Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices. - Portal Embrapa

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Genetics

and

Molecular

Microbiology

Evaluation

of

bacterial

diversity

recovered

from

petroleum

samples

using

different

physical

matrices

Bruna

Martins

Dellagnezze

_,

_Suzan

_Pantaroto

_de

_Vasconcellos

_,

Itamar

Soares

de

Melo

_,

_Eugênio

_Vaz

_dos

_Santos

_Neto

_,

_Valéria

_Maia

_de

_Oliveira

a_{DivisionofMicrobialResources,ResearchCenterforChemistry,Biology,Agriculture(CPQBA),UniversityofCampinas–UNICAMP,}

Campinas,SP,Brazil

b_{FederalUniversityofSãoPaulo(UNIFESP),DepartmentofBiologicalSciences,Diadema,SP,Brazil}

c_{EMBRAPA,NationalCenterforResearchonMonitoringandEvaluatingEnvironmentalImpact,Jaguariúna,SP,Brazil}

d_{PETROBRASRandDCenter,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23February2015 Accepted24November2015 Availableonline22April2016 AssociateEditor:LucySeldin

Keywords: Bacterialdiversity Microbialenrichment Petroleumreservoir Physicalsupport 16SrRNA

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b

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Unravelingthemicrobialdiversityanditscomplexityinpetroleumreservoirenvironments hasbeenachallengethroughouttheyears.Despitethetechniquesdevelopedinorderto improvemethodologiesinvolvingDNAextractionfromcrudeoil,microbialenrichments usingdifferentculture conditionscanbe appliedasa way toincrease therecoveryof DNAfromenvironmentswith lowcellular densityforfurther microbiologicalanalyses. Thisworkaimedattheevaluationofdifferentmatrices(arenite,shaleandpolyurethane foam)assupportmaterialsformicrobialgrowthandbiofilmformationinenrichmentsusing abiodegradedpetroleumsampleasinoculuminsulfatereducingcondition.Subsequent microbialdiversitycharacterizationwascarriedoutusingScanningElectronicMicroscopy (SEM),DenaturingGradientGelElectrophoresis(DGGE)and16SrRNAgenelibrariesinorder tocomparethemicrobialbiomassyield,DNArecoveryefficiencyanddiversityamongthe enrichments.TheDNAfrommicrobialcommunitiesinpetroleumenrichmentswaspurified accordingtoaprotocolestablishedinthisworkandusedfor16SrRNAamplificationwith bacterialgenericprimers.ThePCRproductswerecloned,andpositivecloneswerescreened byAmplifiedRibosomalDNARestrictionAnalysis(ARDRA).Sequencingandphylogenetic analysesrevealedthatthebacterialcommunitywasmostlyrepresentedbymembersofthe generaPetrotoga,Bacillus,Pseudomonas,GeobacillusandRahnella.Theuseofdifferentsupport materialsintheenrichmentsyieldedanincreaseinmicrobialbiomassandbiofilm forma-tion,indicatingthatthesematerialsmaybeemployedforefficientbiomassrecoveryfrom petroleumreservoirsamples.Nonetheless,themostdiversemicrobiotawererecoveredfrom thebiodegradedpetroleumsampleusingpolyurethanefoamcubesassupportmaterial.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:[email protected](B.M.Dellagnezze).

http://dx.doi.org/10.1016/j.bjm.2016.04.004

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Crudeoilbiodegradationinpetroleumreservoirsaffectsthe world productionof fuels, making the recovery and refin-ingprocessesmoreexpensive.Formanyyears,theprevalent occurrenceof biodegradation in petroleum wells has been attributedtotheaerobicbacterial degradationof hydrocar-bons, which can be stimulated by oxygen carried by the infiltrationofmeteoricwatersinthereservoir.1_{However,there} isstrongevidenceforthewidespreadoccurrenceofobligate anaerobesinsubsurfacepetroleumsystems,2–4_andthe flush-ingofmeteoricwaterdoesnotindicatethathighlyreactive oxygensurvivestransportationtodeepreservoirs,sinceeven smallconcentrationsoforganiccompoundscanremove oxy-genfromanaquifer.

