Unveiling mechanisms of nZVI-biochar enhanced nitrogen removal in constructed wetlands: Microbial community assembly

Constructed wetlands (CW) enhanced with nanoscale zero-valent iron supported biochar (nZVI-Biochar) composites showed promise for efficient nitrate-nitrogen (NO₃ - -N) removal, yet the microbial mechanisms and predictive modeling of such systems remained unclear. In this study, four CW configurations were established to evaluate NO₃ - -N removal performance: CK-CW (quartz sand only), C-CW (quartz sand + biochar), FE-CW (quartz sand + nZVI), and CFE-CW (quartz sand + nZVI-Biochar). Denitrification efficiency was assessed under varying operational conditions, including carbon-to-nitrogen (C/N) ratio, hydraulic retention time (HRT), and pH. Microbial community composition and functional gene abundance were analyzed via 16S rRNA high-throughput sequencing and quantitative PCR (qPCR). A random forest model with 10-fold cross-validation was employed to predict NO₃ - -N concentrations and identify key influencing factors, while microbial assembly mechanisms were elucidated using the null model-based iCAMP framework. Results indicated that under optimized conditions (C/N=10, HRT=1 d, pH=7), CFE-CW achieved the highest NO₃ - -N removal, attributed to the enrichment of denitrifying bacteria ( Proteobacteria , Actinobacteriota ) and elevated expression of key nitrogen-cycle genes ( amoA , narG , nirS , nosZ ). A random forest model accurately predicted effluent NO₃ - -N (R²=0.99), identifying TN and NH₄ + -N as dominant factors. The addition of nZVI-Biochar strengthened homogeneous selection indicating a heightened influence of environmental filtering by the amendment on shaping the microbial community structure and promoting phylogenetic clustering within microbial communities. Network analysis revealed that nZVI-Biochar supplementation enhanced microbial community stability and optimized ecological interactions. This integrated analysis demonstrates that nZVI-Biochar optimizes constructed wetlands by enabling accurate performance prediction and revealing underlying microbial mechanisms. • nZVI-Biochar composites drastically boost nitrate removal in constructed wetlands. • Mechanitic insights reveal enhanced microbial diversity and electron exchange. • Eflluent nitrate levels are reliably forecasted by a random forest model. • Ecological analysis identifies drift and homogenizing selection as key processes.