ELSEVIER

Selective removal of acetic acid from

hardwood-spent sulfite liquor using a
mutant yeast*
Henry Schneider

Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario

The hexoses in spent sulfite liquor can be converted to ethanol by yeasts, but conversion to ethanol of the pentose
tr-xylose in lignocellulosic hydrolysates is inhibited generally by the presence of acetic acid. The feasibility of a
yeast-based process for selective removal of the acetic acid in hardwood-spent sulfite liquor was demonstrated.
The process depends on the use of a mutant of Saccharomyces cerevisiae that grows on acetic acid but not on
n--xylose, n-glucose, o-mannose, or n-fructose. The process could be used to decrease the concentration oj
acetic acid in hardwood liquor within 24 h to levels that no longer inhibit bioconversion of xylose to ethanol.
Indications of the conversion of n-xylose to ethanol in the acetic acid-depleted liquor were the ability of several
o-xylose-fermenting yeasts to use essentially all of the sugars and produce as much as 73% of the theoretical
amount of ethanol within 24 h. The process might be generally applicable to obviation of acetic acid inhibition
effects in ethanol production from hemicellulose hydrolysates.

Keywords: Spentsulfite liquor; acetic acid; Saccharomyces cerevisiae; o-xylose; yeast; hemicellulose hydrolysate

Introduction The acetic acid-depletion fermentation depends on sub-

Yeasts are now available that convert hexoses and the pen-
tose D-xylose to ethanol. l4 However, the development of
economic hexose-xylose ethanol fermentations of lignocel-
lulosic hydrolysates using yeasts is hindered, in part, by the
presence of acetic acid5-‘* which inhibits ethanol produc-
tion from hexoses and xylose. The acetic acid is present
because D-xylose is acetylated in many lignocellulos-
ics13-i4 and the acetyl group is released during hydrolysis.
Acetic acid concentrations as low as 0.08% can inhibit xy-
lose fermentation.5
The present paper describes a yeast fermentation that
selectively removes acetic acid from a lignocellulosic hy-
drolysate, hardwood-spent sulfite liquor. The fermentation
has the potential for application to lignocellulosic hydroly-
sates generally. The process reduces acetic acid to very low
levels, causes only small changes in sugar concentration,
and leaves the hydrolysate in a form from which the hexoses
and D-xylose can subsequently be converted to ethanol by
xylose-fermenting yeasts.
*Issued as NRCC publication 395 14
Address reprint requests to Dr. Henry Schneider, Institute of Biological
Sciences. National Research Council of Canada, Ottawa, ON KlA OR6,
Canada
Received 1I July 1995; revised 21 September 1995; accepted 21 Septem-
ber 1995
jecting the lignocellulosic hydrolysate to aerobic fermenta-
tion by a mutant of Saccharomyces cerevisiae that utilizes
acetic acid for growth and does not utilize either the major
hexoses in lignocellulose hydrolysates or o-xylose for
growth. In addition, the mutant does not manifest diauxie in
that it utilizes acetic acid even in the presence of D-glucose.
Wild-type S. cerevisiae utilizes only hexoses in a lignocel-
lulosic hydrolysate such as spent sulfite liquor.
The properties of the mutant on which the acetic acid-
depletion fermentation depends result from a combination
of its inability to phosphorylate the hexoses D-glucose and
o-mannose, the occurrence of metabolic derepression in the
presence of o-glucose and the inability to use D-xylose or
L-arabinose to an appreciable extent. The inability to phos-
phorylate D-glucose and P-mannose results in the inability
to use these sugars, because phosphorylation is the first step
in their metabolism. The inability to phosphorylate these
sugars is the direct result of the presence of mutations in
three genes that render inactive the kinases involved: hxkl
(hexokinase I), hxk2 (hexokinase II), and glcl (glucokinase
I). The hexokinases use b-glucose, o-mannose, and D-fruc-
tose as substrates while glucokinase is specific for D-glu-
cose.i5*i6 The ability of the mutant to use acetic acid in the
presence of D-glucose (metabolic derepression) results from
the presence of the mutant gene for hexokinase. In the pres-
ence of a normally functional hexokinase II gene, enzymes

