MINI-REVIEW

Metabolic effects of furaldehydes and impactson biotechnological processes

João R. M. Almeida & Magnus Bertilsson &

Marie F. Gorwa-Grauslund & Steven Gorsich &

Gunnar Lidén

Received: 30 November 2008 /Revised: 13 January 2009 /Accepted: 14 January 2009 /Published online: 31 January 2009# Springer-Verlag 2009

Abstract There is a growing awareness that lignocellulosewill be a major raw material for production of both fuel andchemicals in the coming decades—most likely throughvarious fermentation routes. Considerable attention has beengiven to the problem of finding efficient means of separatingthe major constituents in lignocellulose (i.e., lignin, hemi-cellulose, and cellulose) and to efficiently hydrolyze thecarbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these willhave to be dealt with in the ensuing microbial processes. Onegroup of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes—mostimportantly 5-hydroxymethyl-2-furaldehyde—are the domi-nant inhibitory compounds found in lignocellulosic hydro-lyzates. The furaldehydes are known to have biologicaleffects and act as inhibitors in fermentation processes. Theeffects of these compounds will therefore have to beconsidered in the design of biotechnological processes usinglignocellulose. In this short review, we take a look at knownmetabolic effects, as well as strategies to overcome problemsin biotechnological applications caused by furaldehydes.

Keywords Furfural . Hydroxymethylfurfural . Reductases .

Bioconversion . Inhibition

Furaldehydes—occurrence and use

Chemically, 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (furfural) comprise a heteroaromatic furan ringand an aldehyde functional group. The chemical propertiesof furaldehydes and the fact that they can be produced fromrenewable sources make them potential alternatives tomany petrochemical derivatives in the production ofplastics and fine chemicals (Moreau et al. 2004). Furfuraland HMF can be formed by dehydration of pentoses andhexoses, respectively (Fig. 1; first half). Furfural isproduced from the hemicellulose of xylose-rich vegetablewaste products (e.g., corncobs (Mains and Laforge 1924)and rice husk (Sangarunlert et al. 2007)), by acid hydrolysisof the pentosan followed by acid-catalyzed dehydration ofpentoses at elevated temperatures. It is a selective solvent,primarily used in processing of petroleum oil and fuel, andits reduced derivative, furfuryl alcohol, is used on industrialscale for furan resin production. Furfural has also been usedas a flavor ingredient in the food industry (Adams et al.1997).

HMF is produced by acid-catalyzed dehydration ofhexoses, mostly fructose and glucose. However, whereasfurfural is produced in several hundred thousand tons peryear, HMF is not yet a major industrial chemical because ofa low selectivity in the conversion or expensive productrecovery (Kuster 1990). As an alternative to productionfrom hexoses, HMF can be produced by hydroxymethyla-tion of furfural (Lecomte et al. 1999). A dicarboxylic acid,2,5-furandicarboxylic acid, is obtained by oxidation ofHMF (Moreau et al. 2004). This is an interesting starting

Appl Microbiol Biotechnol (2009) 82:625–638DOI 10.1007/s00253-009-1875-1

J. R. M. Almeida :M. F. Gorwa-GrauslundDepartment of Applied Microbiology, Lund University,P.O. Box 124, 221 00 Lund, Sweden

M. Bertilsson :G. Lidén (*)Department Chemical Engineering, Lund University,P.O. Box 124, 221 00 Lund, Swedene-mail: gunnar.liden@chemeng.lth.se

S. GorsichBiology Department, Central Michigan University,230 Brooks Hall,Mt. Pleasant, MI 48859, USA

compound for production of polyesters and polyamides,with the potential to partly replace terephthalic acid.Derivates of HMF also occur in for instance the productionof polyurethane foams (Moreau et al. 2004). HMF andfurfural are substances of importance in the food industry.Both furfural and HMF occur naturally in many food items,especially in heat-treated foods. These compounds have, forexample, been suggested as browning indicators in bread(Ramirez-Jimenez et al. 2000), age markers in spirits(Quesada Granados et al. 1996), and as index compoundsof storage temperature abuse in manufacture of juices(Kanner et al. 1981).

Biotechnological relevance

Inhibitor formation during pretreatment and dilute-acidhydrolysis

In the processing of lignocellulose, treatment of thematerial at high temperature, often with an added acid asa catalyst, is an important first step in order to produce abioavailable substrate. The lignocellulose can be treated bya purely chemical hydrolysis, aimed to depolymerize thehemicellulose and cellulose completely (dilute-acid hydro-lysis). Alternatively, the chemical treatment can aim tosolubilize the hemicellulose and break up the rigid cellulosestructures (pretreatment), which requires less harsh con-ditions. After pretreatment, the cellulose can be enzymat-ically digested at mild conditions (Lee et al. 1999; Mosieret al. 2005). The dilute-acid treatment (or pretreatment) willgenerate several inhibitory compounds, which principallycan be divided into three main groups. The first groupcontains the compounds of interest for this review, i.e., thefuraldehydes (furfural and HMF), whereas the other twomain groups are the weak acids (e.g., acetic acid, formicacid, and levulinic acid) and the phenolic compounds (see,e.g., Almeida et al. 2007 or Klinke et al. 2004 for recentreviews on inhibitors in general). Furaldehydes are typical-ly formed from the liberated sugar monomers at high

temperatures, and a fraction of the furaldehydes is alsolikely to be further degraded to organic acids—e.g.,levulinic acid and formic acid (Fig. 1). The formation ofsugar degradation products should obviously be minimizedto avoid loss of sugar but also to minimize the problem ofinhibition of microbial activity caused by the degradationproducts during fermentation. The considerable variation interms of structure and chemical composition betweendifferent raw materials complicates finding the optimalconditions for hydrolytic treatment, and a mostly empiricalexperimental approach is therefore necessary for each rawmaterial (Canettieri et al. 2007; Chen et al. 2007; Nguyen etal. 2000; Roberto et al. 2003; Söderström et al. 2003;Öhgren et al. 2005). However, the fact that the activationenergy is generally higher for hydrolysis than for sugardegradation holds in most investigations (Bhandari et al.1984; Mosier et al. 2002; Ranganathan et al. 1985). Thisimplies that high temperatures and short residence timesfavor hydrolysis more than sugar degradation. Restrictions,such as allowable reactor pressures and heat transfercharacteristics, will put a practical limit on the operatingrange (Lee et al. 1999), which in turn puts a limit on theglucose yield one can hope to obtain from cellulose in a purelyacid-catalyzed hydrolysis process. This limit (in the range of60%) is one main argument for using enzymatic hydrolysis toobtain glucose from the cellulose. Typical composition valuesreported for pretreated material (i.e., material to be enzymat-ically hydrolyzed) and acid-hydrolyzed lignocellulosic mate-rials (i.e., material which has only been hydrolyzedchemically) are given in Table 1.

Biological effects

Aldehydes, in general, have many biological consequencesto eukaryotic cells. More specifically, cellular-reactivealdehydes, such as acrolein, cause oxidative damage andlead to apoptotic events (Tanel and Averill-Bates 2007). Anincrease in aldehyde-induced oxidative damage contributesto many human diseases such as Alzheimer’s disease (Ohtaand Ohsawa 2006) and cardiovascular disease (Uchida

O

CH2OH

OH

OH

OH

OHO

O

OH OH

O

O

OH

OO

OH

OH

OH

OH OO

OH

O

Fig. 1 Schematic representa-tions of the formation andbreakdown of furfural and HMFfrom xylose and glucose,respectively

626 Appl Microbiol Biotechnol (2009) 82:625–638

2000). In both these cases, reactive aldehyde groups causean elevation in reactive oxygen species (ROS), typicallygenerated in the mitochondria. The oxidizing consequencesof ROS are known to cause DNA mutations, proteinmisfolding and fragmentation, membrane damage, andapoptosis (Perrone et al. 2008; Sigler et al. 1999). The cellcan combat aldehydes by various mechanisms. Forinstance, aldehyde dehydrogenases (e.g., ALDH2), whichcan oxidize aldehydes, protects cells against oxidativestress and are linked to various diseases (Ohta et al.2004). Mice with low levels of ALDH2 display character-istic signs of Alzheimer’s disease (Ohta and Ohsawa 2006)and when ALDH2 is induced myocardial infarct size isreduced in rats undergoing cardiac ischemia (Chen et al.2008). The link between aldehydes and diseases has led to thepursuit of drugs that act as aldehyde-sequestering chemicals,such as hydralazine and dihydralazine which were showneffective in sequestering acrolein (Burcham et al. 2002).Whether or not similar strategies could be applied to otherbiological problems linked to aldehydes is not known.

