APPLIED MICROBIAL AND CELL PHYSIOLOGY

Carbon fluxes of xylose-consuming Saccharomyces cerevisiaestrains are affected differently by NADH and NADPH usagein HMF reduction

João R. M. Almeida & Magnus Bertilsson &

Bärbel Hahn-Hägerdal & Gunnar Lidén &

Marie-F. Gorwa-Grauslund

Received: 3 April 2009 /Revised: 20 May 2009 /Accepted: 21 May 2009 /Published online: 9 June 2009# Springer-Verlag 2009

Abstract Industrial Saccharomyces cerevisiae strains able toutilize xylose have been constructed by overexpression ofXYL1 and XYL2 genes encoding the NADPH-preferringxylose reductase (XR) and the NAD+-dependent xylitoldehydrogenase (XDH), respectively, from Pichia stipitis.However, the use of different co-factors by XR and XDHleads to NAD+ deficiency followed by xylitol excretion andreduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolongthe lag phase, and reduce the ethanol productivity. Recently,genes encoding furaldehyde reductases were identified andtheir overexpression was shown to improve S. cerevisiaegrowth and fermentation rate in HMF containing media andin lignocellulosic hydrolysate. In the current study, weconstructed a xylose-consuming S. cerevisiae strain usingthe XR/XDH pathway from P. stipitis. Then, the genesencoding the NADH- and the NADPH-dependent HMFreductases, ADH1-S110P-Y295C and ADH6, respectively,were individually overexpressed in this background. Theperformance of these strains, which differed in their co-factorusage for HMF reduction, was evaluated under anaerobicconditions in batch fermentation in absence or in presence ofHMF. In anaerobic continuous culture, carbon fluxes wereobtained for simultaneous xylose consumption and HMFreduction. Our results show that the co-factor used for HMF

reduction primarily influenced formation of products otherthan ethanol, and that NADH-dependent HMF reductioninfluenced product formation more than NADPH-dependentHMF reduction. In particular, NADH-dependent HMF reduc-tion contributed to carbon conservation so that biomass wasproduced at the expense of xylitol and glycerol formation.

Keywords ADH1 . ADH6 . Furaldehyde reduction . HMF.

Xylose . Lignocellulosic hydrolysate

Introduction

The use of lignocellulosic biomass for sustainable produc-tion of fuels and chemicals with bioconversion is anattractive alternative to fossil raw material (Farrell et al.2006; Hahn-Hägerdal et al. 2006) and baker’s yeastSaccharomyces cerevisiae is the preferred organism in thefermentation industry. However, commercial bioconversionof lignocellulose requires yeast strains capable to utilize bothhexose and pentose sugars (Hahn-Hägerdal et al. 2007a, b;Jeffries 2006), and to do so in presence of metabolicinhibitors produced during lignocellulose pretreatment andhydrolysis (Almeida et al. 2007; Hahn-Hägerdal et al. 2007a;Klinke et al. 2004). For S. cerevisiae to be able to utilize thepentose sugar xylose, the second most abundant sugar innature, heterologous xylose isomerase or xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have beenintroduced in recombinant strains (Chu and Lee 2007;Hahn-Hägerdal et al. 2007a; van Maris et al. 2007).Concurrently, a number of genetic engineering strategies toreduce the influence of lignocellulose derived inhibitors havealso been developed, including construction of S. cerevisiaestrains expressing laccase (Larsson et al. 2001a), phenyl-

Appl Microbiol Biotechnol (2009) 84:751–761DOI 10.1007/s00253-009-2053-1

J. R. M. Almeida : B. Hahn-Hägerdal :M.-F. Gorwa-Grauslund (*)Department of Applied Microbiology, Lund University,P.O. Box 124, S-221 00 Lund, Swedene-mail: Marie-Francoise.Gorwa@tmb.lth.se

M. Bertilsson :G. LidénDepartment of Chemical Engineering, Lund University,P.O. Box 124, S-221 00 Lund, Sweden

acrylic acid decarboxylase (PAD1; Larsson et al. 2001b) anddifferent oxido-reductases (Almeida et al. 2008a; b; Laadanet al. 2008; Liu et al. 2008; Petersson et al. 2006).

Industrial S. cerevisiae strains able to utilize xylose havebeen constructed by overexpression of XYL1 and XYL2 genesencoding XR and XDH respectively, from Pichia stipitis(Chu and Lee 2007; Hahn-Hägerdal et al. 2007a). TheNADPH-preferring XR reduces D-xylose to D-xylitol (Rizziet al. 1988), while XDH oxidizes D-xylitol to D-xyluloseexclusively using NAD+ (Rizzi et al. 1989). Recombinantstrains overexpressing this pathway grow aerobically inxylose and produce ethanol from this sugar under anaerobicconditions (Hahn-Hägerdal et al. 2007a). However, the use ofdifferent co-factors by the two first enzymes in the pathwayleads to NAD+ deficiency followed by xylitol excretion andreduced product yield (Bruinenberg et al. 1983; Kotter andCiriacy 1993; Wahlbom and Hahn-Hägerdal 2002). Conse-quently, several metabolic engineering strategies that increasethe availability of NAD+ have been shown to reduce xylitolexcretion and improve product formation (Anderlund et al.1999; Nissen et al. 2000; Roca et al. 2003; Sonderegger et al.2004; Verho et al. 2003).

Pretreatment and hydrolysis of lignocellulose lead toformation of a vast range of inhibitory compounds, includingfuraldehydes, phenolic derivatives and weak acids (Almeidaet al. 2007; Klinke et al. 2004; Palmqvist and Hahn-Hägerdal2000). The furaldehydes 5-hydroxymethyl-furfural (HMF;Ulbricht et al. 1984) and furfural (Dunlop 1948) areproduced by hexose and pentose sugar breakdown, respec-tively. These compounds inhibit yeast metabolism, prolongthe lag phase and reduce the ethanol productivity (reviewedin Almeida et al. 2009; Klinke et al. 2004). S. cerevisiaestrains are naturally able to reduce furaldehydes to theircorresponding less inhibitory alcohols, however, at low rates(Almeida et al. 2007; Klinke et al. 2004). Once HMF andfurfural are fully reduced by yeast, growth and ethanolproductivity are resumed—albeit at a lower rate than ininhibitor free medium (Liu et al. 2004; Taherzadeh et al.1999, 2000). It has also been shown that fermentation ratescorrelate with the furaldehyde reduction rates, i.e., strainsthat reduce furaldehyde faster also grow faster and fermentlignocellulosic hydrolysates faster (Almeida et al. 2008b;Modig et al. 2008; Nilsson et al. 2005).

