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Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanoge

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MicrobialBiotechnology(2011)doi:10.1111/j.1751-7915.2010.00239.x

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Oligonucleotideprimers,probesandmolecularmethodsfortheenvironmentalmonitoringofmethanogenicarchaea

TakashiNarihiro1andYujiSekiguchi2*1

InternationalPatentOrganismDepositary(IPOD)and2

Bio-medicalResearchInstitute,NationalInstituteofAdvancedIndustrialScienceandTechnology(AIST),Tsukuba,Ibaraki305-8566,Japan.Summary

Fortheidentiﬁcationandquantiﬁcationofmethano-genicarchaea(methanogens)inenvironmentalsamples,variousoligonucleotideprobes/primerstar-getingphylogeneticmarkersofmethanogens,suchas16SrRNA,16SrRNAgeneandthegeneforthea-subunitofmethylcoenzymeMreductase(mcrA),havebeenextensivelydevelopedandcharac-terizedexperimentally.Theseoligonucleotidesweredesignedtoresolvedifferentgroupsofmethanogensatdifferenttaxonomiclevels,andhavebeenwidelyusedashybridizationprobesorpolymerasechainreactionprimersformembranehybridization,ﬂuores-cenceinsituhybridization,rRNAcleavagemethod,genecloning,DNAmicroarrayandquantitativepoly-merasechainreactionforstudiesinenvironmentalanddeterminativemicrobiology.Inthisreview,wepresentacomprehensivelistofsucholigonucleotideprobes/primers,whichenableustodeterminemetha-nogenpopulationsinanenvironmentquantitativelyandhierarchically,withexamplesofthepracticalapplicationsoftheprobesandprimers.Introduction

Methanogenicarchaea(methanogens)arestrictlyanaerobicmicroorganismsproducingmethaneasaresultoftheiranaerobicrespiration(Schink,1997;Thauer,1998).Formethanogenesis,theycanutilizealimitednumberofsubstratessuchascarbondioxide,acetateand

Received6July,2010;accepted12November,2010.*Forcorrespon-dence.E-maily.sekiguchi@aist.go.jp;Tel.(+81)298617866;Fax(+81)2986100.

methyl-group-containingcompoundsunderanoxiccondi-tions(LiuandWhitman,2008).Mostoftheknownmetha-nogensarehydrogenotrophsreducingcarbondioxidetoformmethane;amongthem,formateisalsooftenutilizedastheelectrondonorinsteadofhydrogen.Someofthehydrogenotrophicmethanogenscanalsoutilizesecond-aryalcoholssuchas2-propanolastheelectrondonor.Acetateisanimportantintermediatesubstanceintheanaerobicdecompositionoforganicmatter,andisgener-allyexclusivelyutilizedbylimitedgroupsofmethanogenstoformmethaneunderanoxicconditions,whereexternalelectronacceptorsotherthancarbondioxideareunavail-able.Methyl-group-containingcompounds,suchasmethanolandmethylamines,arealsoutilizedbysomemethanogensthroughdisproportionationofmethylgroups.

Methanogensarefrequentlyfoundinanoxicenviron-ments,suchasricepaddyﬁelds(Iinoetal.,2010;Sakaietal.,2010),wetlands(Cadillo-Quirozetal.,2009;Bräueretal.,2010),permafrost(Krivushinetal.,2010;Shcherbakovaetal.,2010),landﬁlls(Laloui-Carpentieretal.,2006),subsurfaces(Doerfertetal.,2009;Mochi-maruetal.,2009)andruminants(Freyetal.,2009),whichareknowntobethemajorsourcesofatmosphericmethane.Ithasbeenestimatedthattheannualglobalemissionofmethaneis500–600Tg,andatmosphericmethaneconcentrationhasrisenthreefoldoverthepast200years(LiuandWhitman,2008).Withtheincreasedinterestsinglobalclimatechangeandenvironmentalissues,studiesonthediversityandecophysiologicalfunc-tionsofmethanogensinsuchenvironmentshavebeenextensivelyconductedusingcultivation-dependentandcultivation-independentapproaches(LiuandWhitman,2008).Inadditiontosuchenvironments,methanogensplaykeyrolesinﬁeldsofanaerobicdigestiontechnology,whichiswidelyusedasameansfortreatingmunicipalandindustrialwaste/wastewatercontaininghighlevelsoforganiccompounds(Sekiguchi,2006;NarihiroandSekiguchi,2007;Talbotetal.,2008;Tabatabaeietal.,2010).Methanogensareoftencriticalcomponentsofsuchbioconversionsystems,resultingintherecoveryof

2T.NarihiroandY.Sekiguchi

gaseousmethanefromthosewastesasreusableenergyresource.Tobettermanagethebioconversionsystemsandachieveahigherefficiencyinremovingorganiccom-poundsinwastes,methanogensinthesesystemshavebeenextensivelystudiedandthequantitativemonitoringofsuchmethanogenicpopulationsinthesesystemshasbeenconducted(NarihiroandSekiguchi,2007).

Toexploretheecologicalsigniﬁcanceofmethanogensinthesenaturalandengineeredecosystems,identiﬁca-tionandquantiﬁcationtechniquesfordifferentmethano-gengroupsareindispensable.Forthepurpose,analysesofmembranelipid(Weijersetal.,2004;Strapocetal.,2008),autoﬂuorescence(Neuetal.,2002;Tungetal.,2005;Mochimaruetal.,2007),activitymeasurement(Lehmann-Richteretal.,1999;Weijersetal.,2004)andimmunoenzymaticproﬁling(Visseretal.,1991;SorensenandAhring,1997)havebeenused.Inadditiontothesemethods,cultivation-independent,nucleicacid-basedanalysisbyusingoligonucleotideprobe/primers,suchasmembranehybridization,ﬂuorescenceinsituhybridiza-tion(FISH),genecloning,quantitativepolymerasechainreaction(qPCR),andcleavagemethodwithribonucleaseH(RNaseH)weremostwidelyandfrequentlyusedasmeanstodetectandquantifymethanogensmorespeciﬁ-callyandaccurately.Inthisreview,wepresentacata-logueofpreviouslydevelopedoligonucleotideprobes/primerstargetinggenesofmethanogens.Particularemphasisisplacedontheprobes/primersfor16SrRNA,16SrRNAgeneandthegeneforthea-subunitofmethylcoenzymeMreductase(mcrA),whicharegenerallyusedforthetaxonomicclassiﬁcationofmethanogens(Friedrich,2005;LiuandWhitman,2008).Phylogenyofmethanogens

AllthemethanogensisolatedandcharacterizedtodatehavebeenclassiﬁedintothephylumEuryarchaeotaofthedomainArchaea(Garrityetal.,2007).Theyareassignedinto33generaoftheclasses‘Methanomicrobia’,Metha-nobacteria,MethanococciandMethanopyri(Fig.1,Table1).Theclass‘Methanomicrobia’isthemostphylo-geneticallyandphysiologicallydiversegroupofmethano-gensconsistingofthreeorders(Methanosarcinales,MethanomicrobialesandMethanocellales);23generabelongingtosevenfamilies(Fig.1,Table1).WithintheorderMethanosarcinales,thegeneraMethanosarcinaandMethanosaetaareknowntoplayakeyroleintheconver-sionofacetateintomethaneinvariousanaerobicenviron-ments,andtherestareknowntometabolizerelativelybroadrangesofsubstrates,suchashydrogen,methanolandmethylamines(GarrityandHolt,2001).KnownmembersoftheorderMethanomicrobialesareallhydro-genotrophs,andsomeofthemareoftenobservedinanaerobicenvironmentsasimportanthydrogenscaven-gers(LiuandWhitman,2008).MembersoftheclassMethanobacteria,consistingofthefamiliesMethanobac-teriaceaeandMethanothermaceae,arerecognizedasimportanthydrogenotrophsthathavealsobeenwidelyfoundinanaerobicecosystems(GarrityandHolt,2001).Methanobacteriaceaecomprisesfourgenera,Methano-bacterium,Methanosphaera,MethanobrevibacterandMethanothermobacter.TheclassMethanococciincludesthefamiliesMethanococcaceaeandMethanocaldococ-caceae,whicharewidelydistributedinnaturalecosystemssuchasmarinesedimentsanddeepseageothermalsedi-ments(LiuandWhitman,2008).TheclassMethanopyriconsistsofsolelythegenusMethanopyrus,ahyperther-mophilic,hydrogenotrophicmethanogenisolatedfromthedeep-seahydrothermalﬁeld(Takaietal.,2008).

Theisolationandcharacterizationofnovelmethano-gensfromvariousecosystemsareongoing,andthedescriptionsofsuchmethanogenshavebeencarriedoutatanencouragingrate.Recently,hydrogenotrophicmethanogens,whicharenovelathightaxonomiclevels(MethanocellapaludicolaandMethanocellaarvoryzae),havebeenisolated,andthenovelorderMethanocellaleswasproposed(Sakaietal.,2008;2010).Thesemethano-genshavelongbeenconsideredastheuncultivablemethanogengroup(RiceclusterI),andresponsibleforthemajorpartofmethanogenesisinricepaddysoil(Conradetal.,2006).Inaddition,novelhydrogenotrophicmetha-nogensassociatedwithpreviouslyuncultivatedphyloge-neticgroupsoftheorderMethanomicrobiales(formerlyknownasE1/E2orFencluster)wereisolatedfromanaero-bicbioreactors(Imachietal.,2008;Yashiroetal.,2009)andwetlands(Cadillo-Quirozetal.,2009;Bräueretal.,2010).NovelstrainsofthegeneraMethanofollis(Imachietal.,2009),Methanolobus(Doerfertetal.,2009;Mochi-maruetal.,2009),Methanospirillum(Iinoetal.,2010)andMethanobacterium(Krivushinetal.,2010;Shcherbakovaetal.,2010)havealsobeenreportedrecently.

