4种反应器中厌氧氨氧化菌完整梯烷脂的特异性王晗

38120171ENVIＲONMENTALSCIENCEVol．38No．1Jan．2017411*1112211．4000452．400714．EGSB、SBＲ、SBBＲ、EGSB-SBBＲEGSB．EGSBSBＲSBBＲEGSB．SBBＲ5EGSB7．SBBＲCandidatusBrocadiaCandidatusJetteniaAnAOBEGSBCandidatusKueneniaCandidatusJetteniaAnAOB．．X172A0250-3301201701-0238-09DOI10．13227/j．hjkx．2016072212016-07-292016-08-2651278509515785271986～[e-mail]*[e-mail]dderaneLipidsinAnaerobicAmmoniumOxidizingBacteriafromFourＲeactorsWANGHan1FANGFang1*LIKai1XINGHui1GUOJin-song12CHENYou-peng2ZENGQian-song11．CollegeofUrbanConstructionandEnvironmentalEngineeringChongqingUniversityChongqing400045China2．ChongqingInstituteofGreenandIntelligentTechnologyChineseAcademyofSciencesChongqing400714ChinaAbstractIntactladderanelipidsareaspecialkindofphospholipidsintheanammoxosomemembraneofAnAOB．PhospholipidorganicmixturewasextractedrespectivelyfromfoursludgesamplesinanaerobicEGSBreactoraerobicSBＲreactorautotrophicnitrogenremovalSBBＲreactorandanaerobicammoniumoxidationEGSBreactor．PhospholipidextractsofsludgeweredetectedbytheHPLC-ESI-MS/MSmethodtodeterminethetypesofintactladderanelipidsandthespecificityofintactladderanelipidswasalsoanalyzedatthesametimethebacterialcommunitystructureofautotrophicnitrogenremovalSBBＲreactorandanaerobicammoniumoxidationEGSBreactorwasdeterminedbyhigh-throughputsequencingtechnology．AnalyticalresultsofintactladderanelipidsshowedthatintactladderanelipidswerenotdetectedfromanaerobicEGSBreactorandaerobicSBＲreactorandweredetectedfromautotrophicnitrogenremovalSBBＲreactorandanaerobicammoniumoxidationEGSBreactor．FivekindsofintactladderanelipidsweredetectedinthephospholipidextractofsludgefromautotrophicnitrogenremovalSBBＲreactorandsevenkindsofintactladderanelipidsweredetectedinthephospholipidextractofsludgefromanaerobicammoniumoxidationEGSBreactor．Theresultsofhigh-throughputsequencingconfirmedthattheautotrophicnitrogenSBBＲreactorcontainedAnAOBofthegeneraCandidatusBrocadiaandCandidatusJetteniaandtheanaerobicammoniumoxidationEGSBreactorcontainedAnAOBofthegeneraCandidatusKueneniaandCandidatusJettenia．TheseresultsillustratedthatintactladderanelipidswerethepeculiarphospholipidsinAnAOB．KeywordsAnAOBintactladderanelipidsphospholipidhigh-throughputsequencingreactoranaerobicammoniumoxidizingbacteriaAnAOB．12、3～5、PCＲ67、/89/．2002Damsté8/AnAOB3～5ladderanelipids．———14．“”3sn-1、sn-2、sn-3sn-1sn-2sn-3sn-310．phosphocholinePCphosphoethanolaminePEphosphoglycerolPG1011．AnAOB9．11～16AnAOBAnAOBAnAOB．AnAOB．、、、EGSB、SBＲ、SBBＲ、EGSB4．PC、PE、PGPC11PCAnAOB．11.11.1.1、、、Fisher14.8mol·L－1Sigma-Aldrich500mLDNAOMEGA．