Not surprisingly, the furaldehydes furfural and HMF arealso known to cause specific biological effects. The ability offurfural to cause DNA damage has been known for at least30 years. As early as 1978, furfural was shown to induce

DNA mutations in Salmonella typhimurium (Zdzienicka etal. 1978), Escherichia coli (Khan and Hadi 1993), lambdaphage (Hadi et al. 1989), and Drosophila melanogaster(Rodriguez-Arnaiz et al. 1992). Furfural has also beenlinked to the formation of liver tumors in mice (Reynolds etal. 1987) and lung tissue damage (Gupta et al. 1991). HMFdoes have a cytotoxic effect, but its mutagenic effect is lessestablished (Janzowski et al. 2000; Lee et al. 1995).Interestingly, HMF may have some clinical value. In recentstudies, HMF has been proposed to have anticancerproperties (Michail et al. 2007) and HMF derivatives haveanti-sickle-cell-anemia properties (Abdulmalik et al. 2005).

In terms of effect on microorganisms, furfural and HMFcause budding yeasts (Saccharomyces cerevisiae, Kluyver-omyces marxianus, Pichia stipitis, and others; Almeida etal. 2007; Delgenes et al. 1996; Oliva et al. 2003) andbacteria (Zymomonas mobilis, E. coli, and others; Boopathyet al. 1993; Ranatunga et al. 1997; Zaldivar et al. 1999) toreduce fermentation rate and/or stop growing and enter anextended lag phase. S. cerevisiae viability is also reduced(Brandberg et al. 2004; Heer and Sauer 2008). Underanaerobic conditions—and also during respiro-fermentativemetabolism typical of batch processes—the yeast S.cerevisiae will convert furfural and HMF to their less

Table 1 Typical composition of pretreated or dilute-acid-hydrolyzed lignocellulose

Raw material Treatment Glucose(g/L)

Mannose(g/L)

Galactose(g/L)

Xylose(g/L)

Arabinose(g/L)

HMF(g/L)

Furfural(g/L)

Ref

Pretreated lignocelluloseBarley straw 210°C, 15 min 4.6 – 1.3 17.4 1.9 0.2 0.7 (García-Aparicio et al.

2006)Corn stover 3% SO2, 200°C, 5 min 8.3 – 4.0 35.8 6.3 0.06 1.1 (Öhgren et al. 2006)Spruce 3% SO2, 215°C, 5 min 23.4 19.4 4.0 8.9 – 3.0 1.7 (Rudolf et al. 2005)Sugarcanebagasse

2% SO2, 190°C, 5 min 3.5 – 0 32.8 2.7 0 0.9 (Rudolf et al. 2008)

Sugarcanebagasse

−% H2SO4, 205°C, 10 min 23.5c 0.5c – 9.0c 1.8c 0.2c 1.1c (Martín et al. 2002)

Wheat straw −% H2SO4, 210°C, 2.5 min 10.9 – – 51.2 – 0.6 1.7 (Olofsson et al. 2008)Wheat straw 210°C, 5 min 5.7 0.6 1.1 24.7 1.6 0.1 1.4 (Tomás-Pejó et al.

2008)Dilute-acid-hydrolyzed lignocelluloseRice straw 1.6% H2SO4, 121°C, 30 min 21a – – 6a – 0.18 0.13 (Roberto et al. 2003)Spruce 2.4% H2SO4, 180°C, 10 min 21.7 12.7 – 5.0 – 4a 1a (Larsson et al. 1999)Spruce 5 g/L H2SO4, 215°C, 7 min 30.4 19.8 – 4.7 – 5.9 1.3 (Taherzadeh et al.

1997)Spruce I: 0.5% H2SO4, 190°C, 10 min 24.3b 11.8b 3.4b 5.7b 1.5b 2.1b 0.4b (Rudolf et al. 2004)Two step II: <0.5% H2SO4, 215°C, 7 minSpruce/pine I: 0.7% H2SO4, 190°C, 3 min 27.8 33.9 13.4 25.4 9.8 2.4 2.5 (Nguyen et al. 1999)Two step II: 0.4% H2SO4, 215°C, 3 min 70.0 3.0 1.7 0.6 0 5.8 0Sugarcanebagasse

0.34% H2SO4, 160°C, 15 min 3.4 0.6 1.4 31.8 1.9 0.18 2.22 (Neureiter et al. 2002)

a Estimated from a figureb Concentrations concern the mixture of fractions I and IIc After enzymatic hydrolysis of pretreated material

Appl Microbiol Biotechnol (2009) 82:625–638 627

reactive alcohol derivatives using NAD(P)H-dependentreduction reactions during the lag phase, after which growthmay resume (Almeida et al. 2007). During respirativegrowth, however, the conversion products are different. Itwas shown that furfural present in the inlet medium wasconverted exclusively to furoic acid by the examined strainof S. cerevisiae in a fully aerobic chemostat study. Pulseadditions of furfural directly into the chemostat triggered ashift to respiro-fermentative metabolism, and again furfurylalcohol was formed as the major conversion product(Sarvari Horvath et al. 2003).

The observed lag may result from a reduction inavailable cellular energy caused by the inhibition ofenzymes such as alcohol dehydrogenase, aldehyde dehy-drogenase, pyruvate dehydrogenase (Modig et al. 2002),and two key glycolytic enzymes, hexokinase and glyceral-dehyde-3-phosphate dehydrogenase (Banerjee et al. 1981).One may ask if the growth delay in the presence of furfuralis only due to a decrease in available cellular energy or ifthere is a selective advantage for yeast to stop growth untilfuraldehydes are converted? The finding that the pentosephosphate pathway (PPP) is essential for furfural tolerance(Gorsich et al. 2006a) suggested that the NADPH produced inthis pathway may function in more than just furaldehydeconversion since yeast cells lacking a functional PPP haveincreased sensitivity to oxidative damage. This sensitivitymay be due to deficiency of NADPH, which is an essentialcofactor for oxidative stress protection enzymes, includingglutathione reductase and thioredoxin reductase (Carmel-Harel and Storz 2000; Grant et al. 1996; Sigler et al. 1999).Thereby, furaldehydes present in industrial processes couldcause oxidative damage to yeast cells. Under semi-aerobicconditions, a correlation between furfural and ROS wasindeed reported, and the furfural-induced ROS subsequentlycaused tubular mitochondria to aggregate, large vacuoles tofragment, actin cables to lose their structure, and tightlycompacted nuclear chromatin to become diffuse. Oncefurfural was converted to furfuryl alcohol, ROS decreasedand the cellular damage was repaired (Gorsich et al. 2006b).In many fermentation processes, anaerobic conditions wouldprevail. Although not concerning the effects of aldehydes,two separate studies recently reported signs of ROS-inducedoxidative damage in wine and lager brewing yeast, even inthe absence of oxygen (Gibson et al. 2008; Landolfo et al.2008). Together, these results suggested that yeasts do indeedhave a distinct advantage to prevent growth until furfural isconverted. There could be serious consequences if celldivision continued in the presence of mutagenic furaldehydeand furaldehyde-induced cellular damage. Thus, good cellularstrategies to minimize effects of furaldehydes are necessaryand include: (1) chemical conversion of furaldehydes to lessreactive compounds—primarily the corresponding alcohols—and (2) protection against furaldehyde-induced damage and

the repair of any damage caused by furaldehydes (as shownschematically for furfural in Fig. 2).

Understanding the biological effects of furaldehydes willenhance engineering efforts to develop more robust micro-organisms. Whether the product is a therapeutic protein,such as insulin, or an industrially relevant metabolite suchas ethanol, it is imperative that genes that function inmaking these products remain protected without mutations.These mutations could result in either altered product orreduced yield (Wiseman 2005). Maintaining the integrity ofthe cell is also important. If a cell spends energy (i.e.,substrate) repairing cellular damage (e.g., membrane,chromatin, cytoskeleton, and proteins), then less energy(substrate) is available for the desired product.

Affected processes

Due to its presence and biological activity, it is clear thatfuraldehydes constitute a potential problem in all biotech-nical processes that utilize pretreated or acid-hydrolyzedlignocellulose (Saha 2003). The production of fuel ethanolfrom lignocellulosic feedstocks is currently one of thelargest areas of biotechnological research, and inhibitioneffects of the furaldehydes have been extensively studiedfor this application. However, there are several otherprocesses for which the influence of furfural and HMFhas also been examined (Table 2). In general, micro-organisms appear to have the ability to convert both HMFand furfural to less inhibitory compounds and inhibitoryeffects are therefore gradually reduced, as long as initialconcentrations are not too high (Boyer et al. 1992).Nevertheless, substantial inhibition effects on both cellgrowth and ethanol production by both furfural and HMF,at concentrations typical for lignocellulosic hydrolyzates,have been reported (Table 2).

