Pretreatment of lignocellulosic material with fungicapable of higher lignin degradation and lowercarbohydrate degradation improves substrateacid hydrolysis and the eventual conversion toethanol

Sarika Kuhar, Lavanya M. Nair, and Ramesh Chander Kuhad

Abstract: Phanerochaete chrysosporium, Pycnoporus cinnabarinus, and fungal isolates RCK-1 and RCK-3 were tested fortheir lignin degradation abilities when grown on wheat straw (WS) and Prosopis juliflora (PJ) under solid-state cultivationconditions. Fungal isolate RCK-1 degraded more lignin in WS (12.26% and 22.64%) and PJ (19.30% and 21.97%) andless holocellulose in WS (6.27% and 9.39%) and PJ (3.01% and 4.58%) after 10 and 20 days, respectively, than otherfungi tested. Phanerochaete chrysosporium caused higher substrate mass loss and degraded more of holocellulosic content(WS: 55.67%; PJ: 48.89%) than lignin (WS: 18.89%; PJ: 20.20%) after 20 days. The fungal pretreatment of WS and PJwith a high-lignin-degrading and low-holocellulose-degrading fungus (fungal isolate RCK-1) for 10 days resulted in (i) re-duction in acid load for hydrolysis of structural polysaccharides (from 3.5% to 2.5% in WS and from 4.5% to 2.5% in PJ),(ii) an increase in the release of fermentable sugars (from 30.27 to 40.82 g�L–1 in WS and from 18.18 to 26.00 g�L–1 inPJ), and (iii) a reduction in fermentation inhibitors (total phenolics) in acid hydrolysate of WS (from 1.31 to 0.63 g�L–1)and PJ (from 2.05 to 0.80 g�L–1). Ethanol yield and volumetric productivity from RCK-1-treated WS (0.48 g�g–1 and0.54 g�L–1�h–1, respectively) and PJ (0.46 g�g–1 and 0.33 g�L–1�h–1, respectively) were higher than untreated WS(0.36 g�g–1 and 0.30 g�L–1�h–1, respectively) and untreated PJ (0.42 g�g–1 and 0.21 g�L–1�h–1, respectively).

Key words: white-rot fungi, delignification, wheat straw, Prosopis juliflora, Pichia stipitis.

Resume : Les capacites de degradation de la lignine par Phanerochaete chrysosporium, Pycnoporus cinnabarinus et parles isolats fongiques RCK-1 et RCK-3 cultives sur des paillis de paille de ble (PB) ou de Prosopis juliflora (PJ) ont etetestees. L’isolat RCK-1 degradait plus de lignine sur PB (12,26 % et 22,64 %) et sur PJ (19,30 % et 21,97 %) et moins deholocellulose sur PB (6,27 % et 9,39 %) et sur PJ (3,01 % et 4,58 %) apres 10 et 20 jours, respectivement, comparative-ment aux autres champignons testes, alors que Phanerochaete chrysosporium causait une plus grande perte de poids desubstrat et degradait plus de holocellulose (PB : 55,67 %; PJ : 48,89 %) que de lignine (PB : 18,89 % ; PJ : 20,20 %)apres 20 jours. Le pretraitement fongique de la PB et de PJ pendant 10 jours avec le champignon degradant le plus de li-gnine et le moins de holocellulose (l’isolat fongique RCK-1) a resulte en (i) une reduction de la charge acide pour l’hy-drolyse des polysaccharides de structure (de 3,5 % a 2,5 % sur PB et de 4,5 % a 2,5 % sur PJ), (ii) une augmentation dela liberation de sucres fermentables (de 30,27 a 40,82 g�L–1 sur PB et de 18,18 a 26,00 g�L–1 sur PJ) et (iii) une reductionde la quantite d’inhibiteurs de la fermentation (phenols totaux) dans l’hydrolysat acide de PB (de 1,31 a 0,63 g�L–1) et dePJ (2,05 a 0,80 g�L–1). Le rendement en ethanol et la productivite volumetrique de la PB (0,48 g�g–1 et 0,54 g�L–1�h–1, res-pectivement) et de PJ (0,46 g�g–1 et 0,33 g�L–1�h–1, respectivement) traites avec l’isolat RCK-1 etaient superieurs a ceux dela PB (0,36 g�g –1 et 0,30 g�L–1�h–1, respectivement) et de PJ (0,42 g�g–1 et 0,21 g�L–1�h–1, respectivement) non traites.

