Geethanjali et al.

Bull Natl Res Cent (2020) 44:173

https://doi.org/10.1186/s42269-020-00426-5 Bulletin of the National
Research Centre

RESEARCH Open Access

Biodegradation potential of indigenous

litter dwelling ligninolytic fungi on agricultural
wastes
P. A. Geethanjali1*, H. G. Gowtham2 and M. Jayashankar3

Abstract
Background: The present study was focused to study the efficiency of two indigenous litter dwelling ligninolytic
fungi (such as Mucor circinelloides GL1 and Fusarium verticillioides GL5) in degrading the agricultural wastes (areca
husk, coffee husk and paddy straw) through solid-state fermentation.
Results: After fermentation process, the lignocellulosic residues left over were evaluated for their physico-chemical
studies and degradation pattern of cell wall constituents along with the activity of enzymes. In each substrate, the
initial pH was found to change from near-neutral to acidic pH after fungal decomposition. Significantly increased loss
of total organic matter and organic carbon content was observed in each substrate decomposed by the fungal strains
selected. The total nitrogen, crude protein, total phosphorus and total potassium contents of the fungal decomposed
substrates were significantly increased with the progress of time. The study indicated that the degradation patterns
of lignin and holocellulose were more effective from 20 to 120 days after fungal inoculation with respect to their loss
between the different harvesting intervals. During decomposition process, both the strains produced the ligninolytic
enzymes [laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP)] and carboxymethyl cellulase (CMCase) on
each substrate with their remarkably varied activities with respect to different harvesting times.
Conclusions: In concern with the present environmental problems, the present study suggested that these potential
ligninolytic fungi can be utilized successfully for the management of agricultural wastes and reuse of their residues in
the forest soil conservation system to eliminate the harmful effects of the crop residue burning.
Keywords: Agricultural wastes, Biodegradation, Fungi, Holocellulose, Lignin, Lignocelluloses

Background leaf litter in the forest soil. In India, the removal or in

Lignin protects the total polysaccharide fraction of plant situ combustion of forestry and agricultural crop wastes
cell wall that made up of a mixture of cellulose and hemi- is being practiced by most subsistence farmers which
cellulose. It is a large recalcitrant substrate to degrade seriously reduce the soil quality (Kumar et al. 2015; Bhu-
due to its non‐phenolic aromatic nature and heteroge- vaneshwari et al. 2019). The combustion of agricultural
neous structure which make the accumulation of ligno- crop wastes emits the noxious gases, particulate matter
cellulosic materials in the forest soils (Ruiz‐Dueñas and and other reactive hydrocarbons thereby resulting in a
Martínez 2009; Janusz et al. 2017). The accumulated lig- significant disturbance in the global atmospheric chemis-
nocellulosic materials mainly consist of deadwood and try (Jain et al. 2014).
Nowadays, it has attracted a lot of attention of scien-
tists towards the lignocellulosic biomass and waste val-
*Correspondence: geethanjalipa07@gmail.com orization (Arancon et al. 2013; Abdel-Shafy and Mansour
1
Department of Microbiology, Field Marshal K. M. Cariappa College, 2018; Xu et al. 2019). Fungi are the best-known microor-
Madikeri, Karnataka 571201, India
Full list of author information is available at the end of the article ganisms that quickly invade the substrates and capable of

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 2 of 14

degrading cellulose, hemicellulose and lignin efficiently of Western Ghats of Karnataka (India) were selected
by several enzymes. They can be effectively exploited for (Unpublished data) and used to study their efficiency in
in situ decomposition of various crop wastes. Most of the decomposing some agricultural wastes in the laboratory.
extracellular ligninolytic enzymes involved in the deg-
radation of plant litter have been attributed to the fungi
which produce them by adapting their basic metabolism Agricultural wastes
to nutrient availability in the natural habitats (Rineau Three different agricultural wastes such as areca husk,
et al. 2013; Lashermes et al. 2016; Janusz et al. 2017). An coffee husk and paddy straw were used as the solid sub-
eco-friendly strategy has been developed for the enzyme strates to determine the efficiency of selected potential
production by solid-state fermentation process using ligninolytic fungal strains to decompose them. The solid
various agricultural wastes as supporting substrates (Liz- substrates were procured from the local agricultural
ardi-Jiménez and Hernández-Martínez 2017; Sadh et al. farms of Kodagu district in Karnataka (India) during Jan-
2018). The production of hydrolytic (cellulase and hemi- uary, 2017. The substrates were brought to the research
cellulase) and ligninolytic enzymes depends on the mode laboratory and oven dried at 80 °C. The dried substrates
of fermentation process and cultivation of microorgan- were pulverized and sieved through 20 mesh sieve size
isms, especially fungi. and then stored at room temperature in the polyethylene
Inevitably generated agro-industrial wastes are pri- bags for further experiments.
marily composed of cellulose, hemicellulose and lignin,
and found in lignocellulosic nature. They are exploited
Solid‑state fermentation process of agricultural wastes
as solid materials to aid as the nutrients sources and
by ligninolytic fungi
physical supports for the cultivation of microbes during
Solid-state fermentation process was performed to check
solid-state fermentation in absence of free-flowing liquid
the different substrates degradation efficiency of selected
(Krishna 2005). This fermentation process provides the
ligninolytic fungal strains (Takó et al. 2015). Briefly, 10 g
same conditions under which the fungi are being grown
dried substrate was moistened with 40 mL sterile water
and making it an excellent method for the bioconversion
in 250-mL Erlenmeyer flask and autoclaved for 20 min at
of various agro-industrial wastes. The use of these wastes
121 °C. Five mycelial plugs (5 mm of diameter) of 7-day-
supports the excellent fungal growth and increases their
old fungal culture actively growing on malt-extract agar
enzyme activity by making the availability of rich energy
(MEA) medium (2%) were inoculated onto the flasks
nutrients to such fungi (Takó et al. 2015; Sadh et al.
separately containing three different sterilized solid sub-
2018). Besides, the fermentation process also offers the
strates. The flasks separately containing different steri-
new promising possibility of successfully managing and
lized solid substrates without fungal inoculation were
reusing various agro-industrial wastes.
used as the controls. The flasks were then incubated
Generally, several fungi are being employed in the
under static conditions in dark at 28 ± 2 °C for 120 days.
rapid degradation of various agricultural wastes by solid-
For better fungal growth, the moisture content was
state fermentation (Belewu and Babalola 2009; Mussatto
maintained at 70% throughout the incubation period of
et al. 2012; Yoon et al. 2014; Barrios-González and Tar-
decomposition by sprinkling the sterile water regularly
ragó-Castellanos 2017). However, there is a few numbers
in each experiment. The decomposed samples were har-
of reports on the degradation of agro-industrial wastes by
vested at the periodic intervals of 0, 20, 40, 60, 80, 100
the litter decomposing fungal species wherein their activ-
and 120 days after fungal inoculation and used for deter-
ity of ligninolytic enzymes was reported (Hättenschwiler
mining the various parameters as described below. After
et al. 2005). Therefore, in the present study, after success-
harvesting, the decomposed samples were washed twice
ful screening of fifty-eight ligninolytic fungal strains iso-
with sterile water to remove the mycelia, dried and then
lated from litter, two potential strains (M. circinelloides
used for further experiments.
GL1 and F. verticillioides GL5) were selected and used to
study their efficiency in the decomposition of some agri-
cultural wastes. Physico‑chemical analyses
Determination of pH
Methods The decomposed substrate was homogenized with deion-
Microorganisms ized water at the ratio of 1:10 (w/v) by shaking gently for
Two potential ligninolytic fungi such as Mucor circinel- 30 min at 200 rpm. The mixture was filtered through a
loides GL1 (GenBank Accession No.: MF458974) and Whatman filter paper and centrifuged for 10 min at
Fusarium verticillioides GL5 (GenBank Accession No.: 6000 rpm. The pH of filtrate was recorded using a digital
MF458977) which were indigenous to the litter samples pH meter.
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 3 of 14

