Anaerobic degradation of limonene and p-xylene in freshwater enrichment cultures [Elektronische Ressource] / vorgelegt von Amelia-Elena Rotaru
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Anaerobic degradation of limonene and p-xylene in freshwater enrichment cultures [Elektronische Ressource] / vorgelegt von Amelia-Elena Rotaru

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Anaerobic degradation of limonene andp-xylene in freshwater enrichmentculturesAmelia Rotaru2009Anaerobic degradation of limonene and p-xylene infresh-water enrichment culturesDissertationzurErlangung des Grades einesDoktors der NaturwissenschaftenDr. rer. nat.Fachbereich Biologie/Chemieder Universität Bremenvorgelegt vonAmelia-Elena RotaruausRamnicu ValceaRomaniaBremen, 20093Die Untersuchungen zur vorliegenden Doktorarbeit wurden am Max-Planck-Institut für Ma-rine Mikrobiologie in Bremen durchgeführt.Gutachter 1: Prof. Dr. Friedrich Widdel, Universität BremenGutachter 2: PD Jens Harder, Max Planck Institut für Marine Mikrobiologie4Aceasta carte este dedicata parintilor mei care m-au sustinut cudragoste si incredere, de-a lungul acestei „calatorii“ in necunoscut.56ContentsI Anaerobic aliphatic hydrocarbon degradation 151 Anaerobic degradation of saturated aliphatic hydrocarbons 172 Anaerobic of unsaturated aliphatic hydrocarbons 213 Anaerobic limonene degradation: results and discussions 253.1 Scope of the study .................................253.2 Degradation of limonene under methanogenic conditions ...........263.3 Microbial community composition . . .......................303.4 Isolation of novel Deltaproteobacteria ......................373.5 Conclusions and outlook..............................

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Informations

Publié par
Publié le 01 janvier 2009
Nombre de lectures 37
Langue English
Poids de l'ouvrage 6 Mo

Extrait

obicAnaer

p

degradation

-xylene

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Amelia

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2009

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6

Contents

IAnaerobicaliphatichydrocarbondegradation
1Anaerobicdegradationofsaturatedaliphatichydrocarbons
2Anaerobicdegradationofunsaturatedaliphatichydrocarbons
3Anaerobiclimonenedegradation:resultsanddiscussions
3.1Scopeofthestudy.................................
3.2Degradationoflimoneneundermethanogenicconditions...........
3.3Microbialcommunitycomposition.........................
3.4IsolationofnovelDeltaproteobacteria......................
..............................outlookandConclusions3.5erencesRef4

15172125526203739341

IIAnaerobicaromatichydrocarbondegradation49
5Monoaromatichydrocarbondegradation51
6Polyaromatichydrocarbondegradation59
7Anaerobicp-xylenedegradation:resultsanddiscussions61
7.1Scopeofthestudy.................................61
7.2Cultivationofp-xylenedegradingmicroorganismsunderdenitrifyingconditions62
7.2.1Enrichment.................................62
7.2.2Isolationattempts.............................62
7.2.3Growthtestsonotherhydrocarbons...................63
7.3DominanceofnovelBetaproteobacteriainp-xylenedegradingenrichment
36.......................................cultures7.4Quantificationofp-xylenedegradation......................66
7.5Mechanismofp-Xyleneactivation........................67
7.6Conclusionsandoutlook..............................70
73erencesRef8

7

Contents

9

III

10

11

12

13

14

8

utioncontribofExplanation

uscriptsMan

1uscriptMan

2uscriptMan

3uscriptMan

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BssAα-subunitofbenzylsuccinatesynthase

