Fuel Processing Technology 91 (2010) 1807–1811

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Fuel Processing Technology

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Bioconversion of corn stover hydrolysate to ethanol by a recombinant yeast strain

Jing Zhao, Liming Xia ⁎Department of Chemical and Biological 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 © 2010 Elsevier B.V. Aldoi:10.1016/j.fuproc.2010.08.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 February 2010Received in revised form 25 July 2010Accepted 1 August 2010

Keywords:Corn stoverEnzymatic hydrolysateHemicellulosic hydrolysateEthanolRecombinant yeastImmobilization

Three corn stover hydrolysates, enzymatic hydrolysates prepared from acid and alkaline pretreatmentsseparately and hemicellulosic hydrolysate prepared from acid pretreatment, were evaluated in compositionand fermentability. For enzymatic hydrolysate from alkaline pretreatment, ethanol yield on fermentablesugars and fermentation efficiency reached highest among the three hydrolysates; meanwhile, ethanol yieldon dry corn stover reached 0.175 g/g, higher than the sum of those of two hydrolysates from acidpretreatment. Fermentation process of the enzymatic hydrolysate from alkaline pretreatment was furtherinvestigated using free and immobilized cells of recombinant Saccharomyces cerevisiae ZU-10. Concentratedhydrolysate containing 66.9 g/L glucose and 32.1 g/L xylose was utilized. In the fermentation with free cells,41.2 g/L ethanol was obtained within 72 h with an ethanol yield on fermentable sugars of 0.416 g/g.Immobilized cells greatly enhanced the ethanol productivity, while the ethanol yield on fermentable sugarsof 0.411 g/g could still be reached. Repeated batch fermentation with immobilized cells was furtherattempted up to six batches. The ethanol yield on fermentable sugars maintained above 0.403 g/g with allglucose and more than 92.83% xylose utilized in each batch. These results demonstrate the feasibility andefficiency of ethanol production from corn stover hydrolysates.

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1. Introduction

The importance of ethanol, either as a clean and safe transporta-tion fuel or an octane enhancer, has increased with the anticipatedshortage of fossil fuel reserves and increased air pollution. Lignocel-lulose, as a renewable energy source that may alternate the ever-reducing fossil fuels, is of great interest for industrial ethanolproduction [1–3]. Different from sugarcane and starch materials thatwould take up limited agricultural land, lignocellulosic materials aremore and more attractive for ethanol production due to their greatavailability and relatively low cost. Corn stover is one of the mostabundant agricultural wastes, and it is estimated that about250 million tons are produced annually in China. Currently, cornstover is regarded as the most favored lignocellulosic resource forlarge-scale ethanol production in China because it represents a cheap,renewable, widely available feedstock [4].

In the bioconversion of lignocellulose to ethanol, pretreatment isan essential procedure. By altering the structural and chemicalcomposition of lignocellulosic materials, e.g. reducing the crystallinityof cellulose and removing lignin, etc., pretreatment increases thesusceptibility of the carbohydrates to enzyme attack and facilitateshydrolysis of lignocellulosic materials to fermentable sugars effec-tively. Roughly, four categories of pretreatment methods including

physical, chemical, physico-chemical and biological pretreatmentshave been applied over the years [5,6]. Different pretreatmentprocesses have their own advantages and limitations regardingoperation cost, equipment corrosion, formation of byproducts andmaybe efficient for biomass of different composition [7]. In theconsideration of converting lignocellulosic materials to ethanol withhigh efficiency, suitable pretreatment methods that promote efficienthydrolysis to fermentable hexose and pentose utmostly should bechosen.

Glucose and xylose are the main monosaccharides in lignocellu-losic hydrolysate, and bioconversion of glucose and xylose to ethanoleffectively is very important in ethanol production from lignocellu-lose. Several microorganisms could utilize both glucose and xylose inthe hydrolysate of lignocellulosic materials for ethanol production.Pichia stipitis, as well as xylose-utilizing recombinant strains ofSaccharomyces cerevisiae, which introduce genes encoding theenzymes xylose reductase (XR) and xylitol dehydrogenase (XDH)present in P. stipitis, have been proved to have relatively good resultsof ethanol yields with different substrates[8–10]. By far, fermentationof hydrolysates of pretreated corn stover with immobilized cells ofrecombinant S. cerevisiae has rarely been reported.

