Co-metabolism of Norfloxacin by Chlorella pyrenoidosa: Carbon source effects, biotransformation mechanisms, and key driving genes.

Co-metabolism with appropriate carbon sources has been demonstrated to effectively enhance the removal of ubiquitous recalcitrant micropollutant by microalgae. However, the specific impacts of carbon sources on the co-metabolism of antibiotics by microalgae remain insufficiently explored. In this study, transcriptomics, gene network analysis, extracellular polymeric substances (EPS), and enzymatic activity involved in co-metabolic pathways of norfloxacin (NFX), were systematically evaluated to investigate the underlying biological mechanisms involved in NFX co-metabolism by Chlorella pyrenoidosa. Results revealed that glucose, glycine, sodium acetate, and sodium carbonate significantly enhanced NFX removal, with 10 mM glucose being the most effective and achieving a removal efficiency of 61.5 %. Glucose led to notable increase in microalgal biomass production, peroxidase enzyme activity, and EPS protein secretion, thereby accelerating NFX degradation. Mass balance analysis indicated that biotransformation was the primary mechanism for NFX removal as supported by the detection of fluorine element within microalgal cells. Eight major metabolites resulting from defluorination, piperazine ring transformation, decarboxylation, acetylation and oxidation reactions were identified. Furthermore, a transformation pathway was proposed based on mass spectrometry data of extracted NFX intermediates along with their formation dynamics. The four carbon sources exhibited distinct effects on the transcriptome of C. pyrenoidosa. Differentially expressed genes analysis revealed significant influence of these carbon sources on genes related to cytochrome P450 enzyme family, glutathione, and peroxidases, which played major roles in NFX co-metabolism. These findings provide unique insight into the specific impacts of carbon sources on microalgae-based NFX removal, revealing key metabolic genes and underlying biological mechanisms driving NFX co-metabolism by microalgae.