Acetate as a sustainable organic carbon to support mixotrophic growth of Chlorella sorokiniana for alternative protein production

Abstract

Microalgae are well-known for their role as sustainable bio-factories, offering a promising solution to the global food and nutrition crisis. To determine the potential of Chlorella sorokiniana UTEX 1230 for food applications, particularly as an alternative protein source, the study employed a mixotrophic cultivation mode with sodium acetate (NaAc) as a cost-effective organic carbon (NaAc-C) source. Varying levels of NaAc-C and sodium nitrate-sourced nitrogen were investigated to understand the metabolic characteristics of microalgal growth. The designed heterotrophic cultivation confirmed the ability of C. sorokiniana UTEX 1230 to grow on NaAc-C, and then the mixotrophic cultures, when cultured with both NaAc-C and CO₂, exhibited superior growth performance, doubling the biomass concentration compared to the autotrophic control. The addition of nitrogen (750 mg/L NaNO₃) facilitated the thorough metabolism of NaAc-C, resulting in a maximum biomass concentration of 2.82 g/L in the 150 mM NaAc-C group, while excess NaAc-C and nitrogen extended the lag phase, thereby reducing production efficiency. A detailed analysis of nitrogen and protein concentrations over time revealed that higher nitrogen availability led to greater protein accumulation, which was then degraded to support essential life activities under nitrogen starvation. Therefore, a fed-batch strategy with NaAc-C and NaNO₃ supplementation was applied to maximize the positive impact of nutrients on biomass profiles, where 2.63 g/L microalgae were harvested on the 4^th day, with an increase in protein yield to 0.30 g/L/day. These findings offer theoretical guidance for further refining this microalgal strain for use as an alternative protein.