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Biochemical Engineering Journal 49 (2010) 28–32

Contents lists available at ScienceDirect

Biochemical Engineering Journal

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thanol production from corn stover hemicellulosic hydrolysate usingmmobilized recombinant yeast cells

ing Zhao, Liming Xia ∗

epartment of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China

r t i c l e i n f o

rticle history:eceived 15 June 2009eceived in revised form2 November 2009ccepted 14 November 2009

a b s t r a c t

Ethanol production from corn stover hemicellulosic hydrolysate was investigated using immobilizedrecombinant Saccharomyces cerevisiae yeast cells. Detoxification of hemicellulosic hydrolysate by roto-evaporation and lime neutralization was carried out to remove volatile fermentation inhibitors. Allfurfural and more than 50% acetic acid in the hydrolysate were removed, meanwhile the xylose con-centration was enhanced to 71.8 g/L. The fermentability of the detoxified hydrolysate was significantly

eywords:orn stoveremicelluloseydrolysate

improved using immobilized cells of recombinant S. cerevisiae by Ca-alginate. An ethanol concentrationof 31.1 g/L and the corresponding ethanol yield on fermentable sugars of 0.406 g/g were obtained within72 h in batch fermentation of the detoxified hydrolysate with immobilized cells. In addition, repeatedbatch fermentation of immobilized recombinant S. cerevisiae cells was attempted for ethanol productionfor 5 batches. The concentration of ethanol in each batch maintained above 30.1 g/L with the ethanol

ars ocorn

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mmobilized cell

yield on fermentable sugethanol production from

. Introduction

Fossil fuels are finite natural resources and their worldwidese has caused serious global climate and environment problems

n past decades [1,2]. Bio-ethanol, as a clean, safe and renewableesource, has been considered as a potential alternative to thever-reducing fossil fuels [3–5]. At present, commercial bio-ethanols mainly produced from sugarcane or starch materials. How-ver, a dramatic increase of ethanol production from sugarcane ortarch will compete against the limited agricultural land neededor food and feed production. Lignocellulosic materials are gradu-lly considered as more attractive renewable resources for ethanolroduction due to their great availability and relatively low cost.

Among different lignocellulosic raw materials, corn stoverepresents a large amount of agricultural residues with low com-ercial value. Approximately 250 million tons of corn stover are

roduced annually in China [6]. Currently, it is the most favoredignocellulosic resource for large-scale ethanol production. Effi-ient utilization of the hemicellulosic component of lignocelluloseeedstocks offers an opportunity to reduce the cost of fuel ethanol

roduction by 25% [7,8]. After corn stover was subjected to theidely employed dilute acid pretreatment, cellulose fraction of the

esidue was usually further hydrolyzed to glucose and fermentedo ethanol easily [5,9–11]. However, xylose-rich liquor, produced

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

369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2009.11.007

ver 0.393 g/g. These results demonstrate the viability and significance ofstover hemicellulosic hydrolysate.

© 2009 Elsevier B.V. All rights reserved.

by dilute acid hydrolysis of hemicellulose component of corn stover(20–30% of dry weight) [12], was not so easily utilized due to vary-ing amounts of toxic substances in it and the intolerance of yeasttoward these inhibitors.

Recently, an upsurge of interest in immobilization of microor-ganisms on inert supports has been taking place because thistechnique offers many advantages over free cells, such as enhance-ment of productivity, easy cell recovery from fermentation broth,prevention of cell washout, protection of cells against toxic sub-stances and cell recycling in repeated batch operations, etc.[13]. Furthermore, compared with other immobilization methods,immobilization by Ca-alginate seems to be more effective in reduc-ing the inhibitory effect of fermentation inhibitors such as furfuraland acetic acid in hemicellulosic hydrolysate [4].

In this study, utilization of sugars (mainly xylose) in corn stoverhemicellulosic hydrolysate for fuel ethanol production was inves-tigated by using Ca-alginate immobilized cells of recombinantSaccharomyces cerevisiae yeast. This research can provide impor-tant information on the commercial utilization of corn stover forlarge-scale ethanol production.

2. Materials and methods

2.1. Raw material

Corn stover from local farms was milled to pass 2.0 mm screen.The milled corn stover was washed with tap water, filtered andair-dried. The sample was kept at room temperature for use.

