TurkJBiol 26(2002)181-191 ©TÜB‹TAK AlkaliphilicMicro-organismsandHabitats ZeynepULUKANLI KafkasUniversity,FacultyofArtsandScience,DepartmentofBiology,Kars-TURKEY MetinDI⁄RAK KahramanmaraflSütçü‹mamUniversity,FacultyofArtsandScience,DepartmentofBiology,Kahramanmarafl-TURKEY Received:25.06.2001 Abstract: Alkalineenvironmentsaretypicalextremeenvironmentswhichincludenaturallyoccurringsodalakes,deserts,soilsand artificiallyoccurringindustrial-derivedwaters.Micro-organismsthatoccupyextremepHenvironmentshaveresultedinthedefinition ofanunusualgroup,termedalkaliphiles.Inthisreview,thecurrentstatusofthebiodiversityofalkaliphilicmicro-organism sin variousenvironmentsandaspectsoftheirbiotechnologicalpotentialaresummarisedbriefly. KeyWords: Alkaliphiles,extremeenvironments,sodalakes,extremophiles,microbialecology AlkalifilikMikroorganizmalarveHabitatlar› Özet: Alkaliçevreler,geniflflekildesodagölleri,çölleri,topraklargibido¤alformlarsonucuolufltu¤ugibiçeflitliendüstrilerson ucu daoluflmaktad›r.EkstrempHözelli¤igösterençevrelerdeyaflayanmikroorganizmalarAlkalifillerolaraktan›mlanmaktad›r.Bu derlemede,çeflitliçevrelerdeyaflayanalkalifilikmikroorganizmalar›ngünümüzdekibiyolojikçeflitlili¤ivebaz›biyoteknolojik potansiyelleriözetlenmektedir. AnahtarSözcükler: Alkalifiller,ekstremçevreler,sodagölleri,ekstremofiller,mikrobiyalekoloji Introduction AlkalineEnvironments Themostconcentratedandwidespreadoccurrences Themajorgoalofmicrobialecologyistounderstand oforganismsaregenerallyobservedin“moderate” microbialdiversityinnaturalhabitats;therefore, environments.Ithasalsobeenknownthatthereare knowledgeofbothmicro-organismsandhabitatsis “extreme”environmentsonearthwhichwerethoughtto essential.Hypersalinewaterscanbeclassifiedintotwo preventtheexistenceoflife(1).Inthesehabitats, groupings:thefirst,thalassohalinewatersderivedfrom environmentalconditionssuchaspH,temperatureand theevaporationofseawaterandthesecondis salinityconcentrationsareextremelyhighorlow. athalassohalinewaterslargelyderivedfromthesolution Extremeenvironmentsarepopulatedbygroupsof ofevaporativedeposits(2,3).Thedistributionand organismsthatarespecificallyadaptedtotheseparticular abundanceofaspecifictypeofextremeenvironmentsis conditionsandthesetypesofextrememicro-organisms veryimportantforthedegreeofspecialisationofthe areusuallyreferredtoasalkaliphiles,halophiles, biota.Ifonetypeofenvironmentiscommoninthe thermophilesandacidophiles,reflectingtheparticular biosphere,hasawidegeographicaldistributionand typeofextremeenvironmentwhichtheyinhabit(1).In possessesacertainconstancyinitscharacteristics thisreview,theconditionofhighpH,whichoccursin throughoutgeologicalperiods,onecanpresumethata naturenaturallyorartificially,isthebasisoftheextreme longandcomplexevolutionaryprocesscouldhavetaken environmentwhichwillbeconsidered,andsuch place.Inthecaseofhypersalinity,itisclearthatthe environmentsarereferredtoasalkalineenvironments. aboveconsiderationsapplytothalassohalinewaters. Thedatapresentedinthisshortreviewarenotintended Alkaline-carbonatelakesalsopossiblyconformtothese tobeexhaustive,butseektogiveanindicationofthe conditions(3).Indescribingthedistributionand complexityanddiversityofalkalinebiotopes. abundanceofalkaliphilicmicro-organisms,itisessential 