World Journal of Microbiology & Biotechnology 13, 341±346

Fermentation of corn ®bre sugars by an

engineered xylose utilizing Saccharomyces
yeast strain

M. Moniruzzaman, B.S. Dien, C.D. Skory, Z.D. Chen, R.B. Hespell, N.W.Y. Ho, B.E. Dale
and R.J. Bothast*

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and
galactose which are the predominant monosaccharides found in corn ®bre hydrolysates has been examined.
Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding
a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant ef®ciently fermented xylose
alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with
only 4 to 5 g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol
with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80 g/l) and xylose (40 g/l),
this strain produced 52 g/l ethanol, equivalent to 85% of theoretical yield, in less than 24 h. Using a mixture of
glucose (31 g/l), xylose (15.2 g/l), arabinose (10.5 g/l) and galactose (2 g/l), all of the sugars except arabinose were
consumed in 24 h with an accumulation of 22 g ethanol/l, a 90% yield (excluding the arabinose in the calculation
since it is not fermented). Approximately 98% theoretical yield, or 21 g ethanol/l, was achieved using an
enzymatic hydrolysate of ammonia ®bre exploded corn ®bre containing an estimated 47.0 g mixed sugars/l. In all
mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.

Key words: AFEX, corn ®bre, ethanol, Saccharomyces.

The importance of cellulosic biomass as a renewable
energy resource has increased with the anticipated
shortage of fossil fuel reserves and increased air pollu-
tion problems. Corn ®bre residues from the corn wet
milling industry have been proposed as a possible low
cost feedstock for fuel alcohol production (Bothast 1994).
In previous publications, we have reported that pre-
treatment of many types of cellulosic biomass, including
corn ®bre, with the ammonia ®bre explosion (AFEX)
process allows for enhanced enzymatic hydrolysis of the
cellulose, hemicellulose and starch at low enzyme
M. Moniruzzaman is and B.E. Dale was with the Department of Chemical En-
gineering, Texas A&M University, College Station, TX 77843, USA. B.S. Dien,
C.D. Skory, R.B. Hespell and R.J. Bothast are with the Fermentation Bio-
chemistry Research Unit, National Center for Agricultural Utilization Research,
USDA, H Agricultural Research Service, 1815 N. University Street, Peoria, IL
61604, USA. N.W.Y. Ho is with LORRE, 1295 Potter Center, Purdue University,
West Lafayette, IN 47907, USA. B.E. Dale is now with the Department of
Chemical Engineering, Michigan State University, East Lansing, MI 48824, USA.
H Names are necessary to report factually on available data; however, the
USDA neither guarantees nor warrants the standard of the product, and the use
of the name by USDA implies no approval of the product to the exclusion of
others that may also be suitable. *Corresponding author.
(1±5 IU/g substrate) levels (Dale et al. 1996; Moniruzza-
man et al. 1996a). Thus, fermentable sugars can be ob-
tained with minimal use of costly hydrolytic enzymes
(Holtzapple et al. 1991, 1992; Reshamwala et al. 1995;
Moniruzzaman et al. 1996b).
A typical enzymatic hydrolysate of agricultural bio-
mass usually contains a mixture of hexose and pentose
sugars including glucose, xylose, arabinose and galactose.
While the ratios of the sugars may differ depending on the
source of the starting material, ef®cient fermentation of all
sugars is essential for biomass to ethanol fermentations to
be economically competitive with starch based fermen-
tations. Obtaining a microorganism capable of fermenting
the predominant sugars in cellulosic biomass has been
dif®cult because microorganisms used for industrial fer-
mentation of glucose to ethanol (e.g. Saccharomyces cere-
visiae) do not ferment or even metabolize pentoses.
Several yeasts, such as Pichia stipitis (Toivola et al. 1984),
Candida shehatae (de Preez & van der Walt 1983), and
Pachysolen tannophilus (Slininger et al. 1982), are able to

