Accepted Manuscript

Optimization of Cellulase production by a brown rot fungus Fomitopsis sp.
RCK2010 under Solid State Fermentation
Deepa Deswal, Yogender Pal Khasa, Ramesh Chander Kuhad
PII: S0960-8524(11)00374-9
DOI: 10.1016/j.biortech.2011.03.032
Reference: BITE 8265
To appear in: Bioresource Technology
Received Date: 25 January 2011
Revised Date: 10 March 2011
Accepted Date: 11 March 2011
Please cite this article as: Deswal, D., Khasa, Y.P., Kuhad, R.C., Optimization of Cellulase production by a brown
rot fungus Fomitopsis sp. RCK2010 under Solid State Fermentation, Bioresource Technology (2011), doi: 10.1016/
j.biortech.2011.03.032
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1
Optimization of Cellulase production by a brown rot fungus Fomitopsis sp. RCK2010
under Solid State Fermentation

Deepa Deswal, Yogender Pal Khasa and Ramesh Chander Kuhad
*


Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi
South Campus, Benito Juarez Road, New Delhi-110021, India





*
Corresponding Author: Prof. Ramesh Chander Kuhad
Lignocellulose Biotechnology Laboratory
Dept. of Microbiology
University of Delhi South Campus
Benito Juarez Road
New Delhi-110021, India
Email: kuhad85@gmail.com
Tel: +91-11-24112062
Fax: +91-11-24115270


2
Abstract

Culture conditions for enhanced cellulase production from a newly isolated brown rot fungus,
Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.5
and moisture ratio of 1:3.5 (solid:liquid) were found to be optimal for maximum enzyme
production. Of the different carbon sources tested wheat bran gave the maximum production
of CMCase (71.526 IU/g), FPase (3.268 IU/g), and -glucosidase (50.696 IU/g). Among the
nitrogen sources, urea caused maximum production of CMCase (81.832 IU/g), where as
casein and soyabean meal gave the highest FPase (4.682 IU/g) and -glucosidase (69.083
IU/g) production, respectively. Among amino acids tested glutamic acid gave the highest
production for CMCase (84.127 IU/g); however 4-hydroxy-L-proline stimulated maximum
FPase production (6.762 IU/g). Saccharification of pretreated rice straw and wheat straw by
crude enzyme extract from Fomitopsis sp. RCK2010 resulted in release of 157.160 mg/g and
214.044 mg/g of reducing sugar respectively.

Key words: Brown rot fungus, cellulase, optimization, saccharification.



.




3
Introduction

The growing concern for depleting fossil fuel requires a transition from non-renewable carbon
sources to renewable bioresources such as lignocellulose. Regardless of the source,
lignocellulosic materials consist of three main polymers; cellulose, a homopolymer of glucose;
hemicellulose, a heteropolymer of pentoses and hexoses; and lignin, an amorphous polymer of
phenyl propanoid units (Kuhad et al., 1997). Among these cellulose fits in the role perfectly
and thus is also referred as the “biological currency” (Himmel et al., 1999). Every year plants
produce about 180 billion tons of cellulose, making this polysaccharide a huge organic carbon
reservoir on earth. The cellulose synthesis rate is estimated to be equivalent to 70 kg per
person per day (Lutzen et al., 1983). Therefore the importance of cellulose as a renewable
source of energy has become a subject of both, intense research and of commercial interest.
The key step in the utilization of cellulose is its hydrolysis into monomeric sugars and their
eventual conversion into valuable chemicals and energy (Olofsson et al., 2010).
The enzyme, which governs the hydrolysis of cellulose, is known as “cellulase”. Unlike most
of the enzymes cellulase is a complex of enzymes that work synergistically to attack native
cellulose. Cellulase is a family of at least 3 groups of enzymes: firstly endoglucanases (EC
3.2.1.4) which act randomly on soluble and insoluble cellulose chains; secondly exoglucanases
(cellobiohydrolases EC 3.2.1.91) that act to liberate cellobiose from the reducing and non-
reducing ends of cellulose chains and finally, -glucosidases (EC 3.2.1.21) which liberate
glucose from cellobiose. The cellulases give us an opportunity to reap the tremendous benefits
of biomass utilization in an eco-friendly manner (Himmel et al., 1999). Besides this, cellulases
have many other potential applications as well, for example, formulation of washing powder,