Mots-cles : champignon de la pourriture blanche, delignification, paille de ble, Prosopis juliflora, Pichia stipitis.

[Traduit par la Redaction]

Introduction

It has been estimated that global oil reserves will last only

for about another 40 years (Puppan 2002). Hence, there is agreat need to explore new renewable energy sources.Bioethanol is a clean-burning, nonpetroleum liquid fuel.There is a great deal of interest in using bioethanol as atransportation fuel. Advances in technology have allowedthe conversion of biomass into fuel ethanol as a potentialsource of energy. Lignocellulosic biomass, the most abun-dant energy resource available on earth, offers a potentialsource of carbon substrate for the production of ethanol byfermentation. The plant biomass availability in India is huge(Nair 2006), but the presence of lignin makes the access toholocellulose difficult for enzymes and chemicals. There-

Received 1 October 2007. Revision received 14 December 2007.Accepted 10 January 2008. Published on the NRC ResearchPress Web site at cjm.nrc.ca on 29 March 2008.

S. Kuhar, L.M. Nair, and R.C. Kuhad.1 LignocelluloseBiotechnology Laboratory, Department of Microbiology,University of Delhi South Campus, Benito Juarez Road, NewDelhi-110021, India.

1Corresponding author (e-mail: kuhad@hotmail.com).

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fore, it is necessary to break and (or) remove the lignin inplant residues prior to its hydrolysis (Sun and Cheng 2002).Ethanol production from lignocellulosics requires (i) deligni-fication of the substrates to liberate cellulose and hemicellu-lose from lignin, (ii) depolymerization of cellulose andhemicellulose to produce free sugars, and (iii) fermentationof hexose and pentose sugars to produce ethanol (Lee1997). Crystallinity of the cellulose further impedes acidand enzymatic hydrolysis (Bothast and Saha 1997; Kuhad etal. 1997). Hence, pretreatment has become a necessity tomaximize the hydrolysis of cellulosics and eventually theproduction of ethanol. The advantages of biological deligni-fication of lignocellulosics over chemical and mechanicalpretreatment methods include mild reaction conditions,avoids the use of toxic and corrosive chemicals, higherproduct yields, fewer side reactions, less energy demand,and less reactor resistance to pressure and corrosion (Lee1997; Ferraz et al. 2000). In situ microbial delignificationappears to be a plausible strategy to achieve improved depo-lymerization of hemicellulose and cellulose.

The white-rot fungi (WRF) are the most effective basidio-mycetes for biological pretreatment as they degrade ligninmore extensively and rapidly than any other known groupof organisms (Eriksson 1993; Kuhad et al. 1997; Keller etal. 2003; Kuhad and Singh 2007). The common pattern ofattack on lignocellulose by WRF is a simultaneous decay ofcellulose, hemicellulose, and lignin, but a preferentialdecomposition of lignin may also occur (Eriksson 1993;Hatakka 1994; Blanchette 1995; Kuhad et al. 1997; Levinet al. 2005; Kuhad and Singh 2007). Some WRF have beenreported to degrade lignin selectively (Munoz et al. 1997;Watanabe 2003), and this capability of selected WRF canbe exploited for delignification of lignocellulosics (Aleksan-drova et al. 1998) without affecting much of cellulose. Thus,selected lignin-degrading WRF with comparatively low cel-lulase and xylanase activities could be of utmost importancefor efficient delignification and eventually in the reductionof chemical and energy inputs for chemical or enzymatichydrolysis of the substrate(s).