ã 1997 Rapid Science Publishers

World Journal of Microbiology & Biotechnology, Vol 13, 1997 341

M. Moniruzzaman et al.
ferment xylose to ethanol by reduction of xylose to xylitol
by an NADPH/NADH-linked xylose reductase, followed
by oxidation of xylitol to xylulose by a NAD-linked xylitol
dehydrogenase. However, these are constrained by their
rates of ethanol fermentation and the requirement of some
aeration for optimal ethanol productivity. When the fer-
mentations are carried out under anaerobic conditions,
some of the xylose is converted to xylitol instead of
ethanol (de Preez 1994; Jeffries & Kurtzman 1994).
S. cerevisiae has traditionally been used for alcoholic
fermentation because of its ability to produce ethanol
anaerobically in a low pH and a high osmolarity envi-
ronment with unparalleled productivity and ef®cient
yields. A S. cerevisiae that could effectively ferment xylose
would be a signi®cant advancement for the biofuels in-
dustry. The inability to ferment xylose in presumably
due to the lack of xylose reductase or xylitol dehydro-
genase activity. However, S. cerevisiae is able to ferment
xylulose to ethanol since it possesses a xylulose kinase
that is expressed in low amounts (Deng & Ho 1990).
Previous attempts to genetically incorporate the ability to
utilize xylose by using xylose isomerase genes from
bacteria or xylose reductase and xylitol dehydrogenase
from xylose fermenting yeasts have resulted in strains
that produced little or no ethanol (Ho et al. 1983; Sarthy
et al. 1987; Amore et al. 1989; Tantirungkij et al. 1993;
Walfridsson et al. 1995).
In this paper, we characterize fermentation ef®ciency
of a recombinant Saccharomyces strain 1400 (pLNH32)
containing introduced genes encoding not only the
xylose reductase and xylitol dehydrogenase genes from
P. stipitis, but also the xylulose kinase from S. cerevisiae.
Fermentation of the enzymatic hydrolysate from AFEX-
treated corn ®bre was also tested with this strain under
pH-controlled conditions.
Materials and Methods
Yeast Strain
Fermentations were carried out using a Saccharomyces 1400
transformed with the plasmid pLNH32 (Chen & Ho 1993; Ho et al.
1993; Ho & Tsao 1995). Saccharomyces 1400, provided by Labatt
Brewing Company (London Ontario, Canada), is a fusion prod-
uct of S. diastaticus and S. uvarum (syn. carlsbergensis cerevisiae)
that was previously identi®ed as being able to ef®ciently ferment
glucose to ethanol at 40 °C (D'Amore 1989, 1990). Its ability to use
xylose as a sole carbon source was conferred by transformation
with a 2 l replicating plasmid containing the P. stipitis xylose
reductase, xyl1, and xylitol dehydrogenase, xyl2, genes and also
the xylulose kinase gene from S. cerevisiae. These cloned xylose
metabolizing genes have all been fused to promoters not inhib-
ited by the presence of glucose and also not requiring the pres-
ence of xylose for transcription. The P. stipitis promoters for xyl1
and xyl2 were replaced with the S. cerevisiae promoter adh1 and
pyk1, respectively, while the xylulose kinase gene was modi®ed
to be under control of the pyruvate kinase, pyk1, promoter from
342 World Journal of Microbiology & Biotechnology, Vol 13, 1997
S. cerevisiae. The kanamycin resistance gene from TN903 was
utilized for initial selection of yeast transformants on geneticin-
containing media (Gibco BRL, Gaithersburg, MD). Thereafter, the
plasmid was maintained by growth on YEP [10 g yeast extract/l
and 20 g peptone/l (Difco, Detroit, MI)] medium supplemented
with xylose (20 g/l) as a sole carbon source.
AFEX-pretreatment and Enzymatic Hydrolysis of Corn Fibre
Corn ®bre obtained as a by-product using the wet-milling pro-
cess was provided by Pekin Energy Company, Pekin, IL. The
corn ®bre was pretreated by the AFEX process and enzymati-
cally hydrolyzed for 48 h with a mixture of amylase, pectinase,
cellulase and cellobiase as previously described (Moniruzzaman
et al. 1996a). The hydrolysate was centrifuged (18,000 ´ g for
15 min) to remove particulate matter and then sterilized by ®l-
tration through a 0.45 lm membrane ®lter.
Preparation of Inoculum
The recombinant Saccharomyces strain was grown to mid-growth
phase in YEP containing 20 g xylose/l. Approximately 4.0 ml of
culture was then transferred into a 250-ml ¯ask containing
100 ml of YEP medium with 20 g glucose/l. The culture was
incubated for 16 h at 30 °C, while being agitated at 150 rev/min.
Cells were harvested by centrifugation (5000 ´ g, 5 min), sus-
pended in appropriate fermentation medium and used to inoc-
ulate the medium at an initial biomass concentration of either
0.05 g/l (dry weight), referred to as low inoculum level, or 1 g/l
(dry weight), referred to as high inoculum level.
Fermentation Experiments
All fermentations were carried out at 30 °C in ®lter sterilized
YEP supplemented with appropriate sugars or corn ®bre
hydrolysate. Initially, the ability of the recombinant Saccharo-
myces to produce ethanol from individual sugars was examined
in 500-ml shake ¯asks in an orbital shaker (at 200 rev/min) with
a working volume of 100 ml. A low inoculum level was used for
fermentations with individual sugars. In order to estimate the
oxygen requirements for optimal ethanol production, the sugars
glucose, xylose, galactose and arabinose were tested individu-
ally at 80 g/l in aerobic, anaerobic and semi-aerobic conditions.
Aerobic conditions were maintained by covering the ¯asks with
milk ®lter paper (Milkhouse Brand, Janesville, WI). For semi-
aerobic growth, ¯asks were plugged with porous silicone stop-
pers (Cole-Parmer, Niles, IL) that were more restrictive to air
transfer than the ®lter paper. Finally, rubber stoppers were used
to create anaerobic conditions by venting CO2 through a 20
gauge hypodermic needle. Fermentations were also carried out
for YEP containing 80 g glucose/l plus 40 g xylose/l in anaer-
obic shake ¯asks using either low or high inoculum levels.
The sugars glucose, xylose, arabinose and galactose were
then tested in a mixed sugar fermentation since these sugars are
usually present in the hemicellulosic fraction of most biomass.
We chose to simulate a typical composition of sugars that might
be found in a corn ®bre hydrolysate, assuming that the majority
of complex carbohydrates in the AFEX-pretreated corn ®bre
(100 g/l), such as cellulose, hemicellulose and starch are hy-
drolysed into their monomeric forms. Since the protein and oil
portion of the corn ®bre make up nearly 14% of the total dry
weight, we chose to use YEP supplemented with ®lter sterilized
glucose (31.0 g/l), xylose (15.2 g/l), arabinose (10.5 g/l) and
galactose (2.0 g/l). Fermentations were carried out with a high
inoculum level in pH-controlled 500-ml ¯asks (Beall et al. 1991)
with a working volume of 200 ml. Flasks were vented with a 20
gauge hypodermic needle in order to create anaerobic condi-
tions and 2 M KOH was used for pH control. Magnetic stirrers

