Development of an Efficient Algal H2-Producing System

Green algae have the potential to efficiently photoevolve H/sub 2/ from water using the photosynthetic O/sub 2/ - evolving apparatus and the reversible hydrogenase enzyme when CO/sub 2/ is not present. Unfortunately, algal hydrogenases are very sensitive to inactiviation by O/sub 2/, the by-product of the water-splitting process. This problem has been one of the major practical factors limitingthe commerical utilization of green algae for H2 production. The other major limitation, saturation of H2 production by algae at light intensities much lower than normal solar levels, is being addressed by ORNL. The objectives of our project are to generate O/sub 2/ -tolerant mutants of the green alga Chlamydomonas reinhardtii; to test them in a laboratory-scale system for continuous productionof H/sub 2/ under aerobic conditions; and to collaborate with ORNL to improve the overall efficiency of H/sub 2/ production in intact and cell-free systems. The ultimate goal of the work is to configure a photobiological water-splitting process that will lead to a H/sub 2/ -producing system that is cost effective, scalable, non-polluting, and renewable. Our approach to obtain O/sub 2/ -tolerantmutants of Chlamydomonas involves two types of selection techniques. The first depends on the survival of cells under photoreductive conditions, where H/sub 2/ utilization is required, and the second requires the survival of the organisms under H/sub 2/ -producing conditions. In both cases, increasing O/sub 2/ stress is placed on the organisms to select for O/sub 2/ -tolerant strains. Our workthis past year established optimal conditions for the H/sub 2/ -production selection technique in both the wild-type (WT) and the cell-wall-less (cw15) strain of Chlamydomonas. We confirmed the efficacy of the selection technique by demonstrating that variants selected from WT and cw15 cell populations show an increase in up to 2.3 times the I/sub 50/ for O/sub 20/, with no decrease in initialH/sub 2/ -production rate. We also examined the effects of pre-induction of the hydrogenase, electron acceptors, changes in light intensity, and increases in O/sub 2/ concentration on the number of survivors.