Electron partitioning during light- and nutrient-powered hydrogen production by Rhodobacter sphaeroides

The partitioning of reducing power into different electron-accepting pathways was evaluated during growth and stationary phases of H ₂-producing Rhodobacter sphaeroides cultures. For this, an electron balance method was developed using the chemical oxygen demand concept to quantitatively analyze the partitioning of nutrient electrons into H ₂, cell biomass, polyhydroxybutyrate (PHB), and soluble microbial products (SMP). Overall, these four electron sinks were accounted for greater than 85% of the electrons provided by the nutrients. Glucose, lactate, succinate, fumarate, and pyruvate were individually provided as the main carbon source, and in all cases, glutamate was provided as a nitrogen source in order to enhance H ₂ production. About 25-35% of the electrons ended up in H ₂ during growth, while up to 60% of the electrons partitioned into H ₂ in some stationary phase cultures. The other two major electron sinks in the growth phase were cell mass and PHB, while in stationary phase, SMP were accounted for >30% of the substrate electrons utilized. In general, the largest portion of SMP comprised low-molecular weight (<3 kDa) compounds mostly produced during stationary phase, although larger-size molecules were also detected in both phases. Overall, the fractions of electrons that partitioned into H ₂ (0.21 to 0.35) and PHB (0.06 to 0.21) were highly correlated with the standard free energy change of the substrate oxidation half-reaction equation, normalized per electron equivalent. In a PHB(-) mutant, electron redistribution increased H ₂ production, the extent of which depended on the carbon source provided.