4
animal feed production (Han et al., 2010), textile industry, pulp and paper industry, starch
processing, grain alcohol fermentation, malting and brewing, extraction of fruits and vegetable
juices (Bhat, 2000). Therefore, like cellulose, a wide range of applications have made cellulase
one of the most desirable enzyme system. An efficient cellulose hydrolysis requires a high
enzyme loading and except recombinants, the level of cellulase production from majority of
the microorganisms has been generally low. The requirement of high amount of the enzyme
makes the hydrolysis process economically less favorable. In many bioconversion strategies,
the cellulase required for biomass conversion may account for as much as 40% of the total
process cost (Ahamed et al., 2008). Therefore large –scale low cost production of cellulase is
very important for the overall process economics for bioconversion of lignocellulosics into
value added chemicals such as ethanol as biofuel.
A large number of microorganisms such as bacteria, actinomycetes and fungi (Kuhad et al.,
1997; Kalogeris et al., 2003) are known to degrade cellulose. Among fungi, soft rot and white
rot have been extensively studied while brown rots have not been studied much. Cellulolytic
enzymes from soft rot and white rot fungi have been studied in model organisms such as
Trichoderma viride and Phanerochaete chrysosporium respectively. T. viride produces fairly
good amount of exoglucanases and endoglucanases but low level of -glucosidase, which is
insufficient for effective conversion of cellulose to glucose. However, the brown rot fungi
differ substantially from soft-rot and white rot fungi with respect to the cellulolytic enzymes
produced and the pattern of cellulose degradation (Kuhad et al., 1997). These fungi are
generally reported to lack the exoglucanases that can hydrolyze crystalline cellulose (Kuhad et
al., 1997), yet they cause the most destructive type of wood decay and are important
contributors to biomass recycling. Recently brown rot fungi such as Fomitopsis has been

5
reported to hydrolyze microcrystalline cellulose and therefore has been studied in some
laboratories (Yoon et al., 2008).Yoon et al. (2005) have reported the degradation of crystalline
cellulose by Fomitopsis palustris. However, to the best of our knowledge the cellulase
production from Fomitopsis sp. has not been optimized.
Beside the fungus type, the cellulase production is also greatly influenced by media
components, especially carbon and nitrogen sources, minerals and physical factors such as pH,
temperature and moisture (Lynd et al., 2002). In order to obtain maximum enzyme production,
development of a suitable medium and culture conditions is obligatory. Solid-state
fermentation (SSF) conditions have shown to be potential for enzyme production by the
filamentous fungi (Holker et al., 2004). The commercial production of enzymes is carried out
through SSF for its obvious advantages over liquid cultivation (Viniegra-Gonzalez et al.,
2003; Holker et al., 2004). The substrate used in SSF for cellulase production is deterimental
in economizing the enzyme production process. Therefore, various cellulosics substrates such
as sugarcane bagasse, corn stover, wheat straw, and wheat bran have been tested by several
workers for production of cellulases.
In this paper we report the optimization of various physiological and nutritional parameters for
cellulase production from newly isolated brown rot fungus Fomitopsis sp. RCK2010 using
cost effective substrates under solid state fermentation cultivation conditions. Moreover an
attempt has been made to study the application of cellulase(s) in hydrolysis of lignocellulosic
substrates such as wheat straw and rice straw.

Materials and Methods
Raw Materials and their Pretreatment

6
Lignocellulosic substrates like Wheat straw (WS), Rice straw (RS), Wheat bran (WB), Corn
cob (CC), Corn Stover (CS), Prosopis juliflora (PJ), and Lantana camera (LC) were obtained
locally. They were first dried and chopped into small pieces by a chopper, then ground into
smaller particles in a hammer mill (Metrex Scientific Instrumentation Pvt. Ltd., New Delhi,
India) and finally separated by 20 mesh sieve.
The pretreatment of both the substrates (rice straw and wheat straw) was carried out separately
with 0.5% (w/v) H
2
SO
4
and 2.5% NaOH at 121°C for 15 min. The pretreated residues were
washed extensively to neutral pH and dried at 60°C till constant weight.

Microorganism and culture conditions
The fungal isolate RCK2010 procured from the culture collection of Lignocellulose
Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus,
New Delhi, India was grown and maintained on malt extract agar (MEA) composed of (gl
-1
):
malt extract, 20.0; Ca(NO
3
)
2
.4H
2
O, 0.5; MgSO
4
.7H
2
O, 0.5; KH
2
PO
4
, 0.5; and agar, 20.0 (pH
5.5) at 30°C (Dhawan et al., 2002; Vasdev et al., 2005). The fungal cultures were maintained
by periodical subculturing on MEA at 30ºC and stored at 4ºC.

