Conversion of Furfural in Aerobic and Anaerobic Batch Fermentation of Glucose by Saccharomyces Cerevisiae_Taherzadeh, 1999

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JOURNAL OF BIOSCIENCE AND BIOENGINEERINGVol. 87, No. 2, 169-174. 1999

Conversion of Furfural in Aerobic and Anaerobic Batch Fermentationof Glucose by Saccharomyces cerevisiaeMOHAMMAD J. TAHERZADEH, LENA GUSTAFSSON,2 CLAES NIKLASSON, AND GUNNAR LIDl?N*

Department of Chemical Reaction Engineering, Chalmers University of Technology, S-412 96, and Department of Generaland Marine Microbiology, Unive rsity of Giiteborg, Medicinaregatan SC, S-413 9O,2Gliteborg, Sweden

Received 15 Apr i l 199WA ccepted 22 Novemb er 1998The effect of furfural on aerobic and anaerobic batch cultures of SaccharomycescerevisiaeCBS 8066 growingon glucose was investigated. Furfural was found to decrease both the specific growth rate and ethanolproduction rate after pulse additions in both anaerobic and aerobic batch cultures. The specifi c growth rateremained low until the furfural had been completely consumed, and then increased somewhat, but not to theinitial value. The CO2 evolution rate decreased to about 35% of the value before the addition of 4 g-1-l furfural,in both aerobic and anaerobic fermentations. The decreaseof the CO2 evolution rate was rapid at first, and thena more gradual decrease was observed. The furfural was converted mainly to furfuryl alcohol, with a spec ificconversion rate of 0.6 ( f 0.03) g (furfural) -g-l (biomass)- h-l by exponentially growing cells . However, theconversion rate of furfural by cell s n the stationary phase was much lower. A previously unidentified compoundwas detected during the conversion of furfural. This compound was characterized by mass spectrometry and itis suggested that it is formed from furfural and pyruvate.

[Key words: fur fural, ethanol, ye ast, biotransformation]Fuel ethanol can be produced from lignocellulose bychemical hydrolysis of lignocellulosic materials followedby fermentation. One of the major problems with hy-drolyzates produced by acid hydro lysis, is the poor fer-mentability caused by the presence of inhibitors in thehydrolyzates. Furfural is known to be one of the mostimportant of these inhibitors (1). It is a breakdownproduct from pentoses and is formed in a browning reac-tion during hydrolysis in the presence of strong acids(2). It therefore may be impossible to completely avoidfurfural formation in a chemical hydrolysis processdesigned to give a high sugar yield. Since furfural is a

primary breakdown product from pentoses and thereforelikely to be present in hydrolyzates, the effect of furfuralon fermentation by yeast has been the subject of severalinvestigations. For concentrations on the order of 1 g.l-1, clearly negative effects on yeast viability (3), specificgrowth rate (4) and volumetric fermentation rate (5-8)have been shown. Severa l enzymes have been shown tobe sensit ive to furfural, and among the most sensitiveare the glycolytic enzymes glyceraldehyde phosphate de-hydrogenase (EC 1.2.1.12) and alcohol dehydrogenase(EC l.l.l.l), but also hexokinase (EC 2.7.1.1) is inhibit-ed (6). The use of high-level yeast inocula has been sug-gested as a means of overcoming toxicity in batch cul-tures which ferment acid hydrolyzates (3), based on thefact that furfural can be taken up and converted byyeast cells. Conversion of furfural is, in fact, one of thefirst biotransformations known to occur in yeasts (9).The prime product from furfural is furfuryl alcohol(lo), accounting for more than 70% of the converted fur-fural. Villa et al. (11) also report formation of furoicacid, and other metabolic products reported are furoinand furil (12). With respect to the specific ethanolproductivity, Azhar et al. (1) reported that in thepresence of 3 g. 1-l of furfural the specific ethanol forma-tion rate, qe, was only one third of the value without

* Corresponding author.

