carbon cycling by cellulose-fermenting nitrogen-fixing bacteria
TRANSCRIPT
Adv. Space Res. VoL 9, No. 8. pp (8)149—(8)152. 1989 0273—1177/89 S0.0O + .50Printedin Great Britain. All rights reserved. Copyright© 1989 COSPAR
CARBON CYCLING BY CELLULOSE-FERMENTINGNITROGEN-FIXINGBACTERIA
S. B. LeschineandE. Canale-Parola
DepartmentofMicrobiology, UniversityofMassachusetts,Amherst,MA 01003,U.S.A.
ABSTRACF
Themost abundantorganicmaterialson Earth areplant polysaccharidessuchas celluloseandhemicelluloses.Inasmuchasvastquantitiesof thesepolymersarepresentin anaerobicenvironments(e.g.,in soilsandsediments),anaerobicmicroorganismsthat fermentplant polysaccharidesplay acentralrole in carboncycling on theplanetasasourceof CO2 and,indirectly,of CH4. Cellulose-fermentingbacteriafromsoil andpondsedimentwereisolatedinamedium(incubatedin N2) whichlackedasourceof combinednitrogen. The isolateshadtheability to utilizeatmosphericN2 asthenitrogensourcefor cell growth. Nitrogenase(theenzymewhichcatalyzesthereductionofN2to ammonia)wasdemonstratedby meansoftheacetylenereductiontestin theseisolatesandin severalprevious-ly describedanaerobiccellulolytic bacteriaisolatedfromvariousnaturalenvironments.Thus,cellulose-fermentingbacteriathat fix N2 maybewidespreadandmayplay arole in nitrogencycling aswell asin carboncycling on aglobalscale. Knowledgeof thephysiologyandecologyof theseorganismsis crucial to detailingthemechanismsproducinglocal sourcesandsinksof atmosphericgases,interpretingdataobtainedusingspace-basedsensors,andunderstandingtheeffectsof atmosphericwarmingonfermentationsasmajorsourcesof CO2andCH4.
INTRODUCTION
Key processesof biogeochemicalcyclesaremediatedby microorganisms.For example,animportantstepin theglobal carboncycleis the microbialdegradationof cellulose,themost abundantorganicmaterialon Earth/1/.Photosynthesisyieldsannuallyup to 1.5x 1011 tonsof dryplantmaterialworldwide,almosthalfof whichconsistsof cellulose/2/. For life to continueon Earth,thecarbonpresentin thispolymermust bereintroducedinto theatmospherein theformof CO2.
A substantialamountof cellulose(5-10%)is degradedin anaerobicenvironments/2/.Anaerobicactivity occursinproximity to thesurfacein soils,composts,andfreshwater,marine,andestuarinesediments,indicating thataerobicconditionsnormallyprevailonly in athin crust/2/. In anaerobicenvironments,celluloseis initially decomposedbycellulose-fermentingmicroorganismsyielding CO2.H2, organicacids,andethanol. Someproductsof cellulosefermentationserveasgrowthsubstratesfor otherbacteriawhichproduceacetate,C02,andH2. Theseproductsarethenconvertedto CH4by methanogenicbacteria.Thus,asasourceofCO2 and,indirectly,of CH4, anaerobiccellu-lolytic microorganismsplayamajorrolein carboncyclingon theplanet(Fig. 1). Althoughthemicrobiotainvolveddirectly andindirectly in cellulosedegradationin therumenhasbeenstudiedextensively/3,4/, relativelylittle isknownaboutthecomplexinteractionsamongfree-livingmicroorganismsinvolvedin theanaerobicdegradationofcellulosein othernaturalhabitats/2/. Thedistributionof anaerobiccellulolyticbacteriain natureis largelyunknown.
Dueprimarily to recentinterestin thepotentialuseofcellulolytic bacteriafor alcoholfuelproductionfrombiomass,severalspeciesof anaerobiccellulolytic bacteriafrom sediments,compost,andsewagesludgehavebeende-scribed/5-11/. Thelong-termgoalof ourresearchis to advanceunderstandingof thephysiologyandecologyofthesemicroorganisms.Mostly, wehavestudiedstrainsof aClostridium species(referredto as“C strains”)thatweisolatedfrom the sedimentof afreshwaterswamp. Thesestrainsactively fermentnot only cellulosebut alsocomponentsofthehemicellulosicportion ofbiomass(e.g.,xylan,pentoses),formingprimarily ethanol,aceticacid,H2, andCO2/5/. Informationobtainedfrom thesestudiesmayhelpus predicttheresponseof cellulose-fermentingbacteriato currentglobalwarmingandprobablealteredprecipitationpatterns.For example,if anaerobiccellulose
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(8)150 S. B. LeschineandE. Canale-Parola
degradationis enhancedandtherateof CO2 andCH4 productionis increased,therewill be apositive feedbackontherateofclimatechangeby this microbialactivity /12,13/.
