Machine learning-based integration of plant growth regulators and overcompensation enhances microalgal protein production from wastewater

Photoautotrophic wastewater valorization represents a sustainable route for carbon-neutral nutrient upcycling into bioproducts. However, its practical implementation is often constrained by wastewater-induced growth inhibition. Besides, the distribution of metabolic flux across competing pathways may further restrict nutrient allocation towards target product. In this study, we propose a combined strategy integrating plant growth regulators (PGRs) with a nitrogen overcompensation strategy to enhance microalgal performance during potato starch wastewater treatment. Gibberellic acid (GA 3 ) and naphthaleneacetic acid (NAA) both alleviated wastewater inhibition and enhanced nutrient recovery in Chlorella pyrenoidosa , achieving up to 79.9% total nitrogen removal. Notably, supplementation with 10 mg L −1 GA 3 resulted in a 26.2% increase in biomass compared to controls. The random forest model identified candidate high-response concentrations of approximately 10.5 ± 1 mg L −1 for GA 3 and 10.2 ± 1 mg L −1 for NAA and suggested declining biomass responses at higher NAA concentrations, whereas GA 3 showed a broader model-predicted growth-promoting range. Under the nitrogen starvation–repletion condition, PGR-supplemented cultures achieved maximum biomass and protein concentrations of up to 1.58 g L −1 and 594.82 mg L −1 , respectively, together with over 90% removal of COD, total phosphorus, and ammonium. Transcriptomic profiling indicated that GA 3 induced significant upregulation of genes involved in photosynthetic apparatus function, ribosomal biogenesis, and transmembrane transport processes compared to NAA. This work establishes a strategy that integrates PGRs with nitrogen overcompensation to overcome wastewater-induced stress. The proposed approach provides a practical strategy for improving microalgal productivity and nutrient recovery from high-strength wastewater, advancing the development of carbon-neutral biorefinery systems.