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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.

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Calvin Cycle Flux, Pathway Constraints, and Substrate Oxidation State Together Determine the H <sub>2</sub> Biofuel Yield in Photoheterotrophic Bacteria

Calvin Cycle Flux, Pathway Constraints, and Substrate Oxidation State Together Determine the H 2 Biofuel Yield in Photoheterotrophic Bacteria

Calvin Cycle Flux, Pathway Constraints, and Substrate Oxidation State Together Determine the H ₂ Biofuel Yield in Photoheterotrophic Bacteria