Environmentsrichin cellulosearefrequentlydeficientin nitrogen(e.g.,peatsoils, agriculturalwastes,composts).Thus,cellulose-fermentingbacteriathatsatisfytheir nitrogenrequirementsthroughthefixation of N2 would beex-pectedto haveastrongselectiveadvantageoverthosewhichrequireasourceof combinednitrogen.Moreover,suchmicroorganismsmight beexpectedto playamajorrolein nitrogencycling,aswell asin carboncycling,on aglobalscale. However,it hasnot beendeterminedwhethercelluloseis widely usedasan energysourceby nitrogen-fixingbacteria.This is surprisinggiventhe abundanceof celluloseand the suggestion/14/that nitrogen fixation by free-living heterotrophicbacteria in naturalecosystemsis limited by the availability of oxidizablegrowth substrates.Waterburyand coworkers/15/haveshownthat celluloseservesasagrowthsubstratefor anitrogen-fixingaerobicbacteriumwhichexistsin asymbioticrelationshipwith shipworms.Their findingsdemonstratethat thesetwocom-plex physiologicalprocesses,nitrogenfixation and cellulosedegradation,canbe performedby asinglebacterium.Amajor objectiveof our researchwasto determinewhether free-living anaerobiccellulolytic bacteria that are wide-spread in terrestrialenvironmentsfix nitrogenwhen they utilize celluloseas the fermentable substrate for growth.
~
~ H 0’‘~‘2 ~ ~1?. ~
c~ C~f~
] T C0~CH4
6 12 6 x
1. Cellulose 4 OrganIc Acids • Ethanol• CO2 • H2
2. organic Acids. Ethanol 4 Acetate + CO2 + H2
~ fAcetate 4 CO2 + CH4
1,,C02.H2 4 CH4
CELLULOSE 4 Co2 + Cl!4Fig. 1. Roleof cellulose-fermentingbacteria in carboncycling. Cellulose [(C6Hi2O6)~],amajor product ofthephotosynthetic fixation ofCO2. is fermented by anaerobiccellulolytic bacteria(step 1). Ethanol andtheacidicproducts of cellulosefermentation serveas growthsubstratesfor otherbacteriawhich produceacetate,CO2.andH2(step2). MethanogenicbacteriaproduceCH4 from acetateorby reductionof CO2 with H2 (step3).In anaerobicenvironments,thecompletedissimilationofcelluloseresultsin theformationofCO2 andCH4.
ISOLATIONOF NITROGEN-FIXING CELLULOSE-FERMENTINGBACTERIA
Four strains of anaerobiccellulolytic bacteriawere isolatedfrom forest soil andfreshwatermud usingaprocedurethat selectedfor nitrogen-fixing strains/16/. Enrichmentcultureswereprepared by seriallydiluting soil or mudsamplesinto anaerobicculturetubescontainingaliquid growthmedium,designated“MW-C”. Thismedium(whichwas similar to one describedby Daeschand Mortenson /17/) lacked a source of combined nitrogen, includedcellulose(ball-milled WhatmanNo. 1 filter paper;0.6%,thywt/vol) asthefermentablesubstrate,wasprereduced/18/, andwasmaintainedin anN2 atmosphere.After7-14daysof incubationat 30°C,enrichmentculturesshowedsignificantdisappearanceof cellulose. Spent mediumand remainingcellulosefibersfromenrichmentcultureswereseriallydilutedinto meltedcellulosesoft agarmediumin tubes.Thecontentsof thesetubeswerepouredOntoplatesof agar medium within an anaerobic chamber. After 2-4weeksof incubation, coloniessurroundedby zonesofclearingappearedin theotherwiseopaquemedium. Thesecolonieswere transferredby streakingonto platesofcellobioseagarmedium, and restreakedseveraltimes to obtain pure cultures.Colonieswere then transferredintoliquid cellulosemedium (MW-C) to confirm whether the isolatewascellulolytic. Three strains (B1A, B1B, andB lC) were isolatedfrom mud from thebottomof ashallowpond (Beaver’sPond,Shutesbury,Massachusetts)andone (strain B3B) from forest soil nearBeaver’sPond.
CHARACTERIZATION OF NITROGEN-FIXINGCELLULOLYTIC ISOLATES
The isolatesresembledone anothermorphologically (Fig.2). All weremotilecurved rodsmeasuring0.6 to 0.8 jimx 3 to 6 jim. Under thegrowthconditions used,sporeswere never observedeither within cellsor free in culturesupematant fluids. Cellsof all four strainsstainedgram-negative.Electronmicroscopyof thin sectionsof strainBIA cells /16/showedthatthe cytoplasmicmembranewassurrounded by amultilayered cell wall thatdifferedfromtypicalcell wadsof mostgram-negativeanaerobicbacteria(eg.,thoseof mostBacreroidaceae/19/),but resembledthoseof other gram-negativemesophiic cellulolytic bacteria /5,20/. All isolateswereobligatelyanaerobicandfermentedpolysaccharides,hexoses,andpentosesthatarecommonly present in plantmaterials.Noneof thestrainsutilized maltose, glycerol, or amino acids as fermentablesubstrates. Further phenotypicand genotypiccharacterizationoftheisolatesis requiredto determinetheirtaxonomicposition.
