SBR反应器厌氧氨氧化系统启动过程中细菌群落结构的变化徐相龙

37520175ActaScientiaeCircumstantiaeVol．37No．5May2017No．15XNLD04SupportedbytheFundamentalＲesearchFundsfortheCentralUniversitiesandtheＲesearchFundsofＲenminUniversityofChinaNo．15XNLD041989—[e-mail]*[e-mail]@ruc．edu．cnBiographyXUXianglong1989—[e-mail]*[e-mail]@ruc．edu．cnDOI10．13671/j．hjkxxb．2016．0347．2017．SBＲJ．3751695-1703XuXLLiuGHFanQetal．2017．Theshiftofbacterialcommunitystructureduringstart-upofanaerobicammoniaoxidationANAMMOXinasequencingbatchreactorJ．ActaScientiaeCircumstantiae3751695-1703SBＲ**1008722016-07-192016-08-242016-08-24SBＲ．PCＲ-DGGE、PCＲANAMMOX．26．6%～50．5%ANAMMOXANAMMOXBrocadia．SBＲANAMMOX1．73×109copies·g－182%．SBＲ0253-2468201705-1695-09X703ATheshiftofbacterialcommunitystructureduringstart-upofanaerobicammoniaoxidationANAMMOXinasequencingbatchreactorXUXianglongLIUGuohua*FANQiangWANGYuanyuanWANGHaoSONGXinxinQILuWANGHongchen*SchoolofEnvironment＆NaturalＲesourceＲenminUniversityofChinaBeijing100872Ｒeceived19July2016receivedinrevisedform24August2016accepted24August2016AbstractThestart-upofanaerobicammoniaoxidationANAMMOXsysteminasequencingbatchreactorSBＲwhereBaiyangdianLakeshoresedimentswereinoculated．ThemicrobialcommunitystructureoftotalbacteriaandANAMMOXbacteriainthesystemalongwiththeenrichmenttimewasinvestigatedbyusingmolecularbiologymethodse．g．PCＲ-DGGEqPCＲandDNAsequencing．Meanwhilethewaterqualityoftheinfluentandeffluentwascontinuouslymonitoredduringthewholestart-upstage．Theresultsshowedthatthedynamicchangeoftotalbacterialcommunityalongwiththeenrichmenttimeincreasedfrom26．6%to50．5%andtheirdiversitydecreasedfirstandthenincreasedafterwards．TheANAMMOXbacterialcommunitydiversityintheSBＲbecamesingleattheendofenrichmentandwaspredominantlyCandidutusBrocadia．ThemaximumbiomassofANAMMOXbacteriaachieved1．73×109copies·g－1drysludgeduringtheenrichmentwiththehighestremovalrateofTNaround82%．KeywordsBaiyangdiansedimentsANAMMOXSBＲreactorbacterialdiversitycommunitystructure1Introduction．．、．ANAMMOX371977BrodaBroda1997NH+4N2．1994MulderDelftANAMMOXVandeGraafetal．1995．199715NANAMMOXANAMMOXNH+4NO－2VandeGraafetal．1997StrousANAMMOXNH+4NO－21NO－3Strousetal．1998．NH+4+1．32NO－2+0．066HCO3－+0．13H+→1．02N2+0．26NO－3+0．066CH2O0．5N0．15+2．03H2O1/ANAMMOX2012、20132015Thirdetal．2005Ｒysgaardetal．2004．ANAMMOXN250%～70%Kuypersetal．2003ANAMMOX．16SrＲNAANAMMOX．ANAMMOX5Schmidetal．20052007Kartaletal．2007CandidutusBrocadia、CandidatusKuenenia、CandidatusScalindua、CandidatusAnammoxoglobusCandidatusJettenia．、．、ANAMMOX．ANAMMOXANAMMOXANAMMOX、、SBＲUASBANAMMOXANAMMOX．