Os

JD

a

ARR2AA

KSESf

1

bb(lae

ldb2s(

ccSmmMt

0d

Industrial Crops and Products 36 (2012) 584–588

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l homepage: www.e lsev ier .com/ locate / indcrop

ptimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 inimultaneous saccharification and fermentation of delignified sugarcane bagasse

.R.A. Santos, M.S. Lucena, N.B. Gusmão, E.R. Gouveia ∗

epartment of Antibiotics - Federal University of Pernambuco, Cidade Universitária - CEP 50670-901, Recife, PE, Brazil

r t i c l e i n f o

rticle history:eceived 27 July 2011eceived in revised form4 September 2011ccepted 1 October 2011vailable online 13 December 2011

a b s t r a c t

Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous sacchar-ification and fermentation of delignified 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 and5% �-glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzy-matic convertibility of cellulose and volumetric productivity were higher than 25 g/L, 72% and 0.70 g/L h,

eywords:ugarcane bagassenzymatic hydrolysisimultaneous saccharification andermentation

respectively, after 30 h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v �-glucosidase or theculture medium 2 and 10 FPU/g cellulose/5% �-glucosidase were used in SSF at 37 ◦C and 80 rpm. In theSSF 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-efficientsecond generation ethanol processes.

. Introduction

Currently, there is growing interest in the use of lignocellulosesioresources, including agro-industrial residues, such as sugarcaneagasse, in different processes as production ethanol and enzymesCarrilo et al., 2005). Bagasse and sugarcane straw are lignocellu-osic materials that have attracted interest from scientists in Brazils potential sources for lignocellulosic ethanol production (Silvat al., 2010).

There are several technologies available for the conversion ofignocellulosic materials into simple monomeric sugars. The mainifference between those technologies is the catalyst used for thereak-down of polysaccharides in the raw material (Kádár et al.,004). Enzymatic hydrolysis can be used for obtaining fermentableugars from polysaccharides contained in lignocellulosic biomassSun and Cheng, 2002).

Simultaneous saccharification and fermentation (SSF) is a pro-ess scheme for integrating enzymatic hydrolysis into the overallellulose to ethanol bioconversion process (Martín et al., 2002). TheSF is a more efficient process than separate hydrolysis and fer-entation (SHF), since it reduces the accumulation of sugar and

inimizes end-product inhibition (Brethauer and Wyman, 2010).oreover, simultaneous saccharification and fermentation (SSF)

echnique provides the possibility of decreasing the production cost

∗ Corresponding author.E-mail address: estergouveia@gmail.com (E.R. Gouveia).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.10.002

© 2011 Elsevier B.V. All rights reserved.

(Kádár et al., 2004) and to reduce the risk of contamination (Wymanet al., 1992).

The main microorganisms used for industrial ethanol produc-tion are yeasts. Saccharomyces cerevisiae, the yeast traditionallyused for ethanol production, cannot metabolise xylose, thesecond most abundant sugar in lignocellulosic hydrolysates (Hahn-Hägerdal et al., 2001).

Saccharomyces strains require temperature lower than 35 ◦C(Kádár et al., 2004). However, S. cerevisiae UFPEDA 1238 (CultureCollection of Department of Antibiotics of the Federal Universityof Pernambuco, Brazil) performed higher ethanol production at37 ◦C than at 30 ◦C (Santos et al., 2010a). On the other hand, cel-lulases, which are frequently applied in the cellulose hydrolysis,have 50 ◦C as the optimal temperature. At lower temperatures, thesubstantially lower hydrolysis rates would be unfavorable in termsof increased processing time (Kádár 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 forboth the enzymes and the yeast (Öhgren et al., 2007).