Recently, the NADPH-dependent alcohol dehydroge-nase 6 (ADH6; Petersson et al. 2006) and the mutatedNADH-dependent ADH1 (mut-ADH1; Laadan et al.2008) were identified as HMF-reducing enzymes in S.cerevisiae. Strains overexpressing these enzymes dis-played improved growth and fermentation rate in HMFcontaining media and in non-detoxified lignocellulosichydrolysate (Almeida et al. 2008b). It was also observedthat the ADH6 overexpressing strain reduced HMF ap-proximately twice as fast as the mut-ADH1 overexpress-

ing strain, which correlated with lower in vitro HMF-reducing activity of mut-ADH1 (Almeida et al. 2008b). Toincrease NADH-dependent HMF-reducing activity a newset of ADH1 mutants were constructed and among theseADH1-S110P-Y295C (serine 110 mutated to proline andtyrosine 295 to cysteine Y295C) displayed the highest invitro activity (Laadan 2008).

In the current study, we constructed a xylose-consumingS. cerevisiae strain using the XR/XDH pathway fromPichia stipitis. Then, the ADH1-S110P-Y295C and ADH6genes were individually overexpressed in this background.These strains, which differed in their co-factor usage forHMF reduction, now provided a unique pair of strains todistinguish product formation during xylose fermentationwhen HMF is reduced by NADH and NADPH, respective-ly. The performance of the xylose-consuming strainsharboring NADH- or NADPH-dependent HMF reductaseactivity was evaluated in anaerobic batch fermentation inabsence and presence of HMF. In continuous culture,carbon fluxes were obtained for simultaneous xyloseconsumption and HMF reduction. Our results show thatthe co-factor used for HMF reduction primarily influencedformation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formationmore than NADPH-dependent HMF reduction. In particu-lar, NADH-dependent HMF reduction contributed to carbonconservation so that biomass was produced at the expenseof xylitol and glycerol formation.

Materials and methods

Construction of yeast strains

S. cerevisiae strains used in this study were constructed intwo steps. First, a xylose-consuming strain, TMB3320 wasobtained by transformation of TMB3043 (Karhumaa et al.2005; CEN.PK2-1C Δgre3, YIpXK, pB3PGKTAL1,pB3PGKRKI1, pB3PGKTKL1, pB3PGKRPE1, leu2,ura3) with the integrative plasmid YIpOB1-LEU2, harbor-ing the genes for XR and XDH from P. stipitis (Bengtssonet al. 2009). Then, TMB3320 was transformed with themulti-copy plasmid YEplacHXT, carrying either the mutat-ed ADH1-S110P-Y295C gene (Laadan 2008) or the ADH6gene (Petersson et al. 2006). A control strain (TMB3290;control strain) carried the plasmid without any gene forHMF reductase. In these strains, overexpression of mut-ADH1-S110P-Y295C (TMB3291; from now on denotedADH1 strain) and ADH6 (TMB3292; from now on denotedADH6 strain) is regulated by the constitutive truncatedpromoter HXT7 (Hauf et al. 2000). The strains weremaintained on agar plates containing 6.7 g/L YNB and20 g/L glucose.

752 Appl Microbiol Biotechnol (2009) 84:751–761

Enzymatic activity measurements

Cell extracts were prepared with Y-PER reagent (Pierce,Rockford, IL, USA) following the protocol of the supplier.The reducing activity of cells from inoculum cultures wasdetermined prior to each batch fermentation. Cells weregrown overnight at 30°C in 1 L cotton-plugged shake flaskswith 100 mL of two times concentrated defined mineralmedium (Verduyn et al. 1992) supplemented with 40 g/Lglucose and 200 mL/L phthalate buffer (10.2 g/L KHphthalate, 2.2 g/L KOH). Reduction of HMF, furfural(Petersson et al. 2006) and xylose (Smiley and Bolen 1982)was measured in cell free extracts spectrometricallyfollowing NAD(P)H oxidation as described previously,except that 2-mercaptoethanol was not used. The proteincontent in the cell extracts was determined using MicroBCA Protein Assay Kit (Pierce).

Anaerobic batch fermentation

Inoculum cultures were grown aerobically overnight at30°C in 1 L cotton-plugged shake flasks with 100 mL oftwo times concentrated defined mineral medium (Verduynet al. 1992) supplemented with 40 g/L glucose and 200 mL/Lphthalate buffer (10.2 g/L KH phthalate, 2.2 g/L KOH). Thesame medium was used in anaerobic batch fermentation,except that it was supplemented with ergosterol (0.075 g/l),Tween 80 (0.84 g/l), 20 g/L glucose and 50 g/L xylose,and where indicated, 2 g/L HMF. The starting OD620 was0.5. Fermentation was performed in 1-L media at 30°C inBraun Biotech fermentors. pH was maintained at 5.5 with3 M KOH. The stirring rate was 200 rpm. Samples formetabolite and biomass determination were regularly with-drawn from the reactor.

Anaerobic continuous fermentation

The inoculum cultures were grown overnight at 30°C in 1 Lcotton-plugged shake flasks with 100 mL of definedmineral medium (Verduyn et al. 1992) supplemented with20 g/L glucose. The same defined medium supplementedwith ergosterol (0.075 g/L), Tween 80 (0.84 g/L), and 10 g/Lglucose, and 10 g/L xylose was used as carbon and energysource. The starting OD620 in the fermentor was 0.2.Fermentation was carried out in 400 mL media at 30°C inBelach Biotech fermentors at dilution rates of 0.06 h−1 and0.12 h−1. Anaerobic conditions were maintained by contin-uously sparging 0.2 L/min nitrogen gas (containing less than5 ppm oxygen). The stirring rate was 600 rpm and pH 5.5was maintained with 0.75 M NaOH. At the end of the batchphase, i.e., once glucose was completely consumed andcarbon dioxide (CO2) values dropped, the feed was started.The feed was the same as for the batch phase, except when

indicated, 2 g/L HMF were added. Initially the dilution ratewas 0.06 h−1. Samples for analysis of metabolite concentra-tion were withdrawn at steady-state, i.e. when carbon dioxideproduction was constant after a minimum of five residencetimes. Then, the dilution rate was changed to 0.12 h−1 andonce steady-state was reached, samples were withdrawn.Fermentation with and without HMF were carried out inbiological duplicates.

