Untangling metabolic interactions of a nongrowing, hydrogen producing synthetic coculture through a multispecies metabolic flux analysis.

In this study a methodology to elucidate metabolic interactions that enhance hydrogen (H) production in cocultures under nongrowing conditions is presented. Core metabolic models of Rhodopseudomonas palustris and Clostridium butyricum were integrated to perform a multispecies metabolic flux analysis (mMFA), constrained by experimentally measured yields. Flux distributions were clustered, and thermodynamically favorable solutions were identified. The mMFA revealed that, in coculture, R. palustris captures more light energy, producing hydrogen and less reduced compounds from highly reduced, low-energy metabolites supplied by C. butyricum. Increased photosynthetic activity raises the redox state of R. palustris, driving H and lactate production to dissipate excess reducing power. While C. butyricum cannot survive alone under the test conditions, it persists in coculture by consuming acetate and lactate, generating energy via substrate-level phosphorylation and electron bifurcation. Coculture yields higher hydrogen production, primarily from R. palustris. These findings suggest the formation of an energy-coupled association that enables energy conservation, potentially supporting long-term bacterial survival.