Sustained H(2) Production Driven by Photosynthetic Water Splitting in a Unicellular Cyanobacterium

The relationship between dinitrogenase-driven H(2) production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H(2) at rates up to 400 µmol ⋅ mg Chl(−1) ⋅ h(−1) in parallel with uninterrupted photosynthetic O(2) production. Notably, sustained coproduction of H(2) and O(2) occurred over 100 h in the presence of CO(2), with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl(−1) ⋅ h(−1), respectively. Oscillations were not observed when CO(2) was omitted, and instead H(2) and O(2) evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H(2) production displayed a strong dose dependence and lack of O(2) inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H(2) production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H(2) production over that period. Collectively, our results demonstrate that uninterrupted H(2) production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O(2) and have important implications for sustainable production of biofuels.