Determination of total organic matter and organic carbon at 550 °C for about 3 h in muffle furnace, powdered and
content digested with the mixture of tri-acids (sulfuric acid, per-
The total organic matter of the decomposed substrate chloric acid and nitric acid) at 300 °C. After digestion, the
was assessed by using the loss on ignition (LOI) method digest was filtered through a Whatman filter paper and
of Salehi et al. (2011). The decomposed substrate was reacted with fresh mixed reagent (ammonium molyb-
heated to 560 °C by placing it in a ceramic crucible until date, antimony potassium tartrate and 50% sulfuric acid).
constant weight was achieved in the muffle furnace. The reaction mixture was reduced by the acidified ascor-
Then, the dried sample was cooled in the desiccator and bic acid solution (as a reducing agent) to form the molyb-
weighed. Total organic matter was calculated by using denum-blue colored complex. The blue color intensity
the following formula. was measured at 880 nm using spectrophotometer. The

Initial weight of sample − Final weight of dried sample

Total organic matter (mg/g) =
Initial weight of sample

The total organic carbon of the decomposed substrate total phosphorus content was estimated by using the
was then calculated by dividing the total organic matter standard curve plotted with the absorbance against the
by the factor of 1.724. known concentration of standard phosphorous. The con-
tent was expressed as mg/g of the decomposed substrate.
Total organic matter
Total organic carbon (mg/g) =
1.724 Determination of total potassium
where 1.724 is the factor for converting the total organic The total potassium of the decomposed substrate was
matter to total organic carbon. estimated by using the flame emission spectrophotomet-
ric method (Néel et al. 2014). The decomposed substrate
was incinerated at 550 °C for about 3 h in muffle furnace,
Determination of total nitrogen and crude protein powdered and digested with the mixture of tri-acids (sul-
The total nitrogen of the decomposed substrate was ana- furic acid, perchloric acid and nitric acid) at 300 °C. After
lyzed by using the micro-Kjeldahl acid digestion method digestion, the digest was extracted with deionized water
of Rhee (2001). The decomposed substrate was digested and filtered through a Whatman filter paper. Then, the
with concentrated sulfuric acid and distilled into 4% filtrate was fed into the flame emission spectrophotom-
boric acid solution using the micro-Kjeldahl distillation eter and read the absorbance at 766 nm. The total potas-
apparatus. Then, the aliquot was titrated against diluted sium was calculated by using the standard curve plotted
sulfuric acid with the blank. The total nitrogen was calcu- with the absorbance against the known concentration of
lated from the volume of sulfuric acid consumption using standard potassium. The content was expressed as mg/g
the following formula. of the decomposed substrate.
  Titer value × N of H2 SO4 × 14.007
Total nitrogen content mg/g =
1000 × Weight of the sample in g

where the titer value is the difference between the vol- Determination of carbon to nutrient supply (N, P and K) ratio
ume of consumption of sulfuric acid in the test sample Carbon to nutrient supply (N, P and K) ratio of the
and blank, 14.007 is the molecular weight of nitrogen (g/ decomposed substrate was calculated by dividing the
mol), and 1000 is the conversion factor (mL to L). total organic carbon content with each nutrient (such as
The crude protein of each substrate was calculated by total nitrogen, total phosphorus and total potassium).
using the following formula.

Crude protein content (mg/g) = Total nitrogen content × 6.25

Determination of total phosphorus Determination of lignocelluloses content and loss

The total phosphorus of the decomposed substrate was Determination of lignin content and loss
estimated by using the molybdenum-blue spectropho- The lignin content before and after fungal treatment was
tometric method after ascorbic acid reduction (Singh evaluated by measuring Klason lignin and acid soluble
et al. 2015). The decomposed substrate was incinerated lignin of the substrate. The gravimetric measurement of
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 4 of 14

Klason lignin content of the substrate was performed as with sterile water until free of acid, then followed by wash-
per the modified Klason method using hot sulfuric acid ing with acetone. The residue was oven dried at 105 °C for
digestion (Fagerstedt et al. 2015). Briefly, the substrate 4 h, cooled in the desiccator and weighed. Holocellulose
was treated successfully with sulfuric acid (72% v/v) to content was calculated by using the following formula.

Oven dried weight of holocellulose

Holocellulose content (%) = × 100
Oven dried weight of initial sample

depolymerize the crude cellulose and hemicellulose for The change in holocellulose content was measured to
1 h with stirring frequently to assure the complete solu- calculate % of holocellulose loss by using the following
tion at 30 °C. The acid was then diluted with deionized formula.

Initial holocellulose content − Final holocellulose content

Holocellulose loss (%) = ×100
Initial holocellulose content

water to make 3% (v/v) sulfuric acid for the hydrolysis of Enzyme activity
dissolved polysaccharides. The suspension was subse- Extraction of enzymes
quently autoclaved for about 1 h at 121 °C and cooled to The enzymes were extracted from fermented and unfer-
room temperature. The precipitate was filtered and dried mented solid substrates according to the procedure of
for 4 h at 525 °C in the muffle furnace. After cooling, the Leite et al. (2019) with slight modification. The decom-
dried sample was weighed to determine the Klason lignin posed substrate (10 g) was suspended in 50 mL of sodium
content, acid insoluble residue and ash content present. acetate buffer (50 mM pH 5.0) and gently shaken for
Then, Klason lignin is calculated by subtracting the acid 30 min at 150 rpm. The content was transferred to a
insoluble ash from the acid insoluble residue. The acid muslin cloth and filtered. The supernatant was then cen-
soluble lignin was estimated by reading the absorbance trifuged at 10,000 rpm for 15 min at 4 °C. The resultant
spectrophotometrically at 205 nm using the absorption filtrate was served as a source of crude enzyme for the
coefficient (ε = 110 M−1 cm−1). The total lignin content was estimation of protein content and the activity of lignino-
determined by the addition of Klason lignin with the acid lytic enzymes and carboxymethyl cellulase.
soluble lignin. The change in lignin content was measured
to calculate % of lignin loss by using the following formula.