Fig. 2 Overall furfural effects on yeast. Furfural induces ROSaccumulation and cell damage during extended lag phase. Growthresumes and cellular damage is repaired upon furfural conversion

628 Appl Microbiol Biotechnol (2009) 82:625–638

Strategies to overcome inhibition by furaldehydes

Several strategies can be used to overcome inhibitoryeffects of furaldehydes on yeast and bacterial metabolism(Fig. 3). Possibilities include medium detoxification prior tofermentation or use of the bioreduction capability of thefermenting microorganism. Alternatively, the medium may

be augmented with compounds conferring protection to thefuraldehydes.

Detoxification

The first option that comes to mind is perhaps to avoid theinhibition problems altogether by removing the furaldehydes

Table 2 Experimental studies on inhibition caused by furaldehydes in various processes

Fermentationprocess

Microorganism Inhibitor Inhibitorconc. (g/L)

Productivityreduction (%)

Growth ratereduction (%)

Timeconcerned (h)

Inoculum size Ref

Ethanolproduction

S. cerevisiae Furfural 0.5–4.0 21–97 0–80 48 0.7% (v/v) (Banerjee et al. 1981)S. cerevisiae Furfural 1.0–2.0 0a–40 7a–13a 30 0.1 (g/L) (Boyer et al. 1992)

1.0–2.0 4a–12a 6–19 30 2.0 (g/L)S. cerevisiae Furfural 0.9–5.1 18–87 28–100 10 and 5 OD=1.5 (Palmqvist et al. 1999)S. cerevisiae Furfural 1–5 4b–100b 51c–91c – 0.55 (g/L) (Navarro 1994)

1–5 1b–44b 4c–39c – 2.50 (g/L)S. cerevisiae Furfural 4 56 89 12 1.0a (g/L) (Taherzadeh et al. 2000)S. cerevisiae Furfural 1.0–2.0 1d–99d – 144 5% (v/v) (Sanchez and Bautista 1988)S. cerevisiae Furfural 0.5–2.0 43–89 47–90 24 3% (v/v) (Delgenes et al. 1996)S. carlsbergensis Furfural 1–10 35a–100a – 6 10% (w/w) (Pfeifer et al. 1984)S. cerevisiae HMF 2–4 19–41 40–71 12 1.0a (g/L) (Taherzadeh et al. 2000)S. cerevisiae HMF 1.0–2.0 0d–6d – 144 5% (v/v) (Sanchez and Bautista 1988)S. cerevisiae HMF 1.0–5.0 71–95 65–89 24 3% (v/v) (Delgenes et al. 1996)S. cerevisiae HMF 1.5 – 39 2 0.8 (g/L) (Petersson et al. 2006)S. carlsbergensis HMF 5–20 0a–35a – 6 10% (w/w) (Pfeifer et al. 1984)P. stipitis Furfural 0.27–1.5 21b–83b – – 1.0 (g/L) (Nigam 2001)P. stipitis Furfural 0.5–2.0 29–95 25–99 32 3% (v/v) (Delgenes et al. 1996)

HMF 1.0–5.0 17–91 5–99 32 3% (v/v)C. shehatae Furfural 0.5–2.0 20–90 19–90 32 3% (v/v) (Delgenes et al. 1996)

HMF 1.0–5.0 33–96 8–92 32 3% (v/v)P. tannophilus Furfural 0.35–0.7 16a–100a 74a,c–100a,c 168 – (Watson et al. 1984)Z. mobilis Furfural 0.5–2.0 4–44 18–56 12 3% (v/v) (Delgenes et al. 1996)

HMF 1.0–5.0 15–53 49–67 12 3% (v/v)E. coli Furfural 3.7–7.4 −17a–93a 15a–100a 48 OD550=1.0 (Zaldivar et al. 1999)

HMF 4.5–9.0 −6a–51a 10a–45a 48 OD550=1.0Cellulaseproduction

T. reesei Furfural 0.4–1.2 20a–45 – 168 10% (v/v) (Szengyel and Zacchi 2000)

ABE production C. beijerinckii Furfural 2.0 −6 −7 – 5% (v/v) (Ezeji et al. 2007)HMF 2.0 −15 −14 – 5% (v/v)

C. acetobutylicum Furfural 0.5–3.0 21–89 – 11 – (Zverlov et al. 2006)Xylitol production C. guilliermondii Furfural 1.0–2.0 32a–53a 26a–23a 26 and 42 OD600=8

and 0.03a

(g/L)

(Kelly et al. 2008)

C. guilliermondii Furfural 1.0–2.0 – 30–100 72 5% (v/v) (Sanchez and Bautista 1988)HMF 1.0–2.0 – 8–62 72 5% (v/v)

2,3-Butanediol production K. pneumoniae Furfural 3.46 – 50 8 10% (v/v) (Boopathy et al. 1993)HMF 2.27 – 50 8 10% (v/v)

Detoxification E. coli Furfural 3.36 – 50 8 10% (v/v) (Boopathy et al. 1993)HMF 2.65 – 50 8 10% (v/v)

E. aerogenes Furfural 3.75 – 50 8 10% (v/v) (Boopathy et al. 1993)HMF 2.52 – 50 8 10% (v/v)

C. freundii Furfural 3.07 – 50 8 10% (v/v) (Boopathy et al. 1993)HMF 2.65 – 50 8 10% (v/v)

P. vulgaris Furfural 1.63 – 50 8 10% (v/v) (Boopathy et al. 1993)HMF 1.89 – 50 8 10% (v/v)

a Estimated from a figureb Based on maximum volumetric ethanol productivityc Based on maximum specific growth rated Based on CO2 evolution

Appl Microbiol Biotechnol (2009) 82:625–638 629

from the medium before fermentation. Removal of inhibitorsin general from lignocellulosic hydrolyzates has been exten-sively studied, with particular emphasis on dilute-acid hydro-lyzates (see reviews by, e.g., Huang et al. (2008), Sánchez andCardona (2008), and Mussatto and Roberto (2004)). Themethod of choice must be sufficiently cheap and technicallyfeasible. Reported methods include evaporation (boiling),adsorption by activated charcoal, adsorption on ion exchang-ers, solvent extraction, alkaline treatment, or treatment byenzymes (laccases). Most efficient removal of furaldehydesis obtained by alkaline treatment or by ion exchange,whereas laccase treatment has no effect on the furaldehydes.A significant challenge for detoxification is to selectivelyremove inhibitor compounds without removing also thesugars. Alriksson et al. (2005) compared alkaline treatmentswith Ca(OH)2, NaOH, and NH4OH to remove inhibitors fromdilute-acid hydrolyzates of spruce. They found NH4OH to bethe best alkaline reagent, both in terms of removal offuraldehydes and in terms of fermentability of the treatedhydrolyzates. An added advantage in comparison to Ca(OH)2is also that no gypsum will be generated in the process. In asubsequent study (Alriksson et al. 2006), treatment conditionswere varied, and a high removal of the furaldehydes (>90%)was reported for treatments at high pH (>11) and elevatedtemperatures (80°C) for 3 h. However, this was accompaniedby unacceptably large sugar losses (20–25%). Best overallperformance was found by treating the hydrolyzate withNH4OH at pH 9 and 60°C for 3 h. This gave a removal ofboth furfural and HMF (30–40% reduction) at a reasonableloss of sugar yield (about 5%). The problem of a sugar losswhen removing furaldehydes occurs also when anionexchangers are used as reported by Nilvebrant et al. (2001).

From above, it is clear that the detoxification step mayadd significantly to the overall process cost due to capitalcosts, chemical costs, and loss of sugars. Values as high as

20% of the total costs have been given in some estimations(von Sivers et al. 1994).

Bioreduction

With bioreduction, we here understand the ability of themicroorganism to reduce a specific aldehyde compound byan oxido-reductase catalyzed reaction. Reported results(Boopathy et al. 1993; Liu et al. 2004; Nichols et al.2008; Taherzadeh et al. 2000; Villa et al. 1992) show thatmicroorganisms generally are able to reduce the furalde-hydes HMF and furfural to their corresponding alcohols,which are less inhibitory (Fig. 4). However, the observedrates for this reduction vary considerably even betweendifferent strains of the same species. Based on the reductioncapacity, two principal strategies can be conceived tominimize the inhibition by furaldehydes. (1) By processdesign, the fermentation is carried out in such a way thatthe natural reduction capability of the microorganism is notsurpassed. This can be achieved by a fed-batch process, i.e.,a gradual addition of substrate at a rate matching theintrinsic conversion capacity of the culture. The organismmay furthermore obtain an enhanced conversion capacityby a short-term adaptation. (2) By strain development, thebioconversion is improved by screening for new strainsand/or by increasing the reduction capability of a specificstrain using targeted genetic engineering or long-termadaptation (Fig. 3).

