MORPHOLOGY CONTROL OF RHIZOPUS SP.

IN ETHYLENEDIAMINE DELIGNIFIED
CELLULOSE MEDIUM AT DIFFERENT
GROWTH CONDITION FOR MAXIMUM
LACTIC ACID PRODUCTION
AINIL HAWA JASNI (G2013108)
Candidate: Doctor of Philosophy in Engineering
Main Supervisor: Assoc. Prof. Dr. Azlin Suhaida Azmi
Co-supervisors: Assoc. Prof. Dr. Noor Illi Mohamad Puad
Assoc. Prof. Dr. Fathilah Ali
Asst. Prof. Dr. Yusilawati Ahmad Nor
DEPARTMENT OF CHEMICAL ENGINEERING & SUSTAINABILITY
1
KULIYYAH OF ENGINEERING IIUM GOMBAK CAMPUS
 Introduction
Rhizopus, a versatile fungus BUT!! Challenges in LA Production Pretreatment with Ethylenediamine

In the production of LA, Morphological Control - under reported study:

Our focus: Optimizing Lactic Acid Production with Rhizopus sp. Using pretreated cellulose by controlling morphology of
fungi.
2
 Objectives of the study
To determine the maximum transformations of cellulose allomorph,
1 lignin degradation, and re-localization and accessible fermentable
sugars in 5 lignocellulose pretreated samples with different
concentration of EDA and temperature.

To investigate the formation of Rhizopus morphology and their

2 interactions with the delignified cellulose and hemicellulose
under varied process parameters.

To determine maximum LA yield from screened delignified

3 cellulose in the presence of neutralizing agents (CaCO 3, NaHCO3,
NH4(HCO3), NH4NO3 & (NH4)2SO4) in fermentation medium.

3
 Problem Statement

Challenges in Lignocellulosic Biomass Utilization

Optimizing Fungal Morphology for Lactic Acid Production

Urgent Need for Integrative

Approaches

4
 Literature Review

Figure 1: Approximate schematic for lignocellulosic biomass pretreated by EDA

5
 Literature Review
Table 1: Recent findings on LA production by Rhizopus sp.
Fungi species Carbon source Morphology analysis LA production Ref.
R. oryzae Glucose, Filamentous vs. kinetic 80 & 100 g/L (Ajala, 2021)
xylose, ribose, Yam peel growth
hydrolysate
R. oryzae Sophora flavescens residues None 46.78 g/L & 0.97 g/L/h. (Ma, et al., 2020)
R. oryzae LA-UN-1 Glucose Pellet > Filamentous Pellet = 63.5 g/L, Filamentous = 41.5 (Yin, 2020)
form g /L
R. microsporus DMKU Liquefied cassava starch None 84 g/L & 1.25 g/L/h (Trakarnpaiboon, Srisuk,
3 Piyachomkwan, Yang, &
Kitpreechavanich, 2017)
R. oryzae NRRL395 Cassava pulp hydrolysate Immobilized fungal 75.28 g/L (Pimtong, et al., 2017)
cells increase LA yield
R. oryzae NBRC 5384 Paper sludge, hexoses, None 80 g/L (Takano & Hoshino, 2016)
pentoses, sugar alcohol,
disaccharides, and
polysaccharides
R. oryzae AT3 Rotten avocado and guava + None 8.495 g/L (Titik, Retno, & Sardjono, 2014)
CaCO3
R. sp. Cassava starch and leaves None 0.95 g/g (Azmi, Yusuf, Jimat, &
Mohamad Puad, 2016)
R. oryzae NRRL 395 Oil palm trunk None 6.632 ± 0.032 g/L (Lai, Mohd Yahya, Mohd Nor, &
Sulong, 2016)
R.oryzae NRRL 395 EFB Pellet > Clump form 0.12 g/L (Hamzah, Idris, Rashid, &
Ming, 2009)

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 Research Methodology

7
Figure 2: Schematic flow of overall experimental design
 OBJ. 1: FTIR Results
• Chemical Functional Group in Pretreated Substrate

Finding: Treated
EFB samples showed
highly significant
functional group
changes to increased
cellulose content due
to the removal of
lignin and
hemicelluloses before
and after pre-
treatment of EDA
Figure 3: FTIR spectra of raw and EDA pretreated samples at different EDA 8
 OBJ 1: DNS Results
• Total Reducing Sugar Concentration after EDA pretreatment
0.9
M a g n itu d e o f c h a n g e s
0.8
CH BP CP CPH EFB
Reducing Sugar Content (mg/mL)

0.7

0.6

0.5

0.4

0.3

0.2 Findings: Treated

0.1 EFB displayed
0 highest magnitude
0 1 2 3 4 5 6 7 8 9 10
of changes 64.90%,
EDA Concentration (%)
66.50% and 78.3%
of total reducing
Figure 4: Magnitude of changes in total reducing sugar in all samples sugars after EDA
respectively. pre-treatment in
5.5%, 7.75 % and
10% of EDA 9
 • Sugar Component
OBJ1: HPLC Result
BP CH CP CPH

