en

an

Keywords:Cellulosic hydrolysatesBatch fermentation

thevesal yglu

fermentation, the effects of feeding glucose concentrations on ethanol fermentation were studied. The

on noroducn to m

commercialization of biofuel production much more promising[13].

For production of bioethanol, a lower raw material price,together with a high ethanol yield and efcient enzymes, will de-crease the production cost signicantly. Several different pretreat-ment methods have been used to facilitate the enzymatic

tages of this process include the reduction of substrate and end-product inhibition, higher productivity of ethanol, higher dissolvedoxygen rate, decreased fermentation time, and higher saccharica-tion rate [8]. Fed-batch fermentation has been reported as a goodprocess for ethanol production when performed on different rawmaterials such as corn stover [9] and recycled paper-derivedmaterial [10].

Even conversion of corncob hydrolysate to bioethanol either bybatch fermentation [1,11] or by fed-batch fermentation [12,13]separately has been studied in the past, the simultaneous

Corresponding authors. Tel.: +886 4 23590121x37306; fax: +886 4 23599059(C.-L. Hsu), tel.: +886 3 5381183x8482; fax: +886 3 6102342 (H.-D. Jang).

Fuel 97 (2012) 166173

Contents lists available at

ue

.eE-mail address: hungder@mail.ypu.edu.tw (H.-D. Jang).past few decades. Cellulosic biomass is an ideal source of energybecause it is both renewable and available in large quantitiesaround the world. However, the process for the production ofethanol from cellulosic materials is more complicated than itsproduction from sugar or starch-based ones. Specically, thereare technical and economical impediments in regards to the devel-opment of commercial processes that utilize cellulosic biomass.Technologies that will allow for the cost-effective conversion ofcellulosic biomass into fuels and other chemicals are being devel-oped. These technologies include low-cost thermo or chemical pre-treatment, highly effective cellulases and hemicellulases, andefcient and robust fermentative microorganisms, have made the

1,4-exoglucanase and b-glucosidase [5], and b-1,4-endoxylanase.These cellulolytic enzymes have been applied to increase thehydrolysis efciency of cellulosic materials [6].

Many different types of processes for ethanol fermentation havebeen proposed, including batch fermentation, continuous fermen-tation, continuous fermentation with cell recycling, fed-batch cul-tures and repeated-batch cultures [7]. Batch fermentation processis used extensively to convert sugars to ethanol for the productionof beverages and biofuels. As for fed-batch fermentation, the inter-mittent addition of glucose, without the removal of the fermenta-tion broth, into the fed-batch culture is one of the most commonmethods for the production of ethanol in the industry. The advan-Fed-batch fermentationBioethanol

1. Introduction

To lessen the worlds dependenceuse of agricultural biomass for the pbioethanol has drawn much attentio0016-2361/$ - see front matter 2012 Elsevier Ltd. Adoi:10.1016/j.fuel.2012.02.006feeding glucose concentration of 30 g/l resulted in a higher ethanol yield than that of 20 g/l and 10 g/l did.The cell biomass, ethanol yields, and ethanol conversion rate for the fed-batch fermentation, feeding at30 g/l glucose concentration, were 44.5 g/l, 32.3 g/l and 0.64 g ethanol/g glucose, respectively. The resultsindicate that the fed-batch fermentation had a higher ethanol yield than that of the batch fermentation.

2012 Elsevier Ltd. All rights reserved.

n-renewable resources,tion of biofuels such asany researchers in the

hydrolysis of lignocellulosic material [4]. An efcient process forobtaining reducing sugars from lignocellulosic material is to usechemical/physical pretreatment, followed by enzymatic hydroly-sis. The hydrolysis of natural lignocellulose to glucose dependson the synergy of enzymes system, i.e., b-1,4-endoglucanase, b-Available online 18 February 2012Further batch fermentation of cellulosic hydrolysate was performed using batch cultures of Saccharomy-ces cerevisiae BCRC 21812, 23.341.1 g/l biomass and 6.923.0 g/l ethanol was obtained. For the fed-batchComparison of batch and fed-batch fermfor bioethanol production

