Enzyme and Microbial Technology 39 (2006) 11081112

Effect of furfural on carbon metabolism key enzymes of

lactose-assimilating yeasts
Ts. Hristozova a , A. Angelov b, , B. Tzvetkova a , D. Paskaleva a , V. Gotcheva b ,
S. Gargova b , K. Pavlova a
a
b

Institute of Microbiology, Bulgarian Academy of Sciences, Soa 1113, 26 Akad. Bonchev Street, Bulgaria
Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria
Received 9 February 2006; accepted 13 February 2006

Abstract
The metabolic response of lactose-assimilating yeasts to changes of cultivation conditions after addition of furfural into the medium was explored.
Two yeast strains were studiedCandida blankii 35 with an oxidative metabolism, and C. pseudotropicalis 11 with a fermentative metabolism.
Strains were cultivated in a chemostat under carbon limitation and dilution rates D = 0.1 and 0.25 h1 .
During the transition period after a shock treatment with 0.04% furfural, changes of the following enzymes of the carbon metabolism were studied:
hexokinase (HK), glucosephosphate isomerase (GPI) and glyceraldehyde-3P-dehydrogenase (GAPD) of the glycolysis, glucose-6P-dehydrogenase
(GPD) from the pentosophosphate cycle, as well as -galactosidase.
Results show that during cultivation of C. pseudotropicalis 11 at D = 0.1 h1 , furfural concentration of 0.04% caused increase of the glycolytic
enzyme activities, while the level of GPD activity remained rather constant. At the higher dilution rate D = 0.25 h1 the effect of furfural brought to
a significant decrease of all enzyme activities studied, except -galactosidase, for which activity registered at both dilution rates was significantly
higher.
During cultivation of C. blankii 35 at D = 0.1 h1 , furfural brought to lower activities of GPI and GPD, while HK activity was not affected by
the inhibitor, and that of GAPD increased. The furfural shock at D = 0.25 h1 leads to increased activity of the three glycolytic enzymes, while
that of GPD was 33.6% lower as compared to control. Addition of furfural into the cultivation medium of the oxidative strain caused decrease of
-galactosidase activity.
2006 Elsevier Inc. All rights reserved.
Keywords: Candida blankii; Candida pseudotropicalis; Furfural; Glucosephosphate isomerase; Glyceraldehyde-3P-dehydrogenase; -Galactosidase

1. Introduction
Acid wood hydrolysates are strongly inhibiting to yeast and
other fermenting microorganisms. The inhibitors are formed
mainly in the hydrolysis process [9,13]. Furfural, formaldehyde, formic acid, by-products in wood acid hydrolysate
are present in relatively high concentrations. Furfural and 5hydroxymethylfurfural are formed as breakdown products of
pentoses and hexoses, respectively [2]. Several studies have
reported toxicity of furfural on fermentation and growth of yeasts
[2,3,8,17,19,20,2224]. Fireoved and Mutharasan [6] stated that
the presence of furfural did not affect the energetics of Saccha-

Corresponding author. Tel.: +359 23 603 608.

E-mail address: angelov@uft-bio.com (A. Angelov).

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2006.02.015

romyces cerevisiae in chemostat experiments. However, furfural

did inhibit glucose uptake rate at a high dilution rate whereas
the uptake was stimulated at a low dilution rate. In contrast to
these results, furfural has been reported by some authors to be
toxic in batch fermentation processes [1,24,25].
Some enzymes have been reported to be susceptible to furfural [16,29]. Results from our previous studies show the inhibiting effect of furfural on the growth and metabolism of Candida
diddensii and C. tropicalis [14,26]. Nirupama et al. [17] investigated the inhibitory effect of furfural on different glycolytic
enzymes, such as hexokinase, phosphofructokinase, triosephosphate dehydrogenase, aldolase and alcohol dehydrogenase. They
reported that the glycolitic dehydrogenases were most sensitive
to furfural.
The possibility to cultivate yeasts in mixed substrates containing wood hydrolysates and whey requires studying of the growth

