Vitality of cider yeast grown micro-aerobically with added ethanol, butan-1-ol or iso-butanol

Abstract

Vitality and viability of an alcohol-tolerant wine yeast, used in cider production, were assessed after exposure to alkanol(s) during growth. Criteria employed were: methylene blue reduction, ability to form colonies on yeast extract-peptone-glucose agar medium, glucose driven proton efflux rates ("acidification power"), fermentative (CO2 output) rates and adenylate energy charge values. We also monitored the maintenance of transmembrane electrochemical potential across the plasma membrane as measured by flow cytometry and by scanning confocal laser microscopy of oxonol dye exclusion. Growth rates were diminished by a third by 7.5% (v/v) added ethanol, 1% butan-1-ol or 1.4% isobutanol. Exposure to 10% (v/v) ethanol gave 16% loss of "viability", as measured by methylene blue reduction, during the first 20 h of growth. For 1% butan-1-ol, 50% loss of "viability" occurred over 40 h, whereas a similar effect of iso-butanol took 55 h. Adenylate charge values were high (<0.8) in growing cultures, remained high in early stationary phase but declined to 0.4 after 115 h. These values were hardly affected by 5 or 7.5% (v/v) ethanol whereas 10% or 15% (v/v) ethanol gave values of 0.58 and 0.16 after only 5 h exposure. 1% butan-1-ol or iso-butanol decreased adenylate charge values to a greater extent than 10% (v/v) ethanol, with the straight chain alcohol the more potent. Oxonol exclusion indicated that the vast majority of cells with greatly diminished vitality have maintained the plasma membrane potential values required to retain viability, despite extensive exposure to alkanol(s). Thus loss of ability to reduce methylene blue indicates diminished vitality but is not a reliable index of loss of viability. "Acidification power" was a more sensitive indicator of vitality than adenylate charge values. When mixtures of C2 + C4 alcohols were employed effects were generally additive rather than synergistic.