FEMS Yeast Research 4 (2003) 157^164
www.fems-microbiology.org
Xylose and cellobiose fermentation to ethanol by the thermotolerant
methylotrophic yeast Hansenula polymorpha
Olena B. Ryabova a , Oksana M. Chmil a , Andrii A. Sibirny
a
a;b;
Institute of Cell Biology, NAS of Ukraine, Drahomanov Str. 14/16, Lviv 79005, Ukraine
b
Institute of Biotechnology, Rzeszow University, Rzeszwo, Poland
Received 3 January 2003; received in revised form 4 April 2003; accepted 7 April 2003
First published online 28 May 2003
Abstract
Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose
to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37‡C, whereas the well-known xylose-fermenting yeast Pichia
stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up
to 45‡C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A
riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with
riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose,
whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.
0 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Fuel ethanol; Xylose fermentation ; Cellobiose fermentation ; High-temperature alcoholic fermentation; Methylotrophic yeast; Ribo£avin
auxotrophy; Hansenula polymorpha
1. Introduction
During the last decades, much attention has been paid
to fuel ethanol production from cheap and renewable raw
materials, such as plant biomass or lignocellulose (see reviews [1^4]). Bottlenecks in the development of a feasible
and economically pro¢table process appeared to be the
absence of (i) an economic and environmentally friendly
process of cellulose and hemicellulose hydrolysis to monosaccharides ; (ii) organisms able to e¡ectively ferment xylose, other pentoses and cellobiose to ethanol. It is thought
that simultaneous sacchari¢cation and fermentation (SSF),
which combines enzymatic hydrolysis of pretreated lignocellulose by cellulases and hemicellulases with fermentation of the produced hexoses and pentoses to ethanol,
would be the most e⁄cient way to convert lignocellulose
to ethanol [5,6]. Sugars liberated during lignocellulose hydrolysis cause strong product inhibition of the hydrolytic
enzymes. In SSF these sugars are continuously converted
* Corresponding author. Tel. : +380 (322) 740363;
Fax : +380 (322) 721648.
E-mail address : [email protected] (A.A. Sibirny).
to ethanol and are not accumulated in the reaction mixture; remarkably, accumulated ethanol activates the hydrolytic enzymes. However, cellulases operate optimally
at high temperatures (around 50‡C and higher), whereas
the most known organisms able to ferment lignocellulose
sugars, and especially pentose-fermenting natural and recombinant yeasts, are mesophilic organisms with optimal
growth and fermentation temperatures around 30‡C
[3,7,8].
Apparently, the most promising organisms for alcoholic
fermentation of lignocellulose sugars are yeasts. These eukaryotic organisms have much bigger cells than bacteria,
which facilitates their separation from the fermentation
broth. Yeast fermentations are resistant to virus infection
and bacterial contamination and often yeasts are more
resistant to ethanol than bacteria [3]. One of the most
critical features which an industrial ethanol producer
from lignocellulose must have is its ability for e¡ective
xylose fermentation, because xylose accounts for up to
30% of lignocellulose sugars [8]. There are only few natural xylose-fermenting yeasts, such as Pichia stipitis, Candida shehatae and Pachysolen tannophilus. Besides, during
recent years, recombinant strains of Saccharomyces cerevisiae have been constructed which are able to ferment, in
1567-1356 / 03 / $22.00 0 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S1567-1356(03)00146-6
FEMSYR 1578 27-10-03
158
O.B. Ryabova et al. / FEMS Yeast Research 4 (2003) 157^164
addition to glucose and other hexoses, also xylose [9^11].
All these yeast strains, however, cannot ferment lignocellulose sugars at high temperatures, which would be compatible with an SSF process.
There are few reports on thermotolerant yeast species,
e.g. Kluyveromyces marxianus, which is able to ferment
glucose at temperatures up to 45‡C [22]. However, as to
our knowledge, there are no data on xylose fermentation
by this species. Our laboratory for many years has studied
di¡erent aspects of methanol metabolism in the methylotrophic yeast Hansenula polymorpha and possesses a large
collection of natural strains and mutants of this species
[14,15,23^27]. Apparently H. polymorpha is the only
known thermotolerant methylotrophic yeast with optimal
growth temperature of 37‡C and maximal growth temperature up to 48‡C [28,29]. This species is able to ferment
glucose to ethanol and possesses active enzymes of xylulose-5-phosphate metabolism, as methanol is assimilated
in the so-called xylulose monophosphate cycle, a modi¢cation of the pentose phosphate pathway [23,30]. Regarding xylose assimilation, strains of H. polymorpha are designated as positive without or with lag period [31]. Though
the same manual [31] ascertains that H. polymorpha is
unable to ferment xylose, conditions for the fermentation
assay used (Durham tubes) are di¡erent from those used
by us (see Section 2).
