Peculiar H ⁺ Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Intracellular pH (pH<jats:sub>in</jats:sub>) 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<jats:sup>+</jats:sup>homeostasis is accomplished by an industrial wine strain of<jats:named-content content-type="genus-species">Saccharomyces cerevisiae</jats:named-content>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<jats:sub>in</jats:sub>5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H<jats:sup>+</jats:sup>via H<jats:sup>+</jats:sup>-ATPase and of keeping H<jats:sup>+</jats:sup>out by means of an impermeable membrane were evaluated as potential mechanisms of H<jats:sup>+</jats:sup>homeostasis. At 30°C, H<jats:sup>+</jats:sup>efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H<jats:sup>+</jats:sup>. Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H<jats:sup>+</jats:sup>remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H<jats:sup>+</jats:sup>permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H<jats:sup>+</jats:sup>homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.</jats:p>

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

“…An imperative requirement, therefore, is to visualize pH dynamics to understand the basis of pH homeostasis. Such understanding enables the optimization of industrial processes (4,8,35) or the exploitation of pH homeostasis machinery as a therapeutic target. pH i has been measured through a wide range of monitoring techniques, from cell loading with pH-selective fluorescent dyes (4,(7)(8)(9)(10)(11) to the use of green fluorescent protein-based sensors (12)(13)(14)(15).…”

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation

“…Maximum specific growth rate was determined using the DMFit software available on the Combase website (http://www.combase.cc/index.php/en/), where μ max values were obtained after the fit of the growth curves to the model proposed by Baranyi and Roberts (1994). The growth parameter N end is an important checkpoint of alcoholic fermentation and yeast physiology (Viana et al 2012) and a well-known phase where non-Saccharomyces cells normally die-off during alcoholic fermentations (Nissen et al 2003). Growth, glucose and ethanol curves for representative experiments can be seen in Supplementary Figs.…”

Monitoring and Evaluation of Alcoholic Fermentation Processes Using a Chemocapacitor Sensor Array

“…The addition of excess acetic acid at Day 5 of Must 2 fermentation ( Figure 4 ) did not appear to slow down the rate of ethanol production ( Figure 3 ). This is probably due to the fact that Must 2 at Day 5 was already partially fermented, with an ethanol content of 10.36% (v/v) and cell growth was completed, while the toxic effect of acetic acid is expected to be more evident during the cell growth stage of glucose-grown populations of Saccharomyces cerevisiae [ 29 , 36 ]. Thus, according to chemical analysis, the fermentation process of Must 2 is characterized by higher initial ethanol and acetate content and elevated, purposely, higher acidity after 5th day of the fermentation process.…”

“…While cell growth is in progress the presence of ethanol potentiates both the inhibition of fermentation and the internal acidification originated by acetic acid. On the contrary when the cell growth stage is completed (stationary phase) the ethanol production may still proceed even in the presence of acetic acid [ 29 ]. Therefore, acetic acid is potentially responsible for fermentation problems.…”