cellulase28jun2013

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

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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: [email protected]

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) H2SO4 and 2.5% NaOH at 121C for 15 min. The pretreated residues were

washed extensively to neutral pH and dried at 60C 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(NO3)2.4H2O, 0.5; MgSO4.7H2O, 0.5; KH2PO4, 0.5; and agar, 20.0 (pH

5.5) at 30C (Dhawan et al., 2002; Vasdev et al., 2005). The fungal cultures were maintained

by periodical subculturing on MEA at 30C and stored at 4C.

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 30C 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 94C (4 min), 35 cycles of

primer annealing at 58C (40 sec), elongation at 72C (1 min) and denaturation at 94C (1

min). A final elongation step was allowed at 72C 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 30C 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:(NH4)2SO4, 0.5; KH2PO4, 0.5; MgSO4,

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 121C (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 30C. 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 4C 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

(25-40C), 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 50C 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 50C, 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 50C 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 50C 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 25-30C (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 30C. Any increase in temperature beyond 30C 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 30C and thereafter the production of enzyme declined. Wang et

al. (2006) while studying the optimization of multienzyme production by two mixed strains

Aspergillus niger F3 and Aspergillus niger F4 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.

References

Ahamed, A., Vermette, P., 2008. Culture based strategies to enhance cellulase enzyme

production from Trichoderma reesei RUT-30 in bioreactor culture conditions. Biochem. Eng.

J. l40, 399-407.

Badhan, A.K., Chadha, B.S., Kaur, J., Saini, H.S., Bhat, M.K., 2007. Production of multiple

xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099.

Bioresour. Technol. 98, 504-510.

Bhat, M.K., 2000. Cellulase and related enzymes in Biotechnology. Biotechnol. Adv. 18, 355-

383.

Daroit, D.J., Silveria, S.T., Hertz, P.F., Brandelli, A., 2007. Production of extracellular -

glucosidase by Monascus purpureus on different growth substrates. Proc. Biochem. 42, 904-

908.

Dhawan, S., Kuhad, R.C., 2002. Effect of amino acids on laccase production from birds nest

fungus Cythus bulleri. Bioresour. Technol. 84, 35-38.

Entcheva, P., Phillips, D.A., Streit, W.R., 2002. Functional analysis of Sinorhizobium meliloti.

Genes involved in biotin synthesis and transport. Appl. Environ. Microbiol. 68, 2843-2848.

18

Ghose, T.K., 1987. Measurements of cellulase activities. Pure Appl. Chem. 59, 257-268.

Grajek, W., 1986. Temperature and pH optima of enzyme activities produced by cellulolytic

fungi in batch and solid state cultures. Biotechnol. Lett. 8, 587-90.

Gupta, R., Khasa, Y.P., Kuhad, R.C., 2011. Evaluation of pretreatment methods in improving

the enzymatic saccharification of cellulosic materials. Carb. Pol. 84, 1103-1109.

Hamidi-Esfahani, Z., Shojaosadati, S.A., Rinzema, A., 2004. Modeling of simultaneous effect

of moisture and temperature on A.niger growth in solid-state fermentation. Biochem. Eng. J.

21, 265-272.

Hanif, A., Yasmeen, A., Rajoka, M.J., 2004. Induction, production, repression and de-

repression of exoglucanase synthesis in Aspergillus niger. Bioresour. Technol. 94, 311-319.

Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of

lignocellulosic biomass. Bioresour. Technol. 100, 10-18.

Himmel, M.E., Ruth, M.F., Wyman, C.E., 1999. Cellulase for commodity products from

cellulosic biomass. Cur. Opi. Biotechnol. 10,358-364.

Holker, U., Hofer, M., Lenz, J., 2004. Biotechnological advantages of laboratory scale solid

state fermentation with fungi. Appl. Microbiol. Biotechnol. 64, 175-186.

Jecu, L., 2000. Solid-state fermentation of agricultural wastes for endoglucanase production.

Ind. Crops. Prod. 11, 1-5.

Jellison, J., Connolly, J., Goodell, B., Doyle, B., Illman, B., Fekete, F., Ostrofsky, A., 1997.

The role of cations in the biodegradation of wood by the brown rot fungi. Int. Biodeterior.

Biodegrad. 39, 165-179.

19

Kapoor, M., Nair, L.M., Kuhad, R.C., 2008. Cost effective xylanase production from free and

immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis

juliflora. Biochem. Eng. J. 38, 88-97.

Kalogeris, E., Christakopoulos, P., Katapodis, P., Alexiou, A., Vlachou, S., Kekos, D., Macris,

B.J., 2003. Production and characterization of cellulolytic enzymes from the thermophilic

fungus Thermoascus aurantiacus under solid state cultivation of agricultural wastes. Proc.

