For biofuel production using microalgae, it is necessary to mutate the algae to improve the productivity of biomass and oil contents. In this study, we focused on very-long-alkyl ketones so-called alkenone that are thought to be good candidates for biofuels. Previously, we reported dose-dependency of the colony formation after 0-320 Gy of carbon ion irradiation. We screened mutants that showed high alkenone productivity from the irradiated cells. In this study, we irradiated carbon ion beams to the high alkenone-producing mutants to investigate the effects of the second-round mutagenesis on the mutant. The cells cultured for 14 d in a 96-well plate were stained with Nile Red and the amount of lipids was evaluated by a microplate reader. The results suggested that it is possible to further improve the productivity of high alkenone-producing strain by the second-round mutagenesis. In the precious study, 30 Gy was suggested to be a suitable dose to enhance oil productivity, however, it seemed better to use a lower dose for the second-round irradiation
