Fuel 116 (2014) 699702

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Fuel
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Enzymatic hydrolysis of microwave alkali pretreated rice husk for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture
Anita Singh a,b,, Somvir Bajar a, Narsi R. Bishnoi a
a b

Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India Department of Environmental Science, Central University of Jammu, Jammu and Kashmir, India

h i g h l i g h t s
 Rice husk used as feedstock for ethanol production.  Crude unprocessed enzymes were used for saccharication.  Concentrated enzymatic hydrolyzate used for ethanol production by yeast.  Fermentation was accomplished by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture.

a r t i c l e

i n f o

a b s t r a c t
Rice husk is one of the abundant lignocellulosic feed stocks in the world. It contains mixtures of pentose and hexose sugars, so selected for ethanol production. The microwave alkali pretreated rice husk enzymatically hydrolyzed with crude unprocessed hydrolytic enzymes. Concentrated enzymatic hydrolyzate having initial reducing sugar concentrations of 10 g/L, 25 g/L, 50 g/L and 70 g/L were used for ethanol production. Fermentation was accomplished by the action of yeast Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture. Ethanol yield obtained with S. cerevisiae were 0.3 g/g, 0.35 g/g, 0.40 g/g and 0.39 g/g; with S. stipitis were 0.24 g/g, 0.27 g/g, 0.36 g/g and 0.35 g/g; and with co-culture of both were 0.33 g/g, 0.36 g/g, 0.42 g/g and 0.40 g/g with different sugar concentrations. Maximum ethanol yield was found to be 0.42 g/g with co-culture, 0.40 g/g with S. cerevisiae and 0.36 with S. stipitis at initial reducing sugar concentration of 50 g/L. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 6 January 2012 Received in revised form 15 August 2013 Accepted 27 August 2013 Available online 5 September 2013 Keywords: Rice husk Saccharomyces cerevisiae Scheffersomyces stipitis Hydrolyzate Fermentation

1. Introduction Lignocelluloses from agricultural, industrial and forest residuals account for the majority of the total biomass present in the world and are regarded as the largest known renewable carbohydrate for ethanol production [1]. The key requirements for an economical lignocellulosic ethanol process include: efcient pretreatment methods of lignocelluloses, availability of low-cost hydrolytic enzymes, and use of optimal microbial strains capable of converting hexose and pentose sugars to ethanol at high rates, yields, and nal concentrations [2]. Rice husk is agricultural waste, accounting for about one fth of the annual gross rice, 545 million metric tons, of the world [3]. Rice husk predominantly contains hemicellulose
Corresponding author at: Department of Environmental Sciences, Central University of Jammu, Jammu-180011, Jammu and Kashmir, India. Tel.: +91 9354312123/+91 9419294912; fax: +91 1662 276240. E-mail addresses: anitasaharan@gmail.com (A. Singh), nrbishnoi@gmail.com (N.R. Bishnoi).
0016-2361/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fuel.2013.08.072

and cellulose and these can be hydrolyzed into sugars at approximately 0.426 g of sugars per 1 g of dry rice husk [4]. With further process the sugars can be fermented into ethanol at about 0.21 g of ethanol per 1 g of dry rice husk [5]. However, due to the close association of cellulose and hemicellulose with lignin in the plant cell wall, pre-treatment is necessary to make these carbohydrates available for enzymatic hydrolysis and fermentation [6]. A prerequisite to the biological conversion of lignocellulosic biomass to ethanol is to release of the cellulose portion (and subsequently glucose) from the tightly woven lignocellulosic structure [7]. Pretreatment includes physical, chemical, biological and thermal methods and their combinations. High temperature acid treatment can hydrolyze cellulose to yield sugars, this process will result in formation of degradation products like 5-hydroxymethyl furfural which will interfere with microbial fermentation [6]. Pretreatment enhance the accessibility of enzymes and digestibility of holocellulose components because of the salvation and the saphonication during alkaline pretreatment [8]. Alkali pretreatment is a typical chemical pretreatment method and a combination of microwave