Yi-Huang Chang a, Ku-Shang Chang a, Cheng-Wei HuaDepartment of Food Science, Yuanpei University, Hsinchu 300, Taiwanb Institute of Food Science, Yuanpei University, Hsinchu 300, TaiwancDepartment of Food Science, Tunghai University, Taichung 407, Taiwan

a r t i c l e i n f o

Article history:Received 31 January 2011Received in revised form 5 February 2012Accepted 6 February 2012

a b s t r a c t

The optimal conditions forfed-batch cultures were inreducing sugar with maximoverall reducing sugar and

F

journal homepage: wwwll rights reserved.tations using corncob hydrolysate

g b, Chuan-Liang Hsu c,, Hung-Der Jang a,

maximum production of ethanol from cellulosic hydrolysate in batch andtigated and compared. The pretreated corncob could be converted intoields after the enzymemixtures were fed. After 48 h of hydrolytic reaction,cose concentrations reached 0.61 and 0.36 g/g dried substrate, respectively.

SciVerse ScienceDirect

l

lsevier .com/locate / fuel

comparison of the efciencies of both batch and fed-fermentationsby the same cellulosic hydrolysate were rare. Additionally, the im-pact of different fermentation treatments, i.e. batch and fed-batch,of cellulosic hydrolysate on the dynamics of microbial growth andethanol production rate in the respective fermentors were seldomevaluated. In this work, the hydrolysis process of the corncob sub-strate using pretreatment with acid, autoclaving and then hydroly-sis with the enzyme mixtures was examined. The fermentationprocesses and kinetics of the corncob hydrolysate in the batchand fed-batch cultures of Saccharomyces cerevisiae BCRC 21812were also compared.

2. Materials and methods

2.1. Cellulosic material and pretreatment

The corncob materials, purchased from a local market, wereoven-dried for 24 h at 50 C, grounded into particles (diameter210 mm) and stored in pill vials at 25 C. The corncob materialsconsisted mainly of 42% (w/w) cellulose and 28% (w/w) hemicellu-lose, which could be hydrolyzed to reducing sugar. In addition,there was 20% of lignin in the corncob materials. The rest wereashes and minor components. The structural carbohydrate and lig-nin in biomass were determined according to Standard BiomassAnalytical Procedures of National Renewable Energy Laboratory(NREL).

Acid pretreatment was performed with 1% (v/v) sulfuric acid for30 min at a solid-to-liquid ratio of 1:10. The mixture was ltered.Then the ltrate was further hydrolyzed by autoclaving at 121 Cfor 60 min, according to the procedures described in our previousreport [14]. After the pretreatment, the cellulosic residue wassoaked in distilled water and incubated in water bath at 50 C for30 min, and then ltered.

2.2. Microorganisms and cultivation

The strain of S. cerevisiae BCRC 21812, purchased from Biore-sources Collection and Research Center, FIRDI (Hsinchu, Taiwan),was used as an inoculum for ethanol fermentation. S. cerevisiaeBCRC 21812 is traditionally used for alcoholic beverage and bioeth-anol production. It had been found that S. cerevisiae BCRC 21812could grow well in YPD media in the presence of 8% (v/v) ethanolaccording to our previous preliminary study. Yeast cultures weremaintained in a YPD medium containing 2% (w/v) dextrose, 1%(w/v) peptone and 0.5% (w/v) yeast extract at 25 C for 48 h. Theinitial pH was adjusted to 6.5 either 1 N HCl or NaOH prior to ster-ilization at 121 C for 20 min.

2.3. Hydrolysis of cellulosic residue by treatment with the enzymemixture

The mixture of prehydrolysate obtained from the acid and auto-claving pretreatments was collected and ltered with Whatman

TreaSa

tions

(g/ g

DS)

0.6

0.8

1.0

CellobioseGlucoseXyloseOther sugarsTotal sugars

(a)

lysi

(g/ g

Y.-H. Chang et al. / Fuel 97 (2012) 166173 167Hydro200

Tota

l red

ucin

g su

gar

0.0

0.2

0.4

0.6Sample 1

Suga

r con

cent

ra

0.0

0.2

0.4

DS) 0.8

1.0

(b)Fig. 1. (A) Comparison of reducing sugar, glucose, xylose and cellobiose produced from vaand acid treatment for 15 min. Sample 3: autoclaving, acid, and enzymatic hydrolysishydrolysis. The values represent the average of three measurements standard deviatiotmentsmple 2 Sample 3

s time (Hour)806040rious treatments. Sample 1: autoclaving at 121 C for 60 min. Sample 2: autoclaving. (B) Change of total reducing sugar produced of sample 3 for 72 h of enzymaticn.