Biochem. 38, 1099-1104.

Krishna, C., 2005. Solid state fermentation systems-an overview. Crit. Rev. Biotechnol. 25, 1-

30.

Kuhad, R.C., Kumar, M., Singh, A., 1994. A hypercellulolytic mutant of Fusarium

oxysporum. Lett. Appl. Microbiol. 19, 397-400

Kuhad, R.C., Singh, A., Eriksson, K.E.L., 1997. Microorganisms and enzymes involved in the

degradation of plant fiber cell walls. Adv. Biochem. Eng. Biotechnol.57, 46-125.

Kuhad, R.C., Kapoor, R.K., Lal, R., 2004. Improving the yield and quality of DNA isolated

from white-rot fungi. Folia Microbiol. 49, 112-116.

Lee, J.W., Kim, H.Y., Koo, B.W., Choi, D.H., Kwon, M., Choi, I.G., 2008. Enzymatic

saccharification of biologically pretreated Pinus densiflora using enzymes from brown rot

fungi. J. Biosci. Bioeng. 106, 162-167.

Lutzen, N.W., Nielsen, M.H., Oxenboell, K.M., Schulein, M., Stentebjerg-Olesen, B., 1983.

Cellulases and their application in the conversion of lignocellulose to fermentable sugars. Phil.

Ttrans. R. Soc. Lond. B 300, 283-291.

Lynd, L.R., Weimer, P.J., Zyl, W.H., Pretorius, J.S., 2002. Microbial cellulose utilization:

fundamentals and biotechnology. Microbiol. Mol. Rev. 66, 506-577.

20

Membrillo, I., Sanchez, C., Meneses, M., Favela, E., Loera, O., 2008. Effect of substrate

particle size and additional nitrogen source on production of lignocellulolytic enzymes by

Pleurotus ostreatus strains. Bioresour. Technol. 99, 7842-47.

Miller, G.L., 1959. Use of dinitrosalicyclic acid reagent for determination of reducing sugar.

Anal. Chem. 31, 426-428.

Niranjane, A.P., Madhou, P., Stevenson, T.W., 2007. The effect of carbohydrate carbon

sources on the production of cellulase by Phlebia gigantea. Enz. Microb. Technol. 40, 1464-

1468.

Olofsson, K., Wiman, M., Liden, G., 2010. Controlled feeding of cellulases improves

conversion of xylose in simultaneous saccharification and cofermentation for bioethanol

production. J. Biotechnol. 145, 168-175.

Vasdev, K., Dhawan, S., Kapoor, R.K., and Kuhad, R.C., 2005. Biochemical characterization

and molecular evidence of a laccase from the birds nest fungus Cyathus bulleri. Fungal

Genet. Biol. 42, 684-693.

Viniegra-Gonzalez, G., Favela-Torres, E., Aguilar, C.N., Romero-Gomez, S.D.J., Diaz-

Godnez, G., Augur, C., 2003. Advantages of fungal enzyme production in solid state over

liquid fermentation systems. Biochem. Eng. J. 13, 157-167.

Wang, X., J., Bai, J.G., Liang, Y.X., 2006. Optimization of multi enzyme production by 2

mixed strains in solid state fermentation. Appl. Microbiol. Biotechnol. 73, 533-540.

Wood, T. M., and M. K. Bhat. 1988. Methods for measuring cellulase activities, in: W.A.

Wood and S. T. Kellogg (Eds.), Methods in Enzymology, Vol. 160. Academic Press Inc.,

London, U.K., pp 87112

21

Yoon, J.J., Kim, Y.K., 2005. Degradation of crystalline cellulose by the brown-rot

basidiomycete Fomitopsis palustris. J. Microbiol. 43, 487-492.

Yoon, J.J., Cha,C.J., Kim, Y.S., Kim, W., 2008. Degradation of cellulose by the major

endoglucanase produced from the brown-rot fungus Fomitopsis pinicola. Biotech. Lett. 30,

1373-1378.

