The combination sequence effect on nitrogen removal pathway in hybrid constructed wetlands treating raw sewage from multiple perspectives

The combination sequence of traditional hybrid constructed wetlands (HCWs) affects the removal of nitrogen in raw sewage, but the effect of the combination sequence on nitrogen removal pathway have seldom been reported, especially the specific conditions allowing anammox to occur. Three-stage HCWs, namely vertical flow (VF), horizontal flow (HF) and surface flow (SF) constructed wetlands, were arranged in six different sequences to investigate nitrogen removal efficiencies and microbial removal pathways using metagenomic and stable isotope analyses. Results showed that the combination sequence significantly affected nitrogen removal pathways in HCWs. We found the best removal of total nitrogen (~50%) and ammonium (NH₄⁺-N, ~99%) in HCWs with a VFCW in the 1st stage. Metagenomic results and stable isotope analyses further indicated that simultaneous nitrification and heterotrophic denitrification were the main pathways in unsaturated VFCW, which depended on the energy substance and electron donor supplied by chemical oxygen demand (COD_Cr) in raw sewage. Nitrifier, anammox bacteria and autotrophic denitrifies prevailed in the subsequent saturated CWs, which tend to nitrogen loss by partial nitrification and anammox in HFCW when fed with NH₄⁺-N wastewater with low COD_Cr. Providing NH₄⁺-N and oxygen in low COD_Cr wastewater was the essential step to facilitate anammox process in HFCW. It implied that the problem of poor nitrogen removal due to carbon limitation could be overcome by optimizing conditions in anammox's favor.