Bioresource Technology 100 (2009) 6679–6681

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Bioresource Technology

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Short Communication

Hydrolysis of cellulose derived from steam exploded bagasse by Penicilliumcellulases: Comparison with commercial cellulase

Rajkumar Singh a, A.J. Varma b, R. Seeta Laxman a,*, Mala Rao a,*

a Biochemical Sciences Division, National Chemical Laboratory, Pune 411008, Indiab Polymer Sciences and Engineering Division, National Chemical Laboratory, Pune 411008, India

a r t i c l e i n f o

Article history:Received 15 May 2009Received in revised form 20 July 2009Accepted 21 July 2009Available online 15 August 2009

Keywords:Sugarcane bagassePenicillium cellulaseHigh b-glucosidaseEnzymatic hydrolysisAccelleraseTM 1000

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.07.060

* Corresponding authors. Tel.: +91 20 25902720; faLaxman), tel.: +91 20 25902228 (M. Rao).

E-mail addresses: r.seeta@ncl.res.in (R. Seeta LaxRao).

a b s t r a c t

A complete cellulase from Penicillium pinophilum was evaluated for the hydrolysis of a-cellulose derivedfrom steam exploded sugarcane bagasse and other cellulosic substrates. a-Cellulose at 1% substrate con-centration was completely hydrolyzed by Penicillium cellulase within 3 h wherein at 10% the hydrolysiswas 100% within 24 h with an enzyme loading of 10 FPU/g. The hydrolysate yielded glucose as major endproduct as analyzed by HPLC. Under similar conditions, hydrolysis of Sigmacell (microcrystalline cellu-lose), CP-123 (pulverized cellulose powder) and ball milled Solka Floc were 42%, 56% and 52%, respec-tively. Further the hydrolysis performance of Penicillium sp. cellulase is compared with Trichodermareesei cellulase (AccelleraseTM 1000) from Genencore. The kinetics of hydrolysis with respect to enzymeand substrate concentration will be presented.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lignocellulosic biomasses are considered as significant sourcefor the generation of sugar streams, organic products and fuel/eth-anol. Cellulases, a group of enzymes which catalyze the hydrolysisof cellulose are considered as a potential tool for industrial sac-charification of biomass. Sugarcane bagasse a byproduct of sugar-cane industry is the most abundant lignocellulosic feed stock inIndia, second after Brazil, the largest producer with 27% of totalglobal production. Approximately 179 million tons of bagasse isannually produced in India, cultivated on 4.3 million hectare areawith the yield of 41498.0 kg/hectare (Kapoor et al., 2006). Mostof the bagasse is burnt for generating power for boilers and is usedas a fuel directly by sugar industry (Pandey et al., 2000).

Within the context of production of fuels from biomass, pre-treatment has come to denote as one of the processes necessaryto render cellulosic biomass susceptible to the action of cellulases.Several pretreatment processes have been developed for the pre-treatment of sugarcane bagasse including steam explosion, liquidhot water process, acid hydrolysis, alkali pretreatment and wetoxidation. Few reports are available on the steam explosion pro-cess with minor modifications for the pretreatment of sugarcane

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x: +91 20 25902648 (R. Seeta

man), mb.rao@ncl.res.in (M.

bagasse (Hendriks and Zeeman, 2009). In principle steam explosion(SE) is one of the attractive pretreatment methods that can causedisintegration of the material, thereby creating a large surface areaon which cellulase enzyme complex can act upon. Simultaneouslyhemicellulose is separated during the steam explosion processthereby improving the accessibility to the enzymes and enhance-ment of the over all lignocellulose degradation (Wei et al., 2006).In the current report, steam explosion a proprietary process devel-oped at National Chemical Laboratory (NCL) is used as a pretreat-ment procedure for sugar cane bagasse. The NCL process is basedon steam explosion of sugar cane bagasse to separate lignin, cellu-lose and hemicellulose along with a relevant downstream process-ing (patent application 1893 DEL 2007, 27th Aug) to yield purecellulose, lignin and hemicellulosic hydrolysate as the otherproducts.

