Nitrogenase-mediated H-2 accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H-2 uptake and indirectly by energy-demanding metabolic processes such as poly-P-hydroxybutyrate (PHB) formation. H-2 accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m(2). Mutations in either hupSL (H-2-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m(2), however, was 70% higher than that of cells grown at 3 W/m(2), while the H-2-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H-2 uptake by hydrogenase, monitored by measuring the difference in H-2 accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m(2). On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H-2 accumulation independent of light intensity, reflecting the fact that the cellular PHB; content was not changed significantly depending on light intensity. Therefore, H-2 uptake by hydrogenase should be suppressed to achieve higher H-2 accumulation of R. sphaeroides, especially at 15 W/m(2).