Figure 1. Effect of low inoculation level on fermentation of glucose
and xylose mixtures. Fermentations were carried out anaerobically
using YEP medium supplemented with 80 g glucose/l and 40 g
xylose/l. d, ethanol; n, glucose; s, xylose; h, xylitol.
Figure 2. Effect of high inoculation level on fermentation of glucose
and xylose mixtures. Fermentations were carried out as in Figure 1
with 80 g glucose/l and 40 g xylose/l. d, ethanol; n, glucose; s,
xylose; h, xylitol.
were employed to evenly distribute the base. Finally, corn ®bre
hydrolysate, supplemented with 10 g yeast extract/l and 20 g
peptone/l, was utilized as a fermentation medium using the
same parameters for fermentation as that described for the pH
controlled ¯asks. For all fermentations, 1 ml aliquots were
withdrawn periodically until 120 h to determine cell growth,
ethanol and residual sugar concentrations.
Fermentation of corn ®bre sugars
Analytical Procedure
Ethanol concentrations were determined by gas chromatography
equipped with a ¯ame ionization detector and a Porapak Q col-
umn. Sugar and sugar alcohol concentrations were analysed by
HPLC using an HPX-87C column (Bio-Rad Laboratories) and a
differential refractometer. For comparison purposes, sugars
concentrations in the corn ®bre hydrolysate were also analysed
by HPLC as described by Cotta (1993) using a Dionex LC20 sys-
tem (Sunnyvale, CA) equipped with a Carbopac PA100 column.
Results and Discussion
Fermentation of Individual Sugars
Saccharomyces strain 1400 (pLNH32) was assessed for its
ability to ferment the sugars glucose, xylose, galactose
and arabinose separately under aerated, micro-aerated
and anaerobic culture conditions. The sugars glucose
and galactose were completely consumed within 12 h
regardless of aeration conditions (data not shown). Under
anaerobic condition, maximum ethanol concentrations
from glucose and galactose were 36.3 g/l and 34.2 g/l
respectively, equivalent to 89 and 84% of the theoretical
yield (Table 1). The yeast consumed xylose at a slower
rate than the hexose sugars and only produced 26.9 g
ethanol/l, corresponding to 66% of the theoretical yield
(Table 1). At 96 h, all of the xylose was consumed and
approximately 4±5 g xylitol/l was present under all
aeration conditions. No arabinose was consumed during
the course of the experiment (data not shown).
Fermentation of Glucose and Xylose Mixtures
Fermentations of a mixture of glucose and xylose were
carried out using both a low inoculum (0.1 g cell dry
weight/l) and a high inoculum level (2.0 g cell dry
weight/l). Cultures inoculated at both levels completely
consumed the glucose in less than 12 h (Figures 1 and 2).
However, the low inoculum culture failed to consume
almost half the xylose by 5 d and produced a maximum
ethanol concentration of only 45.3 g/l. By comparison,
the high inoculum culture consumed more than 90% of
the xylose in this time and produced a maximum ethanol
concentration of 52.5 g/l. Therefore, the ethanol yield of
the low inoculum culture was 74% of the theoretical yield
as compared with 86% for the other culture (Table 1).
The large amount of xylose left unused in the low in-
oculum culture might suggest that the 2 l plasmid har-
bouring the xylose utilization genes may not be as stable
in the presence of glucose as we had originally thought.
Preliminary studies had shown that growing S. cerevisiae
1400 (pLNH32) up to 20 generations on glucose had very
little effect on ethanol production with subsequent fer-
mentations using a mixture of glucose and xylose (data
not shown). However, these data suggest that in the ab-
sence of selective pressure such as the antimicrobial agent
geneticin in the medium or xylose as a sole carbohydrate
World Journal of Microbiology & Biotechnology, Vol 13, 1997 343
M. Moniruzzaman et al.

