好氧同时硝化反硝化菌的分离鉴定及系统发育分析张光亚

　2005,11(2):226～228　　ChinJApplEnvironBiol=ISSN1006-687X　2005-04-25　-*张光亚**　方柏山　闵航1　陈美慈1(　　362011)(1　　310029)　　-,SDN,,,.;,.16SrDNA,SDNRhodococcusruber16SrDNA99%.PHYLIPS.4113　-;Rhodococcussp.SDN;16SrDNA;CLC　Q344.3IDENTIFICATIONANDPHYLOGENETICANALYSISOFANISOLATEDAEROBICSIMULTANEOUSNITRIFIER-DENITRIFIER*ZHANGGuangya**,FANGBaishan,MINHang1CHENMeici1(DepartmentofBiotechnologyandBioengineering,HuaqiaoUniversity,Quanzhou　362011,Fujian,China)(1DepartmentofBioscience,CollegeofLifeSciences,ZhejiangUniversity,Hangzhou　310029,China)Abstract　Anewstrain,aerobicsimultaneousnitrifieranddenitrifiernamedSDN,wasisolatedfromsoi.lThestrain'sgrowthcyclewashypha-rod-coccianditscloneswereorange-red.Thestraincoulddenitrifysodiumnitrateinaerobicenvironmentandnitrifyintheculturemediawithsodiumacetateascarbonsourceandammoniumsulfateasnitrogensource,aswellasac-etamideasthesolecarbonandnitrogensources.Thephylogenticanalysisbasedonpartial16SrDNAandperformedbyPHYLIPshowedthatthestrainwastheclosestrelativeofRhococcusruberwith99%sequencesimilarity.Andtheunrootedphy-logenetictreeofthestrainanditsneighboringdenitrifierswasgiven.Fig4,Tab1,Ref13Keywords　aerobicsimultaneousnitrificationdenitrification;Rhodococcussp.SDN;16SrDNAsequence;phylogenetica-nalysisCLC　Q344.3　　,.,.,.,[1～3],,-.-:(Alcaligenesfaecalis)[4],(Thiosphaerapantotropha)[5],(Paracoccusdenitrificans)[6],.-:、/,;、pH,;、:2003-11-10　　:2004-02-19*　SupportedbytheFundofHuaqiaoUniversity**　Correspondingauthor(E-mail:[e-mail])[6].-.-,16SrDNA..1　1.1　1.1.1　[7]　　2g,NaOH1.6g,KH2PO48.2g,MgSO47H2O0.5g,KCl0.5g,CuSO45H2O0.5mg,CaSO42H2O0.5mg,ZnSO47H2O0.5mg,FeCl36H2O,0.5mg,H2O1000mL,pH7.0.1.1.2　　　,,,,,.1.2　(0.2%),,5min20mL120mL,,.3.1.3　,.3.1.4　1.4.1　　　OlympusBH-2,,1/4,,.1.4.2　16SrDNAPCR　　,100μL,,7min,12000r/min5min,PCR.16SrDNAPCR():BSF8/20:5'-AGAGTTTGATCCTGGCTCAG-3';BSR1541/20:5'-AAGGAGGTGATCCAGCCGCA-3'..PCR(50μL)[8]:10×PCR5μL,MgCl2(25mmolL-1)4μL,dNTP2μL,BSF8/20BSR1541/201μL,DNA1μL,Taq(10000U/mL)0.5μL,35.5μL.PCR:1.94℃2min;2.94℃1min,56℃1min,72℃2min;3.229;4.72℃10min;5.60℃10min.PCRQIA-GeneDNA.,BSF8/20.1　、Table1　Names,strainnumberandaccessionnumberofsequencesofstrainsandtherelatedbacteriausedintheconstructionofunrootedphylogenetictreeNamesofbacteriaStrainnumberAccessionnumberAlcaligenesdenitrificansDSM12141TAJ005447ParacoccusdenitrificansATCC19367Y16930ParacoccusversutusATCC25364Y16932AlcaligenesfaecalisB3/IU71008ArthrobacterglobiformisDSM20124TX80736ArthrobacterramosusDSM20546T.X80742BacilluscoagulansIAM12463T.D16267BacillusdenitrificansDSM466Z26927NitrosovibriotenuisAY123803Nitrosospirasp.NpAVY10127NitrosomonaseuropaeaM103AF037106PseudomonasaeruginosaATCC25330.M34133PseudomonasputidaATCC11172.D85992RhodococcuscoprophilusATCC29080T.X81928Rhodococcussp.SDNAJ459106RhodococcusruberDSM43338T.X80625Rhodococcus.fasciansATCC35014TX819321.4.3　　　