sources of energy. Ethanol is one of the most important renewable
fuels contributing to the reduction of negative environmental impacts generated by
the worldwide utilization of fossil fuels (Cardona and Sánchez 2007). Application
of agro-industrial residues in bioethanol not only provides alternative substrates but
also helps solve their disposal problem.
Several researchers have successfully hydrolyzed citrus waste using commercial cellulase and pectinase enzymes to glucose, galactose, fructose, arabinose,
xylose, rhamnose, and galacturonic acid (Grohmann and Baldwin 1992; Grohmann
et al. 1994a, 1995a; Wilkins et al. 2007a). According to Grohmann et al. (1994a),
glucose, fructose and galactose from hydrolyzed citrus peel waste can be fermented to ethanol by Saccharomyces cerevisiae yeast. Galacturonic acid from pectin
hydrolysis, arabinose, and xylose as well as the sugars mentioned above can be
fermented by Escherichia coli K011 to produce ethanol and acetic acid (Grohmann
et al. 1994b, 1995b). E. coli KO11 is a recombinant bacterial strain developed to
ferment arabinose and xylose as well as hexoses to ethanol. However, in order to
14 Biotechnological Potential of Fruit Processing Industry Residues
279
ferment these sugars, orange peel oil concentration in the hydrolysate must be reduced prior to fermentation (Grohmann et al. 1994a). The inhibitory effect on yeast
growth due to orange peel oil and/or D-limonene, a monoterpene that makes up
more than 90% of citrus peel oils, has been observed by several researchers (Wilkins
et al. 2007b, c). Yields obtained by the previous researchers were quite promising
in using citrus wastes for bioethanol production.
It should be noted that due to the high amounts of citrus wastes available in
the US, researchers of the US Department of Agriculture worked with commercial enzymes to economically hydrolyze pectin, cellulose and hemicellulose from
citrus peel wastes. The goal was to optimize the process and develop a model refinery that would also extract marketable by products (Predd 2006). Florida Power
& Light Energy LLC (FPL Energy LCC) planned to develop a commercial scale
cellulosic ethanol plant that can produce ethanol using waste citrus peel as feedstocks (O’Sullivan and Stewart 2007), while the southeast Biofuels LLC subsidiary
has filed an application with the Florida Department of agriculture and Consumer
Services for a $500.000 grant in concerting citrus peel waste to ethanol (Ames
2008).
A SSF process for the production of ethanol from apple pomace by different strains of S. cerevisiae was described by several researchers (Ngadi and Correia 1992; Joshi and Sandhu 1996; Sandhu and Joshi 1997; Khosravi and Shojaosadati 2003). The results indicated that alcohol fermentation from apple pomace is
an efficient method to reduce waste disposal, with the concomitant production of
ethanol. Nogueira et al. (2005) evaluated the alcoholic fermentation of the aqueous
extract of apple pomace with satisfactory yields showing that it is a suitable substrate
for alcohol production.
Finally, grape pomace has been evaluated by Hang et al. (1986) as potentially
feedstock for bioethanol production under SSF by the naturally occurring yeast
flora. The yields obtained were 80% of the theoretical based on the fermentable
sugar consumed.
14.3.1.3 Organic Acids
Among the various products obtained through microbial cultivation on agroindustrial residues, organic acids are particularly important. The ratio of carboxylic
acids microbiologically manufactured in the bulk of biotechnological products is
very high. These compounds are valuable building blocks for chemical synthesis,
which can be used in several applications. Among organic acids, citric acid production has been well studied and reported. The amount of citric acid manufactured
annually exceeds 800000 MT, and its production is increasing at 5% a year. Citric
acid is widely used in several industrial processes, such as in the food and pharmaceutical industries. It is produced mainly by submerged fermentation using A. niger
or Candida sp. from different sources of carbohydrates, such as molasses and starch
based media (Vendruscolo et al. 2008).
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D. Mamma et al.
Several researchers have evaluated different types of citrus waste as carbon
source for citric acid producton under both SmF (Aravantinos-Zafiris et al. 1994;
Rivas et al. 2008) and SSF (Zhang 1988; Kang et al. 1989) conditions using different A. niger strains. Hang and Woodams (1985) used grape pomace as substrate for
citric acid production under SSF.
The use of apple pomace as substrate for the production of citric acid by A. niger
in SSF (column reactors) was reported by Shojaosadati and Babaeipour (2002).
Apple pomace has also been used for fatty acid production. Stredansky et al. (2000)
evaluated the γ -linolenic acid (GLA) production in Thamnidium elegans by SSF.
14.3.1.4 Heteropolysaccharides
Long-chain, high-molecular-mass polymers that dissolve or disperse in water to give
thickening or gelling properties are indispensable tools in food product formulation.
Such food polymers are also used for secondary effects, which include emulsification, stabilization, suspension of particulates, control of crystallization, inhibition
of syneresis, encapsulation, and film formation (Vuyst and Degeest 1999). Xanthan
gum is the most important microbial polysaccharide from the commercial point of
view, produced by bacteria of the genus Xanthomonas, with a worldwide production
of about 30000 tons per year. It has widespread commercial applications as a viscosity enhancer and stabilizer in the food, pharmaceutical and petrochemical industries
(Galindo 1994). The utilization of agro-industrial by-products for the production of
polysaccharides by microorganisms has many advantages, such as reducing production costs and recycling natural resources.
In studies of Bilanovic et al. (1994) and Green et al. (1994) four different fractions of citrus waste were compared as substrates for xanthan fermentation: whole
citrus waste, pectic, hemicellulosic and cellulosic extracts, in SmF. Stredansky and
Conti (1999) studied the SSF as an alternative strategy for the production of xanthan
by X. campestris. The choice was based on the observation that solid substrates
reproduce the natural habitat of this phytopathogenic bacterium. This technique
allows problems connected with broth viscosity to be overcome and utilizes cheap
substrates. The exopolysaccharide was produced on a number of agro-industrial
residues or by-products such as spent malt grains, apple pomace, grape pomace,
and citrus peels. With most of the substrates, the gum production was comparable
to those obtained with SmF (Stredansky and Conti 1999).
14.3.1.5 Aroma compounds
Most of the flavouring compounds are presently produced via chemical synthesis
or extraction from natural materials. However, recent market surveys have demonstrated that consumers prefer foodstuff that can be labelled as natural. Plants have
been major sources of essential oils and flavours but their use depends on natural
factors difficult to control, such as weather conditions and plant diseases. An alternative route for flavour synthesis is based on microbial biosynthesis or bioconversion
(Janssens et al. 1992). Several micro-organisms including bacteria and fungi, are
14 Biotechnological Potential of Fruit Processing Industry Residues
281
currently known for their ability to synthesise different aroma compounds. Fungi
from the genus Ceratocystis produce a large range of fruit-like or flower-like aromas
(peach, pineapple, banana, citrus and rose) depending on the strain and the culture
conditions (Christen et al. 1997; Meza et al. 1998). Among the genus, C. fimbriat
has a great potential for ester synthesis. It grows rapidly, has a good ability to sporulate and produces a wide variety of aromas.
Bromarski et al. (1998) evaluated the potential of several agro-industrial residues
such as cassava bagasse, apple pomace, amaranth and soybean using a strain of
C. fimbriat . All media supported fungal growth. While amaranth medium produced
pineapple aroma, the medium containing apple pomace produced a strong fruity
aroma. This same medium was used by Christen et al. (2000) for the production
of volatile compounds by Rhizopus strains. Authors found that the production of
volatile compounds was related mainly to the medium used, and no difference was
observed among the strains studied. The odors detected have a slight alcoholic note,
and the apple pomace produced intermediate results, compared with the amaranth
grain supplied with mineral salt solution.
Medeiros et al. (1999) cultivated a strain of Kluyveromyces marxianus in SSF
using different solid substrates such as cassava bagasse, giant palm bran, apple
pomace, sugarcane bagasse and sunflower seeds. The feasibility of using cassava
bagasse and giant palm bran as substrates to produce fruity aroma was confirmed.
14.3.1.6 Protein Enriched Feeds
Cells of algae, fungi, yeasts, and bacteria are composed of up to 60% high-quality
protein. These organisms multiply quickly under different conditions, being able
to consume diverse types of industrial residues. Considering that traditional animal
protein sources, such as meat and milk, have a higher cost and, as such, are not
accessible to a large part of the global population, the production of alternative protein sources, such as those originated by microorganisms, appears to be an attractive
solution for raising protein intake.
Furthermore, the use of agro-industrial residues for growing microbial cells as a
suitable protein source for human consumption is an interesting approach for adding
value to industrial by-products. The use of biotechnological manipulated ingredients
for the production of animal feed has been growing each year. During microbial processing, along with the conversion of lignocellulosic waste into foods, an increase in
protein content and an improvement in the digestibility of the substrate are observed
(Vendruscolo et al. 2008).
Citrus fruit peel and apple pomace have been used for protein enrichment and
single cell protein (SCP) by the different fungal species of Penicillium (Scerra
et al. 1999; Vendruscolo et al. 2008), Neurospora, Chaetomium, Sporotrichum
(Shojaosadati et al. 1999), Aspergillus (Vendruscolo et al. 2008), Rhizopus (Soccol
and Vandenberghe 2003) and Trichoderma (De Gregorio et al. 2002; Vendruscolo
et al. 2008) and yeasts, such as S. cerevisiae, Torula utilis Candida utilis
(Vendruscolo et al. 2008). The recycling of viticulture residues through SSF by
Pleurotus has great potential to produce human food and yields an available highfiber feed for limited use in ruminants (Nchez et al. 2002).
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14.3.2 Enzymatic Modifications
Numerous studies have been made in the exploitation of fruit waste ingredients
to generate several value-added products through enzymatic modification such as
prebiotic oligosaccharides and modified biologically active molecules.
14.3.2.1 Prebiotic Oligosaccharides
In recent years a number of oligomers termed prebiotics have been described. These
resist digestion in the upper gastrointestinal (GI) tract and are able to modulate the
gut microbiota by stimulating indigenous beneficial flora components while suppressing, or not affecting, less desirable bacteria, such as proteolytic bacteroides and
clostridia (Gibson and Roberfroid 1995). Prebiotics have also been reported to indirectly lead to a reduction in serum triglyceride levels (Williams and Jackson 2002).
In addition, there is evidence showing that prebiotics may indirectly affect mineral absorption in the large bowel and show beneficial effects against inflammatory
bowel diseases by stimulating butyrate production and thus accelerating the mucosal
cell proliferation and healing processes (Bamba et al. 2002).
Although any dietary material that enters the large intestine can be considered as
potentially prebiotic, currently, the most well known prebiotics are non-digestible
oligosaccharides (Gibson and Roberfroid 1995). Different oligosaccharides with
prebiotic properties are commercially available, such as inulin, fructooligosaccharides (FOS), galacto-oligosaccharides and lactulose, but currently there is increasing interest in the identification and development of new prebiotic compounds,
perhaps with added functionality (Menne et al. 2000; Rao 2001; Tuohy et al. 2002).
Pectic substances are hydrolysed by the action of pectinases or pectolytic enzymes that are widely distributed in higher plants and microorganisms (Jayani et al.
2005). Pectic oligosaccharides (POS) were manufactured from commercial pectin
in an enzyme membrane reactor (Olano-Martin et al. 2001) and then evaluated for
their prebiotic properties (Olano-Martin et al. 2002). These pectic oligosaccharides
had a low prebiotic potential compared to FOS, although they were more selectively
fermented than were the parent pectins (Olano-Martin et al. 2002). Pectic oligosaccharides also protected colonocytes against Escherichia coli verocytotoxins (OlanoMartin et al. 2003a) and stimulated apoptosis in human colonic adenocarcinoma
cells (Olano-Martin et al. 2003b).
Recently, it has been demonstrated that POS from orange peel showed prebiotic properties increasing the bifidobacterial and E. rectale numbers (Manderson
et al. 2005). Orange peel albedo (white part) was also a good source of pectic
oligosaccharides with prebiotic properties produced by a microwave and autoclave
extraction (Hotchkiss et al. 2003). Incubating bergamot peel for 2 h with a commercial enzyme preparation from Aspergillus sp. (pectinase 62 L) produced a material
rich in oligosaccharides. The prebiotic effect of a POS rich extract enzymatically
derived from bergamot peel was studied using pure and mixed cultures of human
faecal bacteria. Addition of the bergamot oligosaccharides (BOS) resulted in a high
increase in the number of bifidobacteria and lactobacilli, whereas the clostridial
14 Biotechnological Potential of Fruit Processing Industry Residues
283
population decreased. A prebiotic index (PI) was calculated for both FOS and BOS
after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h
incubation, with BOS showing a greater value (6.90) than FOS (6.12) (Mandalari
et al. 2007).
Furthermore the potential of apple pomace for lactic acid production and oligomeric carbohydrates by simultaneous saccharification and fermentation (SSF) was
evaluated (Gulloä et al. 2007). It was found that operating at low cellulase (Celluclast 1.5 L, cellulases from T. reesei) and cellobiase (NS50010, ß-glucosidase
from A. niger) charges (1 FPU/g and 0.25 IU/FPU, respectively) and short reaction
times (10 h), 18.3 kg of oligosaccharides (which can be used as prebiotics) can be
produced from 100 kg of dry apple pomace. The distribution of total oligosaccharide
components was as follows: 5.8 kg of glucooligosaccharides, 7.4 kg of xylooligosaccharides, and 5.1 kg of arabinooligosaccharides.
14.3.2.2 Esterification of Flavonoids, Phenolic Acids and Terpenoids
Flavonoids (aglycon, glycosylated) are widely used in pharmaceutic, cosmetic and
food preparation. They have several physico-chemical properties and biological activities but they are characterized by a low solubility and stability. In order to take
advantage of these properties, their enzymatic acylation with fatty and aromatic
acids under different operating conditions has been suggested as a promising route
by several authors.
Various types of enzymes have been tested for acylation of flavonoids, such
as proteases, acyl transferases and lipases, subtilisin (the first enzyme used for
flavonoid ester synthesis) and mostly lipase B of Candida antarctica (CAL-B)
(Chebil et al. 2006). Some reactions were also catalyzed by acyl transferases, but
they required the use of either an activated acyl donor (ester of coenzyme A) or
the presence in situ of a system allowing the generation of these derivatives (Chebil
et al. 2006). Different acyl donors (aliphatic acids, aromatic acids and vinyl esters)
were used for the enzymatic esterification of isoquercitrin, quercitrin, luteolin7 glucoside, naringin, rutin, catechin-7-O-␣-D-glucoside, phloridzin, hesperidin,
epigallocatechin, 3-glucoside anthocyanin (delphinidin, cyaniding, pelargonidin),
quercetin, catechin in solvent free or added-solvent systems with the reaction time
and temperature ranging from a few hours to several days and from 30 to 60◦ C,
respectively (Chebil et al. 2006). These studies include the investigation of several factors that affect the acylation reactions such as the type, origin and concentration of the enzyme, the nature of the reaction, the operating conditions, the
composition of the reaction media, and the nature of substrates on regioselectivity
(Chebil et al. 2006).
Phenolic acids possess interesting biological properties (antioxidant, chelator,
free radical scavenger, UV filter, antimicrobial). Generally, such natural antioxidants are partially soluble in aqueous media. This is limiting their usefulness in
oil-based food processing and has been reported as a serious disadvantage if an
aqueous phase is also present. Therefore, the modification of these compounds
via esterification with fatty alcohols results in the formation of more lipophilic
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derivatives. There are several reports on the enzymatic lipophilisation of phenolic acids in non-conventional reaction media, such as organic solvent mixtures,
ionic liquids and solvent-free systems. Although lipases are the most commonly
used biocatalysts for this kind of reactions, feruloyl esterases, tannases, cutinases
are also reported (Figueroza-Espinoza and Villeneuve 2005). Several studies report
the enzymatic lipophilisation of phenolic acids present in citrus wastes, apple and
grape pomace, such as caffeic, cinnamic, ferulic, p-hydroxybenzoic, gentistic, gallic, vanillic, syringic, o- and m-coumaric, p-coumaric, chlorogenic and sinapic acid
(Figueroza-Espinoza and Villeneuve 2005; Karboune et al. 2005; Safari et al. 2006;
Stevenson et al. 2007; LopezGiraldo et al. 2007).
Active components of the essential oils are mostly constituted by alcohols of terpenic nature (e.g. citronellol, carvone, etc.). These compounds, with similar chemical characteristics such as terpenic esters, are being used mainly as fragrances and
flavor agents of great industrial importance. These compounds, when chemically
synthesized, are not considered as natural products, so enzymes, and especially
lipases, can be used as biocatalysts to carry out their esterification and transesterification reactions (Perea et al. 2007). Recent reports on the enzymatic ersterification of terpenoids refer to D-limonene, menthol, geraniol, citronellol, nerol (Kahlow
et al. 2001; Dominguez de Maria et al. 2006; Perea et al. 2007) esterification with
different acyl donors, using microbial lipases, often leading to optically active products of great importance.
14.3.2.3 Glycosylation of Flavonoids
Functional properties, such as solubility, physicochemical stability, bioactivity, pharmacokinetics and cellular localization of natural products, such as flavonoids, are
greatly affected by glycosylation, which is, therefore, an important factor to be considered in industrial applications of these products. Recently, for example, it has
been reported that the efficiency of absorption of a flavonoid glucoside, quercetin
glucoside, was greater than that of quercetin itself (Morand et al. 2000). Glycosylation occurs widely in plant cells and is considered to be an important method for
the conversion of water-insoluble and unstable organic compounds into the corresponding water-soluble and stable compounds (Shimoda et al. 2006, 2007a, b). This
reaction can be carried out through enzymatic or chemical methods. Enzymatic synthesis of glycoconjugates has many advantages over conventional chemical methods. The regio- and enantio-selectivity of GTs provide a simple means to synthesise
stereospecific glycosides without the requirement of protection and deprotection of
other functional groups. These processes are often difficult or impossible for chemical synthesis.
Enzymatic glycosylation usually employs glycosidases or “glycosynthases” and
glycosyltransferases (GTs). The use of glycosidases provides an alternative method
for biocatalytic glycosylation (Ly and Withers 1999). One of the most powerful
approaches to the enzymatic synthesis of glycosides is Withers’s “glycosynthase”
technology (Jakeman and Withers 2002). Glycosynthases are genetically engineered
nucleophile-less mutant glycosidases that can catalyze the formation of glycosidic
14 Biotechnological Potential of Fruit Processing Industry Residues
285
linkages, primarily but not exclusively by using glycosyl fluoride donors, yet are
incapable of hydrolyzing the product. Recently, a non-natural glycosidase mutant
(Cel7B-E197S glycosynthase) has been identified that has novel regiospecificity
(O4’, O6) and activity (disaccharide transfer to flavonoids) with catalytic efficiencies comparable with those of natural GT counterparts (Yang et al. 2007).
Unlike glycosidases and glycosynthases, GTs are enzymes that have evolved
naturally for glycosylation reactions. Many mammalian and microbial GTs have
been employed for the synthesis of oligosaccharides and antibiotic glycosides
(Lim 2005). In contrast, due to the small number of plant GT sequences that were
available, their use in biocatalysis has been limited. A GT (UGT73A10) isolated
from cDNA of Lycium barbarum L. fruits effectively catalyzed the regiospecific
glucosylation of (+)-catechin towards to 4-O-D-glucopyranoside of (+)-catechin
(Noguchi et al. 2008). The reaction product was more stable than aglycon under
acidic conditions and at elevated temperatures. Moreover, two Arabidopsis cluster
F GTs are capable of glycosylating the 3-OH of quercetin in vitro. While these
two closely related GTs have been found to recognize the same acceptor molecule
displaying the same regioselectivity, they catalyse the glycosidic linkage by means
of different sugar donors. GT 78D2 uses UDP-Glc while GT 78D1 prefers UDP-Rha
as the sugar donor (Jones et al. 2003; Limet et al. 2004). Another GT in cluster F,
ACGaT from Aralia, forms quercetin-3-O-galactoside with UDP-Gal as the sugar
donor (Kubo et al. 2004), indicating that all three GTs have a common ancestor,
they have evolved to recognize different sugar donors.
Whilst many plant GTs glycosylate the aglycone moiety of glycosides to form
products such as quercetin-3,7-di-O-glucoside (Limet et al. 2004), some other GTs
(Family 1, CAzY) are found to glycosylate the sugar moiety of glycosides. Citrus
Cm1,2RhaT is able to transfer a rhamnose molecule onto the 2-OH group of the
glucose moiety attached to the 7-OH group of naringenin, resulting in the formation
of a bitter tasting glycoside in some Citrus species (Frydman et al. 2004). There
is also a GT in Citrus species such as mandarin, that transfers rhamnose onto the
6-OH group of the glucose moiety of naringenin-7-O-glucoside forming a tasteless
glycoside (Frydman et al. 2004).
14.3.2.4 Halogenation of Flavonoids
The effects of flavonoids, on the central nervous system have been considered.
They process anxiolytic activity and low sedative or myorelaxant effects (Medina
et al. 1997). Among the most active compounds, a number of halogenated flavones
have been reported; in particular, 6-bromoflavone and 6-bromo- 3-nitroflavone
showed activities close to or higher than that of diazepam, a benzodiazepine
derivative which is a classical anxiolytic, anticonvulsant, sedative and skeletal muscle relaxant drug. In order to show these activities, the presence of electro-donating
or withdrawing substituents on the aromatic ring of the flavonoids seems to be
essential (Sternbach 1978). In the literature, several methods for halogenating aromatic compounds are reported. Direct bromination, e.g. with elemental bromine, is
a highly polluting method which, in addition, involves serious difficulties connected
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D. Mamma et al.
with the handling of a highly corrosive agent. Other methods, including NBSamberlyst (Goldberg and Alper 1994), metal-oxo-catalysed KBr–H2 O2 (Clague and
Butler 1995) and KBr–NaBO3 (Roche et al. 2000) suffer from harsh conditions or
require complex or laborious work-up.
One of the enzymatic reactions that have been widely studied is a chloroperoxidase-catalyzed halogenation (Franssen et al. 1987). Chloroperoxidase from
Caldariomyces fumago (CPO; EC 1.11.1.10) is a well-known enzyme, capable of
halogenating a great variety of organic compounds such as b-ketoacids, cyclic bdiketones, steroids, alkenes, activated aromatic compounds and heterocylcic compounds (Yaipakdeea and Robertsonb 2001). The reaction mechanism of CPO
involves the formation of a halogenium ion (X+ ) or hypohalous acid (HOX) as an
intermediate which can effect electrophilic substitution with electron-rich substrates
(Libby et al. 1992).
The whole cells and the chloroperoxidase enzyme of Caldariomyces fumago
were capable of halogenating the flavanones, naringenin and hesperetin, at C-6 and
C-8 in the presence of either Cl– or Br– (Yaipakdeea and Robertsonb 2001). The
biohalogenated products of naringenin and hesperetin were isolated and found to
be identical to those obtained from chemical reactions using molecular halogen and
hypohalous acid.
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Chapter 15
Wine Industry Residues
Bo Jin and Joan M. Kelly
Contents
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
15.2 Description of Chemical Characteristics
of Winery Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
15.2.1
Production of Winery Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
15.2.2
Characteristics of Residues from Winery Industry . . . . . . . . . . . . . . . . . . . . . . 296
15.2.3
Chemical Characteristics of Winery Wastewater . . . . . . . . . . . . . . . . . . . . . . . . 296
15.3 Advances in Molecular Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
15.3.1
Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
15.3.2
Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
15.3.3
Prospects Arising from Fungal Genome Projects . . . . . . . . . . . . . . . . . . . . . . . 299
15.4 Biotechnological Processes for Bioconversion of Winery Residues to Bioenergy and
Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
15.4.1
Process Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
15.4.2
Process Configuration and Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
15.5 Renewable Energy and Biomaterials from Winery Residues . . . . . . . . . . . . . . . . . . . . . . 305
15.5.1
Renewable Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
15.5.2
Organic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
15.5.3
Microbial Biomass Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
15.5.4
Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
15.5.5
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Abstract The wine industry produces a substantial quantity of organic residues that
are both highly polluting and costly to treat. These residues are mainly carbohydraterich organics such as sugars and cellulose, which are biodegradable and naturally
rich in nutrients, making them suitable substrates for biotechnological production.
This chapter briefly introduces potential utilization of winery residues for producB. Jin (B)
School of Earth and Environmental Sciences, School of Chemical Engineering, The University of
Adelaide, Adelaide, SA 5005, Australia
e-mail: bo.jin@adelaide.edu.au
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 15,
C Springer Science+Business Media B.V. 2009
293
294
B. Jin and J.M. Kelly
tion of bioenergy and biomaterials through bioconversion processes. We highlight
advanced molecular biotechnology for strain development and integrated biotechnological processes, which can lead the bioconversion of winery residues into renewable bioenergy and biomaterials as a sustainable solution.
Keywords Winery residues and effluent · Simultaneous saccharification and fermentation · Bioenergy · Organic acid · Molecular biotechnology · Microbial
biomass
15.1 Introduction
Wine production is one of the most important industries in many countries worldwide. Distilled grape marc, a complex lignocellulosic material, is one of the most
abundant organic residues. The winery residue is produced after pressing the crushing grapes in white wine processing or after fermentation and maceration in red wine
production. Grape marc is usually distilled in wineries to recover ethanol which is
further used to produce spirituous liquors, leaving huge amounts of distilled grape
marc unused after the winemaking process. The treatment of these waste residues
requires many successive and costly steps, which pose increasing disposal and pollution problems. Landfill and incineration are popular methods to deal with winery
residues, but both contribute to production of greenhouse gas. Most of the existing
winery waste treatment processes cause large losses of nutrient resources. Grape
marc contains cellulosic and hemicellulosic material that can be hydrolyzed to produce liquors containing monomers of glucose from cellulose and xylose and other
sugars from hemicellulose, which are biodegradable and naturally rich in nutrients,
making them suitable substrates for biotechnological production by microorganisms.
Bioconversion of the carbohydrate wastes to value-added products is of great
importance for the production of renewable resources in a sustainable society. With
industrial development growing rapidly, there is general agreement that sustainable
environmental protection can only be achieved by integrating a general environmental awareness into a company’s business functions. Recently, bioconversion of
carbohydrate wastes is receiving increased attention in view of the fact that these
wastes can act as a substrate for the production of useful biomaterials and chemical
intermediates.
In this Chapter, the winery residues are regarded as a valuable biomass or potential substrates for biotechnological production. Therefore, chemical constituents
of the winery residues and effluent will be described. Recently developed biotechnological processes which integrate saccharification with fermentation steps will
be introduced as potential bioprocesses for bioconversion of winery residues into
bioenergy and biomaterials. Advanced molecular biotechnology for strain development to meet specific bioprocess and bioproducts will be discussed. As potential
bioproducts from winery residues, representative bioenergy (ethanol and hydrogen),
15 Wine Industry Residues
295
organic acid (lactic acid and citric acid), enzyme, polymers and microbial biomass
will be briefly discussed. It is expected that this work gives guidance for development of sustainable technology to make winery residues suitable for development of
renewable energy and bioproducts, thereby offering biotechnology an opportunity
to assist in maintaining environmental quality.
15.2 Description of Chemical Characteristics
of Winery Residues
15.2.1 Production of Winery Residues
The main solid by-products and residues from the winery production are grape stalk,
grape marc, wine lees and winery sludge (biosolids). Figure 15.1 shows the process
flow sheet for generation of winery residues and wastewater from the winery and
distillery industries. The main wastes from the viticulture activities are the vine
stalks generated during the pruning of the grapevine. The principle by-product is the
grape marc, which comprises grape stalks, seeds and skins left after the crushing,
draining and pressing stages of wine production. Grape marc is commonly processed to produce alcohol and tartaric acid, which results in a new lignocellulosic
by-product, spent grape marc. The wine lees are accumulated in the bottom of grapejuice or wine fermentation tanks. The distillation of the alcohol from low-quality
wine, wine lees and grape marc produces a large quantity of a viscose and acidic
wastewater known as vinasse. In many winery industries, an aerobic depuration
process is operated after the distillation to treat the winery effluents, vinasse and
winery wastewater, therefore, generating waste biosolids. A large proportion of the
wastewater comes from cleaning and wine production and cooling processes.
Vineyards
Vine stalk
Grape vines
Crushing
and pressing
Grape
marc
Vinasses
Alcohol
Fig. 15.1 Wine and
distillation waste products.
Sketch flow sheet showing
the generation of
by-products, residues and
wastewater from the wine and
distillation industries
Fermentation
Spent grape
marc
Alcohol/ tartrates
Clarification
Wine lees
Vinasses
Wine
Distillation
Biosolids
Wastewater
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B. Jin and J.M. Kelly
15.2.2 Characteristics of Residues from Winery Industry
Grape marc (pomace) typically contains approximately 50% skins, 25% seeds and
25% stalks. Most grape marc is processed to extract commercial products such as
alcohol by fermentation and distillation, grape tannins, and anti-oxidants. Grape
marc is also used as feedstock for cattle and livestock but is limited to 30% of
the total food for ruminants due to the very low nutritional value, or composted
and returned to mulch vines. Winery residues are lingocellulosic biomass, which
typically consists of three basic polymers: 50% cellulose (C6 H10 O5 )x -glucose polymer, 35% hemicellulose (C5 H8 O4 )m -xylose polymer and 15% lignin [C9 H10 O3 .
(OCH3 )0.9–1.7 ]n . Table 15.1 summarizes the typical chemical compositions of grape
stalk, grape marc, wine lees and winery biosolids. These data represent the analytical results from 87 samples of winery and distillery residues from different Spanish
wineries and distilleries (adapted from Bustamante et al. 2008).
15.2.3 Chemical Characteristics of Winery Wastewater
The chemical composition of winery wastewater collected in vintage and nonvintage periods in Adelaide, Australia are shown in Table 15.2 (adapted from
Chapman 1995; Chapman et al. 2001). The winery wastewater is rich in organic
compounds, mainly carbohydrate-rich organics including sugars and organic acids,
which are ideal substrates for biotechnological production.
15.3 Advances in Molecular Microbiology
As outlined above, much of the waste produced in the wine industry, like other food
and beverage industries, is high in nutrients, and this represents a problem if the
Table 15.1 Typical chemical composition of winery residues
Parameter
Vine stalk
Organic matter
Organic carbon∗
Soluble carbon
Total nitrogen
P
K
Na
Ca
Mg
Fe
Mn
Cu
Zn
Polyphenols
920,000
316,000
74,500
12,400
940
30,000
1,250
9,500
2,100
128
25
22
26
23,900
Grape marc
915,000
280,000
37,400
20,300
1,150
24,200
1,200
9,400
1,200
136
12
28
24
10,600
Wine lees
759,000
300,000
87,800
35,200
4,940
72,800
1,385
9,600
1,600
357
12
189
46
8,900
Winery sludge
669,000
257,000
26,900
44,300
10,900
20,700
3,390
81,600
2,700
3,128
91
262
227
1,100
Amounts are expressed on a dry weight basis, in mg per Kg (Adapted from Bustamante et al. 2008.
The ∗ indicates oxidisable organic carbon.
15 Wine Industry Residues
297
Table 15.2 Nutrient composition of wastewater produced by the wine industry
After separation of solids in
storage lagoons
Fresh effluent
Parameter (mg l−1 )
Vintage
Non-vintage
Vintage
Non-vintage
Total organic carbon
Total K-nitrogen
Total P
C:N:P
Sulfur
Fe
Na
K
Ca
Mg
Tartaric acid
Lactic acid
Acetic acid
Ethanol
Glycerol
Glucose
Fructose
pH
Total solids∗
Suspended solids∗
1,400
47
0.020
30:2.8:0.3
349
2.8
250
131
26
29
530
350
100
3,130
190
300
530
4–8
1,570
190
900
31
0.010
30:6.1:1.3
209
2.3
328
204
30
15
350
120
50
800
31
0.002
30:1.4:0.1
6.7
0.95
148
98
25
10
200
150
220
1,490
60
trace
80
4–6
2,430
230
300
19
0.002
30:5.2:0.5
30.6
1.2
219
55
22
10
20
10
370
120
280
270
6–10
1,640
410
20
Trace
trace
4–8
2,800
410
The apparent increase in solids after separation (∗ ) is due to algal biomass growth during storage
(adapted from Chapman 1995).
waste is released into the environment, but opens up an opportunity for the waste
to be turned into valuable secondary products. Recent reviews of methods for treatment of wine industry waste (Arvanitoyannis et al. 2006b), as well as potential uses
and applications for the waste materials (Arvanitoyannis. 2006a), have concentrated
on engineering and on classical microbiological approaches. Microorganisms have
been used in a number of different treatments, either to decontaminate the waste by
removing either toxic compounds or nutrients, or to produce valuable products such
as enzymes and organic acids including citric and lactic acid, ethanol, or biomass
for use in animal production. Recent advances in genomics have opened up new
and exciting possibilities for genetic and metabolic engineering of microorganisms
to achieve desirable outcomes (Abe et al. 2006; Martinez et al. 2008).
15.3.1 Bacteria
Many bacteria can ferment sugar to ethanol, and thus can potentially convert cellulosic biomass to ethanol. Strains of Zymomonas mobilis ferment only sucrose,
glucose and fructose to ethanol, and this restricts their use with more complex
substrates. However, strains of Escherichia coli have been engineered such that
they contain genes to express Z. mobilis pyruvate decarboxylase and alcohol
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B. Jin and J.M. Kelly
dehydrogenase, and these show enhanced ethanol production. Klebsiella oxytoco
has also been manipulated to express the Z. moblis ethanol fermentation enzymes,
and further changed to express a celliobiose uptake system and cellulase genes from
Erwinia chrysanthemi (Zhou et al. 2001).
However, considerable mechanical and chemical pre-treatments of lignocellulosic compounds are required, as the bacteria do not directly degrade lignin
(Alterthum and Ingram 1989). In none of these cases has the ethanol production
reached levels that can be achieved using yeast, however, further engineering may be
possible now that genome sequences are available for a wide range of prokaryotes,
and a greater range of pathways, as well as the genes encoding enzymes for steps in
the pathways, become known.
Many anaerobic bacteria, including thermophilic Clostridium thermocellum, can
degrade cellulose and hemicellulose via a multi-enzyme complex, the cellulosome,
which performs a variety of cellulolytic and hemicellulolytic activities (Masai
et al. 2007). However C. thermocellum cannot use glucose (Lynd et al. 2002), and
thus this species has potential for mixed culture fermentations.
15.3.2 Filamentous Fungi
Although the yeast, Saccharomyces cerevisiae, has been extensively used in the
wine, brewing and baking industries, it is not effective for use with lignocellulosic substrates, as it does not produce a large range of enzymes necessary to break
down lignin and cellulose, it does not have a highly expressing secretory pathway,
and pre-treatments of lignocellulosic material often produces inhibitors to growth.
However, progress has been made in engineering yeast to ferment pentose sugars
(Hahn-Hagerdal et al. 2007). Filamentous fungi, on the other hand, can produce
a large range of enzymes necessary to break down lignin and cellulose, and have
highly efficient secretory pathways, and thus have more potential for winery waste
conversion.
There are numerous examples where filamentous fungi have been used to produce useful end-products in industries that produce cellulosic and other potentially
nutrient rich waste materials, and these have been the subject of a number of recent
reviews (Kumar et al. 2008). The naturally occurring filamentous fungi and yeasts
have been enhanced by generations of mutagenesis and selection, or by using recombinant DNA techniques to alter one or a few enzymes, such as various cellulolytic
enzymes, or by taking advantage of synergistic reactions by cultivating a number of
different microorganisms with complementary properties together.
However, several significant problems remain and must be overcome to make
the process ready for large-scale use for the production of products for the food
and pharmaceutical industries, and for the production of biomass and bio-fuels
(Somerville 2006). At present these problems include the accessibility of the cellulose in lignin requiring pre-treatment that is both expensive and potentially polluting, the amounts of enzymes required to convert cellulose to sugars, and in the case
of bio-fuel production, the fact that highly cellulolytic fungi do not convert sugars
15 Wine Industry Residues
299
to ethanol at high levels in aerobic conditions, and fungi that do convert sugars to
ethanol are not naturally highly cellulolytic. Because filamentous fungi are better
able to access and degrade lignin and cellulosic material, these represent the best
prospect for the treatment of solid winery wastes.
There are some recent examples, specifically in the wine industry, where filamentous fungi have been used to convert wastes to valuable products. Investigations
using Aspergillus awamori with grape marc as the substrate showed that it provided all the nutrients necessary to produce hydrolytic enzymes including cellulases,
pectinases and xylanases, in a solid state fermentation process. Further, the solid
grape waste was competitive with other agricultural wastes when used as the substrate in such processes (Botella et al. 2005). Phanerochaete chrysosporium, a white
rot fungus that degrades lignin but leaves the cellulose of the wood un-degraded and
thus useful for further processing, has been used with grape seed and stem waste
for laccase production (Couto et al. 2006; Moldes et al. 2007). Trichoderma viride
is used as a bio-control agent against phytopathogens, and solid state fermentation
using grape marc and wine lees as a substrate resulted in high yields of T. viride
conidia (Bai et al. 2008). Rhizopus oligosporus has been used in solid state phenolic antioxidant production using cranberry pomace as a substrate (Vattem and
Shetty 2002). Strains of T. viride, A. niger and A. oryzae were assessed for efficiency
in biomass mass production and nutrient clearing of winery waste water (Zhang
et al. 2008). Efficiencies of 5 gm per litre were found for T. viride without nutrient supplementation, accompanied by 84–90% chemical oxygen demand reduction,
indicating that a biotechnological treatment process integrated with fungal biomass
production from the winery waste streams could be effectively developed.
Pre-treatments can increase the efficiency of bioreactors, and Trametes pubescens
has been used effectively with wine distillery waste water to provide additional
nutrients for altered flux and the removal of components of wastewater leading
to a lower overall contamination measured by chemical oxygen demand (Melamane et al. 2007). Notwithstanding these early successful applications, full adoption
of fungal conversion of winery waste to useful end-products is technically problematic and still some way off. The information emerging from fungal genomic,
metabolomic and proteomic projects will provide the necessary platform to enable
the next step towards successful biological waste conversion in the wine and other
industries.
15.3.3 Prospects Arising from Fungal Genome Projects
With the advent of publicly available whole genome sequences, new approaches to
strain development and assessment are being developed.
15.3.3.1 Aspergillus oryzae
Aspergillus oryzae has a long history of use in the food industry, and more recently it has been modified by using recombinant DNA techniques to overproduce
both native and heterologous proteins of importance in the food, chemical and
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B. Jin and J.M. Kelly
pharmaceutical industries (Machida 2002). This fungus has a very high secretory
ability, and there are a large number of examples where the safety of such products for human use has been verified, and GRAS (Generally Regarded As Safe)
status has been granted (Abe et al. 2006). Much of the production of high value
added products has been from growth on valuable compounds including glucose and
starch (Ward et al. 1997), and thus modification of these processes to utilize waste
materials will be beneficial. Analysis of highly expressed genes identified highly
expressed pathways (Maeda et al. 2004), but a full description of the metabolic
capability of the organism was uncovered by whole genome sequencing. Analysis of the genome identified that A. oryzae contains a greater number of metabolic
and transporter genes than in other fungi, including the closely related Aspergillus
nidulans and Aspergillus fumigatus, andNeurospora crassa (Kobayashi et al. 2007;
Machida et al. 2005), particularly the hydrolytic and proteolytic enzymes that degrade high molecular weight bimolecular such as carbohydrates, polypeptides and
nucleic acids, consistent with its use in applied processes (Abe et al. 2006).
The full sequence of the genome has accelerated the development of oligonucleotide DNA microarray technology, and arrays are commercially available
(FarmLab Inc, Tokoyo Japan). This microarray technology opened up a number
of new and promising areas for development. At the production level, microarrays
have been used to monitor gene expression levels on a genome wide scale, both
during liquid fermentations and on solid media including refuse material (Maeda
et al. 2004), allowing a full understanding of the metabolic flux during the fermentation, and the tweaking of growth conditions to achieve maximum production. This
full understanding of the metabolic flux at all stages of fermentations will provide
the impetus for further genetic engineering, with the possibility to more cleanly
analyze all the effects of changes made. At the research level, knowledge of the A.
oryzae genome sequence has led to the discovery of new proteins and biochemical pathways for further analysis. One example is in the degradation of biodegradable plastics, where a theoretical analysis of the A. oryzae genome combined with
microarray analysis uncovered genes that were responsible for degrading one such
compound (Maeda et al. 2005).
15.3.3.2 Aspergillus niger
Aspergillus niger has been used as a biological factory in the production of chemicals, commercial enzymes and pharmaceuticals. A. niger has been used for the production of organic acids, and citric acid fermentation has been honed over the years
to such an extent that 90% of sugar is converted. A. niger is also highly efficient for
secreted protein production, with yields up to 10 gm per litre. Metabolic engineering
has proved a very powerful approach to optimising industrial fermentation processes
by making specific genetic changes using recombinant DNA technology for the
construction of improved production strains (Patil et al. 2004). In a 2001 review,
Nielsen has set out examples of such approaches, including where heterologous
proteins were produced, where substrate range was extended, where new products
were formed, where xenobiotics were degraded, where overall cellular physiology
15 Wine Industry Residues
301
was improved, where by-products were reduced or eliminated, and where yield or
productivity was improved (Nielsen 2001).
Mathematical models have been used to design optimal metabolic network structures, and the post-genomic era has led to genome-scale stoichiometric models of
microorganisms (Patil et al. 2004). A comprehensive analysis of A. niger central
carbon metabolism has been developed, and used to quantify the flux through the
branches of the metabolic network (David et al. 2003). This framework allows the in
silico analysis of environmental and genetic changes, an important predictive tool in
the investigation of metabolite production in newly designed production processes
and strains. The availability of the full genome sequence in the model filamentous
fungus, A. nidulans, has allowed the full potential of this approach to become apparent (David et al. 2006, 2005).
A genome wide transcriptional analysis of gene expression in glucose, glycerol
and ethanol growth conditions has been established (David et al. 2006), and a complete metabolic network has been defined. In addition, this approach has allowed the
genome-wide examination of the effects of a mutation in creA, which encodes the
major repressor protein in carbon catabolite repression (Dowzer and Kelly 1991), by
the quantification of the fluxes in the central carbon metabolism for different conditions of glucose repression (David et al. 2005). These conditions included growth
on xylose in the presence of glucose, and as these are components of lignocellulose,
an abundant and renewable carbon source, they are relevant to industrial production
processes.
15.3.3.3 Trichoderma reesei
Trichoderma reesei is adapted to a nutrient poor environment, using extracellular
hydrolyases to extract glucose from polysaccharides, and enzymes produced from
T. reesei are used in the paper, textile and food industries. Intensive analysis of the
regulation of transcription of cellulase and hemicellulase genes has been undertaken
(Stricker et al. 2008), and these have been the target of genetic manipulation to
increase production.
15.3.3.4 Other Fungi
The genome sequencing efforts have increased exponentially as methods of sequencing and data analysis have been refined, and it can be expected that the
genomes of more esoteric non-model fungi with a widely diverse metabolic repertoires will be determined. The future of value adding industrial waste materials,
including those of the wine industry, lies with applying these techniques of genetic and metabolic engineering to a broad range of microorganisms, as their DNA
sequences rapidly become available. For example, P. chrysosporium has superior lignin degrading properties, and its genome sequence is about to be released
(Martinez et al. 2005). Value-added products including fermentable sugars, organic
acids, solvents and bio-fuels may efficiently be produced from lignocellulosic
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biomass using the understandings that arise from genome sequences of presently
intractable microorganisms and advances in engineering technology.
15.4 Biotechnological Processes for Bioconversion of Winery
Residues to Bioenergy and Biomaterials
15.4.1 Process Synthesis
The bioconversion of cellulose and hemicellulose materials such as winery residues
to bioproducts generally takes place in four operation phases: pre-treatment, hydrolysis, fermentation and product separation/distillation. According to Cardona and
Sanchez’s report (Cardona et al. 2007), a schematic flow sheet for the bioconversion
process for winery residues is proposed in Fig. 15.2. The first step is size reduction and pre-treatment by delignification of the lignocellulosic feedstock to liberate
cellulose and hemicellulose from lignin. The pre-treatment is to alter or remove
structural and compositional impediments to hydrolysis in order to improve the rate
of enzyme hydrolysis and increase yields of fermentable sugars from cellulose and
hemicellulose. The second phase is depolymerization of the carbohydrate polymers
(cellulose and hemicellulose) using acid and enzymatic hydrolysis to produce free
sugars. In the fermentation operation, the sugars from the pre-treatment and hydrolysis steps are fermented by bacteria, yeast or filamentous fungi. Depending on the
operation system and products involved in the fermentation, the target products will
be separated and recovered in a downstream process. To develop a viable biotechnological process for producing market products from winery residues, the following
features need to take into a consideration (Hahn-Hagerdal et al. 2006):
r
r
r
r
Efficient de-polymerization of cellulose and hemicellulose to soluble sugars.
Efficient fermentation of a mixed-sugar hydrolysate containing six-carbon
(hexoses) and five-carbon (pentoses) sugars as well as fermentation inhibitory
compounds.
Advanced process integration to minimize process energy demand.
Cost-efficient removal of lignin.
The sequential bioprocess configuration employed to convert winery residues
into bioproducts implies that the solid fraction of pre-treated lingocellulosic materials undergo acid or enzymatic hydrolysis (saccharification). Once the hydrolysis
is completed, the resulting cellulose hydrolysate is fermented and converted into
the target bioproducts. This process is called separate hydrolysis and fermentation
(SHF). In recent decades, advanced biotechnological processes have been developed to minimize the process steps and enhance the production efficiency. The most
promising of these is to combine the hydrolysis and fermentation into a single stage.
The solid fraction from the pre-treatment that contains cellulose and lignin undergoes a simultaneous saccharification and fermentation (SSF) process or a simultaneous saccharification and co-fermentation (SSCF), as described in Fig. 15.2.
15 Wine Industry Residues
Fig. 15.2 Process diagram
for bioconversion of winery
residues to bio products.
SSF: simultaneous
saccharification and
fermentation; SSCF:
simultaneous saccharification
and co-fermentation; CF:
co-fermentation; CBP:
consolidated bio processing;
DSR: downstream separation
and recovery
303
Winery residues
Pretreatment
cellulose +lignin
Cellulase
production
pentose +lignin
SSCF
Cellulose
hydrolysis
SSF
Hexose
Fermentation
CF
Pentose
Fermentation
CBP
DSR
Microbial biomass
Biomass, Metabolites
(target products
and by-products)
Target product
By-product
15.4.2 Process Configuration and Integration
15.4.2.1 Pre-treatment
The main processing challenge in the bioproduction from winery residues is the
pre-treatment step. The lignocellulosic biomass is made up of a matrix of cellulose
and lignin bound by hemicellulose chains. During the pre-treatment, this matrix
should be broken in order to reduce the degree of crystallinity of the cellulose and
increase the fraction of amorphous cellulose, which is the most suitable form for
enzymatic attack. A successful pre-treatment must meet the following requirements
(Patel et al. 2007): (1) to improve formation of sugars or the ability to subsequently
form sugars by hydrolysis, (2) to avoid loss of carbohydrate, (3) to avoid formation
of by-products inhibitory to subsequent hydrolysis and fermentation processes, and
(4) to be cost effective.