The task of hydrolyzing lignocellulose to fermentable monosac-charide is still technically problematic because the digestibility ofcellulose is hindered by many physical–chemical, structural andcompositional factors. The pretreatment is a necessary step to altersome structural characteristics of lignocelluloses, increasing glucan

and xylan accessibility to the enzymatic attack. The combination ofthe composition of the substrate, type of pretreatment, and loadand efficiency of the enzymes used for the hydrolysis have a greatinfluence on biomass digestibility, although the individual impacts

ops an

oe

bfomcm

2

2

7BP1Rl1

wiaPqm

2

l(ABP(a

2

wAcg

2

t1aawttm

2

i9

J.R.A. Santos et al. / Industrial Cr

f these factors on the enzymatic hydrolysis are still unclear (Alvirat al., 2010).

The aim of this study was to evaluate the ethanol productiony S. cerevisiae UFPEDA 1238 in simultaneous saccharification andermentation of delignified sugarcane bagasse (8%, w/v). The effectsf temperature (32 ◦C and 37 ◦C), agitation (80 and 100 rpm), enzy-atic load (20 FPU/g cellulose and 10%, v/v �-glucosidase; 10 FPU/g

ellulose and 5%, v/v �-glucosidase) and composition of cultureedium were evaluated.

. Material and methods

.1. Raw material and delignification

Sugarcane bagasse, pretreated by steam explosion at 200 ◦C formin on the pilot scale, was kindly provided by Department ofiotechnology of Engineering College of Lorena (University of Saoaulo). A portion of the pretreated material was delignified with% (w/v) NaOH. The delignification reaction was made in a reactoregmed (AUE/20), fitted with mixing and heating systems, using a

iquid–solid 1:10 (w/v). The operation was carried out at 100 ◦C forh.

The content of polysaccharides and lignin in the raw materialas determined by two-step analytical acid hydrolysis, accord-

ng to the analytical procedure recommended Rocha et al. (1997)nd validated for sugarcane bagasse by Gouveia et al. (2009).olysaccharide content was calculated after the chromatographicuantification of sugars in the hydrolysates, and lignin was deter-ined as the hydrolysis residue.

.2. Enzymes and activities

A commercial preparation of Trichoderma reesei cellulases (Cel-uclast 1.5L: 42.40 FPU/mL and 21.10 CBU/mL) and a �-glycosidase1340 CBU/mL) preparation (Novozym 188), both from Novozymes/S (Bagsværd, Denmark), kindly donated by the Department ofiotechnology of Engineering College of Lorena (University of Sãoaulo) were added. Enzyme activities expressed in filter paper unitsFPU)/mL and in unit of cellobiose (CBU)/mL, were determinedccording to the method described by Ghose (1987).

.3. Microorganism

The industrial strain S. cerevisiae UFPEDA 1238 was used, whichas kindly provided by the Culture Collection of Department ofntibiotics of the Federal University of Pernambuco, Brazil. Thisulture was maintained on the medium culture containing (in g/L):lucose (20), yeast extract (5), peptone (3) and agar (15), at pH 7.0.

.4. Inoculum culture

Pure yeast culture growth in culture medium described in Sec-ion 2.3, was added to a 500 mL Erlenmeyer flask, which contained00 mL of following medium: glucose (20 g/L), yeast extract (5 g/L)nd peptone (3 g/L) at pH 7.0. The Erlenmeyer flask was incubated inrotary shaker at 30 ◦C and 250 rpm. After 12 h, the cells suspensionas filtrate through a 0.45 �m filter. The filtrate was discarded and

he cells were re-suspended in 10 mL sterile water and transferredo a 250 mL Erlenmeyer flask, containing 90 mL of fermentation

edium.

