Bioresource Technology 100 (2009) 66796681

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Bioresource Technology
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Short Communication

Hydrolysis of cellulose derived from steam exploded bagasse by Penicillium cellulases: Comparison with commercial cellulase
Rajkumar Singh a, A.J. Varma b, R. Seeta Laxman a,*, Mala Rao a,*
a b

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

a r t i c l e

i n f o

a b s t r a c t
A complete cellulase from Penicillium pinophilum was evaluated for the hydrolysis of a-cellulose derived from steam exploded sugarcane bagasse and other cellulosic substrates. a-Cellulose at 1% substrate concentration was completely hydrolyzed by Penicillium cellulase within 3 h wherein at 10% the hydrolysis was 100% within 24 h with an enzyme loading of 10 FPU/g. The hydrolysate yielded glucose as major end product as analyzed by HPLC. Under similar conditions, hydrolysis of Sigmacell (microcrystalline cellulose), CP-123 (pulverized cellulose powder) and ball milled Solka Floc were 42%, 56% and 52%, respectively. Further the hydrolysis performance of Penicillium sp. cellulase is compared with Trichoderma reesei cellulase (AccelleraseTM 1000) from Genencore. The kinetics of hydrolysis with respect to enzyme and substrate concentration will be presented. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 15 May 2009 Received in revised form 20 July 2009 Accepted 21 July 2009 Available online 15 August 2009 Keywords: Sugarcane bagasse Penicillium cellulase High b-glucosidase Enzymatic hydrolysis AccelleraseTM 1000

1. Introduction Lignocellulosic biomasses are considered as signicant source for the generation of sugar streams, organic products and fuel/ethanol. Cellulases, a group of enzymes which catalyze the hydrolysis of cellulose are considered as a potential tool for industrial saccharication of biomass. Sugarcane bagasse a byproduct of sugarcane industry is the most abundant lignocellulosic feed stock in India, second after Brazil, the largest producer with 27% of total global production. Approximately 179 million tons of bagasse is annually produced in India, cultivated on 4.3 million hectare area with the yield of 41498.0 kg/hectare (Kapoor et al., 2006). Most of the bagasse is burnt for generating power for boilers and is used as a fuel directly by sugar industry (Pandey et al., 2000). Within the context of production of fuels from biomass, pretreatment has come to denote as one of the processes necessary to render cellulosic biomass susceptible to the action of cellulases. Several pretreatment processes have been developed for the pretreatment of sugarcane bagasse including steam explosion, liquid hot water process, acid hydrolysis, alkali pretreatment and wet oxidation. Few reports are available on the steam explosion process with minor modications for the pretreatment of sugarcane

* Corresponding authors. Tel.: +91 20 25902720; fax: +91 20 25902648 (R. Seeta Laxman), tel.: +91 20 25902228 (M. Rao). E-mail addresses: r.seeta@ncl.res.in (R. Seeta Laxman), mb.rao@ncl.res.in (M. Rao). 0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.060

bagasse (Hendriks and Zeeman, 2009). In principle steam explosion (SE) is one of the attractive pretreatment methods that can cause disintegration of the material, thereby creating a large surface area on which cellulase enzyme complex can act upon. Simultaneously hemicellulose is separated during the steam explosion process thereby improving the accessibility to the enzymes and enhancement of the over all lignocellulose degradation (Wei et al., 2006). In the current report, steam explosion a proprietary process developed at National Chemical Laboratory (NCL) is used as a pretreatment procedure for sugar cane bagasse. The NCL process is based on steam explosion of sugar cane bagasse to separate lignin, cellulose and hemicellulose along with a relevant downstream processing (patent application 1893 DEL 2007, 27th Aug) to yield pure cellulose, lignin and hemicellulosic hydrolysate as the other products. Trichoderma sp. is an extensively studied organism for cellulase production and hydrolysis of differently pretreated diverse lignocelluloses (Tabka et al., 2006). After screening for a large number of cultures at NCL, a Penicillium strain has been selected as a source of complete cellulase with high b-glucosidase activity. The present paper reports the hydrolysis of cellulose derived from sugarcane bagasse by steam explosion and other cellulosic substrates such as CP-123, Sigmacell and Solka Floc by Penicillium cellulase. Further the comparison of hydrolysis performance of Penicillium cellulase with commercial cellulase (AccelleraseTM 1000) from genetically modied Trichoderma reesei will also described. By virtue of the high b-glucosidase activity in the Penicillium cellulase complex, the hydrolysis yielded glucose as the major end product.