Initial lignin content − Final lignin content

Lignin loss (%) = × 100
Initial lignin content

Determination of holocellulose content and loss Estimation of protein content

The holocellulose (i.e., the total carbohydrate fraction con- The protein content was estimated at the wavelength
taining both cellulose and hemicellulose) content of the of 595 nm by Bradford method (He 2011) using bovine
substrate before and after fungal treatment was determined serum albumin (BSA) as a standard. The protein content
(Rabemanolontsoa and Saka 2012). The sample was ground was expressed as mg/mL.
and extracted with the mixture of alcohol:benzene (1:1 v/v)
at 70 °C for 8 h to remove the extractives. One gram of the Estimation of activity of enzymes
sample was suspended in 150 mL of sterile water. The solu- The activity of ligninolytic enzymes [laccase, manga-
tion was mechanically stirred. Ten drops of glacial acetic nese peroxidase (MnP) and lignin peroxidase (LiP)] and
acid and sodium chlorite (1.5 g) were added to the solution carboxymethyl cellulase (CMCase) was determined.
with vigorous shaking. The mixture was then incubated in Laccase activity was determined by monitoring the oxi-
a water bath for about 1 h at 70 °C. After incubation, the dation of guaiacol as per the method described by Arora
same quantity of reagent was added and the same process and Sandhu (1985). The MnP activity was determined
was repeated until the total reaction time was 4 h. The solu- by monitoring the oxidation of phenol red as per the
tion was cooled to room temperature, filtered and washed method described by Orth et al. (1993). The LiP activity
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 5 of 14

was determined by monitoring the oxidation of dye azure carbon contents in all the substrates by the selected fun-
B as per the methods described by Archibald (1992), and gal strains, but the reducing effect was varied depend-
Arora and Gill (2001). The CMCase activity was deter- ing on the substrate type (Table 1). There was the higher
mined as per the method described by Miller (1959) loss of initial total organic matter of 65.1 to 21.5, 72.7 to
using 1% (w/v) carboxymethyl cellulose (CMC) as a sub- 18.3 and 68.2 to 16.5 mg/g in areca husk, coffee husk and
strate and 3,5-dinitrosalicylic acid (DNS) as a coupling paddy straw, respectively, decomposed by M. circinel-
reagent. The enzyme activity was expressed as Interna- loides GL1 at the end of fermentation period. Also, when
tional Units (IU) per mL. treated with F. verticillioides GL5, the initial total organic
matter was reduced to 39.5, 37.5 and 35.8 mg/g in areca
Statistical analysis husk, coffee husk and paddy straw, respectively. A simi-
For each solid substrate, all the assays were performed lar trend of increase in loss of initial total organic carbon
by using the randomized complete block design (RCBD) was observed from 37.7 to 12.4, 42.1 to 10.6 and 39.5
with triplicates. Each experiment was repeated three to 9.5 mg/g in areca husk, coffee husk and paddy straw,
times. The experimental data were analyzed statistically respectively, decomposed by M. circinelloides GL1. The
and subjected to one-way ANOVA by using IBM SPSS reduction in initial organic carbon content to 22.9, 21.7
Statistics, version 23 (Wagner 2016). The significant dif- and 20.8 mg/g was also observed in areca husk, coffee
ferences observed between the treatment means were husk and paddy straw, respectively, when treated them
determined by the highest significant difference which with F. verticillioides GL5.
was calculated by using Tukey’s test at level p ≤ 0.05.
Determination of total nitrogen and crude protein content
Results An improvement in the total nitrogen and crude protein
Growth of ligninolytic fungi on the solid substrates contents of all three substrates decomposed by both the
All the three substrates (such as areca husk, coffee husk fungal strains was observed as the fermentation proceeds
and paddy straw) used were provided the first glimpse when compared to the un-inoculated control (Table 1).
of primary mechanism of spread of selected ligninolytic The initial total nitrogen content was increased from
fungal strains from 2 days after inoculation. Both M. cir- 0.58 to 1.63 mg/g (areca husk), 0.59 to 1.92 mg/g (coffee
cinelloides GL1 and F. verticillioides GL5 were able to husk) and 0.56 to 1.25 mg/g (paddy straw) which caused
grow extensively on all the three tested substrates. The by the inoculation with M. circinelloides GL1 compared
extensive mycelial growth and hyphal infiltration of both to F. verticillioides GL5 and control. In addition, the ini-
the fungal strains into the substrates were clearly evident tial crude protein of areca husk (3.6 mg/g), coffee husk
from the bottom of the inoculated flasks from 5 days after (3.7 mg/g) and paddy straw (3.5 mg/g) was increased to
inoculation which indicates the suitable environmen- 10.1, 12.0 and 7.8 mg/g, respectively, in treatment with
tal conditions for the growth of inoculated fungi. How- M. circinelloides GL1 and followed by F. verticillioides
ever, paddy straw was well supported the fastest mycelial GL5.
growth and spread of both the fungal strains compared to
other substrates. Determination of total phosphorus and potassium content
The periodical changes of both phosphorus and potas-
Physico‑chemical analyses sium contents during the fermentation of the substrates
Determination of pH indicated the increased total phosphorus and potassium
The initial pH of each substrate was changed from near- contents were varied significantly over that of initial con-
neutral to acidic pH at the end of 120 days fermentation tents by the treatment with selected fungi (Table 1). The
period (Table 1), which supports the extensive fungal initial total phosphorus content was increased from 0.49
growth and their abundance. The initial pH was changed to 1.5 mg/g (areca husk), 0.52 to 1.49 mg/g (coffee husk)
from 6.2 to 5.0, 5.8 to 4.2 and 6.5 to 4.4 in areca husk, cof- and 0.5 to 1.21 mg/g (paddy straw) in the treatment with
fee husk and paddy straw, respectively, decomposed by M. circinelloides GL1 compared to F. verticillioides GL5
M. circinelloides GL1. The treatment with F. verticillioides and control. Also, the initial total potassium content of
GL5 was also changed the initial pH to 4.5, 4.3 and 4.8 in areca husk (0.68 mg/g), coffee husk (0.93 mg/g) and
areca husk, coffee husk and paddy straw, respectively. paddy straw (0.88 mg/g) was significantly increased to
6.85, 3.55 and 5.3 mg/g, respectively, in treatment with
Determination of total organic matter and organic carbon M. circinelloides GL1 and followed by F. verticillioides
content GL5.
Decomposing time had a significant reducing
effect on both the total organic matter and organic
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 6 of 14

