This study focusses on the assimilation of carbon in concentrated municipal wastewater rich in organic carbon using the mixotrophic microalga Auxenochlorella protothecoides UMN280 with the addition of supplemental CO2. The entire growth period of A. protothecoides UMN280 can be characterized by three phases: first, a phase where algae grew in a mixotrophic-dominated mode; second, a transition phase; and last, a phase where algae grew in a photoautotrophic-dominated mode. In this study, it was found that light intensity had a strong effect on algal biomass production; the culture system would transfer from a mixotrophic-dominated mode to a photoautotrophic-dominated mode quicker under higher light intensities. The addition of CO2 exhibited an important role in the photoautotrophic-dominated cultivation stage. At certain level of irradiance and certain range of CO2 injection rate, higher CO2 injection rate would result in a higher level of carbon fixation. It is clearly beneficial to inject exogenous CO2 in the mixotrophic wastewater algae production system when a light source is available, such as during daylight hours.