• algae;
  • hydrogen production;
  • photobioreactor;
  • sulfur deprivation;
  • hydrogenase;
  • oxygen sensitivity


The green alga, Chlamydomonas reinhardtii, is capable of sustained H2 photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O2-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10–15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H2 gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O2-consumption phase, an anaerobic phase, a H2-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H2-production phase; (3) re-addition of small quantities of sulfate (12.5–50 μM MgSO4, final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H2 production, an increase in the length of the H2-production phase, and an increase in the total amount of H2 produced; and (4) increases in the culture optical density in the presence of 50 μM sulfate result in a decrease in the initial specific rates of H2 production and in an earlier start of the H2-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H2 production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H2-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 731–740, 2002.