Isolation And Identification Of Heterotrophic Nitrifying Bacteria And Their Bacteriophages In Daqing Wetland Waters

Heterotrophic Nitrifying Bacteria（HNB）have the characteristics of simultaneous nitrification and denitrification using organic compounds,and can proliferate rapidly in a short time in the reactor.Therefore,HNB has great potential in the field of biological nitrogen removal of wastewater.As an engineering bacterial agent,heterotrophic nitrifying bacteria are often used in ammonia-nitrogen wastewater treatment system,and their growth and metabolic activity have a great influence on the effect of ammonia-nitrogen wastewater treatment.However,in the actual treatment process,the bacterial activity is not only affected by environmental factors such as temperature and carbon source,but also interfered by bacteriophage infection,which leads to the sudden cleavage of bacteria resulting in the loss of metabolic function of the strain,affecting the process performance and the quality of wastewater treatment.Therefore,in this experiment,the water samples of Daqing wetland were collected,the heterotrophic nitrifying bacteria and their corresponding bacteriophages were screened,and the denitrification characteristics of the most efficient heterotrophic nitrifying bacteria and the biological characteristics of bacteriophages were determined.The whole genome of representative bacteriophages was determined to provide basic data and theoretical basis for reducing or even eliminating bacteriophage interference in sewage treatment.The main results are as follows:（1）A total of 15 strains of heterotrophic nitrifying bacteria were isolated from wetland water.According to molecular biological identification,they belong to 7 genera:Pseudomonas,Pannonibacter,Brucella,Stenotrophomonas pavanii,Delftia,Serratia and Acinetobacter.Among them Pseudomonas sp.LLM-5 strain can remove 81.81%and 77.95%ammonia nitrogen and total nitrogen within 24 hours,and accumulate only 1.94 mg/L of nitrite nitrogen,which is significantly better than other strains（P<0.05）.（2）The strain LLM-5 grew well when NH₄⁺-N,NO₂^--N and NO₃^--N were the only nitrogen sources,and the maximum removal rates were 80.27%,51.47%and 65.98%,respectively.The results showed that heterotrophic nitrifying bacteria LLM-5 had the characteristics of aerobic denitrification.The strain showed the best nitrogen removal performance when carbon source was sodium succinate,C/N ratio was 16:1,p H value was 9.0,salinity was 2%,temperature was40℃and initial ammonia nitrogen concentration was 1000 mg/L.Under the optimum culture conditions,LLM-5 strain had the maximum removal rate of 87.06%of NH₄⁺-N and 81.27%of TN,and only accumulated a small amount of HN-AD intermediate metabolites.（3）Fifteen strains of HNB were used as potential hosts to isolate bacteriophages.Finally,six corresponding bacteriophages were isolated by LWB-2,LWB-3,LLM-4,LLM-5,NYS-4 and LFW-4 bacteria.The morphological analysis of transmission electron microscope showed that PPP1,PSP1,DTP1 and AOP1 belonged to podoviridae family,while SOP1 and BPP1 belonged to siphoviridae family.The host specificity of PPP1 and PSP1 was lower than that of the other four strains.The results of biological characteristics showed that the bacteriophages of SOP1 and PSP1 were 151,108 CFU/cell,while those of BPP1,PPP1,DTP1 and AOP1 were 94,66,37 and49 min,respectively.The latent stage was short（except for 10 min of SOP1 and PSP1 phages,the rest was 20 min）.The results of temperature,p H and UV stability tests showed that six strains bacteriophages were isolated and all had the ability of resistant high-temperature,p H and ultraviolet light.（4）Through genome sequencing and analysis,the genome of PSP1 infecting LLM-5 phage is 267 842 bp in length,including 292 open reading frames（ORF）,including 40 ORF known function.PSP1 only has homology with Ralstonia phage RP31（similarity is 75.34%,coverage of the same gene is 0%）.In addition,the bacteriophage does not contain genes related to bacterial virulence and drug resistance.The annotation of known functional genes of bacteriophages involves the processes of bacteriophage infection,adsorption,lysis of bacteria and self-replication.Combined with the results of phylogenetic analysis of phage PSP1 and the comparison of functional proteins,it is suggested that the PSP1 obtained in this study is a new phage strain.To sum up,among 15 strains of heterotrophic nitrifying bacteria isolated from Daqing wetland water,one strain of HNB LLM-5 with high nitrification efficiency under high temperature and high ammonia nitrogen stress was screened,and 6 strains bacteriophage infecting HNB were obtained.Biological characteristics and genomic analysis found a novel bacteriophage,which provides data reference and theoretical basis for biological treatment and phage control of high temperature and high ammonia nitrogen wastewater.