Maximizing Nitrogen Removal and Lipid Production by Microalgae under Mixotrophic Growth Using Response Surface Methodology: Towards Enhanced Biodiesel Production

Abstract

The present study aimed to optimize synthetic wastewater composition as a mixotrophic medium for enhanced growth and lipid accumulation coupled with high nitrogen removal by the green microalga Chlorella sp. Individual effects of the three main independent variables (nitrate concentration, seawater ratio, and glycerol supplementation) were tested initially, then response surface methodology (RSM) was subsequently performed to explore the optimum combined conditions. The highest lipid productivity of 37.60 mg/L day was recorded at 25% seawater. Glycerol supplementation enhanced both lipid content and biomass production, which resulted in the highest recorded lipid productivity of 42.61 mg/L day at 4 g/L glycerol. Central composite design followed by numerical optimization was further applied which suggested NaNO3 concentration at 101.5 mg/L, seawater ration of 23.8%, and glycerol supplementation of 0.25 g/L as the optimum conditions for dual maximum lipid productivity and nitrogen removal of 46.9 mg/L day and 98.0%, respectively. Under the optimized conditions, dry weight and lipid content increased by 31.9% and 20.3%, respectively, over the control, which resulted in increase in lipid productivity by 71.5%. In addition, optimization process resulted in pronounced changes in fatty acid proportions where saturated fatty acids increased by 7.4% in the optimized culture with simultaneous reduction of polyunsaturated fatty acids. The estimated biodiesel characteristics calculated from the fatty acid methyl ester (FAMEs) profile showed agreement with the international standards, while optimized cultures showed an 8.5% lower degree of unsaturation, which resulted in higher cetane numbers and lower iodine values. This study provides economical approach for optimization and efficient nutrient recycling through cultivation of Chlorella sp. for further enhanced biodiesel production.