Inrecentwork,researchershavesuggestedthat biodegra-dationprocessescanoccurattheoil–watertransitionzone, in which microbial life should be possible within water dropletscontainingactivemicrobialcommunities.5_Data gath-ered from several studies indicate that oil biodegradation in deep subsurface petroleum reservoirs occurs through anaerobicmicrobialmetabolismratherthanaerobic mecha-nisms,resultinginadecreaseoflighthydrocarbonsandan increaseofoildensity,acidity,viscosityandsulfurcontent.6–8 Inaddition, viableanaerobic hydrocarbon degradation pro-cesseshaverecentlybeenestablishedforbothsaturatedand aromatichydrocarbons.9–11_{Studiesaimingtoevaluate} inter-mediatemetabolites,characteristicofanaerobichydrocarbon degradation,havebeencarried outand allowedthe identi-ficationofcompoundssuchasreduced2-naphthoicacids,12 2-methylnaphthalene,tetralin,aswellasnaphthoicacidsin petroleumreservoirs.13

Sulfate reduction and methanogenesis are the most likely processes responsible for in-reservoir hydrocarbon oxidation.14 _{Oil degradation linked to sulfate reduction} wouldexplaintheconsistenthydrocarboncompositional pat-ternsseen inmany degradedoilsworldwide.Sulfatearises from geological sources, suchas evaporitic sediments and limestone,orfromtheinjectionofseawaterforpressure sta-bilization, and may leadto significant oil degradation and increasedresidual-oilsulfurcontent.15_{Souringinoilfield} sys-temsismostcommonlyduetotheactionofsulfate-reducing prokaryotes,adiversegroupofanaerobicmicroorganismsthat respire sulfateand produce sulfide(the key souring agent) whileoxidizingdiverseelectrondonors.8

Inthissense,effortshavebeenmadebyseveralresearchers inordertorecoverandcharacterizetheanaerobicmicrobial community inhabiting the deep petroleum biosphere.2,16,17 Thestudyofgenomesofuncultivatedmicrobeshavebecome possible through metagenomics, a cultivation-independent approachthatallowstoexplorethemetabolicpotentialofthe unseenbiodiversitybycloninglargeDNAfragmentsdirectly isolatedfromtheenvironment.18_{Withtheuseofthe} metage-nomic approach, bacteria capable of degrading petroleum hydrocarbons,includinganaerobes, havebeen moredeeply investigatedandtheirmetabolicroutesunraveled.19,20 How-ever,theextremelylowamountofDNAobtainedfromsamples derived from petroleum reservoirs using direct nucleic acid extraction procedures is often a restraint when the

phylogeneticand/ormetabolicdiversityofmicrobial commu-nitiesareinvestigated,21,22_{becauseofthelowcellulardensity} and activity found in such hostile environment. Microbial enrichments using different cultureconditions, simulating the chemical and physical parameters of natural environ-ments,canbeappliedtoovercomethislimitationandincrease the recovery ofDNA from environments with low cellular density.23 _{Althoughcultivation underlaboratory conditions} candiminishthebiodiversityrecovered,thistechniqueallows theselectionofmicroorganismsthathavesomefunctionof particularinterest,suchasenzymaticactivityor biodegrada-tionability.24

This work aimed to evaluate the efficiency of different matrices, used asphysical supports, inrecovering anaero-bicbacterialdiversityfromabiodegradedoilsamplederived from a petroleum reservoir in Campos Basin (Brazil). The matrices were usedinorder toevaluatetheir effectin the increaseofbiomass,aswellasasupportforbiofilm forma-tion.Therelativeabundanceanddiversityoftheanaerobic microbiotarecoveredfrom theenrichmentswerecompared byusingmicroscopicandmolecularanalysis(DGGEand16S rRNAlibraries).

Material

and

methods

Sampling

PetroleumsampleswereobtainedinJuly2005fromfive pro-ductionwellsatthePampoPlatform,CamposBasin(Macaé, RJ, Brazil), withlogistic supportfrom CENPES/Petrobras, as described in details byVasconcellos et al.22 _Sampleswere collected intriplicateusing500mLsterilizedSchott bottles, which were completely filledwith the samplesinorder to avoidoxygeninflux.Sampleswerekeptoniceduring trans-portationtothelaboratoryandstoredatroomtemperaturefor subsequentanaerobicbacterialenrichmentassays.

Anaerobicenrichments

The biodegraded petroleum sample (P2) usedin this work as inoculum (10% v/v) for the anaerobic enrichments was collected from oilreservoir2inCamposBasin, RJ, Brazil.22 Thiswellwascharacterizedashighlybiodegraded,level5–6, according toPetersand Moldowan,25 _withaverage temper-ature71◦Cand approximately2000mdeep.Thepetroleum samplewashomogenizedinwaterbathat50◦C.The enrich-mentsweresettledinSchott bottles(1L)containing500mL ofZindermedium26 _{supplementedwith organicsubstrates} (sodium acetate, sodium formate, sodium lactate, yeast extract,methanol)tostimulatethegrowthofsulfate reduc-ingbacteria,accordingtomethodsdescribedbyDubourguier et al.27 _{andSilva etal.}28_{. Thecysteine–HClsolution (2mM)} was added to the enrichments (1%, v/v) as final electron acceptor.

Three different matrices were independently appliedas physical supportsto allow bacterial biofilm formation and increasebiomassrecoveryundersulfatereducingcondition: (1) polyurethanefoamcubes(PF) (1cm2_{), (2)slices ofshale}

(S), and (3) arenite (A). Bacterial enrichment using P2 as inoculumbutwithoutanymatrixwassettledascontrol(WS). Eachconditionwasevaluatedintriplicate.