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irradiation with alkali may be an alternative pretreatment approach for lignocellulosic materials at lower temperature to solve the pretreatment problem [8]. The cellulose component resulted from the pre-treatment process can be converted to ethanol in a two-step process where the cellulose is rst converted to glucose sugars by hydrolysis; the resulting sugars can in turn be converted to ethanol by fermentation [9]. The hydrolysis of polysaccharides is usually catalyzed by hydrolytic enzymes, because enzymatic hydrolysis produces better yields than acid-catalyzed hydrolysis [7]. Cellulases are mixture of at least three different enzymes: endoglucanase (EG, endo-1,4d-glucanohydrolase, or EC 3.2.1.4.) which attacks regions of low crystallinity in the cellulose ber, creating free chain-ends; exoglucanase or cellobiohydrolase (CBH, 1,4-b-d-glucan cellobiohydrolase, or EC 3.2.1.91.) which degrade the molecule further by removing cellobiose units from the free chain-ends; and b-glucosidase (EC 3.2.1.21.) which hydrolyzes cellobiose to produce glucose, in much smaller amounts [10]. However, the most widely used cellulase from Trichoderma reesei is poor in cellobiase, and thus restricts the conversion of cellobiose to glucose [9]. The accumulation of cellobiose will cause severe feedback inhibition to the cellulase reaction [11]. Therefore, cellulase complex system is crucial to raise enzymatic hydrolysis yield. Glucose and xylose are the two dominant sugars in lignocellulosic hydrolyzates after saccharication. Both need to be fermented efciently, but current approaches are inefcient, since no native microorganisms can convert all sugars into ethanol at high yield [1]. The lack of industrially robust microbes for co-fermentation of glucose and xylose has been a major technical barrier. Researchers have taken two different approaches to solve this problem either by using genetically engineered microbes or co-culture of microbes [2]. The present study conducted with enzymatic hydrolysis of microwave alkali pretreated rice husk biomass by crude enzymes produced in-house by Aspergillus heteromophus. Enzymatic hydrolyzate was fermented by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture for ethanol production. 2. Methods 2.1. Material and microorganism

buffer was supplemented with crude enzyme (activities of exoglucanase 8.2 FPU/g, endoglucanase 198.2 IU/g, xylanase 134.3 IU/g and b-glucasidase 147.8 IU/g [8]). Samples were withdrawn at various intervals, centrifuged and supernatant analyzed for total reducing sugar released. The hydrolyzate was concentrated by evaporation in rotatory evaporator to reducing sugar content of 10 g/L, 25 g/L, 50 g/L and 70 g/L. 2.3. Fermentation Concentrated enzymatic hydrolyzate of different sugar concentration was used for ethanol production. The hydrolyzate was inoculated with 2% v/v of a 48 h old seed culture of S. cerevisiae, S. stipitis and co-culture (1:1) of both yeast. Incubation was carried out in stoppered asks at room temperature (28 2 C) without agitation. 5 ml samples were withdrawn after 36 h (optimized fermentation time) [9], centrifuged for 10 min at 4 C at 10,000 rpm and supernatant was analyzed for ethanol and residual reducing sugar contents. 2.4. Analytical methods Total reducing sugar in enzymatic hydrolyzate of biomass was estimated by DNS method [12]. The enzymatic hydrolyzate was concentrated by the methods of Dehkhoda et al. [13]. The estimation of ethanol was done by Spectrophotometer [14]. 2.5. Fermentation parameters The ethanol volumetric productivity (Qp) (gp/l/h) was calculated as the ratio of ethanol concentration (g/L) at the end of the run to the fermentation time (t, h). The yield of ethanol (Yp/s) to consumed sugar (gp/gs) was dened as ratio of ethanol concentration to the sugar consumption (SoSf, S0 initial sugar concentration and Sf nal sugar concentration). Sugar conversion (%) calculated as a ratio of sugar consumption to the initial sugar concentration. The efciency of sugar conversion to ethanol (g, %) has been estimated by the ratio of ethanol yield to theoretical value of ethanol yield (0.51 g/g). 3. Results and discussion

Reducing sugar relaesed (g/L)