furfural in batch cultures. In contrast, Fireoved andMutharasan (13) did not find a significant change in thebiomass yield on ATP due to furfural in a chemostat cu l-ture operating at low dilution rates. The speci fic ethanolformation rate, thus, did not change in chemostat cul-ture, although some outliers were reported for which anincreased value of qe was found. The authors suggestedadaptation effects to be the explanation for these obser-vations.In a previous work (14), we found a clear relationbetween the fermentation rate of dilute acid hydrolyzatesfrom several kinds of wood, and the sum of the con-centration of furfural and 5-hydroxymethyl-furfural(HMF). When the sum of furfural and HMF concentra-tions exceeded 20 mmol. 1-l (about 2 g . I-), the fermenta-tion rate decreased strongly. Furthermore, in the hy-drolyzates that fermented well, the concentration of fur-fural and HMF decreased during the fermentation, inagreement with what has previously been reported foracid hydrolyzates from oak (3). Presumably, the conver-sion of furfural and HMF is of great importance for ena-bling fermentation of the hydrolyzates.In the present work, the uptake of furfural and theeffects on the fermentation rate of Saccharomycescerevisiae were studied by making pulse additions of fur-fural into a synthetic medium. In previously reportedstudies, furfural has normally been present in the mediumfrom the beginning of the batch cultivat ions. However,by using pulse addition the transient metabolic responsecan be followed. Furthermore, in comparison to che-mostat cultures, the effects of a higher concentration offurfural in the broth can be studied. On-line measure-ments were made of the exhaust gas-composition as wellas the biomass concentration to provide response timeswith high resolution. The uptake rate of furfural andthe product distribution were determined for both growingcultures and stationary phase cells, under both aerobicand anaerobic conditions.

169



170 TAHERZADEH ET AL. J. BIOSCI. BIOENG.,

MATERIALS AND METHODSYeast strain and medium Saccharomyces cerevkiaeCBS 8066, obtained from Centraalbureau voor Schim-melcultures, Delft, the Netherlands, was used in all ex-periments. The strain was maintained on agar platesmade from yeast extract [ lo g.l-1, soy peptone [20 g. i-l],and agar [20 g.1.-I] with D-glucose [20 g.l-] as an ad-ditional carbon source. Inoculum cultures were grown in

300-ml cotton-plugged-conical flask s on a shaker with ashaking diameter of 12mm at 30C for 24 h. The liquidvolume was 100 ml and the shaker speed 170rpm. Thegrowth medium was a defined medium as previouslyreported (15).Cultivation conditions Anaerobic and aerobicbatch cultiva tions were carried out in a BioF lo IIIbioreactor (New Brunswick Scientific, Edison, NJ, USA)with a working volume of 2.5 1 at a temperature of 30Cand a stirring rate of 500rpm. The pH value in the me-dium was controlled at 5.00 (tO.01) by addition of 2 MNaOH. Nitrogen or air was continuously spargedthrough the reactor at a flow rate of 0.80 1 min l (atNTP) controlled by a mass flow controller (Hi-Tech,Ruurlo, the Netherlands). The nitrogen gas had a guaran-teed oxygen content of less than 5 ppm (ADR clas s 2,l(a), A GA, Sweden). An oxygen probe was used tocheck the dissolved oxygen saturation in the aerobicexperiments in order to avoid oxygen limitation.The experiments were carried out both aerobically andanaerobically. In the aerobic experiments 5 (or 10) gof furfural was introduced into the medium, duringexponential growth in the respiro-fermentative phase. Theexperiments continued up to a few hours after completeuptake of ethanol in the respiratory phase. In the anaero-bic experiments 5 (or 10) g of furfural was introducedinto the medium, while the cel ls were anaerob ically grow-ing on glucose in the exponential phase. The conditionswere switched to aerob ic after the complete uptake ofglucose (seen from measurements of the CO2 evolutionrate) and were continued until the end of the ethanoluptake. In an additional experiment, 10 g of furfural wasintroduced into an anaerobic culture which had enteredthe stationary phase, i.e. after the complete uptake ofglucose. This experiment ran anaerobically for 18 h afterthe addition, and then the conditions were changed toaerobic for 3 h.Analytical methodsGas analysis The carbon dioxide in the outlet gaswas continuously measured with an acoustic gas monitor(Bruel&K jaer 1308) (16), and gas measurement signalswere averaged fo r 30 s. The instrument was calibratedwith a gas of the following composition: 5% C02, 20%O2 with nitrogen as inert gas.Biomass concentration Cell dry weight was deter-mined from duplicate lo-ml samples, which were cen-trifuged, washed with disti lled water and dried for 24 hat 103C. At least three samples were taken for eachbatch cultivation. An on-line flow injection analysis(FIA ) system designed according to Benthin et al. (17)was used for determination of the optical density every30min. Addition of furfural to the medium was foundto have no significant effect on the accuracy of the FIAmeasurements. The optical density measured by FIA wascalibrated against the dry weight samples. The concentra-tions of protein and total carbohydrates of the biomasswere measured before and after furfural conversion

