A two-stage strategy was developed to improve the microalgal carbohydrate accumulation for advanced biofuel production. In the first stage, Chlamydomonas reinhardtii CC125 was cultivated in photo-bioreactors by repeated fed batch operation to improve the biomass production. Optimal culture conditions achieved in the batch operation were applied to the repeated fed batch operation. Biomass productivity reached 0.47 g L-1 d(-1) with 70% medium replacement ratio and 5% CO2 under continuous light of 135 mu mol m(-2) s(-1). In the second stage, reducing CO2 content to 0.04% led to a high carbohydrate content of 71%, showing more than 9 times improvement compared to that in the biomass from the first stage culture. These results suggest that photoautotrophic two-stage cultivation is an effective approach to accumulate microalgal carbohydrate as a feedstock for biofuel production. (C) 2015 Elsevier Ltd. All rights reserved.