Antibiotic residues in reclaimed wastewater irrigation: Implications for nitrogen metabolism and microbial ecology in rice paddy soils

Rice cultivation is highly water-intensive, and reclaimed wastewater (RW) irrigation presents a sustainable solution to water scarcity. However, this practice potentially introduces residual antibiotics in RW into agroecosystems, disrupting microbial-mediated nitrogen cycling and promoting the dissemination of antibiotic resistance genes (ARGs). In this study, we assessed how long-term irrigation with RW containing mixed antibiotics (0–100 μg/L) affects soil microbial communities, nitrogen functional genes, ARGs, as well as its potential implications for nitrous oxide (N₂O) emissions in rice paddies. A five-year mesocosm experiment was conducted, integrating metagenomic sequencing, quantitative PCR, and network analysis. Results showed that low antibiotic concentrations (0–10 µg/L) did not alter microbial community composition but increased microbial diversity and network complexity. Proteobacteria, Actinobacteria, Nitrospirae, and Chloroflexi were the dominant phyla involved in nitrogen cycling, with Proteobacteria harboring the majority of ARGs and nitrogen functional genes. The abundance of key nitrification ( amoA , hao ) and denitrification ( nar , nirK , nirS , norB , nosZ ) genes was not inhibited by antibiotics; instead, low concentrations stimulated their expression, indicating a hormetic response. Proteobacteria-dominated genera (e.g., Sulfuricella , Geobacter, Acidovorax ) were identified as major co-hosts of both ARGs and nitrogen functional genes, highlighting their dual roles in nitrogen transformation and ARG dissemination. Importantly, the increased abundance of nosZ , which catalyzes N₂O reduction to N₂, suggests a lower potential for N₂O (a potent greenhouse gas) accumulation under RW irrigation. Overall, our findings suggest that long-term RW irrigation containing environmentally relevant antibiotic concentrations can maintain nitrogen cycle functionality without exacerbating N₂O emissions, providing important insights for the safe and sustainable reuse of RW in agroecosystems.