Dynamics of antibiotic resistance genes and bacterial community structure within substrate biofilms

Biofilms that develop on the surface of substrates are critical for treating wastewater. The accumulation of antibiotic resistance genes (ARGs) within these biofilms is particularly noteworthy. Despite their importance, studies that focus on biofilms attached to substrate surfaces remain scarce. This investigation explored the prevalence and succession of ARGs and microbial dynamics in biofilms on different substrates (ceramic, biomass filter, and steel slag) versus water biofilms over a year. Results showed distinct differences in ARG profiles between water and substrate biofilms. Multidrug ARGs constituted 39.14-46.73% of all ARGs in the substrate biofilms, with macrolide ARGs making up 11.98-14.52%. Seasonal variations influenced the diversity of the ARGs, notably increasing during the spring. The neutral community model suggested that the ARG assembly was dominantly driven by stochastic process. Proteobacteria, Actinobacteria and Campylobacter emerged as the predominant phyla within these biofilms. The microbial community distribution was predominantly influenced by ammonium nitrogen (NH₄⁺-N) (R² = 0.4113), temperature and total nitrogen (TN). Notably, temperature exerted a critical impact on the microbial community distribution (P = 0.001), identifying it as the principal factor for spatial arrangement. Furthermore, the structural variations of ARGs were primarily driven by total organic carbon (TOC) (R² = 0.3988), temperature, oxidation-reduction potential (ORP) and NH₄⁺-N. Our findings provided new insights into the optimization of substrate selection and ecological management to manage ARG enrichment, offering a promising strategy for aquatic ecological restoration and pollution control.