反硝化基因工程菌株YP4S的构建及对污水除氮特性研究

ResearchReportYP4S1112111*1,,510642;2,,510642*,[e-mail]416SrDNA(Klebsiellasp.)。NCBI(Nir)nirSPAOIDNAPCRnirS、pYP4SYP4YP4S。YP4SYP4。YP4SCOD、TN、NH4+-NNO3-NYP4SYP4NO2-N(32.44±3.96)%NO2-N。YP4SC/N=10、T=30℃、r=200r/minpH=7.0TN。YP4SCOD、TN、TP、NH4+-NNO3-N(95.87±0.82)%、(76.38±3.84)%、(97.13±0.54)%(75.35±2.57)%NO2-N(3.31±1.24)mg/LYP4S。nirS,,,ConstructionofDenitrifyingGeneticallyEngineeredStrainYP4SandStudyonItsNitrogenRemovalCharacteristicsofWastewaterZengMei1TuYupei1ZhouXimei1ZhanFujian2HuangMinghui1LiuLijie1WuGuanghong1*1CollegeofLifeScience,SouthChinaAgriculturalUniversity,Guangzhou,510642;2CenterofExperimentalTeachingforBasicCourses,SouthChi-naAgriculturalUniversity,Guangzhou,510642*Correspondingauthor,[e-mail]:10.13417/j.gab.038.000670AbstractThestrainYP4wasscreenedfromthefarmsludge.ItwasidentifiedasKlebsiellasp.throughthehom-ologoussequencealignmentwitha16SrDNAphylogenetictree.ThegenesequenceofnirSencodingnitritereductase(Nir)wasobtainedbysearchingNCBIdatabase,andtheprimersweredesigned.TakingthegenomicDNAofPseudomonasaeruginosaPAOIastemplate,thetargetfragmentnirSwasamplifiedbyPCR,andtherecombinantplasmidpYP4Swasobtainedbydoubleenzymedigestion,cloningandtransformation.Finally,wildstrainYP4wastransformedtoconstructdenitrifyinggeneticengineeringstrainYP4S.TheidentificationresultofstraingrowthcurveshowedthatthegrowthcharacteristicsofengineeringstrainYP4SandYP4werebasicallythesame.TheengineeringstrainYP4ShadahighremovalrateofCOD,TN,NH4+-N,NO3-Ninsimulatedwastewater.ComparedwithstrainYP4,stainYP4ScouldsignificantlyreduceNO2-Naccumulationby(32.44±3.96)%,whichsignificantlyreducedtheaccumulationofNO2-N.StainYP4S,whichwasobtainedbymeansoforthogonaltest,hadahigher2016(201610564158)(2011B031000019)ZengM.,TuY.P.,ZhouX.M.,ZhanF.J.,HuangM.H.,LiuL.J.,andWuG.H.,2019,ConstructionofdenitrifyinggeneticallyengineeredstrainYP4Sandstudyonitsnitrogenremovalcharacteristicsofwastewater,JiyinzuxueYuYingyongShengwuxue(GenomicsandAppliedBiology),38(2):670-676(,,,,,,,2019,YP4S,,38(2):670-676)2019382670676removalrateofTNinsimulatedwastewaterundertheoptimumconditionswithC/N=10,T=30℃,r=200r/minandpH=7.ApplyingtheengineeringstrainYP4Stotreattheactualwastewaterinpigfarmsedimentationtank,theremovalratesofCOD,TN,TP,NH4+-N,NO3-NofstrainYP4Swere(95.87±0.82)%,(76.38±3.84)%,(97.13±0.54)%,(75.35±2.57)%respectively.TheaccumulationofNO2-Nwas(3.31±1.24)mg/L,indicatingthattheengineeringstrainYP4Shadahigh-efficientdenitrificationandithadpotentialapplicationprospectsfortheactualsewageremediationwithhighnitrogencontent.KeywordsDenitrificationgenenirS,Geneticallyengineeredstrain,Denitrification,Biologicalnitrogenremoval。