Industrial Crops and Products 36 (2012) 584588

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Industrial Crops and Products

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Optimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 in simultaneous saccharication and fermentation of delignied sugarcane bagasse
J.R.A. Santos, M.S. Lucena, N.B. Gusmo, E.R. Gouveia
Department of Antibiotics - Federal University of Pernambuco, Cidade Universitria - CEP 50670-901, Recife, PE, Brazil

a r t i c l e

i n f o

a b s t r a c t
Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous saccharication and fermentation of delignied sugarcane bagasse. Temperature (32 C, 37 C), agitation (80; 100 rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v -glucosidase or 10 FPU/g cellulose and 5% -glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzymatic convertibility of cellulose and volumetric productivity were higher than 25 g/L, 72% and 0.70 g/L h, respectively, after 30 h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v -glucosidase or the culture medium 2 and 10 FPU/g cellulose/5% -glucosidase were used in SSF at 37 C and 80 rpm. In the SSF with culture medium 2 (supplemented with ammonium, phosphate, potassium and magnesium), 150 L ethanol/t bagasse was achieved, with minimum enzyme loading (10 FPU/g cellulose and 5%, v/v -glucosidase) for 8%, w/v of solids, which is often an important requirement to provide cost-efcient second generation ethanol processes. 2011 Elsevier B.V. All rights reserved.

Article history: Received 27 July 2011 Received in revised form 24 September 2011 Accepted 1 October 2011 Available online 13 December 2011 Keywords: Sugarcane bagasse Enzymatic hydrolysis Simultaneous saccharication and fermentation

1. Introduction Currently, there is growing interest in the use of lignocelluloses bioresources, including agro-industrial residues, such as sugarcane bagasse, in different processes as production ethanol and enzymes (Carrilo et al., 2005). Bagasse and sugarcane straw are lignocellulosic materials that have attracted interest from scientists in Brazil as potential sources for lignocellulosic ethanol production (Silva et al., 2010). There are several technologies available for the conversion of lignocellulosic materials into simple monomeric sugars. The main difference between those technologies is the catalyst used for the break-down of polysaccharides in the raw material (Kdr et al., 2004). Enzymatic hydrolysis can be used for obtaining fermentable sugars from polysaccharides contained in lignocellulosic biomass (Sun and Cheng, 2002). Simultaneous saccharication and fermentation (SSF) is a process scheme for integrating enzymatic hydrolysis into the overall cellulose to ethanol bioconversion process (Martn et al., 2002). The SSF is a more efcient process than separate hydrolysis and fermentation (SHF), since it reduces the accumulation of sugar and minimizes end-product inhibition (Brethauer and Wyman, 2010). Moreover, simultaneous saccharication and fermentation (SSF) technique provides the possibility of decreasing the production cost

Corresponding author. E-mail address: estergouveia@gmail.com (E.R. Gouveia). 0926-6690/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.10.002

(Kdr et al., 2004) and to reduce the risk of contamination (Wyman et al., 1992). The main microorganisms used for industrial ethanol production are yeasts. Saccharomyces cerevisiae, the yeast traditionally used for ethanol production, cannot metabolise xylose, the second most abundant sugar in lignocellulosic hydrolysates (HahnHgerdal et al., 2001). Saccharomyces strains require temperature lower than 35 C (Kdr et al., 2004). However, S. cerevisiae UFPEDA 1238 (Culture Collection of Department of Antibiotics of the Federal University of Pernambuco, Brazil) performed higher ethanol production at 37 C than at 30 C (Santos et al., 2010a). On the other hand, cellulases, which are frequently applied in the cellulose hydrolysis, have 50 C as the optimal temperature. At lower temperatures, the substantially lower hydrolysis rates would be unfavorable in terms of increased processing time (Kdr et al., 2004; Adsul et al., 2005). The optimal temperature for the yeast and the enzymes used differ, which means that the conditions used in SSF cannot be optimal for both the enzymes and the yeast (hgren et al., 2007). The task of hydrolyzing lignocellulose to fermentable monosaccharide is still technically problematic because the digestibility of cellulose is hindered by many physicalchemical, structural and compositional factors. The pretreatment is a necessary step to alter some structural characteristics of lignocelluloses, increasing glucan and xylan accessibility to the enzymatic attack. The combination of the composition of the substrate, type of pretreatment, and load and efciency of the enzymes used for the hydrolysis have a great inuence on biomass digestibility, although the individual impacts