Microwave alkali pretreated rice husk were used for present study [8]. Yeast S. cerevisiae MTCC 174 was purchased from Institute of Microbial Technology, Chandigarh (IMTEC), India and S. stipitis NCIM No. 3497 from National Collection of Industrial Microorganism (NCIM) National Chemical laboratory, Pune, Maharashtra, India. The yeast cultures was maintained on YEPD agar slants and sub-cultured weekly. Yeast for inoculation was grown in 1 L Erlenmeyer asks lled with 400 mL of medium containing 50 g/L glucose, 20 g/L peptone and 10 g/L yeast extract. After incubation at 30 C and 150 rpm for 48 h, the cell suspension was aseptically collected by centrifugation (7200 g; 10 min) at 4 C and suspended in 0.9% NaCl for further use. 2.2. Enzymatic hydrolysis Enzymatic hydrolysis was done by crude unprocessed enzyme [9]. Enzymatic hydrolysis of microwave alkali pretreated rice husk (50 g) was carried out in 3 L bioreactor (BioAge, Mohali, India) (equipped with agitator for stirring and temperature controller) containing 1.5 L citrate buffer (50 mM, pH 5.0 0.2, 50 0.5 C) at 100 rpm. The cellulosic substrate was soaked in citrate buffer for 2 h before adding enzymes. Sodium azide was also added at a concentration of 0.005% to restrict any microbial growth during the course of enzymatic hydrolysis. The substrate soaked in citrate

3.1. Enzymatic hydrolysis and its concentration Pretreated rice husk used in present study contains 46% cellulose and 20% hemicelluloses [8]. The sugar content production depends on the enzyme used for hydrolysis. The data of the reducing sugar concentration resulted from the enzyme hydrolysis of the alkali pre-treated rice husk using hydrolytic enzymes from A. heteromorphus was shown in Fig. 1 Enzymatic hydrolysis using
16 14 12 10 8 6 4 2 0 12 24 48 72 96 120

Incubation time (h)

Fig. 1. Reducing sugar production during enzymatic hydrolysis over time.

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cellulases does not generate inhibitors like in acid hydrolysis and the enzymes are very specic for cellulose [7]. Maximum reducing sugar 13.5 g/L was obtained at 72 h after that decrease observed. The microwave pretreated biomass with alkali has a lower degree of crystallinity which enhances the enzymatic hydrolysis and hence facilitates the conversion of the cellulose into reducing sugars [8]. This higher increase of reducing sugars in alkali pretreated substrates may also be attributed to the release of lignin moieties during alkali treatment which in turn enhanced accessibility of cellulose to the enzymes [15]. Saha et al. [16] studied enzymatic saccharication of alkaline peroxide pretreated rice hulls and found sugar yield 428 12 mg/g (90% yield). Saha et al. [17] studied enzymatic saccharication of lime pretreated rice hulls and found the maximum yield of monomeric sugars 154 71mg g1 (32% yield). Concentration of rice husk hydrolyzate was carried out to increase the sugar concentration for ethanol fermentation. Concentration of rice husk hydrolyzate was carried out by rotatory evaporator (vacuum distillation unit) from 13.5 g/L to 70 g/L. The hydrolyzate was evaporated to with the main purpose of increasing the sugar content [18]. The concentrated hydrolyzate was used for ethanol production with initial reducing sugar concentrations of 10 g/L, 25 g/L, 50 g/L and 70 g/L. 3.2. Ethanol production by mono-culture of yeast Glucose and xylose are two main dominant sugars in lignocellulosic hydrolyzate. 1070 g/L reducing sugar concentration was used for fermentation by S. cerevisiae, S. stipitis and co-culture of both for ethanol production. The sugar consumptions seemed to depend on the yeasts abilities to utilize the different sugars in the enzymatic hydrolyzates [2]. Ethanol production by S. cerevisiae with variation of reducing sugar concentration was shown in Table 1. Hexose and pentose sugars are the primary reactant in yeast metabolism. When reducing sugar concentration increased from 10 to 25 g/L, ethanol conc. enhanced by 57%, and when sugar conc. increased from 25 to 50 g/L, ethanol enhanced by 56% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 42 %. Under fermentative condition, the rate of ethanol production is related to the available sugar concentration [19,20]. At very low substrate concentration, the yeast starved and productivity decreases [21]. An important secondary effect of higher sugar content is catabolite repression of the oxidative pathways [22]. Ethanol yield to consumed sugar was shown in Table 1 and highest yield was obtained with reducing sugar level 50 g/L. The result of ethanol yield of this study was in agreement with previous report on fermentation of lignocellulosic hydrolyzates using yeast. Meghawati et al. [4] studied maximum ethanol concentration 2.63% (v/v) indicating 92.18% ethanol yield from rice husk using Bakers yeast. Latif and Rajoka [23] conducted simultaneous and saccharication and fermentation of corn cobs to produce ethanol with yield of 0.42 g/g (2.1% v/v and 82.20 % ethanol yield)