Biomass and metabolites measurements

Cell concentration was determined as absorbance at 620 nmand as dry weight. For cell dry weight, 5 mL samples of cellsuspension were filtered under vacuum through pre-weighedGelman filters (Ø 47 mm Supor-450, 0.45 μm). Filters werewashed with water and dried in a microwave oven (350 W) for8 min. Cell weight was the weight difference of filters with andwithout cells. Dry weight and OD measurements correlatedwell. Samples for metabolite measurement were immediatelycentrifuged and filtered through 0.2 μm filters and stored at−20°C until analysis. Glucose, xylose, ethanol, xylitol, glycer-ol, acetic acid, pyruvate, succinate and HMF were analyzedusing an HPLC system (Waters, Milford, Massachusetts, USA)equipped with an Aminex HPX-87H column (Bio-Rad,Hercules, California) at 65°C. The mobile phase was 5 mMsulfuric acid at a flow rate of 0.6 mL/min. All compounds weredetected with a refractive index detector, except for HMFwhich was also detected with a UV-detector (210 nm).

Carbon dioxide measurements

The carbon dioxide evolution rate was monitored on-linemeasuring concentrations of carbon dioxide and oxygen inoutgoing gas from the fermentor with a CP460 gas analyzer(Belach Bioteknik AB, Solna, Sweden). The gas analyzerwas calibrated using a gas mixture containing 20.0%oxygen and 5.0% carbon dioxide.

Metabolic flux analysis

Metabolic flux analysis of ADH1 and ADH6 strains infermentation with and without HMF was used in order toillustrate the results and facilitate the discussion. This analysiswas based on the continuous fermentations. The metabolic fluxmodel was based on a previous model (Wahlbom et al. 2001),which already comprises the most important reactions foranaerobic metabolism of xylose and glucose. One reactionand one component (HMF) were added, representing HMFreduction. In strain ADH6, the HMF reduction was assumedto be totally NADPH-dependent and in strain ADH1 NADHwas assumed to be the only co-factor in this reaction. Basedon Wahlbom et al. (2001), measured biomass production wascalculated to molar fluxes by assuming a cell composition of

Appl Microbiol Biotechnol (2009) 84:751–761 753

50% proteins, 40% polysaccharides and 5% nucleotides byweight. The fluxes were normalized against the total amount(moles) of carbon atoms taken up (from glucose and xylose).

Results

HMF-reducing enzymes

The natural ADH1 enzyme from S. cerevisiae is not able toreduce HMF. However, a mutant ADH1 enzyme from theindustrial S. cerevisiae strain TMB3000 displayed HMF-reducing activity (Laadan et al. 2008). This mutant ADH1carried six amino acid mutations and displayed a uniqueNADH-dependent HMF reductase activity (Laadan et al.2008). Recently, a new set of point mutations in the ADH1have been introduced and the kinetic parameters of theresulting mutant enzymes have been determined (Laadan2008). The mutant ADH1-S110P-Y295C enzyme displayedthe highest NADH-dependent HMF-reducing activity and thisactivity was in the same range as the NADPH-dependentHMF-reducing activity of ADH6 (Petersson et al. 2006).Contrary to the mutated ADH1, ADH6 is a native S.cerevisiae enzyme, which specifically requires NADPH asco-factor for the reduction of several compounds (Larroy et al.2002), including HMF and furfural (Petersson et al. 2006).

The experimental model—in vitro furaldehydeand xylose reduction

Xylose-consuming strains were constructed by integrating theXYL1 and XYL2 genes from P. stipitis into the genome of S.cerevisiae. Then, genes encoding for either mutated ADH1-S110P-Y295C (ADH1 strain) or ADH6 (ADH6 strain) were

expressed in this background. A control strain (control) wasobtained by transformation with a plasmid without any HMFreductase encoding gene. Furaldehyde and xylose reducingactivity was determined in crude cell extracts of the threestrains. Almost identical in vitro HMF and furfural reductaseactivity was confirmed for the ADH1 and the ADH6 strains.In contrast—and as expected—the co-factor preference ofthe strains determined from in vitro experiments was entirelydifferent; while the ADH1 strain almost exclusively usesNADH, the ADH6 strain uses NADPH (Fig. 1). The controlstrain displayed only low HMF and furfural reducing activitywith either co-factor (Fig. 1), which verified that host strainhad low natural ADH6 activity. The three strains had thesame XR activity and the activity measurement confirmedthe NADPH preference of XR (Fig. 1).

Since the ADH6 strain in addition to NADPH-dependentHMF reduction also displayed high background NADH-dependent furfural reduction (Fig. 1), and since HMF andfurfural inhibition of yeast metabolism is qualitativelysimilar (Almeida et al. 2009), HMF was chosen as modelfuraldehyde in the following experiments. This generatedan experimental set-up, which allowed discrimination of theinfluence of NADH- and NADPH-dependent furaldehydereduction on xylose metabolism. The influence of furalde-hyde reduction with different co-factors was investigated inboth anaerobic batch and continuous cultures in the absenceor presence of HMF in the medium.

HMF reduction in anaerobic batch culture

Product formation for the control, ADH1, and ADH6 strainswas first followed in anaerobic batch cultivation without andwith HMF (Fig. 2 and Table 1). Glucose and xyloseconsumption and product formation were similar for the

0.0

1.0

2.0

3.0

4.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

0.0

1.0

2.0

3.0

4.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

0.0

0.5

1.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

0.0

1.0

2.0

3.0

4.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

0.0

1.0

2.0

3.0

4.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

0.0

0.5

1.0

Control ADH1 ADH6

Spec

. Act

. (U

/mg)

A C

D F

B

E

Fig. 1 HMF (a and d), furfural (b and e), and xylose (c and f)reduction activity measured in crude cell extracts using NADH (a, b,and c) and NADPH (d, e, and f) as co-factor. Cells were grown

aerobically overnight in defined mineral medium. The values areaverage of two independent measurements. The standard error fromthe average was less than 10%

754 Appl Microbiol Biotechnol (2009) 84:751–761

0

20

40

60G

lu. X

yl. (

g/L

)