Enzyme and Microbial Technology 19:94-98, 1996

Published 1996 by Elsevier Science Inc. 0141-0229/96
655 Avenue of the Americas, New York, NY 10010 SSDI 0141-0229(95)00241-3
 Spent
for the metabolism of carbon sources other than D-glucose,
such as acetic acid and r+galactose, are repressed to low
levels when D-glucose is present in the medium. Thus, these
carbon sources are not used by yeasts with a functional
hexokinase II gene in the presence of u-glucose unless the
concentration of u-glucose is very low. The inability of the
mutant to use P-xylose and t_--arabinose is important to
ensure that these sugars are not utilized by the derepressed
mutant yeast.
Materials and methods
Strains
The mutant yeast employed, S. cerevisiae YGSCD 308.3 (hnkl.
hxk2, glkl, adel, trpl, hisl. metll), was maintained on slants of
1% peptone, 0.5% yeast extract, 0.5% ethanol, and 1% agar. The
u-xylose-fermenting yeasts employed, Pachysolen tannophilus
NRRL Y-2460, Pichia stipitis CBS No. 5776, and Yamadazyma
stipitis ATCC No. 66278 were maintained on nutrient agar.
Production of mutant yeast for acetic acid removal
Growth Medium. The medium used to grow large quantities of
cells of the mutant for use in the removal of acetic acid from
hardwood-spent sulfite liquor, referred to as Medium A, had the
following composition: 2% peptone, 0.5% yeast extract, 1% n-ga-
lactose, O&0.8% (w/v) acetic acid as specified, 0.4 M monobasic
ammonium phosphate, and 0.06% (w/v) 2-deoxyglucose. The ini-
tial pH was 5.5. Inocula were grown from slants in the same
medium.
Growth conditions. Medium A (15 ml) was placed in a 25-m]
loosely-capped Erlenmeyer flask, inoculated, and kept on a gyra-
tory shaker at 300 rpm and 30°C. Inocula consisted of aliquots of
cultures in medium A with cell densities between 5-9 x 10’ cells
ml-‘.
Acetic acid removal by fermentation
Preparation of liquor. Steam-stripped hardwood-spent sulfite li-
quor supplied by Tembec Inc. (Temiskaming, Canada) was diluted
with water to 20% (w/w) solids and supplemented with phosphate
(0.5% w/v monobasic ammonium phosphate) and 40 fig ml-i of
the auxotrophic requirements of the mutant. The liquor was ster-
ilized using tyndallization by heating the supplemented liquor to
70°C for 1 h. The pH decreased on tyndallization, and in order to
obtain a final pH of 5.8-5.85, the pH was adjusted to 6.3 with
concentrated ammonia prior to heating.
Fermentation protocol. Cells of the mutant grown in Medium A
were centrifuged at 12,000 g for 10 min at room temperature in a
50-m] plastic centrifuge tube. The supematant was poured off and
the cells resuspended in 15 ml of supplemented liquor. The resus-
pended cells were transferred to a 250-m] Erlenmeyer flask that
was loosely capped and then incubated for 24 h on a gyratory
shaker at 300 rpm at 30°C. Cell-free aliquots of the supematant for
analysis were obtained by centrifugation. For cell recycle experi-
ments, the supematant was drained as completely as possible from
the centrifuged cells, the cells were resuspended in 15 ml of
supplemented liquor, and the incubation repeated.
Enzyme Microb. Technol.,
sulfite liquor acetic acid removal yeast: H. Schneider
Performance of xylose-fermenting yeasts in acetic
acid-depleted hardwood liquor
For ethanol production experiments, liquor that had been subject to
fermentation by the mutant was centrifuged at 12,000 g to remove
