Accumulation on the Viability of Saccharomyces

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1976, p. 158-162 Copyright 1976 American Society for Microbiology Vol. 31, No. 2 Printed in U.SA. Influence of the Rate of Ethanol Production and Accumulation on the Viability of Saccharomyces cerevisiae in "Rapid Fermentation" I TILAK W. NAGODAWITHANA AND KEITH H. STEINKRAUS* Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456 Received for publication 6 June 1975 Whereas "rapid fermentation" of diluted clover honey (25 Brix) fortified with yeast nutrients using 8 x 10' brewers' yeast cells per ml resulted in an ethanol content of 9.5% (wt/vol; 12% vol/vol) in 3 h at 30 C, death rate of the yeast cells during this period was essentially logarithmic. Whereas 6 h was required to reach the same ethanol content at 15 C, the yeast cells retained their viability. Using a lower cell population (6 x 107 cells/ml), a level at which the fermentation was no longer "rapid," the yeast cells also retained their viability at 30 C. Ethanol added to the medium was much less lethal than the same or less quantities of ethanol produced by the cell in "rapid fermentation." It was considered possible that ethanol was produced so rapidly at 30 C that it could not diffuse out of the cell as rapidly as it was formed. The hypothesis was postulated that ethanol accumulating in the cell was contributing to the high death rate at 30 C. It was found that the intracellular ethanol concentration reached a level of approximately 2 x 1011 ethanol molecules/cell in the first 30 min of fermentation at 30 C. At 15 C, with the same cell count, intracellular ethanol concentration reached a level of approximately 4 x 101" ethanol molecules/cell and viability remained high. Also, at 30 C with a lower cell population (6 x 107 cells/ml), under which conditions fermentation was no longer "rapid," intracellular ethanol concentration reached a similar level (4 x 10"' molecules ethanol/cell) and the cells retained their viability. Alcohol dehydrogenase (ADH) lost its activity in brewers' yeast under conditions of"rapid fermentation" at 30 C but retained its activity in cells under similar conditions at 15 C. ADH activity was also retained in fermentations at 30 C with cell populations of 6 x 107/ml. It would appear that an intracellular level of about 5 x 101" ethanol molecules/cell is normal and that this level does not damage either cell viability or ADH activity. Higher intracellular ethanol concentrations, such as 2 x 1011 molecules ethanol/cell (a fourfold increase in intracellular ethanol concentration), are accompanied by inactivation of ADH and loss of cell viability. By using 7 x 108 cells of brewers' yeast per ml, 25 Brix honey solutions fortified with nitrogen, phosphate, and minerals were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C (6). Under these conditions of"rapid fermentation," death rate of the cells was high, with only 2.1% of the cells surviving at the end of the fermentation. When the fermentation temperature was decreased to 15 C and the content of dissolved oxygen in the medium was maintained at 13%, fermentation time to 9.5% (wt/vol) ethanol increased to 6 h, but the yeast cells retained their viability (6). Rapid fermentations using high cell populations require Journal Paper no. 2128, New York State Agricultural Experiment Station. higher levels of vitamins and minerals than slower fermentations at lower temperatures or those using smaller populations (Nagodawithana and Steinkraus, unpublished data). However, even in the presence of the higher levels of vitamins and minerals, brewers' yeast, the most rapid fermenting yeast we have encountered, loses its viability much more rapidly at 30 C than it does at lower temperatures. The effect of ethanol on yeast metabolism has been studied (1, 3, 9; F. F. Pironti, Ph.D. thesis, Cornell Univ., Ithaca, N.Y., 1971). Under Pironti's conditions, ethanol appeared to inhibit yeast cell growth at relatively low concentration, whereas the fermentative activity of the cells seemed to tolerate ethanol until its concentration approached approximately 20% (vol/ 158