Ts. Hristozova et al. / Enzyme and Microbial Technology 39 (2006) 11081112

and productivity of yeast populations. Yeast cultivation in such

mixed substrates can decrease the content of inhibitors in the
medium, which could contribute to more effective assimilation
of reducing sugars from the two substrates. It has been found that
strain Candida blankii 35 effectively assimilates whey lactose
[27]. The effect of dilution rate on biomass and protein production by C. blankii 35 and C. pseudotropicalis 11 was studied [27].
Also, our previous results regarding the effect of furfural on the
growth and biosynthesis abilities of the same yeast strains were
reported [28]. However, the effect of furfural on the metabolic
activity of the whey assimilating strains has not been studied.
The present work presents a study on the effect of furfural on key enzymes from carbon metabolism: -galactosidase,
the glycolytic enzymes hexokinase, glucosephosphate isomerase, glyceraldehyde-3P-dehydrogenase, as well as glucose6-phosphate dehydrogenase from the pentosophosphate cycle of
the lactose-assimilating yeasts Candida blankii 35 and C. pseudotropicalis 11.
2. Materials and methods
2.1. Microorganisms and media
All experiments were performed with the lactose-assimilating yeast strains
Candida blankii 35 and C. pseudotropicalis 11 from the collection of the Institute
of Microbiology, BAS (registered at the National Bank for Industrial Microorganisms and Cell Cultures as #160 and 161, respectively). The cultures were
maintained on malt agar slopes at 4 C.
Control medium contained (g/dm3 ): lactose10, (NH4 )2 SO4 3.0,
MgSO4 7H2 O0.7, NaCl0.5, CaCl2 (50% w/v)0.2 ml, KH2 PO4 1.0,
K2 HPO4 0.1, yeast extract (Difco)0.2. The medium was sterilized at 120 C
for 30 min. For the purpose of the study, 0.04% (v/v) furfural was added to the
medium.

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by a mechanical disintegrator with glass beads (d = 1.5 mm) at 0 C for 15 min.

Biomass suspension was then centrifuged at 16,000 g for 30 min. Supernatant
obtained was used for determination of the enzyme activities.
2.3.4. Enzyme assays
Enzyme activities were assayed immediately after the preparation of the
extracts. Spectrophotometric assays were carried out with a model 100-60 spectrophotometer (Hitachi) at 340 nm for HK, GPI, GAPD and GPD and at 420 nm
for -galactosidase and 30 C. The reaction velocities were proportional to the
amount of enzyme added. The assay mixtures for the individual enzymes are
described below.
2.3.5. -Galactosidase (EC 3.2.1.23)
Tubes containing 3.5 ml 0.02N potassium phosphate (pH 7.25) and 0.5 ml
0.01 M nitrophenyl--d-galactoside were incubated in water bath at 30 C. 1 ml
of the enzyme preparation was added to start the process. Reaction was stopped
after 10 min by the addition of 2 ml Na2 CO3 . The o-nitrophenol released was
determined via spectrophotomer [7].
2.3.6. Hexokinase (EC 2.7.1.1)
The reaction mixture contained: 0.2 ml of each ATPMg mixture (0.075 M
ATP and 0.04 M MgCl2 ), the buffer mixture (0.1 M histidine, 0.1 M Tris, 0.01 M
EDTA and 0.01 M MgCl2 ) and 0.01 M glucose and 0.3 ml of water. The reaction
started with 0.1 ml of diluted enzymes [4].
2.3.7. Glucosephosphate isomerase (EC 5.3.1.9)
The reaction mixture contained: 1 ml 0.1 M Tris buffer (pH 9.0), 0.2 ml TMN
(0.001 M), 0.2 ml 0.1 M MgCl2 , 0.1 ml glucose 6-phosphate dehydrogenase,
0.1 ml 0.1 M glucose 6-phosphate and 0.1 ml of enzyme [21].
2.3.8. Glyceraldehyde-3P-dehydrogenase (EC 1.2.1.12)
The reaction mixture contained: 2.6 ml pyrophosphatecysteine buffer (pH
8.5), 0.1 ml DPN (7.6 103 M), 0.1 ml enzyme. The initial absorption of the
reaction mixture was observed, and the component, 0.2 ml of 1:1 mixture of
glyceraldehydes-3-phosphate (7.6 103 M) and sodium arsenate (0.17 M) was
added [12].

