Iron-rich tidal flow constructed wetlands (ITCW) have emerged as effective and eco-friendly systems for achieving high-efficiency total nitrogen (TN) removal in tailwater. However, the microbial removal mechanism in ITCW, particularly the microbial assembly processes, remains unclear, which limits understanding of the relationship between micro-level microbial activities and macro-level performance of ITCW. In this study, ITCW was constructed to investigate its performance, microbial responses, and community assembly mechanisms using metagenomics and the neutral community model (NCM). Results demonstrated that total nitrogen removal in ITCW increased by 52.53 ± 9.03 % and 21.20 ± 6.26 %, respectively, compared to conventional constructed wetlands and iron-based constructed wetlands. The combination of tidal flow and iron substrate established an active iron cycle in ITCW. The iron cycle improved electron transfer capacity and supported the enrichment of nitrifiers (e.g., Nitrospira) and denitrifiers (e.g., Hydrogenophaga, Thauera), enhancing nitrogen removal efficiency in ITCW. Metagenomic analysis confirmed an increased relative abundance of functional genes associated with nitrogen metabolism (e.g., amoA, nirSKABD) and the iron cycle (e.g., korB). NCM analysis indicated that stochastic processes influenced community assembly in all three CWs. Nonetheless, tidal flow operations in ITCW reduced stochastic influences, fostering niche specialization of functional nitrogen microorganisms, which supported their growth while minimizing functional redundancy, thereby enhancing nitrogen treatment performance. These findings offer significant insights into microbial mechanisms for nitrogen removal and provide theoretical guidance for ITCW design and operation.
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