J. Zhao, L. Xia / Biochemical Engineering Journal 49 (2010) 28–32 29

Table 1Chemical composition of corn stover hemicellulose hydrolysate before and after concentration and detoxificationa.

Hemicellulose hydrolysate Glucose (g/L) Xylose (g/L) Arabinose (g/L) Acetic acid (g/L) Furfural (g/L)

Before concentration and detoxification 0.9 ± 0.1 12.1 ± 0.5 2.9 ± 0.2 0.40 ± 0.09 0.08 ± 0.01After concentration and detoxification 4.8 ± 0.2 71.8 ± 1.3 14.3 ± 0.7 1.16 ± 0.18 –

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.2. Preparation of hemicellulosic hydrolysate

Corn stover sample, after being steeped in 10% ammonia for4 h and washed, was pretreated at 108 ◦C with 1.5% sulfuric acidor 6 h, with an initial solid–liquid ratio of 1:10 (w/v). The mixtureas separated by vacuum filtration before the hemicellulosicydrolysate was collected and analyzed.

.3. Detoxification of hemicellulosic hydrolysate

The hemicellulosic hydrolysate was detoxified and concen-rated by evaporation. The evaporation system comprises a rotatoryvaporator and a vacuum pump. The hydrolysate was concentratedo one sixth of the original volume. Exceptions were pointed out inhe text. The concentrated hemicellulosic hydrolysate was regu-arly stirred at 75 ◦C in a water bath shaker and lime solution waslowly added. Lime solution was used in order to neutralize resid-al sulfuric acid and adjust the pH of hydrolysate to 6.5–7.0. Theeaction ceased at the above mentioned pH range. CaSO4 precipi-ate was removed by vacuum filtration after 1 h, and the chemicalomposition of the liquor was analyzed.

.4. Microorganism and inoculum preparation

The recombinant xylose-utilizing yeast strain S. cerevisiae ZU-10xpresses xylose reductase (XR) and xylitol dehydrogenase (XDH)rom the chromosomally integrated Pichia stipitis genes XYL1 andYL2, respectively, and over-expresses the homologous XKS1 genencoding xylulokinase (XK). The recombinant yeast could utilizeoth glucose and xylose simultaneously, while the consumptionate of glucose was obviously faster than xylose. In the separate fer-entation process of 80.0 g/L pure glucose and 80.0 g/L pure xylose,

thanol yield reached 0.492 g/g glucose and 0.366 g/g xylose corre-pondingly. S. cerevisiae ZU-10 was maintained on YPX-agar slantsontaining 10 g/L yeast extract, 20 g/L peptone, 20 g/L xylose and0 g/L agar [14].

A loopful of cells was transferred to 250 mL Erlenmeyer flasksontaining 50 mL of medium consisting of 5 g/L yeast extract,0 g/L peptone, 10 g/L xylose and 10 g/L glucose. The cultures were

ncubated for 24 h in an orbital shaker (180 rpm, 30 ◦C). 10 mLnoculum from the above culture was further propagated in 250 mLrlenmeyer flasks containing 100 mL of medium. The medium com-osition was as follows: 5 g/L yeast extract, 10 g/L peptone, 20 g/Lylose, 20 g/L glucose, 2.5 g/L KH2PO4, 0.25 g/L CaCl2 and 0.25 g/LgCl2. The cultures were incubated for 36 h in an orbital shaker

180 rpm, 30 ◦C).

.5. Cell immobilization

Cells grown in a propagation medium were collected by cen-rifugation for 10 min at 4000 rpm, 4 ◦C. The collected cells were

dded into 2% (w/v) sodium alginate solution, well mixed with% (w/v) diatomite. The mixed solution was extruded through ayringe into a 2% (w/v) CaCl2 solution. Alginate drops solidifiedpon contact with CaCl2, forming beads (2–3 mm in diameter)ntrapping yeast cells. The beads were allowed to harden for 12 h

at 4 ◦C. The initial mean density of the yeast cells in the Ca-alginateimmobilized carrier reached 8.9 × 109 cells/mL of gel beads at thetime of immobilization.

