Selective removal of acetic acid from hardwood-spent sulfite liquor using a mutant yeast

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: Spent sulfite liquor; acetic acid; Saccharomyces cerevisiae; o-xylose; yeast; hemicellulose hydrolysate

Introduction Yeasts are now available that convert hexoses and the pentose D-xylose to ethanol. l4 However, the development of economic hexose-xylose ethanol fermentations of lignocellulosic hydrolysates using yeasts is hindered, in part, by the presence of acetic acid5-‘* which inhibits ethanol production 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 xylose fermentation.5

The present paper describes a yeast fermentation that selectively removes acetic acid from a lignocellulosic hydrolysate, hardwood-spent sulfite liquor. The fermentation has the potential for application to lignocellulosic hydrolysates 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 1 I July 1995; revised 21 September 1995; accepted 21 Septem- ber 1995

The acetic acid-depletion fermentation depends on sub- jecting the lignocellulosic hydrolysate to aerobic fermentation 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 lignocellulosic 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 phosphorylate 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-fructose as substrates while glucokinase is specific for D-glucose.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 presence of a normally functional hexokinase II gene, enzymes

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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-galactose, O&0.8% (w/v) acetic acid as specified, 0.4 M monobasic ammonium phosphate, and 0.06% (w/v) 2-deoxyglucose. The initial 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 gyratory 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 liquor 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 resuspended 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 experiments, 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.

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 tyndallization, 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-arabinose was omitted because it was present in relatively low concentration 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 experiments with spent sulfite liquor within reasonable time pe- riods in Medium A containing 0.4-0.8% acetic acid required 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-

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ties by about one-third, as estimated from growth experiments of the mutant in defined n-galactose media without ammonium phosphate supplementation. Inocula from slants used to store the mutant (1% peptone, 0.5% yeast extract, 0.5% ethanol, 1.5% agar) were unsuitable for growing large amounts of cells when time was a factor because of lag times of a week or more.

All acetic acid removal experiments described used cells from cultures with cell densities of 8-10 x IO’ cells ml-‘. Cells from cultures with higher cell densities were not used because such cultures were in stationary phase, and preliminary experiments suggested that stationary phase cells did not remove acetic acid as effectively.

Removal of acetic acid from spent sulfite liquor

The acetic acid concentration in 20% (w/w) hardwood liquor 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 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 phosphate-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 acid mixtures simulating the composition of hardwood- spent sulfite liquor.

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 chromatograms are in Figure 1. The concentrations of o-galactose decreased from 0.37 to 0.21% (w/v) or to 57% of the original value, a decrease expected because the mutant grows on

Table 1 Sugar concentrations before and after acetic acid removal with the mutant yeast

Sugar Before f% w/v) After (% w/v)

o-glucose 0.42 0.47 o-mannose 0.88 0.87 o-galactose 0.37 0.21 o-xylose 2.33 2.23 Xylitol 0.0 0.1

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 formation of 0.1% (w/v) xylitol. The decrease in n-xylose concentration reflects the ability of some yeast strains to

I;(!.. I& E

WV_ _ _

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-galactose; D, o-mannose; E, xylitol

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

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 associated with the evaluation of small changes in sugar concentration 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 concentration of nonsugar components in the liquor that con- tribute to the sloping baseline. These small changes in baseline 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 occurred 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 fermentation 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 ability 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

Yeast

Acetic-acid Untreated liquor depleted liquor

(No. of doublings) (No. of doublings)

P. tannophilus NRRL Y-2460

Y. stipitis CBS 6776 Y. stipitis CBS 66278

4 7.4 6.4 9.2 2.5 7.5

The production of ethanol from both hexoses and D-xylose 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 theoretical 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 maximum as 0.51 g ethanol g-’ hexose. Ethanol produced in excess of 0.8% (w/v) must therefore come from the fermentation 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. cerevisiae mutant to remove acetic acid from hardwood-spent sulfite liquor without appreciably altering sugar concentration. 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 optimization, 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--galactose would preserve a sugar with the potential for fermentation to ethanol. These changes can readily be carried out by classical genetic methods.

The mutant used converted a small fraction of the D-xylose 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 because the biochemistry of acetic acid and o--xylose metabolism are probably independent. It might also be feasible to transfer the mutations that give the acetic acid depletion

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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 fermentation rate and ethanol yield3*4,‘7 depend on pH. Lower pH values are preferable in obviating bacterial contamination. Cell density and the aeration rate1,3 are extremely important because they play a key role in determining the rates and yield of ethanol production as well as the rate of o-xylose 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 nutrients 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. tannophilus 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 appreciable effect.21 Issues relating to nutrient requirements during the ethanol production step were obviated in the present

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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