High-yield hydrogen production from glucose in the presence of ammonia by genetically engineered Cereibacter...

The anoxygenic phototrophic bacterium Cereibacter sphaeroides produces molecular hydrogen (H₂) by nitrogenase using light energy and electrons derived from organic compounds, the process of which is known as photo-fermentative H₂ production. An objective of photo-fermentative H₂ production is inhibition by ammonia since expression of nitrogenase is repressed under nitrogen-sufficient conditions. Therefore, we have previously constructed the ΔhupcbbP∗ strain that can produce H₂ from acetate in the presence of 10 mM ammonium chloride. In this study, we observed that the ΔhupcbbP∗ strain can also produce H₂ during photoheterotrophic growth with glucose and 10 mM ammonium chloride. For further genetic manipulation, we constructed a plasmid-free strain CsHydtrc2, which produced similar amounts of H₂ with those of the ΔhupcbbP∗ strain during photoheterotrophic growth with glucose in the presence of ammonia. Furthermore, supplementation of sodium bicarbonate to culture medium stabilized culture pH and markedly enhanced H₂ production of the CsHydtrc2 strain. Gene deletion analyses revealed that the Entner-Doudoroff pathway, but not the Embden-Meyerhof pathway, is essential for H₂ production by the CsHydtrc2 strain with glucose. Plasmid expression of genes involved in glucose metabolism suggested that the glucose incorporation is the rate-limiting step in the glucose metabolism in the CsHydtrc2 strain under photoheterotrophic growth conditions.