Despitetheseeffortsincultivatingasyetuncultivablemethanogenspresentinenvironments,therearestillavastnumberofuncultivablearchaealtaxathatmayhavesimilarmetabolicfunctionsasthoseofknownmethano-gens.Forexample,16SrRNAgenetypesassignedintotheWSA2(orArcI)groupwerefrequentlyretrievedfrommethanogenicwaste/wastewatertreatmentsystems(SekiguchiandKamagata,2004;Chouarietal.,2005).TheWSA2groupisconsideredtobeanarchaealtaxonattheclasslevelwithnoculturedrepresentatives(Hugen-holtz,2002).However,ChouariandcolleagueshavefoundthatWSA2-relatedcellscanbeenrichedusingformate-orhydrogen-containingculturemedia,suggest-ingthattheyharbourmethanogenicactivity(Chouarietal.,2005).AnotherexamplesimilartotheRiceClusterIgroupisRiceClusterII(RC-II).MembersoftheRC-IIgroupwerealsoconsideredtobemethanogens,because

Probesformethanogens3

Genus

Methanoculleus marisnigri, M59134Methanoculleus submarinus, AF531178Methanoculleus chikugoensis, AB038795Methanoculleus thermophilus, AB065297Methanoculleus bourgensis, AY196674Methanoculleus palmolei, Y16382Methanoculleus receptaculi, DQ787476Methanolacinia paynteri, AY196678Methanoplanus petrolearius, U76631Methanoplanus limicola, M59143Methanomicrobium mobile, M59142Methanogenium marinum, DQ177344Methanogenium organophilum, M59131Methanofollis aquaemaris, AF262035Methanofollis formosanus, AY1862Methanofollis liminatans, Y128Methanofollis tationis, AF095272Methanofollis ethanolicus, AB371073Methanoregula boonei, DQ282124Methanolinea tarda, AB162774Methanosphaerula palustris, EU156000Methanospirillum hungatei, AY196683Methanocalculus halotolerans, AF033672Methanocalculus pumilus, AB008853Methanocalculus ensis, AF411470Methanocalculus chunghsingensis, AF347025Methanocorpusculum labreanum, AY260436Methanocorpusculum parvum, M59147Methanocorpusculum bavaricum, AY196676Methanolobus bombayensis, U20148Methanolobus vulcani, U20155Methanolobus tindarius, M59135Methanolobus profundi, AB370245Methanolobus oregonensis, U20152Methanolobus taylorii, U201Methanolobus zinderi, EU711413Methanomethylovorans hollandica, AF120163Methanomethylovorans thermophila, AY672821Methanococcoides alaskense, AY941801Methanococcoides burtonii, CP000300Methanohalophilus mahii, M59133Methanohalophilus portucalensis, AY290717Methanohalobium evestigatum, U20149Methanosalsum zhilinae, FJ224366Methanosarcina siciliae, U20153Methanosarcina acetivorans, M59137Methanosarcina thermophila, M59140Methanosarcina barkeri, AJ012094Methanosarcina mazei, AJ012095Methanosarcina lacustris, AF432127Methanosarcina baltica, AJ2388Methanosarcina semesiae, AJ012742Methanimicrococcus blatticola, AJ238002Methanosaeta thermophila, AB071701Methanosaeta concilii, NR_028242Methanosaeta harundinacea, AY817738Methermicoccus shengliensis, DQ787474Methanocella paludicola, AB196288Methanobacterium palustre, AF093061Methanobacterium subterraneum, X99044Methanobacterium formicicum, AF169245Methanobacterium alcaliphilum, DQ9335Methanobacterium bryantii, AY196657Methanobacterium ivanovii, AF095261Methanobacterium oryzae, AF028690Methanobacterium beijingense, AY350742Methanobacterium congolense, AF233586Methanobacterium aarhusense, AY386124Methanothermobacter thermoflexus, X99047Methanothermobacter thermophilus, X99048Methanothermobacter defluvii, X99046Methanothermobacter wolfeii, AB104858Methanothermobacter marburgensis, X153Methanothermobacter thermautotrophicus, AY196660Methanobrevibacter arboriphilus, AY196665Methanobrevibacter cuticularis, U41095Methanobrevibacter curvatus, U62533Methanobrevibacter filiformis, U82322Methanobrevibacter acididurans, AF242652Methanobrevibacter gottschalkii, U55238Methanobrevibacter thaueri, U55236Methanobrevibacter millerae, AY196673Methanobrevibacter smithii, U55233Methanobrevibacter woesei, U55237Methanobrevibacter ruminantium, AY196666Methanobrevibacter olleyae, AY615201Methanobrevibacter wolinii, U55240Methanosphaera stadtmanae, CP000102Methanocaldococcus fervens, AF056938Methanocaldococcus jannaschii, L77117Methanocaldococcus vulcanius, AF051404Methanocaldococcus indicus, AF7621Methanocaldococcus infernus, AF025822Methanotorris formicicus, AB100884Methanothermococcus okinawensis, AB057722Methanococcus aeolicus, DQ1951Methanococcus vannielii, AY196675Methanothermococcus thermolithotrophicus, M59128Methanothermus sociabilis, AF095273Methanothermus fervidus, M59145Methanopyrus kandleri, AE009439FamilyOrderClass

Methanoculleus

0.10MethanolaciniaMethanoplanusMethanomicrobiumMethanogenium

Methanomicrobiales

MethanofolisMethanoregulaMethanolinea

MethanosphaerulaMethanospirillumMethanocalculus

Methanomicrobiaceae

incertae sedisMethanospirillaceaeincertae sedis

MethanocorpusculumMethanocorpusculaceae

Methanomicrobia

Methanolobus

MethanomethylovoransMethanococcoidesMethanohalophilusMethanohalobiumMethanosalsum

Methanosarcinaceae

Methanosarcinales

Methanosarcina

MethanimicrococcusMethanosaetaMethermicoccusMethanocella

MethanosaetaceaeMethermicoccaceaeMethanocellaceae

Methanocellales

Methanobacterium

Methanothermobacter

Methanobacteriaceae

Methanobacteriales-1

Methanobacteria-1

Methanobrevibacter

MethanosphaeraMethanocaldococcusMethanotorris

Methanococcus

MethanothermococcusMethanothermusMethanopyrus

MethanococcaceaeMethanothermaceaeMethanopyraceae

Methanobacteriales-2Methanopyrales

Methanobacteria-2Methanopyri

Methanocaldococcaceae

Methanococcales

Methanococci

Fig.1.Phylogenyandtaxonomyofmethanogens.Theneighbour-joiningtreewasconstructedonthebasisof16SrRNAgenesequencesusingtheARBpackage(Ludwigetal.,2004)withthedataset(Yarzaetal.,2008)providedfromsilvadatabases(http://silva.mpi-bremen.de/),showingrepresentativespeciesofmethanogensthathavebeendescribedtodate.

the16SrRNAgeneclonesaffiliatedwiththisgroupwerefrequentlyobservedinmethanogenicenrichmentculturescontainingethanolasanelectrondonor,andbecausetheRC-IIgroupisalineagewithinthephylogeneticradiationoftheordersMethanosarcinalesandMethanomicrobiales(Lehmann-Richteretal.,1999).Ascanbenotedfromtheseexamples,thereisnodoubtthattheactualbiodi-versityofmethanogenswillbemuchexpandedinthe

4T.NarihiroandY.Sekiguchi

Table1.Oligonucleotideprobesandprimerstargetingthe16SrRNAgeneofmethanogens.

Probelength(mer)20182016151615201617181820171719182015152021182020161820

TargetgroupMostmethanogens

ProbenameArchf2bArchr1386A1fA1100rMet83FMet86FMet448FMet1027FMet1340R109f146f1324rARC344f25f1391rA24fA357fA329rA348rA693rMet630FMet803RA1040fARC344ARC915MER11068R

CMSMM1068m

Probesequence(5′–3′)a

TTCYGGTTGATCCYGCCRGAGCGGTGTGTGCAAGGAGCTCYGKTTGATCCYGSCRGAGTGGGTCTCGCTCGTTGACKGCTCAGTAACACGCTCAGTAACACGTGGGGTGCCAGCCGCCGC

GTCAGGCAACGAGCGAGACCCGGTGTGTGCAAGGAGACKGCTCAGTAACACGTGGSATAACCYCGGGAAACGCGAGTTACAGCCCWCRAACGGGGYGCAGCAGGCGCGACYGGTYGATYCTGCCRGGACGGGCGGTGTGTRCATCYGKTTGATCCYGSCRGACCCTACGGGGCGCAGCAGTGTCTCAGGTTCCATCTCCGCCCCRTAGGGCCYGGGGATTACARGATTTC

GGATTAGATACCCSGGTAGTGTTGARTCCAATTAAACCGCAGAGAGGWGGTGCATGGCCTCGCGCCTGCTGCICCCCGTGTGCTCCCCCGCCAATTCCTGGGCACGGGTCTCGCTATGCTTCACAGTACGAACGGATGCTTCACAGTACGAAC

Application

PCR(forward)PCR(reverse)

PCR(forward),DGGEPCR(forward),DGGEPCR(forward)PCR(forward)sequencingsequencingPCR(reverse)PCR(forward)PCR(forward)PCR(reverse)

PCR(forward),DGGEPCR(forward)PCR(reverse)

PCR(forward),DGGEPCR(forward),DGGEPCR(reverse),DGGEPCR(reverse),DGGEPCR(reverse),DGGEqPCR(forward),DGGEqPCR(reverse),DGGEPCR(forward),DGGEMH