Table 1. Summary of fermentation performance of Saccharomyces strain 1400 (pLNH32) on various substrates under different
conditions.

Initial substrate Fermentation Maximum Ethanol Ef®ciencyd Vpe Final

concentration conditionsa ethanolb yieldc (%) (g/l/h) biomass
(g/l) (g/l) (g/g S) concentrationf
(g/l)

glucose (80) ¯ask, anaerobic, low inocu- 36.3 0.46 89 2.91 2.3
lum, without pH control
xylose (80) ¯ask, anaerobic, low inocu- 26.9 0.34 66 0.53 1.9
lum, without pH control
arabinose (80) ¯ask, anaerobic, low inocu- 0.0 0.00 0 0.00 ±
lum, without pH control
galactose (80) ¯ask, anaerobic, low inocu- 34.2 0.43 84 2.80 2.7
lum, without pH control
glucose (80) and xylose (40) ¯ask, anaerobic, low inocu- 45.3 0.38 74 3.34 2.5
lum, without pH control
glucose (80) and xylose (40) ¯ask, anaerobic, high inocu- 52.5 0.44 86 3.30 3.0
lum, without pH control
glucose (80) and xylose (40) ¯ask, anaerobic, high inocu- 52.0 0.44 85 3.44 2.4
lum, with pH (5.0) control
synthetic sugar mixture glu- ¯ask, anaerobic, high inocu- 22.0 0.46 90 1.60 2.2
cose (31), xylose (15.2), galac- lum, with pH (5.0) control
tose (2.0), arabinose (10.5)
corn ®bre enzymatic hydrolyzate ¯ask, anaerobic, high inocu- 21.0 0.50 98 1.60 1.7
glucose (33.5), xylose (7.5), lum, with pH (5.0) control
galactose (1), arabinose (5.0)

a
See materials and methods for details for these 5 d fermentations; b ethanol values were corrected (when necessary) for dilution by base
addition during fermentation; c ethanol yield in g/g of substrate available for fermentation (arabinose not included); d calculated as: ethanol
produced/theoretical maximum (0.5 g ethanol/g sugar) from sugar (arabinose not included) substrate ´ 100; e maximum volumetric productivity
during batch fermentation; f dry weight.

source, the 2 l plasmid may be diluted or lost completely. appears that pH control of the fermentation reduces the
When a high inoculum was used, this effect would have time for complete fermentation from 120 h to less than
been minimized because of the smaller number of cell 24 h. It may be possible that pH control is allowing more
generations occurring in the non-selective medium. Ef-
forts are currently directed at studying the effects of non-
selective growth conditions on plasmid stability and
integrating the xylose utilizing genes into the genome.

