Bicarbonate species in the aqueous phase is the primary source for CO2 for the growth of microalgae. The potential of carbon dioxide (CO2) fixation by Chlorella pyrenoidosa in enriched bicarbonate medium was evaluated. In the present study, effects of parameters such as pH, sodium bicarbonate concentration and inoculum size were assessed for the removal of CO2 by C. pyrenoidosa under mixotrophic condition. Central composite design tool from response surface methodology was used to validate statistical methods in order to study the influence of these parameters. The obtained results reveal that the maximum removal of CO2 was attained at pH 8 with sodium bicarbonate concentration of 3.33 g/l, and inoculum size of 30 %. The experimental results were statistically significant with R 2 value of 0.9527 and 0.960 for CO2 removal and accumulation of chlorophyll content, respectively. Among the various interactions, interactive effects between the parameters pH and inoculum size was statistically significant (P