Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high-energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA-2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO4 center dot 7H2O, EDTA-2Na, and trace metal solution, respectively. These values were validated using a 5-L jar fermenter. Lutein concentration was increased by almost 80% (139.64 +/- 12.88 mg/L to 252.75 +/- 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 +/- 3.23 mg/L) and 240,000 L (263.13 +/- 2.72 mg/L). These observations suggest C. vulgaris as a potential lutein source. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:736-743, 2014