Few studies have been reported on the pretreatment ofplant biomass with WRF for its effect on cellulose hydroly-sis. According to Hatakka (1983), 35% of the wheat straw isconvertible to reducing sugars when pretreated with Pleuro-tus ostreatus for 5 weeks. Taniguchi and co-workers (2005)also observed a similar conversion rate in rice strawpretreated with Pleurotus ostreatus for 60 days. Keller andco-workers (2003) observed a 3- to 5-fold improvement inenzymatic cellulose digestibility in corn stover pretreatedwith Cyathus stercoreus for 29 days. Zhang et al. (2007)found that biological pretreatment with Coriolus versicoloris effective in improving the yield of reducing sugars andthat it is feasible to produce reducing sugars from bambooresidues. Most of these fungal pretreatments have sufferedbecause of long incubation periods. Therefore, to economizemicrobial pretreatment of lignocellulosics to improve the hy-drolysis of carbohydrates to reducing sugars and to eventu-ally improve ethanol yield, there is a need to test more andmore basidiomycetous fungi for their ability to delignify theplant material quickly and efficiently.

In the present study, we have attempted to examine thecapabilities of 4 basidiomycetous fungi: Phanerochaete

chrysosporium (simultaneous WRF), Pycnoporus cinnabari-nus (selective WRF), and fungal isolates RCK-1 and RCK-3(both new WRF), to delignify wheat straw (WS) and Proso-pis juliflora (PJ) and to improve their hydrolysis to yieldhigher reducing sugars. An attempt has also been made toferment the acid hydrolysates from fungal-pretreated ligno-cellulosic materials by Pichia stipitis to ethanol.

Materials and methods

Organisms and growth conditionsPhanerochaete chrysosporium K3 and Pycnoporus cinna-

barinus PB (ATCC 2004378) were kindly donated byK.-E.L. Eriksson, Professor Emeritus, Department of Bio-chemistry and Molecular Biology, University of Georgia,Athens, USA; and unidentified basidiomycetous fungal iso-lates RCK-1 and RCK-3 were procured from Lignocellu-lose Biotechnology Laboratory, Department ofMicrobiology, University of Delhi South Campus, NewDelhi, India. Pichia stipitis NCIM-3498 was obtained fromNational Chemical Laboratory, Pune, India. The fungi weregrown and maintained on malt extract agar containing thefollowing (g�L–1): malt extract, 20.0; KH2PO4, 0.5;MgSO4�7H2O, 0.5; Ca(NO3)2�4H2O, 0.5; agar, 20.0(pH 5.5) (Vasdev and Kuhad 1994; Vasdev et al. 2005).Pycnoporus cinnabarinus and fungal isolate RCK-1 weregrown at 30 8C, while Phanerochaete chrysosporium andfungal isolate RCK-3 were at 37 8C. Fungal cultures werestored at 4 8C and subcultured every fortnight.

Pichia stipitis was maintained on agar slants containingthe following (g�L–1): xylose, 20; malt extract, 3; yeast ex-tract, 1.5; peptone, 5; agar, 20; at 30 8C and pH 5.0 (Nigam2001a). Stock cultures were stored at 4 8C and subculturedevery fortnight.

Preparation of inoculumFungal inoculum was prepared by growing each fungus in

250 mL Erlenmeyer flasks having 50 mL of sterile malt ex-tract broth containing the following (g�L–1): malt extract,20.0; KH2PO4, 0.5; MgSO4�7H2O, 0.5; Ca(NO3)2�4H2O, 0.5(pH 5.5). Each flask was inoculated with 2 fungal discs(8 mm diameter each) from the periphery of the 7-day-oldfungal culture growing on malt extract agar plates (Vasdevand Kuhad 1994; Vasdev et al. 2005). Pycnoporus cinnabar-inus and fungal isolate RCK-1 were incubated at 30 8C,while Phanerochaete chrysosporium and fungal isolateRCK-3 were at 37 8C. Seven-day-old fungal cultures from 2flasks (562.5 ± 3.50 mg dry mass) were pooled together andhomogenized and used as inoculum for each enamel tray.