in all experiments. The determination of the cellu lar pro-teins were carried out by the Biuret method with modi-fications according to Verduyn et al. (18), and the totalcellular carbohydrate content was determined accordingto Herbert et al. (19).Metabolite analyses Samples for HPLC analysiswere withdrawn from the broth via 0.45-pm sterilefilters. Glycerol and furfuryl alcohol were analyzed onan Aminex HPX-87P column (Bio-Rad, USA) as previ-ously described (14). The amount of glucose, ethanol,acetic acid, fumaric acid, furoic acid, furfural, pyruvicacid and succinic acid was determined by an AminexHPX-87H column (Bio-Rad) as described in (14).Mass spectrometry In order to characte rize anunknown compound found by HPLC, mass spectromet-ry was used. Samples were obtained from preparativeHPLC, and subsequently analyzed using a high resolu-tion mass spectrometer (Zab-Spec, VG Analytical, Fis-ons instrument, England) operating in CI-mode, withNH3 as the ionizing gas, and operating in EI-mode.Calculations Evolved carbon dioxide was calculatedfrom measurements of the gas composition and the gasflow rate, and other metabolite yields were obtained fromHPLC data. A biomass composition of CH1.7600.56N0.17was used in the carbon balance calculat ions (18). Themetabolite and biomass yields were calculated from theconcentrations determined at the end of the exponentialgrowth phase. The average carbon balance determinedat the end of the exponential growth phase was 96%(SD 3%). Specific growth rates and specific productivitieswere determined based on the biomass concentrationsobtained from the FIA measurements.

RESULTSAnaerobic pulse addition experiments Anaerob icconversion of furfural was studied by the injection offurfural (2 or 4 g . I-*) to exponentially growing batch cul-tures. The results of such an experiment are summarized

in Fig. 1. When compared to the control experiment(Fig. la), the most obvious effect seen immediate ly afterpulse addition was a decrease of the COz evolution rate,CER (Fig. lb). The decrease of CER was rapid the firstfew minutes after the addition, e.g. 34% (24%) within5 min, followed by a more gradual decrease. The CER iscoupled to the ethanol production rate, and measuredethanol concentrations clearly showed that the ethanolproduction rate also fell after addition of furfural (Fig.lb). The specific ethanol production rate fell from 1.6(t-O.l)g.g-*h-l to about 0.5 (+0.2)g.g -.h- afteraddition of 4 g . I-* of furfural. The specif ic growth rate,,u, decreased even further, from 0.4 to 0.03 (t-0.02) h.-l,and remained at this low value until the furfural hadbeen complete ly consumed (c f Fig. 3a). After exhaustionof furfural in the medium p increased to somewherebetween 0.13 (kO.03) h-l, i.e. to less than half of theinitial value (Fig. lc).Furfural addition caused an increased pyruvate forma-tion rate, and for some time an increased uptake ofacetate from the medium (Fig. lc). However, as the ad-ded furfural was consumed, the rate of formation ofpyruvate decreased, and acetate accumulation resumed.Also , compounds of the TCA-cycle were affected (Fig.Id). The formation rate of succinate decreased and theconcentration of fumarate actua lly decreased (notshown) after addition of furfural to the medium. Fur-