Effect of pH on Fermentation
Studies were conducted to see if pH control of the culture
medium would effect the rate of sugar utilization and
ethanol production. Fermentations of glucose and xylose
mixtures under anaerobic condition with high inoculum
level (2.0 g cell dry wt/l) were automatically maintained
at pH 4.5, 5.0, 5.5 and 6.0. Although the results were
nearly identical for the range of pH tried, maintaining the
pH at a constant level seemed to increase the rate of
sugar consumption and ethanol production dramatically
(Figure 3, data only shown for pH 5.0). Both sugars in a
glucose/xylose mixture (60 g/l + 60 g/l) were simulta-
neously and completely utilized within 24 h and pro-
duced a maximum of 52 g ethanol/l, equivalent to 85%
of theoretical yield. Only 14 mmol/l base was needed to Figure 3. Fermentation of glucose and xylose mixtures in pH con-
trolled ¯asks. Fermentations were carried out anaerobically using
maintain the pH at 5.0 while the sugars were being
YEP medium supplemented with 80 g glucose/l and 40 g xylose/l.
consumed. Comparing these results with that from the Medium was maintained at pH 5.0 with 2 M KOH. d, ethanol; n,
anaerobic shake ¯asks without pH control (Figure 2), it glucose; s, xylose; h, xylitol.