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2. Methods 2.1. Chemicals All chemicals were of analytical grade. The following chemicals were obtained from as follows: cellulose powder CP-123 (Pulverized) was obtained from Schleicher and Schull GmbH, D-3354 Dassel, W. Germany. p-Nitro phenyl b-D glucoside (PNPG), carboxy methyl cellulose (CMC), 3,5-dinitrosalicylic acid (DNSA) and Sigmacell were obtained from SigmaAldrich Co. St. Louis, MO, USA. 2.2. Preparation of sugarcane bagasse cellulose NCL has developed a proprietary process for the extraction of 93% a-cellulose from sugarcane bagasse and the process is under patenting (Varma A.J., 2007 Indian patent application 1893/DEL/ 2007 dated 27th August 2007). Sugarcane bagasse was obtained from Tamil Nadu Pulp and Paper Mills, Chennai, India. This bagasse contains about 43% cellulose, 30% xylan, and 20% lignin, in addition to some silica and other constituents. It was cut into small shreds of 13 mm size and then pretreated with steam and alkali by a proprietary 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 Potato Dextrose Agar (PDA). Enzyme production was carried out in 500 ml Erlenmeyer ask for 5 days on modied Mandels and Weber medium (Mandels and Weber, 1969) except that the levels of ammonium sulphate and urea were ve time higher and 2.5% cellulose powder and 1% wheat bran were used as carbon source. The culture ltrate was centrifuged at 7000 rpm and the clear supernatant obtained was used as the source of enzyme. In some cases, the culture ltrate was concentrated by ultraltration through PM-10 membrane (Amicon Corp.). The concentrated preparation had carboxyl methyl cellulase (CMCase)-130 U/ml, lter paper activity (FPAase)-10 U/ml and p-nitro phenyl-b-glucosidase (PNPGase)-56 U/ml. AccelleraseTM 1000 from a genetically modied T. reesei was a kind gift from Genencore USA and had carboxyl methyl cellulase (CMCase)-3150 U/ml, lter paper activity (FPAase)-100 U/ml and p-nitro phenyl-b-glucosidase (PNPGase)-450 U/ml. 2.4. Enzyme assays Carboxyl methyl cellulase (CMCase) and lter paper activity (FPAase) were measured according to standard procedure recommended by Commission on Biotechnology, IUPAC (Ghose, 1987) p-nitro phenyl-b-glucosidase (PNPGase) was determined according to (Ghose and Bisaria, 1987). One unit of enzyme activity is dened as the amount of enzyme required to liberate one lM of reducing sugar per minute under the assay conditions. 2.5. Cellulose hydrolysis The hydrolysis of a-cellulose derived from bagasse (after steam explosion), pulverized cellulose (CP-123), microcrystalline cellulose (Sigmacell), Solka Floc (ball milled for 8 h) were carried out using Penicillium cellulase and commercial cellulase from Genencore (Accellerase) in 50 ml of stoppered ask in 10 ml reaction volume. About 1 g of cellulosic substrate was incubated with 5, 10 and 20 FPU of cellulase at 50 C in 10 ml of 50 mM sodium acetate buffer pH 4.8 under stationary condition. Hydrolysis was terminated