EFB
Findings: EFB has highest
sugars content of intensity of
refraction index signals (mV)
versus retention time with
more than 99.2% sugar yield
after EDA pre-treatment
among all other substrates.
Figure 5: HPLC peaks of sugar analysis of samples a) before & b)
10
after EDA pre-treatment.
 OBJ 1: XRD Results
Transformation of Allomorph & Crystal Structure
CP
BP CH CP

CPH EFB

Findings:
XRD analysis indicated a
substantial transformation to
cellulose III in EFB, accompanied
by a decrease in the CI/cellulose
ratio from 1.82 to 1.18. This shift is
considered the optimal condition
for enzymatic hydrolysis at 30 ℃
Figure 5: XRD patterns of raw and EDA pretreated samples with 5.5% EDA. 11
 OBJ1: SEM Result
Findings: SEM images (Figure
a b 6(b), Figure 6(d), and Figure
6(e)) of pretreated EFB, a
wave-like pattern is evident,
possibly caused by lignin
redistribution (Zhu et al.,
2018). Similarly, the surface of
pretreated EFB in Figure 6(f)
shows a corrugated appearance,
suggesting lignin removal and
decreased crystallinity.
Comparable morphology has
been noted in EDA-pretreated
corn stover (Qin L., 2017).

Figure 6: Morphological features of EFB treated under different EDA concentrations at a) 0%, b) 1.0%, c) 2.25%, d) 5.5
%, e) 7.75% and f) 10% at constant optimal selected temperature of 30 ℃ under 400x magnification magnitude. 12
 SUMMARY FINDINGS of OBJ 1
Table 4: Selected experimental conditions for pretreatment of EDA of this study

Optimized substrate Temperature (℃) EDA Concentration

(%)

EDA conditions
EFB 30 5.5

• EFB substrate treated under the parameter of 30 ℃ & 5.5% of EDA of

pretreatment was chosen for subsequent experiment of fungal fermentation for
LA production

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 OBJ2: Morphology Results

Figure 7: a) R. microsporus fungi under naked eye on PDA Figure 8: Stained Rhizopus microsporus under a light
with the substrate. b)-d) R. microsporus 10x magnification microscope of 40x magnification. a) Germination of
of light microscope. spores b)-e) Vegetative Growth of Zygospores with
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unequal suspensors.
 OBJ 2: Morphology Results
Table 5: Morphological Characteristics of Rhizopus sp. in Different Flask Sizes at 100 rpm.
FLASK SIZE: Sample: Control Sample: EFB

LA yield rate: 0.0012 g/L/hr LA yield rate: 0.0014 g/L/hr

150 mL
Diameter: 4.5 cm Diameter: 4.0 cm

LA yield rate: 0.0020 g/L/hr LA yield rate: 0.0026 g/L/hr

200 mL Diameter: 3.2 cm Diameter: 2.0cm

LA yield rate: 0.0031 g/L/hr LA yield rate: 0.0036 g/L/hr

500 mL Diameter: 0.5 cm Diameter: 0.9 cm

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 OBJ 3: Effects of N.A. on morphology
6
2.5
NaHCO3
(NH4)2SO4
5 NH4NO3
2

Fungi Growth Diameter (cm)

4
Diameter size (cm)

1.5
3

Without N.A. 1
NH4(HCO3)
2 CaCO3

1 0.5

0 0 Without N.A CaCO3 NaHCO3 NH4(HCO3) NH4NO3 (NH4)2SO4

0 1 2 3 4 5 6 7 Average Diameter

Time (Day)

Figure 9: Average Rhizopus sp. growth diameter with and without the presence of different with and without N.A.
Findings: The use of CaCO3 as N. A. appeared to promote the formation of compact fungal pellets with relatively smaller diameter sizes. This
morphology is favorable in bioprocessing, as it facilitates easier solid-liquid separation during downstream processes (Boodhoo, Flickinger, Woodley,
& . Emanuelsson, 2022). 16
 OBJ 3: Effects of N.A. on LA production
1.8 Without N.A. CaCO3 NaHCO3
NH4(HCO3) NH4NO3 (NH4)2SO4
1.6
1.4
1.4
1.2

Reducing Sugar content (wt %)

LA Concentration (g/L)

1.2 Without N.A.

1
1 CaCO3
0.8 NaHCO3 0.8

0.6 NH4(HCO3) 0.6

0.4 NH4NO3 0.4

0.2 (NH4)2SO4
0.2
0
0 1 2 3 4 5 6 7 0
0 1 2 3 4 5 6 7
Time (Day)
Time (Day)
Figure 10: LA yield and Reducing sugar content in the Rhizopus sp. fermentations over 7 days incubation with varying N.A

- CaCO3 appeared to lead to higher LA concentrations.