using S. cerevisiae. Fermentation of glucosexylose mixtures of lignocellulosic biomass using S. cerevisiae by Govindaswamy and Vane [24] produced ethanol yield of 0.49 g/g (2.45% v/v and 95.89% ethanol yield). Ethanol yield obtained from Lantana camara hydrolyzate (34.75 1.54 g/L sugars) using yeast was 17.7 0.96 g/L (1.87% v/v and 93.93% ethanol yield) [25]. Saha et al. [16] studied enzymatic saccharication of rice hulls and found ethanol concentration was 8.0 0.2 g/L with a yield of 0.20 g/g hulls in the case of simultaneous saccharication and fermentation by the E. coli strain. Xylose fermentation has been identied as an important factor for economically viable bioethanol production due to the large proportion of xylose in lignocellulosic material [26]. The yeasts used for industrial fermentations (Saccharomyces sp.) are not capable of fermenting xylose. The successful fermentation of xylose would increase the ethanol production [1]. So, S. stipitis used for the production of ethanol from lignocellulosic hydrolyzate. Table 2 showed that ethanol production from rice husk hydrolyzate by S. stipitis. Table 2 shows that when reducing sugar concentration increased from 10 to 25 g/L, ethanol conc. enhanced by 60%, and when sugar conc. increased from 25 to 50 g/L, ethanol enhanced by 57% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 41 %. Ethanol yield to consumed sugar was shown in Table 2 and highest yield was obtained with reducing sugar level 50 g/L. 3.3. Ethanol production by co-culture of yeast Co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature [1,2]. According to a US Department of Energy (DOE) study, co-culture bioconversion is a very plausible and potentially high-payoff opportunity for ethanol production [27]. Table 3 shows that ethanol production from rice husk hydrolyzate by co-culture of S. cerevisiae and S. stipitis. Table 3 showed that when reducing sugar concentration increased from 10 to 25 g/L, ethanol conc. enhanced by 54.5%, and when

Table 2 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with S. stipitis. Initial sugar (gs/l) Residual sugar (gs/l) Sugar consumption (%) Ethanol (gp/l) Qp (gp/ l/h) Yp/ s (gp/ gs) 0.24 0.27 0.36 0.35 Efciency of sugar conversion to ethanol (%) 47.6 52.9 70.5 68.6

10 25 50 70 Time 36 h.

5 14 30 36

50 44 40 48.5

1.2 0.1 3.0 0.5 7.1 0.9 12.2 1.2

0.03 0.08 0.20 0.36

Table 1 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with S. cerevisiae. Initial sugar (gs/l) Residual sugar (gs/l) Sugar consumption (%) Ethanol (gp/l) Qp (gp/ l/h) Yp/ s (gp/ g s) 0.3 0.35 0.40 0.39 Efciency of sugar conversion to ethanol (%) 58.8 68.6 78.4 76.5

Table 3 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with co-culture of S. cerevisiae and S. stipitis. Initial sugar (gs/l) Residual sugar (gs/l) Sugar consumption (%) Ethanol (gp/l) Qp (gp/ l/h) Yp/ s (gp/ g s) 0.33 0.36 0.42 0.40 Efciency of sugar conversion to ethanol (%) 64.7 70.5 82.3 78.4

10 25 50 70 Time 36 h.

