TurkJBiol 31(2007)59-66 ©TÜB‹TAK LifeintheAbsenceofOxygen:AlternativeElectronAcceptorsfor AnaerobicMicroorganismsinaPetroleumEnvironment MelikeBALK LaboratoryofMicrobiology,WageningenUniversity,HesselinkvanSuchtelenweg4,6703CT,Wageningen,TheNetherlands Received:01.12.2006 Abstract: Anaerobicmicroorganismsderiveenergybytransferringelectronsfromanexternalsourceordonortoanexternal electronsinkorterminalacceptorandoftenhavethecapacitytoreduce2ormoreterminalelectronacceptors.Thewell-knowntype ofmicrobialrespiration,inwhichoxygenservesasanelectronacceptorfortheoxidationoforganiccarbonand/orhydrogen,h as beenstudiedelsewhereindetail.Anaerobicmicroorganismsarewidelydistributedinoil-producingvents,hydrothermalvents, volcanichotsprings,non-volcanicgeothermallyheatedsubsurfaceaquifers,andsoil.Inthisstudy,anaerobic,thermophilic,a nd fermentingmicroorganismsinapetroleumsamplefromtheAd›yamanregionofTurkeywereexaminedfortheirabilitytouse differentelectronacceptors.Thetemperaturerangeforgrowthoftheenrichmentculture(TP1)wasbetween40and65°Cand theoptimumpHrangedfrom4.5to8.0.TP1hadtheabilitytouseawidevarietyofmono-,di-,andpolysaccharidestoform acetate,lactate,ethanol,H 2,andCO 2.Nosulfate-reducingormethanogenicmicroorganismswerefound.Asanelectronacceptor, TP1reducesthiosulfate,elementalsulfur,sulfite,Fe(III),anthraquinone-2,6-disulfonate(AQDS),arsenake,andMnO 2,butnot sulfate,nitrate,(per)chlorate,orselenate.Herein,weshowthattheenrichmentculturefromthepetroleumenvironmentwasab le toreducemultipleelectronacceptors.TheutilizationoftheseelectronacceptorsbyTP1alsoindicatedtheirpresenceinthis area. TheresultspresentedsuggestthatTP1mayoccupyanicheasanenvironmentalopportunistbytakingadvantageofdiverseelectron acceptors. KeyWords: Anaerobicbacteria,electronacceptorutilization,petroleumenvironment,degradationoforganiccompounds OksijensizYaflam:BirPetrolBölgesindekiAnaerobikMikroorganizmalar IçinAlternatifElektronAkseptörleri Özet: Anaerobikmikroorganizmalarenerjilerinielektronlar›nbirbaflkakaynaktanveyabirdonördenbirterminalelectron akseptörünetransferedilmesiyoluylakazanmaktad›rlarveço¤unluklaikiveyadahafazlaelektronakseptörünüindirgemeözelli¤ ine sahiptirler.Oksijeninelektronakseptörüolarakkullan›ld›¤›veböyleceorganikkarbonunyadahidrojeninoksideedildi¤itür olan mikrobiyelaerobikmetabolizmakonusundapekçokçal›flmabulunmaktad›r.Anaerobikmikroorganizmalarpetrolüretimiyap›lan bölgelerde,hidrotermalkaynaklarda,volkanikveyajeotermalbölgelerdekisuveyatopraklardayayg›nolarakbulunmaktad›rlar.B u araflt›rmadaTürkiye’ninAd›yamanilindebulunanbirpetrolbölgesindenörnekal›nm›flveanaerobik,termofilik,fermentasyon yapabilenmikroorganizmalar›nçeflitlielektronakseptörlerinikullanmayetenekleriincelenmifltir.Zenginlefltirilenkültürün(TP 1) optimumgeliflmeiçins›cakl›karal›¤›40-60°CvepHoptimumuise4,5-8,0aral›¤›ndabulunmufltur.TP1oldukcafarkl›mono-,di - vepolisakkaritlerikullanmayetene¤indeolupasetat,laktat,etanol,H 2 veCO2 oluflturmaktad›r.Kültürdesülfatindirgeyenvemetan oluflturanmikroorganizmalarbulunmam›flt›r.TP1elektronakseptörüolaraktiyosulfat,elementelsülfür,sülfit,Fe(III),antraki non- 2,6-disülfonat(AQDS),arsenatveMnO 2’iindirgeyebiliyorken,sülfat,nitrat,(per)kloratveselenat›kullanamad›¤›gözlenmifltir.Bu