Antibiotic resistance gene control coupled with enhanced denitrification in a pilot-scale biochar-based biofiltration...

Biofiltration systems can synergistically remove multiple pollutants from paddy field drainage, including nutrients and emerging contaminants such as antibiotic residues. However, their application is often limited by insufficient denitrification, which can also create nitrosative stress that induce horizontal transfer of antibiotic resistance genes (ARGs) among denitrifying bacteria, thereby posing potential secondary environmental risks. In this study, a pilot-scale innovative biochar-based biofiltration (IBBF) system amended with goethite was developed and operated over two rice-growing cycles. Biochar promoted the colonization of denitrifying bacteria, while goethite released Fe²⁺ through dissimilatory iron reduction of Geobacter and Deferrisoma, thereby further driving autotrophic denitrification by Acidovorax and Comamonas. The iron cycling enabled more stable and efficient electron transfer, increasing the abundance of genes involved in nitrogen metabolism by 1.4-fold. The total nitrogen removal efficiency of the IBBF system remained above 85% even when the treatment capacity increased from 1.87 to 4.75 m³/d. Furthermore, the system alleviated nitrosative stress and the associated imbalance in tryptophan and methionine metabolism, thereby suppressing ARG conjugative transfer via the regulation of global repressor genes. As a result, the overall removal efficiency of ARGs exceeded 99.0%, and the number of detected ARG types decreased by 44.6%. Therefore, the secondary environmental risks from ARGs in the IBBF system were effectively controlled.