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Euglena gracilis , a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of  E. gracilis  that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich  E. gracilis  with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY 505/515 , and FACS-based isolation of top 0.5% lipid-rich  E. gracilis  cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich  E. gracilis  mutant strain, named B 1 ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting