La lecture à portée de main
Découvre YouScribe en t'inscrivant gratuitement
Je m'inscrisDécouvre YouScribe en t'inscrivant gratuitement
Je m'inscrisDescription
Sujets
Informations
Publié par | universitat_bremen |
Publié le | 01 janvier 2009 |
Nombre de lectures | 37 |
Langue | English |
Poids de l'ouvrage | 6 Mo |
Extrait
obicAnaer
p
degradation
-xylene
in
of
freshwater
cultures
Amelia
uRotar
2009
limonene
and
hmentenric
Anaerobicdegradationoflimoneneandp-xylene
cultureshmentenricfresh-water
tationDisser
zur
ErlangungdesGradeseines
derDoktorsNaturwissenschaften
nat..rer.Dr
Biologie/ChemieachbereichF
UnivderBremenersität
novorgelegtv
uRotarAmelia-Elena
aus
alceaVRamnicu
Romania
2009Bremen,
in
3
4
DieUntersuchungenzurvorliegendenDoktorarbeit
rineMikrobiologieinBremendurchgeführt.
hterGutachterGutac
1:2:
.rD.ProfJensPD
wurden
am
FriedrichWiddel,UniversitätBremen
Harder,MaxPlanckInstitutfürMarine
k-InstitutMax-Planc
Mikrobiologie
für
Ma-
Aceasta
dragoste
car
si
te
este
dedicata
,incredere
de-a
par
intilor
lungul
mei
acestei
care
m-au
„calator
ii“
in
utsustin
cu
necunoscut.
5
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
sexAnne
wledgmentsknoAc
83
85
78
113
129
151
153
viationsabbreofList
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