Diversity Of Denitrifiers And Key Denitrification Functional Genes In Sediments Of Nine Estuary Of Dianchi Lake

Nitrogen cycle is one of the most important cycles in the geochemical cycle,and denitrification is an important part of the nitrogen cycle,which plays an important role in agriculture,industry and water.The cause of eutrophication in water is mainly caused by the accumulation of chemical elements such as nitrogen and phosphorus.The denitrification process can reduce nitrate and nitrite in water to gaseous forms such as nitric oxide,nitrous oxide,and nitrogen,which is of great significance for controlling eutrophication of water.The estuary area has become one of the important areas for studying plate due to its special geographical location and important ecological functions.The rivers passing through Dianchi Lake are distributed along shore of Dianchi Lake,and the areas passing through them vary.It can provide some new ideas for the biological treatment of Dianchi Lake eutrophication.In this study,the estuary sediments of 9 lakes in Dianchi Lake basin were used as the research object.The diversity and community structures of denitrifying microbes and denitrification key functional genes were analyzed in different estuaries of Dianchi Lake by using high-throughput amplicon sequencing and pure culture with denitrifiction functional genes nirS,nirK and nosZ as molecular markers.The community analysis results of different denitrification functions at the sampling points of Dianchi Lake show that the main environmental factors affecting nirS-type,nir K-type,and nosZ-type denitrifer are TP,TN,and NO₃^--N,respectively.There are differences in physical and chemical factors between each sample point,these differences depend on the geographical location of the different rivers entering the lake,the flow area,the length of the stream,and the source of pollution.Using nirS-type,nirK-type and nosZ-type genes as molecular markers,the numbers of sequence obtained was 180,155,14,061,111,and 111,713,and the numbers of OTU obtained were 1,417,1316,and 1633,respectively.This result indicates that the species in the Dianchi estuary sediments are rich and high diversity.At the same time,it can be showed that although the nosZ gene has the least numbers of sequence,there are more microbial groups containing this gene than other genes.The diversity of the three genotypes of denitrifying bacteria was significantly different in each site.The sites with the highest diversity of nirS,nirK,and nosZ genes were Baiyuhe,Chaihe,and Baiyuhe.Except for the Chaihe site,the diversity of nir K-type denitrifying bacteria in other sites was significantly lower than that of the other two genotypes.This indicates that the diversity of nirK genes in other sites except the Chaihe site is relatively low.In addition,the samples with high diversity of three genotypes were all inflow rivers with severe agricultural non-point source pollution in the southeast of Dianchi Lake.Studies on the communities of various gene type denitrification groups show that the dominant groups of the three gene type denitrifying microorganisms are Proteobacteria.In addition,Dechloromonas and Thauera,Bradyrhizobium and Bosea,Leptothrix and Thiobacillus are the dominant genu in nirS type denitrifier,nirK type denitrifier,and nosZ type denitrifier at the genus level,respectively.But none of these strains were isolated in pure culture.For isolation of aerobic denitrifying bacteria from estuarine sediments,a total of439 strains of denitrifying bacteria were isolated from 9 samples of Dianchi Lake.These strains belong to 2 phylum（Proteobacteria,Firmicutes）and 3 classes（Gammaproteobacteria,Betaproteobacteria,Bacilli）,4 orders,6 genera,33 species.Among these strains,Pseudomonas is the dominant genus,total 267 strains are obtained,accounting for 60.8%of the total strains;Acinetobacter is the second dominant genus（total 151 strains）,accounting for 34.3%.Amplification of three key denitrification functional genes of these strains showed that Pseudomonas was the dominant denitrification group among pure culture strains,of which 17 strains of Pseudomonas amplified all three denitrification genes;In addition,Acinetobacter is one of the important denitrifying bacteria.Potential new taxonomic strain YIM MLB12 was studied in multi-phase classification.Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM MLB12^Tformed a tight phylogenic lineage with members of the genus Lampropedia and was closely related to‘Lampropedia puyangensis’2-bin with 98.3%16SrRNA gene sequence similarity.Strain YIM MLB12 is a Gram-negative bacterium with short rod-shaped cells,no flagella,and no endospores.The physiological and biochemical characteristics show that the optimal temperature for the growth of the strain is 25-30℃,the optimal pH is pH 7.0-8.0,and the optimal salinity（NaCl）is 0.5-2%.The results of cytochemical analysis showed that the main quinotype of the strain was ubiquinone 8;the main fatty acid components were summed feature 3,C_16:0,C_10:00:0 3-OH,sumed feature 8,C_12:02:0 3-OH and C_14:0;The mass spectrum of polar lipids is mainly composed of diphosphatidyl glycerol,phosphatidyl monomethylethanolamine,phosphatidylethanolamine and phosphatidylglycerol;the main polyamine is spermidine.Comprehensive phenotype and genotype,strain YIM MLB12 is a new species of the genus Lampropedia,named Lampropedia aestuarii sp.nov YIM MLB12^T（YIM MLB12^T=KCTC 42886^T=CGMCC 1.17071^T）.