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10 20 30Time (h)FIG. 1. Anaerobic batch cul t ivation of S. cerevisiae growing onglucose (50 g. I- r ) a s a carbon and energy sou rce. (a) CER for a controlbatch cul ture in which no fur fural was added. Figures (b-d) resul ts

from a pulse exper iment, in which fur fural(4 g. I- i) was introduced atthe t ime indicated by the arrow. (b) CER ( line) and ethanol concen-tration ( n ) , (c) concentrations of biomass (A, note: logar ithmic scale) ,pyruvic acid (0) and acetic acid (O), (d) concentrations of glycerol( n ) and succinic acid ( q ) . The arrows show the t ime of fur furaladdition.

fural also leads to a decreased glycerol formation rate(Fig. Id), although the formation of glycerol per con-sumed glucose was increased (Fig. 2). Protein and totalcarbohydrate levels were measured, but no majorchanges in biomass composition could be found. The

CONVERSION OF FURFURA L BY S. CEREVZSIAE 171

5s>4-75 3- FurfuralzB 2-30

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0 10 20 30 40 50Glucose (g 1-l)

FIG. 2. Glycerol concentration vs. glucose concentration in apulse addition expe riment. Furfural (4 g.l-I) wa s introduced intoan anaerobic batch cul t ivation of S. cerevisiae growing on glucose (50g . - *) as a carbon and energy source.protein content was 55(+-6)% and the total carbohydratecontent was 25( f 6)%, with no systematic change regard-less of the concentration of furfural present.The specific uptake rate of furfural, qf, was 0.6 g .g-.h-l and was constant during the uptake period (notshown). No appreciable lag was seen between effects onCER and uptake of furfural. Furfuryl alcohol was themain product of furfural degradation (Fig. 3a), but alsofuroic acid (< 1% of added furfural) was identified as aminor product. However, from a plot of the sum of fur-fural, furfuryl alcohol and furoic acid it can be deducedthat at least one additional product is intermittently

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Time (h)FIG . 3 . (a) Conve rsion of furfural (0) to furfuryl alcohol (0)in anaerobic batch cul t ivation of S. cerevisiae growing on glucose (50g.l - I) as a carbon and energy source. (b) The sum of the concentra-tions of furfural, furfuryl alcohol and furoic acid ( w , left-hand scale)and the peak area of the unknown compound ( 0, r ight-hand scale)during a pulse addition expe rimen t.



172 TAHERZADEH ET AL. J . BIOSCI. BIOENO.,

Respirative /phase / Stationaryphase

&-----% i 0 80Time (h)FIG. 4. Conversion of furfuryl alcohol ( w ) to furoic acid ( 0) byS. cerevbiue dur ing respirative growth on ethanol, and in a stationaryphase cul ture.

