Insights into iron-enhanced denitrification coupled with antibiotic resistant genes control in biochar-based...

In biofiltration (BF) systems, biochar can enhance pollutant removal by promoting biofilm formation. Its abundant pore structure can also sequester antibiotics away from microbial cells, thereby reducing its bioavailability and accumulation of antibiotic resistance genes (ARGs). However, dense biofilms favor horizontal ARG transfer, especially among denitrifying bacteria, which are prone to stress under low influent C/N conditions. In this study, a strategy combining iron minerals was proposed to alleviate the ARG accumulation in BF systems. Compared with magnetite, goethite and siderite released Fe²⁺ through microbial dissimilatory iron reduction and chemical dissolution respectively, thereby driving iron‑autotrophic denitrification and enhancing the activity of electron‑transfer mediators (cytochrome c and Fe-S proteins). As a result, the level of nitrosative stress was reduced with significant downregulation of related genes (hmp, hcp, norR, and etc.), which was a driving force for conjugative transfer of ARGs. Specifically, the excessive accumulation of tryptophan and shortage of methionine were thus alleviated, which contributed to the regulation of global repressor gene expression and the mitigation of ARG conjugative transfer. With the combination of goethite or siderite in BF systems, the abundance of resistance genome in biofilm exhibited a reduction of 52.68 ± 3.80% and 41.26 ± 4.20%, respectively, which could effectively reduce the environment-ecological risk of antibiotic and ARGs.