Isolation And Identification Of SND Strains And Denitrification Efficiency Research Of The NirS Genetic Engineering YP1S Constructed

Sewage denitrification has always been a matter of concern.In recent years,a kind of denitrifying bacteria with different functions of traditional autotrophic nitrifying and denitrifying bacteria had been found.Simultaneous nitrification and denitrification could be realized by heterotrophic nitrification and nitrification and denitrification.Denitrification,the discovery and research of these bacteria have important application significance and economic value to the denitrification treatment.In order to construct a highly efficient and stable denitrification SND engineering strain,the identification and identification of SND bacteria,the construction and identification of engineering bacteria and the denitrification efficiency were systematically studied.First,six kinds of SND strains were obtained by simultaneous enrichment,isolated and purificated from the pig farm wastewater.The results showed that:YP1,YP2,YP3,YP4,YP5 and YP6.The results showed that the removal rate of NH₄⁺-N was 94%,which indicated that the two strains had better effect on the removal rate of NH₄⁺-N and COD,and the removal rate of NH₄⁺-N was higher than that of YP1 and YP4 strains.Heterotrophic nitrification.（67.98±0.53）%、（39.07±0.96）%、（52.30±0.48）%、（56.92±0.58）%、（44.92±0.63）%,respectively,and the removal rate of TN was the main difference between the six strains.（52.91±0.59）%,YP1 and YP4 were the highest.At the same time,YP4 strain also had the largest amount of NO₂^--N accumulation of 28.98 mg/L,followed by YP1,NO₂^--N accumulation also reached25.12 mg/L.YP1 and YP4 were identified by physiological and biochemical analysis of strain and 16S rRNA homology ratio.YP1 was identified as Acinetobacter and YP4 was Klebsiella.Nitrite Reductases（NirS）was the most critical rate-limiting step in the simultaneous nitrification and denitrification process to accelerate the denitrification process.In order to improve the denitrification efficiency of the strain,we modified the denitrification by transgenic technology The SND bacteria YP1 was cloned and expressed in the nuclear genome of YP1 strain by using the pUC18-mini-Tn7T-Gm-Lac Z20 vector and the pTNS2helper plasmid.The nitrite reductase gene NirS was cloned and expressed in the nuclear genome of YP1 strain.High efficiency denitrification NirS engineering strain YP1S.According to bioinformatics analysis,NirS enzyme was a stable protein with a molecular mass of about 98.48 kD.The activity of NirS was higher than that of wild strain YP1,and the activity of NirS was（301±18.63）U/(mg·L^-1),and the activity of NirS was higher than that of wild strain YP1.Promote the degradation of NO₂^--N by strain,and reduce its accumulation in water.YP1S and YP1 were treated in the actual sewage for 36h,YP1S removed the COD and NH₄⁺-N in the actual wastewater,and the removal rate of TN was89.23%,which was higher than that of the original strain YP1S.YP1 strain increased TN removal rate by up to 25%,YP1S decreased NO₂^--N content from 25.12 mg/L to 3.1 mg/L.The denitrification conditions of engineering bacteria YP1S were further explored and optimized.The results showed that the primary and secondary effects of the factors on the SND denitrification of YP1S were:temperature>pH>C/N>speed.And the response surface were analyzed.The optimal conditions for the engineering strain YP1S were:C/N=10、T=30°C、pH=7 and r=200 rpm.Compared with the commercially available nitrifying strain B（sub-nitroprusside）,the engineering bacteria YP1S had a 27.57%higher clearance rate for TN and NH₄⁺-N in the actual sewage of the farm.At the same time,the genomic DNA of YP1S was extracted and amplified by NirS gene.The denitrification efficiency of engineering bacteria YP1S was detected by 10 generations.The results showed that the denitrification efficiency stability of TN,NH₄⁺-N,NO₃^--N and COD were better than those of the transgenic plants,which indicated that the amplification of NirS gene had no loss of exogenous gene.In summary,the constructed engineering strain YP1S had the genetic stability and denitrification stability of nitrite reductase gene NirS,which was in accordance with the requirements of biological nitrogen removal on water nitrogen level.In the future,Will continue to observe,in-depth discussion to meet and meet the requirements of large-scale sewage treatment of denitrification,could be used to control water eutrophication and other water pollution problems,is a promising application of high efficiency denitrification bacteria.