0

5

10

15

20

25

Eto

h. X

ol. (

g/L

)

0

1

2

3

4

5

Gly

. Ace

t. B

iom

ass

(g/L

)

0

20

40

60

Glu

. Xyl

. (g/

L)

0

20

40

60

Glu

. Xyl

. (g/

L)

0

20

40

60

Glu

. Xyl

. (g/

L)

0.0

0.5

1.0

1.5

2.0

2.5

HM

F (g

/L)

0

20

40

60

Glu

. Xyl

. (g/

L)

0.0

0.5

1.0

1.5

2.0

2.5

HM

F (g

/L)

0

20

40

60

0 20 40 60 80 100

Time (h)

Glu

. Xyl

. (g/

L)

0.0

0.5

1.0

1.5

2.0

2.5

HM

F (g

/L)

0

5

10

15

20

25

Eto

h. X

ol.(

g/L

)

0

1

2

3

4

5

Gly

. Ace

t. B

iom

ass

(g/L

)

0

5

10

15

20

25

Eto

h. X

ol. (

g/L

)

0

1

2

3

4

5

Gly

. Ace

t. B

iom

ass

(g/L

)

0

5

10

15

20

25

Eto

h. X

ol. (

g/L

)

0

1

2

3

4

5

Gly

. Ace

t. B

iom

ass

(g/L

)

0

5

10

15

20

25

Eto

h. X

ol.

(g/L

)

0

1

2

3

4

5

Gly

. Ace

t. B

iom

ass

(g/

L)

0

5

10

15

20

25

0 20 40 60 80 100

Time (h)

Eto

h. X

ol.

(g/L

)

0

1

2

3

4

5G

ly. A

cet.

Bio

mas

s (

g/L

)

A

B

D

F

C

E

Fig. 2 Fermentation profile of control (a and d), ADH1 (b and e) andADH6 (c and f) strains in anaerobic batch fermentation without (a, b,and c) and with HMF (d, e, and f). Substrates are shown in the leftcolumn while products in the right one. Symbols: glucose (closesquares), xylose (closed triangles), HMF (gray diamonds), ethanol

(open squares), xylitol (open triangles), biomass (open circles),glycerol (open diamonds circles), acetate (asterisks). Experimentsdone in duplicate showed similar results and the figure shows one ofthe representative profile for each fermentation

Appl Microbiol Biotechnol (2009) 84:751–761 755

three strains in the absence of HMF in the medium (Fig. 2a–c and Table 1) confirming that the presence of the plasmidscarrying HMF reductase encoding genes did not influencesubstrate consumption and product formation under theexperimental conditions. In contrast fermentation profileschanged significantly in presence of HMF (Fig. 2d–f). Thecontrol strain required 50 h for complete conversion of HMF,while the ADH1 and ADH6 strains only required approxi-mately 15 h (Fig. 2d–f). Xylose consumption by the ADH1and ADH6 strains was not affected by HMF and wascompleted after 90–100 h, while it was reduced by as muchas 60% for the control strain. As a result, the control strainhad only produced 15 g/L ethanol at the end of theexperiment, compared to 20 g/L for the ADH overexpressingstrains (Fig. 2d–f). Ethanol yields were similar for the ADH1and ADH6 strains and were not affected by the presence ofHMF (Table 1). HMF reduced the biomass yield of thecontrol strain, but did not affect the ADH6 strain, andslightly increased the yield of the ADH1 strain (Table 1).HMF significantly increased acetate yields in the control,ADH1 and ADH6 strains (Table 1). In contrast, HMFreduced the glycerol yield of the ADH1 strain with 40%,from 0.20 to 0.12 g/g consumed glucose. Similar increase inacetate yields and reduced glycerol yield was previouslyobserved for strains overexpressing either a mutated ADH1or ADH6 during glucose fermentation (Almeida et al.2008b). Xylitol formation was not affected by HMF in anyof the ADH overexpressing strains, whereas HMF reducedthe xylitol yield of the control strain (Table 1).

The results showed that neither of the ADH overexpressingstrains was inhibited byHMF—in contrast to the control strainwhere xylose consumption was significantly reduced. This re-confirmed that ADH6 overexpression overcomes HMFinhibition (Almeida et al. 2008b) and also showed, for the

first time, that overexpression of the mutated ADH1-S110P-Y295C does the same. The presence of the mutated ADH1and ADH6 enzymes and, their difference in co-factor usagealso altered product formation in batch fermentation ofglucose/xylose mixtures in presence of HMF.

Simultaneous HMF reduction and xylose consumptionin continuous culture fermentation

In batch culture, the glucose and xylose consumption phaseswere separated, with xylose only being consumed whenglucose concentrations had decreased below a threshold level(Fig. 2). HMF was primarily converted during the glucoseconsumption phase, i.e., mainly before the xylose consump-tion (Fig. 2). To specifically assess the influence of co-factorusage during concurrent xylose metabolism and HMFreduction, a continuous fermentation set-up was used. Thecontrol, ADH1 and ADH6 strains were cultivated at twodilution rates, 0.06 h−1 and 0.12 h−1, in defined mineralmedium supplemented with 10 g/L glucose, 10 g/L xyloseand in the absence or presence of 2 g/L HMF (Table 2).

Similar to the results in batch fermentation, sugar con-sumption and product formation for the three strains weresimilar in absence of HMF (Tables 2 to 4), and as previouslyobserved the residual concentration of xylose increased withincreased dilution rate (Wahlbom and Hahn-Hägerdal 2002).The presence of HMF brought about substantial changes insugar consumption and product formation at both dilutionrates. At 0.12 h−1 the residual concentration of HMF wassignificantly higher, 0.7 g/L compared to 0.2 g/L, for thecontrol strain than for the ADH strains (Table 2) reflectingthe higher specific HMF uptake rate for the ADH over-expressing strains (Table 3). Faster HMF reduction allowedthe ADH1 strain to slightly increase its xylose consumption

Table 1 Yields in anaerobic batch fermentation with 20 g/L glucose and 50 g/L xylose as carbon and energy source in presence or not of 2 g/L HMF

HMF Strain Yields (g/g)

