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

Abstract

ABSTRACT 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.