çal›flmailebirpetrolbölgesindeneldeedilenzenginlefltirilmiflkültürüncokfarkl›özelliklerdekielektronakseptörlerinikul lanabilme yetene¤indeolduguortayaç›kar›lm›flt›r.ElektronakseptörlerininTP1taraf›ndankullan›lmas›,buminerallerinsözkonusubölge deki varl›¤›n›daiflaretetmektedir.Buradakisonuçlar,TP1kültürününbulundu¤uortamdade¤iflikelektronakseptörlerikullanabilmes i aç›s›ndanavantajl›veoportunistolabilece¤inigöstermektedir. AnahtarSözcükler: Anaerobikbakteriler,elektronakseptörkullan›m›,petrolbölgesi,organikbilefliklerinparçalanmas› Introduction fromtheirchemicalenvironmentsbytransferring RespiringmicroorganismsinhabitmuchoftheEarth’s electronsfromreducedtooxidizedchemicalspecies. hydrosphereandlithosphere.Suchmicroorganisms Anaerobicmetabolismoftenoccursintheabsenceof derivetheenergytheyneedtolive,grow,andreproduce oxygenandthenorganiccontaminantscanbetransferred 59 LifeintheAbsenceofOxygen:AlternativeElectronAcceptorsforAnaerobicMicroorganismsinaPetroleumEnvironment orbiodegradedanaerobically.Someanaerobicbacteria remineralizing organiccarbon.Mostremarkably,they usenitrate,sulfate,iron,manganese,andcarbondioxide performthesefunctions ineverynookandcranny,from astheirelectronacceptorsforthebreakdownoforganic theEarth’snearsurfacetoitsdepths, includingeventhe chemicals,andcarbondioxideandmethanearethemain mostextremeenvironments(18). finalproducts.Anaerobicmicroorganismshave Manyaquifersandsedimentaryenvironments dominatedlifeonEarthforperhapsmorethan2billion contaminatedwithhydrocarbonshavelimitedoxygen. yearsbeforetheincreasingoxygencontentofthe Thus,theresidentmicroorganismsmustusealternative atmosphereforcedthemtoretreatintonicheshostileto electronacceptorswhenoxidizingthereducedmolecules. oxygenicorganisms.Theseenvironmentswereprovided Microbialcommunitiesassociatedwithpetroleum bygeologicalconditions(1). environmentshaveattractedbroadinterestbecauseof In1926,thefirstbacteriawereisolatedfromoil theuniquepropertiesoftheconstituentorganisms. fields(2).Moderatetoextremethermophilicanaerobes However,littleattentionhasbeengiventopetroleum belongingtoBacteriaandArchaeainhabitoilreservoirs environmentcommunitiesascompletemicrobial andhavebeenisolatedfromorbeendetectedinoilfield ecosystems.Inthisresearch,inordertoaddressthe samples(3-8).Thepresenceofcloselyrelatedbacteriain relativecontributionsofdifferentgeochemistriestothe remoteoilfields(9,10)supportstheexistenceofawide- energydemandsoftheseecosystems,anenrichment spreadanaerobicbiosphereinoilreservoirs.Temperature culturefromapetroleumenvironmentintheAdıyaman isthemainlimitingfactorformicrobialgrowthinoil regionofTurkeywasinvestigated.Asarelativelysimple reservoirsbecausetemperatureincreaseswithdepthata andconfinedecosystemsetting,apetroleumenvironment meanrateof3°Cper100m.Differenttypesofdata wasconsideredandasimplequestionwasposed:what suggestthatthepresenceofindigenousbacteriainoil arethemainelectronacceptorsformetabolicenergythat fieldscouldbelimitedtoathresholdtemperatureof80- drivesuchcommunities? 