.5. Simultaneous saccharification and fermentation

Simultaneous saccharification and fermentation was performedn 250 mL Erlenmeyer flasks. Each Erlenmeyer flask contained0 mL of fermentation medium (with the nutrients dissolved in

d Products 36 (2012) 584–588 585

a sodium citrate buffer at 50 mM and pH 4.8) and 8 g of delig-nified bagasse. The Erlenmeyer flasks were incubated in a rotaryshaker at 50 ◦C and 150 rpm. After a 6 h prehydrolysis, each Erlen-meyer flask was inoculated with yeast cells (described in Section2.4) and incubated at 37 ◦C and 80 rpm. Initial cell concentrationwas 1 g/L. Nutrients added were: (NH4)2SO4 1 g/L; K2HPO4 0.5 g/L;MgSO4·7H2O 0.25 g/L; yeast extract 2 g/L; peptone 1 g/L (culturemedium 1) and (NH4)2SO4 2 g/L; KH2PO4 2 g/L; MgSO4·7H2O0.75 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 at37 ◦C/80 rpm, 32 ◦C/80 rpm and 37 ◦C/100 rpm. These three condi-tions were chosen according to our previous study (Santos et al.,2010b). The experiments were performed in duplicates. The enzy-matic convertibility of cellulose (ECC) was calculated based inethanol concentration (Martín et al., 2008).

ECC = Ef − Ei

Ci × 0.57

where Ef is the final ethanol concentration (g/L); Ei is the initialethanol concentration (g/L); Ci, initial cellulose concentration (g/L).

The factor 0.57 is the stoichiometric yield of ethanol from cellu-lose.

2.6. Chromatographic analysis

Sugars, carboxylic acids, ethanol and furan aldehydes werequantified by HPLC (Agilent HP 1100, Germany). All sampleswere filtered through a 0.45 �m filter. Cellobiose, glucose, arabi-nose, xylose, acetic acid, formic acid and ethanol were separatedon an Aminex HPX-87H+ (Bio-Rad, Hercules, CA, USA) columnat 50 ◦C, using 5 mM H2SO4 at a flow rate of 0.6 mL/min asmobile phase, and detected RI-detector (Agilent). Furfural and 5-hydroxymethylfurfural (HMF) were separated on a C-18 column(Beckman) at 25 ◦C, using 11.2/88.8 acetonitrile/1% (v/v) acetic acidmixture at a flow rate of 0.8 mL/min as mobile phase, and detectedby their UV absorbance at 274 nm (Agilent).

3. Results and discussion

Sugarcane bagasse, pretreated by steam explosion contained49.89% cellulose, 7.99% hemicellulose and 34% lignin (Gouveia et al.,2009). Steam pretreated sugar cane bagasse was delignified foravoiding the influence of lignin, since this compound forms a barrierto enzymatic attack (Chang and Holtzapple, 2000).

The yield of cellulosic pulp recovered after the alkaline delig-nification was 50%. The pulp contained 81.8% cellulose, 6.4%hemicelluloses and 3.0% lignin. The content of cellulose in the solidfraction increased as a result of the solubilisation of lignin. In addi-tion to the increase of cellulose content and decrease of lignincontent, an enhancement of its enzymatic convertibility is expectedsince it has previously been reported that NaOH increased hard-wood digestibility from 14 to 55% concomitantly with a reductionof lignin content from 24–55% to 20% (Kumar et al., 2009).

Steam explosion is the most widely employedphysical–chemical pretreatment for lignocellulosic biomass.The auto-hydrolysis of acetyl groups present in hemicellulose(Alvira et al., 2010) is observed. The lignin is redistributed and tosome extent removed from the material (Pan et al., 2005). Removalof hemicelluloses and the redistribution of lignin probably mayhave exposed the material and increased the delignification

(91.10%).

Ethanol concentration, volumetric productivity and the enzy-matic convertibility of cellulose (ECC), were higher at 37 ◦C and80 rpm (Fig. 1), while in the other two SSF (32 ◦C/80 rpm and

586 J.R.A. Santos et al. / Industrial Crops and Products 36 (2012) 584–588

F1C

3Ehiie(

c3bcf

ctso1i

ta�l8a

atwu

druatr

c2lcEi

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

ig. 1. Ethanol concentration, ECC and QP, in each SSF run at 20 FPU/g cellulose,0%, v/v �-glucosidase and culture medium 1: A (32 ◦C, 80 rpm); B (37 ◦C, 80 rpm);(37 ◦C, 100 rpm).

7 ◦C/100 rpm), were found similar values. Ethanol concentration,CC and volumetric productivity, after 30 h (considering a 6 h pre-ydrolysis), reached 26.03 g/L, 74.30% and 0.88 g/L h, respectively,

n SSF at 37 ◦C and 80 rpm. Decreasing temperature (37–32 ◦C) orncreasing agitation (80–100 rpm) decreased the ECC (21.62%), thethanol concentration (23.08%) and the volumetric productivity22.70%).

According to Öhgren et al. (2007), the temperature used in SSFannot be optimal for enzymes and yeast. However, in our studies,7 ◦C was optimal temperature found to SSF of sugar cane bagassey S. cerevisiae UFPEDA 1238. This strain presented higher ethanoloncentration at 37 ◦C than at 30 ◦C or 45 ◦C, when was performedermentation with sucrose (Santos et al., 2010a).

Analysis of variance by Origin 6.0 was performed with ethanoloncentrations obtained at 28 h in the SSF runs, when the tempera-ure and agitation were varied (SSF: A, B and C). These results wereignificantly different (F = 247.93; ˛ = 0.05). However, the analysisf variance between the A SSF (32 ◦C, 80 rpm) and C SSF (37 ◦C,00 rpm), showed that the ethanol concentrations were not signif-

cantly different (F = 0.0969; ˛ = 0.05).For evaluating decreasing enzyme load and of the composi-

ion of culture medium, experiments were carried out at 50 ◦Cnd 150 rpm with 10 or 20 FPU/g cellulose and 5 or 10%, v/v-glucosidase, respectively. After 6 h, yeast suspension was inocu-

ated and the temperature and agitation were reduced to 37 ◦C and0 rpm, respectively. SSF were carried out with two culture mediaccording to composition described in Section 2.5.

As can be seen in Fig. 2, decreasing enzyme loads (20–10 FPU/gnd 10–5%, v/v �-glucosidase) also decreased (8.40%) the ECC, whenhe culture medium 1 was used in both SSF (B and E). However,hen the enzyme load was reduced and the culture medium 2 wassed, the ECC decreased 0.86% only (B SSF and D SSF).

Decreasing enzyme loads also decreased the volumetric pro-uctivity about 20% (B SSF and E SSF) and 12% (B SSF and D SSF),espectively, when culture medium 1 or the culture medium 2 wassed. On the other hand, the ethanol concentration increased 6.45%nd decreased 8.45%, when the enzyme loads were reduced andhe culture medium 2 (B SSF and D SSF) or 1 (B SSF and E SSF),espectively, were used.

The enzymatic convertibility of cellulose (ECC) and the ethanoloncentration were higher than 72% and 25 g/L, respectively, at8 h, 37 ◦C and 80 rpm, when the culture medium 1 (20 FPU/g cel-

ulose and 10% �-glucosidase) or the culture medium 2 (10 FPU/gellulose and 5% �-glucosidase) was used. Martín et al. (2008) foundCC higher than 80%, after 120 h, in SSF with S. cerevisiae or Mucor

ndicus strains. However, the ethanol concentrations achieved were

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 con-tent of the raw material, although these authors have utilized highwater insoluble solids content (10%).

In ethanol production from lignocellulosic materials, ethanolconcentration should be as high as possible in order to minimize theenergy consumption in evaporation and distillation (Wingren et al.,2003). Increasing water insoluble solids content, in SSF, increasesthe glucose and ethanol concentrations. A water insoluble solidcontent of 8% was high enough to obtain reasonable ethanol con-centration (higher than 25 g/L).

Analysis of variance was performed with the ethanol concen-tration obtained at 28 h in the SSF runs, when the enzyme loadand culture medium were varied (SSF: B, D and E). These resultswere significantly different (F = 15.75; ˛ = 0.05). However, the anal-ysis 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 maximumethanol concentrations were not significantly different (F = 7.08;˛ = 0.05).