The pre-treatment stage provides the physical disruption of the lignocellulose
matrix in order to facilitate acid-or enzymatic-catalyzed hydrolysis. The pretreatment can have significant implications on the configuration and efficiency of
the process and, ultimately the economics. The pre-treatment can be carried out
by physical (mechanical attack, steam explosion), chemical (ammonia fibber explosion, supercritical CO2 treatment) and biological methods. Depending on the
pre-treatment and hydrolysis processes involved, the lignocellulosic biomass of the
winery residues can be saccharified into mainly glucose followed by pentoses and
other hexoses, and lignin (van Wyk 2001). The glucose and pentose and other hexose
sugars can be used for bioconversion processes.
15.4.2.2 Simultaneous Saccharification and Fermentation Process
Alternatively, microbial conversion of lignocellulosic materials to bioproducts can
be made much more effective by coupling the enzymatic hydrolysis of lignocellulosic
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substrates and fermentation of the derived glucose into a single step, known as ‘Simultaneous Saccharification and Fermentation’. This method eliminates the need
for a complete hydrolysis step prior to the fermentation step. In the SSF process,
enzymatic hydrolysis, cell growth and microbial production occur simultaneously.
The SSF process is more attractive than the SHF process due to higher yields and
less energy consumption. The glucose formed during the enzymatic hydrolysis of
cellulose can be immediately consumed by the microbial cells converting it into
bioproducts (Cardona et al. 2007). Therefore, a direct benefit of SSF is a decrease in
the inhibition caused by glucose accumulation, leading to an increase in the saccharification rate, consequently increasing productivity and reducing reactor volume and
capital costs.
15.4.2.3 Simultaneous Saccharification and Co-fermentation
The SSCF process is a promising integration alternative, in which co-fermentation
of mixed cultures is employed. The use of mixed culture has advantages for
allowing the microorganisms access multi-carbon sources, enhances enzymatic
saccharification and fermentation efficiency, and consequently promotes product
yield and productivity. In a SSCF configuration, it is necessary that both fermenting microorganisms be compatible in terms of operating condition and substrate
consumption.
15.4.2.4 Consolidated Bio-Processing
The consolidated bio-processing (CBP), known as direct microbial conversion, is
a reaction-reaction integration for the transformation of biomass into bioproducts.
The key advance of CBP over other process strategies for biomass conversion is
that only one microbial community is employed in the processing system for the
production, cellulose hydrolysis, and fermentation, which are carried out in a single
step. The important consequence of the CBP operation is that no-capital or operation
expenditures are required for enzyme production within the process. Lynd et al.
(2005) reported the comparative simulation of SSCF and CBP processes assuming
aggressive performance parameters intended to be representative of a mature technology. The feasibility of a CBP process can be established when a microorganism
or microbial consortium can be developed according to enzymatic and metabolic
activities and process strategies (Sun and Cheng 2002).
The process design has been boosted due to process reaction-reaction integration.
The development of a simultaneous process is a promising approach to make the
biotransformation of winery residues technically and economically viable as an industrial process. The simultaneous processes such as SSCF and CBP allow synergistic interactions as well as the development of more compact technological schemes.
In general, the reaction-reaction integration can be proposed for the integration of
different microbial transformation taking place during the metabolic production.
This integration is oriented to the complete assimilation by the microorganisms of
all the sugars released during the pre-treatment and hydrolysis of lingocellulosic
biomass. Besides the understanding of the enzymatic and metabolic aspects related
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to the molecular biology of these microorganisms and process integration, there is
a need to study the relationships of this co-fermentation process through process
modelling and simulation in order to define the optimal cultivation conditions for
increasing yield and productivity of the target product. One of the major challenges
is to optimize the integration of process engineering, fermentation technology, enzyme engineering and metabolic engineering (Hahn-Hagerdal et al. 2006).
15.5 Renewable Energy and Biomaterials
from Winery Residues
15.5.1 Renewable Energy
Energy demand is increasing continuously due to speedy growth in population and
industrial development. Two significant challenges associated with using conventional fuels, such as coal, oil and natural gas, are depletion of fossil fuels and
deterioration of the environment. As concerns increase over the supply of oil and
changes in global climate, there is an urgent need to develop clean and renewable
alternative energy resources. Ethanol and hydrogen are the two most important fuels
contributing to the reduction of negative environmental impacts. Ethanol has already
been introduced on a large scale, and it is expected to be one of the dominating renewable bio-fuels in the transport sector within the coming years. Due to its environmentally friendly nature and high-energy yield, hydrogen (H2 ) offers a tremendous
candidate as an ideal clean and sustainable energy in the future. Hydrogen has the
highest gravimetric energy density of any known fuel without producing polluting
emissions. In addition, H2 has great potential for chemical synthesis or for electrical storage and generation with fuel cells. H2 fuel cells and related technologies
provide the essential link between renewable energy sources and sustainable energy
services.
15.5.1.1 Ethanol
Ethanol production utilizing lingocellulosic materials such as winery residues, generally takes place in three phase: (1) delignification of the lingocellulosic feedstock
to liberate cellulose and hemicellulose from lignin, (2) depolymerization of the carbohydrate polymers (cellulose and hemicellulose) to produce free sugars, and (3)
fermentation of mixed hexose and pentose sugars to produce ethanol. Alzate and
Toro (2006) and Hamelinck et al. (2005) summarized the most commonly used and
proposed process configurations, including SHF, SSF, SSCF and CBP. The SHF
has been employed in most industrial processes. SSF has been recognized as an
effective process configuration, which effectively removes glucose, an inhibitor to
cellulase activity, thereby increasing the yield and rate of cellulose hydrolysis (Sun
and Cheng 2002).
As the centre of the ethanol production process, fermentation is carried out under anaerobic conditions. According to the biochemical reactions, the theoretical
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maximum yield is 0.51 kg ethanol and 0.49 kg CO2 produced from 1 kg of xylose
and glucose. However, in practice, the microorganisms use some of the glucose for
growth and the actual yield is less than 100% theoretical yield. Xylose-fermenting
microorganisms for ethanol production are found among bacteria, yeast and filamentous fungi. Bacteria include both native and genetically engineered organisms,
and many have characteristics useful for SSF process. The ethanologenic bacteria
that demonstrate the most promise for industrial exploitation are E. coli, K. oxytoca
and Z. mobilis (Dien et al. 2003). Z. mobilis is well recognized for its ability to
produce ethanol rapidly and efficiently from glucose-based feed stocks. One of the
most effective ethanol producing yeasts, S. cerevisiae, has several advantages owing
to its high yield from hexoses and high tolerance to ethanol and other inhibitory
compounds (Hamelinck et al. 2005).
15.5.1.2 Hydrogen
H2 production through bioprocesses represents an exiting new area of technology
development for bioenergy generation. Hydrogen can be produced by anaerobic
bacterial growth on carbohydrate rich substrates giving organic fermentation end
products, hydrogen and CO2 . The bacteria found to produce hydrogen from carbohydrate containing organic materials include species of Enterobactor, Bacillus and
Clostridium. Most biologically produced H2 in the biosphere is produced by microbial fermentation processes. Many microorganisms contain enzymes, known as
hydrogenases, that either oxidize H2 to protons and electrons or reduce protons and
thus release molecular H2 . The physiological role and biochemical characteristics of
these hydrogenases are variable. The metabolic flexibility of these microorganisms
allows the production of a range of end products. It has been widely accepted that the
highest theoretical yield of 4 mol H2 /mol glucose can be obtained if acetate is the
sole by-product (Eq. 15.1), while a maximum 2 mol H2 /mol glucose is associated
with butyrate as the end by-product (Eq. 15.2) (Hallenbeck and Benemann 2002).
In practice, however, high H2 yields are associated with a mixture of acetate and
butyrate fermentation products, and low H2 yields are associated with propionate
and reduced end products.
C6 H12 O6 + 2H2 O = 2CH3 COOH + 2CO2 + 4H2
C6 H12 O6 = CH3 CH2 CH2 COOH + 2CO2 + 2H2
(15.1)
(15.2)
Clostridium thermocellum is a thermophilic bacterium that utilizes cellulose as a
sole carbon source and carries out mixed product fermentation, synthesizing various
amounts of 36 lactate, formate acetate, ethanol, H2 , and CO2 , under different growth
conditions (Demain et al. 2005). Hydrogen and soluble end-product synthesis patterns by C. thermocellum in batch cultures were investigated by Levin et al. (2006)
in batch cultures, using either cellobiose or cellulosic substrates, providing an average yield of 1.6 mol H2 mol/glucose equivalent. Fermentative H2 production is an
exciting R&D area that offers a potential means of producing H2 from a variety of
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renewable resources or wastewaters via a low energy required continuous bioprocess. However, a typically low H2 yield associated with the H2 fermentation process
continues to be a challenge for developing a viable industrial process. Research is
needed to update advance knowledge of bioprocess engineering, providing a better understanding of the regulation of metabolisms underlying the metabolic flux
network of the H2 fermentative system, consequently improving H2 productivity
and yield.
15.5.2 Organic Acids
Microbial production of organic acids is a promising approach for obtaining buildingblock chemicals from renewable carbon sources. Most of the organic acids are natural products of microorganisms, or at least natural intermediates in major metabolic
pathways. Because of their functional groups, organic acids are extremely useful as
starting materials for the chemical industry. Here, we introduce the recent developments exemplified by two organic acid products: lactic acid and citric acid.
15.5.2.1 Lactic Acid
Lactic acid (2-hydroxypropionic acid) is the most widely occurring multifunctional
organic acid, and has been widely used in food and pharmaceutical industries. One
of its most promising applications is its use for biodegradable and biocompatible
polylactate polymers, an environmentally friendly alternative to non-biodegradable
plastics derived from petrochemicals. Lactic acid can be produced using bacteria
and fungi. Lactic acid producing bacteria (LAB) have received wide interest because of their high growth rate and product yield. However, LAB have complex
nutrient requirements because of their limited ability to synthesize B-vitamins and
amino acids, making supplementation of sufficient nutrients such as yeast extracts
to production media necessary. In addition, the difficulty in separating fermentation
broth containing lactic acid from bacterial biomass increases the overall cost of production process. Fungal Rhizopus species have attracted attention in recent decades,
and have been recognized as suitable candidates for lactic acid production. Unlike
the LAB, Rhizopus strains generate L-lactic acid as a sole isomer of lactic acid (Jin
et al. 2003; Yin et al. 1997). Rhizopus strains grow better under nitrogen-limited
environments than the LAB. Lactic acid production from lignocellulosic agricultural biomass using lactic acid bacteria has been explored (Venkatesh 1997), but a
SSF process for lactic acid production using lignocellulosic material and Rhizopus
species has not been reported. However, Ruengruglikit and Hang (2003) and Miura
et al. (2004) developed a process which integrated lignocellulose hydrolysis with
lactic acid fermentation. Ruengruglikit and Hang carried out the fermentation in
the presence of carboxyl methyl cellulase and xylanase, while Miura et al. used
Acremonium thermophilus ATCC 24622.
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B. Jin and J.M. Kelly
15.5.2.2 Citric Acid
The production of citric acid (also known as the tricarboxylic acid), by the filamentous fungus A. niger represents the oldest microbial process for production of a
high volume and low cost organic acid. A fermentation process for the production of
citric acid can be operated in either a solid state fermentation system or a submerged
bioreactor process. A. niger can access glucose, starch and cellulose-based carbohydrate materials as carbon sources. The crucial parameters resulting in efficient production of citric acid by A. niger have been determined empirically and include high
substrate concentration, low and finite content of nitrogen and certain trace metals,
through maintenance of high dissolved oxygen and low pH. The exact definition
of these parameters enabled the development of a highly efficient biotechnological process, such that citric acid concentration of 140 g/L are now easily reached
(Forster et al. 2007). In addition to the well-established filamentous fungal species,
yeast Yarrowia lipolytica has been recently developed as a microbial cell factory for
citric acid production (Papanikolaou et al. 2006). Y. lipolytica proved efficient in the
production of citric acid from carbon sources such as glucose and sucrose.
15.5.3 Microbial Biomass Protein
Winery wastes are suitable substrates for production of microbial biomass protein
(MBP). The MBP products could be used for human or animal consumption, which
are marketable products and may offset the operating costs of the treatment process
(Jin et al. 2002). Both fungi and yeast can be used to produce MBP. However, fungi
seem more attractive because filamentous or pellet morphology of fungi permits low
cost separation and recovery of the MBP from the culture media, which makes up
a significant fraction of the capital and operating costs for MBP production. Fungi
can be grown using almost any organic waste products that contain carbohydrates,
such as confectionery and distillery waste, vegetable waste and wood processing
effluents.
A biotechnological treatment process integrated with fungal biomass production
(FBP) from the winery waste streams was investigated by Zhang et al. (2008). They
reported that it could be possible to develop a hybrid biotechnological process, integrating the production of fungal biomass protein with treatment of winery wastewater. Three filamentous fungi, T. viride WEBL0702, A. oryzae WEBL0401 and
A. niger WEBL090, demonstrated a high capability for over 80% COD reduction
and fungal biomass production. T. viride had a lower nitrogen requirement compared
to A. oryzae and A. niger, indicating that T. viride could tolerate the fluctuation of
nutrient change in winery wastewater.
15.5.4 Polymers
There has been a considerable interest in using low cost carbon substrates for the
production of polymers including Poly(3-hydroxybutyrate), and food industry waste
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water has been shown to be a viable alternative carbon source in high cell density
PHB production (Nath et al. 2008). There are no examples to date where winery
wastes have been used, but the prospect is that further development of strains and
processes will allow winery wastes to be converted to polymers.
15.5.5 Enzymes
There have been few examples to date where winery wastes have been used as
the substrate for enzyme production, but it is likely that this waste stream will
become increasingly important as economic and environmental necessities drive
the development of techniques for processing a broad range of nutrient rich industrial waste products. Winery wastes have been used with P. chrysosporium, a white
rot fungus, for lignolytic enzyme production (Rodriguez-Navarro et al. 2004), and
grape seed and stem waste was the substrate for T. versicolor in laccase production
(Couto and Toca-Herrera 2007). Grape pomace has been used withA. awamori to
produce hydrolytic enzymes including cellulases, pectinases and xylanases (Botella
et al. 2005).
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Chapter 16
Biotechnological Potential
of Brewing Industry By-Products
Solange I. Mussatto
Contents
16.1 By-Products Generation During the Brewing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.2 Spent Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.2.1
Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.2.2
Potential Uses of Spent Grains in Biotechnological Processes . . . . . . . . . . . .
16.3 Spent Hops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.3.1
Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.3.2
Potential Uses of Spent Hops in Biotechnological Processes . . . . . . . . . . . . .
16.4 Surplus Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.4.1
Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.4.2
Potential Uses of Surplus Yeast in Biotechnological Processes . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The manufacture of beer inevitably involves generation of various residues
and by-products. The most common by-products are spent grains, spent hops and
surplus yeast, which are generated from the main raw materials used for beer elaboration, the barley malt, hop and yeast, respectively. These three brewery by-products
are available in large quantities throughout the year, but their use has still been
limited, being basically sold to local dairy farmers to be used as cattle feed, or
simply as a land fill. However, they represent large potential resources for use in
biotechnological processes, in consequence of their complex compositions containing carbon, nitrogen and minerals. Several attempts have been made to use them in
biotechnological processes, as for example in fermentative processes for the production of value-added compounds (xylitol, arabitol, ethanol, lactic acid, among
others); as substrate for microorganisms cultivation, or simply as raw material for
extraction of compounds such as sugars, proteins, acids and antioxidants. From an
environmental viewpoint, the elimination of industrial by-products represents a solution to pollution problems, and merits thus large attention. In this chapter, the main
S.I. Mussatto (B)
Institute for Biotechnology and Bioengineering, Centre of Biological Engineering,
University of Minho. Campus de Gualtar, 4710-057, Braga, Portugal
e-mail: solange@deb.uminho.pt
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 16,
C Springer Science+Business Media B.V. 2009
313
314
S.I. Mussatto
characteristics and potential applications of the three main brewery by-products are
reviewed focusing on their use in biotechnological processes.
Keywords Brewing · Spent grains · Spent hops · Trub · Surplus yeast · Fermentation
16.1 By-Products Generation During the Brewing Process
Brewing process consists initially mixing of the milled barley malt with water in
a mash tun, and the temperature is slowly increased (from 37 to 78◦ C) to promote
enzymatic hydrolysis of malt constituents. During this process, malt starch is converted to fermentable sugars (mainly maltose, and maltotriose) and non-fermentable
sugars (dextrins), and proteins are partially degraded to polypeptides and amino
acids. This enzymatic conversion stage (mashing) produces a sweet liquid called
wort. The insoluble, undegraded part of the malted barley grain is allowed to settle
to form a bed in the mash tun and the sweet wort is filtered through it (lautering).
The residual solid fraction obtained after this stage is constituted by the spent grains
(Mussatto et al. 2006a). In some cases, due to economic reasons or aiming to produce beers of distinct qualities, part of the barley malt – usually 15 to 20% – is
replaced by unmalted cereal like corn (maize), rice, wheat, oats, rye or sorghum,
called adjuncts (Kunze 1996). In these cases, at the end of the mashing process, the
insoluble part of these grains is separated with the undegraded part of the malted
barley grain (spent grains).
After filtered, the wort is transferred to the brewing kettle (also known as cooper)
where it is boiled during at least one hour with the addition of hops. During this
process, the bitter and aromatic hop components are transferred into the wort. Such
substances will confer typical beer qualities, such as bitter taste, flavour, and foam
stability. At the boiling end, the medium is cooled and the liquid extract is then
separated from the hop residues to be further processed (Keukeleire 2000). The hop
residues (spent hops), which are then useless are dumped directly as being of no
further value. A fraction of the hop components end up in the trub (a precipitation product of the wort boiling process that may include insoluble hop materials,
condensation products of hop polyphenols and wort proteins, and isomerized hop
acids adsorbed onto trub solids (Huige 2006)). Hops can be used in the natural form
(cone), or in the forms of powder (hammer-milled whole hops), pellets (powered
hops packed as pellets), or extract (the main components of hops to the beer quality
are extracted by means of a suitable solvent) (Kunze 1996).
After removal of the precipitate produced during boiling, the cooled hopped wort
is pitched with yeast in a fermentation vessel, where the yeast cells will convert
the fermentable sugars to ethanol and carbon dioxide. During fermentation, cell
mass increases three- to six fold. At the end of this stage, most of the cells are
collected as surplus yeast, at the top or at the bottom of the fermentor (according
to the nature of yeast used) (Keukeleire 2000). Figure 16.1 is a schematic representation of the brewing process and the points where the main by-products are
generated.
16 Biotechnological Potential of Brewing Industry By-Products
BARLEY
MALT
315
Adjuncts
(optional)
Milling
Mashing
Water
HOPS
Liquid
fraction
Boiling
Filtration
(Wort)
Cooling
Solid
fraction
SPENT
GRAINS
Solid
fraction
SPENT
HOPS
Liquid
fraction
Fermentation
YEAST
Cells
collection
SURPLUS
YEAST
Fig. 16.1 Schematic representation of the brewing process and points where the main by-products
are generated
16.2 Spent Grains
16.2.1 Chemical Composition
Spent grains are the most abundant brewing residue, corresponding to around 85%
of total generated. It is estimated that about 200 t of wet spent grains with 70 to
80% water content are produced per 10,000 hl of produced beer (Kunze 1996). It
316
S.I. Mussatto
is therefore of particular interest to sell these spent grains or reuse them for the
obtainment of added-value products.
Spent grains are basically composed by the barley grain husks (Figure 16.2),
but can present some residual starch endosperm as a function of the brewing
regime used. They can also present some residues from other cereals eventually
used during mashing together with barley, such as wheat, rice, corn (maize) and
others.
Since the barley malt husk is a lignocellulosic material, spent grains are a residue
rich in cellulose, hemicellulose and lignin, and also contain high protein content.
Cellulose and hemicellulose together comprise almost 50% (w/w) of the spent grain
composition, revealing the presence of a large amount of sugars in this material,
with xylose, glucose and arabinose being the most abundant (Mussatto and Roberto
2006). Lignin contains numerous phenolic components, mainly acids such as ferulic, p-coumaric, syringic, vanillic and p-hydroxybenzoic (Mussatto et al. 2007a).
Protein bound amino acids include leucine, valine, alanine, serine, glycine, glutamic acid and aspartic acid in the largest amounts, and tyrosine, proline, threonine,
A
B
Fig. 16.2 Appearance
of spent grains (A) and
micrograph by scanning
electron microscopy (B) with
45-fold magnification
(Mussatto et al. 2006b)
1 mm
16 Biotechnological Potential of Brewing Industry By-Products
317
arginine, and lysine in smaller amounts. Cystine, histidine, isoleucine, methionine,
phenylalanine, and tryptophan can also be present (Huige 2006, Mariani 1953).
Spent grains also contain considerable amounts of minerals present in ashes
(specially silicon); and extractives, a fraction consisting of waxes, lipids, gums,
starches, resin, tannins, essential oils and various other cytoplasmatic constituents.
Lipids include triacylglycerols, diacylglycerols, fatty acids (palmitic, oleic and
linoleic acids), sterols, sterol esters and sterol glycosides, plus various hydrocarbons
(including alkanes and carotenoids) (Briggs et al. 1986). Vitamins such as: biotin,
choline, folic acid, niacin, pantothenic acid, riboflavin, thiamine and pyridoxine, are
also present in this material.
Nevertheless, the chemical composition of spent grains vary according to barley
variety, harvest time, malting and mashing conditions, and the quality and type of
adjuncts (other cereal grains) added in the brewing process (Huige 2006). Some
analysis of spent grains chemical composition is shown in Table 16.1
Table 16.1 Chemical composition of spent grains
Reference a
Components (% dry weight basis)
Cellulose (glucan)
Hemicellulose
xylan
arabinan
Lignin
Acetyl groups
Proteins
Ashes
Extractives
1
2
3
4
16.8
28.4
19.9
8.5
27.8
1.3
15.3
4.6
5.8
25.4
21.8
nr
nr
11.9
nr
24.0
2.4
nr
21.9
29.6
20.6
9.0
21.7
1.1
24.6
1.2
nr
25.3
41.9
nr
nr
16.9
nr
nr
4.6
9.5
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
Minerals (mg/kg dry weight basis)
Calcium
Sodium
Potassium
Magnesium
Aluminum
Iron
Barium
Strontium
Manganese
Copper
Zinc
Phosphorus
Sulfur
Chromium
Silicon
a
3515.0
309.3
258.1
1958.0
36.0
193.4
13.6
12.7
51.4
18.0
178.0
5186.0
1980.0
5.9
10740.0
1 From Mussatto and Roberto et al. 2006; 2 From Kanauchi et al. 2001; 3
From Carvalheiro et al. 2004; 4 From Silva et al. 2004. nr = non reported
318
S.I. Mussatto
16.2.2 Potential Uses of Spent Grains in Biotechnological
Processes
The main application of spent grains until nowadays has been as animal feed, due
to its high content of protein and fibre. However, because the chemical composition
is rich in sugars, proteins and minerals, several attempts have been made to use
them in biotechnological processes. When compared to the other two main brewery
by-products, the spent grains are the most evaluated for reuse in biotechnological
purposes. The great interest in this material is probably due to the large amount
generated and rich chemical composition that permit its reuse in different areas.
16.2.2.1 Substrate for Microorganisms Cultivation
and Enzyme Production
Wet spent grains from a lauter tun contain high moisture 70–80% (w/w) and fermentable sugar contents, characteristics that make it liable to deteriorate rapidly due
to microbial activity. Owing to these characteristics and the high protein content,
several studies have been performed aiming to use the spent grains as substrate for
cultivation of microorganisms. The fungus species Pleurotus, Agrocybe, Lentinus,
Aspergillus and Trichoderma, and the bacteria Bacillus and Streptomyces (a soil
actinobacteria), are some of the microorganisms successfully cultivated in spent
grains (Wang et al. 2001, Szponar et al. 2003, Bartolomé et al. 2003, Bogar et al.
2002, Sim and Oh 1990).
The production of several enzymes was also verified from cultivation of microorganisms in spent grains, including xylanase by Aspergillus awamori (Bhumibhamon
1978) and Streptomyces avermitilis (Bartolomé et al. 2003), feruloyl esterase by
Streptomyces avermitilis (Bartolomé et al. 2003), alpha-amylase by Bacillus subtilis (Duvnjak et al. 1983), Bacillus licheniformis (Okita et al. 1985) and Aspergillus
oryzae (Bogar et al. 2002, Francis et al. 2003), and cellulase by Trichoderma reesei
(Sim and Oh 1990).
16.2.2.2 Source of Value-Added Compounds
As before mentioned, spent grain is a lignocellulosic material and thus, it is constituted by several polysaccharides, which can be degraded into their corresponding constituents by hydrolytic procedures (hydrothermal, enzymatic or acidic). On
hydrolysis, cellulose yields glucose, while the hemicellulose yields xylose, arabinose, mannose, galactose and the acids acetic and hydroxycinnamic (ferulic and
p-coumaric) (Mussatto and Roberto 2004, Palmqvist and Hahn-Hägerdal 2000).
Additionally, a wide variety of arabino-oligoxylosides with different structural features can be obtained according to the hydrolysis process used (Kabel et al. 2002).
As a whole, the compounds obtained by hydrolysis of spent grain are of industrial
interest, mainly in the food industry. Such compounds can be purified for use as
is, or as substrate in different fermentative processes for obtainment of value-added
16 Biotechnological Potential of Brewing Industry By-Products
319
products. Considering also the low cost, spent grains appears to be an attractive raw
material for industrial purposes.
16.2.2.3 Use in Fermentative Processes
Due to the composition rich in sugars and nutritional factors, hydrolysates produced
from spent grains can be used in fermentative processes to produce several compounds of industrial interest. Some examples include the use of the sugar rich hydrolysate as fermentation medium for the production of ethanol by Saccharomyces
cerevisiae (Laws and Waites 1986), xylitol by Candida guilliermondii (Mussatto
et al. 2008), xylitol, arabitol, ethanol and glycerol by Debaryomyces hansenii
(Carvalheiro et al. 2005, Duarte et al. 2004), and lactic acid by Lactobacillus delbrueckii (Mussatto et al. 2007b), Lactobacillus pentosus or Lactobacillus rhamnosus (Cruz et al. 2007).
Besides the use as substrate in fermentative processes, spent grain can also be
employed as carrier for cell immobilization. It has been suggested its use as cell
immobilization carrier during the production of pectinase by Kluyveromyces marxianus CCT 3172 (Almeida et al. 2003), straightdough and sourdough bread using
baker’s yeast, kefir and Lactobacillus casei (Plessas et al. 2007), or during beer
elaboration by Saccharomyces yeast (Brányik et al. 2001, Dragone et al. 2007). In
the last case, the material must be initially pre-treated by a sequence of HCl and
NaOH solutions to obtain a cellulose-based carrier, which due to the irregular shape
and non-homogeneous chemical composition, provide “active sites” that are readily
colonized by brewing yeasts.
There are also other alternatives for the spent grains reuse in breweries, such as
the use of an extract obtained from spent grain pressing as an antifoaming agent
in the fermentor. Besides effective for such purpose, the extract addition improves
the hop utilization and not affects the properties of the final beer (Roberts 1976).
When neutralized and added to wort, the spent grains extract enhances the yeast
performance and also not affects the quality of the produced beer.
16.2.2.4 Other Uses
A range of other uses for spent grains has also been proposed, as an adsorbent for
removing VOC emissions or organic material from effluent, as a source of biogas
and soil conditioner produced by anaerobic digestion, as a medium for growing
earthworms to use in poultry food, and as raw material for the production of charcoal
bricks or bleached cellulose pulps that can be used in the manufacture of specific
kind of papers (Mussatto et al. 2006b, 2008, Briggs et al. 1986).
Additionally, the water pressed from spent grains has a high biological demand
(BOD) and constitutes an undesirable effluent. Some efforts to utilize this liquor
include: (1) recovering its extract by incorporating it in a subsequent mash (with or
without prior treatments to remove lipids and polyphenols); (2) using it as an antifoaming agent in deep fermentors; (3) using it as a nutrient medium for supporting
useful microorganisms; (4) collecting the solids from it by centrifugation and adding
to animal feeds (Briggs et al. 1986).
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16.3 Spent Hops
16.3.1 Chemical Composition
The hop, Humulus lupulus, is an agricultural crop essentially used for brewing. It
is rich in bitter constituents (␣-acids (humulones), -acids (lupulones), soft and
hard resins) and ethereal oils, which supply bittering and aroma components to
beer. However, only 15% of the hops constituents end up in the beer, 85% will
became spent hop material (Huige 2006). The lupulones, for example, are insoluble
at the normal pH value of the wort and not isomerizes during boiling, being largely
removed with the spent hops and the trub. The hop phenolic components (e.g.,
p-hydroxycoumaric, gallic, ferulic, protocatechinic, and caffeic acids; catechins,
flavones, and anthocyanidines, among others) are also precipitated with proteins
during wort boiling (Esslinger and Narziss 2005).
Spent hop is a material with high amounts of nitrogen free extract, fibres and
proteins (Table 16.2). Crude fibre is constituted by several sugars (rhamnose, arabinose, xylose, mannose, galactose and glucose), among of which, glucose and xylose
are the most abundant. Pectic sugars, uronic acid, rhamnose, arabinose and galactose account for 46% of the polysaccharides in spent hops (Oosterveld et al. 2002).
Mono- and multifunctional aliphatic carboxylic acids in spent hops include oxalic,
glucaric, gluconic, threonic, glyceric, glycolic, lactic and acetic acid (Fischer and
Bipp 2005).
As previously mentioned, a fraction of the hop components end up in the trub,
mainly when hop powdered, pellets or extracts are used in the brewing process.
Hot trub consists of protein (40–70%), bitter substances (7–15%), other organic
compounds, such as polyphenols, and mineral substances (20–30%); while the cold
trub consists of protein (50%), polyphenols (15–25%) and carbohydrates of high
molecular mass (20–30%) (Esslinger and Narziss 2005).
Table 16.2 Chemical composition of spent hops
Reference a
Component (% dry matter basis)
Protein
Lipid
Ash
Crude fiber b
Nitrogen free extract
a
b
1
2
23.0
4.5
6.5
26.0
40.0
22.4
nr
6.0
23.6
nr
1 From Huige 2006; 2 From Briggs et al. 1982
cellulose, hemicellulose and lignin nr = non reported
16.3.2 Potential Uses of Spent Hops
in Biotechnological Processes
In spite of the chemical composition rich in nitrogen, carbon and protein, spent
hops have been few explored as substrate in biotechnological processes. Basically,
16 Biotechnological Potential of Brewing Industry By-Products
321
the addition to spent grains is the most prevalent method for disposal of spent hops
at the brewery site, but it compromises quality of the mixture, mainly if it is desired
the spent grains use as animal feed. Unlike spent grains, the direct use of spent
hops as feed supplement is not desirable due to the presence of bitter substances
in this residue. Animals unwillingly eat bitter fodder and they are discouraged by
sedative–hypnotic properties of 2-methyl-3-buten-2-ol, which is the product of bitter
acid degradation. Therefore, to be used as feed supplement, it is firstly necessary to
remove or degrade the spent hops bitter acids that can be made by selected fungi
or yeasts like Candida parapsilosis (Huszcza and Bartmanska 2008, Huszcza et al.
2008).
When obtained separately from spent grains, an alternative frequently used for
spent hops disposal is as mulch or as soil conditioner and fertilizer, due to the high
nitrogen content (Table 16.2) (Huige 2006). Additionally, some alternative ways of
utilizing spent hops in biotechnological processes have been proposed, which are
listed below.
16.3.2.1 Source of Value-Added Compounds
Several compounds of industrial interest can be recovered from spent hops, such
as flavours, saccharides and organic acids, which can be obtained after oxidation
or hydrolysis of this material (Oosterveld et al. 2002, Fischer and Bipp 2005,
Laufenberg et al. 2003, Vanderhaegen et al. 2003, Krishna et al. 1986). Among
these compounds, the hop acids, particularly, have potential as natural antibacterial
in distillery mashes for alcoholic fermentation, being a safe alternative to control
bacteria in ethanol fermentations, able to efficiently replace antibiotics in ethanol
production (Ruckle and Senn 2006).
Pectins, compounds widely used as ingredient for the food industry as gelling
agent and thickening agent, represent a large part of the polysaccharides in spent
hops and can be recovered from this material by acid extraction conditions. Spent
hops pectins include homogalacturonans, and arabinogalactan-proteins with a protein part rich in cystein, threonin, serinin, alanin, and hydroxyprolin (Oosterveld
et al. 2002). Residual hops resins can also be recovered by extraction with acetone,
obtaining an unsaturated drying oil for paints (Huige 2006).
16.3.2.2 Use in Fermentative Processes
Trub addition to the pitching wort was found to increase yeast vitality and yield
as well as fermentation performance of Saccharomyces cerevisiae, and the more
trub was added, the greater the effect. The influence of hot trub on yeast vitality
and fermentation performance is associated with several components of hot trub,
e.g. lipids, zinc and particulate properties. Although some authors underline the
nutritive importance of lipids and particularly of unsaturated long-chain fatty acids,
others believe zinc might be the most effective component of trub (Kühbeck et al.
2007).
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16.4 Surplus Yeast
16.4.1 Chemical Composition
The surplus yeast is another brewing by-product that merits considerable attention,
due to the large quantity produced (is the second largest by-product from breweries)
and rich chemical composition.
The most abundant element in yeast cells is carbon, which accounts for just
under 50% of the dry weight. Other major elemental components are oxygen
(30–35%), nitrogen (5%), hydrogen (5%) and phosphorus (1%). The most abundant classes of macromolecules are proteins and carbohydrates (Table 16.3). However, the precise composition of each class of macromolecules within a given
cell varies as a function of physiological condition and phase in growth cycle
(Briggs et al. 2004).
The protein content present several bound amino acids, including arginine, cystine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine; leucine, lysine and tyrosine being the most
Table 16.3 Chemical composition of surplus yeast
Reference a
Components (% dry weight basis)
1
2
3
Protein
Lipid
Ash
Crude fiber b
Carbohydrates
48
nr
7
3
nr
nr
1
8
nr
36
50
nr
7
nr
42
0.12
0.12
0.01
0.24
1.43
1.71
0.09
0.38
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
50
15
10
7
4
3
0.2
Minerals in ash (%)
Calcium
Chlorine
Iron
Magnesium
Phosphorus
Potassium
Sodium
Sulfur
Vitamins (mg/100 g)
Niacin
Thiamin
Panthotenate
Riboflavin
Folic acid
Pyridoxine
Biotin
a
1 From Huige 2006; 2 From Lamoolphak et al. 2006; 3 From Lewis
and Young 1995
b
glucans, mannans, and polymeric hexosamines. nr = non reported
16 Biotechnological Potential of Brewing Industry By-Products
323
abundant. It is also rich in vitamins (biotin, choline, folic acid, niacin, pantothenic
acid, riboflavin, thiamin, and vitamin B-6), mainly niacin (Lewis and Young 1995;
Huige 2006).
The total mineral content of yeast is approximately 5–10% of the cell dry weight.
This fraction comprises a multitude of elements, specially potassium and phosphorus. The composition of some of them is shown in Table 16.3. Besides the mentioned
minerals, cobalt, copper, manganese, and selenium can also be found in lower extends (ppm) (Huige 2006).
16.4.2 Potential Uses of Surplus Yeast
in Biotechnological Processes
When compared to spent grains, surplus yeast has a much higher content of protein,
vitamins and amino acids; reason by which it has been currently utilized for animal
feed and nutritional supplement after drying (Chae et al. 2001). However, the inclusion of yeast in food products is limited by the amount of nucleic acid, primarily
ribonucleic acid (RNA); since in humans, RNA is metabolized to uric acid, which
can lead to gout (Huige 2006). Due to the composition rich in protein, amino acids,
minerals, and other compounds of interest, several attempts have been done aiming
to reuse the surplus yeast in biotechnological processes.
16.4.2.1 Source of Value-Added Compounds
Several compounds of industrial interest can be isolated from brewer’s yeast, such as
enzymes, proteins, vitamins, amino acids, cytochromes, the purine components of
DNA and RNA, among others (Huige 2006). Protein and amino acids, for example,
can be recovered by employing various processes such as autolysis, plasmolysis
in organic salt solution or non-polar organic solvent, acid or alkali catalyzed hydrolysis, enzymatic hydrolysis, or hydrothermal decomposition (Lamoolphak et al.
2006).
-glucan, a hydrocolloid of large interest by the pharmaceutical and functional
food industries, can also be extracted from brewer’s yeast. This compound is of
great interest because it has potential in improving the functional properties of food
products, being used as a thickening, water-holding, or oil-binding agent, and emulsifying or foaming stabilizer (Romero and Gomez-Basauri 2003, Thammakiti et al.
2004). It has also, a potentially valuable addition to starch-based foods to restrict
retrogradation of the starch (Satrapai and Suphantharika 2007). It is expected that
-glucan from brewer’s yeast, with low cost of production, simple extraction technology, and potential infinite supply will dominate the market for the foreseeable
future (Zekovic et al. 2005).
Yeast extract (a mixture of amino acids, peptides, nucleotides and other soluble
components of yeast cells) is produced by the breaking down of yeast cells using
endogenous or exogenous enzymes. Such compound is of particular interest for use
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S.I. Mussatto
in the food industry as a flavouring agent in soup, sauces, gravies, stews, snack food
and canned food. Other application is as vitamin supplements in health food (Chae
et al. 2001).
16.4.2.2 Use in Fermentative Processes
The use of brewer’s yeast autolysate during fermentation of vegetable juices by
Lactobacillus acidophilus, favourably affects the increase of the number of lacticacid bacteria, reduction of fermentation time and enrichment of vegetable juices
with amino acids, vitamins, minerals and antioxidants (Rakin et al. 2007). The use
of yeast extract in microbiological media is also well known and largely used as
source of nutrients (Chae et al. 2001).
References
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immobilized yeast cells on spent grains. J Biosci Bioeng 96:513–518
Bartolomé B, Gómez-Cordovés C, Sancho AI et al. (2003) Growth and release of hydroxycinnamic
acids from brewer’s spent grain by Streptomyces avermitilis CECT 3339. Enzyme Microb Technol 32:140–144
Bhumibhamon O (1978) Production of acid protease and carbohydrate degrading enzyme by Aspergillus awamori. Thai J Agr Sci 11:209–222
Bogar B, Szakacs G, Tengerdy RP et al. (2002) Production of ␣-amylase with Aspergillus oryzae
on spent brewing grain by solid substrate fermentation. Appl Biochem Biotechnol 102–103:
453–461
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Chapter 17
Biotechnological Potential of Cereal
(Wheat and Rice) Straw and Bran Residues
Hongzhang Chen, Ye Yang and Jianxing Zhang
Contents
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2 The Utilization of Cereal Straw and Bran Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2.1 Firewood Fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2.2 Paper Making . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2.3 Animal Feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2.4 Returning Straw to Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.2.5 Lignocellulose Chemical Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.3 Fractionated Conversion of Cereal Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.3.1 Bringing Out the Concept of Fractionated Conversion Process . . . . . . . . . . . . .
17.3.2 Flow Diagram of Ecological Industry Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.3.3 Fractionated Conversion for Various Products . . . . . . . . . . . . . . . . . . . . . . . . . . .
17.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Cereal straw, one of the most abundant renewable lignocellulose resources which possess valuable components, has gradually become the research hot
spot as a promising substitute for both the fossil fuel resource and petroleum-based
industry with the increasing calling for bio-fuel and green chemistry. However, existing technologies of straw utilization unilaterally emphasize the primary utilization of the whole plant or some certain components, which not only result in low
technical content of corresponding products but also fail to make full use of the
lignocellulose resources. Based on the decades of research work, we find out that
the bio-structural inhomogeneities of straw, both in the chemical composition and
molecular structure between each part of straw, are the ultimate reasons why straw
can not be utilized in a whole.Thus, the concept of fractionated conversion of straw
emerges as the time requires. In this chapter, this innovative concept is explained
H. Chen (B)
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,
Chinese Academy of Sciences, Beijing 100190, PR China
e-mail: hzchen@home.ipe.ac.cn
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 17,
C Springer Science+Business Media B.V. 2009
327
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in detail by taking the fractionated conversion of the corn straw, rice straw and rice
husk as examples. Only through utilizing different parts of straw in the guidance of
its structures and characteristics we can make full use of the straw resources.
Keywords Fractionated conversion · Biomass total utilization · Cereal straw ·
Steam explosion · Inhomogeneity · Biorefinery
17.1 Introduction
Along with the fast growing of world’s population and great development of social
economy, though the petroleum-based agriculture and industry have greatly boosted
the development of both the society and people’s living standard, in less than 400
years, we human beings have almost used up the fossil fuels such as coal, oil, and
natural gas formed and accumulated through 2.5 billion years and caused a series of
problems such as energy crisis, resource exhaustion and environment deterioration
which not only threaten the living of ourselves but also confront the chemical industry with new challenge of human beings’ sustainable development. Meanwhile, the
traditional starch–based fermentation industry, however, cost as many as several billion tons of foodstuff per year, hence the limitation of both arable land and foodstuff
set huge barrier to its development (Wyman 2007).
Energy shortage, food shortage and the call for developing biomass resource as
chemical raw material in Green Chemistry starting from 1990s stimulate the countries worldwide to notice that the utilization of natural cellulose material like straw
holds the strategic significance to their development. European Union has brought
forward a short-term goal of alleviating the fossil energy dependence on each member country by cutting down 20–30% cost of bio-fuel production and actualizing
27–48% motor vehicle using bio-fuel (Council 2006).
The United States has invested several billion US dollars in the research work of
substitutable energy and clean energy since the announcement of “Advanced Energy
Initiative” (Milliken et al. 2007) in 2006, including non-grain crop based ethanol
production, and reducing the technical costs of renewable energy such as wind
energy, solar energy, geothermal energy and biomass energy. They have already
invested 354 million US dollars, to reach the final goal of replacing 75% petroleum
imported from Middle East by 2025 (President Bush 2006; Schell et al. 2008). Chinese government has pointed out clearly to strengthen the utilization of biomass
resource and exploit the technology of biomass-based clean liquid fuel production
in the “China’s Agenda 21 —White Paper on China’s Population, Environment, and
Development in the 21st Century”.
Cereal straw is one of the most abundant, annually renewable resources in the
world. According to a valid data, there are as many as 2.9 billion tons of cereal
straw produced per year all over the world, and only in China there is 0.7 billion tons
cereal straw produced per year. However, such abundant resource has not attracted
enough attentions and thus has not been utilized reasonably. In fact, cereal straw is
the production of plants’ photosynthesis, which is constituted by high percentage
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
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of macromolecule compounds such as cellulose, hemi-cellulose and lignin. Both
cellulose and hemi-cellulose are polymers made up of fermentable sugar which can
be fermented into chemical materials and liquid fuel such as ethanol, acetone, acetic
acid, as well as be used as the fermenting materials of antibiotics, organic acid and
enzyme after hydrolysis. Lignin, comprised of phenylpropane derivatives, can be
further transformed to other chemicals used as the raw material in organic chemistry
industry (Chen 2005).
17.2 The Utilization of Cereal Straw and Bran Residues
Cereal straw is one of the most abundant renewable resources in the world, long
before human have been utilizing it in various different forms.
17.2.1 Firewood Fuel
As a traditional energy transforming mode, direct combustion is economic, low cost
and easy to promote. However, according to the research, using natural straw as
the fuel to combust directly has pretty low combustive efficiency because it is very
difficult to be combusted completely. As a result, the heat loss usually varies from
30% to 90%, which not only wastes the resource but also causes serious pollution
problem to the environment. Presently, relative research of straw as one kind of
fuel is concentrated on the improvement of the low caloric value, and researches
such as central gas supply of straw gasification, technology of methane or ethanol
production from straw fermentation are on the way.
17.2.2 Paper Making
The utilization history of lignocellulose and fiber material has much to do with
paper making industry which can date back to 3rd Century BC (Kamm et al. 2005;
Kamm et al. 2007). Plant fiber is the raw material in the pulp and paper industry.
Nowadays, wood fiber accounts for more than 90% of the world paper, nevertheless,
to those countries which lack in wood fiber, fiber material such as straw is a good
substitute. China is the largest straw pulp -producing country in the world, providing more than 75% of the world’s non-wood pulp (Chen 2008). Pulp and paper
industry all focus on the utilization of the cellulose component in the fiber material
and removal of both the hemi-cellulose and lignin components which accounts for
the formation of black liquor. This process not only wastes the hemi-cellulose and
lignin components, but the removal step dramatically generates the increment of
the cost, and the black liquor pollutes the nearby environment especially the water
resources. Obviously, it is urgent to develop new technology for straw utilization
in solving the problems mentioned above. Fortunately, there are researchers who
bring out biotechnologies such as bio-pulping (Chen et al. 2002), bio-bleaching and
enzymatic deinking (Chen 2005) to tackle the pollution problem.
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17.2.3 Animal Feed
The development of animal husbandry depends on the sufficient supply of feedstuff.
Countries like New Zealand, Australia have abundant meadow which can afford
forage-based animal husbandry while other developed countries like America can
afford grain-based animal husbandry. China is a country which has a large population and relatively less cultivatable land, in other words, neither forage nor grain is
sustainable for the country’s fast growing animal husbandry. The abundant straw resource is undoubtedly the most suitable choice considering the national conditions.
In fact, the crop straw occupies about 30% of the livestock feedstuff. However,
limited by the straw structure itself, natural straw has high percentage of lignin and
ash components while only low percentage of raw protein, and poor palatability,
which together lead to the low digestion and insufficient amount of nourishment.
Ruminant such as cattle and sheep can digest about 40–50% of the straw on average
while pig can only digest 3–25% and chicken are the worst, almost can’t digest it at
all (Liu 2006). Therefore, it is necessary to develop straw processing technology in
order to decrease or even eliminate its limitation of digestion and nourishment. Now,
the feed industry has made great progress due to the straw processing technologies
like silage and straw ammoniation.
17.2.4 Returning Straw to Soil
It is a tradition in the agriculture history to use organic fertilizer, and the easiest
and most traditional method is returning straw which mainly contains both straw
mulching and straw incorporation to the soil. According to straw returning application, the soil can gain more organism and nutrient which can bring soil fertility
betterment as well as adjustment of the physical properties, and finally optimize the
environment of farmland. However, the problem is that returning natural straw to
soil directly needs multiple kinds of microorganisms in the soil to function together
to decompose which may take as long as several years. Obviously this kind of returning is so slow to the extent that the undecomposed straw can not function as fertilizer
but also set some barriers to the new shoots. As a result, nowadays returning the
manure of ruminant to soil and other compost are more commonly used.
17.2.5 Lignocellulose Chemical Industry
Up to now, it has been more than 200 years since the lignocellulose and cellulose
fiber chemical industry was founded (Kamm et al. 2005). Utilizing the complex
biorefinery technologies, through corresponding chemical processing, the lignocellulose can be decomposed into different fractions such as cellulose, hemi-cellulose,
lignin, extraction and ash, from which the product line is based (Kamm et al. 2007).
For example, to produce vanillin from lignin, gain carbohydrates from cellulose, and
prepare furfural from hemi-cellulose. Wood has gained wide research and industry
utilization because of its high content of homogeneous component of cellulose. As
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
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the substitute material of wood, straw catches more and more attention from worldwide researchers and becomes the research hotspot.
