Community Structure And Denitrification Characteristics Of NirS-type Denitrifiers In Typical Water Bodies

The micro-ecological structure of denitrifying bacterial populations is closely related to the nitrogen cycle in water bodies,driving nitrogen flow and material circulation in the hydrosphere.It is of great scientific significance to study the characteristics of denitrifying bacteria population in different eutrophic waters to reveal the nitrogen pollution load of typical water sources,and research of microbial remediation technology.In this study,micro-polluted（drinking water reservoir）,moderate pollution（urban inner lake）and heavy pollution（wastewater treatment plant）water bodies were selected.Based on the analysis of water quality parameters of different pollution levels,high-throughput DNA sequencing technology was used to diagnose nirS type denitrifying bacterial population structure,in order to explore the coupling mechanism of water quality of different pollution types and population structure diversity of nirS-type denitrifying bacteria.Based on selective medium coupled ultrasonic pretreatment technology,high-efficiency aerobic denitrifying bacteria were successfully domesticated and screened.The denitrification characteristics of high-efficiency bacterium were studied and ¹³C-MFA metabolic flow technology to diagnose a high-efficiency aerobic denitrifying bacteria carbon metabolism pathway,which provided scientific basis and theoretical support for the engineering application of aerobic denitrifying bacteria.The findings are follows:（1）The results based on drinking reservoir show that:The proportion of unclassified genus is larger,which is more than 65%.The dominant denitrifying bacteria are Paracoccus sp.（10.7%）and Azoarcus sp.（9.6%）in Jinpen reservoir and Dechloromonas sp.（10.8%）and Azoarcus sp.（8.1%）in Lijiahe reservoir.Redundancy analysis（RDA）showed that water quality in different months had significant difference on the population structure of nirS denitrifying bacteria（P<0.01）.The results showed that environmental factors comprehensively regulated the composition and distribution of nirS denitrifying bacteria.（2）The results based on urban inner lake show that:There are significant differences in the water quality in the water quality of the different urban lakes（P<0.01）.High throughput DNA sequencing showed that the dominant bacteria were Proteobacteria（79.8%）and Firmicutes（1.8%）.A co-occurrence network analysis indicated that keystone taxa were Alicycliphilus sp.,Dechloromonas sp.,and Paracoccus sp.,etc.Denitrifying bacterial communities inhabited the different urban lakes are influenced by ammonia nitrogen,manganese and algal cell concentrations.A variance partitioning analysis（VPA）also showed that geographic location was more important than water quality factors in structuring the denitrifying bacterial communities.（3）The results based on typical wastewater treatment plants show that:In different typical wastewater treatment system,the abundance of nirS gene per ng DNA ranged from 4.6×10² to 2.4×10³ copies per ng DNA.Among WWTPs,total nitrogen removal efficiencies varied from 25.8 to 84%,which was positively correlated with diversity indices,whereas abundance-based coverage estimator index decreased with an increase in latitude.The dominant phyla across all samples were proteobacteria,accounting for46.23%of the sequences.The dominant genus was Thauera sp.,Alicycliphilus sp.,and Pseudomonas sp.,etc.Based on network analysis,the coexistence and interaction between dominant genera may be vital for regulating the nitrogen and carbon removal behaviors.Multivariate statistical analysis revealed that both geographic location and wastewater factors concurrently govern the distribution patterns of nirS denitrifying bacterial community harbored in WWTPs.（4）Three novel Mix-CADB consortiums were isolated from the sediment using ultrasonic processor pretreatment.The results showed that the nitrate and total organic carbon（TOC）removal efficiencies of Mix-CADB were higher than 99%and 97%,respectively.The nirS gene sequence observed that the dominant phyla were Firmicutes and Proteobacteria.Network analysis demonstrated that total nitrogen（TN）and chemical oxygen demand（COD）removal performance were driven by the co-occurrence and interaction of Bacillus subtilis,Pseudomonas stutzeri,Rhodococcus sp.,etc.Mix-CADB inoculation can remove 86%TN and 93%COD of real wastewater.（5）A highly efficient aerobic denitrifying bacteria was isolated and screened successfully.Its metabolic pathway was diagnosed based on ¹³C-MFA stable isotope labeling,and identified as Paracoccus denitrificans by 16S rRNA technology.Nitrogen balance analysis showed that about 71.88%of the initial nitrogen was removed in the form of gas,and 21.53%of the nitrogen was converted into biomass.The optimum conditions for aerobic denitrification were as follows C/N with 7.47,shaking speed with108 r/min,temperature with 31℃and initial pH with 8.02.The average TN removal rate and COD removal rate of the three wastewater treatment plants are 89%and 91%,which are significantly higher than WWTPs denitrification process.