In the present study, the effect of two commonly-used chemicalpretreatment methods, dilute acid pretreatment and alkaline pre-treatment, on the composition and fermentability of corn stoverhydrolysates was compared. Both free and immobilized cells of arecombinant S. cerevisiae yeast were used for fermentation of cornstover enzymatic hydrolysate obtained by alkaline pretreatment. This

1808 J. Zhao, L. Xia / Fuel Processing Technology 91 (2010) 1807–1811

research can provide important information on the commercialutilization of corn stover for large-scale ethanol production, whichis of far-reaching significance in both new energy resourcesdevelopment and environmental protection.

2. Materials and methods

2.1. Raw material and pretreatment

Corn stover sample was subjected to two pretreatment methods toget different hydrolysates. The main composition of raw corn stoverwas as follows (dry weight basis): cellulose 38.7%, hemicellulose21.7%, lignin 19.3%, and others 20.3%. For acid pretreatment, cornstover sample was pretreated at 108 °C with 1.5% sulfuric acid for 6 h,with an initial solid–liquid ratio of 1:10 (w/v). For alkalinepretreatment, corn stover was pretreated at 80 °C with 2% sodiumhydroxide for 75 min with an initial solid–liquid ratio of 1:8 (w/v).The mixture was subjected to vacuum filtration to separate the solidresidues and the filtrate fraction. The corn stover residue andhydrolysate was collected and analyzed separately. The maincomposition of the acid-pretreated corn stover residue was as follows(dry weight basis): cellulose 62.4%, hemicellulose 12.0%, lignin 24.9%,and others 0.7%. Themain composition of the alkaline-pretreated cornstover residue was as follows (dry weight basis): cellulose 64.1%,hemicellulose 24.6%, lignin 8.6%, and others 2.7%. The recovery forcellulose and hemicellulose was 92.3% and 32.2% for acid pretreat-ment, 97.2% and 66.5% for alkaline pretreatment, respectively.

2.2. Preparation of hydrolysates

The hemicellulosic hydrolysate obtained from acid-pretreatmentwas detoxified and concentrated by evaporation. The concentratedhemicellulosic hydrolysate was regularly stirred at 75 °C in a waterbath shaker and lime solution was slowly added to neutralize residualsulfuric acid and adjust hydrolysate to pH 6.5–7.0. The hemicellulosichydrolysate was adjusted to pH 6.5–7.0 by adding lime solution toremove the residual SO4

2− by forming CaSO4 precipitate andmeanwhile to adjust the pH suitable for the fermentation of thehemicellulosic hydrolysate. CaSO4 precipitate was removed byvacuum filtration after 1 h, and the chemical composition of theliquor was analyzed.

Corn stover residues obtained after two pretreatments were bothhydrolyzed by the cellulase and cellobiase mixture with enzymeloadings of 20 filter paper activity units (FPU)/g substrate and 10cellobiase units (CBU)/g substrate as previously described [4]. Thehydrolysates were concentrated and analyzed respectively.

The hydrolysates were concentrated by roto-evaporation methodat 60 °C. The evaporation system comprises a rotatory evaporator anda vacuum pump.

2.3. Microorganism

The recombinant xylose-utilizing yeast strain S. cerevisiae ZU-10expresses xylose reductase (XR) and xylitol dehydrogenase (XDH)from the chromosomally integrated P. 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 at4 °C [11].

2.4. Inoculum preparation

A loopful of cells were transferred to each 250 mL Erlenmeyer flaskcontaining 50 mL of sterile culture medium consisting of 5 g/L yeastextract, 10 g/L peptone, 10 g/L xylose and 10 g/L glucose, and thecultures were incubated for 24 h in an orbital shaker (180 rpm, 30 °C).