ThepolyurethanefoamcubesweresubmittedtoUV steril-izationfor20minfollowedbyimmersioninautoclavedZinder medium,underanaerobiccondition(N2flow).Approximately

80cubeswereaddedtoeachflaskofmicrobial enrichment undercondition(1).Samplesofshaleandarenite,whichare natural components ofoil reservoirs, were kindly donated byDr.EugenioV.dosSantosNeto(CENPES/Petrobras).Shale wasslicedinsmallpieces,whilearenitewasgratedin pow-der.Bothmaterialswere autoclavedtwiceinSchott bottles (250mL)containing200gofslicesorpowder.After steriliza-tion,10gofeachmatrixwereinoculatedinthecorresponding microbialenrichments.

Allanaerobicenrichmentswereincubatedat55◦C,during 60days,inarotaryshakerat100rpm.

Microscopicanalysis

Scanningelectronicmicroscopy(SEM)wasusedtoevaluate theabundanceanddiversityofcellmorphologyofallbacterial enrichmentsunderstudy.Theanalysesweredevelopedwith aZeissmicroscopemodelLEO982,atEmbrapa/CNPMA,using protocolsdescribedbyMeloetal.29_.

DNAextraction

TheDNAextractionfromthebacterialenrichmentswas car-ried out using a protocol based on Gro␤kopf et al.30 _and Neria-Gonzálesetal.,31_{withadaptationstopetroleum} sam-ples. Firstly, 50mL of a sterilized Tween 80 solution (10%) (Sigma–Aldrich) were added into enrichments in order to promote homogenization of oil/water phases, as well as improvingtherecoveryofcellsadheredonthephysical sup-ports (foams, shale and arenite).Total enrichment volume was distributed in sterile tubes (50mL) and centrifuged at 10,000rpm,for20min,4◦C.Supernatantswerediscardedand cellsweretransferredtomicrotubes(2mL).Afterwards, micro-bialpelletsretrievedfromtheenrichments(3×500mL)were suspendedin600␮LPBSbuffer,homogenizedbyvortexand lysozyme was added atafinal concentration of17mg/mL. Afterincubationat37◦Cfor2h,proteinaseKandSDSwere added(finalconcentrationof0.7mg/mLand2%,respectively) andthesolutionwasincubatedat60◦Cfor90min.The micro-tubeswere submittedtothreefreeze–thawcycles (2minin liquidnitrogenfollowedby2minat65◦C).Glassbeadswere addedtothetubesandmanualagitationwasperformedfor 1min. Thesolutionwas extractedoncewith equalvolume ofsaturatedphenol(pH8.0)andoncewithequalvolumeof chloroform:isoamylalcohol(24:1).ForDNAprecipitation,5M NaCl(10%)and2volumesofcoldethanolwereaddedtothe solution.Thepelletwaswashedoncewithethanol70%,dried and suspendedinMilli-Q water.The yieldand integrityof theDNAobtainedwereconfirmedthroughNanoVuePlusTM

Spectrophotometer(GEHealthcare)andelectrophoresisin1% agarosegelstainedwithethidiumbromideanddocumented usingaUVPBioImagingSystemGDS-8000(UVP,Upland,CA, USA).

Constructionof16SrRNAgenelibraries,ARDRAand phylogeneticanalyses

Fortheconstructionofthe16SrRNAgenelibraries, amplifi-cationwasperformedfromtotalcommunityDNA,obtained fromeachenrichment,byusingthebacterialprimerset27f and1100r.32_{Onlyonelibrarywasassembledforeach} enrich-ment.Fiftymicroliter-reactionmixturesweremadecontained 50ngoftotalDNA,2UofTaqDNApolymerase(Invitrogen), 0.2mM ofdNTP mixand 0.4␮M ofeach primer,in 1X Taq

buffer.ThePCRamplificationswereperformedusing10cycles of1minat94◦C,30sat60◦C,decreasing 0.5◦Ceach cycle, and 3min at72◦C, followed byanother 10 cycles of 1min at 94◦C, 30s at 56◦C and 3min at 72◦C. Amplicons were pooled from five reactions (∼500ng), purified using GFXTM

PCR-DNA andgel band purification kit (GE Healthcare)and

cloned using the pGEM-T cloning vector kit, according to the manufacturer’s instructions(Promega, Madison,Wisc.). Insert-containingclonesweresubmittedtoARDRAby diges-tionofM13ampliconswiththeenzymesHaeIII,HhaIandMsp

I,independently,at37◦Cfor2.5h.Clonesrepresentingdistinct ribotypeswereselectedforDNAsequencingandphylogenetic affiliation.