Identification of the fungus
Isolation of Genomic DNA
Fungal isolate RCK2010 was grown in malt extract broth (MEB) as described elsewhere
(Dhawan et al., 2002; Vasdev et al., 2005) at 30˚C under static cultivation conditions for 168
h. The cultures were harvested by filtering through Whatman No.1 filter paper. The fungal
mycelium was thoroughly washed with Milli Q water and ground in liquid nitrogen. Genomic

7
DNA of fungal mycelium was isolated using method modified by Kuhad and coworkers as
reported earlier (Kuhad et al., 2004).

Phylogenetic studies of the fungus
ITS sequence of fungal isolate RCK 2010 was amplified by PCR using pITS-1 (5’-
TCCGTAGGTGAACCTGCGG – 3’) and pITS-4 (5’- TCCTCC GCTTATTGATATGC-3’)
primer pair. Following cycling parameters: an initial denaturation at 94˚C (4 min), 35 cycles of
primer annealing at 58˚C (40 sec), elongation at 72˚C (1 min) and denaturation at 94˚C (1
min). A final elongation step was allowed at 72˚C for 8 min. The PCR product was eluted
using gel extraction kit (Quigen Sciences, Maryland, USA) and was sequenced at The Centre
for Genomic Application (TCGA) Okhla, New Delhi, India. Sequence obtained was compared
with ITS sequences available in GenBank, using Clustal W and a dendrogram was constructed
to establish the taxonomic rank of the fungus (DNA STAR, Madison, WI, USA).

Inoculum preparation
Each Erlenmeyer flask (250 ml) containing 50 ml of MEB was inoculated with four mycelial
discs (0.8 cm dia each) and incubated at 30ºC under static cultivation conditions for 7 days.
The mycelial mat thus obtained was homogenized with pestle and mortar under sterile
conditions and used as primary inoculum for further experiments.


Cellulase production under solid state fermentation

8
Solid state fermentation was carried out in 250 ml Erlenmeyer flasks, each having 5.0 g of dry
Wheat bran moistened with mineral salt solution (gl
-1
:(NH
4
)
2
SO
4
, 0.5; KH
2
PO
4
, 0.5; MgSO
4
,
0.5 and pH 5.5) to attain the final substrate-to-moisture ratio of 1:3.5. The flasks were
sterilized by autoclaving at 121˚C (15psi), and thereafter cooled to room temperature and
inoculated with desired volume of inoculums to obtain 0.25 g ± 0.013 of fungal dry mass
(0.5% w/w). The contents of the flasks were mixed well with sterilized glass rod to distribute
the inoculum through out the substrate and incubated at 30˚C. The fungal fermented wheat
bran (myco-bran) was aseptically removed from flasks after an appropriate interval, suspended
in 50 ml citrate buffer (100 mM, pH 5.5) and shaken gently for 45 min. The extrudates were
squeezed through muslin cloth for maximizing the enzyme extraction and centrifuged at
10,000 rpm at 4°C for 10 min. The enzyme solution thus obtained was assayed for various
cellulase activities to study their time course production.

Optimization of cellulase production
The cellulase production by the fungus was optimized following one factor at a time (OFAT)
approach. The effect of various factors such as initial pH (3.0-10.0), incubation temperature
(25-40˚C), substrate to moisture ratio (1:1-1:4), metal ions (2mM), and different carbon and
nitrogen sources, as described in the text, was tested. In addition effect of amino acids (0.2 %
w/w), vitamins (0.2% w/w) and surfactants (0.2% w/w) to enhance cellulase production was
also investigated.


Application of cellulase in cellulose hydrolysis:

9
Enzymatic hydrolysis of untreated and pretreated substrates (rice straw and wheat straw) was
carried out at 2% (w/v) consistency in 50mM citrate phosphate buffer (pH 4.8) containing
0.005% (w/v) sodium azide. The flasks were added with crude enzyme solution equivalent to
FPase activity of 25 IU/g pretreated substrate and incubated at 50°C and 150 rpm for 24 h.
Samples were withdrawn at regular intervals, centrifuged at 8000 rpm for 10 min and the
supernatant was analyzed for reducing sugars released.

Analytical methods
The total cellulase (filter paper cellulase, FPcellulase), Carboxymethyl cellulase (CMCase)
and -glucosidase activities were determined in accordance with the International Union of
Pure and Applied Chemistry procedures as reported by Ghose (1987). FPcellulase (FPase)
activity was assayed by measuring the release of reducing sugars in a reaction mixture
containing Whatman No.1 filter paper (1.0 cm x 6.0 cm 50.0 mg) as substrate in 50mM
sodium citrate buffer (pH 4.8) at 50°C, after 60 min. Carboxymethlycellulase (CMCase)
activity was assayed by measuring the release of reducing sugars in a reaction mixture
containing 0.5 ml of crude enzyme and 0.5 ml of 2% (w/v) of CMC solution in 50mM sodium
citrate buffer (pH 5.5) incubated at 50˚C for a period of 30 min. Reducing sugars were assayed
by dinitrosalicyclic acid (DNSA) method of Miller (1959). -Glucosidase activity was
determined by assaying the release of p-nitrophenol (pNP) at 430 nm from a reaction mixture
containing 1 ml p-nitrophenyl glucopyranoside (pNPG) (1 mM), 1.8 ml acetate buffer and 0.2
ml suitably diluted enzyme, incubated at 50°C for 30 min (Wood et al., 1969). One unit of
enzyme activity was defined as the amount of enzyme required to liberate 1mole of glucose
or p-nitrophenol, from the appropriate substrate, per ml per minute under the assay conditions.

