DOMBEK AND INGRAM
J 100 _ 0
z 80 0
10 15 ETHANOL (%V/V)
FIG. 6. Effects of added ethanol on ApH. Cells were removed from various stages of batch fermentation (indicated on graph), h a r v e s t e d , a n d s u s p e n d e d i n f r e s h m e d i u m c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l a t 3 0 ° C f o r 1 0 m i n , a n d A p H determined. was
Why then does the rate of glycolysis in viable yeast cells decline during batch fermentation? Two nutritional factors have been identified previously which reduced but did not eliminate the ethanol-associated decline in activity (7, 11, 12). Our results with added and accumulated ethanol indi- cate that physiological changes such as ethanol damage, rather than an immediately reversible effect of ethanol, appear responsible. Added ethanol inhibited fermentation, but washing did not restore full activity. Similarly, the replacement of ethanol-containing broth from the middle to later stages of fermentation with fresh medium did not immediately restore fermentative activity. The exposure of cells to ethanol in some way damaged their ability to produce ethanol. The extent of this damage appears related to both ethanol concentration and the duration of exposure.
We have examined cell viability, internal pH, and individ- ual enzymes involved in glycolysis and alcohologenesis as sites for changes (including ethanol damage) which could be responsible for the loss of ethanol productivity during batch fermentation. No appreciable loss of cell viability was ob- served during 48-h batch fermentations. The activities of glycolytic and alcohologenic enzymes measured in vitro remained high and did not appear limiting, consistent with earlier reports of the persistence of hexokinase and alcohol dehydrogenase activity (16). The specific activities of many of these continued to increase even after increases in total cell protein had ended, suggesting that they may be prefer- entially synthesized. Only a modest loss of total activity (per milliliter) was observed during the latter stages of fermenta- tion, consistent with a low rate of turnover of these en-
The internal pH of the cell was maintained near neutrality despite acidification of the broth and the accumulation of over 12% ethanol. This latter observation was contrary to expectation based upon earlier studies with cells suspended in water (6). These earlier studies had demonstrated that ethanol enhanced the leakage of protons (6), with an acidi- fication of the cytoplasm below the optimal pH for glycolytic and alcohologenic enzymes. Although such enhanced leak- age may also occur in fermentation broth, the maintenance of a high internal pH in broth containing ethanol indicates that such leakage must be offset by the action of hydrogen ion pumps such as ATPase.
Cells from the later stages of fermentation resistant to inhibition by ethanol and to the disruptive effects of ethanol on membrane integrity (as measured by proton leakage). During batch fermentation, cells may be undergo- were more
APPL. ENVIRON. MICROBIOL.
ing progressive adaptations to accumulated ethanol. Changes in the lipid composition of yeast cell membranes have been observed in response to accumulated ethanol and have been proposed as an important factor involved in such adaptation (2, 7, 15).
The results of our investigations do not identify the cause for the decline in fermentative activity during batch fermen- tation but rather narrow the range of remaining factors. Although the activities of glycolytic and alcohologenic en- zymes assayed in vitro under substrate-saturating conditions remained high during batch fermentation, the in vivo activ- ities of these enzymes within the cell cannot be accurately predicted. The activities of some of these are subject to modulation by allosteric effectors in addition to constraints imposed by the availability of individual substrates, cofac- tors, and coenzymes. Further studies are now under way to explore the levels of these low-molecular-weight intracellu- lar constituents.
This research has been supported in part by the Florida Agricul- tural Experiment Station (publication no. 7838), by grants from the Department of Energy, Office of Basic Energy Sciences (FG05- 86ER3574) and the National Science Foundation (DMB 8204928), and by the Department of Agriculture, Alcohol Fuels Program (86-CRCR-1-2134).
1. Andreason, A. A., and T. J. B. Stier. 1954. Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid re- quirement for growth in defined medium. J. Cell. Comp. Phys- iol. 43:271-281. 2. Beavan, M. J., C. Charpentier, and A. H. Rose. 1982. Produc- tion and tolerance of ethanol in relation to phospholipid fatty- acyl composition in Saccharomyces cerevisiae NCYC 431. J. Gen. Microbiol. 128:1447-1455. 3. Bernt, E., and I. Gutman. 1971. Ethanol determination with alcohol dehydrogenase, p. 1499-1502. In H. U. Bergermeyer (ed.), Methods of enzymatic analysis, vol. 3. Academic Press, Inc., New York. 4. Buttke, T. M., S. D. Jones, and K. Bloch. 1980. Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae. J. Bacteriol. 144:124-130. 5. Buttke, T. M., and A. L. Pyle. 1982. Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 152:747-756. 6. Cartwright, C. P., J.-R. Juroszek, M. J. Beavan, F. M. S. Ruby, S. M. F. De Morais, and A. H. Rose. 1986. Ethanol dissipates the proton-motive force across the plasma membrane of Saccharo- myces cerevisiae. J. Gen. Microbiol. 132:369-377. 7. Casey, G. P., and W. M. Ingledew. 1986. Ethanol tolerance in yeasts. Crit. Rev. Microbiol. 13:219-290. 8. Casey, G. P., C. A. Magnus, and W. M. Ingledew. 1984. High-gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appi. Environ. Microbiol. 48:639-646. 9. Clifton, D., S. B. Weinstock, and D. G. Fraenkel. 1978. Glycolysis mutants in Saccharomyces cerevisiae. Genetics 88: 1-11. 10. Dombek, K. M., and L. 0. Ingram. 1985. Determination of intracellular concentration of ethanol in Saccharomyes cerevi- siae during fermentation. Appl. Environ. Microbiol. 51:197- 200. 11. Dombek, K. M., and L. 0. Ingram. 1986. Nutrient limitation as a basis for the apparent toxicity of low levels of ethanol during fermentation. J. Ind. Microbiol. 1:219-225. 12. Dombek, K. M., and L. 0. Ingram. 1986. Magnesium limitation and its role in the apparent toxicity of ethanol during yeast fermentation. Appl. Environ. Microbiol. 52:975-981. 13. Guijarro, J. M., and R. Lagunas. 1984. Saccharomyces cerevi-
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