344 World Journal of Microbiology & Biotechnology, Vol 13, 1997


Figure 4. Fermentation of mixed sugars simulating a typical corn
®bre hydrolysate. Fermentations were carried out anaerobically in
¯asks maintained at pH 5.0 using YEP medium supplemented with
31 g glucose/l and 15.2 g xylose/l, 10.5 g arabinose/l and 2 g ga-
lactose/l. d, ethanol; n, glucose; j, xylose plus galactose; h, xylitol.
Figure 5. Fermentation of AFEX pretreated corn ®bre hydrolysate.
Fermentations were carried out anaerobically in ¯asks maintained at
pH 5.0 using YEP medium supplemented with corn ®bre hydroly-
sates. d, ethanol; n, glucose; j, xylose and galactose; h, xylitol.
ef®cient uptake of xylose and further study of this effect
is currently in progress.
Xylose was ef®ciently converted to ethanol by Sac-
charomyces 1400 (pLNH32) and very little xylitol accu-
mulated during these fermentations. In Pichia, it has been
suggested that xylitol accumulates in the culture medium
Fermentation of corn ®bre sugars
due to the apparent redox imbalance which develops as a
result of difference in cofactor (NADPH and NAD+) re-
quirements of the xylose reductase and xylitol dehydrog-
enase enzymes (Bruinenberg et al. 1983, 1984). A slight
amount of oxygen seems to help balance the oxido-
reduction reaction to minimize xylitol accumulation in the
culture medium without providing enough oxygen for
utilization of the ethanol. A similar phenomenon may be
occurring in our recombinant yeast although little differ-
ence in xylitol accumulation was observed by varying
oxygen conditions. This suggests that xylitol accumula-
tion is more likely to be the result of something else such as
limiting xylitol dehydrogenase or xylulose kinase activity.
Fermentation of Corn Fibre Enzymatic Hydrolysate
For comparison purposes, conditions were used to du-
plicate a similar mixture of sugars that would be present
in a corn ®bre hydrolysate. The fermentation was carried
out in a pH-controlled ¯ask (pH 5.0), with an inoculum
level of 2.0 g/l dry weight. The recombinant Saccharo-
myces strain 1400 (pLNH32) ef®ciently and completely
utilized glucose, xylose and galactose within approxi-
mately 24 h and produced 22.0 g/l of ethanol or 90% of
theoretical yield (Table 1 and Figure 4). It should be noted
that arabinose was not included for theoretical ethanol
yield calculations, because neither the parent or recom-
binant strain can ferment arabinose. However, it is inter-
esting that some arabinose (less than 25%) was also
consumed in the presence of mixed sugars, but was con-
verted into arabitol (detected by HPLC, data not shown).
The yeast was next used to ferment corn ®bre enzy-
matic hydrolysate. The AFEX-pretreated corn ®bre en-
zymatic hydrolysate contained the free sugars glucose
(33.5 g/l), xylose (7.5 g/l), arabinose (5.0 g/l) and ga-
lactose (1.0 g/l). The fermentations were carried out in
pH 5.0 controlled ¯ask, under anaerobic conditions with
an inoculum level of 2.0 g/l (dry weight). The recombi-
nant strain ef®ciently utilized the hydrolysate sugars,
glucose, xylose and galactose and produced 21.0 g/l
ethanol which is equivalent to 98% of the theoretical
yield (Table 1 and Figure 5). As before, less than 25% of
the arabinose consumed could be accounted for by the
accumulating arabitol (data not shown).
The higher yield of ethanol from the hydrolysate
compared to the sugar mixture might be the result of
proteins from either the corn ®bre or the enzymes used in
hydrolysis increasing the available nitrogen in the culture
medium. Additionally, residual ammonia (approximate-
ly 0.5%) from the AFEX-treatment could be improving the
fermentation by providing nitrogen. D'Amore et al. (1989)
also showed that the parent Saccharomyces 1400 produced
higher levels of ethanol when the nutrient components of
the fermentation medium, which included ammonium
sulphate, were doubled. However, additional experi-
World Journal of Microbiology & Biotechnology, Vol 13, 1997 345
M. Moniruzzaman et al.
ments are needed to determine the effect of ammonia on
cell growth and ethanol production by this strain.
The results presented in this paper demonstrate that
the recombinant strain ef®ciently utilizes the sugars
glucose, xylose and galactose and produces ethanol at
high yields. This paper also demonstrates that ethanol
production from AFEX-pretreated corn ®bre hydrolysate
by this recombinant yeast is a promising alternative for
ethanol fermentation. Since arabinose is also present in
corn ®bre hydrolysates in signi®cant quantities, further
efforts to engineer this recombinant Saccharomyces strain
to allow arabinose fermentation, could improve ethanol
accumulation by up to 20%.
Acknowledgement
This work was supported under a Speci®c Cooperative
Agreement (#58-3620-4-131) from the Fermentation Bio-
chemistry Research Unit, ARS, USDA, Peoria, IL. The
authors thank Patricia O'Bryan for technical assistance.
References
Amore, R., Wilhelm, M. & Hollenberg, C.P. 1989 The fermen-
tation of xyloseÐan analysis of the expression of Bacillus and
Actinoplanes xylose isomerase genes in yeast. Applied Mi-
crobiology and Biotechnology 30, 351±357.
Beall, D.S., Ohta, K. & Ingram, L.O. 1991 Parametric studies of