In general, state of the art of straw utilizing mainly focuses on the primary utilization of straw which are weak in foundation, integrity and system. The reports unilaterally emphasize on the utilization of the whole plant or some certain component
and fail to reach the goal of making full use of the three main components (cellulose,
hemi-cellulose and lignin) of the lignocellulose resources which accounts for the
problems of the low technical content on the using of biomass resource and the poor
quality of the corresponding products. In order to solve problems mentioned above
fundamentally, the most crucial point is to realize that the bio-structural inhomogeneity of straw, that is to say, the differences, both in the chemical composition
and structure between each part of straw, are the ultimate reason that straw can not
be used in a whole.
The three main components—cellulose, hemi-cellulose and lignin crosslink
tightly in the unpretreated straw, and due to their totally different chemical structures
and properties, none of them can be utilized efficiently. Therefore, it is necessary to
separate each component apart while maintaining the macromolecule’s integrality
as much as possible. Only through this processing, the different fractions can be
utilized in an optimized way and fulfill their greatest value. Meanwhile, we should
cultivate a clear sense that not only the cellulose component is a valuable resource,
the other components are also potential resources rather than wastes which await
the future industrial utilization. Secondly, the breakthrough of straw transformation
technology calls for new development of corresponding process engineering theory
on the solid phase complex materials.
Finally, it is more important to investigate the characteristics of straw utilization
and technical bottle-neck, and build up systematic theory on the straw transformation. As for the utilization of biomass resource, applying mechanically existing
knowledge is far from enough to solve the technical problems and it is necessary to
build up more updating, comprehensive and systematic theory to guide the development of cellulose science.
In this chapter we will discuss about corn straw, rice straw and rice husk as examples to introduce the progress of research work done for the fractionated conversion
of cereal straw in our laboratory.
17.3 Fractionated Conversion of Cereal Straw
17.3.1 Bringing Out the Concept of Fractionated
Conversion Process
Since the 1970s, the transformation and utilization technology of biomass have
made great progress, however, from the aspect of current utilization and development of biomass resource, there are still large amount of barriers and problems, one
of which is the unilateral emphasis on the biological or thermal chemical technology
without the sense of total utilization of natural solid phase organic material through
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fractionating and recycling. The single utilization technology not only wastes much
of the biomass resource but also causes pollutions to the environment, therefore,
looking for new effective technology of biomass utilization becomes the inevitable
trend. Under such circumstances, the concept of fractionated conversion emerges as
the time requires.
Based on the summarization of our many years research experience, we bring
out the concept of fractionated conversion of cereal straw. Now the question arises
what is this so-called fractionated conversion of cereal straw? In fact it is a process
in which preparation of biomass products depends on the compositions and characteristics of the raw materials. The main route is biotransformation and thermal
chemical transformation, biotechnology and other physical or chemical methods
will be used as well if necessary in this conversion.
17.3.2 Flow Diagram of Ecological Industry Chain
Technical process scheme (shown in Fig. 17.1) is as follows:
(1) Cutting up straw into 5 cm or so pieces, and adding water to adjust the moisture
to about 35% (Chen and Liu 2007).
Straw
Straw explosion
Water washing
Solid fiber
Fiberboard
modified materials
Water washed
liquid
Long fiber
Xylooligosaccharide
Purification
Paper making
Cellulose acetate
Carboxymethyl cellulose
Cellulose derivatives
Utilization of
lignin
Short fiber
Solid state
fermentation
Simultaneous
saccharification
& fermentation
Cellulase
Ethanol, hydrogen
acetone, butanol
Fermented
residue
Pyrolysis
Ash
Bio-oil fuel
Nano-silica
dioxide
Fig. 17.1 Brief introduction of flow diagram of ecological industry chain
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
333
(2) Using steam explosion pre-treatment to activate the raw straw, and the condition
of steam explosion is 1.5 MPa, maintaining for 3.5 min (Chen and Liu 2007).
(3) Fibers separated from the steam exploded (SE) production can produce medium
and high density strawboard directly. SE production can also be washed with
water. The water washed liquor can be used as stuff to produce xylooligosaccharide, and the solid fiber can be separated into two main parts: the
long fiber part and the short fiber part through the carding equipment.
(4) Long fiber part can be used in ethanol autocatalytic pulping, preparation of
cellulose acetate (Zhang and Chen 2007) and carboxymethyl cellulose.
(5) The separated short fiber part can be the appropriate substrate to produce cellulase in solid state fermentation (Xu et al. 2002) which can be used in the later
fermentation of ethanol (Chen et al. 2007), hydrogen (Li and Chen 2007) and
acetone butanol (Qureshi et al. 2007).
(6) Simultaneous saccharification and fermentation (SSF) (Han and Chen 2008) for
fuel ethanol: use the short fiber as the substrate, adding cellulase obtained from
the solid state fermentation and activated yeast (Chen and Jin 2006; Rudolf
et al. 2005) to conduct SSF.
(7) Utilization of fermentation residues: they can be used in generating electricity
or preparing bio-fuel (Luo et al. 2004).
(8) Utilization of straw ash: reclaim the valuable nano-silicon dioxide from ash.
17.3.3 Fractionated Conversion for Various Products
17.3.3.1 Fractionated Utilization of SE Corn Straw
Cut up the corn straw into 5 cm or so pieces and then add 30% (wt. %) water to
the material and mix them up. Put them into a 0.5 m3 steam explosion reactor, keep
the steam pressure at 1.5 MP in the reactor and maintain for 3, 4, 5 min respectively,
and then release the pressure in a quick shot. Then the steam exploded straw is
extracted with 5 times water (wt. %) and both the water washed liquor and the solid
material are collected. The sugar content of water washed liquor can be determined
by HPLC. The solid material then is separated into long fiber part and short fiber
part through the carding equipment. The utilization of each fraction are as follows:
xylo-oligosaccharide can be distilled from the water washed liquor and then prepare
levulinic acid; long fiber part can be used as paper pulping and the short fiber part
can ferment ethanol and acetone butanol.
Content of the long fiber part of the SE products decreases along with the intensity of steam explosion. In the same steam pressure of 1.5 MPa, maintaining as
long as 3 min, 4 min and 5 min, the contents of long fiber are 69.71% , 46.53% ,
45.39%, respectively. And the weight proportion of long fiber and short fiber are
1:0.45, 1:1.15, 1:1.17, consequently.
Compared with the natural corn straw, the cellulose content of SE product can
reach 40% or so which is higher than that of natural corn straw; the content of
hemi-cellulose decreases from 30% to 20%. Under the same SE condition, the
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Fig. 17.2 The ethanol yield
of fractionated corn straw
Long fiber fraction
Short fiber fraction
16
Ethanol yield rate / %
14
12
10
8
6
4
2
0
1.5 MP ax3m in
1.5 MP ax4m in
1.5 MP ax5m in
content of neutral washing component in long fiber part and short fiber part is different and the former one has a lower percentage; the contents of ash and lignin have
less discrepancy.
The ethanol fermentation test using fractionated SE corn straw as substrate was
conducted. In general, all ethanol yields (shown in Fig. 17.2) were above 10% and
the short fiber part had the highest yield of above 14%.
17.3.3.2 Fractionated Conversion at Morphological Organic
Level of Rice Straw
a. Inhomogeneity of rice straw structure
In the angle of configuration, rice straw can be divided into four main different
organs: root (seed root, adventitious root, branch root), stalk (node and internode), leaf (leaf blade and leaf sheath) and panicle (threshing panicle), and the
straw roots (Jin and Chen 2006) are usually kept in the soil after harvest. In
Fig. 17.3, it is clearly shown that the differences in chemical compositions result
in the different transforming capacity of straw’s morphological fractions; therefore the utilization of rice straw in a fractionated way is not only important but
also necessary.
b. Inhomogeneity of capacity of ethanol fermentation of rice straw’s morphological
fractions
Using abundant agricultural cereal straw to prepare fuel ethanol in order to partly
replace fossil fuel is a significant developmental trend. However, the structural inhomogeneity and large amount of non-fiber cells of straw directly lead to poor
ethanol yield and high cost. Under such circumstances, investigation on the capacity
of ethanol fermentation of rice straw’s morphological fractions will turn out to be
the pertinent basic guidance of appropriate transformation of fractionated rice straw.
Under the same SSF condition, the different ethanol yields of leaf sheath, leaf blade,
node, internode and panicle respectively are shown in the Fig. 17.4 (Jin 2007).
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
335
Fig. 17.3 Chemical composition of each morphological fraction of rice straw lower than 40 mesh
Cellulose component of the straw will firstly be hydrolyzed into glucose which
then transformed to ethanol via microorganism fermentation, the biochemical reaction is as formula (17.1):
(C6 H10 O5 )n → n C6 H12 O6 → 2n C2 H5 OH + 2n CO2
Fig. 17.4 Ethanol yield of each morphological fraction of rice straw (Jin 2007)
(17.1)
336
H. Chen et al.
According to the formula, the theoretical yield of ethanol from glucose is 0.51
g-ethanol/g-glucose.
In Fig. 17.4, the vertical coordinate is the ethanol yield (wt.%) which denotes
the ethanol yield per gram of substrate in the SSF test. Obviously each fraction has
distinguished transforming capacity. Under the same SSF condition, the sequence
of capacity of ethanol fermentation is: internode > node >leaf sheath > leaf blade
> panicle, and when the fermenting time reaches as much as 48 hours, the ethanol
yield can reach 11.5%, 9.0%, 6.9%, 6.9% and 6.3%, respectively. Internode has the
highest ethanol yield which towers above that of leaf sheath and leaf blade 66.7%.
This law has positive correlativity with the enzymatic capacity of each morphological fraction of rice straw basically. Via the investigation of different transformation
capacity of each morphological fraction of rice straw, it is more testified that to
utilize rice straw in a fractionated way is very necessary.
17.3.3.3 Fractionated Conversion of Cereal Husk
Due to the abundant published papers on the research using bran and chaff, we will
mainly introduce the total utilization of rice husk based on our research work.
Rice husk is characteristic of small cumulus density, hard husk and poor degradation capacity which directly lead to the ignorance of its utilization. The main
components of rice husk are cellulose, lignin and silicon derivatives which may
vary with the different breeds and producing areas. The average contents are: raw
fiber 35.5% ∼ 45% (polycondensed pentose 16% ∼ 22%), lignin 21% ∼ 26%, ash
11.4% ∼ 22%, and silicon dioxide 10% ∼ 21%.
Based on the characteristics of each component of rice husk and our research
work, we bring forward the new technical process of multilevel utilization of rice
husk which includes classifying rice husk into rich in silicon part, hemi-cellulose
part and short fiber part and then adopting corresponding feasible means to utilize
each part. The detailed processing is as follows: the rich in silicon part can prepare
nano-silicon dioxide directly, and the hemi-cellulose part can prepare furfural while
the short fiber part can be hydrolyzed to ferment fuel ethanol, together these can
realize the effective total utilization of rice husk.
1. Project scheme
Technical process scheme (shown in Fig. 17.5) is as follows:
(1) SE pre-treatment of rice husk.
(2) Mechanical carding of rice husk: using mechanical carding equipment to separate SE rice husk into 3 parts, hemicellulose, component rich in silicon and
short fiber.
(3) Preparation of fuel ethanol in SSF: using cellulose enzymatic hydrolysisfermentation coupling method to prepare fuel ethanol.
(4) Preparation of furfural: to prepare furfural either by catalyzed with solid superacid or in acid free autocatalysis.
(5) Preparation of nano-silicon dioxide with component rich in silicon.
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
337
Rice Husk
Steam Explosion
Extraction
Mechanical
Carding
Component
rich in silicon
Short fiber
Acid free
autocatalysis
Fluidized
pyrolysis
Ball milling &
crushing
Heat recovery
Catalyzed by
solid superacid
Simultaneous
Saccharifiction and
Fermentation
Distillation
Dilute Acid
Extraction
Furfural
Fermentation
Residue
Fuel Ethanol
Nano-Silicon
Dioxide
Fig. 17.5 Flow diagram of rice husk’s utilization
2. Preparation of nano-silicon dioxide from SE rice husk’s rich in silicon part
Compared with other biomass resources like wood, straw has much higher ash
content, above 60% of which is silicon dioxide. Among all the straws, rice straw
has the highest content of both the ash content and the percentage of silicon dioxide
in the ash. In the plant, silicon dioxide usually appears as amorphous global nanoconglomeration (10 nm or so) which is comprised of SiOn (OH)4–2n . For a long time,
the effect of ash on the cellulose enzymatic hydrolysis has always been ignored,
however, according to the research into the correlation between the physicochemical
characteristic of silicon and cellulose hydrolysis in the cell wall of rice straw, a
new discovery emerges that the content of lignin and insoluble silicon has distinct
synergistic relation which uncovers their synergistic effect on blocking the cellulose
from hydrolysis.
Using steam explosion to pretreat straw, then to separate fiber tissue from
parenchyma cell in carding to obtain most of the ash part, in this way the decrease
of ash amount can reduce its inhibiting effect on hydrolysis as well as collect the
silicon for better utilization. Research result shows that silicon dioxide still exists
in the residues after enzymatic hydrolysis and fermentation, and SE pre-treatment
causes no effect on the configuration of silicon dioxide which is still at amorphous
state. Besides, the extremely low impure heavy metal ion content in the straw facilitates the preparation of high purity amorphous nano-silicon dioxide which not
only cuts down the production cost but also enhances the added value of straw.
Such obtained nano-silicon dioxide is white powder which has an average granular
diameter of 50 nm, and the total impure ion content is below 5.5 mg/kg (< 10 ppm)
which reaches the high purity level.
338
H. Chen et al.
X-ray Diffraction (XRD) study on the structure of silicon dioxide in the rice husk
under different SE pretreating conditions, comparing with crystalloid SiO2 map,
indicates some important discoveries. From the comparison map, except the position
and intensity coincidence of several apices, the positions and intensities of other
apices are distinct, in other words, the produced SiO2 still existed in amorphous state
which testifies that the SE pre-treatment and carding process have no effect on the
SiO2 structure. SiO2 obtained after the acid bath and combustion is not nano-level
SiO2 yet, and it needs further disposal with Ball Mill.
3. Fermentation of SE rice husk’s short fiber part to ethanol in SSF
Using plant cellulose as the raw material to ferment ethanol is an effective approach to solve the current ethanol fermentation problems, such as the high cost
and limitations of raw material resources. The fermentation results of the SE rice
husk are shown in the Table 17.1 clearly in which the steam explosion and later
carding cause similar effects on the fermentation yield as enzymatic hydrolysis,
and the short fiber parts obtained from the mechanical carding are more suitable to
ferment ethanol and also gain much higher yield because of the increase of material
accessibility to enzyme.
Table 17.1 Effects of steam explosion and carding on the ethanol fermentation yield
Carded SE material
∗
1.5 MPa 6 min
1.6 MPa 5 min∗
∗
Ethanol yield(%)
Rich in silicon part
Short fiber part
Rich in silicon part
Short fiber part
7.08
11.46
7.85
12.48
the SE condition of raw material.
4. Preparation of fufural from SE rice husk hemi-cellulose part
Presently, the main industrial raw materials for industrial furfural production are
corncob and bagasse. And the industrial process is to add sulfuric acid as catalyst, and then after two reactions the hemi-cellulose can be hydrolyzed to pentose
which can be finally hydrolyzed into furfural. Steam explosion pre-treatment can
decompose most of the hemi-cellulose into xylose and part of them can be further
hydrolyzed into furfural, meanwhile there is some acid produced in the steam explosion process which can act as catalyst in the later furfural preparation.
A series of tests were conducted on the preparation of furfural from SE rice
husk’s hemi-cellulose especially on the preparation process from SE extraction
liquor and optimize the reaction conditions. The results show great prospect in developing innovative process for the industrial furfural production and perfect the
total utilization of rice husk.
17 Biotechnological Potential of Cereal (Wheat and Rice) Straw and Bran Residues
339
17.4 Conclusion
Agricultural straws may vary in component contents with the breeds and environmental conditions, and even the same breed of straws can have distinct differences
on the chemical compositions and structures on each morphological fraction. Therefore, unless utilize the straw resource by combining multiple subjects of knowledge,
technology and industrial information, none of one single technology can realize the
development and utilization of straw resource.
To optimize cellulose processing by refining biomass pre-treatment and utilizing the residues is an advisable method to reduce cost and enhance comprehensive
utilization of straw. According to genetic engineering technology, we can design
or cultivate specific microorganism which can ferment both C-5 and C-6 sugars
and bear some inhibitions to promote current fermentation technology. And with
the increasing knowledge of the relation between straw’s cell wall structure and
inner mechanism of enzymatic hydrolysis, the gene modified straw can be designed
to meet the wanted needs(Energy 2006; Sedlak et al. 2003). The natural macromolecule and high polymer, and those high polymers obtained from microorganism
fermentation, can also be the ideal raw material for the production of bio-based
material and chemicals.
In a word, to establish the biomass transforming and utilizing technology network with the fractionated conversion of lignocellulose in the core can not only set
new ecological balanced system but also can form new biorefining product chain,
thereby initiate an innovative way to realize the farthest utilization of lignocellulose
and ultimately substitute the petroleum product chain.
Acknowledgments This work was financially supported by National Basic Research Program of
China (973 Program, 2004CB719700), National key technology R&D program (2007BAD39B01)
and Knowledge Innovation Program of Chinese Academy of Sciences (KSCX1-YW-11A1).
Abbreviations
SE:
SSF :
HPLC:
Steam Exploded
Simultaneous saccharification and fermentation
High Pressure Liquid Chromatography
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Chapter 18
Palm Oil Industry Residues
Mynepalli K.C. Sridhar and Olugbenga O. AdeOluwa
Contents
18.1 The Oil Palm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2 Oil Palm Residues and Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2.1 Utilization of Empty Fruit Bunch (EFB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2.2 Utilization of Palm Kernel Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2.3 Miscellaneous Organic Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2.4 Palm Oil Mill Effluent (POME) Management . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.2.5 Wastes to Energy and the Carbon Credit Schemes . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
342
343
343
348
350
350
353
354
Abstract Oil palm industry generates a large quantity of residues and wastes in
the form of empty fruit bunch, palm kernel shells, trunk of the plant, fibre, leaves
and others. When palm oil is extracted and processed, it also produces effluents
with high organic matter, suspended matter and oil and grease. These wastes cause
ecosystem degradation and affects health of the communities. This chapter describes
the methods of managing or treating the residues and wastes as these are untapped
resources. Some of the byproducts derivable from these wastes when appropriately
managed are energy, mulch, compost or organic fertilizer from empty fruit bunches
shells and sludges from effluent treatment. Empty fruit bunches and palm kernel
shells were successfully converted into compost by enriching with goat manure or
poultry manure and were useful in developing oil palm nurseries and other food
crops. Biogas and electricity are generated from effluent management, and several
biochemicals such as ethanol, fatty acids, waxes and others which could be obtained
through application of biotechnology. Palm oil wastes contribute to Green House
Gases (GHG) and conversion to energy is a good means of obtaining carbon credit
facility for sustainable management. The spent materials are also used in cultivating
mushrooms. These technologies find wider application in developing African and
Asian countries where oil palm plantations are major economic resource.
M.K.C. Sridhar (B)
Division of Environmental Health Sciences, Faculty of Public Health, University of Ibadan,
Ibadan, Nigeria
e-mail: mkcsridhar@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 18,
C Springer Science+Business Media B.V. 2009
341
342
M.K.C. Sridhar and O.O. AdeOluwa
Keywords Palm oil wastes · Energy · Mulch · Fertilizer · Palm kernel shells · Elaeis
guineensis Jacq
18.1 The Oil Palm
Oil palm cultivation originated in West Africa, where oil palm trees were originally
interplanted in traditional agricultural production systems along with other annual
and perennial crops. Some 5,000 years ago it was said to have been domesticated in
Nigeria. Production was for subsistence or trade within the region. By 1961 trade
in palm oil had increased substantially, and Nigeria had 74 per cent of the world’s
plantations (Poku 2002). By the early 1970s monocrop plantations of oil palm had
increased dramatically in Malaysia and Indonesia and by 2000, these two countries accounted for just over half of the world’s total plantation area, and Nigeria
accounted for just over 30 per cent.
Oil palm (Elaeis guineensis Jacq.) is the most productive oil producing plant in
the world, with one hectare of oil palm producing between 10 and 35 tonnes of
‘fresh fruit bunch (FFB)’ per year which is the harvested part. FFB can be harvested
generally after three years from planting. Largest amount of FFB is harvested about
10 years after planting. There are three main varieties of the West African oil palm:
Dura, Pisifera, and Tenera. The Tenera palm produces the highest oil content of
the three, but is actually a hybrid between the Dura and Pisifera. Over 40 per cent
of an individual palm fruit and over 20 per cent of a fruit bunch from a typical
Tenera palm can be extracted as palm oil. In its lifespan of 200 years, oil palm’s
economic life is 20–25 years. Of this, the plant spends its first 11–15 months in the
nursery, first harvest comes up in 32–38 months from planting, and peak yield is
5–10 years from planting. Normally, Oil palm grows in the lowlands of the humid
tropics, 15◦ N–15◦ S with evenly distributed rainfall of 1800–5000 mm/year. According to Hartley (1988), palm has a wide adaptability range of soils and low pH, but
sensitive to high pH (> 7.5), and to stagnant water. Oil palms are cultivated on large
plots of land with planting density of 128–148 plants per hectare. They are largely
dependent on the planting materials, soil, temperature and climate.
Oil is obtained from the fleshy mesocarp of the fruit and the yield is at least
45–56% of fresh fruit bunch, while in kernel, it accounts for at least 40–50%
(Kittikun et al. 2000). The theoretical potential yield for oil palm from both mesocarp and kernel has been estimated at 17 t oil ha–1 yr–1 (Corley 1983). At present,
average plantation yields in favourable environments are about 6 t oil ha–1 yr–1
which is considerably greater than yields from any other oil crop (Mutert and
Fairhurst 1999). Red palm oil is increasing in popularity, as it contains large quantities of carotenoids. Palm wine is made by tapping the male inflorescence of the oil
palm and fermenting the resulting sap. Alternatively, entire trees can be felled and
the meristem tapped, which is often done where old plantations are being replanted.
Palm wine has been an important part of West African culture, and is still made
today in large quantities, fetching good prices.
Usually large quantities of fertilizers are required to cultivate oil palm. Palm oil
is used as a cooking oil, is the ingredient for most margarine, is the base for most
18 Palm Oil Industry Residues
343
liquid detergents, soaps, and shampoos, and in its most dense form, serves as the
base for lipstick, waxes, and polishes. It is also used to reduce friction during the
manufacture of steel. Oil palm plantations are known in most countries for their
wide range of negative environmental impacts. These are land and water contamination, loss of land and resources to local inhabitant and loss of biodiversity brought
about by cultivation and management.
18.2 Oil Palm Residues and Wastes
Processing of Fresh Fruit Bunches (FFB) for oil extraction involves several steps:
i. Steam sterilization of bunches (inactivates lipase enzymes and kills microorganisms that produce free fatty acids, reducing oil quality)
ii. Stripping fruit from bunches
iii. Crushing, digestion, and heating of the fruit
iv. Oil extraction from macerated fruit (hydraulic pressing)
v. Palm oil clarification
vi. Separating fibre from the endocarp
vii. Drying, grading, and cracking of the endocarp
viii. Separating the endocarp from the kernel
ix. Kernel drying and packing
Oil palm industry produces a wide variety of wastes in large quantities. Liquid
wastes arise from oil extraction and processing. The solid wastes are the leaves,
trunk, empty fruit bunches, seed shells and fibre from the mesocarp. Many byproducts are made from theese wastes with a view of conservation of resources and to
safeguard environment (Figs. 18.1 and 18.2). Composting and biogas have been
produced for many years. In recent years, some of the notable biotechnological
ventures have been production of organic chemicals, ethanol and other organic solvents, fatty acids, polysaccharides, and polymers. In many developing countries,
every part of oil palm is utilized either for housing and fencing, energy source
or making local handicrafts (e.g. Raphia palm). Trunks of mature trees that have
been cut to allow replanting should be recycled more effectively. They contain up
to 1,000 Kg per hectare of potassium. Windrowing the trunks gives a slow breakdown of the material. This is the best way to release the nutrients. This chapter
reviews some of the methods and practices of oil palm residue utilization and waste
management.
18.2.1 Utilization of Empty Fruit Bunch (EFB)
EFB is a major waste product produced in oil palm plantations which need to be
managed. Every 25 metric tonnes of full fruit bunches yield 16 metric tonnes of
empty fruit branches. This can be returned to the fields. If applied at 6 metric tons per
hectare per year, given average yields, eventually it can return half of the nutrients
originally harvested in the bunches once it decomposes. In the past, EFB was often
344
M.K.C. Sridhar and O.O. AdeOluwa
Fig. 18.1 Wastes, residues and byproducts from palm tree
used as fuel to generate steam at the palm oil mills and many farmers still practice
it (Fig. 18.3). Currently, burning is prohibited globally to prevent air pollution (Ma
et al. 1993) and alternate ways of utilization are sought. The ash, with a potassium
content of about 30 per cent (Lim 2000) was used as fertilizer. The EFB is now
used mainly as mulch. Empty fruit branches and trunks can be chipped and used
as mulch if they are free of diseases (Hamdan et al. 1998). When used in circles
around mature trees, the mulch can reduce herbicide requirements, but it may be a
less efficient way to recycle nutrients than when spread over a larger area. Placed
around young palms, EFB helps to control weeds, prevent erosion and maintain
soil moisture. However, due to the current labour shortage, the transportation and
distribution of EFB in the field is getting more expensive. Empty fruit bunches have
also been used successfully in a joint Finnish/Indonesian project to produce paper.
These efforts should be evaluated and, to the extent that they are appropriate, should
be encouraged. Studies have shown the production of edible mushrooms is also a
financially viable possibility (WWF 2005).
There is a growing interest in composting EFB, in order to add value, and also
to reduce the volume to make application easier (Yusri et al. 1995, Thambirajah
et al. 1995, Danmanhuri 1998). Aisueni and Omoti (1999) reported that oil palm
18 Palm Oil Industry Residues
345
Fig. 18.2 (1) Palm tree; (2) Palm fruits for oil extraction of red palm oil and palm kernel oil;
(3) Empty Fruit Bunch (EFB) used for energy or composting; (4) Pulp from palm fruits after
extractopm pf po; ised as emergu spirce’ (5–7) Raphia palm leaves and fronds used for fencing,
roofing or weaving of mats
industry is one of the best sources of agricultural wastes that can be used as organic fertilizers. According to the authors, the palm industry in Nigeria produces 40
million metric tonnes FFB (fresh fruit bunch) annually of which about 16 million
tonnes can be gotten for composting into organic manure. Apart from the EFB, palm
kernel also has great potential for organic fertilizer production.
346
M.K.C. Sridhar and O.O. AdeOluwa
30
25
Fuel
24.24
Refuse
21.21
20
Compost
15.15
15
Soap making
12.12
12.12
10
selling
Multiple
choices
5
0
Fig. 18.3 Percentage of oil palm farmers in Nigerial managing the EFB
EFB is composed of 45–50 per cent cellulose and about 25–35 per cent of hemicellulose and lignin (Deraman 1993). It is fibrous, and the fibres stick together to
form vascular bundles. Short, uniform, fibres can be obtained by mechanical cutting
of the EFB. The fibrous nature of the material promotes aerobic conditions and
therefore is considered suitable for the production of good quality compost (Suhaimi
and Ong 2001). During the composting process, nitrogen is immobilized.
The natural decomposition of EFB in oil palm plantations after the fruits have
been harvested was studied by Hamdan et al. (1998). The EFB was spread in the
field as mulch on top of nylon net, at a rate of 30, 60 and 90 mt/ha/year. At each
EFB application rate, spots were selected for nitrogen supplementation to meet a
C: N ratio of 15, 30 and 60 (Control). Decomposition was estimated by the weight of
EFB remaining in the nylon net. The EFB was found to be completely decomposed
after 10 months of application.
Composting of EFB is being extended to farmers by the Department of Agriculture of Malaysia (Danmanhuri 1998). The initial method adopted was to mix the
EFB with 20% chicken manure, heap it in 3 × 3 × 0.7 m boxes and cover with
plastic. It took 11 to 12 months to mature. In 1995, this method was modified by
first exposing big piles of EFB in the open for two months. Then mixed with 20%
chicken manure and heaped in sheds measuring 12 × 36 × 3 m, the heap was mixed
regularly at monthly intervals. It took about 4 months to reach maturity. Maturity
was determined when the temperature of heap stabilized at 30◦ C and the pH reading
4.5 to 6.0. Agharan (1984) composted EFB of varying fractions (bunches cut into
2, 4, 8 portions, and whole bunch as control), mixed with chicken dung at a ratio
of 3 parts of EFB to 1 part of chicken dung. The moisture content, pH, C: N ratio,
Mg, Ca, and P content of finished compost showed a rise in every heap when the 1st
week’s value was compared with the 10th week’s value. For the K content, heaps
showed a difference in % value, no difference in nutrient levels between the different
fractions of cuts studied. After the 10th week, the heap made up of bunches cut into
8 became crumbly and loose and the colour turned slightly black, while the others
partly disintegrated with dark brown colour.
18 Palm Oil Industry Residues
347
Danmanhuri (1998) reported that about 15 m3 of compost could be turned in
one hour, using a tractor equipped with a backhoe to mix the compost. Composted
material was shredded at the end of composting period and was left for 1 to 2 weeks
prior to packing. The final product contained (%) total nitrogen 3.3, phosphate 0.05,
K 0.2, Ca 1.0 and Mg 0.2. Different organic N sources like the manure of goat,
cattle and chicken, have also been evaluated as N additives for the composting of
EFB (Thambirajah et al. 1995) adding 25 kg of manure per 90 kg shredded EFB
reduced the C: N ratio. EFB composted with goat, cattle and chicken manure had a
C:N ratio of 14:1, 18:1, and 12:1 respectively, after 60 days of composting, while
the control without manure had C:N ratio of 1:24.
Zaharah and Lim (2002) investigated the decomposition of nutrients released
from EFB component parts (spikelet, stalk and mixture of stalk and spikelet) with
and without mineral N fertilizers under field condition. First part of the experiment
was observed for a period of 9 months and in the second part, a field experiment
was also conducted where 17 year-old palms were treated with different N and K
inorganic fertilizer rates, with and without EFB mulching. It was observed that the
EFB component parts decomposed at a significantly different rates in the order of
stalk > mixture > spikelet Lignin, polyphenol, C and N contents in EFB showed
good correlation with soil N dynamics. In mature oil palm, application of inorganic
N, EFB and N + EFB significantly increased the yield. The soil chemical characteristics, pH value, organic C and exchangeable K were significantly improved with
EFB application, making EFB a suitable ameliorant in improving soil quality for
sustainable oil palm production.
The composition of EFB from Nigerian studies by AdeOluwa (2005) is shown
in Table 18.1. Here, EFB was converted to compost and used the product to grow
oil palm nurseries. EFB, EFB soaked in water (for 5 days) and cow dung were used
in the development of a composting process. The EFB and soaked EFB were mixed
with varying proportions of cow dung and composted aerobically. The mixing of
Table 18.1 Composition of unsoaked and soaked EFB, and Cow dung used as raw materials
to produce compost
Raw materials used for compost
Composition
Unsoaked EFB
Soaked EFB
Cow dung
N, %
C, %
C: N
P, %
K, %
Ca, %
Mg, %
S, %
0.97
45.0
46.48
0.05
0.97
0.3
0.32
0.14
1.08
44.7
41.39
0.05
0.43
0.27
0.26
0.15
2.26
36.05
15.99
0.3
2.96
1.28
0.8
0.37
Zn, mg kg–1
Cu, mg kg–1
Mn, mg kg–1
Fe, mg kg–1
49
17
118
706
40
12
107
597
98
25
323
1842
348
M.K.C. Sridhar and O.O. AdeOluwa
Table 18.2 Composition of the compost obtained after 5 weeks from unsoaked and soaked EFB
Composition
60% EFB + 40% cow dung
60% SEFB + 40% cow dung
N, %
C, %
C: N ratio
P, %
K, %
Ca, %
Mg, %
S, %
1.19
44.45
37.89
0.11
1.19
0.47
0.40
0.21
1.50
43.75
30.45
0.13
1.10
0.44
0.37
0.21
Zn, mg kg–1
Cu, mg kg–1 ,
Mn, mg kg–1 ,
Fe, mg kg–1
99
19
144
650
85
18
153
644
EFB and cow dung in the ratio of 60:40 yielded good quality comost which has
matured within 5 weeks (Table 18.2).
The unsoaked EFB treatments had higher nutrient elements than the soaked EFB,
except for N, P and S. Lower nutrients in soaked pre-treatments should have been
caused by the leaching of these nutrients in water used for soaking the EFB.
Application of EFB composts at the rate of 100 g/10 kg pot indicated significant
growth of palm seedlings in th enursery. The results showed: girth (0.71 cm) and
index leaf dry weight (0.33 g) of oil palm seedlings under the application of soaked
oil palm EFB and cow dung (60:40) were significantly (p < 0.05) higher than those
from the urea treatment (0.44 cm girth and 0.13 g index leaf dry weight) in the prenursery. Unsoaked EFB and cow dung also in the same ratio 60:40 resulted in the
best seedling growth in the nursery (p < 0.05). Oil palm seedling growth in the nursery was not significantly influenced by the quantity of EFB combined with mineral
fertilizer. However, combination of 21 g plant–1 NPKMg (1:1:1:1) with unsoaked
EFB: cow dung (60:40) significantly (p < 0.05) increased the number of leaves of
oil palm seedling compared to other levels of mineral fertilizer investigated.
18.2.2 Utilization of Palm Kernel Wastes
Palm kernel oil (white palm oil) is obtained from the seed known as kernel or endosperm. When the oil has been extracted, the residue known as ‘palm kernel cake’
(PKC) is rich in carbohydrate (48 per cent) and protein (19 per cent) and is used as
cattle feed (Onwueme and Sinha 1991). The ash contains large amounts of potassium. When the PKC is further solvent extracted to remove oil, it becomes ‘palm
kernel de-oil cake’ which has little or no nutritional value (carbon 42.73%, nitrogen
0%, volatile matter 67.71% and calorific value 4031 Kcal/Kg) and is mostly used
as fuel source in industry. As PKC is deficient in nitrogen, there is need to amend it
with additional nitrogen if it has to be converted into compost. In the West African
communities livestock wastes are readily available and thus become a rich source
18 Palm Oil Industry Residues
349
Table 18.3 Composition of wastes used in the composting (Dry weight basis)
Waste
Moisture
content, %
Carbon
(C%)
Nitrogen
(N %)
Phosphorus Potassium
(P)
(K)
Palm Kernel Cake
Goat dung
Poultry droppings
58.92
29.79
49.11
96.21
75.94
78.83
2.88
3.62
2.83
0.60
0.51
3.29
0.19
0.18
0.16
for nitrogen amendment. The most common livestock which are reared around the
farms and residences are goats and sheep, poultry and to some extent piggery. Cows
are common in certain parts.
Kolade et al. (2006) developed a process of converting PKC into compost using
poultry manure, and goat manure as supplements. Composting was carried out using combinations of PKC and poultry manure (3:1 ratio) and PKC and goat/sheep
manure (3:1 ratio). The composting was carried out in locally made woven baskets
which facilitate natural aeration of the composting material. The amount of waste in
each basket was 10 Kg (7.5 Kg PKC and 2.5 Kg livestock waste) and kept in a green
house at the University. A clean plastic sheet was spread under each basket to collect
the leachates and putting back on to the composting material. The composting was
carried out for six weeks. The composition of the raw materials and the finished
product are given in Tables 18.3 and 18.4.
The quality of the finished composts was assessed by following the nutrient
levels, C: N ratio, moisture level and texture. The compost quality was within
the acceptable limits (Table 18.2). The compost made from PKC and goat manure
however showed higher nitrogen and phosphorus levels which are needed for crop
production in Nigeria. Increasing the nitrogen levels in the composts prepared from
wastes is a challenge and supplementing with natural sources of nitrogen is more
environmentally friendly than opting for mineral sources (Sridhar et al 2001). The
results of growth experiments using the test crop Amaranthus spp indicate that the
performances of composts prepared from PKC + poultry manure and PKC + goat
manure were comparable when applied at 4 tons/Ha with those of Organo-mineral
fertilizer or chemical fertilizer (NPK 15-15-15).
Referring to the paper by Kolade et al. (2006), Jim Vlahos, a businessman
(jimvlahos@comcast.net) commented thus: “Creating charcoal from spent (or waste)
palm kernels may have various forms of new benefit and value. Two interesting applications I can see for this charcoal is, first, a soil amendment instead of
Table 18.4 Composition of the composts prepared from palm kernel cake (Composting period of
6 weeks)
Treatment
PKC+ poultry manure
(3:1 ratio by dry weight)
PKC + goat manure
(3: 1 ratio by dry weight)
pH
value
Carbon
(C %)
Nitrogen
(N %)
Phosphorus Potassium
(P%)
(K%)
7.35
81.56
3.52
0.188
0.277
7.46
80.64
4.63
0.195
0.149
350
M.K.C. Sridhar and O.O. AdeOluwa
composting and, second, activated carbon. For the soil application, a great deal
of information can be found at this website: http://www.css.cornell.edu/faculty/
lehmann/index.htm. In addition to improving crop yields by putting the charcoal
in the soil, the CO2 that would have been released by burning the palm kernels in a
boiler would be sequestered as charcoal in the soil. . . . palm kernel is very dense and
can be used to create a quality activated carbon, which is used around the globe in
filtering pollutants from water as well as power-plant gaseous emissions. These also
represent significant global challenges. As a businessman, I am trying to identify an
abundant, low-cost source of palm kernels that have already had the oils extracted
from them. In addition to working to solve the global challenges I mentioned, your
paper indicated that there is also a more specific problem around palm kernel waste”
(Jim Vlahos 2008, Personal communication).
18.2.3 Miscellaneous Organic Chemicals
Kouichi Miura (2001) reported researches on the utilization of oil palm wastes in
which a variety of chemicals were derived:
(a) molecular sieving carbon by the phrolysis of oil palm shell impregranted with
ZnCl2 ,
(b) treatment with acetosolv- and ethanosolv-processes to obtain pulp of high quality,
(c) recovery of 30% of small molecule fatty acids and pure cellulose were successfully recovered from oil palm shell through hot water and solvent extraction,.
(d) using a new catalyst, zirconia supporting FeOOH, developed recovery of valuable chemicals from the waste water containing ligno-cellulose, and
(e) developed new methods to produce a bacterial biodegradable plastic from Palm
Oil Mill Effluent (POME).
18.2.4 Palm Oil Mill Effluent (POME) Management
Wastewater effluent from palm oil mills is a mixture of water, crushed shells, and
fat residue resulting from initial processing of crude palm oil from fresh palm fruits,
which must be crushed within 24 hours of harvest. For every one tonne of Crude
Palm Oil (CPO) produced, 2.5 m3 of POME is generated which is a universally
accepted figure. POME generation rate per FFB will be 0.5 m3 -POME/t-FFB. In
Malaysia some 10 million tonnes of POME is produced as against 9.9 million tones
of solid waste from palm oil processing annually.
Igwe and Onyegbado (2007) reviewed the existing technologies that are found
applicable in treating high BOD effluents such as POME.
1. Tank digestion and facultative ponds: In this system, raw effluent after oil
trapping is pumped to a closed tank which has a retention time of about 20 days.
The liquid is mixed by means of horizontal stirrers. The methane gas (CH4 )
18 Palm Oil Industry Residues
2.
3.
4.
5.
351
generated is flared off into the atmosphere. Digested liquid is discharged into a
holding pond before it is disposed on to land (Songeha 1974).
Tank digestion and mechanical aeration: This includes digestion tank and an
aeration pond. Raw effluent after oil trapping is pumped to the acidification pond
through a cooling tower and retained for one to two days. It is then mixed with
an equal volume of liquid from the anaerobic digester before it is fed back to the
digester. The hydraulic retention time of the digester is about twenty days. The
digested liquid is discharged to an aeration pond with two floating aerators. The
liquid is aerated for twenty days before it is discharged.
Decanter and facultative ponds: In a few mills, decanters are used to separate
the fruits juice after pressing into liquid and solid phase, the liquid which is
mainly oil is fed to the conventional clarification process. The water resulting
from the clarification station is recycled. The solid is either disposed off on land
or is dried in a rotary drier to about 10 per cent moisture and then used as fuel.
The effluent thus consists of only the sterilizer condensate and waste from the
hydro cyclone and is greatly reduced in volume to be treated in a series of ponds
(Wood 1984).
Anaerobic and facultative ponds: This system consists of a series of ponds
connected in series for different purposes. The effluent after oil trapping is retained in an acidification buffering pond for about two or three days and the
resultant effluent is treated in an anaerobic pond with a hydraulic retention time
of thirty to eighty days depending on the mills. This digested liquid is further
treated in a series of facultative ponds before it is discharged. In some cases, part
of the digested liquid is recycled to the acidification and buffering pond. The
total hydraulic retention time of the system ranges from 75 to 120 days.
Antra system: The treatment consists of a combination of mechanical, chemical
process and ponds. The raw effluent after oil trapping is separated into water and
solid phases using a three- phase decanter. The oil is returned to the main line
while the solid is dried in a rotary drier after the filter press. The water containing dissolved and suspended solids is treated with coagulants and flocculants to
remove as much solids as possible before it is fed to an anaerobic digester which
has a hydraulic retention time of about ten days. The digested liquid is further
treated in an aeration tower and then oxidized (Sinnappa 1978).
Some of the more recent tested technologies to manage the POME are anaerobic digestion, anaerobic digestion with methane tapping, conversion of methane
to electricity generation, aerobic lagooning, conventional waste treatment using biological methods e.g. activated sludge process, extended aerobic process and bio
reactor system. Some of these practices are:
(A) Up-flow anaerobic sludge fixed film (UASFF) reactor: Zinatizadeh et al.
(2006) studied the interactive effects of feed flow rate (Q F ) and up-flow
velocity (Vup ) on the performance of an ‘Up-flow Anaerobic Sludge Fixed
Film (UASFF) reactor treating palm oil mill effluent (POME). Long-term
performance of the UASFF reactor was first examined with raw POME at a
352
(B)
(C)
(D)
(E)
M.K.C. Sridhar and O.O. AdeOluwa
hydraulic loading rate (HRT) of 3 d and an influent COD concentration of
44,300 mg/l. Twelve dependent parameters were either directly measured or
calculated as response. These parameters were total COD removal, soluble
COD removal, effluent pH, effluent total volatile fatty acid, effluent bicarbonate
alkalinity, effluent total suspended solids, CH4 percentage in biogas, methane
yield (YM ), specific methanogenic activity (SMA), food-to-sludge ratio (F/M),
sludge height in the UAS Bioreactor portion and solids retention time (SRT).
The optimum conditions for POME treatment were found to be 2.45 l/d and
0.75 m/h for Q F and Vup , respectively (corresponding to HRT of 1.5 d and recycle ratio of 23.4:1). The present study provides valuable information about
interrelations of quality and process parameters at different values of the operating variables.
Pond system: Chin et al. (1996) treated POME containing COD in the range
of 45,000 to 65,000 mg/l, 5-day BOD 18,000 to 48,000 mg/l and oil and grease
greater than 2,000 mg/l. The COD:N:P ratio is around 750:7.3:1. The nutrient content is low for aerobic treatment processes but sufficient for anaerobic
treatment processes. They evaluated the treatment efficiency of a pond system
consisting of 8 ponds in series treating 600 cu m/day of waste water. The
pond system has been in operation since the mid 1980’s. Effluent COD was
1,725 mg/l, BOD 610 mg/l, ammonia-N 115 mg/l, nitrate nitrogen 5 mg/l, TKN
200 mg/l and phosphate 60 mg/l. However, the effluent was not able to meet the
discharge standard of 50 mg/l BOD.
Use of a marine yeast: A study carried out by Oswal et al. (2002) showed that
palm oil mill effluent (POME), from a factory site in India contained about
250, 000 mg l(–1) chemical oxygen demand (COD), 11, 000 mg l (–1) biochemical oxygen demand, 65 mg l(–1) total dissolved solids and 9000 mg l(–1) of
chloroform-soluble material. Treatment of this effluent using Yarrowia lipolytica NCIM 3589, a marine hydrocarbon-degrading yeast isolated from Mumbai, India, gave a COD reduction of about 95% with a retention time of two
days. Treatment with a chemical coagulant further reduced the COD and a
consortium developed from garden soil clarified the effluent and adjusted the
pH to between 6 and 7. The complete treatment reduced the COD content to
1500 mg l(–1) which is a 99 per cent reduction from the original.
Anaerobic digestion: In Lepar Hilier Palm Oil mill, Malaysia, at present there
are two systems used in treating POME, firstly lagoon system second is the
combination of lagoon and open digesting tanks. The Mills have 24,600 Ha of
oil palm plantation which produces 54 t/Ha FFB. Annually, 259,890 tonnes of
FFB is processed with the production of 300 to 4000 tonnes/month of CPO.
The effluent produced has COD of 40,000 to 60,000 mg/l which has to be
treated to the National standards of < 100 BOD before discharging into river.
Anaerobic lagoon method (30, 000 m 3 × 4 ponds) was adopted.
Activated sludge process: The efficiency of the activated sludge process was
evaluated by treating anaerobically digested and diluted raw POME obtained
from Golden Hope Plantations, Malaysia (Vijayaraghavan et al. 2006). The
treatment of POME was carried out at a fixed biomass concentration of
18 Palm Oil Industry Residues
353
3900 ± 200 mg/L, whereas the corresponding sludge volume index was found
to be around 105±5 mL/g. The initial studies on the efficiency of the activated
sludge reactor were carried out using diluted raw POME for varying the hydraulic retention time, viz: 18, 24, 30 and 36 h and influent COD concentration,
viz: 1000, 2000, 3000, 4000 and 5000 mg/L, respectively. The results showed
that at the end of 36 h of hydraulic retention time for the above influent COD,
the COD removal efficiencies were found to be 83%, 72%, 64%, 54% and 42%
whereas at 24 h hydraulic retention time they were 57%, 45%, 38%, 30% and
27%, respectively. The effectiveness of aerobic oxidation was also compared
between anaerobically digested and diluted raw POME having corresponding
CODs of 3908 and 3925 mg/L, for varying hydraulic retention time, viz: 18, 24,
30, 36, 42, 48, 54 and 60 h. The dissolved oxygen concentration and pH in the
activated sludge reactor were found to be 1.8–2.2 mg/L and 7–8.5, respectively.
The scum index was found to rise from 0.5% to 1.9% during the acclimatizing
phase and biomass build-up phase.
18.2.5 Wastes to Energy and the Carbon Credit Schemes
In recent years, a novel closed tank methane recovery system and conversion to
electricity generation was being planned for Lepar Hilier Palm Oil mill (Kyushu
Institute of Technology 2003). Scientifically, it has been proved that anaerobic digestion of the bio-waste POME will produce biogas which is a mixture of CH4
(65%) and CO2 (35%), which are Green House Gases (GHG). It has been shown that
CH4 emitted from this process has a good potential in the power generation using
a gas engine. Biogas yield from POME is approximately 20 to 28 (m3 − CH4 /m3 Biogas). 1 m3 biogas has the potential to generate approximately 1.8 kWh, which is
about 25% power generation efficiency of its heat value. The power generated then
can be supplied to Power Company by grid connection or can be consumed locally
by the mill, small/medium scale industries or settlers in the residential areas. This
proposal has the environmental component and carbon credit investment through
reducing the GHG from reducing the emissions as well as saving from conventional
power generation.