The16SrRNAgenesequencesweredeterminedbydirect amplificationoftheinsertsfromovernightgrownclone cul-tureswithM13forwardandreverseprimersandsequencing

with the DYEnamic ET Dye Terminator Cycle Sequencing Kit

for the automated MegaBace 500 system (GE Healthcare) using the primers 10f, 1100r, 765f and 782r,32 _{according to} themanufacturer’srecommendations.Partial16SrRNAgene sequencesobtainedfromcloneswereassembledina contigu-ous sequenceusing the phred/Phrap/CONSED program.33,34 Phylogeneticaffiliationwasachievedasdescribedpreviously byVasconcellosetal.22_.

Thenucleotidesequencesdeterminedinthisstudywere depositedattheGenbankdatabaseundertheaccession num-bers:GenBankID:JN998802toJN998890.

DGGEanalyses

The PCR targeting 16S rDNA for the DGGE analyses was performed using the universal primers 968f (attached to a 40-nucleotide GC-rich sequence) and 1401r,35 _{which are} homologoustotheconservedbacterial16SrDNAregions.The PCR amplificationswere performed in 50␮Lreactions con-taining 50ng of total community DNA recovered from the microbialenrichments,5␮Lof10× Tris–HClreactionbuffer, 1.5mMMgCl2, 0.4␮Mprimers968f and1401r,0.2mMdNTP

mixand2UTaqDNAPolymerase(Invitrogen,GrandIsland, N.Y.,USA).ThePCRamplificationswereperformedusingan initialdenaturationstepof5minat94◦C,10cyclesof1min at94◦C,30sat58◦C,decreasing1◦Ceachcycle,and2minat 72◦C,followedbyanother25cyclesof1minat94◦C,30sat 53◦Cand2minat72◦C.Theampliconswerefirstcheckedon 1.2%agarosegelspriortotheDGGEanalyses.

TheDGGEanalyseswerecarriedoutintheD-Code Univer-salMutationDetectionSystem(Bio-Rad,USA)usingalinear denaturing gradient of urea and formamide ranging from 50%to70%(100%denaturantcorrespondingto7Mureaand 40% (v/v) deionized formamide). Gels (6% polyacrylamide)

Table1–AmountofDNAextractedfromanaerobic enrichmentsusingdifferentsupportsandwithout support.

Anaerobicenrichment DNAamount(ng/␮L)

Shale 15±1.52

Arenite 14±0.57

Polyurethanefoams 25±2.64

Withoutsupport 20±2.44

containing6␮LofPCRproductsforeachsample,intriplicate, wererunat50Vand60◦Cfor14hin0.5×TAEbuffer.Gelswere stainedwithSYBRGreen1×solutionanddocumentedunder UVlight.

Results

Microscopicanalysesofbacterialenrichments

Adensecellularbiomass(upto108cells/mL)was observed whenusingpolyurethanefoamsasmatricesinthe anaero-bicenrichmentsafter60daysofincubation.Theenrichments without physicalsupports exhibited low mediumturbidity (104_{cells/mL)whencomparedtotheothersinwhichmatrices} wereemployed.

The SEM analyses demonstrated that bacterial enrich-mentswithoutphysicalsupportsyieldedlowabundanceof cellsandnobiofilmformation(Fig.1aandb).Actually,inthis conditioncellswereshowntobesparselydistributed.Onthe otherhand,adensebiomassyieldandbiofilmformationcould beobservedaround(shale)orinsidetheporous(areniteand polyurethanefoam)oftheothermatrices(Fig.1c–h).

A predominance of coco rods was detected in the enrichments without physical supports (Fig. 1a and b), whereasstraightrodsformingpolymericstructures(EPS)were observedwhenusingpolyurethanefoams(Fig.1candd).The useofareniteandshaleassupportsallowedanintensebiofilm formationinvolvedbyEPS,andthusthedeterminationofthe microbialmorphologywasnotpossible(Fig.1e–h).

DNAextractionandDGGEanalyses

After60daysofincubation,therecoveryofcommunityDNA waspossibleforall the petroleum-basedanaerobic enrich-mentsperformedinthis study.Itisworthtomentionthat differentcontrolsweresetup, usingonlythe matricesand sterilized medium. The controls did not show any turbid-ity,demonstratingthatnocontaminationoccurred.Although theenrichmentwithoutphysicalsupportexhibitedlower tur-bidityofcells,theenrichmentscontainingshaleandarenite assupportshowedlower DNArecoverywhen comparedto theenrichmentswithoutsupportandtotheonecontaining polyurethanefoams(Table1).Thiswasprobablyduetothe strongadsorptionofbacterialcellstothepiecesofshaleand arenite granules,making it difficultthe recovery ofall the biomassdevelopedintheseenrichments.