Fast dissolution and ECC almost 40%, after a 6 h pre-hydrolysisat 50 ◦C and 150 rpm was achieved for both enzyme loads. In SSF oftrebol (Martín et al., 2008), was also found rapid dissolution after6 h at 50 ◦C. Santos et al. (2010b) observed that SSF of sugar canebagasse without pre-hydrolysis is a slower process.

Ethanol concentrations and volumetric productivities (QP) in allconditions were higher than that found by Öhgren et al. (2007)at 35 ◦C in isothermal SSF and Kádár et al. (2004) in isothermal SSF(40 ◦C) or non-isothermal SSF (50 ◦C during 24 h pre-hydrolysis and30 ◦C after inoculation of yeast). Martín et al. (2008) also obtainedlower ethanol concentration and volumetric productivity in non-isothermal SSF (50 ◦C during 6 h pre-hydrolysis and 32 ◦C afterinoculation of yeast). Table 1 shows a comparison between someethanol concentrations and volumetric productivities found in lit-erature and in the present work.

Supplementation of culture medium with higher concentrationsof ammonium, potassium, phosphorus and magnesium may havefavorably influenced the fermentation. S. cerevisiae uses nitrogenas ammonium, amide (urea) or amine (amino acids). Phosphorus isabsorbed as H2PO−

4 , and the sulfur can be assimilated as sulfate(Lima et al., 2000). Martín et al. (2008) reported that high con-

tent of potassium and phosphorus in the culture medium was alsofavorably for the fermentation.

In SSF, after 12 h, glucose was completely consumed in allfive conditions (20 FPU/g cellulose and 10%, v/v �-glucosidase:

J.R.A. Santos et al. / Industrial Crops and Products 36 (2012) 584–588 587

Table 1Ethanol concentration using various substrates, microorganisms and enzyme load in isothermal and non-isothermal SSF.

Microorganism SSFa T (◦C) N-SSFb T (◦C) Pre-hydrolysis (h) Ethanolc (g/L) QPd (g/L h) Reference

K. marxianus 40 – – 17.8 0.25 Kádár et al. (2004)– 50 and 30 24 16.0 0.22

S. cerevisiae 40 – 16.6 0.23– 50 and 30 24 15.1 0.21

S. cerevisiae 35 – – 20.5 0.17 Öhgren et al. (2007)S. cerevisiae – 50 and 32 6–8 23.7 0.20 Tomás-Pejó et al. (2008)S. cerevisiae UFPEDA 1238 – 50 and 37 6 27.71 0.77 Present work (Fig. 2 – SSF D)

a Isothermal.b Non-isothermal.c Ethanol concentration.d Volumetric productivity.

A B C D E0

50

100

150

200

Yie

ld (L

Eth

anol

/ton

baga

sse)

SSF

Fig. 3. Yield (L ethanol/t bagasse) under five 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/gc ulose,v -gluc

3l3Fimhct

uSoyaat

rwlc�ao

ellulose, 5%, v/v �-glucosidase, culture medium 1); C (37 ◦C, 100 rpm, 20 FPU/g cell/v �-glucosidase, culture medium 2); E (37 ◦C, 80 rpm, 10 FPU/g cellulose, 5%, v/v �

2 ◦C/80 rpm; 37 ◦C/80 rpm; 37 ◦C/100 rpm - 10 FPU/g cellu-ose and 5%, v/v �-glucosidase: 37 ◦C/80 rpm/culture medium 1;7 ◦C/80 rpm/culture medium 2). In isothermal (40 ◦C) SSF of Solkaloc 2000 the glucose concentration stayed at around 3–5 g/L dur-ng 72 h (Kádár et al., 2004), when S. cerevisiae or Kluyveromyces

arxianus were utilized. These authors reported that the cell mayave been suffered with this temperature and that it was espe-ially unexpected with the thermo tolerant K. marxianus, which washought to be performing much better at 40 ◦C than S. cerevisiae.

