The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO3 medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.