TheDGGEanalysesrevealeddistinctbandprofilesbetween samplesoriginatedfrombacterialenrichmentswithand with-outphysicalsupports,reflectingdifferencesinthebacterial communitycomposition(Fig.2a).Thetypeofphysicalsupport

usedinthebacterialenrichmentsalsoresultedindifferences, in particular dominant populations reflected by the DGGE bands.Thesedifferenceswereobservedintermsofnumber andintensityofbands.Mostbands observedintheprofiles correspondingtotheenrichmentwithoutanyphysical sup-port were not observed in the enrichments with support, indicatingthatthebacteriarelatedtothosebandswerenot favoredundertheconditionssettledfortheotherenrichments (Fig.2a,blue arrowsinlanes 1–3).Onthe other hand, pro-files derivedfromthe enrichmentscontainingsupportsfor anincreaseofbiomassrevealedspecificbandsnotobserved intheenrichmentwithout anyphysicalmatrix(Fig.2a,red andgreenarrowsinlanes4–12).Enrichmentswithareniteand shaleasmatricesweremoresimilartoeachotherwhen com-paredtotheenrichmentwithpolyurethanefoams(Fig.2b). Inaddition,bandpatternscorrespondingtotheareniteand shaleenrichmentsshowedthelowestrelativerichness (num-berofbands);whereas,theenrichmentswithpolyurethane foamshowedthehighestcomplexbandpatterns(Fig.2a,lanes 4–6).

16SrRNAgenelibraries,ARDRAandphylogenetic analysis

Thebacterialdiversityofthefourdifferentenrichment cul-turesevaluatedinthisworkwasdeterminedbyanalysisof 16SrRNAgeneclonelibraries.

Atotalof95clonesfromtheWS(withoutsupport) enrich-ment,46clonesfromthePF(polyurethanefoams)enrichment, 63clonesfromtheS(shale)enrichmentand50clonesfrom the A (arenite) enrichmentwere screenedby ARDRA aim-ing toselectdifferent ribotypesforsubsequentsequencing andphylogeneticanalysis.CombineddatafromARDRAand sequencinganalysesallowedtounravelthebacterial diver-sityrecoveredinthepetroleumenrichments(Fig.3).Clones wererelatedtosequencesavailableattheGenbankandRDP (RibosomalDatabaseProject)publicdatabase.

TheARDRAanalysisofthe95clonesfromtheWS enrich-mentshowedsevendistinctrestrictionprofiles.Cloneswere affiliatedtothegeneraPetrotoga(48.4%)(PhylumThermotogae)

andBacillus(51.6%)(PhylumFirmicutes)(Fig.3a).

FifteendistinctribotypesweredetectedinthePF enrich-ment. Sequencing of clones representing such ribotypes revealed a more diversified microbiota, which included the generaRahnella(13%),Pseudomonas(15.2%), Achromobac-ter (6.5%) and Acinetobacter (2.2%) (Phylum Proteobacteria),

Geobacillus(13%),Paenibacillus(10.8%),Thermicanus(6.5%),

Weis-sella (4.4%), Leuconostoc (6.5%) and Bacillus (2.2%) (Phylum

Firmicutes),Petrotoga(4.4%)(PhylumThermotogae)and Kocu-ria(8.7%)(PhylumActinobacteria).Someclones(6.5%)were related to sequences from uncultured bacteria, and thus considered unaffiliated (Figure 3b). The ARDRA screening of the 63 clonesfrom the S enrichment yieldedthree dis-tinct ribotypes. Analyses of the clone sequences revealed thatthebacterialcommunitywascomposedbasicallybythe phylaThermotogae,representedbythegenusPetrotoga(94%), and Proteobacteria, represented bythe genus Rahnella(6%) (Fig.3c).

Finally,ARDRAscreeningalsorevealedthreedistinct ribo-types from 50 clones recovered from the A enrichment.

Fig.1–SEManalysesofdifferentbacterialenrichmentsfromCamposBasinoil.(a,b)Oilwithoutsupport;(c,d)oiladded withpolyurethanefoams;(e,f)oiladdedwithshale;(g,h)oiladdedwitharenite.Arrowsindicatesbiofilmformation.

SimilarlytotheSenrichment,theexperimentsusing aren-iteasphysicalsupportrevealedthe massivepredominance ofthegenusPetrotoga(PhylumThermotogae)(96%).Besides Thermotogae,thePhylumFirmicuteswas alsoidentifiedin

this microbiota,representedbyclonesrelated tothegenus

Thermicanus(4%)(Fig.3d).

Phylogeneticanalysisallowedtheidentificationofmany bacterial membersatthespecie level(Fig.4).Themajority

85 90 95 100

b

a

Fig.2–(a)DGGEanalysesshowingthedistinctband profiles.M:marker;Lanes1–3:WSenrichments;lanes4–6: PFenrichments;lanes7–9:Senrichments;lanes10–12:A enrichments.(b)Dendrogramshowingthegroupingof differentprofilesamongtheenrichmentsanddistinct matrices.

oftheclonesrelatedtoPetrotogapresentinthelibrariesfrom theA,S,PFandWSenrichmentswererelatedtothree Petro-togaspecies,beingPetrotogahalophilatheclosestone.These clone sequences exhibited high sequence similarity (99%)

withPetrotoga strains isolated from oilreservoirs (Table 2).