No. 4 lter paper. A commercial cellulase mixture, 1.5 ml (1000 IU/ml) Cellulase (Sigma, St Louis, MO, USA) supplemented with0.52 ml (250 IU/ml) Novozyme 188 (Sigma, St Louis, MO, USA),was used to hydrolyze the cellulosic residue. Enzymatic hydrolysiswas performed with a 100 ml prehydrolysate and the commercialcellulase solution. The protocols of enzymatic hydrolysis of prehy-drolysate to produce reducing sugar were also according to ourprevious report [14]. The mixtures were incubated at 50 C in anorbital shaker with a speed of 160 rpm for 72 h. Samples werewithdrawn and analyzed for levels of total reducing sugar, glucose,xylose, and cellobiose concentration.

2.4. Batch and fed-batch fermentation of cellulosic hydrolysate

Inoculum was prepared by transferring 5% (v/v) of the cells(108/ml) of S. cerevisiae BCRC 21812 into fermentation media.The medium in the batch ethanol fermentation was (%, w/v): cellu-losic hydrolysate, 14; peptone 0.5; yeast extract 0.25 at pH 6.0.The medium for fed-batch fermentation was 2% cellulosic hydroly-sate, 0.5% peptone and 0.25% yeast extract. After 24 h, the cellulosichydrolysate containing 13% (w/w) glucose was fed. The cultureswere shaken at 150 rpm for 2 d, then adjusted to 100 rpm at25 C. Samples were collected regularly and ltered through a0.45 lm Millipore membrane. Glucose, xylose, cellobiose and eth-anol concentrations were analyzed by HPLC (Waters Co., MA, USA).

2.5. Analysis methods

The dry weight content of the raw materials was determined by

pro spectrophotometer set at 600 nm (GE Healthcare Co., IL,USA). The reducing sugars liberated by these reactions were mea-sured using the 3,5-dinitrosalicylic acid method [15], with glucoseas standard. Reducing sugar was calculated as g/g dried substrate(DS).

Glucose, xylose, cellobiose and ethanol were analyzed by HPLC(Waters Co., MA, USA) with a cation exchanger Sugarpak column(300 6.5 mm i.d.). Secondary de-ionized water, at a ow rate of0.5 ml/min, was used as the mobile phase. The injection volumewas 20 ll and the column temperature was maintained at 90 C.All samples were ltered through a 0.22 lm lter before undergo-ing HPLC analysis. The eluate out of HPLC was detected by a refrac-tive index detector at 50 C.

3. Results and discussion

3.1. Pretreatment and enzymatic hydrolysis of cellulosic material

The corncob substrate samples were pretreated with autoclaveat 121 C for 60 min and either with 1.0% (w/v) sulfuric acid for15 min (Sample 2 in Fig. 1A) or without sulfuric acid (Sample 1).For Sample 2, after the pretreatment, approximately 0.43 g/g DSof reducing sugars was recovered (Fig. 1A). The results show that61.4% (w/w) of the cellulosic substrate was converted to reducingsugar after pretreatment with autoclave and acid. No toxic effectsfrom furfurals and HMFs were observed during the fermentationstudies as conrmed by Sumphanwanich et al. [16]. Their resultsindicated the corncob waste with acid-treatment generated non-toxic levels of furfurals (0.7 g/l) and HMFs (0.8 g/l) in the hydroly-

/l) 40

(b)

168 Y.-H. Chang et al. / Fuel 97 (2012) 166173Biom

ass c

once

ntra

tion

(g

0

10

20

30

0 1 2 350

pH v

alu

es

4.0

4.5

5.0

5.5drying samples for 24 h at 110 C. Samples were withdrawn fromthe fermentation broth, and yeast biomass was determined bymeasuring cell optical density recorded with a Ultrospec 2100

6.0Fermentatio

Fig. 2. Change of (A) pH values, and (B) concentration of biomass, during the time coHydrolysate w/ 1% glucoseHydrolysate w/ 2% glucoseHydrolysate w/ 3% glucoseHydrolysate w/ 4% glucose

4 5 6 7sates for fermentation.The prehydrolysate was further hydrolyzed with the reaction

of enzyme mixtures at pH 6.0 and 50 C for 72 h (Sample 3).