In this paper, we describe that the thermotolerant methylotrophic yeast H. polymorpha is able to ferment xylose,
glucose, cellobiose and other lignocellulose sugars at elevated temperatures, 37‡C and higher. Ethanol yield and
productivity from glucose and xylose as well as ethanol
tolerance were determined for several H. polymorpha
strains at di¡erent temperatures. The accumulated data
show that H. polymorpha is a promising organism for
fermentation of lignocellulose sugars at high temperatures,
compatible with an SSF process. The preliminary data of
this report have been published before as conference reports [12,13].
H. polymorpha A3, defective in alcohol dehydrogenase,
was isolated as allyl alcohol-resistant strain [16]. Mutants
of the ribo£avin-overproducing yeasts Pichia guilliermondii
rib3, defective in reductase, and Candida famata rib7, defective in ribo£avin synthase were described by us before
[17,18].
Yeasts were cultivated in semisynthetic yeast nitrogen
base (YNB) medium supplemented with the carbon source
(1^6%) and yeast extract (0.05%), without shaking (standing cultures) or with shaking (100 rpm; limited aeration
cultures), at di¡erent temperatures (30^45‡C). Cultivation
of standing cultures occurred in test tubes (15 cm height,
1.5 cm diameter, cotton closures) containing 5 ml of the
medium. Limited aeration cultures were cultivated in conditions similar to those described earlier by T. Je¡ries’
group (see [32]), with the modi¢cation that the medium
did not contain peptone and that (NH4 )2 SO4 (3.4 g l31 )
was used as nitrogen source instead of urea. We found
that use of the Je¡ries’ medium with peptone and urea
signi¢cantly diminished ethanol accumulation by H. polymorpha (data not shown). Cells were grown in 125-ml
Erlenmeyer shake £asks containing 50 ml medium. Cultivation was conducted at 37‡C for H. polymorpha and at
30‡C for P. stipitis (unless speci¢ed otherwise), with shaking at 100 rpm during 20, 40 and 60 h. The starting cell
density after inoculation was 1^2 mg dry weight ml31 .
Media were inoculated from cultures pregrown in the
same media (with 2% glucose or 2% xylose) and at the
same cultivation conditions as used for the main culture
(with the exception that the inoculation culture was grown
in 500-ml £asks containing 100 ml medium with shaking
at 200 rpm until middle-exponential growth phase). Cells
for inoculation were harvested by centrifugation, washed
with water and concentrated to achieve the starting density mentioned above.
Media for cultivation of ribo£avin-de¢cient mutants additionally contained ribo£avin (10^100 Wg ml31 ).
2.2. Assays
2. Materials and methods
2.1. Strains and media
The following wild-type yeast strains were used : P. stipitis CBS 6054 (kindly provided by Dr. T. Je¡ries, Madison, WI, USA), S. cerevisiae (baker’s yeast of Lviv yeast
factory, Ukraine), H. polymorpha ML3, ML8, N95 [14],
CBS 4732, 356 [15], ML6, ML9 (provided by Dr. Y. Kapultsevich, Moscow, Russia). Strains H. polymorpha ML3,
ML6, ML8, ML9 and N95 were isolated in the former
Soviet Union during a search for methylotrophic yeasts
as the potential source of single-cell protein [14]. The ribo£avin-de¢cient mutant H. polymorpha rib7, defective in
ribo£avin synthase, was isolated by us earlier (A.A. Sibirny and B.V. Kshanovska, unpublished). The mutant
Cell biomass was determined turbidimetrically at 600
nm; dry weight was calculated according to the corresponding calibration curves. Ethanol was assayed using
the alcohol oxidase/peroxidase-based enzymatic kit ‘Alcotest’ [19], glucose was determined enzymatically using glucose oxidase/peroxidase [20]. The xylose concentration in
the medium was determined by the Somogyi^Nelson photometric method [21].