181 AlkaliphilicMicro-organismsandHabitats - toconsidertheiroriginunderwhatconditionsandhow mustbeabsentsothatgroundwaterscontainingHCO 3 - thealkaline-typebodiesarose. areproducedwherethemolarconcentrationsofHCO 3 - 2+ 2+ Therearetwokindsofnaturallyoccurringstable /CO3 greatlyexceedthoseofCa andMg .Through alkalineenvironmentsintheworld.First,highCa 2+ evaporativeconcentration,suchwatersrapidlyachieve environments(groundwatersbearinghighCaOH)and saturationwithrespecttoalkalineearthcationswhich + - 2+ precipitateasinsolublecarbonates,leavingNa ,Cl and second,lowCa environments(sodalakesanddeserts - - aredominatedbysodiumcarbonate)(4,5). HCO3 /CO3 asthemajorionsinsolution(4,7,8,10). - 2- GroundwatersbearingCa 2+ havebeenidentifiedin AlkalinitydevelopsduetoashiftintheCO 2/HCO3 /CO3 → - ↑ variouspartsoftheworld.Theseincludelocationsin equilibriumas:2HCO 3 CO2 +CO 2 +2H 2O.Alkalinity California,Oman,theformerYugoslavia,Cyprus,Jordan evolvesconcomitantwiththeprecipitationofotherions, + - andTurkey(6,7).ThegenesisofalkalinityinhighCa 2+ especiallyNa andCl ,leadingtothedevelopmentof springs,whichisextremelycomplexanddependentona alkalineandsalineconditions.Therelativesalinityofany particulargeologicalprocess,isproducedasaresultof lakeisdependentonthelocalgeologicandclimatic lowtemperatureweatheringofsilicateminerals conditions,resultinginsaline,alkalinelakes.Inlakesof 2- lowersalinity,theconcentrationofCO 3 usuallyexceeds containingcalciumandmagnesiumolivine(MgFeSiO 4) - - andpyroxene(MgCaFeSiO ).Thesesilicatescontaining thatofCl ,butinbrinesofhighersalinityCl exceeds 3 2- CO3 concentrations(7,8,10).LakeMagadi,WadiNatrun calciumandmagnesiumdecomposeonexposuretoCO 2 chargedsurfacewaters,releasingCa 2+ andOH - into andtheDeadSeaareexamplesofathalassohalinelakes solution.Duringthisprocess,Mg 2+ isremovedfrom worldwide.Itisnotthepurposeofthisreviewtodiscuss atlengththegenesisofhypersalineenvironmentsandthe solutionbyimmobilisationasserpentine(Mg3Si2O5(OH)4), readerisreferredtoarticlesbyEugsterandHardie(11) orbyprecipitationasbrucite(Mg(OH) 2),magnesite andHardie(12). (MgCO3),ordolomite(MgCa(CO 3)2).CO 3 isfurther ⇔ 2+ removedascalcite(CaCO3),leadingtoaCa(OH)2 Ca Diverseindustralactivitiesincludingfoodprocessing +OH - equilibrium,producinganextremelyalkaline (KOHmediatedremovalofpotatoskins),cement environmentaroundpH11.Theprocessknownas manufacture(orcasting),alkalineelectroplating,leather serpentinisationalsoproducesahighlyreducingcondition tanning,paperandboardmanufacture,indigo duetothereleaseofFe 2+.Hydrogengasisalsoevolved fermentationandrayonmanufacture,andherbicide bytheoxidationofH 2Obytransientmetalhydroxides manufacturegenerateanthropogenicsourcesofalkaline (7,8). typeenvironments(13-16). Sodalakesandsodadesertsrepresentthemost Types,CultivationandPreservation stable,naturallyoccurringalkalineenvironmentsfound Alkaline-adaptedmicro-organismscanbeclassified worldwide.Theseenvironmentsarecharacterisedbyhigh intotwomaingroupings,alkaliphiles(alsocalled concentrationsofNa 2CO3 (usuallyasNa 2CO3.10H2Oor alkalophiles)andalkalitolerants.Thetermalkaliphiles Na2CO3.NaHCO3.2H2O).Thedistinguishingfeatureof (alcali