The application of hydrogen as a clean and efficient energy carrier in the near future becomes more and more evident. Within the process of photobiological hydrogen production, purple non-sulfur bacteria are an interesting subject of study because of their high hydrogen producing capacity. In a previous study, the used Rhodopseudomonas sp. had proven to efficiently produce hydrogen from acetic acid and light energy. We constructed a pneumatically agitated flat-panel photobioreactor as a model system for optimization of photoheterotrophic hydrogen production. Batch experiments and a chemostat experiment were performed to investigate the proper functioning of the new photobioreactor. During the first experiments, argon gas was sparged through the system for mixing and inhibition of growth was observed. Experimental results indicate that the stripping of carbon dioxide from the culture liquid caused this inhibition of growth. Possibly, the Rhodopseudomonas sp. used requires carbon dioxide during growth on a highly reduced substrate like acetate. Recirculating the gas prevented the carbon dioxide from being stripped from the system. In this mode of operation, growth was supported. (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.