The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon-to-nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L glucose, 2.5 g L KNO ) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500-mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g and 305 mg L day , respectively. A repeated fed-batch culture with four cycles of feeding with the fivefold concentrated medium in a 3-L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L was obtained with a TFA productivity of 223 mg L day . The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589-1600, 2017.