cells, the liquor supplemented with solid Yeast Nitrogen Base
(Difco) to 0.67% (w/v), and the pH adjusted so that after tyndal-
lization, the pH was 5.c5.8.
Experiments with P. tannophilus employed 15 ml of acetic
acid-depleted liquor in a loosely capped 125ml Erlenmeyer Bask
shaken at 100 rpm, 3O”C, and an initial pH of 5.0 for 24 h. Ex-
periments with P. stipitis used 10 ml of liquor in a 100-m] flask
filled with air, sealed and shaken at 180 rpm, 30°C and an initial
pH of 5.8 for 16 h. Experiments with Y. stipitis employed 10 ml of
liquor in a 40-ml vial sealed with a rubber septum through which
air was pumped at the rate of 0.1 ml min.’ while the vial was
shaken at 215 rpm, 3O”c, and an initial pH of 5.85. The inocula
used with all three organisms were grown to late log phase in
0.67% Yeast Nitrogen Based containing 4% o-xylose. The cells
were removed from the growth medium by centrifugation and then
resuspended in liquor to the density in the growth medium prior to
harvesting.
In experiments for measuring the growth of the wxylose-
fermenting yeasts in acetic-acid depleted liquor, IO ml of acetic
acid-depleted liquor was supplemented with solid Yeast Nitrogen
Base to 0.67%, the pH adjusted to 5.8, tyndallized, inoculated, and
placed in 250-m] loosely capped Erlenmeyer flasks that were then
kept on a gyratory shaker at 250 rpm at 30°C. Cell density was
measured after inoculation with an approximately 1% inoculum of
late log phase cells and at daily intervals afterwards. The inoculum
was grown in 2% o-xylose in 50 ml of 0.67% Yeast Nitrogen Base
for 3 days in 250-ml loosely capped Erlenmeyer flask kept at 30°C
on a gyratory shaker at 300 rpm.
Analytical methods
Acetic acid concentrations were determined by HPLC using an
Aminex HPX-87H column (Bio-Rad, Hercules, Calif. USA) with
0.01 N sulfuric acid as eluent with detection by refractive index.
Monosaccharides were determined by HPLC using two Aminex
HPX-87P columns (Bio-Rad, Hercules, Calif. USA) in series with
detection by refractive index. Quantification was carried out only
for o-glucose, o-galactose, pmannose, and o-xylose. L-arabi-
nose was omitted because it was present in relatively low concen-
tration and was not used to a significant extent by the organisms
employed. Precolumns for monosaccharide analysis consisted of
Aminex Garbo-C and the Aminex deashing system (Bio-Rad, Her-
cules, Calif. USA). Ethanol was determined using the carbohydrate
columns.
Results
Growth of mutant in Medium A
The production of quantities of cells required for experi-
ments with spent sulfite liquor within reasonable time pe-
riods in Medium A containing 0.4-0.8% acetic acid re-
quired relatively large inocula of cells grown on such media.
For example, a culture with a cell density of 9 x lo7
cells ml-’ could be grown in 3-4 days using a 3-5% (v/v)
inoculum from cultures in Medium A with cell densities of
5-9 x 10’ cells ml-‘. Experiments leading up to those re-
ported indicated that factors which increased the rate of
growth were increases in aeration rate, larger inoculum size,
and decreases in acetic acid concentration. The presence of
the relatively high concentration of ammonium phosphate
(0.5 M) added to minimize changes in pH associated with
acetic acid use increased the time to attain high cell densi-
1996, vol. 19, August 95
Papers

