Multi-metabolism regulation insights into nutrients removal performance with adding heterotrophic nitrification-aerobic denitrification bacteria in tidal flow constructed wetlands

Constructed wetlands (CWs) usually exhibit limits in functional redundancy and diversity of microbial community contributing to lower performances of nutrients removal in decentralized domestic sewage treatment. To address this quandary, heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria was added in tidal flow CWs (TFCWs) developing for nitrogen (N) and phosphorus (P) removal. With addition of HN-AD bacteria, TFCWs could be setup more rapidly and obtained better removal efficiencies of 66.9%–70.1% total nitrogen (TN), and 88.2%–92.4% total phosphorus (TP) comparing with control systems (TN: 53.9%; TP: 83.9%) during stable operation. Typical-cycles variations showed that TFCWs with addition of HN-AD bacteria promoted NO₃⁻-N and NH₄⁺-N removal respectively under hydraulic retention time (HRT) of 14 h and 8 h with slight NO₂⁻-N accumulation. Activated alumina (AA) coupled with HN-AD bacteria decreased P release and relieved its poor removal performance in CWs. Based on metagenomic taxa and functional annotation, Pseudomonas and Thauera played pivotal roles in N removal in TFCWs. Furthermore, gradient oxic environments by 8 h-HRT promoted co-occurrence of heterotrophic nitrifiers (mostly Pseudomonas stutzeri) and autotrophic nitrifiers (mostly Nitrosomonas europaea. and Nitrospira sp.) which potentially accelerated NH₄⁺-N transformation by elevated nitrification and denitrification related genes (e. g. amoABC, hao, napA and nirS genes). Meanwhile, the addition of HN-AD bacteria stimulated nirA and gltD genes of N assimilation processes probably leading to NH₄⁺-N directly removal. The conceptual model of multi-metabolism regulation by HN-AD process highlighted importance of glk, gap2 and PK genes in glycolysis pathway which were vital drivers to nutrients metabolism. Overall, this study provides insights into how ongoing HN-AD bacteria-addition effected microbial consortia and metabolic pathways, serving theoretical basis for its engineered applications of TFCWs in decentralized domestic sewage treatment.