Fuel Processing Technology 90 (2009) 1193–1197

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Fuel Processing Technology

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Simultaneous saccharification and fermentation of alkaline-pretreated corn stover toethanol using a recombinant yeast strain

Jing Zhao, Liming Xia ⁎Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China

⁎ Corresponding author. Tel./fax: +86 571 8795 1840E-mail address: xialm@zju.edu.cn (L. Xia).

0378-3820/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.fuproc.2009.05.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 February 2009Received in revised form 8 May 2009Accepted 18 May 2009

Keywords:Simultaneous saccharificationand fermentationCorn stoverEthanolCelluloseHemicelluloseRecombinant yeast

Bio-ethanol converted from cheap and abundant lignocellulosic materials is a potential renewable resourceto replace depleting fossil fuels. Simultaneous saccharification and fermentation (SSF) of alkaline-pretreatedcorn stover for the production of ethanol was investigated using a recombinant yeast strain Saccharomycescerevisiae ZU-10. Low cellobiase activity in Trichoderma reesei cellulase resulted in cellobiose accumulation.Supplementing the simultaneous saccharification and fermentation system with cellobiase greatly reducedfeedback inhibition caused by cellobiose to the cellulase reaction, thereby increased the ethanol yield. 12 h ofenzymatic prehydrolysis at 50 °C prior to simultaneous saccharification and fermentation was found to havea negative effect on the overall ethanol yield. Glucose and xylose produced from alkaline-pretreated cornstover could be co-fermented to ethanol effectively by S. cerevisiae ZU-10. An ethanol concentration of 27.8 g/L and the corresponding ethanol yield on carbohydrate in substrate of 0.350 g/g were achieved within 72 h at33 °C with 80 g/L of substrate and enzyme loadings of 20 filter paper activity units (FPU)/g substrate and10 cellobiase units (CBU)/g substrate. The results are meaningful in co-conversion of cellulose and hemi-cellulose fraction of lignocellulosic materials to fuel ethanol.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Recently, the use of bio-ethanol as a clean, safe alternative sourceof fuel has raised considerable interest due to the diminishing fossilfuel reserves and increased air pollution [1,2]. Currently industrial bio-ethanol is mainly produced from sugarcane or starch materials; how-ever, the limited quantity of food stuff in China and their compara-tively high prices greatly restrict large scale production of bio-ethanol.Lignocellulosic materials are cheap, abundant renewable resourcesand promising raw materials for ethanol production, yet, they areusually disposed or directly burned due to lack of effective utilization,thus often causing serious environmental pollution. Therefore, studyonbio-ethanolproduction from lignocellulosicmaterials is of far reach-ing importance in both new energy resources development and envi-ronmental protection [3,4].

In the process of ethanol production from lignocellulosic materials,enzymatic hydrolysis and fermentation can be performed separatelyor simultaneously. Compared with separate hydrolysis and fermenta-tion (SHF), simultaneous saccharification and fermentation (SSF) ismore favored because in SSF glucose released by the action of cellulaseis converted quickly to ethanol by the fermentingmicroorganism, thusminimizing end-product inhibition to cellulase caused by glucose andcellobiose accumulation. The improved saccharification rates andhigher

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yield of ethanol are observed in SSF. Furthermore, the presence ofethanol in the culture broth helps to avoid undesired microbial con-tamination [5–7].

Lignocellulosic materials are the largest sources of hexose andpentose sugars. The conversion of hexose (mainly glucose) to ethanolhas been the research focus for the past decades. However, exploita-tion of lignocellulosics can be enhanced by the efficient utilization ofpentose alongside the hexose fraction. Xylose is the most abundantpentose sugar in hemicellulose (20–30% of dry weight of lignocellu-losic biomass), and it is second only to glucose in natural abundanceof all monosaccharides in lignocellulosic hydrolysate. Saccharomycescerevisiae, which is one of the most prominent ethanol-producingmicroorganisms utilizing hexose, has been found unable to utilizexylose due to lack of the key enzymes in the xylose-metabolisingpathway [8]. Thus, the efficient utilization of xylose in hemicellulosein addition to glucose in cellulose by a recombinant xylose-fermentingS. cerevisiae strain would offer an opportunity to reduce the produc-tion cost of bio-ethanol significantly [9,10].

In China, concentrated agricultural residue corn stover is producedannually. Corn stover contains high contents of cellulose (around 39%of dry weight) and hemicellulose (around 22% of dry weight); there-fore, it is an ideal feedstock for the production of fuel ethanol. In thiswork, the SSF of alkaline-pretreated corn stover for ethanol produc-tionwas investigated using a recombinant xylose-utilizing S. cerevisiaestrain. The main objective of the article was to evaluate the feasibilityof co-conversion of cellulose and hemicellulose to ethanol by a re-combinant yeast strain using SSF.

