Unveiling the power of COD/N on constructed wetlands in a short-term experiment: Exploring microbiota co-occurrence patterns and assembly dynamics

Constructed wetlands (CWs) are a cost-effective and environmentally friendly wastewater treatment technology. The influent chemical oxygen demand (COD)/nitrogen (N) ratio (CNR) plays a crucial role in microbial activity and purification performance. However, the effects of CNR changes on microbial diversity, interactions, and assembly processes in CWs are not well understood. In this study, we conducted comprehensive mechanistic experiments to investigate the response of CWs to changes in influent CNR, focusing on the effluent, rhizosphere, and substrate microbiota. Our goal is to provide new insights into CW management by integrating microbial ecology and environmental engineering perspectives. We constructed two groups of horizontal subsurface flow constructed wetlands (HFCWs) and set up three influent CNRs to analyse the microbial responses and nutrient removal. The results indicated that increasing influent CNR led to a decrease in microbial α-diversity and niche width. Genera involved in nitrogen removal and denitrification, such as Rhodobacter, Desulfovibrio, and Zoogloea, were enriched under medium/high CNR conditions, resulting in higher nitrate (NO-3-N) removal (up to 99 %) than that under lower CNR conditions (<60 %). Environmental factors, including water temperature (WT), pH, and phosphorus (P), along with CNR-induced COD and NO- 3-N play important roles in microbial succession in HFCWs. The genus Nitrospira, which is involved in nitrification, exhibited a significant negative correlation (p < 0.05) with WT, COD, and P. Co-occurrence network analysis revealed that increasing influent CNR reduced the complexity of the network structure and increased microbial competition. Analysis using null models demonstrated that the microbial community assembly in HFCWs was primarily driven by stochastic processes under increasing influent CNR conditions. Furthermore, HFCWs with more stochastic microbial communities exhibited better denitrification performance (NO- 3-N removal). Overall, this study enhances our understanding of nutrient removal, microbial co-occurrence, and assembly mechanisms in CWs under varying influent CNRs.