The Asian Palm Oil Company in Thailand is a producer of crude palm oil, with
a production capacity of 40,000 tonnes of fresh palm per hour (total capacity is
50,000 tonnes). The company has 62 employees. The company converted its boilers
to be able to utilize palm shells and fibre, rather than fuel oil, while the palm kernels are sold to manufacturers of low-grade palm oil. Using a ‘Completely Stirred
Tank Reactor “(CSTR) for energy generation With support from the Energy for
Environment Foundation, the company also installed an anaerobic wastewater treatment system. The major piece of equipment is CSTR that uses microorganisms to
digest the organic substances in the wastewater under anaerobic conditions. This
process reduces BOD in the wastewater at the same time as it produces biogas. The
biogas is then used to generate electricity. It was concluded from the experience
that waste-to-energy practices require intensive technology development and high
354
M.K.C. Sridhar and O.O. AdeOluwa
investment costs, and operating personnel must be qualified to handle the system
(Waranusantikule 2003).
From the studies reviewed, POME management is a difficult task. A combination
of treatment techniques should be used and there is need for adequate land availability. Biological treatment is the solution and the partially treated effluent and the
sludge obtained makes an excellent soil additive. There is evidence that nitrogen
may be lost if POME is stored for long periods in effluent ponds.
References
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Danmanhuri M A (1998). Hands-on experience in the production of empty fruit bunches (EFB)
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Igwe J C and Onyegbado C C (2007) A Review of Palm Oil Mill Effluent (POME) Water Treatment. Global Journal of Environmental Research, 1(2): 54–62
Kittikun A H, Prasertsan P, Srisuwan G and Krause A (2000) Environmental Management of Palm
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Kolade O O, Coker A O, Sridhar M K C, and Adeoye G O (2006). Palm kernel waste management
through composting and crop production. The Journal of Environmental Health Research, UK,
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Part IV
Enzymes Degrading Agro-Industrial
Residues and Their Production
Chapter 19
Amylolytic Enzymes
Dhanya Gangadharan and Swetha Sivaramakrishnan
Contents
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.2 Amylolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.2.1 Exo Acting Amylases – Glucoamylases and β-Amylases . . . . . . . . . . . . . . . . . .
19.2.2 Endo Acting Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.2.3 Debranching Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.2.4 Cyclodextrinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.3 Production of Amylolytic Enzymes – Effective Utilisation of Agro Residues . . . . . . . .
19.3.1 Cereals and Cereal Bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.3.2 Oil Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.3.3 Other Starchy and Non Starchy Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
360
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361
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362
363
364
365
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Abstract Amylolytic enzymes act on starch and related oligo- and polysaccharides.
The recent wealth of information on the DNA sequence, structural analysis and
catalytic mechanism led to the extensive research on starch hydrolyzing enzymes
which led the concept of the alpha amylase family. Amylolytic enzymes are extensively used in starch liquefaction, paper industries, food, pharmaceutical and sugar
industries which demands a specific hydrolysis profile. To fulfill the industrial requirements, the primary concern is the formulation of a simple indigenous and cost
effective system for producing high titers of amylases. One alternative low cost and
feasible production method is the use of agro-industrial residues as fermentation
substrates. These residues represent one of the best reservoirs of fixed carbon in nature. Considerable research has been carried out in the effective utilization of these
residues in large scale production of enzymes. This chapter gives a brief overview
on the wide range of naturally occurring agricultural by products explored so far for
the production of amylolytic enzymes.
D. Gangadharan (B)
Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST),
CSIR, Trivandrum-695 019, Kerala, India
e-mail: dhanya 28@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 19,
C Springer Science+Business Media B.V. 2009
359
360
D. Gangadharan and S. Sivaramakrishnan
Keywords Amylases · Agro-industrial residues · Starch hydrolyzing enzymes · Oil
cakes · Bran · Solid-state fermentation
19.1 Introduction
Starch represents one of the most abundant storage polysaccharides in nature and
the most popular ingredient in food. It is composed exclusively of ␣-glucose units
that are linked by ␣-1,4- or ␣-1,6-glycosidic bonds. The two high-molecular-weight
components of starch are amylose (15–25%), a linear polymer consisting of ␣1,4-linked glucopyranose residues with their molecular weights varying from hundreds to thousands, and amylopectin (75–85%), a branched polymer containing, in
addition to ␣-1,4 glycosidic linkages, ␣-1,6-linked branch points occurring every
17–26 glucose units with molecular weight as high as 100 million (Bertoldo and
Antranikian 2002). Because of the complex structure of starch, cells require an appropriate combination of enzymes for its depolymerization to oligosaccharides and
smaller sugars, such as glucose and maltose. Amylolytic enzymes play an important
role in the degradation of starch and are produced in bulk from microorganisms
representing about 25–33% of the world enzyme market. Microbial enzymes are
preferred for their stability over plant and animal enzymes which increases their
spectrum of industrial applications. They also have the advantages of cost effectiveness, consistency, less time and space required for production and ease of process
modification and optimization. The amylolytic enzymes find a wide spectrum of
applications in food industry for production of glucose syrups, crystalline glucose,
high fructose corn syrups, maltose syrups, reduction of viscosity of sugar syrups, reduction of haze formation in juices, solubilization and saccharification of starch for
alcohol fermentation in brewing industries, retardation of staling in baking industry, in detergent industry used as an additive to remove starch based dirts, in paper
industry for the reduction of viscosity of starch for appropriate coating of paper, in
textile industry for warp sizing of textile fibers and in pharmaceutical industry they
are used as a digestive aid (Sivaramakrishnan et al. 2006).
The vast research on whole genome sequencing and the accumulated protein
sequence databases since the last two decades led to the study of a full range of
starch hydrolyzing enzymes. The homology among alpha amylases from different
origins was first studied by (Friedberg 1983). The detailed study of the amylolytic
enzymes proved the existence of four highly conserved regions in eleven different
␣-amylases which is related to the catalytic and substrate binding sites (Nakajima
et al. 1986). Thus the structural similarity and common catalytic mechanism among
most of the amylases led to the concept for one enzyme family, ‘the alpha amylase
family’. The family included enzymes acting on ␣-glucosidase linkage to produce
␣-anomeric mono and oligosaccharides or form ␣-glucosidic linkages by transglycosylations, they possess four conserved regions containing the catalytic (Asp-206,
Glu-230 and Asp-297) and substrate binding sites and a (/␣)8 or TIM barrel catalytic domain (Kuriki and Imanaka 1999). Thus the amylolytic and related enzymes
19 Amylolytic Enzymes
361
have been classified into the families of glycoside hydrolases (GHs) and almost one
hundred GH families have been reported. Amylolytic enzymes of microbial origin
are divided into exo-acting, endo-acting, debranching and cyclodextrin producing
enzymes.
19.2 Amylolytic Enzymes
19.2.1 Exo Acting Amylases – Glucoamylases and β-Amylases
Glucoamylases (1,4-␣-D-glucan glucohydrolase, EC 3.2.1.3) catalyse hydrolysis of
␣-1,4 and ␣-1,6 glucosidic linkages to release -D-glucose from the non-reducing
ends of starch and related poly- and oligosaccharides. They have widely been reported to occur in a large number of microbes, including bacteria, yeast and fungi.
Filamentous fungi, however, constitute the major source among all microorganisms
and strains of genera Aspergillus and Rhizopus are mainly used for commercial
production (Pandey 1995). -amylases are known to be produced only by plants
and certain bacteria mostly by several species of the genus Bacillus, including
B. polymyxa, B. cereus, B. megaterium, and also by Clostridium thermosulfurogenes
(Selvakumar et al. 1998). They hydrolyze ␣-1,4 bonds but cannot bypass ␣-1,6
linkages in amylopectin and glycogen and they produce maltose from amylose and
maltose and a -limit dextrin from amylopectin and glycogen.
19.2.2 Endo Acting Amylases
␣-amylases (E.C. 3.2.1.1.) hydrolyse ␣-1,4 bonds and bypass ␣-1,6 linkages in amylopectin and glycogen. In spite of the wide distribution of amylases in microbes,
animals and plants, microbial sources, namely fungal and bacterial amylases are
preferred in industries. Among bacteria, Bacillus sp. is widely used for thermostable
␣-amylase production while fungi belonging to the genus Aspergillus are most common (Sivaramakrishnan et al. 2006). They are classified in two categories depending
on the extent to which they hydrolyze starch. Liquefying ␣-amylases hydrolyze 30
to 40% of starch and saccharifying ␣-amylases hydrolyze 50 to 60%.
19.2.3 Debranching Amylases
Isoamylases and pullulanases are debranching enzymes that hydrolyze only ␣-1,6
linkages. On the basis of substrate specificity and product pattern, pullulanase (pullulan ␣-glucano-hydrolase; EC 3.2.1.41) have been classified into two groups: type
I and type II. As they hydrolyze the ␣-glucosidase-resistant ␣-1,6 linkages in dextrins, they improve the starch saccharification rate and yield when used in combination with ␣-glucosidases. Many mesophilic (Aerobacter aerogenes, B. macerans,
B. acidopullulyticus and Bacillus sp), thermophilic and hyperthermophilic bacteria
362
D. Gangadharan and S. Sivaramakrishnan
and archae (B.stearothermophilus, Clostridium thermosulfurogenes, Pyrococcus
and Thermococcus genus) have been reported to produce pullulanase (Gomes
et al. 2003, Kunamneni and Singh 2006).
19.2.4 Cyclodextrinases
Cyclodextrin glycosyltransferase (␣-1,4-D-glucan,␣-4-D-(␣ − 1,4-D-glucano)transferase, EC 2.4.1.19) produces a series of non-reducing cyclic dextrins (␣-,
- and ␥-cyclodextrins) from starch, amylose, and other polysaccharides. ␣-, - and
␥-cyclodextrins contain six, seven and eight glucose units, respectively, that are
linked by ␣-1,4-bonds. Thermococcus sp., B. coagulans, C. thermohydrosulfuricum
39E, B. sphaericus and alkalophilic Bacillus sp are the most reported microorganisms producing these enzymes.
19.3 Production of Amylolytic Enzymes – Effective
Utilisation of Agro Residues
Commercial production of enzymes is generally carried out by submerged (SmF)
and solid state fermentation (SSF). The physico-chemical and nutritional requirements are unique for a particular microorganism. The composition and concentration of media and fermentation conditions greatly affect the growth and production
of extracellular enzymes from microorganisms. The production of amylolytic enzymes in submerged fermentation employing synthetic media have been largely
exploited (Tigue et al. 1995 and Hamilton et al. 1999) but is limited by the high
cost of production. In the case of Smf the abundance of water gives more control of
environmental factors such as temperature, oxygen concentration and pH and also
provides ease in handling. However few reports have suggested agro residues as
an alternative for synthetic basal media for the production amylase (Crueger and
Crueger 2000, Hernandez et al. 2006, Gangadharan et al. 2008). Large scale production of enzymes would require formulation of a cost effective media and the
commercial success of amylases is linked to the utilization of starchy biomass as an
industrial raw material. The most inexpensive and highly energy rich substrates for
fermentation is represented by agro-industrial residues.
The utilization of agro residues for the production of enzymes has gained renewed interest from researchers for the use of SSF as it solves solid waste disposal
problem and also produce lesser waste water (Pandey 2003). Initially SSF was considered to be suitable for fungi and yeast considering the low water activity but there
has been continous exploitation of bacterial cultures (Nampoothiri and Pandey 1996,
Gangadharan et al. 2006 and Selvakumar 1999). Several naturally occurring agricultural by products such as wheat bran, coconut oil cake, groundnut oil cake, rice bran,
wheat and paddy straw, sugar beet pulp, fruit pulps and peels, corn cobs, saw dust,
maize bran, rice husk, soy hull, sago hampas, grape marc, coconut coir pith, banana
19 Amylolytic Enzymes
363
waste, tea waste, cassava waste, aspen pulp, sweet sorghum pulp, apple pomace,
peanut meal, cassava flour, wheat flour, corn flour, steamed rice, steam pre-treated
willow, starch etc. could be used in one or the other industrial bioprocess for the
production of value added products through SSF (Pandey et al. 2001).
Among the above substrates amylolytic enzyme production have been carried
out mainly with wheat bran, rice bran, rice husk, oil cakes, tea waste, cassava,
cassava bagasse, sugarcane bagasse (Mulimani et al. 2000). Banana waste, corn
flour, saw-dust, soybean meal, sweet potato, potato, rice hull, sugar beet pulp have
also been tried by some of the researchers. The utilization of agro residues such
as wheat bran, molasses bran, maize meal, millet cereal, wheat flakes, barley bran,
crushed maize, corncobs and crushed wheat have been exploited for the production alpha amylase by thermophilic fungus Thermomyces lanuginosus under solid
state fermentation. Among the amylolytic enzymes commercial production of alpha
amylase and glucoamylase utilizing agro residues are well studied and production
of  amylases and pullulanase have been studied to a some extent. There are limited
reports on the production of cyclodextrinases and research on the utilization of agro
residues are yet to be explored in detail Thus the agro residues popularly employed
for amylolytic enzyme production can be broadly classified as cereal brans, oil cakes
and other starchy and non starchy substrates.
19.3.1 Cereals and Cereal Bran
Cereals are the fruits of cultivated grasses belonging to the genus Gramineae. The
main cereals grown are wheat, rice, corn, barley, oats, sorghum and millet. Cereal
grains contain 60–70% starch (Dendy and Dobraszczyk 2001). They have similar structure which include a hull (husks) and a kernel (caryopsis) and the kernel
contains three components – bran, germ and endosperm. The bran is separated
from cleaned and scoured cereals during milling. Among different agricultural
by-products evaluated, wheat bran was found to be the best basal and standardized medium for optimal production of alpha amylase (Haq et al. 2003, Baysal
et al. 2003, Balkan and Ertan 2007, Sivaramakrishnan et al. 2007). The strains of
Bacillus sp. AS-1 and Aspergillus sp. AS-2 colonized well on the wheat bran based
solid media and exhibited high production of ␣ amylase and glucoamylase (Soni
et al. 2003).
Table 19.1 Chemical composition of wheat bran and rice bran
Content
Moisture
Protein
Fat
Ash
Total dietary fibre
Starch
Wheat-bran (%)
6.4
16.4
6.8
6.5
44.5
11.1
Rice bran (%)
12
16
22
10
25
10–20
364
D. Gangadharan and S. Sivaramakrishnan
The physico- chemical properties of wheat bran (soft white) and rice bran are
tabulated (Table 19.1). Corn gluten meal (CGM), a by-product of corn wet milling
which are traditionally used for animal feed was found to be a promising substrate for the production of ␣ amylase by B. amyloliquefaciens due to its high
content of proteins (≥ 60%), vitamin and other minerals (Tanyildizi et al. 2007).
Agricultural raw starches such as pearl millet, rice, gram, hordium, corn and
wheat starches at 1% levels were tested for the production of alpha amylase by
B. licheniformis (Haq et al. 2005). Barley and oat brans are chiefly composed of
beta glucans.Comparatively higher production was found in the case of pearl millet,
which is represented by 56–65% starch, 20–22% amylose, free sugars ranging from
2.6–2.7% and a total protein content of 8–19% (Dendy and Dobraszczyk 2001).
Spent brewing grain was found to be a good substrate for the production of ␣amylase by A.oryzae under solid-state fermentation (Francis et al. 2003). Spent
grain is the by product of breweries left after the grain (barley, corn, wheat, rice,
and other grains) is fermented and the alcoholic solution drawn off. It is normally
wet, with 80 to 85% moisture content and relatively high protein content (27–30%).
19.3.2 Oil Cakes
It is the solid residue that are usually extracted from various types of oily seeds
like soya bean, pea nuts, linseed, cotton seed, cotton seed and sunflower by being
pressed and removing the oil. They are valued for being rich in minerals and protein.
They are rich in fibre and have high concentration of non-starch polysaccharides
(NSP). Their chemical composition varies due to the differences in the extraction
methods of oil. They are obtained by extraction of oil by means of a solvent from
the expeller pressed oil cake. The meal may also be obtained directly from seeds
after a preliminary treatment. The expeller pressed oil cake used for extraction are
obtained by pressing clean and sound seeds. The meal are subjected to heat and
steam treatment under controlled and regulated conditions so as to prevent denaturation of the protein and removal of traces of solvent. The material are in the form
of either flakes or powder and are free from harmful constituents and castor cake
or husk, rancidity, adulterants, insects or fungus infestation and from musty odour.
The moisture content, crude protein and crude fibre (weight percent) of ground nut,
cotton seed, linseed, mustard, sesame, coconut and safflower oil cakes have been
tabulated (Table 19.2). Soya bean are also included under oil seeds and they are
composed of oil and protein accounting to 60%, 35% carbohydrate and 5% ash.
The production of a thermostable pullulanase of Thermoactinomyces thalpophilus
was studied in shake-flask cultures. Maximum production of pullulanase was
obtained with 5% (w/v) soybean meal, 2% (w/v) yam starch (Odibo and Ob 1990).
Oil cakes such as coconut oil cake (COC), sesame oil cake (SOC), groundnut oil
cake (GOC), palm kernel cake (PKC) and olive oil cake (OOC) were screened to be
used as substrate for the alpha amylase production by A. oryzae and they were also
compared with wheat bran (WB). It was found that GOC and its combination with
WB (1:1) resulted in higher enzyme titres (Ramachandran et al. 2004). Arasarat-
19 Amylolytic Enzymes
365
nam et al. have reported glucoamylase production by A. niger using rice bran and
paddy husk as alternative substrates against wheat bran. Paddy husk was reported to
enhance the nutrient utilization when mixed with the substrates like rice bran, corn
flakes, soya flour and soy meal powder by A. niger CFTRI 1105 during SSF thereby
increasing glucoamylase production (Arasaratnam et al. 2001).
19.3.3 Other Starchy and Non Starchy Substrates
Cassava (Manihot esculenta Crantz) is a root crop of tropical American origin,
and the fourth most important staple crop in the tropics. Its starchy roots produce
more raw starch per unit of land than any other staple crop. It is grown almost
exclusively in arid and semiarid tropics, where it accounts for approximately 10
percent of the total caloric value of staple crops. Cassava starch is composed of
unbranched amylose (20±5%) and branched amylopectin (20±5%. Cassava fibrous
residue (CFR) contains about 10–15% crude fibre, 55–65% starch and very low
ash content (1–1.2%) (dry weight basis) (Jyothi et al. 2005). Because of its low
ash content, CFR could offer numerous advantages in comparison to other crop
residues such as rice straw and wheat straw, which have 17.5% and 11.0% ash
contents, respectively, for uses in bioconversion processes using microbial cultures
(Pandey et al. 2000a, Pandey et al. 2000b). A study was carried out to investigate
the ␣-amylase production by B. subtilis strain CM3 in SSF using CFR as the substrate (Swain and Ray 2007). Gonzalez et al. determined the optimal nutritional
and operative conditions for amylolytic enzymes (␣ amylase and glucoamylase)
production by S. fi uligera DSM-70554, cultured with cassava starch as the sole
carbon source under different fermentation strategies, in order to improve cassava
starch utilization. They described 97% degradation of cassava starch with a remaining 3% likely related to limit dextrins when grown under batch culture mode
(Gonzalez et al. 2008).
Molasses, a by-product of sugar industry, is one of the cheapest sources of
carbohydrates. Besides a large amount of sugar – 50% (sucrose 33.5%, invert
sugar 21.2%), molasses contain nitrogenous substances (0.4–1.5%), vitamins such
as thiamine (830g per 100 g dry weight), pyridoxine (650g per 100 g), folic acid
(3.8g per 100 g), biotin (120g per 100 g), pantothenic acid (2140g per 100 g),
and trace elements (CaO 0.1–1.1%; MgO 0.03–0.1%; K2 O 2.6–5.0%) (Pandey 2003).
Table 19.2 Chemical composition of oil cakes
Oil cake
Moisture (%)
Crude protein (%) Crude fat(%) Crude fibre (%)
Groundnut
Cotton seed
Linseed
Mustard
Sesame
Coconut
Safflower
10
8.0
10
10
10
10
8.0
51
40
29
35
37
21
41
1.0
8.0
8.0
8.0
8.0
8.0
8.0
10
10
10
9.0
7.0
12
13
366
D. Gangadharan and S. Sivaramakrishnan
The enzyme titre and cost of ␣-amylase production by Geobacillus thermoleovorans
using cane molasses and synthetic media has been compared. The enzyme titres
were found to increase by 2.5 fold and cost reduced by nearly 22 fold when
molasses was employed (Uma Maheswar Rao and Satyanarayana 2007). Cane molasses served as an excellent carbon and energy source for the economical production of glucoamylase by alginate-immobilized Thermomucor indicae-seudaticae,
which was almost comparable with that in sucrose yeast-extract broth (Kumar and
Satyanarayana 2007).
Potato is grown and consumed all over the world, and a large number of processed food industries market potato-based products. Although potato peel does
not pose serious disposal and environmental problems, meaningful utilization of
this nutrient-rich waste has not drawn much attention. Interestingly potato peel was
found to be a superior substrate for solid state fermentation, compared to wheat bran,
for the production of ␣-amylase by two thermophilic isolates of B. licheniformis and
B. subtilis (Shukla and Kar 2006). Potato starch was found to be superior to other
starch grains and tubers (amaranthus, wheat, sago, cassava, rice, maize etc) for the
production of alpha amylase by B. licheniformis SPT 27 (DharaniAiyer 2004). High
titres of  amylase production with 16.5% potato starch was reported in the case of
C. thermosulfurogenes (Reddy et al. 2003). The composition of starch from Amaranthus paniculatas was reported to be 66.4%, which was utilized in the production
of alpha amylase by A. flavu under SSF (Viswanathan and Surlikar 2001).
Brewery (BW) and meat processing (MPW) wastewaters, were used as a base of
the culture media in the production of amylase by A.niger UO – 1 under submerged
fermentation. BW contained (g/L): total sugars- 1.98, reducing sugars – 1.46, total
nitrogen – 0.095, total phosphorous – 0.034 and MPW (g/L) was- total sugars – 1.82,
reducing sugars – 0.99, total nitrogen – 0.172, total phosphorous – 0.028 (Hernandez et al. 2006). Tea waste is composed of approximately 19% crude protein, 5.4%
calcium and 0.84% of phosphorous. They have been popularly used as cattle feed.
SSF experiments with A. niger for the synthesis of glucoamylase production concluded tea waste, enriched with minerals, as a potential solid substrate (Selvakumar
et al. 1998).
Banana is one the most consumed fruits in the world and India is one of the
largest producing countries of this fruit. Each hectare of banana crop generates
nearly 220 ton of plant residual waste that consists mainly of lignocellulose material and the waste disposal often causes serious environmental problem. The main
residual wastes of the banana crop are leaves and pseudostem, both containing high
levels of lignocelluloses (Shah et al. 2005). A. oryzae, produced amylase when banana fruit stalk was used as substrate in a solid state fermentation system. Banana
waste has been exploited as an SSF substrate for ␣ amylase production by B. subtilis
(Krishna and Chandrasekaran 1996). An attempt was also made to utilize the food
waste, kind of organic waste discharged from households, cafeterias and restaurants,
which accounts for a considerable proportion of municipal solid garbage in China
for the production of glucoamylase by A. niger. Wang et al. characterized the food
waste and carbon content 53.68%, nitrogen 2.54%, reducing sugar 13.65%, total
19 Amylolytic Enzymes
367
sugar 50.23%, starch 46.12%, crude protein – 15.56%, crude lipid – 18.06%, crude
fiber – 2.26% (Wang et al. 2008).
19.4 Conclusion
The cost and availability of the substrates play an important role in the development
of efficient processes. The feasibility of agricultural by products for the commercial
production of amylolytic enzymes has been well explored. The effective use of these
residues has served dual purpose of value addition and waste management. Eventhough wheat bran was given the prime position among agro residues as substrate,
extensive research on the compositional analysis of the individual substrates has
proved their high nutritive and productive value.
Abbreviations
SSF:
SmF:
CGM:
NSP:
COC:
SOC:
GOC:
PKC:
OOC:
WB:
CFR:
BW:
MPW:
Solid state fermentation
Submerged fermentation
Corn gluten meal
Non-starch polysaccharides (NSP)
Coconut oil cake
Sesame oil cake
Groundnut oil cake
Palm kernel cake
Olive oil cake
Wheat bran
Cassava fibrous residue
Brewery wastewater
Meat processing wastewater
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Sivaramakrishnan S, Gangadharan D, Nampoothiri KM et al. (2006) ␣-amylases from microbial
sources – An overview on recent developments. Food Technol Biotechnol 44 (2):173–184
Sivaramakrishnan S, Gangadharan D, Nampoothiri KM et al. (2007) Alpha amylase production by
Aspergillus oryzae employing solid state fermentation. J Sci Ind Res India 66:621–626
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and fungal glucoamylase system and its suitability for the hydrolysis of wheat starch. Process
Biochem 39:185–192
Swain MR, Ray RC (2007) Alpha-amylase production by Bacillus subtilis CM3 in solid state
fermentation using cassava fibrous residue. J Basic Microbiol 47:417–425
Tanyildizi MS, Ozer D, Elibol M (2007) Production of bacterial ␣-amylase by B. amyloliquefaciens
under solid substrate fermentation. Biochem Eng J 37:294–297
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sp.IMD 370. Enzyme Microb Technol 17:570–573
Uma Maheswar Rao JL, Satyanarayana T (2007) Improving production of hyperthermostable and
high maltose-forming ␣-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its application. Bioresour Technol 98:345–352
Viswanathan P, Surlikar NR (2001) Production of ␣ amylase with A. flavu on Amaranthus grains
by solid state fermentation. J Basic Microbiol 1:57–64
Wang Q, Wang X, Wang X (2008) Glucoamylase production from food waste by Aspergillus niger
under submerged fermentation. Process Biochem 43:280–286
Chapter 20
Cellulolytic Enzymes
Reeta Rani Singhania
Contents
20.1
20.2
20.3
20.4
20.5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cellulase Production Employing Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . .
Pre-Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microbial Source of Cellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulation of Cellulases in Cellulolytic Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . .
20.5.1 Beta-Glucosidase: Bottleneck in Cellulase Complex . . . . . . . . . . . . . . . . . . . . . .
20.6 Application of Cellulase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.6.1 Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.7 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract A world-wide interest has been emerged in the commercial potential
of using cellulolytic enzymes to generate glucose feedstock using lignocellulosic
biomass which could further be converted into value added products. Employing cellulolytic enzymes, for bio-ethanol production from biomass has led to the
development of an environmentally safe and sustainable technology. Concept of
bio-refinery is emerging to replace already existing petro-refinery as the later is supposed to exhaust in near future. In this chapter, importance of cellulolytic enzymes
for biomass conversion and its production aspects have been discussed.
Keywords Cellulase · β-glucosidase · Bio-ethanol · Agro-industrial residues ·
Pre-treatment · Tricoderma reesei
R.R. Singhania (B)
Biotechnology division, National Institute for Interdisciplinary Science and Technology,
Trivandrum 695 019, Kerala, India
e-mail: reetasinghania 123@rediffmail.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 20,
C Springer Science+Business Media B.V. 2009
371
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20.1 Introduction
Cellulolytic enzymes are the third most important industrial enzyme due to its versatile applications in various industries such as paper and pulp, textile and detergent
industry. The resurgence in utilization of biomass for bio-ethanol and other value
added organic compounds production has attracted major attention of researchers
globally towards cellulases. Renewable plant resources could be used as a way to
supplement hydrocarbon resources and meet increasing worldwide needs for consumer goods, which emphasizes on need of an environmentally safe and sound
bio-refinery. Lignocellulosic biomass is more attractive for the purpose as it does
not compete with food availability unlike starchy biomass. Cellulose is the most
abundant and ubiquitous biopolymer on earth, considered to be almost inexhaustible
raw material. At molecular level, it is a linear polymer of glucose composed of
anhydroglucose units coupled to each other by -1-4 glycosidic bonds. The number
of glucose units in cellulose molecule varies from 250 to 10000 depending on the
source and pre-treatment. Cellulose and hemicelluloses are the principle sources
of fermentable sugars in lignocellulosic feedstock; however, nature has designed
woody tissue for effective resistance to microbial attack. This is the reason that
cellulose is relatively inaccessible not only to bigger molecules like protein but also
to small molecules as water in some cases. There are crystalline and amorphous
regions, in the polymer and also several types of surface irregularities exist. Due to
the compact and stringent structure as well as its complex association with other
component, very few reactive sites are available for enzyme attachment, which necessitates an appropriate pre-treatment method (Pandey and Soccol 2000). Suitable
pre-treatment methods disrupt lignin coating and make the fibers accessible to enzyme action.
Cellulase is not a single enzyme but is a complex of three major types of cellulase: Cellobiohydrolase (CBH or 1,4--D-glucan cellobiohydrolase, EC 3.2.1.91),
Endo -glucanase (EG or endo-1,4 -D glucan glucanohydrolase, EC 3.2.14) and
-glucosidase (BG, EC 3.2.1.21), which acts synergistically to produce oligosacchaGlucose
Cellobiohydrolase
(CBH)
BGL
Endoglucanase
(EG)
EG
Cellobiose
β -glucosidase
(BGL)
Fig. 20.1 Action of different component of cellulase
CBH
20 Cellulolytic Enzymes
373
rides and glucose. Cellulase hydrolyzes cellulose and produce glucose, cellobiose
and other oligo-saccharides as primary products. Bioconversion of cellulose can
yield several useful bio-based products.
Figure 20.1 shows action of different component of cellulase, acting synergistically to hydrolyse cellulose. Endoglucanase acts first on amorphous cellulose fibers
and attacks in between the chain randomly, to release small fibers with free reducing
and non reducing ends. Then exoglucanase acts on free ends to release cellobiose,
which is finally hydrolyzed by -glucosidase to get the final end product as glucose.
20.2 Cellulase Production Employing Agro-Industrial Residues
Agro-industrial residues have been widely used for cellulase production employing
variety of microorganisms, as are the rich source of cellulose. Majority of the reports
on commercial production of cellulases available utilizes submerged fermentation
because of ease of controlling the conditions. However, in nature, the growth and
cellulose utilization of aerobic microorganisms elaborating cellulases probably resembles solid-state fermentation than a liquid culture. While the production cost in
the crude fermentation by SmF was about $ 20/kg, by SSF it was only $ 0.2/kg if
in situ fermentation was used. Ten fold reduction in production cost has been indicated by using SSF than SmF (Tengerdy 1996). Processing of agricultural wastes
in SSF systems for cellulolytic enzyme production has been reviewed (Nigam and
Singh 1996). They have emphasized with the appropriate technology, improved
bioreactor design, and operation controls; SSF may become a competitive method
for the production of cellulases. Detailed review of the application of SSF technology for cellulase production are available (Pandey et al. 1999, Xia and Cen 1999).
Solid substrate fermentation can be proposed as a better technology for commercial
production of cellulases considering the low cost input and ability to utilize naturally
available sources of cellulose as substrate. T reesei has been exclusively studied as a
microbial source of extracellular cellulase capable of hydrolyzing native cellulose.
Solid-state fermentation is rapidly gaining interest as a cost effective technology for
production of enzyme. The major technical limitation in fermentative production
of cellulases remains the longer fermentation time and low productivity. Carbon
sources in majority of commercial cellulase fermentations are cellulosic biomass
including straw, spent hulls of cereals and pulses, rice or wheat bran, rice or wheat
straw, sugarcane bagasse, water hyacinth, paper industry waste and other cellulosic
biomass (Belghith et al. 2001 and Tengerdy 1996). Table 20.1 shows the various
agro-industrial residues utilized by various microorganisms and the mode of cellulase production. Lactose, cellobiose, sophorose, gentibiose, are the known inducer
of cellulase production but only lactose can be used for economically feasible industrial production.
SSF has been proved to be an effective technology for cellulase production for
bio-ethanol applications. Though there is a limit to purify the cellulase, mainly
because almost all the membranes are cellulosic in nature which serves as natural
substrate for enzyme due to which the enzyme molecule get adhere onto the surface.
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Table 20.1 Cellulase production-substrate, microorganisms and bioprocess employed
Substrate
Microorganisms
Method
Wheat bran/corn cob
Soybean industry residue
Banana waste
Rice chaff/wheat bran(9:1)
Aspergillus niger NRRL3
Bacillus subtilis
B. subtilis
Mixed culture (T. reesei and A. niger)
A. niger
Neurospora crassa
Thermoascus auranticus
Penicillium decumbans
P. janthinellum
Trichoderma viride
T. reesei
H. grisea
Strptomyces drodowiczii
T. reesei
T. reesei ZU 02
T. reesei ZU 02
A. flavu
P. chrysosporium
SSF
SSF
SSF
SSF
Rice straw
Wheat straw
Wheat straw/bran (8:2)
Sugar cane bagasse
Wheat bran
Stream treated willow
Corn cob residue
Corn stover residue
Saw dust/bagasse and corn cob
Soy hull
Smf-fed batch
SmF
SSF
SSF
SmF
SmF
SmF
SmF
SmF
SmF
SSF
SSF
SmF
SSF
Though, for hydrolyzing biomass, cellulase does not need high degree of purity.
Concentrated crude extract can be used directly.
Even though T. reesei, Penicillium, Aspergillus and Humicola can hydrolyze
native cellulose, the reaction may be sometime very slow due to recalcitrance of
biomass. Very rarely cellulose can be found in pure state in nature, usually is
embedded in matrix of lignin and is bound with hemicelluloses. It is necessary to
remove lignin from cellulose with proper pre-treatment method to make cellulose
accessible for the microorganisms. It is an important and a necessary step for commercial hydrolysis of lignocellulosic biomass.
20.3 Pre-Treatment
Cellulose and hemicelluloses are the principle source of C6 and C5 fermentable
sugars in lignocellulosic feedstock; nature has designed woody tissue for effective
resistance to microbial attack. It emphasizes on use of proper pre-treatment method
for making these accessible for microorganisms. Pre-treatment of lignocellulosic
biomass has been an active field for of research for several decades, and a wide variety of thermal, mechanical, chemical and biological pre-treatment approaches (and
combinations thereof) have been investigated and reported in the scientific literature
(McMillan 1994). It is important that the selected pre-treatment technology includes
number of requirements;
1. Should improve the enzymatic accessibility of the lignocellulosic compound
2. Result in the minimum loss of the potential sugars
20 Cellulolytic Enzymes
375
3. Prevent the formation of molecules which are inhibitory to microbial degradation
or enzymatic action
4. Pre-treatment technology should be economically sound in order to make the
overall process i.e. conversion of biomass to simple sugar a feasible technology
Pre-treatment involves delignification of the feedstock in order to make cellulose
more accessible during hydrolysis. It results in separation of lignin and hemicellulose components from cellulose, as well as enlarges the inner surface area of fibers
thus paving a way for enhanced enzymatic hydrolysis. Steam explosion, alkali and
acid-pre-treatment are some of the common methods of pre-treatment. Steam explosion is most commonly used and alkali pre-treatment has been found to be better
in lignin removal (Carrillo et al. 2005). Solid concentration is the key factor significantly affecting the process economics for a dilute acid pre-treatment/enzymatic hydrolysis based process. Solid loading of 30% have been also investigated for dilute
acid pre-treatment. Still the relationship between enzymatic digestion and structural
properties of pretreated material has to be explored for better understanding of the
factors affecting cellulose hydrolysis.
20.4 Microbial Source of Cellulolytic Enzymes
Cellulolytic microbes are primarily cellulose degraders but generally do not utilize
lipids or protein as energy source (Lynd et al. 2002). Many of them can utilize other
carbohydrates in addition to cellulose but few anaerobic cellulolytic species have
restricted carbohydrate range, limited to cellulose and their hydrolyzed product.
Several fungi, bacteria and even actinomycetes have been involved in cellulase production. Certain fungi having characteristic ability to produce extracellular proteins
in large amount have been studied extensively. Such fungal strains are most suited
for the extracellular cellulase production. These organisms produce cellulases when
grown on cellulose containing medium but in presence of easily utilizable sugars its
production is inhibited. Major cellulase producers have been enlisted (Table 20.2).
Fungi can grow and utilize agro-industrial residues better than other microbes as
it closely resemble to their natural habitat. Several fungi have been extensively
employed for commercial production of cellulases depending upon their ultimate
application.
Filamentous fungi are well known as a cost effective resource for industrial cellulases. One of the most extensively studied fungi is T. reesei which is capable of
hydrolyzing native cellulose (Reczey et al. 1996, Singhania et al. 2006, Singhania
et al. 2007). Inspite of being a prolific natural producer of extracellular cellulases,
it may not be the most effective cellulase system for use in biomass conversion
processes that essentially require complete hydrolysis of the feedstock for economic viability. As a result of extensive studies of 40 years, more than 60 cellulolytic fungi have been reported, representing the soft-rot, brown-rot and white-rot
fungi. Fungi such as Humicola, Aspergillus and Penicillium, bacteria such as Cellulomonas, Pseudomonads and actinomycetes such as, streptomyces are actively
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Table 20.2 Major microorganisms employed in cellulase production
Major groups
Fungi
Genus
Aspergillus
Fusarium
Humicola
Melanocarpus
Neorospora
Phanerochaete
Penicillium
Talaromyces
Trichoderma
Bacteria
Acidothermus
Bacillus
Clostridium
Pseudomonas
Rhodothermus
Cellulomonas
Actinomycetes
Streptomyces
Thermomonospora
Species
A. niger
A. nidulans
A. oryzae
A. aculeatus
F. solani
F. fusosporium
H. insolens
H. griesa
M. albomyces
N. crassa
P. chrysosporium
P. brasilianum
P. occitanis
P. decumbans
P. purpurogenum
P. janthinellum
T. emersonii
T. reesei
T. harzianum
T. longibrachiatum
A. cellulolyticus
Bacillus sp.
B. subtilis
C. thermocellum
C. acitobutylicum
C. cellulovorans
P. cellulose
R. marinus
C. fim
C. uda
S. drodzowiczii
S. lividans
T. fusca
T. curvata
involved in cellulase production. Several other fungi are capable of utilizing cellulose but only few of them are capable of secreting extracellular cellulase complex,
which could have practical applications in biomass hydrolysis. Besides T. reesei;
Penicillium, Aspergillus and Humicola have the ability to hydrolyze native cellulose.
20.5 Regulation of Cellulases in Cellulolytic Microorganisms
Cellulase system of microbes can be generally regarded as complexed or noncomplexed. Non complexed cellulase systems from aerobic fungi and bacteria have
components of cellulase system free and almost secretable. Cellulase production in
cellulolytic microbes is tightly regulated by catabolite repression. The whole mech-
20 Cellulolytic Enzymes
377
anism of induction and repression thus helps the microbes to save energy which is
not desired when it has to be used as an industrial source of cellulases. The cellulolytic system of Trichoderma has been extensively studied. It is inducible and
the repression is very strong when cellobiose accumulates in the medium beyond a
threshold, though at basal levels it acts as an inducer. Since the CBH enzymes from
T. reesei generate cellobiose, it tends to accumulate in the medium unless BGL
(-glucosidase) converts it into glucose immediately. In T. reesei, the BGL is produced in lowest quantities compared to the other two classes of enzymes, and it
is slow acting, making BGL the rate limiting component in the hydrolysis of cellulose. The BGL from T. reesei is also subject to feed back inhibition by glucose
and cellobiose. The commercial strains of fungi currently used for cellulase production, specifically T. reesei produces a cellulase mixture containing very little of
BGL enzyme which is inhibited by excess glucose in the medium. Increasing the
-glucosidase production as well as its glucose tolerance is therefore highly desired
for obtaining an enzyme preparation, which is efficient in biomass hydrolysis. There
can be several approaches for this issue. This can be achieved by transforming the
T. reesei with heterologus BGL genes that can produce efficient glucose tolerant
enzyme. If the BGL gene expressed from a stronger promoter the yield can be
increased several fold. Several foreign proteins have been successfully expressed
in Trichoderma using the cbh1 promoter to control their expression.
20.5.1 Beta-Glucosidase: Bottleneck in Cellulase Complex
Most of the cellulase complex is deficient in -glucosidase or it contains small
amount thereby making hydrolysis inefficient. Though Aspergillus and few Penicillium strains have been reported to produce high amount of -glucosidase, isolation
of potent -glucosidase having glucose tolerance property producing fungal strain,
is still the major bottleneck. Efficiency of cellulose complex can be increased by
preparing a cocktail of enzyme supplementing -glucosidase from heterogamous
source (Sukumaran et al. 2009). Another strategy could be the over expression of
BGL gene, which can be effected by use of the CBH1 promoter to drive its expression. The gene for glucose tolerant BGL can then be isolated and cloned into
suitable vector and engineered further to express it from the CBH1 promoter. There
have been limited reports of isolation of glucose tolerant BGL from filamentous
fungi but there is lot of potential for isolating glucose tolerant BGL from unexplored
filamentous fungi.
20.6 Application of Cellulase
Cellulases have occupied a major portion of enzyme market after amylase and
protease. It has wide applications in various industries including, detergent industry, textile industry, paper and pulp industry and biofuel. The lignocellulosic plant
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biomass is renewable and can be used for producing several compounds which are
currently being sourced from petroleum. This potential has led to the development of
a “biorefinery” concept where plant biomass is the raw material for generating fuel
and chemicals. With the imminent depletion of petroleum, renewable lignocellulosic
feedstock could be one of the sources to supplement hydrocarbon resources and to
meet the increasing worldwide needs for fuel, chemicals and materials. There is
a compelling need for commercializing economically viable and environmentally
safe biorefineries capable of making a whole range of bio-based products, especially ethanol for fuel applications, using carbohydrates and lignin present in the
biomass.
Fuel ethanol production from lignocellulosic biomass is emerging as one of the
most important technologies for sustainable production of renewable transportation
fuels. In this chapter, bioethanol production has only been discussed as application
of cellulolytic enzymes.
20.6.1 Bioethanol
The idea of generation of ethanol from lignocellulosic residues has been conceived
by NREL (Northern renewable energy laboratory) in USA. In order to make it competitive with gasoline by the turn of the century, an extensive programme is going
on with a strategy that will reduce the cost of bioconversion of biomass to ethanol,
in countries like Canada, Denmark and Brazil. It was proposed to be done in two
steps i.e. hydrolysis of lignocellulosic material into their monomers and thereby its
further conversion into ethanol by fermentation. The crux of the technology lies in
the cellulose hydrolysis and the latter one is well stabilized. The major requisite
for exploitation is the efficient hydrolysis of the relatively recalcitrant cellulose to
produce glucose which is the building block for all other chemicals and metabolites.
While chemical methods do exist for biomass hydrolysis, they are inefficient and
generate toxic byproducts and effluents that can create pollution. The most efficient
method of biomass hydrolysis is through enzymatic saccharification where cellulases and hemicellulases are utilized. Significant research work has been done on
cellulases and is still being carried out, with a major thrust on microbial cellulases.
Due to the apparent advantages of ethanol having high octane rating and also
being a renewable alternative to existing transport fuels, there is now an increased interest in commercializing technologies for its production from inexpensive biomass
(Schell et al. 2004). Most of the fuel ethanol produced in the world is currently
sourced from starchy biomass or sucrose (molasses or cane juice), but the technology for ethanol production from non food plant sources is being developed rapidly
so that large scale production will be a reality in the coming years. The process of
converting low value biomass to ethanol via fermentation depends on the development of economically viable cellulolytic enzyme to achieve effective depolymerisation of the cellulosic content of the biomass. Reduction in cost of “biomass-ethanol”
may also be achieved by efficient technologies for saccharification which includes
the use of better “enzyme cocktails” and conditions for hydrolysis (Sukumaran
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379
et al. 2009). Cellulase preparation used in this process must hydrolyze crystalline
cellulose completely, operate effectively at mild pH, withstand process stress and
they need not be derived from microbes that are generally regarded as safe (GRAS).
The ability to engineer cellulase systems in anticipation of each application is key
to successful optimization and commercialization.
Agro-residues could be used as raw material for bioethanol production. Advances
in industrial biotechnology, offers ample opportunities on economic utilization of
agro-industrial residues. According to Indian scenario rice straw and sugarcane
bagasse can be the probable feedstock for long term motive. A part of it can be used
in the site itself for generating energy, but still a major part of it constitutes waste.
Disposal of these residues itself is a major problem, causing pollution. Bioethanol
production involves several steps starting from selection of proper feedstock, its
pre-treatment, cellulase production, hydrolysis of feedstock using cellulases and finally fermentation of hydrolysate to obtain ethanol. This bioconversion of cellulose
(enzymatic hydrolysis) is the costliest step in overall process which could be brought
down by employing multifaceted approach as cheaper raw material for enzyme
production, cheaper technology as solid-state fermentation, appropriate feedstock
for bioconversion as well as appropriate pre-treatment method. Artificial cellulase
preparation and engineering cellulases can help to modify cellulase to suit for the
particular application. Expression cassettes, site directed mutagenesis and antisense
technology have been successfully employed in designing cellulase. Potent cellulase gene from different filamentous fungi can be isolated, cloned and expressed
in the host organism to get better combination or synergism. Enzyme cocktail can
be prepared using cellulases from different sources to achieve maximum efficiency
which otherwise is not possible due to lack of one or the other component of native
cellulase. Cellulase from T. reesei can be supplemented by -glucosidase from A.
niger to overcome repression and feed back inhibition of -glucosidase in T. reesei
(Sukumaran et al. 2006).
20.7 Future Perspectives
Research has shed light into the mechanisms of microbial cellulase production
and has led to the development of technologies for production and application
of cellulose degrading enzymes. Lignocellulosic biomass is the potential source
of biofuels besides biofertilizers, animal feed, chemicals and the raw material for
paper industry. Exploitation of this renewable resource needs either chemical or
biological treatment, and in the latter context cellulases have gained wide popularity over the past several decades. However use of the current commercial preparations of cellulase for bioconversion of lignocellulosic waste is economically not
feasible.
Reduction in the cost of cellulase production and improvement in the efficiency
of cellulase are major goals for future cellulase research. The former task may include optimizing growth conditions or processes, whereas, the latter required direct
efforts in protein engineering and microbial genetics to improve the properties of
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the enzymes. Optimization of growth conditions and processes has been attempted
to a large extent in improving cellulase production. Many of the commercial production of cellulases involve submerged fermentation technology and employ hyper
producing mutants. In spite of several efforts directed at generating hyper producer
by directed evolution, the cost of enzyme has remained high. Imparting desired
features to enzyme by protein engineering are the area where cellulase research
has to advance. Solid-state fermentation on lignocellulosic biomass particularly by
using host/substrate specific microorganisms could be an alternative strategy. Filamentous fungi are capable of producing optimal cellulase complex for degradation
of host lignocellulose (Tengerdy and Szakacs 2003). Enzyme complex prepared
from same lignocellulosic material have been proved to perform better (Chahal
et al. 1996). Mixed culture gives improved production and enzyme complexes with
high efficiency. Thus, SSF may be considered as a cost effective means for large
scale production of cellulases which probably would be several fold cheaper, compared to the commercial cellulase preparations.