Cellobiose concentrations were lower than 0.2 g/L, after 12 h,tilizing 10 or 5%, v/v �-glucosidase. Öhgren et al. (2007) performedSF with 25%, v/v �-glucosidase to hydrolysis all cellobiose. On thether hand, Chen et al. (2007), when not supplementing the hydrol-sis with �-glucosidase, observed a severe inhibition of cellulasesctivity due to the accumulation of cellobiose (7.4 g/L). When theseuthors supplemented with �-glucosidase (6.5 CBU/g substrate),he concentration of cellobiose decreased to 0.6 g/L.

Fig. 3 shows a comparison of ethanol yield from raw mate-ial. Ethanol volume (in L) in relation to bagasse mass (in ton)as calculated considering the recovering of solids after the alka-

ine delignification (50%) and steam explosion (68%). In SSF with

ulture medium 2, enzyme load 10 FPU/g cellulose and 5%, v/v-glucosidase (D), yield higher than 150 L EtOH/t bagasse waschieved. This represents 100 L EtOH/t sugarcane, since each tonf cane generates 2/3 of bagasse.

5%, v/v �-glucosidase, culture medium 1); D (37 ◦C, 80 rpm, 10 FPU/g cellulose, 5%,osidase, culture medium 1).

4. Conclusions

The adequate combination of conditions of pre-hydrolysis (6 h at50 ◦C and 15 rpm), temperature (37 ◦C), agitation (80 rpm), compo-sition of culture medium (with higher concentration of nutrients)and enzyme load (10 FPU/g cellulose and 5%, v/v �-glucosidase) wasa successful method to ethanol production by S. cerevisiae UFPE1238 in SSF of delignified sugarcane bagasse. High yield ethanolwith minimum enzyme loads and lower time (34 h, consideringa 6 h pre-hydrolysis) was achieved, which is often an importantrequirement to provide cost-efficient second generation ethanolprocesses.

Acknowledgements

The authors acknowledge the financial support from ConselhoNacional de Desenvolvimento Científico e Tecnológico, Brasilia DF,Brazil (CNPq) and from Fundacão de Amparo à Ciência e Tecnologiado Estado de Pernambuco (FACEPE).

References

Adsul, M.G., Ghuleb, J.E., Singhb, R., Shaikhb, H., 2005. Polysaccharides from bagasse:applications in cellulase and xylanase production. Carbohydr. Polym. 57, 67–72.

5 ops an

A

B

C

C

C

GG

H

K

K

L

M

M

ing ethanol from softwood: comparison of SSF and SHF and identification of

88 J.R.A. Santos et al. / Industrial Cr

lvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment tech-nologies for an efficient bioethanol production process based on enzymatichydrolysis: a review. Bioresour. Technol. 101, 4851–4861.

rethauer, S., Wyman, C.E., 2010. Review: continuous hydrolysis and fermentationfor cellulosic ethanol production. Bioresour. Technol. 101, 4862–4874.

arrilo, F., Lis, M.J., Colom, X., Lopez-Mesas, M., Valdeperas, J., 2005. Effect of alkalipretreatment on cellulase hydrolysis of wheat straw: kinetic study. ProcessBiochem. 40, 3360–3364.

hang, V.S., Holtzapple, M., 2000. Fundamentals factors affecting biomass reactivity.Appl. Biochem. Biotechnol. 84, 5–37.

hen, M., Xia, L., Xue, P., 2007. Enzymatic hydrolysis of corncob and ethanol produc-tion from cellulosic hydrolysate. Int. Biodeterior. Biodegr. 59, 85–89.

hose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268.ouveia, E.R., Nascimento, R.T., Souto-Maior, A.M., Rocha, G.J.M., 2009. Validation

of methodology for the chemical characterization of sugar cane bagasse. Quim.Nova 32, 1500–1503.

ahn-Hägerdal, B., Wahlbom, C.F., Gardonyi, M., Van Zyl, W.H., Cordero Otero, R.,Jönsson, L.J., 2001. Metabolic engineering of Saccharomyces cerevisiae for xyloseutilisation. Adv. Biochem. Eng. Biotechnol. 73, 53–84.

