Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase

ABSTRACT The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH 4 ) from carbon dioxide (CO 2 ) and hydrogen (H 2 ) from protons (H + ) when it expresses a variant form of molybdenum (Mo) nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH 4 and H 2 . As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH 4 and H 2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH 4 and H 2 production by R. palustris is inhibition of nitrogenase by ADP. Increasing the amounts of electrons available to nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H 2 uptake resulted in higher yields of H 2 and, in some cases, CH 4 . Our results provide a more complete understanding of the constraints on nitrogenase-based production of biofuels. IMPORTANCE A variant form of Mo nitrogenase catalyzes the conversion of CO 2 and protons to the biofuels CH 4 and H 2 . A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH 4 and H 2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of nitrogenase activity by ADP. In general, the amount of CH 4 produced by the variant nitrogenase in vivo was affected by electron availability much less than was the amount of H 2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo nitrogenase.