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10.1128/AEM.70.12.7378-7387.2004.2004,70(12):7378.DOI:Appl.Environ.Microbiol. IgorTiago,AnaPaulaChungandAntónioVeríssimo PopulationsEnvironment:HeterotrophicAerobicBacterialDiversityinaNonsalineAlkaline:Theseinclude:REFERENCES:Thisarticlecites27articles,15ofwhichcanbeaccessedfreeCONTENTALERTSmore»articlescitethisarticle),Receive:RSSFeeds,eTOCs,freeemailalerts(whennew::onNovember3,2011byNATIONALSCIENCELIBRARY,CAS–7387Vol.70,No.120099-2240/04/$08.000DOI:10.1128/AEM.70.12.7378–7387.2004Copyright©2004,AmericanSocietyforMicrobiology.AllRightsReserved.BacterialDiversityinaNonsalineAlkalineEnvironment:HeterotrophicAerobicPopulationsIgorTiago,AnaPaulaChung,andAnto´nioVerı´ssimo*DepartamentodeZoologiaandCentrodeNeurocieˆncias,UniversidadedeCoimbra,Coimbra,PortugalReceived23March2004/Accepted16August2004Heterotrophicpopulationswereisolatedandcharacterizedfromanalkalinegroundwaterenvironmentgeneratedbyactiveserpentinization,whichresultsinaCa(OH)2-enriched,extremelydilutedgroundwaterwithpH11.4.Onehundredeighty-fivestrainswereisolatedindifferentmediaatdifferentpHvaluesduringtwosamplingperiods.Toassessthedegreeofdiversitypresentintheenvironmentandtoselectrepresentativestrainsforfurthercharacterizationofthepopulations,wescreenedtheisolatesbyusingrandomamplifiedpolymorphicDNA-PCRprofilesandgroupedthembasedonsimilaritiesdeterminedbyfattyacidmethylesteranalysis.Phenotypiccharacterization,determinationsofGCcontent,phylogeneticanalysesbydirectse-quencingof16SrRNAgenes,anddeterminationsofpHtolerancewereperformedwiththeselectedisolates.Although38differentpopulationswereidentifiedandcharacterized,thevastmajorityoftheisolatesweregrampositivewithhighGCcontentsandwereaffiliatedwiththreedistinctgroups,namely,strainscloselyrelatedtothespeciesDietzianatrolimnae(32%oftheisolates),toFrigoribacterium/Clavibacterlineages(29%oftheisolates),andtothetypestrainofMicrobacteriumkitamiense(20%oftheisolates).OtherisolateswerephylogeneticallyrelatedtostrainsofthegeneraAgrococcus,Leifsonia,Kytococcus,Janibacter,Kocuria,Rothia,Nesterenkonia,Citrococcus,Micrococcus,Actinomyces,Rhodococcus,Bacillus,andStaphylococcus.Onlyfiveiso-latesweregramnegative:onewasrelatedtotheSphingobacterialineageandtheotherfourwererelatedtothe-Proteobacterialineage.DespitethepHoftheenvironment,thevastmajorityofthepopulationswerealkalitolerant,andonlytwostrainswereabletogrowatpH11.Inaworldwidesense,naturalalkalineenvironmentsareuncommon.Sodalakesandsodadesertsarethemostsystem-aticallystudiedoftheseandrepresentthemoststablenaturallyoccurringalkalineenvironmentsonearth,withpHvaluesgen-erallyhigherthan10andoccasionallyreaching12(18,19).Theformationoftheseenvironmentsisdependentonagroupofconditionsthatincludethetopologyandtheclimaticcharac-teristicsofaparticularlocationaswellasthegeologicalcom-positionoftheterrain.Conditionssuitablefortheformationofsodalakesarefoundinclosedhydricbasinswithalow-calcium,high-sodiumgeologicalcompositionlocatedinarid,semiarid,tropical,orsemitropicalareaswhereevaporativeconcentra-tionratesexceedthewaterinflowratessuchthatsaltsaccu-mulate.ThealkalinityisduetoashiftintheCO2/HCO3/CO32equilibriumtowardsapredominanceofCO32thatintheabsenceofCa2orMg2canremaininsolution(inthepresenceofthoseions,carbonatewillprecipitateasCaCO3orMgCO3).Therefore,theselakesarecharacterizedbyhighcon-centrationsofsodiumcarbonateandalsorepresentsaline,andsometimeshypersaline,environmentsduetotheconcomitantincreaseintheClconcentration(18,19).Themicrobialdiversityofthistypeofsalinelakeshasbeenstudiedprimarilybyfocusingontheisolationandcharacter-izationofindividualorganismswithpotentialindustrialappli-cations(15,16,18).ThephylogeneticdiversityofBacteriaandArchaeahasalsobeeninvestigated(8),andvariationsinthebacterialcommunitycompositionwiththedepthofalakewererecentlyanalyzed(17).Nonsalinealkalineenvironmentsaremuchmorerare;theirgenesisisdependentoncomplexgeologicalcontexts,buttheyareprobablyallrelatedtoasinglegeochemicalprocessknownasserpentinization.Briefly,thisprocesscanbeenvisagedastheweathering,byCO2-chargedwaters,ofsilicatemineralspresentinmaficand/orultramaficrocks.Thisprocessisim-portantforthedevelopmentofthecharacteristicsofavarietyofenvironments,suchasgroundwaterassociatedwithkimber-liteformation,asintheLakeTimiskamingandKirklandLakekimberlitefieldsinCanada(30),andgroundwaterandsurfacewaterassociatedwithsomeophiolitesinnorthernCalifornia(33),theSemailinOman(2),theTroodosophiolicticcomplexinCyprus(3),andtheMaqarinsiteinJordan(1).Recently,serpentinizationhasbeenimplicatedintheformationofanewclassofseafloorhydrothermalsystemcomposedofvariablemixturesofcalcite,aragonite,andbrucite,knowastheLostCityField,whichisconsi