Active research in the field of cellulase regulation has led to genetic improvement of cellulase production by various methods including over expressing cellulases from the cbh1 promoter of T. reesei and generation of desired variation
in the cellulase production profile of organism. Feedback inhibition of cellulase
biosynthesis is another major problem to be encountered. Trichoderma and other
cellulase producing microbes produces very little of -glucosidase compared to
other fractions of cellulolytic enzymes. Cellobiose is the potent inhibitor of CBH
and EG and it gets accumulated due to low -glucosidase production and thereby
inhibits the hydrolysis of cellobiose into glucose. This issue has been addressed
by various means like addition of exogenous -glucosidases to remove cellobiose
and engineering -glucosidase genes into the organism so that it is overproduced.
More and more research has been oriented towards genetic manipulation as process
design and medium formulation has come to an age and future definitely requires
controlled genetic intervention into the physiology of cellulase producers to improve
production and thereby make cellulase production more cost effective. The major
tasks ahead include overriding the feedback control by glucose and development of
integrated bioprocesses for the production of cellulases.
20.8 Conclusion
Cellulase production with improved profile and efficiency is the crux in bioconversion of lignocellulosic biomass. Though, large number of research papers has
been published and still publishing on all aspects of cellulase, its application for
bioethanol production still has a long way to go to resume as economically feasible
technology in near future. Improving its cellulose hydrolyzing efficiency and getting
higher sugar concentration in the hydrolysate without any energy input, are still an
area which has to be seriously addressed and solved. Protein engineering could serve
to increase specific activities of enzyme as well as to increase process tolerance and
stability. It needs a concerted effort in understanding the basic physiology of cel-
20 Cellulolytic Enzymes
381
lulolytic microbes and the utilization of this knowledge coupled with engineering
principles to achieve a better utilization of lignocellulose, the most abundant natural
resource.
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Schell DJ, Riley CJ, Dowe N et al. (2004) A bioethanol process development unit: initial operating
experiences and results with a corn fiber feedstock. Bioresour Technol 91:179–188
Singhania RR, Sukumaran RK, Pillai A et al. (2006) Solid-state fermentation of lignocellulosic
substrates for cellulase production by Trichoderma reesei NRRL 11460. Ind J Biotechnol
5:332–336
Singhania RR, Sukumaran RK, Pandey A (2007) Improved cellulase production by Trichoderma
reesei RUT C30 under SSF through process optimization. Appl Biochem Biotechnol 142:60–70
Sukumaran RK, Singhania RR, Pandey A (2006) Microbial cellulases-Production, applications and
challenges. J Sci Ind Res 64:832–844
Sukumaran RK, Singhania RR, Mathew G M, Pandey A et al. (2009) Cellulase production using
biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production. Renew Energ 34:421–424
Tengerdy RP (1996) Cellulase production by solid substrate fermentation. J Sci Ind Res
55:313–316
Tengerdy RP, Szakacs G (2003) Bioconversion of lignocelluloses in solid-state fermentation.
Biochem Eng J 13:169–179
Xia L, Cen P (1999) Cellulase production by solid state fermentation on lignocellulosic waste from
the xylose industry. Process Biochem 34:909–912
Chapter 21
Pectinolytic Enzymes
Nicemol Jacob
Contents
21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.2 Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.2.1 Primary Structure of Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.2.2 Secondary Structure of Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.3 Pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.3.1 Production of Pectinases Using Agro-Industrial Residues . . . . . . . . . . . . . . . . . .
21.3.2 Purification of Pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21.4 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract Pectic substances are prominent structural constituents of primary cell
walls and middle lamella in non-woody plant tissues. Pectinases are a group of
enzymes that contribute to the degradation of pectin by various mechanisms. In
nature, pectinases are important for plants as they help in cell wall extension and
fruit ripening. They have a significant role in maintaining ecological balance by
causing decomposition and recycling of plant materials. The industrial applications
of pectinolytic enzymes include fruit juice clarification, tissue maceration, wine
clarification, plant fiber processing, oil extraction, coffee and tea fermentation etc.
Microbial production of pectinolytic enzymes is mainly from filamentous fungi,
yeasts and filamentous and non-filamentous bacteria and is produced in two different techniques viz; submerged fermentation (SmF) and solid-state fermentation
(SSF). SSF permits the use of agricultural and agro-industrial residues as substrates
for enzyme production. As these residues are renewable and in an abundant supply,
they represent a potential low cost raw material for microbial enzyme production.
N. Jacob (B)
Biotechnology Division, National Institute for Interdisciplinary Science and Technology (CSIR),
Trivandrum 695019, Kerala, India
e-mail: nicemariacyril@yahoo.co.in
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 21,
C Springer Science+Business Media B.V. 2009
383
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N. Jacob
The significance of various agro-industrial residues as raw materials for pectinolytic
enzyme production is highlighted in this article.
Keywords Pectin · D-Galacturonic acid · Pectinases · Fruit juice clarification ·
Wine clarification · Degumming of plant fibers
21.1 Introduction
Ecofriendly biotechnological processes seem to be very important as far as the modern society is concerned for which microbial enzymes are recognized as efficient
tools. Pectinases are a group of enzymes that contribute to the degradation of pectin,
which is a complex acidic polysaccharide present in the primary cell wall and middle
lamella of higher plant tissues. The significance of these enzymes for the development of environment friendly industrial processes has already been established.
Pectinolytic enzymes can be applied in various industrial sectors wherever the
degradation of pectin is favourable for a particular process. Pectinases can be divided into two groups based on the optimum activity pH of these enzymes: acidic
and alkaline. Fruit juice clarification/extraction is one among the important applications of acidic pectinases (Rai et al. 2004; Sun et al. 2006; Lee et al. 2006; Sin
et al. 2006). Fruit juices contain colloids that may lead to fouling problem during
filtration process and these colloids are basically polysaccharides such as pectin
and starch (Rai et al. 2004). Pre-treatment of juices with pectinases is performed to
lower the amount of pectin present and to decrease the viscosity of the juice, which
in turn accelerates the subsequent filtration process. Also, it helps to increase the
clarity of the juice. Besides fruit juice clarification, pectinases can be used for fruit
juice extraction to increase the juice yield. Wine processing industry also recognizes
the importance of acidic pectinases (Roldan et al. 2006), where the enzyme can be
applied at different stages. The addition of pectinases during crushing of the fruits
increases the juice yield and also accelerates the release of anthocyanins into the
juice. Pectinase treatment at the pre-fermentation or fermentation stage, settles out
suspended particles. After fermentation, enzyme is added to the wine to increase
its clarity and filtration rate (Kashyap et al. 2001). Tissue maceration is another
important application of acidic pectinases in which organized tissue is transformed
into a suspension of intact cells and it is significant in the food industry as well as
in the field of biotechnology. The process can be applied for the liquefaction and
saccharification of biomass, isolation of protoplasts (Balestri and Cinelli 2001) etc.
Alkaline pectinases are important for retting and degumming of plant fibers
(Ossola and Galante 2004; Sharma et al. 2005) as an ecofriendly alternative to the
traditional chemical processes. Plant fibers contain gum after decortication, which
necessitates a degumming process, for textile purpose, in which the non-cellulosic
gummy material is removed from the surface of the fibers. Pectinases have a leading
role in the degumming of natural fibers by removing interlamellar pectin which acts
as a cementing substance between the fibers. Alkaline pectinases are also applied in
paper and pulp (Viikari et al. 2001; Ricard and Reid 2004) industry. Pre-treatment
21 Pectinolytic Enzymes
385
of pulp with pectinases is recommended to lower the cationic demand (Reid and
Ricard 2000) and to decrease the cost of the process. Vegetable oil extraction can be
augmented by applying enzymes which liquefy the structural cell wall components
of oil containing crop which eliminates the use of carcinogenic organic solvents.
Coffee and tea fermentation (Angayarkanni et al. 2002; Jayani et al. 2005) and treatment of pectic waste water are some other fields which utilize pectinolytic enzymes.
21.2 Pectin
The plant cell wall is a very dynamic structure as new material is constantly being
laid down and old material degraded and removed. The cell wall lends strength
and support to plants and the modern view of the plant cell wall is that it is a
cellular compartment rather than a rigid and inert network involved in protection
and structural support. Throughout plant development, the cell wall is subjected
to many chemical and physical changes such as loosening during cell expansion
and enzymatic degradation during fruit ripening (Mollet et al. 2003). The structural constituents of a young plant cell wall are cellulose, hemicellulose and pectic
substances. The cellulose microfibrils provide strength to the cell wall, while hemicelluloses and pectic substances act as the cementing substance for the cellulose
network.
Pectins or pectic substances contribute to complex physiological processes like
cell growth and cell differentiation and so determine the integrity and rigidity of
plant tissue. It is one of the most complex biomacromolecules in nature and it can
be composed of as many as 17 different monosaccharides, with at least seven different polysaccharides. Pectic substances are the sole polysaccharides in the middle
lamella responsible for cell cohesion. The texture of vegetables and fruits during
growth, ripening and storage is strongly influenced by the type of pectin present.
The structure of pectin present in fruits and vegetables depends on enzymatic and
chemical modifications occurring during these processes. One of the most characteristic changes during the ripening of fleshy fruits is softening. The change is
attributed to enzymatic degradation and solubilization of pectic substances.
21.2.1 Primary Structure of Pectin
The predominant structure of pectin consists of homogalacturonan (HG) which is
an essentially unbranched molecule composed of poly ␣-1,4 D-galacturonic acid
(PGA). The basic unit of PGA is shown in Fig. 21.1. The galacturonic acid (GA)
residues can be methyl esterified at C-6 and some of the hydroxyl groups on C2 or C3
can be acetylated. Blocks of more than 10 unesterified GA residues generally yield
pectin molecules, which are sensitive to calcium cross linking (Daas et al. 2001).
Recent reports points to the fact that pectin is not an extended back bone consisting
of homogalacturonan and rhamnogalacturonan regions, but rather a rhamnogalacturonan with neutral sugar and homogalacturonan side chains (Vincken et al. 2003).
HG with -D-xylose side chains is referred to as xylogalacturonan (XGA). Its pres-
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N. Jacob
Fig. 21.1 The basic unit
of pectin. Poly ␣-1,4
D-galacturonic acid is the
basic unit of pectin. Blocks of
this simple polymer alternate
with hairy, non-gelling
regions containing side
chains with other unusual
sugars
ence in plants seems to be confined to reproductive organs (fruits and seeds). HGs
can contain clusters of four different side chains with very peculiar sugar residues
such as apiose, aceric acid, Dha (3-deoxy-D-lyxo-2-heptulosaric acid) and Kdo
(3-deoxy-D-manno-2-octulosonic acid). These side chains, together with the HG
fragment of ∼9 galacturonosyl residues to which they are attached, are referred to
as rhamnogalacturonan II (RG-II) (Ridley et al. 2001). Two molecules of RG-II
can complex with boron, forming a borate-diol ester, which can crosslink two HG
molecules. Only the apifuranosyl residues of the 2-O-methyl-D-xylose containing
side chains in each of the subunits of the dimer participate in the cross-linking (Ishii
et al. 1999). The backbone of rhamnogalacturonan I (RG-I) is composed of as many
as 100 repeats of the disaccharide, [→2)-␣-L-Rhamnose-(1→4)-␣-D-Galacturonic
acid-(1→] (Albersheim et al. 1996). The rhamnosyl residues can be substituted at
O-4 with neutral sugars. The side chains are mainly composed of galactosyl and/or
arabinosyl residues. They can be single unit, but also polymeric such as arabinogalactan I (AG-I) and arabinan (50 glycosyl residues or more). AG-I is composed of
a 1,4-linked -D-galactose back bone. Arabinose residues can be attached to the O-3
of the galactosyl residues. The arabinans consist of a 1,5-linked ␣-L-arabinose back
bone. Complexes of RG-I, AG-I and arabinan are often referred to as pectic hairy regions (HR), in which AG-I and arabinan are the hairs. Another type of arabinogalactan, arabinogalactan-II (AG-II), is mainly associated with proteins (arabinogalactan
proteins or AGPs) and so it is unclear whether AG-II is part of the pectin complex.
The major part of AGPs (>90 %) consists of polysaccharides. Even though the fine
structure of the constituent polysaccharides of pectin is known in much detail, new
structural details are added to the collection of elements already known, with the
analysis of pectic polysaccharides from different plant sources (Vincken et al. 2003).
21.2.2 Secondary Structure of Pectin
Although there is a rather detailed picture on the primary structure of pectic
constituent polysaccharides, surprisingly little is known on how they assemble
into a macromolecular network (Vincken et al. 2003). Selective cleavage of the
galacturonosyl residues of the RG-I back bone by treatment with lithium in ethylene
diamine has been resulted in a collection of fragments in which neutral side chains
are attached to rhamnitol at the reducing end (Lau et al. 1987). Also, oligosaccharides with small neutral sugar side chains attached to rhamnosyl residues have been
found in the digests of pectic material obtained with either dilute acid or particular pectic enzymes. These observations suggest that the neutral sugar containing
21 Pectinolytic Enzymes
387
hairs are covalently linked to RG-I. There is also evidence that HG and RG-I are
covalently attached to each other (Vincken et al. 2003). When plant tissues are
treated with a rather crude mixture of enzymes including pectinases, cellulases
and hemicellulases, the native pectic polysaccharides can be partially degraded and
therefore the forthcoming material is referred to as modified hairy regions (MHR).
A detailed structural characterization of the so called MHR has been extremely
valuable in defining the various pectic subunits, and in providing clues on how
the various subunits are connected to each other. The MHR has been subsequently
fragmented at specific sites with novel enzymes. Based on these studies a tentative
structure is put forward. Pectin is represented as one molecule containing smooth
and hairy regions. These regions are assembled from three subunits: stretches of
RG-I, containing single-unit galactosyl side chains, XGA and RG, containing long
arabinan and AG-I side chains. The smooth regions are mainly composed of HG.
The HR consist of RG-I, XGA, arabinan, AG-I and AG-II. Also, it is believed that
XGA can be a continuation of HG and that the pectic back bone consists of regions
with varying GA:Rhamnose ratios (Vincken et al. 2003). The schematic structure of
pectin is given in Fig. 21.2.
Fig. 21.2 Schematic structure of pectin. It is one of the most complex biomacromolecules in nature
and it can be composed at least seven different polysaccharides. The major polysaccharides are HG,
XGA, RG II and RG I and AG I, arabinan and AG II are side chains of RG I. The smooth regions
are mainly composed of HG. The HR consist of RG-I, arabinan, AG-I, AG-II and XGA
21.3 Pectinases
Pectinases can be classified as esterases, eliminative depolymerases and hydrolytic
depolymerases with respect to their role in the degradation of pectin. Pectinesterases
or pectin methyl hydrolases catalyze hydrolytic removal of the methyl ester group
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N. Jacob
of pectin, forming pectic acid. The target of the enzyme is a methyl ester group
next to a non-esterified galacturonate unit. Depolymerases degrade the ␣-1,4 glycosidic linkages uniting adjacent GA residues of pectin. Among the depolymerases,
hydrolytic depolymerases act by hydrolysis, whereas eliminative depolymerases effect depolymerization of pectin by transelimination, which results in galacturonide,
with an unsaturated bond between C4 and C5. Hydrolytic depolymerases are classified into two, based on the substrate of preference. Polymethylgalacturonases act
upon pectin, while polygalacturonases degrade PGA. Similarly, eliminative depolymerases or lyases are either polymethylgalacturonate lyases/pectin lyases or polygalacturonate lyases depending upon the substrate preferred. All the depolymerizing
enzymes occur in two forms according to their site of action. Endoform of an enzyme catalyzes random cleavage of the substrate, whereas exoform of an enzyme
catalyzes sequential cleavage of the substrate from the non-reducing end.
Pectinases are produced by plants, insects and saprophytic microorganisms such
as bacteria and fungi. The major sources of plant pectinases are tomatoes and oranges (Torres et al. 2005). Among the animal sources, polygalacturonase obtained
from whole-body extracts of the rice weevil, Sitophilus oryzae has been purified to
apparent homogeneity (Shen et al. 1996). In contrast to plant and animal sources,
microorganisms are attractive sources of enzymes because of their diversity and the
possibility of yield enhancement by environmental and genetic manipulations and
also due to the short life span of microorganisms.
Microbial production of pectinolytic enzymes is mainly from filamentous fungi,
yeasts and filamentous and non-filamentous bacteria. Generally, fungal enzymes are
acidic in nature, while alkaline enzymes are produced by bacterial strains. The most
common source of commercial pectinolytic enzymes is the filamentous fungi especially Aspergillus sp. and is produced in two different techniques viz; submerged
fermentation (SmF) and solid-state fermentation (SSF). The advantages of SSF over
SmF include high volumetric productivity, relatively higher concentration of the
products, less effluent generation, requirement for simple fermentation equipments
etc. (Pandey et al. 1999). The growing condition in SSF approximates the natural
habitat of filamentous fungi more closely than in liquid culture, so these microorganisms are able to grow well on solid substrate and excrete large quantities of enzyme
(Castilho et al. 2000).
Bacterial pectinases are generally alkaline in nature and are found suitable for
applications like plant fiber processing, treatment of pectic waste water, paper pulping etc. Bacterial pectinase production is usually carried out by SmF, since SSF is
generally believed to be suitable for fungi which require a low water activity (0.6)
compared to bacteria (0.95). High moisture content in the fermentation medium
increases the possibility of contamination by other microbes. However, several researchers have reported enhanced enzyme production by bacterial strains under SSF,
proving the fact that SSF process is a better option than SmF for bacterial strains
also. Improved production of alkaline and thermotolerant pectinase has been reported by Bacillus sp. DT7 under SSF using wheat bran (Kashyap et al. 2003). An
alkalophilic Streptomyces sp. RCK-SC, which is able to produce a thermostable
alkaline pectinase, has been isolated from soil samples and in an immobilized cell
21 Pectinolytic Enzymes
389
system containing polyurethane foam, enzyme production has been enhanced by
32 % as compared to shake flask cultures, using wheat bran as solid substrate, at
substrate-to-moisture ratio of 1:5 after 72 h of incubation (Kuhad et al. 2004).
21.3.1 Production of Pectinases Using
Agro-Industrial Residues
SSF permits the use of agricultural and agro-industrial residues as substrates which
are converted into bulk chemicals and fine products with high commercial value.
Agro-industrial waste materials can be used both as source of energy for growth and
as carbon for synthesis of cell biomass and other products (Mahmood et al. 1998).
The selection of a substrate for enzyme production in an SSF process depends on
several factors, mainly related with cost and availability of the substrate (Pandey
et al. 1999). As agro-industrial residues are renewable and in an abundant supply
(∼3.5 billion tonnes/year), they represent a potential low cost raw material for microbial enzyme production (Robinson and Nigam 2003). Solid substrate not only
supplies the nutrients to the microbial cultures growing in it but also serves as an
anchorage for the cells. The ideal solid substrate is one that provides all the necessary nutrients for the microorganism. However, some of the nutrients in the solid
substrate may be available in sub optimal concentrations, or even not present in
the substrates. In such cases, it would be necessary to supplement them externally
(Pandey et al. 1999) to enhance growth and subsequently enzyme production.
As there is an increasing demand for pectinases for various applications, its cost
effective production using low value substrates is becoming important for industries.
Agro-industrial residues like sugar beet pulp, citrus pulp, apple pomace, sugar cane
bagasse, wheat bran, orange bagasse, grape pomace etc. are used for the production
of pectinolytic enzymes by different fungi and bacteria. Many of the substrates used
for SSF are unrefined and is of agricultural origin making complete characterization
and exact reproducibility difficult (Mitchell and Lonsane 1992). However, a general
picture of the composition of various agro industrial residues and their significance
in pectinolytic enzyme production is presented below.
21.3.1.1 Sugarcane Bagasse
Sugarcane bagasse is the fibrous residue of cane stalks left over after the crushing and extraction of the juice from the sugar cane. It is a ligno-cellulosic residue
(by-product) of the sugar industry and is almost completely used by the sugar factories themselves as fuel for the boilers. A sugar factory produces nearly 30 % of
bagasse out of its total crushing. Several processes and products have been reported
that utilize sugarcane bagasse as raw material. These include electricity generation,
pulp and paper production, and products based on fermentation like animal feed,
bioethanol, enzymes etc. (Pandey et al. 2000a). One of the successful attempts is the
utilization of bagasse as raw material for the fermentative production of microbial
enzymes employing potent microbial cultures.
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N. Jacob
Bagasse consists of approximately 50 % cellulose and 25 % each of hemicellulose and lignin. Chemically, bagasse contains about 50 % ␣-cellulose, 30 % pentosans and 2.4 % ash (Pandey et al. 2000a). Since the pectin content of sugar cane
bagasse is low, it acts as only a solid inert support during SSF processes for pectinolytic enzyme production. The high fibre content is an advantage to be used as
substrate for SSF, when it is used alone or with other low fiber substrates such as
wheat bran, as it can increase the interparticle spacing with a possible increase in the
aeration and nutrient diffusion, supporting enzyme production (Martins et al. 2002).
Pectinases are inducible enzymes, and the inducers are GA, its polymer (pectin
and polypectate) and structural relatives (mucic acid, tartonic acid and dulcitol)
(Maldonado and Saad 1998). It is suggested to use a pectin rich inducer when sugar
cane bagasse is used as the sole substrate. Sugar cane bagasse has been successfully
used for the production of pectinases by Aspergillus niger, Thermoascus aurantiacus,Moniliella sp.,Penicillium sp. etc (Table 21.1).
Table 21.1 Utilization of agro-industrial residues for pectinolytic enzyme production
Microorganism
Substrate
Reference
Aspergillus niger
Aspergillus carbonarius
Thermoascus aurantiacus
Sugar cane bagasse
Wheat bran
Wheat bran or orange bagasse or
sugar cane bagasse
Wheat bran
Wheat bran and orange pulp
Sugar cane bagasse, orange
bagasse and wheat bran
Sugar cane bagasse, orange
bagasse and wheat bran
Monosodium glutamate waste
water and sugar beet pulp
Wheat bran
Orange bagasse and wheat bran
Sugar beet pulp
Apple pomace
Grape pomace
Wheat bran
Wheat bran
Maldonado and Saad 1998
Singh et al. 1999
Martins et al. 2002
Bacillus sp. DT7
Fusarium moniliforme
Moniliella sp.
Penicillium sp.
Aspergillus niger
Streptomyces sp. RCK-SC
Penicillium viridicatum
Bacillus gibsoni
Aspergillus niger
Aspergillus awamori
Aspergillus niger
Streptomyces lydicus
Kashyap et al. 2003
Niture and Plant 2004
Martin et al. 2004
Martin et al. 2004
Bai et al. 2004
Kuhad et al. 2004
Silva et al. 2005
Li et al. 2005
Joshi et al. 2006
Botella et al. 2007
Dinu et al. 2007
Jacob and Prema 2008
21.3.1.2 Sugar Beet Pulp
Sugar beet, alongside sugar cane, is the main source of sugar across the world. It is
grown widely in Europe, North and South America, Asia and parts of North Africa
and the crop is at the core of a multi-billion dollar global industry. After the sugar
has been extracted, the remaining pulp contains very little sugar, but is valuable
as a fiber and energy source. The pulp is composed of (% on dry basis) pectin,
28.7; cellulose, 20; hemicellulose, 17.5; protein, 9.0; lignin, 4.4; fat, 1.2; ash, 5.1
(Xue et al. 1992). Since the pectin content of beet pulp is high it can be used for
21 Pectinolytic Enzymes
391
the microbial production of pectinolytic enzymes without adding any pectinaceous
materials as enzyme inducer. It has been used as raw material for pectinase production by Aspergillus niger (Bai et al. 2004). Sugar beet pulp has also been used as
the carbon source as well as the pectinase inducer to produce extracellular alkaline
pectinase, by Bacillus gibsoni, under SSF (Li et al. 2005).
21.3.1.3 Citrus Bagasse
Citrus bagasse is the biomass remaining after juice extraction from citrus fruits on an
industrial scale. Citrus bagasse consists of peel, pulp and seeds, which correspond to
half of the fresh fruit weight (Garzon and Hours 1992). Lemon and orange pulps are
the major waste products released by citrus juice industry. Lemon pulps, essentially
constituted of pectins, are similar to a homogenous cream from which no further
juice can be mechanically recovered. The pulps can be disposed only after drying
because pulps with high moisture content may undergo spontaneous fermentation
causing environmental problems. The disposal cost of lemon pulps is high since it
cannot be easily and cheaply dried to a stable product with moisture content lower
than 10 %. In this respect, an alternative practical use of pulps is slurry state fermentation of the material for the production of pectinases (De Gregorio et al. 2002), as
a value addition of the material and as an economical way of enzyme production.
Orange bagasse, composed of peel, seed and pulp, is the waste material released
after juice extraction and is usually dried and marketed as a component of animal
feed. Since the production cost of dried bagasse is high and its selling price is
low, the process can be considered only as a waste disposal method (Mahmood
et al. 1998). The pellet of orange bagasse contains 11.8 % fibre, 6.4 % protein, 63
% nitrogen, 6.7 % ash, 19 % total sugar (9 % reducing sugar) and 0.1 % pectin
(Martins et al. 2002). The microbial cultures which are proved to be suitable for
the production of pectinases utilizing orange bagasse as raw material are Thermoascus aurantiacus, Penicillium sp., Moniliella sp., Penicillium viridicatum etc.
(Table 21.1).
21.3.1.4 Apple and Grape Pomaces
Pomace (ultimately from Latin pomum) or marc is the solid remains of grapes or
other fruits after pressing for juice or oil. It is essentially the pulp, peel, seeds and
stalks of the fruit after the oil, water or other liquid has been pressed out. One of the
main agro-industrial by products, abundantly produced in Europe, is apple pomace,
from the apple juice and cider industries. Apple pomace is often used to produce
pectin. It has high moisture content (∼80 %) which poses disposal problems for the
pulping industry (Robinson and Nigam 2003). Apple pomace, composed of peel,
seed and pulp, contains high levels of pectic substances and it could be used as a
substrate for the microbial biosynthesis of pectinases. Apple pomace has been effectively used for the production of pectinases by Aspergillus niger (Joshi et al. 2006).
Grape pomace is produced in large qualities in wine production with the issue of
disposal being an important environmental consideration. Grape pomace has tradi-
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N. Jacob
tionally been used to produce grape seed oil, a practice that continues to this day in
small amounts, and Pomace brandy, such as grappa. Today, pomace is most commonly used as fodder or fertilizer. Some wineries will reuse the material as fertilizer
while others are exploring options of selling the used pomace to biogas companies
to be used in the creation of renewable energy. Grape pomace has been found to be
suitable for pectinase production by Aspergillus awamori (Botella et al. 2007).
21.3.1.5 Wheat Bran
Wheat is an ancient grain and thought to have originated in southwestern Asia.
It has been consumed as a food for more than 12,000 years. As it was looked
upon as the Staff of Life, it played an important role of religious significance and
was part of the sacred rituals of many cultures. As rice has been the dietary staple of Asia, wheat has served this role for many of other regions of the world.
Wheat is a cereal grass of the Graminaceae (Poaceae) family and of the genus
Triticum and it is estimated that approximately one-third of the world’s people depend upon wheat for their nourishment. Today, the largest commercial producers of
wheat include the Russian Federation, the United States, China, India, France and
Canada.
Wheat bran is the hard outer layer of wheat grains, and consists of combined
aleurone and pericarp. Along with germ, it is an integral part of whole grains, and is
often produced as a by-product of milling in the production of refined grains. Wheat
bran is composed of 8.12 % fibre, 15.7 % protein, 65 % nitrogen, 4.57 % ash and
16.7 % total sugar (5.22 % reducing sugar) (Martins et al. 2002).
Among the various agro-industrial residues, wheat bran however holds the key,
as it is produced worldwide in enormous quantities as an important by-product
of the cereal industry, and has most commonly been used as substrate for various biotechnological processes (Pandey et al. 1999). Moreover, as wheat bran is
a cheap and readily available byproduct, the production of pectinase using wheat
bran may be a cost-effective affair (Kashyap et al. 2003). Pectinolytic enzyme
production using wheat bran is achieved using various microbial cultures such
as Aspergillus niger, Aspergillus carbonarius, Streptomyces sp., Streptomyces lydicus, Thermoascus auriantacus, Penicillium viridicatum, Fusarium moniliforme,
Bacillus sp., etc. as it is evident from Table 21.1. According to recent reports,
the leading fungal pectinase producer is Aspergillus carbonarius with an enzyme
yield of 480 U/g (Singh et al. 1999) under SSF using wheat bran as the substrate.
The highest reported polygalacturonase production in SSF is from Bacillus sp.
(23076 U/g) followed by Streptomyces sp. (4857 U/g) (Kapoor and Kuhad 2002;
Kuhad et al. 2004) with wheat bran as the solid substrate. As it is evident from
recent reports, wheat bran is the most potent solid substrate for pectinolytic enzyme
production by both fungi and bacteria, with or without the presence of enzyme
inducers.
21 Pectinolytic Enzymes
393
21.3.1.6 Coffee Pulp and Husk
Coffee pulp and husk had been used previously for the production of pectinases
since it contains considerable proportion of pectin in it. Coffee pulp or husk is a
fibrous mucilagenous material acquired during the processing of coffee cherries by
wet or dry process, respectively. The organic nature of the material makes it an
ideal substrate for microbial processes for the production of value-added products
(Pandey et al. 2000b).
21.3.2 Purification of Pectinases
An improved awareness of the properties of pectinases is important in commercialization of these enzymes in various fields. Enzyme inactivation and stability are
considered to be the major constraints in the rapid development of biotechnological
processes and the stability of pectinases is affected by both physical parameters (pH
and temperature) and chemical parameters (inhibitors or activators). Stability studies provide valuable information regarding the structure and function of enzymes
(Gummadi and Panda 2003). Purification of the enzyme is necessary before characterization because crude enzyme may contain different stabilizing components
and so the properties may vary greatly. Purification of pectinases has been effected
by combinations of different chromatography procedures (Celestino et al. 2006) or
single step procedures like aqueous two phase systems (Lima et al. 2002) or affinity
precipitation (Mondal et al. 2004).
When the raw material for enzyme production is an inert support, like sugar
cane bagasse with inducers, the enzyme obtained will be less contaminated than in
the case protein rich substrates like wheat bran. Purification requires more steps,
when the enzyme is more contaminated, increasing the cost of the process. So the
fermentation substrate should be selected carefully depending upon the end use of
the enzyme and the growth requirements of a particular organism. If only crude enzyme is required for an application (fiber processing), purification is not necessary,
but when it is applied for the quality improvement of a food material (fruit juice
clarification), purification is compulsory.
21.4 Conclusions and Perspectives
Agro-industrial by-products can be successfully utilized for microbial pectinolytic
enzyme production and as these residues are locally abundant low cost raw materials, enzyme production will be a cost effective affair. Also, the approach prevents
the accumulation of these by-products as an environmental threat. Identification
and bioconversion of new locally available agro-wastes is advantageous as it not
only leads to the value addition of these residues, but also helps to keep the
environment clean.
394
N. Jacob
Abbreviations
AG I
AG II
AGPs
Dha
GA
HG
HR
Kdo
MHR
PGA
RG I
RG II
SmF
SSF
XGA
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Arabinogalactan-I
Arabinogalactan-II
Arabinogalactan proteins
(3-deoxy-D-lyxo-2-heptulosaric acid)
Galacturonic acid
Homogalacturonan
Hairy regions
(3-deoxy-D-manno-2-octulosonic acid).
Modified hairy regions
Polygalacturonic acid
Rhamnogalacturonan-I
Rhamnogalacturonan-II
Submerged fermentation
Solid-state fermentation
Xylogalacturonan
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Chapter 22
Ligninolytic Enzymes
K.N. Niladevi
Contents
22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.2 Structure of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.3 Lignin Degrading Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.3.1 Lignin Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.3.2 Manganese Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.3.3 Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.4 Sources of Lignin Degrading Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.5 Production of Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6 Ligninolytic Enzyme Production from Agro-Industrial Residues . . . . . . . . . . . . . . . . . . .
22.6.1 Sugarcane Bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.2 Wheat Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.3 Rice Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.4 Wheat Bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.5 Rice Bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.6 Coffee Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.6.7 Fruit Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.7 Purification of Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
398
398
400
401
402
403
403
404
405
405
406
407
407
407
408
408
409
410
410
Abstract Ligninolytic enzymes are involved in the degradation of the complex and
recalcitrant polymer lignin. This group of enzymes is highly versatile in nature and
they find application in a wide variety of industries. The biotechnological significance of these enzymes has led to a drastic increase in the demand for these enzymes
in the recent time. Production of enzymes/metabolites from microbial sources is
a costly affair and the only alternate to minimize the production cost is the use
of inexpensive raw materials. The utilization of agro-industrial residues in this asK.N. Niladevi (B)
Biotechnology Division, National Institute for Interdisciplinary Science and Technology (CSIR),
Trivandrum 695019, Kerala, India
e-mail: nilanandini@yahoo.co.in
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 22,
C Springer Science+Business Media B.V. 2009
397
398
K.N. Niladevi
pect is much appreciated due to their low cost and ease in availability. Adopting
solid-state fermentation for enzyme production may add to the benefit of reducing
the production costs. The studies have proved that huge quantities of lignocellulosic
residues are available world wide for the production of ligninolytic enzymes. The
current trend is to make use of every such locally available agro-industrial residue
for enzymes production to meet the demand for the same from the industrial sectors.
Keywords Ligninolytic enzymes · Lignin peroxidase · Manganese peroxidase ·
Laccase · Agro-industrial residues · Solid-state fermentation
22.1 Introduction
The term lignin degrading enzymes encompasses mainly three oxidative enzymes;
lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. The demand for
these enzymes has increased in the recent years due to their potential applications
in diverse biotechnological areas. Lignin degrading enzymes are widely used in
the pollution abatement, especially in the treatment of industrial effluents that contains hazardous compounds like dyes, phenols and other xenobiotics. The role of
these enzymes in textile dye decolourization and degradation of phenolic and nonphenolic aromatic compounds has been extensively studied (Wesenberg et al. 2003;
Crecchio et al. 1995). The industrial preparation of paper requires separation and
degradation of lignin in wood pulp. Pre-treatments of wood pulp with ligninolytic
enzymes might provide milder and cleaner strategies of delignification that are also
respectful of the integrity of cellulose (Kuhad et al. 1997). Ligninolytic enzymes
are commonly used for biobleaching of kraft pulp and a laccase mediator system
R
is already in commercial application (Call and
with the trade name Lignozym
Mücke 1997). The ligninolytic enzymes also find application in the stabilization of
wine and fruit juices (Minussi et al. 2002), denim washing (Pazarlioglu et al. 2005),
cosmetic industry (Aaslyng et al. 1996) and Biosensors (Ferry and Leech 2005).
The ever increasing demand for these enzymes in the industrial sectors necessitates the production of enzymes from inexpensive raw materials. The ligninolytic enzymes can be produced from a wide variety of agro-industrial residues
that are lignocellulosic in nature. It is a well known fact that majority of the agroindustrial residues are lignocellulosic in nature. The production of enzymes from
agro-industrial residues is of great significance owing to two reasons; (i) the process
helps in the reuse of industrial wastes and thereby reduces the problems arising out
of their disposal and (ii) the enzymes produced from the residues can be utilized for
various industrial applications.
22.2 Structure of Lignin
The significance of ligninolytic enzymes lies in the fact that they are capable of
degrading the highly complex recalcitrant polymer, lignin. Lignin serves as the sec-
22 Ligninolytic Enzymes
399
ond major reservoir of fixed carbon sources in nature, next to cellulose, comprising
15% of the earth’s biomass (Hammel 1992). True lignin is distributed widely but
not universally throughout the plant kingdom. It is found in all vascular plants,
where it is deposited in cell walls along with cellulose and hemicellulose. Lignin
fills the spaces in the cell wall between different plant polysaccharides by covalent linking (Fig. 22.1) and thereby conferring mechanical strength to the cell wall
and by extension the plant as a whole (Chabannes 2001). Lignin is synthesized
in plants from the phenyl propanoid precursors such as coniferyl, synapyl, and pcoumaryl alcohols. These precursors (Fig. 22.2) are consisted of an aromatic ring
and a 3-carbon side chain. Free radical copolymerization of these alcohols produces
the heterogeneous, optically inactive, cross-linked, and highly polydisperse polymer (Kirk and Farrell 1987). In the lignin molecule the precursors form three types
of subunits: hydroxyphenol- (H-type), guaiacyl- (G-type) and syringyl subunits
(S-type). Unlike other biopolymers, lignin contains no readily hydrolysable bond
recurring at periodic intervals along a linear backbone. Instead, lignin is a three
Fig. 22.1 Schematic structure of a lignin molecule
Source: www.research.uky.edu/.../green energy.html
400
K.N. Niladevi
Fig. 22.2 Structure of the
lignin precursors 2a.
p-Coumaryl alcohol; 2b.
Coniferyl alcohol; 2c.
Sinapyl alcohol
Source: www.steve.gb.com/
science/ molecules.html
dimensional amorphous polymer containing many different stable C–C, C–O–C,
-O-4 linkages etc; the most common being the -aryl ether (β-O-4) bond (Argyropoulos and Menachem 1997). It consists of an apparently random complex of
phenolic and non- phenolic compounds.
The structural complexity of lignin makes it one of the most recalcitrant molecule
and its breakdown involves multiple biochemical reactions, that has to take place
more or less simultaneously; cleavage of inter monomeric linkages, demethylations, hydroxylations, side chain modifications and aromatic ring fission followed
by dissimilation of the aliphatic metabolites produced (Vicuna 1988). Under natural
conditions this task is achieved by the ligninolytic enzymes produced by different
groups of microorganisms. The molecular mass of lignin is high, about 100 kDa
or more, which prevents its uptake inside the microbial cell (Eriksson et al. 1990).
Thus, the biological degradation of macromolecular lignin must occur through the
activity of extracellular enzymes. The ability of these enzymes to act upon different phenolic and non-phenolic compounds in lignin is exploited in the commercial
sector for different applications.
22.3 Lignin Degrading Enzymes
The extremely complex nature of lignin requires an array of oxidative enzymes to
be involved in its complete degradation. Lignin peroxidase, manganese peroxidase
and laccase are the major lignin degrading enzymes. The characteristics of these enzymes differ widely with the microbial source. The ability of an organism to produce
one or more of these enzymes also varies greatly among different microbial groups.
Apart from LiP, MnP and laccase, a wide range of other enzymes such as veratryl alcohol oxidase (Bourbonnais and Paice 1988), Aryl alcohol dehydrogenase, Quinone
oxidoreductase, aromatic acid reductase, vanillate hydroxylase, dioxygenase, catalase (Leisola and Fietcher 1985; Buswell and Eriksson 1988) aromatic aldehyde
oxidase (Deobald and Crawford 1989) and Glyoxal oxidase (Kersten 1990) are
also believed to be involved in the tedious process of lignin degradation. However,
these enzymes are of less importance as they could either act as mediators of lignin
degradation by producing H2 O2 required for the activity of peroxidases or catalyze
22 Ligninolytic Enzymes
401
the breakdown products of lignin degradation that is effected by the activities of
enzymes like LiP, MnP and laccase.
Much attention has been paid in the recent time to commercialize these enzymes,
especially laccases for different industrial purposes. Novozyme has commercialized
laccases from Trametes villosa and Trametes pubescens (Novozym 51003), which is
very efficient for lignin modification processes. The commercial laccase preparation
Zylite (Zytex Pvt Ltd, Mumbai) is used for denim washing while the laccase meR
finds application in bleaching processes. Laccase from
diator system Lignozym
Aspergillus niger has also been commercialized by Novo Nordisk under the trade
name DeniliteTM , which is specifically meant for textile industry.
22.3.1 Lignin Peroxidase
Lignin peroxidase, commonly known as ligninase is one of the most important enzyme involved in the degradation of lignin. It was discovered in 1983 from the WRF,
P. chrysosporium. Since then, this enzyme has been demonstrated in wide variety of
organisms including brown rot fungi, soft rot fungi and filamentous bacteria. LiPs
are oligomannose type glycoprotein with a molecular weight range of 38 KDa to
43 KDa (Schmidt et al. 1990). LiP is having relatively high redox potential, so the
compounds with high redox potentials that are not oxidized by other enzymes are
also oxidized by LiP. It is this particular character of LiP that makes it an important
part of ligninolytic system. LiP can oxidize both phenolic and non-phenolic compounds. This enzyme employs free radical chemistry to cleave the propyl side chain
of lignin substructures (Schoemaker et al. 1985) and have been shown to depolymerize lignin invivo (Hammel et al. 1993). LiPs have the unusual ability to cleave the
recalcitrant nonphenolic units that comprise approximately 90% of lignin (Glenn
et al. 1983; Tien and Kirk 1983). The reactions catalysed by LiP include C␣-C
cleavage of the propyl side chains of lignin and lignin models, hydroxylation of
benzylic methylene groups, oxidation of benzyl alcohols to the corresponding aldehydes or ketones, phenol oxidation and even aromatic ring cleavage of non-phenolic
lignin model compounds (Renganathan et al. 1985; Umezawa and Higuchi 1987;
Chung and Aust 1995).
This heme peroxidase has a classical peroxidase catalytic mechanism for which
H2 O2 is required (Fig. 22.3). The native enzyme is oxidized by H2 O2 and generates
two-electron deficient compound I. Compound I can oxidize a compound and can be
reduced to compound II, which is one electron deficient. A subsequent oxidation of
another molecule by compound II returns the peroxidase to its native resting stage.
When there is excess H2 O2 , it will combine with compound II of LiP, generating
compound III, which is an inactive form of the enzyme. In many cases, the substrates
are not directly accessible to heme of LiP and thus direct oxidation of substrate does
not occur. In such cases involvement of redox mediator plays an important role.
Veratryl alcohol is an excellent substrate for LiP and it acts as the redox mediator
for indirect oxidation of other substrates. Veratryl alcohol stimulates oxidation by
preventing enzyme inactivation (Valli et al. 1990) and it is oxidized by LiP to VA
402
K.N. Niladevi
Fig. 22.3 Catalytic cycle of peroxidases
cation radical, which is a strong oxidant, and it acts as an electron transfer mediator
in the catalytic reaction of LiP.
22.3.2 Manganese Peroxidase
Manganese peroxidase is another important enzyme produced by the lignin degraders. It is also a heme peroxidase and requires H2 O2 for its activity. The redox potential of the MnP – Mn system is lower than that of LiP and normally it
does not oxidize non-phenolic lignin models. However, it has been reported that the
MnP from Panus tigrinus is able to degrade nonphenolic lignin model compounds
(Maltseva et al. 1991). MnP shows a strong preference for Mn (II) as its reducing
substrate (Glenn and Gold 1985). MnP oxidizes Mn2+ to Mn3+ , which is stabilized
by organic acid chelators viz; oxalate, malonate, glyoxylate etc and acts in turn as
a low molecular mass, diffusible, redox mediator that attacks organic molecule and
oxidizes various compounds nonspecifically via hydrogen and one electron abstraction. The organic acids also facilitate the release of Mn (III) from the active site
of the enzyme. The one electron oxidation of Mn (II) to Mn (III) in a multi step
reaction cycle is as follows:
MnP + H2 O2 → MnP compound I + H2 O (Reaction 1)
MnP compound I + Mn (II) → MnP compound II + Mn (III) (Reaction 2)
MnP compound II + Mn (II) → MnP + Mn (III) + H2 O(Reaction3).
This enzyme is mostly reported in WRF, where it is produced in combination
with LiP or laccase.
22 Ligninolytic Enzymes
403
Fig. 22.4 Catalytic cycle of
laccase
O•
OH
4x
4x
R
R
4e– + 4H+
T2
T1
T3
His-Cys-His
(Cu2+→Cu+)
O2
T2
2 H2O
22.3.3 Laccase
Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducingsubstrate molecules concomitant with the four-electron reduction of molecular oxygen to water (Piontek et al. 2002). Laccases oxidize a broad range of substrates,
preferably phenolic compounds. In the presence of mediators, laccases exhibit an
enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Laccases differ from LiP and MnP in that it does not
require H2 O2 to oxidize its substrates. The active site of each laccase molecule has
four copper ions: one type-1 (T1), one type-2 (T2) and two type-3 (T3) coppers
(Fig. 22.4). The two copper ions at T3 site are antiferromagnetically coupled. The
T2 and T3 sites are arranged in a unique trinuclear cluster that is capable of binding
oxygen, which is the final electron acceptor. Laccase activity is widely distributed
among different groups of fungi, bacteria and also in few plants and insects.
22.4 Sources of Lignin Degrading Enzymes
Lignin degrading enzymes have been demonstrated in both plants and animals.
However, the microorganisms remain the important source for lignin degrading enzymes. The production of ligninolytic enzymes from different microbial sources has
been well documented.
Fungi are the most potent source of lignin degrading enzymes. The saprophytic
fungi secrete these enzymes to degrade the lignin polymer. Among fungi, the
white rot fungi are the best known producer of these enzymes, followed by the
404
K.N. Niladevi
brown rots and the soft rots. The most studied lignin degrading system is that of
P. chrysosporium. Lignin degradation by P. chrysosporium is a classical secondary
metabolic activity induced particularly by nitrogen starvation. This organism secretes LiP, MnP and laccase for lignin degradation (Tien and Kirk 1983; Glenn and
Gold 1985; Srinivasan et al. 1995). Different species of white rot fungus Trametes
viz; T. Versicolor, T. hirsuta, and T. ochracea have been reported to be producing
LiP and MnP along with laccase (Tomsovsky and Homolka 2003). There are also
other fungal species such as P. ostreatus and P. radiata (Vares et al. 1995; Pradeep
and Datta 2002) that secrete all the three major ligninolytic enzymes. There are
many efficient delignifying fungi that secrete MnP as the sole extracellular peroxidase, including Lentinula edodes (Leatham 1986), Bjerkandera adusta (Wang
et al. 2002), Ceriporiopsis subvermispora (Lobos et al. 1994), Dichomitus squalens
(Perie et al. 1996) and Rigidoporus lignosus (Galliano et al. 1991). The commercial preparations of ligninolytic enzymes have been carried out mainly from fungal
sources. The ligninolytic enzyme titer of fungi is much higher than that from the
bacterial counterparts and this could be the main reason for the dependence on fungi
for commercial enzyme production.
Among bacteria, the actinomycetes are the potent producers of ligninolytic enzymes. Extracellular lignin peroxidase has been demonstrated in different Streptomyces strains such as S. viridosporus, S. chromofuscus andS. psammoticus
(Ramachandra et al. 1987; Pasti et al. 1990; Niladevi and Prema 2005). Apart
from LiP, the Streptomyces strains remain as a good source for laccase too (Arias
et al. 2003; Suzuki et al. 2003). Even though a large number of bacterial strains
have been suggested in lignin degradation, the production of lignin degrading enzymes, especially lignin peroxidase is restricted to few strains of Pseudomonas
(Yang et al. 2006) while laccase activity is established in bacteria like Azospirillum
lipoferum and Bacillus subtilis (Givaudan et al. 1993; Martins et al. 2002). The details on bacterial production of MnP are scanty with only a few reports. Esposito et
al. has reported the production of MnP by a Streptomyces strain CCT 4916 that was
able to degrade the herbicide diuron by oxidative reactions (Esposito et al. 1998).
MnP activity was also detected in the actinobacterium S. psammoticus (Niladevi and
Prema, 2005).