Ethanola Ethanolb Xylitolc Glycerold Acetated Biomassd

Absent Control 0.28±0.00 0.27±0.00 0.40±0.01 0.21±0.02 0.034±0.005 0.14±0.01

ADH1 0.28±0.02 0.27±0.02 0.39±0.04 0.20±0.00 0.032±0.001 0.16±0.00

ADH6 0.28±0.00 0.25±0.00 0.41±0.01 0.20±0.01 0.033±0.003 0.14±0.01

Present Control 0.35±0.01 0.18±0.01 0.18±0.04 0.22±0.00 0.053±0.001 0.06±0.01

ADH1 0.30±0.00 0.27±0.01 0.38±0.05 0.12±0.00 0.049±0.002 0.17±0.01

ADH6 0.28±0.00 0.26±0.01 0.44±0.01 0.18±0.02 0.043±0.002 0.13±0.01

All values are based in grams product by grams consumed sugar (glucose plus xylose), except where indicated contrarily. Values are average ofduplicate experiments.a Yields based on total consumed sugarb Yields based on total added sugarc Yields based on consumed xylosed Yields based on consumed glucose

756 Appl Microbiol Biotechnol (2009) 84:751–761

while the opposite was observed for the control strain, whereHMF reduced the xylose consumption (Table 2).

Both the control and the ADH strains consumed all glucosein presence of HMF. Similarly, ethanol yields were notaffected by HMF and were not significantly different for thethree strains at both dilution rates (Table 4). However,especially at the higher dilution rate, HMF altered produc-tivity and yield of all other products (Table 3 and 4). For theADH1 strain, the specific productivity of xylitol andglycerol, products related to oxidation of NADH, decreasedwith 23% and 60%, respectively, at dilution rate of 0.06 h−1

and with 26% and 70%, respectively, at dilution rate of

0.12 h−1 (Table 3). The reduced specific productivityreflected the decreased yield of these products (Table 4). Incontrast, the biomass yield on glucose of the ADH1 strainincreased significantly in presence of HMF at 0.12 h−1

(Table 4). For the ADH6 strain, the biomass yield wasslightly increased in the presence of HMF (Table 4). For thecontrol strain, reduced xylose consumption (Table 2) resultedin slightly reduced productivity of xylitol (Table 3) while thexylitol yield was not affected (Table 4). For the ADH6 strain,neither xylitol production rate (Table 3) nor yield (Table 4)were affected. HMF increased acetate formation significantlyand equally for all three strains (Table 4).

Table 2 Feed composition, residual concentrations, xylose and HMF uptake and xylitol and ethanol production in anaerobic continuous culture atdifferent dilution rates

Dilution rate (h−1) Strain Fed concentration (g/L) Residual concentration (g/L) Uptake (g/L) Production (g/L)

Glucose Xylose HMF Glucose Xylose HMF Xylose HMF Xylitol Ethanol

0.06 Control 10.2±0.1 10.4±0.1 – 0.0±0.0 6.3±0.1 – 4.1±0.2 – 1.3±0.0 4.1±0.1

ADH1 10.2±0.0 10.5±0.0 – 0.0±0.0 6.5±0.2 – 3.9±0.2 – 1.3±0.0 4.0±0.1

ADH6 10.2±0.3 10.5±0.2 – 0.0±0.0 6.5±0.2 – 3.9±0.2 – 1.3±0.1 4.1±0.1

Control 9.7±0.4 10.1±0.2 2.1±0.0 0.0±0.0 6.1±0.0 0.4±0.1 4.0±0.2 1.7±0.1 1.4±0.0 3.8±0.3

ADH1 10.2±0.0 10.4±0.1 2.0±0.0 0.0±0.0 6.2±0.0 0.1±0.0 4.2±0.1 1.9±0.0 1.2±0.0 4.1±0.0

ADH6 10.3±0.2 10.7±0.1 2.1±0.1 0.0±0.0 6.5±0.5 0.1±0.0 4.3±0.4 2.0±0.1 1.6±0.1 4.2±0.2

0.12 Control 10.2±0.1 10.4±0.1 – 0.0±0.0 7.1±0.1 – 3.3±0.0 – 1.3±0.0 4.2±0.1

ADH1 10.2±0.0 10.5±0.0 – 0.0±0.0 7.2±0.1 – 3.2±0.1 – 1.2±0.0 3.9±0.1

ADH6 10.2±0.3 10.5±0.2 – 0.0±0.0 7.4±0.4 – 3.1±0.2 – 1.2±0.1 4.2±0.1

Control 9.7±0.4 10.1±0.2 2.1±0.0 0.0±0.0 7.1±0.1 0.7±0.0 3.0±0.1 1.4±0.0 1.1±0.1 3.7±0.4

ADH1 10.2±0.0 10.4±0.1 2.0±0.0 0.0±0.0 6.9±0.1 0.2±0.0 3.6±0.1 1.9±0.0 1.1±0.0 4.1±0.0

ADH6 10.3±0.2 10.7±0.1 2.1±0.1 0.0±0.0 7.3±0.2 0.2±0.0 3.5±0.2 1.9±0.1 1.4±0.0 4.2±0.2

Values are average of duplicate experiments.

Table 3 Specific uptake rates (glucose, xylose and HMF) and specific productivity rates (others; mmol/g biomass/h) in anaerobic continuousculture at different dilution rates.

Dilution rate (h−1) HMF Strains Glucose Xylose HMF Ethanol Xylitol Glycerol Acetate

0.06 Absent Control 2.8±0.0 1.4±0.0 – 5.2±0.0 0.44±0.02 0.67±0.04 0.05±0.01

ADH1 3.0±0.1 1.4±0.0 – 5.2±0.3 0.44±0.02 0.79±0.01 0.04±0.00

ADH6 2.9±0.1 1.4±0.0 – 5.5±0.2 0.46±0.04 0.79±0.00 0.05±0.02

Present Control 2.4±0.1 1.2±0.1 0.59±0.04 4.3±0.5 0.42±0.02 0.49±0.01 0.15±0.04

ADH1 2.5±0.0 1.2±0.0 0.68±0.01 4.6±0.1 0.34±0.00 0.31±0.01 0.14±0.05

ADH6 2.6±0.1 1.3±0.2 0.72±0.05 4.9±0.1 0.48±0.04 0.62±0.08 0.07±0.10

0.12 Absent Control 5.1±0.3 2.0±0.1 – 8.9±0.7 0.78±0.01 1.10±0.03 0.09±0.01

ADH1 5.2±0.1 2.0±0.1 – 8.5±0.1 0.74±0.02 1.26±0.13 0.09±0.01

ADH6 5.3±0.4 1.9±0.0 – 9.2±0.6 0.77±0.02 1.27±0.10 0.08±0.03

Present Control 4.6±0.3 1.7±0.1 0.93±0.02 7.5±1.0 0.65±0.03 0.88±0.10 0.24±0.05

ADH1 4.1±0.2 1.7±0.1 1.08±0.03 7.0±0.2 0.55±0.03 0.38±0.01 0.24±0.06

ADH6 4.5±0.1 1.8±0.1 1.18±0.07 7.9±0.4 0.74±0.00 0.94±0.05 0.28±0.06

The fed contained 10 g/L glucose and 10 g/L xylose for carbon and energy source and where indicated 2 g/L HMF. Values are average ofduplicate experiments.