10

Results and Discussion:
Identification of Fungus RCK2010:
The fungal isolate RCK 2010 was identified based on the sequence variation present in
internal transcribing spacer (ITS) region. Sequence analysis suggested that RCK 2010 was
phylogenetically related to members of the genus Fomitopsis, where it showed maximum
similarity of 93% with the Fomitopsis genus (Figure 1). Based on this sequence homology, the
fungus RCK2010 was identified as Fomitopsis and hereafter it is named as Fomitopsis sp.
RCK2010. The sequence had been deposited in NCBI Genbank database with accession
number: GU991381.

Time course of cellulase production:
Fomitopsis sp. RCK2010 grown under SSF started producing all the three cellulases on day 2
of incubation. The CMCase production peaked (71.699 IU/g substrate) on eleventh day,
whereas maximum -glucosidase (53.679 IU/g substrate) and FPase (3.492 IU/g substrate)
production was observed on the fifteenth and sixteenth day, respectively (Figure 2). Any
further increase in incubation did not favor any increase in the enzyme production. The exact
comparison of cellulase production by different microorganisms reported in literature may not
be possible because many a times different laboratories estimate the enzyme under different
conditions. However, the comparison of cellulase production by Fomitopsis sp. RCK2010
under SSF with ones earlier reported showed that the fungus tested in this study produced
fairly good amount of cellulases (Table 1).


11
Effect of Physiological Parameters on cellulase production
Temperature is one of the most important physical variable affecting solid-state fermentation
(Krishna, 2005). The optimization of incubation temperature for production of cellulases from
Fomitopsis sp. RCK2010 under SSF conditions revealed that the enzyme production gradually
increased from 25-30°C (Figure3) and maximal enzyme production of all the three enzymes
viz. CMCase (70.784 IU/g), FPase (3.247 IU/g) and -glucosidase (50.989 IU/g) was observed
at 30°C. Any increase in temperature beyond 30°C had a drastic adverse affect on the enzyme
production. Hanif and co-workers (2004) also reported an increase in cellulase production
from Aspergillus niger up to 30°C and thereafter the production of enzyme declined. Wang et
al. (2006) while studying the optimization of multienzyme production by two mixed strains
Aspergillus niger F
3
and Aspergillus niger F
4
in solid state fermentation also reported the
similar temperature optima.
The pH of the medium is one of the most critical environmental parameter affecting the
mycelial growth, enzyme production and the transport of various components across the cell
membrane (Kapoor et al, 2008). The cellulase production by Fomitopsis sp. RCK2010 was
also tested at different pH ranging from 3.0 to 10.0. The fungus produced maximum CMCase
(72.696 IU/g), FPase (3.307 IU/g) and -glucosidase (50.951 IU/g) at initial medium pH 5.5
(Figure 4). Increasing the initial pH of the medium from 5.5 to 10.0 showed a significant
decrease in the production of cellulases. A loss of more than 50% in enzyme production was
observed at initial medium pH of 10.0. The decrease in initial pH of the medium from 5.5 to
3.0 also caused a slight decrease in the CMCase and FPase production. The optimum initial
pH for maximum fungal cellulase production has been reported to be variable in majority of
the cases (Niranjane et al., 2007). The -glucosidase produced by the fungus retained more

12
than 90% of its optimal activity at pH ranging from 3.0 to 7.0. Although further increase in pH
caused a decrease in enzyme production. Kalogeris and co-workers (2003) also reported a
decrease in the -glucosidase by Thermoascus aurantiacus, when the pH of the production
medium was shifted from acidic to alkaline.
The moisture content of the growth medium is critical variable affecting the solid-state
fermentation. The optimal moisture content in solid-state fermentation depends on the nature
of substrate, the requirements of microorganism and the type of end product (Kalogeris et al.,
2003). The result of this study has shown that an increase in the initial moisture ratio from 1:1
to 1:3.5 greatly enhanced the enzyme production (Table 2) of all the three cellulases. The
substrate to moisture ratio of 1:3.5 resulted in maximum production of CMCase (72.071 IU/g),
FPase (3.191IU/g), -glucosidase (66.594IU/g). However, any further increase in moisture
level in Solid State fermentation showed lower enzyme production which may be attributed to
particles sticking, limited gas exchange and higher vulnerability to bacterial contamination,
while low moisture leads to reduced solubility of nutrients, substrate swelling (Hamidi-
Esfahani et al, 2004).