22.5 Production of Ligninolytic Enzymes
Microorganisms are the best choice for the production of enzymes and other commercially important metabolites. This is because the micro and macro environment
of the microorganisms can be controlled easily for enhancing the enzyme production
as per the needs. Moreover, genetic manipulation is also easy with the microbial system. Production of ligninolytic enzymes from different fungal and bacterial sources
has been carried out in both submerged fermentation (SmF) and solid-state fermentation (SSF) methods. However, the enzyme production by SSF is more prevalent,
probably because of the advantages of SSF over SmF, which include higher product
22 Ligninolytic Enzymes
405
titers, lower wastewater output, reduced energy requirements, simpler fermentation
media, etc (Pandey et al. 2001). Moreover, this technique offers the possibility of
using by-products and wastes from food and agricultural industries as the raw material for enzyme production, making the process much more efficient from both
economical and environmental standpoints. Solid-state fermentation is generally
defined as the growth of microorganisms on solid materials in the absence or near
absence of free water (Pandey et al. 2000a). In SSF, apart from supplying nutrients,
the solid substrate also serves as an anchorage for the microorganisms, facilitating
their growth and enzyme production.
22.6 Ligninolytic Enzyme Production
from Agro-Industrial Residues
Selection of suitable substrate is one of the key factors determining the success of
any fermentation process. The substrate must be easily available in the local area
in surplus amount and of low cost to make the entire process cost effective. It is
at this particular scenario the significance of agro-industrial residues blooms. The
annual production of agro-industrial residues has been estimated to about 3.5 billion
tonnes and they represent a potential low cost raw material for microbial enzyme
production (Robinson and Nigam 2003). Lignocelluloses are the most abundant
renewable organic matter on earth and they contribute to the majority of the agroindustrial residues available all over the world. The utilization of lignocelluloses
for ligninolytic enzyme production has been studied extensively. Production of LiP,
MnP and laccase from a wide variety of agro-industrial residues has been reported.
Most of the works on ligninolytic enzyme production from agro-industrial residues
deal with fungi (Rodriguez Couto and Sanroman 2005). The list of agro-industrial
residues used for the production of ligninolytic enzymes by different microbial
strains is given in Table 22.1.
22.6.1 Sugarcane Bagasse
The bagasse (or the crushed cane fibres), which results from the milling of sugar
cane, is among the world’s most widely used and available non wood fibres. It
is used in the boilers for steam production which is used to power the process.
The surplus bagasse is used in industry, to produce power, make paper, building
materials, as a fuel and even as stock feeds. It contains about 50% cellulose, 25%
hemicellulose and 25% lignin (Pandey et al. 2000b). The utilization of this lignocellulosic residue for the production of various industrially important enzymes has
been a field of interest to the researchers. The fibrous nature of bagasse makes it
more suitable for solid- state fermentation technique.
Sugar cane bagasse has been widely used for the production of ligninolytic enzymes in SSF. The production of laccase and manganese peroxidase in SSF by
Trametes versicolor and laccase alone by Flammulina velutipes using bagasse has
406
K.N. Niladevi
Table 22.1 Microbial production of ligninolytic enzymes from agro-industrial residues
Support
Microbial strain
T. versicolor,
Flammulina velutipes
Pleurotus ostreatus,
Phanerochaete
chrysosporium
Coffee pulp
Streptomyces
psammoticus
Pleurotus ostreatus,
P. pulmonarius
Wheat bran
Ganoderma sp. Fomes
sclerodermeus
Wheat straw
Phlebia radiata,
Trametes versicolor
Streptomyces cyaneus
Rice straw
Streptomyces
psammoticus
Orange peelings Trametes hirsuta
Banana Skin
Trametes pubescens
Rice bran
Coriolus versicolor
Grape seeds
T. hirsuta
Bagasse
Kiwi fruit waste
Trametes hirsuta
Ligniolytic enzymes
Reference
MnP, laccase laccase
MnP, LiP, laccase
Pal et al. 1995; Pal
et al. 1995 Pradeep
and Datta 2002
Laccase MnP, laccase
Niladevi et al., 2007
Marnyye et al. 2002
Laccase MnP, laccase
Revankar et al. 2007
Papinutti et al. 2003
Vares et al. 1995;
Schlosser et al. 1997
Berrocal et al. 1997
Niladevi et al. 2007
Lip, MnP, laccase
MnP,laccase Laccase
Laccase
Laccase
Laccase
Laccase
Laccase
Laccase
Rosales et al.,2007
Osma et al., 2007
Chawachart et al. 2004
Rodriguez-Couto et al.,
2006
Rosales et al., 2005
been reported by Pal et al. (Pal et al. 1995). The authors have reported that a preferential degradation of non-condensed (syringyl-type) lignin units was observed
during the fermentation of bagasse by these organisms. Bagasse has been successfully used for producing LiP, MnP and laccase in semi-solid-state fermentation also
(Gonçalves et al. 1998). Bagasse powder has been found to have enhancing effect
on ligninolytic enzyme production by T. versicolor in shake liquid culture (Masud
Hossain and Anantharaman 2006).
22.6.2 Wheat Straw
Wheat is an annual agricultural crop grown for the grain that is a valuable food product. It is the staple food for majority of the human population and hence cultivated
world wide. By-products from growing wheat have been used for many years for
a variety of applications. The use, for example, of wheat straw as structural filler
for mud bricks dates back several hundred years, for pulp back to 1827, and for
building panels back into the early 1900s. Wheat straw is the stem and leaf of the
wheat plant that is left after the harvest of grains. It consists of about 36% cellulose,
31% hemicellulose and 7% lignin. The current researchers have new approaches
in utilizing the bulk quantity of wheat straw produced annually. This includes the
production of ethanol and various enzymes from this agro-industrial residue.
Wheat straw is one of the best substrates for the production of ligninolytic enzymes. Laccase and MnP activity has been reported during the growth of Trametes
22 Ligninolytic Enzymes
407
versicolor on wheat straw (Schlosser et al. 1997). Wheat straw has served as a
best substrate for the production of LiP, MnP and laccase from several other fungi
too (Vares et al. 1995; Arora et al. 2002). Wheat straw has also been used for the
production of ligninolytic enzymes under SSF by Streptomyces strains (Berrocal
et al. 1997).
22.6.3 Rice Straw
Rice is the world’s second largest cereal crop after wheat and produces the largest
amount of crop residues, about 330 million metric tonnes. Ninety percent of the
world’s production is in developing countries of East and Southeast Asia (Van
Soest 2006). A major portion of the rice straw is used as cattle feed and packaging material and not many other uses have been assigned to this agro-industrial
waste. The production of ligninolytic enzymes from rice straw is of much relevance
considering the higher lignin content of rice straw as compared to wheat straw (Rodriguez Couto and Sanroman 2005). However, the information on utilization of rice
straw for this purpose is scanty. Production of all the three ligninolytic enzymes
by Streptomyces psammoticus using rice straw in SmF has been reported (Niladevi
and Prema, 2005) and it was the best substrate for laccase production in SSF as
compared to other agro-industrial residues (Niladevi et al. 2007).
22.6.4 Wheat Bran
Wheat Bran is the portion of the grain immediately under the husk of the wheat kernel. During the milling process of the wheat grains for the production of wheat flour,
large quantities of wheat bran is produced as by-product. Wheat bran is extensively
used as feed for farm animals. Among the various agro-industrial residues, wheat
bran however holds the key, as it is produced worldwide in enormous quantities as
an important by-product of the cereal industry, and has most commonly been used
as substrate for various biotechnological processes (Pandey et al. 1999). Similar to
the production of other industrially important enzymes, wheat bran serves as one
of the most suitable substrates for ligninolytic enzyme production. Wheat bran has
been reported to be the best substrate for laccase production in SmF by Ganoderma
lucidum (Songulashvili et al. 2007). Wheat bran has been used for the production
of laccase by Ganoderma strain under SSF and very high laccase activity of 10,050
U/gds has been achieved (Revankar et al. 2007). Production of MnP and laccase
from wheat bran has also been reported (Papinutti et al. 2003).
22.6.5 Rice Bran
Rice bran is a by-product of the rice milling process, and it contains various antioxidants that impart beneficial effects on human health. The major rice bran fraction
408
K.N. Niladevi
contains 12%–13% oil and highly unsaponifiable components (4.3%). it also contains 4-hydroxy-3-methoxycinnamic acid (ferulic acid), which is also a component
of the structure of non-lignified cell walls. The presence of lignin related compounds
in rice bran is a factor that favours ligninolytic enzyme production. Laccase production has been carried out from rice bran in SmF and SSF using the basidiomycete
fungus Coriolus versicolor and it was observed that rice bran was a better substrate
for laccase production in both SmF and SSF as compared to other substrates like
wheat bran and rice straw meal (Chawachart et al. 2004). However, it remains a fact
that the nutritional requirement of the organism is the main factor that determines
the suitability of a particular substrate.
22.6.6 Coffee Pulp
The processing of coffee cherries involves the removal of shell and mucilaginous
part from the cherries. The processing is done by two methods: dry and wet processing. Depending upon the method of coffee cherries processing, i.e. wet or dry
process, the solid residues (sub-products) obtained are termed as pulp or husk, respectively (Pandey et al. 2000c). Coffee pulp contains 50% carbohydrates, 10%
proteins, 18% fibers, fat 2.5%, caffeine 1.3% and tannins 1.8–8.56% on dry weight
basis (Elias 1979). Traditionally, coffee pulp had found only a limited application as
fertilizers, livestock feed, compost, etc (Pandey et al. 2000c). In many of the coffee
producing countries the disposal of coffee pulp causes significant environment problems and assigning new uses for this agro-industrial waste is gaining more attention.
The production of ligninolytic enzymes from coffee pulp could be an efficient
method for the successful utilization of coffee pulp since, the presence of tannins and other phenolic compounds might facilitate lininolytic enzyme production.
One of the popular uses of coffee pulp is for mushroom cultivation (Salmones
et al. 2005). It has been established that the growth of white rot fungi on coffee
pulp results in the production of various extracellular enzymes, including the ligninolytic enzymes such as laccase and mangaese peroxidase (Marnyye et al. 2002).
Apart from white rot fungi, the production of laccase from coffee pulp (both SmF
and SSF) by filamentous bacteria (Streptomyces) has also been reported (Niladevi
et al. 2007; Niladevi and Prema 2008).
22.6.7 Fruit Wastes
Fruit wastes are another category of agro-industrial wastes that could be utilized
successfully for the production of ligninolytic enzymes. It needs emphasis that the
wastes generated from fruit industries are not widely used or accepted substrates
for ligninolytic enzyme production. However, the available literature indicates that
liginolytic enzymes could very well be produced from the residues obtained during
the processing of different fruits and it is a step ahead of the conventional substrates
those have been used commonly for the purpose.
22 Ligninolytic Enzymes
409
Banana waste is one of the important fruit wastes available world wide as it is
used as the common table fruit. The term banana waste includes mainly the leaf
and pseudo stem that are commonly used for paper making. The banana peel, which
is the waste from banana processing, is also termed as banana waste. Both these
wastes can be utilized for the production of ligninolytic enzymes. Banana skin has
been used as a support-substrate for the production of extracellular laccase by the
white-rot fungus Trametes pubescens (Osma et al. 2007). Production of cellulolytic
and ligninolytic enzymes from banana waste under SSF conditions has been investigated by Shah et al. and they have reported that the level of ligninolytic enzymes produced from banana waste was higher than the cellulolytic enzymes (Shah
et al. 2005). The reports suggest the suitability of banana waste for ligninolytic
enzymes production under SSF.
Grape seeds are the major waste from the grape processing units. The uitilization
of grape seeds as support substrate for laccase production in laboratory-scale SSF
bioreactors has been suggested (Rodriruez Couto et al. 2006). The lignin content
of grape seeds is much higher (around 44%) than many of the other substrates and
hence it can be utilized for the production of other ligninolytic enzymes like LiP
and MnP.
The production of kiwi fruit in the world is around 926,008,000 kg per year. Part
of this production is rejected because the kiwi fruit does not have the right shape.
The possibility of utilizing rejected kiwi fruits and peelings for laccase production
has been investigated (Rosales et al. 2005). The same group has also studied the
utility of another fruit waste; the orange peelings, for laccase production under SSF
(Rosales et al. 2007). The studies indicate that the same approach can be extended
to other fruit wastes for ligninolytic enzymes production.
22.7 Purification of Ligninolytic Enzymes
The end use of the enzyme is the key factor that determines the extent of purity
required. Many of the industrial applications of the ligninolytic enzymes (viz; dye
decolourization, phenol degradation, biobleaching etc) require only crude preparations of enzyme and in such cases the enzyme purification is not obligatory. However in certain cases, purified enzymes are also used for application purposes (Kokol
et al. 2007). Ion exchange and gel permeation chromatographic techniques are the
commonly adopted methods for the purification of ligninolytic enzymes. The number of purification steps required depends largely upon the nature of the substrate
from which the enzyme is produced and the mode of fermentation. In general, the
enzymes produced by SSF are more contaminated than those produced by SmF
and hence the enzyme purification becomes more tedious in SSF, partly depending
on the nature of the substrate. Substrates like wheat and rice straw are deficient
in protein that makes enzyme purification easier whereas, enzymes produced from
cereal brans need more steps for purification. Purification and characterization of
the ligninolytic enzymes (LiP, MnP and laccase) have been carried out from large
numbers of microbial strains including the basidiomycetes and ascomycetes fungi,
410
K.N. Niladevi
bacteria and actinobacteria. Purified ligninolytic enzymes with unusual properties
like thermo stability, alkaline stability, salt tolerance etc are of much importance
to the industries. Many reports are available on ligninolytic enzymes with novel
properties (Nozomi et al. 2002; Hoshino et al. 2002; Suzuki et al. 2003; Niladevi
et al. 2008) and these enzymes can be considered as an asset, owing to their significance in application sectors.
22.8 Conclusions
The concern towards minimizing the environmental problems is increasing all
around and it necessitates the need for a green technology in every industrial sector. A large number of industries use agro-based natural materials as raw materials
and generate considerable quantity of residues, which usually accumulates in the
environment. The current trend in biotechnology research is oriented towards the
utilization of these residues for the production of enzymes and other metabolites
and great success has been attained by many researchers in this aspect, mostly with
the aid of solid-state fermentation technology. In this scenario, bioconversion of
lignocellulosic agro-industrial residues to ligninolytic enzymes is of immense importance while considering the demand for these enzymes in the global market.
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Part V
Bioconversion of Agro-Industrial
Residues
Chapter 23
Anaerobic Treatment of Solid
Agro-Industrial Residues
Michael Ward and Poonam Singh nee’ Nigam
Contents
23.1 History of Anaerobic Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.2 Biogas Production from Agricultural Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.2.1 Hydrolysis of Agricultural Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.2.2 Acidogenesis of Hydrolysed Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.2.3 Acetogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.2.4 Methanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.3 Biogas Production Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.3.1 Small Scale Production – Rural . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.3.2 Large Scale Production – Industrial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.4 Feed Stocks for Anaerobic Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.4.1 Cattle Manure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.4.2 Agricultural Residues (Case Study Turkey) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.4.3 Agro-Wastes: (Case Study Taiwan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.4.4 Canadian Biomass Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.5 Cropgen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.5.1 Biogas Production from Maize in Differing Vegetation Periods . . . . . . . . . . . . .
23.5.2 Finnish Canary Grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.5.3 Biogas Production in Ireland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.5.4 Biomethane Versus Bioethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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429
Abstract Solid agro-industrial wastes can be recycled in a cheaper eco-friendly
bioconversion way using the anaerobic treatment method. With the depletion of
fossil fuels the identification of bio-renewable fuel replacements is well underway.
Biomethane derived from Biogas is one possible answer to this dilemma. Biogas
production technology has been used for decades in developing countries for cheap
M. Ward (B)
Centre for Vision Science, Queens University of Belfast, Royal Victoria Hospital, Belfast
BT12 6BA, Northern Ireland, UK
e-mail: michaelward@hotmail.co.uk
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 23,
C Springer Science+Business Media B.V. 2009
417
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M. Ward and P. Singh nee’ Nigam
production of fuel for heating and cooking from agro-residues, specifically animal
manure. More developed countries have followed suit with production from a wide
range of agro-wastes. With the convening of the EU, the 6th framework program
and publication lead to the investigation of crops specifically for possible energy
production. Varieties such as Maize, Sugar Beet and Wheat etc have been studied
intensively. This route of investigation has not only applied to the EU but also across
the world.
Keywords Agricultural residues · Anaerobic treatment · Biogas
Methanogenesis · Acetogenesis · Hydrolysis · Cattle manure · Biomethane
·
23.1 History of Anaerobic Digestion
The anaerobic digestion of agricultural and agro-industrial residues now has been
studied in detail by researchers in various countries. This technology has been in
practice on small rural scale or industrial scale utilizing locally produced waste
industrial substrates and residual biomass. But the process of methanogenesis or
methane production as a result of anaerobic digestion of organic matter was known
much earlier than any publication on this subject came. Although Italian physicist Alessandro Volta (1745–1827) discovered methane as a type of combustible
air, over 200 years ago. As we know it, Methanogenesis is the terminal step in
the biodegradation of organic matter in many natural anaerobic (anoxic) habitats,
such as swamps. Volta collected gas from marsh sediments and showed that it could
burn into flames, still it was not until 1933 that the first publication highlighted an
organism which produced methane as a catabolic end product (Thauer 1998). This
publication by Stephenson and Stickland was pre-empted by the discovery of the
enzyme “Hydrogenase” in a number of Archaea species two years previous. The
enzyme allowed the micro-organism to thrive in an inorganic medium by use of
formate as the sole carbon source. Due to the publication being the first to isolate, in
pure culture, a methanogen and study the aforementioned hydrogenase, it has been
marked as the beginning of the modern era for study of Methanogenesis.
Marjory Stephenson is heralded as the discoverer of hydrogenase and is recognized by her peers for her outstanding contribution to science as a whole but specifically Microbiology. Her lengthy contributions included cofounding of the Society
for General Microbiology and her eventual Presidency (Postgate 1995).
23.2 Biogas Production from Agricultural Residues
Methanogenesis is methane production by methanogens via microbial decomposition of organic matter in anaerobic environments, such as river and lake beds. It is
estimated that 1% of plant material formed per year by photosynthesis is remineralized via methane with an estimated 109 tons of combustible gas being produced
(Thauer 1998). This is methane which is naturally produced and not harvested.
23 Anaerobic Treatment of Solid Agro-Industrial Residues
419
Instead it is oxidized, buried leading to methane deposits or more critically diffuses
into the atmosphere. Atmospheric methane leads to an increase in the green house
effect as it is a potent green house gas.
But it is important to note the process is not carried out solely by a single microorganism but by syntrophic associations. There are a number of stages in the production of methane from agricultural residues. These stages are not always clearly
defined. These stages include Hydrolysis, Acidogenesis, Acetogenesis and finally
afore mentioned Methanogenesis. Typically, in a freshwater environment, plant material in the form of glucose from cellulose is completely decomposed to CO2 and
CH4 . This is carried out primarily by the fermentation of large complex polymers,
such as carbohydrates or proteins, to Carbon Dioxide (CO2 ), Hydrogen (H2 ) acetate
or formate, followed by the conversion of these substrates to methane. Methanogens
have a wide range of optimum temperatures for gaseous production. But research
has highlighted increased temperature not to be an advantage in production times
and cost. The end product of these stages is termed Biogas.
The constituents of the Biogas are methane (CH4 ), carbon dioxide (CO2 ), making up approximately 90%. Other impurities such as hydrogen sulphide, nitrogen, hydrogen, methylmercaptans and oxygen complete the unrefined fuel source
(Zinoviev et al. 2007). With increasing pressure to produce biofuels on a par with
fossil based fuels, refinement and upgrading techniques allow the level of methane
content within the gas to spike at around 97%. The refinement to almost total
methane introduces the term Biomethane and increases the value in terms of energy
of the end product. Processes involved can be Water Scrubbing, Pressure Swing
Absorption Technologies, Chemical Absorption, Membrane Separation etc. These
processes facilitate the introduction of Biomethane to the national gas grids for
home use.
23.2.1 Hydrolysis of Agricultural Residues
The initial stage of depolymerisation of the residues is carried out by a large number
of bacteria including obligate anaerobes such as Clostridia and facultative anaerobes
such as Streptococci and Enteric bacteria (Nigam and Singh 2004). Complex polymers including carbohydrates, protein and lipids are broken down into monomers
by enzymes produced by these micro-organisms. These enzymes include cellulase,
amylase and protease. The hydrolases are either secreted from or anchored on the
cell and the enzymes may be endohydrolases or exohydrolases (Parawira 2004).
This extensive variety of hydrolyses allows for the degradation of organic particles
to that of transportable molecules. Understanding of this stage is vital when dealing
with solid residues where hydrolysis is usually the rate limiting step.
23.2.2 Acidogenesis of Hydrolysed Residues
The less complex end products of Hydrolysis are used as substrates by fermentive
micro-organisms in this stage. These allow for the production of organic acids such
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M. Ward and P. Singh nee’ Nigam
as acetic, propionic, butyric and other short-chain fatty acids, alcohols, H2 , CO2 .
Most of the products formed in the metabolism of glucose have, as an intermediate,
pyruvic acid. Pyruvic acid fermentation can lead to the production of a number of
C1 and C4 compounds such as Volatile Fatty Acids (VFAs) such as acetic, propionic
and butyric acids. This is generally the fastest reaction in the anaerobic conversion
from solid organic residues to liquid phase digestion. Due to the large numbers of
species of bacteria involved in both stages, several organic acids and alcohols are
produced. The concentration and proportion of individual VFAs produced in the
acidogenic stage is important in the overall performance of the anaerobic digestion
system since, acetic and butyric acids are the preferred precursors for methane formation (Hwang et al. 2001).
23.2.3 Acetogenesis
Obligate hydrogen-producing bacteria further degrade propionic, butyric and valric acids to acetate, formate, CO2 and H2 . This intermediate depolymerisation is
key, because prior to this degradation the acids are unable to be processed by
methanogens. It is at this advanced point that definition between stages becomes
blurred. With the breakdown to acetate, formate, CO2 and H2 occurring in both Acidogenic and Acetogenic stages. Acetogens are very slow growing and particularly
sensitive to environmental change. Long lag periods have been of particular determent when it comes to the bacteria adjusting to new conditions (Xing et al. 1997).
Acetogens need for low hydrogen partial pressure makes its syntrophic association
with hydrogen-consuming methanogens ideal, especially when such reactions are
thermodynamically unfavorable.
23.2.4 Methanogenesis
The final stage of this total process that of methane production in the form of Biogas.
Utilization of mainly H2 /CO2 and acetic acid form methane and CO2 . A limited
number of other substrates can be used, such as methanol, methylamines, alcohols
plus CO2 and formate can also be used by methanogens.
Methanogens use a number of specialized enzymes in order to produce methane.
Specifically reduction of CO2 to methane utilizes Co-enzyme M (CoM) and
Co-enzyme B (CoB). The reduction of CO2 to the formyl level and consequent
further reduction to methylene allows for the transfer of the methyl group to an
enzyme containing CoM to create methyl-CoM. This methyl group consequently is
removed by Co-enzyme F430 forming a Ni+2 –CH3 complex. This complex finally
is reduced by electrons from CoB and generates methane and a disulphide complex
of CoM and CoB. The complex is reduced by hydrogen to generate CoM and CoB.
This reaction allows for energy conservation in the process of Methanogenesis.
The microbiology and the biochemistry of methane synthesis have been described in detail by Nigam and Singh (2004).
23 Anaerobic Treatment of Solid Agro-Industrial Residues
421
23.3 Biogas Production Technology
Anaerobic installations for waste and residual biomass bioconversion are known
as digesters. The primary process in biogas production is the digestion of organic
material to soluble and gaseous products as a result of microbial metabolism. The
advanced design for such installations presently in use are various types, such as
two-phase, plug-flow, packed-bed and fluidized-bed digesters. The biomethanation
plants and digesters types used for anaerobic digestion are significantly important
for the overall economy of the process, these have been discussed in detail by Nigam
and Singh (2004).
23.3.1 Small Scale Production – Rural
The technology for production of Biogas is not that of only recent development,
quite the opposite. In developing countries such as India and rural China , other
Asian and South American countries, the technology has been used for decades
only on that of a small scale. In rural parts of India anaerobic digestion of manure in
small digestion facilities produce what is known as “Gobar/Gober Gas”. There have
been approximations of over 2 million of these home facilities which provide energy
for cooking or possible on-site electric generation. The importance of the facilities
in countries which are otherwise totally dependant on outsourcing for energy need
cannot be overstated. The design of the facility is that of an airtight circular pit
made of concrete with a pipe connection. The manure is usually directed to the
digester directly from the cattle shed. The cost efficiency and simplicity of process
makes it one of the most environmentally sound energy sources for rural needs.
Other facilities which can be operated on both small and large scale include plugflow or covered lagoon digesters.
In Colombia experiments with diesel engines-generator sets partially fuelled by
biogas demonstrated that biogas could be used for power generation, reducing electricity costs by 40% compared with purchase from the regional utility. These examples showcase potential on an obvious small scale, but it is that of large scale
production that may redress the problems caused by fossil fuel dependence viewed
not only in the obvious sky rocketing of cost, but also and more importantly environmental damage.
23.3.2 Large Scale Production – Industrial
In a number of European countries including Denmark and Sweden digester facilities are in place with the processing abilities of 25,000 to 100,000 gallons of manure
per day. These facilities also handle other organic wastes collected from individual
farms. Treatment of sewage stabilization and the removal of odour has in the past
produced Biogas almost as a by-product with its use for reinvestment in treatment,
but without optimization of the Methanogenesis (Nigam and Singh 2004).
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M. Ward and P. Singh nee’ Nigam
Industrial sized digester facilities can have a number of different structures including One-Phase, Two-Phase, previously mentioned Plug-Flow, Batch Systems,
Packed-Bed and Fluidized-Bed digesters (Yang et al. 2004). About 90% of the fullscale plants currently in use in Europe for anaerobic digestion of agro-residues rely
on One-Phase systems (Vandevivere et al. 2002). This system is viewed by industrialists as more beneficial than Two-Phase or Batch-System due to reduce risk of
malfunction and reduction of starting capital. But the trade off is that of a reduction in quality of methane percentage in the harvested Biogas. This is highlighted
with the vast number of publications on the Multi-Phase systems which are viewed
by researchers as the clear pathway for development. Research has provided data
highlighting the limitations on the One-Phase system to be the delicate balancing
acts between the stages leading to and including Methanogenesis. These include pH
balance, substrate build up etc.
With Two-Phase systems a separation of the Hydrolysis and Acidogenesis from
Methanogenesis stages allow for the elimination of the limitations of stability and
control cause during One-Phase processing. But with this system comes the added
costs of start up and maintenance. The advantages of a Two-Phase reactor over a
One-Phase reaction are listed in Table 23.1. Batch Systems by design recirculate
leachate from the feed stocks in order to continually disperse inoculants, nutrients
and acids. Some Batch Systems operate the Methanogenesis stage at a higher temperature in order to decrease production times. But the failure for the system to gain
recognition within the industry with little development of these facilities highlights
its high use of end product in turn reducing its feasibility.
Table 23.1 Advantages of the two-stage system over the one-stage system when treating the same
agro-industrial wastes. (Parawira 2004)
• Two-stage systems can treat three times the organic loading of a one-stage process, and
therefore have shorter hydraulic retention time for rapidly degradable waste. The
volumetric capacity of the two-stage system is theoretically higher than that of a
single-stage system.
• Significantly higher biomass conversion efficiency and higher COD removal efficiency.
• Higher methane concentration (80–85%) in the biogas produced because the specific
activity of methanogens is increased.
• Better process reliability, resilience and stability, especially with variable waste
conditions and readily degradable waste, which causes unstable performance in
one-stage systems.
• Physical separation of the acidogenic and methanogenic phases allows maintenance of
appropriate densities of the acid- and methane-producing microbes enabling
maximisation of their rates.
• The acid phase and methane phase can be started much more easily and quickly than in
conventional, single-stage digesters.
• The acidification reactor can serve as a buffer system when the composition of the
wastewater is variable and can help in the removal of compounds toxic to the
methanogens.
• Based on information from full-scale operating systems, two-stage systems produce less
and better quality Class A biosolids. This is the main reason for using the two-stage
process.
23 Anaerobic Treatment of Solid Agro-Industrial Residues
423
23.4 Feed Stocks for Anaerobic Digestion
With vast numbers of grass species, crops, vegetable wastes and also live stock manure there are numerous feed stocks for production of Biogas. Primarily manure has
been the main stay of these digesters in the developing world, but now also in industrialized countries (Tsai et al. 2003). With vast production of agro-wastes, such as
corn husk, silage, plant stalks, fruit stones etc. there is another opportunity in which
to not only provide waste disposal but environmentally beneficial energy production
(Levin et al. 2006; Demirbas 2005). Research is also being directed toward specific
growth of termed “Energy Crops” not for that of food consumption, but for that of
specific use in digesters. Driven by the Biofuels Directive in Europe, countries such
as Ireland have invested in feasibility studies into growth of these Energy Crops on
arable lands once used in sugar production (Murphy and Power 2008), other studies
include Reed Canary grass in Finland (Paavola et al. 2007).
23.4.1 Cattle Manure
Amon et al. carried out research highlighting levels of Biogas, methane yield and
nutrient qualities obtained from Austrian Alpine dairy cows. Within this study the
levels of milking were controlled along with the animal’s dietary composition. The
milk yield ranged from 11.2 to 29.2 liters (l) of milk per cow, with differing levels
of hay, grass silage and maize silage comprising the feed viewed in Table 23.2.
These studies were carried out at small laboratory scales using 1l eudiometer batch
digesters.
The composition of the manure was determined in an effort to detect key constituents in the production of methane in the form of Biogas. The results highlighted
manures with higher crude protein levels gave higher methane yield during digestion. Lignin, an integral part of the cell walls of plant cells, in the manure reduced
the specific methane yield. This was highlighted in the higher feeding intensities
and milk yield Table 23.3.
The treatment Dairy-3 yielded that of the highest methane content of 166.3 Nl
(kg VS)−1 . Previous research has highlighted more favorable biogas return derived
from pig or poultry waste due to most of the biodegradable carbon in the cattle feed
being digested in the animal’s rumen and gut. With cattle feeds varying through
out the year recorded levels of biogas production fluctuate. This highlights variation
Table 23.2 Use of agri-residues in Diet of Dairy cattle that delivered the manure for anaerobic
digestion experiments. (Adapted from Amon et al. 2007)
Treatment
Milk yield [l/day]
Grass silage
Hay
Maize silage
Concentrate [kg Dry Mass]
Dairy 1
Dairy 2
Dairy 3
Dairy 4
Dairy 5
Dairy 6
11.2
11.2
17.6
16.0
29.2
29.2
10.4
6.4
4.8
10
3.8
6.2
5.2
5.4
4.0
5.0
3.2
3.0
0
5.8
5.2
0
3.6
0
0
0
4.6
5.8
11.0
10.0
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M. Ward and P. Singh nee’ Nigam
Table 23.3 Effect of Composition of dairy cow manure on the yield of specific biogas and methane
(Adapted from Amon et al. 2007)
Gas yielda [Nl(kg/VS)]
Composition of manure [g (kg DM)−1 ]
Treatment
DMb
XP
Cel
ADL
GE[MJ]
Biogas
Methane
Dairy 1
Dairy 2
Dairy 3
Dairy 4
Dairy 5
Dairy 6
143.7
128.8
135.0
159.6
148.5
157.3
162.6
154.3
156.6
150.6
180.2
296.5
194.7
227.3
250.8
164.1
161.8
210.1
162.1
128.2
124.7
183.3
190.4
121.7
15.8
17.3
14.6
19.3
15.6
16.8
208.2
213.1
245.8
222.5
238.9
267.7
136.5
131.8
166.3
143.1
125.5
159.2
DM = Dry Matter; XP = Crude Protein; Cel = Cellulose; ADL Lignin; GE = Gross Energy
Nl = norm litre (273 K, 1.013 bar).
b
[g (kg FM)−1 ].
a
of crop feed sources have an effect on the production of the Biogas, with other
research determining differing level of crop development adding to this problem
(Amon et al. 2007).
23.4.2 Agricultural Residues (Case Study Turkey)
As with all but a select few countries, Turkey has a high dependence on importing to
sustain its energy needs. Animal husbandry and agricultural development are both at
high levels within the country, with both comes the obvious potential for production
of renewable energy from agrowastes. This is highlighted with the extensive effort
of honing biogas production projects since the 1960s (Demirbas 2005). Construction of a number of facilities has stemmed from this research. Tables 23.4 and 23.5
highlight the possible recoverable bio-energy from animals (1997) in Kilotonne of
oil equivalent (Ktoe) and selected crops (2001) in Million tons of oil equivalent
(Mtoe).
With the vast potential at its disposal Turkey is at the forefront among Organization for Economic Co-operation and Development (OECD) countries, with a total
potential for 9.5 Mtoe from crop residues. With Biogas production being quotes
Table 23.4 Recoverable bioenergy potential of animal wastes (case study Turkey, 1997) (Adapted
from Demirbas et al. 2006)
Animal
Number of
animalsa
Coefficient of
conversionb
Energy
potential
(Kote)
Recoverable
energy potential
(Kote)
Sheep & Goats
Donkey, horse, mule & camel
Poultry
Cattle & Buffalo
75,095
1370
311,500
12,121
0048
0235
0003
0245
3604
322
935
2970
1081
97
281
891
a
b
Thousand head.
Kote per thousand head.
23 Anaerobic Treatment of Solid Agro-Industrial Residues
425
Table 23.5 Agricultural residue potential (case study Turkey, 2001) (Adapted from Demirbas
et al. 2006)
Residue
Annual Production (million tons)
Energy Potential (Mtoe)
Wheat Straw
Barley Straw
Maize Stalk
Cotton Hull
Sunflower Head
Sugar Beet Waste
Hazelnut Shell
Oat Straw
Rye Straw
Rice Husk
Fruit Peel
Total
26.4
13.5
4.2
2.9
2.7
2.3
0.8
0.5
0.4
0.3
0.3
54.4
7.2
3.9
1.2
0.9
0.8
0.7
0.3
0.2
0.1
0.1
0.1
15.5
as 1.5–2 Mtoe of this total. The Turkish city of Izmit boasts the country’s only
waste-to-energy facility with a capacity of 5.4 MW. This highlights the shortfall in
putting research into practice possibly due to further funding being necessary to
facilitate the conclusive results being published.
23.4.3 Agro-Wastes: (Case Study Taiwan)
The 1970s energy crisis highlighted to the world that biomass utilization for energy production was a necessity. With a small island country such that of Taiwan
importing more than 95% of its energy supplies, a collapse in distribution would
be catastrophic. These possible dilemmas lead to the convening of the National
Energy Conference (NEC). The aim of which was to boost the countries renewable energy supply to that of 3% of its total usage. Previously agro-residues had
been used through processes as combustion for heat or electrical generation. But
anaerobic digestion highlighted the possibility of gaseous product for a number of
energy solutions including possible vehicle transport. Another aspect of Taiwanese
industry is that of the every growing hog farms. With estimates of over 7 million
animals these also highlighted a major possibility in feed stock for digesters.
23.4.3.1 Hog Manure
Hog manure and urine primarily posed the problem of environmental safety during
disposal. By striving to identify a sustainable fuel lead to not only the production of
Biogas, but also that of high grade fertilizers for rice crop growth. This industrial
crop growth in turn produced agro-waste in the form of Rice Husk. Previous to
Biofuel technology development, husk had been used industrially for combustion.
But with this development production of hydrogen-rich synthesized gas has become
possible. At the date of publication Biogas production based upon estimations of
7 million hogs could have reached the levels below:
426
M. Ward and P. Singh nee’ Nigam
Biogas production capacity: 6 : 4 × 108 m3 = year (based on 0.25 m3 /day/head)
Electricity generation: 9 : 1 × 108 kW-h=year (based on 0.7 m3 biogas/kW-h)
Equivalent electricity charge: US$ 6 : 7×107 = year (based on US$ 0.074/kW-h)
Equivalent methane mitigation: 2 : 5 × 105 metric ton=year (based on 60%
methane in the biogas) (Tsai et al. 2004)
23.4.3.2 Rice Husk
A byproduct of the aforementioned Biogas production was that of a crop fertilizer.
This was in turn used in rice crop productions which lead to the accumulation of rice
husk. Again driven by the NEC Hydrogen gas production, in the form of Synthesis
gas (Syngas), was investigated in the processing of these agro-wastes. This Syngas
would in turn be converted to electrical power. Based upon preliminary evaluations
by Tsai et al. the following figures for energy production and power saving were
reached in favor of Syngas production:
Electricity generation: 4 : 5 × 108 kW-h=year (based on 3600 kcal/kg heating
value, 30% energy efficiency)
Equivalent electricity charge saving: US$ 3 : 3 × 107 = year (based on US$
0.074/kW-h)
23.4.4 Canadian Biomass Residues
As with most, if not all developed countries, Canada is almost totally dependant
upon fossil fuel imports to sustain its energy needs. With not only an agricultural but
forestry industry with the extent of the Canadian, a virtual untapped natural energy
resource was being disposed of with no regard for is possibly enormous monetary
and environmental benefit. This discarded waste biomass is also responsible of 10%
of the countries green house gas emissions.
Throughout the country’s provinces studies were carried out to discover the
possible value in the production of methane and hydrogen as fuels, with a comparison of the two also undertaken. Taking into account residues such that of
Agricultural residues, live stock manure agro-residues etc. the two gaseous fuels
were compared. Potential methane yield from these feed stocks are summarised in
Table 23.6.
Table 23.6 Potential methane production from a selection of residual biomass in Canada. (Adapted
from Levin et al. 2007)
Total biomass (ODT/year)
Substrate used (t/year)
Volume methane (m3 /year)
Agricultural
residues
Livestock
manure
OMSW
Municipal
biosolids
1.29×107
4.52×106
2.10×109
1.61×107
6.59×106
2.31×109
1.50×107
5.25×106
2.06×109
3.87×105
1.36×105
6.45×107
23 Anaerobic Treatment of Solid Agro-Industrial Residues
427
Table 23.7 Comparison of total energy potentials from Methane and Hydrogen Biofuels (Levin
et al. 2006)
Biomass
Agricultural crop
Residues
Livestock manure
OMSW
Municipal
Biosolids
1
Energy recovery
efficiency of methane1
Energy recovery
efficiency of hydrogen1
41.5
36.4
34.8
8.2
4.9
8.2
42.4
7.8
Heating value of biofuels/theoretical energy value of biomass
In this study methane production was highlighted to be superior within the test
study due to its higher energy yield than hydrogen (Table 23.7). But it is important
to note these are preliminary studies and not on an industrial scale.
23.5 Cropgen
Funded by the European Union’s (EU) 6th framework program involving 11 partners in 6 European countries, the research aimed to develop a biomass sustainable
fuel source to be integrated into the existing energy infrastructure in the medium
term. Long term aims are development of a renewable fuel economy via use of
agro-residues and energy crops via anaerobic digestion.
From this body many different crops and plants have been studied as for their possible benefits over one another for biogas production. (Salter et al. 2007) highlights
many values for methane potential from these feed stocks. It has been recorded
that each crop or plant can be used at different stages of growth to yield greater
amounts of methane. This has provided the opportunity of not only a mono-crop for
production, but many crops selected to enhance a cropping system.
23.5.1 Biogas Production from Maize in Differing
Vegetation Periods
As part of the Amon study Maize at three different developmental periods were
harvested to identify the optimum for biogas production. These three stages were
Milk, Wax and Full ripeness. Correlations between harvesting technology and biogas production yield were investigated. Analysis of the composition of the maize
allowed estimates to be made as to the highest yield of biogas. Not only was the
composition of the crop under scrutiny, but pre-storage and harvesting technique.
The study summated the crop to be stored as silage prior to digestion to increase
yield, with late ripening varieties obtaining superior results. Optimum results were
gained from whole maize crop again highlighting the possible production of energy
crops solely for energy production.
428
M. Ward and P. Singh nee’ Nigam
23.5.2 Finnish Canary Grass
Another study stemming from the 6th framework program was that into Biogas
production from canary grass. Investigation was undertaken to determine optimum
harvesting periods to maximize gaseous production. The study used two plots of
land from 2005–2006 encompassing three growth periods Generative, Flowering
and Vegetative. The yield from each period was compared with the generative in
both years reaping the greatest dividend 340–350 lCH4 /kg of volatile solid.
23.5.3 Biogas Production in Ireland
Decline of the sugar production industry in Ireland has lead to the availability of
arable land previously used to grow sugar beet. Murphy and Power (2008) investigated the use of Wheat, Barley and Sugar Beet in crop rotation to produce methane.
During three scenarios different crop variations were used as feed stocks for the
digesters. These scenarios were:
Scenario 1: Wheat, Barley and Sugar Beet
Scenario 2: Wheat, Wheat and Sugar Beet
Scenario 3: Wheat alone
The afore mentioned crops have previously been shown to produce different amounts
of Biogas with Wheat producing significantly more, but this is on a weight basis.
On an area of crop production, i.e. larger area for crops to be produced, Sugar Beet
is the superior crop. The study was limited to 48 Kilo Hectares (kha).
As a possible end product vehicle fuel was used to highlight the differences in
each methane yield. Firstly the harvested Biogas would be cleansed of impurities
to attain a 97% methane composition by removal of carbon dioxide to produce
Biomethane. Finally by processes of compression, to 200 bar, the Biomethane could
be used as transport fuel. Scenario 2 proved to be vastly superior with the production
of Biomethane possibly allowing for the operating of 104, 591 cars or 3377 buses,
converted to Biomethane as fuel, for an average year.
23.5.4 Biomethane Versus Bioethanol
The study also revealed a comparable view on the methane production against that of
a paper previously written by the authors on Bioethanol production. In this comparison it was revealed gross energy output of the Biomethane system was 17% higher
than that of the Bioethanol. Maybe of even greater significance was that of the cost
of Biomethane production dipping to 76% of its counterpart. This suggested the
production of Biomethane from such energy crops is superior to that of Bioethanol.
Table 23.8 summates these findings.
23 Anaerobic Treatment of Solid Agro-Industrial Residues
429
Table 23.8 Comparison of Bioethanol and Biomethane from the crop rotation scenarios. (Murphy
and Power 2008)
Scenario
M3N /a × 106
PJ/a
e/l
e/MJ
1
2
3
Biomethane
106.49
118.19
90.48
3.9
4.33
3.31
0.93
0.90
0.83
0.025
0.027
0.023
Scenario
l/a × 106
PJ/a
e/MN 3
e/MJ
1
2
3
Ethanol
158.28
175.53
150.93
3.33
3.7
3.18
0.70
0.69
0.60
0.033
0.033
0.028
23.6 Concluding Remarks
With the obvious enormity in the gulf that shall be left by the loss of fossil fuels
in 50, 100 or 150 years depending upon the literature, it is vital that a sustainable,
reliable fuel source or sources be identified. A number of biofuels such as Biogas
and Biomethane may well be the fuel that could fit bill. But as with all energy
sources it is exactly that of fitting the bill that it shall be held accountable to. Even
with the dwindling supplies of fossil fuels, it is still the cost of Biofuels compared
with these which is the major point of contention.
Biogas has its uses in developing countries as a cheap energy sources especially
on a small scale, but it is that of the larger scale production of possibly Biomethane
which truly has the attention of organizations such as the EU (CROPGEN 2007).
From such directives as the 6th Framework possible production of Biomethane
could lead to the powering of cars, buses etc. It is of paramount importance to stress
that this is a possibility that will only become a reality through hard work, stress,
endeavor and funding which at times seems to have a limit. But at what point will
that limit be surpassed pre or post fossil fuel?
References
Amon T, Amon B, Kryvoruchko V, Zollitsch W, Mayer K, Gruber L (2007) Biogas production
from maize and airy cattle manure-Influence of biomass composition on the methane yield.
Agriculture, Ecosystems and Environments 118:173–182
CROPGEN (2007). European Union 6th Framework Program
Demirbas A (2005) Potential applications of renewable energy sources, biomass combustion problems in boiler power systems and combustion related environmental issues. Prog. Eng. Comb.
Sci. 31:171–192
Demirbas A, Pehlivan E, Altun T (2006) Potential evolution of Turkish agricultural residues as
bio-gas, bio-char and bio-oil sources. International Journal of Hydrogen Energy. Elsevier 31:
613–620
Hwang S, Lee Y, Yang K (2001) Maximisation of acetic acid production in partial Acidogenesis
of swine wastewater. Biotechnology and Bioengineering 75:521–529
430
M. Ward and P. Singh nee’ Nigam
Levin DB, Sparling R, Islam R, Cicek N (2006) Hydrogen production by Clostridium thermocellum 27405 from cellulosic biomass substrates. Int. J. Hydrogen Energ. 31:1496–1503
Levin D B, Zhu H, Beland M, Cicek N, Holbein B E (2007) Potential for hydrogen and ethane
production from biomass residues in Canada. Bioresource Technology 98:654–660
Murphy J D, Power N (2008) Technical and economic analysis of biogas production in Ireland
utilising three different crop rotations. Applied Energy 86:1–11
Nigam P, Singh D (2004) Biomethanogenesis In: Ashok Pandey (ed) Concise Encyclopedia of
Bioresource Technology, The Haworth Press Inc., NY
Paavola T, Lehtomaki A, Seppala M, Rintala J (2007) Methane Production from Red Canary Grass.
European Union 6th Framework Program
Parawira W (2004) Anaerobic Treatment of Agricultural Residues and Wastewater. University of
Lund Department of Biotechnology
Postgate J (1995) Society for General Microbiology: Fifty Years On. Special publication of the
Society for General Microbiology
Salter A, Delafield M, Heaven S, Gunton Z (2007) Anaerobic digestion of verge cuttings for transport fuel. Waste and Resource Management 160:105–112
Thauer R K (1998) Biochemistry of Methanogenesis: A Tribute to Marjory Stephenson. Microbiology 144:2377–2406
Tsai W T, Chou Y H, Chang Y M (2004) Progress in energy utilization from agrowastes in Taiwan.
Renewable and Sustainable Energy Reviews 8:461–481
Vandevivere P, De Baere L, Verstraete W (2002) Types of anaerobic digester for solid wastes In:
Mata-Alvarez J, editor. Biomethanization of the Organic Fraction of Municipal Solid Wastes.
