Biohydrogen production relationship to biomass composition, growth, temperature and nitrogenase isoform in the anaerobic photoheterotrophic diazotroph Rhodopseudomonas palustris

Hydrogen (H₂) gas is an obligatory byproduct of nitrogen (N₂) reduction during biological nitrogen fixation by the metalloenzyme nitrogenase. Despite significant efforts, diazotrophic H₂ production rates remain too low to compete with fossil fuel-derived H₂. Here, we investigate the role of temperature (14, 19, 30 °C), carbon metabolism (acetate or succinate as carbon source), and nitrogenase isoform (molybdenum, vanadium, iron-only nitrogenase) in controlling N₂ reduction and H₂ production rates in the model anaerobic photoheterotroph Rhodopseudomonas palustris. Rates of H₂ production are primarily controlled by growth rate and secondarily by nitrogenase enzymology. The iron-only nitrogenase exhibits the highest H₂:N₂ stoichiometries (6.3–12.7); H₂:N₂ stoichiometries for molybdenum and vanadiumnitrogenases are similarly lower (2.5–4.1 and 2.6–4.3, respectively) and uncorrelated with growth rate or temperature. Hydrogen inhibition of growth is lower than H₂ inhibition of purified nitrogenase. These results help provide a framework for optimizing physical and metabolic conditions in diazotroph-based biohydrogen production efforts.