Substrate filling ratio affects nitrogen removal and antibiotic resistance risk in modular moving bed constructed wetland: Biofilm-mediated microbial community succession and resistome profiles reshaping.

Constructed wetlands (CWs) are widely used for advanced treatment of wastewater treatment plant effluents and their nitrogen (N) removal performance is often inhibited by antibiotics. Biofilms on CW substrates play a fundamental role in pollutant biodegradation, microbial community stability and antibiotic resistance gene (ARG) dissemination. This study investigated the effects of substrate filling ratios (90% and 60%) in modular moving bed constructed wetlands (MMB-CWs) on operational performance, biofilm properties and antibiotic resistance risks. The MMB-CW with higher substrate filling ratio exhibited a better N removal efficiency of 83.7% and a significant reduction of nitrous oxide emission by 72.6%. The higher substrate filling ratio increased the protein/polysaccharide ratio of extracellular polymeric substances (EPS), potentially forming a hydrophobic barrier and structured a highly modular microbial network with pronounced niche differentiation. Genome-centric analysis revealed that core taxa carrying denitrification and anammox genes (narG, narH, nirS, nosZ, hzs, hdh) enriched by 1.5- to 12.6-fold in abundance in the MMB-CW with 90% substrate filling ratio. Notably, Desulfobacillus increased by 1.7-fold in abundance, which served as a keystone species driving denitrification, EPS construction, oxidative stress adaptation and energy production. The elevated abundances of enzymes catalyzing key electron- and energy-generating steps in the tricarboxylic acid cycle and denitrification enzymes drove a more complete denitrification process. The highly modular network restricted horizontal gene transfer of ARGs, mitigated the enrichment of pathogenic antibiotic-resistant bacteria (PARBs) and occurrence of high-risk ARGs in MMB-CW. The findings provide an optimization strategy for MMB-CW in view of treatment performance and ecological risk.