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of these factors on the enzymatic hydrolysis are still unclear (Alvira et al., 2010). The aim of this study was to evaluate the ethanol production by S. cerevisiae UFPEDA 1238 in simultaneous saccharication and fermentation of delignied sugarcane bagasse (8%, w/v). The effects of temperature (32 C and 37 C), agitation (80 and 100 rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v -glucosidase; 10 FPU/g cellulose and 5%, v/v -glucosidase) and composition of culture medium were evaluated. 2. Material and methods 2.1. Raw material and delignication Sugarcane bagasse, pretreated by steam explosion at 200 C for 7 min on the pilot scale, was kindly provided by Department of Biotechnology of Engineering College of Lorena (University of Sao Paulo). A portion of the pretreated material was delignied with 1% (w/v) NaOH. The delignication reaction was made in a reactor Regmed (AUE/20), tted with mixing and heating systems, using a liquidsolid 1:10 (w/v). The operation was carried out at 100 C for 1 h. The content of polysaccharides and lignin in the raw material was determined by two-step analytical acid hydrolysis, according to the analytical procedure recommended Rocha et al. (1997) and validated for sugarcane bagasse by Gouveia et al. (2009). Polysaccharide content was calculated after the chromatographic quantication of sugars in the hydrolysates, and lignin was determined as the hydrolysis residue. 2.2. Enzymes and activities A commercial preparation of Trichoderma reesei cellulases (Celluclast 1.5L: 42.40 FPU/mL and 21.10 CBU/mL) and a -glycosidase (1340 CBU/mL) preparation (Novozym 188), both from Novozymes A/S (Bagsvrd, Denmark), kindly donated by the Department of Biotechnology of Engineering College of Lorena (University of So Paulo) were added. Enzyme activities expressed in lter paper units (FPU)/mL and in unit of cellobiose (CBU)/mL, were determined according to the method described by Ghose (1987). 2.3. Microorganism The industrial strain S. cerevisiae UFPEDA 1238 was used, which was kindly provided by the Culture Collection of Department of Antibiotics of the Federal University of Pernambuco, Brazil. This culture was maintained on the medium culture containing (in g/L): glucose (20), yeast extract (5), peptone (3) and agar (15), at pH 7.0. 2.4. Inoculum culture Pure yeast culture growth in culture medium described in Section 2.3, was added to a 500 mL Erlenmeyer ask, which contained 100 mL of following medium: glucose (20 g/L), yeast extract (5 g/L) and peptone (3 g/L) at pH 7.0. The Erlenmeyer ask was incubated in a rotary shaker at 30 C and 250 rpm. After 12 h, the cells suspension was ltrate through a 0.45 m lter. The ltrate was discarded and the cells were re-suspended in 10 mL sterile water and transferred to a 250 mL Erlenmeyer ask, containing 90 mL of fermentation medium. 2.5. Simultaneous saccharication and fermentation Simultaneous saccharication and fermentation was performed in 250 mL Erlenmeyer asks. Each Erlenmeyer ask contained 90 mL of fermentation medium (with the nutrients dissolved in

a sodium citrate buffer at 50 mM and pH 4.8) and 8 g of delignied bagasse. The Erlenmeyer asks were incubated in a rotary shaker at 50 C and 150 rpm. After a 6 h prehydrolysis, each Erlenmeyer ask was inoculated with yeast cells (described in Section 2.4) and incubated at 37 C and 80 rpm. Initial cell concentration was 1 g/L. Nutrients added were: (NH4 )2 SO4 1 g/L; K2 HPO4 0.5 g/L; MgSO4 7H2 O 0.25 g/L; yeast extract 2 g/L; peptone 1 g/L (culture medium 1) and (NH4 )2 SO4 2 g/L; KH2 PO4 2 g/L; MgSO4 7H2 O 0.75 g/L; yeast extract 4 g/L (culture medium 2). Enzyme loads of 10 or 20 FPU/g cellulose (Celluclast 1.5L) and 5 or 10%, v/v (of the volumetric Celluclast 1.5L addition) glucosidase were used. Temperature and agitation were kept at 37 C/80 rpm, 32 C/80 rpm and 37 C/100 rpm. These three conditions were chosen according to our previous study (Santos et al., 2010b). The experiments were performed in duplicates. The enzymatic convertibility of cellulose (ECC) was calculated based in ethanol concentration (Martn et al., 2008). ECC = Ef Ei Ci 0.57