Hydrolysate w/ 1% glucoseHydrolysate w/ 2% glucoseHydrolysate w/ 3% glucoseHydrolysate w/ 4% glucose

(a)n time (days)urse of batch fermentation in the hydrolysate medium with 14% (w/w) glucose.

The reducing sugar concentration reached 0.61 g/g DS after 48 hof hydrolysis (Fig. 1B). However, extending the hydrolysis timebeyond 48 h did not help further in increasing the reducing su-gar concentration. From the reducing sugar concentration afterhydrolysis, it indicates that 87.1% (w/w) of the cellulosic/hemi-cellulosic components were hydrolyzed and converted to reduc-ing sugar after treatment with the enzyme mixture. Xylose wasdetected in the hydrolysate, showing that the presence of b-1,4-endoxylanase assisted in the hydrolysis of xylan in the substrate.Additionally, the b-1,4-endoglucanase and b-1,4-exoglucanasehydrolyze cellulose chains resulted in the formation of cellobi-ose, which can be further cleaved into glucose by cellobiase. Itwas found that a signicantly low amount of cellobiose existedin the cellulosic hydrolysate, indicating that cellobiase increasedthe hydrolysis of cellobiose in the resulting prehydrolysate. Inaddition, a high amount of glucose and a comparatively loweramount of cellobiose existed in the cellulosic hydrolysate, indi-cating good activities of b-glucosidase in enzyme mixtures.Thereby, with the aid of enzymatic hydrolysis, higher yields oftotal reducing sugar (0.61 g/g DS), glucose (0.36 g/g DS) and xy-lose (0.17 g/g DS) in the resulted hydrolysates were achieved.These ndings indicate that the enzyme mixtures helped to in-crease the hydrolysis efciency of the cellulosic hydrolysatesand were necessary to produce the monosaccharides for furtherethanol fermentation.

3.2. Batch fermentation of cellulosic hydrolysate for bioethanolproduction

Batch fermentation for bioethanol production was performed inthe cellulosic hydrolysate-based media containing various concen-trations of glucose as the main carbon source. Due to the concernthat the high concentration of glucose in the hydrolysate would in-hibit the growth of yeast, the maximum concentration of glucose(40 g/l) was used. To determine the effect of glucose concentrationon the growth prole of S. cerevisiae BCRC 21812, batch experi-ments were performed in conical asks with glucose concentrationin the hydrolysates ranging from 10 to 40 g/l. Fig. 2 shows the plotsof cell biomass and pH values against fermentation time. For thehydrolysate medium, the pH decreased slowly and remained above5.6 throughout the rst ve days of the fermentation and de-creased rapidly from 5.7 to 5.0 after 6 d of cultivation (Fig. 2A).As reported by Palmqvist and Hahn-Hagerdal [17], cell growth incellulosic hydrolysates strongly depended on pH, due to the largeconcentration of dissociated weak acids at low pH. The pH, around5.0 during the entire fermentation process, did not inuence thegrowth of yeast cells and thus favored the ethanol production.The yeast cell biomass increased from 23.3 to 41.1 g/l with the in-creased concentration of glucose from 1% to 4% in the hydrolysate(Fig. 2B). In addition, after the inoculation of yeast cells, the micro-bial biomass began to increase, reached the maximal values after

15

20

25

30

GlucoseXyloseCellobioseEthanol

(a) (b)

Y.-H. Chang et al. / Fuel 97 (2012) 166173 169Con

cent

ratio

ns (g

/l)

0

5

10

0

10

20

30

40

50(c)

0 1 2 3 4 5 6Fermentati

Fig. 3. Change of glucose, xylose, cellobiose and ethanol during batch culture in th(d)

0 1 2 3 4 5 6 7on time (days)e hydrolysate medium with (A) 1%, (B) 2%, (C) 3% and (D) 4% (w/w) glucose.

2 d of incubation, and then remained steady thereafter. These re-sults indicated that the yeast grew well on the cellulosic hydroly-sate with glucose at a concentration up to 4%. However, cell growthwas greatly repressed when the glucose concentration reached 4%.Specically, when the glucose concentration increased from 3 to4%, the yeast cell biomass did not show obvious increase, i.e. from40.2 to 41.1 g/l. This fact showed that the concentration of glucoseat 4% would inhibit the growth of yeast, due to the halt in cellbiomass.

