The impact of severe nitrogen limitation and microaerobic conditions on extended continuous cultivations of Saccharomyces cerevisiae with cell recirculation

Continuous cultivations of Saccharomyces cerevisiae ATCC 96581 with severe nitrogen limitation (C/N ratios 200 and 400 g g−1) and cell recirculation were carried out under anaerobic and microaerobic conditions for more than 300 h. With a dilution rate of 0.06 h−1 and 90% recirculation in combination with an estimated 70% biomass sedimentation rate in the bleed flow, specific growth rates of 0.002–0.006 h−1 were obtained. Under these conditions, ethanol yields of 0.46–0.48 g g−1 were achieved. The biomass yields on ATP were only 1.6–2.9 g mol−1, indicating metabolic uncoupling or high maintenance energy requirements. Viability levels, measured by FUN® staining and fluorescence microscopy, usually varied between 100 and 80%. However, under anaerobic conditions at C/N ratio 400, a reproducible drop to 25% viability occurred between 250 and 300 h of fermentation, after which the culture recovered again. Under anaerobic conditions, an increase in the C/N ratio from 200 to 400 resulted in a three-fold higher specific glycerol production, in spite of lower biomass formation and lower cellular protein and RNA content. A low oxygen addition eliminated the large drop in viability and the increased glycerol production observed at C/N 400, and caused viability and glycerol levels similar to the anaerobic C/N 200 case. A S. cerevisiae W303-1A gpd1Δgpd2Δ mutant, completely deficient in glycerol production, could ferment a nitrogen-limited medium under RQ-controlled microaerobic conditions with an ethanol yield of 0.45 g g−1, indicating that the increased glycerol production under nitrogen limitation is not necessary, as long as there is sufficient oxygen transferred to the culture.