For fermentation with free cells, cells were harvested by centrifuga-tion (4800 rpm, 5 min), suspended in sterilized water and used toinoculate the fermentation medium. For fermentation with immobi-lized cells, 5 mL inoculum from the above culture was furtherpropagated in 250 mL Erlenmeyer flasks containing 100 mL ofmedium. Themedium composition was as follows: 5 g/L yeast extract,10 g/L peptone, 20 g/L xylose, 20 g/L glucose, 2.5 g/L KH2PO4, 0.25 g/LCaCl2 and 0.25 g/L MgCl2. The cultures were incubated for 36 h in anorbital shaker (180 rpm, 30 °C).

2.5. Cell immobilization

Cells grown in a propagation medium was collected by centrifu-gation for 10 min at 4000 rpm, 4 °C. The collected cells were addedinto 2% (w/v) sodium alginate solution, well mixed with 1% (w/v)diatomite. Themixed solution was extruded through a syringe into 2%(w/v) CaCl2 solution. Alginate drops solidified upon contact withCaCl2, forming beads (2–3 mm in diameter) and thus entrapping yeastcells by stirring continuously. The beads were allowed to harden for12 h at 4 °C. The mean density of the yeast cells in the Ca-alginateimmobilized carrier reached 8.9×109 cells/mL of gel beads [12].

2.6. Fermentation

Fermentation with free cells was carried out at 30 °C underanaerobic conditions, and 1.5 mL of the cell suspension wasinoculated into 250 mL Erlenmeyer flasks with a working volume of100 mL. Fermentation with immobilized cells was carried out in250 mL Erlenmeyer flasks containing 30 mL immobilized cells and150 mL hydrolysate as the production medium. Concentrated hemi-cellulosic hydrolysate and enzymatic hydrolysate, supplemented with2 g/L yeast extract, 2.5 g/L KH2PO4, 0.25 g/L CaCl2 and 0.25 g/L MgCl2andwith initial pH 5.5, were utilized as the fermentationmedium. Theflasks were sealed with rubber stoppers equipped with needles forCO2 venting and then placed in an incubator shaker with an agitationrate of 120 rpm at 30 °C. The gel beads for immobilization werecollected and washed with sterile water for three times afterfermentation before theywere reused in the newproductionmedium.

2.7. Analytical methods

Filter paper activity and cellobiase activity were determinedaccording to standard IUPAC (International Union of Pure and AppliedChemistry) procedures [13]. One FPU is defined as the amount ofenzyme that releases 1 μmol of glucose equivalents from WhatmanNo. 1 filter paper per minute. One CBU is the amount of enzyme thatconverts 1 μmol of cellobiose to 2 μmol of glucose per minute.

The cellulose was determined by HNO3-ethanol method, lignin by72% H2SO4 method, and hemicellulose by two-brominating method[14].

Samples from the fermentation flasks at different time intervalswere centrifuged at 10,000 rpm for 10 min, and then the supernatantwas filtered through 0.45 μm membrane filters. Sugars, ethanol andother fermentation byproducts were analyzed using 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 columntemperature 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=concentration ofproduced ethanol / initial concentration of fermentable sugars (i.e.,the sum of glucose and xylose) in the hydrolysate.

The ethanol yield on dry corn stover=the amount of producedethanol / the amount of raw corn stover used.

The fermentation efficiency=the ethanol yield on fermentablesugars / theoretical ethanol yield.

Table 2Results of the fermentation of different hydrolysates with S. cerevisiae ZU-10.

Hydrolysate

I II III

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Three parallel samples were used in all analytical determinations,and data are presented as themean of three replicates. Themean andstandard deviation of a data set were calculated using MicrosoftOffice Excel.