ties by about one-third, as estimated from growth experi- Table 1 Sugar concentrations before and after acetic acid re-
ments of the mutant in defined n-galactose media without moval with the mutant yeast

ammonium phosphate supplementation. Inocula from slants

used to store the mutant (1% peptone, 0.5% yeast extract, Sugar Before f% w/v) After (% w/v)

0.5% ethanol, 1.5% agar) were unsuitable for growing large

amounts of cells when time was a factor because of lag o-glucose 0.42 0.47
o-mannose 0.88 0.87
times of a week or more.
o-galactose 0.37 0.21
All acetic acid removal experiments described used cells o-xylose 2.33 2.23
from cultures with cell densities of 8-10 x IO’ cells ml-‘. Xylitol 0.0 0.1
Cells from cultures with higher cell densities were not used
because such cultures were in stationary phase, and prelimi-
nary experiments suggested that stationary phase cells did
not remove acetic acid as effectively. n-galactose. The concentration of D-xylose decreased from
2.33 to 2.23% (w/v), or to 96% of the original value. The
decrease in n-xylose concentration was accompanied by the
Removal of acetic acid from spent sulfite liquor
formation of 0.1% (w/v) xylitol. The decrease in n-xylose
The acetic acid concentration in 20% (w/w) hardwood li- concentration reflects the ability of some yeast strains to
quor is 0.68% (w/v). The target concentration chosen for
removal of acetic acid from hardwood liquor was 0.1%
(w/v) or less and the target time chosen was 24 h. Both
targets were achieved. Acetic acid concentrations of 0.04%
(w/v) or less were obtained within 24 h using either lx or 2x
the density of cells of the mutant grown in Medium A.
The rate of acid removal from hardwood-spent sulfite
liquor depended on the rate of aeration as demonstrated by
changing the rate of oxygen transfer by altering the ratio of
liquor to flask volume. For example, the use of a 250-ml
flask, 100 ml of liquor, and 2x cells required 3 days to
achieve the levels of acetic acid achieved after 24 h with 15
ml of liquor.
Acetic acid removal could be carried out on a 24 h cell
recycle basis, indicating that cells of the mutant could be
used repeatedly. Recyclability was demonstrated with 2x
cells and 15 ml portions of liquor for ten cycles by consis-
tent removal of acetic acid to levels of 0.04% or less. In the
course of some recycle experiments, acetic acid would be
removed incompletely in one of the cycles, as indicated by
acetic acid values up to 0.17% (w/v) but the next cycle
would give a low value of 0.04% or less. The transient high

I;(!
values were traced to inadvertent too tight closure of the
shake flasks caps, an event that decreased the access of
oxygen to the culture and hence the rate of acetic acid
removal.
During the course of acetic acid removal, the pH of the
liquor increased from 5.8-5.85 to 6.9-7.0. The magnitude
of the pH change depended on the amount of acetic acid
removed as determined in preliminary experiments. The pH
change associated with acetic acid removal was limited to
no more than 1.2 units by the buffer capacity of the phos-
phate-supplemented liquor. In the absence of such buffer
capacity, the pH change expected was greater than 2.2 units,
as indicated in preliminary experiments with sugar-acetic E
acid mixtures simulating the composition of hardwood-
spent sulfite liquor.
WV_ _ _

Small changes in sugar concentration occurred in the
course of the acetic acid removal fermentation. Typical
changes are shown in Table 1 for cycle #5 in a recycle
experiment using 2x cells. The corresponding chromato-
grams are in Figure 1. The concentrations of o-galactose
decreased from 0.37 to 0.21% (w/v) or to 57% of the origi-
nal value, a decrease expected because the mutant grows on
I&
Figure 1 Typical HPLC elution profiles for sugars before and
after treatment of hardwood-spent sulfite liquor for removal of
acetic acid with the mutant yeast. The upper chromatogram was
obtained before treatment and the lower after treatment (24 h
fermentation time, cycle #5). The dashed line was drawn to ap-
proximate the true baseline. A, o-glucose; B, o-xylose; C, o-ga-
lactose; D, o-mannose; E, xylitol

