An oxygen limitation strategy based on dynamic enzyme activity was applied to improve glycerol accumulation and decrease the residual sugar level in a fermentation of Candida krusei in a bioreactor. By applying oxygen limitation at 88 h when the activities of two glycerol synthetic enzymes cytosolic glycerol-3-phosphate dehydrogenase (ctGPD) and glycerol-3-phosphatase (GPP) were low and the activity of mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD) which catalyzes the glycerol dissimilation was high, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 51.8 g l(-1) at 96 h and 54.9 g l(-1) at 116 h, which was 18 and 60% higher than the control (without oxygen limitation), respectively. The residual sugar was consumed completely while it was 11.2 g l(-1) at the end of fermentation in the control. Under oxygen limitation, ethanol production was detected at a final concentration of 3.6 g l(-1), This work suggests a metabolic flux shift by oxygen limitation in the bioreactor. (C) 2004 Elsevier Ltd. All rights reserved.