bssAgeneencodingtheα-subunitofbenzylsuccinatesynthase

BTEXbenzene,toluene,ethylbenzene,xylenes

CARD-FISHcatalyzedreporterdeposition-fluorescenceinsituhybridization

APID

0’GΔ

FISH

GC

HAP

RNA

rRNA

(4’,6-diamidino)ylindol2-phen

7.00)pHatm,1,C(25°conditionsstandardunderenergy)(freeGdelta

fluorescentinsituhybridization

yaphrchromatoggas

ydrocarbonsharomaticpoly

ucleicibonracid

RNAibosomalr

SDS-PAGEsodiumdodecylsulfate-polyacrylamidegelelectrophoresis

SR

SRB

TEA

atesulfreduction

iabacterreducingatesulf

electronminalteracceptor

9

Contents

10

ySummar

Theanaerobicdegradationofhydrocarbonshasbeenintensivelyexploredinthelastdecade
yieldinginsightsintonewphysiologicalcapabilitiesandbiochemicalpathways.However,for
afewhydrocarbons,e.g.p-xylene,itprovedtobemoredifficulttoenrichmicroorganisms
andtoisolatepurestrains.Thermodynamically,themineralizationofhydrocarbonsisleast
favorableunderanoxicconditionsandespeciallyundermethanogenicconditions.Inmy
thesis,twoenrichmentcultureswerecharacterized,amethanogenicfreshwaterenrichment
culturegrownonlimonene,themostabundantmonoterpeneinnature,andadenitrifying
freshwaterenrichmentculturegrownonp-xylene.
Themethanogenicenrichmentcultureconsumedlimonenewithproportionalformationof
methane.Thefullcycle16SrRNAgeneapproachrevealedthepresenceofArchaearelated
toMethanosaetaandMethanoculleusandBacteriarelatedtoSyntrophobacteraceae,Bac-
teroidetes,andtheCandidateDivisionOP3.ThiscandidatephylumlieswithinthePlancto-
mycetes,Chlamydiae,Verrucomicrobia,Lentisphaeraesuperphylumandhasnomember
inculture.HencenucleicacidprobesweredevelopedtotargettheOP3-phylotype.The
probedetectedverysmallsphericalcells,whichlivedaloneorattachedtolargercellsand
represented18%ofthetotalDAPI-stainedpopulation.Thustheymayplayanimportant
roleinlimonenedegradation.TheotherBacteriaweremainlyDeltaproteobacteria(13%),
andonly1%wereBacteroidetes.TogetherwithanewEUB-338probespecificfortheOP3
cells,thebacterial338probemixturedetected40%ofthetotalcellswhereastheArchaea
probedetected33%.Thepresenceofseveralphylotypessuggeststhatmorethenonesyn-
trophicbacteriumandonemethanogenicarchaeonareinvolvedinlimonenedegradation
tomethanegasandcarbondioxide.
Syntrophicbacteriawereisolatedusingfumarateandlactateasorganicsubstratesfor
fermentation.AllsevenstrainsbelongedphylogeneticallytoDeltaproteobacteria,with11%
16SrRNAgenedissimilaritytoDesulfoarculusbaarsii.Initialco-cultureexperimentswith
Methanosarcinamazeishowedgrowthonlactateorfumarateinthepresenceoflimonene.
However,theirroleinlimonenedegradationcouldnotdemonstrated.
Denitrifyingenrichmentcultureswereestablishedwithafreshwatersedimentmixand
p-xyleneassoleelectrondonorandcarbonsource.Severalbatchtransfersandliquid
dilution-to-extinctionseriesenrichedacurvedrodmorphotype,0.5×2μminsize.This
dominantmorphotype(96%ofallcells)wasaswellidentifiedasthedominantphylotype
(91-95%)usingthe16SrRNAfullcycleapproach.Theorganismaffiliatedphylogeneti-
callywithBetaproteobacteriaandnotwithotheranaerobichydrocarbondegradingmicroor-
ganismsfromtheAzoarcus-Thaueraclade.Theirclosestrelativewasagroupofsteroid-

