- Many conditions affecting hydrogen (H₂) production by the cyanobacterium, Synechocystis sp. PCC 6803, were optimized to yield maximum H₂ accumulation. Biological H₂ production from photosynthetic species is a promising form of renewable energy since an abundant supply of sunlight hits the Earth every day, and photosynthetic bacteria can harness this solar energy and efficiently split water to produce H₂ in a safe, clean manner. The H₂ could then be used in fuel cells in a closed cycle, with water and heat as the only byproducts. There are many techniques currently in development to maximize H₂ production. We chose to use statistical optimization procedures to identify the factors which have the greatest impact on H₂ production, and simultaneously optimize them. Initially we optimized concentrations of NH₄⁺, HCO₃⁻, and SO₄²⁻, and achieved a 148-fold increase in H₂ production over sulfur deprived cultures, which have been shown to produce more H₂ than cultures grown on complete BG-11 media. With 0.52 mM NH₄⁺, 20.1 μM SO₄²⁻, and 46 mM HCO₃⁻, 0.81±0.36 μmol H₂ mg Chl⁻¹ h⁻¹ was obtained. This increase was associated with a 44-fold increase in glycogen concentration over cultures grown on BG-11. Glycogen breakdown provides substrate to the hydrogenase enzyme under dark, anaerobic conditions. Since interaction effects are strong, we then optimized pH and NH₄⁺ simultaneously, and achieved another 1.94-fold increase over the previously optimized media. This was achieved with an advanced optimization algorithm, which had never been applied to biotechnological applications. Both of these increases in H₂ production were accomplished under optimal glycogen accumulation conditions, which include acclimation to the media formulation over an extended light period, followed by immediate anaerobic, dark fermentative conditions. In an additional study we explored 24-hour H₂ production under natural, diurnal light/dark cycling, and examined glycogen accumulation dynamics as well as electron availability to the hydrogenase. Electron availability was manipulated by exposing the cultures to various inhibitors of enzymes in the photosynthetic and respiratory electron transport chains. Over 3 days, with 9.4 mM KCN and 1.5 mM malonate in the previously optimized media we were able to increase H2 production 30-fold over standard BG-11 without inhibitors.