Phylogenetic Of NirS Type Denitrifiers And Ecological Distribution Characteristics In The Pearl River Estuary

With the increasing population density in coastal areas and the development of modern industry,a large number of industrial and agricultural wastewater and domestic sewage are discharged into the ocean,resulting in the migration of excessive active nitrogen from the land to the sea.This phenomenon has become the main cause of eutrophication in estuaries,causing a series of environmental hazards.Denitrification is an important process in the microbial nitrogen cycle,which is very important for the removal of excess nitrogen from estuarine sediments.The microorganisms acting on the denitrification process are called denitrifiers,among which denitrifiers are various and widely distributed,so the conventional 16S rRNA sequencing method cannot be used to study their ecological characteristics.In this study,nirS gene was used as a molecular marker to study the diversity of denitrifiers,a total of 30 stations were sampled along the main stream of the Pearl River Estuary?PRE?in the winter and summer of 2016.From upstream to downstream,the five stations including PRE-1,PRE-3,PRE-7,PRE-13 and PRE-18,and each station comprising surface,middle and bottom layers.We analyzed the activity,abundance and community structure of nirS-type denitrifiers using ¹⁵N-labeled incubation quantitative-PCR and high throughput sequencing techniques in sediment cores from Pearl River Estuary?PRE?.The specific results are as follows:1.We calculated similarity threshold of nirS gene and 16S rRNA gene,respectively.As far as the relationship between the two types of genes have carried on the regression analysis,it is concluded that the correlation of denitrification nirS gene and 16S rRNA genes is not very good,and the nirS gene did not evolve with the evolution of the 16S rRNA gene and nirS genes that not only exists the phenomenon of vertical transfer,there is the trend of horizontal transfer,this is ultimately a diversity of denitrifiers,widely distributed,so can not get clear taxonomy and thus it is difficult to further analyze the diversity of denitrifiers in the environment.2.Based on the collected sequences of denitrifiers in pure culture,this study conducted OTU clustering on the sequences of denitrifiers in pure culture through MOTHUR software,and finally established the similarity threshold of OTU at the genus level with the sequence similarity of 93%.At the same time,based on the sequence of pure culture and part of the collection quality control the sequence of environmental samples after the sequence file?Template file?,and each sequence is form the taxonomy information file?Taxonomic file?,template file and taxonomic file constituted the reference taxonomy database of functional genes,and it contains284 sequence,and the sequence length were greater than 390 bp.Finally,the determined similarity threshold and the constructed database were used to analyze the community ecological distribution characteristics of denitrifiers in the sediments of PRE.3.Compared with the surface sediment of the PRE,the subsurface sediment has the same potential activity of denitrification.The abundance of nirS gene in the PRE sediments decreased sharply from the surface to the bottom,but its potential activity did not show significant temporal and spatial changes.No significant difference in the structure of denitrifier community from the surface layer to the subsurface layer,but there was significant difference in the diversity between different sites.The redundancy analysis indicated that redox potential,nutrient content and organic matter and other environmental factors jointly affected the diversity and community distribution of nirS type denitrifying microorganisms in the sediments of PRE.Our evidence showed that nirS-type denitrifers played important roles in the nitrogen removal in subsurface sediments of PRE.