formed (Fig. 3b), accounting for at most 20% of the con-verted furfural. An unidentified peak could be found inthe HPLC chromatograms, which matched the timeprofile of the missing compound well. No anaerobic con-version of furfuryl alcohol after the complete conversionof furfural was obtained with the investigated strain.The effects of aeration To be able to make a com-parison between aerobic and anaerobic conditions, 2 or4 g-1-l furfural was introduced into an aerobically grow-ing culture in the exponential growth phase. Respiro-fermentative growth normally gives lower glycerol andhigher biomass yields than anaerobic growth, which wasalso observed in the presence of furfural. The specificconversion rate of furfural was, within experimentalerrors, identical to the anaerobic case (0.6kO.03 g. g-l.h-l), and the presence of oxygen did not affect theproduct yields from furfural. The main products underaerobic conditions were also furfuryl alcohol and thepreviously mentioned unknown compound. The specificgrowth rate after furfural addition was, furthermore, thesame for both aerobic and anaerobic experiments.In aerobic batch cultivations of S. cerevisiue therespiro-fermentative phase is, however, followed byrespirat ive growth on ethanol (20). Furfuryl alcohol wasconverted to furoic acid during the respirative phase(Fig. 4). The conversion had already started duringrespiro-fermentative growth after complete conversion offurfural, but at low rate. It also continued after the com-plete consumption of ethanol, although, at a low rate.Shortly after the start of conversion of furfuryl alcoholto furoic acid, furfural was detected in the medium at aconcentration of up to 0.06g.I-r, but later became onceagain undetectable. This suggests that the conversion offurfuryl alcohol to furoic acid occurs via furfural.Stationary phase cells Since furfural reduction tofurfuryl alcohol requires reducing power, it is probable

that the glycolytic rate influences the conversion rate.For this reason, furfural was also introduced into ananaerobic batch culture in stationary phase, immediatelyafter the complete uptake of glucose. The experimentcontinued anaerobically for 18 h after the addition offurfural. The rate of furfural degradation was clear lylower (0.07 g.g-l. h-l), than that found for exponential-ly growing cells . The main product of furfural degrada-tion was furfuryl alcohol. The yield of the previouslymentioned unidentified compound was less than 1% ofconverted furfural, i.e. much smaller than in the case ofexponentially growing cells.

TABLE 1. High-resolution mas s spectrome try data from analysisof the unknown compound using chemical ionization (CI) wi thNH4+ as the ionizing gas and using electron impact ionization (EI)m/z

100.105124.075125.058129.066138.052139.037140.072141.056142.085143.091156.065157.070158.082159.086195.175212.200

Relative Suggestedintensi tya fragmentChemical ionization (CI)1.221.122.26 b H 9 0 2 1 A1.862.961.43 WW312.201.29 IGWM14.98 lGH&Nl+1.52100.00 K+MWl+8.8322.07 PXhO~N1.852.26 [C&z10zNsl-4.61 lG~z&zN~l+

m/z(calculated)

125.060

139.039141.055142.086156.066158.082195.169212.196

27.18328.18329.15134.09236.04239.05941.05843.02543.99344.99881.03895.01697.030

Electron impact ionization (EI)5.816.1815.166.355.6814.9610.1728.80100.00 co29.436.88 C&O26.68 C S H 30212.92 C S H 502

43.99081.03495.01397.029

a Only peaks with relative intensi t ies higher than 1% and with mas sto charge ratio (m/z) higher than 100 are shown for the CI spectrum,and only peaks with relative intensi t ies higher than 5% are shown forthe EI spectrum.Characterization by mass-spectrometry of the unknowncompound The previously mentioned unknown com-pound (Fig. 3b) was detected by HPLC on both RI andUV detectors. It had a comparat ively long residence timein the hydrogen ion exchange column, and the ratio be-tween the UV response (at 210nm) and RI response wasvery high. Palmqvist et al. (21) reported formation of anunknown compound from furfural in S. cerevisiae, andpreviously Shvets et al. (22) also reported conversion offurfural to an unidentified compound characterized byhigh absorption of UV light at wavelengths less than240nm. In order to identify the compound in thepresent work, the eluted peak was collected from theHPLC system and subsequently analyzed by mass spec-trometry. Unfortunately, the compound was not found

in available spectral libraries. However, based on iden-tified mass fragments and known metabolic effects, a ten-tative candidate can be suggested. From a careful con-sideration of the fragments of EI and CI (Tables 1 and2) in combination with physiological considerations, thefollowing molecule is suggested to be the unknown com-pound.f : P-C-y--H,

