ABSTRACTHydrogen gas (H2) is a possible future transportation fuel that can be produced by anoxygenic phototrophic bacteria via nitrogenase. The electrons for H2are usually derived from organic compounds. Thus, one would expect more H2to be produced when anoxygenic phototrophs are supplied with increasingly reduced (electron-rich) organic compounds. However, the H2yield does not always differ according to the substrate oxidation state. To understand other factors that influence the H2yield, we determined metabolic fluxes inRhodopseudomonas palustrisgrown on13C-labeled fumarate, succinate, acetate, and butyrate (in order from most oxidized to most reduced). The flux maps revealed that the H2yield was influenced by two main factors in addition to substrate oxidation state. The first factor was the route that a substrate took to biosynthetic precursors. For example, succinate took a different route to acetyl-coenzyme A (CoA) than acetate. As a result,R. palustrisgenerated similar amounts of reducing equivalents and similar amounts of H2from both succinate and acetate, even though succinate is more oxidized than acetate. The second factor affecting the H2yield was the amount of Calvin cycle flux competing for electrons. When nitrogenase was active, electrons were diverted away from the Calvin cycle towards H2, but to various extents, depending on the substrate. When Calvin cycle flux was blocked, the H2yield increased during growth on all substrates. In general, this increase in H2yield could be predicted from the initial Calvin cycle flux.IMPORTANCEPhotoheterotrophic bacteria, likeRhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H2as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H2. Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H2yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO2-fixing Calvin cycle flux that competes against H2production for electrons. We show that the H2yield can be improved on all substrates by using a strain that is incapable of Calvin cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H2or fix CO2even on relatively oxidized substrates.
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