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VOL. 53, 1987

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FIG. 1. Alcohol production by strain KD2 during batch fermen- tation with 20% glucose and 0.5 mM magnesium sulfate.

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vested by centrifugation at 10,000 x g for 30 s at 4°C and washed in an equal volume of 50 mM potassium phosphate buffer (pH 7.4). All subsequent steps were carried out at 4°C. The pellet was suspended in the same buffer containing 2 mM mercaptoethanol and 2 mM EDTA and disrupted with 0.1-mm glass beads using a Mini-Bead Beater (Biospec Products, Bartlesville, Okla.; five periods of disruption, 1 min each, with cooling on ice between treatments). Cell debris was removed by centrifugation at 10,000 x g for 5 min, and the supernatant was assayed immediately for enzymatic activities. Only two enzymes at a time were assayed in each batch fermentation experiment to avoid potential problems which could result from storage of cells or extracts.

Pyruvate decarboxylase and all glycolytic enzymes were assayed spectrophotometrically by the methods of Maitra and Lobo (21) as modified by Clifton et al. (9). All enzymes were assayed under substrate-saturating conditions except triose phosphate isomerase, which was assayed with 1 mM substrate. The amounts of coupling enzymes were adjusted as needed to ensure a linear reaction rate. Alcohol dehydro- genase was assayed by measuring the oxidation of ethanol as described by Maitra and Lobo (21) but using a buffer at pH 8.7 containing 75 mM sodium pyrophosphate, 75 mM semicarbazide hydrochloride, and 21 mM glycine (3).

Determination of internal pH and membrane energization. The measurements of internal pH and At were performed using 7-['4C]benzoic acid and [3H-phenyl]tetraphenyl phosphonium bromide, respectively. Protocols were similar to those described by Cartwright et al. (6) except that cells were incubated in their native growth medium rather than distilled water and 0.4-,um-pore-size polycarbonate filters were used instead of mixed cellulose ester filters. Cell volumes were determined as previously described (10). These ranged from 2.23 ,il/mg of cell protein for cells removed from the 12-h stage of batch fermentation to 0.86 [lI/mg of cell protein for cells removed from the 48-h stage. As a control for adventitious binding of radioactive com- pounds, cells were permeabilized with a combination of ethanol, toluene, and Triton X-100 as described by Salmon (25), washed with 50 mM phosphate buffer, resuspended in native broth, and processed. This treatment resulted in a

ETHANOL PRODUCTION BY S. CEREVISIAE

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complete collapse of ApH and loss of membrane potential. Calculations were performed as described by Rottenberg ( 2 4 ) . M a t e r i a l s . Y e a s t e x t r a c t , p e p t o n e , a n d a g a r w e r e o b t a i n e d from Difco Laboratories, Detroit, Mich. Glucose, coupling enzymes, coenzymes, and substrates were purchased from Sigma Chemical Co., St. Louis, Mo. Inorganic salts were obtained from Fisher Scientific Co., Orlando, Fla. Absolute ethanol was supplied by AAPER Alcohol and Chemical Co., Shelbyville, Ky. Radioactive compounds were purchased from New England Nuclear Corp., Boston, Mass.

RESULTS

Reversibility of the decline in fermentative activity. Figure 1 shows a representative batch fermentation with 20% glucose beginning with a low inoculum. Growth as measured by cell protein was exponential for the first 12 h and became stationary between 18 and 24 h. During these growth peri- ods, relatively low concentrations of ethanol had accumu- lated (<5% [vol/vol]), well below the minimum inhibitory concentration of added ethanol for growth (8% [vol/vol]). Ethanol production proceeded exponentially for the initial 12 h (1% [vol/vol] accumulated ethanol).

Cells were removed at various times during batch fermen- tation, and the rate of ethanol production per milligram of cell protein was determined (Fig. 2). Cells were most active at the earliest times measured, 12 h, and declined by 50% after 24 h (6.5% [vol/vol] accumulated ethanol). Approxi- mately 40% of the fermentative activity remained after the accumulation of 10% (vol/vol) ethanol (30 g of remaining glucose per liter). The abrupt, final decline in activity reflects the near-complete exhaustion of glucose, the substrate. Removal of ethanol from cells by washing and suspending in fresh mcdium resulted in only a modest increase in fermen- tative activity in all but the highest level of accumulated ethanol. The apparent increase in activity in the cells which had accumulated 12.1% (vol/vol) ethanol was primarily due to the restoration of fermentable substrate glucose.

Loss of viability was examined as a possible cause for the failure of washing to restore full fermentative activity (data not shown). Cell number paralleled the increase in cell protein (Fig. 1) and increased exponentially for the initial 12 h, reaching a maximum of 3 x 108 cells per ml after 18 h. Cell number remained constant for the remaining period of fer-

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FIG. 2. Changes in fermentative activity of cells during batch fermentation. Fermentative activity is expressed as micromoles of carbon dioxide evolved per hour per milligram of cell protein. Symbols: 0, activity measured in native broth; 0, activity measured after cells were suspended in fresh medium containing 20% glucose.

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