HC-CH COOH



VOL. 87, 1999This molecule has a molecular weight of 184. It can easi-ly loose the carboxylic group as CO2 during ionization,both with CI and EI (cf. Table 1). The remaining parthas a molecular weight of 140, and will give the frag-ments m/z= 158.082 with NH4+ and m/z= 141.055 withH+. These peaks are both found in the CI spectrumwith high accuracy. A further loss of two hydrogensgives the fragments m/z=156.066 with NH4+ and139.039 with H+ in the CI spectrum. Moreover, C4H30-CO-CH3 could be the fragment giving m/z=142.086with NH4+ and 125.060 with H+. The furfuryl group(C4H30-CO-) is, after COZ, the strongest peak in the EIspectrum. Some other hydrogenated or deoxygenatedfurfuryl fragments can also be identified in the EIspectrum. Another peak, m/z= 110.041, with a low intensityis also found (not shown in Table 1). It corresponds tothe fragment C6H602 with the calculated m/z of110.037. This fragment is interesting since, if combinedwith five or six ammonia molecules, it will give rise tothe two peaks with the largest m/z in the CI spectrum(Table 1).

DISCUSSIONIt is clear that the glycolysis of S. cerevisiae is affectedby addition of furfural, and that the effect occurs rapid-ly. Furthermore, as is evident from the much lower q f instationary culture, the reduction of furfural is dependenton an active glycolysis. According to Kang and Okada(23), the aldehyde group of either acetaldehyde or fur-fural may be reduced by alcohol dehydrogenase (ADH)according to the reactions below.

CH,-CHO + NADHADH+H+ c---f CH3-CH20H+NAD+ (1)

C4H30-CHO + NADH+ H+ &y C4H30-CH20H + NAD+ (2)

It was easily confirmed with an ordinary kit for enzymat-ic determination of ethanol (cat. nr. 176 290, BoehringerMannheim, Germany) that furfuryl alcohol was rapidlyconverted by alcohol dehydrogenase. It is reasonable tobelieve that reduction of furfural competes with thereduction of acetaldehyde and therefore occupies partof the glycolytic capacity of the cells. This should leadto a lower flux of glucose to ethanol, but does not fullyexplain the decreased flux. The specific formation rate ofethanol prior to the addition of furfural was about1.6g.g-.h- corresponding to 35mmol.g-1-h-1. Thesum of the specific formation rate of ethanol (0.5 g.g-1.h--l) and the specific formation rate of furfuryl alcohol(0.5 g.g-. h-l) after the furfural addition was onlyabout 15 mmol.gpl.h-. Thus, when compared to fur-fural-free medium, the total turnover of ADH for reduc-tion reactions has been decreased to less than half theoriginal value. This may be caused by direct inhibitioneffects of other enzymes, e.g. hexokinase (6), but mayalso be the result of cellu lar regulation in response to adecreased speci fic growth rate.The increased formation of pyruvate after addition offurfural (Fig. lc) may be caused by a reduced capaci tyfor reduction of acetaldehyde downstream of pyru-vate, but it may also be caused by a restricted capacityof pyruvate decarboxylase. The observed net consump-tion of acetate (Fig. lc) may occur by conversion to

CONVERSION OF FURFURA L BY S. CEREVISZAE 173acetyl-CoA (24), and may be partly explained by adecreased acetaldehyde dehydrogenase activ ity. Apparent-ly, some oxidation of furfural to furoic acid takes place,although the yield of furoic acid from furfural is verylow. Presumably its formation is catalyzed by aldehydedehydrogenase, but this reaction is less favored than thereduction of furfural.Biomass synthesis is the main source of surplusNADH resulting in glycerol formation during anaerobicconditions (18, 25). One could therefore expect that theglycerol yield should be lowered in the presence of fur-fural, since the biomass growth yield is lowered. Thatwas, however, not observed. On the contrary, theglycerol yield increased after introduction of furfural,which indicates another source of NADH production.The production of the new compound, discussed in theresults section, could be this source. The compound maybe produced from pyruvate and furfural, probably in-volving the action of thiamine pyrophosphate. Since for-mation of pyruvate produces 1 NADH, the formation ofthe new compound will also yield 1 NADH per formedmolecule.Conclusion Furfural is a strong inhibitor of the fer-mentation of glucose by S. cerevisiae. However, furfuralis reduced to the less inhibiting compound furfuryl alco-hol by the yeast. The results therefore suggest that insitu detoxification may possib ly allow fermentation ofstrongly inhibiting hydrolyzates. However, a complica-tion to be kept in mind is that the degradation rate offurfural is correlated with the rate of glycolysis.