2.6. Fermentation

Fermentation was carried out in 250 mL Erlenmeyer flaskscontaining 30 mL immobilized cells and 150 mL detoxified hemi-cellulosic hydrolysate as the production medium. The medium wassupplemented with 2 g/L yeast extract, 2.5 g/L KH2PO4, 0.25 g/LCaCl2 and 0.25 g/L MgCl2. The flasks were sealed with rubber stop-pers equipped with needles for CO2 venting and then placed inan incubator shaker with an agitation rate of 120 rpm at 30 ◦C.The beads were collected and washed with sterile water for threetimes after batch fermentation before they were reused in the newproduction medium.

2.7. Analytical methods

Samples from the fermentation flasks at different time inter-vals were centrifuged at 10000 rpm for 10 min, and the supernatantwas filtered through 0.45 �m membrane filters. Sugars, ethanol andother fermentation byproducts were analyzed on a HPLC system(Model 500, Syltech, USA) equipped with an organic acid column (ICSep ICE-Coregel 87H3, Transgenomic, USA) and a refractive indexdetector (Model 6040 XR, Spectra-Physics, USA). The column tem-perature was fixed at 60 ◦C. Pure water was used as the mobilephase at a flow rate of 0.5 mL/min.

The ethanol yield on fermentable sugars was calculated as con-centration of produced ethanol divided by initial concentration offermentable sugars (i.e., the sum of glucose and xylose) in the hemi-cellulosic hydrolysate.

Three parallel samples were used in all analytical determina-tions, and data are presented as the mean of three replicates. Themean and standard deviation of a data set were calculated usingMicrosoft Office Excel.

3. Results and discussion

3.1. Concentration and detoxification of corn stover hemicellulosehydrolysate

Acetic acid and furfural formed in the dilute acid hydrolysis ofthe hemicellulose are the main inhibitiors to the metabolic activ-ity of yeast cells [15]. In ethanol production from lignocellulose,the efficient recovery of ethanol seems to require higher ethanolconcentration [16], which in turn requires a relatively high concen-tration of initial fermentable sugar in hydrolysate. The hydrolysateis usually concentrated prior to fermentation to achieve a highersugar concentration; however, the inhibitors would also accumu-late to a higher level and threaten the fermentability of yeast cells.

Therefore, detoxification of hemicellulosic hydrolysate to removeinhibitors such as acetic acid and furfural is of great significance tosubsequent fermentation.

As seen in Table 1, 0.40 g/L of acetic acid and 0.08 g/L of fur-fural were detected in the undetoxified hemicellulosic hydrolysate,

30 J. Zhao, L. Xia / Biochemical Engineering Journal 49 (2010) 28–32

Table 2Effects of xylose concentration in detoxified hemicellulosic hydrolysate on ethanol production by immobilized S. cerevisiae ZU-10 cells.

Initial xylose (g/L) Concentration multiple (fold) Fermentation time (h) Residual xylose (g/L) Ethanol (g/L) Ethanol yield (g/g)

12.1 ± 0.5 1.0 18 0.0 ± 0.0 4.8 ± 0.5 0.369 ± 0.01430.1 ± 0.9 2.5 30 0.6 ± 0.1 12.4 ± 0.4 0.386 ± 0.01250.4 ± 1.1 4.2 48 1.7 ± 0.2 21.8 ± 0.9 0.407 ± 0.017

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3.2.3. Time course of ethanol production from detoxifiedhemicellulose hydrolysate

The detoxified hemicellulosic hydrolysate, supplemented withnitrogen source and inorganic salts, was fermented by recombinantS. cerevisiae ZU-10 cells. As shown in experiments using free cells

71.8 ± 1.3 6.0 7290.5 ± 1.4 7.5 96

110.0 ± 1.1 9.1 120

ach value corresponds to the mean of three replicates ±standard deviation.

nd the concentration of xylose and glucose was 12.1 g/L and.9 g/L, respectively. Arabinose and glucose seemed to be moreasily absorbed on CaSO4 after concentration and detoxificationnd suffered larger losses than xylose in the hydrolysate. Afteroncentration (six-fold) and detoxification, the concentration ofcetic acid was 1.16 g/L, equivalent to 0.19 g/L in the initial vol-me of the undetoxified hydrolysate, indicating that more than0% acetic acid was removed. Furfural was undetectable in theetoxified hemicellulosic hydrolysate. Most acetic acid and fur-ural were removed due to their volatile characteristics. Meanwhile,oncentration of the fermentable sugars could also be achieved,hich makes the following fermentation more economic. Study

