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Microalgae with their improved growth rates and accumulation of high-value-added products make their commercial production attractive. Among them, lutein, which is a carotenoid, plays a very important role due to its various applications in the food and pharmaceutical industry. Induction of its biosynthesis can be triggered by various stress conditions like light. In this study, three different light intensities (50,150 and 300 µmol photons/m²s) and aeration rates (1, 3, and 5 L/min) were utilized to induce the lutein biosynthesis and biomass productivity in Scenedesmus obliquus. Lutein was isolated by preparative chromatography using a semiprep C30 column (10 × 250 mm, 5µm) and its confirmation was made by LC-MS/MS. According to the results, Scenedesmus obliquus synthesized the maximum lutein (8.01 ± 0.1 mg/g) with biomass productivity of 1.698 g/L at 150 µmol photons/m²s light intensity using 3 L/min as aeration rate. To the best of the authors' knowledge, this was the first study that the lutein was isolated by preparative chromatography using semiprep C30 carotenoid column with a simple and rapid separation, which can be used as a reference methodology for the isolation of other carotenoids. Scenedesmus obliquus can be an important alternative source for commercial production of lutein, as it is indicated from the results of this study. [ABSTRACT FROM AUTHOR]