Fungal treatment of WS and PJ under solid-statecultivation conditions

Enamel trays (25 � 20 cm2) containing 75 g of either WS(1.5–2.0 cm) or PJ (40–60 mesh size) were moistened withmineral salt solution containing (g�L–1) the following:KH2PO4, 0.5; MgSO4�7H2O, 0.5; Ca(NO3)2�4H2O, 0.5(pH 5.5); and were autoclaved at 15 psi (1 psi = 6.89 kPa)and 121 8C for 15 min. The substrate to moisture level waskept at ratio of 1:4 and 1:3 for WS and PJ, respectively.Each enamel tray was inoculated with 7.5 ± 0.047 mg fun-gal dry mass�(g WS or PJ)–1. The trays with fungal isolate

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RCK-1 and Pycnoporus cinnabarinus were incubated at30 8C, while Phanerochaete chrysosporium and fungal iso-late RCK-3 were at 37 8C for 20 days. The trays were man-ually agitated carefully after a regular interval of 3 days.Samples (1 g each) from enamel trays containing either WSor PJ were withdrawn on the 10th and 20th day of incuba-tion, suspended in 3 mL of citrate–phosphate buffer(100 mmol�L–1 and pH 5.5), and vortexed for 20 min atroom temperature to recover maximum enzymes. The en-zyme extracts were centrifuged at 10 000g for 20 min at4 8C and were assayed for various enzymes activities.

Acid hydrolysisUntreated and fungal-pretreated WS and PJ samples were

washed 3 times with tap water and dried at 60 8C until aconstant mass was obtained. Thereafter, these untreated andtreated samples of plant material were hydrolyzed with vari-ous concentrations of sulfuric acid (0.5%, 1.5%, 2.5%, 3.5%,4.5% v�v–1) at 121 8C for 45 and 60 min with an initial sub-strate to liquid ratio of 1:10. Thereafter, the hydrolysate ob-tained was separated by vacuum filtration and analyzed forreducing sugars and total phenolics.

Ethanol fermentationAcid hydrolysates of untreated WS and PJ and of WS and

PJ pretreated with fungal isolate RCK-1 for 10 days wereused for ethanol fermentation using Pichia stipitis underbatch cultivation conditions. Inoculum of Pichia stipitis wasprepared as described previously (Nigam 2001a, 2001b).Fermentation was carried out in a 250 mL Erlenmeyer flaskcontaining 100 mL of neutralized acid hydrolysates of eitheruntreated or treated WS or PJ supplemented with the follow-ing (g�L–1): (NH4)2SO4, 0.5; KH2PO4, 2.0; MgSO4�7H2O,1.0; yeast extract, 1.5 (pH 5.5). Each flask was inoculatedwith 3.5% (v�v–1) inoculum of Pichia stipitis and incubatedat 30 8C in a New Brunswick Scientific shaker (New Jersey,USA) at 150 r�min–1. Ethanol concentration was estimatedby gas chromatography.

Analytical methodsSubstrate mass loss was determined by calculating the dif-

ference in the dry mass of the substrate at the beginning andof mycoresidue (substrate + fungal mass) at the end of thefungal treatment. The holocellulose and lignin fractions ofuntreated and fungal-pretreated WS and PJ were determinedaccording to Tappi Test Methods (1992).

Total phenolics were determined by the method of Single-ton and co-workers (1999) using vanillin as a standard. Thereducing sugars released were measured by the dinitrosali-cylic acid method of Miller (1959).