VOL. 31, 1976 VIABILITY LOSS IN "RAPID FERMENTATION" 159 vol; 16.26% wt/vol). The effect of ethanol on growth rates and ethanol production was shown to be noncompetitive inhibition, confirmed by Lineweaver-Burk plots (1). Inhibition by ethanol was investigated after the concentration of yeast cells reached maximum value in the shochu-molded rice fermentation (5). The decrease in the rate of ethanol production was shown to be directly related to the decrease in the number of viable yeast cells. Added thiamine enabled yeast enzymes to tolerate the amount of ethanol the cell would normally produce but not higher concentrations (9) Ṫhe experiments reported were undertaken to attempt to determine the part rate of ethanol production or accumulation contributes to loss of viability in "rapid fermentations" with brewers' yeast at 30 C. MATERIALS AND METHODS Yeast strains. A brewers' strain ofsaccharomyces cerevisiae was kindly supplied by Mario Frati, Genesee Brewing Co., Rochester, N.Y. The brewers' yeast was obtained as a solid paste containing from 3.85 x 109 to 4.04 x 109 cells/g. The yeast was washed with citrate-phosphate buffer, ph 6.8, and centrifuged to recover the cells to be used in the experiments. Yeast counts. Based upon the number of yeast cells desired in the inoculated medium, a given weight, generally 1 kg of yeast paste, was slurried with 1 liter of the medium. After thorough dispersion, a 1-ml sample was diluted serially and plated in duplicate to obtain the initial viable count. The culture medium used for pour plates contained 1.5% maltose, 1.5% malt extract (Difco), and 1.5% agar (Difco). The plates Wvere incubated at 30 C for 48 h, and the final colony count was taken as the average of the two plates for the dilution containing 30 to 300 colonies/plate. Calculations involving cell populations were based upon viable plate counts. The percentage of viability was determined by dividing the viable count at the desired fermentation time of the initial viable count. If multiplication occurred, the percentage of viability thus rose above 100%. Fermentation medium. Clover honey stored at 1 C was diluted with water to provide the 250 Brix sugar substrate. A basal level of 0.25 g of Actiferm per liter (Budde & Westermann, New York, N.Y.), a yeast vitamin mixture, was added to the fermentation medium and this level is referred to as X. The 0.25 g of Actiferm per liter contained 12.5 ug of biotin, 250 Mg of pyridoxine, 1.87 mg of meso-inositol, 2.5 mg of calcium pantothenate, 5 mg of thiamine, 25 mg of peptone, and 215 mg of ammonium sulfate. The medium was also supplemented with 1.0 g of (NH4)2SO4, 0.5 g of K:1P04, 0.2 g of MgCl2, 0.05 g of NaHSO4, and 5.0 g of citric acid per liter as per formula I (10). The basic level of mineral saltscitric acid supplement was referred to as Y. Certain fermentations in this study were conducted using a 2Y level of mineral salt-citric acid supplement and 4X concentration (1 g of vitamins [Actiferm] per liter). The ph of the fermentation medium was adjusted to 4.2. It then was pasteurized at 76.5 C for 30 min and cooled to fermentation temperature before inoculation. Apparatus. A Microferm laboratory fermentor model 214 (New Brunswick Scientific Co., New Brunswick, N.J.) with 14-liter fermentors (working capacity, 7 liters) was used. The fermentations were carried out at 15 or 30 C and with 13% dissolved oxygen (unless otherwise specified) with agitation at 300 rpm. A fermentor sampler (New Brunswick model S21) was used to sample medium during fermentation. A dissolved oxygen controller model DO-60 by New Brunswick Co., was used in conjunction with the above to measure and control oxygen level. Preparation of cell-free extract. Samples (25 ml) were withdrawn from the fermenting medium at desired time intervals and chilled immediately using a dry ice-ethanol mixture; all subsequent operations were carried out at 0 to 4 C. Cells were harvested by centrifugation at 9,000 x g for 10 min in Sorvall centrifuge, model SS-3, washed twice with 12.5-ml portions of 0.1 M citrate-phosphate buffer (ph 6.8), and centrifuged at 9,000 x g for 10 min. The volumes of all three supernatants were determined, and 1-ml samples were set aside for ethanol analysis. The cell pellet was then suspended in 12.5 ml of 0.1 M citrate-phosphate buffer, ph 6.8, and 1 ml of the thoroughly mixed suspension was suitably diluted for determination of cell number by direct microscopic count using an American Optical Bright Line hemacytometer. The remaining yeast suspension was disintegrated by sonic oscillation (Sonifier cell disruptor, model W 185D, Heat Systems-Ultrasonics Inc., Plainview, N.Y.) for a total of 6 min, subjecting the cell to a period of 30 s of sonic oscillation