Table 1 Changes in physico-chemical properties of solid substrates treated with ligninolytic fungi during solid-state
fermentation in different time intervals
Time pH (1–14) Total OM Total OC Total N (mg/g) Crude protein Total P (mg/g) Total K (mg/g)
interval (mg/g) (mg/g) (mg/g)
(day)

Areca husk
Control 6.2 ± 0.115a 65.1 ± 1.66a 37.7 ± 0.96a 0.58 ± 0.014g 3.6 ± 0.08g 0.49 ± 0.034i 0.68 ± 0.09e
ab a a f f gh
  M. circinel- 20 6.0 ± 0.115 62.8 ± 1.14 36.4 ± 0.66 0.72 ± 0.020 4.4 ± 0.12 0.67 ± 0.020 1.20 ± 0.11e
loides GL1 40 5.8 ± 0.173 abc
55.9 ± 1.49 bc
32.4 ± 0.86 bc
0.89 ± 0.020 e
5.5 ± 0.12 e
0.93 ± 0.013 e
2.85 ± 0.13d
bcd ef ef e e d
60 5.6 ± 0.115 45.0 ± 1.15 26.1 ± 0.66 0.93 ± 0.026 5.8 ± 0.16 1.07 ± 0.020 4.75 ± 0.14c
cde g g d d c
80 5.4 ± 0.057 31.0 ± 0.86 17.9 ± 0.50 1.13 ± 0.013 7.0 ± 0.08 1.23 ± 0.017 5.74 ± 0.15b
def g g b b b
100 5.2 ± 0.115 27.5 ± 1.60 15.9 ± 0.93 1.38 ± 0.011 8.6 ± 0.07 1.38 ± 0.044 6.09 ± 0.23b
efg h h a a a
120 5.0 ± 0.173 21.5 ± 1.04 12.4 ± 0.60 1.63 ± 0.023 10.1 ± 0.14 1.50 ± 0.031 6.85 ± 0.18a
  F. verticil- 20 5.8 ± 0.115abc 63.2 ± 1.60a 36.6 ± 0.93a 0.69 ± 0.02f 4.3 ± 0.13f 0.56 ± 0.026hi 0.83 ± 0.12e
lioides GL5 40 5.6 ± 0.057bcd 56.8 ± 0.62b 32.9 ± 0.36b 0.74 ± 0.017f 4.6 ± 0.10f 0.68 ± 0.043g 1.18 ± 0.15e
60 5.4 ± 0.100cde 50.8 ± 1.66cd 29.4 ± 0.96cd 0.88 ± 0.017e 5.4 ± 0.10e 0.80 ± 0.026f 2.78 ± 0.17d
80 5.1 ± 0.115def 46.7 ± 1.15de 27.0 ± 0.66de 0.96 ± 0.023e 6.0 ± 0.14e 0.94 ± 0.020e 4.60 ± 0.10c
100 4.8 ± 0.173fg 43.0 ± 1.60ef 24.9 ± 0.93ef 1.25 ± 0.028c 7.8 ± 0.17c 1.08 ± 0.017d 4.99 ± 0.22c
120 4.5 ± 0.115g 39.5 ± 0.86f 22.9 ± 0.50f 1.36 ± 0.023b 8.5 ± 0.14b 1.21 ± 0.026c 5.94 ± 0.18b
Coffee husk
Control 5.8 ± 0.115a 72.7 ± 1.87a 42.1 ± 1.08a 0.59 ± 0.014i 3.7 ± 0.09i 0.52 ± 0.037h 0.93 ± 0.04e
ab a a gh gh fg
  M. circinel- 20 5.5 ± 0.208 68.7 ± 2.22 39.8 ± 1.29 0.72 ± 0.018 4.5 ± 0.11 0.70 ± 0.041 1.14 ± 0.06de
loides GL1 40 5.3 ± 0.115 abc
54.9 ± 1.26 bc
31.8 ± 0.72 bc
0.85 ± 0.020 fg
5.3 ± 0.12 fg
0.82 ± 0.049 ef
1.64 ± 0.15cd
bcd ef ef e e de
60 5.0 ± 0.057 41.6 ± 1.71 24.1 ± 0.99 1.08 ± 0.037 6.7 ± 0.23 0.97 ± 0.043 1.86 ± 0.13c
bcd g g c c bc
80 5.0 ± 0.152 30.9 ± 1.70 17.9 ± 0.98 1.45 ± 0.026 9.1 ± 0.16 1.16 ± 0.026 2.18 ± 0.10c
de h h b b b
100 4.6 ± 0.100 24.5 ± 1.35 14.2 ± 0.78 1.72 ± 0.027 10.7 ± 0.17 1.28 ± 0.044 2.85 ± 0.15b
e h i a a a
120 4.2 ± 0.115 18.3 ± 1.32 10.6 ± 0.76 1.92 ± 0.023 12.0 ± 0.14 1.49 ± 0.031 3.55 ± 0.18a
abc a a hi hi gh
  F. verticil- 20 5.4 ± 0.173 69.3 ± 1.48 40.2 ± 0.85 0.68 ± 0.046 4.2 ± 0.28 0.62 ± 0.026 1.10 ± 0.08de
lioides GL5 40 5.1 ± 0.100 bcd
59.2 ± 1.54 b
34.3 ± 0.89 b
0.78 ± 0.017 gh
4.9 ± 0.10 gh
0.70 ± 0.026 fg
1.14 ± 0.11de
cd bc bc f f ef
60 4.9 ± 0.057 54.4 ± 1.28 31.5 ± 0.74 0.92 ± 0.015 5.7 ± 0.09 0.83 ± 0.020 1.67 ± 0.14cd
de cd cd d d de
80 4.7 ± 0.100 50.4 ± 1.21 29.2 ± 0.70 1.28 ± 0.043 8.0 ± 0.27 0.97 ± 0.029 1.82 ± 0.14c
de de de cd cd cd
100 4.6 ± 0.115 45.3 ± 1.66 26.3 ± 0.96 1.33 ± 0.040 8.3 ± 0.25 1.07 ± 0.034 2.06 ± 0.20c
e f f c c bc
120 4.3 ± 0.057 37.5 ± 0.93 21.7 ± 0.54 1.45 ± 0.028 9.0 ± 0.17 1.17 ± 0.027 2.95 ± 0.16b
Paddy straw
Control 6.5 ± 0.115a 68.2 ± 1.12a 39.5 ± 0.65a 0.56 ± 0.023i 3.5 ± 0.14h 0.50 ± 0.037h 0.88 ± 0.08e
ab b b fg fg fg
  M. circinel- 20 6.2 ± 0.152 61.3 ± 1.18 35.5 ± 0.68 0.68 ± 0.026 4.2 ± 0.16 0.69 ± 0.026 1.36 ± 0.15e
loides GL1 40 6.0 ± 0.115 abc
53.8 ± 1.09 c
31.2 ± 0.63 c
0.83 ± 0.029e 5.2 ± 0.18 e
0.84 ± 0.023 de
2.62 ± 0.15d
bcd c c cd cd cd
60 5.8 ± 0.057 48.3 ± 1.72 28.0 ± 0.99 1.04 ± 0.037 6.4 ± 0.23 0.91 ± 0.035 3.24 ± 0.14cd
de d d bc bc bc
80 5.4 ± 0.173 35.8 ± 1.41 20.7 ± 0.82 1.11 ± 0.020 6.9 ± 0.12 1.00 ± 0.023 3.98 ± 0.26bc
ef e e ab ab ab
100 5.1 ± 0.100 26.0 ± 1.61 15.0 ± 0.93 1.19 ± 0.034 7.4 ± 0.21 1.10 ± 0.029 4.66 ± 0.17ab
g e f a a a
120 4.4 ± 0.115 16.5 ± 1.32 9.5 ± 0.76 1.25 ± 0.028 7.8 ± 0.17 1.21 ± 0.028 5.30 ± 0.17a
ab ab ab gh gh gh
  F. verticil- 20 6.3 ± 0.115 66.5 ± 0.85 38.5 ± 0.49 0.63 ± 0.026 3.9 ± 0.16 0.60 ± 0.029 1.04 ± 0.18e
lioides GL5 40 6.1 ± 0.057 ab
63.5 ± 1.65 ab
36.8 ± 0.95 ab
0.76 ± 0.020 ef
4.7 ± 0.12 ef
0.76 ± 0.023 ef
1.26 ± 0.19e
bcd b b e e de
60 5.9 ± 0.100 61.2 ± 1.58 35.5 ± 0.92 0.83 ± 0.034 5.2 ± 0.21 0.85 ± 0.028 2.52 ± 0.22d
cde c c d d cd
80 5.5 ± 0.115 53.8 ± 1.09 31.2 ± 0.63 0.98 ± 0.034 6.1 ± 0.21 0.91 ± 0.040 3.15 ± 0.35cd
ef c c cd cd bc
100 5.2 ± 0.115 48.3 ± 1.41 28.0 ± 0.82 1.05 ± 0.017 6.5 ± 0.10 1.03 ± 0.040 3.96 ± 0.31bc
fg d d ab ab ab
120 4.8 ± 0.173 35.8 ± 1.25 20.8 ± 0.72 1.18 ± 0.015 7.3 ± 0.09 1.09 ± 0.034 5.21 ± 0.17a
‘ ± ’ indicates standard error (SE) calculated by using triplicates
Values (mean of triplicates) with different letters within the same vertical column indicate the significant difference at p ≤ 0.05
A italicized cell represents the maximum value
Total OM total organic matter, Total OC total organic carbon, Total N total nitrogen, Total P total phosphorus, Total K total potassium
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 7 of 14