Fig. 1. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 withT. reesei cellulase (20 FPU/g substrate; 1.64 CBU/g substrate). (○) glucose; (●) xylose;(□) cellobiose; (◊) arabinose; (▲) ethanol.

1194 J. Zhao, L. Xia / Fuel Processing Technology 90 (2009) 1193–1197

2. Materials and methods

2.1. Microorganism

The xylose-utilizing yeast strain S. cerevisiae ZU-10 expressesxylose reductase (XR) and xylitol dehydrogenase (XDH) from thechromosomally integrated Pichia stipitis genes XYL1 and XYL2,respectively, and over-expresses the homologous XKS1 gene encodingxylulokinase (XK). It was maintained on YPX-agar slants containing10 g/L yeast extract, 20 g/L peptone, 20 g/L xylose and 20 g/L agar[11].

2.2. Lignocellulosic material and pretreatment

Corn stover from local farms was milled to pass a 2.0 mm screen.The milled corn stover was washed thoroughly with tap water toremove sticky clay, then filtered and air-dried, and had the followingchemical composition (dry weight basis): cellulose 38.7%, hemicellu-lose 21.7%, lignin 19.3% and others 20.3%.

Corn stover sample was pretreated with 0.5 M NaOH solution at80 °C for 75 min with a solid-to-liquid ratio of 1:8 (w/v). The solidcellulosic residues were collected and washed to neutral pH, filteredand stored for later use. The chemical composition of corn stoverresidue was as follows (dry weight basis): cellulose 64.1%, hemi-cellulose 24.6%, lignin 8.6% and others 2.7%.

2.3. Enzymes

Cellulase and cellobiase were produced according to Xia andCen [12] and Shen and Xia [13], respectively. Each gram of dry kojiproduced by Trichoderma reesei ZU-02 contained 146 filter paperactivity units (FPU), 12 cellobiase units (CBU) and 1458 units ofxylanase activity. Each gram of dry koji produced by Aspergillus nigerZU-07 contained 376 CBU and no detectable filter paper activity.

Filter paper activity (FPA) and cellobiase activity (CBA) weremeasured according to the method recommended by Ghose [14] andexpressed as international units (IU). One unit of filter paper activity(FPU) is the amount of enzyme that forms 1 µmol glucose (reducingsugar as glucose) per minute. One unit of cellobiase activity (CBU)is the amount of enzyme that forms 2 µmol glucose per minutefrom cellobiose. Xylanase activity was assayed using the Baileymethod [15].

2.4. Inoculum preparation

For the inoculum preparation of recombinant S. cerevisiae ZU-10, aloopful of cells were transferred into each 250 mL E-flask with 50 mLof sterile culture medium containing 10 g/L glucose, 10 g/L xylose, 5 g/Lpeptone and 3 g/L yeast extract. The flasks were sealed with gauze andincubated in a rotary shaker at 30 °C and 180 rpm for 24 h. Cells wereharvested by centrifugation (4800 rpm, 5 min), suspended in sterilizedwater and used as inoculum in the SSF process.

2.5. Simultaneous saccharification and fermentation (SSF)

SSF experiments were performed in 250 mL E-flasks in a rotaryshaker at 120 rpm and 33 °C. The flasks were sealed with rubberstoppers equipped with needles for CO2 venting. Each 100 mL ofSSF reaction mixtures containing alkaline-pretreated corn stover 8 g(dry weight), peptone 0.5 g, yeast extract 0.3 g, CaCl2 0.025 g, MgCl20.025 g and KH2PO4 2.5 g was previously autoclaved at 110 °Cfor 20 min. After cooling down, the initial pH was adjusted to 5.0 withCa(OH)2. SSF experiments were started by inoculation with 2 mL cellsuspension corresponding to 1 g/L dry yeast and addition of cellulasekoji (20 FPU/g substrate) and Tween 80 (0.5 g). Exceptions are pointedout in the text.

2.6. Analytical methods

Cellulose was determined by HNO3–ethanol method, lignin by 72%H2SO4 method, and hemicellulose by two-brominating method [16].Reducing sugars were estimated by the 3, 5-dinitrosalicylic acid (DNS)method [14].

Samples for analysis of glucose, xylose, cellobiose, arabinose andethanol contents were centrifuged at 10,000 rpm for 10 min. Thesupernatant was filtered through 0.45 μm membrane filters and thenanalyzed on a HPLC system (Model 500, Syltech, USA) equipped withan organic acid column (IC Sep ICE-Coregel 87H3, Transgenomic, USA)and a refractive index detector (Model 6040 XR, Spectra-Physics,USA). 5 mM H2SO4 solution was used as the mobile phase at a flowrate of 0.5 mL/min. The column temperature was fixed at 60 °C.

