Development of an Efficient Algal H2-Production System

Photosynthetic hydrogen evolution by green algae is catalyzed by a reversible hydrogenase enzyme that is extremely sensitive to inactivation by O2, a by-product of photosynthetic water oxidation. Oxygen sensitivity has been one of the major barriers to the development of a commercial algal H2-production system. The objective of our project is to generate O2-tolerant, H2-producing mutants from thegreen alga Chlamydomonas reinhardtii to be used in a photobiological water-splitting, H2-producing systems that is cost effective, renewable, scalable, and non-polluting. We have developed two approaches to obtain desired C. reinhardtii mutants, based either on the H2-producing or H2-uptake activity of the enzyme. The first approach depends on the ability of algal cells to produce H2 incompetition with a drug that, when reduced, releases toxice products. H2-production selective pressure is applied in the presence of increasing O2 stress to enrich for O2-tolerant organisms. The second approach requires that algal cells grow, using H2 as a source of electrons for CO2 fixation. Addition of O2 during the H2-uptake selects for O2-tolerant organisms. Our previous work consisted ofdeveloping an assay for simultaneous measurement of H2 and O2 production, as well as establishing a reliable way of measuring O2 tolerance by different algal strains based on estimating an O2I50 for H2 evolution. In addition, we investigated the conditions required for application of H2-production selective pressure. The efficacy of this selection was demonstrated by the isolation of a variant(D5) out of the parental cw15 (cell wall-less) population with an increased O2I50 for H2 evolution. Finally, several laboratory-scale photobioreactors were built and tested using a mutant of Anabaena variabilis that lacks H2-uptake activity, as a model system that would mimic an O2-tolerant algal strain. We were able to demonstrate H2 production efficiencies of 1-2% and continuous H2-productioncapability for 8 months using the cyanobacterial mutant.