Efficient biotransformation of norfloxacin by Castellaniella defragrans HL-1: Kinetics, pathways, toxicity assessment, and genomic insights

A novel strain, Castellaniella defragrans HL-1, was isolated from activated sludge acclimated to high norfloxacin (NOR) concentrations (5–45 mg/L) for 123 days. This strain exhibited efficient NOR biotransformation with sodium acetate (NaAc) as the primary carbon source. The optimal conditions (pH 7.5, 35 ℃, 0.987 g L^-1 NaAc, OD₆₀₀ = 0.335) were determined using single-factor experiments and response surface methodology (RSM), achieving 78.4–89.2% NOR removal (1–5 mg L^-1) within 24 h. The first-order kinetic constant (k) ranged from 0.070–0.093 h⁻¹, surpassing most reported NOR-degrading strains. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) identified 15 degradation intermediates, including seven unreported phenolic hydroxylated metabolites derived from the benzene ring of NOR. EAWAG Biocatalysis/Biodegradation Database (EAWAG-BBD) predicted the complete metabolic pathways for converting these intermediate products into glycine. ECOSAR toxicity assessment indicated low toxicity risks of NOR metabolites. Luminous bacteria bioassays demonstrated a 56.1 ± 3.3% reduction in acute toxicity after 24 h compared to the initial 40 mg L⁻¹ NOR toxicity. Genomic analysis revealed a complete NOR degradation enzymatic system in C. defragrans HL-1, including cytochrome P450-mediated hydroxylation, N-acetylation, and piperazine ring cleavage. This study highlights the bioremediation potential of C. defragrans HL-1 against fluoroquinolone antibiotics and provides novel insights into their biotransformation mechanisms.