Fig. 3 shows the change of glucose, xylose and cellobiose con-centration, and ethanol yield by S. cerevisiae culture after 6 daysas compared to the initial glucose concentration in the hydrolysate.In the fermentation using 12% glucose in the hydrolysate, the glu-cose was exhausted after 2 d, whereas the ethanol production yieldincreased rapidly after the rst day of fermentation. This resultindicates that the glucose consumption was consistent with thetime period of ethanol production. As shown in Figs. 3A and B,the glucose was rapidly used up by the yeast within 2 d, with 12% glucose in the hydrolysate. However, 1.24.1 g/l xylose and1.73.3 g/l cellobiose were detected and could not be utilized bythe yeast cells after 2 d. The fermentation was completed after2 d. The maximal concentrations of ethanol were 6.9 and 8.5 g/lfor the cultures of 1% and 2% glucose in the hydrolysate, respec-tively, when the glucose were used up. The fermentation resultssuggest that S. cerevisiae could grow well in the hydrolysate med-ium and achieve virtually complete conversion to ethanol fromglucose in the hydrolysate. However, 810 g/l of glucose was not

utilized by the yeast strain after 6 d of fermentation when the glu-cose concentrations in the hydrolysate were 34% (Fig. 3C and D).Furthermore, considerable quantities of xylose and cellobiose werealso detected, which could not be utilized by the yeast cells. Whenthe substrate concentration reached 4%, yeast biomass and ethanolyield were not signicantly increased, suggesting that a consider-able inhibitory effect had occurred. In addition, the ethanol yieldswere 18.3 and 23.0 g/l when the initial glucose concentrations inthe hydrolysate were 3% and 4%, respectively. Besides, rate of theconversion of glucose to ethanol was 0.580.61 g ethanol/g glu-cose, using 34% glucose in the hydrolysate, a signicant higheramount than that (0.45 g ethanol/g glucose) of Yu and Zhang [1].To develop an improved culture method for ethanol productionwith S. cerevisiae, batch ask cultures were rstly carried out todetermine the suitable substrate concentration of the initial media.It was found that S. cerevisiae grew with a similar pattern in glu-cose concentrations up to 4%, indicating a good ability to deal withosmotic stress. This made it possible to feed concentrated glucosesolution in a discontinuous way during the fed-batch fermentation.

3.3. Fed-batch fermentation of cellulosic hydrolysate for bioethanolproduction

It requires a high initial sugar concentration in the cellulosichydrolysate to obtain high concentrations of ethanol in the fer-mentation broth. However, higher sugar concentration in thehydrolysate often causes mixing and heat transfer problems, due

(g/l) 20

25

30

5.6

5.8

6.0

ion

(a)

3

170 Y.-H. Chang et al. / Fuel 97 (2012) 166173Biom

ass c

once

ntra

tion

0

5

10

15

Fermentat

Con

cent

ratio

n (g/

l)

0

5

10

15

20

25

Hydrolysate supplementation

(b)

0 1 2Fig. 4. Time course of (a) biomass and pH, and (b) concentration of sugars and ethanol, duw) glucose and was fed with 1% glucose after 1 d of fermentation.pH v

alu

e

4.6

4.8

5.0

5.2

5.4

time (day)

GlucoseXyloseCellobioseEthanol

4 5 6ring the fed-batch fermentation in the hydrolysate medium, which contained 2% (w/

to the rheological properties of a very dense brous suspension [7].Such problems could be effectively avoided in the fed-batch fer-mentation process, where the substrate is added gradually andthe viscosity of the reaction mixture can be kept at a low level.The glucose concentration in the hydrolysate increased from aninitial 2% to 3% by addition of 1% on the rst day in the fed-batchprocess (Fig. 4). It was observed that the cell biomass concentra-tion reached 22.3 g/l on the second day of fermentation and thatthe pH of the fermentation broth slightly decreased from 5.8 to5.5 during the 5 d of fermentation. The residual glucose concentra-tion in the hydrolysate was 1.8 g/l, which was much lower thanthat of the batch culture. After the addition of the hydrolysate,the concentration of xylose increased from 6 to 15.2 g/l. This indi-cates that S. cerevisiae could readily ferment the glucose in hydro-lysate to ethanol but could not metabolize xylose, due to the lack ofxylose-degrading enzymes. In addition, the results show that mostof the glucose in the hydrolysate was used in fed-batch cultures,therefore, higher concentrations of ethanol (19.0 g/l) were pro-duced than in batch cultures with 3% glucose in the hydrolysate.Fed-batch cultures shortened the reaction time degrading anequivalent substrate, therefore enhancing the efciency of utilizingthe cellulosic substrate.