5 15 30 34

50 40 40 51.4

1.5 0.1 3.5 0.5 8.1 1.1 14.0 1.3

0.04 0.10 0.22 0.36

10 25 50 70 Time 36 h.

2.5 10 20 18

75 60 60 74.3

2.5 0.1 5.5 0.5 12.6 1.1 20.8 1.5

0.06 0.15 0.31 0.54

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A. Singh et al. / Fuel 116 (2014) 699702 [8] Singh A, Tuteja S, Singh N, Bishnoi NR. Enhanced saccharication of rice straw and hulls by microwave-alkali pretreatment and lignocellulolytic enzyme production. Bioresour Technol 2011;102:177382. [9] Singh A, Bishnoi NR. Optimization of Ethanol production from microwave alkali pretreated rice straw using statistical experimental designs by Saccharomyces cerevisiae. Ind Crops Prod 2012. http://dx.doi.org/10.1016/ j.indcrop.2011.12.033. [10] Gottschalk LMF, Oliveira RO, Bon EPS. Cellulases, xylanases, b-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse. Biochem Eng J 2010;51:728. [11] Fang H, Zhao C, Song XY. Optimization of enzymatic hydrolysis of steamexploded corn stover by two approaches: Response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02. Bioresour Technol 2010;101:41119. [12] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:4268. [13] Dehkhoda A, Tomas B, Taherzadeh MJ. Comparison of vacuum and high pressure evaporated wood hydrolyzate for ethanol production by repeated fed batch using occulating Saccharomyces cerevisiae. BioResources 2008;4(1): 30920. [14] Caputi AJ, Ueda M, Brown T. Spectrophotometric determination of ethanol in wine. Am. J. Enol. Vitti. 1968;19:1605. [15] Gupta R, Khasa YP, Kuhad RC. Evaluation of enzymatic saccharication of cellulosic materials. Carbohyd polym 2011;84:11039. [16] Saha BC, Cotta MA. Enzymatic saccharication and fermentation of alkaline peroxide pretreated rice hulls to ethanol. Enzyme Microb Technol 2007;41:52832. [17] Saha BC, Cotta MA. Lime pretreatment, enzymatic saccharication, and fermentation of rice hulls to ethanol. Biomass Bioenerg 2008;32:9717. [18] Yadav KS, Naseeruddin S, Prashanthi GS, Sateesh L, Rao LV. Bioethanol fermentation of concentrated rice straw hydrolyzate using co-culture of Saccharomyces cerevisiae and Pichia stipitis. Bioresour Technol 2011;102: 64738. [19] Park YK, Sato HH. Fungal invertase as an aid for fermentation of cane molasses into ethanol. Appl Environ Microbiol 1982;44:9889. [20] Atiyeh H, Duvnjak Z. Production of fructose and ethanol from cane molasses using Saccharomyces cerevisiae ATCC 36858. Acta Biotechnol 2001;23(1): 3748. [21] Levenspiel O. The Monod equation: a revisit and a generalization and product inhibition situations. Biotechnol Bioeng 1980;22:1671. [22] Moss FJ, Rickard PAD, Bush FE, Caiger P. The response by microorganisms to steady-state growth in controlled concentrations of oxygen and glucose. II. Saccharomyces carlsbergensis. Biotechnol Bioeng 1971;13:6375. [23] Latif F, Rajoka MI. Production of ethanol and xylitol from corn cobs by yeasts. Bioresour Technol 2001;77:57. [24] Govindaswamy S, Vane LM. Multi-stage continuous culture fermentation of glucosexylose mixtures to fuel ethanol using genetically engineered Saccharomyces cerevisiae 424A. Bioresour Technol 2010;101:127784. [25] Kuhad RC, Gupta R, Khasa YP, Singh A. Bioethanol production from Lantana camara (red sage): Pretreatment, saccharication and fermentation. Bioresour Technol 2010;101:834854. [26] Von Sivers M, Zacchi G. Ethanol from lignocellulosics: a review of the economy. Bioresour Technol 1996;56:13140. [27] Department of Energy. From biomass to biofuels: a roadmap to the energy future, based on a workshop, Rockville; 79 December 2005. ski Z. Ethanol production on the media [28] Kordowska-Wiater M. Targon containing glucose and xylose by coculture of Pichia stipitis CCY39501 and respiratory decient mutant of Saccharomyces cerevisiae V30. Electr J Pol Agric Univ Food Sci Technol 2001;4(2):1528. [29] Rouhollah H, Iraj N, Giti E, Sorah A. Mixed sugar fermentation by Pichia stipitis, Saccharomyces cerevisiae, and an isolated xylose-fermenting Kluyveromyces marxianus and their cocultures. Afr J Biotechnol 2007;6(9):11104. [30] Taniguchi M, Itaya T, Tohma T, Fujii M. Ethanol production from a mixture of glucose and xylose by co-culture of Pichia stipitis and a respiratory-decient mutant of Saccharomyces cerevisiae. J Ferment Bioeng 1997;83:36470. [31] Taniguchi M, Itaya T, Tohma T, Fujii M. Ethanol production from a mixture of glucose and xylose by a novel coculture system with two fermentors and two microltration modules. J Ferment Bioeng 1997;84(1):5964.

