Microalgae are currently being recognized as a sustainable resource for valuable products such as pigments for their superior antioxidant activities. Chlorella sorokiniana MB-1- M12 is a lutein-rich mutant capable of growth and lutein production in photoautotrophic, mixotrophic, heterotrophic, and photoheterotrophic metabolism. Despite the capacity of heterotrophic growth, light and CO2 were found to be critical for lutein accumulation in MB-1- M12. Thus, an innovative two-stage heterotrophic-mixotrophic process (TSHM) performed under optimal conditions brought about 10 g/L biomass and 7.42 mg/g lutein. Integration of fed-batch and semi-batch operational strategies and scale-up in a 5L bioreactor increased biomass production, lutein content, and lutein production to reach 20 g/L, 8.71 mg/g, and 181.11 mg/L, respectively. This work demonstrated that integration of the TSHM process with appropriate operational strategies (i.e., integrated fed-batch and semi-batch operation) could enhance the lutein accumulation in MB-1-M12, making it apt for marketable lutein production.