Techniques for Isolation of Oxygen-Tolerant Mutants of Hydrogen-Producing Chlamydomonas reinhardtii

The hydrogen metabolism of photosynthetic bacteris and cyanobacteria involves the coordinated action of three enzymes: nitrogenase, reversible hydrogenase, and uptake hydrogenase. Green algae, on the other hand, contain only the reversible hydrogenase, which is responsible for both hydrogen production and uptake in this organism. The quantum yield for hydrogenase-catalyzed hydrogen production ismuch higher than that for nitrogenase. Algal hydrigenases, however, are extremely sensitive to oxygen. For this reason, green algae cannot be utilized commercially for hydrogen production. We have investigated two types of selective pressure to isolate mutants of Chlamydomonas reinhardtii that produce hydrogen in the presence of oxygen. The first is bassed on competition between hydrogenase andmetronidazole for electrons from light-reduced ferredoxin. Since reduction of metronidazole results in the release of toxic products that eventually kill the organism, cells with an active oxygen-tolerant hydrogenase will survive a short treatment with the drug in the light in the presence of oxygen. Using this technique, we have isolated a variant of C. reinhardtii that evolves hydrogen with anI 50 for oxygen three times higher than the wild type strain. The second selective pressure depends on growth of algal cells under photoreductive conditions. Algal cells must fix carbon dioxide in the presence of oxygen with reductants derived from hydrogen uptake by the reversible hydrogenase. We will describe in detail both selective pressures, as well as the characteristics of the mutantsisolated by application of these selective pressures to a population of mutagenized wild type cells. This work was supported by the U.S. DOE Hydrogen Program.