followed by a period of 1 min of cooling. A 1-ml amount of the sonicated suspension was suitably diluted to determine the population of unruptured cells by direct microscopic count. The treatment ruptured approximately 60% of the cells in the suspension. The cell debris was removed by centrifuging at 32,800 x g for 15 min. Having measured the volume of the cell extract, it was held at 1 C until further use. The extract was subsequently analyzed for ethanol by gas-liquid chromatography. The ethanol content of the extract was used to calculate the ethanol content of the ruptured cells. Moles of ethanol were converted to molecules of ethanol per cell by multiplying by Avogadros number and dividing by the total number of ruptured cells. A Carle gas chromatograph, model 9000, equipped with a flame ionization detector was used for the separation and quantitation of ethanol using procedures described earlier (6). ADH assay. Alcohol dehydrogenase (ADH) activity was measured by a procedure based on previous methods (2, 7), modified as follows. A 3-ml amount of buffer, ph 8.8 (2), containing 10 Ml of 1.5 mg of ethanol per ml and 0.05 ml of the cell-free extract,

160 NAGODAWITHANA AND STEINKRAUS was transferred directly to a cuvette (1-cm light path), and the optical density at 340 nm was determined. Twenty microliters of 0.15 M nicotinamide adenine dinucleotide (NAD) was transferred to the cuvette with a syringe and mixed immediately with a plastic rod, and the change in optical density at 340 nm was monitored every 30 s during the first 3 min. From the standard curve for reduced NAD (NADH), the optical density reading at 340 nm was directly converted to the amount of NADH plus H+ formed in the cuvette with time for the particular run. The initial rate of NADH plus H+ formed per minute was calculated from the graph, showing the increase of NADH plus H+ with time for the particular run. Since NAD and ethanol were added in excess, the reaction rate was dependent on the activity of ADH in the 0.05-ml cell-free extract added to the cuvette. Protein in the cell-free extract was determined by the method of Lowry et al. (4) using bovine albumin serum as the standard. The specific activity of ADH = micromoles of NADH plus H+ formed per minute/milligram of protein. RESULTS AND DISCUSSION Whereas "rapid fermentation" (defined as fermentation in which the ethanol content rises from 0 to 9.5% [wt/vol; 12% vol/vol] in 6 h or less) using 8 x 108 cells/ml at 30 C resulted in an ethanol content of 9.5% (wt/vol; 12% vol/vol) in 3 h, death rate of the yeast cells 9 0I5'C during this period was essentially logarithmic (Fig. 1). Although it required 6 h to reach the same ethanol content at 15 C, the yeast cells retained their viability. Using a lower cell population (6 x 107 cells/ml), a level at which the fermentation was no longer "rapid" at 30 C, the yeast cells also retained their viability. It was found that 9.4% (wt/vol) ethanol produced by the cells during fermentation at 30 C was much more lethal than even 13.8% (wt/vol) ethanol added to a similar cell population in the diluted honey base (Fig. 2). Only 15.5% of the cells inoculated survived 3 h of fermentation to 9.4% ethanol (wt/vol) at 30 C, whereas 57.5% of the cells inoculated survived 3 h in the same medium in which the ethanol content was increased to 12% (wt/vol) by addition of ethanol. The internal ethanol content of cells was determined during fermentation using 8 x 108 cells/ml at 30 C. It was found that the higher concentration of brewers' yeast cells (8 x 108 cells/ml) contained a higher ethanol content (1.6 x 101" ethanol molecules/cell) after 30-min fermentation at 30 C than the same concentration of cells fermenting at 15 C, which contained 3.9 x 10"' ethanol molecules/cell (Table 1). The lower population of cells (6.3 x 107 100 90 APPL. ENVIRON. MICROBIOL. Downloaded from http://aem.asm.org/ Ua 8 2 3 O~ E \0 10 30 C 7~@ @ 30 C 80 8% ETHANOL WN 70 60 V 3 0 C \ ~~~~~~~~(EXTERNAL) on March 16, 2019 by guest 94% ETHANOI W/V 10 (FERMENTATLON) FIG. 1. Effect of initial cell population on viable cell count during fermentation using brewers' yeast at 15 and 30 C. 13% dissolved oxygen (D.O.). 0 0 1 2 3 FIG. 2. Loss of viability of brewers' yeast cells at 30 C in 25 Brix honey solutions containing increasing concentrations of added (external) ethanol compared with loss of viability during rapid fermentation when ethanol is being produced by the cells.