by boiling at 100 C for 5 min at the end of stipulated time intervals and reducing sugar was assayed by dinitrosalicylic method. Extent of hydrolysis was calculated and expressed as percentage based on initial cellulose taken as 100%. The control experiments for hydrolysis including enzyme, substrate, reagent blanks and heat inactivated enzyme have been carried out. 2.6. End product analysis by HPLC The end products were analyzed by Waters HPLC system using Waters Sugar Pack Column with a mobile phase of Milli Q water with 100 lM EDTA and 200 lM CaCl2 with a ow rate of 0.4 ml/ min. 3. Results and discussion The Penicillium strain used in the present investigation was isolated from soil sample collected near decaying wood and was identied as Penicillium pinophilum based on ITS sequence homology (99%) (Unpublished data). The cellulases from Penicillium sp. show a high ratio of lter paper activity to CMCase activity. At a given units of lter paper activity, it is evident that CMCase activity of Accellerase is double that of Penicillum enzyme with marginally lower b-glucosidase. (Table 1). a-Cellulose was completely converted into soluble sugars forming a transparent solution within 3 h at 1% substrate concentration by Penicillium cellulase at 10 FPU/g (data not shown) suggesting that cellulose without lignin can be hydrolyzed rapidly. The hydrolysis patterns of different cellulosic substrates at 10% substrate concentration by cellulases from Penicillium and commercial Accellerase enzyme is compared in Table 2. It was observed that the percentage hydrolysis of a-cellulose increased with increased enzyme loading and with Penicillium enzyme, a maximum hydrolysis of 100% occurs at 10 FPU/g of substrate in 48 h. In comparison, Accellerase enzyme showed 57% hydrolysis which increased to 60% after 96 h. At lower enzyme loadings of 5 FPU/g, hydrolysis by Penicillium cellulase and Accellerase reached maximum of 69.47% and 21.25%, respectively. However with increased enzyme loading (20FPU/g) a maximum hydrolysis of 86% was obtained in 96 h by Accellerase. The percentage hydrolysis of Solka Floc by Penicillium cellulase and Accellerase enzyme were comparable at all enzyme concentrations tested with a maximum saccharication of 59.96% and 52.39% at 96 h respectively. The hydrolysis pattern of CP-123 using cellulase from Penicillium and Accellerase enzyme shows that the rate of hydrolysis increased with increased enzyme concentration. Maximum hydrolysis of 60.18% and 32.57% were obtained for Penicillium cellulase and Accellerase enzyme at 20 FPU/g substrate in 96 h. The percentage hydrolysis of Sigmacell by Penicillium cellulase and Accellerase cellulase was 48.38% and 27.51%, respectively under similar experimental conditions with an enzyme substrate loading of 20 FPU/g. The end product analysis of the hydrolysate obtained after saccharication of cellulose derived from bagasse shows glucose as the major end product for both Penicillium cellulase (9.7%) and Accellerase enzyme (8.5%) with traces of xylose (data not shown).

Table 1 Different components of cellulase complex of Penicillium sp. and AccelleraseTM 1000. IU/ml FPU CMC PNPGase Penicillium 5 65 28 10 130 56 20 260 112 AccelleraseTM 1000 5 157.5 22.5 10 315 45 20 630 90

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

R. Singh et al. / Bioresource Technology 100 (2009) 66796681 Table 2 Enzymatic hydrolysis of cellulosic substrates by Penicillium sp. and Accellerase. Time (h) Substrate Penicillium sp. (FPU/g) 5 16 10 75.23 28.72 40.15 36.31 86.67 33.46 44.77 39.76 98.99 37.27 47.01 40.39 100.00 39.09 48.57 41.34 100.00 40.19 50.57 43.15 20 98.99 43.82 51.26 42.77 100.00 52.07 56.26 45.58 100.00 55.07 58.04 46.39 100.00 58.02 59.27 47.61 100.00 59.96 60.18 48.38 Accellerase (FPU/g) 5 13.83 18.32 13.25 6.72 15.28 23.32 15.60 8.51 20.15 25.05 15.90 9.13 20.33 26.72 16.16 10.08 21.25 29.02 17.26 11.16 10 39.05 27.22 16.71 11.03 45.95 30.21 21.08 13.12 56.90 32.82 22.48 13.45 58.21 35.55 23.87 13.91 60.38 37.66 25.23 14.52

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20 58.64 41.71 20.72 23.44 66.01 45.22 25.46 25.46 77.42 48.03 30.03 26.07 85.70 49.86 31.28 26.96 86.45 52.39 32.57 27.51

a-Cellulose
Solka Floc CP-123 Sigmacell

51.72 20.86 15.74 23.87 60.29 25.55 18.16 26.49 67.28 28.48 21.13 25.89 68.36 30.09 23.98 26.94 69.47 32.07 25.86 27.49