2.2. Cultivation
The fermenter with a work volume of 1.5 dm3 was inoculated with 100 ml
20-h culture. Cultivation was carried out in a chemostat under carbon limitation
and at two dilution rates 0.1 and 0.25 h1 . Experiments were performed at
38 C, aeration 1.5 dm3 /min dm3 and stirring rate of 600 rpm. Dissolved oxygen
tension was above 40% air saturation at all dilution rates. pH was automatically
controlled at 4.04.2 by the addition of 2 M KOH.
Furfural was added by impulse introduction, so that its concentration in the
fermenter to be 0.04% (v/v) at a dilution rate of 0.1 h1 or 0.25 h1 . Fresh
medium with the same furfural concentration was fed into the fermenter to
maintain a certain dilution rate. Parameter observation commenced immediately
after the addition of furfural and continued up to the 36th hour of cultivation,
e.g. to the establishment of a new steady state.
Cell dry weight was determined in duplicate of 10 ml samples, which were
centrifuged, washed with distilled water, and dried for 24 h at 105 C.

2.3. Analyses
2.3.1. Residual lactose
Residual lactose in the supernatant was measured by the phenolsulfuric acid
method [5].
2.3.2. Protein determination
Protein was determined by the Lowry method with bovine serum albumin
as the standard [15].
2.3.3. Preparation of cell extracts
Samples of cultures were harvested after centrifugation and triple washing,
biomass was re-suspended in Tris buffer (0.1 M, pH 7.5). Cells were destructed

2.3.9. Gucose-6-phosphate dehydrogenase (EC 1.1.1.49)

The assay mixture contained: Tris hydrochloride buffer (pH 8.0) (50 mM),
MgCl2 (5 mM), and NADP+ (0.4 mM). The reaction was started with 5 mM
glucose 6-phosphate [11].
One unit of enzyme activity is defined as the amount of enzyme catalyzing
the conversion of 1 mol of substrates per min at 30 C. Activities reported
are the average of at least six to eight (manual assays) repeated assays on each
sample extract.
Results represent data of three independent replicates of each experiment
subjected to statistical analysis. Standard deviations are presented in all figures.

3. Results and discussion

Our previous studies showed the effect of dilution rate (from
0.1 to 0.5 h1 ) on the growth of yeast strains C. blankii 35 and
C. pseudotropicalis 11 [27]. Based on the results, two dilution
rates0.1 and 0.25 h1 were selected for the present study, as
clear differences were observed in the physiological response
under those rates. Also, the effect of furfural in concentrations
0.02, 0.04 and 0.08% on the growth and biosynthesis abilities
of these lactose-assimilating strains was explored [28]. For our
further work, furfural concentration of 0.04% was selected as
appropriate for studying the effect of the inhibitor on the enzyme
activities monitored.
Results show that after treatment with 0.04% furfural during
cultivation of C. blankii 35 in a chemostat at a dilution rate of

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Ts. Hristozova et al. / Enzyme and Microbial Technology 39 (2006) 11081112

Fig. 3. Effect of 0.04% furfural on residual lactose, -galactosidase and hexokinase (HK) activities of Candida blankii 35 at D = 0.25 h1 .
Fig. 1. Effect of 0.04% furfural on residual lactose, -galactosidase and hexokinase (HK) activities of Candida blankii 35 at D = 0.1 h1 .