London: IWA Press; p. 112–140
Xing J, Criddle C, Hickey R (1997) Effects of a long-term periodic substrate perturbation on an
anaerobic community. Water Research 31:2195–2204
Yang Y, Tsukahara K, Yagishita T, Sawayama S (2004) Performance of a fixed-bed reactor packed
with carbon felt during anaerobic digestion of cellulose. Biosource Technology 94:197–201
Zinoviev S, Arumugam S, Miertus S (2007) Biofuel Production Technologies. Background paper,
Dubrovik, Croatia
Chapter 24
Vermicomposting of Agro-Industrial
Processing Waste
V.K. Garg and Renuka Gupta
Contents
24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.2 Vermicomposting Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.2.1 Basic Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.2.2 Potential Agro-Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.2.3 Process Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.2.4 Major Steps in Vermicomposting Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.3 Suitable Earthworm Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.3.1 Eisenia fetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.3.2 Eudrilus eugeniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.3.3 Perionyx excavatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.4 Vermicompost Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.5 Vermiwash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.6 Types of Vermicomposting Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7 Vermicomposting of Different Agro-Industrial Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.1 Sugar Industrial Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.2 Winery Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.3 Crop Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.4 Textile Industry Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.5 Coir Pith . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.6 Cassava Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.7 Pulp and Paper Mill Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.8 Coffee Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.9 Woodchips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.10 Oil Industry Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.7.11 Food Industry Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
432
433
433
434
435
437
438
439
439
439
439
440
441
442
442
448
448
449
450
450
451
451
451
452
452
454
454
Abstract Agro-industrial wastes- wastes from agriculture, food processing or any
cellulose based industries- remain largely unutilized and often cause environmental
V.K. Garg (B)
Department of Environmental Science and Engineering, Guru Jambheshwar University of Science
and Technology, Hisar 125001, Haryana, India
e-mail: vinodkgarg@yahoo.com
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7 24,
C Springer Science+Business Media B.V. 2009
431
432
V.K. Garg and R. Gupta
problems like dispersing foul odors, occupying vast areas, ground and surface water
pollution etc. These wastes could be converted into potential renewable source of
energy, if managed sustainably and scientifically. In the last few decades, vermicomposting technology has been arising as a sustainable tool for the efficient utilization
of the agro-industrial processing wastes and to convert them into value added products for land restoration practices. The product of the process, i.e., vermicompost is
humus like, finely granulated and friable material which can be used as a fertilizer
to reintegrate the organic matter to the agricultural soils. The usability of the process depends upon several factors like raw material, various process conditions- pH,
temperature, moisture, aeration etc., type of vermicomposting system and earthworm species used. The chapter briefly discusses with the process technology of
vermicomposting and the present state of research in the vermicomposting of agroindustrial processing wastes.
Keywords Vermicomposting · C:N ratio · Agricultural waste · Eisenia fetida ·
Vermiwash · Textile industry sludge
24.1 Introduction
In recent years, there is a marked trend towards the use of novel technologies,
mainly based on biological processes, for recycling and efficient utilization of organic residues. With this, it is possible to conserve the available resources and to
recover the natural products, and in some cases, to combat the disposal problems
and minimize the pollution effects. Vermicomposting has been arising as an innovative and low cost biotechnology for the conversion of agro-industrial wastes
into value added products, which can be utilized for improving the soil structure
and fertility in organic farming. The product of the process, i.e., vermicompost is
humus like, finely granulated and friable material which can be used as a fertilizer
to reintegrate the organic matter to the agricultural soils. With the adverse impacts
of agrochemicals on crop production, it is now well understood that optimum crop
yield and maintenance of soil fertility can only be achieved by the balanced use of
mineral fertilizers and organic manures. Vermicomposting of agro-industrial wastes
can help mitigating these issues, as these residues, if utilized efficiently, represent a
vast resource of plant nutrients. The process can be considered as a pre-treatment in
order to obtain a stabilized material which may respond more efficiently and safely
than the raw material to agricultural soils.
On the other hand, the traditional methods for disposal of these agro-industrial
processing wastes such as burning in situ, open dumping, land filling are now-a-days
experiencing unacceptability in many countries mainly due to scarcity of landfill
sites and increasing public awareness about the impacts that land disposal and mass
burning of the unsorted wastes can have on the environment. Vermicomposting technology can be a suitable tool to convert a significant proportion of these wastes into
useful products, thereby reducing the associated environmental impacts.
24 Vermicomposting of Agro-Industrial Processing Waste
433
This chapter describes in brief the process technology of vermicomposting as
well as the present state of research in the vermicomposting of agro-industrial processing wastes.
24.2 Vermicomposting Technology
Vermicomposting is generally defined as the solid phase decomposition of organic
residues in the aerobic environment by exploiting the optimum biological activity
of earthworms and micro-organisms. The process depends upon the earthworms
to fragment, mix and promote microbial activity in the organic waste material.
The earthworms ingest organic solids and convert a portion of it into earthworm
biomass and respiration products and egests peat like material termed as vermicompost (Loehr et al. 1985). As compared to the thermal composting, vermicomposting
generates a product with lower mass, high humus content, processing time is lower,
phytotoxicity is less likely, fertilizer value is usually greater, and an additional product (earthworms) which can have other uses is produced.
24.2.1 Basic Process
The vermicomposting process takes place in the mesophilic temperature range
(35–40◦ C). The different phases during the process are as follows:
r
r
r
Initial pre-composting phase: The organic waste is pre-composted for about 15
days before being fed to earthworms. During this phase, readily decomposable
compounds are degraded and the potential volatile substances are eliminated
which may be toxic to earthworms.
Mesophilic phase: During this phase, earthworms, through their characteristic
functions of breaking up organic matter, combine it with the soil particles and
enhance microbial activities and condition organic waste materials for the formation of organic manures.
Maturing and stabilization phase
In the vermicomposting process, the action of the earthworms is both physical/
mechanical and biochemical. Physical participation in degrading the organic substances results in fragmentation, thereby increasing the surface area for further
microbial colonization. Biochemical changes in organic matter decomposition are
carried out through enzymatic digestion, enrichment by nitrogen excrement and
transport of organic and inorganic material.
The passage of material through the earthworm intestine rapidly converts the
locked up minerals of nitrogen, potassium, phosphorus, calcium etc. into the forms
that are much more soluble and available to plants than the parent material. This
is made possible by various enzymes present in their gut as well as enzymes of
certain type of ingested microorganisms, viz., proteases, lipases, amylases, cellulases, chitinases etc which degrade the cellulosic and proteinaceous materials in
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V.K. Garg and R. Gupta
organic waste (Hand et al. 1988). The earthworms seem to have developed mutualistic relationship with microorganisms ingested for decomposition of organic matter
present in their food (Satchell 1983; Lattuad et al. 1999). Thus, the final quality of
the vermicompost is the result of combined efforts taken by earthworms and the
microorganisms. Also, it has been found that earthworms release coelomic fluids
in which mucocytes, vacuolocytes, granulocytes and lymphocytes are present (Kale
and Krishnamoorthy 1981) which kill the bacteria and the parasites present in the
waste, thus, making the vermicompost odor and pathogen free. Significantly, the
vermicompost is considered an excellent product of homogeneous and odor-less
nature, has reduced levels of contaminants, rich in microbial population and tends
to hold more nutrients over a longer period, without adversely impacting the environment.
24.2.2 Potential Agro-Industrial Residues
Virtually, any organic waste material of biological origin and biodegradable in nature may be used as a substrate material for the vermicomposting process, provided
that it does not contain any material potentially toxic to earthworms. Since, the
agro-industrial processing wastes are the byproduct or end product of the processing of agricultural materials; they offer potential opportunities to be used as
a substrate for the earthworms and micro-organisms. The agro-industrial wastes
are huge source of plant nutrients and their disposal means the ultimate loss of
the resourceful material. At present, these wastes are either grossly underutilized
or completely unutilized due to in situ burning in the fields or land disposal to
the surrounding areas. These individually and cumulatively agro-industrial wastes
could effectively be tapped for resource recovery through vermicomposting technology for use in sustainable land restoration practices. A wide variety of agroindustrial processing wastes explored for vermicomposting are encapsulated in
Table 24.1.
Table 24.1 Potential agro-industrial processing wastes
Agricultural wastes
rice husk, cereal residues, wheat bran, millet straw etc.
Food processing waste
canning industry waste, breweries waste, dairy industry waste, sugar industry waste press mud and
trash, wine industry waste, oil industry waste- non edible oil seed cake, coffee pulp, cotton
waste etc.
Wood processing waste
Wood chips, wood shavings, saw dust
Other industrial wastes
fermentation waste, paper and cellulosic waste, vegetal tannery waste
Local organic products
Coco fiber dust, tea wastes, rice hulls etc.
Fruits and vegetable processing waste
24 Vermicomposting of Agro-Industrial Processing Waste
435
24.2.3 Process Control
The optimization of vermicomposting process at a desired efficiency requires knowledge of control parameters that govern the continuity of the process. The following
parameters are considered to be of great importance for an efficient process management as well as favorable for the effective growth and reproduction of the earthworms: (a) Temperature (b) Moisture (c) Aeration (d) pH (e) Carbon to nitrogen
ratio (f) Food source
24.2.3.1 Temperature
Temperature is probably the most important factor affecting the metabolism, growth
and reproduction of earthworms. It has been observed experimentally that, during
vermicomposting process, most of earthworm species require moderate temperatures in the range of 10–35◦ C. At temperature in excess of 35◦ C, metabolic activity
of earthworms begins to decline and sometimes, mortality also occurs. It is, therefore, desirable to maintain the temperature of the treatment process as constant as
possible. Care should be taken especially when highly putrescible waste is processed
as it generates lots of heat during initial stage of decomposition. In such conditions,
to maintain the temperature within the optimal range, it is advisable to provide substrate biomass aeration for a few days before inoculation of earthworms. The key to
successful vermicomposting lies in adding the materials to the surface of the piles or
beds in thin, successive layers so that heating does not become excessive. Avoidance
of such overheating, requires careful management. Earthworms are active and consume organic materials in a relatively narrow layer of 6–9 inches below the surface
of compost or heap (Ismail 1997).
24.2.3.2 Moisture
Suitable moisture condition is an important requirement of earthworms. They require moisture in the range of 60–70% (Dominguez and Edwards 1997). The
rmoisture content during vermicomposting process depends on many factors like
physical status of the wastes, its porosity, type of vermicomposting system used etc.
The excessive wet biomass may become anaerobic with consequent production of
unpleasant odors, while lower moisture content in substrate biomass may dry up
the earthworms. Optimum metabolic rates in the process can be achieved by using
the suitable water content that does not restrict oxygen transfer and utilization in the
feed mixture. The moisture content in the vermicomposting process is maintained
through periodic sprinkling of water in the feed mixture.
24.2.3.3 Aeration
The adequate oxygen supply is the pre-requisite for a vermicomposting system.
Factors such as high levels of fatty/oily substances in the feedstock or excessive moisture combined with poor aeration may render anaerobic conditions in
436
V.K. Garg and R. Gupta
vermicomposting system. Worms suffer severe mortality partly because they are
deprived of oxygen and partly because of toxic substances (e.g. ammonia and other
phytotoxic metabolites) produced under such conditions. Proper aeration could
be achieved by periodic turning manually or by mechanical mixing of substrate
biomass.
24.2.3.4 pH
The earthworms operates efficiently at pHs in much wide range of 5 to 9, however,
a range of 7.5 to 8 is considered to be optimum. Although adjustment of pH in
the starting biomass is rarely required, but, when the substrate biomass with high
nitrogen is processed, pH should be conditioned appropriately. At high pH, release
of ammonia may results in unpleasant odors from the initial feed mixtures. It is to be
noted, that pH of the substrate undergoes considerable changes during vermicomposting process. In the initial stages, formation of CO2 and organic acids lower the
pH values, and as the process progresses, the pH value rises due to decomposition
of proteins.
24.2.3.5 Carbon to Nitrogen Ratio
The carbon to nitrogen ratio of the organic waste affects substrate decomposition
throughout vermicomposting process. Nevertheless, these two elements have to be
not only available, but necessarily in a balanced ratio. The optimum C: N ratio for
faster organic matter stabilization should be in the range of 25 to 30. Excessive
nitrogen of a specific compostable substrate will allow rapid decomposition causing nitrogen loss through volatilization, while excessive carbon presence slows the
biological activity. However, substrates with C: N ratios exceeding 40:1 decompose
at a very slow rate; it is advisable to add some nitrogen supplement to ensure reequilibrium of the C: N ratio and effective decomposition. For instance, the mixing
of lignocellulosic residues (C: N ratio 100–200) with some bulking agent having low
C: N ratio like sludge (10–15) allows the optimal condition for the biological transformation process. The C: N ratio keeps on changing during the vermicomposting
process. The loss of carbon as CO2 in the process of respiration and production of
mucous and nitrogenous excrements by earthworms enhance the level of nitrogen,
thus lowering the C: N ratio. It is to be noted that a decline in C: N ratio to < 20
indicate an advanced degree of organic matter stabilization and reflect a satisfactory
degree of maturity of organic wastes (Senesi 1989).
24.2.3.6 Food Source
Concerning the biological aspects, it is necessary that initial feed material should be
qualified physically, chemically and biologically. Following points should be taken
care of before the onset of vermicomposting process:
(a) Physical characteristics of Initial Feed Material: Physical characteristics of
the initial feed material has a marked influence on the stabilization process.
24 Vermicomposting of Agro-Industrial Processing Waste
437
For example, the substrate biomass should always maintain adequate porosity to favor the movement of air, and, hence sufficient oxygen supply within
the feed. Thus, prelim size reduction or shredding of the substrate material
to such an extent should be done that represents a good compromise between the goal of increasing the surface area-to volume ratio of the substrate
to enhance the microbial decomposition rates and the need of preventing exaggerated compaction because of excessive shredding of the initial substrate.
Organic residues that are poorly structured and too wet (e.g. food residues,
sludges) require mixing with proper bulking agents (e.g. saw dust, dried manure, wood chips etc.) to improve porosity and mitigate excessive moisture
content.
(b) Composition of Starting Material: Composition of the starting material greatly
influences the quality of the finished product. If the aim is the production of
biofertilizers for the agricultural soils from the organic waste, it should not
contain any non-biodegradable or toxic substance (e.g. inert materials, plastics,
glass, metal objects, detergents, pharmaceuticals etc.), which pose a risk either
directly to the earthworms or through their metabolic products. So, the sourceseparated organic fractions should, therefore, be selected for the vermicomposting process so that they will not contaminate the vermicompost produced and
pollute the soil.
(c) Salt content: Worms are very sensitive to salts, preferring salt contents less
than 0.5% in feed (Gunadi et al. 2002). If seaweed is used as a feed, then it
should be rinsed so as to reduce salt content. If farmyard manures are to be
used as feed material, they can be leached first to reduce the salt content. This is
done by simply running water through the material for some time (Gaddie and
Douglas 1975). If the manures are pre-composted outdoors, salts will not be a
problem.
24.2.4 Major Steps in Vermicomposting Process
Vermicomposting process involves following steps:
r
r
r
r
r
r
r
Collection of waste biomass.
Segregation of biodegradable and non biodegradable waste biomass.
Selection of biodegradable waste biomass for vermicomposting.
Shredding of waste biomass to crush it into a homogeneous mixture and to increase surface area for biological action.
Pre-composting to remove any volatile compounds potentially toxic to earthworms.
Inoculation of earthworms and maintenance of proper conditions such
as temperature, moisture, pH, aeration etc throughout the vermicomposting
process.
Screening and sorting of worms and cocoons after vermistabilization of
waste.
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V.K. Garg and R. Gupta
During the decomposition, vermicomposting process can be marked complete
by earthworms when the waste mixtures become fine, granulated and brown or dark
brown in colour. Vermicompost can be collected in layer by layer so as to separate the intermingled earthworms. Then earthworms and cocoons can be separated
from the remaining material by a dynamic separation method involving a sieve and
photo/thermal stimulus.
24.3 Suitable Earthworm Species
The earthworm species should possess a few characteristics, in order to attain the
objectives of verrmicomposting. These are as detailed below:
1. wide adaptability (tolerance) to environmental factors (capability to live in varying temperature and moisture conditions);
2. feeding preference and adaptability for wide range of organic material (high and
rich organic matter);
3. high growth rate, low incubation period, high reproduction and cocoon production rate;
4. high consumption, digestion and assimilation rate for organic matter
decomposition;
5. Easy to culture.
The vermicompost produced using different species of earthworms show variation in nutrient composition. So, the selection of the suitable species for particular
vermicomposting application is important. It is well established that epigeic species
of earthworms are used widely for the purpose of vermicomposting of different
organic wastes (Ismail 2005). Among these, Eudrilus eugeniae, Perionyx excavatus and Eisenia fetida have a great potential as wastes decomposers (Hartenstein
et al. 1979). These species are prolific breeders, maintaining a high reproduction
rate under favorable, moisture and food availability. They show high metabolic
activity and hence are particularly useful for vermicomposting. The vermicultural
characteristics of these earthworm species have been given in Table 24.2.
Table 24.2 Vermicultural characteristics of some earthworms
Name of
earthworm
Upper limit
of soil
Vermitemp.
tolerance stabilization
time (weeks)
(◦ C)
No. of
young/
cocoons
Incubation
period
Av. size
(weeks) (g)
5–9
25
6–8
2–4
3–4
0.5
7–10
30
3–4
2–3
4
1
15–18
30
4–5
1
4
1
Age for
cocoon
Optimum production
temp.(◦ C) (weeks)
Eisenia
18–25
fetida
Eudrilus
20–25
eugeniae
Perionyx
25–30
excavatus
Source: Dash and Senapati (1985).
24 Vermicomposting of Agro-Industrial Processing Waste
439
24.3.1 Eisenia fetida
Eisenia fetida, popularly known as red wriggler, red worm, tiger worm etc is perhaps
the most widely used earthworm for vermicomposting. The species has also in wide
usages for various toxicological studies as test worm. Mature individuals can attain
up to 1.5 g body weight. Each mature worm on average produces one cocoon every
third day and from each cocoon emerge from 1 to 3 individuals on hatching within
23 days. Average life of a worm is 1–2 years.
24.3.2 Eudrilus eugeniae
Eudrilus eugeniae, a native of Equatorial West Africa, is commonly known as
Night Crawler. It grows faster than other species accumulating mass at the rate of
12 mg day−1 . Mature individuals can attain body weight up to 4.3 g/individual. Maturity is attained over a period of 40 days, and, a week later, individuals commence
cocoon production (on average one cocoon day−1 ). Life span in laboratory has been
estimated from 1 to 3 years. The temperature tolerance of Eudrilus eugeniae is lesser
than that of E. fetida. This species is widely used as vermicomposting worm in
tropical and sub-tropical regions.
24.3.3 Perionyx excavatus
This species is highly adaptable and can tolerate a wide range of moisture and quality of organic matter. Average growth rate of Perionyx excavatus is 3.5 mg day−1
and body weight (maximum) 600 mg. Maturity is attained within 21–22 days and
reproduction commences by 24th day, with 1 to 3 hatchlings per cocoon. Scientists opine that species is amongst the best suited for vermicomposting in tropical
climates.
24.4 Vermicompost Quality
Vermicompost is a peat like material containing most nutrients in plant available
forms such as nitrates, phosphates, calcium, potassium, magnesium etc. It has high
porosity, water holding capacity and high surface area that provides abundant sites
for microbial activity and for the retention of nutrients. The plant growth regulators
and other plant growth influencing materials i.e. auxins, cytokinins and humic substances etc. produced by the microbes have been found in vermicomposts (Atiyeh
et al. 2002). The nutrients status of the vermicompost obtained from different organic materials is given in Table 24.3.
The vermicompost along with finely divided organic residues, partially digested
by the earthworms, living microorganisms, nitrogenous and other excretory products of earthworm metabolism forms vermicast. The casts possess high moisture
content and aerobic conditions and hence provide an extraordinary favorable microenvironment for wide range of decomposing microorganisms (Lee 1985). In fact,
the most important role of earthworm on the soil may be the stimulation of the
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V.K. Garg and R. Gupta
Table 24.3 Chemical composition of vermicompost
Characteristics
Value
Organic carbon%
Total Nitrogen %
Phosphorus%
Potassium%
Sodium%
Calcium & magnesium (Meq/100 g)
Copper; mg kg−1
Iron, mg kg−1
Zinc, mg kg−1
Sulphur, mg kg−1
9.15 to 17.88
0.5 to 0.9
0.1 to 0.26
0.15 to 0.256
0.055 to 0.3
22.67 to 47.6
2.0 to 9.5
2.0 to 9.3
5.7 to 9.3
128.0 to 548.0
microbial activity in cast (Nowak 1975). Many investigators have shown that there
is increased microbial population in earthworm casts than in the surrounding soil
(Arthur 1965).
Various bacterial species of Pseudomonas, Micrococci, Acromobacter and fungal
strains of Aspergillus niger, Penicillium sp were found in the vermicompost obtained
from coir waste (Gobi et al. 2001). Rodriguez et al. (1996) investigated the bacterial
pathogens of poultry litter in the vermicompost and their survival in the intestinal
tract of earthworms cultivated. They found that the fecal coliforms like Salmonella
and Pseudomonas species and other bacteria, which were present in poultry manure,
were significantly reduced in the vermicompost prepared from it. They concluded
that E. fetida presents an antibacterial response especially against gram negative
bacteria starting from gizzard and continuing along intestine.
Vermicompost can not be described as being nutritionally superior to other organic manures but unique in the way in which it is produced, even right in the field
and at low cost makes it very attractive for practical application. Various benefits of
using vermicomposts are given below:
r
r
r
r
r
Improves the physical structure and natural fertility of soil.
Increases the water holding capacity of soil.
Decreases the external inputs as chemical fertilizers reducing soil and water pollution.
Helps in restoring the microbial population for nitrogen fixation and phosphate
solubilization.
Produces superior quality of food and yield is enhanced.
24.5 Vermiwash
In the vermicomposting process, the bed filled with organic wastes, bedding materials and earthworms is fitted with a drainage and collection system. Vermicomposting
produces a leachate as a result of addition of moisture contents through the column
of worm action. Draining of this water or leachate is important to prevent saturation
24 Vermicomposting of Agro-Industrial Processing Waste
441
of the vermicomposting unit and attraction of pests. The leachate so obtained is
termed as vermiwash. It is beneficial in the sense that when collected it can be used
as a liquid fertilizer as it contains large amounts of plant nutrients. It is a collection
of excretory products and mucous secretion of the earthworms, along with the micronutrients from the organic molecules. Vermiwash, if collected properly, is clear
and transparent, honey brown colored fluid. It should be noted; however, that plant
bioassay test of vermiwash should be done prior to its use as foliar spray in order
to explore the presence of pathogens and phytotoxic compounds. If used as fertilizer, the vermiwash is better diluted to avoid plant damage, but this automatically
decreases its nutrient content so it has to be combined with other mineral fertilizers.
Commercial formulations of liquid fertilizers are sometimes complemented with
certain chemical compounds, such as polyoxyethylene tridecyl alcohol as dispersant
and polyethylene nonylphenol as adherent, to increase nutrient availability for plants
(Eibner et al. 1984).
24.6 Types of Vermicomposting Systems
Application of vermicomposting as an environmental biotechnology for the management of organic residues relies on the systems that lead to a satisfactory control
of the process. These systems should be designed with the motto to achieve high
decomposition rates within relatively short stabilization time. The type of the system
to be adopted depends upon the land area available, the characteristics and amount
of waste to be treated, estimated time required for the stabilization of substrate material and local climate. The common vermicomposting systems include Windrow
system, Beds and bins systems and Reactor system.
Windrow systems are extensively used both in the open air and under cover,
but require either a lot of land or large buildings. The two most common types of
windrow include (a) Static pile windrows (batch): These windrows are simply piles
of mixed bedding and feed that are inoculated with worms and allowed to stand
until the processing is complete. These piles have a height of < 60 cm (at the time
of settling).
(b) Top-fed windrows (continuous flow): These windrows are set up as a
continuous-flow operation which means the bedding is placed first, then inoculated
with worms, and then covered repeatedly with usually < 10 cm thick layers of
food. Worms tend to consume food at the food/bedding interface, and then drop
their castings near the bottom of the windrow. Unlike the batch windrows described
above, these windrows require continuous feeding and are difficult to operate during
winter due to excessive moisture content in the bed. In addition, if windrow covers
are used, they must be removed and replaced every time the worms are fed, creating
extra work for the operator. The advantages of top-feeding have mainly to do with
the greater control the operator has over the worms’ environment: since the food
is added on a regular basis, the operator can easily assess conditions at the same
time and modify such things as feeding rate, pH, moisture content, etc., as required.
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V.K. Garg and R. Gupta
This tends to result in a higher-efficiency system with greater worm production and
reproduction.
In Bed and bin system, bins are used to breed and harvest the vermicompost and
also in some cases beds are made on the ground for the same purpose. This method
is labour intensive as vermicompost has to be separated manually.
The Reactor systems have raised beds with mesh bottom. Feed stocks are added
daily in layers on top of the mesh. Finish vermicompost is collected by scrapping a
thing layer just above the mesh, and then it falls into the chamber below. The reactor
systems are rectangular boxes and not more than three meters in width. The worms
are never disturbed in their beds – the material goes in the top, flows through the
reactor (and the worms’ guts), and comes out the bottom. For maximum efficiency
these should be established under cover.
24.7 Vermicomposting of Different Agro-Industrial Wastes
Various agro-industrial wastes have been tested for their potential use as feed stock
in vermicomposting process (Tables 24.4 and 24.5). A brief review of these studies
is presented in this section
24.7.1 Sugar Industrial Waste
Sugar industry belongs to the most important agro-processing industries all over the
world. During the production process, considerable amounts of byproducts such as
(a) sugarcane bagasse (b) filter cake or press mud (c) sugarcane trash are produced.
Sugarcane bagasse is the fibrous residue left over after the crushing and extraction
of juice from the sugarcane stalk. Because of its high ligno-cellulose content and
low ash content, sugarcane bagasse is efficiently utilized by the industry itself as a
fuel to the boilers. In addition to this, bagasse has a significant market price to add
the economical benefits to the industry.
The second byproduct, filter cake (or sometimes called as press mud) originates
from the activated sludge process applied for the wastewater treatment in the sugar
mills. For about 134 million tones of sugarcane crushed, 4.0 million tones of filter
cake are generated (Yadav 1995). Filter cake has a great fertilizer value as it is a
rich source of organic matter, organic carbon, sugar, cane wax, protein, enzymes,
macronutrients (N, P and K), micronutrients (Zn, Fe, Cu, Mn etc) and microbes.
Physically, filter cake is a soft and spongy, light weight, amorphous, dark brown
to black material. It has been estimated that filter cake consists of about 1.0–3.1%
N; 0.6–3.6%P and 0.3–1.8% K. Farmers are reluctant to apply it directly to the
soils due to its mal-odor, immaturity and fear that its application may lead to crust
formation, pH variation and pollution problem. Wax content of filter cake (8.15%)
affects the soil properties by direct application and its high rate of application (up
to 100 tones/acre) leads to soil sickness and water pollution. Filter cake generates
S. No. Agro-industrial waste
Organic amendment
Earthworm species
Reference
1.
2.
3.
4.
Filter cake, trash, bagasse
Distillery Sludge
Post harvest crop residues
Solid textile mill sludge
Eudrilus eugeniae
Perionyx excavatus
Eudrilus eugeniae
Eisenia fetida
5.
6.
Guar gum industry waste
Paper-pulp mill sludge
Sen and Chandra (2007)
Suthar and Singh (2008)
Suthar (2008)
Kaushik and Garg (2003) Garg
et al. (2006) Kaushik and
Garg (2004)
Suthar (2006)
Banu et al. (2001)
7.
8.
Cattle manure
–
Cow dung
Eisenia Andrei
Eisenia fetida
Eisenia fetida
12.
13.
Viticulture and winery waste
Wood chips (from platinum ore
extraction process)
Olive pomace
Wheat straw
Mustard residues and Sugarcane
trash
Filter cake
Rubber leaf litter
Cow dung
Cow dung
Cattle shed manure
(a) Cow dung, (b) Poultry droppings,
(c) Biogas plant slurry, (d)
Agricultural wastes
–
(a) Mangifera indica, (b) cow dung,
(c) Saw dust
–
Sewage sludge
(a) Cow dung, (b) Horse dung
–
14.
Paper Waste
Eisenia fetida
Sangwan et al. (2007, 2008)
(a) Perionyx excavatus, (b) Eudrilus Chaudhuri et al. (2003)
eugeniae, (c) Eisenia fetida
Eisenia fetida
Gupta and Garg (2009)
9.
10.
11.
Cow dung
Perionyx excavatus
(a) Eudrilus eugeniae, (b) Eisenia
fetida, (c) Lampito mauritii
Eisenia andrei
Eisenia fetida
Nogales et al. (2005)
Maboeta and Rensburg (2003)
Plaza et al. (2008)
Singh and Sharma (2002)
Bansal and Kapoor (2000)
24 Vermicomposting of Agro-Industrial Processing Waste
Table 24.4 Different Agro-industrial processing wastes which have been tested for vermicomposting in yesteryears
443
444
V.K. Garg and R. Gupta
Table 24.5 Different Agro-industrial processing wastes which have been tested for vermicomposting in yesteryears
S. No. Agro-industrial waste
Organic amendment Earthworm species
Eisenia andrei
9.
a) Paper-pulp mill
Cattle manure
sludge b) Dairy
sludge
Paper-pulp mill sludge Brewery yeast
Paper-pulp mill sludge Primary sewage
sludge
Agricultural industrial –
waste
Coffee pulp
–
Lignocellulosic waste Municipal biosolids
from
Olive oil industry
–
Paper-pulp mill sludge (a) Pig slurry, (b)
Poultry slurry, (c)
Sewage sludge
Dairy sludge
(a) Cereal straw, (b)
wood shavings
Cotton waste
Cattle dung
10.
Lignocellulosic wastes –
Eisenia fetida
1.
2.
3.
4.
5.
6.
7.
8.
Reference
Elvira et al. (1998)
Lumbricus terrestris Butt (1993)
Eisenia andrei
Elvira et al. (1996)
–
Viel et al. (1987)
Eisenia fetida
Eisenia andrei
Orozco et al. (1996)
Benitez et al. (1999)
–
Eisenia andrei
–
Elvira et al. (1997)
Eisenia andrei
Nogales et al. (1999)
–
Zajonc and
Sidor (1990)
Vinceslas-Akpa and
Loquet (1996)
immense heat (65◦ C) and its natural decomposition takes long time and also does
not remove the foul odor completely. The compost so obtained has lesser nutritive
value and more compactness. However, the application of non-matured materials i.e.
those with an incomplete stabilization of their organic fraction may lead to harmful
effects to the soils.
Filter cake have been utilized significantly in the vermicomposting process for
the production of stabilized and nutrient rich manure. Pre-treatment of filter cake is
necessary prior to its utilization in vermiconversion processes to improve its palatability to the earthworms. It is kept in shade and turned for about 10–15 days till
drying. This will reduce the foul odor as well as the volatile substances present in
fresh filter cake.
The feasibility and adaptability of filter cake with various bulking agents like
cow dung and horse waste have been tested earlier (Sangwan et al. 2008). It was
hypothesized that different feed composition could affect the viability of process.
The earthworms were fed to a given range of feed composition i.e. from 0% to 50%
of filter cake amended with cow dung and horse waste agents in different set of
experiments (composition given by vermireactor no. 1–6). The C: N ratio, which
is the indicator of maturity of organic wastes, decreased with time in all the feed
mixtures. A glimpse of Figs. 24.1 and 24.2 provides an insight of the degree of
stabilization of organic matter in terms of reduction in C: N ratio. The C: N ratio
is important because plants cannot assimilate mineral nitrogen unless this ratio is
in the order of 20: 1 or less. Initial C: N ratios were in the range of 21.9–42.2
in filter cake amended with cow dung. It decreased significantly after 90 days of
24 Vermicomposting of Agro-Industrial Processing Waste
Fig. 24.1 Effect of
Vermicomposting on C: N
ratio in Filter cake amended
with cow dung
445
45
Initial C:N ratio
With worms
Without worms
40
35
C:N ratio
30
25
20
15
10
5
0
1
2
3
4
5
6
Vermireactor No.
worm activity and final C: N ratios were in the range of 14.4–16.3, depicting a high
degree of stabilization (Fig. 24.1). In another set of experiment, initial C: N ratios
for different feed mixtures in filter cake amended with horse dung were in the range
of 22.5–34.9. While, the C: N ratios of vermicompost obtained were in the range of
15.8–18.1 (Fig. 24.2). Also, the C: N ratio in worm-inoculated units was lower than
wormless units.
It was found that earthworms promote such microclimatic conditions in the vermireactors that increases the loss of organic carbon from substrates through microbial respiration. Addition of nitrogen in the form of mucus, nitrogenous excretory substances, growth stimulating hormones and enzymes from earthworms has
also been reported (Tripathi and Bhardwaj 2004). These nitrogen rich substances
were not originally present in feed and might have contributed additional nitrogen
content.
Initial C:N ratio
With worms
Without worms
40
35
C:N ratio
30
25
20
15
10
5
Fig. 24.2 Patterns of C: N
ratio during vermicomposting
of filter cake amended with
horse dung
0
1
2
3
4
Vermireactor No.
5
6
446
V.K. Garg and R. Gupta
Table 24.6 The composition of Different potting media
Treatment
no.
Composition
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Soil – control
compost (10%) + soil
compost (20%) + soil
compost (30%) + soil
compost (40%)+ soil
commercial vermicompost (10%) + soil
commercial vermicompost (20%) + soil
commercial vermicompost (30%)+ soil
commercial vermicompost (40%)+ soil
filter cake (10%) & cow dung (90%) vermicompost + soil
filter cake (20%) & cow dung (80%)vermicompost + soil
filter cake(30%) & cow dung (70%)vermicompost + soil
filter cake (40%) & cow dung (60%)vermicompost + soil
filter cake (10%) & horse dung (90%)vermicompost + soil
filter cake (20%) & horse dung (80%) vermicompost + soil
filter cake (30%) & horse dung (70%)vermicompost + soil
filter cake (40%) & horse dung (60%)vermicompost + soil
Further in the extension of these experiments, assessment of the quality of vermicompost produced from filter cake mixed with cow and horse waste on the growth
and productivity of an ornamental plant, namely, marigold was done. A total of
seventeen potting media were prepared by various combinations of vermicomposts,
compost and soil. The composition of different potting media is presented in Table 24.6. The filter cake + cow dung and horse dung vermicomposts have higher
manurial value and affects the growth and productivity of plants synergistically.
Addition of vermicomposts in appropriate quantities had improved growth and
flowering of plants including leaf areas, plant shoot biomass, root biomass, plant
height (Fig. 24.3) and flower numbers (Fig. 24.4). The vermicompost addition also
improved the physical, chemical and biological properties of the potting soil. The
results also revealed that composts are lesser effective than vermicomposts. This
shows that vermicomposts have a great potential in horticulture and agriculture in
sustainable organic farming so that growth and productivity can be increased with
maintenance of natural fertility of soil. But still prior to vermicompost application
at large scale, further research involving its effects on other crops is desired.
The molecular structure of humic acids(HA) extracted from the vermicompost
made from press mud, trash and sugarcane bagasse for 60 days was investigated
by FT-IR and 13 C CP/MAS NMR spectroscopy (Sen and Chandra 2007). A rapid
decrease in C: N ratio and lignocellulosic (lignin, cellulose and hemicellulose) content was observed during early phases of vermicomposting process. The solid state
spectroscopy (FT-IR and 13 C NMR) is considered as the most powerful tool for
examining the carbon composition of the organic material. The spectra of HA indicated a high rate of change in structure with increase in the alkyl C/O- alkyl C
ratio during the process. Aromatic structures and carboxyl groups decreased after
40 days indicating extensive mineralization during final stages of vermicomposting.
24 Vermicomposting of Agro-Industrial Processing Waste
30
447
40 days
101 days
130 days
25
Height (cm)
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Treatment No.
Fig. 24.3 Height of marigold plants grown in different treatments (treatment no. details in
Table 24.6)
40 days
70 days
101 days
130 days
18
16
Number of flowers
14
12
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Treatments No.
Fig. 24.4 Total number of flowers produced in different treatments (treatment no. details in
Table 24.6)
448
V.K. Garg and R. Gupta
Sugarcane trash has been suggested as an efficient soil conditioner. Moreover,
sugarcane trash as an amendment material, in vermicomposting of some nontraditional material such as sewage sludge, not only supply a considerable amount
of organic matter, but at the same time, enhances the nutritive value of end product.
24.7.2 Winery Waste
The major byproduct of winery industry is grape marc (GM), containing grape
seeds, stalks and skins left over after crushing, draining and pressing stages of
wine production. Grape marc is usually processed to produce alcohol and tartaric
acid, resulting in formation of new lingo-cellulose by-product spent grape marc
(SGM). The latter may be used as fuel for heating, as soil mulches and as organic
amendments. In addition, wine lee, the material that accumulates in the bottom of
wine fermentation tanks is produced. This by-product is used to make alcohol and
tartarates, resulting in soild lees cakes (LC). Finally, the alcohol production from
the above by-products leads to the generation of huge quantities of viscose, acidic
wastewaters known as vinasse. Also, the main waste of viticulture activity is vine
shoot (VS) produced during pruning of grapevines. The potential of these winery
wastes (i.e. SGM, LC, VS, vinasse biosolids) in vermicomposting has been investigated using Eisenia andrei (Nogales et al. 2005). The evolution of the earthworm
biomass and enzyme activity was tracked for 16 weeks of vermicomposting in a
lab scale experiment. Changes in the hydrolytic enzymes and overall microbial activities during the vermicomposting process indicated the decomposition of winery
wastes. The C: N ratio, conductivity, phytotoxicity was reduced, while humic materials, nutrient contents and pH were increased in all the cases, thereby, increasing
the agronomic value of the winery waste. Thus, winery wastes show potential as
raw substrate in vermicomposting technology and feasibility of such wastes in large
scale studies can be explored.
24.7.3 Crop Residues
It is to be noted that a great percentage of the crop nutrient input is returned back
in the form of plant residues during cultivation. For instance, it has been estimated
that 30–35% nitrogen and phosphorus and 70–80% of potassium applied as the nutrient input remain available in the crop residues. Various efforts have been made
to convert these nutrient rich crop residues into value added product i.e. vermicompost. The suitability of crop residues like soybean straw, wheat straw, maize stover,
chickpea straw etc. for vermicomposting has been assessed. Vermicomposting of
mustard residues and sugarcane trash mixed with cattle dung using the earthworm
Eisenia fetida have been studied (Bansal and Kapoor 2000). There was an increase
in mineral nitrogen after 90 days and microbial activity was also increased up to
60 days.
24 Vermicomposting of Agro-Industrial Processing Waste
449
Preliminary studies were carried out on wheat straw to test the technical viability of an integrated system of composting, with bioinoculants and subsequent
vermicomposting, to overcome the problem of lignocellulosic waste degradation,
especially during the winter season (Singh and Sharma 2002). Wheat straw was
pre-composted for 40 days by inoculating it with Pleurotus sajor – caju, Trichoderma harzianun, Aspergillus niger and Azotobacter chroococcum in different
combinations. This was followed by vermicomposting for 30 days. The results
indicated a significant decrease in cellulose, hemicellulose and lignin content during
pre-decomposition and vermicomposting. The best quality compost was obtained
when the substrate was treated with all the four bio-inoculums together followed by
vermicomposting. Seasonal variations in vermicomposting of red gram pod husk,
black gram pod husk, green gram pod husk, wheat straw, rice straw, sorghum
straw, grass and parthenium waste and Cicer arietinum spiked with cattle dung
were tested (Pulikeshi et al. 2003). The results revealed that seasonal changes directly affected the activities of E. fetida and indirectly the compostability of the
waste.
The post-harvest residues of some crops, e.g. wheat (Triticum aestivum), millets
(Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were utilized to recycle through vermicomposting by Eudrilus eugeniae (Suthar 2008). The
crop residues were amended with animal dung; and four types of initial fed materials were prepared: (a) millet straw (S. vulgare + Pennisenum typhoides in equal
quantity) + sheep manure (1:2 ratio) (MS), (b) pulse bran (Vigna radiata) + wheat
straw (Triticum aestivum) + cow dung (1:1:2 ratio) (PWC), (c) mixed crop residues
(mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR
+ CD) and (d) cattle shed manure (CSM). The ready vermicompost obtained from
MCR+CD vermibed showed the maximum increase in total N(143.4%), available
P (111.1%) and exchangeable K (100.0%). The end product showed reduction in
C: N ratio between the ranges of 60.7% (CSM) and 70.3% (MCR + CD). During
experimentation, the maximum mortality for E. eugeniae was recorded in MS followed by CSM > PWC > MCR + CD. Results indicated that the C: N ratio of the
substrate significantly influenced the growth parameters of E. eugeniae. This study
clearly indicates that vermicomposting of crop residues and cattle dung can not only
produce a value added product but also acts as good culture medium for large-scale
production of earthworms.
24.7.4 Textile Industry Sludge
On an industrial scale the sludge resulting from the dyeing and printing operations
of textile mills is managed through destructive methods: land filling practices and
incineration. This sludge is characterized by high BOD, COD, sodium and other
dissolved solids as well as micro-nutrients and heavy metals. Investigations have
been made to transform textile mill sludge spiked with poultry droppings in to
value added product, i.e., vermicompost by vermicomposting technology (Garg
and Kaushik 2005). The growth and reproduction of E. fetida was monitored in
450
V.K. Garg and R. Gupta
a range of different feed mixtures for 77 days in the laboratory under controlled
experimental conditions. The maximum growth was recorded in 100% cow dung
(CD). Replacement of poultry droppings by cow dung in feed mixtures and vice
versa had little or no effect on worm growth rate and reproduction potential. Worms
grew and reproduced favourably in 70% poultry droppings (PD) + 30% solid textile
mill sludge (STMS) and 60% PD + 40% STMS feed mixtures. Greater percentage
of STMS in the feed mixture significantly affected the biomass gain and cocoon
production. Net weight gain by earthworms in 100% CD was 2.9–18.2 fold higher
than different STMS containing feed mixtures. The mean number of cocoon production was between 23.4 ± 4.65 (in 100% CD) and 3.6 ± 1.04 (in 50% PD +
50% STMS) cocoons earthworm−1 for different feed mixtures tested. Vermicomposting resulted in significant reduction in C: N ratio and increase in nitrogen and
phosphorus contents. Total potassium, total calcium and heavy metals (Fe, Zn, Pb
and Cd) contents were lower in the final product than initial feed mixtures. This
demonstrated vermicomposting as an alternate technology for the recycling and
environmentally safe disposal/management of textile mill sludge using an epigeic
earthworm E. fetida, so avoiding its disposal in open dumps, agricultural fields etc.
24.7.5 Coir Pith
Coir pith is the byproduct of the coconut farm and the coir industry. A wide C:
N ratio coupled with low N content, high lignin content and presence of soluble
tannin related phenolic compounds (8–12%) cause slow decomposition of these
agro-wastes. However, vermicomposting of coir pith has been successfully carried
out by Lumbricus rubellus at pilot scale level (Kavian et al. 1998). Gobi et al. (2001)
studied the vermicomposting of coir pith by Eudrilus eugeniae and found that the
NPK values increased significantly from its original value after vermicomposting.
The lignin and cellulose content were lesser than initial amount.
24.7.6 Cassava Roots
The peels of bitter cassava root, a major source of food carbohydrate in tropics, form
toxic waste which is lethal to the soil invertebrates and inhibit the root growth of the
plants. Investigations by Mba (1996) highlighted the ability of Eudrilus eugeniae
to partially detoxify the wastes and convert the toxic cassava peels into valuable
vermicompost. Further, in the field studies, it was found that cassava vermicompost
enhanced cowpea aerial biomass production, but acidified the soil. Thus, the usefulness of the resources needs to be optimized in order to eliminate the toxin effects
and increase the bio-fertilizing ability during vermicomposting. The optimization
was done by adding three agricultural wastes, viz., poultry dropping, cow dung and
guava leaves. Of the three, the guava leaves treatment increased the soil CEC, soil
buffering capacity, eliminated the acidifying effect of cassava and promoted earthworms diversity and activities in cowpea plots.
24 Vermicomposting of Agro-Industrial Processing Waste
451
24.7.7 Pulp and Paper Mill Sludge
The solid or semi-solid sludges obtained from the pulp and paper industry are
great source of organic matter which can be effectively used in bioprocesses.
However, two factors may limit the biooxidative processes: difficulty in degradation
of structural polysaccharides and the low nitrogen content of the sludge. Both the
problems could be solved by mixing these wastes with some nitrogen rich material
acting as a natural inoculant of microbial populations.
The paper mill sludge was considered a suitable feed for Lumbricus terrestris
(Butt 1993). This sludge had no deleterious effects on the earthworms, although
worm growth rate was poor. The low level of nitrogen (< 0.5%) was considered a
limiting factor. By the addition of spent yeast from the brewing industry, the C:N
ratio of this sludge could be adjusted according to the requirements. In one such feed
mixture, containing paper mill waste and spent yeast from the brewing industry in
66:1 ratio, Lumbricus terrestris grew from the hatchling stage (50 mg biomass) to
maturity (3–4 g biomass) with in 90 days with an acceptable low level of mortality.
Kavian et al. (1998) studied bio-management of paper mill sludge using Lumbricus
rubellus in order to convert solid effluents or semi-solid sludge from paper mill into
a value added product, i. e., vermicompost using vermiculture biotechnology. The
vermicomposting of paper mill sludge mixed with sewage sludge, pig slurry and
poultry slurry in different ratios was studied by Elvira et al. (1997) by E. andrei.
Solid paper-mill sludge mixed with sewage sludge in 3: 2 ratio resulted in the highest growth rate and the lowest mortality of E. andrei, whereas paper mill sludge
mixed with pig slurry exhibited a high mortality. High mortality was not due to lack
of food but the degradation process might have resulted in change of the environmental characteristics, the polysaccharides breakdown could modify the structure of
the substrate so the water retention capacity decreases and this fact would increase
worm mortality.
24.7.8 Coffee Pulp
The ability of E. fetida pre-adapted to coffee pulp was tested to transform coffee pulp
into vermicompost under different experimental conditions in outdoor containers
(Orozco et al. 1996). The results showed that C and N content were not affected
by the depth of bed, whereas time affected both. After ingestion of pulp by the
earthworms, an increase in available P, Ca, Mg but a decrease in K was detected.
24.7.9 Woodchips
Woodchips and sewage sludge that were produced as waste product by platinum
mines were tested to vermicompost by E. fetida to examine the growth and reproductive success of the worms over 84 days for commercial use of vermitechnology (Maboeta and Rensburg 2003). The results revealed that there was no effect
on worm growth but reproductive success decreased and aluminum, copper, nickel
452
V.K. Garg and R. Gupta
were bio-concentrated in the worms. Earthworms with an addition of microorganism
inoculum (consisting of Pseudomonas, Lactobacillus and Saccharomyces spp) did
not bio-concentrate any heavy metal in their body tissue and had a significantly
higher reproductive success than their counterpart treatments without microbial inoculum. It was concluded that only economically feasible way to convert woodchip
and sewage sludge from platinum mines would be with the addition of microorganism inoculate.
24.7.10 Oil Industry Waste
Olive oil mills use a two-phase centrifugation system for oil separation after pressing
of olives. The process generates a semisolid waste, i.e., the two-phase olive pomace
called “alperujo”. Various chemical changes occurring in a mixture of two-phase
olive pomace and cattle manure after vermicomposting with E. andrei for eight
months were assessed (Plaza et al. 2008). Humic acid (HA)-like fractions were
isolated from the substrate material before and after the vermicomposting process,
and then analyzed for elemental and acidic functional group composition, by ultraviolet/visible, FT-IR and fluorescence spectroscopies. Prior to vermicomposting, the
HA-like fractions contained a prevalent aliphatic character, large C contents, small
O and acidic functional group contents. In addition to this, there was a marked
presence of proteinaceous materials and polysaccharide-like structures, extended
molecular heterogeneity and small degrees of aromatic ring polycondensation, polymerisation and humification. After vermicomposting, the total extractable C and
HA–C contents in the bulk substrates increased, and the C and H contents, aliphatic
structures, polypeptidic components and carbohydrates decreased in the HA-like
fractions, whereas O and acidic functional group contents increased. Further, an
adequate degree of maturity and stability was achieved after vermicomposting, and
the HA-like fractions approached the characteristics typical of native soil HA. Vermicomposting was thus able to promote organic matter humification in the mixture
olive pomace and cattle manure, thus enhancing the quality of these materials as soil
organic amendments.