PCR(reverse),DGGE,MH,FISHPCR(reverse)PCR(reverse)RNaseH

Reference

Skillmanetal.(2004)Skillmanetal.(2004)Embleyetal.(1992)Embleyetal.(1992)WrightandPimm(2003)WrightandPimm(2003)WrightandPimm(2003)WrightandPimm(2003)WrightandPimm(2003)Grosskopfetal.(1998)Marchesietal.(2001)Marchesietal.(2001)Casamayoretal.(2001)Dojkaetal.(1998)Barnsetal.(1994)Yuetal.(2008)Yuetal.(2008)Yuetal.(2008)Yuetal.(2008)Yuetal.(2008)Hooketal.(2009)Hooketal.(2009)

ReysenbachandPace(1995)Raskinetal.(1994b)Raskinetal.(1994b)Halesetal.(1996)Banningetal.(2005)Narihiroetal.(2009b)

ClassMethanomicrobiaOrderMethanocellalesFamilyMethanocellaceaeGenusMethanocella

OrderMethanomicrobiales

SANAE1136MG1200MG1200mM(SA/MI)355MMB282FMMB749FMMB832R298F586RF2SC668F2SC732AR934F

MCU1023TAQMG1200bMc412fMc578r

SMCUT1253F3SC984GMG1128GMM829SMPL623cSMPL623cSMPP1252dSMPP1252dF7SC1260MSP1025TAQNOBI109fNOBI633F6SC393eGMCP4F6SC393eGMCL4886A84SMSP129MSMX860MSSH859MSrr859MSMX860mMSL812FMSL860FMSL1159R

GTGTACTCGCCCTCCTCG

CGGATAATTCGGGGCATGCTGCCGGATAATTCGGGGCATGCTGGTAAAGTTTTCGCGCCTGATCGRTACGGGTTGTGGG

TYCGACAGTGAGGRACGAAAGCTGCACCTAACGCRCATHGTTTACGGAGCAAGAGCCCGGAGTCCAAGAGACTTAACAACCCATCCTACCCCCGAAGTACCCCTCTCGAAGCCGTTCTGGTGAGGCGAGGAATTGGCGGGGGAGCAC

GAATGATTGCCGGGCTGAAGACTCCCGGATAATTCGGGGCATGCTGCTGGGTGTCTAAAACACACCCAAATTGCCAGTATCTCTTAG

GCCTTTCGGCGTCGATACCCCATATCGCTGTCCTACCCGGCGTTCCGGAGAACAAGCTAGCTCGTAGTTACAGGCACACCTTCTCTTAAACGCCTGCAGGTTCTCTTAAACGCCTGCAGGCTTCTCAGTGTCGTTGCTCACTTCTCAGTGTCGTTGCTCA

TATCCTCACCTCTCGGTGTC

GAATGATAGTCGGGATGAAGACTCTAACTGCTCAGTAACACGTGATTGCCAGTTTCTCCTGGACAGGCACTCAGGGTTTCCGCCCTGCCCTTTCTTCACATGACAGGCACTCAGGGTTTCCCCCCGCCCTTTCTCCTGGTGTCTTCCGGTCCCTAGCCTGCCATATCCCCTTCCATAGGGTAGATTGGCTCGCTTCACGGCTTCCCTTCGCTTCACGGCTTCCCTTCGCTTCACGGCTTCCCTGGCTCGCTTCACGGCTTCCCTGTAAACGATRYTCGCTAGGTAGGGAAGCCGTGAAGCGARCCGGTCCCCACAGWGTACC

FISH

MH,FISHRNaseHMH

qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)qPCR(reverse)RNaseHRNaseH

qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)qPCR(reverse)RNaseHRNaseHRNaseH

FISH,RNaseHRNaseRNaseRNaseRNase

HHHH

1821221818242118202222202422231820202020202020202026171820202020222321181920202117

Sakaietal.(2007)Raskinetal.(1994b)Narihiroetal.(2009b)Ovreåsetal.(1997)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Franke-Whittleetal.(2009a)Franke-Whittleetal.(2009a)Narihiroetal.(2009b)Narihiroetal.(2009b)Shigematsuetal.(2003)Shigematsuetal.(2003)Shigematsuetal.(2003)Horietal.(2006)Horietal.(2006)

Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Yanagitaetal.(2000)NarihiroNarihiroNarihiroNarihiro

etal.etal.etal.etal.

(2009b)(2009b)(2009b)(2009b)

FamilyMethanomicrobiaceaeGenusMethanoculleus

SpeciesM.thermophilus

GenusMethanofollisGenusMethanogeniumGenusMethanomicrobiumGenusMethanoplanusSpeciesM.limicola

SpeciesM.endosynbiosusSpeciesM.petroleariusGenusMethanolacinia

FamilyMethanospirillaceaeGenusMethanospirillumGenusMethanosphaerulaGenusMethanolinea

FamilyMethanocorpusculaceaeGenusMethanocorpusculumFamilyincertaesedisGenusMethanocalculusGenusMethanoregulaSpeciesM.booneiSpeciesM.formicicaOrderMethanosarcinales

RNaseH

qPCR(probe)qPCR(forward)

qPCR(reverse),FISHRNaseHRNaseHRNaseHRNaseHFISHFISHMHFISH

PCR(reverse)RNaseH

qPCR(forward)qPCR(probe)qPCR(reverse)

Narihiroetal.(2009b)Tangetal.(2005)Imachietal.(2008)Imachietal.(2008)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Bräueretal.(2006)Yashiroetal.(2009)Raskinetal.(1994b)Boetiusetal.(2000)Skillmanetal.(2004)Narihiroetal.(2009b)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Probesformethanogens5

Table1.cont.

Probelength(mer)

Targetgroup

FamilyMethanosaetaceaeGenusMethanosaeta

ProbenameProbesequence(5′–3′)aApplicationReference

SpeciesM.concilii

SpeciesM.thermophila

FamilyMethanosarcinaceae

GenusMethanimicrococcusGenusMethanosarcina

GenusMethanococcoidesGenusMethanohalobiumGenusMethanohalophilusGenusMethanolobusSpeciesM.psychrophilusGenusMethanomethylovoransGenusMethanosalsumFamilyMethermicoccaceaeGenusMethermicoccusClassMethanobacteriaOrderMethanobacteriales

MX825MX825mixMX1361

S-G-Msae-0332-a-A-22Mst702FMst753FMst862RMS1b

SAE761TAQSAE835R

S-F-Msaet-0387-S-a-21S-F-Msaet-00-A-a-31S-F-Msaet-0573-A-a-17Rotcl1MS1MS2MS5

MMX1273GTMS393mMs413fMs578rTMX745MS1414EelMS240fMsc380FMsc492FMsc828RR15Fg

FMSC394MS821mhGMIB12MS821SARCI551SARCI5MB1MB3MB4240F5RMB1b

SAR761TAQSAR835R

S-G-Msar-0450-S-a-19S-P-Msar-00-A-a-31S-G-Msar-05-S-a-20GMCO441GMHB842GMHP1258GMLB834R15FR15RGMSS261

TCGCACCGTGGCCGACACCTAGCTCGCACCGTGGCYGACACCTAGCACGTATTCACCGCGTTCTGTTTAGGTCCGGGATGCXCCACGTTAATCCTYGARGGACCACCA

ACGGCAAGGGACGAAAGCTAGGCCTACGGCACCRACMACCCGGCCGGATAAGTCTCTTGA

ACCAGAACGGACCTGACGGCAAGGGACAACGGTCGCACCGTGGCCGATAAGGGRAYCTCGAGTGCY

AGACCCAATAAHARCGGTTACCACTCGRGCCGGCCGRCTACAGACCCT

CTCCCGGCCTCGAGCCAGACCCGGATAAGTCTCTTGA

CTGAATGAGAGCGCTTTCTTTGGCCACGGTGCGACCGTTGTCGGGTTTTAGGAGATTCCCGTCACCCAGCACTCGAGGTCCCCCAGATGTGTAAAATACATCTGTTTCTGGCAGTATCCACCGACCCTTGCCGTCGGATCCGTTCTCACCCATACCTCACTCGGGCTATCAGGTTGTAGTGGGGAAACCGYGATAAGGGGATTAGCAAGGGCCGGGCAATAGCGARCATCGTTTACG

GCTACACGCGGGCTACAATGAATGCTGGCACTCGGTGTCCCGCCATGCCTGACACCTAGCGCACCTTTCGGTGTAGTTGCC

CGCCATGCCTGACACCTAGCGAGCGACCCAATAATCACGATCACTCCCGGTTCCAAGTCTGGCTTTGGTCAGTCCTCCGGCCAGACTTGGAACCGTTTATGCGTAAAATGGATT

CCTATCAGGTAGTAGTGGGTGTAATCCCGGAGGACTGACCAAACGGTTTGGTCAGTCCTCCGG

ACCAGAACGGGTTCGACGGTGAGGAGACACGGTCGCGCCATGCCTTAGCAAGGGCCGGGCAAGA

AGACCCAATAATCACGATCACCACTCGGGCCATCCCGGAGGACTGACCAAAACATGCCGTTTACACATGTGTCGGCACTAGGAACGGCCGTCCGTCACTTTTCAGTGTAGGTGAAACGGTCGCACCGTCCCAGGCTACACGCGGGCTACAATGA

AATTTAGGTTCGAACACGGCATGAAGTCGGCTAGCAGGTACCTTG

MH,FISHRNaseHFISH

MH,FISH

qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)qPCR(probe)qPCR(reverse)FISHMHMH

MH,FISHRNaseHRNaseH

qPCR(forward)qPCR(reverse)RNaseHMH,FISHFISH

qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)RNaseHRNaseHRNaseHMH,FISHFISHFISHMHMH