Process design

Mode of fermentation

Lignocellulosic inhibitors reduce yeast viability and fer-mentation rates considerably during the first hours of the

F

Fig. 3 Schematic representationof strategies to overcome inhi-bition by furaldehydes

630 Appl Microbiol Biotechnol (2009) 82:625–638

process (Brandberg et al. 2004; Larsson et al. 1999;Taherzadeh et al. 2000). A number of studies indicate thata high specific reduction rate of HMF and furfural is wellcorrelated with a high fermentation rate of spruce hydroly-zate (Almeida et al. 2008a; Modig et al. 2008; Nilsson et al.2005). The ability to maintain viability in the presence offuraldehydes as well as the in situ detoxification capacity ofthe microorganism is therefore likely to determine themaximum production rate. Ideally, the microorganismshould maintain the same specific substrate conversion ratethroughout the process. A feed strategy in which theintrinsic capacity of furaldehyde reduction is not exceededallows a continuous in situ detoxification to take place,without accumulation of the furaldehydes in the medium.Obviously, the highest allowable feed rate is desirable inorder to reach the highest ethanol productivity, and severalfeeding strategies based on measured carbon dioxideevolution rate have been designed for this purpose (Nilssonet al. 2001; Rudolf et al. 2004).

A recent study comparing the performance of S.cerevisiae strains in spruce hydrolyzate underlined theimportance of comparing strains under representativeconditions (Modig et al. 2008). When six industrial strainswere subjected to a pulse addition (mimicking batchcultivation) of spruce hydrolyzate, growth was onlyobserved for the strain TMB3000 (μ=0.03 h−1). Inaddition, the fermentation rates decreased strongly uponaddition of hydrolyzate. The furfural present in thehydrolyzate was converted, whereas approximately 50%of the HMF added remained at the end of the fermentation.In contrast, when the same six strains were compared infed-batch fermentation mode, furfural was not detected atall, and the HMF levels were significantly lower or almostzero for all strains. Furthermore, all six strains presentedclearly higher specific ethanol productivities than in thepulse addition of hydrolyzate, also when comparing onlythe initial most productive 8 h following the pulse addition(Modig et al. 2008).

A different approach to improve yeast performance is touse encapsulated cells (Talebnia et al. 2005). A lowdiffusion rate of inhibitors through the membrane (orimmobilization matrix) may provide a less stressfulenvironment. However, the use of encapsulated cells canbe hampered by the gradual deactivation of cells and costaspects.

Short-term adaptation

The fed batch is probably advantageous not only due to thefact that the furaldehyde conversion rate is optimized butalso since it allows an adaption to the furaldehydes (andother inhibitory compounds). Adaptation of the yeast has,for instance, in simultaneous saccharification and fermen-

tation (SSF) experiments, been shown to be important(Alkasrawi et al. 2006). Fermentation rates in SSF weresignificantly higher for yeast produced in spruce hydroly-zate than for yeast produced on a pure glucose medium. Inaddition, HMF conversion rate during fermentation ofspruce hydrolyzate is higher for the yeast exposed tohydrolyzate in the cultivation step (Alkasrawi et al. 2006).These results indicated that the yeast is able to develop ashort-term adaptative response towards the lignocellulosicinhibitors. The induction of HMF and furfural reductionactivities during fed-batch cultivation might play animportant role during yeast acclimatization to the hydroly-zate (Modig et al. 2008). However, the role of specificenzymatic activities is still not entirely clear.

Strain development

Long-term adaptation

The short-term adaptation is primarily related to theincrement of gene expression and enzyme activities and istherefore to be regarded as a process design strategy. Long-term adaptation, on the other hand, is based on selection ofadvantageous cellular properties through iterative geneticdiversification and selection—a true strain development.The preferred properties should be obtained by a suitablyapplied selective pressure. In contrast to the short-termadaptation, the improved character derived from long-termadaptation strategies should remain even when the selectivepressure is lifted. Yeast strains with increased tolerancetowards furfural and HMF have been obtained by employ-ing such a concept (Heer and Sauer 2008; Liu et al. 2005).The yeast strain TMB3400 was grown in minimal mediumcontaining 3 mM furfural. Cells from cultures in lateexponential phase were then transferred to a fresh mediaamended with furfural. Upon shorter lag phases, thefurfural concentration was increased continuously. Finally,populations were streaked out and single colonies wereobtained. The best strain isolated after approximately 300generations showed a lag phase of 17 h instead of 90 h forparental strain in media supplemented with 17 mM furfural.Furthermore, viability tests in medium containing furfuralshowed that the evolved strain remained viable, whereas theparental strain showed continuously decreasing colony-forming unit capacity after 10 h (Heer and Sauer 2008).

Similar results were obtained with adaptation of P.stipitis NRRL Y-7124 and S. cerevisiae NRRL Y-12632 tomedia amended either with HMF or furfural (Liu et al.2005). Cells grown in a liquid medium were sequentiallytransferred into fresh media supplemented with the inhibitorat higher and higher concentrations once logarithmicgrowth was reached. For each strain, at least 100 transferswere necessary to obtain stable populations. The derivative

Appl Microbiol Biotechnol (2009) 82:625–638 631

strains S. cerevisiae 307-12H60 and 307-12H120 and P.stipitis 307 10H60 showed enhanced ability to reduce HMFto 2,5-bis-hydroxymethylfuran (FDM or HMF alcohol) atconcentrations as high as 30 and 60 mM in the fermentationbroth. S. cerevisiae 307-12-F40 converted furfural intofurfuryl alcohol (FM) at significantly higher rates comparedto the parental strain.

Finally, S. cerevisiae strain TMB3001 was adapted tosugarcane bagasse hydrolyzate instead of to a specificfuraldehyde (Martín et al. 2007). The adaptation wasperformed in a continuous culture system with a graduallyincreasing inhibitor concentration in the hydrolyzate feed. Anadapted strain was selected after approximately 350 h ofcultivation. The evolved strain showed better ethanol yieldand furfural conversion rate than the parental strain inhydrolyzate with lower levels of inhibitors (50% hydrolyzate),but no advantages were seen with more concentratedhydrolyzate (100% hydrolyzate). HMF reduction capabilitiesof the parental and evolved strains were not reported. Theimproved S. cerevisiae strains obtained by the long-termadaptation strategies discussed here showed improvedtolerance but in different ways. While the evolved strainobtained from Heer and Sauer (2008) showed improvedviability in presence of furfural, the S. cerevisiae strainsobtained by Liu et al. (2005) and Martín et al. (2007)showed increased furfural and or HMF reduction rate. Theseresults highlight the importance of the choice of the selectivepressure applied in long-term adaptation strategies.

Targeted genetic engineering

Long-term adaptation of strains to furaldehydes or ligno-cellulosic hydrolyzates is an efficient way to improvetolerance of the selected strain. A major drawback is,however, that the genetic modifications responsible for theimprovement are not easily found and are not easilytransferable to another strain. Genetic engineering strate-

gies, on the other hand, are based on known traits and canbe easily transferable from one strain to another. Asdiscussed above, the rate of furaldehyde conversion is akey factor for obtaining a high fermentation rate. Both thespecific conversion rate and also the cofactor utilization areknown to be strain dependent (Almeida et al. 2007). As anincreased rate of furaldehyde conversion is a key factor forobtaining a high fermentation rate, several oxido-reductasesresponsible for furaldehyde conversion have been identified(Fig. 4) and used to construct strains of S. cerevisiae withincreased tolerance towards lignocellulosic hydrolyzateinhibitors (Table 3). Microarray analysis showed that morethan 15 reductases were overexpressed when S. cerevisiaewas cultivated in presence of HMF, including alcoholdehydrogenases 6 and 7 (ADH6 and ADH7). The NADPH-dependent ADH6 (Larroy 2002a) was the first enzymeidentified to catalyze both HMF and furfural reduction in S.cerevisiae (Petersson et al. 2006).

A mutated alcohol dehydrogenase 1 (mut-ADH1) genehas been isolated from the strain TMB3000 (Linden et al.1992). The mut-ADH1 enzyme possesses a unique NADH-dependent HMF-reducing activity (Laadan et al. 2008;Modig et al. 2008; Nilsson et al. 2005). Almeida et al.(2008b) recently reported that also xylose reductase from P.stipitis (Ps-XR) possesses a furaldehyde-reducing activity.Ps-XR is a well-characterized enzyme, but previous kineticstudies have been focused solely on sugars and keto-alcohols (Jeppsson et al. 2003; Rizzi et al. 1988).