Trichoderma sp. is an extensively studied organism for cellulaseproduction and hydrolysis of differently pretreated diverse ligno-celluloses (Tabka et al., 2006). After screening for a large numberof cultures at NCL, a Penicillium strain has been selected as a sourceof complete cellulase with high b-glucosidase activity. The presentpaper reports the hydrolysis of cellulose derived from sugarcanebagasse by steam explosion and other cellulosic substrates suchas CP-123, Sigmacell and Solka Floc by Penicillium cellulase. Furtherthe comparison of hydrolysis performance of Penicillium cellulasewith commercial cellulase (AccelleraseTM 1000) from geneticallymodified Trichoderma reesei will also described. By virtue of thehigh b-glucosidase activity in the Penicillium cellulase complex,the hydrolysis yielded glucose as the major end product.

Table 1Different components of cellulase complex of Penicillium sp. and AccelleraseTM 1000.

IU/ml Penicillium AccelleraseTM 1000

FPU 5 10 20 5 10 20CMC 65 130 260 157.5 315 630PNPGase 28 56 112 22.5 45 90

The enzyme activities were determined by IUPAC method (Ghose, 1987; Ghose andBisaria, 1987) as described in Section 2.

6680 R. Singh et al. / Bioresource Technology 100 (2009) 6679–6681

2. Methods

2.1. Chemicals

All chemicals were of analytical grade. The following chemicalswere obtained from as follows: cellulose powder CP-123 (Pulver-ized) was obtained from Schleicher and Schull GmbH, D-3354 Das-sel, W. Germany. p-Nitro phenyl b-D glucoside (PNPG), carboxymethyl cellulose (CMC), 3,5-dinitrosalicylic acid (DNSA) and Sig-macell were obtained from Sigma–Aldrich Co. St. Louis, MO, USA.

2.2. Preparation of sugarcane bagasse cellulose

NCL has developed a proprietary process for the extraction of93% a-cellulose from sugarcane bagasse and the process is underpatenting (Varma A.J., 2007 Indian patent application 1893/DEL/2007 dated 27th August 2007). Sugarcane bagasse was obtainedfrom Tamil Nadu Pulp and Paper Mills, Chennai, India. This bagassecontains about 43% cellulose, 30% xylan, and 20% lignin, in additionto some silica and other constituents. It was cut into small shredsof 1–3 mm size and then pretreated with steam and alkali by a pro-prietary process to remove the xylan, lignin, and other impurities.The cellulose thus obtained by this process contains a-cellulose(93%), b-cellulose (4.1%), c cellulose considered as hemicellulose(2.22%) and traces of lignin (0.18%).

2.3. Microorganism and culture media

Penicillium strain used in present study was maintained on Po-tato Dextrose Agar (PDA). Enzyme production was carried out in500 ml Erlenmeyer flask for 5 days on modified Mandels and We-ber medium (Mandels and Weber, 1969) except that the levels ofammonium sulphate and urea were five time higher and 2.5% cel-lulose powder and 1% wheat bran were used as carbon source. Theculture filtrate was centrifuged at 7000 rpm and the clear superna-tant obtained was used as the source of enzyme. In some cases, theculture filtrate was concentrated by ultrafiltration through PM-10membrane (Amicon Corp.). The concentrated preparation had car-boxyl methyl cellulase (CMCase)-130 U/ml, filter paper activity(FPAase)-10 U/ml and p-nitro phenyl-b-glucosidase (PNPGase)-56U/ml. AccelleraseTM 1000 from a genetically modified T. reeseiwas a kind gift from Genencore USA and had carboxyl methyl cel-lulase (CMCase)-3150 U/ml, filter paper activity (FPAase)-100 U/mland p-nitro phenyl-b-glucosidase (PNPGase)-450 U/ml.