ethanol production from xylose and other sugars by recom-
binant Escherichia coli. Biotechnology and Bioengineering 38, 296±
303.
Bothast, R.J. 1994 Genetically engineered microorganisms for the
conversion of multiple substrate to ethanol. Proceedings Corn
Utilization Conference V, June 8±10, St. Louis, MO, USA.
Bruinenberg, P.M., de Bot, P.H.M., van Dijken, J.P. & Scheffers,
W.A. 1983 The role of redox balances in the anaerobic fer-
mentation of xylose by yeasts. European Journal of Applied
Microbiology and Biotechnology 18, 287±292.
Bruinenberg, P.M., de Bot, P.H.M., van Dijken, J.P. & Scheffers,
W.A. 1984 NADH-linked aldose reductase: the key to anaer-
obic alcoholic fermentation of xylose by yeasts. Applied Mi-
crobiology and Biotechnology 19, 256±260.
Chen, Z.D. & Ho, N.W.Y. 1993 Cloning and improving the ex-
pression of Pichia stipitis xylose reductase gene in Saccharomyces
cerevisiae. Applied Biochemistry and Biotechnology 39/40, 135±147.
Cotta, M.A. 1993 Utilization of xylooligosaccharides by selected
ruminal bacteria. Applied and Environmental Microbiology 59,
3557±3563.
Dale, B.E., Leong, C.K., Pham, T.K., Esquivel, V.M., Rioss, I. &
Latimer, V.M. 1996 Hydrolysis of lignocellulosics at low
enzyme levels: application of the AFEX process. Bioresource
Technology (in press).
D'Amore, T., Celotto, G., Russell, I. & Stewart, G.G. 1989 Selec-
tion and optimization of yeast suitable for ethanol production
at 40 °C. Enzyme Microbiology and Technology 11, 411±416.
D'Amore, T., Panchal, C.J., Russell, I. & Stewart, G.G. 1990 A
study of ethanol tolerance in yeast. Critical Reviews in Bio-
technology 9, 287±304.
Deng, X.X. & Ho, N.W.Y. 1990 Xylulokinase activity in various
yeasts including Saccharomyces cerevisiae containing the cloned
346 World Journal of Microbiology & Biotechnology, Vol 13, 1997
xylulokinase gene. Applied Biochemistry and Biotechnology 24/
25, 193±199.
de Preez, J.C. 1994 Process parameters and environmental fac-
tors affecting D-xylose fermentation by yeasts. Enzyme Mi-
crobiology and Technology 16, 944±956.
de Preez, J.C. & van der Walt, J.P. 1983 Fermentation of D-xylose
to ethanol by a strain of Candida shehatae. Biotechnology Letters
5, 357±362.
Ho, N.W.Y. & Tsao, G.T. 1995 Recombinant yeasts for effective fer-
mentation of glucose and xylose. PCT Patent No. WO95/13362.
Ho, N.W.Y., Chen, Z.D. & Brainard, A. 1993 Genetically engi-
neered yeasts capable of effective fermentation of xylose to
ethanol. In Proceedings of Tenth International Conference on
Alcohol Fuels, p. 738. Colorado Springs, CO, USA.
Ho, N.W.Y., Stevis, P., Rosenfeld, S., Huang, J.J. & Tsao, G.T.
1983 Expression of the E. coli xylose isomerase gene by a yeast
promoter. Biotechnology and Bioengineering Symposium 13, 245±
250.
Holtzapple, M.T., June, J.H., Ashok, G., Patibandla, S.L. & Dale,
B.E. 1991 The ammonia freeze explosion (AFEX) process: a
practical lignocellulose pretreatment. Applied Biochemistry and
Biotechnology 28/29, 59±74.
Holtzapple, M.T., Lundeen, J.E., Sturgis, R., Lewis, J.E. & Dale,
B.E. 1992 Pretreatment of lignocellulosic municipal solid
waste by ammonia ®ber explosion (AFEX). Applied Biochem-
istry and Biotechnology 34/35, 5±21.
Jeffries, T.W. & Kurtzman, C.P. 1994 Strain selection, taxonomy,
and genetics of xylose-fermenting yeasts. Enzyme Microbiology
Technology 16, 922±932.
Moniruzzaman, M., Dale, B.E., Hespell, R.B. & Bothast, R.J.
1996a Enzymatic hydrolysis of high moisture corn ®ber pre-
treated by AFEX and recovery and recycle of the enzyme
complex. Applied Biochemistry and Biotechnology (in press).
Moniruzzaman, M., Dien, B.S., Ferrer, B., Hespell, R.B., Dale,
B.E., Ingram, L.O. & Bothast, R.J. 1996b Ethanol production
from AFEX pretreated corn ®ber by recombinant bacteria.
Biotechnology Letters (in press).
Reshamwala, S., Shawky, B.T. & Dale, B.E. 1995 Ethanol pro-
duction from enzymatic hydrolysates of AFEX-treated coastal
bermudagrass and switchgrass. Applied Biochemistry and Bio-
technology 51/52, 43±55.
Sarthy, A.V., McConaughy, B.L., Lobo, Z., Sundstrom, J.A.,
Furlong, C.E. & Hall, B.D. 1987 Expression of the Escherichia
coli xylose isomerase gene in Saccharomyces cerevisiae. Applied
and Environmental Microbiology 53, 1996±2000.
Slininger, P.J., Bothast, R.J., van Cauwenberge, J.E. & Kurtzman,
C.P. 1982 Conversion of D-xylose to ethanol by the yeast
Pachysolen tannophilus. Biotechnology and Bioengineering 24,
371±384.
Tantirungkij, M., Nakashima, N., Seki, T. & Yoshida, T. 1993
Construction of xylose-assimilating Saccharomyces cerevisiae.
Journal of Fermentation and Bioengineering 75, 83±88.
Toivola, A., Yarrow, D., van den Bosch, E., van Dijken, J.P. &
Scheffers, W.A. 1984 Alcoholic fermentation of D-xylose by
yeasts. Applied and Environmental Microbiology 47, 1221±1223.
Walfridsson, M., Hallborn, J., Penttila, M., Keranen, S. & Hahn-
Hagerdal, B. 1995 Xylose-metabolizing Saccharomyces cerevisiae
strains overexpressing the TKL1 and TAL1 genes encoding
the pentose phosphate pathway enzymes transketolase and
transaldolase. Applied and Environmental Microbiology 61,
4184±4190.
(Received in revised form 5 September 1996; accepted 11
September 1996)