Screening of a S. cerevisiae gene disruption library formutants with growth deficiencies in presence of furfuralrevealed the PPP gene zwf1 involvement in furaldehydetolerance (Gorsich et al. 2006a). The overexpression ofzwf1, which encodes glucose-6-phasphate dehydrogenase(Zwf1p), allowed S. cerevisiae growth at high furfuralconcentrations. Contrary to adh6, mut-adh1, and xyl1(encodes xylose reductase), the improved toleranceachieved by overexpression of ZWF1 cannot be directly

NADPH

NADP+

NADH

ADH1*

ADH6ADH7 FFRXR

NAD+

NADH

NAD+

Furfural

O

OH

OO

FM

ADH1*XR

HMF

FDM

O

OH O H

OO

O H

ADH6ADH7

XR

Fig. 4 Reduction of furfural and HMF to their corresponding alcohols(FM and FDM), respectively. Enzymes and cofactors involved in thereduction are indicated. Enzymes: ADH1* mutated alcohol dehydro-genase 1 (Laadan et al. 2008) ADH6 and ADH7 alcohol dehydroge-

nase 6 (Petersson et al. 2006) and 7 (Larroy 2002b; Petersson et al.2006), FFR furfural reductase (Gutiérrez et al. 2006), XR xylosereductase (Almeida et al. 2008b)

632 Appl Microbiol Biotechnol (2009) 82:625–638

related with furaldehyde reduction rate (Table 3). Instead, itis proposed that higher abundance of reducing equivalentsgiven by Zwf1p activity would favor the activities offurfural reductases (FFR) and/or NADPH-dependent stressresponse enzymes.

There have been few reports on identification of genesresponsible for furaldehyde tolerance in microorganismsother than yeasts (Table 3). An FFR from E. coli LYO1 waspurified and characterized (Gutiérrez et al. 2006). However,to the best of our knowledge, the overexpression of theFFR gene to develop E. coli strains with increasedtolerance has still not been attempted.

In addition to the furaldehyde reduction capacity, theprotection against ROS-induced damage caused by furalde-hydes is of interest. There are several known genesinvolved in the function of oxidative stress protection (e.g.,SOD1, GPX1, TPS1, GSH1, GLR1, TRR1/2, and SIR2).However, their role in protecting against oxidative stress,specifically caused by furaldehydes, has not yet beeninvestigated (Carmel-Harel and Storz 2000; Grant et al.1996; Sigler et al. 1999).

Selection

As discussed previously, strains with an innate highfuraldehyde reduction rate show a higher specific glucoseconsumption rate and ethanol production rate in HMF- and/or furfural-containing media. It thus appears favorable toselect for strains with a high innate tolerance, as indicatedby a high furaldehyde conversion rate. Such strains may bedirectly employed in a given fermentation process, used indetoxification steps, or be the host for strain improvements.In fact, several screening experiments, using hydrolyzate(Brandberg et al. 2004; Modig et al. 2008) or a mixture ofcomponents present in hydrolyzates, including furaldehydes(Martin and Jonsson 2003), have been carried out in orderto identify candidate strains to be used in ethanolicfermentation. A common characteristic found in thesestudies was that the best strain indeed had the highestfuraldehyde conversion rate.

Screening of 12 strains of S. cerevisiae for anaerobicgrowth in spruce, barley, and wheat straw hydrolyzateshighlighted the importance of strain selection when using

Table 3 Strain improvement by targeted genetic engineering

Gene—enzyme Origin Identification Experimental conditions Strain improvementa Ref

ADH6—alcoholdehydrogenase 6

S. cerevisiae Microarrayanalysis

Pulse addition of 1.5 g/LHMF in (an)aerobicbatch cultivation inmineral medium

Four times higher specificuptake rate of HMF

(Petersson et al. 2006)

Batch fermentation ofspruce hydrolyzate

Four times higher specificuptake rate of HMF and20% higher specificethanol productivity

(Almeida et al. 2008a)

MUT-ADH1—mutated alcoholdehydrogenase 1

S. cerevisiaestrain TMB3000

Proteinpurification,MS, and geneisolation

Semi-aerobic growth inmineral mediumsupplementedwith 20 mM HMF

Reduction of lag phaseand two times faster HMFconversion

(Laadan et al. 2008)

Batch fermentation ofspruce hydrolyzate

Four times higher specificuptake rate of HMF and18% higher specificethanol productivity

(Almeida et al. 2008a)

ADH7—alcoholdehydrogenase 7

S. cerevisiae Microarrayanalysis

Growth in minimalmediumsupplementedwith 40 mM HMF

Exit lag phase after 94 h,while the control failed togrow even after 156 h

(Larroy 2002b;Liu et al. 2008;Petersson et al. 2006)

XYL1—xylosereductase

P. stipitis Proteinpurification

Growth in minimalmedium supplementedwith 2 g/L HMF

Increased in vivo HMFconversion rate by 20%

(Almeida et al. 2008b)

ZWF1—glucose-6-phasphatedehydrogenase

S. cerevisiae Screening of a S.cerevisiaegene disruptionlibrary

Growth in SD-complete mediumsupplemented with50 mM furfural

Allowed growth at thisfurfural concentration,which was lethal forthe control strain

(Gorsich et al. 2006a)

FFR—furfuralreductase

E. coli strainLYO1

Proteinpurification

Data not available (Gutiérrez et al. 2006)

a Strain improvement was obtained by overexpression of the corresponding gene in S. cerevisiae strains

Appl Microbiol Biotechnol (2009) 82:625–638 633

different lignocellulosic hydrolyzates (Almeida et. al.personal communication). The screening showed a greatvariation between the hydrolysates and the strains. Hydro-lyzed barley straw, which contains the highest furfuralconcentration, was most inhibitory, i.e., the hydrolyzatewith the lowest minimum inhibitory concentration, fol-lowed by spruce and wheat straw hydrolyzates. In addition,it was shown that a strain that is the most efficient in onehydrolyzate is not necessarily the most suitable in anotherhydrolyzate. It is therefore suggested that the strains whichperformed well in all three hydrolyzates have a more globalresponse to the hydrolyzate inhibitors than strains thatperformed well in only one hydrolyzate. In the latter case,the adaptation may be directed towards a specific inhibitor(Almeida et. al. manuscript in preparation).

Selections of furaldehyde-converting microorganismsother than S. cerevisiae have also been carried out. Lopezet al. (2004) applied a sequential enrichment strategy toisolate microorganisms from furfural-contaminated soil ableto convert HMF, furfural, and ferulic acid. In theirscreening, the initial selection was based on stable growthon solid medium containing furfural, HMF, and ferulic acidindividually or in their mixture as carbon and energysource. Thereafter, isolated microorganisms were trans-ferred into a dilute-acid hydrolyzate from corn stover.Finally, from 12 selected isolates, five bacteria (related toMethylobacterium extorquens, Pseudomonas sp., Flavobac-terium indologenes, Acinetobacter sp., and Arthrobacteraurescens) and one fungus (Coniochaeta ligniaria) thatwere able to reduce HMF and furfural from defined mineralmedium were identified. However, only the fungus C.ligniaria was able to reduce these compounds in cornstover hydrolyzate (Lopez et al. 2004). The better perfor-mance of C. ligniaria in the hydrolyzate may be associatedwith its ability to metabolize and remove furaldehydes as wellas organic acids and other aldehydes from the corn stoverdilute-acid hydrolyzate (Nichols et al. 2008).

Addition of compounds to the medium

Instead of removing inhibitory compounds from themedium (which may be difficult as discussed), one couldinstead potentially add compounds, which confer protectionagainst stress. Possibilities include, e.g., trehalose andglutathione. Trehalose is known to protect yeast fromROS by protecting membranes and proteins from oxidativedamage (Alvarez-Peral et al. 2002; Arguelles 2000;Benaroudj et al. 2001; Wiemken 1990), and glutathione isa major cellular reductant that can interact directly withhydroxyl radicals converting them to water (Grant et al.1996; Sigler et al. 1999). Moreover, glutathione whenadded exogenously can protect yeast against ROS andincrease yeast life span (Heeren et al. 2004). Whether or not

trehalose or glutathione will protect yeast from furalde-hydes is not yet known.

Influence of bioreduction on product distribution

As discussed previously, furaldehydes reduce growth andmetabolic activity by directly inhibiting enzymes and ordamaging cellular structures. In addition, important secondaryeffects of furaldehydes on yeast metabolism are the changes incofactor balance of the cell, which may appear whenfuraldehydes are reduced. As HMF and furfural reduction iscoupled with NADH and/or NADPH oxidation (Fig. 4), themetabolic fluxes related to cofactor regeneration may changedepending on the enzyme used in the reduction. S. cerevisiaestrains overexpressing ADH6 or mut-ADH1 gene, togetherwith a control strain, were compared in anaerobic batchfermentation of glucose in the absence and presence of HMF(Almeida et al. 2008a). In the presence of the inhibitor, allthree strains produced twice as much acetate duringfermentation than in its absence. The increased acetateproduction could be explained by the need for generationof NADPH—in case of HMF reduction with ADH6p—andavoidance of acetaldehyde accumulation due to depletion ofNADH, in case of reduction via mut-ADH1p. The glycerolyields per biomass did not change for the control and mut-ADH1 strain, but the ADH6-overexpressing strain produced24% more glycerol (Almeida et al. 2008a).

