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Industrial Crops and Products 41 (2013) 283– 288

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me page: www.elsev ier .com/ locate / indcrop

ustard stalk and straw: A new source for production of lignocellulolyticnzymes by the fungus Termitomyces clypeatus and as a substrate foraccharification

wagata Pala,1, Samudra Prosad Banikb,2, Suman Khowalaa,∗

Drug Development Diagnostics and Biotechnology, CSIR-Indian Institute of Chemical Biology 4, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, IndiaMaulana Azad College, Department of Microbiology, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, India

r t i c l e i n f o

rticle history:eceived 21 February 2012eceived in revised form 4 April 2012ccepted 14 April 2012

eywords:ustard stalk and straw

a b s t r a c t

Agro residue of mustard obtained as mustard stalk and straw (MSS) was investigated for the first timefor production of lignocellulolytic enzymes by Termitomyces clypeatus and also for use as substrate forsaccharification. MSS with high cellulose and hemicellulose content was utilized as sole source of carbonby the fungus for productions of enzymes such as (CMcase, �-glucosidase, xylanase and �-xylosidase) insubmerged fermentation. Production of enzymes were further increased by 2–10 folds on supplementa-tion with common agro-residues such as wheat bran and rice straw (MWR) in 1:1:1 ratio and by using

ermitomyces clypeatusretreatmentellulolytic enzymesemicellulolytic enzymesaccharification

alkali treated MSS (TMSS). The enzymes obtained from MWR and TMSS media could saccharify 10% (w/v)wheat bran up to 53% and 58% in 24 h, and xylan up to 52% and 81% in 12 h, respectively. MSS was usedfor saccharification by enzymes of T. clypeatus grown in cellulose media after pretreatment with hotwater and NaCl respectively, where extent of saccharification was doubled to 80% by salt treatment ascompared to that with hot water. The results indicated that MSS can be used as a potential and cheaprenewable raw material from India for production of bio-ethanol.

. Introduction

Lignocellulosic biomasses such as agricultural crop residuesrovide a low cost feedstock for biological production of fuels andhemicals, and offer economic, environmental and strategic advan-ages (van Wyk, 2001). In India mustard stalk and straw (MSS),evoid of the seed, constitute to about 70% of the total plant and

s considered as agricultural waste and which do not use as cat-le feed also, either left in the field for natural decay or is burntithout serving any purpose rather than adding to environmentalollution. The cellulose (48.5%) and hemicellulose (29.6%) con-ent of MSS (Maiti et al., 2007) is higher than other commonlysed agro-wastes, rice straw (cellulose 28.5%, hemicellulose 24.7%),heat bran (cellulose 30%, hemicellulose 27.2%) (Gawande andamat, 1999), whereas economically it is cheaper. The world pro-

uction of mustard–rapeseed increased from ∼26.8 million tons in993–1994 to ∼36.54 million tons in 2001–2002 and ∼55.2 million

n 2011. After Canada – China, India and EU are the major producers

∗ Corresponding author. Tel.: +91 33 2499 5813/3491; fax: +91 33 2473 5197.E-mail addresses: sumankhowala@iicb.res.in, sumankhowala@yahoo.com

S. Khowala).1 Tel.: +91 33 2499 5813/3491; fax: +91 33 2473 5197.2 Tel.: +91 33 2249 3737.

926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2012.04.022

© 2012 Elsevier B.V. All rights reserved.

and large consumers of the crop and products. Mustard is cultivatedin 6.81 million ha in India and produced 6.43 million tons of oilseedin 2010 and contributed to 28.3 and 19.8% as shares in the world.On crushing mustard seeds, after recovery of oil, the remaining isobtained as MSS (lignocellulosic agro-waste) and approximately22 MT MSS (annually) was available in India (Tripathi et al., 2008).Therefore utilization of agro-waste of MSS for alternate use willhelp to meet the recent demand of bio-ethanol and reduce envi-ronmental pollution.

