Biochar for mitigating the oxytetracycline stress of Nitrite-DAMO system: Microbial metabolic mechanisms and metagenomics research.

Denitrifying anaerobic methane oxidation (DAMO) serves as a critical biogeochemical nexus linking the global carbon and nitrogen cycles to mitigate greenhouse gas emissions. However, ubiquitous antibiotics in DAMO habitats and wastewater systems presents a severe ecological threat, exacerbating methane emissions, nitrogen accumulation, and biotoxicity. Investigating mitigation strategies and mechanisms is essential for addressing these real-world environmental challenges. This study focused on the nitrite-dependent anaerobic methane oxidation (Nitrite-DAMO) system to investigate the comprehensive effects of biochar on denitrification performance and microbial metabolic characteristics under long-term oxytetracycline (OTC) stress (1 mg/L and 10 mg/L), along with the potential mechanisms. Results indicated that biochar significantly mitigated OTC toxicity and effectively enhanced both denitrification and methane oxidation performances. Average denitrification rates in biochar-amended groups reached 0.86 and 0.73 mg/(L·d), while the methane oxidation capacities increased to 2.27 and 1.76 times those of the non-biochar groups. Biochar established physicochemical barriers against antibiotic stress by stimulating extracellular polymeric substances (EPS) and enhancing electron transport system activity (ETSA). High-throughput sequencing and metagenomic analysis revealed that biochar drove microbial community succession, enriching functional bacteria (Candidatus Methylomirabilis and Thauera), while significantly upregulating the abundance of functional genes involved in nitrogen and carbon metabolism pathways (nirK, pmoA/B/C). Crucially, biochar suppressed the proliferation of potential hosts and disrupted transposons-mediated horizontal gene transfer (HGT), thereby substantially mitigating the accumulation and dissemination risks of antibiotic resistance genes (ARGs). The synergistic mitigation mechanisms elucidated herein provide theoretical guidance for in-situ regulation strategies to reduce methane emissions in antibiotic-contaminated wetlands, paddy fields, and river sediments.