Table 2 Changes in carbon to nutrient supply ratios in solid substrates treated with ligninolytic fungi during solid-state
fermentation in different time intervals
Time interval (day) C/N ratio C/P ratio C/K ratio

Areca husk
Control 64.4 ± 0.26a 77.6 ± 3.56a 57.2 ± 6.59a
b c
  M. circinelloides GL1 20 50.7 ± 2.25 54.5 ± 2.42 30.7 ± 3.19b
d de
40 36.3 ± 0.36 34.6 ± 0.46 11.4 ± 0.81c
e fg
60 27.9 ± 1.24 24.2 ± 0.51 5.5 ± 0.27c
fg hi
80 15.8 ± 0.60 14.6 ± 0.61 3.1 ± 0.08c
gh hi
100 11.5 ± 0.72 11.5 ± 0.95 2.6 ± 0.25c
i i
120 7.6 ± 0.30 8.2 ± 0.31 1.8 ± 0.13c
b b
  F. verticillioides GL5 20 53.1 ± 1.75 65.6 ± 2.21 46.1 ± 6.35a
c c
40 44.5 ± 1.24 48.6 ± 3.22 28.9 ± 4.07b
d d
60 33.4 ± 1.04 36.9 ± 1.82 10.6 ± 0.31c
e ef
80 28.2 ± 1.37 28.8 ± 1.30 5.8 ± 0.23c
f fg
100 19.9 ± 1.07 22.9 ± 0.76 5.0 ± 0.24c
f gh
120 16.8 ± 0.33 18.9 ± 0.73 3.8 ± 0.17c
Coffee husk
Control 71.2 ± 3.52a 82.2 ± 7.86a 45.4 ± 2.81a
b bc
  M. circinelloides GL1 20 54.8 ± 0.68 57.1 ± 2.55 35.2 ± 2.61b
d de
40 37.3 ± 0.31 38.9 ± 3.13 19.8 ± 2.40c
e fg
60 22.2 ± 0.17 25.0 ± 1.83 13.1 ± 1.28cde
gh gh
80 12.3 ± 0.90 15.3 ± 0.50 8.3 ± 0.86def
hi h
100 8.2 ± 0.49 11.1 ± 0.94 5.0 ± 0.56ef
i h
120 5.5 ± 0.35 7.0 ± 0.36 3.0 ± 0.37f
b b
  F. verticillioides GL5 20 59.2 ± 3.25 64.8 ± 4.05 37.0 ± 3.38ab
c cd
40 43.6 ± 0.16 49.1 ± 1.77 30.8 ± 4.04b
d de
60 34.2 ± 0.41 38.0 ± 0.09 19.1 ± 2.04c
e ef
80 22.8 ± 1.19 30.0 ± 0.94 16.2 ± 1.28cd
ef fg
100 19.7 ± 1.16 24.6 ± 1.70 12.8 ± 0.80cde
fg fgh
120 15.0 ± 0.59 18.5 ± 0.07 7.4 ± 0.57def
Paddy straw
Control 71.0 ± 4.11a 80.3 ± 7.65a 45.5 ± 3.64a
  M. circinelloides GL1 20 52.2 ± 2.98c 51.2 ± 2.69c 26.7 ± 3.50c
40 37.4 ± 1.41ef 37.2 ± 1.78def 11.9 ± 0.48d
60 27.0 ± 1.97gh 30.8 ± 2.23efg 8.6 ± 0.08d
80 18.6 ± 0.65hi 20.6 ± 0.36gh 5.2 ± 0.48d
100 12.6 ± 0.42ij 13.6 ± 1.20hi 3.2 ± 0.32d
120 7.6 ± 0.52j 7.9 ± 0.73i 1.8 ± 0.20d
b b
  F. verticillioides GL5 20 61.4 ± 3.29 64.1 ± 2.55 39.4 ± 6.80ab
cd cd
40 48.1 ± 2.14 48.5 ± 1.52 30.4 ± 4.47bc
de cde
60 42.8 ± 2.40 41.9 ± 2.24 14.3 ± 1.63d
fg ef
80 31.6 ± 0.93 34.1 ± 1.29 10.1 ± 1.26d
gh fg
100 26.7 ± 0.65 27.3 ± 1.70 7.1 ± 0.67d
i ghi
120 17.6 ± 0.82 19.0 ± 1.20 4.0 ± 0.27d
‘ ± ’ indicates standard error (SE) calculated by using triplicates
Values (mean of triplicates) with different letters within the same vertical column indicate the significant difference at p ≤ 0.05
A italicized cell represents the maximum value
C/N carbon/nitrogen, C/P carbon/phosphorus, C/K carbon/potassium
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 8 of 14