Actually,themajorityoftheanalyzedclonesinall libraries were affiliated with the genus Petrotoga, amounting to155 clones, 48 from A, 59 from S, 46 from WS and 2from PF enrichment.ClonesrelatedtoBacilluspresentinthelibraries fromtheWS(49clones)andPF(1clone) enrichmentswere relatedtoBacillusginsengihumi,showing99%sequence simi-larity(Table2).TenclonespresentintheSandPFlibraries wererelatedtoRahnellaaquatilisandRahnellasp.(sequence similarity between 80% and 99%). Five clones were closely relatedtothespeciesThermicanusaegyptius,twofromtheA libraryandthreefromthePFlibrary,showing99%ofsequence similarity.

Otherbacterial specieswereidentified onlyfromthe PF library(Fig.4).OneclonerelatedtoAcinetobacterursingiiwas found,showing99%sequencesimilarity.Threecloneswere relatedtoLeuconostocmesenteroidesandtwoclonestoWeissella

confusa,bothspeciesisolatedfromfermentedfood(Table2),

with99%sequencesimilarity.Sixcloneswereclusteredwith highbootstrapvaluewiththetypestrainsofGeobacillus

ther-moglucosidasiusandGeobacilluscaldoxylosidasius,showing99%

sequencesimilarity.Inthiscase,itwasnotpossibletodefine the identification of the clones at the species level, since they were recovered in a tight cluster with both species. FivecloneswerecloselyrelatedtoPaenibacillus naphthalenovo-rans(99%sequencesimilarity).Inaddition,fourcloneswere clustered with the actinobacterium Kocuria kristinae (100% bootstrapvalue),threecloneswithAchromobacterxylosoxidans

(99%sequencesimilarity;100%bootstrapvalue)and,finally, sevencloneswiththetypestrainofPseudomonasputida.

Discussion

Inthisstudy,threedifferenttypesofmatrices(polyurethane foam, shale and arenite) were evaluated as supports for biomass immobilization and increase in anaerobic enrich-mentsfromabiodegradedpetroleumsample(CamposBasin). Shale,inparticular,constitutesnearly70%oftherockspresent inasedimentarybasin.Geochemicalanalysesshowedthat almostallhydrocarbonsofthepetroleumfromCamposBasin arefromshale,belongingtotheLowerCretaceousLagoaFeia Formation.36_{Inthissense,thismaterialisprobablyofgreat} relevanceinprovidingasubstrate forthemicrobialgrowth inpetroleumreservoirs.Theuseofshaleinbacterial enrich-mentfrom thepetroleumsampleallowedthecellstogrow aroundtheshaleslicesgeneratingatypeofbiofilm.Infact, cellaggregates,aswellastheEPSstructureresponsibleforthe maintenanceofcellcohesion,wereobservedforthe enrich-ments employing the two other physical supports,arenite and polyurethanefoams,but notfortheWS (without sup-port) enrichment. These results confirm the usefulness of thesetypesofphysicalsupportstoenablebiofilmformation andincreasethemicrobialbiomassfromlowcellular abun-dancesamples,corroboratingpreviousfindings.28,37_However, intermsofDNArecovery,polyurethanefoamswerethemost efficientmaterial.

Itisknownintheliteraturethatbacteria cansurvivein associations, named as biofilms, producing dense biomass and polymeric substances able to keep them together as a unit.38 _{In petroleum reservoirs the presence of} micro-bialbiofilmsaremostlyassociatedwithcorrosionprocess.39 In case of specificgroups as sulfate reducing bacteria, for instance,itisgenerallyacceptedthatsouringmicrobiotacan form mixed community biofilms on the reservoir mineral matrix.8

Immobilization often simulates what occurs naturally when cells grow on surfaces or within natural structures. Numerousbiotechnologicalprocessesareincrementedbythe useofmicrobialimmobilizationtechniques.Thesetechniques canbedividedintofourtypes,basedonthephysical character-isticsofthesupportsemployed:(1)attachmentoradsorption on solid carrier surfaces, (2) entrapment within a porous matrix,(3)self-aggregationbyflocculation(naturalorinduced) and (4) cell containmentbarriers.40 _{However,the choiceof} amaterialastheidealphysicalsupportcanbedeterminant intheselectionofamicrobialcommunity,28_whichcouldbe observedalsointhiswork.Theauthorsdemonstrated that