NOMENCLATUREADH : alcohol dehydrogenaseCER : carbon dioxide evolution rate, mmol.Z-l. h-lCI : chemical ionizationEI : electron impact ionizationFIA : flow injection analysisTCA : tricaboxylic acid cycleqe : speci fic ethanol product ivity, g . g-l. h- 4f : speci fic furfural uptake rate, g-g-l. h-lP : speci fic growth rate, h-

AC KN OWLED GMEN T SThis work was f inancially supported by the Swedish NationalBoard for Technical Development. The authors are grateful toDr. Gunnar Stenhagen for help with the MS analyses and to Dr.Ni ls-Olof Ni lvebrant for comm ents on the manu scr ipt.

1.

2.3.

4.

5.

REFERENCESAzhar, A.F., Bery, M . K., Colcord, A. R., Roberts, R. S.,aad Corbi tt , G. V.: Factors affecting alcohol ferme ntation ofwood acid hydrolyzate. Biotechnol. Bioeng. Sym p., 11, 293-300(1981).Sj i istr i im, E.: Wood chem istry. Fundamental and appl ications,2nd ed. Academic Press, San Diego (1993).Cbung, I. S. and Lee, Y. Y.: Ethanol fermentation of crudeacid hydro lyzate of cellulose using high-level y eas t inocula.Biotechn ol. Bioeng., 2 7, 308-315 (1985).Boyer, L. J., V ega, J. L., Klasson, K. T., Clausen, E. C., andGaddy, J. L.: The effects of fur fural on ethanol production bySaccharom yces cerevisiae in batch cul ture. Biomass Bioenergy,3, 41-48 (1992).Zauner, E., Bronn, W. K., De l lweg, H ., and Tress& R.: Inhibi-tory activi ty of impur i t ies of beet molasses and plant pro-tectants on yeast (Saccharomyce s cerevisiae) respiration and



174 TAHERZADEH ET AL. J . BIOSCI. BIOENG..fermentation. Branntweinwir tschaft, 119, 154-163 (1979).6. Baner jee, N., Bhatnagar, R., and Viswanathan, L.: Inhibi t ionof glycolysis by fur fural in Saccharomyces cerevisiae. Eur. J.Appl. Microbial. Biotech nol., 11, 226-228 (1981).7. Baner jee, N., Bhatnagar, R., and Viswanathan, L.: Develop-ment of resistance in Saccharomyw cerevisiae against inhibito-ry effects of browning reaction products. Enzyme Microb. Tech-nol., 3, 24-28 (1981).8. Sanchez, B. and Bautista, J.: Effects of fur fural and 5-hydrox-ymethyl fur fural on the fermentation of Saccharomyces cere-visiae and biomass production from Candida guilliermondii.Enzym e Microb. Technol., 10, 315-318 (1988).9. Windisch, W. : Uber die Bi ldung und den Verbleib des Fur-furols, sowie dessen Bedeutung im Brauereibetr iebe. Chemis-ches Central-Blatt, 69, 1213-1214 (1898).10. Diaz de Vi l legas, M. E., Vi l la, P., Guerra, M., Rod r iguez, E.,Redondo, D., and Martinez, A.: Conversion of fur fural intofurfuryl alcohol by Saccharomyces cerevisiae 354. ActaBiotech nol., 12, 351-354 (1992).11. Vi l la, G. P., Bartroli , R., Lopez, R., Guerra, M ., Enr ique, M.,Penas, M., Rodr iguez, E., Redondo, D., Iglesias, I . , and Diaz,M.: Microbial transformation of fur fural to fur furyl alcoholby Saccharomyces cerevisiae. Acta Biotechnol., 12, 509-512(1992).12. Mor imoto, S., Hirashima, T., and Matutani , N.: Studies onfermentation product from fur fural by yeast, I I I . Identi f icationof furoin and fur i l. J. Fermen t. Te chnol., 47, 486-490 (1969).13. Fireoved, R .L. and Mutharasan, R.: Effect of fur fural andethanol on the growth and energetics of yeast under microaero-bi t condit ions. Ann. N. Y. Acad. Sci ., 469,433-446 (1986).14. Taherzadeh, M. J., Ekluod, R., Gustafsson , L., Nlklasson, C.,and Lid&n, G.: Character ization and fermentation of di lute-acid hydrolyzates from wood. Ind. Eng. Che m. Res., 36, 4659-4665 (1997).15. Taherzadeh, M. J., Liden, G., Gustafssoo, L., and Niklasson,C.: The effects of pantothenate deficiency and acetate addi t ionon anaerobic batch fermentation of glucose by Saccharomyces