Xylanase, total cellulolytic activity, and endoglucanaseactivity were quantified by measuring the release of xyloseand glucose as reducing sugar groups by the dinitrosalicylicacid method (Miller 1959) at 50 8C, using 1% (m�v–1) birchwood xylan, Whatman filter paper No. 1, and 1% (m�v–1)carboxymethyl cellulose as substrate, respectively, in100 mmol�L–1 citrate–phosphate buffer (pH 5.0). One unitof xylanase or cellulase (total cellulolytic and endoglucanaseactivities) was defined as the amount of enzyme required torelease 1 mmol xylose�min–1 or 1 mmol glucose�min–1 fromthe substrates under assay conditions.

Laccase activity was assayed in a reaction mixture(1.0 mL) containing 10 mmol�L–1 guaiacol in 100 mmol�L–1

citrate–phosphate buffer, pH 5.0 (Vasdev and Kuhad 1994;Sharma et al. 2005). One unit (U) of laccase is defined asthe change in absorbance of 0.01�mL–1�min–1 at 470 nm.

Ethanol was estimated by Gas Chromatography (GC,PerkinElmer, Clarus 500) with an elite-wax (crossbond-PEG) column at 120 8C, flame ionization detector at210 8C, and injector at 180 8C using isopropanol as stand-ard. The carrier gas was nitrogen.

To determine the dry mass of yeast cells, the homogen-ized cell suspension (1 mL) was centrifuged (10 000 rpm)in preweighed Eppendorf tubes at 4 8C. The cell pellet wasrinsed with sterilized distilled water, recentrifuged at10 000 rpm and dried till constant mass at 60 8C. All theexperiments were performed in triplicates.

Results and discussion

Solid-state cultivation conditionsThe untreated WS and PJ were found to contain 18.3% ±

0.22% and 26.1% ± 0.48% lignin, respectively, and 75.4% ±0.56% and 69.2% ± 0.22% holocellulose, respectively. All 4fungi colonized WS and PJ fairly well within 3 days of in-cubation. However, fungal isolate RCK-1 and Pycnoporuscinnabarinus colonized both substrates faster than Phanero-chaete chrysosporium and fungal isolate RCK-3. All 4 fungiexhibited different preferences for lignin and holocellulosecontents of WS and PJ. The difference in the degradation ofcell wall components by different fungi could be attributedto their different enzyme profiles (Table 1). Lignin degrada-tion is a complex process and the enzymes involved prob-ably have synergistic effects on each other (Tuomela et al.2000). Among the fungi studied, Phanerochaete chrysospo-rium was the most efficient cellulase producer along withlignin-degrading enzymes. Fungal isolate RCK-1 producedminimum levels of cellulases but considerably largeamounts of laccase (50.17 and 102.50 U�g–1 in WS and PJ,respectively) compared with other fungi. Irrespective of thesubstrates, Pycnoporus cinnabarinus produced maximumlaccase (137.85 and 110.20 U�g–1 in WS and PJ, respec-tively) after 10 days. Maximum xylanase production was ob-served from Phanerochaete chrysosporium and Pycnoporuscinnabarinus grown on WS and PJ, respectively, for10 days. Phanerochaete chrysosporium caused maximummass loss of WS and PJ, which could be due to maximumdegradation of lignin and holocellulosic fraction of the sub-strate (Table 1), which is in accordance with the observa-tions of Karunanandaa and co-workers (1992). After10 days, fungal isolate RCK-1 exhibited comparatively lessdegradation of polysaccharides but, interestingly, degradedmore lignin in comparison with other fungi tested (Table 1).Similarly, several strains of Cereporiopsis subvermisporahave been shown to cause selective lignin degradation withmoderate mass losses in several wood species (Blanchette etal. 1992) and bermuda grass (Akin et al. 1995).

Reducing sugars released from WS and PJ degraded byPhanerochaete chrysosporium were higher than the sub-strates degraded by fungal isolate RCK-1 (Table 1). The dif-ference in sugar release levels may be attributed to thedifferential level of cellulases and xylanases produced by

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the basidiomycetous fungi studied here (Table 1). Phanero-chaete chrysosporium also produced higher levels of cellu-lase and xylanase than fungal isolate RCK-1, and it causeda maximum release of phenolics in WS and PJ after20 days of fermentation compared with the rest of the fungi(Table 1), which may be due to higher lignin degradation.

