ABSTRACTRhodobacter sphaeroidesproduces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such asl-glutamate.R. sphaeroidesutilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2yield from acetate in the presence ofl-glutamate has been reported to be low. In this study, the deletion ofccrencoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway inR. sphaeroides, revealed that the EMC pathway is essential for H2production from acetate andl-glutamate but not for growth and acetate consumption in the presence ofl-glutamate. We introduced a plasmid expressingaceBAfromRhodobacter capsulatusencoding two key enzymes for the glyoxylate bypass intoR. sphaeroides, which resulted in a 64% increase in H2production. However, compared with the wild-type strain expressing heterologousaceBAgenes, the strain withaceBAintroduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2production, although its H2yield was comparable to that of the wild-type strain expressing heterologousaceBAgenes. These results suggest that PHB production is important for fitness to the culture during H2production from acetate andl-glutamate when both acetate-assimilating pathways are present.IMPORTANCEAs an alternative to fossil fuel, H2is a promising renewable energy source. Although photofermentative H2production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2yields from acetate andl-glutamate byR. sphaeroideshave been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass inR. sphaeroidesmarkedly increased H2yields from acetate andl-glutamate. Therefore, this study provides a novel strategy for improving H2yields from acetate in the presence ofl-glutamate and contributes to a clear understanding of acetate metabolism inR. sphaeroidesduring photofermentative H2production.