Glucose conversion (%) 100.00±0.00 100.00±0.00 100.00±0.00Xylose conversion (%) 57.99±1.08 70.20±0.73 88.47±1.46Final ethanol (P, g/L) 34.3±0.9 20.9±1.1 41.2±1.2Ethanol yield onfermentable sugars(YE/FS, g/g)

0.413±0.012 0.273±0.010 0.416±0.006

Ethanol yield on dry cornstover (YE/C, g/g)

0.091±0.011 0.043±0.008 0.175±0.013

Volumetric ethanolproductivity (Qp, g/L/h)

0.953±0.025 0.218±0.011 0.572±0.017

Fermentation efficiency (E, %) 82.73±1.13 58.91±1.62 84.28±1.22

Each value corresponds to the mean of three experiments±standard deviation.I, concentrated enzymatic hydrolysate from acid-pretreated corn stover;II, concentrated hemicellulosic hydrolysate from acid pretreatment of corn stover;III, concentrated enzymatic hydrolysate from alkaline-pretreated corn stover.

3. Results and discussion

3.1. Comparison of ethanol production from different corn stoverhydrolysates

Concentrated enzymatic hydrolysate and hemicellulosic hydro-lysate from acid-pretreated corn stover, as well as enzymatichydrolysate from alkaline-pretreated corn stover were obtainedand evaluated for composition and fermentability (Tables 1 and 2).As shown in Table 1, enzymatic hydrolysates of corn stover containhigh levels of glucose and xylose but little arabinose, hemicellulosichydrolysate of corn stover contains a large amount of xylose andcertain arabinose but little glucose. Fufural, as one of the maininhibitors to yeast cells and its fermentative performance, was notdetected in all hydrolysates. Acetic acid, as another main inhibitor tothe physiological activity of yeast cells, varied in its amount indifferent hydrolysates. The concentration of acetic acid ranged from0.66 to 1.16 g/L, which may be related to the concentration multipleof initial hydrolysate.

Results of the fermentation of different hydrolysate withrecombinant S. cerevisiae ZU-10 can be seen from Table 2. As forenzymatic hydrolysate from acid-pretreated corn stover, the ethanolyield on fermentable sugars was relatively high, reaching 0.413 g/g;meanwhile the volumetric ethanol productivity of 0.953 g/L/h wasthe highest among three hydrolysates, since glucose was morequickly consumed than xylose in the hydrolysate. However, only57.99% xylose could be consumed. Hemicellulosic hydrolysate fromacid pretreatment of corn stover contained mostly xylose and asmall amount of glucose, so the ethanol yield on fermentable sugarswas quite low, only 0.273 g/g. The prolonged fermentation cycle dueto mainly xylose constitutes in the hydrolysate dedicated to lowvolumetric ethanol productivity and fermentation efficiency. As foralkaline-pretreated enzymatic hydrolysate, 88.47% of initial xylosecould be consumed, much more improved compared to that in acid-pretreated enzymatic hydrolysate. It is possibly because the alkalinepretreatment was relatively mild compared with acid pretreatment,fermentation inhibitors such as acetic acid and some other phenoliccompounds were low in concentration, affecting the recombinantyeast's fermentability to a lower extent [15]. Thus, xylose conversionwas more rapid and efficient in alkaline-pretreated enzymatichydrolysate than that in acid-pretreated enzymatic hydrolysate.The ethanol yield on fermentable sugars reached 0.416 g/g and thefermentation efficiency was 84.28%, relatively high due to thecomprehensive utilization of glucose and xylose in the enzymatichydrolysate.

Table 1Chemical composition of different corn stover hydrolysate.

Hydrolysate

I II III

Glucose (g/L) 66.1±1.1 4.8±0.2 66.9±0.9Xylose (g/L) 16.9±0.8 71.8±1.3 32.1±0.8Arabinose (g/L) 1.2±0.2 14.3 ±0.7 4.1±0.4Acetic acid (g/L) 0.78±0.16 1.16±0.18 0.66±0.12Furfural (g/L) - - -

Each value corresponds to the mean of three experiments±standard deviation.I, concentrated enzymatic hydrolysate from acid-pretreated corn stover;II, concentrated hemicellulosic hydrolysate from acid pretreatment of corn stover;III, concentrated enzymatic hydrolysate from alkaline-pretreated corn stover.