- The fungi actively metabolized sugar in both the substrate and media to produce lactic acid, with CaCO 3 exhibiting the highest
rate among all conditions.
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 OBJ 3: Effects of N.A.
Table 6: Values of LA yield of each group tested.
10
9 Sample Growth max Max production of
time (hr) LA (g/L)
8
7
6
5
PH

4
Without N.A. 120 1.035
3
2 Without N.A. CaCO3 NaHCO3 NH4(HCO3) NH4NO3
1 (NH4)2SO4 (NH4)2SO4 168 0.048

0
0 1 2 3Time (Day) 4 5 6 7
NH4NO3 24 0.005
Figure 11: pH levels in the Rhizopus sp. fermentation media over 7 days
incubation with varying N.A.
NaHCO3 72 0.242

- CaCO3 prevents fermentation medium from becoming too acidic,

maintaining an optimal pH for fungi in lactic acid (LA) production. NH4(HCO3) 120 0.068
- Ammoniacal N.A. is unsuitable, resulting in significantly lower LA
concentration and slower sugar digestion compared to other nitrogen
sources. CaCO3 96 1.66
- Excessively high ammonia concentration is detrimental to cell
growth, contributing to reduced cell growth and LA production in
batches using ammoniacal solutions NH 4(HCO3), NH4NO3, and
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(NH4)2SO4 (Yen, Chen, & Pan, 2010).
 CONCLUSION
• The effects of fermentation incubation time on fungal growth and LA yield in
Rhizopus are multifaceted.

• The maximum LA yield result showed significant higher results of 1.66 g/g
production than a study done by (Hamzah, F., & A. Idris, R. R. (2009) which
recorded 0.12 g/L of LA yield in pellet form of Rhizopus Oryzae after 96 hr.

• He also reported pellet form produced 30% more LA than clump form with the
addition of CaCO3 .

• CaCO3 likely contributed to maintaining an optimal pH level in the fermentation

process, enhancing microbial activity and, subsequently, LA yield.

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 PUBLICATIONS & CONFERENCES
1. Identification Of Rhizopus Sp. Fungi For Alternative Lactic Acid Production Source – In submission IIUM
Engineering Journal • 1st Middle East Chemical Engineering
2. Enhancing Enzymatic Digestibility through Alkaline Ethylenediamine Treatment of Empty Fruit Bunch Symposium (MEChES 2023), 18th -19th Oc-
Crystalline Cellulose – Compulsory in submission Chemical Engineering Research and Design Elsevier Q1 tober 2023, Abu Dhabi, UAE
(3.9 IF).
3. Transformation of Cellulose Allomorph of Musa acuminata balbisiana Peel Using Ethylenediamine –
• KERICE 2022 BRONZE AWARD
Accepted in Journal of Physics: Conference Series (JPCS), IOP. Q4 (0.3)
4. Book Chapter: “Engineered Biomaterials: Synthetic Approach and Applications, Section 1: Approach for • 2nd International Conference on Material
Synthesis of Biomaterials: Naturally Derived Biomaterials: Advances and Opportunities – Published
Springer Nature asni, A.H., Azmi, A.S., Puad, N.I.M., Ali, F., Nor, Y.A. (2023). Naturally Derived Biomaterials: Processing & Tech 2023, 4-5 Feb 2023,
Advances and Opportunities. In: Malviya, R., Sundram, S. (eds) Engineered Biomaterials. Engineering Avana Hotel, Bangkok
Materials. Springer, Singapore. https://doi.org/10.1007/978-981-99-6698-1_1
5. Book Chapter: Agrowaste Lignocellulose Valorization for Sustainable Circular Bioeconomy – In Submission • KERICE 2024 (0.3)
SpringerNature Journal
6. Current Advance Techniques of Treating Organic Pollutants in Waterbodies. In Submission Journal of
Applied Research in Water and Wastewater

7. Book Chapter: Compatibility Study of Nanofillers-Based Polymer Composites, Ismail M. Fareez, Ramli M. Zaki,
Ainil Hawa Jasni, Azirah Akbar Ali, Suresh Sagadevan, Zaharah WahidBook: Nanofillers for Sustainable
Applications, Edition1st Edition First Publishedby Taylor & Francis, ImprintCRC Press 0.1201/9781003400998-5
Published

8. Book chapter: Silver Nanoparticles in Various New Applications, Ainil Hawa Jasni, Azirah Akbar Ali, Suresh
Sagadevan and Zaharah Wahid Submitted: 26 October 2020 Reviewed: 19 January 2021 Published: 15
September 2021 DOI: 10.5772/intechopen.96105 Published
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