sugar conc. increased from 25 to 50 g/L, ethanol enhanced by 56.3% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 39%. Ethanol yield to consumed sugar was shown in Table 3 and highest yield was obtained with reducing sugar level 50 g/L. Kordowaska-Wiater et al. [28] studied fermentation of glucose/xylose mixture by co-culture of P. stipitis CCY39502 and S. cerevisiae V30 under batch fermentation ethanol yield 0.39 g/g and ethanol productivity 0.39 g/L/h. Rouhollah et al. [29] studied fermentation of glucose/ xylose mixture by co-culture of P. stipitis CCUG18492 and S. cerevisiae under batch fermentation and obtained ethanol productivity 0.41 g/L/h. Taniguchi et al. [30] found ethanol productivity 0.39 g/L/h under batch fermentation by co-culture of P. stipitis CBS5773 and S. cerevisiae No. 7 using glucose/ xylose mixture. Taniguchi et al. [31] reported ethanol production by co-culture of P. stipitis and a respiratorydecient mutant of S. cerevisiae of variable concentrations in synthetic medium containing glucose and xylose under batch fermentation and found ethanol productivity of 0.50 g/L/h. Co-culture fermentation provides the opportunity to achieve simultaneous conversion of glucose and xylose, maximize substrate utilization rate, increase ethanol yield and production rate, and reduce process costs [2].

4. Conclusions Crude unprocessed on-site produced enzymes were used for hydrolysis of microwave alkali pretreated rice husk. The reducing sugar produced during hydrolysis were concentrated and used for ethanol production by S. cerevisiae and S. stipitis and their coculture. Highest ethanol production with co-culture was 20.8 g/L and co-culture of S. cerevisiae and S. stipitis produced 32% more ethanol than S. cerevisiae alone and 41% more ethanol than S. stipitis alone. Rice husk is an abundant agricultural residue with low commercial value, can be used for ethanol production.

References
[1] Singh A, Bishnoi NR. Enzymatic hydrolysis optimization of microwave alkali pretreated wheat straw and ethanol production by yeast. Bioresour Technol 2011. http://dx.doi.org/10.1016/j.biortech.2011.12.084. [2] Chen Y. Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review. J Ind Microbiol Biotechnol 2011;38:58197. [3] Abbas A, Ansumali S. Global Potential of Rice husk as a renewable feedstock for ethanol biofuel production. Bioenerg Res 2010;3:32834. [4] Megawati, Sediawan WB, Sulistyo H, Hidayat M. Kinetics of sequential reaction of hydrolysis and sugar degradation of rice husk in ethanol production: Effect of catalyst concentration. Bioresour Technol 2011;102:20627. [5] Demirbas A. Bioethanol from cellulosic materials: a renewable motor fuel from biomass. Energy Sour 2005;2005(27):32737. [6] Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:67386. [7] El-Zawawy WK, Ibrahima MM, Abdel-Fattahb YR, Soliman NA, Mahmoud MM. Acid and enzyme hydrolysis to convert pretreated lignocellulosic materials into glucose for ethanol production. Carbohyd Polym 2011;84:86571.