The experiments have been repeated two to four times;
the data of typical experiments are presented in the ¢gures.
3. Results
We possess six strains of H. polymorpha isolated in the
former Soviet Union in the seventies of the 20th century
FEMSYR 1578 27-10-03
O.B. Ryabova et al. / FEMS Yeast Research 4 (2003) 157^164
during a search for potential single-cell protein producers
from methanol [14], as well as two collection strains of this
species, used for study of methanol metabolism [15,27].
Preliminary testing of the available strains of H. polymorpha for their ability to ferment xylose and other carbon sources was conducted using standing cultures (see
Section 2). All eight strains were cultivated in tubes for
48 and 96 h without shaking at 37‡C in media containing
di¡erent carbon sources at 2%, and growth and ethanol
accumulation were determined. All tested strains grew
and accumulated ethanol from glucose, mannose, galactose, maltose and xylose (with exception of strain N95,
which grew but did not accumulate su⁄cient amounts of
ethanol in xylose medium). Most strains (with exception of
CBS 4732) accumulated ethanol also from cellobiose (the
last strain practically did not grow on this sugar) (Table
1).
After 96 h, strain CBS 4732 did not show any accumulation of ethanol in media with xylose and galactose
though this alcohol was found after 48 h of cultivation.
Strains KT2 and ML8 did not show any ethanol accumulation in the media with galactose and cellobiose, respectively. Apparently cells consumed ethanol, accumulated in
the ¢rst stage of cultivation. Most strains grew very poorly
in the medium with L-arabinose, with exception of strain
N95 which grew and accumulated small amounts of ethanol in the medium with this sugar (Table 1).
H. polymorpha strains 356, KT2, ML3 and CBS 4732
were studied in more detail. Xylose and glucose fermentation by these strains was compared with those of the type
strain CBS 6054 of one of the best xylose-fermenting
yeasts, P. stipitis [7], in conditions of limited aeration
(shaking at 100 rpm, for detailed conditions of cultivation
159
see Section 2) after 20, 40 and 60 h of incubation. In the
medium with 4% xylose at 30‡C, ethanol accumulation in
P. stipitis was much higher than that in all tested strains of
H. polymorpha (Fig. 1); however, at 37‡C strains of the
methylotrophic yeast showed much higher ethanol accumulation than P. stipitis (Fig. 2). It is interesting to note
that the maximal ethanol accumulation from xylose in the
best H. polymorpha strain 356 was even higher than in
P. stipitis (3 mg ml31 versus 2.3 mg ml31 after 60 h of
cultivation, at the corresponding optimal temperatures for
each organism, 37 and 30‡C, respectively; see Figs. 2 and
1). In the medium with 4% glucose the maximal ethanol
accumulation in H. polymorpha at 37‡C was practically
equal to that in P. stipitis at 30‡C (Figs. 1 and 2). Growth
of H. polymorpha strains in xylose medium was slightly
weaker than that of P. stipitis. Amounts of ethanol accumulated in the medium with xylose were much lower than
in glucose medium and lower in comparison to amounts of
consumed xylose (Figs. 1 and 2). It suggests that xylose
partially is converted to some intermediate, apparently
xylitol, which can accumulate in the medium. More vigorous aeration (shaking in 125-ml £asks containing 50 ml
medium at 200 rpm) led to a signi¢cant drop in ethanol
accumulation by H. polymorpha 356 and ML3 from both
xylose (5- and 4.9-fold, respectively), and to a weak decrease for glucose (1.8- and 2.1-fold, respectively), compared with conditions of limited aeration (shaking at
100 rpm). Thus, in H. polymorpha, as in P. stipitis and
other xylose-fermenting yeasts, oxygen depresses alcoholic
fermentation [7].
