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Production of CO2-tolerant microalgae have received much attention as well as physicochemical fixation of CO2 in industrial flue gas. Although many microalgae that are tolerant to high levels of CO2 have been found and evaluated, the CO2 concentration for their good growth is generally lower than their maximum tolerable CO2 level. In the present study, we attempted to isolate microalgae capable of growing in high levels of CO2 (high-level-CO2-preferring microalgae, HCP-microalgae). We used a CO2-permeable polystyrene bottle for the enrichment of HCP-microalgae in environmental samples.