In the context of hydrogen production by microalgae, the growth of Chlamydomonas reinhardtii was characterized under autotrophic and mixotrophic conditions in a fully controlled photobioreactor (PBR). The combined effect of light transfer conditions, as represented by the illuminated fraction gamma, with acetate consumption was observed upon establishment of anoxia. Anoxia was reached in batch cultures when gamma was close to 1 (almost fully illuminated culture) in mixotrophic conditions while a value of gamma approximate to 0.46 in autotrophic conditions was not sufficient. Based on these results, continuous hydrogen production was established in a cylindrical PBR operated in luminostat with constant illumination and in mixotrophic conditions. Maximum hydrogen gas production was equal to 1.4 +/- 0.1 ml(H2)l(-1)h(-1) for photon flux density of 110 mu mol m(-2)s(-1) and reactor illuminated fraction of gamma = 0.5. Carbon mass balance was realized, emphasizing the necessity to work in strictly autotrophic conditions for hydrogen production with no concomitant CO2 release. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.