Two-stage continuous cultivation of microalgae overexpressing cytochrome P450 improves nitrogen and antibiotics removal from livestock and poultry wastewater

Improper treatment of livestock and poultry wastewater (LPWW) rich in ammonium nitrogen (NH₄-N) and antibiotics leads to eutrophication, and contributes to the risk of creating drug-resistant pathogens. The design-build-test-learn strategy was used to engineer a continuous process using Chlorella vulgaris to remove NH₄-N and antibiotics. The optimized system removed NH₄-N at a rate of 306 mg/L/d, degraded 99 % of lincomycin, and reduced the hydraulic retention time to 4 days. The physiological, metabolic, and genetic mechanisms used by microalgae to tolerate LPWW, remove NH₄-N, and degrade antibiotics were elucidated. A new cytochrome P450 enzyme important for NH₄-N and antibiotic removal was identified. Finally, application of synthetic biology improved the NH₄-N removal rate to 470 mg/L/d, which is the highest removal rate using microalgae reported to date. This research contributes to the mechanistic understanding of wastewater detoxification by microalgae, and the goal of achieving a circular bioeconomy for nutrient and water recycling.