96 Enzyme Microb. Technol., 1996, vol. 19, August

 Spent
convert D-xylose to xylitol’ even though neither sugar is
used for growth.
The HPLC chromatograms also indicated that small
changes occurred in the concentration of D-glucose and
D-mannose. The concentration of &glucose increased from
0.42 to 0.47% (w/v) while the concentration of D-mannose
decreased from 0.88 to 0.87% (w/v). These small changes
are suggested as only apparent and due to inaccuracies as-
sociated with the evaluation of small changes in sugar con-
centration in spent sulfite liquor by HPLC. The inaccuracies
appear to arise from a combination of two major factors.
One is that the sugars elute as fused peaks on a sloping
baseline, The recording integrator detected the beginning of
peaks when the rate of change in apparent baseline values
exceeded preset values. The second factor is that it is likely
that changes occurred during the fermentation in the con-
centration of nonsugar components in the liquor that con-
tribute to the sloping baseline. These small changes in base-
line would slightly alter the point at which peaks were
judged to begin and end with consequent small changes in
apparent sugar concentration. Indications that changes oc-
curred in nonsugar components of the liquor during the 24-h
fermentation period consisted of darkening of the liquor
color. Additional evidence for the occurrence of changes in
nonsugar components was a decrease in pH when the liquor
was incubated and shaken as for a fermentation but with
cells omitted. The pH decreased from 5.8 to 5.6 within 24 h
and to 5.3 after 3 days.
Fermentation of xylose and hexoses in acetic
acid-depleted liquor
The inhibitory effects of acetic acid on D-xylose fermenta-
tion decreased greatly on treatment of the liquor with the
mutant as indicated by enhanced growth of D-xylose-
fermenting yeasts in the acetic acid depleted-liquor, the abil-
ity of the n-xylose-fermenting yeasts to utilize both hexoses
and n-xylose in the acetic acid-depleted liquor, and the
production of ethanol in association with the use of these
sugars.
Enhanced growth of D-xylose-fermenting yeasts in the
acetic acid-depleted liquor was indicated by increases in cell
density with time. Three or more additional doublings in
cell density occurred in the acetic acid-depleted liquor than
in the liquor that had not been pretreated with the mutant.
The results of typical experiments are shown in Table 2. The
additional doublings are highly significant in indicating
growth in acetic acid-depleted liquor, since their occurrence
is responsible for most of the biomass produced and the use
of most of the sugar.
Table 2 Effect of removal of acetic acid from hardwood liquor
on growth of xylose-fermenting yeasts
Acetic-acid
Untreated liquor depleted liquor
Yeast (No. of doublings) (No. of doublings)
P. tannophilus NRRL
Y-2460 4 7.4
Y. stipitis CBS 6776 6.4 9.2
Y. stipitis CBS 66278 2.5 7.5
Enzyme Microb. Technol.,
sulfite liquor acetic acid removal yeast: H. Schneider
The production of ethanol from both hexoses and D-xy-
lose in the acetic acid-depleted liquor was indicated by com-
parison of the amount of ethanol produced with the amount
expected on the basis of the sugar composition. The theo-
retical maximum amount of ethanol that can be produced
from the D-glucose, n-mannose, and o-galactose in acetic
acid-depleted liquor is 0.8%, taking the theoretical maxi-
mum as 0.51 g ethanol g-’ hexose. Ethanol produced in
excess of 0.8% (w/v) must therefore come from the fermen-
tation of b-xylose. Ethanol concentrations in excess of
0.8% (w/v) were obtained within 24 h; for example, 1.25%
(w/v) with Y. stipitis CBS No. 5776 in 16 h and 1.2% (w/v)
with P. tunnophilus NRRL Y-2460 in 23 h. Sugar use in all
of these experiments was 95% or greater of the amount
present originally. Taking sugar use in all cases to be 100%
and the theoretical maximum for ethanol from o-xylose to
be 0.51 g g-‘, the ethanol yields were 73, 65, and 62% of
theoretical with Y. stipitis ATCC No. 66278, P. stipitis CBS
No. 5776, and P. tunnophilus NRRL Y-2460. respectively.
Discussion
The results demonstrate the feasibility of using the S. cer-
evisiae mutant to remove acetic acid from hardwood-spent
sulfite liquor without appreciably altering sugar concentra-
tion. The results also show that the approach can be used to
remove acetic acid on a cell recycle basis within a 24-h time
frame and that both the hexoses and wxylose can be fer-
mented in acetic acid-depleted hardwood liquor within a
24-h time frame. The acetic acid removal process and the
subsequent xylose-fermentation process both require opti-
mization, factors of which are discussed below.
Acetic acid removal from hardwood liquor
Optimization requires attention to factors that affect the rate
of acetic acid use, such as cell density, aeration rate, and pH.
The aeration rate is particularly important because acetic
acid utilization requires oxygen and the utilization rate of
acetic acid will depend on the aeration rate. Hydrogen ion