Determination of carbon to nutrient (N, P and K) ratios circinelloides GL1 produced the maximum activity of lac-
After solid-state fermentation, the selected fungal strains case (12.15 IU/mL), MnP (0.98 IU/mL) and LiP (2.47 IU/
reduced the initial carbon to nutrient (N, P and K) ratios mL) on above-mentioned cultivation periods in coffee
compared to untreated control (Table 2). Mucor circinel- husk (Fig. 2a–c). In case of paddy straw, M. circinelloides
loides GL1 rapidly decreased the initial C/N ratio of areca GL1 inoculation showed the significantly maximum
husk (64.4), coffee husk (71.2) and paddy straw (71.0) to activity of laccase (15.83 IU/mL), MnP (0.75 IU/mL) and
7.6, 5.5 and 7.6, respectively, compared to F. verticillioides LiP (4.5 IU/mL) on above-mentioned cultivation peri-
GL5 and control. Likewise, the treatment of M. circinel- ods (Fig. 3a–c). However, it was also observed that M.
loides GL1 significantly decreased the initial C/P ratio circinelloides GL1 offered the maximum CMCase activ-
of areca husk (77.6), coffee husk (82.2) and paddy straw ity of 9.95, 8.9 and 11.73 IU/mL on 20 days of incubation
(80.3) to 8.2, 7.0 and 7.9, respectively, when compared in areca husk, coffee husk and paddy straw, respectively
with the treatment of F. verticillioides GL5 and control. (Fig. 4a–c). The enzyme activity decreased gradually as
It was also observed the reduction in initial C/K ratio the fermentation time increased.
of areca husk (57.2), coffee husk (45.4) and paddy straw
(45.5) to 1.8, 3.0 and 1.8, respectively, which offered by Discussion
M. circinelloides GL1 treatment. The success of fungal decomposition process primarily
depends on the selection of an adequate solid substrate
(as a supporting material) for performing the solid-state
Determination of the content and loss of lignin fermentation. It is a traditional cultivation practice to
and holocellulose use the agro-industrial lignocellulosic wastes for small
Both the fungal strains were able to effectively degrade and larger scale production of major lignin-degrading
the lignin and holocellulose in all three substrates tested enzymes. The responsible use of indigenous microbes
in the study. The degradation pattern of lignin and holo- for the degradation of agro-industrial wastes at a faster
cellulose was more effective after fungal inoculation with rate leads to improve soil organic matter and nutrients
respect to their loss between the different harvesting availability (Anwar et al. 2014). In the present study,
times (Table 3). Inoculation with M. circinelloides GL1 three agricultural wastes (such as areca husk, coffee husk
was offered a significant reduction in initial lignin con- and paddy straw) as the lignocellulosic substrates were
tent of areca husk (19.9%), coffee husk (18.7%) and paddy used to study the patterns of production of major ligni-
husk (16.0%) to 6.5, 6.4 and 3.0%, respectively. Thus, M. nolytic and cellulolytic enzymes under fermentation.
circinelloides GL1 was causing a significant higher lignin Ligninolytic fungi have been most extensively studied
loss of 67.1, 65.7 and 80.8% in areca husk, coffee husk and due to their apparent ligninolytic properties as well as
paddy husk, respectively. their faster growth and more easily handling in the fields
Similarly, the inoculation with M. circinelloides GL1 (Levin et al. 2004; Singh and Singh 2014).
also exhibited a significant reduction in initial holo- The substrates used in this study were well supported
cellulose content of areca husk (55.1%), coffee husk for the extensive mycelial growth and hyphal infiltra-
(58.8%) and paddy husk (57.5%) to 28.0, 26.3 and 15.2%, tion of selected fungal strains. However, paddy straw
respectively. Besides, M. circinelloides GL1 caused a sig- supported the fastest mycelial extension of both strains,
nificantly higher degree holocellulose loss of 49.0, 55.2 followed by coffee husk and areca husk. The rapid colo-
and 73.5% in areca husk, coffee husk and paddy husk, nization of fungi on substrates may be due to their high
respectively. competitive saprophytic nature. This remarkable result
was in agreement with the studies of Jonathan et al.
Estimation of activity of enzymes (2008) and Adejoye and Fasidi (2009). In the solid-state
The fermentation profiles of the selected fungal strains fermentation process, it is very much essential to opti-
had shown significant differences in the studied enzymes mize the initial moisture content as it greatly affects the
(laccase, MnP, LiP and CMCase) activity in decomposi- substrate utilization and enzyme production. Therefore,
tion of agricultural wastes. Both strains produced all approximately about 70% moisture content was main-
enzymes, but their activities were remarkably varied tained throughout the experimental periods. The mois-
with respect to fermentation time (Figs. 1, 2, 3, 4). The ture content varies between 30 and 85% for the optimum
distinguished higher activity of laccase (9.4 IU/mL), fungal growth and their nature of utilization of sub-
MnP (1.45 IU/mL) and LiP (3.5 IU/mL) recorded by strates. However, it depends upon the organisms and
M. circinelloides GL1 on 80, 60 and 60 day of cultiva- types of substrates used for the cultivation (Raimbault
tion, respectively, in areca husk compared to F. verticil- 1998). An increase in moisture content decreases the
lioides GL5 and untreated control (Fig. 1a–c), while M. substrate porosity and limits oxygen and mass transfer.
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 9 of 14

Table 3 Changes in lignocellulose content and loss of solid substrates treated with ligninolytic fungi during solid-state
fermentation in different time intervals
Time interval Lignin content (%) Lignin loss (%) Holocellulose Holocellulose loss (%)
(day) content (%)

