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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 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 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

a b s t r a c t

A complete cellulase from Penicillium pinophilum was evaluated for the hydrolysis ofa-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 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 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 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.

1. Introduction

Lignocellulosic biomasses are considered as significant source

for the generation of sugar streams, organic products and fuel/eth-

anol. Cellulases, a group of enzymes which catalyze the hydrolysis

of 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 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, pre-

treatment 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 pre-

treatment 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 pro-

cess with minor modifications for the pretreatment of sugarcane

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 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 based

on 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 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 ligno-

celluloses (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

modified 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.

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2009.07.060

* Corresponding authors. Tel.: +91 20 25902720; fax: +91 20 25902648 (R. Seeta

Laxman), tel.: +91 20 25902228 (M. Rao).

E-mail addresses: [email protected] (R. Seeta Laxman), [email protected] (M.

Rao).

Bioresource Technology 100 (2009) 66796681

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h
http://dx.doi.org/10.1016/j.biortech.2009.07.060mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.biortech.2009.07.060


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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 (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 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 underpatenting (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 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 in

500 ml Erlenmeyer flask for 5 days on modified Mandels and We-

ber medium (Mandels and Weber, 1969) except that the levels of

ammonium sulphate and urea were five time higher and 2.5% cel-

lulose powder and 1% wheat bran were used as carbon source. The

culture 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-10

membrane (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)-56

U/ml. AccelleraseTM 1000 from a genetically modified T. reesei

was a kind gift from Genencore USA and had carboxyl methyl cel-

lulase (CMCase)-3150 U/ml, filter 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 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 according

to (Ghose and Bisaria, 1987). One unit of enzymeactivity is defined

as the amount of enzyme required to liberate one lM of reducingsugar per minute under the assay conditions.

2.5. Cellulose hydrolysis

The hydrolysis ofa-cellulose derived from bagasse (after steamexplosion), pulverized cellulose (CP-123), microcrystalline cellu-

lose (Sigmacell), Solka Floc (ball milled for 8 h) were carried out

using 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 and

20 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 stipulatedtime 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 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 using

Waters Sugar Pack Column with a mobile phase of Milli Q water

with 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. show

a high ratio of filter paper activity to CMCase activity. At a given

units of filter 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 con-verted 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 cel-

lulosic substrates at 10% substrate concentration by cellulases from

Penicillium and commercial Accellerase enzyme is compared in Ta-

ble 2. It was observed that the percentage hydrolysis ofa-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 enzyme

loadings 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 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 concentra-

tions tested with a maximum saccharification 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 Pen-

icillium cellulase and Accellerase enzyme at 20 FPU/g substrate in

96 h. The percentage hydrolysis of Sigmacell by Penicillium cellu-

lase and Accellerase cellulase was 48.38% and 27.51%, respectively

under 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 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 ofPenicillium sp. and AccelleraseTM 1000.

IU/ml Penicillium AccelleraseTM 1000

FPU 5 10 20 5 10 20

CMC 65 130 260 157.5 315 630

PNPGase 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) 66796681


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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 pre-

treated pure cellulose and lignocellulosic materials using non-

volatile cellulose solvent (phosphoric acid). Pretreated Avicel

and 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 wood

lignocellulosics the pretreated cellulosic samples were hydro-

lyzed to 9697% 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 pure

celluloses 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 to

monosaccharides. The comparative studies using the Penicillium

cellulase and AccelleraseTM 1000 have shown that the saccharify-

ing potencies are comparable towards the treated substrates such

as steam exploded bagasse and ball milled cellulose powder. How-

ever 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 ex-

ploded bagasse to major end product as glucose by using a single

enzyme preparation from P. pinophilum having high b-glucosidase

activity.

Acknowledgements

MR thanks Dr. Raj Lad and Dr. S. Bade, Genencore for the Accel-

lerase 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.

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combined cellulase xylanase and feruloyl esterase treatment. Enzyme Microb.

Technol. 39, 897902.

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Table 2

Enzymatic 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 41.71

CP-123 15.74 40.15 51.26 13.25 16.71 20.72

Sigmacell 23.87 36.31 42.77 6.72 11.03 23.44


CP-123 18.16 44.77 56.26 15.60 21.08 25.46

Sigmacell 26.49 39.76 45.58 8.51 13.12 25.46


CP-123 21.13 47.01 58.04 15.90 22.48 30.03

Sigmacell 25.89 40.39 46.39 9.13 13.45 26.07


CP-123 23.98 48.57 59.27 16.16 23.87 31.28

Sigmacell 26.94 41.34 47.61 10.08 13.91 26.96


CP-123 25.86 50.57 60.18 17.26 25.23 32.57

Sigmacell 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) 66796681 6681

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