0.1 h1 , the rate of lactose assimilation in the first few hours

strongly decreased and residual lactose content increased, while
after the 12th hour the process is gradually regulated (Fig. 1). A
6% biomass decrease was registered and the substrate utilization
rate at these conditions after shock with 0.04% furfural increased
by 17% [28]. This showed that the presence of furfural in the
medium impeded processes within yeast cells. The strain reacted
quickly and the activity of -galactosidase, which is responsible
for lactose assimilation decreased (Fig. 1). The enzyme activity
remained stable until the 4th hour after the furfural shock. When
the new steady state was reached, -galactosidase activity was
three times lower compared to control.
For this strain, furfural had a different effect on the glycolytic enzymes studied. At these cultivation conditions, hexokinase activity was not affected by furfural; glucosephosphate isomerase activity decreased 3.7 times, and the activity of glyceraldehyde-3P-dehydrogenase increased almost three
times (Fig. 2). After the addition of furfural, glucose-6Pdehydrogenase activity started to decrease and on the 4th hour,
it is about 3.8 times lower, but when the new steady state was
established, the culture overcame the inhibiting effect of furfural and the activity of this enzyme almost reached control
levels.

Fig. 2. Effect of 0.04% furfural on glucosephosphate isomerase (GPI),

glyceraldehydes-3P-dehydrogenase (GAPD) and glucose-6P-dehydrogenase
(GPD) activities of Candida blankii 35 at D = 0.1 h1 .

At the higher dilution rate (0.25 h1 ) furfural also influenced

the rate of lactose assimilation and residual lactose content
increased (Fig. 3). Under these conditions, the decrease tendency
of -galactosidase activity after addition of furfural remained
unchanged. At this flow rate, hexokinase was not affected by the
inhibitor, and the activities of glucosephosphate isomerase and
glyceraldehyde-3P-dehydrogenase decrease 1.9 and 1.7 times,
respectively (Fig. 4).
Experiments show that during cultivation of C. blankii 35
at dilution rate of 0.25 h1 , addition of 0.04% furfural did not
influence the activity of glucose-6P-dehydrogenase (Fig. 4).
Some authors reported that furfural was inhibiting for hexokinase and glyceraldehyde-3P-dehydrogenase of Saccharomyces
cerevisiae [1719]. -Galactosidase activity and glycolysis of
C. blankii 35 was affected by addition of furfural, and the
effect occurred rapidly. According to Kang and Okada [10] the
aldehyde group of furfural may be reduced by alcohol dehydrogenase. The inhibition effect shown in our results is probably
caused by direct inhibition of other enzymes, e.g. glucosephosphate isomerase and glyceraldehydes-3P-dehydrogenase, but
may also be the result of cellular regulation in response to a
decreased specific growth rate. Similar opinion is expressed by
Taherzadeh et al. [25].
During cultivation of the fermenting strain C. pseudotropicalis 11 at D = 0.1 and 0.25 h1 , a significant increase of galactosidase activity was observed after addition of 0.04% fur-

Fig. 4. Effect of 0.04% furfural on glucosephosphate isomerase (PGI),

glyceraldehydes-3P-dehydrogenase (GAPD) and glucose-6P-dehydrogenase
(GPD) activities of Candida blankii 35 at D = 0.25 h1 .

Ts. Hristozova et al. / Enzyme and Microbial Technology 39 (2006) 11081112

Fig. 5. Effect of 0.04% furfural on residual lactose, -galactosidase and hexokinase (HK) activities of Candida pseudotropicalis 11 at D = 0.1 h1 .

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Fig. 7. Effect of 0.04% furfural on residual lactose, -galactosidase and hexokinase (HK) activities of C. pseudotropicalis 11 at D = 0.25 h1 .

