Cometabolic degradation of ofloxacin by aerobic methane oxidation coupled with denitrification: Identification of degraders, helper bacteria, and metabolic networks.

The aerobic methane oxidation coupled with denitrification (AME-D) system enables simultaneous nitrogen removal and antibiotics cometabolic degradation, yet the underlying microbial ecological mechanisms remain poorly understood. This work took ofloxacin (OFL) as a typical antibiotic pollutant and established long-term stable sequencing-batch AME-D reactors to explore their nitrogen removal efficiency and OFL degradation sustainability under antibiotic stress. A multi-omics approach combining metagenomics, metaproteomics, and metabolomics was adopted to identify the core degraders and functional helper bacteria, and unravel the synergistic metabolic interactions sustaining the system's performance. Results indicate that the AME-D cometabolic system maintains high-efficiency nitrogen removal capacity and achieves effective OFL degradation under OFL stress. The piperazine ring is the primary reactive site of OFL, undergoing ring cleavage to form intermediate products. Multi-omics results demonstrate that microbial community structure is significantly reshaped by OFL pressure. Aerobic methane-oxidizing bacteria (MOB) are identified as core degraders, which mediate OFL cometabolism via methane monooxygenase (pMMO/sMMO) and supply available electron donors. Denitrifiers and stress-tolerant auxiliary bacteria form synergistic networks by optimizing nitrogen metabolism, activating efflux pumps and regulating antioxidant defenses, which maintains system functional stability under high OFL stress. Fluorescence in situ hybridization (FISH) verification confirms that MOB, nitrifiers and denitrifiers form compact spatial interaction structures in sludge, which provide favorable conditions for interspecific substance exchange and electron transfer. This study clarifies the multi-scale functional maintenance mechanism of AME-D cometabolic system, offering theoretical support for the treatment of antibiotic-laden wastewater and ecological risk control of antibiotic resistance.