| Photosynthetic microorganisms are becoming a major agricultural venue for renewable energy production. A novel concept of photosynthetic co-cultivation was proposed for the functional integration of complementary light-absorption and metabolism in purple photosynthetic bacteria and unicellular green algae, for enhanced hydrogen production. It was shown that the purple bacterium Rhodospirillum rubrum and green alga Chlamydomonas reinhardtii can grow harmoniously in the same medium. Furthermore, the light intensity perceived was shown to influence the constituency, favoring green algae at increasing intensities, and vice versa. With higher light, significant algal-derived oxygen was found to inhibit photosynthesis in purple bacteria, which instead flourished aerobically. Hydrogen was not evolved on account of the oxygen produced by algal photosynthesis and from ammonia nutrients required for the algae. Ammonia repression of the bacterial nitrogenase/hydrogenase was circumvented through use of a mutant of R. rubrum with constitutive expression of nitrogenase. An algal mutant with a decreased light-harvesting antenna was found to be more amenable for co-cultivation, as a balanced constituency was obtainable at higher light intensities.;Additional experiments were performed to investigate the merit of a fed-batch technique for purple bacterial hydrogen production, whereby stationary phase cells are treated as non-growing industrial catalysts. Inoculated cultures evolved hydrogen during exponential growth and continued to evolve at high rates for 70 h after growth had ceased. Upon replenishment of the electron donor, succinate, cultures resumed hydrogen evolution without further growth. However, rates and yields were lower with successive succinate additions than those observed during the initial growth phase. Supplementation of cultures with varying amounts of growth medium, given with the succinate replenishment, partially restored the hydrogen production rate and yield, and caused a proportional increase in biomass. Thus, while growth is not required for hydrogen production, this work establishes the necessity for cell growth in order to maintain maximal rates, suggesting the industrial suitability of a semi-continuous culture strategy.;The oxygen self-repression of hydrogen production in green algae can be addressed by sulfur-deprivation; thus it was investigated what effect such sulfur-deprivation might have upon hydrogen-producing purple bacterial cultures. The removal of sulfur nutrients halted growth in R. rubrum, with reductant and energy redirected toward storage polymer formation (poly-beta-hydroxybutyrate (PHB) and polysaccharides), some accumulating extracellularly. Hydrogen production ceased altogether, and the nitrogenase enzyme was shown to exhibit a prompt decline in activity, protein levels, and mRNA transcripts. This shift from hydrogen to polymer production was shown to be reversible upon replenishment of sulfur nutrients, suggesting the design of a photobiological system for alternating the processes of H2 and PHB production. |
