Accumulation of O2-Tolerant Phenotypes in H2-Producing Strains of Chlamydomonas reinhardtii by Sequential Applications of Chemical Mutagenesis and Selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H₂-evolving activities of up to 10 times the O₂-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H₂-metabolism clones following exposure to increasing amounts of O₂, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H₂ following exposure to O₂ concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H₂-production activity once O₂ is removed. These results suggest that O₂-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O₂ when it is actively catalyzing H₂ evolution. Published by Elsevier Science Ltd. on behalf of the International Association for Hydrogen Energy.