Enzymatic conversion of waste cellulose and hemicelluloseto sugars for bioethanol is commonly used for productionof bioethanol (Verma et al., 2011). Micro-organisms synthe-size cellulolytic and xylanolytic enzymes to release the lockedmonosaccharide from different agro wastes and can fulfill theneed for biofuel production. These enzymes are produced by sev-eral fungi using different agro wastes such as Myceliophthora sp.(Badhan et al., 2007) and Aspergillus niger (Kang et al., 2004) inrice straw, Trichoderma reesei (Knob et al., 2010), Thermoascusaurantiacus (da Silva et al., 2005) using wheat bran, sugarcanebagasse, etc., A. niger, Fusarium oxysporum, Neurospora crassa andPandanus decumbens using orange peels (Mamma et al., 2008). To

achieve maximum saccharification from lignocellulolytic enzymesthe biomass must be pretreated. Pretreatment allows removal oflignin, reduces cellulose crystallinity, and increases the porosity ofthe materials. Pre-treatment should ideally involve requirement

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f low energy with no recycling or environmental costs. Severalttempts were made to enhance cellulose saccharification by usingellulose dissolution as a pretreatment step and different celluloseissolving agents were used including alkali, salts, acids, etc. (Kuond Lee, 2009).

The present study evaluated MSS as a substrate for produc-ion of lignocellulolytic enzymes by Termitomyces clypeatus and totudy the potential of the agroresidue as additional source for enzy-atic saccharification. MSS (with and without alkali pretreatment)as used as a sole source of carbon for production of cellulolytic

CMCase, �-glucosidase) and hemicellulolytic (endoxylanase and-xylosidase) enzymes by the fungus. MSS was supplemented withheat bran and rice straw for increased enzyme production. Inext phase MSS (with and without pretreatments) was sacchari-ed by enzymes of T. clypeatus. The study showed that MSS canerve as an important alternative cheap substrate for productionf lignocellulolytic enzymes as well as can be used as a source foraccharification.

. Materials and methods

Chemicals such as p-nitro phenyl �-d-glucosidase (pNPG), p-itrophenyl-�-xylopyranoside (pNPX), carboxy methyl celluloseCMC), DNSA and xylan were purchased from Sigma Aldrich. Allther chemicals and salts (analytical grade) were purchased locally.

Mustard stalk and straw residue (without leaves and mustardeeds) was collected from field, washed and dried overnight in ovent 50–55 ◦C and was cut into small pieces (1 in.). The chopped MSSas powdered in mixer and sieved to ∼1 mm size. Rice straw andheat bran were obtained from local market. Rice straw was cut,

round, sieved into small pieces and dried similar to that of MSS.heat bran was washed in stream of distilled water to remove

tarch and was dried overnight in oven at 50 ◦C.

.1. Media preparation

Edible fungus T. clypeatus (MTCC 5091) was used in the study.or enzyme production mustard stalk and straw (MSS) (w/v), 1%,SS1; 2%, MSS2; 4%, MSS4; 8%, MSS8 and alkali treated mustard

talk and straw (TMSS, 1%, w/v) were used as substrates for growthedia. In supplemented media MSS was mixed with wheat bran

MW: 1:1), rice straw (MR: 1:1) and (MWR: 1:1:1) by dry weight.est of media composition were, (%, w/v) – yeast extract 0.5, ammo-ium di hydrogen phosphate 2.5, and other micronutrients such asaCl2, 2H2O 0.037; KH2PO4, 0.087; MgSO4, 7H2O 0.05; boric acid.057; FeSO4, 7H2O 0.025; MnCl2, 4H2O 0.0036; NaMoO4, 4H2O.0032; ZnSO4, 7H2O 0.03 and 10% inoculum was used as given ear-

ier (Pal et al., 2010). The experiments were carried out with threeets of 50 ml 250 ml−1 flasks for 10 days at pH 5.0, 30 ± 2 ◦C underhaking condition (150 rpm) in orbital shaker. Aliquots at intervalsere withdrawn, centrifuged and the clear supernatants were used

or analyses.Fungal growth was determined by separating the mycelia along

ith the residual mustard/agro waste of each medium (50 ml in50 ml flask, in triplicate) with filter paper, washed thrice withistilled water, and subsequently pressed the biomass (agrowastend mycelia) with blotting paper to soak the excess water. Theet biomass (1 g) was then transferred to pre-weighted centrifuge

ubes and 5 ml of sodium sulphate (150 g 1−1) was added to eachube to separate the mycelia from the agro wastes (Augustine et al.,

006). The tubes were centrifuged at 12,000 rpm for 15 min. Theupernatant containing only the mycelia was collected, filteredith filter paper, washed with distilled water and excess water was

emoved by soaking with blotting paper. The wet mycelia obtained

roducts 41 (2013) 283– 288

were then kept in hot air oven (at 50 ◦C, 24–48 h) until a constantdry weight was observed.

For saccharification experiments enzyme preparations wereobtained from cellulose medium (1%, w/v) of T. clypeatus describedas earlier (Mukherjee et al., 2001).

2.2. Pre-treatment of MSS

Alkali treated mustard stalk and straw (TMSS) was prepared bysuspending the sieved agro residue in 1 N NaOH for 3 h at roomtemperature. The alkali was then removed by thorough washingwith distilled water. Ground and sieved MSS (50 g 100 ml−1) wasautoclaved with distilled water or salt (0.1–2 N NaCl) at 121 ◦C for1 h. After treatment, the residual fibers were separated by filtration,washed with excess water. Excess water of treated MSS prepara-tions were soaked on blotting paper and dried overnight in oven asabove.

2.3. Assay of enzyme activities

�-Glucosidase (E.C.3.2.1.21) assay was carried out in the reac-tion mixture (1 ml) containing 2 mM pNPG in 0.1 M sodium acetatebuffer, pH 5.0 and an appropriate amount of the enzyme. Incuba-tion was carried out at 45 ◦C for 10 min. Reaction was terminatedby the addition of 0.5 ml Na2Co3 (1 M). Intensity of the yellowcolor developed by liberation of pNP was measured at 400 nm.Unit of enzyme activity was expressed by the enzyme that pro-duced 1 �mol of pNP per minute under the assay conditions(Pal et al., 2010). �-Xylosidase (E.C.3.2.1.37) was assayed simi-larly using p-nitrophenyl-�-d-xylopyranoside (pNPX) as substrate(Bhattacharyya et al., 1997). Carboxy methyl cellulase (CMCase)activity in the culture filtrate was assayed by using carboxymethylcellulose. 0.5 ml of properly diluted enzyme preparation in 50 mMacetate buffer (pH 5.0) incubated with 0.5 ml of 2% CMC in samebuffer at 50 ◦C for 30 min. Released reducing sugar was estimatedby DNS method as glucose equivalent (Miller, 1959). Xylanase wasassayed using birch wood xylan as substrate (Bailey et al., 1992).The solution of 0.9 ml xylan (1%, w/v) and the 0.1 ml of enzymeat appropriate dilution were incubated at 50 ◦C for 10 min, and thereducing sugar was determined by the DNS method at 540 nm withxylose as standard. Enzyme activity unit was defined as 1 �mol ofreducing sugar released per min under the assay conditions.

2.4. Estimation of protein and carbohydrate content

Protein was assayed using coomassie blue (Bradford) proteinassay reagent according to the technical instruction manual, withBSA as standard. The carbohydrate content and reducing sugarswere determined by orcinol-sulphuric acid (Brown and Anderson,1971) and DNS methods respectively with glucose as standard.

2.5. Saccharification of MSS, wheat bran and xylan by enzymepreparations of T. clypeatus

Wheat bran (10%, w/v), xylan (10%, w/v) and MSS preparations(1%, w/v) with and without pretreatments were saccharified usingculture filtrate enzyme preparations (xylanase equivalent 40 U g−1

substrate) of the fungus T. clypeatus grown in different media.Substrates (10 ml in 50 ml stoppered flask) were incubated withenzymes in 50 mM sodium acetate buffer, pH 5.0 at 50 ◦C. Reac-tion was terminated by keeping the digests at 100 ◦C for 5 min atdifferent time intervals and centrifuged. Extent of hydrolysis was

calculated and expressed as:

saccharification (%) = reducing sugarcarbohydrates in substrate

× 0.9 × 10

S. Pal et al. / Industrial Crops and Products 41 (2013) 283– 288 285

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ig. 1. Production profile of CMCase (A), �-glucosidase (B), xylanase (C) and �-xylo ), MSS4 ( ), MSS8 (-�-) and TMSS ( ). 1, 2, 4 and 8 denoted respective concen-xylosidase were done as earlier using pNPG and pNPX as substrates (Pal et al., 20

The control saccharification experiments for enzyme, substrate,eagent blanks and heat inactivated enzymes were also carried out.

.6. Analyses of saccharification digests by HPLC

Total soluble carbohydrates obtained after saccharificationigests of wheat bran and xylan were analyzed by HPLC usingreheated (80 ◦C) Rezex RCM carbohydrate column (Phenomenex,00 mm × 7.8 mm) with double distilled water as mobile phase at

flow rate of 0.3–0.6 ml min−1. Standard sugars were used as ref-rence.

All experiments were performed in triplicate and the analyticaleasurements were done in duplicate and average results ±S.D.ere presented.