51.6% 6% 4% 96% 94% Petrotoga Petrotoga Thermicanus Rahnella 48.4% 6.5% 6.5% 2.2%6.5% 15.2% Pseudomonas Geobacillus Rahnella Weissella Paenibacillus Kocuria Acinetobacter Petrotoga Achromobacter Thermicanus Leuconostoc Bacillus Uncultured 13% 13% 4.4% 10.8% 8.7% 2.2% 4.4% 6.5% Petrotoga

a

b

d

c

Fig.3–Occurrenceofbacterialgenerainthepetroleumenrichments:(a)Enrichmentwithoutsupportmaterial(WS)(95 clones,7ribotypes);(b)enrichmentwithpolyurethanefoams(PF)assupportmaterial(46clones,15ribotypes);(c) enrichmentwithshale(S)assupportmaterial(63clones,3ribotypes);and(d)enrichmentwitharenite(A)assupport material(50clones,3ribotypes).

Table2–BacterialdiversityofanaerobicenrichmentsfromabiodegradedpetroleumsamplefromCamposbasin revealedbyculture-independentmethods.

Bacterialgenus Library No.oftotalclones Closestrelatives Source %similarity

Petrotoga A 48 Petrotogahalophila Oilwell 99

S 59 Petrotogahalophila Oilwell 99

PF 2 Petrotogahalophila Oilwell 98

WS 46 Petrotogahalophila Oilwell 99

Bacillus PF 1 Bacillusginsengihumi Spacecraftcleanrooms 99

WS 49 Bacillusginsengihumi OrchardsinChina/Spacecraft

cleanrooms

99

Thermicanus A 2 Thermicanusaegyptius Oxicsoil 99

PF 3 Thermicanusaegyptius Oxicsoil 99

Rahnella S 4 Rahnellaaquatilis Fruitsandvegetables 99

PF 6 Rahnellasp. 99

Pseudomonas PF 7 Pseudomonasputida Riverwaterpollutedwith

phenoliccompounds/soil

99

Geobacillus PF 5 G.caldoxylosidasius Coolsoilenvironments 99

1 G.thermoglucosidasius 99

Paenibacillus PF 5 P.naphthalenovorans Rhizosphereofsaltmarsh plants

99

Kocuria PF 3 K.kristinae PrincessElisabethStation

Antarctica

99

Achromobacter PF 3 A.xylosoxidans Non-rhizobialplant 99

Leuconostoc PF 1 L.mesenteroides Fermentedfood 100

2 Leuconostoclactis Dairyproducts 99

Acinetobacter PF 1 Acinetobactersp. Spacecraftassociatedclean rooms

99

1B_PF(JN998848) (5)

Geobacillus thermoglucosidasius DSM2542T_(AY608981) Geobacillus caldoxylosidasius DSM12041T (AF067651) 2F_PF (JN998858)

12C_WS (JN998810 ) (48)

Bacillus ginsengihumi DSM18134T (AB245378) 3E_PF (JN998856 ) (4)

Paenibacillus naphthalenovorans DSM 14203 T

(

Weissella confusa DSM20196T(AB023241) 1F_PF (JN998851) (2)

Leuconostoc mesenteroides DSM 20484T(AB023244) 7B_PF (JN998864 ) (1)

Leuconostoc lactis JCM 6123T(AB023968) 11E_A (JN998836) (1)

2F1_PF(JN998854 ) (2)

Thermicanus aegyptius DSM12793T(AJ242495) 1D_ PF (JN998849) (2)

Achromobacter xylosoxidans DSM2402T(Y14908) 10F_ PF (6)

Pseudomonas putida DSM291T_(D84020) Pseudomonas fluorescens DSM 50090T(D84013)

11H_ PF (JN998890 ) (5) 6B_S (JN998845 ) (3)

Rahnella aquatilis DSM 4594T (AJ233426) Acinetobacter ursingii CNCTC 6735T(AJ275038) 2C_PF (JN998853)

Acinetobacter schindleri CNCTC 6736T(AJ278311) 5B_PF (JN998857) (3)

Kocuria kristinae DSM20032T(X80749) 1E_PF(JN998850 ) (1)

Petrotoga olearia DSM13574T(AJ311703) Petrotoga mexicana DSM14811T (AY125964) 2C_ WS (JN998805) (45)

7C_S (JN998847) (58)

Petrotoga halophilaDSM 16923T _(AY800102) 1B_A (JN998828) (47)

Methanohalophilus portucalensis FDF-1T

88 82 85 100 100 100 76 100 100 100 100 99 98 100 100 100 100 86 100 100 100 76 84 100 97 100 0.05 Thermotogae Actinobacteria Proteobacteria Firmicutes

Fig.4– Phylogeneticanalysisbasedonpartialbacterial16SrRNAsequencesofclonesfromdiverseenrichmentsamplesand relatedspecies.Bootstrapvaluesgreaterthan70%arelisted.GenBankaccessionnumbersarelistedafterspeciesnames. Numbersinbracketscorrespondtoadditionalclonespresenting.97%sequencesimilaritywiththeclonesrepresentedin thetree.LettersPF,WS,AandScorrespondtothesamplelibraries.Methanohalophilusportucalensiswasusedasoutgroup.

differentmicrobialgenerawererecovereddependingonthe typeofsupportmaterialused,suggestingthatthe appropri-atenessofthesupportmaterialinamicrobialenrichmentwill dependonthemicrobialgroupofinterest.