Furaldehyde reduction also affected redox balance inxylose-consuming S. cerevisiae strains which overexpressthe NAD(P)H-dependent xylose reductase and NAD+-dependent xylitol dehydrogenase from P. stipitis (Wahlbomand Hahn-Hägerdal 2002; Almeida et. al. manuscript inpreparation). In the strain TMB3001, furfural and HMFreductions are coupled with NADH and NADPH, respec-tively. Xylose-fermenting batch cultures were subjected topulse additions of 3 g/L furfural or 1.5 g/L HMF. TheNADPH-dependent HMF reduction did not affect xylitolexcretion, but the NAD+ produced during furfural reductiondecreased the xylitol excretion (Wahlbom and Hahn-Hägerdal 2002). Similarly, regeneration of NAD+ duringHMF reduction by mut-ADH1 in continuous culturecultivations resulted in reduced glycerol and xylitol yields(Almeida et. al. manuscript in preparation).

All together, these results point that overexpression ofHMF and furfural reductases not only helps in thedetoxification of lignocellulosic hydrolyzates, but they canalso be beneficially used for changing product distributionduring xylose fermentation, most probably contributing toreduce xylitol and increase ethanol yields (Wahlbom andHahn-Hägerdal 2002). Thus, a fermentation setup whichfavors increased detoxification and flux for desired path-ways may be developed.

634 Appl Microbiol Biotechnol (2009) 82:625–638

Concluding remarks

Effects of furaldehyde compounds in biotechnologicalprocesses are clearly to be counted on—particularly inlignocellulose-based processes. Proper strategies for avoid-ing a negative impact on the process performance includeoptimized pretreatment—to minimize the formation of thecompounds—and careful strain selection and adaption, aswell as an appropriate fermentation technology—to opti-mize the in situ conversion of the furaldehydes to lessinhibitory products. Since reduction appears to be a keycellular strategy of detoxification, genetic engineering interms of overexpression of reductases, such as ADH6,offers interesting possibilities for strain improvement.

Acknowledgements JA, MB, GL, and MFGG were financiallysupported by the Swedish Energy Agency. SG was financiallysupported by the Research Excellence Funds, ORSP, Central Michi-gan University.

References

Abdulmalik O, Safo MK, Chen Q, Yang J, Brugnara C, Ohene-Frempong K, Abraham DJ, Asakura T (2005) 5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibitssickling of red blood cells. Br J Haematol 128:552–561

Adams TB, Doull J, Goodman JI, Munro IC, Newberne P, PortoghesePS, Smith RL, Wagner BM, Weil CS, Woods LA, Ford RA(1997) The FEMA GRAS assessment of furfural used as aflavour ingredient. Food Chem Toxicol 35:739–751

Alkasrawi M, Rudolf A, Lidén G, Zacchi G (2006) Influence of strainand cultivation procedure on the performance of simultaneoussaccharification and fermentation of steam pretreated spruce. EnzMicrob Technol 38:279–286

Almeida JR, 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 JR, Röder A, Modig T, Laadan B, Lidén G, Gorwa-Grauslund MF (2008a) NADH- vs NADPH-coupled reductionof 5-hydroxymethyl furfural (HMF) and its implications onproduct distribution in Saccharomyces cerevisiae. Appl Micro-biol Biotechnol 78:939–945

Almeida JR, Modig T, Roder A, Lidén G, Gorwa-Grauslund MF(2008b) Pichia stipitis xylose reductase helps detoxifyinglignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfu-ral (HMF). Biotechnol Biofuels 1:12

Alriksson B, Horváth I, Sjöde A, Nilvebrant N-O, Jönsson L (2005)Ammonium hydroxide detoxification of spruce acid hydro-lysates. Appl Biochem Biotechnol 124:911–922

Alriksson B, Sjöde A, Nilvebrant N-O, Jönsson L (2006) Optimalconditions for alkaline detoxification of dilute-acid lignocellulosehydrolysates. Appl Biochem Biotechnol 130:599–611

Alvarez-Peral FJ, Zaragoza O, Pedreno Y, Arguelles JC (2002)Protective role of trehalose during severe oxidative stress causedby hydrogen peroxide and the adaptive oxidative stress responsein Candida albicans. Microbiol 148:2599–2606

Arguelles JC (2000) Physiological roles of trehalose in bacteriaand yeasts: a comparative analysis. Arch Microbiol 174:217–224

Banerjee N, Bhatnagar R, Viswanathan L (1981) Inhibition ofglycolysis by furfural in Saccharomyces cerevisiae. Appl Micro-biol Biotechnol 11:226–228

Benaroudj N, Lee DH, Goldberg AL (2001) Trehalose accumulationduring cellular stress protects cells and cellular proteins fromdamage by oxygen radicals. J Biol Chem 276:24261–24267

Bhandari N, MacDonald DG, Bakhshi N (1984) Kinetic studies ofcorn stover saccharification using sulphuric acid. BiotechnolBioeng 26:320–327

Boopathy R, Bokang H, Daniels L (1993) Biotransformation offurfural and 5-hydroxymethyl furfural by enteric bacteria. J IndMicrobiol Biotechnol 11:147–150

Boyer LJ, Vega JL, Klasson KT, Clausen EC, Gaddy JL (1992) Theeffects of furfural on ethanol production by Saccharomycescerevisiae in batch culture. Biomass Bioenergy 3:41–48

Brandberg T, Franzén CJ, Gustafsson L (2004) The fermentationperformance of nine strains of Saccharomyces cerevisiae in batchand fed-batch cultures in dilute-acid wood hydrolysate. J BiosciBioeng 98:122–125

Burcham PC, Kaminskas LM, Fontaine FR, Petersen DR, Pyke SM(2002) Aldehyde-sequestering drugs: tools for studying proteindamage by lipid peroxidation products. Toxicol 181–182:229–236

Canettieri EV, Rocha GJdM, de Carvalho JJA, de Almeida e Silva JB(2007) Optimization of acid hydrolysis from the hemicellulosicfraction of Eucalyptus grandis residue using response surfacemethodology. Biores Technol 98:422–428

Carmel-Harel O, Storz G (2000) Roles of the glutathione- andthioredoxin-dependent reduction systems in the Escherichia coliand Saccharomyces cerevisiae responses to oxidative stress.Annu Rev Microbiol 54:439–461

Chen S-F, Mowery RA, Chambliss CK, van Walsum GP (2007)Pseudo reaction kinetics of organic degradation products indilute-acid-catalyzed corn stover pretreatment hydrolysates. Bio-technol Bioeng 98:1135–1145

Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD,Mochly-Rosen D (2008) Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Sci 321:1493–1495

Delgenes JP, Moletta R, Navarro JM (1996) Effects of lignocellulosedegradation products on ethanol fermentations of glucose andxylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichiastipitis, and Candida shehatae. Enz Microb Technol 19:220–225

Ezeji T, Qureshi N, Blaschek HP (2007) Butanol production fromagricultural residues: impact of degradation products on Clos-tridium beijerinckii growth and butanol fermentation. BiotechnolBioeng 97:1460–1469

García-Aparicio M, Ballesteros I, González A, Oliva J, Ballesteros M,Negro M (2006) Effect of inhibitors released during steam-explosion pretreatment of barley straw on enzymatic hydrolysis.Appl Biochem Biotechnol 129:278–288

Gibson BR, Lawrence SJ, Boulton CA, Box WG, Graham NS,Linforth RST, Smart KA (2008) The oxidative stress response ofa lager brewing yeast strain during industrial propagation andfermentation. FEMS Yeast Res 8:574–585

Gorsich S, Dien B, Nichols N, Slininger P, Liu Z, Skory C (2006a)Tolerance to furfural-induced stress is associated with pentosephosphate pathway genes ZWF1, GND1, RPE1, and TKL1 inSaccharomyces cerevisiae. Appl Microbiol Biotechnol 71:339–349

Gorsich SW, Slininger PJ, McCaffery JM (2006b) The fermentationinhibitor furfural causes cellular damage to Saccharomycescerevisiae. Biotechnology for Fuels And Chemicals SymposiumProceedings Paper No. 4–17

Grant CM, Collinson LP, Roe J-H, Dawes IW (1996) Yeastglutathione reductase is required for protection against oxidativestress and is a target gene for yAP-1 transcriptional regulation.Mol Microbiol 21:171–179

Appl Microbiol Biotechnol (2009) 82:625–638 635

Gupta GD, Misra A, Agarwal DK (1991) Inhalation toxicity offurfural vapours: an assessment of biochemical response in ratlungs. J Appl Toxicol 11:343–347

Gutiérrez T, Ingram LO, Preston JF (2006) Purification andcharacterization of a furfural reductase (FFR) from Escherichiacoli strain LYO1-An enzyme important in the detoxification offurfural during ethanol production. J Biotechnol 121:154–164

