Sustained Hydrogen Photoproduction by Chlamydomonas reinhardtii: Effects of Culture Parameters

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.