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

MC

ase

IU

/g

beta

-gl

uco

sida

se IU

/g

0

0.5

1

1.5

2

2.5

3

3.5

FP

ase

IU

/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)

Suga

r re

leas

ed (m

g/g)

Control RSNaOH treated RSAcid treated RSControl WSNaOH treated WSAcid 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 3.2850.094 0.1880.007 6.4310.154 1:1.5 4.0490.092 0.3190.024 26.0551.051 1:2 31.5441.415 1.2410.050 44.6750.953

1:2.5 71.0291.216 3.3080.010 51.8630.644 1:3 71.8760.460 3.2370.088 58.7770.776

1:3.5 72.5710.810 3.1910.035 66.5940.078 1:4 70.6690.942 2.4980.005 66.6080.151

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 2.2150.040 0.1910.009 2.6580.067 Corn stover 3.6980.048 0.2430.032 2.7730.154 Wheat straw 2.4100.099 0.2390.013 3.2290.560 Prosopis juliflora 1.0520.071 0.7320.037 1.3930.098 Lantana camera 2.8950.097 0.4470.030 3.2270.086 Wheat bran 71.5260.910 3.2680.067 50.6960.662

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% N2 equivalent)

CMCase FPase -glucosidase

Control 71.6990.665 3.3190.004 51.4210.273 Inorganic

NaNO2 52.7781.095 4.4640.025 37.4120.800 NaN3 13.1660.398 0.000 0 KNO3 53.2650.596 3.4510.019 41.5490.725 Fe(NO3)3 46.3530.781 3.3590.024 41.9030.426 NH4Cl 46.8441.068 3.1500.023 47.6840.621 Ca(NO3)2 41.1250.955 3.1310.037 43.7791.094 (NH4)2C2O4 66.9740.489 4.5280.034 46.0760.954 NaNO3 57.8640.588 3.1730.018 47.5220.713 Pb(NO3)2 60.7990.325 2.6290.038 57.1140.889

Organic

Peptone 71.6500.636 4.3810.050 63.6930.668 Yeast extract 74.1631.015 4.3630.058 56.9770.409 Trytone 77.2580.873 4.1450.021 64.1260.936 Casein 71.5440.676 4.6820.037 64.9461.015 Soya been meal 74.9960.520 4.1550.021 69.0830.904 Urea 81.8320.812 4.5620.029 54.9281.029 Corn steep liquor 70.8090.848 4.6430.032 52.1490.406

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 71.6990.665 3.2690.067 51.9210.434

Aminoacids

L-Alanine 63.1600.443 5.0310.037 60.5240.713 L-Arginine 73.7561.063 4.9410.028 57.1160.893 L-Asparagine 77.8441.068 4.8340.011 65.9810.528 L-Aspartic Acid 79.0990.901 5.8340.011 61.0970.517 L-Cysteine 74.1670.397 5.1590.024 63.8890.839 L-Cystine 76.7991.042 6.1770.016 66.2110.100 L-Glutamic acid 84.1270.965 5.1090.071 61.7560.356 L-Glutamine 74.2480.826 4.8540.021 63.1040.991 Glycine 62.8250.561 6.2640.025 64.5430.680 L-Isoleucine 58.2910.853 5.0180.004 77.5460.733 L-Leucine 51.8480.543 3.4880.006 54.8150.384 L-lysine hydrochloride 56.3680.774 5.2730.013 65.2720.552 L-Methionine 35.0170.468 3.7480.018 51.9430.392 L-serine 64.1670.397 5.4470.030 66.2660.822 L-Threonine 72.0530.922 5.4470.022 62.0260.908 L-valine 61.7701.051 5.0460.022 61.6810.674 L-Phenylalanine 38.1150.893 4.0870.006 60.9680.928

4-hydroxy-L-proline 56.6940.670 6.7620.056 69.9950.898 L-Tryptophan 61.9581.015 3.6720.013 62.1050.422 L-Tyrosine 63.1330.940 4.0670.027 60.2630.590 L-Histidine hydrochloride 61.1230.477 5.9540.021 61.9970.941

Vitamins

Ascorbic acid 64.7791.099 5.6680.124 92.2512.244

34

Riboflavin 65.7160.610 5.3770.107 73.0220.508 Retinol 51.0500.919 4.3650.172 73.4861.190 Biotin 52.5610.725 5.2250.158 86.6611.155 Folic acid 68.9780.529 2.6130.100 41.6641.153 Pyridoxine 33.7991.042 1.3660.177 20.6560.926 Thiamine 39.2470.878 1.5610.081 21.1260.607 Cyanocobalamine 35.9960.525 1.7840.088 22.2280.887

Surfactant

Tween-20 67.9231.498 4.7960.134 55.1951.322 Tween-40 74.9011.461 5.5510.324 56.3911.277 Tween-60 87.7911.560 5.5510.165 58.5681.618 Tween-80 89.5671.619 6.1180.149 61.7911.565 TritonX-100 98.2681.764 6.7110.156 65.3611.244 PEG-400 67.4551.200 3.5650.172 55.6621.725 PEG-4000 71.7191.551 4.2730.086 56.3601.245 PEG-6000 79.5441.210 5.1370.175 61.5021.650 PEG-8000 74.8651.478 4.6010.141 61.3651.242

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