Hadi SM, Shahabuddin, Rehman A (1989) Specificity of theinteraction of furfural with DNA. Mutat Res 225:101–106

Heer D, Sauer U (2008) Identification of furfural as a key toxin inlignocellulosic hydrolysates and evolution of a tolerant yeaststrain. Microb Biotechnol 1:497–506

Heeren G, Jarolim S, Laun P, Rinnerthaler M, Stolze K, Perrone GG,Kohlwein SD, Nohl H, Dawes IW, Breitenbach M (2004) Therole of respiration, reactive oxygen species and oxidative stress inmother cell-specific ageing of yeast strains defective in the RASsignalling pathway. FEMS Yeast Res 5:157–167

Huang H-J, Ramaswamy S, Tschirner UW, Ramarao BV (2008) Areview of separation technologies in current and future biorefi-neries. Sep Pur Technol 62:1–21

Janzowski C, Glaab V, Samimi E, Schlatter J, Eisenbrand G (2000) 5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-dam-aging potential and reactivity towards cellular glutathione. FoodChem Toxicol 38:801–809

Jeppsson M, Träff K, Johansson B, Hahn-Hägerdal B, Gorwa-Grauslund MF (2003) Effect of enhanced xylose reductaseactivity on xylose consumption and product distribution inxylose-fermenting recombinant Saccharomyces cerevisiae.FEMS Yeast Res 3:167–175

Kanner J, Harel S, Fishbein Y, Shalom P (1981) Furfural accumulation instored orange juice concentrates. J Agric Food Chem 29:948–949

Kelly C, Jones O, Barnhart C, Lajoie C (2008) Effect of furfural,vanillin and syringaldehyde on Candida guilliermondii growthand xylitol biosynthesis. Appl Biochem Biotechnol 148:97–108

Khan QA, Hadi SM (1993) Effect of furfural on plasmid DNA.Biochem Mol Biol Int 29:1153–1160

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

Kuster BFM (1990) 5-Hydroxymethylfurfural (HMF). A reviewfocussing on its manufacture. Starch Stärke 42:314–321

Laadan B, Almeida JR, Radstrom P, Hahn-Hägerdal B, Gorwa-Grauslund MF (2008) Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Sac-charomyces cerevisiae. Yeast 25:191–198

Landolfo S, Politi H, Angelozzi D, Mannazzu I (2008) ROSaccumulation and oxidative damage to cell structures in Saccha-romyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochim Biophys Acta 1780:892–898

Larroy C, Fernandez MR, Gonzalez E, Pares X, Biosca JA (2002a)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

Larroy C, Pares X, Biosca JA (2002b) Characterization of aSaccharomyces cerevisiae NADP(H)-dependent alcohol dehy-drogenase (ADHVII), a member of the cinnamyl alcoholdehydrogenase family. Eur J Biochem 269:5738–5745

Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K,Zacchi G, Nilvebrant N-O (1999) The generation of fermentationinhibitors during dilute acid hydrolysis of softwood. EnzMicrobial Technol 24:151–159

Lecomte J, Finiels A, Moreau C (1999) A new selective route to 5-hydroxymethylfurfural from furfural and furfural derivatives overmicroporous solid acidic catalysts. Ind Crop Prod 9:235–241

Lee YC, Shlyankevich M, Jeong HK, Douglas JS, Surh YJ (1995)Bioactivation of 5-hydroxymethyl-2-furaldehyde to an electro-philic and mutagenic allylic sulfuric acid ester. Biochem BiophysRes Commun 209:996–1002

Lee Y, Iyer P, Torget R (1999) Dilute-acid hydrolysis of lignocellu-losic biomass. In: Scheper T (ed) Recent progress in bioconver-sion of lignocellulosics. Springer, Berlin, pp 93–115

Linden T, Peetre J, Hahn-Hägerdal B (1992) Isolation and character-ization of acetic acid-tolerant galactose-fermenting strains ofSaccharomyces cerevisiae from a spent sulfite liquor fermenta-tion plant. Appl Environ Microbiol 58:1661–1669

Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, GorsichSW (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 Z, Slininger P, Gorsich S (2005) Enhanced biotransformation offurfural and hydroxymethylfurfural by newly developed etha-nologenic yeast strains. Appl Biochem Biotechnol 121:451–460

Liu ZL, Moon J, Andersh JB, Slininger PJ, Weber S (2008) Multiplegene-mediated NAD(P)H-dependent aldehyde reduction is amechanism of in situ detoxification of furfural and 5-hydrox-ymethylfurfural by Saccharomyces cerevisiae. Appl MicrobiolBiotechnol 81:743–753

Lopez MJ, Nichols NN, Dien BS, Moreno J, Bothast RJ (2004) Isolationof microorganisms for biological detoxification of lignocellulosichydrolysates. Appl Microbiol Biotechnol 64:125–131

Mains GH, Laforge FB (1924) Furfural from corncobs. Ind Eng Chem16:356–359

Martin C, Jonsson LJ (2003) Comparison of the resistance ofindustrial and laboratory strains of Saccharomyces and Zygosac-charomyces to lignocellulose-derived fermentation inhibitors.Enz Microbial Technol 32:386–396

Martín C, Galbe M, Wahlbom CF, Hahn-Hägerdal B, Jönsson LJ(2002) Ethanol production from enzymatic hydrolysates ofsugarcane bagasse using recombinant xylose-utilising Saccharo-myces cerevisiae. Enzyme and Microb Technol 31:274–282

Martín C, Marcet M, Almazán O, Jönsson LJ (2007) Adaptation of arecombinant xylose-utilizing Saccharomyces cerevisiae strain toa sugarcane bagasse hydrolysate with high content of fermenta-tion inhibitors. Biores Technol 98:1767–1773

Michail K, Matzi V, Maier A, Herwig R, Greilberger J, Juan H, KunertO, Wintersteiger R (2007) Hydroxymethylfurfural: an enemy or afriendly xenobiotic? A bioanalytical approach. Anal BioanalChem 387:2801–2814

Modig T, Lidén G, Taherzadeh MJ (2002) Inhibition effects of furfuralon alcohol dehydrogenase, aldehyde dehydrogenase and pyruvatedehydrogenase. Biochem J 363:769–776

Modig T, Almeida JR, Gorwa-Grauslund MF, Lidén G (2008)Variability of the response of Saccharomyces cerevisiaestrains to lignocellulose hydrolysate. Biotechnol Bioeng100:423–429

Moreau C, Belgacem MN, Gandini A (2004) Recent catalyticadvances in the chemistry of substituted furans from carbohy-drates and in the ensuing polymers. Top Catal 27:11–30

Mosier NS, Ladisch CM, Ladisch MR (2002) Characterization of acidcatalytic domains for cellulose hydrolysis and glucose degrada-tion. Biotechnol Bioeng 79:610–618

Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M,Ladisch M (2005) Features of promising technologies forpretreatment of lignocellulosic biomass. Biores Technol96:673–686

Mussatto SI, Roberto IC (2004) Alternatives for detoxification ofdiluted-acid lignocellulosic hydrolyzates for use in fermentativeprocesses: a review. Biores Technol 93:1–10

636 Appl Microbiol Biotechnol (2009) 82:625–638

Navarro AR (1994) Effects of furfural on ethanol fermentation bySaccharomyces cerevisiae: mathematical models. Curr Microbiol29:87–90

Neureiter M, Danner H, Thomasser C, Saidi B, Braun R (2002)Dilute-acid hydrolysis of sugarcane bagasse at varying con-ditions. Appl Biochem Biotechnol 98–100:49–58

Nguyen Q, Tucker M, Keller F, Beaty D, Connors K, Eddy F (1999)Dilute acid hydrolysis of softwoods. Appl Biochem Biotechnol77:133–142

Nguyen Q, Tucker M, Keller F, Eddy F (2000) Two-stage dilute-acidpretreatment of softwoods. Appl Biochem Biotechnol 84–86:561–576

Nichols NN, Sharma LN, Mowery RA, Chambliss CK, van WalsumGP, Dien BS, Iten LB (2008) Fungal metabolism of fermentationinhibitors present in corn stover dilute acid hydrolysate. EnzMicrob Technol 42:624–630

Nigam JN (2001) Ethanol production from wheat straw hemicellulosehydrolysate by Pichia stipitis. J Biotechnol 87:17–27

Nilsson A, Taherzadeh MJ, Lidén G (2001) Use of dynamic stepresponse for control of fed-batch conversion of lignocellulosichydrolyzates to ethanol. J Biotechnol 89:41–53

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

Nilvebrant N-O, Reimann A, Larsson S, Jönsson L (2001) Detoxifi-cation of lignocellulose hydrolysates with ion-exchange resins.Appl Biochem Biotechnol 91–93:35–49