Appl Microbiol Biotechnol (2009) 84:751–761 757

Intracellular fluxes for the ADH1 and ADH6 strainsduring anaerobic growth in the absence and presence ofHMF were calculated using a previously described stoi-chiometric model (Wahlbom et al. 2001). HMF reductionwas included in the model as one reaction with NADH-dependent HMF reduction for the ADH1 strain andNADPH-dependent HMF reduction for the ADH6 strain(Fig. 3). Since the difference between the two strains wasmost pronounced at the higher dilution rate 0.12 h−1

(Table 3), these data were used to calculate the intracellularfluxes (Fig. 3). In absence of HMF, the xylose flux throughthe NADPH-dependent branch of XR was higher thanthrough the NADH-dependent branch for both strains.Acetate formation was negligible and the oxidative pentosephosphate pathway (ox-PPP) indicated as glucose 6-P toribulose 5-P in Fig. 3 was the main source of NADPH inboth strains. In the presence of HMF, fluxes throughNADH/NAD+ coupled reactions, i.e., formation of glycerol,xylitol, xylulose (xylulose-5-P), and biomass, were eithersignificantly reduced or enhanced, especially for the ADH1strain (Fig. 3). In addition, both strains displayed increasedfluxes through the NADH-dependent branch of XR andthrough aldehyde dehydrogenase, which leads to NADPHand acetate formation. Contrarily, the fluxes through theTCA cycle were reduced. The flux through the ox-PPP wasnot affected in the ADH1 strain; however, it slightlyincreased for the ADH6 strain.

Discussion

The current investigation made use of a unique experimentalmodel consisting of two recombinant xylose-consumingstrains of S. cerevisiae, each expressing a furaldehyde

reducing alcohol dehydrogenase (ADH). In one strain theenzyme, a mutated ADH1, ADH1-S110P-Y295C, exclusivelyused NADH (Laadan 2008) and in the other strain theenzyme ADH6 (Petersson et al. 2006) exclusively usedNADPH. Anaerobic fermentation of glucose/xylose mixturesin batch and continuous culture demonstrated that NADH-dependent furaldehyde reduction (i.e., the ADH1 strain) ledto carbon conservation with increased xylose uptake andbiomass production at the expense of xylitol and glycerolformation. Reduced glycerol formation is accompanied byincreased ATP availability for biomass production, whichconsumes NAD+ and regenerates NADH for HMF reduc-tion. In contrast, NADPH-dependent furaldehyde reductiononly marginally influenced the glucose/xylose metabolism.

Xylose metabolism in natural an recombinant strains ofyeast harboring the XR/XDH pathway results in xylitolexcretion (Hahn-Hägerdal et al. 2007b; Jeffries 2006) becausethe two enzymes use NADPH and NAD+, respectively,which creates a deficiency of NAD+ for the XDH reaction.For natural xylose-utilizing strains, it was early recognizedthat electron acceptors such as acetoin and diacetyl increasethe intracellular pool of NAD+ reducing xylitol excretion andincreasing carbon conservation during xylose utilization(Bruinenberg et al. 1983). It was subsequently shown thatfurfural, a compound formed during lignocellulose pretreat-ment and hydrolysis, acted as an electron acceptor in arecombinant xylose-consuming strain and reduced xylitolexcretion (Wahlbom and Hahn-Hägerdal 2002). Further-more, no xylitol was formed when a non-detoxifiedlignocellulose hydrolysate was fermented with a recombinantxylose-consuming yeast strain (Karhumaa et al. 2007). Thecurrent results indicate that absence of xylitol excretion andcarbon conservation are primarily governed by the ability toregenerate NAD+.

Table 4 Yields of xylitol (mol/mol xylose consumed), ethanol, glycerol, acetate (mol/mol glucose + xylose consumed), and biomass (c mol/molglucose consumed) in anaerobic continuous culture at dilution rate 0.12 h−1

HMF Strains Yields (mol/mol)

Xylitola Ethanolb Glycerolb Acetateb Biomassc

Absent Control 0.39±0.01 1.25±0.03 0.16±0.01 0.01±0.00 0.96±0.06

ADH1 0.37±0.01 1.19±0.05 0.18±0.01 0.01±0.00 0.93±0.02

ADH6 0.41±0.01 1.27±0.02 0.18±0.01 0.01±0.00 0.92±0.06

Present Control 0.38±0.04 1.17±0.08 0.14±0.01 0.04±0.01 1.05±0.07

ADH1 0.32±0.00 1.20±0.02 0.06±0.00 0.04±0.01 1.18±0.04

ADH6 0.41±0.02 1.24±0.10 0.15±0.01 0.04±0.01 1.08±0.02

The fed contained 10 g/L glucose and 10 g/L xylose for carbon and energy source and where indicated 2 g/L HMF. Values are average ofduplicate experiments.a Yields based on consumed xyloseb Yields based on total consumed sugarc Yields based on consumed glucose; molecular weight of 1c-mol biomass was 24.6

758 Appl Microbiol Biotechnol (2009) 84:751–761

Carbon conservation has been extensively investigatedfor ethanolic hexose fermentation, where the production ofglycerol constitutes a significant reduction of product yield(Oura 1977). The common denominator for metabolicengineering strategies has been to reduce glycerol forma-tion by redirecting carbon fluxes from NADPH- to NADH-consuming reactions (Anderlund et al. 1999; Nissen et al.2000). These strategies have also applied to recombinantxylose-utilizing strains (Roca et al. 2003). When theammonium assimilation pathway was engineered by delet-ing GDH1, which encodes an NADPH-dependent gluta-mate dehydrogenase, and by overexpressing GDH2, which

encodes an NADH-dependent glutamate dehydrogenasecarbon conservation was achieved with an increasedspecific xylose consumption rate and reduced xylitol yield(Roca et al. 2003). Similarly, net re-oxidation of NADH wasachieved by channeling carbon fluxes through a recombinantphosphoketolase pathway in a xylose-consuming strain(Sonderegger et al. 2004). This in combination with thedeletion of ALD6 contributed to conserve carbon byincreasing rate of xylose fermentation and reducing xylitolformation.