cerevisiae. Appl. Microbial. Biotech nol., 46, 176-182 (1996).16. Chr istensen, L., Schulze, U., Nielsen, J., and Vll ladsen, J.:Acoustic o ff-gas analyser for bioreactors. Precision, accuracyand dynamics of detection. Chem . Eng. S ci ., 50, 2601-2610(1995).17. Benthin, S., Nielsen, J., and Vil ladsen, J.: Character izationand application of precise and robust flow-injection analyse rsfor on- line measurement dur ing ferme ntations. Anal. Chim .Acta, 247, 45-50 (1991).18. Verduyn, C., Postma, E., Scheffers, W. A., and van-Di jken,J. P.: Physiology of Saccharomyces cerevisiae in anaerobicglucose- limited chem ostat cul tures. J. Gen. Microb., 136, 395-403 (1990).19. Herbert, G., Phi l lips, P. J., and Strange, R. E.: Chemical anal-ysis of microbial cel ls, p. 209-344. In Norr is, J. R. and Rib-bons, D. W. (ed.) , Methods in microbiology, 5B. Academ icPress, London (1971).20. Fiechter, A., Fuhrmann, G. F., and Ki tppel i, 0.: Regulation ofglucose metabol ism in growing yeast cel ls. Adv. Microbial .Physiol ., 22, 123-183 (1981).21. Palmqvist, E., AImeida, J., and Hahn-Hi igerdal , B.: Inf luenceof fur fural on anaerobic glycolyt ic kinetics of Saccharomycescerevisiae in batch culture. Biotechno l. Bioeng. (1999). (inpress)22. Shvets, V. N., Nlkolalchuk, T.V., Slyusarenko, T. P., andUsenko , V. A.: Effec t of fur fural on alcohol fermentation, alco-hol qual ity, and bakers yeasts. Izv. Vyssh. Uchebn. Zaved.,Pishch. Tekhnol., 2, 48-52 (1977).23. Kang, S. S. and Okad a, H.: Alcohol dehydrogenase ofCephalosporium sp. induced by fur furyl alcohol. J. Fermen t.Technol., 51, 118-124 (1973).24. Taherzadeh, M. J., Niklasson, C., and Lid&, G.: Aceticacid- fr iend or foe in anaerobic batch conversion of glucose toethanol by Saccharomyces cerevisiae? Chem. Eng. Sci ., 52,2653-2659 (1997).25. Nordstrom, K.: Yeast growth and glycerol formation. ActaChem . Stand., 20, 1016-1025 (1966).

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Conversion of Furfural in Aerobic and Anaerobic Batch Fermentation of Glucose by Saccharomyces Cerevisiae_Taherzadeh, 1999