2.4. Enzyme assays

Carboxyl methyl cellulase (CMCase) and filter paper activity(FPAase) were measured according to standard procedure recom-mended by Commission on Biotechnology, IUPAC (Ghose, 1987)p-nitro phenyl-b-glucosidase (PNPGase) was determined accordingto (Ghose and Bisaria, 1987). One unit of enzyme activity is definedas the amount of enzyme required to liberate one lM of reducingsugar per minute under the assay conditions.

2.5. Cellulose hydrolysis

The hydrolysis of a-cellulose derived from bagasse (after steamexplosion), pulverized cellulose (CP-123), microcrystalline cellu-lose (Sigmacell), Solka Floc (ball milled for 8 h) were carried outusing Penicillium cellulase and commercial cellulase from Genen-core (Accellerase) in 50 ml of stoppered flask in 10 ml reaction vol-ume. About 1 g of cellulosic substrate was incubated with 5, 10 and20 FPU of cellulase at 50 �C in 10 ml of 50 mM sodium acetate buf-fer pH 4.8 under stationary condition. Hydrolysis was terminated

by boiling at 100 �C for 5 min at the end of stipulated time intervalsand reducing sugar was assayed by dinitrosalicylic method. Extentof hydrolysis was calculated and expressed as percentage based oninitial cellulose taken as 100%. The control experiments for hydro-lysis including enzyme, substrate, reagent blanks and heat inacti-vated enzyme have been carried out.

2.6. End product analysis by HPLC

The end products were analyzed by Waters HPLC system usingWaters Sugar Pack Column with a mobile phase of Milli Q waterwith 100 lM EDTA and 200 lM CaCl2 with a flow rate of 0.4 ml/min.

3. Results and discussion

The Penicillium strain used in the present investigation was iso-lated from soil sample collected near decaying wood and was iden-tified as Penicillium pinophilum based on ITS sequence homology(99%) (Unpublished data). The cellulases from Penicillium sp. showa high ratio of filter paper activity to CMCase activity. At a givenunits of filter paper activity, it is evident that CMCase activity ofAccellerase is double that of Penicillum enzyme with marginallylower b-glucosidase. (Table 1). a-Cellulose was completely con-verted into soluble sugars forming a transparent solution within3 h at 1% substrate concentration by Penicillium cellulase at 10FPU/g (data not shown) suggesting that cellulose without lignincan be hydrolyzed rapidly. The hydrolysis patterns of different cel-lulosic substrates at 10% substrate concentration by cellulases fromPenicillium and commercial Accellerase enzyme is compared in Ta-ble 2. It was observed that the percentage hydrolysis of a-celluloseincreased with increased enzyme loading and with Penicillium en-zyme, a maximum hydrolysis of 100% occurs at 10 FPU/g of sub-strate in 48 h. In comparison, Accellerase enzyme showed 57%hydrolysis which increased to 60% after 96 h. At lower enzymeloadings of 5 FPU/g, hydrolysis by Penicillium cellulase and Accel-lerase reached maximum of 69.47% and 21.25%, respectively. How-ever with increased enzyme loading (20FPU/g) a maximumhydrolysis of 86% was obtained in 96 h by Accellerase.

The percentage hydrolysis of Solka Floc by Penicillium cellulaseand Accellerase enzyme were comparable at all enzyme concentra-tions tested with a maximum saccharification of 59.96% and52.39% at 96 h respectively. The hydrolysis pattern of CP-123 usingcellulase from Penicillium and Accellerase enzyme shows that therate of hydrolysis increased with increased enzyme concentration.Maximum hydrolysis of 60.18% and 32.57% were obtained for Pen-icillium cellulase and Accellerase enzyme at 20 FPU/g substrate in96 h. The percentage hydrolysis of Sigmacell by Penicillium cellu-lase and Accellerase cellulase was 48.38% and 27.51%, respectivelyunder similar experimental conditions with an enzyme substrateloading of 20 FPU/g.

The end product analysis of the hydrolysate obtained after sac-charification of cellulose derived from bagasse shows glucose asthe major end product for both Penicillium cellulase (9.7%) andAccellerase enzyme (8.5%) with traces of xylose (data not shown).