MH,FISH

qPCR(forward)qPCR(reverse)qPCR(forward)qPCR(probe)qPCR(reverse)qPCR(forward)qPCR(probe)qPCR(reverse)RNaseHRNaseHRNaseHRNaseH

qPCR(forward)qPCR(reverse)RNaseH

23232022202217212421213117201721222020231820211818181821202020242019171519251820242119312020202022212520

Raskinetal.(1994b)Narihiroetal.(2009b)Crocettietal.(2006)

ZhengandRaskin(2000)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Shigematsuetal.(2003)Shigematsuetal.(2003)Shigematsuetal.(2003)Sawayamaetal.(2004)Sawayamaetal.(2004)Sawayamaetal.(2004)ZeppFalzetal.(1999)Rocheleauetal.(1999)Rocheleauetal.(1999)Rocheleauetal.(1999)Narihiroetal.(2009b)Narihiroetal.(2009b)Horietal.(2006)Horietal.(2006)

Narihiroetal.(2009b)Raskinetal.(1994b)Boetiusetal.(2000)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Zhangetal.(2008a)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Raskinetal.(1994b)

SorensenandAhring(1997)SorensenandAhring(1997)Rocheleauetal.(1999)Rocheleauetal.(1999)Rocheleauetal.(1999)

Franke-Whittleetal.(2009a)Franke-Whittleetal.(2009a)Shigematsuetal.(2003)Shigematsuetal.(2003)Shigematsuetal.(2003)Sawayamaetal.(2006)Sawayamaetal.(2006)Sawayamaetal.(2006)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Zhangetal.(2008a)Zhangetal.(2008a)Narihiroetal.(2009b)

FamilyMethanobacteriaceaeGenusMethanobrevibacterSpeciesM.ruminantiumSpeciesM.smithii

GenusMethanobacterium

MB310MB311Mbacf331MB1174MBT857FMBT929FMBT1196R1401RMB1175mMEB859fMbb1GMB406MBR1001ForwardReverseProbefMbiumGMBA755

S-F-Mbac-0398-S-a-20S-G-Mbac-0526-A-a-33S-G-Mbac-0578-A-a-22GMSP838

GenusMethanosphaera

CTTGTCTCAGGTTCCATCTCCGACCTTGTCTCAGGTTCCATCTCCCTTGTCTCAGGTTCCATCTCTACCGTCGTCCACTCCTTCCTCCGWAGGGAAGCTGTTAAGTAGCACCACAACGCGTGGATACCGTCGTCCACTCCTT

KTTTGGGTGGYGTGACGGGCCCGTCGTCCACTCCTTCCTCAGGGAAGCTGTTAAGTCCCTCCGCAATGTGAGAAATCGGCCATCCCGTTAAGAATGGCTCAGCCTGGTAATCATACACCGGGTATCTAATCCGGTTCCTCCCAGGGTAGAGGTGAAACCGTCAGAATCGTTCCAGTCAGCGTTCGTAGCCGGCYTGATGGCTTTCGTTACTCACCCCCAAGTGCCACTCTTAACGAAYGGCCACCACTTGAGCTGCCGGTGTTACCGC

AGACTTATCAARCCGGCTACGACCGGAACAACTCGAGGCCAT

MHFISHPCR

MH,FISH

qPCR(forward)qPCR(probe)qPCR(reverse)PCR(reverse)RNaseHFISHPCR

RNaseHFISH

qPCR(forward)qPCR(reverse)qPCR(probe)PCR

RNaseH

qPCR(forward)qPCR(reverse)qPCR(probe)RNaseH

22232022191818202018202019202022181820332220

Raskinetal.(1994b)Crocettietal.(2006)Skillmanetal.(2004)Raskinetal.(1994b)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Banningetal.(2005)Narihiroetal.(2009b)Boetiusetal.(2000)Skillmanetal.(2004)Narihiroetal.(2009b)Yanagitaetal.(2000)Armougometal.(2009)Armougometal.(2009)Armougometal.(2009)Skillmanetal.(2004)Narihiroetal.(2009b)Sawayamaetal.(2006)Sawayamaetal.(2006)Sawayamaetal.(2006)Narihiroetal.(2009b)

6T.NarihiroandY.Sekiguchi

Table1.cont.

Probelength(mer)211920192220322522192116222020202422

Targetgroup

GenusMethanothermobacter

Probename

Mt392fMt578r410F667R

GMTB1

S-F-Mbac-0398-S-a-20S-G-Mthb-09-S-a-32S-G-Mthb-05-A-a-25FMTH1183

M(CO/BA)377Mccr

MCC495FMCC686FMCC832R1202R

S-F-Mcc-1109-b-A-20MC504

FMCMT1044i

Probesequence(5′–3′)a

ACTCTTAACGGGGTGGCTTTTTCATGATAGTATCTCCAGCCTCTTAACGGGGTGGCTTTTCCCTGGGAGTACCTCCAGCAAAAGCGGCTACCACTTGAGCTCCCAAGTGCCACTCTTAACG

CGGACGCTTTAGGCCCAATAAAAGCGGCTACCGGGATTTCACCAGAGACTTATCAGTACGGACCTACCGTCGCCCGCACCCCCGTCGCACTTKCGTGWASTVGCAACATAGGGCACGGTAAGGGCTGGGCAAGT

TAGCGGTGRAATGYGTTGATCCCACCTAGTYCGCARAGTTTACCAGGRGATTCGGGGCATGCGCAACATGGGGCRCGGGTCT

GGCTGCTGGCACCGGACTTGCCCAGTCAACCTGGCCTTCATCCTGC

ApplicationqPCR(forward)qPCR(reverse)qPCR(forward)qPCR(reverse)RNaseH

qPCR(forward)qPCR(probe)qPCR(reverse)RNaseHMH

PCR(reverse)qPCR(forward)qPCR(probe)qPCR(reverse)PCR(reverse)MHFISHRNaseH

Reference

Horietal.(2006)Horietal.(2006)

Franke-Whittleetal.(2009a)Franke-Whittleetal.(2009a)Narihiroetal.(2009b)Sawayamaetal.(2006)Sawayamaetal.(2006)Sawayamaetal.(2006)Narihiroetal.(2009b)Ovreåsetal.(1997)Skillmanetal.(2004)Yuetal.(2005)Yuetal.(2005)Yuetal.(2005)

Banningetal.(2005)Nercessianetal.(2004)Crocettietal.(2006)Narihiroetal.(2009b)

FamilyMethanothermaceaeGenusMethanothermusClassMethanococciOrderMethanococcales

Family

MethanocaldococcaceaeGenusGenusFamilyGenus

MethanocaldococcusMethanotorris

MethanococcaceaeMethanococcus

GenusMethanothermococcusClassMethanopyriOrderMethanopyralesFamilyMethanopyraceaeGenusMethanopyrus

MC1109GMC728GMC231GMC416GMTL416GCAACATAGGGCACGGGTCTACCCGTTCCAGACAAGTGCCTTACTACCTAATCGAGCGCAGTCCTTGATAAAAGCCCATGCTGTGCTAGAAAAGCCTACGCAGTGCMHRNaseRNaseRNaseRNase

HHHH

2022222220Raskinetal.(1994b)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)Narihiroetal.(2009b)

FMCMT1044i

S-G-Mp-0431-a-A-20GMPK1331GTCAACCTGGCCTTCATCCTGCTTACACCCCGGTACAGCCGCGGTTACTACCGATTCCACCTTCRNaseHMH

RNaseH222022Narihiroetal.(2009b)Nercessianetal.(2004)Narihiroetal.(2009b)

a.IUPACAmbiguityCodes:Y=CorT,R=AorG,K=GorT,S=CorG,W=AorT,M=AorC,H=AorCorT,V=AorCorGb.Archf2probecoversmembersoftheordersMethanomicrobiales,MethanosarcinalesandMethanococcales.c.SMPL623probecoversmembersoftheMethanoplanuslimicolaandM.endosynbiosus.d.SMPP1252probecoversmembersoftheMethanoplanuspetroleariusandMethanolacinia.e.F6SC393probecoversmembersofthegeneraMethanocorpusculumandMethanocalculus.

f.EelMS240probetargetsformembersofthegeneraMethanolobus,Methanohalophilus,MethanococcoidesandMethanomethylovorans.g.R15FprobecoversmembersofthegeneraMethanomethylovoransandMethanosarcinaandMethanolobuspsychrophilus.h.MS821mprobecoversmembersofthegeneraMethanimicrococcusandMethanosarcina.

i.FMCMT1044probecoversmembersofthefamilyMethanocaldococcaceaeandgenusMethanopyrus.MH,membranehybridization.

futureasthenumberofisolatedanddescribedmethano-genscontinuestoincrease.However,inthisreview,wemainlyfocusonthequantitativemonitoringtoolsforpre-viouslyculturedmethanogens.