At 40‡C, ethanol accumulation from glucose by strain
356 was approximately the same as at 37‡C (Table 2). In
xylose medium, however, ethanol accumulation was 3-fold
Table 1
Growth (mg dry weight ml31 ) and ethanol accumulation (mg ml31 ) by various strains of H. polymorpha cultivated for 48 h or 96 h with di¡erent carbon sources (2%) in tubes containing 5 ml of medium without shaking at 37‡C
Strain
Carbon source
Glucose
Growth
CBS 4732
356
KT2
N95
ML3
ML6
ML8
ML9
CBS 4732
356
KT2
N95
ML3
ML6
ML8
ML9
48 h
1.3
0.8
0.4
0.3
1.0
0.4
0.3
0.6
96 h
2.4
1.6
0.9
1.8
1.6
0.8
0.9
1.3
Xylose
Maltose
Mannose
Cellobiose
L-Arabinose
Galactose
Ethanol Growth
Ethanol Growth
Ethanol Growth
Ethanol
Growth
Ethanol Growth
Ethanol Growth
Ethanol
4.5
3.9
2.5
4.2
3.2
1.6
1.7
2.0
0.2
0.3
0.3
0.3
0.2
0.2
0.1
0.2
1.3
1.2
1.2
0.1
0.9
1.0
1.3
1.1
1.0
0.3
0.3
0.5
0.4
0.2
0.2
0.8
1.5
2.2
2.2
1.8
2.4
1.4
2.2
1.6
1.2
0.9
0.5
0.8
1.1
0.4
0.3
2.8
10.3
4.8
3.2
4.7
7.0
2.9
2.4
4.4
0.4
0.4
0.5
0.3
0.4
0.2
0.2
0.3
0.0
3.2
4.2
3.8
3.4
2.5
2.4
2.5
0.4
0.3
0.3
0.3
0.2
0.2
0.3
0.3
1.9
3.2
2.9
1.2
2.7
2.4
2.5
3.1
0.05
0.1
0.3
0.3
0.2
0.2
0.2
0.1
0.0
0.02
0.0
0.2
0.0
0.0
0.0
0.0
7.8
7.6
9.1
9.1
7.4
3.6
4.5
8.2
1.5
0.6
0.8
0.9
0.6
0.5
0.6
0.5
0.0
2.1
3.4
0.2
2.1
1.9
2.1
2.3
1.7
0.9
0.7
0.8
1.6
0.9
0.8
0.6
1.3
3.3
2.9
0.2
3.2
3.6
1.7
2.6
1.4
1.8
1.2
1.2
1.7
2.3
1.8
1.6
6.1
8.0
7.0
3.7
4.7
9.4
7.7
9.8
0.6
0.9
1.2
1.0
1.0
0.8
0.8
0.8
0.0
3.6
5.3
2.1
2.2
2.1
0.0
1.4
0.9
0.6
0.7
0.3
0.6
0.6
0.6
0.6
0.0
1.4
0.0
0.06
0.6
0.5
2.0
0.2
0.6
0.1
0.3
0.3
0.4
0.4
0.4
0.1
0.1
0.2
0.0
0.06
0.04
0.06
0.0
0.0
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O.B. Ryabova et al. / FEMS Yeast Research 4 (2003) 157^164
Fig. 1. Growth in the media with 4% glucose (a), 4% xylose (c) and ethanol accumulation in the media with glucose (b), xylose (d) by di¡erent strains
of H. polymorpha and P. stipitis CBS 6054 cultivated in shake £asks (limiting aeration conditions) at 30‡C. O, P. stipitis CBS 6054; R, H. polymorpha
KT2; E, H. polymorpha CBS 4732 ; F, H. polymorpha 356; a, H. polymorpha ML3.
suggest that H. polymorpha can be considered as potential
xylose-fermenting organism at high temperatures (40‡C
and higher), more compatible with an SSF process than
the well-known yeast species.
For determining tolerance towards exogenous ethanol in
H. polymorpha compared with S. cerevisiae and P. stipitis,
these yeasts were cultivated in liquid media with 1% glucose and di¡erent concentrations of exogenous ethanol at
their own optimal growth temperatures (30‡C for P. stipitis
and S. cerevisiae and 37‡C for H. polymorpha). H. polymorpha was more resistant to exogenous ethanol than
P. stipitis, though more susceptible than baker’s yeast
(Fig. 3).
