DHA production by Schizochytrium sp. S31 was studied in batch cultures on glycerol with stepwise dissolved oxygen strategy. Three growth stages were identified as cell growth, lipid accumulation and lipid turnover. It was revealed that fatty acid (FA) shifts during the three growth stages involved the activity changes of glycerol kinase (GK), FAD(+)-dependent glycerol-3-phosphate dehydrogenase (FAD(+)-G-3-PDH), malic enzyme (ME), ATP citrate lyase (ACL) and NAD+-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Glycerol dissimilation in Schizochytrium sp. S31 was suggested via a phosphorylation by GK and a following oxidation by FAD(+)-G-3-PDH. Lipid accumulation of this strain was a growth-associated process, but the assimilable nitrogen depletion enhanced the accumulation of lipids. The exhaustion of glycerol induced the lipid turnover stage, where the short chain fatty acids were preferentially degraded and converted into lipid-free biomass (X-f) which was correlated to the increase of DHA content in biomass. (C) 2013 Elsevier Ltd. All rights reserved.