Effect of nutritional parameters on cellulase production
Extracellular enzyme production depends greatly on the composition of the medium. Various
lignocellulosic substrates were tested as carbon source for their effect on cellulase production.
The influence of the carbon sources on cellulase production by Fomitopsis sp. RCK2010 is
depicted in Table 3. Among the carbon sources tested, wheat bran was found to induce
maximum production of CMCase (71.526 IU/g), FPase (3.268 IU/g) and -glucosidase
(50.696 IU/g). It is well known that the type and composition of the carbohydrates present in

13
wheat bran are suitable for the induction of cellulases in filamentous fungi under solid-state
fermentation (Jecu, 2000). The cellulase production by Fomitopsis sp. RCK2010 was found to
be at low level when grown on other lignocellulosic materials like corncob and Prosopis
juliflora. The unsuitability of the lignocellulosic substrates to support enzyme production
under solid-state fermentation might be due to inappropriate physical properties like particle
size, geometry and compactness of the substrate (Krishna, 2005).
Different nitrogen sources have shown variable effects on cellulase production by Fomitopsis
sp. RCK2010 (Table 4). Among various organic nitrogen sources, urea caused maximum
CMCase (81.832 IU/g) production, whereas casein and soyabeen meal resulted in maximum
FPase(4.682 IU/g) and -glucosidase(69.083 IU/g) production respectively. The inorganic
nitrogen sources did not exhibit any significant effect on increase in enzyme production. Our
results are in agreement with the observations of Daroit et al. (2007). However, some studies
have also shown that inorganic nitrogen source resulted in improved enzyme production
compared to organic nitrogen source (Kalogeris et al., 2003).
Presence of metal ions was shown to influence enzyme production by microorganism in
culture. Various metal ions (2mM) were used as additives in the basal medium to determine
their stimulatory or inhibitory effect on enzyme production (Figure 5). An increase in CMCase
production was observed when the production medium was supplemented with zinc (92.979
IU/g) and nickel ions (89.059 IU/g), while mercury (40.817 IU/g) and calcium (41.125 IU/g)
exhibited an inhibitory effect. The addition of sodium (6.388 IU/g), manganese (6.831 IU/g)
and copper (6.795 IU/g) had a stimulatory effect on FPase production. The fungus was
observed to produce maximum -glucosidase (98.971 IU/g) in presence of barium ions but the
addition of potassium (41.549 IU/g), calcium (44.779 IU/g) and ferric ions (41.903 IU/g)

14
inhibited the -glucosidase production. According to observations made by us, zinc and nickel
supplemeted medium enhanced the production of all the three cellulase enzymes. These results
can be attributed to the fact that the metals might be acting as cofactors for many enzymes
involved in intermediatory metabolism (Jellison et al., 1997).
Among various amino acids and their analogues studied, the maximum CMCase production
(84.127 IU/g) was observed in the presence of L-glutamic acid, whereas, aspartic acid (79.099
IU/g), asparagine (77.844 IU/g) and L-cystine (76.799 IU/g) comparatively brought about a
lesser increase in CMCase production (Table 5). L-methionine and L-phenylalanine
completely inhibited CMCase production. There was a significant increase in FPase
production on addition of 4-hydroxy-L-proline (6.762 IU/g), glycine (6.264 IU/g) and L-
cystine (6.177 IU/g). Our observations are in accordance with earlier reports where increase in
laccase production has been reported in presence of glycine (Dhawan et al., 2002). Changes in
the membrane permeability in presence of glycine had been reported due to release of
enzymes extracellularly (Dhawan et al., 2002). Addition of L-isoleucine gave the maximum -
glucosidase production (77.546 IU/g).
The presence of vitamins usually affects the rate of biosynthesis of many metabolites.
Ascorbic acid supplementation gave maximal FPase (5.668 IU/g) and -glucosidase (92.251
IU/g) (Table 5). Riboflavin and biotin also exhibited a stimulatory effect on FPase and -
glucosidase production. Biotin is well documented for playing important role in functioning of
several physiological and metabolic enzymes including pyruvate carboxylase and several
tricarboxylic acid cycle auxillary enzymes (Entcheva et al., 2002). Our results are in
accordance with the earlier reports where the effect of vitamins was studied on laccase

15
production (Dhawan et al., 2002). Interesting to note that none of the vitamin tested here did
not induce the CMCase production.
The use of surfactants in the production of hydrolytic enzymes is well known (Kapoor et al.,
2008). PEG of different molecular weights were used to study their effect on cellulase
production. It was observed that there was a gradual increase in cellulase production with
increase in molecular weight of PEG upto 6000, which gave maximum CMCase (79.544
IU/g), FPase (5.137 IU/g) and -glucosidase (61.502 IU/g) production. These results are in
accordance with our previous studies, where the addition of Tween 80 enhanced the cellulase
production by 30% (Kuhad et al., 1994). In the present study, Triton X-100 maximally
enhanced CMCase (98.268 IU/g), FPase (6.711 IU/g) and -glucosidase (65.361 IU/g)
production from Fomitopsis sp. RCK2010 (Table 5). Such compounds probably increase the
permeability of the cell membrane, allowing more rapid secretion of enzymes (Ahamed et al,
2008).

Enzymatic hydrolysis
The efficacy of crude cellulases from Fomitopsis sp. RCK2010 in hydrolyzing the untreated
and pretreated rice straw and wheat straw was evaluated. The time course of enzymatic
saccharification revealed that irrespective of the substrate and pretreatment used, the release of
sugars increased with increase in the saccharification time studied here (Figure 6). However,
the maximum sugars were released on enzymatic hydrolysis of alkali pretreated wheat straw
(214.044 mg/g of substrate), more than from alkali pretreated rice straw (157.160 mg/g of
substrate) after 24h (Figure 6). This higher increase of reducing sugars in alkali pretreated
substrates may be attributed to the release of lignin moieties during alkali treatment which in

16
turn enhanced the accessibility of cellulose to the enzymes (Gupta et al., 2010). While,
comparatively the lower release of sugars in acid treated substrate may be due to the
relocalization of acid hydrolyzed lignin to the surface of substrate (Hendriks et al., 2009).
Earlier there were several reports with commercial enzymes from T. reesei, a soft rot fungus,
to release higher amount of glucose during enzymatic saccharification of cellulosics, however,
the saccharification of cellulosic substrates with the enzymes from brown rot fungi has been
comparatively lower (Table 6). The higher saccharification of cellulosics by commercial
preparations could be because these contain different cellulases in pure form and also because
the delignified (chlorite pretreated) substrate used in these studies were of high cellulose
purity (~90% w/w) (Gupta et al 2011), when compared with the hydrolysis of alkali treated
wheat straw and rice straw, the partially delignified cellulosic substrates, with crude cellulase
from Fomitopsis sp RCK2010 (Table 6). Interestingly, the enzyme from Fomitopsis sp.
RCK2010 has shown comparatively higher saccharification efficiency than the enzymes from
brown rots reported earlier (Table 6). Yoon et al. (2005) have reported a release of only 32
mg/g sugar from avicel after 43h, while Lee and coworkers (2008) when enzymatically
hydrolyzed Pinus densiflora observed only 3.53 mg/g sugar release.

Conclusion
Our study indicates that Fomitopsis sp. RCK2010 has good potential in producing complete
cellulase and hydrolyzing the cellulosics into fermentable sugars. Moreover, if the cellulase
production form this fungus is further optimized using statistical methods such as response
surface methodology (RSM) which uses combinatorial interactions of culture conditions, it
may result in improved production of cellulases.

17

Acknowledgement
The authors are grateful to Council of Scientific and Industrial Research, Government of
India, New Delhi, India, for the financial support during the progress of this work.


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21
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Legend to figures

Figure 1: The phylogenetic dendogram for Fomitopsis sp. RCK2010 and related fungal
strains based on ITS sequence
Figure 2: Time course of cellulase production by Fomitopsis sp. RCK2010 under Solid state
fermentation
Figure 3: Effect of temperature on cellulase production by Fomitopsis sp. RCK2010 under
SSF
Figure 4: Effect of pH on cellulase production by Fomitopsis sp. RCK2010 under SSF
Figure 5: Effect of different ions on cellulase production by Fomitopsis sp. RCK2010 under
SSF
Figure 6: Enzymatic hydrolysis profile of alkali and acid treated rice straw and wheat straw








23
Fomitopsis cf. meliae KYO
Fomitopsis cf. meliae 1P 1 1
Fomitopsis sp. RCK2010
Fomitopsis cf. meliae 2IV 7 1
Fomitopsis sp. 9V 3 1 isolate 9V 3 1
Fomitopsis ostreiformis isolate BCC23382
Fomitopsis palustris...
0.01

