It is known that ethanol accumulation in P. stipitis is
lower than that at 37‡C. We also observed that H. polymorpha strains can grow and ferment both xylose and
glucose at much higher temperatures, 43 and 45‡C, though
growth rate and ethanol accumulation were lower than at
37‡C. Ethanol was produced from both glucose and xylose
even at 48‡C, though cells did not grow at this temperature. At 43 and 45‡C, strains 356 and ML3 accumulated
16 and 33 times less ethanol from xylose than at 37‡C
(Table 2). Such di¡erences were much smaller in glucose
medium. Apparently, thermotolerance of ethanol production in H. polymorpha depends on the carbon source: the
substrate rendering better growth, glucose, provides less
marked dependence of ethanol production on temperature
than the poor substrate, xylose. The accumulated data
Table 2
Maximal biomass and ethanol accumulation (g l31 ) from glucose and xylose by H. polymorpha 356 at di¡erent temperatures
Carbon source
Incubation temperature
30‡C
Glucose
Xylose
37‡C
40‡C
43‡C
45‡C
48‡C
Biomass
Ethanol
Biomass
Ethanol
Biomass
Ethanol
Biomass
Ethanol
Biomass
Ethanol
Biomass
Ethanol
2.6
2.3
7.4
0.23
4.2
2.8
13.2
2.98
3.2
2.0
13.2
1.0
2.7
1.6
7.9
0.18
1.3
1.0
2.4
0.09
1.1
0.8
2.2
0.03
Cultivation was carried out under conditions of limited aeration (see Section 2). Inoculation biomass dry weight was 1 mg ml31 .
FEMSYR 1578 27-10-03
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161
Fig. 2. Growth in the media with 4% glucose (a), 4% xylose (d), ethanol accumulation in the media with glucose (b) and xylose (e) and glucose (c) and
xylose (f) consumption by di¡erent strains of H. polymorpha and P. stipitis CBS 6054 cultivated in shake £asks (limiting aeration conditions) at 37‡C.
O, P. stipitis CBS 6054; R, H. polymorpha KT2; E, H. polymorpha CBS 4732; F, H. polymorpha 356; a, H. polymorpha ML3.
considerably enhanced in the mutant with deleted cytochrome c gene PsCYC1, apparently due to redirection of
reductants produced in glycolysis into ethanol production
[32]. We supposed that it also would be possible to enhance ethanol accumulation by regulation of pyruvate
partitioning between pyruvate decarboxylase (leading to
ethanol) and pyruvate dehydrogenase complex (leading
to tricarboxylic acid cycle and terminal oxidation to
CO2 ). Since the pyruvate dehydrogenase complex is £avin
adenine dinucleotide (FAD) dependent, we hypothesized
that starvation of H. polymorpha for ribo£avin, which can
be achieved in the corresponding ribo£avin-de¢cient mutant, might redistribute the £ux of pyruvate towards ethanol. Growth and ethanol production by the ribo£avin-defective mutant H. polymorpha rib7 were studied in test
tubes in standing cultures. It was found that starvation
for ribo£avin increased the productivity of alcoholic fermentation in H. polymorpha both from glucose and xylose
FEMSYR 1578 27-10-03
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O.B. Ryabova et al. / FEMS Yeast Research 4 (2003) 157^164
Fig. 3. E¡ect of exogenous ethanol on the growth of H. polymorpha 356
(E), P. stipitis CBS 6054 (R) and S. cerevisiae wild-type strain (O) in
the liquid synthetic media containing 1% glucose. Cultivation at 37‡C
(H. polymorpha) and 30‡C (other cultures) in tubes containing 5 ml of
medium without shaking (standing cultures) for 48 h.
Fig. 5. Productivity of ethanol by H. polymorpha wild-type strain 356
and alcohol dehydrogenase-defective mutant A3 during cultivation in
media with 2% glucose (O, 356; E, A3) and 2% xylose (R, 356; F, A3)
in tubes containing 5 ml medium without shaking (standing cultures) at
37‡C.
(Fig. 4). It is interesting to note that cultivation under
conditions of ribo£avin limitation led to a similar increase
in ethanol accumulation from glucose in ribo£avin-de¢cient mutants of other yeast species, P. guilliermondii
and C. famata (not shown). Cultivation of H. polymorpha
rib7 in media with glucose under conditions of limited
aeration (100 rpm) also led to an increase of ethanol accumulation under ribo£avin de¢ciency, though the di¡erence between maximal ethanol levels at suboptimal ribo£avin concentration and at optimal ribo£avin supply was
similar to that in standing (almost anaerobic) cultures.