16SrD-NA(602bp),EMBL1.BlastGenBank(ht-tp://),ClustalX1.8,PHYLIP3.6,Treeview.1.2　2.1　16SrDNA,,SDN.,H-R-C(hypha-rod-cocci,--),.DNA,16SrRNA()PCR,,,602bp16SrDNA(EMBLAJ459106).BLAST,(Rhodococcus.ruber)16SrDNA99%,(1)(,),RhodococcusruberRhodo-coccussp.SDN.1　SDN(1200×)Fig.1　Phase-contrastphotomicrographofstrainSDN(1200×)2.2　SDN(0.2%),(0.2%),5min20mL120mL,,(2),,2.,,,.,Rhodococcussp.,,C/N5,[9].C/N2/1,C/N,C/N,.2.3　SDNRobertson[10],Pseudomonasaureofaciens,A.faecalis,P.denitrificans.Rhodococcussp.SDN,NO-23.24h,48h,,,227　2:-　　,,.W.Verstraete[7].LesleyA.R.[5],,,,.2　Fig.2　DenitrificationofstrainSDN3　Fig.3　NitrificationofstrainSDN2.4　Rhodococcussp.SDN,4.(Nitrosomonaseuropaea).,Rhodococcussp.SDN(R.ruber),.(N.tenuis)Nitrosovibriosp..-:、C/N、pH.;,-、(amoA[12,13]),、、,,.4　16SrDNASDNFig.4　Unrootedphylogenetictreebasedonthepartial16SrDNAsequencesofstrainSDNandrelatedbacteriaReferences1　DingAZ,FuJM,ShengGY.Evidenceofaerobicdenitrification0.ChinScieBull,2000,45(3):2779～27822　XieSG,ZhangXJ,WangZS.Integratedstudyonbiochemicalmecha-nisminbiofiltersystem.ActaSciCircumst,2002,22(5):557～5613　RobertsonLA,KuenenJG,Aerobicdenitrification:acontroversyre-vived.ArchMicrobiol,1984,139:351～3544　Ottes,SckalkJ,KuenenJG.HydeoxylamineoxidationandsubsequentnitrousoxideproductionbytheheterotrophicammoniaoxidizerAlcali-genesfaecalis.ApplMicrobiolBiotechnol,1999,51(2):255～2615　LesleyAR,vanNielEWJ,ROBAMTorremans,PatriciaAMA.Simul-taneousnitrificationanddenitrificationinaerobicchemoststculturesofThiosphaerapantotropha.ApplEnvironMicrobiol,1988,54:2812～28186　LukowT,DiekmannH.Aerobicdenitrificationbyanewlyisolatedhet-erotrophicbacteriumstrainTL1.BiotechnolLett,1997,11(19):　1157～11597　VerstraeteW,AlexanderM.HeterotrophicnitrificationbyArthrobactersp.JBacteriol,1972,110:955～9618　HanRY,MinH,ChenZQ,LiuLH.Phenotypiccharacteristicsandphylogeneticanalysisofcrudeoildegradingbacteria.BiodiversSci,2002,10(2):202～2079　HuangHK,TsengSK.NitratereductionbyCitrobacterdiversusunderaerobicenvironmen.tApplMicrobiolBiotechnol,2001,55:90～9410　RobertsonLA,CornelisseR,deVosP,HadinrtomoR,KuenenJG.Aerobicdenitrificationinvariousheterotrophicnitrifiers.AntonievanLeeuwenhoek,1989