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Osmotic stress represents one of the major environmental challenges experienced by yeast during industrial fermentations. This stress is particularly associated with high-gravity processes, which utilize concentrated substrates to yield products with elevated concentrations of ethanol. The aims of this work were to quantitatively measure factors affecting extracellular osmotic pressure (osmolality) during brewing fermentations and to determine their effects on yeast at the physiological and molecular level. Osmolality was observed to increase during fermentation owing predominantly to ethanol production, indicating a strong relationship between these environmental parameters. High osmolality was shown to have a negative impact on yeast physiology, viability, and vitality, and although genome integrity was unaffected, cell membrane fluidity became altered. These data not only demonstrate the occurrence of an increase in osmotic pressure during fermentation but also provide an explanation for the decrease in yeast quality typically observed under high-gravity conditions. The results presented here are directly relevant to all brewery fermentations worldwide and have applications within associated industries in which microorganisms are used for ethanol production, including food products, alcoholic beverages, and biofuels. Keywords: Genome integrity, High gravity, Membrane fluidity, Osmotic stress, Yeast physiology