The NADPH-dependent HMF reduction encoded byoverexpression of ADH6 was expected to reduce the

Fig. 3 Flux distribution duringanaerobic growth ofS. cerevisiae strains ADH1(left values) and ADH6 (rightvalues) calculated on the basisof steady-state data obtained inHMF-free medium (top values)and in presence of the inhibitor(bottom values) at a dilution rateof 0.12 h−1. Gray boxes indicatemain substrate and productsmeasured in the fermentationmedia. HMF reaction wasexclusively NADH-dependentin the ADH1 strain andNADPH-dependent in theADH6 strain. Biomass fluxrepresents the sum ofpolysaccharides, proteins, andRNA fluxes. The data areexpressed in millimoles pergram biomass × h × c moleof consumed glucose and xylose(mmol/g × h × c mol consumedglu + xyl)

Appl Microbiol Biotechnol (2009) 84:751–761 759

NADPH availability in HMF medium and lead to NADH-dependent xylose reduction by XR. Then NAD+ would beavailable for XDH and prevent xylitol excretion. Themetabolic flux analysis supported the expectation of anincreased NADH usage by XR and increased flux throughXDH. However, xylitol yields were not significantlyreduced by the presence of HMF (Table 4). Instead, thexylose consumption rate increased and glycerol yielddecreased. Furthermore, the demand for NADPH forHMF reduction was met partially by increased acetateformation (Fig. 3 and Table 4) via the NADP+-dependentaldehyde dehydrogenase ALD6 (Saint-Prix et al. 2004).

In conclusion the present investigation demonstrated that theNADH-dependent HMF reductase, a mutated ADH1, efficient-ly detoxified an HMF supplemented medium similar to whathas previously been demonstrated for an NADPH-dependentHMF reductase (Almeida et al. 2008b). Furthermore, and incontrast to the NADPH reductase, the NADH-dependentreductase was able not only to conserve carbon by reducingglycerol formation, but also conserve carbon by reducingxylitol formation during xylose fermentation. In view of thefact that lignocellulose hydrolysates contain furaldehydes aswell as xylose, the overexpression of the mutated ADH1offers the possibility to simultaneously detoxify the substrateand to conserve carbon in its bioconversion.

Acknowledgment This work was supported by the Swedish EnergyAgency.

References

Almeida JRM, Modig T, Petersson A, Hahn-Hägerdal B, Lidén G,Gorwa-Grauslund MF (2007) Increased tolerance and conversionof inhibitors in lignocellulosic hydrolysates by Saccharomycescerevisiae. J Chem Technol Biotechnol 82:340–349

Almeida JRM, Modig T, Röder A, Lidén G, Gorwa-Grauslund MF(2008a) Pichia stipitis xylose reductase helps detoxifyinglignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF). Biotechnol Biofuels 1:12

Almeida JRM, Röder A,Modig T, Laadan B, Lidén G, Gorwa-GrauslundMF (2008b) NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on productdistribution in Saccharomyces cerevisiae. Appl Microbiol Biotech-nol 78:939–945

Almeida JRM, Bertilsson M, Gorwa-Grauslund MF, Gorsich S, Lidén G(2009) Metabolic effects of furaldehydes and impacts on biotech-nological processes. Appl Microbiol Biotechnol 82:625–638

Anderlund M, Nissen TL, Nielsen J, Villadsen J, Rydstrom J,Hahn-Hägerdal B, Kielland-Brandt MC (1999) Expression of theEscherichia coli pntA and pntB genes, encoding nicotinamidenucleotide transhydrogenase, in Saccharomyces cerevisiae and itseffect on product formation during anaerobic glucose fermentation.Appl Environ Microbiol 65:2333–2340

Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF (2009) Xylosereductase from Pichia stipitis with altered coenzyme preferenceimproves ethanolic xylose fermentation by recombinant Saccha-romyces cerevisiae. Biotechnol Biofuels 2:9

Bruinenberg PM, Debot PHM, Vandijken JP, Scheffers WA (1983)The role of redox balances in the anaerobic fermentation ofxylose by yeasts. Eur J Appl Microbiol 18:287–292

Chu BC, Lee H (2007) Genetic improvement of Saccharomycescerevisiae for xylose fermentation. Biotechnol Adv 25:425–441

Dunlop AP (1948) Furfural formation and behavior. Ind Eng Chem40:204–209

Farrell AE, Plevin RJ, Turner BT, Jones AD, O'Hare M, Kammen DM(2006) Ethanol can contribute to energy and environmental goals.Science 311:506–508

Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF, Lidén G, Zacchi G(2006) Bio-ethanol—the fuel of tomorrow from the residues oftoday. Trends Biotechnol 24:549–556

Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I,Gorwa-Grauslund MF (2007a) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953

Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF(2007b) Metabolic engineering for pentose utilization in Saccha-romyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177

Hauf J, Zimmermann FK, Müller S (2000) Simultaneous genomicoverexpression of seven glycolytic enzymes in the yeastSaccharomyces cerevisiae. Enzyme Microb Technol 26:688–698

Jeffries TW (2006) Engineering yeasts for xylose metabolism. CurrOpin Biotechnol 17:320–326

Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF (2005)Investigation of limiting metabolic steps in the utilization ofxylose by recombinant Saccharomyces cerevisiae using metabol-ic engineering. Yeast 22:359–368

Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-GrauslundMF(2007) Comparison of the xylose reductase-xylitol dehydrogenaseand the xylose isomerase pathways for xylose fermentation byrecombinant Saccharomyces cerevisiae. Microb Cell Fact 6:5

Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products producedduring pre-treatment of biomass. Appl Microbiol Biotechnol66:10–26

