Peculiar H⁺ homeostasis of Saccharomyces cerevisiae during the late stages of wine fermentation

Abstract

Intracellular pH (pH(in)) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H(+) homeostasis is accomplished by an industrial wine strain of Saccharomyces cerevisiae while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH(in) 5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H(+) via H(+)-ATPase and of keeping H(+) out by means of an impermeable membrane were evaluated as potential mechanisms of H(+) homeostasis. At 30°C, H(+) efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H(+). Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H(+) remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H(+) permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H(+) homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.

Fig 1

Fermentation kinetics of S. cerevisiae ISA1000 in white grape must at 15°C (A) or 30°C (B). Dashed lines (X, Y, and Z) indicate sample points for pH_in determination and H⁺ movement assays, corresponding to exponential and early and late stationary phases, respectively. Abs, absorbance.

Fig 2

Intracellular pH of S. cerevisiae ISA1000 during white grape must fermentation at 15, 25, and 30°C. Cells were collected at representative sample points for exponential and early and late stationary phases.

Fig 3

Rates of net H⁺ efflux in S. cerevisiae ISA1000. Cells were collected at exponential and early and late stationary phases during white grape must fermentation at 15, 25, and 30°C. The assays were performed at the same temperatures at which the cells were grown.

Fig 4

Rates of passive H⁺ influx in S. cerevisiae ISA1000. Cells were collected at exponential and early and late stationary phases during white grape must fermentation at 15, 25, and 30°C. Each assay was performed at the same temperature at which the cells were grown.

Fig 5

Effect of ethanol on the rates of passive H⁺ influx in S. cerevisiae ISA1000 cells collected at exponential and late stationary phases during white grape must fermentation performed a 15°C (A) or 30°C (B). Each assay was performed at the same temperature at which the cells were grown.