Analysis Of Denitrifying Microorganisms Diversity And Isolation Of Efficient Strains In Myriophyllum Aquaticum Constructed Wetlands

With the development of social economy,the phenomenon of excessive oxygen-consuming pollutants ammonia nitrogen in our country is becoming more and more serious,which threatens the safety of the water environment.Constructed wetlands(CWs)have received increasing attentions for their N removal performances,which are mostly determined by microorganism.Different influent ammonia nitrogen concentration may influence N removal efficiency in practice,while the microbiological mechanism of effects of different ammonia nitrogen concentrations on microorganisms enabling N removal in CWs are poorly understood.In this study,Myriophyllum aquaticum CWs were established to investigate the influences of different ammonia nitrogen concentrations(A,0 mg/L;B,2 mg/L;C,24mg/L;D,105 mg/L;E,124 mg/L)on the N removal efficiency,microbial community structure and response to ammonia nitrogen concentration of CWs.Our findings suggested 105 mg/L ammonia nitrogen concentration CWs achieved highest N removal rate,removing 89.30%NH₄⁺-N and 92.34%TN from the influent.The study found the abundance of nitrifying bacteria and denitrifying bacteria in CWs with high ammonia nitrogen concentrations was significantly higher than that control group,revealing that the biological mechanisms of N removal in CWs with different ammonia nitrogen concentrations may be different.Different ammonia nitrogen concentrations were proved to alter composition structure of microbial communities via 16S high-throughput sequencing,e.g.denitrifiers Rhodococcus sp.was obviously enriched with increasing ammonia nitrogen concentrations.In addition,interactions among microbial populations shifted and enhanced by increasing NH₄⁺-N concentrations.These results indicated different effects of different ammonia nitrogen concentrations on microbial community,which make the interaction across microbial communities more complicated.Redundancy analysis showed that the ammonia nitrogen concentration affected the abundance of denitrifying bacteria.These results can provide theoretical basis and support for elucidating the microbiological mechanism of nitrogen removal in CWs of different ammonia nitrogen concentrations.Microorganisms dominate the denitrification process.Therefore,it is very important to isolate microorganisms that can efficiently remove NO₃^-,whose removal is the most central step in biological N removal.A total of 33 strains were isolated in this study,among which M3-1,L3-2 and C1-3 had a faster degradation rate of NO₃^--N,identified as Pantoea sp.,Klebsiella sp.,Enterobacter sp.,respectively.The quantitative real-time PCR results showed that the abundance of nar G gene encoding the nitrate reductase of three strains were 24,25 and 12 times more than nir S gene encoding the nitrite reductase,respectively.The results indicated that the three strains can rapidly degrade NO₃^--N but there is accumulation of NO₂^--N.The optimal conditions of strain C1-3 proved to have higher NO₃^-removal capacity was C/N ratio of10,glucose as the carbon source,temperature of 30°C,p H of 7.0,initial inoculum of 2%by single-factor experiments.In addition,the strain C1-3 achieved 9.12 mg/L/h NO₃^--N removal rate under NO₃^--N concentration of 800 mg/L.These results indicate that strain C1-3 can efficiently degrade and tolerate nitrate with high concentrations,which can provide a theoretical basis for strain applications.