Cultivation under vigorous aeration (250-ml Erlenmeyer
£asks containing 50 ml medium, shaking at 200 rpm)
did not lead to signi¢cant ethanol accumulation from either glucose or xylose, even under conditions of ribo£avin
starvation (4.7 times less ethanol under ribo£avin starvation in glucose medium than in standing cultures, and no
ethanol in xylose medium). As changes of aeration intensity directly a¡ect cell respiration, our data suggest that
respiration is not the primary point of the observed e¡ect
of £avin starvation. One may assume that ribo£avin de¢ciency redistributes pyruvate £ux through the £avin coenzyme-independent enzymes pyruvate decarboxylase and
alcohol dehydrogenase to ethanol. Mechanisms of £avin
e¡ects on alcoholic fermentation need to be studied in
more detail.
Earlier, we had isolated mutants of H. polymorpha defective in alcohol dehydrogenase [15,16,23]. Mutant A3
had a 30^40 times decreased activity of this enzyme due
to absence of the major alcohol dehydrogenase isoenzyme.
This mutant accumulated ethanol from glucose much
slower than the wild-type strain, whereas the rates of ethanol accumulation in xylose medium were identical in the
wild-type strain and the alcohol dehydrogenase-defective
mutant (Fig. 5). One may assume that alcohol dehydrogenase is the limiting enzyme in H. polymorpha for ethanol
accumulation from glucose, when the rate of the process is
relatively high. In xylose medium, the rate of ethanol production is signi¢cantly lower and apparently is limited not
by alcohol dehydrogenase but by some other transport or
enzymatic steps.
Fig. 4. E¡ect of exogenous ribo£avin on the growth (a) and Productivity (b) of ethanol in the ribo£avin-defective mutant H. polymorpha in
media with 2% glucose (O) or 2% xylose (R) cultivated in tubes containing 5 ml medium without shaking at 37‡C (standing cultures) for
48 h.
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163
WI, USA) and Dr. C.A. Abbas (Decatur, IL, USA) for
valuable discussions.
4. Discussion
This article is apparently the ¢rst full report on the
ability of the thermotolerant methylotrophic yeast H. polymorpha to ferment xylose and cellobiose to ethanol (see
our conference reports [12,13]). There is some discrepancy
between our data on sugar fermentation and assimilation
patterns presented in the characteristics and identi¢cation
manual [31], apparently, due to di¡erence in experimental
conditions. In this manual, the yeast Pichia angusta (synonym of H. polymorpha) is indicated as unable to ferment
xylose. Fermentation of cellobiose is indicated as absent or
delayed (longer than 7 days) and elsewhere as variable.
Growth on xylose and cellobiose is indicated as variable
[31]. Strains tested by us grow and ferment xylose and
cellobiose (except strains N95 and CBS 4732 which cannot
utilize xylose and cellobiose, respectively). The ability of
H. polymorpha to ferment xylose is very poor at standard
temperature 30‡C (at which such experiments have apparently been conducted with other xylose-fermenting yeasts).
However, at 37‡C the rate of xylose fermentation by
H. polymorpha is comparable to that of P. stipitis at
30‡C. On the other hand, P. stipitis practically cannot
ferment xylose or glucose at 37‡C. The ability of the available strains of H. polymorpha to ferment xylose and cellobiose at elevated temperatures makes this organism very
promising for development of lignocellulose fermentation
technology, compatible with simultaneous enzymatic hydrolysis of lignocellulose polymers (SSF process). It is interesting to note that the amounts of ethanol produced
from cellobiose are quite similar to those produced from
glucose.
We have found that ethanol productivity from glucose
and xylose can be substantially (up to 40^50%) elevated in
a ribo£avin-de¢cient mutant of H. polymorpha under suboptimal supply with £avins. Apparently under £avin limitation pyruvate is redistributed via a £avin-independent
pathway to ethanol production. It is important that such
stimulation of ethanol accumulation could be obtained
during cultivation without shaking, i.e. practically under
anaerobic conditions. These data show potential for further improvement of ethanol productivity in H. polymorpha.
We plan to identify the bottleneck(s) limiting growth
and ethanol production from xylose. As methods of molecular genetics and gene engineering, including that of
gene ampli¢cation, are well developed for H. polymorpha
[29,33,34], it is possible to eliminate identi¢ed bottleneck(s) and to create very active and industrially feasible
fuel ethanol producers in this species.
Acknowledgements
The authors are grateful to Dr. T.W. Je¡ries (Madison,
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