Regulation of alcoholic fermentation in batch and chemostat cultures ofKluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation ('Crabtree effect'). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostate cultures (D = 0.05-0.40 h-1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolities. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D = 0.10 h-1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at mu = 0.10 h to growth at mu max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation.