Hydrogen gas (H(2)) is a possible future transportation fuel that can be produced by anoxygenic phototrophic bacteria via nitrogenase. The electrons for H(2) are usually derived from organic compounds. Thus, one would expect more H(2) to be produced when anoxygenic phototrophs are supplied with increasingly reduced (electron-rich) organic compounds. However, the H(2) yield does not always differ according to the substrate oxidation state. To understand other factors that influence the H(2) yield, we determined metabolic fluxes in Rhodopseudomonas palustris grown on (13)C-labeled fumarate, succinate, acetate, and butyrate (in order from most oxidized to most reduced). The flux maps revealed that the H(2) yield 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. palustris generated similar amounts of reducing equivalents and similar amounts of H(2) from both succinate and acetate, even though succinate is more oxidized than acetate. The second factor affecting the H(2) yield was the amount of Calvin cycle flux competing for electrons. When nitrogenase was active, electrons were diverted away from the Calvin cycle towards H(2), but to various extents, depending on the substrate. When Calvin cycle flux was blocked, the H(2) yield increased during growth on all substrates. In general, this increase in H(2) yield could be predicted from the initial Calvin cycle flux.
Importance: Photoheterotrophic bacteria, like Rhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H(2) as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H(2). 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 H(2) yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO(2)-fixing Calvin cycle flux that competes against H(2) production for electrons. We show that the H(2) yield 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 H(2) or fix CO(2) even on relatively oxidized substrates.