The results of fed-batch culture with initial 2% glucose in cellu-losic hydrolysate and an addition of 2% glucose were showed inFig. 5. The cell biomass increased rapidly after the inoculation ofS. cerevisiae and reached the maximum (42.5 g/l) on the third dayof fermentation (Fig. 5A). The glucose in the hydrolysate was goingto be used up after 1 d of fermentation, and the ethanol yields were

only 15 g/l (Fig. 5B). After the addition of the hydrolysate, the eth-anol yields increased and reached the maximum on the second dayof fermentation. Thus, the overall ethanol yields and glucose con-version to ethanol rate were estimated to be 24.0 g/l and0.60 g ethanol/g glucose. The results of fed-batch culture with ini-tial 2% glucose in hydrolysate and an addition of 3% glucose areshowed in Fig. 6. The cell biomass increased rapidly after the inoc-ulation of S. cerevisiae and reached the maximum (44.5 g/l) on thesecond day of fermentation; moreover, the pH of the broth de-creased steadily from 5.8 to 5.2 during the fermentation process(Fig. 6A). After feeding the hydrolysate to the media, the ethanolyields increased and reached 32.3 g/l at the second day of fermen-tation. Thus, rate of the conversion of glucose to ethanol was esti-mated to be 0.64 g ethanol/g glucose. As proposed in this study,these resulting data were higher than those from the batch culturesystem. Furthermore, rate of the conversion of glucose to ethanolfrom the fed-batch fermentation in this study was signicantlyhigher than that (0.44 g ethanol/g glucose) of the study using thebatch culture of Candida tropicalis [18].

4. Conclusions

As an economical way to produce ethanol from cellulosic sub-strate, synergetic hydrolysis of cellulase and xylanase mixturescreates a feasible process that can be used in the production of bio-ethanol. Bioethanol production with fed-batch fermentation offersadvantages over that with batch fermentation. The conversion rate

)

30

35

4.8

5.6

5.8

6.0

ion3

(a)

Y.-H. Chang et al. / Fuel 97 (2012) 166173 171Biom

ass c

once

ntra

tion

(g/l

0

5

10

15

20

25

Fermentat0 1 2

Con

cent

ratio

n (g/

l)

0

5

10

15

20

25(b) Hydrolysate supplementationFig. 5. Time courses of (a) biomass and pH, and (b) concentration of sugars and ethanol,(w/w) glucose and was fed with 2% glucose after 1 d of fermentation.4.6

time (day)4 5 6

GlucoseXyloseCellobioseEthanolpH v

alu

e

5.0

5.2

5.4during the fed-batch fermentation in the hydrolysate medium, which contained 2%

trat

tat

uel 90

)

30

35(b) Hydrolysate supplemenBiom

ass c

once

n

10

20ion

(g/l)

30

40

50

(a)

172 Y.-H. Chang et al. / Fof ethanol from glucose was higher in fed-batch fermentation thanit was in batch fermentation. Moreover, the substrate inhibition ef-fects on cell biomass and yields of ethanol were less pronouncedfor fed-batch fermentation than batch fermentation. Further workshould be focused on scale-up of fed-batch fermentation to makethe process industrially feasible.

Acknowledgment

The authors would like to thank the National Science Council ofthe Republic of China (Taiwan) for nancially supporting this re-search under Contract No. NSC 97-2313-B-264-001-MY3.

References

[1] Yu Z, Zhang H. Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysatewith Saccharomyces cerevisiae. Bioresour Technol 2004;93:199204.

[2] Tabka MG, Herpol-Gimbert I, Monod F, Asther M, Sigoillot JC. Enzymaticsaccharication of wheat straw for bioethanol production by a combinedcellulase xylanase and feruloyl esterase treatment. Enzyme Microb Technol2006;39:897902.

[3] hgren K, Vehmaanpera J, Siika-Aho M, Galbe M, Viikari L, Zacchi G. Hightemperature enzymatic prehydrolysis prior to simultaneous saccharicationand fermentation of steam pretreated corn stover for ethanol production.Enzyme Microb Technol 2007;40:60713.