Figure 1








24




Figure 2










25





0
10
20
30
40
50
60
70
80
25 30 35 37 40 45
Temperature (°C)

C
M
C
a
s
e

I
U
/
g

b
e
t
a
-
g
l
u
c
o
s
i
d
a
s
e

I
U
/
g
0
0.5
1
1.5
2
2.5
3
3.5

F
P
a
s
e

I
U
/
g
CMCase
Beta-glucosidase
FPase






Figure 3



26











Figure 4







27










Figure 5





28





0
50
100
150
200
250
0 4 8 12 16 20 24
Time (h)
S
u
g
a
r

r
e
l
e
a
s
e
d

(
m
g
/
g
)
Control RS
NaOH treated RS
Acid treated RS
Control WS
NaOH treated WS
Acid treated WS

Figure 6










29




Table 1: Comparisons of cellulase production by Fomitopsis sp. RCK2010 with other
fungi under SSF


Enzyme Strain Activity (IU/g) Carbon Source Reference
CMCase Humicola lanuginosa 1.7 Beet pulp Grajek, W., 1986
Myceliophthora sp. 26.6 Whaet bran Badhan et al., 2007
Pleurotus ostreatus IE8
Fomitopsis sp.RCK2010
0.18
71.699
Sugarcane bagasse
Wheat bran
Membrillo et al., 2008
Present work
FPase Myceliophthora sp. 0.74 Wheat bran Badhan et al., 2007
Pleurotus ostreatus IE8 0.013 Sugarcane bagasse Membrillo et al., 2008
Thermoascus auranticus 4.4 Wheat straw Kalogeris et al., 2003
Fomitopsis sp.RCK2010 3.492 Wheat bran Present work
-glucosidase Humicola lanuginosa 46.8 Beet pulp Grajek, W., 1986
Sporotrichum thermophile 12.1 Beet pulp Grajek, W., 1986
Myceliophthora sp. 3.83 Wheat bran Badhan et al., 2007
Fomitopsis sp.RCK2010 53.679 Wheat bran Present work










30








Table 2: Effect of substrate to moisture ratio on cellulase production by Fomitopsis sp.
RCK2010 under SSF


Enzyme Activity (IU/g) Substrate:Moisture
CMCase FPase -glucosidase
1:1 3.285±0.094 0.188±0.007 6.431±0.154
1:1.5 4.049±0.092 0.319±0.024 26.055±1.051
1:2 31.544±1.415 1.241±0.050 44.675±0.953
1:2.5 71.029±1.216 3.308±0.010 51.863±0.644
1:3 71.876±0.460 3.237±0.088 58.777±0.776
1:3.5 72.571±0.810 3.191±0.035 66.594±0.078
1:4 70.669±0.942 2.498±0.005 66.608±0.151













31










Table 3: Effect of different carbon sources on cellulase production by Fomitopsis sp.
RCK2010 under SSF


Enzyme Activity (IU/g) Carbon source (5g)
CMCase FPase -glucosidase
Corn cob 2.215±0.040 0.191±0.009 2.658±0.067
Corn stover 3.698±0.048 0.243±0.032 2.773±0.154
Wheat straw 2.410±0.099 0.239±0.013 3.229±0.560
Prosopis juliflora 1.052±0.071 0.732±0.037 1.393±0.098
Lantana camera 2.895±0.097 0.447±0.030 3.227±0.086
Wheat bran 71.526±0.910 3.268±0.067 50.696±0.662












32




Table 4: Effect of different nitrogen source on cellulase production by Fomitopsis sp.
RCK2010 under SSF


Enzyme Activity (IU/g) Nitrogen source
(0.05% N
2

equivalent)
CMCase FPase -glucosidase
Control 71.699±0.665 3.319±0.004 51.421±0.273
Inorganic
NaNO
2 52.778±1.095 4.464±0.025 37.412±0.800
NaN
3 13.166±0.398 0.000 0
KNO
3 53.265±0.596 3.451±0.019 41.549±0.725
Fe(NO
3
)
3 46.353±0.781 3.359±0.024 41.903±0.426
NH
4
Cl 46.844±1.068 3.150±0.023 47.684±0.621
Ca(NO
3
)
2 41.125±0.955 3.131±0.037 43.779±1.094
(NH
4
)
2
C
2
O
4 66.974±0.489 4.528±0.034 46.076±0.954
NaNO
3 57.864±0.588 3.173±0.018 47.522±0.713
Pb(NO
3
)
2 60.799±0.325 2.629±0.038 57.114±0.889