CarbonCycling (S)151
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/ ~ l~t.• ~ ~ —
/ ~‘r~,’ ~j•;_” ~
‘.•, ~ ..~. ~
FIG. 2 A-C. Phase-contrastphotomicrographsof nitrogen-fixingcellulolytic isolates (wet-mountpre-parations),strains(A) BIA, (B)BIB, (C) B 1C,and(D) B3B. Cellswereculturedto late-exponentialphaseina cellobiose-containingmedium. Cells of strainsBIC (C) and B3B (D) areentangledin cellulosefibersintroducedinto theculturealongwith theinoculum(a7-dayculture in cellulosemedium).Scalebar,10 p.M.
Thepresenceof nitrogenasein the isolateswasdemonstratedby meansof the acetylenereductiontest/21/. Cellsreducedacetyleneto ethylene(e.g., 1310nmol/h/mgcell protein)when growingin mediumMW-C but not whengrowingin thesamemediumsupplementedwith 0.2%NH4C1. Inasmuchastheisolatesgrewin amediumlackingcombinednitrogenandcontainingcelluloseasthefermentablesubstrate,it wasconcluded/16/ thattheyutilized thispolysaccharideastheenergysourcefor N2fixation.
N1TROGENASEACTIVITY IN OTHERCELLULOSE-FERMENTINGBACTERIA
Severalpreviouslydescribedanaerobiccellulolytic species[Clostridium strainC7, from mud of afreshwaterswamp in Woods Hole, Massachusetts,U.S.A. /5/;Clostridium papyrosolvens,from estuarinesedimentsof theRiver Don in Aberdeenshire,Scotland/6/; strainJW-2, from wetwoodof an elm in Amherst,Massachusetts,U.S.A. 120/] were culturedin a defmed,cellulose-containingmedium/16/. Thepreviouslydescribedcellulolyticspecies,aswell asthenewisolates,reducedacetyleneto ethylenewhengrownin this medium,butnot whengrownin thesamemediumsupplementedwith NH4CI, aresultthat indicatedthepresenceof nitrogenasein thesebacteria.Moreover,growth in a cellobiose-containingmediumlacking combinednitrogenwasdependenton N2 /16/. Nogrowthoccurredwhencultureswereincubatedin anargonatmosphere.TheseobservationsindicatedthatN2 servedasthenitrogensourcefor thegrowingcells. Thus,cellulolytic bacteriafrom avarietyof environmentssynthesizedactive nitrogenaseandapparentlyincorporatedN2 into cell materialduringgrowth. Although othercellulolyticbacteriamaypossessnitrogenase/15,22/,this is the first demonstrationof nitrogenfixation duringthe anaerobicdegradationofcellulose. Ourfinding of nitrogenaseactivity in cellulose-fermentingbacteriaisolatedfrom avarietyof environments is consistentwith the view /16/thatnitrogen-fixingcellulolytic bacteriaarewidespreadin nature.
DISCUSSION
In the study describedabove,we did not attempt to enumeratenitrogen-fixing cellulolytic bacteriain specificterrestrialenvironments.Theresultsof suchexperiments,if performedusingclassicalmethodsinvolving viablecounts,probablywould not bemeaningfulbecausethe~bacteriaoftenadhereto or areentangledin cellulosefibers(e.g., Fig. 2C,D). Furthermore,the growthrequirementsof diversenitrogen-fixingcellulolytic bacteriaare notknown. Futurestudiesaimedat identifying cellulolytic speciesin their naturalenvironmentsandestimatingtheiractivitieswill beconductedwith theuseof oligonucleotideprobescomplementaryto ribosomalribonucleicacid(rRNA) sequencesuniqueto membersof groupsof cellulose-fermentingorganisms.Sincetheprobesarecomple-mentaryto rRNAs,andactively growingcellsmaycontainmore than l0~ribosomes/23/, eachapotentialprobetarget,singlecellsmaybelabeledandidentifiedby in situ hybridizationandmicroautoradiographicprocedures/24/.This methodpermitstheidentificationof singlecellsmicroscopically(cultivation is not required)andoffersaverypowerful tool to identifythepresenceofmembersof particularbacterialgroupsin naturalecosystems.Themetabolicactivity ofpopulationsof cellulolyticbacteriain naturalsamplesmaybeestimatedby measuringtheamountofprobeboundto bulk extractedrRNA. Sincethe rRNAcontentof cells is proportionalto growthrateoverawide rangeofgrowthrates/25/, theamountof group-specificprobehybridizedperunit of biomasswouldprovide anestimateofthe metabolicactivity of thatgroup/24,26/. Resultsof thesestudieswill contributeto ourunderstandingof thediversityof cellulose-fermentingnitrogen-fixingbacteria,their quantitativecontributionto cellulosebreakdownandnitrogenfixation, their potentialfor improvingsoil fertility, andtheeffectof atmosphericwarmingon their activity.
ACKNOWLEDGMENTS
We thankT.A. Warnickfor experttechnicalassistance.This work wassupportedby NationalScienceFoundationgrantBSR-8708469andby U. S. Departmentof EnergycontractDE-FGO2-88ER13898.
(3)152 S. B. Leschineand E. Canale-Parola
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