where Ef is the nal ethanol concentration (g/L); Ei is the initial ethanol concentration (g/L); Ci , initial cellulose concentration (g/L). The factor 0.57 is the stoichiometric yield of ethanol from cellulose. 2.6. Chromatographic analysis Sugars, carboxylic acids, ethanol and furan aldehydes were quantied by HPLC (Agilent HP 1100, Germany). All samples were ltered through a 0.45 m lter. Cellobiose, glucose, arabinose, xylose, acetic acid, formic acid and ethanol were separated on an Aminex HPX-87H+ (Bio-Rad, Hercules, CA, USA) column at 50 C, using 5 mM H2 SO4 at a ow rate of 0.6 mL/min as mobile phase, and detected RI-detector (Agilent). Furfural and 5hydroxymethylfurfural (HMF) were separated on a C-18 column (Beckman) at 25 C, using 11.2/88.8 acetonitrile/1% (v/v) acetic acid mixture at a ow rate of 0.8 mL/min as mobile phase, and detected by their UV absorbance at 274 nm (Agilent). 3. Results and discussion Sugarcane bagasse, pretreated by steam explosion contained 49.89% cellulose, 7.99% hemicellulose and 34% lignin (Gouveia et al., 2009). Steam pretreated sugar cane bagasse was delignied for avoiding the inuence of lignin, since this compound forms a barrier to enzymatic attack (Chang and Holtzapple, 2000). The yield of cellulosic pulp recovered after the alkaline delignication was 50%. The pulp contained 81.8% cellulose, 6.4% hemicelluloses and 3.0% lignin. The content of cellulose in the solid fraction increased as a result of the solubilisation of lignin. In addition to the increase of cellulose content and decrease of lignin content, an enhancement of its enzymatic convertibility is expected since it has previously been reported that NaOH increased hardwood digestibility from 14 to 55% concomitantly with a reduction of lignin content from 2455% to 20% (Kumar et al., 2009). Steam explosion is the most widely employed physicalchemical pretreatment for lignocellulosic biomass. The auto-hydrolysis of acetyl groups present in hemicellulose (Alvira et al., 2010) is observed. The lignin is redistributed and to some extent removed from the material (Pan et al., 2005). Removal of hemicelluloses and the redistribution of lignin probably may have exposed the material and increased the delignication (91.10%). Ethanol concentration, volumetric productivity and the enzymatic convertibility of cellulose (ECC), were higher at 37 C and 80 rpm (Fig. 1), while in the other two SSF (32 C/80 rpm and

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Fig. 1. Ethanol concentration, ECC and QP , in each SSF run at 20 FPU/g cellulose, 10%, v/v -glucosidase and culture medium 1: A (32 C, 80 rpm); B (37 C, 80 rpm); C (37 C, 100 rpm).