Ethanol yield on dry corn stover was 0.175 g/g for alkaline-pretreated enzymatic hydrolysate, more than the sum of that for bothacid-pretreated enzymatic hydrolysate and hemicellulosic hydroly-sate. For alkaline-pretreated enzymatic hydrolysate, ethanol yield onfermentable sugars and fermentation efficiency were also highestamong the results for three hydrolysates. Considering the operationcost and cycle as well, alkaline-pretreated enzymatic hydrolysate waschosen as a suitable source for further ethanol production.

3.2. Fermentation of enzymatic hydrolysate with free cells ofrecombinant S. cerevisiae ZU-10

Fermentation of the enzymatic hydrolysate from alkaline-pre-treated corn stover was carried out by using the free cells ofrecombinant S. cerevisiae ZU-10. As shown in Fig. 1, recombinant S.cerevisiae ZU-10 could utilize both glucose and xylose in thehydrolysate, yet the consumption rate was different. Glucose wasconsumed much faster than xylose, and totally utilized within 12 h;while xylose was consumed at a slow rate of 0.307 g/L/h within 96 h.66.9 g/L glucose and 32.1 g/L xylose were converted to 41.2 g/Lethanol within 72 h with an ethanol yield on fermentable sugars of0.416 g/g. The concentration of byproducts as xylitol and glycerolwere both increased gradually, reaching 2.9 g/L and 6.5 g/L at 96 h,respectively.

Fig. 1. Time course of ethanol production from enzymatic hydrolysate of corn stover byfree cells of recombinant S. cerevisiae ZU-10. Glucose (○); xylose (●); ethanol (▲);xylitol (◊); glycerol (□). Error bars represent the standard deviation.

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3.3. Fermentation of enzymatic hydrolysate with immobilized cells ofrecombinant S. cerevisiae ZU-10

3.3.1. Batch fermentation of enzymatic hydrolysateIn recent years, immobilization of microbial cells in different

carriers attracts increasing interest due to its advantages over freecells, such as enhancement of productivity, easy cell recovery fromfermentation broth, protection of cells against toxic substances andcell recycling in repeated batch operations, etc.[16,17]. However,fermentation of enzymatic hydrolysate from alkaline-pretreated cornstover with Ca-alginate immobilized cells was rarely reported in theliterature yet.

To study the influence of acetic acid in enzymatic hydrolysate onthe fermentability of immobilized S. cerevisiae ZU-10 cells, mixedsugars with the initial composition of 70.0 g/L glucose and 30.0 g/Lxylose were used as the simulating medium for ethanol production(Fig. 2(A)). In the pure sugar system, glucose was totally utilizedwithin 6 h and 65.0% of xylose was quickly utilized; within 18 h,xylose was decreased to 1.1 g/L with the consumption ratio of 96.3%.Ethanol concentration reached 42.5 g/L with the yield on ferment-able sugars of 0.425 g/g. Fermentation profile of enzymatic hydro-lysate from alkaline-pretreated corn stover was shown in Fig. 2(B).Glucose was also totally consumed within 6 h. Only 1.2 g/L xyloseremained in the fermentation broth at 24 h with the consumptionratio of 96.3%, meanwhile the utilization rate of xylose in the

Fig. 2. Time course of ethanol production from (A) mixed sugars and (B) enzymatichydrolysate of corn stover by immobilized cells of recombinant S. cerevisiae ZU-10.Glucose (○); xylose (●); ethanol (▲); xylitol (◊); glycerol (□). Error bars represent thestandard deviation.

hydrolysate was 1.29 g/L/h. The high density of immobilized yeastcells contributed to the enhancement of glucose/xylose convertingspeed. In addition, immobilization of microorganisms on inertsupports could protect cells against toxic substances better thanfree cells, the relatively high tolerance of immobilized yeast cells toinhibitors would also be the reason for the enhancement of glucose/xylose converting speed.

