Abstract
Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.
Highlights
Cyanothece produces H2 catalyzed by hydrogenase or nitrogenase
Carbohydrate serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2)
By utilizing monochromatic excitation sources and detection of dissolved hydrogen, we monitor the kinetics of hydrogen evolution from both light and dark pathways within the same experimental incubation, allowing visualization of the independent responses of each pathway to applied stresses. We show that both intracellular reductant and ATP availability are limiting factors for maximal photo-H2 production, and that by increasing reductant availability via dark anaerobic incubation, or by channeling the flow of reductant through one of the specific NADH dehydrogenases to increase ATP availability, we can substantially increase the rate of photo-H2 production
Summary
Cyanothece produces H2 catalyzed by hydrogenase or nitrogenase. Results: Photo-H2 is nitrogenase mediated (via PSI) with reductant originating from catabolism and ATP from photophosphorylation. Carbohydrate serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2) Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H2 production rate by 2-fold (at the expense of the dark-H2 rate), due to production of additional ATP (via the proton gradient). Cyanothece species accumulate carbohydrate, primarily in the form of glycogen granules, during the day and subsequently degrade this carbohydrate at night to provide the energy and reductant for nitrogenase function and O2 respiration [19] This natural diurnal cycling, efficient conversion of intracellular carbohydrate to energy, efficient respiration and intracellular anoxia, and presence of both classes of hydrogen producing enzymes makes the Cyanothece species among the best candidates for H2 production [20, 21]. We show that both intracellular reductant and ATP availability are limiting factors for maximal photo-H2 production, and that by increasing reductant availability via dark anaerobic incubation, or by channeling the flow of reductant through one of the specific NADH dehydrogenases to increase ATP availability, we can substantially increase the rate of photo-H2 production
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