The ethanol yield was calculated as concentration of producedethanol (in g/L) divided by carbohydrate (monosaccharides, i.e. thesum of glucose and xylose) concentration in the substrate (in g/L).The theoretical weight of glucose and xylose released during thehydrolysis is (due to the addition of water when the glycosidic bondsare hydrolysed) 1.11 times the weight of cellulose and 1.14 times theweight of hemicellulose, respectively.

Three parallel samples were used in all analytical determinations,and data are presented as the mean of three replicates.

3. Results and discussion

3.1. SSF process by S. cerevisiae ZU-10 with T. reesei ZU-02 cellulase

SSF process of alkaline-pretreated corn stover was performed withcellulase from T. reesei ZU-02 (20 FPU/g substrate) at 80 g/L of sub-strate (dry weight basis). As shown in Fig. 1, results indicated thatglucose and xylose released from enzymatic hydrolysis can be co-fermented to ethanol by recombinant S. cerevisiae ZU-10, yet withdifferent consumption rates. Glucose produced was quickly convertedto ethanol with no accumulation, while xylose was utilized slowerthan glucose.

It was found that cellobiose increased gradually to 10.3 g/L within72 h, indicating relatively low cellobiase activity in T. reesei cellulase.At the T. reesei cellulase dosage of 20 FPU/g substrate, only 1.64 CBU/gsubstrate of cellobiase was present in the SSF system. Accumulatedcellobiose caused feedback inhibitory effect to enzymatic hydrolysis ofcellulase, as the enzyme is more susceptible to end-product inhibition

Fig. 2. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10with T. reesei cellulase and A. niger cellobiase (20 FPU/g substrate; 10 CBU/g substrate).(○) glucose; (●) xylose; (□) cellobiose; (◊) arabinose; (▲) ethanol.

1195J. Zhao, L. Xia / Fuel Processing Technology 90 (2009) 1193–1197

caused by cellobiose than glucose [17,18]. Meanwhile, cellobiose couldnot be utilized to produce ethanol by recombinant S. cerevisiae ZU-10,thus restricting the final ethanol concentration to 17.9 g/L at 72 h withthe ethanol yield of 0.226 g/g.

3.2. Effects of cellobiase supplement

To weaken the feedback inhibition caused by cellobiose accumula-tion, cellobiase produced by A. niger ZU-07 was supplemented to theSSF system to enhance the total cellobiase activity to 10 CBU/gsubstrate. The time course of SSF with T. reesei cellulase and A. nigercellobiase (20 FPU/g substrate; 10 CBU/g substrate) was shown inFig. 2. Due to the improvement of cellobiase activity in the SSFsystem, the concentration of cellobiose was a relatively low level(within 1 g/L) throughout the SSF process without obvious accumula-tion. Therefore, feedback inhibition by cellobiose to cellulase wasgreatly reduced, resulting in more effective saccharification of cellu-lose and a higher ethanol concentration. At 72 h, the ethanol con-centration was 27.8 g/L, with the ethanol yield of 0.350 g/g. This wasgreatly improved compared with SSF without cellobiase supplement.In the following SSF experiments, T. reesei cellulase was supplementedwith A. niger cellobiase (2 FPU:1 CBU) to reduce feedback inhibitioncaused by cellobiose accumulation.

3.3. Effects of enzyme loadings

Enzyme loading is considered as one of the most important fac-tors in ethanol production of lignocellulosic materials [19]. Effects ofenzyme loadings (presented as FPU/g substrate) on SSF was inves-tigated. Synergetic hydrolysis by a more balanced cellulase complex

Table 1Effects of enzyme loadings (presented as filter paper activity per gram of substrate, FPU/gsubstrate) on SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10.

Enzyme loading(FPU/g substrate)

Glucose(g/L)

Xylose(g/L)

Arabinose(g/L)

Ethanol(g/L)

Ethanol yield(g/g)

5 ND 6.9 1.1 14.7 0.18510 ND 4.6 2.2 20.5 0.25815 ND 1.8 2.3 25.8 0.32520 ND 1.6 2.7 27.8 0.35025 ND 1.2 2.8 28.1 0.354

ND — not detected.