howed that free cells of recombinant S. cerevisiae ZU-10 were sen-itive to inhibitors such as acetic acid and furfural. In fermentationf 80.0 g/L of pure xylose with free cells of recombinant S. cere-isiae ZU-10, the ethanol concentration within 72 h decreased from8.9 g/L to 27.9 g/L and 24.5 g/L with the addition of 0.25 g/L and.50 g/L of acetic acid respectively, and to 27.9 g/L and 22.1 g/L withhe addition of 0.08 g/L and 0.12 g/L of furfural respectively. As theoncentration of acetic acid and furfural further increased, ethanolermentability of free cells was harmed more seriously (data nothown). In contrast, the tolerance of Ca-alginate immobilized cellso acetic acid and furfural was greatly enhanced to 1.20 g/L and.12 g/L, respectively. When immobilized cells of recombinant S.erevisiae ZU-10 was used to ferment 80.0 g/L of pure xylose solu-ion containing 1.20 g/L of acetic acid, over 95.3% of xylose wasonsumed and the ethanol yield was over 0.378 g ethanol/g xyloseithin 60 h (data not shown). Thus, the concentrated and detoxi-ed hemicellulosic hydrolysate was suitable for ethanol productionsing immobilized yeast cells.

.2. Ethanol production from detoxified hemicellulosicydrolysate by immobilized recombinant S. cerevisiae ZU-10 cells

.2.1. Influence of initial pHFermentation processes of detoxified hemicellulosic

ydrolysate within 72 h at different initial pH were investi-ated. As can be seen in Fig. 1, when pH was below 4.5, theoncentration of residual xylose was quite high; ethanol concen-ration was below 29.8 g/L with the ethanol yield below 0.389 g/g.he yeast cells failed to convert xylose to ethanol effectively at lowH, which was possibly due to the toxicity of undissociated aceticcid. As the previous publication explained, the undissociatedrganic acid was more easily to pass through cell membraneshan its dissociated form, thus would cause inhibition to theermentability of microorganisms [17]. When the pH of the extra-ellular environment is low in a certain extent, acetic acid wouldainly exist in its undissociated form and could be toxic to theetabolic activity of yeast cells. Therefore, increasing the initial

H would reduce the toxicity of the detoxified hydrolysate as more

cetic acid dissociated. The results showed that pH 5.5 was mostuitable for fermentation of detoxified hemicellulosic hydrolysatey immobilized recombinant S. cerevisiae ZU-10 cells. At pH 5.5,he concentration of residual xylose was only 2.1 g/L and ethanoloncentration reached 31.1 g/L with the ethanol yield of 0.406 g/g.

2.1 ± 0.2 31.1 ± 0.7 0.406 ± 0.01123.7 ± 0.9 28.4 ± 0.7 0.294 ± 0.01561.8 ± 1.3 19.7 ± 0.5 0.168 ± 0.012

3.2.2. Influence of different xylose concentration in thehemicellulosic hydrolysate

Through the roto-evaporation procedure, hemicellulosichydrolysate of different concentration multiples could be obtained.With the increase of the concentration multiple, both the inhibitorsand fermentable sugars in the hydrolysate would accumulate.Concentrated fermentable sugars would be economic for ethanolproduction; yet, the excessive concentration would inducenegative effects: the metabolic activity of yeast cells would beinhibited by high concentration of toxic inhibitors; besides, highconcentration of fermentable sugar (mainly xylose) might alsocause a substrate inhibitory effect to yeast cells. Table 2 discussedthe relationship between xylose concentration and fermenta-tion results of detoxified hemicellulosic hydrolysate. With theincrease of xylose concentration, residual xylose accumulatedand more time was consumed in the ethanol production. Whenthe xylose was below 71.8 g/L, more than 96.7% xylose couldbe utilized; ethanol concentration could reach 31.1 g/L within72 h at the concentration multiple 6. As the xylose concentrationincreased over 90.5 g/L (concentration multiple 7.5), both thexylose utilization rate and the final ethanol concentration wereobviously decreased. Previous study showed that immobilizedrecombinant S. cerevisiae ZU-10 cells could tolerate a quite highconcentration of pure xylose. For the initial concentration ofxylose 120.0 g/L, more than 99.3% of xylose could be utilized andethanol yield was over 0.373 g/g xylose (data not shown). Thus,the high concentration of inhibitors could be the main factor forthe inefficient fermentation of highly concentrated hemicellulosichydrolysate.