. Results and discussion

.1. Production of cellulolytic and xylanolytic enzymes using MSSnd TMSS

Production of CMCase, �-glucosidase, xylanase and �-ylosidase were studied using MSS as sole source of carbonnd increased with increasing concentration of MSS (Fig. 1).ptimum production (∼2 U ml−1) of CMCase (Fig. 1A) and �-lucosidase activity (Fig. 1B) was found in MSS8 medium on 7thnd 9th day, respectively. The activity of the CMCase in MSS8edium was 4 fold higher than Trichoderma lignorum grown on

anana agro-waste (Baig, 2005) and 2 fold higher than Aspergilluserreus using bagasse (Youssef and Berekaa, 2009). In T. clypeatusMCase production has been associated with the production ofnother cellulolytic enzyme �-glucosidase which is a constitutivenzyme of the fungus (Mukherjee et al., 2006). In this studyroduction of the �-glucosidase in MSS as sole source of carbonas almost 9 times higher than that produced respectively in

ugar cane bagasse by fungus Phanerochaete chrysosporium (Khalil,002) and respectively 12 times higher than bacteria Paenibacillusurdlanolyticus (Waeonukul et al., 2008) using bagasse and cornull in the medium. So MSS may be a good source for production of-glucosidase, which is an important enzyme in cellulase enzymeomplex.

In MSS8 medium, xylanase activity of 14.12 U ml−1 (day 5)as observed whereas 8.65–9.76 U ml−1 activity was obtained inSS1-4 media between 2nd and 4th day (Fig. 1C). Production of �-

ylosidase was observed between 0.041 U ml−1 and 0.066 U ml−1

(D) in MSS media. The fungus was grown in medium containing MSS1 (-�-), MSS2ns (%, w/v) of MSS in the media. Media preparation and assay of �-glucosidase andMCase and xylanase were estimated by DNS method (Miller, 1959).

on 8–9th day in the same media (Fig. 1D). It was observed thatactivity of xylanase and �-glucosidase in 8% MSS (MSS8) were 1.6fold and 4 fold higher than 1% MSS (MSS1) present in the medium.Production of enzymes increased with MSS concentration, due toincreased metabolic activities and growth of the fungus in presenceof higher substrate. The production range was almost identical tothat of the enzyme produced by mesophilic fungus F. oxysporum incorn stover (Panagiotou et al., 2003).

Production of all the enzymes were enhanced in alkali treatedmustard (TMSS, 1% w/v) medium by T. clypeatus (Fig. 1A–D).CMCase activity was increased by 1.7 fold (2.95 U ml−1) in TMSSmedium than untreated MSS1 medium (Fig. 1A). Production of �-glucosidase and xylanase in TMSS medium were also increasedrespectively by 2.3 and 4.2 times as compared to MSS1 medium(Fig. 1B and C). Production of xylanase by Trichoderma viride, usingvarious lignocellulosic substrates in submerged culture fermenta-tion was improved by using alkali treated lignocellulosics maizestraw and jowar straw as substrate (Goyal et al., 2008). FPase activ-ity was also produced in all MSS media (0.3–0.5 U ml−1) [data notshown here]. Presence of lignin (20–35%) in agricultural biomass,inhibits the production of the lignocellulolytic enzymes as well astheir action on cellulose and hemicellulose (Gawande and Kamat,1999). By alkali treatment the lignin was removed and accessibil-ity and surface area of the treated substrate was increased (Goyalet al., 2008), as a result enzyme production was 2–4 fold high inalkali treated MSS.

3.2. Effect of addition of wheat bran and rice straw to MSS onenzyme production

Enzyme activities were further increased by supplementingMSS with other common agro-wastes such as wheat bran (MW)and rice straw (MR) and in combination with the both (MWR)(Fig. 2A–D). The CMCase activity increased by 1.1, 1.24 and1.42 times, respectively in MW, MR and MWR media as com-pared to that in MSS (Fig. 2A). Activity of �-glucosidase (Fig. 2B)also increased by 2.9, 1.62 and 3.2 fold in MW, MR and MWRmedia respectively than MSS1 media. On supplementation, pro-duction of xylanase increased significantly from 14 U ml−1 (MSS8)to 99 U ml−1, 84 U ml−1 and 138 U ml−1 in MW, MR and MWR

media, respectively (Fig. 2C). Better production of CMCase and �-glucosidase activities were reported using rice straw and wheatbran in media at the ratio of 4:1 and 1:4 for respectively by T. viridein comparison to each of the individual medium (Qia et al., 2007).

