The Mechanism Of Enterobacter And Klebsiella Bacteria To Purify Nitrogen Pollution In Wastewater And Promote Biological Nitrogen Fixation In Glycine Max L.

Nitrogen(N)is one of the most common elements in nature.Free nitrogen cannot be directly utilized by plants,and available nitrogen in soil is scarce,thus nitrogen becomes a key factor limiting agricultural development.Excessive application of nitrogen fertilizer will lead to nutrient loss,soil fertility decline,groundwater serious nitrogen pollution and other adverse environmental problems.Therefore,it is the focus of current research to study efficient functional biological agents to treat nitrogen pollution in water and promote green and sustainable development instead of chemical nitrogen fertilizer application.In the early stage of this study,7 strains with heterotrophic nitrification-aerobic denitrification(HNAD)ability were isolated from the underground sedimentary water of Wuhan Agricultural Pharmaceutical Plant and the nitrogen removal ability was tested.It was found that one of the strains Enterobacter sp.Z1 and Klebsiella sp.Z2 had strong nitrogen removal ability,but they could not completely remove nitrogen from the water.The co-cultured strains “Z1+Z2” showed significant nitrogen removal ability,with the removal rates of ammonia,nitrate and nitrite in the medium reaching more than 97%,and the average removal rates were 18.20 mg/L*h,11.7 mg/L*h and 5.18 mg/L*h,respectively.In order to explore the nitrogen removal mechanism of the mixed strains“Z1+Z2”,we conducted metabolomics,proteomics,fluorescence quantification and RNA interference analysis.Metabolomics analysis showed that Indoleacetic acid(IAA)and its secondary metabolites catechol and o-aminobenzoate were up-regulated 105 times,2.55 times and 5.09 times,respectively,under co-cultured conditions.Analysis of experimental data showed that the promotion of IAA metabolism on HNAD process was the result of multiple regulation: 1)The strain Z2 promoted the autologous decarboxyl-degrading metabolic pathway,provided IAA for the strain Z1,and activated the hydrolyzation-redox metabolic pathway for strain Z1.2)IAA induced the expression of key enzymes in denitrification in the nitrogen removal process.3)The metabolic pathway of IAA in strain Z1 required nitrate and nitrite as electron acceptors.At the same time,in order to further verify the application potential of mixed microbial agents,we designed a small bioreactor,and selected industrial wastewater with low organic matter,pig farm wastewater with high organic matter and wastewater with combined pollution of pesticide and nitrogen for automatic treatment experiments.The results showed that the nitrogen removal rates of strains “Z1+Z2” in natural wastewater could reach more than 96%.More importantly,it can simultaneously remove the accompanying phosphorus,pesticide and organic matter pollution in the wastewater,showing a strong ability to control and repair water pollution.Both Enterobacter sp.Z1 and Klebsiella sp.Z2 belonged to HNAD bacteria and could produce a large amount of IAA,the latter having high value in soil environment.Therefore,in this study,the mixed microbial strains “Z1+Z2” were used as the model,aiming to explore the relationship between rhizosphere bacteria and symbiotic nitrogen fixation of soybean and rhizobia in pot experiment.Red and green fluorescent proteins were used to label strains Z1 and Z2,and the experiments of soybean hydroculture and soil culture were carried out.The results showed that the mixed microbial agents “Z1+Z2”could well colonize in soybean rhizosphere soil and significantly promote the biological nitrogen fixation system of soybean.The results showed that the aboveground fresh weight,the nitrogen-fixation enzyme activity,the number and weight of root nodules increased by 50%,94%,71% and 54%,respectively.Then,we used rhizosphere metabolomics analysis,soybean nitrogen-fixing phenotype detection,fluorescence quantification(qRT-PCR),Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis to reveal the significant accumulation of flavonoids,plant hormones and sulfonates after the addition of strains “Z1+Z2”.Through molecular level analysis and verification of experimental data,we found that the promotion mechanisms of the biological nitrogen fixation process were the result of multiple regulation,mainly summarized in two aspects:1)Strain Z1 could produce a large amount of Salicylicacid(SA),while strain Z2 mainly accumulated IAA in the soil,which jointly promoted the early growth and development of the nodules.2)Taurine produced and secreted by soybean can be absorbed by strain Z1 and Z2 and catabolic to produce acetyl-Co A,which is then absorbed by soybean to synthesize flavonoids.In conclusion,this study found that the co-cultured strains “Z1+Z2” had super high nitrogen removal capacity,and the metabolism of IAA was closely related to the process of nitrogen removal.At the same time,the mixed microbial agents “Z1+Z2” can secrete abundant small molecular chemicals and regulate the biological nitrogen fixation process of soybean from multiple angles.In this study,the interaction mechanism between efficient nitrogen removal by mixed microbial agents and promoting biological nitrogen fixation system of soybean was analyzed,which provided a new theoretical basis for biological nitrogen removal and promoting nitrogen cycle by rhizosphere microorganisms,and also provided a new possibility for microbial treatment of natural pollution and environmental remediation.