90°C(8).Thermophilicandhyperthermophilic fermentativemicroorganismsalsoconstitutean MaterialsandMethods importantmicrobialcommunityofoilfieldenvironments. Themembersoftheorder Thermotogales (11-13), Studysiteandsamplecollection family Thermoanaerobiaceae,whichincludethegenera Theanaerobicsamplewasobtainedfromthe Thermoanaerobacter and Thermoanaerobacterium formationwaterproducedfromthepetroleumwell, (9,14),andfermentative Archaea (15),wereisolatedin Ikizce-2,whichislocatedintheAdıyamanregionof differentmicrohabitatswithinoilreservoirs.Natural southeastTurkey(lat38°11’0”N,long38°29’0”E)and environmentsforthermophilicmicroorganismsare operatedbytheTurkishPetroleumCorporation(TPAO), widespreadonEarth’ssurface.Theexistenceof inMay2005.Theculture wasenrichedfromthe microorganismsinthedeepterrestrialsubsurfacehas formationwaterbyserialdilution.Samplingdepthwas beennotedforalongtime,butonlyinthepastdecade 2398-2443m. hastherebeenanincreasinginterestinexploring anaerobicconsortia(16). Enrichment MicroorganismshavechangedEarthinanumberof ways.Theyhavealteredthechemistryoftheatmosphere Thesample(5ml)wastransferredto50-mlsterilized (17)andmodifiedthecompositionsofoceans,rivers, bicarbonate-bufferedmediumina117-mlserumvial petroleumenvironments,andporefluids throughcontrol sealedwithabutylrubberstopperunderagasphaseof ofmineralweatheringratesorbyinducingmineral N2/CO2 (80/20,v/v).Pyruvate(20mM)wasusedasthe precipitation.Theyhavechangedthespeciationofmetals electrondonor.Theenrichmentculturewasgrownat20- and metalloidsinwater,soils,andsedimentsbyreleasing 90°Candaftergrowth,culturesweretransferredto complexing agentsandbyenzymaticallycatalyzingredox liquidmediacontaining20mMofpyruvateasa reactions.Theyhave shapedthephysicalworldbybinding substrate. sediments,precipitating oredeposits,andweathering Thecompositionofbasalmediumusedforroutine rocks,andhavesustainedcommunities ofhigher growthandsubstrateutilizationexperimentscontained organismsthroughprimaryproductionandby -1 thefollowing(gl );Na2HPO4.2H2O:0.53;KH2PO4:0.41; 60 M. BALK NH4Cl:0.3;CaCl 2.2H2O:0.11;MgCl 2.6H2O:0.10;NaCl: at60°C.Hydrogenandmethaneweremeasuredona 0.3;NaHCO 3:4.0;Na 2S.9H2O:0.48.Additionally,the ChrompackCP9001gaschromatographfittedwitha basalmediumcontainedacidandalkalinetraceelements TCDdetector.Theinjectoranddetectortemperatures (1mll-1)andvitamins(0.2mll-1).Theacidtraceelement were60and130°C,respectively(19,20).Benzene, solutioncontainedthefollowing(millimolar):FeCl 2:7.5; toluene,andxyleneweredeterminedwithgas H3BO4:1;ZnCl 2:0.5;CuCl 2:0.1;MnCl 2:0.5;CoCl 2:0.5; chromatographybyinjecting0.2mlofheadspacegasinto a436Chrompackgaschromatograph(GC)equipped NiCl2:0.1;HCl:50.Thealkalinetraceelementsolution withaflameionizationdetector(FID)connectedtoaSil wascomposedofthefollowing(millimolar):Na 2SeO3: 5CBcolumn(25m× 0.32mm× 1.2µm)andwithsplit- 0.1;Na 2WO4:0.1;Na 2MoO4:0.1;NaOH:10.0.The vitaminsolutionhadthefollowingcomposition(gl -1): injection(ratio:1:50).Operatingtemperaturesofthe biotin:0.02;niacin:0.2;pyridoxine:0.5;riboflavin:0.1; injectoranddetectorwere250and300°C,respectively. thiamine:0.2;cyanocobalamine:0.1;p-aminobenzoic Theoventemperaturewas70°C.CarriergaswasN 2 acid:0.1;panthothenicacid:0.1.Themediumwas withaninletpressureof50kPa.Thiosulfate,nitrate, supplementedwith0.01%BBLyeastextract. (per)chlorate,arsenate,selenate,andsulfatewere analyzedbyanHPLCsystemequippedwithanIonpac