fromArabic,sodaash, phile,loving)isgenerally 2+ sodalakesisdepletedMg andthepresenceofthis restrictedtothosemicro-organismsthatactuallyrequire carbonateprovidesbufferingcapacitytothelakewaters alkalinemediaforgrowth.Theoptimumgrowthrateof (9).Theformationofalkalinityinthesodalake thesemicro-organismsisobservedinatleasttwopH environmentrequiresacombinationofgeographical, unitsaboveneutrality.Organismscapableofgrowingat topographicalandclimaticconditions:firstly,the pHvaluesmorethan9or10,butwithoptimumgrowth presenceofgeologicalconditionswhichfavourthe ratesataroundneutralityorless,arereferredtoas formationofalkalinedrainagewaters;secondly,suitable alkalitolerant(7,17,18).Inthemediausedtoisolate topographywhichrestrictssurfaceoutflowfromthe alkaliphilicbacteria,asamplecouldbeenrichedwith drainagebasin;andthirdly,climaticconditionsconducive differentsubstratessuchaspeptones,glucose,oxbile, toevaporativeconcentration.Suchconditionsarefound casaminoacidsandcaseine(19).ThepHofthesmall- inaridandsemi-aridzonesoftropicalorsubtropical scaleculturesgrowninmediaiscontrolledbyNa 2CO3 or areas.Avitalconditionnecessaryfortheformationofa anequivalentamountofNa 2CO3.10H2O(maintainingthe 2+ 2+ sodalakeisthatsignificantamountsofCa andMg pHvaluesat10-11),and/orBorax/NaOH,Na2HPO4/NaOH 182 Z.ULUKANLI,M.DI⁄RAK buffersystems(bufferingcapacityovertherangeofpH (10-15%w/v)hasbeenusedinsomelaboratories.Inthis 9-12invariousmedia).Buffersystemsascomparedwith methodtheuseoffrozensuspensionwitha Na2CO3 orNa 2CO3.10H2Oarelessaffectedby cryoprotectantonglassbeadsat–76ºCallowsindividual atmosphericCO2.ThepHoflarge-scaleculturesgrownin beadstoberemovedwithoutthawingthecultures bioreactorsisusuallycontrolledbyanNaOHsystem.This completelyforrecovery(26).Foralkaliphiles,themethod necessitatestheuseofapHcontrolsystemtopreventpH canbesuccessfullyusedinlaboratorieswithlimited changesandkeepthepHover11(17).Alkaliphilic equipment. strains’liquidculturesmaybepreparedwithoutany MicrobialEcologyofAlkalineEnvironments specialprecautions,sincethehighpHofthealkaliphile mediumappearstohaveanadverseeffectonthe NaturallyoccurringenvironmentswithextremepH constituentsofliquidmedia.Whenpreparingsolidmedia valueswhichsupportmicrobialgrowtharewidely distributed.Often,organismsgrowinginthese usingagaritisessentialtoautoclavetheNa 2CO3 separatelyfromtheagar,otherwisetheagarwilldarken environmentsexperiencefarmoreneutralpHvaluesthan andnotsolidifyproperly.Inpreparingthealkalinemedia theaveragevalueoftheirecosystemowingtothenature withoutNa2CO3 careshouldbetakenthatthepHofthe oftheirmicroenvironment.Sodalakesareprobablythe carbonate-freemediumisnottooacidbeforeautoclaving mostproductivenaturallyoccurringenvironmentsinthe theagar,otherwisetheagarisalsoadverselyaffected. worldwithmeangrossprimaryproductivitiesonaverage Thesamplesandculturesshouldbeincubatedat37ºC. beingatleastanorderofmagnitudeabovethatofan Althoughtherearesomestrainswhichrequirelower averageaquaticenvironment.Thisremarkable growthtemperatures,theoptimumofthemajorityofthe productivityispresumablyaconsequenceofrelatively otherstrainsisabove40ºC.Theenrichedsamplesshould highsurfacetemperatures(30-45ºC),highlight thenbetransferredtoagarmedia.Inthelaboratory,