| The promotion of sponge city construction in China has promoted the wide use of low-impact development facilities-bioretention.However,its unstable removal effect on nitrogen pollutants limited the further application.Clarifying the mechanism of nitrogen removal in this system,especially the principle of microbial nitrogen removal,can fundamentally solve this problem.The discovery of complete ammonia oxidation process overturned the previous academic cognition of nitrogen cycle theory.According to the existing studies,complete ammonia-oxidizing microorganisms were likely to exist in the bioretention and affected the nitrification process.Research on the distribution characteristics of complete ammonia-oxidizing microorganisms in bioretention is helpful to clarify the mechanism of microbial nitrogen removal in this system.However,there has been no relevant research evidence at present,let alone the discussion of their contribution in the process of ammonia-oxidizing,which restricted the updating and improvement of the theory on microbial nitrogen removal in bioretention.In this study,field sampling and simulation experiment were carried out respectively.With the technology of metagenomic sequencing,high-throughput sequencing and real-time fluorescent quantitative PCR,critical metabolism pathways in microbial nitrogen were investigated,and so with the absolute abundance and diversity of Comammox amoA genes,to valid its widespread existence in bioretention,summary its spatial and temporal distribution characteristics,evaluate its contribution potential in the process of microbial ammonia oxidation,and discuss the relationship between its characteristics and engineering impact factors.（1）Metagenomic sequencing implied that there was a complete ammonia oxidation pathway in bioretentions.Results of field sampling experiments exhibited that the detection rates of Comammox amoA genes in 18 bioretentions of Shenzhen was100%,and were detected in different seasons,which proved the universal existence of Comammox in this system.The abundance distribution of Comammox amoA genes reflected significant spatial and temporal differences.On temporal scale,the abundance of Comammox amoA genes in dry season was two orders of magnitude higher than that in wet season;On spatial scale,the land-used types with high vegetation coverage and good maintenance often showed higher abundance of Comammox amoA genes;The abundance of Comammox amoA genes was higher in soil matrix with higher concentrations of organic matter.The alpha diversity index of Comammox amoA genes in bioretentions was similar to the natural ecology such as agricultural land,forest soil and wetland,but different from the artificial systems such as sewage treatment plant and wastewater treatment plant.The colony structure of Comammox in the same land-used system was similar.Comammox Clade B and Nitrospira nitrosa were not detected in this study.All the detected gene sequences were divided into 20 OTUs,and strain with the most abundance was Comammox Clade A2.According to the results of phylogenetic analysis,the gene sequences of the dominant strains in bioretentions were highly similar to those found in wetlands and intertidal zones.The concentrations of soil organic matter would promote the overall growth of Comammox amoA genes,but the effects of soil physical and chemical properties on each dominant strains were different:the abundance of Comammox Clade A2 was significantly positively correlated with the concentrations of NH4+-N and NO3--N;the abundance of Nitrospira inopinata was significantly negatively correlated with the concentrations of NH4+-N;the abundance of Nitrospira nitrificans was positively correlated with concentrations of OM.The land-used types of bioretentions would affect the diversity of Comammox colony.The colony structure of Comammox in bioretentions with similar land-used types showed similarity.（2）After a rainfall event,there was an obvious dynamic response process of Comammox in bioretentions.The abundance of Comammox amoA genes increased continuously with the times after rain,and reached the peak in 24 or 36 hours after rain,and then began to decline.In the simulated experiment columns,the average abundance of Comammox amoA genes was similar to that of AOA,and the growth rates of genetic abundance in the rainfall cycle was higher than that of AOB;In a practical bioretention,the relative abundance of Comammox amoA genes was always more than 50%,and its genetic abundance was higher than that of AOB.These suggested that Comammox had a significant potential for ammonia oxidation in bioretention systems,and might play a major role in the process of nitrogen transformation.Soil depth had a significant effect on the abundance distribution of Comammox.The abundance of Comammox amoA genes in root layer was about one order of magnitude lower than that in non-root layer,but its relative abundance in root layer was higher than that in non-root layer.At the same time,in the whole rainfall cycle,the peak time for genetic abundance in root layer was significantly earlier than that in non-root layer.（3）The abundance and diversity of Comammox in bioretentions were different under various plant species.The absolute abundance of Comammox amoA genes was the lowest in bioretentions with Canna,but its relative abundance was the highest.Meanwhile,the diversity of Comammox amoA genes in bioretentions with Canna was the lowest,and the colony structure and dominant strains were significantly different from that in other plant species;The overall abundance of denitrification genes decreased in bioretentions with Canna,but Comammox promoted the denitrification process most.Plant species had a significant effect on the distribution of Comammox in bioretentions.The reason for this phenomenon might be that plant species regulated the physical and chemical properties of soil matrix,thus indirectly affect the microbial function.The results of this study can fill in the research blank of complete ammonia oxidation reaction in bioretention system,reveal the distribution characteristics,impact factors and nitrification function of Comammox,help to improve the study on the mechanism of microbial nitrogen removal in bioretention,and provide a theoretical basis for improving its effectiveness on nitrogen removal. |