Oligonucleotideprobes/primersfor16SrRNAanditsgene

16SrRNAanditsgenearethemostfrequentlyusedbiomarkersforthedeterminationofmethanogenicpopu-lationsinenvironments.16SrRNAgene-targetedprobes/primersfrequentlyusedforidentifyingmethanogensarelistedinTable1.Toentirelydescribemethanogenicpopu-lationsinecosystemsofinterest,16SrRNAgene-targetedprimersetsforawiderangeofmethanogentaxa,suchas146f/1324r(Marchesietal.,2001)andMet83F(Met86F)/Met1340R(WrightandPimm,2003),weredeveloped.Inaddition,anumberofoligonucleotideprobes/primersforspeciﬁcallyandhierarchicallydetectingmethanogensatdifferenttaxonomiclevelsweredesignedtoresolvediffer-entmethanogenpopulationsinwaste/wastewatertreat-

mentanaerobicsludges(Rocheleauetal.,1999;ZhengandRaskin,2000;Horietal.,2006;Ariesyadyetal.,2007;Franke-Whittleetal.,2009a;Narihiroetal.,2009a,b),therumen(Yanagitaetal.,2000;Skillmanetal.,2004),subseaﬂoorsediments(Boetiusetal.,2000;Nercessianetal.,2004),sediments(Falzetal.,1999),thehumangut(Armougometal.,2009)andwetlands(Bräueretal.,2006;Zhangetal.,2008a,b)(Table1).Nowadays,almostalloftheknownculturablemethanogenscanbedetectedusingtheseprobes/primersattheclass,order,familygenusandevenspecieslevels;atthegenuslevel,itshouldbenotedthattheprobes/primerstargetingforthegeneraMethermicoccus,Methanomethylovorans,Metha-nocaldococcusandMethanotorrisarelacking.Oligonucleotideprobes/primersformcrAgeneThe16SrRNAgenehasbeenbestusedfortheidentiﬁ-cationofmethanogensinenvironments.However,becausearchaeal16SrRNAgenesotherthanthoseofmethanogenscanalsooftenbedetectedusingPCR

Probesformethanogens7

Table2.OligonucleotidePCRprimersandprobestargetingthemcrAgene.

Probelength(mer)

Probe/primernamePCRprimerSet1Set2Set3Set4Set5Set6Set7Set8

Name

Direction/Application

Probesequence(5′–3′)ReferenceSpeciﬁcity

MCRfMCRrME1ME2MLfMLrME1ME2bMrtA_forMrtA_revMETH-fMETH-rmlasmcrA-revME3MFME3MF-eME2r’ME3

SAE716TAQSAR716TAQMCU716TAQmbac-mcrAmrtAmcpmspFenmsamsarMcvME0McvME3rMcvME1r

ForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseForwardForwardReverseClonescreeningTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeTaqManprobeFISHFISHFISH

TAYGAYCARATHTGGYTACRTTCATNGCRTARTT

GCMATGCARATHGGWATGTCTCATKGCRTAGTTDGGRTAGT

GGTGGTGTMGGATTCACACARTAYGCWACAGCTTCATTGCRTAGTTWGGRTAGTTGCMATGCARATHGGWATGTC

TCCTGSAGGTCGWARCCGAAGAAAAACAATCAACCACGCACTCGTGAGCCCAATCGAAGGARTRYTMTWYGACCARATMTGYTGDGAWCCWCCRAAGTG

GGTGGTGTMGGDTTCACMCARTACGTTCATBGCGTAGTTVGGRTAGTATGTCNGGTGGHGTMGGSTTYACATGAGCGGTGGTGTCGGTTTCACTCATBGCRTAGTTDGGRTAGT

GGTGGHGTMGGWTTCACACA

AGGCCTTCCCCACTCTGCTTGAGGATAGAAATTCCCAACAGCCCTTGAAGACAGCAGTACCCGACCATGATGGAGGACARGCACCKAACAMCATGGACACWGTCCAACTCYCTCTCMATCAGRAGCGAGCCGAAGAAACCAAGTCTGGACCTGGTWCMACCAACTCACTCTCTGTCAAVCACGGYGGYMTCGGMAAGCCTTGGCRAATCCKCCGWACTTG

TCTCTCWGGCTGGTAYCTCTCCATGTACGGAAAAATTCGAAGAAGATCTGTGTGAAACCTACGCCACCGACATTCCAATCTGCATTGC

17172021322320232018201824242323212026262625242425212328202020

Springeretal.(1995)Halesetal.(1996)Lutonetal.(2002)Halesetal.(1996)

Shigematsuetal.(2004)Scanlanetal.(2008)Colwelletal.(2008)SteinbergandRegan(2008)Nunouraetal.(2008)

MostmethanogensMostmethanogensMostmethanogensMostmethanogens

MethanosphaerastadtmanaeMostmethanogensMostmethanogensMostmethanogens

Probe

Halesetal.(1996)

Shigematsuetal.(2004)Shigematsuetal.(2004)Shigematsuetal.(2004)SteinbergandRegan(2009)SteinbergandRegan(2009)SteinbergandRegan(2009)SteinbergandRegan(2009)SteinbergandRegan(2009)SteinbergandRegan(2009)SteinbergandRegan(2009)Kubotaetal.(2006)Kubotaetal.(2006)Kubotaetal.(2006)

MostofmethanogensGenusMethanosaetaGenusMethanosarcinaGenusMethanoculleus

FamilyMethanobacteriaceaeFamilyMethanobacteriaceaeFamilyMethanocorpusculaceaeFamilyMethanospirillaceaeGenusMethanoregulaFamilyMethanosaetaceaeGenusMethanosarcinaMethanococcusvannieliiMethanococcusvannieliiMethanococcusvannielii

Theprobes/primerslistedhere.

primersetsforawiderangeofmethanogentaxa,ithaslimitationinexclusivelydescribingthepopulationstruc-tureofmethanogens.Therefore,thereisaneedtodetectmethanogensonthebasisoffunctionalgenesthatarefoundtobeuniqueinmethanogenesis.SuchafunctionalgenefrequentlyusedismcrA.MethylcoenzymeMreduc-tase(mcr)istheterminalenzymeinvolvedinmethano-genesis,whichreducesthemethylgroupbondofmethylcoenzymeMwiththereleaseofmethane(Friedrich,2005).Becausethea-subunitofmcr(mcrA)anditsisoen-zymegene(mrtA)arehighlyconservedamongmethano-gens,andthatthesegenesarealmostexclusivelyfoundinmethanogens,mcrA/mrtA-baseddetectionofmethano-genshasbeenused.ThephylogenyofmethanogensdeterminedusingmcrA/mrtA(ortranslatedaminoacid)sequencesisingoodaccordancewiththosedeterminedusing16SrRNAgenesequences(Friedrich,2005).Pre-viouslyreported,frequentlyusedprobes/primersformcrA/mrtAarecategorizedintothreeprimersets,namely,MCR(Springeretal.,1995),ME(Halesetal.,1996)andML(Lutonetal.,2002)(Table2).Thetargetedregionsoftheforwardprimersofthesesetsareconsiderablydiffer-ent,whereasthoseofthereverseprimersarealmostthesame.TheMCRprimersetwasoriginallydesignedtodeterminethephylogenyofthefamilyMethanosarci-naceae(Springeretal.,1995).TheMEprimersetwasdesignedtodescribemethanogenicpopulationsinwet-

lands(Halesetal.,1996),forwhichthedifficultyinampli-fyingmcrA/mrtArelevanttoMethanosarcinaceaeandMethanobacteriaceaewaspointedoutlater(Luedersetal.,2001;Juottonenetal.,2006).TheMLprimersetwasdevelopedonthebasisofthemcrAsequencesobtainedfromﬁveorders,comprisingMethanosarcinales,Methanomicrobiales,Methanobacteriales,Methanococ-calesandMethanopyrales(Lutonetal.,2002).Fourotherprimersetsandprobesforspeciﬁctaxonomicgroupshavealsobeendevelopedrecently(Table2).

Assessingthebiodiversityofmethanogensin

complexcommunitiesbyPCRdetectionandcloningofmethanogengenes

Someofthenotedprimersfor16SrRNAandmethylcoenzymeMreductasegeneshaveoftenbeenusedforthedetectionandidentiﬁcationbyPCRtoexplorethediversityofmethanogensinenvironmentalsamples(Table3).Forexample,the146f/1324rprimersetformostofalltheknownmethanogenswasdesignedforthe16SrRNAgenecloneanalysisofdeepsedimentgashydratedeposit,andtheresultsshowedthatgeneclones(phylo-types)affiliatedwithMethanosarcinaandMethanobrevi-bacterpredominatedinthesediments(Marchesietal.,2001).Similarly,someoftheseprimersshowninTable1havebeenusedforPCRtoproﬁlemethanogenpopula-

8T.NarihiroandY.Sekiguchi

Table3.ExamplesofoligonucleotideprimersetsforPCR-basedanalysesformethanogens.

TypeofsampleAnaerobicprocess

ApplicationqPCR

Targetgene16SrRNA

Targetgroup

MethanomicrobialesMethanosarcinalesMethanobacterialesMethanococcalesMethanosarcinaceaeMethanosaeta

MethanocorpusculaceaeMethanospirillaceaeMethanosaetaceaeMethanobacteriaceaeMethanobacteriaceaeMethanoregulaMethanosarcinaMethanoculleusMethanosarcina

Methanothermobacter

MethanoculleusthermophilusMethanosaetathermophilaMethanothermobacterMethanosaetaMethanosarcinaMethanoculleusMethanosaetaMethanosarcinaMethanoculleusMethanosaeta

Probeset(forward/reverse/probe)aMMB282F/MMB832R/MMB749FMSL812F/MSL1159R/MSL860FMBT857F/MBT1196R/MBT929FMCC495F/MCC832R/MCC686FMsc380F/Msc828R/Msc492FMst702F/Mst862R/Mst753Fmlas/mcrA-rev/mcpmlas/mcrA-rev/mspmlas/mcrA-rev/msa

mlas/mcrA-rev/mbac-mcrAmlas/mcrA-rev/mrtAmlas/mcrA-rev/Fenmlas/mcrA-rev/msar298F/586R240F/5R410F/667RMc412f/Mc578rMs413f/Ms578rMt392f/Mt578r

MS1b/SAE835R/SAE761TAQMB1b/SAR835R/SAR761TAQAR934F/MG1200b/MCU1023TAQME1/ME2b/SAE716TAQME1/ME2b/SAR716TAQME1/ME2b/MCU716TAQS-F-Msaet-0387-S-a-21/S-F-Msaet-00-A-a-31/S-F-Msaet-0573-A-a-17S-G-Msar-0450-S-a-19/S-P-Msar-00-A-a-31/S-G-Msar-05-S-a-20S-F-Mbac-0398-S-a-20/S-G-Mbac-0526-A-a-33/S-G-Mbac-0578-A-a-22S-F-Mbac-0398-S-a-20/S-G-Mthb-09-S-a-32/S-G-Mthb-05-A-a-25