(2 FPU:1 CBU) could effectively avoid cellobiose accumulation andimprove SSF (Table 1). At 72 h, ethanol concentration and yield sig-nificantly increased with an increase in the enzyme loadings up to20 FPU/g substrate, beyond which the increase leveled off. Therefore,the suitable enzyme loading for the SSF process was identified as20 FPU/g substrate, i.e. an enzyme complex including cellulase of20 FPU/g substrate and cellobiase of 10 CBU/g substrate. This enzymeloading was therefore chosen in the following experiments.

3.4. Effects of temperature

The optimal temperature for enzymatic hydrolysis by cellulasecomplex is 45–50 °C; while for fermentation the optimal temperatureis generally 30–35 °C. SSF experiments at three different temperatureswere performed (Fig. 3). At 33 °C, sugars (mainly glucose and xylose)produced by enzymatic hydrolysis were quickly fermented to ethanolby recombinant S. cerevisiae ZU-10, the reducing sugar concentrationwas maintained at a low level and ethanol concentration increasedsteadily during the SSF process. At 36 °C, the vitality of S. cerevisiaeZU-10 was weakened. The production rate of reducing sugars fromhydrolysis was higher than its consumption rate, thereby the reducingsugars accumulated continuously and the ethanol concentration

Fig. 3. SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 at temperatures of33 °C, 36 °C, and 40 °C, respectively. (A) reducing sugar concentration in SSF; (B) ethanolconcentration in SSF. (♦) 33 °C; (●) 36 °C; (▲) 40 °C.

1196 J. Zhao, L. Xia / Fuel Processing Technology 90 (2009) 1193–1197

leveled off from 24 h to 72 h.When the temperature reached 40 °C, thefermentation performance of S. cerevisiae ZU-10 was badly inhibitedand ethanol production almost ceased. Only enzymatic hydrolysis ofcorn stover residue was carried out effectively. The results indicatedthat S. cerevisiae ZU-10 was not thermo-resistant and the suitabletemperature for the SSF is 33 °C. This temperature was thereforechosen in the following experiments.

3.5. Effects of enzymatic prehydrolysis

Enzymatic prehydrolysis prior to SSF may help to decrease theviscosity of the slurry for easier stirring and better mass and heattransfer, making the subsequent SSF possible even with high water-insoluble substances and shortening SSF cycle. In this consideration,12 h of prehydrolysis was performed at 50 °C with a higher substrateconcentration of 120 g/L (dry weight basis) to investigate the effect ofenzymatic prehydrolysis on SSF. Yeast cells were added afterwards toinitiate SSF process. The concentration profiles of glucose, xylose,arabinose and ethanol in SSF with 12 h prehydrolysis compared withpure SSF were shown in Fig. 4. In the SSF process preceded by 12 h ofprehydrolysis, ethanol concentration reached 21.2 g/L and 35.8 g/L at

Fig. 4. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10at 120 g/L of substrate concentration. (A) 12 h of enzymatic prehydrolysis prior to SSF;(B) pure SSF. (○) glucose; (●) xylose; (◊) arabinose; (▲) ethanol.

12 h and 108 h respectively. The corresponding highest ethanol yieldwas 0.301 g/g. However, in pure SSF process, ethanol concentrationwas 16.5 g/L and 38.2 g/L at 12 h and 108 h respectively with thehighest ethanol yield of 0.321 g/g. Enzymatic prehydrolysis increasedthe ethanol productivity during the initial 12 h in SSF process, but hada negative effect on the overall ethanol yield. Similar results were alsoobtained in SSF process at 80 g/L and 200 g/L of substrate content(dry weight basis) (data not shown). The reasons for the lowerethanol yield due to prehydrolysis were not clear now. Maybe suddenchanges in the osmotic pressure in the surroundings caused yeast cellsto make physiological changes and produced less ethanol in the ex-periments with prehydrolysis [20].

4. Conclusions

Both the glucose from cellulose and the xylose from hemicellulosein corn stover could be converted to ethanol in SSF process by arecombinant yeast strain. Synergetic enzymatic hydrolysis by cellulasefrom T. reesei ZU-02 and cellobiase from A. niger ZU-07 (20 FPIU/gsubstrate; 10 CBIU/g substrate) greatly reduced the feedback inhibi-tion caused by cellobiose accumulation, thereby effectively improveSSF performance. Ethanol concentration reached 27.8 g/L in SSF at33 °C within 72 h, with the ethanol yield of 0.350 g/g. Enzymaticprehydrolysis prior to SSF increased the ethanol productivity duringthe initial 12 h in SSF process, but had a negative effect on the overallethanol yield.

Acknowledgments

Support from Hi-tech Research and Development Program ofChina (2007AA05Z401) and Major Project of Natural Science Founda-tion of Zhejiang Province (Z407010) is gratefully acknowledged.

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