The contribution ratio of autotrophic and heterotrophic metabolism in the mixotrophic culture of Chlorella sorokiniana (C. sorokiniana) was investigated. At the early stage of mixotrophic growth (day 0–1), autotrophy contributed over 70% of the total metabolism; however, heterotrophy contributed more than autotrophy after day 1 due to the rapid increase in cell density, which had a shading effect in the photo-bioreactor. Heterotrophy continued to have a higher contribution until the available organic carbon was depleted at which point autotrophy became dominant again. Overall, the increase in algal biomass and light conditions in the photo-bioreactor are important factors in determining the contribution of autotrophy and heterotrophy during a mixotrophic culture.