fural (Figs. 5 and 7). When dilution rate of 0.1 h1 was applied,
in the first 3 h after the shock the enzyme activity kept decreasing, but after that period went up and when the new steady state
was reached, the enzyme activity was about 3.8 times higher
than control (Fig. 5).
Activities of the glycolytic enzymes also increased as a result
of the furfural addition. For hexokinase the increase registered
was 4.6 times, for glucosephosphate isomerase2.3 times, and
for glyceraldehyde-3P-dehydrogenase1.4 times. Activity of
glucose-6P-dehydrogenase during the transition state after the
furfural shock varied, reaching a highest value on the 24th hour
(Fig. 6). However, after culture growth was stabilized and a
steady state was reached, the enzyme activity is within the range
of control.
The higher growth rate of the culture in the presence of furfural lead again to increased -galactosidase activityabout
1.7 times (Fig. 7). Furfural suppressed the glycolytic enzymes
activity of C. pseudotropicalis 11: for hexokinase4.4 times
decrease, for glucosephosphate isomerase3.8 times, and for
glyceraldehyde-3P-dehydrogenase2.9 times (Figs. 7 and 8).
Furfural had an inhibitory effect on the enzyme from the
penthosephosphate cycle as well. Activity of glucose-6Pdehydrogenase decreased about two times. Modig et al. [16]
observed inhibiting effect of furfural on the metabolism of Saccharomyces cerevisiae, and in particular on the aldehyde dehydrogenase, alcohol dehydrogenase and pyruvate dehydrogenase.

Taherzadeh et al. [25] established that the reduction of furfural to

furfuryl alcohol was vital for overcoming the toxic effects of furfural, since growth, although at a reduced specific growth rate,
is possible in the presence of both furfuryl alcohol and furoic
acid. The strong demand for NADH in the reduction of furfural
may leave insufficient NADH for terminal respiration, and thus
resulting in insufficient ATP generation to sustain growth. The
strong inhibition effects of furfural on the growth and glycolysis
of C. pseudotropicalis 11 at higher dilution rate can be explained
partly by this NADH demand, and partly by direct inhibition at
the enzymatic level.
The results show that the growth rate is very important for
the inhibiting effect of furfural. At the lower growth rate, the
fermenting strain C. pseudotropicalis 11 overcame the influence of furfural. Activities of -galactosidase, hexokinase,
glucosephosphate isomerase and glucose-6P-dehydrogenase
increased, which proved that furfural in concentration 0.04% did
not have an inhibitory effect. However, when the growth rate
was higher (0.25 h1 ), furfural in the same concentration was
inhibitory to the enzymes from the glycolysis and the penthosephosphate cycle.
The oxidative strain C. blankii 35 was more sensitive
towards furfural and at both dilution rates activities of galactosidase and glucosephosphate isomerase were inhibited.
Hexokinase was not affected by furfural, as well as glucose-6Pdehydrogenase at a dilution rate of 0.25 h1 .

Fig. 6. Effect of 0.04% furfural on glucosephosphate isomerase (GPI),

glyceraldehydes-3P-dehydrogenrase (GAPD) and glucose-6P-dehydrogenase
(GPD) activities of C. pseudotropicalis 11 at D = 0.1 h1 .

Fig. 8. Effect of 0.04% furfural on glucosephosphate isomerase (GPI),

glyceraldehydes-3P-dehydrogenase (GAPD) and glucose-6P-dehydrogenase
(GPD) activities of C. pseudotropicalis 11 at D = 0.25 h1 .

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Ts. Hristozova et al. / Enzyme and Microbial Technology 39 (2006) 11081112

4. Conclusions
Strain C. blankii 35 was more susceptible to furfural. At both
growth rates, -galactosidase activity significantly decreased,
while for C. pseudotropicalis 11 after a few hours of inhibition, the enzyme activity increased up to 3.8 times compared to
control.
The effect of furfural was found to be in close relation with
the physiological state of the culture studied, which is predetermined by the different growth rates. Present results show that
the cultures with higher growth rate (0.25 h1 ) are more susceptible to the effect of furfural and the glycolytic enzymes are
significantly inhibited. Similar conclusions are made regarding
the effect of furfural on the production of biomass and protein
[28].
The study showed that glucose-6P-dehydrogenase from the
pentosephosphate cycle of C. blankii 35 was not inhibited by
0.04% furfural, while for C. pseudotropicalis 11 significant
inhibitory effect was observed at growth rate of 0.25 h1 .
Results show that the inhibitory effect of 0.04% furfural on
the enzyme activities explored is related with the growth rate as
well as with the specific properties of each strain.
Acknowledgement
The study was supported by Fund Science of the University
of Food Technologies, Plovdiv, Bulgaria.
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