Areca husk
Control 19.9 ± 0.58a – 55.1 ± 1.24a –
b e
  M. circinelloides GL1 20 17.0 ± 0.64 14.5 ± 3.23 48.4 ± 1.27bc 12.1 ± 2.30ef
bc de bc
40 15.0 ± 0.63 24.2 ± 3.19 47.8 ± 1.33 13.1 ± 2.42ef
cd cd cd
60 13.0 ± 0.76 34.6 ± 3.83 44.6 ± 1.32 18.9 ± 2.41de
d c de
80 11.9 ± 0.59 40.0 ± 2.97 41.0 ± 1.58 25.5 ± 2.88cd
ef ab fg
100 8.7 ± 0.52 56.2 ± 2.66 32.0 ± 1.60 41.9 ± 2.91ab
f a g
120 6.5 ± 0.44 67.1 ± 2.21 28.0 ± 1.73 49.0 ± 3.14a
b e ab
  F. verticillioides GL5 20 17.5 ± 0.32 12.0 ± 1.61 53.9 ± 0.74 2.0 ± 1.34f
bc de bc
40 15.5 ± 0.54 21.9 ± 2.75 49.0 ± 1.32 11.0 ± 2.41ef
cd cd bc
60 13.7 ± 0.66 31.1 ± 3.34 47.8 ± 1.30 13.1 ± 2.36ef
d c cd
80 12.3 ± 0.49 38.1 ± 2.48 45.1 ± 1.81 18.1 ± 3.30de
e b ef
100 9.2 ± 0.55 53.7 ± 2.76 37.5 ± 1.44 31.9 ± 2.61bc
ef ab fg
120 7.2 ± 0.52 63.8 ± 2.66 32.9 ± 1.21 40.1 ± 2.20ab
Coffee husk
Control 18.7 ± 0.51a – 58.8 ± 1.27a –
bc ef
  M. circinelloides GL1 20 16.2 ± 0.40 13.1 ± 2.16 52.3 ± 1.15bc 11.0 ± 1.96fg
c e cd
40 15.5 ± 0.51 17.1 ± 2.77 47.4 ± 1.35 19.2 ± 2.31ef
60 11.5 ± 0.58de 38.3 ± 3.10cd 41.1 ± 1.61ef 30.1 ± 2.75cd
80 9.8 ± 0.40ef 47.2 ± 2.18bc 36.9 ± 1.45fg 37.1 ± 2.46bc
100 8.3 ± 0.46fg 55.6 ± 2.47ab 33.0 ± 1.04g 43.8 ± 1.76b
120 6.4 ± 0.69g 65.7 ± 3.70a 26.3 ± 1.08h 55.2 ± 1.84a
  F. verticillioides GL5 20 18.1 ± 0.32ab 3.2 ± 1.71f 56.5 ± 1.16ab 3.9 ± 1.98g
40 16.0 ± 0.57bc 14.4 ± 3.08ef 51.2 ± 1.72bc 12.9 ± 2.92fg
60 12.6 ± 0.92d 32.6 ± 4.93d 46.9 ± 1.24cd 20.1 ± 2.11ef
80 11.5 ± 0.72de 38.4 ± 3.86cd 43.1 ± 1.34de 26.6 ± 2.28de
de cd fg
100 11.0 ± 0.40 41.1 ± 2.15 36.5 ± 1.24 37.8 ± 2.11bc
ef bc g
120 9.1 ± 0.58 51.3 ± 3.13 32.6 ± 1.48 44.4 ± 2.51b
Paddy straw
Control 16.0 ± 0.55a – 57.5 ± 1.12a –
bc gh
  M. circinelloides GL1 20 14.3 ± 0.31 10.4 ± 1.98 52.5 ± 1.24ab 8.6 ± 2.16gh
d f cd
40 12.3 ± 0.38 22.7 ± 2.40 43.6 ± 1.55 24.1 ± 2.71ef
ef de cd
60 9.7 ± 0.43 39.3 ± 2.72 41.8 ± 1.42 27.1 ± 2.47ef
g c e
80 7.6 ± 0.37 52.2 ± 2.34 32.1 ± 1.27 44.1 ± 2.21d
h b g
100 5.2 ± 0.34 67.5 ± 2.16 21.2 ± 1.21 63.1 ± 2.11b
i a h
120 3.0 ± 0.34 80.8 ± 2.17 15.2 ± 0.98 73.5 ± 1.70a
ab h a
  F. verticillioides GL5 20 15.6 ± 0.17 2.2 ± 1.10 54.4 ± 1.51 5.3 ± 2.63h
cd fg bc
40 13.2 ± 0.46 17.5 ± 2.88 47.5 ± 1.07 17.3 ± 1.86fg
e e c
60 10.4 ± 0.31 34.5 ± 1.98 44.7 ± 1.53 22.1 ± 2.66f
fg cd d
80 8.7 ± 0.40 45.6 ± 2.52 38.1 ± 1.64 33.7 ± 2.86e
g c ef
100 7.5 ± 0.43 53.1 ± 2.72 30.0 ± 1.32 47.7 ± 2.31cd
h b fg
120 5.4 ± 0.32 66.2 ± 2.01 25.0 ± 1.26 56.5 ± 2.20bc
‘ ± ’ indicates standard error (SE) calculated by using triplicates
Values (mean of triplicates) with different letters within the same vertical column indicate the significant difference at p ≤ 0.05
A italicized cell represents the maximum value
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 10 of 14

Fig. 1 Changes in ligninolytic enzyme activity of areca husk treated Fig. 2 Changes in ligninolytic enzyme activity of coffee husk treated
with ligninolytic fungi during solid-state fermentation in different with ligninolytic fungi during solid-state fermentation in different
time intervals. a laccase, b MnP and c LiP time intervals. a laccase, b MnP and c LiP

The relatively high moisture content may also result in Arora (2010) wherein the loss of total organic mat-
limited fungal growth within the substrate and their sur- ter could be ascribed to use as the energy and available
face growth. carbon source during the degradation of lignocellulosic
The fungal growth favors at lower pH and resulting materials by white-rot fungi. Study on the effect of car-
in 50-fold increase in their relative abundance between bon (C) and nitrogen (N) source on the production of
high pH (7.4) and low pH (3.3) (Rousk et al. 2011). We ligninolytic enzymes is getting much importance. The C
have observed the reduction in initial pH from neutral to and N sources have proved as the powerful nutritional
acidic pH in all substrates decomposed by both strains factors that regulating the production of ligninolytic
for the period of 120 days after inoculation. The change enzymes by wood rotting basidiomycetes (Mikiashvili
in initial pH in the fungal decomposed agricultural et al. 2006). The release of glucose from the lignocellu-
wastes as the period of fermentation increased may be loses has promoted the rapid fungal growth within the
linked to the increase in metabolic products within the raw materials as well as fungi need readily metaboliz-
substrates. Initial pH of the straw-mycelium mixture was able carbon sources (such as glucose) for lignin degrada-
considerably changed during the growth of fungi, and tion (Wu et al. 2005). The organic carbon is converted
the pH change was directly correlated with the fungal into carbon dioxide (­CO2) and energy as the metabolic
decomposition (Zadražil 1977). end products; subsequently, the carbon content of sub-
The loss of initial total organic matter was significantly strate reduces as the degradation process progresses. We
increased in all the substrates decomposed by selected have also noticed a significantly decreasing trend in ini-
strains. This result was in agreement with Sharma and tial organic carbon during decomposition of substrates
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 11 of 14