24.7.11 Food Industry Waste
Yadav and Garg (2008) investigated the effect of food industry sludge (FIS) amended
with cow dung (CD) on the growth and fecundity of E. fetida. Nine waste mixtures of CD and increasing contents of FIS over a total amount of 150 g (100, 90,
80, 70, 60, 50, 40, 30 and 20% CD). It was inferred from the study that addition
of 30% of FIS with CD had no adverse effect on the growth and fecundity of
E. fetida (Fig. 24.5). There was a significant decrease in pH, total organic carbon
and C: N ratio, but increase in nitrogen, potassium and phosphorus contents was
recorded in the final vermicast than the initial feed mixture. After 84 day’s of worm’s
activity, final C: N ratio was in the range of 9.58 to 23.33 in vermicompost and in the
24 Vermicomposting of Agro-Industrial Processing Waste
25
Growth rate worm –1 day –1 (mg)
Fig. 24.5 Growth rate per
worm per day (mg) in
different vermireactors
containing Food Industry
Sludge
453
20
19.6
19.05 18.57
16.19
15.82 15.97
15
10
6.9
3.67
5
0
0
1
2
3
4
5
6
7
8
9
Vermireactor number
23.33
37.12
33.55
38.62
34.28
30.58
16.06
30.63
15.34
14.42
13.8
10.94
9.58
10
12.64
20
28.12
29.76
30.38
26.73
30
26.57
C:N ratio
40
21.11
41.18
41.8
44.84
45.02
50
46.61
48.38
50.23
60
0
1
2
3
Initial feed
4
5
6
Vermireactor number
Compost (without worms)
7
8
9
Vermicompost
Fig. 24.6 C: N ratio of initial feed, Compost and Vermicompost in different vermireactors containing Food Industry sludge
454
V.K. Garg and R. Gupta
range of 26.57–33.55 in compost (without worms). As evident from Fig. 24.6 that
C: N ratio reduction was higher in vermicompost (37.14%–80.9%) than in compost
(without worms) (9.61%–47%). A high degree of organic matter stabilization was
achieved in all the vermireactors fed with FIS and CD. This demonstrates the role
of earthworms in much more rapid decomposition and rates of mineralization of
organic matter.
24.8 Conclusions
Vermicomposting technology is a suitable tool for efficient conversion of agroindustrial processing wastes, which serves as a rich source of plant nutrients.
These waste materials are packed with a tremendous source of energy, protein and
nutrients, which would otherwise be lost if they are disposed as such in the open
dumps and landfills. Moreover, with the use of vermicompost as organic amendments in the agriculture, recycling of the nutrients back to the soil takes place, in
turn, maintaining the sustainability of the ecosystem. Therefore, the vermicomposting technology has enormous potential in agro-industrial waste management in a
sustainable and decentralized manner, as it yields rich organic fertilizer, safely disposes of organic waste and helps tackle environmental problems such as landfill and
the expense of collecting and transporting this waste.
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Index
A
Aceric acid, 386
Acetogenesis, 65, 419, 420
Acidogenesis, 53, 65, 419, 422
Acidogenic and hydrogenic bacteria, 62, 67
Acidulant, 38, 42, 53
Actinomycete, 135, 209, 375, 376, 404
Added-value products, 316
Aeration, 39, 46, 47, 55, 56, 137–149, 174,
202–204, 212–215, 349, 351, 390, 432,
435–437
Aerobacter aerogenes, 113, 361
Agaricus bisporus, 117, 164, 166, 169,
171–178
Agastache rugosa, 111, 112
Agricultural crops, 4, 5, 163, 166, 320,
406, 427
Agricultural residues, 24, 28, 29, 38, 39, 43,
51, 70, 130, 133, 135, 164, 166–168,
197, 199, 207, 210, 216, 226, 240, 242,
418, 419, 424–426
Agricultural waste, 4, 14, 38, 44, 79, 81, 82,
91, 92, 120, 130, 180, 184, 203, 226,
299, 345, 373, 434, 443, 450
Agroindustrial residues, 3–10, 13–29, 37–58,
61–63, 68, 70, 73, 74, 77–92, 117, 129,
130, 132, 135, 139, 140, 143, 148, 159,
163–187, 199, 225, 226, 229, 233, 239,
278–281, 359, 360, 362, 373, 375, 379,
383, 384, 389, 390, 392, 397, 398,
405–407, 410, 417–429, 434
Agroindustrial wastes, 6, 20, 41, 62, 64, 66,
71, 74, 118, 119, 143, 198, 232, 389,
407, 408, 417, 422, 431, 432, 434,
442–444, 454
Agroresidues, 84, 130, 137, 141–143, 165,
198, 201, 205–207, 216, 232, 242, 253,
362, 363, 367, 379, 418, 422, 425–427
Alcohols, 16, 20, 72, 107, 117, 154, 155, 234,
241, 279, 295–297, 360, 400, 401,
441, 448
Alkalinity, 67, 352
Allium cepa, 111, 112
Allium sativum, 111
Amaranthus spp., 349
Amylases, 6, 7, 43–45, 69, 71, 154, 200, 204,
209, 210, 234, 235, 243, 261, 262, 278,
318, 359–366, 377, 419, 433
␣-amylases, 6, 154, 200, 209, 210, 234, 243,
261, 262, 278, 318, 359–361, 363–366
-amylases, 7, 361, 363, 366
Amylolytic enzymes, 46, 359–367
Anaerobic, 18, 48, 62, 65, 68, 298, 305, 306,
351, 353, 375, 418, 421, 435
digestion, 62–67, 69, 81, 319, 351–353,
418, 420–423, 425, 427
sludge, 69
treatment, 266, 352, 417–429
Animal
feed, 14, 17, 24, 26, 78–82, 84, 86, 88, 90,
91, 135, 163–165, 186, 198, 200, 203,
210, 228, 239, 241, 248, 253–255, 258,
259, 264–266, 281, 318, 319, 321, 323,
330, 364, 379, 389, 391
nutrition, 77, 78, 82, 83, 86–92
Ankaflavin, 155, 156
Antibacterial, 131, 165, 277, 321, 440
Antibiotics, 7–9, 130, 131, 134, 135, 141, 143,
203, 205, 207, 210, 263, 285, 321, 329
Apiose, 386
Apple(s), 15, 273, 274, 276
pomaces, 45, 52, 63, 81, 84, 90, 118, 119,
184, 226, 232, 273, 274, 276–281, 283,
363, 389–391
Arabinan, 256, 317, 386, 387
Arabinogalactan I (AG-I), 386, 387
Arabitol, 313, 319
457
458
Aroma, 85, 106, 109–115, 117–120, 199, 209,
225, 226, 232, 244, 247, 281
application, 105
compounds, 63, 105–120, 231, 232, 273,
280, 281
production, 105, 106, 109, 111, 112, 118,
119, 209, 232, 244
Aromatic compounds, 63, 105–120, 231, 232,
273, 280, 281, 320
Arthrobacter globiformis, 116
Artificial (synthetic) logs, 184
Ascomycetous, 25, 114, 150, 153, 210, 409
Aspergillus niger, 24, 25, 40–45, 47, 48, 50–53,
84, 85, 116, 117, 120, 135, 198–201,
209, 210, 231, 233, 242, 243, 247,
261, 262, 264, 279, 280, 283, 299–301,
308, 365, 366, 374, 379, 390–392, 401,
440, 449
Aspergillus oryzae, 41, 55, 56, 85, 89, 119,
131, 210, 212, 261, 299, 300, 308, 318,
364, 366, 376
Aspergillus sp., 47, 118, 151, 204, 266, 278,
282, 363, 388
Azaphilones, 147, 151, 155, 156
B
Bacillus subtilis, 119, 131, 200, 243, 318, 365,
366, 374, 376, 404
Bacteria, 5, 19, 24, 25, 57, 62, 67, 68, 80, 81,
83, 84, 89, 112–115, 119, 133, 134,
150, 151, 177, 203, 208, 209, 231, 234,
241, 247, 260, 277, 278, 280–282, 297,
298, 302, 306, 307, 318, 321, 324, 361,
375, 376, 383, 388, 389, 392, 401, 403,
404, 408, 410, 419, 420, 434, 440
Bacterial endotoxins, 131, 134
Bagasse, 14, 20, 41, 45, 46, 51, 52, 54, 55, 63,
82, 84, 85, 117–119, 131, 159, 164,
166–168, 170, 180, 183–186, 198–200,
210, 225–235, 239–248, 254, 262, 281,
338, 363, 373, 374, 379, 389–391, 393,
405, 406, 441, 443, 446
Banana waste, 28, 118, 363, 366, 374, 409
Barley, 14–18, 25, 28, 82, 131, 138, 166–168,
184, 198, 199, 201, 202, 313–317, 363,
364, 425, 428
Basidiomata, 176, 184, 185
Basidiomycetes, 84, 114, 150, 163, 166,
171–173, 177, 182, 186, 210, 242, 264,
408, 409
Beer, 53, 110, 112, 113, 155, 211, 313–331,
319, 320
Beverages, 38, 41, 42, 54, 110, 112, 114–116,
155, 233, 278, 296
Index
BGL gene, 377
Bioactive compounds, 129, 130, 132, 134,
263, 285
Biocatalysis, 5, 10, 285
Biocatalyst, 5, 69, 72, 117, 284
Bio-chemicals versus petrochemicals, 3
Bioconversion, 5, 20, 22, 38, 44, 70, 109, 112,
115, 116, 136, 177, 182, 197–199, 209,
210, 216, 226, 230, 240, 245, 248, 266,
280, 294, 302, 303, 365, 373, 378–380,
393, 410, 417, 421
Biodegradation, 26, 27, 80, 164, 165, 172, 182,
186, 418
Biodiesel, 4, 62, 71–74
Bioenergy, 4, 294, 302, 306, 424
Bioethanol, 4, 18, 24, 62, 63, 69–71, 73, 233,
245, 248, 273, 278, 279, 371–373,
378–380, 389, 428, 429
Biofuels, 4, 10, 61–74, 198, 199, 233, 241,
279, 298, 301, 305, 327, 328, 333, 377,
379, 419, 423, 425, 427, 429
Biogas, 4, 18, 62–66, 68, 69, 266, 319, 341,
343, 352, 353, 392, 417–429, 443
Biohydrogen, 62, 68, 69, 73
Biological efficiency (BE), 164–166, 179,
181–184
Biologically active compounds, 130
Biomass, 4, 5, 10, 13, 15, 17, 18, 57, 63,
68–71, 77, 78, 80–88, 90–92, 130,
140–142, 158, 164, 166–170, 197–200,
204, 206, 210, 216, 226, 234, 242, 266,
267, 294–299, 302–304, 307, 308, 328,
331, 332, 337, 339, 352, 353, 362,
371–380, 384, 389, 391, 399, 418, 421,
422, 425–427, 433, 435–437, 446, 448,
450, 451
Biomass total utilization, 328
Biomethane, 62, 64, 69, 73, 417–419, 428, 429
Biopigments, 159, 232
Bioplastics, 7, 246–248
Bioprocess technology, 225, 253
Bioreactor, 39, 46, 47, 56, 113, 115, 118,
136–140, 142, 143, 198, 211–213, 308,
352, 373, 409
Biorefinery, 53, 57, 330, 371, 372, 378
Bio-renewable fuel, 417
Biotechnological applications, 172, 174, 277
Biotransformation, 80, 111, 113, 114, 116,
165, 198, 304, 332
of cassava bagasse for feed or food, 234
Bitter substances, 320, 321
Bjerkandera adusta, 19, 24, 26, 28, 117, 198,
201, 404
Index
Bran, 4, 25, 28, 38, 41, 43, 45, 51, 52, 61, 63,
64, 73, 84, 85, 117, 119, 130, 131, 136,
137, 141, 169, 172, 173, 180, 182, 199,
200, 226, 233, 241, 242, 254, 258, 259,
262, 266, 281, 327–339, 360, 362–367,
373, 374, 388–390, 392, 393, 406–408,
434, 449
Brewery, 63, 313, 314, 318, 321, 366, 444
Brewing, 167, 261, 298, 313–324, 360,
364, 451
Brown rot fungi, 24, 25, 171, 206, 375, 401
Buffalo, 424
Buffering, 38, 42, 67, 351, 450
Bulk-chemicals, 4, 5, 10, 118, 389
By-products, 8, 14, 38–41, 44, 51, 66, 87,
116, 117, 163, 164, 166–169, 175, 198,
199, 203, 234, 239, 240, 246, 253, 254,
266, 273–277, 279–281, 295, 301, 303,
306, 313–324, 359, 362–365, 367, 389,
391–393, 405–407, 421, 448
C
Candida tropicalis, 113, 247
Capsicum frutescens, 112
Carbon credit, 341, 353
-carotene, 150, 152, 263
Casing, 177, 178
Cassava, 20, 41, 45, 46, 51, 52, 54, 63, 82, 84,
88, 89, 117–119, 131, 159, 198, 199,
225–235, 244, 281, 363, 365, 366, 450
bagasse, 20, 41, 46, 51, 52, 63, 82,
117–119, 159, 225–235, 281, 363
roots, 230, 450
Castor cake, 258, 364
Cattle, 15, 63, 66, 86, 87, 241, 296, 313, 330,
347, 348, 366, 407, 418, 421, 423, 424,
443, 444, 448, 449, 452
manure, 66, 423, 443, 444, 452
CBH1, 377, 380
Cellobiose, 116, 171, 206, 306, 373, 377, 380
Cellulase, 23, 24, 71, 83, 91, 171, 173, 186,
199, 200, 204, 210, 230, 234, 241–243,
247, 264, 278, 283, 298, 299, 301,
303, 305, 307, 309, 318, 333, 372–380,
387, 433
Cellulose, 14–17, 19–26, 29, 45, 53, 67, 69, 71,
73, 80, 82, 85, 90, 118, 168, 170–173,
183, 200, 206, 234, 239, 240, 243, 246,
249, 256, 258, 264, 276, 279, 293, 294,
296, 298, 299, 302–306, 308, 316–320,
328–333, 335–339, 346, 350, 372–380,
385, 390, 398, 399, 405, 406, 419, 424,
431, 442, 446, 448–450
459
Cellulosic, 15, 70, 82, 90, 115, 233, 240, 279,
280, 294, 297–299, 306, 373, 384, 389,
433, 434
Ceratocystis f mbriata, 85, 118, 119, 231, 232,
247, 281
Ceratocystis moniliformis, 114, 115
Ceratocystis variospora, 114
Cereals, 4, 14, 64, 159, 164, 166, 316, 363, 373
bran, 25, 117, 172, 173, 363, 409
grains, 5, 25, 43, 130, 135, 137, 167, 169,
175, 184, 317, 363, 392
straw, 167, 176, 180, 184, 327–329, 331,
332, 334, 444
Charcoal, 19, 112, 319, 349, 350
Cheese Whey (CW), 66
Chelation, 38, 41
Chemical composition, 165, 166, 214, 229,
255, 277, 296, 315, 317–320, 322, 327,
331, 334, 335, 339, 363–365, 440, 441
Chemical oxygen demand (COD), 66, 299,
308, 352, 353, 422, 449
Cinnamomum tenuipilum, 115
Citric acid, 6, 7, 38–53, 84, 89, 199, 231, 233,
244, 245, 277, 279, 280, 295, 300,
307, 308
Citrus, 45, 81, 84, 90, 198, 200, 201, 233,
274–281, 284, 285, 389, 391
bagasse, 391
Cladosporium suaveolens, 113
Cloud point, 71
C/N ratio, 67, 173, 184, 246, 346–349, 436,
444–446, 448–454
Co-digestion, 62, 66, 67
Co-enzyme, 420
Coffee pulp and husk, 84, 117, 164, 170, 393
Coir pith, 167, 362, 450
Colonization, 137, 164, 169, 172–174,
176–178, 180, 183, 184, 433
Compost, 80, 131, 164, 165, 172, 173,
176–178, 198, 216, 330, 341, 346–349,
408, 435, 444, 446, 449, 453, 454
Composting, 63, 91, 172, 176–178, 202, 209,
216, 226, 343–347, 349, 350, 433,
437, 449
Confectionery, 42, 308
Continuously Stirred Tank Reactor (CSTR),
69, 353
Copra cake, 254–256, 261
Corn steep liquor, 8, 70, 243
Corn wet milling, 354
Corynebacterium glutamicum, 87, 114, 116
Corynebacterium sp., 116
Cottonseed cake, 254, 260, 263–266
460
Cow dung, 63, 347, 348, 443–446, 449,
450, 452
Crop-based residues, 168
Cropgen, 427, 429
Crop residues, 5, 14, 15, 54, 80, 163, 164, 166,
167, 169, 176, 199, 225, 229, 254, 365,
407, 424, 427, 443, 448, 449
Cultivation, 6, 26, 43, 55, 56, 62, 63, 79, 80,
83, 87, 88, 115, 118, 119, 130, 132,
133, 135, 137, 140–142, 159, 163–166,
169, 170, 172–178, 180, 182–186, 198,
201, 203, 206–211, 214, 215, 230, 232,
233, 242, 279, 304, 305, 313, 318, 342,
343, 408, 448
Cyclodextrinases, 362, 363
D
Dairy, 8, 42, 54, 112–114, 119, 313, 423, 424,
434, 444
Degumming of plant fibers, 278, 384
D-Galacturonic acid, 385, 386
Dha (3-deoxy-D-lyxo-2-heptulosaric
acid), 386
Diacetyles, 105
Drum bioreactor, 46, 118, 138–140, 142, 212
Dry residues, 14, 167
Dura, 342
E
Eco-friendly bioconversion, 417
Edible oil cakes, 253–267, 434
Eisenia fetida, 438–440, 443, 444, 448–452
Elaeis guineensis Jacq., 342
Empty fruit bunch, 341, 343–348
Energy, 3–5, 7, 8, 14, 16, 20, 22, 42, 46, 62,
66, 68, 78, 82, 83, 86, 89, 130, 133,
134, 167, 168, 170, 201–203, 205, 207,
225, 226, 230, 235, 240, 256, 263, 266,
278, 279, 295, 302, 304, 305, 307, 328,
329, 341–343, 345, 353, 362, 366, 375,
377–380, 389, 390, 392, 405, 418–421,
423–429, 432, 454
Ensiling, 77, 80, 81, 86, 87, 90, 92, 209
Enteric bacteria, 419
Environmental parameters, 173, 174, 209
Enzymatic, 7, 8, 10, 14, 16, 19, 23, 25, 27–29,
71–73, 83, 116, 171, 201, 205, 230,
233, 234, 245, 246, 248, 263, 265, 273,
274, 278, 282–284, 286, 302–304, 314,
318, 323, 329, 336–339, 374, 375, 378,
379, 385, 433
modifications, 273, 274, 282
Enzyme(s), 5, 6, 9, 14, 20, 24–26, 28, 42–46,
48–50, 53, 57, 63, 68–72, 77, 79, 80,
Index
83, 85, 91, 92, 105, 106, 109, 110, 112,
113, 115, 117, 118, 134, 136, 150,
154–156, 163, 165, 167, 169, 171–173,
176, 183, 184, 186, 198–200, 203–207,
209, 210, 212–216, 226, 230, 233, 235,
239, 242, 243, 245, 248, 253, 254, 256,
259–262, 265, 267, 273, 275, 277–279,
282–286, 295, 297–302, 304–306, 309,
318, 323, 329, 338, 343, 359–367,
371–381
production, 24, 28, 92, 167, 173, 186, 200,
210, 215, 242, 260, 261, 304, 309, 318,
363, 373, 379, 383, 384, 388–393, 398,
404–408
Ergot alkaloids, 130, 131, 244, 247
Escherichia coli, 113, 115, 247, 278, 282,
297, 306
Ethanol, 6, 18, 20, 22, 24, 47, 48, 57, 63, 65,
68–71, 107, 115, 116, 118, 141, 165,
210, 211, 225, 226, 232–234, 240, 245,
247, 276–279, 294, 297–299, 301, 305,
306, 313, 314, 319, 321, 329, 333–338,
341, 343, 371–373, 378, 379, 406, 429
fermentation, 24, 298, 321, 334, 336, 338
Eudrilus eugeniae, 438, 439, 443, 449, 450
F
Feedstock, 4–7, 9, 69, 199, 245, 253, 254, 259,
279, 296, 302, 305, 371, 372, 374, 375,
378, 379, 435
Fermentation, 3–10, 17–20, 24–26, 29, 38–51,
53–56, 62, 68–71, 73, 77–82, 84,
88, 89, 91, 92, 109, 110, 112, 113,
117–119, 129–143, 148, 153, 155, 158,
159, 163, 164, 166, 175, 186, 197–216,
225, 226, 230–235, 241–245, 253–255,
260, 263–267, 277, 279, 280, 283,
294–296, 298–300, 302–308, 314, 315,
319, 321, 324, 328, 329, 332–339, 359,
360, 362–366, 372, 378–380, 383–385,
388, 389, 391, 393, 398, 404–406, 409,
410, 419, 420, 434, 448
residues, 333
Fermentative process, 90, 313, 318, 319,
321, 324
Fermenter design, 211
Fertilizer, 18, 26, 71, 186, 241, 258, 273, 321,
330, 341, 342, 344, 345, 347–349, 408,
425, 426, 432, 433, 440–442
Fiber, 5, 14, 18, 19, 22, 38, 63, 71, 85, 118,
137, 229, 230, 245, 246, 255, 256, 258,
259, 265, 266, 276–278, 281, 318, 320,
322, 329, 330, 331, 334, 336–338, 341,
343, 346, 353, 360, 363–365, 367, 372,
Index
373, 375, 383, 384, 388, 390–393, 405,
408, 434
Filamentous fungi, 40, 79, 116, 130, 150, 151,
156, 175, 199, 200, 209, 210, 232, 241,
242, 298, 299, 301, 302, 306, 308, 361,
375, 377, 379, 380, 383, 388
Finnish canary grass, 428
Fixed film reactor, 351
Flavonoids, 275, 276, 283–285
Food acidulant, 38, 233
Food industry residues, 77, 78
Food industry sludge (FIS), 452–454
Fossil
fuel, 18, 64, 73, 226, 278, 327, 328, 334,
417, 421, 426, 429
resources, 3, 4
Fractionated conversion, 327, 328, 336, 339,
331–338
Fresh fruit bunch (FFB), 342, 343, 345,
350, 352
Fructification, 165, 172, 178, 180, 182,
184, 185
Fruit industry wastes, 90
Fruiting body, 164, 165, 172–176, 179, 181,
182, 185, 186, 264
Fruit juice
clarification, 278, 282, 284, 393
extraction, 278, 384
Fruit processing industry residues, 273–286
Fruit wastes, 90, 277, 282, 406, 408, 409
Fumaric acid, 56, 57, 231, 233
Fungal cellulase, 230
Furfural, 246, 330, 336–338
G
Gamma-linolenic acid, 73, 280
Gaseous phase, 202
Gas exchange, 175, 205, 206, 214
General microbial cultivation systems, 230
General properties, 228
Generation of bagasse, 228
Geotrichum candidum, 109, 200, 210
Gibberllic acid, 245, 247
Glucoamylases, 6, 44, 53, 69, 200, 210, 261,
361, 363, 365, 366
Glucose syrups, 6, 7, 360
Glucosidase, 200, 210, 234, 242, 278,
283, 360, 361, 371–373, 377,
380, 389
Glutamyl-monascorubrine, 157
Glutamylrubropunctatine, 157
Glycoside hydrolases, 361
Glycyrrhiza glabra glandulifera, 111
461
Goat
dung, 349
manure, 341, 349
Grape(s), 15, 115, 273, 274, 276, 277, 294, 391
pomaces, 45, 52, 90, 273, 274, 277–280,
284, 309, 389–392
GRAS (generally recognized as safe), 40, 41,
78, 92, 232, 300, 379
Green house gas, 68, 70, 71, 74, 294, 341, 353,
419, 426
Groundnut cake, 254
Growth-promoter, 131, 134
Growth rate, 173, 184, 185, 206, 209, 307, 438,
439, 450, 451, 453
H
Hanseniaspora guilliermondii, 114
Hansenula anomala, 115
Heme peroxidase, 401, 402
Hemicellulose, 14–17, 19–24, 26, 29, 71, 73,
80, 82, 90, 168, 170, 171, 200, 239,
240, 242, 243, 245, 246, 248, 258, 264,
276, 279, 294, 296, 298, 302, 303, 305,
316–318, 320, 336, 346, 372, 374, 375,
385, 390, 399, 405, 406, 446, 449
Hemicellulosic, 21, 70, 246, 280, 294
Heteropolysaccharides, 15, 168, 242, 273, 280
Heterotrophic, 68
Hog manure, 425
Homogalacturonan, 321, 385
Hydrogen, 67–69, 294, 305, 306, 322, 333,
402, 419, 420, 425–427
Hydrolysis, 7, 8, 19, 21, 29, 43, 44, 67, 87,
155, 198, 201, 209, 214, 230, 234, 241,
243, 245, 246, 248, 278, 302–305, 307,
314, 318, 321, 323, 329, 337–339, 359,
361, 374–380, 388, 417–419, 422
Hydrolysis/liquefaction, 65
I
Idiophase, 49, 130
Immuno-suppressive drugs, 131, 134
Indigoids, 152
Industrial enzymes, 6, 80, 239, 372
Industrial fermentation substrates, 5
Inhomogeneity, 328, 331, 334
Inoculum (spawn), 44, 47, 51, 55, 56, 164,
175, 178, 212–214, 234, 449, 452
Inulinase, 243
Invertase, 69, 70
Isoamylases, 361
J
Jute seed, 258
462
K
Kapok cake, 258
Karanja
kernel, 259
seed, 258
Kdo (3-deoxy-D-manno-2-octulosonic
acid), 386
Kernel, 85, 117, 205, 254–259, 262, 265, 266,
341–343, 345, 348–350, 353, 363,
364, 407
Kluyveromyces marxianus, 85, 115, 116, 119,
231, 232, 243, 281, 319
Kusum
kernel, 259
seed, 258
L
Laccase, 14, 19, 24–26, 28, 171, 173, 184, 247,
299, 309, 398, 400–409
Lactic acid, 7, 38, 39, 41, 46, 53–57, 80, 81,
86, 89, 112–114, 119, 199, 208, 230,
231, 235, 244, 247, 264, 283, 295, 297,
307, 313, 319
Lactobacillus acidophilus, 119, 265, 324
Lactobacillus amylophilus, 119
Lactobacillus casei, 41, 55, 231, 264, 319
Lactobacillus helveticus, 41, 55
Lactobacillus paracasei, 41, 43, 119
Lactobacillus rhamnosus, 113, 319
Lactobacillus sp., 54, 112
Lactococcus lactis, 112–114
Laying Hen Litter (LHL), 66
Lentinula edodes, 25, 28, 164–166, 169,
171–174, 176, 177, 180–182,
184–186, 404
Leuconostoc mesenteroides, 112, 231
L-glutamic acid, 244
Lignin, 14–17, 19–29, 67, 71, 82, 90, 116,
168–173, 183, 186, 199, 200, 204, 206,
234, 239, 240, 243, 245, 246, 258, 276,
296, 298, 299, 301–303, 305, 316, 317,
320, 329–332, 334, 336, 337, 346, 347,
372, 374, 375, 378, 390, 397–409, 423,
424, 446, 449, 450
degradation, 19, 22, 24, 26, 171, 172, 199,
210, 401, 404
peroxidase, 19, 26, 171, 398, 400, 401
Ligninolytic enzymes, 26, 397–410
Lignocellulose, 15, 16, 20, 21, 24, 26, 28, 29,
67, 83, 84, 165, 166, 168, 171, 172,
177, 184, 186, 198, 199, 210, 246, 301,
303, 307, 327, 329–331, 339, 366, 380,
381, 405
Index
Lignocellulosic biomass, 63, 166–168, 296,
303, 304, 371, 372, 374, 378–380
Lignocellulosic material, 4, 18, 21, 71, 77,
82, 163, 165, 171, 175, 183, 198, 240,
294, 298, 302, 303, 305, 307, 316, 318,
378, 380
Lignocellulosic residues, 26, 165–169, 186,
241, 378, 389, 398, 405, 436
Lignocellulosic wastes, 55, 82, 83, 85, 200,
281, 379, 434, 444, 449
Lindera strychnifolia, 111
Linseed cake, 153, 254, 258
Lipase, 8, 43, 72, 110, 160, 171, 200, 210, 243,
261, 262, 283, 284, 343, 433
Lipids, 7, 49, 73, 74, 91, 106, 158, 201, 229,
266, 275, 317, 319–322, 367, 375, 419
Liquid process, 78
Litter-decomposing fungi (LDF), 171
M
Mahua
kernel, 258
seed, 258
Maize germ, 259
Malus silvestris, 111
Manganese peroxidase, 14, 19, 24–26, 28, 171,
173, 398, 400–407, 409
Mango kernel, 258, 259
Marine yeast, 352
Mechanical carding, 336–338
Mesophilic, 66, 67, 69, 361, 433
Methanogenesis, 65, 67, 419–422
Methanogenic, 62, 66, 68, 352, 422
Methanogens, 418–420, 422
Methyl ketones, 109, 117, 120
Microbial biomass, 81–83, 87, 88, 91, 164,
200, 206, 216, 295, 303, 308
Microbial-nutrition, 3
Microbial transformations, 115, 132, 201, 273,
274, 277, 304
Microorganisms, 5–7, 10, 14, 19, 22, 24, 26,
29, 40, 45–50, 53, 55, 79, 84, 85, 89, 90,
109, 112, 114, 115, 117–119, 129–135,
137–139, 141, 143, 147, 149–153, 158,
163, 186, 198–203, 209, 210, 214–216,
231, 235, 241, 246, 280–282, 294, 297,
298, 301, 302, 304–307, 306, 313, 318,
319, 330, 335, 339, 343, 353, 360–362,
373, 374, 376, 380, 388–390, 400,
403–405, 419, 433, 434, 439, 452
Minerals, 6, 8, 18, 48, 202, 258, 267, 275, 276,
313, 317, 318, 322–324, 364, 366, 433
Index
Mixing, 23, 47, 135, 137, 138, 140, 141, 182,
203, 212, 214–216, 314, 436, 437,
449, 451
Moisture, 17, 43, 47, 51, 55, 56, 80, 81, 132,
137, 139, 140, 167, 169, 174, 182,
184, 198, 201–205, 207–209, 211,
214–216, 228, 229, 234, 242, 244,
258, 318, 332, 344, 346, 349, 351,
363–365, 388, 389, 391, 432, 435,
437–441
content, 47, 51, 55, 56, 81, 132, 137, 139,
140, 142, 167, 174, 182, 202, 204,
205, 207, 208, 214–216, 228, 242,
346, 349, 364, 388, 391, 435, 437,
439–441
Molasses, 6, 8, 9, 14, 28, 40, 41, 45, 52, 63, 68,
73, 87, 169, 240, 254, 276, 279, 363,
365, 366, 378
Molecular biotechnology, 294
Monascorubrin, 151, 155–158
Monascus, 147, 148, 150–160, 232
Mono alkyl esters, 72
Morphological fraction, 334–336, 339
Mulch, 296, 321, 330, 341, 342, 344, 346,
347, 448
Mushrooms (cultivated, medicinal, wild), 63,
83, 85, 91, 163, 166–187, 230, 232,
241, 264, 341, 344
cultivation, 163–165, 169, 170, 174, 175,
177, 181, 184, 186, 232, 408
production, 163–187, 232, 253
Musk melon kernel, 259
Mustard oil cake, 256, 262, 264, 266
Mycelium growth, 164, 165, 172–174, 176,
177, 185
Mycotoxins, 131, 134–136, 139, 199, 210
N
Nano-silicon dioxide, 333, 336, 337
Natural insecticide, 234
Natural SSF, 209
Neem
kernel, 259
seed, 258
Neurospora sp., 118, 119, 281
Nicotiana tabacum, 111, 258
Nitrogen, 6, 8, 9, 45, 47, 50, 53, 73, 81, 91,
114, 150, 155, 156, 158
content, 169, 228, 321, 445, 451
NREL (Northern renewable energy
laboratory), 378
O
Obligate anaerobes, 419
463
Oil
cakes, 4, 20, 62, 74, 243, 253–267, 348,
362–365
industry waste, 434, 452
palm, 166, 341–344, 346–348, 350, 352
Oligomers, 21, 71, 248, 282
Oligopeptides, 155
Olive mill wastes (OMW), 25, 28, 66, 68, 91
Olive oil cake, 20, 243, 256–258, 262,
266, 364
Opuntia ficu indica, 119
Organic acids, 6, 9, 37–57, 63, 68, 80, 83, 165,
204, 213, 216, 225, 226, 230, 233, 235,
239, 244, 246, 254, 273, 279, 294–296,
300, 301, 307, 308, 321, 329, 402, 419,
420, 436
Oryza sativa, 111, 112, 258
Oxidase, 26, 113, 400, 403
Oxidoreductase, 400
P
Packed bed column, 46, 211, 213, 245, 262
Palm kernel
cakes, 85, 256, 265, 266, 348, 349, 364
shells, 341
Palm oil
mill effluent, 350–354
wastes, 341, 342, 353
Particle size, 51, 137, 205, 206, 214, 241, 244
Pasteurization, 81, 120, 178
Pectinases, 71, 91, 171, 199, 200, 204, 234,
245, 247, 277, 278, 282, 309, 319,
383–385, 387–393
Pectinesterases, 387
Pectin(s), 200, 256, 276, 277, 282, 321,
385, 391
lyases, 388
Pediococcus pentosaceus, 119
Penicillin, 7, 9, 131, 199, 210, 244
Penicillium glaucum, 40
Penicillium roqueforti, 117, 137, 201
Percentage conversion, 72
Perilla frutescens, 111, 112
Perionyx excavatus, 438, 439, 443
Petroleum
diesel, 63, 71
prices, 3
Phanerochaete chrysosporium, 24, 26, 71, 84,
117, 204, 206, 299, 301, 309, 374, 376,
401, 404, 406
Pharmaceutical(s), 42, 116, 134, 175, 300, 437
industries, 38, 41, 53, 54, 133, 279, 298,
300, 307, 360
Phenol, 22, 25, 26, 401, 409
464
Phenolic, 109, 116, 186, 258, 265, 277, 283,
284, 299, 311, 316, 320, 398, 400–403,
408, 450
acids, 277, 283, 284
compounds, 109, 132, 403, 408, 450
Photoheterotroph, 68
Phycocianin, 152
Physical, 4, 10, 14, 19, 20, 26, 28, 52, 171,
176, 202, 205, 211, 214, 230, 231, 264,
303, 330, 332, 385, 393, 422, 433, 435,
436, 440, 446
Pichia anomala, 114
Pigments, 147, 148, 150–153, 156–159, 232
Pisifera, 342
Plant oils, 5, 6, 78
Pleurotus sp., 47, 84, 85, 164–166, 169–174,
176–178, 180, 182–185, 198
Polygalacturonases, 245, 388, 392
Polygalacturonic acid (PGA), 385, 388, 392
Polygonum hydropiper, 111
Polyketides, 136, 148, 156
Polymers, 4, 23, 24, 42, 65, 214, 280, 295,
296, 302, 305, 307, 308, 309, 329, 339,
343, 419
Polyporus tuberaster, 117
Polysaccharides, 9, 15, 16, 25, 26, 63, 168,
185, 186, 200, 228, 245, 246, 263, 280,
301, 318, 320, 321, 343, 359, 360, 362,
364, 384–387, 399, 451, 452
Poultry
droppings, 88, 349, 443, 449, 450
manure, 63, 86, 177, 341, 349, 440
Pour point, 71
Prebiotics, 282, 283
Preservative, 42, 53, 86, 154, 233
Pretreatment, 6, 8–10, 214, 246, 303
Primordia, 172, 173, 182
Protein, 8, 18, 26, 50, 57, 63, 65, 67, 71, 77–92,
130, 135, 154–157, 163, 165, 169, 173,
175, 186, 200, 201, 204, 207, 210, 211,
225, 229, 234, 239, 241, 247, 253, 255,
256, 258–260, 263, 265–267, 273, 276,
281, 299–301, 308, 313, 314, 316–318,
320–323, 330, 348, 360, 363–367, 372,
375, 377, 379, 380, 386, 390–393, 408,
409, 419, 423, 424, 436, 442, 454
enrichment, 77–79, 81–90, 92, 210,
211, 281
Pseudomonas putid, 116, 117
Psychrophilic, 67
Pullulanases, 361–364
Pulp and paper mill sludge, 451
Pure culture (SSF), 210
Index
Pycnoporus cinnabarinus, 116
Pyrizines, 105
R
Rapeseed cake, 254, 256, 260
Redox potential, 401–403
Refuse, 77–79, 81, 82, 92, 227, 300, 346
Renewable energy, 4, 240, 295, 305, 328, 378,
424, 425
Renewable-resources, 3, 5, 13, 38, 235, 294,
307, 328, 329, 379
Residue to product ratio (RPR), 167
Residues, 3–10, 13–29, 37–57, 61–64, 66, 68,
70, 73, 74, 77–92, 117, 118, 129–133,
135, 137, 139–143, 147, 148, 150,
159, 163–187, 197–210, 216, 225–230,
232–234, 239–242, 254, 256, 273–286,
293–309, 313–316, 321, 339–354, 359,
362–365, 367, 373–375, 378, 379,
383–386, 388–390, 392, 393, 397, 398,
405–408, 410, 417
Resins, 51, 54, 114, 320, 321
Rhamnogalacturonan, 385, 386
Rhizopus oryzae, 41, 53–56, 84, 119, 198, 199,
231, 232, 244, 247, 261
Riboflavin, 151, 152, 317, 322, 323
Rice
bran, 25, 28, 45, 52, 63, 73, 85, 117, 130,
131, 137, 169, 180, 182, 226, 233, 242,
258, 259, 266, 362–365, 397, 406–408
hulls, 130, 363, 434
husk, 131, 167, 170, 328, 331, 336–338,
362, 363, 425, 426, 434
straw, 17, 18, 20, 24, 29, 63, 68, 70, 82,
166, 167, 170, 186, 200, 229, 242, 264,
327–339, 365, 374, 379, 406–409, 449
Rubber seed kernel, 259
Rubropunctatin, 151, 155–158
S
Saccharification, 7, 46, 71, 230, 231, 234, 235,
248, 283, 294, 302–304, 333, 360, 361,
378, 384
Saccharomyces cerevisiae, 85, 115, 116, 198,
211, 245, 278, 279, 281, 298, 306,
319, 321
Safflower oil cake, 254, 255, 257, 258, 364
Secondary metabolites, 109, 129–143, 147,
155, 165, 206, 207, 213, 214, 263, 267
Serratia marcescens, 116
Sesame oil cake, 255, 257, 258, 261, 263,
265, 364
Silage making, 80, 92
Index
Simultaneous saccharification and fermentation, 71, 235, 283, 302–307, 333, 334,
337, 338
Single cell oil, 73, 83, 84
Slaughter house Wastewater (SW), 64, 66, 86
Slurry process, 77, 78, 81
Smooth and hairy regions of pectin, 387
Solid agro-industrial wastes, 417
Solid matrix particles, 202
Solid phase, 201, 248, 331, 351, 433
Solid state (substrate) fermentation (SSF),
10, 24–26, 29, 38, 39, 40, 42, 44–48,
51, 53–57, 78–81, 83, 89, 90, 92, 105,
118–120, 131–137, 141, 143, 148, 158,
159, 163–166, 175, 186, 197–216,
230–233, 235, 241–245, 260–262, 266,
277–281, 283, 299, 308, 333, 362–364,
366, 373, 379, 380, 383, 388–392, 398,
404–410
Solid substrates, 24, 38, 44, 51, 77, 79, 80, 84,
89–92, 119, 135–142, 144, 158, 159,
172, 176, 199, 201, 202, 204, 206, 207,
209, 210, 212–215, 242, 260, 280, 281,
366, 373, 388, 389, 392, 405
Soybean cake, 183, 254, 256, 258, 260, 261,
263–265
Spawn, 164, 175, 177, 178, 180, 182, 183
run, 178, 180, 182
Spent coffee, 258, 259
Spent grains, 313–319, 321, 323, 364
Spent hops, 313–315, 320, 321
Starch, 4–9, 41–45, 47, 48, 51–54, 63, 71, 82,
84, 88, 89, 134, 136, 150, 154, 168,
169, 182, 200, 207, 226–230, 232–235,
243, 279, 300, 308, 314, 316, 323, 328,
359–367, 384
hydrolyzing enzymes, 359, 360
Stationary phase, 130, 133
Steam explosion, 19, 20, 83, 246, 248, 303,
333, 337, 338, 375
Sterilization, 29, 43, 86, 133, 176, 182,
234, 343
Streptococci, 419
Streptococcus cremoris, 112
Streptococcus diacetilactis, 112
Streptococcus lactis, 112
Streptococcus thermophilus, 41, 45, 112
Submerged fermentation, 10, 24, 25, 41, 48,
88–90, 118, 133, 136, 140, 142, 201,
203, 215, 230, 231, 233, 245, 260, 279,
362, 366, 373, 380, 383, 388, 404
Substrates, 4–10, 16, 17, 23–26, 29, 38, 39,
43–47, 49, 51, 52, 54–57, 61, 62, 66,
465
68, 71, 74, 77–92, 106, 109, 110,
116–120, 130–133, 135–143, 155, 158,
159, 163–187, 198–215, 225, 229–235,
239, 241–245, 248, 253, 260, 264, 266,
267, 273, 278–281, 283, 286, 293, 294,
296–300, 304, 308, 309, 313, 318–320,
323, 324, 334, 336, 359–367, 373, 374,
380, 383, 388–393, 401–403, 405–409,
406, 418–420, 422, 426, 434–437, 441,
445, 448, 449, 451, 452
Sugar beet pulp, 84, 85, 226, 362, 363,
389–391
Sugarcane, 41, 44, 69, 164, 167, 233, 442,
443, 448
bagasse, 17, 45, 51, 63, 119, 131, 164, 167,
168, 170, 180, 183–186, 198, 226, 232,
233, 239–248, 262, 281, 363, 373, 379,
389, 405, 442, 446
Sugar Industrial waste, 77, 82, 434, 442
Sugars, 4–6, 8
Sulfite waste liquor, 6, 7
Sunflower cake, 131, 254, 256
Supplements, 48, 91, 151, 154, 169, 172, 180,
182, 183, 185, 186, 206, 207, 242, 260,
263, 264, 321, 323, 349, 372, 378,
389, 436
Surface area, 22, 47, 137, 203, 205, 375, 433,
437, 439
Surplus yeast, 313, 314, 322, 323
Switchgrass, 70, 71, 73
T
Taiwan, 425
Tamarind kernel, 259
Tank digestion, 350, 351
Tapioca seed, 259
Teak kernel, 259
Tea seed kernel, 259
Tenera, 342
Terpenes, 114, 115
Terpenoids, 283, 284
Textile industry sludge, 449
Theobromo cacao, 111
Thermal chemical transformation, 332
Thermophilic digestion, 66
Thippi, 70, 71
Tissue maceration, 383, 384
Tobacco
seed, 258, 259
waste hydrolysate, 73
Trametes suaveolens, 117
Transesterification, 72, 73, 156, 284
Tray fermentation, 211
466
Trichoderma viride, 43, 85, 113, 117, 198–201,
243, 299, 308, 374
Tricoderma reesei, 24, 85, 198, 242, 243,
247, 283, 301, 318, 373–377,
379, 380
Triglycerides, 71, 72, 282
Trub, 314, 320, 321
Turkey, 274–276, 424, 425
Tyromyces sambuceus, 113
U
Up-flow, 351
Upflow Anaerobic Sludge Blanket (UASB)
reactor, 69
Up flow Fixed Bed Reactor (UFBR), 69
V
Valorization, 77, 78, 87, 91, 92, 163, 185, 243
Value-added products, 38, 79, 88, 130, 134,
164, 198, 199, 225, 226, 231, 235, 239,
244, 254, 259, 273, 277, 282, 294,
300, 301, 363, 371, 393, 432, 448,
449, 451
Value addition, 51, 82, 118, 165,
231, 235, 239, 255, 266, 367,
391, 393
Vanilla planifolia, 111, 116
Vanillin, 107, 111, 112, 116, 117
Vegetable oil, 71, 73, 74
extraction, 278, 385
Vermicomposting, 431–434
systems, 432, 435, 436, 441
Vermicompost quality, 439
Vermiwash, 440, 441
Viscosity, 6, 71, 133, 243, 280, 360, 384
Vitamins, 6, 8, 9, 18, 82, 84, 91, 150, 152,
202, 244, 275, 276, 307, 317, 322–324,
364, 365
Vitis vinifera, 115, 274
Volatile Fatty Acids (VFA), 67, 106, 352, 420
W
Water activity, 45, 47, 133, 173, 176, 207, 208,
215, 245, 362, 388
factor, 208, 209
Water melon
kernel, 259
oil cake, 258
West African oil palm, 342
Wet oxidation, 246, 248
Wet residues, 15
Index
Wheat
bran, 25, 28, 38, 41, 43, 45, 52, 63, 84,
119, 131, 136, 137, 169, 180, 182, 199,
200, 226, 233, 241, 242, 261, 262,
362–367, 373, 374, 388–390, 392, 393,
406–408, 434
straw, 17, 21, 22, 24, 25, 28, 38, 70, 81,
82, 130, 131, 165, 167, 168, 170, 173,
174, 178, 183–186, 198, 199, 201, 229,
242, 265, 373, 374, 406, 407, 425, 443,
448, 449
Whey concentrate, 73
White rot fungi, 19, 24–26, 85, 117, 171, 172,
206, 241, 247, 299, 309, 375, 403, 404,
408, 409
mushroom forming, 171
Windrow system, 441
Wine clarification, 383, 384
Wine Grape Residues (WGR), 64, 66
Winery
residues and effluent, 294
waste, 294–296, 298, 299, 308, 309,
443, 448
Wood-degrading fungi (WDF), 171
Workhorse, 5
Wort, 314, 315, 319–321
X
Xanthan gum, 231, 232, 280
Xylanase, 25, 91, 186, 242, 243, 247, 262, 278,
299, 307, 309, 318
Xylitol, 246, 247, 313, 319
Xylogalacturonan (XGA), 385, 387
Y
Yarrowia lipolytica, 43, 50, 113, 151, 308, 352
Yield, 4, 5, 9, 13, 16, 17, 21, 26, 38–40, 43–49,
53–56, 64, 66, 67, 69–71, 88, 89, 109,
113–116, 118, 120, 130, 133, 135–143,
150, 153, 158, 159, 165, 167, 169,
173, 175, 177, 180, 182–185, 203, 206,
208, 209, 212, 226, 230, 233–235, 240,
242–244, 246, 248, 254, 260, 264, 274,
279–281, 299–302, 304–307, 318, 321,
334–336, 338, 342, 343, 347, 348, 350,
352, 353, 361, 373, 377, 384, 385, 388,
392, 423, 424, 426–429, 432, 440, 454
Z
Zygosaccharomyces rouxii, 118, 119, 261
Zymase, 69, 70