Improving Chlorella protein production under heterotrophic high cell density fed-batch cultivation with a two-stage nitrogen nutrient supply strategy

Heterotrophic cultivation has been proved to be an effective and industrially scalable route for the economical production of Chlorella biomass. Nevertheless, the low protein content of heterotrophic Chlorella biomass hinders its utilization as a superior protein source. In this study, the influences of various C/N ratios on cell growth and protein production were evaluated for a previously isolated protein-rich alga Chlorella sorokiniana CMBB276. The results indicated that a high C/N ratio was favorable for cell growth, whereas a low C/N ratio was advantageous for protein synthesis. A proposed two-stage nitrogen supply strategy by shifting the C/N ratio from 18 to 6 was found to be capable of simultaneously improve the protein content and yield of C. sorokiniana CMBB276, achieving the maximum protein content of 58.6% of dry cell weight (DCW) and a final protein yield of 87.0 g L⁻¹, respectively. We further verified that the fast accumulated ammonium in C. sorokiniana CMBB276 cultivated with the switched C/N ratio contributed to the remarkable enhancement of protein content, while concurrently causing cytotoxicity. Moreover, under high ammonium stress, while maintaining a high protein content, C. sorokiniana CMBB276 persisted in growing with a decreased level of cellular reactive oxygen species (ROS) and cell membrane damage through exogenous addition of acetate, which lays the foundation for further improvement of protein production by applying the two-stage nitrogen supply strategy in combination with acetate addition.