Abstract: The present application overcomes the issue of the low yields of biological hydrogen production by cells by utilizing purified hydrogenase enzymes in vitro. Two methods are considered. The major advantage of these approaches is that they by-pass the mechanisms that limit the rates of intact organisms, thus achieving rates much closer to the actual capacity of the enzyme.

Abstract: A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

Abstract: The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H2 in a light catalyzed reaction having water as the reactant.

Abstract: Provided herein are methods for measuring H2 production by a hydrogenase. Also provided are methods for evolving a hydrogenase or hydrogen-sensing system by comparing the level of production of H2 by the hydrogenase to the level Of H2 produced by a wild-type hydrogenase cultured under the same conditions and selecting a microorganism with increased H2 production over the H2 production by a microorganism having a wild-type hydrogenase. Further provided herein are microorganisms and plasmids comprising a hupSL promoter coupled with a reporter gene.

Abstract: Provided herein are systems and methods for generating photocurrent and systems and methods for generating hydrogen. The systems comprise (a) isolated biological photosystem II (PSII) complexes; (b) an anode selected from the group consisting of graphite anode, glassy carbon anode, and FTO anode, wherein the PSII complexes are deposited onto the anode; (c) a source of red or white light, wherein red or white light illumination of the PSII complexes deposited on the anode generates a photoresponse comprising a photocurrent; (d) a working solution; and (e) a cathode.

Abstract: The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H2. Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H2 in a light catalyzed reaction having water as the reactant.

Abstract: A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.