Continuous cultivations of Sacchaivinyces certvisiae ATCC 96581 with severe nitrogen limitation (C/N ratios 200 and 400g g(-1)) and cell recirculation were carried out under anaerobic and microaerobic conditions for more than 300h. 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 0.46-0.48g g(-1) were achieved. The biomass yields on ATP were only 1.6-2.9gmol(-1), indicating metabolic uncoupling or high maintenance energy requirements. Viability levels, measured by FUNO 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 300h 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. certvisiae W303-1A gpdI Delta gpd2 Delta 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. (c) 2006 Elsevier Inc. All fights reserved.