Hydrogen (H2) gas is an obligatory byproduct of nitrogen (N2) reduction during biological nitrogen fixation by the metalloenzyme nitrogenase. Despite significant efforts, diazo- trophic 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 nitroge-nase) in controlling N2 reduction and H2 production rates in the model anaerobic photo-heterotroph 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 stoichi-ometries (6.3e12.7); H2:N2 stoichiometries for molybdenum and vanadiumnitrogenases are similarly lower (2.5e4.1 and 2.6e4.3, respectively) and uncorrelated with growth rate or temperature. Hydrogen inhibition of growth is lower than H2 inhibition of purified nitro-genase. These results help provide a framework for optimizing physical and metabolicconditions in diazotroph-based biohydrogen production efforts.