24

a-Cellulose
Solka Floc CP-123 Sigmacell

48

a-Cellulose
Solka Floc CP-123 Sigmacell

72

a-Cellulose
Solka Floc CP-123 Sigmacell

96

a-Cellulose
Solka Floc CP-123 Sigmacell

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

A number of lignocellulose pre-pretreatment technologies are under intensive investigations on both laboratory and at pilot plant scales (Wyman et al., 2005). Zhang et al. (2007) have pretreated pure cellulose and lignocellulosic materials using nonvolatile cellulose solvent (phosphoric acid). Pretreated Avicel and a-cellulose were completely converted to soluble sugars within 3 h at 10 g/litre substrate concentration wherein for herbaceous cellulose corn stower and Switch grass and hard wood lignocellulosics the pretreated cellulosic samples were hydrolyzed to 9697% at 24 h using a mixture of commercial cellulase (Genencore Spezyme) and b-glucosidases (Novozymes 188 bglucosidases).

enzyme preparation from P. pinophilum having high b-glucosidase activity. Acknowledgements MR thanks Dr. Raj Lad and Dr. S. Bade, Genencore for the Accellerase enzyme. Mr. Gyan Prakashs help in HPLC experiments is thankfully acknowledged. References
Ghose, T.K., 1987. Measurement of cellulase activities (recommendations of commission on biotechnology IUPAC). Pure Appl. Chem. 59 (2), 257268. Ghose, T.K., Bisaria, V.S., 1987. Measurement of hemicellulase activities, part 1: xylanases (recommendations of commission on biotechnology IUPAC). Pure Appl. Chem. 59 (12), 17391752. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100 (2), 1018. Kapoor, R.K., Chandel, A.K., Kuhar, S., Gupta, R., Kuhad, R.C., 2006. Bioethanol from crop residue, production forecasting and economics: an Indian perspective. In: Kuhad, R.C., Singh, A. (Eds.), Lignocellulosic Biotechnology: Current and Future Prospects. I.K. International, New Delhi, India, pp. 3244. Mandels, M., Weber, J., 1969. The production of cellulases. Adv. Chem. 95, 391414. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., 2000. Biotechnological potential of agro-industrial residues I: sugarcane bagasse. Bioresour. Technol. 74, 6980. Tabka, M.G., Herpoel-Gimbert, I., Monodb, F., Asther, M., Sigoillot, J.C., 2006. Enzymatic saccharication of wheat straw for bioethanol production by a combined cellulase xylanase and feruloyl esterase treatment. Enzyme Microb. Technol. 39, 897902. Wei, Sun Zhan, Chen, Zhang Hang, Hue, Yan Wang, Yu, Run Ma, 2006. Study on enzymatic hydrolysis of steam treated straw using a ball mill shaker. J. Beijing Univ. Chem. Technol. 33 (6), 2630. Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R., Lee, Y.Y., 2005. Coordinated development of leading biomass pretreatment technologies. Bioresour. Technol. 96, 19591996. Zhang, Y.-H.P., Ding, You Shi, Jonathan Meilenz, R., Cui, Bia Jing, Richard Elander, T., Laser, Mark, Michael Himmel, E., James McMillan, R., Lee Lynd, R., 2007. Fractionating recalcitrant lignocellulose at modest reaction condition. Biotechnol. Bioeng. 97 (2), 214223.

4. Conclusion The present studies were carried out to investigate the hydrolysis of cellulose derived from bagasse by a steam explosion pretreatment proprietary process developed at NCL and other pure celluloses by Penicillium cellulase. It was also of interest to compare its potential with commercially available cellulase (AccelleraseTM 1000) from Genencor. AccelleraseTM 1000 is a cellulase blend product with a high bglucosidase, capable of hydrolyzing lignocellulosic biomass to monosaccharides. The comparative studies using the Penicillium cellulase and AccelleraseTM 1000 have shown that the saccharifying potencies are comparable towards the treated substrates such as steam exploded bagasse and ball milled cellulose powder. However in case of microcrystalline cellulose and untreated cellulose powder (CP-123), the hydrolysis by Penicillium cellulase was much superior to that of Accellerase. It has been also demonstrated that the quantitative conversion of cellulose derived from steam exploded bagasse to major end product as glucose by using a single