Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid CycleExperimental aspects of this research were supported equally by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S. Department of Energy (DOE), through Grant DE-FG02-05ER15707 and by the Office of Science (BER), U.S. Department of Energy, through Grant DE-FG02-07ER64482. Experimental work was a

When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked 13C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield. Background: The metabolism of non-growing microbes is poorly understood. Results: In a nitrogen-starved and non-growing photoheterotrophic bacterium, metabolic flow was diverted to mobilize electrons for H2 production. Conclusion: During starvation bacteria decouple their metabolism from biosynthesis. Significance: An understanding of metabolic activities of non-growing cells can be used to engineer improved biocatalysts.