Fig. 1. Effects of initial pH value on ethanol fermentation of detoxified hemicel-lulosic hydrolysate by immobilized recombinant S. cerevisiae ZU-10 cells. Residualxylose (�); ethanol (�). Error bars represent the standard deviation.

J. Zhao, L. Xia / Biochemical Engineering Journal 49 (2010) 28–32 31

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ig. 2. Time course of ethanol production from detoxified corn stover hemicelluloseydrolysate by recombinant S. cerevisiae ZU-10 (a) free cells; (b) immobilized cells.lucose (©); xylose (�); ethanol (�); xylitol (♦); glycerol (�). Error bars represent

he standard deviation.

o ferment detoxified hydrolysate (Fig. 2(a)), the concentration ofesidual xylose was still quite high after 120 h, reaching 15.6 g/Ln the fermentation broth; 78.3% xylose was utilized and 21.6 g/Lthanol was produced with the ethanol yield of 0.282 g/g. In con-rast, the fermentation process with immobilized cells proved toe more effective: after 72 h, only 2.1 g/L xylose remained in theermentation broth, 97.1% xylose was utilized and 31.1 g/L ethanolas produced with the ethanol yield of 0.406 g/g (Fig. 2(b)). Again,

he results proved that immobilized cells had a greater tolerance tohe inhibitors and better fermentability of detoxified hemicellulosicydrolysate, which was concurrent with the simulation experiment

n Section 3.1. More xylose in the hydrolysate was effectively con-erted to ethanol with immobilized cells. The fermentation courseas shortened, leading to the increase of both ethanol yield androductivity.

Meanwhile, the concentration of glycerol (byproduct) was alsoncreased in the fermentation process with immobilized cells,eaching 8.1 g/L within 72 h, compared with 3.1 g/L within 120 hsing free cells. In ethanol production, approximately 4–10% carbonource is converted into glycerol considering different yeast strains,

ubstrates and techniques. If the production of glycerol could beffectively inhibited to improve ethanol yield, more than 1.3 bil-ion liters of ethanol could be produced annually on earth withoutncreasing the overall cost of carbon sources. The disadvantagesf high glycerol production could be eliminated through genetic

Fig. 3. Repeated batches fermentation of hemicellulose hydrolysate by immobilizedrecombinant S. cerevisiae ZU-10 cells. Residual xylose (white); ethanol (dashed);ethanol yield (�). Error bars represent the standard deviation.

and metabolic strategies for construction of yeast strains of higherethanol yield [18,19]. The glycerol yields for free and immobilizedcells were 0.041 g/g and 0.106 g/g fermentable sugars respec-tively at the end of fermentation process. Glycerol is producedfrom the glycolytic intermediate dihydroxyacetone phosphate cat-alyzed by NAD+-dependent glycerol-3-phosphate dehydrogenaseand glycerol-3-phosphate phosphatase. Expression of genes GPD2and GPP1 encoding the two enzymes is simulated under anaerobicconditions [20]. Possibly due to in a more anaerobic environmentcreated by the Ca-alginate immobilized carrier, immobilized cellstended to choose the metabolic pathway of glycerol productionmore than free cells, thus more glycerol was produced with immo-bilized cells.

Ethanol production from hemicellulosic hydrolysate treatedwith dilute sulfuric acid has been examined using several pentose-utilizing yeasts. Previously reported results with respect to arelatively high ethanol yield were listed below. In terms of sugarcane bagasse hydrolysate, Van Zyl et al. [21] obtained the result of0.38 g/g with Pichia stipitis CBS 7126; Roberto et al. [22] obtainedthe result of 0.35 g/g (ethanol concentration of 24 g/L) with P. stipi-tis CBS 5773; Cheng et al. [23] got 0.34 g/g (ethanol concentrationof 19 g/L) using Pachysolen tannophilus DW06. The result of 0.46 g/gwith the ethanol concentration of about 10 g/L was achieved withP. stipitis CBS 5776 and red oak hydrolysate [24]. Ethanol yieldreached 0.35 g/g (ethanol concentration of 12.9 g/L) and 0.41 g/g(ethanol concentration of 19.1 g/L) with a parent strain of P. stipitisNRRL Y-7124 and its adapted strain using wheat straw hydrolysate[25]. Water-hyacinth (Eichhornia crassipes) hydrolysate was alsotaken as a substrate, and 18 g/L of ethanol with an ethanol yieldof 0.35 g/g was obtained with P. stipitis NRRL Y-7124 [26]. Com-paring our observations with those above, it can be concluded thatthe present study was of significance for biomass conversion in theproduction of fuel ethanol.

