SUMMARY Estimates of intracellular ethanol concentration in batch culturesof Saooharomyoes cerevisiae were significantly affected by continued fermentation during processing of the sample. Precooling of samples to 4°C and reduction of centrifugation time significantly reduced apparent intracellular ethanol concentration.Intracellular ethanol concentrations were substantially lower than in some previous reports and fell below the extracellular ethanol concentrations during the later stages of fermentation. I NTRODUCTI ONA number of recent reports suggest that the concentration of ethanol within fermenting yeast cells may be many times higher than the extracellular ethanol concentration

By feeding ethanol at various high rates to low cell density cultures of Saccharomyces cerevisiae it was shown that the sharp fall in viability when ethanol is produced during rapid fermentations is in part a direct consequence of the high rate of change of extracellular ethanol concentration. Nevertheless, the fall in viability in high cell density rapid fermentations which produced 98 g L(-1) ethanol in 3 h considerably exceeded that of control low cell density cultures to which ethanol was added at the same rate. This difference was shown to be not due to intracellular ethanol accumulation or to differences in glucose concentration between the cultures. The concentrations of a range of potentially toxic fatty acids, higher alcohols, and esters were measured during rapid fermentations, but when added at these concentrations to control cultures in the presence of ethanol they had no significant toxic effect. However, when rapid fermentations were conducted in rich medium containing 80 g L(-1) yeast extract, the apparent difference in toxicity of produced and added ethanol virtually disappeared. Magnesium was shown to be the component of yeast extract primarily responsible for this effect. The high rate of fall of viability when ethanol is rapidly produced is suggested to be partly due to the inability of the cells to adapt quickly enough to the rising ethanol concentration and partly to an increased demand for magnesium at higher ethanol concentrations which cannot be met in Mg-unsupplemented high cell density fermentations.

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