Acid hydrolysisThe sugar content reached maximum in untreated WS and

PJ (30.27 and 18.18 g�L–1, respectively) when hydrolyzedwith 3.5% and 4.5% acid concentration at 121 8C and60 min (Fig. 1). On the other hand, WS and PJ pretreatedby fungal isolate RCK-1 for 10 days, when subjected toonly 2.5% acid hydrolysis, interestingly yielded more reduc-ing sugars (40.82 and 26.00 g�L–1, respectively) than otherfungi (Fig. 1). The higher reducing sugar yields in substratespretreated with fungal isolate RCK-1 after acid hydrolysiscould be because isolate RCK-1 caused maximum lignin re-

Fig. 1. Maximum reducing sugars obtained from acid hydrolysis (2.5%) of wheat straw and Prosopis juliflora treated with fungi for 10 and20 days (121 8C, 60 min). The sugar content reached maxima in untreated wheat straw and P. juliflora (30.27 and 18.18 g�L–1, respectively)at 3.5% and 4.5% acid concentration.

Table 1. Changes in lignocellulosic components, reducing sugars, and total phenolics released after fungal treatment of wheat straw andProsopis juliflora for 10 and 20 days under solid-state fermentation.

Phanerochaete chry-sosporium

Pycnoporuscinnabarinus

Fungal isolateRCK- 1

Fungal isolateRCK- 3

Change brought about afterfermentation Substrate 10 days 20 days 10 days 20 days 10 days 20 days 10 days 20 days

Substrate loss (%) Wheat straw 31.29 53.57 12.32 25.62 11.61 16.05 15.63 20.05Prosopis juliflora 34.70 55.93 15.04 28.49 8.5 12.04 10.03 16.93

Lignin degradation (%) Wheat straw 10.23 18.89 11.67 19.12 12.26 22.64 11.14 20.17Prosopis juliflora 15.23 20.21 8.08 16.06 19.30 21.97 12.64 19.38

Holocellulose degradation (%) Wheat straw 17.56 55.67 3.34 10.58 6.27 9.39 14.21 17.83Prosopis juliflora 10.01 48.89 3.58 6.55 3.01 4.58 6.45 12.02

Reducing sugars* (g�L–1) Wheat straw 1.98 3.34 0.86 1.28 0.18 0.98 0.68 1.52Prosopis juliflora 0.24 1.06 0.07 0.56 0.05 0.49 0.29 0.73

Phenolics{ (g�L–1) Wheat straw 0.15 0.33 0.00 0.00 0.00 0.00 0.00 0.00Prosopis juliflora 0.16 1.12 0.00 0.00 0.00 0.00 0.00 0.00

*Reducing sugars in untreated wheat straw and Prosopis juliflora were undetectable.{Total phenolics in untreated wheat straw and Prosopis juliflora were 0.56 and 1.21 g�L–1, respectively.

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moval and minimum loss of structural polysaccharides(Table 1), which eventually increased the accessibility toacid hydrolysis. Fungal pretreatment of WS and PJ for20 days showed a decrease in sugar yield when comparedwith pretreatment for 10 days (Fig. 1). This might be due toincreased cellulase and xylanase production when each traywas incubated for 20 days (Table 1). Therefore, the reducingsugars released from these treated samples were higherwhich were washed away from WS and PJ during substratepreparation for acid hydrolysis (Table 1).

The hydrolysates from fungal-treated plant materials hadlow levels of phenolics as compared with the untreated ones(Fig. 2). The presence of these phenolics in the fermentationmedium affects sugar uptake by the yeast, which leads todecreased ethanol productivity (Mussatto and Roberto2004). The considerable reduction in total phenolics in fun-gal-treated samples avoided the need of detoxification of hy-drolysates, and fermentation was carried out directly afterneutralization of acid hydrolysate. Irrespective of the

substrates, the release of total phenolics after chemical hy-drolysis of WS and PJ fermented with Phanerochaete chrys-osporium, Pycnoporus cinnabarinus, and fungal isolatesRCK-3 was higher in comparison with the hydrolysatesfrom the substrates treated with fungus RCK-1 (Fig. 2).This may be attributed to the difference in ligninolytic abil-ity of the different fungi studied (Table 1).