AR934F/MG1200b/MSP1025TAQNOBI109f/NOBI633109f/UNIV1492rb25f/1391r

25f/UNIV1492rb109f/UNIV1492rbmlas/mcrA-rev

ME1/ME2R15F/R15R

Met630F/Met803RA357f/A693rA24f/A329rA24f/A348r

Archf2/Archr1386Archf2/MSrr859

Mbacf331/Archr1386fMbium/Archr1386Archf2/Mccr

fMbb1/Archr1386forward/reverse/probeMrtA_for/MrtA_rev146f/1324r

METH-f/METH-r

ME3MFandME3MF-e/ME2r’355Fc/1068R109f/1401R344Fd/1202R

ARC344f/ARC915MLf/MLrA1f/A1100r109f/ARC915

Met83F(orMet86F)/Met1340RMCRf/MCRr

ReferenceYuetal.(2005)

qPCRmcrASteinbergandRegan(2009)

qPCR16SrRNAFranke-Whittleetal.(2009a)

qPCR16SrRNAHorietal.(2006)

qPCR16SrRNAShigematsuetal.(2003)

qPCRmcrAShigematsuetal.(2004)

qPCR16SrRNASawayamaetal.(2004)

qPCR16SrRNAMethanosarcinaSawayamaetal.(2006)

Methanobacterium

Methanothermobacter

qPCRqPCR

PCR-cloningPCR-cloning16S16S16S16SrRNArRNArRNArRNAMethanospirillumMethanolinea

MostmethanogensMostmethanogensTangetal.(2005)Imachietal.(2008)Narihiroetal.(2009a)Ariesyadyetal.(2007)

Anaerobicprocess,wetlandWetlandRumen

PCR-cloningPCR-cloningqPCR

qPCR,DGGEPCR,DGGE

mcrAmcrA

16SrRNA16SrRNA16SrRNA

Mostmethanogens

MethanolobuspsychrophilusMostmethanogensMostmethanogens

SteinbergandRegan(2008)Halesetal.(1996)Zhangetal.(2008a)Hooketal.(2009)Yuetal.(2008)

PCR-typing16SrRNA

GastrointestinaltractDeepseasediments

Lakesediment

qPCR

PCR-cloningPCR-cloningqPCRqPCR

PCR-cloning16SrRNAmcrA

16SrRNAmcrAmcrA

16SrRNA

SulfurouslakeLandﬁll

CiliateendosymbiontRicepaddysoilPurecultures

PCR,DGGEPCR-cloningPCR,DGGEPCR,DGGEPCR-ribotypingPCR-cloning16SrRNAmcrA

16SrRNA16SrRNA16SrRNAmcrA

MostmethanogensMethanosarcinalesMethanobacterialesMethanobacteriumMethanococcalesMethanobrevibacter

MethanobrevibactersmithiiMostmethanogensMostmethanogensMostmethanogensMostmethanogensMethanomicrobiaMethanobacterialesMethanococcalesMostmethanogensMostmethanogensMostmethanogensMostmethanogensMostmethanogensMostmethanogens

Skillmanetal.(2004)

Armougometal.(2009)Scanlanetal.(2008)Marchesietal.(2001)Colwelletal.(2008)Nunouraetal.(2008)Banningetal.(2005)

Casamayoretal.(2001)Lutonetal.(2002)Embleyetal.(1992)Grosskopfetal.(1998)WrightandPimm(2003)Springeretal.(1995)

a.b.c.d.TheprimersequenceswereshowninTables1and2.

UNIV1492rreverseprimerwasoriginallyreferredfromLane(1991)asanuniversalprimer.

355FforwardprimerwasoriginallyreferredasM(SA/MI)355probedevelopedbyOvreåsetal.(1997)asshowninTable1.

344FforwardprimerwasoriginallyreferredasARC344probedevelopedbyRaskinandcolleagues(1994b)asshowninTable1.

Probesformethanogens9

tionsbydenaturinggradientgelelectrophoresis(DGGE)(e.g.(Casamayoretal.,2001;2002;Yuetal.,2005;2006;2008).Asexamples,WrightandPimm(2003)developedPCRandsequencingprimersforthe16SrRNAgeneofmethanogens,andusedthemfortheribotypingofmembersoftheclasses‘Methanomicrobia’andMethano-bacteria.ThedetectionofmethanogensbyPCRinlambrumensampleswasperformedusingmethanogen-speciﬁcprimerstargetingdifferenttaxonomiclevels(Skill-manetal.,2004).Banningandcolleagues(2005)designednovelreverseprimerstoprovidespeciﬁcampli-ﬁcationofthe16SrRNAgenesof‘Methanomicrobia’(MethanomicrobialesandMethanosarcinales),Methano-bacterialesandMethanococcales,andsuccessfullyusedthemfortheidentiﬁcationofmethanogenicpopulationstructuresinlakesediments.

MassiveparallelsequencingofPCR-ampliﬁed16SrRNAgenesusingnextgenerationsequencers(suchastheFLXpyrosequencers)allowsustoobtainahugenumberofcommunitysequencetags(forexamplec.10000–10000016Spyrotagsforeachsample),whichismorethananySanger-basedcloningstudytodate,andhavebeenusedforcharacterizingarchaealpopulations(includingmethanogens)inhydrothermalchimneys(Bra-zeltonetal.,2010a,b).Themethodologicaladvancementsof16SrRNAgenepyrosequencingincludehigherresolu-tion(moresequences)forgene-basedcommunitystruc-tureanalysis,analysisofmultiplerelatedsamplesanduseofmetadata(TringeandHugenholtz,2008).Becauseoftheseadvancements,aswellasrecentdevelopmentofanalyticaltoolsformassivesequencedatasuchasQIIME(Caporasoetal.,2010),themethodmaybefurtherusedforcharacterizingdiversityofmethanogensinecosystems.Similarly,theprimersformethylcoenzymeMreductasegeneshaveoftenbeenusedforPCRdetectionandiden-tiﬁcationtoexclusivelyexplorethediversityofmethano-gensinsamples.Forexample,theMCRsetwasusedtoelucidatethediversityofmethanogensinvariousenviron-mentswithPCR-basedcloning(Kemnitzetal.,2004;Dhillonetal.,2005;Alainetal.,2006)andT-RFLPanaly-ses(Ramakrishnanetal.,2001;Kemnitzetal.,2004).SuchcloninganalyseswerealsoconductedusingtheME(Halesetal.,1996;Nercessianetal.,1999;Galandetal.,2002;2005;Tatsuokaetal.,2004)andMLprimersets(Lutonetal.,2002;Castroetal.,2004;Juottonenetal.,2005;Nercessianetal.,2005;Ufnaretal.,2007;Smithetal.,2008).ComparativestudiesusingthesethreeprimersetshaveindicatedthattheMLprimersetismoreefficientforretrievingphylogeneticallydiversemethanogensinthewetlandthanothers(Juottonenetal.,2006;Jermanetal.,2009).Owingtothisadvantage,theMLsethasbeenusedextensivelytodeterminethediversityofmethanogensinvariousanaerobicecosystems.Inaddition,ithasbeennotedthatthesemcrA-targetedprimersets(especially

ME-relatedprimerset)werealsousedforthequantitativedetectionofanaerobicmethanotrophicarchaea(ANME)inmethaneseepsediments(Inagakietal.,2004;Nunouraetal.,2006;2008).ThisisduetothefactthatanaerobicmethaneoxidationrepresentedbytheANMEgroupisconsideredtoproceedwithmcr-typeenzymes(ThauerandShima,2008).DetailedinformationaboutthemcrA-basedqPCRforANMEsisdescribedbelow.

Polymerasechainreaction-basedmoleculartech-niques,suchasPCR-cloning,pyrosequencing,DGGEandT-RFLPareadequatetogainentirecommunitycom-positionanddiversityofmethanogensinecosystems.Basedonthefrequencyofretrievalofphylotypesingenelibrary(orrelativeintensityofDGGEorT-RFbandsinelectropherogram),relativeabundanceofphylotypesofinterestcanbeinferred.However,itshouldbenotedthatentiremicrobialcommunitystructureanalysisbasedonbulkcelllysis,DNAextraction,PCRandcloningareoftensuspectbecauseofseveralbiasesinvolvedineachofthesteps(Dahllof,2002).Therefore,oneshouldbecarefultodiscussontheabundanceofphylotypesinsamplesbasedsolelyonthedataobtainedbythesemethods.Morereliablemethodstocarryoutquantitativedetectionofdifferentgroupsofmethanogensinsampleswouldbetousethefollowingquantitativemoleculartechniques.Identiﬁcationandquantiﬁcationofmethanogensincomplexcommunitiesbymembranehybridizationmethod

QuantitativemembranehybridizationoflabelledDNAprobestocommunityrRNAshasbeenappliedtovariousenvironmentalrRNAsforthequantitativedetectionofspe-ciﬁcgroupsofmicrobespresentincomplexcommunities(Stahletal.,1988;Raskinetal.,1994a).RNA-dependentcommunityanalysisisknowntoindicatetheinsituactivityofindividualmembersinecosystems,becauseofthereasonsthatRNAsynthesisisknowntoreﬂecttheinsitugrowthratesoforganisms(Poulsenetal.,1993;Amannetal.,1995),andthattheturnoverofRNAisthoughttobemuchhigherthanthatofDNA.Therefore,rRNA-dependentmoleculartechniqueslikethepresentoneprovidepreciseinformationaboutthedynamicnatureofindividualmicrobesinsystems.In1994,Raskinandcolleaguescarriedouttheﬁrstleadingstudiesonthedevelopmentofeightoligonucleotideprobesforthequantitativedetectionofmethanogensinanaerobicwastewatertreatmentsludges(StahlandAmann,1991;Raskinetal.,1994a,b).Inthesestudies,theyestablishedthegroup-speciﬁcoligo-nucleotideprobestargetingMethanomicrobiales(probesMG1200andMSMX860),Methanobacteriaceae(probesMB310andMB1174)andMethanococcales(probeMC1109).Becauseoftheimportanceofmethaneproduc-tionfromacetateinanaerobicbioreactors,speciﬁcprobes

10T.NarihiroandY.Sekiguchi

foraceticlasticmethanogens,suchasthemembersofMethanosarcinaceae(probesMS1414andMS821)andMethanosaeta(probeMX825),werealsodeveloped.