11

Contents

degraders:Denitratisomaoestradiolicum,Sterolibacteriumdenitrificansandstrain72Chol.
Thisenrichmentculturescoupledcompletemineralizationofp-xylenetodenitrificationof
nitratetodinitrogengas.Bygaschromatographymassspectrometricanalysisofmetabo-
litesfoundincellextracts,(4-methylbenzyl)succinateand(4-methylbenzyl)itaconatewere
identified,supportinganactivationmechanismbyadditiontofumarate.Agenefragmentfor
abenzylsuccinylsynthasecouldbesequenced,andrevealedaminoacidsimilaritieswith
TutDfromthewellknowntoluenedegradingdenitrifier,Thaueraaromatica.
Thisstudyhasrevealedthatanaerobichydrocarbondegradationinvolvesabroaddiver-
sityofmicroorganism,evenoutsidethephylumProteobacteria.Mythesisestablishedthe
participationofnovelorganismstoanaerobichydrocarbondegradationandrevealedforthe
firsttimethemorphologyofCandidatephylumOP3cells.

12

Introductoryremarksonanaerobic
degradation

Deepsedimentlayersoflakes,riversandsoils,aswellaquifersandundergroundwaters,
hostorganismscapabletodegradeorganicmatter.Inthesehabitatsoxygenislimiting,
soanaerobiclifeistheonlyalternative.Today,twotypesareknown,fermentationand
respirationofalternativeelectronacceptors.Nitrate,metals(Fe3+orMn4+),sulfateor
carbondioxidearethemajorelectronacceptorsrespiredbymicroorganisms.
InmythesisIwillfocusontwodifferentanaerobicrespiratoryprocesses,havingastermi-
nalelectronacceptors1)nitrateand2)carbondioxide.Inbothcasesthemicroorganisms
gainedenergybydegradationofhydrocarbons.
Hydrocarbonsareanubiquitousclassofnaturalcompoundsthatconsistsolelyofhy-
drogenandcarbonatoms.Therearetwomajorcategoriesofhydrocarbons,aliphaticand
aromatic.Aliphatichydrocarbonsaresaturated(alkanes)andunsaturated(alkenesand
alkynes),linear(e.g.n-alkanes),branchedorcyclic,whereasaromatichydrocarbonsare
characterizedbythepresenceofoneormorebenzenerings(mono-orpolycyclic)which
canbesubstitutedbyaliphaticalkylgroups(e.g.alkylbenzenes).Somehydrocarbons
areproducedbyplants(CheesbroughandKolattukudy,1984;Singeretal.,2003),animals
(HylemonandHarder,1999),insects(Pageetal.,1997),andmicroorganisms(Birchand
Bachofen,1988;HarderandFoss,1999).Saturatedaliphaticandaromatichydrocarbons
arealsoformedatveryhightemperaturesandpressures,afterdepositionandsedimenta-
tionofdeadorganicmatter.Thesearethemainconstituentsofoilandnaturalgas(Tissot
andWelte,1984).Besides,numeroushydrocarbonsareextensivelyproduced,orrefinedin
industryfordifferentpurposes.Becausehydrocarbonsarewidespreadintheenvironment,
themicrobesdegradingthemareexpectedtobeasubiquitous(SpormannandWiddel,
2000).Inthelastthreedecades,anaerobicmicrobescapableofhydrocarbondegradationwere
studiedextensively(Widdeletal.,2006)especiallytheonesmediating„useful“processes.
Bioremediationofpetroleumpollutedenvironmentsorobtainingnovelindustrialbiocata-
lystsaresomeexamples.Beyondtheapplicationsofanaerobichydrocarbondegradation,
theultimategoalistoidentifyunusualenzymesandreactionscatalyzedbymicrobes,to
theirdiscovenerhowvironment,interandactionswhatareroleestablishedanaerobicbetwheenydrocarbonorganismsdegrandadersbetwplayeenintheorganismsglobalcar-and
.cyclebon

13

Contents

14

obicAnaer

trPa

I

aliphatic

ydrhocarbon

degradation

15

1Anaerobicdegradationofsaturated
ocarbonsydrhaliphatic

Saturatedaliphatichydrocarbonsoralkanesarenonreactivehydrocarbonswhichconta

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