concentration is of interest because it could influence the
rate of acetic acid use, and lower pH values are preferred
because they are less prone to bacterial contamination. A
suitable pH study might indicate conditions that would al-
low the acetic acid removal step to be carried out at an
initial pH of about 5.8 followed by controlled decreases in
pH as acetic acid is removed. The mutant used in the present
study was employed to demonstrate proof of principle. For
industrial purposes, several changes in its properties are
desirable. Elimination of auxotrophic requirements would
decrease media costs. Elimination of the ability to use o--ga-
lactose would preserve a sugar with the potential for fer-
mentation to ethanol. These changes can readily be carried
out by classical genetic methods.
The mutant used converted a small fraction of the D-xy-
lose to xylitol which is effectively nonfermentable under the
conditions used. Decreasing the time for acetic acid removal
by fermentation might minimize the loss of D-xylose be-
cause the biochemistry of acetic acid and o--xylose metabo-
lism are probably independent. It might also be feasible to
transfer the mutations that give the acetic acid depletion
1996, vol. 19, August 97
Papers
properties of interest to a strain of S. cerevisiae that is a very
poor converter of o-xylose to xylitol.
For industrial purposes, the revertability of the kinase
mutations employed’6 need to be studied. Long-term culture
of the mutant on hexose-containing media would result in
the accumulation and eventual overgrowth by revertants
which would cause the strain to lose its desired properties.
Difficulties due to reversion were obviated in the present
study by growing cultures for inocula in the presence of
2-deoxyglucose which kills cells that metabolize o-glucose
or o-mannose.i6 The absence of difficulties from reversion
in the present study was demonstrated by the lack of use of
significant amounts of o-glucose and o-mannose even in
cell-recycle experiments. The use of 2deoxyglucose to
grow inocula cultures on an industrial scale is undesirable if
only because of increased production costs. The reversion
problem could be eliminated by the use of nonreverting
mutants which can be constructed using molecular genetic
methods.
Xylose fermentation
Optimization of the o-xylose fermentation requires consid-
eration of two interrelated issues: conditions that yield high
ethanol concentration within chosen fermentation times and
nutritional factors for recycling xylose-fermenting yeasts in
spent sulfite liquor. While ethanol could be obtained from
o-xylose, higher yields are of interest and elucidation of
conditions that give such yields will require information
about the effects of strain, pH, cell density, and aeration
rate. Hydrogen ion effects are important because the fer-
mentation rate and ethanol yield3*4,‘7 depend on pH. Lower
pH values are preferable in obviating bacterial contamina-
tion. Cell density and the aeration rate1,3 are extremely im-
portant because they play a key role in determining the rates
and yield of ethanol production as well as the rate of o-xy-
lose use. The present study used different cell density and
aeration conditions for the various o-xylose-fermenting
yeasts employed. These conditions were selected on the
basis of preliminary experiments that identified conditions
that would produce ethanol from the o-xylose and hexoses
in the acetic acid-depleted liquor within 24 h.
Nutritional factors might enter into considerations for
obtaining high ethanol yield during cell recycle because a
fairly dense suspension of cells might be required to provide
appropriate rates of ethanol production, and the liquor might
not have all of the nutrients required to maintain optimal
fermentation. Deficiency of nutrients during the ethanol
production step might be caused by utilization of these nu-
trients during the acetic acid-removal step.Candidates for
nutrients depleted during the acetic acid-removal step are
trace metals such as manganese and zinc which are present
in wood18 and are required in minute amounts for yeast
growth and function. I9 Other nutrients that will have to be
considered are the vitamins biotin and thiamine. P. tan-
nophilus requires both vitamins for growth,2o Cundidu she-
hatue requires only biotin,21 and Y. stipitis requires only
biotin.20 A deficiency in these vitamins can have negative
effects on ethanol productivity and the rate of sugar use2o,21
and supplementation with biotin alone can have an appre-
ciable effect.21 Issues relating to nutrient requirements dur-
ing the ethanol production step were obviated in the present
98 Enzyme Microb. Technol., 1996, vol. 19, August
study by supplementing the acetic acid-depleted liquor with
Yeast Nitrogen Base.
Acknowledgements
This work was supported in part by Temfibre, Inc. and
Energy, Mines and Resourses Canada (DSS Contract File
No. 2344OO-9002/01-SZ).
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