Theseprobeshavebeensuccessfullyappliedtothequantiﬁcationofmethanogensinlaboratory-andfull-scaleanaerobicbioreactorsbasedonrRNA(Raskinetal.,1995;Griffinetal.,1998;Liuetal.,2002;McMahonetal.,2004;Zhengetal.,2006).Althoughmembranehybridiza-tionenablesthesensitivequantiﬁcationofindividualspeciesofrRNAmolecules,thismethodrequiresseverallaboriousexperimentalsteps,oftenradioactivelylabelledDNAprobes,andreferencerRNAsamplesasexternalstandardsforeachexperiment.Thus,themethoditselfmaybereplacedbysimilarbutmuchrapidandsimplermethods,suchasreal-timeRT-PCRandRNaseHmethods.However,theprobesusedformembranehybridizationexperimentsmaybealsousedasprobes/primersinotherexperimentsshownbelow.FISHformethanogens

Whole-cellFISHbasedon16SrRNAisnowcommonlyusedtodetectspeciﬁcgroupsofmicrobesandtoquantifypopulationsofinterestinenvironmentsbydirectcountingunderamicroscope(Amannetal.,1995).Inaddition,FISHisusedforvisualizingthespatialdistributionofthepopu-lationofinterestinbioﬁlms,suchasthoseofmethanogensinsludgegranulesinmethanogenicwastewatertreatmentsystems(Sekiguchietal.,1999).Basically,theprobesdevelopedformembranehybridizationofmethanogen16SrRNAsorreverseprimersforPCRampliﬁcationofmethanogen16SrRNAgenescandirectlybeusedasoligonucleotideprobesforinsituhybridizationstudies,theprobespreviouslydesignedbyRaskinandcolleagues(Raskin,etal.,1994b)havefrequentlybeusedforthepurposeofFISHstudiesaswell.TheseprobeshavebeenusedforthequantitativedetectionofmethanogensusingtheFISHtechniqueinvariousanaerobicecosystems,suchaspeatbog(e.g.Hornetal.,2003),aquifer(e.g.Kleikemperetal.,2005),landﬁlls(e.g.Laloui-Carpentieretal.,2006)andanaerobicwastewatertreatmentpro-cesses(e.g.Sekiguchietal.,1999;Plumbetal.,2001;Boonapatcharoenetal.,2007;Chenetal.,2009).Recently,theimprovementofthespeciﬁcityandsensitivityoftheprobesdesignedbyRaskinandcolleagues(1994b)hasbeenreported.Crocettiandcolleagues(2006)reﬁnedtheexperimentalconditionsofsuchprobesforFISHanaly-sistoaccuratelyandsensitivelydetectmethanogens.Inadditiontothequantiﬁcation,theprobes(Table1)havealsobeenusedforinvestigatingthelocalizationofmethanogensinbioﬁlms(sludgegranules)[e.g.(Roch-eleauetal.,1999;Sekiguchietal.,1999;Plumbetal.,2001;Zhengetal.,2006;Vavilinetal.,2008;Chenetal.,2009)].Inanaerobicsludgegranules,hydrogenotrophic

methanogensareoftenjuxtaposedwithsyntrophicsubstrate-degradingbacteria,suchassyntrophicpropionate-oxidizingbacteriasuchasmembersofthegeneraSyntrophobacterandPelotomaculum;suchcloseproximitybetweensyntrophicbacteriaandmethanogenshasbeenobservedbyFISHwithconfocallaserscanningmicroscopy(Harmsenetal.,1995;1996;Sekiguchietal.,1999;Imachietal.,2000).Anaerobicciliatesoftenpossesendosymbioticmethanogenswithintheircells,andthedistributionofsuchmethanogensineukaryoticcellshasbeenobservedbytheFISHmethod[e.g.(Embleyetal.,1992;Shinzatoetal.,2007)].

AlthoughFISHisapowerfulmethodforvisualizingthecellsofinterest,therearesomedrawbacksindetectingcells;oneofsuchproblemsisconcernedwiththepenetra-tionofoligonucleotideprobesintothecells(Amannetal.,1995).Formethanogens,FISHstainingisoftendifficultforsomeMethanobacteriumandMethanobrevibactercells,forwhicholigonucleotideprobesdonotpenetrateintotheircells(Sekiguchietal.,1999;Yanagitaetal.,2000;Naka-muraetal.,2006).Tosolvethisproblem,ﬁxedcellsweresubjectedtofreeze-thawcyclesbeforehybridization,resultingintheimprovementofprobepenetration(Sekigu-chietal.,1999).Anotherwaytosolvethisproblemistheuseofrecombinantpseudomureinendoisopeptidase,whichincreasesthepermeabilityofoligonucleotideprobesintocells,andallowsabettervisualizationofmethanogensinanaerobicgranularsludgeandtheendosymbioticmethanogensintheanaerobicciliateTrimyemacompres-sum(Nakamuraetal.,2006).Animprovedprotocolofcatalysedreporterdeposition-FISHformethanogenswithrecombinantpseudomureinendoisopeptidasehasalsobeenreported,whichcanincreaseﬂuorescencesignalintensityinFISHfordetectingcellswithalowrRNAcontent(Kubotaetal.,2008).

Recently,mcrA-basedinsitudetectionofmethanogenshasbeenperformedusingthetwo-passtyramidesignalampliﬁcation-FISHapproachcombinedwithlockednucleicacids(Kubotaetal.,2006;Kawakamietal.,2010).Theseattemptswere,atthispoint,onlypartiallysuccessfulindetectingmethanogencells,becausemcrAisgenerallypresentasasinglecopygeneontheirchro-mosome,whichresultsinalowsensitivityofdetection.qPCR

QuantitativePCRof16SrRNAgeneandmcrAhasalsobeenusedtoquantifytheabundanceofmethanogensinrecentyears.ExamplesofqPCRprimerandprobesetsfordifferenttaxaofmethanogensarelistedinTable3.Forexample,theprimersMet630F/Met803RweredevelopedfortheSYBRgreen-basedreal-timeqPCRforalmostalltheknownmethanogensintherumenofthedairycow(Hooketal.,2009).Yuandcolleagues(2005)designed

Probesformethanogens11

TaqMan-basedqPCRprobes/primersets(sixsetsintotal)foreachoftheordersMethanomicrobiales,Methanosarci-nales,MethanobacterialesandMethanococcales,aswellasthefamiliesMethanosaetaceaeandMethanosarci-naceae.Theyappliedapartofthesesetstoquantifyingaceticlasticmethanogensinmethanogenicsludgesfortreatingsewagesludges,cheesewheywastewaterandsyntheticwastewater,andrevealedthatthepopulationofaceticlasticmethanogensisaffectedbytheacetatecon-centrationinthewastewaters(Yuetal.,2006).qPCRdetectionusingspeciﬁcprimersforparticulargroupsofmethanogensofinterest,suchasMethanoculleus(Shige-matsuetal.,2003;Horietal.,2006;Franke-Whittleetal.,2009a),Methanolinea(Imachietal.,2008),Methanospir-illum(Tangetal.,2005),Methanosaeta(Shigematsuetal.,2003;Sawayamaetal.,2004;Horietal.,2006),Metha-nosarcina(Shigematsuetal.,2003;Sawayamaetal.,2006;Franke-Whittleetal.,2009a),Methanolobus(Zhangetal.,2008a,b),Methanobrevibacter(Armougometal.,2009),Methanobacterium(Sawayamaetal.,2006)andMethanothermobacter(Horietal.,2006;Sawayamaetal.,2006;Franke-Whittleetal.,2009a)havealsobeenreportedtodate(Table3).

FortheqPCRdetectionofmcrA,theMEprimersetwasusedforthequantiﬁcationofmethanogenicandmethan-otrophicpopulationsinmethaneseepsediments(Inagakietal.,2004;Nunouraetal.,2006).Afterwards,Nunouraandcolleagues(2008)slightlymodiﬁedtheMEprimerseries,andshowedthatthemixtureoftheME3MFandME3MF-eforwardprimersandtheME2’reverseprimerismostsuitablefortheqPCRdetectionofthemethanogensandANMEsintheenvironments.TheresultsshowedthatasigniﬁcantamountofmethanogensandANMEswasfoundinanaerobicallydigestedsludgeandmethaneseepsediments.TheMLprimersetwasalsousedforthequantitationofmethanogenicarchaealpopulationsintherumen(Denmanetal.,2007)andhumansubgingivalplaque(Viannaetal.,2008).Moreover,SteinbergandRegan(2008;2009)developedthemlas/mcrA-revprimerset,whichisaderivativeoftheMLprimerset,fortheclonelibraryconstructionandqPCRanalysesofmetha-nogensinoligotrophicfenandanaerobicdigestersludge.Inaddition,thegenus-speciﬁcTaqManprobesforthemcrA-basedquantitativedetectionoftheMethanosaeta,MethanosarcinaandMethanoculleusresidentinacetate-fedchemostats,andtheresultsshowedthatdilutionrateisakeyfactorintheacetatebioconversionpathway(Shigematsuetal.,2004).

