This study's primary purpose is to produce high-value astaxanthin and canthaxanthin using microalga Chlorella zofingiensis performing a two-stage cultivation mode (mixotroph followed by simultaneous nitrogen and high light stress conditions). In this study, glycerol and sodium acetate were used as organic carbon sources, added to the culture medium. On the other hand, vinasse, known as ethanol industry waste was used as an alternative for the culture medium. At the end of the first stage of cultivation (after seven days), the optimum concentrations were considered as 1.2, 2, and 0.1 g/L, among different concentrations performed by vinasse, glycerol, and sodium acetate, respectively. In addition, 2% CO2 aeration increased the productivity and biomass concentration of the cultures compared to the atmospheric air aeration mode. The highest lipids, carbohydrates, and proteins accumulation of 30.7 ± 1.5, 30.2 ± 0.1, and 37.6 ± 0.7% dry weight happened when glycerol, vinasse, and sodium acetate were used as carbon sources, respectively. Compared to the autotrophic cultures (control), in mixotrophic cultures, the protein content decreased, while the number of lipids and carbohydrates increased. The highest biomass productivity (161.9 ± 7.8) was related to glycerol. At the end of the second stage of cultivation, the protein content decreased significantly while the carbohydrates and lipids increased. The highest amount of synthesized astaxanthin (7.7 ± 0.1 mg/g dry weight) and canthaxanthin (1.2 ± 0.1% mg/g dry weight) was obtained using glycerol and vinasse, respectively.
