Contributing towards the realization of photobiological hydrogen production
Photobiological H2-production represents an 'ideal' system for renewable energy production if only sunlight is required to catalyze the conversion of water into H2 and O2. Ignoring nitrogenase-based systems, several photobiological organisms exist with such native capabilities, at least on paper. Considerable development will be needed to realize such a future system. We are interested in addressing the following known and potential issues in order to contribute towards a working solution:
Thermodynamic limitations of NADP(H):H2-pathways
Photobiological H2-production (either by direct or indirect biophotolysis) that does not depend solely on nitrogenase-catalyzed H2-synthesis (i.e. in the complete absence of H2-evolving hydrogenases) will to some degree involve a reversible NAD(P)H:H2-pathway. Such pathways are catalyzed either by a multimeric NiFe-hydrogenase (ex. HOX in Synechocystis) or by a combination of ferredoxin/flavodoxin-dependent hydrogenase and ferredoxin/flavodoxin:NAD(P)H-oxidoreductase (ex. HydA1 and FNR in Chlamydomonas). If the 'biochemically effective' intracellular NAD(P)H/NAD(P)+-ratio to some degree is regulated ('fixed') in these organisms, it is likely that net H2-accumulation will be limited by the near-equilibrium NAD(P)H:H2-pathway as the reaction is highly unfavourable in the direction of interest (H2-synthesis) at co-factor ratios reported in most organisms (in the range of 1:10 - 10:1). We have experimentally demonstrated such a limitation with a defined E. coli model system. As far as I am aware, both the thermodynamic limitations of NAD(P)(H):H2 and NADPH-metabolism in general have not been sufficiently studied in phototrophic H2-producers to allow us to conclude that such pathways pose a limitation to phototrophic H2-production, or not.
Stimulation of hydrogenase O2-tolerance through modification of the protein-environment
The rapid reversible or irreversible inactivation of Hox and HydA1, respectively, by O2 (or O2-derived metabolites) represent one of the greatest challenges for phototrophic H2-production. In parallel, or as one underlying reason for inactivation, Fe-S clusters present in most enzymes of H2-pathways and hydrogenase assembly may also be damaged by exposure to O2. The irreversible inactivation of HydA1 by O2-exposure has been successfully mitigated by strategies that effectively have raised the 'compensation point'.
Whether such strategies make sense or not is still unsolved in our view given that any electrons that are utilized to reduce O2 to water, eventually would have to originate also from water (in a fully light-dependent system). The stoichiometry of this approach does not make sense unless O2 is removed from the otherwise closed system, for example by energetically costly gas-sparging (which coincidentally may also resolve thermodynamic issues associated with H2-accumulation). Such solutions may work in a laboratory, but will they function in practice?
When such strains or conditions have been utilized under closed conditions, at sufficiently low light intensity, anoxic conditions have developed resulting in hydrogenase activity. A future research target by some research groups is also to introduce a native or engineered hydrogenase that displays enhanced tolerance to O2. As a complement to both of the above strategies, I propose to stimulate hydrogenase-tolerance and H2-production through further modification of the protein environment. One such strategy is to enhance the assembly and/or repair of Fe-S clusters. We recently demonstrated this approach in E. coli and showed that it also can be beneficial in the absence of O2, and that the effect is even greater following exposure to O2. The exact mechanism of stimulation remains unknown and demands further research (see Fe-S repair).
The above issues will be approached using experience (NADPH) and solutions (Fe-S) developed and verified in a fermentative E. coli model-system. To transfer solutions to a different host represents a great challenge on it's own. In addition, the solutions developed earlier, are not fully understood (Fe-S), and therefore require further in-depth fundamental study (see Fe-S projects). A starting point for both studies (NADPH and Fe-S) is to develop a functional model system.