286 S. Pal et al. / Industrial Crops and Products 41 (2013) 283– 288

Fig. 2. Kinetics of CMCase (A), �-glucosidase (B), xylanase (C) and �-xylosidase (D)production using supplemented MSS medium. MSS (-�-) was supplemented withwbc

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Fig. 3. Kinetics of extracellular protein (A) production and fungal growth (B) in MSS(-�–), MSS2 ( ), MSS4 ( ), MSS8 ( ), MW ( ), MR ( ) MWR ( ) and TMSS(-�–) media. Data represented an average of three independent experiments ±S.D.shown by error bar.

Table 1ASaccharification of wheat bran.

Incubation time Saccharification (%)

MSS MW MR MWR TMSS

sugars like, xylose, arabinose and glucose along with other oligosac-charides. With increasing time (in 24 h) intensity of all monosugarsincreased indicating efficient and synergistic saccharification of

Table 1BSaccharification of xylan by enzymes produced from MMS, MWR and TMSS media.

Incubation time Saccharification (%)

heat bran (MW) (-�-), rice straw (MR) (-�-) (in ratio of 1:1) and with both wheatran and rice straw (MWR) (-�-) (in ratio of 1:1:1) by maintaining total biomassoncentration at 1% (w/v) respectively in all media.

arginal increase in �-xylosidase activity was also observed in sup-lemented MSS medium (Fig. 2D). Xylanase activity increased upo 17 and 4 fold in presence of MWR (Fig. 2) and TMSS (Fig. 1)espectively as compared to MSS medium. High cellulosic com-osition and sufficient nitrogen sources are known to be ideal forood growth of fungal cultures and lignocellulolytic enzyme pro-uctions (Brijwani et al., 2010; Dobermann and Fairhurst, 2002).heat bran and rice straw being good sources of protein on sup-

lementation to MSS favor growth and lignocellulolytic enzymeroduction by the fungus. Using wheat bran as a supplementingaterial with soybean hulls resulted in increased production of

ellulase enzyme (Brijwani et al., 2010), whereas supplementationy rice straw effectively improved the CMCase production from A.iger in submerged fermentation (Irfan et al., 2011)

.3. Kinetics of extracellular protein production and fungalrowth in MSS media

Kinetics of protein content was measured in MSS media. Extra-ellular protein production was maximum in MWR medium at.88 mg ml−1 on 9th day (Fig. 3A). Optimum protein production inhe range of 0.34–0.37 mg ml−1 on 4th day was observed in MSS,

W and MR media. In TMSS medium protein content was esti-ated as 0.46 mg ml−1 on 4th day. Fungal growth (biomass) wasaximum on 4th day in all the media (Fig. 3B). Maximum growthas obtained in TMSS media (0.128 g 50 ml−1) due to delignifi-

ation of the agro waste by alkali treatment. Earlier in presencef soluble (cellobiose, Pal et al., 2010) and insoluble (cellulose,ukherjee and Khowala, 2002) substrates growth was optimum

n 4th day. It was observed that kinetics of enzyme production inhe all MSS media was not related to those on growth of the fungusxcept in TMSS medium.

.4. Saccharification of xylan and wheat bran by enzymesroduced in MSS media

Among the enzymes tested, titer of xylanase was highest in alledia. Enzyme preparations were concentrated in terms of uni-

orm xylanase activity (xylanase equivalent 40 U g−1 substrate) foresting the saccharification efficiencies on wheat bran (Table 1A)

6 h 30.8 35.7 31.9 44.7 48.224 h 34.9 40.0 41.2 52.9 58.5

and xylan (Table 1B). About 58% saccharification of wheat bran wasobtained in 24 h by enzyme preparation TMSS, whereas about 35%and 53% saccharification was obtained by using enzyme prepara-tions from MSS and MWR media. The TMSS enzyme preparationcould saccharify birch wood xylan up to 78% in only 6 h, whichincreased to 81.3% in 12 h. Saccharification efficiency of enzymesproduced in MSS, MW, MR and MWR media were 15–22 foldshigher than enzymes of T. lignorum investigated in saccharificationof steam treated agro-waste after 24 h (Baig et al., 2004). Hydrolysisof wheat bran up to 25% in 144 h was reported by enzymes fromMacrophomina phaseolina (Roy et al., 1993). Rate of wheat bran sac-charification (44.7% in 6 h) by MWR enzyme preparation was betteras compared to xylan (37.5%), which was due to involvement of allthe cellulolytic and hemicellulolytic enzymes in saccharificationof wheat bran, whereas only xylanolytic enzymes participated inxylan hydrolysis.