VOL. 31, 1976 cells/ml) at 30 C had an internal ethanol content similar to the cells fermenting more slowly at 15 C (3.9 x 101' molecules ethanol/cell). This may be related to the relatively greater volume of free liquid with the lower cell population, which would facilitate diffusion of ethanol from the cells during fermentation. At 15 C the intracellular ethanol concentration did not increase above 5.3 x 10"' molecules/ cell during the first 2 h of rapid fermentation, whereas ethanol in the high cell population fermenting at 30 C remained at the 2 x 1011 molecules/cell level. Nor was there any significant increase in the number of ethanol molecules within the cells at the lower cell population level during the fermentation. The TABLE 1. Comparison of the molecules of ethanol found within yeast cells at different stages of fermentation Molecules of ethanol per average cell Time (h) 8 x 101 cells/ 8 x 10' cells/ 6.3 x 107 cells/ ml at 15 C ml at 30 C ml at 30 C (cells remain (cells die rap- (cells remain viable) idly) viable) 0 3.7 x 101"' 6.4 x 10"' 4.4 x 10"' 0.5 3.6 x 10"' 1.6 x 10' 3.9 x 10"' 1 4.9 x 10"' 2.0 x 10" 4.5 x 10"' 2 5.3 x 10"' 1.6 x 1011 5.0 x 10"' "Initial viable cell count of brewers' yeast cells..20.18i.16.14.12 U.10 Q.08 06o.04.02 0 1 2 3 FIG. 3. ADH activity of brewers' yeast as influenced by internal ethanol molecules per cell at 15 and 30 C using an initial cell count of 8 x 101 cellsl ml in a 250 Brix medium, 13% dissolved oxygen, with adjunct nutrients added at 2Y + 4X levels. VIABILITY LOSS IN "RAPID FERMENTATION" 161 1- u u; U) 0 4.x1011 10 20 30 FIG. 4. ADH activity of brewers' yeast as influenced by internal ethanol molecules per cell at 30 C using an initial cell count of 6.3 x 107 cellslml. fermentation, however, at this cell concentration was no longer rapid. As the ethanol concentration increased inside the cells in the 30 C fermentation carried out with initial cell count of 8 x 10' brewers' yeast cells/ml, the ADH activity rapidly decreased (Fig. 3). The concentration of ethanol was much lower in the cells at 15 or 30 C using a cell population of 6.3 x 107/ml, and there was no inactivation of ADH (Fig. 3 and 4). Thus, it can be concluded that ethanol accumulates within the brewers' yeast cells under conditions of "rapid fermentation" using a high cell population at 30 C. It would appear that an intracellular level of approximately 5 x 10"' ethanol molecules/cell is in the normal range in brewers' yeast cells. This level does not inactivate ADH or damage cell viability. Higher intracellular ethanol concentrations such as 2 x 1011 molecules of ethanol per cell (a fourfold increase in intracellular ethanol concentration) are accompanied by inactivation of ADH and loss of cell viability. LITERATURE CITED 1. Aiba, S., M. Shoda, and M. Nagatani. 1968. Kinetics of product inhibition in alcohol fermentation. Biotechnol. Bioeng. 10:845-864. 2. Bonnichsen, R. 1965. Ethanol determination with alcohol dehydrogenase and DPN, p. 285-287. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press Inc., New York. 3. Holzberg, I., R. K. Finn, and K. H. Steinkraus. 1967. A kinetic study of alcoholic fermentation of grape juice. Biotechnol. Bioeng. 9:413-427. z au

162 NAGODAWITHANA AND STEINKRAUS 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 5. Nagatani, M., Y. Kuba, and S. Sugania. 1969. Kinetics of product inhibition in shochu making. J. Ferment. Technol. 47:723-728. 6. Nagodawithana, T. W., C. Castellano, and K. H. Steinkraus. 1974. The effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations. Appl. Microbiol. 28:383-391. APPL. ENVIRON. MICROBIOL. 7. Racker, E. 1955. Alcohol dehydrogenase from Baker's yeast, p. 500-503. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York. 8. Rahn, 0. 1929. The decreasing rate of fermentation. J. Bacteriol. 18:207-226. 9. Rahn, 0. 1952. Acid and alcohol tolerance imparted by thiamine. Growth 16:59-63. 10. Steinkraus, K. H., and R. A. Morse. 1966. Factors influencing the fermentation of honey and mead production. J. Apic. Res. 5:17-26.