Bicarbonate source for cultivation of microalgae is an alternate method mainly to avoid CO2 loss. The main aim of the work is to assess the bio-fixation ability of CO2 from ammonium bicarbonate by Chlorella pyrenoidosa under mixotrophic condition. Furthermore, statistical optimization has been carried out to study the influence of pH, concentration and inoculum size and identify best conditions for CO2 removal. The results revealed that maximum removal of CO2 was obtained at pH 6.0; with ammonium bicarbonate concentration, 6.66 g L-1 and inoculum size, 36.81%. The obtained results were statistically analyzed and the results were obtained with regression co-efficient R2 value of 0.94 for CO2 removal and 0.86 for corresponding chlorophyll content. From the study, it can be concluded that microalgae could able to grow in ammonium bicarbonate source which indicates that microalgae could assimilate ammonium and CO2 in to their cells even at high concentration. Bicarbonate captured CO2 proves to be a significant method for cultivation of microalgae supports commercial production. © 2015 Asian Network for Scientific Information.