Fig. 3 Changes in ligninolytic enzyme activity of paddy straw treated Fig. 4 Changes in CMCase enzyme activity of different solid
with ligninolytic fungi during solid-state fermentation in different substrates treated with ligninolytic fungi during solid-state
time intervals. a laccase, b MnP and c LiP fermentation in different time intervals. a areca husk, b coffee husk
and c paddy straw

by the selected fungi. Pramanik (2010) has reported that

the initial organic carbon content of organic wastes was incubation. An increment of 51.6% in crude protein was
found to decrease as C­ O2 during vermicomposting by the reported in another study where the fermentation of
ligninolytic fungi. rice husk was conducted by Trichoderma viridii for the
Our results showed that the total nitrogen content period of 10 days (Zaid and Ganiyat 2009). In addition,
was in an upward trend with decomposing time which it was observed that there was an increase in initial total
caused by the loss of total organic carbon associated phosphorus and potassium contents of substrates after
with the mineralization of total organic matter. The decomposition by the selected fungi. The results were
increase in nitrogen content might be due to the forma- similar to the findings of Pramanik (2010) who reported
tion of new cell structures, enzymes and hormones by that the total nitrogen, phosphorus and potassium
the soil microbes (Zhu 2007). However, there was a raise contents in organic wastes were consistently found to
in the crude protein content of areca husk, coffee husk increase during the vermicomposting by the ligninolytic
and paddy straw inoculated with the fungal strains with fungi.
increasing the incubation period. It may be due to the The elemental carbon to nutrient supply (N, P and K)
non-proliferation of fungi on the solid substrates during ratio is used to estimate the availability of associated
the decomposition process. Belewu and Babalola (2009) nutrients in the agricultural crop residues. As the plant
have reported that an increase of 16% in crude protein litter decomposes, the labile organic carbon substrates
content of rice husk in an in vitro digestibility experi- decrease and decline in the C/N and C/P ratios as N
ment conducted with Rhizopus oligosporus for 7 day of and P compounds are incorporated to form the humic
Geethanjali et al. Bull Natl Res Cent (2020) 44:173 Page 12 of 14

substances (Grandy and Neff 2008). The data showed be very efficient producers of ligninolytic and cellulolytic
that a sharp decrease in the C/N, C/P and C/K ratios enzymes in the solid-state fermentation of agricultural
was observed in the decomposition of the tested sub- wastes. Therefore, the present study suggested that these
strates by the selected fungi with respect to the initial potential ligninolytic fungal strains can be successfully
ratios. The C/N ratio is playing an important role in used for managing and reusing the agricultural crop resi-
bioconversion and establishment of the compost matu- dues in the conservation system of forest soils to elimi-
rity level of agricultural wastes (Bernal et al. 1998). The nate the harsh effects of the crop residue burning.
C/N ratio was declined rapidly in the composted sub-
strates, as the C content was lost in the form of ­CO2
Abbreviations
through the microbial respiration and the N content BSA: Bovine serum albumin; M. circinelloides: Mucor circinelloides; F. verticil-
was recycled during fermentation (Ryckeboer et al. lioides: Fusarium verticillioides; MEA: Malt-extract agar; LOI: Loss on ignition;
2003). Osono and Takeda (2001) have reported the CMC: Carboxymethyl cellulose; DNS: 3,5-Dinitrosalicylic acid; IU: International
Units; MnP: Manganese peroxidase; LiP: Lignin peroxidase; CMCase: Carboxy-
decrease in the initial C/P and C/K ratios during the methyl cellulose; OM: Organic matter; OC: Organic carbon; C: Carbon; N: Nitro-
leaf litter decomposition by fungi. gen; P: Phosphorus; K: Potassium; CO2: Carbon dioxide; RCBD: Randomized
The agro-industrial wastes are principally composed of complete block design.
a complex mixture of cellulose, hemicellulose and lignin Acknowledgements
which are used for the production of biofuels and other The authors are acknowledged to the Field Marshal K. M. Cariappa College,
value-added products (Sadh et al. 2018; Tsegaye et al. Madikeri, and the University Grants Commission (UGC), New Delhi, for provid-
ing necessary facilities.
2019). Several fungal species have the ability to decom-
pose lignocellulosic wastes by the successive production Authors’ contributions
of a mixture of extracellular hydrolytic and oxidative GPA and JM conceptualized and designed the research work. GPA and GHG
performed the experiments, analyzed and interpreted the data. All authors
(ligninolytic) enzymes (Navarro et al. 2014). During the read and approved the final manuscript.
degradation process, cellulose and other labile substrates
are first degraded easily and then followed by lignin depo- Funding
Not applicable.
lymerization with increased ligninolytic enzyme activity
(Baldrian 2009). In our study, the selected potential fun- Availability of data and materials
gal strains reduced the lignin and holocellulose contents Not applicable.
of the substrates with respect to increase in incubation Ethics approval and consent to participate
time. Li et al. (2008) have reported that an efficient ligni- Not applicable.
nolytic fungus F. concolor was significantly degraded and
Consent for publication
removed 13.07% of lignin and 7.62% of holocellulose con- Not applicable.
tents of wheat straw.
Moreover, all the three substrates used were well sup- Competing interests
The authors declare that they have no competing interests.
ported for the production of ligninolytic and cellulolytic
enzymes by the selected fungi. The enzyme activities Author details
1
presented have shown that the enzyme production pat- Department of Microbiology, Field Marshal K. M. Cariappa College, Madikeri,
Karnataka 571201, India. 2 Department of Studies in Biotechnology, University
terns are dependent on fungi and type of substrates. The of Mysore, Manasagangotri, Mysuru, Karnataka 570006, India. 3 Department
fungal enzyme production levels could be ascribed to the of Studies and Research in Microbiology, Post Graduate Centre, Jnana Kaveri
different chemical compositions of the substrates. The Campus, Mangalore University, Kodagu, Karnataka 571232, India.
statement agreed with the results reported by Shah et al. Received: 3 August 2020 Accepted: 21 September 2020
(2005), who reported the increased ligninolytic and cel-
lulolytic enzymes production patterns in the bioprocess-
ing of banana waste by Aspergillus spp. and Phylosticta
spp.
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