Saccharification digests of wheat bran by MWR enzymes in 6 h(Fig. 4A) and 24 h (Fig. 4A′) were analyzed by HPLC using standard

MSS MWR TMSS

6 h 30.1 37.5 78.012 h 45.6 52.5 81.3

S. Pal et al. / Industrial Crops and Products 41 (2013) 283– 288 287

Fig. 4. Analysis of saccharification digests. Saccharified wheat bran digests obtained at intervals of 6 h (A) and 24 h (A′) were loaded on HPLC [Rezex RCM carbohydratecolumn (Phenomenex, 300 mm × 7.8 mm) preheated (80 ◦C)] with double distilled water at 0.6 ml min−1 along with standard sugars glucose (12.7 min), xylose (14.1 min),arabinose (17.5 min). Similarly xylan digests after 12 h (from Table 1B) from enzyme preparation of MWR (B) and TMSS (B′) were analyzed on HPLC (at 0.3 ml min−1) alongw ylotrit

wf(Hxt

3

bp1sbdea

Fchi

ith standard sugars glucose (34.5 min), xylose (37.17 min), xylobiose (28.3 min), xime.

heat bran by xylanolytic and cellulolytic enzymes (Fig. 4A′). Dif-erence in hydrolytic efficiency of enzyme preparations from MWRFig. 4B) and TMSS (Fig. 4B′) media on xylan was also apparent inPLC analysis of the digests which showed increased peak area ofylose, xylobiose and xylotriose in TMSS digests as compared tohose observed from MWR digests.

.5. Evaluation of pre-treated MSS for saccharification

It was observed that after pretreatment saccharification of MSSy fungal enzymes increased considerably (Fig. 5). By using sim-le hot water treatment MSS saccharification increased from about6 to 40% and further to 82% by salt (NaCl) treatment. Extent ofaccharification using TMSS was observed at 86% similar to that

y salt treated MSS. Numerous pretreatment methods have beeneveloped in search of ways to remove the lignin barrier andnhance the accessibility of cellulose to hydrolytic degradation butgents without any hazards are advantageous (Taherzadeh and

ig. 5. Efficiency of pretreatment of MSS in deferent concentration of NaCl and sac-harification efficiency of pretreated mustard straw. Saccharification efficiency ofot-water (HW), salt (NaCl) pretreated MSS and TMSS (NaOH) was measured. C,

ndicated saccharification without pretreated MSS.

ose (24.75 min), arabinose (43.1 min). E, enzyme preparation; S, substrate used; T,

Karimi, 2008). Among several methods acid or alkali treatmentwere commonly known methods for liberating the lignin contentfrom lignocellulosic wastes though chemical pretreatments haveserious disadvantages in terms of the requirement for specializedcorrosion resistant equipment, extensive washing and proper dis-posal of chemical wastes (Mahalakshmi et al., 2011). The alternativemethod of hot water pretreatment was reported as the most costeffective method (Dien et al., 2006) and enhanced the saccharifi-cation of MSS by about 2.6 fold than the untreated residue. It wasalso observed that addition of salt in hot water treatment enhancedthe rate of saccharification by additional 2 fold than hot water pre-treatment. Comparable hydrolysis was obtained with TMSS and saltpretreated MSS by the enzyme preparations. The choice of sodiumchloride as a pretreating agent seemed to be suitable due to lowtoxicity and low economics.

4. Conclusions

Search for newer sources of cheap agrowaste are in hugedemand for production of bio-energy. In India energy crisis is acuteand use of crops like mustard stalk and straw (MSS) having highersugar content, is only limited to burning as fuel and pollute the envi-ronment. The present study proved that MSS can be a good choice asa cheap agro-residue for production of lignocellulolytic enzymes byT. clypeatus and can have an alternative use to generate bioethanol.However better results were obtained after pretreatment of MSS. Infuture MSS will be used for enzyme productions by solid state fer-mentations and also by other organisms and to use the agro residueas a substrate for direct saccharification into fermentable sugars bycommercial enzymes.

Acknowledgements

This research was supported by grants from Dept. of Biotech-nology, Govt. of India and Labonya Prova Bose Trust, Kolkata. Myspecial thanks are extended to Dr. Gouriprosad Datta (Dept. of

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hysiology, Rammohan College, Kolkata) for his constant encour-gement and support.

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