Organic
Peptone 71.650±0.636 4.381±0.050 63.693±0.668
Yeast extract 74.163±1.015 4.363±0.058 56.977±0.409
Trytone 77.258±0.873 4.145±0.021 64.126±0.936
Casein 71.544±0.676 4.682±0.037 64.946±1.015
Soya been meal 74.996±0.520 4.155±0.021 69.083±0.904
Urea 81.832±0.812 4.562±0.029 54.928±1.029
Corn steep liquor 70.809±0.848 4.643±0.032 52.149±0.406




33
Table 5: Effect of amino acids, vitamins and surfactants on cellulase production by
Fomitopsis sp. RCK2010 under SSF

Enzyme Activity (IU/g) Additives (0.2%,)w/w
CMCase FPase -glucosidase
Control
71.699±0.665 3.269±0.067 51.921±0.434
Aminoacids

L-Alanine
63.160±0.443 5.031±0.037 60.524±0.713
L-Arginine
73.756±1.063 4.941±0.028 57.116±0.893
L-Asparagine
77.844±1.068 4.834±0.011 65.981±0.528
L-Aspartic Acid
79.099±0.901 5.834±0.011 61.097±0.517
L-Cysteine
74.167±0.397 5.159±0.024 63.889±0.839
L-Cystine
76.799±1.042 6.177±0.016 66.211±0.100
L-Glutamic acid
84.127±0.965 5.109±0.071 61.756±0.356
L-Glutamine
74.248±0.826 4.854±0.021 63.104±0.991
Glycine
62.825±0.561 6.264±0.025 64.543±0.680
L-Isoleucine
58.291±0.853 5.018±0.004 77.546±0.733
L-Leucine
51.848±0.543 3.488±0.006 54.815±0.384
L-lysine hydrochloride
56.368±0.774 5.273±0.013 65.272±0.552
L-Methionine
35.017±0.468 3.748±0.018 51.943±0.392
L-serine
64.167±0.397 5.447±0.030 66.266±0.822
L-Threonine
72.053±0.922 5.447±0.022 62.026±0.908
L-valine
61.770±1.051 5.046±0.022 61.681±0.674
L-Phenylalanine
38.115±0.893 4.087±0.006
60.968±0.928
4-hydroxy-L-proline
56.694±0.670 6.762±0.056 69.995±0.898
L-Tryptophan
61.958±1.015 3.672±0.013 62.105±0.422
L-Tyrosine
63.133±0.940 4.067±0.027 60.263±0.590
L-Histidine hydrochloride
61.123±0.477 5.954±0.021 61.997±0.941
Vitamins

Ascorbic acid
64.779±1.099 5.668±0.124 92.251±2.244

34
Riboflavin
65.716±0.610 5.377±0.107 73.022±0.508
Retinol
51.050±0.919 4.365±0.172 73.486±1.190
Biotin
52.561±0.725 5.225±0.158 86.661±1.155
Folic acid
68.978±0.529 2.613±0.100 41.664±1.153
Pyridoxine
33.799±1.042 1.366±0.177 20.656±0.926
Thiamine
39.247±0.878 1.561±0.081 21.126±0.607
Cyanocobalamine
35.996±0.525 1.784±0.088 22.228±0.887
Surfactant

Tween-20
67.923±1.498 4.796±0.134 55.195±1.322
Tween-40
74.901±1.461 5.551±0.324 56.391±1.277
Tween-60
87.791±1.560 5.551±0.165 58.568±1.618
Tween-80
89.567±1.619 6.118±0.149 61.791±1.565
TritonX-100
98.268±1.764 6.711±0.156 65.361±1.244
PEG-400
67.455±1.200 3.565±0.172 55.662±1.725
PEG-4000
71.719±1.551 4.273±0.086 56.360±1.245
PEG-6000
79.544±1.210 5.137±0.175 61.502±1.650
PEG-8000
74.865±1.478 4.601±0.141 61.365±1.242




35





Table 6: Comparison of enzymatic hydrolysis of different pretreated substrates by
cellulases from different fungi and commercial preparations



Source Substrate Reducing sugar
(mg/g of substrate)
Reference
L. sulphureus Pinus densiflora 70.9 Lee et al., 2008
Trichoderma reesei and Novozyme 188
Trichoderma reesei and Novozyme 188
Trichoderma reesei and Novozyme 188
Corn cob
Prosopis
Lantana
826.2
838.9
777.6
Gupta et al., 2011
Gupta et al., 2011
Gupta et al., 2011
Fomitopsis pinicola Pinus densiflora 3.5 Lee et al., 2008
Fomitopsis palustris
Fomitopsis sp.RCK2010
Fomitopsis sp. RCK2010
Avicel
Rice straw
Wheat straw
32
157.2
214.1
Yoon et al., 2005
Present work
Present work