好氧活性污泥中自养硝化细菌分离方法研究蒋进元

4　12环境工程学报Vol.4,No.12201012ChineseJournalofEnvironmentalEngineeringDec.2010蒋进元1,2　陈梅娟2,3　赵　鑫4　周岳溪2　刘鸿亮1,2(1.,100875;2.,100012;3.,100029;4.,100012)　　、、。,;,;。NorB,,。　　　　X703　　　A　　　1673-9108(2010)12-2653-04StudyonmethodsforisolationofnitrobacteriainaerobicsludgeJiangJinyuan1,2　ChenMeijuan2,3　ZhaoXin4　ZhouYuexi2　LiuHongliang1,2(1.CollegeofEnvironment,BeijingNormalUniversity,Beijing100875,China;2.ResearchCenterofWaterpollutionControlTechnologies,ChineseResearchAcademyofEnvironmentalSciences,Beijing100012,China;3.CollegeofChemicalEngineering,BeijingUniversityofChemicalandTechnology,Beijing100029,China;4.CenterofEnvironmentalImpactAssessment,ChineseResearchAcademyofEnvironmentalSciences,Beijing100012,China)Abstract　Inthisstudy,agarplate,agaroseplate,washedagarplateandsilicagelplatewereusedtoiso-lateandculturenitrobacteriafromaerobicactivatedsludgeofmunicipalsewageplant.Theresultsshowedthat,whencultureconditionswerethesame,therewerenon-targetbacteriagrowingbothontheagarandwashedagarplate,whilethatdidnothappenontheagaroseplate,itwascumbersometomakesilicagelplate,anddehydra-tionandcleftappearedinthelatecultures.Thenitrobacteriawassuccessfullyseparatedoutfromagaroseplate,whichwasidentifiedbymicroscopeobservationandNorBgenedetection,soagaroseplatecouldbeusedforiso-latingandculturingtheautotrophicnitrifyingbacteriawhichhasalonggenerationtime.Keywords　nitrobacteria;isolateandculture;agaroseplate:(863)(2008AA10Z406);(2007KYYW46):2009-12-06;:2010-01-03:(1974～),,,。E-mail:[e-mail]　　,,2,,。,,,,[1]。,、[2-5],,[6-9]:(1)、,;(2),;(3),。,,、,。A2/O,28℃,、、4,NorB。1　1.1　　A2/O。1.2　:NaNO21g,Na2CO31g,NaCl0.5g,K2HPO40.5g,MgSO40.5g,FeSO40.4g,1000mL,pH7.8。121℃30min。1.3　:NaNO21g,NaCl0.5g,K2HPO40.15g,MgSO40.05g,CaCO33mg,FeSO40.15mg,(NH4)6Mo7O240.05mg,1000mL,pH7.8。121℃30min[10]。、、、。1.3.1　琼脂平板2%,。,30mL,。1.3.2　琼脂糖平板2%,,。,30mL,。1.3.3　水洗洋菜平板2%。,30mL,。:2～3d,,,,,[11]。1.3.4　硅胶平板,,。:155mL5.5mL1.84,185mL22mL1.19,;500mL82.59g,。12mL8mL,,,60min,,,。5～8,15～25min。1%AgNO3Cl-,。Cl-,1,。,30min,。1.4　1.4.1　硝化细菌的富集培养50mL,28℃、150r/min,。10%。,NO-3,NO-3[12],NO-3,。NO-390%,,,,8。。1.4.2　硝化细菌的分离培养,4。,,,“”、。,,“”。:600μL,,。,“”:,,。28℃、14～21d,,,265412:,3。,。。1.4.3　分离菌株的初步鉴定NorB。1.4.3.1　菌株形态的观察(OLYMPUSBX51TF),[13]。1.4.3.2　PCR对NorB基因的检测,NorB:,,[14]。7～14d。TIANGENDNADNA,PCR。GenbankNorB,Oligo,1500bp。:F:5′GGTGAAT-TCATGGACATCCGAGCTCAAGTT,R:5′GCAAAGCTTTTACTAGAAGCCTATGGTCCTCT,()。PCR(50μL):10×PSBuffer:5μL;10mmol/LdNTP(Mg2+):1μL;DNA:1μL;(10pmo1/L)1μL;primestar(5U/μL):0.5μL;40.5μL。PCR[15]:94℃5min;94℃50s;54℃50s;72℃1min30s;35;72℃10min。PCR1%,marker(DL2000),,,。2　2.1　8,,。1～8,,。8,:0;1～3,;4,;5～8,,。,。2.2　14～21d,4。2.2.1　琼脂平板4d,6～7,“”。14,3“”,3,NA-1NA-3。2.2.2　琼脂糖平板12～14,“”,21“”,3“”,7,NB-1NB-7。2.2.3　水洗洋菜平板6,,“”。14“”,3“”,3,NC-1NC-3。2.2.4　硅胶平板6～7“”,10,,,。。3,2,ND-1、ND-2。4,,,,,。,。,,、。。2.3　2.3.1　分离菌株的表观形态观察结果1。[13],ND-1ND-213。265541　Table1　FormofisolatedstrainsNA-1、、、NA-2、、、NA-3、、、NB-1、、、NB-2、、、NB-3、、、NB-4、、、NB-5、、、NB-6、、、NB-7、、、NC-1、、、NC-2、、、NC-3、、、ND-1、、、ND-2、、、2.3.2　PCR对NorB基因的检测结果DNAPCR,NB-3NB-4,1。PCR,NB-3NB-4NorB,13NorB,,15NB-3NB-4。1　NB-3NB-4NorBFig.1　ElectrophoretogramofNorBgenebyPCRdetectionwithprimersforNB-3andNB-4,,4,2。3　　,,,。,,10d,。,。[1],,..,2008,25(4):60-64[2],,,..,2009,26():160-162[3],,..,1999,27(3):351-354[4],,,.-.,2009,22(5):511-515[5],,,..,2009,31(2):15-18[6]HanakiK.,WantawinC.,OhgakiS.,etal.Effectsoftheactivityofheterotrophsonnitrificationinasuspendedgrowthreactor.WaterResearch,1990,24(3):289-296[7]FerayC.,MontuelleB.Competitionbetweentwonitrite-oxi-dizingbacterialpopulations:Amodelforstudyingtheim-pactofwastewatertreatmentplantdischargeonnitrificationinsediment.MicrobiologyEcology,2002,42(1):15-23[8]ZhuS.M.,ChenS.L.Effectsoforganiccarbononnitrifi-cationrateinfixedfilmbiofilters.AquaculturalEngineer-ing,2001,25(1):1-11[9]HuJ.,LiD.,TaoY.Effectofacetateonnitriteoxidationinmixed-populationbiofilms.JournalofBioscienceandBioen-gineering,2008,106(6):580-586[10]BockE.,KoopsH.P.,RudertM.,etal.Anewfaculta-tivelynitriteoxidizingbacterium,Nitrobactervulgarissp.nov.Arch.Microbiol.,1990,153(2):105-110[11],,..,2002,14(6):348-350[12],..,2009,22(1):114-118[13]GarrityG.M.,BellJ.A.,LilburnT.G.Bergey'sManu-alofSystemaicBacteriology.9nded.NewYork,Ameri-can:Springer-VerlagKG,2004.60-61[14],.NorB.,2005,22(1):31-32[15]VanparysaB.,SpieckbE.,HeylenaK.,etal.Thephylog-enyofthegenusnitrobacterbasedoncomparativerep-PCR,16SrRNAandnitriteoxidoreductasegenesequenceanalysis.SystematicandAppliedMicrobiology,2007,30(4):297-3082656