QuantitativePCRmethodprovidessensitive,quantita-tivedataofgeneofinterestwithasufficientlyhighdynamicrangeofquantiﬁcation(ZhangandFang,2006).Therefore,inadditiontotheuseofdigitalPCR(Ottesenetal.,2006),qPCRmaybefurtherusedforquantitativemonitoringofmethanogentaxaofinterestsincomplex

microbialcommunities.However,itshouldbenotedthatthemethodisPCR-basedandhencetheirdatacanbesuspectbecauseofbiasesinvolvedinDNAextractionandprimer/probemismatches.

AssessingmethanogenpopulationbyRNaseHmethod

Althoughtheabove-mentionedquantitativemethodssuchasmembranehybridizationandqPCRarebecominggeneralmeanstodeterminetheabundanceofthepopu-lationofinterestinacomplexmicrobialcommunity,thereisaneedtodevelopmoresimpleandrapidtechniquesthatmeettheneedsforreal-timemonitoringofthepopulationofinterestinacomplexcommunity.Recently,asimpleandrapidquantiﬁcationmethod,namely,theRNaseHmethod,hasbeendeveloped(Uyenoetal.,2004).Thismethodisbasedonthesequence-speciﬁccleavageof16SrRNAwithribonucleaseH(RNaseH)andoligonucleotide(scissor)probes.RNAsfromacomplexcommunitywereﬁrstmixedwithanoligonucleotideandsubsequentlydigestedwithRNaseH.BecauseRNaseHspeciﬁcallydegradestheRNAstrandofRNA:DNAhybridheterodu-plexes,thetargetedrRNAsarecleavedatthehybridizationsiteinasequence-dependentmannerandareconse-quentlycutintotwofragments.Incontrast,non-targetedrRNAsremainintactunderthesameconditions.ForthedetectionofcleavedrRNAs,theresultingRNAfragmentpatternscanberesolvedbygelelectrophoresisusingRNA-stainingdyes.Therelativeabundanceofthetargetedspeciesof16SrRNAfragmentsintotal16SrRNAcanalsobequantiﬁedbydeterminingthesignalintensityofindi-vidual16SrRNAbandsinanelectropherogram(withouttheuseofexternalstandards).BecausethismethoddoesnotrequireanexternalRNAstandardforeachexperiment,asisrequiredinmembranehybridization,andbecausethepresentmethodisrelativelyeasytoperformwithinashorttime(i.e.within2–3h),thistechniquemayprovidedirect,rapidandeasymeansofthequantitativedetectionofparticulargroupsofanaerobesbasedontheirrRNA,suchasthoseofmethanogensaswell.

Thismethodhasbeensuccessfullyappliedtothequan-tiﬁcationofactivemethanogensinanaerobicbiologicaltreatmentprocesses(Uyenoetal.,2004;Sekiguchietal.,2005;Narihiroetal.,2009b).Ingeneral,oligonucleotideprobesusedinFISHandmembranehybridizationmethodscandirectlybeusedasscissorprobesintheRNaseHmethod.Recently,atotalof40probes,includingnewlydesignedandpreviouslyreportedprobeslistedinTable1,havebeenoptimizedforthespeciﬁcquantiﬁca-tionofmethanogensatdifferenttaxonomiclevelsforuseintheRNaseHmethodandhavebeenappliedtoquan-titativeandcomprehensivedetectionofmethanogensinvarioustypesofanaerobicbiosystems(Narihiroetal.,

12T.NarihiroandY.Sekiguchi

2009b).Asaresult,methanogenpopulationswereiden-tiﬁedatdifferenttaxonomiclevelsandwereinﬂuencedbytheprocesstemperatureandwastewatercompositions.BecauseofthereasonsthatthismethodisbasedonrRNAandthattheRNA(rRNA)levelisoftendependentontheinsituactivityofindividualcellsasdescribedabove,thismethodmaybeusedforreal-timemonitoringofactivemethanogensandotherimportantbacteriainengineeredecosystemssuchaswaste/wastewatertreat-mentsystemstobettercontrolsuchbioreactors.Stableisotopeprobing(SIP)-baseddetectionofactivemethanogenpopulationsinenvironmentsToidentifymetabolicallyactivepopulationsinenviron-ments,SIPofDNA(Radajewskietal.,2000)andRNA(Maneﬁeldetal.,2002)hasbeenusedinrecentyears.Inprinciple,SIPtechnologyisbasedontheincorporationof13

C-labelledsubstratesintothenucleicacids.Thesepara-tionofisotopicallylabelled(active)fractionsfromunla-beled(inactive)fractionsisgenerallyperformedwithdensitygradientcentrifugation.Thesubstrate-assimilatedmicroorganismsinthelabelledfractionsareidentiﬁedbyasetofPCR-basedmoleculartechniquessuchasgenecloning,DGGEandothermethods.Therefore,forthepurposeofidentifyingactivemethanogensthatarerespon-sibleforparticularmetabolismsinenvironments,theprobes/primerslistedinTables1and2canbeused.

Asexamples,activemethanogenpopulationsinvolvedinthesyntrophicpropionateoxidationinanoxicsoilwereanalysedonthebasisofrRNA-SIP,anditwasfoundthatthemembersofthegeneraMethanobacterium,Metha-nosarcinaandMethanocellaplayakeyroleinscavenginghydrogen/formate/acetateinsyntrophicassociationwithpropionate-oxidizingbacteria(Luedersetal.,2004).ConradandcoworkershavestudiedthedetectionofactivemethanogenpopulationsusingDNA-SIPcombinedwith13C-labelledCO2,andtheresultsofT-RFLPproﬁlingandphylogeneticanalysisforclonal16SrRNAgenefrag-mentssuggestthatmembersoftheRC-Igroup(Metha-nocellales)serveasimportantmethanogensinricepaddyﬁelds(LuandConrad,2005;Luetal.,2005).Theactivemethanogenicpopulationsinenrichmentcultureofmunicipalsolidwastedigesterresiduesspikedwith13C-labelledsubstrates(suchascellulose,glucoseandsodiumacetate)weredeterminedbyDNA-SIPfollowedbycloninganalysis(Lietal.,2009).Othermethodsandfutureperspectives

DNAmicroarrayplatform,likePhyloChip,isbecominganimportanttoolforparalleldetectionofdifferentcommunitymembersofmicrobesinecosystems.Forhighthrough-putandcomprehensivedetectionofmethanogensinparallel,ANAEROCHIP(Franke-Whittleetal.,2009b)and

GeoChip(Wangetal.,2009)havebeendevelopedrecently.Theprimers/probessummarizedinthisreviewmaybeintegratedintosuchaplatformforparallelandhierarchicaldetectionofmethanogens.Theseprimer/probesformethanogenscanalsobeusedinnovelPCR-basedtechniques,suchasthehierarchicaloligonucleotideprimerextensionmethod(WuandLiu,2007),whichhasrecentlybeendevelopedforquantitative,multiplexdetec-tionoftargetedmicrobialgenesamongPCR-ampliﬁedgenes.SIPtechnologyhasbeennotedasanimportantpretreatmentstepforfunctionalmicrobialcommunityanalyses,suchasRamanmicroscopy-FISH(Huangetal.,2007;2009)andmetagenomicapproaches(Kalyuzhnayaetal.,2008;Suletal.,2009).Moreover,recentadvancesinanalyticalchemistry,suchasisotoperatiomassspectrom-etry(Penningetal.,2006;Vavilinetal.,2008)andsecond-aryionmassspectrometry(Orphanetal.,2001),holdgreatpromiseforthehighlysensitivedeterminationoftargetedmicrobes.Thus,inadditiontodescribingthediversityofmethanogensinparticularenvironmentsofinterestonthebasisofDNAandRNA,suchfunction-relatedanalysesofmethanogensmaybecomeimportantintheﬁeldsofenvironmental,determinativeandappliedmicrobiology.

Asdescribedinthisminireview,avastnumberofprobe/primershavebeendevelopedfordescribingandquanti-fyingmethanogenpopulations,coveringmostpartsoftheknownculturablemethanogensdescribedsofar.Avarietyofmolecularmethodshavealsobeendevelopedthatareusedincombinationwiththeprobe/primers.Becausethesemolecularmethodshavetheirownadvancementsanddrawbacks,researchersneedtoselectappropriatecombinationsofmethodsandprobe/primersdependingonwhattheresearchersneedtoknow.Fordetails,recentreviewsmaybehelpfulfortheselectionofmoleculartechniquestobeused(Talbotetal.,2008;Tabatabaeietal.,2010).Inmolecularecology,multipleapproachesarebesttogainacompletepictureofmethanogenpopu-lationsinenvironments.Therefore,theuseofappropriate(multiple)moleculartechniquesincombinationswithothernon-molecularbasedmethodslikemembranelipid,autof-luorescence,activitymeasurementandimmunoenzy-maticproﬁlingshouldbeconsidered.Itshouldalsobenotedthattherearestillanumberofuncultivatedmetha-nogensinvariousenvironments,andthattheyshouldbefurtherisolatedandcharacterizedindetail.Monitoringtoolsforsuchunculturedmethanogensremaintobedevelopedtofurtherincreaseinthecoverageofmetha-nogenspresentinenvironments.Acknowledgements

ThisworkwassupportedbytheEnvironmentResearchandTechnologyDevelopmentFund(S2-03)andtheGlobal

Probesformethanogens13

EnvironmentResearchFund(RF-076)oftheMinistryoftheEnvironment,Japan.

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