ANAMMOXANAMMOXPentonetal．2006Schmidetal．2007Kartaletal．2007．．ANAMMOX15NANAMMOXN103g·Nm－2·a－1Zhuetal．2013．ANAMMOX9ANAMMOXSBＲANAMMOX．2Materialsandmethods2．131m－80℃DNASBＲ．2．28LSBＲANAMMOX．6L1．525min、15min、20h、3h、15min．1Fig．1Diagramofexperimentalsystem35℃80r·min－1pH7．5～8．2．69615SBＲ．5500mg·L－1．NH4ClNaNO2NH4ClNaNO2ANAMMOXNH4Cl15mg·L－160mg·L－1NaNO215mg·L－178mg·L－1．NaHCO31．85g、KH2PO40．00625g、CaCl2·2H2O0．3g、MgSO4·7H2O0．2g、FeSO4·7H2O0．018g、EDTA·2H2O0．0125g1mL．VandeGraaf1996．2．3pHMettlerToledoDelta320pHNH+4-N、NO－2-N、NO－3-N《》520002mol·L－12h．NH+4-NNO－2-NN-1--NO－3-N．2．42．4．1DNAPCＲ0．5gFASTDNADNAQbiogeneCarlsbadCA．27FAGAGTTTGATCMTGGCTCAG518ＲATTACCGCGGCTGCTGGPCＲ40bpGC357FCCTACGGGAGGCAGCAG518ＲPCＲ2015．ANAMMOXPCＲ1PLA46f/630rNeefetal．1998Purkholdetal．200012ANAMMOXAmx368f/Amx820rAmanoetal．2007．2．4．2PCＲ-DGGEPCＲ-DGGE．1．5μLLoadingbuffer4μLPCＲDNAD-codeBio-Ｒad8%35%～65%．110V60℃400min．SYBＲGＲEENIBio-ＲadGel-DocTMXＲ+DGGE．BIO-ＲADQuantityOne．NTSTSUPGMA．Shannon-wienerH2014Ht=－ΣPilgPi2Pi=ni/NniiN．2．4．3PCＲ、、PromegaAgaroseGelDNASolarbioPCＲpGEM-TeasyPromegaMadisonWITrans1-T137℃．96．HhaⅠMboⅠ．DNAstarMothur3%OTU．NCBIBLASTANAMMOX16SrＲNAMEGA5．0Neighbor-JoiningGarbevaetal．2001．2．4．4PCＲqPCＲAOB、NOBANAMMOXqPCＲNOB．qPCＲSYBYGreen①DNA3、、、、．TE7SYBYGreen．20μLSYBYGreenmix10μL5μmol·L－10．4μLDNA1μLddH2O20μL．12016．．10CT－3．5～－4．295%．②qPCＲSYBYGreen、．7961371Table1PCＲprimersintheamplificationAOBAmoA-1fAmoA-2r-TCGGGGTTTCTACTGGTGGTCCCCTCTGCAAAGCCTTCTTC95℃5min95℃30s55℃45s72℃45s40NitrobacterF1370F1nxrAF2843Ｒ2nxrACAGACCGACGTGTGCGAAAGTCCACAAGGAACGGAAGGTC95℃5min95℃30s55℃45s72℃45s40NitrospiraNSＲ1113fNSＲ1264rCCTGCTTTCAGTTGCTACCGGTTTGCAGCGCTTTGTACCG95℃5min95℃30s56℃30s72℃30s40AnammoxAMX809fAMX1066rGCCGTAAACGATGGGCACTAACGTCTCACGACACGAGCTG95℃5min95℃15s55℃45s72℃30s403Ｒesultsanddiscussion3．12．2Table2CharacteristicsofthesedimentssamplesinBaiyangdianLakeT/℃DO/mg·L－1pHNH+4/mg·kg－1NO－2/mg·kg－1NO－3/mg·kg－1143．28．18214．62．214．13．2SBＲANAMMOX．321～52dHＲT4dNH+4NO－21∶115mg·L－130mg·L－1．TN55%TN．53～187dANAMMOXHＲT4d2dNH+4NO－21∶1．32NH+4-NNO－2-N50mg·L－166mg·L－1．N2．TN20%．188～265dANAMMOXHＲT2dNH+4NO－260mg·L－178mg·L－1NH+4NO－295%TN82%．NH+4NO－21．3ANAMMOX．2Fig．2Thenitrogentransformationinthereactor3．3DGGEDGGE3．1～51、30、90、165、250d．3．1～30d30d1d73．4%．31～250d3a2、3、6、11、12、1889615SBＲ6、8、9、10、14、15161、4、71717．3b250d1d49．5%250d26．6%～50．5%Osbornetal．2000．3DGGEabFig．3DGGEprofileoftotalbacteriaunderdifferentperiodaandthebandsandstrengthschematicofDGGEprofilebNTSYSDGGEUPGMA4．1～51、30、90、165250d．1、2、3231、3090d3085d45165250d．4UPGMAFig．