Glucose (66.9 g/L) and 32.1 g/L xylose were converted to 40.7 g/Lethanol within 24 h, with an ethanol yield on fermentable sugars of0.411 g/g. The results showed that 0.42 g/L acetic acid contained inenzymatic hydrolysate caused no obvious inhibitory effect to thefermentability of immobilized S. cerevisiae ZU-10 cells. The concen-tration of byproducts as xylitol and glycerol were 2.8 g/L and 5.3 g/L,respectively, both lower than that produced with free cells. Comparedwith fermentation of enzymatic hydrolysate with free cells, thefermentation cycle with immobilized cells was greatly shortened from72 h to 24 h and ethanol productivity was effectively enhanced to1.70 g/L/h from 0.572 g/L/h.

As previously mentioned [12], detoxified hemicellulosic hydroly-sate with initially 71.8 g/L xylose and 4.8 g/L glucose was alsofermented with immobilized S. cerevisiae ZU-10 cells. The ethanolyield on fermentable sugars was 0.406 g/g within 72 h and the ethanolproductivity was 0.432 g/L/h. Compared with the current research,obviously, enzymatic hydrolysate of alkaline-pretreated corn stoverhad better fermentability, which led to higher ethanol yield andproductivity, also a shortened fermentation cycle. The results alsoindicated that the operation process would be much easier inhandling raw materials and preparation of fermentable sugars afterusing alkaline pretreatment than acid pretreatment.

3.3.2. Repeated batch fermentation of enzymatic hydrolysateRepeated batch fermentation of enzymatic hydrolysate from

alkaline-pretreated corn stover was carried out using immobilizedyeast cells in shake flasks (Fig. 3), 24 h for each batch. The resultsshowed that after repeated use of immobilized cells for six batches,the conversion of glucose and xylose could be maintained at 100%and more than 92.83%, respectively. The average concentration ofethanol was 40.4 g/L with an average ethanol yield on fermentablesugars of 0.414 g/g. Immobilized yeast cells could be used forsustainable ethanol production from corn stover enzymatic hydro-lysate. The vitality and stability of immobilized yeast cells was to beproved by further repeated usage in fermentation of enzymatichydrolysate.

Fig. 3. Repeated batch fermentations of corn stover enzymatic hydrolysate byimmobilized cells of recombinant S. cerevisiae ZU-10. Residual xylose (white); ethanol(dashed); ethanol yield (●). Error bars represent the standard deviation.

1811J. Zhao, L. Xia / Fuel Processing Technology 91 (2010) 1807–1811

4. Conclusions

Three corn stover hydrolysates were used for ethanol productionwith recombinant S. cerevisiae ZU-10. For alkaline-pretreated enzy-matic hydrolysate, the ethanol yield on fermentable sugars andfermentation efficiency were highest among the results for threehydrolysates. The ethanol yield on dry corn stover for enzymatichydrolysate from alkaline pretreatment was also higher than the sumof those of enzymatic hydrolysate and hemicellulosic hydrolysatefrom acid-pretreated corn stover. The results demonstrated that itwould be more effective to use enzymatic hydrolysate from alkaline-pretreated corn stover for ethanol production rather than thehydrolysates from acid pretreatment. In fermentation with free cellsof S. cerevisiae ZU-10, 41.2 g/L ethanol was obtained within 72 h fromenzymatic hydrolysate from alkaline-pretreated corn stover contain-ing 66.9 g/L glucose and 32.1 g/L xylose. Fermentation of the samehydrolysate was greatly improved in productivity with immobilizedcells of S. cerevisiae ZU-10, reaching 1.70 g/L/h compared with0.572 g/L/h obtained by free cells. All glucose and 96.3% xylose wereconsumed within 24 h and 40.7 g/L ethanol was obtained with theethanol yield on fermentable sugars of 0.411 g/g. The immobilizedcells were also proved to be reusable in six batches of fermentation.More than 92.83% xylose was utilized in each batch by the sameimmobilized cells, and ethanol yield on fermentable sugars could bemaintained above 0.403 g/g.

Acknowledgement

Financial support from Hi-tech Research and DevelopmentProgram of China (2007AA05Z401) and Major Project of NaturalScience Foundation of Zhejiang Province (Z407010) is gratefullyacknowledged.

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