A challenge for photobiological production of hydrogen gas (H2) as a potential biofuel is to find suitable
electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron
donor for nitrogenase-catalyzed H2 production by the purple nonsulfur phototrophic bacterium (PNSB)
Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is
also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate
and bicarbonate and produced H2 when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions).
In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate
to H2. H2 production with acetate and succinate as electron donors was less efficient (40 to 60%), partly
because nongrowing cells excreted the intermediary metabolite -ketoglutarate into the culture medium. The
fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth
using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of
H2 production. The possibility to use thiosulfate expands the range of electron-donating compounds for H2
production by PNSBs beyond biomass-based electron donors.