For biofuel production by micoralgae, it is important to improve the algae to high biomass or oil contents strain. In this study, we focused on alkenones, as biofuel candidate, that are very-long-alkyl ketones and are only known in five haptophyte species. We selected one of the alkenone-producing haptophytes, Tisochrysis lutea, to mutate by heavy ion beam irradiation that is known as a good method to obtain useful mutants. In addition, the mutants obtained by this method can be applicable in open system as non-GMO. So far, we have selected high oil-producing strains from fluorescence intensity of lipids by lipid staining with Nile red regent. It is considered that these high oil-producing strains will have features such as, high photosynthetic activity, high growth rate, high ratio to lipid in intracellular carbon or suppression of lipid degradation. Here, we screened and analyzed the strains which are suppressed alkenone degradation.Alkenone is known to degrade as energy source like polysaccharides under dark condition. Therefore, we stained intracellular neutral lipids with Nile red regent and monitored fluorescence intensity of stained lipids under dark condition by using microplate reader. First, we cultured selected high oil-producing strains with 96-well plate for 2 weeks and then put them in dark condition for 1 week. When we compared fluorescence intensity of lipids before and after 1 week dark condition, we observed little decrease in fluorescence intensity from 72 strains. Second, we cultured selected 31 of 72 strains with plastic flask for the analysis of alkenone amounts by GC-FID. From the results, we obtained 4 candidate strains as which are suppressed alkenone degradation under dark condition. Finally, we checked time course of several component, such as polysaccharides, protein, polar and non-polar lipids under dark condition. After dark condition, polysaccharides were immediately degraded and over 80% were degraded within 1 week. Degradation of alkenone was started 2 days after dark condition in wild-type but 7 days or later after dark condition in candidate strains. It is suggested the possibility that not only alkenone degrading enzyme but also dark-acclimation to lipid metabolism are changed in these strains.Marine Biotechnology Conference 201
