MICROBIAL COMMUNITY COMPOSTS BASED ON 16S AND 18S

1101EnvironmentalTechnology,Vol.26.pp1101-1113©SelperLtd.,2005MICROBIALCOMMUNITYDYNAMICSINMANURECOMPOSTSBASEDON16SAND18SrDNAT-RFLPPROFILESS.M.TIQUIA115FScienceBuilding,DepartmentofNaturalSciences,TheUniversityofMichigan,Dearborn,MI48128,USA(Received20March.;Accepted28April2005)ABSTRACTCompostprocessingisassumedtoberelatedtothemicrobialcommunitiespresent.However,methodsthatwillevaluatetheserelationshipsarenotwellunderstood.Inthisstudy,terminalrestrictionfragmentlengthpolymorphism(T-RFLP)analysiswasusedtoevaluatethediversityofPCR-amplifiedbacterial16Sandfungal18SrDNAcommunitiesfrommanurecompostsatdifferentstagesofcomposting(initial[day0],thermophilic[day24],andmature[day104]).Resultsshowedthatthebacterialandfungalcommunityprofileschangedoverthecompostingprocess,withbacterialcommunitiesshowingahigherdiversitycomparedwiththefungalcommunities.Duringthethermophilicstage(day24),thediversityofthebacterialcommunitiesincreased,whilethefungalcommunitiesdecreased.Asthecompostreachedmaturity(day104),areversepatternwasobservedbetweenthediversityofbacterialandfungalcommunities.Thatis,the18SrDNAT-RFLP-baseddiversityindicesincreased,whilethe16SrDNAT-RFLP-baseddiversitydecreased.Differencesintemperatureprofilesatdifferentstagesofcompostingimpactedthechemicalpropertiesandthediversityofthemicrobialcommunities.Theday104compost(mature)hadlowerwater,organicmatterandCcontentsandhigherCandOMlosscomparedwiththeday0(initial)andday24(thermophilic)composts,whichaffectedthediversityofthemicrobialcommunities.TheresultspresentedheredemonstratedthatdistinctivecommunitypatternsfrommanurecompostscouldberapidlygeneratedusingT-RFLPanalysis.ThesuccessionofpeaksincombinationofincreasinganddecreasingpeakheightsatdifferentstageofcompostingindicatesthehighpotentialofT-RFLPtechniquetomonitorthedynamicsofmicrobialcommunities,andtheirvariationqualitativelyandquantitatively.Keywords:T-RFLP,geneticdiversity,principalcomponentsanalysis,manurecomposting,compostmaturity,organicmatterINTRODUCTIONCharacterizingandunderstandingofmicrobialcommunitystructureanddiversitymayprovideinformationneededtoimproveandevaluatecompostprocessingandquality.Whilechangesinphysico-chemicalproperties(temperature,bulkdensity,cation-exchangecapacity,C:Nratio,pHandorganicC)duringthecompostingprocesshavebeenextensivelystudied[1–7],informationonmicrobialcommunitiesduringcompostingisunderexplored.Moreoever,thereisaneedforarapidmethodofassessmentofmicrobialdiversityduringcomposting.Manyearlystudiesonthemicrobiologyofcompostingdealtwiththeisolationanddescriptionofvariousmicrobesincompostusingclassicalculturemethod[8-14].Sincethismethodcannotdetectnon-culturablespecies,thereisapossibilitythatorganismswhicharedifficulttoisolatearepredominant.Recently,moleculartoolshavebeenusedtodetectcompostmicrobesindependentofculture[15-17].Blancetal.[15]usedtheamplifiedribosomalDNArestrictionanalysis(ARDRA)tocharacterizethermotolerantpopulationssuchasThermusstrainsandBacillus-relatedbacteriaisolatedfromcomposts.Kowalchuketal.[16]characterizedammonia-oxidisingbacteriaincompostusingdenaturinggradientgelelectrophoresis(DGGE)fromreversetranscriptase-PCR(RT-PCR)amplified16SrRNAgenes.Petersetal.[17]analyzedmicrobialcommunitysuccessionduringcompostingatabroaderphylogeneticlevelusingsingle-strand-conformationpolymorphisms(SSCP).Muchoftheseworks,however,werebasedontheorganismsthatcouldbefoundandutilizedtospeedupdecomposition.Attemptshavenotbeenmadetoassessthediversityofthemicrobialcommunitiesatdifferentstagesofcomposting.Inthisstudy,terminalrestrictionfragmentlengthpolymorphisms(T-RFLP)ofbacterial16Sandfungal18SrDNAgeneswereusedtocharacterizemicrobialcommunitystructureanddiversityinmanurecompostsatdifferentstagesofcomposting.Thisanalysisisbasedontherestrictionendonucleasedigestionoffluorescentlyend-labelledPCRproducts.ThedigestedproductismixedwithaDNAsizestandard,itselflabelledwithadistinctfluorescentdye,andthefragmentsarethenseparatedbygelelectrophoresisusing1102anautomatedsequencer[18].Uponanalysis,onlytheterminalend-labelledrestrictrionfragmentsaredetected.Anelectropherogramisproduced,whichshowsaprofileofcompostmicrobialcommunityasaseriesofpeaksofvaryingheight.Thistechniquehasbeeneffectivelyusedintheexplorationofcomplexmicrobialenvironments[19-22].Bruce[19]exploitedthistechniquetoanalysethediversityofmercuryresistancegenesinpollutedsoils.T-RFLPhasalsobeenusedtostudybacterial[20],archeal[21]andeukaryal[22]populationsinnaturalhabitats.Thispaperaddressesanareaofappliedmicrobiologythathasgrowingimportance.Thereisaneedforarapidmethodtogaininsightsaboutthestructureanddiversityofmicrobialcommunitiesduringcomposting.Theuseof16Sand18SrRNAT-RFLPprofilesmayprovideonesuchroute.Inthisstudy,T-RFLPwasfoundtobeusefulininvestigatingthediversityofcomplexcompostcommunities.TheT-RFLPprofilesshoweddistinctcharacteristicsofthemicrobialcommunitiesunderdifferentcompostingconditionsandmay,therefore,beusefulinevaluatingtheprogressofthecompostingprocess.MATERIALSANDMETHODSCompostingSet-upCowandhorsemanuresweremixedhomogeneouslyatarati