3.2.4. Repeated batch fermentation by immobilized recombinantS. cerevisiae ZU-10 cells

Repeated batch fermentation was carried out using immo-bilized yeast cells in shake flasks for 72 h each (Fig. 3). Theresults showed that after repeated use of immobilized cells forfive batches, the utilization rate of xylose could be maintained

more than 92.1%, and the concentration of ethanol was steadilyabove 30.1 g/L with the ethanol yield over 0.393 g/g. The vitalityand stability of immobilized yeast cells against toxic inhibitorswas to be proved by further repeated usage in hydrolysatefermentation.

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. Conclusions

This study demonstrates that it is feasible to use Ca-alginatemmobilized cells of recombinant S. cerevisiae ZU-10 for ethanolroduction from detoxified corn stover hemicellulosic hydrolysate.fter detoxification, the concentration of fermentation inhibitorsas effectively controlled within an acceptable level: furfural was

ompletely removed; 1.16 g/L acetic acid in the six-fold concen-rated hemicellulosic hydrolysate was proved to be tolerated byhe immobilized recombinant S. cerevisiae ZU-10 cells. Detoxifiedemicellulosic hydrolysate could be converted to ethanol by immo-ilized cells both efficiently and economically. At the initial pH 5.5nd xylose concentration 71.8 g/L, 97.1% xylose and 100% glucose inhe hydrolysate were utilized within 72 h; 31.1 g/L ethanol was pro-uced with the ethanol yield on fermentable sugars 0.406 g/g. The

mmobilized cells were also proved to be reusable in five batchesf fermentation. More than 92.1% xylose was utilized in each batchy the same immobilized cells, and ethanol yield on fermentableugars could maintain above 0.393 g/g.

cknowledgement

Financial support from Hi-tech Research and Development Pro-ram of China (2007AA05Z401) and Major Project of Naturalcience Foundation of Zhejiang Province (Z407010) is gratefullycknowledged.

eferences

[1] C.J. Campbell, J.H. Laherrere, The end of cheap oil, Sci. Am. 3 (1998) 78–83.[2] W. Bach, Fossil fuel resources and their impacts on environment and climate,

Int. J. Hydrogen Energy 6 (1981) 185–201.[3] Z. Kádár, Z. Szengyel, K. Réczey, Simultaneous saccharification and fermenta-

tion (SSF) of industrial wastes for the production of ethanol, Ind. Crop Prod. 20(2004) 103–110.

[4] Y. Yamashita, A. Kurosumi, C. Sasaki, Y. Nakamura, Ethanol production frompaper sludge by immobilized Zymomonas mobilis, Biochem. Eng. J. 42 (2008)314–319.

[5] K. Öhgren, A. Rudolf, M. Galbe, G. Zacchi, Fuel ethanol production fromsteam-pretreated corn stover using SSF at higher dry matter content, Biomass

Bioenergy 30 (2006) 863–869.

[6] M. Chen, J. Zhao, L.M. Xia, Enzymatic hydrolysis of maize straw polysaccha-rides for the production of reducing sugar, Carbohydr. Polym. 71 (2008) 411–415.

[7] T.W. Jeffries, C.P. Kurtzman, Strain selection, taxonomy, and genetics of xylose-fermenting yeasts, Enzyme Microb. Technol. 16 (1994) 922–932.

[

[

ing Journal 49 (2010) 28–32

[8] M.H. Toivari, A. Aristidou, L. Ruohonen, M. Penttila, Conversion of xylose toethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase(XKS1) and oxygen availability, Metab. Eng. 3 (2001) 236–249.

[9] V.S. Chang, W.E. Kaar, B. Burr, M.T. Holtzapple, Simultaneous saccharifica-tion and fermentation of lime-treated biomass, Biotechnol. Lett. 23 (2001)1327–1333.