1.1.2Agilent1200Agilent6410FD-1-50SCIENTZⅡDCT15ＲTMD200-1FastPrep-1MPBiomedicalsQuickchem8500S2DＲB200YSIProDSS．1.21.2.12dEGSB、SBＲSBBＲEGSBTN、NH+4-N、NO－2-N、COD．1.2.245g50mL5000r·min－116～24h．1.31.3.1DNASBBＲEGSB0.2gFastPrep-1SoilDNAExtractKitOMEGADNA．1.3.217～23、、、4．①10mL、5mL4mL2∶1∶0.815min．②5mL5mL、、1∶1∶0.95000r·min－110min．③．①～③5．④1mL4∶10.45μm－80℃PC．932381.41.4.1Quickchem8500S2TN、NH+4-N、NO－2-NDＲB200CODDO、pH、．1.4.2DNA．IlluminaMiSeqPE250338F/806ＲSBBＲEGSB．1.4.3PCPC1011181924．-HPLC-ESI-MS/MS5020-05616250mm×2.1mm5μm30℃20μL．A///14.8mol·L－179∶20∶0.12∶0.04B///14.8mol·L－188∶10∶0.12∶0.040.25mL·min－1．100%A45min35%A+65%B20min100%A20min．184m/z100～1000．．N235V300℃．AgilentMassHunterWorkstationsm/z184．m/zPC．1.5AgilentMassHunterWorkstationsB.03.01MicrosoftOfficeExcel2013MicrosoftOfficeWord2013．22.11．1．4．11Table1StatusofeachreactorEGSBSBＲSBBＲEGSB/mg·L－1≤0.14.00.2～2.0≤0.05/h2.512.024.06.0/d—20——pH6.8～7.57.0～7.58.0+0.27.5～8.0/℃20+1.025+1.030+2.033+1.0/m·h－15.0——4.4COD/mg·L－1350～450350～400＜20＜20COD/mg·L－1＜40＜50＜20＜20/mg·L－130+5—200+5200+5/mg·L－1——＜20＜15/mg·L－1——＜5200+5/mg·L－1——＜15＜10/mg·L－130+545～50200+5400+10/mg·L－1—＜15＜35＜30COD/%89.0～93.678.9～92.1——/%——＞90.0＞90.0/%———＞95.0/%——＞80.0＞80.01“—”04214EGSBCOD1COD89.0%～93.6%AnAOBAnAOB．SBＲCODCOD78.9%～92.1%4.0mg·L－1AnAOBAnAOB．SBBＲDO0.2～2.0mg·L－1．200mg·L－190.0%80.0%AnAOB．EGSBAnAOB90.0%95.0%AnAOB．2.22.1SBBＲEGSBAnAOB12．12SBBＲEGSBAnAOBSBBＲCandidatusBrocadiaCandidatusJetteniaEGSBCandidatusJetteniaCadidatusKuenenia．SBBＲ21＞1%1．、、．AnAOBCandidatusBrocadiaCandidatusJetteniaCandidatusBrocadia11.2%AnAOB．Nitrospira7.9%．1%others1SBBＲFig．1BacterialcommunitystructureofautotrophicnitrogenremovalSBBＲreactorongenuslevelNitrosomonasNitrosomonadaceaeuncultured4.1%1.8%．、、、25．EGSB2．AnAOBCandidatusKueneniaCandidatusJetteniaCandidatusJettenia55.8%AnAOB．γ-Protebacteriaunclassified7.2%EGSB．Anaerolineaceaeuncultured8.26%1423826．SM1A02PhycisphaeraceaunclassifiedAnAOB．3184Fig．3Masschromatogramofphospholipidextractfromsludgeineachreactorbasedonm/z184precursorionscan2EGSBFig．2BacterialcommunitystructureofanaerobicammoniumoxidationEGSBreactorongenuslevel2.3PC2.3.1PC．3m/z184．3EGSBSBＲSBBＲ2～3．EGSB43EGSB3．2.3.2PC1011．1EGSB4EGSBa、b3aA、B．4EGSBAm/z787m/z722760m/z708、736748Bm/z523．1011A、BPC．242144EGSBFig．4MassspectracorrespondingtothepeaksofmasschromatograminphospholipidextractofanaerobicEGSBreactor2SBＲ5SBＲa、b、c3bA、B、C．5SBＲFig．5MassspectracorrespondingtothepeaksofmasschromatograminphospholipidextractofaerobicSBＲreactor5SBＲAm/z732m/z730、760787m/