ádár, Z., Szengyel, Z., Réczey, K., 2004. Simultaneous saccharification and fermen-tation (SSF) of industrial wastes for the production of ethanol. Ind. Crop Prod.20, 103–110.

umar, R., Mago, G., Balan, V., Wyman, C.E., 2009. Physical and chemical character-izations of corn stover and poplar solids resulting from leading pretreatmenttechnologies. Bioresour. Technol. 100, 3948–3962.

ima, U.A., Basso, L.C., Amorim, H.V., 2000. Ethanol production. Industrial Biotech-nology, vol. 3, cap. 1, pp. 16–17.

artín, C., Galbe, M., Wahlbom, C.F., Hahn-Hägerdal, B., Jönsson, L.J., 2002. Ethanolproduction from enzymatic hydrolysates of sugarcane bagasse using recombi-

nant xylose –utilising Saccharomyces cerevisiae. Enzyme Microb. Technol. 31,274–282.

artín, C., Thomsen, M.H., Hauggaard-Nielsen, H., Thomsen, A.B., 2008. Wet oxida-tion pretreatment, enzymatic hydrolysis and simultaneous saccharification andfermentation of clover–ryegrass mixtures. Bioresour. Technol. 99, 8777–8782.

d Products 36 (2012) 584–588

Öhgren, K., Bura, R., Lesnicki, G., Saddler, J., Zacchi, G., 2007. A comparison betweensimultaneous saccharification and fermentation and separate hydrolysis andfermentation using steam-pretreatment corn stover. Process Biochem. 42,834–839.

Pan, X., Xie, D., Gilkes, N., Gregg, D.J., Saddler, J.N., 2005. Strategies to enhance theenzymatic hydrolysis of pretreated softwood with high residual lignin content.Appl. Biochem. Biotechnol. 124, 1069–1079.

Rocha, G.J.M., Silva, F.T., Araújo, G.T., Curvelo, A.A.S., 1997. A fast and accurate methodfor determining the cellulose and polyoses through HPLC. In: V Brazilian Sym-posium Proceedings on the Chemistry of Lignin and Other Wood Components,Curitiba, PR, Brazil, vol. 5, pp. 113–115.

Santos, J.R.A., Gusmão, N.B., Gouveia, E.R., 2010a. Selection of industrial strains ofSaccharomyces cerevisiae with potential performance for ethanol production inadverse conditions of temperature and agitation. Braz. J. Agric. Ind. Prod. 12,75–80.

Santos, J.R.A., Souto-Maior, A.M., Gouveia, E.R., Martín, C., 2010b. Comparison of SHFand SSF processes from sugar cane bagasse for ethanol production by Saccha-romyces cerevisiae. Quim. Nova 33, 904–908.

Silva, A.S., Inoue, H., Endo, T., Yano, S., Bon, E.R.S., 2010. Milling pretreatment of sug-arcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation.Bioresour. Technol. 101, 7402–7409.

Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol produc-tion: a review. Bioresour. Technol. 83, 1–11.

Tomás-Pejó, E., Oliva, J.M., Ballesteros, M., Olsson, L., 2008. Comparison of SHFand SSF processes from steam-exploded wheat straw for ethanol productionby xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiaestrains. Biotechnol. Bioeng. 100, 1122–1131.

Wingren, A., Galbe, M., Zacchi, G., 2003. Techno-economic evaluation of produc-

bottleneck. Biotechnol. Prog. 19, 1109–1117.Wyman, C.E., Spindler, D.D., Grohmann, K., 1992. Simultaneous saccharification and

fermentation of several lignocellulosic feedstocks to fuel ethanol. Biomed. Bio-eng. 3, 301–307.