37 C/100 rpm), were found similar values. Ethanol concentration, ECC and volumetric productivity, after 30 h (considering a 6 h prehydrolysis), reached 26.03 g/L, 74.30% and 0.88 g/L h, respectively, in SSF at 37 C and 80 rpm. Decreasing temperature (3732 C) or increasing agitation (80100 rpm) decreased the ECC (21.62%), the ethanol concentration (23.08%) and the volumetric productivity (22.70%). According to hgren et al. (2007), the temperature used in SSF cannot be optimal for enzymes and yeast. However, in our studies, 37 C was optimal temperature found to SSF of sugar cane bagasse by S. cerevisiae UFPEDA 1238. This strain presented higher ethanol concentration at 37 C than at 30 C or 45 C, when was performed fermentation with sucrose (Santos et al., 2010a). Analysis of variance by Origin 6.0 was performed with ethanol concentrations obtained at 28 h in the SSF runs, when the temperature and agitation were varied (SSF: A, B and C). These results were signicantly different (F = 247.93; = 0.05). However, the analysis of variance between the A SSF (32 C, 80 rpm) and C SSF (37 C, 100 rpm), showed that the ethanol concentrations were not significantly different (F = 0.0969; = 0.05). For evaluating decreasing enzyme load and of the composition of culture medium, experiments were carried out at 50 C and 150 rpm with 10 or 20 FPU/g cellulose and 5 or 10%, v/v -glucosidase, respectively. After 6 h, yeast suspension was inoculated and the temperature and agitation were reduced to 37 C and 80 rpm, respectively. SSF were carried out with two culture media according to composition described in Section 2.5. As can be seen in Fig. 2, decreasing enzyme loads (2010 FPU/g and 105%, v/v -glucosidase) also decreased (8.40%) the ECC, when the culture medium 1 was used in both SSF (B and E). However, when the enzyme load was reduced and the culture medium 2 was used, the ECC decreased 0.86% only (B SSF and D SSF). Decreasing enzyme loads also decreased the volumetric productivity about 20% (B SSF and E SSF) and 12% (B SSF and D SSF), respectively, when culture medium 1 or the culture medium 2 was used. On the other hand, the ethanol concentration increased 6.45% and decreased 8.45%, when the enzyme loads were reduced and the culture medium 2 (B SSF and D SSF) or 1 (B SSF and E SSF), respectively, were used. The enzymatic convertibility of cellulose (ECC) and the ethanol concentration were higher than 72% and 25 g/L, respectively, at 28 h, 37 C and 80 rpm, when the culture medium 1 (20 FPU/g cellulose and 10% -glucosidase) or the culture medium 2 (10 FPU/g cellulose and 5% -glucosidase) was used. Martn et al. (2008) found ECC higher than 80%, after 120 h, in SSF with S. cerevisiae or Mucor indicus strains. However, the ethanol concentrations achieved were

Fig. 2. Ethanol concentration, ECC and QP , in each SSF run at 37 C and 80 rpm: B (20 FPU/g cellulose, 10%, v/v -glucosidase, culture medium 1); D (10 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 2); E (10 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 1).

not higher than 20 g/L as a consequence of the low cellulose content of the raw material, although these authors have utilized high water insoluble solids content (10%). In ethanol production from lignocellulosic materials, ethanol concentration should be as high as possible in order to minimize the energy consumption in evaporation and distillation (Wingren et al., 2003). Increasing water insoluble solids content, in SSF, increases the glucose and ethanol concentrations. A water insoluble solid content of 8% was high enough to obtain reasonable ethanol concentration (higher than 25 g/L). Analysis of variance was performed with the ethanol concentration obtained at 28 h in the SSF runs, when the enzyme load and culture medium were varied (SSF: B, D and E). These results were signicantly different (F = 15.75; = 0.05). However, the analysis of variance between the B SSF (20 FPU/g cellulose, 10%, v/v -glucosidase, culture medium 1) and D SSF (10 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 2), showed that the maximum ethanol concentrations were not signicantly different (F = 7.08; = 0.05). Fast dissolution and ECC almost 40%, after a 6 h pre-hydrolysis at 50 C and 150 rpm was achieved for both enzyme loads. In SSF of trebol (Martn et al., 2008), was also found rapid dissolution after 6 h at 50 C. Santos et al. (2010b) observed that SSF of sugar cane bagasse without pre-hydrolysis is a slower process. Ethanol concentrations and volumetric productivities (QP ) in all conditions were higher than that found by hgren et al. (2007) at 35 C in isothermal SSF and Kdr et al. (2004) in isothermal SSF (40 C) or non-isothermal SSF (50 C during 24 h pre-hydrolysis and 30 C after inoculation of yeast). Martn et al. (2008) also obtained lower ethanol concentration and volumetric productivity in nonisothermal SSF (50 C during 6 h pre-hydrolysis and 32 C after inoculation of yeast). Table 1 shows a comparison between some ethanol concentrations and volumetric productivities found in literature and in the present work. Supplementation of culture medium with higher concentrations of ammonium, potassium, phosphorus and magnesium may have favorably inuenced the fermentation. S. cerevisiae uses nitrogen as ammonium, amide (urea) or amine (amino acids). Phosphorus is absorbed as H2 PO 4 , and the sulfur can be assimilated as sulfate (Lima et al., 2000). Martn et al. (2008) reported that high content of potassium and phosphorus in the culture medium was also favorably for the fermentation. In SSF, after 12 h, glucose was completely consumed in all ve conditions (20 FPU/g cellulose and 10%, v/v -glucosidase:

J.R.A. Santos et al. / Industrial Crops and Products 36 (2012) 584588 Table 1 Ethanol concentration using various substrates, microorganisms and enzyme load in isothermal and non-isothermal SSF. Microorganism K. marxianus S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae UFPEDA 1238
a b c d

587

SSFa T ( C) 40 40 35

N-SSFb T ( C) 50 and 30 50 and 30 50 and 32 50 and 37

Pre-hydrolysis (h) 24 24 68 6

Ethanolc (g/L) 17.8 16.0 16.6 15.1 20.5 23.7 27.71

QP d (g/L h) 0.25 0.22 0.23 0.21 0.17 0.20 0.77

Reference Kdr et al. (2004)

hgren et al. (2007) Toms-Pej et al. (2008) Present work (Fig. 2 SSF D)

Isothermal. Non-isothermal. Ethanol concentration. Volumetric productivity.

200

Yield (L Ethanol/ton bagasse)

150

100

50

0 A B C D E

SSF
Fig. 3. Yield (L ethanol/t bagasse) under ve different conditions: A (32 C, 80 rpm, 20 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 1); B (37 C, 80 rpm, 20 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 1); C (37 C, 100 rpm, 20 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 1); D (37 C, 80 rpm, 10 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 2); E (37 C, 80 rpm, 10 FPU/g cellulose, 5%, v/v -glucosidase, culture medium 1).

32 C/80 rpm; 37 C/80 rpm; 37 C/100 rpm - 10 FPU/g cellulose and 5%, v/v -glucosidase: 37 C/80 rpm/culture medium 1; 37 C/80 rpm/culture medium 2). In isothermal (40 C) SSF of Solka Floc 2000 the glucose concentration stayed at around 35 g/L during 72 h (Kdr et al., 2004), when S. cerevisiae or Kluyveromyces marxianus were utilized. These authors reported that the cell may have been suffered with this temperature and that it was especially unexpected with the thermo tolerant K. marxianus, which was thought to be performing much better at 40 C than S. cerevisiae. Cellobiose concentrations were lower than 0.2 g/L, after 12 h, utilizing 10 or 5%, v/v -glucosidase. hgren et al. (2007) performed SSF with 25%, v/v -glucosidase to hydrolysis all cellobiose. On the other hand, Chen et al. (2007), when not supplementing the hydrolysis with -glucosidase, observed a severe inhibition of cellulases activity due to the accumulation of cellobiose (7.4 g/L). When these authors supplemented with -glucosidase (6.5 CBU/g substrate), the concentration of cellobiose decreased to 0.6 g/L. Fig. 3 shows a comparison of ethanol yield from raw material. Ethanol volume (in L) in relation to bagasse mass (in ton) was calculated considering the recovering of solids after the alkaline delignication (50%) and steam explosion (68%). In SSF with culture medium 2, enzyme load 10 FPU/g cellulose and 5%, v/v -glucosidase (D), yield higher than 150 L EtOH/t bagasse was achieved. This represents 100 L EtOH/t sugarcane, since each ton of cane generates 2/3 of bagasse.

4. Conclusions The adequate combination of conditions of pre-hydrolysis (6 h at 50 C and 15 rpm), temperature (37 C), agitation (80 rpm), composition of culture medium (with higher concentration of nutrients) and enzyme load (10 FPU/g cellulose and 5%, v/v -glucosidase) was a successful method to ethanol production by S. cerevisiae UFPE 1238 in SSF of delignied sugarcane bagasse. High yield ethanol with minimum enzyme loads and lower time (34 h, considering a 6 h pre-hydrolysis) was achieved, which is often an important requirement to provide cost-efcient second generation ethanol processes.

Acknowledgements The authors acknowledge the nancial support from Conselho Nacional de Desenvolvimento Cientco e Tecnolgico, Brasilia DF, Brazil (CNPq) and from Fundac o de Amparo Cincia e Tecnologia do Estado de Pernambuco (FACEPE).

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