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Selective Removal of Acetic Acid From Hardwood-Spent Sulfite Liquor Using A Mutant Ye
Selective Removal of Acetic Acid From Hardwood-Spent Sulfite Liquor Using A Mutant Ye
Selective Removal of Acetic Acid From Hardwood-Spent Sulfite Liquor Using A Mutant Ye
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
for the metabolism of carbon sources other than D-glucose, Performance of xylose-fermenting yeasts in acetic
such as acetic acid and r+galactose, are repressed to low acid-depleted hardwood liquor
levels when D-glucose is present in the medium. Thus, these
For ethanol production experiments, liquor that had been subject to
carbon sources are not used by yeasts with a functional fermentation by the mutant was centrifuged at 12,000 g to remove
hexokinase II gene in the presence of u-glucose unless the cells, the liquor supplemented with solid Yeast Nitrogen Base
concentration of u-glucose is very low. The inability of the (Difco) to 0.67% (w/v), and the pH adjusted so that after tyndal-
mutant to use P-xylose and t_--arabinose is important to lization, the pH was 5.c5.8.
ensure that these sugars are not utilized by the derepressed Experiments with P. tannophilus employed 15 ml of acetic
mutant yeast. 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
Materials and methods 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
Strains 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
The mutant yeast employed, S. cerevisiae YGSCD 308.3 (hnkl. shaken at 215 rpm, 3O”c, and an initial pH of 5.85. The inocula
hxk2, glkl, adel, trpl, hisl. metll), was maintained on slants of used with all three organisms were grown to late log phase in
1% peptone, 0.5% yeast extract, 0.5% ethanol, and 1% agar. The 0.67% Yeast Nitrogen Based containing 4% o-xylose. The cells
u-xylose-fermenting yeasts employed, Pachysolen tannophilus were removed from the growth medium by centrifugation and then
NRRL Y-2460, Pichia stipitis CBS No. 5776, and Yamadazyma resuspended in liquor to the density in the growth medium prior to
stipitis ATCC No. 66278 were maintained on nutrient agar. harvesting.
In experiments for measuring the growth of the wxylose-
fermenting yeasts in acetic-acid depleted liquor, IO ml of acetic
Production of mutant yeast for acetic acid removal 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
Growth Medium. The medium used to grow large quantities of
kept on a gyratory shaker at 250 rpm at 30°C. Cell density was
cells of the mutant for use in the removal of acetic acid from
measured after inoculation with an approximately 1% inoculum of
hardwood-spent sulfite liquor, referred to as Medium A, had the
late log phase cells and at daily intervals afterwards. The inoculum
following composition: 2% peptone, 0.5% yeast extract, 1% n-ga-
was grown in 2% o-xylose in 50 ml of 0.67% Yeast Nitrogen Base
lactose, O&0.8% (w/v) acetic acid as specified, 0.4 M monobasic
for 3 days in 250-ml loosely capped Erlenmeyer flask kept at 30°C
ammonium phosphate, and 0.06% (w/v) 2-deoxyglucose. The ini-
on a gyratory shaker at 300 rpm.
tial pH was 5.5. Inocula were grown from slants in the same
medium. Analytical methods
Acetic acid concentrations were determined by HPLC using an
Growth conditions. Medium A (15 ml) was placed in a 25-m] Aminex HPX-87H column (Bio-Rad, Hercules, Calif. USA) with
loosely-capped Erlenmeyer flask, inoculated, and kept on a gyra- 0.01 N sulfuric acid as eluent with detection by refractive index.
tory shaker at 300 rpm and 30°C. Inocula consisted of aliquots of Monosaccharides were determined by HPLC using two Aminex
cultures in medium A with cell densities between 5-9 x 10’ cells HPX-87P columns (Bio-Rad, Hercules, Calif. USA) in series with
ml-‘. 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-
Acetic acid removal by fermentation 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-
Preparation of liquor. Steam-stripped hardwood-spent sulfite li-
cules, Calif. USA). Ethanol was determined using the carbohydrate
quor supplied by Tembec Inc. (Temiskaming, Canada) was diluted
columns.
with water to 20% (w/w) solids and supplemented with phosphate
(0.5% w/v monobasic ammonium phosphate) and 40 fig ml-i of
Results
the auxotrophic requirements of the mutant. The liquor was ster-
ilized using tyndallization by heating the supplemented liquor to Growth of mutant in Medium A
70°C for 1 h. The pH decreased on tyndallization, and in order to The production of quantities of cells required for experi-
obtain a final pH of 5.8-5.85, the pH was adjusted to 6.3 with
ments with spent sulfite liquor within reasonable time pe-
concentrated ammonia prior to heating.
riods in Medium A containing 0.4-0.8% acetic acid re-
quired relatively large inocula of cells grown on such media.
Fermentation protocol. Cells of the mutant grown in Medium A For example, a culture with a cell density of 9 x lo7
were centrifuged at 12,000 g for 10 min at room temperature in a cells ml-’ could be grown in 3-4 days using a 3-5% (v/v)
50-m] plastic centrifuge tube. The supematant was poured off and inoculum from cultures in Medium A with cell densities of
the cells resuspended in 15 ml of supplemented liquor. The resus-
5-9 x 10’ cells ml-‘. Experiments leading up to those re-
pended cells were transferred to a 250-m] Erlenmeyer flask that
ported indicated that factors which increased the rate of
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
growth were increases in aeration rate, larger inoculum size,
analysis were obtained by centrifugation. For cell recycle experi- and decreases in acetic acid concentration. The presence of
ments, the supematant was drained as completely as possible from the relatively high concentration of ammonium phosphate
the centrifuged cells, the cells were resuspended in 15 ml of (0.5 M) added to minimize changes in pH associated with
supplemented liquor, and the incubation repeated. acetic acid use increased the time to attain high cell densi-
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
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_ _ _
I&
Small changes in sugar concentration occurred in the
course of the acetic acid removal fermentation. Typical Figure 1 Typical HPLC elution profiles for sugars before and
changes are shown in Table 1 for cycle #5 in a recycle after treatment of hardwood-spent sulfite liquor for removal of
acetic acid with the mutant yeast. The upper chromatogram was
experiment using 2x cells. The corresponding chromato-
obtained before treatment and the lower after treatment (24 h
grams are in Figure 1. The concentrations of o-galactose fermentation time, cycle #5). The dashed line was drawn to ap-
decreased from 0.37 to 0.21% (w/v) or to 57% of the origi- proximate the true baseline. A, o-glucose; B, o-xylose; C, o-ga-
nal value, a decrease expected because the mutant grows on lactose; D, o-mannose; E, xylitol
convert D-xylose to xylitol’ even though neither sugar is The production of ethanol from both hexoses and D-xy-
used for growth. lose in the acetic acid-depleted liquor was indicated by com-
The HPLC chromatograms also indicated that small parison of the amount of ethanol produced with the amount
changes occurred in the concentration of D-glucose and expected on the basis of the sugar composition. The theo-
D-mannose. The concentration of &glucose increased from retical maximum amount of ethanol that can be produced
0.42 to 0.47% (w/v) while the concentration of D-mannose from the D-glucose, n-mannose, and o-galactose in acetic
decreased from 0.88 to 0.87% (w/v). These small changes acid-depleted liquor is 0.8%, taking the theoretical maxi-
are suggested as only apparent and due to inaccuracies as- mum as 0.51 g ethanol g-’ hexose. Ethanol produced in
sociated with the evaluation of small changes in sugar con- excess of 0.8% (w/v) must therefore come from the fermen-
centration in spent sulfite liquor by HPLC. The inaccuracies tation of b-xylose. Ethanol concentrations in excess of
appear to arise from a combination of two major factors. 0.8% (w/v) were obtained within 24 h; for example, 1.25%
One is that the sugars elute as fused peaks on a sloping (w/v) with Y. stipitis CBS No. 5776 in 16 h and 1.2% (w/v)
baseline, The recording integrator detected the beginning of with P. tunnophilus NRRL Y-2460 in 23 h. Sugar use in all
peaks when the rate of change in apparent baseline values of these experiments was 95% or greater of the amount
exceeded preset values. The second factor is that it is likely present originally. Taking sugar use in all cases to be 100%
that changes occurred during the fermentation in the con- and the theoretical maximum for ethanol from o-xylose to
centration of nonsugar components in the liquor that con- be 0.51 g g-‘, the ethanol yields were 73, 65, and 62% of
tribute to the sloping baseline. These small changes in base- theoretical with Y. stipitis ATCC No. 66278, P. stipitis CBS
line would slightly alter the point at which peaks were No. 5776, and P. tunnophilus NRRL Y-2460. respectively.
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
Discussion
fermentation period consisted of darkening of the liquor The results demonstrate the feasibility of using the S. cer-
color. Additional evidence for the occurrence of changes in evisiae mutant to remove acetic acid from hardwood-spent
nonsugar components was a decrease in pH when the liquor sulfite liquor without appreciably altering sugar concentra-
was incubated and shaken as for a fermentation but with tion. The results also show that the approach can be used to
cells omitted. The pH decreased from 5.8 to 5.6 within 24 h remove acetic acid on a cell recycle basis within a 24-h time
and to 5.3 after 3 days. frame and that both the hexoses and wxylose can be fer-
mented in acetic acid-depleted hardwood liquor within a
Fermentation of xylose and hexoses in acetic
24-h time frame. The acetic acid removal process and the
acid-depleted liquor subsequent xylose-fermentation process both require opti-
The inhibitory effects of acetic acid on D-xylose fermenta- mization, factors of which are discussed below.
tion decreased greatly on treatment of the liquor with the
mutant as indicated by enhanced growth of D-xylose- Acetic acid removal from hardwood liquor
fermenting yeasts in the acetic acid depleted-liquor, the abil-
ity of the n-xylose-fermenting yeasts to utilize both hexoses Optimization requires attention to factors that affect the rate
and n-xylose in the acetic acid-depleted liquor, and the of acetic acid use, such as cell density, aeration rate, and pH.
production of ethanol in association with the use of these The aeration rate is particularly important because acetic
sugars. acid utilization requires oxygen and the utilization rate of
Enhanced growth of D-xylose-fermenting yeasts in the acetic acid will depend on the aeration rate. Hydrogen ion
acetic acid-depleted liquor was indicated by increases in cell concentration is of interest because it could influence the
density with time. Three or more additional doublings in rate of acetic acid use, and lower pH values are preferred
cell density occurred in the acetic acid-depleted liquor than because they are less prone to bacterial contamination. A
in the liquor that had not been pretreated with the mutant. suitable pH study might indicate conditions that would al-
The results of typical experiments are shown in Table 2. The low the acetic acid removal step to be carried out at an
additional doublings are highly significant in indicating initial pH of about 5.8 followed by controlled decreases in
growth in acetic acid-depleted liquor, since their occurrence pH as acetic acid is removed. The mutant used in the present
is responsible for most of the biomass produced and the use study was employed to demonstrate proof of principle. For
of most of the sugar. industrial purposes, several changes in its properties are
desirable. Elimination of auxotrophic requirements would
Table 2 Effect of removal of acetic acid from hardwood liquor decrease media costs. Elimination of the ability to use o--ga-
on growth of xylose-fermenting yeasts lactose would preserve a sugar with the potential for fer-
mentation to ethanol. These changes can readily be carried
Acetic-acid out by classical genetic methods.
Untreated liquor depleted liquor The mutant used converted a small fraction of the D-xy-
Yeast (No. of doublings) (No. of doublings)
lose to xylitol which is effectively nonfermentable under the
conditions used. Decreasing the time for acetic acid removal
P. tannophilus NRRL
by fermentation might minimize the loss of D-xylose be-
Y-2460 4 7.4
Y. stipitis CBS 6776 6.4 9.2 cause the biochemistry of acetic acid and o--xylose metabo-
Y. stipitis CBS 66278 2.5 7.5 lism are probably independent. It might also be feasible to
transfer the mutations that give the acetic acid depletion
properties of interest to a strain of S. cerevisiae that is a very study by supplementing the acetic acid-depleted liquor with
poor converter of o-xylose to xylitol. Yeast Nitrogen Base.
For industrial purposes, the revertability of the kinase
mutations employed’6 need to be studied. Long-term culture Acknowledgements
of the mutant on hexose-containing media would result in This work was supported in part by Temfibre, Inc. and
the accumulation and eventual overgrowth by revertants Energy, Mines and Resourses Canada (DSS Contract File
which would cause the strain to lose its desired properties. No. 2344OO-9002/01-SZ).
Difficulties due to reversion were obviated in the present
study by growing cultures for inocula in the presence of References
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