Kotter P, Ciriacy M (1993) Xylose fermentation by Saccharomycescerevisiae. Appl Microbiol Biotechnol 38:776–783

Laadan B (2008) Yeasts as cellular factories. PhD Thesis. LundUniversity

Laadan B, Almeida JRM, Rådström P, Hahn-Hägerdal B, Gorwa-Grauslund M (2008) Identification of an NADH-dependent5-hydroxymethylfurfural-reducing alcohol dehydrogenase inSaccharomyces cerevisiae. Yeast 25:191–198

Larroy C, Fernandez MR, Gonzalez E, Pares X, Biosca JA (2002)Characterization of the Saccharomyces cerevisiae YMR318C(ADH6) gene product as a broad specificity NADPH-dependentalcohol dehydrogenase: relevance in aldehyde reduction. Bio-chem J 361:163–172

Larsson S, Cassland P, Jönsson LJ (2001a) Development of aSaccharomyces cerevisiae strain with enhanced resistance tophenolic fermentation inhibitors in lignocellulose hydrolysates byheterologous expression of laccase. Appl Environ Microbiol 67:1163–1170

Larsson S, Nilvebrant NO, Jönsson LJ (2001b) Effect of over-expression of Saccharomyces cerevisiae Pad1p on the resistanceto phenylacrylic acids and lignocellulose hydrolysates underaerobic and oxygen-limited conditions. Appl Microbiol Biotech-nol 57:167–174

Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW(2004) Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMFconversion to 2, 5-bis-hydroxymethylfuran. J Ind MicrobiolBiotechnol 31:345–352

Liu ZL, Moon J, Andersh BJ, Slininger PJ, Weber S (2008) Multiplegene-mediated NAD(P) H-dependent aldehyde reduction is a

760 Appl Microbiol Biotechnol (2009) 84:751–761

mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. ApplMicrobiol Biotechnol 81:743–753

Modig T, Almeida JRM, Gorwa-Grauslund MF, Lidén G (2008)Variability of the response of Saccharomyces cerevisiae strains tolignocellulose hydrolysate. Biotechnol Bioeng 100:423–429

Nilsson A, Gorwa-Grauslund MF, Hahn-Hägerdal B, Lidén G (2005)Cofactor dependence in furan reduction by Saccharomycescerevisiae in fermentation of acid-hydrolyzed lignocellulose.Appl Environ Microbiol 71:7866–7871

Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J (2000)Optimization of ethanol production in Saccharomyces cerevisiaeby metabolic engineering of the ammonium assimilation. MetabEng 2:69–77

Oura E (1977) Reaction products of yeast fermentations. ProcessBiochem 12:19–21

Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosichydrolysates. II: inhibitors and mechanisms of inhibition. Bio-resource Technol 74:25–33

Petersson A, Almeida JRM, Modig T, Karhumaa K, Hahn-Hägerdal B,Gorwa-Grauslund MF, Lidén G (2006) A 5-hydroxymethylfurfural reducing enzyme encoded by the Saccharomyces cerevi-siae ADH6 gene conveys HMF tolerance. Yeast 23:455–464

Rizzi M, Erlemann P, Bui-Thanh N-A, Dellweg H (1988) Xylosefermentation by yeasts. 4. Purification and kinetic studies ofxylose reductase from Pichia stipitis. Appl Microbiol Biotechnol29:148–154

Rizzi M, Harwart K, Erlemann P, Buithanh NA, Dellweg H (1989)Xylose fermentation by yeasts. 6. Purification and properties ofthe Nad + -xylitol-dehydrogenase from the yeast Pichia stipitis. JFerment Bioeng 67:20–24

Roca C, Nielsen J, Olsson L (2003) Metabolic engineering ofammonium assimilation in xylose-fermenting Saccharomycescerevisiae improves ethanol production. Appl Environ Microbiol69:4732–4736

Saint-Prix F, Bonquist L, Dequin S (2004) Functional analysis of theALD gene family of Saccharomyces cerevisiae during anaerobicgrowth on glucose: the NADP+-dependent Ald6p and Ald5p

isoforms play a major role in acetate formation. Microbiology150:2209–2220

Smiley K, Bolen P (1982) Demonstration of D-xylose reductase andD-xylitol dehydrogenase in Pachysolen tannophilus. BiotechnolLett 9:607–610

Sonderegger M, Schumperli M, Sauer U (2004) Metabolic engineer-ing of a phosphoketolase pathway for pentose catabolismin Saccharomyces cerevisiae. Appl Environ Microbiol 70:2892–2897

Taherzadeh MJ, Gustafsson L, Niklasson C, Lidén G (1999)Conversion of furfural in aerobic and anaerobic batch fermenta-tion of glucose by Saccharomyces cerevisiae. J Biosci Bioeng 87:169–174

Taherzadeh MJ, Gustafsson L, Niklasson C, Lidén G (2000)Physiological effects of 5-hydroxymethylfurfural on Saccharo-myces cerevisiae. Appl Microbiol Biotechnol 53:701–708

Ulbricht RJ, Northup SJ, Thomas JA (1984) A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. FundamAppl Toxicol 4:843–853

van Maris AJA, Winkler AA, Kuyper M, de Laat WTAM, van Dijken JP,Pronk JT (2007) Development of efficient xylose fermentation inSaccharomyces cerevisiae: xylose isomerase as a key component.Adv Biochem Eng Biot 108:179–204

Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect ofbenzoic acid on metabolic fluxes in yeasts: a continuous-culturestudy on the regulation of respiration and alcoholic fermentation.Yeast 8:501–517

Verho R, Londesborough J, Penttilä M, Richard P (2003) Engineeringredox cofactor regeneration for improved pentose fermentation inSaccharomyces cerevisiae. Appl Environ Microbiol 69:5892–5897

Wahlbom CF, Hahn-Hägerdal B (2002) Furfural, 5-hydroxymethylfurfural, and acetoin act as external electron acceptors duringanaerobic fermentation of xylose in recombinant Saccharomycescerevisiae. Biotechnol Bioeng 78:172–178

Wahlbom CF, Eliasson A, Hahn-Hägerdal B (2001) Intracellularfluxes in a recombinant xylose-utilizing Saccharomyces cerevi-siae cultivated anaerobically at different dilution rates and feedconcentrations. Biotechnol Bioeng 72:289–296

Appl Microbiol Biotechnol (2009) 84:751–761 761