10] M.P. Tucker, K.H. Kim, M.M. Newman, Q.A. Nguyen, Effects of temperatureand moisture on dilute acid steam explosion pretreatment of corn stover andcellulase enzyme digestibility, Appl. Biochem. Biotechnol. 105 (2003) 165–177.

11] K. Öhgren, J. Vehmaanpera, M. Siika-Aho, M. Galbea, L. Viikari, G. Zacchi, Hightemperature enzymatic prehydrolysis prior to simultaneous saccharificationand fermentation of steam-pretreated corn stover for ethanol production,Enzyme Microb. Technol. 40 (2007) 607–613.

12] J.M. Dominguez, N. Cao, C.S. Gong, G.T. Tsao, Dilute acid hemicellulosehydrolysates from corn cobs for xylitol production by yeast, Bioresour. Technol.61 (1997) 85–90.

13] Y. Kourkoutas, A. Bekatorou, I.M. Banat, R. Marchant, A.A. Koutinas, Immo-bilization technologies and support materials suitable in alcohol beveragesproduction: a review, Food Microbiol. 21 (2004) 377–397.

14] M. Chen, Z.F. Wang, L.M. Xia, Ethanol fermentation on xylose by immobilizedrecombinant yeast, J. Zhejiang Univ. (Eng. Sci.) 42 (2008) 290–293.

15] E. Palmqvist, H. Grage, N.Q. Meinander, B. Hahn-Hägerdal, Main and interac-tion effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth andethanol productivity of yeasts, Biotechnol. Bioeng. 63 (1999) 46–55.

16] A. Wingren, M. Galbe, G. Zacchi, Techno-economic evaluation of producingethanol from softwood: comparison of SSF and SHF and identification of bot-tlenecks, Biotechnol. Prog. 19 (2003) 1109–1117.

17] F. Lü, P.J. He, L.M. Shao, D.J. Lee, Stress of pH and acetate on product formationof fermenting polysaccharide-rich organic waste, Biochem. Eng. J. 39 (2008)97–104.

18] S. Bjorkqvist, R. Ansell, L. Adler, G. Liden, Physiological response to anaerobicityof glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae,Appl. Environ. Microbiol. 63 (1997) 128–132.

19] T.L. Nissen, M.C. Kielland-Brandt, J. Nielsen, J. Villadsen, Optimization of ethanolproduction in Saccharomyces cerevisiae by metabolic engineering of the ammo-nium assimilation, Metab. Eng. 2 (2000) 69–77.

20] A.L. Zhang, X. Chen, Improved ethanol yield through minimizing glycerol yieldin ethanol fermentation of Saccharomyces cerevisiae, Chin. J. Chem. Eng. 16(2008) 620–625.

21] C. Van Zyl, B.A. Prior, J.C. Du Preez, Production of ethanol from sugar canebagasse hemicellulose hydrolyzate by Pichia stipitis, Appl. Biochem. Biotechnol.17 (1988) 357–369.

22] I.C. Roberto, L.S. Lacis, M.F.S. Barbosa, I.M. de Manchilla, Utilization of sugarcane bagasse hemicellulosic hydrolyzate by Pichia stipitis for the production ofethanol, Process Biochem. 26 (1991) 15–21.

23] K.K. Cheng, B.Y. Cai, J.A. Zhang, H.Z. Ling, Y.J. Zhou, J.P. Ge, J.M. Xu, Sugarcanebagasse hemicellulose hydrolysate for ethanol production by acid recoveryprocess, Biochem. Eng. J. 38 (2008) 105–109.

24] A.V. Tran, R.P. Chambers, Red oak wood derived inhibitors in ethanol fermen-

tation of xylose by Pichia stipitis CBS 5776, Biotechnol. Lett. 7 (1985) 841–846.

25] J.N. Nigam, Ethanol production from wheat straw hemicellulose hydrolysateby Pichia stipitis, J. Biotechnol. 87 (2001) 17–27.

26] J.N. Nigam, Bioconversion of water-hyacinth (Eichhornia crassipes) hemicel-lulose acid hydrolysate to motor fuel ethanol by xylose–fermenting yeast, J.Biotechnol. 97 (2002) 107–116.