Enhanced Antibiotic Dissipation in Swine Wastewater Facilitated by Heavy Metals through a Transcriptionally Upregulated Microalgal Metallohydrolase

Swine wastewater represents a complex pollution matrix laden with antibiotics, heavy metals, and ammonia, demanding integrated remediation strategies. While microalgae offer a sustainable solution, their efficacy is often limited by low stress tolerance and degradation capacity. Here, we applied adaptive evolution to Chlorella sorokiniana, yielding an evolved strain with significantly enhanced simultaneous removal of ammonia, Cu 2+, Zn 2+, and antibiotics from real swine wastewater. The evolved strain maintained stable performance across multiple treatment cycles under both microbe-rich and sterile conditions, accompanied by reproducible enrichment of specific bacterial taxa. Transcriptomic analysis identified a novel and highly upregulated metallohydrolase (MHO), which was functionally validated as a key mediator of coremediation through overexpression and mutagenesis. Structural modeling and docking revealed that Cu 2+ /Zn 2+ jointly stabilize the active conformation of MHO, enabling metal-dependent degradation of enrofloxacin and sulfadiazine into less toxic derivatives. The enzyme and the evolved strain exhibited broad pH and temperature tolerance, along with broad-spectrum degradation ability toward multiple fluoroquinolones and sulfonamides. This study unveils a previously unrecognized microalgal detoxification mechanism and demonstrates adaptive evolution as a powerful tool for engineering robust strains for complex wastewater bioremediation.