[4] Sun Y, Cheng JY. Hydrolysis of lignocellulosic materials for ethanol production:a review. Bioresour Technol 2002;83:111.

Fermentation t0 1 2 3

Con

cent

ratio

n (g/

l

0

5

10

15

20

25

Fig. 6. Time courses of (a) biomass and pH, and (b) concentration of sugars and ethanol, dw) glucose and was fed with 3% glucose after 1 d of fermentation.pH v

alu

e

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

GlucoseXyloseCellobioseEthanol

ion

7 (2012) 166173[5] Tolan JS, Foody B. Cellulase from submerged fermentation. Adv Biochem EngBiotechnol 1999;65:4167.

[6] Sreenath HK, Koegel RG, Moldes AB, Jeffries TW, Straub RJ. Ethanol productionfrom alfalfa ber fractions by saccharication and fermentation. Proc Biochem2001;36:1199204.

[7] Yoshida F, Yamane T, Nakamoto K. Fed-batch hydrocarbon fermentations withcolloidal emulsion feed. Biotechnol Bioeng 1973;15:25770.

[8] Stanbury PF, Whitaker A, Hall SJ. Principles of fermentation technology. 2nded. Oxford (UK): Pergamon Press; 1995.

[9] Varga E, Klinke HB, Reczey K, Thomsen AB. High solid simultaneoussaccharication and fermentation of wet oxidized corn stover to ethanol.Biotechnol Bioeng 2004;88:56774.

[10] Ballesteros M, Oliva JM, Manzanares P, Negro MJ, Ballesteros I. Ethanolproduction from paper material using a simultaneous saccharication andfermentation system in a fed-batch basis. World J Microbiol Biotechnol2002;18:55967.

[11] Chen M, Xia M, Xue P. Enzymatic hydrolysis of corncob and ethanol productionfrom cellulosic hydrolysate. Int Biodeterior Biodegr 2007;59:859.

[12] Olofsson K, Rudolf A, Liden G. Designing simultaneous saccharication andfermentation for improved xylose conversion by a recombinant strain ofSaccharomyces cerevisiae. J Biotechnol 2008;134:11220.

[13] Toms-Pej E, Oliva JM, Gonzlez A, Ballesteros I, Ballesteros M. Bioethanolproduction from wheat straw by the thermotolerant yeast Kluyveromycesmarxianus CECT 10875 in a simultaneous saccharication and fermentationfed-batch process. Fuel 2009;88:21427.

[14] Hsu CL, Chang KS, Lai MZ, Chang TC, Chang YH, Jang HD. Pretreatment andhydrolysis of cellulosic agricultural wastes with a cellulase-producingStreptomyces for bioethanol production. Biomass Bioenergy2011;35:187884.

[15] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducingsugar. Anal Chem 1959;31:4268.

[16] Sumphanwanich J, Leepipatpiboon N, Srinorakutara T, Akaracharanya A.Evaluation of dilute-acid pretreated bagasse, corn cob and rice straw for

ime (day)4 5 6

uring the fed-batch fermentation in the hydrolysate media, which contained 2% (w/

ethanol fermentation by Saccharomyces cerevisiae. Ann Microbiol2008;58(2):21925.

[17] Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates. I.Inhibition and detoxication. Bioresour Technol 2000;74:1724.

[18] Garca Martn JF, Cuevas M, Bravo V, Snchez S. Ethanol production from oliveprunings by autohydrolysis and fermentation with Candida tropicalis. RenewEnergy 2010;35:16028.

Y.-H. Chang et al. / Fuel 97 (2012) 166173 173

Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production1 Introduction2 Materials and methods2.1 Cellulosic material and pretreatment2.2 Microorganisms and cultivation2.3 Hydrolysis of cellulosic residue by treatment with the enzyme mixture2.4 Batch and fed-batch fermentation of cellulosic hydrolysate2.5 Analysis methods

3 Results and discussion3.1 Pretreatment and enzymatic hydrolysis of cellulosic material3.2 Batch fermentation of cellulosic hydrolysate for bioethanol production3.3 Fed-batch fermentation of cellulosic hydrolysate for bioethanol production

4 ConclusionsAcknowledgmentReferences