Diversity And Nitrogen Removal Characteristic Of Denitrifying Bacteria In The Sediments Of Estuary Around Dianchi Lake

Nitrogen pollution is the main cause of deterioration of water bodies. With the development of industry and the improvement of living standard, the problem of nitrogen pollution is s increasingly more serious. As a result of conventional methods for nitrogen removal can’t meet the demand of application, people pay more attention to the research of biological nitrogen removal technology. The discovery of aerobic denitrification and the isolation of aerobic denitrifiers provide a promising approach for biological nitrogen removal and also lay a new theory foundation for the application of biological nitrogen removal. Aerobic denitrification is the co-respiration or co-metabolism of O2and nitrate, which microbial can use organic substrate as electron donor, and simultaneously utilize oxygen and nitrate as electron acceptors to convert nitrate to nitrogen gas under aerobic conditions. Without the limitation of oxygen, aerobic denitrification has more advantages than traditional anaerobic nitrogen removal on application. While there have been many studies on the relationship between isolation of aerobic denitrifiers and their physiological characteristics, and the presence and abundance of genes involved in the denitrification process, only a few recent reports combine these aspects.The objective of this research was to study the bacterial diversity and give prominence to community composition of the denitrifiers in sediment of estuarine around Dianchi lake and soil of lakeside area by using denaturing gradient gel electrophoresis (DGGE), Miseq pyrosequencing, and cultivation method. To provide some theoretical basis for revealing bacterial populations and improving nitrogen pollution in Dianchi lake. Then screeed a number of strains which have high aerobic denitrifing. Results are important to the rhizosphere bacterial diversity research in phosphorus-rich environment and solve the phosphorus pollution of Dianchi Lake. Providing strain resources and research methods that have potential application value for biological denitrification.According to the sampling location to analysis the sediment and soil characteristics, we found that organic carbon and total nitrogen in the estuary aroound Caohai and the northern coast of Waihai samples are higher than eastern, southern and western coast of Waihai samples, and overall the trend of two factors are decreasing from north to south.For the diversity comparisons of bacteria in spring, summer and winter sample, DGGE analysis results showed that, by using bacterial16S rRNA gene V3 hypervariable region as study section, no matter the DGGE of different sample in same season or same sample in different season the profiles displayed a higher band number, the number and distribution of bands in all sediment samples were significantly different. Reflect a high species richness and diversity of samples. While they were no significant differences in soil samples. It can be concluded that seasons and sampling sites are impacting on bacteria composition and community structure by changing physical and chemical factor in environment. Reflect there were no significant differences in bacteria composition and community structure between different soil samples, this may be due to the soil environment and colony structure is stable than sediment.For the diversity comparisons of denitrifying bacteria in sample, DGGE analysis results indicate that, by using the nitrite reductase gene nirK and nirS as study section, DGGE profiles display a high band number and diversity as well as the analysis results of16S rRNA genes. nirS and nirK sequences are all belong to uncultured bacterium after sequencing, but show high richness. By way of comparison, the diversity and abundance of nirS gene is higher than nirK in the sediment, and it is just the opposite in the soil. And nirS gene is more sensitive to abiotic factors influence.In the isolation experiment of aerobic denitrifying bacteria,484strains were isolated. These isolates belong to4bacteria phyla (Proteobacteria、Actinobacteria、 Bacleroidetes、Firmicutes),13families (Comamonadaceae、Moraxellaceae、 Aeromonadaceae、Rhodospirillaceae%Bacillaceae、Enterobacteriaceae、 Comamonadaceae、Ralstonia_f、Rhizobiaceae-Flavobacteriaceae、 Pseudomonadaceae、 Nocardiaceae、Sphingobacteriaceae、Rhodobacteraceae),22genera (Paracoccus、Gemmobacter、Rhizobium、Sphingobacterium、Rhodococcus、 Raoultella、Pseudomonas、Myroides、Lampropedia、Klebsiella、Flavobacterium、 Ensifer、Diaphorobacter、Delftia、Cupriavidus、Comamonas、Citrobacter、Bacillus、 Azospirillum、Aeromonas、Acinetobacter、Acidovorax),77species. There are467strains belong to Proteobacteria, accounting for97%of the total number of bacteria, belonging to Alpha-, Beta-, Gamma-proteobacteria, And321strains belong to Pseudomonas, accounting for76%of the Gammaproteobacteria. Five strains were isolated as potential novel species. The multiple classification study of two strains found that the two strains represent a potential new species. Through the nitrate reduction test, functional genes (nirS, nirK, nosZ) detection and denitrifying activity determination screened4strains with high nitrate nitrogen removal efficiency under aerobic conditions. The rates of nitrate removal by the4strains could above92%, the highest reached99%in48h. And the accumulation of nitrite are below0.3mg/L, the minimum is0.049mg/L. After phylogenetic analysis base on16S rRNA gene indicated that the3strains belong to Aeromonas and one belong to Delftia, in wihch has been successfully isolated aerobic denitrifying bacteria have high denitrifying ability. As the optimum C/N ratio of four strains are20, indicated they are suited for denitrification with high C/N.