NO3→NO2→NO→N2(ni-tratereductase,Nar)、(nitritereductase,Nir)、(nitrousoxidereductase,Nor)(nitrousoxidereductase,Nos)(Baietal.,2013;Azzizetal.,2017)。(Nir)nirSnirK(Hasegawaetal.,2001)。nirS。(Beuckelsetal.,2015;Chenetal.,2015)。。、(Barnardetal.,2015;Beuckelsetal.,2015)。(Barnardetal.,2005;Heetal.,2016)。YP4。Tn7nirSYP4nirSYP4YP4S。11.1YP4、。DNAPCRDNA(1A)。16SrRNABLASTMEGA6.0YP4YP4(1B)YP4(Kleb-siellasp.)99%YP4Klebsiellasp.YP4。1.2nirSYP4SDNAPCR1700bp(2A)。T4DNAPCRnirSpUC18mini-Tn7T-Gm-LacZ20,DH5αGm301YP416SrRNA:A:PCR;B:16SrRNAFigure1The16SrRNAidentificationandphylogenetictreeofstrainYP4Note:A:PCRproductelectrophoresis;B:16SrRNAmoleculardevelopmenttreeYP4S671HindⅢBamHⅠ2.7kb2kb(2B)pUC18mini-Tn7T-Gm-LacZ20()。pYP4Sptns2YP4Gm30LB2~3PCR1.7kb(2C)nirSYP4YP4S。1.3YP4SYP4SYP4OD600YP4SYP42h28hYP4S(3)。YP4SnirSYP4S。1.4YP4SYP4SYP4YP4S2nirSYP4S:A:;B:;C:PCRFigure2Electrophoresisidentificationfordenitrifyinggene%nirSofdenitrificationgenenirSNote:A:Targetgeneacquisition;B:Doubledigestion;C:ColonyPCRidentificationCOD、TN、NH4+-NNO3-N(94.4±0.85)%、(76.03±1.35)%、(93.17±0.25)%(64.6±1.69)%(4)YP4。YP4SYP4NO2-N(32.44±3.96)%NO2-NYP4S(Nir)。1.5YP4SYP4S36hCOD90%(4)。L9(43)YP4S(TN)A2B2C3D2(1;2)。1(,2002)C/N=10、T=30℃、r=200r/minpH7.0。(R)TN(2)YP4STN3Figure3Determinationofgrowthcurveofstrains4Figure4Determinationofvariousindexesfortreatmentofsimu-latedsewagebystrains672YP4S1YP4STNTable1OrthogonaltestofTNremovalratebyYP4SNumber123456789C/N555101010151515(℃)Tempera-ture(℃)203040203040203040(r/min)Rotationrate(r/min)100150200150200100200100150pHpHvalue6.07.08.08.06.07.07.08.06.0TN(%)RemovalrateofTN(%)29.8260.9159.0948.7167.8160.7344.4258.1860.972YP4STNTable2TherangeanalysisoforthogonaltestresultsofTNremovalratesforYP4SbyvariousfactorsFactorsofcodeA(C/N)B()B(Temperature)C()C(Rotationrate)D(pH)K1149.82122.95148.73158.6K2177.25186.9170.59166.06K3163.57180.79171.32165.98k149.9440.9849.5852.87k259.0862.356.8655.35k354.5260.2657.1155.33R9.1421.327.532.48B()＞A(C/N)＞C()＞D(pH)。1.6YP4SYP4SYP4SCOD。C/N=10、T=30℃、pH7.0r=200r/minYP4S36hCOD、TN、TP、NH4+-NNO3-N(95.87±0.82)%、(76.38±3.84)%、(97.13±0.54)%、(75.35±2.57)%NO2-N(3.31±1.24)mg/L(5)。YP4SNO2-N。25YP4SFigure5Determinationofthevariousindexesofactualwastewa-tertreatmentbyengineeringstrainYP4S、(Heetal.,2016)。(simultane-ousnitrificationanddenitrificaion,SND)(NO3)NO3、NO2(Beuckelsetal.,2015;Chenetal.,2015)。(NO2)(Nir)(HallinandLind-gren,1999;HuangandTseng,2001)。Nir