Table 2Enzymatic hydrolysis of cellulosic substrates by Penicillium sp. and Accellerase.

Time (h) Substrate Penicillium sp. (FPU/g) Accellerase (FPU/g)

5 10 20 5 10 20

16 a-Cellulose 51.72 75.23 98.99 13.83 39.05 58.64Solka Floc 20.86 28.72 43.82 18.32 27.22 41.71CP-123 15.74 40.15 51.26 13.25 16.71 20.72Sigmacell 23.87 36.31 42.77 6.72 11.03 23.44

24 a-Cellulose 60.29 86.67 100.00 15.28 45.95 66.01Solka Floc 25.55 33.46 52.07 23.32 30.21 45.22CP-123 18.16 44.77 56.26 15.60 21.08 25.46Sigmacell 26.49 39.76 45.58 8.51 13.12 25.46

48 a-Cellulose 67.28 98.99 100.00 20.15 56.90 77.42Solka Floc 28.48 37.27 55.07 25.05 32.82 48.03CP-123 21.13 47.01 58.04 15.90 22.48 30.03Sigmacell 25.89 40.39 46.39 9.13 13.45 26.07

72 a-Cellulose 68.36 100.00 100.00 20.33 58.21 85.70Solka Floc 30.09 39.09 58.02 26.72 35.55 49.86CP-123 23.98 48.57 59.27 16.16 23.87 31.28Sigmacell 26.94 41.34 47.61 10.08 13.91 26.96

96 a-Cellulose 69.47 100.00 100.00 21.25 60.38 86.45Solka Floc 32.07 40.19 59.96 29.02 37.66 52.39CP-123 25.86 50.57 60.18 17.26 25.23 32.57Sigmacell 27.49 43.15 48.38 11.16 14.52 27.51

Solka Floc (8 h ball milled), CP-123 (pulverized) and Sigmacell (microcrystalline).

R. Singh et al. / Bioresource Technology 100 (2009) 6679–6681 6681

A number of lignocellulose pre-pretreatment technologies areunder intensive investigations on both laboratory and at pilotplant scales (Wyman et al., 2005). Zhang et al. (2007) have pre-treated pure cellulose and lignocellulosic materials using non-volatile cellulose solvent (phosphoric acid). Pretreated Aviceland a-cellulose were completely converted to soluble sugarswithin 3 h at 10 g/litre substrate concentration wherein for her-baceous cellulose corn stower and Switch grass and hard woodlignocellulosics the pretreated cellulosic samples were hydro-lyzed to 96–97% at 24 h using a mixture of commercial cellulase(Genencore Spezyme) and b-glucosidases (Novozymes 188 b-glucosidases).

4. Conclusion

The present studies were carried out to investigate the hydroly-sis of cellulose derived from bagasse by a steam explosion pretreat-ment proprietary process developed at NCL and other purecelluloses by Penicillium cellulase. It was also of interest to com-pare its potential with commercially available cellulase (Acceller-aseTM 1000) from Genencor.

AccelleraseTM 1000 is a cellulase blend product with a high b-glucosidase, capable of hydrolyzing lignocellulosic biomass tomonosaccharides. The comparative studies using the Penicilliumcellulase and AccelleraseTM 1000 have shown that the saccharify-ing potencies are comparable towards the treated substrates suchas steam exploded bagasse and ball milled cellulose powder. How-ever in case of microcrystalline cellulose and untreated cellulosepowder (CP-123), the hydrolysis by Penicillium cellulase was muchsuperior to that of Accellerase. It has been also demonstrated thatthe quantitative conversion of cellulose derived from steam ex-ploded bagasse to major end product as glucose by using a single

enzyme preparation from P. pinophilum having high b-glucosidaseactivity.

Acknowledgements

MR thanks Dr. Raj Lad and Dr. S. Bade, Genencore for the Accel-lerase enzyme. Mr. Gyan Prakash’s help in HPLC experiments isthankfully acknowledged.

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