Öhgren K, Galbe M, Zacchi G (2005) Optimization of steampretreatment of SO2-impregnated corn stover for fuel ethanolproduction. Appl Biochem Biotechnol 124:1055–1067

Öhgren K, Rudolf A, Galbe M, Zacchi G (2006) Fuel ethanolproduction from steam-pretreated corn stover using SSF at higherdry matter content. Biomass Bioenergy 30:863–869

Ohta S, Ohsawa I (2006) Dysfunction of mitochondria and oxidativestress in the pathogenesis of Alzheimer’s disease: on defects inthe cytochrome c oxidase complex and aldehyde detoxification. JAlzheimers Dis 9:155–166

Ohta S, Ohsawa I, Kamino K, Ando F, Shimokata H (2004)Mitochondrial ALDH2 deficiency as an oxidative stress. Ann NY Acad Sci 1011:36–44

Oliva J, Sáez F, Ballesteros I, González A, Negro M, Manzanares P,Ballesteros M (2003) Effect of lignocellulosic degradationcompounds from steam explosion pretreatment on ethanolfermentation by thermotolerant yeast Kluyveromyces marxianus.Appl Biochem Biotechnol 105:141–153

Olofsson K, Rudolf A, Lidén G (2008) Designing simultaneoussaccharification and fermentation for improved xylose conver-sion by a recombinant strain of Saccharomyces cerevisiae. JBiotechnol 134:112–120

Palmqvist E, Almeida JS, Hahn-Hägerdal B (1999) Influence offurfural on anaerobic glycolytic kinetics of Saccharomycescerevisiae in batch culture. Biotechnol Bioeng 62:447–454

Perrone GG, Tan SX, Dawes IW (2008) Reactive oxygen species andyeast apoptosis. Biochim Biophys Acta 1783:1354–1368

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

Pfeifer PA, Bonn G, Bobleter O (1984) Influence of biomass degradationproducts on the fermentation of glucose to ethanol by Saccharomy-ces carlsbergensis W 34. Biotechnol Lett 6:541–546

Quesada Granados J, VillalonMir M, Lopez Garcia-Serrana H, LopezMartinez MC (1996) Influence of aging factors on the furanicaldehyde contents of matured brandies: aging markers. J AgricFood Chem 44:1378–1381

Ramirez-Jimenez A, Guerra-Hernandez E, Garcia-Villanova B(2000) Browning indicators in bread. J Agric Food Chem48:4176–4181

Ranatunga T, Jervis J, Helm R, McMillan J, Hatzis C (1997) Identificationof inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation. Appl Biochem Biotechnol 67:185–198

Ranganathan S, Douglas GM, Narendra NB (1985) Kinetic studies ofwheat straw hydrolysis using sulphuric acid. Can J Chem Eng63:840–844

Reynolds SH, Stowers SJ, Patterson RM, Maronpot RR, AaronsonSA, Anderson MW (1987) Activated oncogenes in B6C3F1mouse liver tumors: implications for risk assessment. Sci237:1309–1316

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

Roberto IC, Mussatto SI, Rodrigues RCLB (2003) Dilute-acidhydrolysis for optimization of xylose recovery from rice strawin a semi-pilot reactor. Ind Crop Prod 17:171–176

Rodriguez-Arnaiz R, Romas Morales P, Zimmering S (1992)Evaluation in Drosophila melanogaster of the mutagenicpotential of furfural in the mei-9a test for chromosome loss ingerm-line cells and the wing spot test for mutational activity insomatic cells. Mutat Res 280:75–80

Rudolf A, Galbe M, Lidén G (2004) Controlled fed-batch fermenta-tions of dilute-acid hydrolysate in pilot development unit scale.Appl Biochem Biotechnol 114:601–617

Rudolf A, Alkasrawi M, Zacchi G, Lidén G (2005) A comparisonbetween batch and fed-batch simultaneous saccharification andfermentation of steam pretreated spruce. Enz Microb Technol37:195–204

Rudolf A, Baudel H, Zacchi G, Hahn-Hägerdal B, Lidén G (2008)Simultaneous saccharification and fermentation of steam-pre-treated bagasse using Saccharomyces cerevisiae TMB3400 andPichia stipitis CBS6054. Biotechnol Bioeng 99:783–790

Saha B (2003) Hemicellulose bioconversion. J Ind MicrobiolBiotechnol 30:279–291

Sanchez B, Bautista J (1988) Effects of furfural and 5-hydroxyme-thylfurfural on the fermentation of Saccharomyces cerevisiae andbiomass production from Candida guilliermondii. Enz MicrobTechnol 10:315–318

Sánchez ÓJ, Cardona CA (2008) Trends in biotechnological produc-tion of fuel ethanol from different feedstocks. Biores Technol99:5270–5295

Sangarunlert W, Piumsomboon P, Ngamprasertsith S (2007) Furfuralproduction by acid hydrolysis and supercritical carbon dioxideextraction from rice husk. Kor J Chem Eng 24:936–941

Sarvari Horvath I, Franzen CJ, Taherzadeh MJ, Niklasson C, Lidén G(2003) Effects of furfural on the respiratory metabolism ofSaccharomyces cerevisiae in glucose-limited chemostats. ApplEnviron Microbiol 69:4076–4086

Sigler K, Chaloupka J, Brozmanová J, Stadler N, Höfer M (1999)Oxidative stress in microorganisms—I. FoliaMicrobiol 44:587–624

Söderström J, Pilcher L, Galbe M, Zacchi G (2003) Two-step steampretreatment of softwood by dilute H2SO4 impregnation forethanol production. Biomass Bioenergy 24:475–486

Szengyel Z, Zacchi G (2000) Effect of acetic acid and furfural oncellulase production of Trichoderma reesei RUT C30. ApplBiochem Biotechnol 89:31–42

Taherzadeh MJ, Eklund R, Gustafsson L, Niklasson C, Lidén G(1997) Characterization and fermentation of dilute-acid hydro-lyzates from wood. Ind Eng Chem Res 36:4659–4665

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

Appl Microbiol Biotechnol (2009) 82:625–638 637

Talebnia F, Niklasson C, Taherzadeh MJ (2005) Ethanol productionfrom glucose and dilute-acid hydrolyzates by encapsulated S.cerevisiae. Biotechnol Bioeng 90:345–353

Tanel A, Averill-Bates DA (2007) Activation of the death receptorpathway of apoptosis by the aldehyde acrolein. Free Radic BiolMed 42:798–810

Tomás-Pejó E, Oliva JM, Ballesteros M, Olsson L (2008) Comparisonof SHF and SSF processes from steam-exploded wheat straw forethanol production by xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng100:1122–1131

Uchida K (2000) Role of reactive aldehyde in cardiovascular diseases.Free Radic Biol Med 28:1685–1696

Villa GP, Bartroli R, Lopez R, Guerra M, Enrique M, Penas M,Rodriquez E, Redondo D, Iglesias I, Diaz M (1992) Microbialtransformation of furfural to furfuryl alcohol by Saccharomyces-cerevisiae. Acta Biotechnol 12:509–512

von Sivers M, Zacchi G, Olsson L, Hahn-Hägerdal B (1994) Costanalysis of ethanol production from willow using recombinantEscherichia coli. Biotechnol Prog 10:555–560

Wahlbom CF, Hahn-Hägerdal B (2002) Furfural, 5-hydroxymethylfurfural, and acetoin act as external electron acceptors during

anaerobic fermentation of xylose in recombinant Saccharomycescerevisiae. Biotechnol Bioeng 78:172–178

Watson NE, Prior BA, Lategan PM, Lussi M (1984) Factors inacid treated bagasse inhibiting ethanol production from D-xylose by Pachysolen tannophilus. Enz Microb Technol 6:451–456

Wiemken A (1990) Trehalose in yeast, stress protectant rather thanreserve carbohydrate. Antonie Leeuwenhoek 58:209–217

Wiseman A (2005) Avoidance of oxidative-stress perturbation in yeastbioprocesses by proteomic and genomic biostrategies? Lett ApplMicrobiol 40:37–43

Zaldivar J, Martinez A, Ingram LO (1999) Effect of selectedaldehydes on the growth and fermentation of ethanologenicEscherichia coli. Biotechnol Bioeng 65:24–33

Zdzienicka M, Tudek B, Zielenska M, Szymczyk T (1978) Mutagenicactivity of furfural in Salmonella typhimurium TA100. Mutat Res58:205–209

Zverlov V, Berezina O, Velikodvorskaya G, Schwarz W (2006)Bacterial acetone and butanol production by industrialfermentation in the Soviet Union: use of hydrolyzedagricultural waste for biorefinery. Appl Microbiol Biotechnol71:587–597

638 Appl Microbiol Biotechnol (2009) 82:625–638