Sawada and co-workers (1995) investigated the effect offungal and steam explosion pretreatment on enzymatic sac-charification of beech wood meal and indicated that fungalpretreatment followed by mild steaming maximizedenzymatic saccharification. Further, they suggested that thelignin network covering the holocellulose (cellulose andhemicellulose) is broken down by successive fungal pre-treatment and steam explosion pretreatments, which togethermaximized subsequent saccharification of beech wood.

Ethanol fermentationUntreated and treated WS and PJ hydrolysates were in-

Fig. 2. Total phenolics released from acid hydrolysis (2.5%) of wheat straw and Prosopis juliflora treated by fungi for 10 and 20 days(121 8C, 60 min).

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vestigated for reducing sugars utilization, biomass, and etha-nol production by Pichia stipitis as a function of time(Figs. 3 and 4). Irrespective of the hydrolysates, either fromtreated or untreated substrates, tested for ethanol fermenta-tion, the yeast biomass as well as ethanol were observed toincrease regularly until 36 h of fermentation and, thereafter,it declined (Figs. 3 and 4). According to Ferrari et al.(1992), Pichia stipitis NRRL Y-7124 when grown on Euca-lyptus wood hydrolysates produced maximum ethanol(12.60 g�L–1) after 75 h. Nigam (2001a, 2001b) also re-ported maximum ethanol production from hardwood(14.5 g�L–1) and wheat straw (19.1 g�L–1) hydrolysates us-ing an adapted strain of Pichia stipitis NRRL Y-7124 after

100 and 60 h, respectively. Earlier studies from our labora-tory have shown maximum ethanol production (28.0 g�L–1)by Candida shehatae at 72 h, which remains nearly con-stant until the end of fermentation, when xylose was usedas the sole carbon source (Abbi et al. 1996). Interestingly,reducing sugars in the fermentation medium in our studywere not found to be completely utilized by the yeasteven after 44 h. Similar observations have been reportedby Roberto et al. (1991), Amartey and Jeffries (1996), andNigam (2001b). The incomplete consumption of sugarsmight be attributed to the intolerance of the fermentingyeast to ethanol and inhibitors.

Maximum ethanol yield and volumetric productivity from

Fig. 3. Fermentation profile of acid hydrolysate of untreated and RCK-1-treated wheat straw for 10 days. For untreated samples: &,biomass (g�L–1); !, ethanol produced (g�L–1); *, reducing sugars (g�L–1). For treated samples: &, biomass (g�L–1); ~, ethanol produced(g�L–1); *, reducing sugars (g�L–1).

Fig. 4. Fermentation profile of acid hydrolysate of untreated and RCK-1-treated Prosopis juliflora for 10 days. For untreated samples: &,biomass (g�L–1); !, ethanol produced (g�L–1); *, reducing sugars (g�L–1). For treated samples: &, biomass (g�L–1); ~, ethanol produced(g�L–1); *, reducing sugars (g�L–1).

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Table 2. Fermentation profile of acid hydrolysate of untreated and RCK-1-treated wheat straw and Prosopis juliflora for 10 days.

Ethanol yield (g�g–1) Ethanol productivity (g�L–1�h–1) Biomass yield (g�g–1)

Wheat straw Prosopis juliflora Wheat straw Prosopis juliflora Wheat straw Prosopis juliflora

Time (h) Untreated Treated Untreated Treated Untreated Treated Untreated Treated Untreated Treated Untreated Treated0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

12 0.03 0.07 0.05 0.07 0.03 0.25 0.07 0.15 0.14 0.19 0.06 0.1916 0.07 0.11 0.06 0.14 0.07 0.27 0.07 0.22 0.18 0.24 0.17 0.2320 0.16 0.14 0.13 0.21 0.16 0.28 0.11 0.28 0.23 0.24 0.22 0.2524 0.21 0.22 0.24 0.27 0.21 0.37 0.18 0.29 0.25 0.25 0.27 0.2728 0.26 0.26 0.29 0.34 0.26 0.38 0.19 0.31 0.27 0.30 0.31 0.3032 0.31 0.31 0.35 0.39 0.31 0.40 0.20 0.32 0.30 0.33 0.32 0.3436 0.36 0.48 0.42 0.46 0.36 0.54 0.21 0.33 0.32 0.36 0.33 0.3740 0.27 0.42 0.35 0.44 0.27 0.43 0.16 0.29 0.31 0.29 0.31 0.3144 0.17 0.37 0.22 0.38 0.17 0.34 0.09 0.22 0.23 0.28 0.22 0.30

Table 3. Production of ethanol from different sources of hydrolysates by Pichia stipitis.

Sample No. Source of hydrolyzate Pretreatment CultureTime(h)

Ethanolyield(g�g–1)

Ethanolproductivity(g�L–1�h–1) Reference

1 Corn cob Alkali, enzyme P. stipitis CBS 6054 48 0.41 ND Amartey and Jeffries 19962 Wheat straw Alkali, enzyme P. stipitis Y-7154 72 0.43 ND Awafo et al. 19983 Hardwood Dilute acid P. stipitis A* 100 0.40 0.21 Nigam 2001a4 Wheat straw Dilute acid P. stipitis A* 60 0.41 0.54 Nigam 2001b5 Wheat straw Dilute acid P. stipitis NRRL Y-7154 80 0.35 0.30 Nigam 2001b6 100% Glucose — P. stipitis CBS 6054 96 0.42 0.24 Agbogbo et al. 20067 25% Glucose/75% xylose mixture — P. stipitis CBS 6054 96 0.45 0.20 Agbogbo et al. 20068 Wheat straw Fungus, dilute acid P. stipitis 36 0.48 0.54 This report9 Prosopis juliflora Fungus, dilute acid P. stipitis 36 0.46 0.33 This report

Note: ND, not determined. —, no pretreatment.*Adapted culture of P. stipitis NRRL Y-7124.

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anada

fungal-treated WS (0.48 g�g–1 and 0.54 g�L–1�h–1, respec-tively) and PJ (0.46 g�g–1 and 0.33 g�L–1�h–1, respectively)hydrolysates were higher than that of untreated WS(0.36 g�g–1 and 0.30 g�L–1�h–1, respectively) and PJ (0.42 g�g–1

and 0.21 g�L–1�h–1, respectively) hydrolysates after 36 h(Table 2). Since last one decade various workers have fer-mented hydrolysates rich in sugars obtained as a result of var-ied treatments and reported the ethanol production bydifferent strains of Pichia stipitis, as depicted in Table 3. Theethanol yield (0.48 g�g–1) obtained in our study is higher ascompared to other studies (Amartey and Jeffries 1996;Awafo et al. 1998; Nigam 2001a, 2001b; Agbogbo et al.2006) (Table 3). Ethanol productivity was observed to be0.54 g�L–1�h–1 from fermentation of wheat straw hydroly-sates by Pichia stipitis, which was very much in agreementwith Nigam (2001b). Our study demonstrates the potentialof in situ pretreatment of lignocellulosic biomass with se-lective lignin-degrading fungi before its chemical or enzy-matic hydrolysis in increasing sugar yield and in turnproducing ethanol as a biofuel.

AcknowledgementsThe authors thank the Department of Biotechnology

(DBT), Government of India, for funding a nationwide proj-ect on production of fuel ethanol from lignocellulosic feed-stock. The technical support rendered by Mr. Manwar Singhduring this work is duly acknowledged.

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