TheDGGE analyseswere performed as anefficient tool for the comparison of the microbial diversity recovered from the different bacterial enrichments implemented in thisstudy.Theresultsdemonstratedthat thepolyurethane foamsallowedthedevelopmentofthemostcomplex bacte-rialcommunity,reflectedbythehighernumberofdominant populations in the DGGE profiles, differently from the

other enrichments, which yielded the lowest number of bands.

Thecompositionofphytotypes atthe phylumlevel was similarbetweenthelibrariesderivedfromtheWS(without support)andA(arenite)bacterialenrichments,bothshowing ThermotogaeandFirmicutesasthepredominantphyla.The phylum Thermotogae was the most predominant in the arenitelibrary(96%),whileintheWSclonelibrarythe Ther-motogaeandFirmicuteswerefoundatthesimilarproportion (48.4% and 51.6%respectively). In manymicrobial commu-nity studies from oil reservoirs, injection water or other

petroleum-associated environments, members of these two phyla are common inhabitants and, not rarely, predominant.2,41,42 _{Members of the family} Thermoto-gaceae (order Thermotogales) belong to two physiological groups: extreme thermophiles that grow at temperatures above70◦C andmoderate thermophilesthatgrowatlower temperatures.43 _{ThegenusPetrotoga, as the generaGeotoga}

and Thermotoga, is described as occurring exclusively in

petroleumreservoirs.44,45

TheS(shale)bacterialenrichmentalsoyieldeda commu-nity composed mainly bythe phylum Thermotogae(94%), representedbythegenusPetrotoga,followedbythephylum Proteobacteria(6%),representedonlybythegenusRahnella.

Although not commonly described in petroleum environ-ments, this bacterium was previously reported as being involvedinhydrocarbonbiodegradationinAntarcticsoils con-taminatedwithpolycyclicaromatichydrocarbons(PAHs)and inenrichmentsfromBrazilian petroleumsamples.46,47 _{In a} recentstudyofourresearchgroup,16 _{thebacterialdiversity} oftheaerobic(AER)andanaerobic(ANA)enrichmentsofoil samplesfromPotiguarBasin(RN,Brazil)wasevaluatedby16S rRNAclonelibraryanalysis,and38.4%oftheclonesfromthe anaerobicenrichmentwereaffiliatedtothegenusRahnella.

The clone library originated from the PF (polyurethane foams)enrichmentswasmorediverseatbothgenusand phy-lumlevels.Theuseofpolyurethanefoamassupportforthe immobilizationprocesshasbeenalreadyreportedin litera-ture,withwideapplicationinstudiesofmoleculessuchas enzymes,48_dyes49_{andevenbuildingmaterial.}50_Polyurethane foam presents somefeaturessuchas porosity, whichdoes notonlyincreasethesurfaceareabutalsominimizethe dif-fusion limitation forsubstrate and product.48 _{Silva et al.}28 alsodescribedtheuseofpolyurethanefoamsasanefficient supportmaterialforanaerobicbiomassimmobilizationand incrementofmicrobialgrowth,especiallyforsulfatereducing bacteria.

PhylotypeanalysisoftheclonelibraryderivedfromthePF enrichmentsrevealedthatthecloneswereaffiliatedtofour differentphylaindistinctabundances(Proteobacteria, Ther-motogae, Firmicutes and Actinobacteria). Differences were morepronouncedwhencomparingthemicrobiotarecovered fromtheenrichmentsatthegenuslevel.ThePFenrichments allowedtherecoveryofrepresentativesof12differentgenera, corroborating the efficiency of polyurethanefoams forthe improvementofthemicrobialdiversityrecovery.Manyofthe generafoundusingpolyurethanefoamasphysicalsupport,

suchasGeobacillus,Bacillus,Achromobacter,Acinetobacter,

Pseu-domonas,KocuriaandPaenibacillus,aredescribedasorganisms

livingin petroleum-associated environments, and some of themareinvolvedinhydrocarbondegradationprocesses.The

Geobacillus spp. constitute a thermophilic group, classified

into the order Bacillales, described as being isolated from petroleum reservoirs.51 _{Liu and co-workers}52 _{studied the}

alkBgenesinspeciesofthisgroup,suggestingtheirpotential as hydrocarbon degraders. The Achromobacter species have been previouslydescribed inthe literatureas hydrocarbon degradersand/orassociatedwithoilfieldenvironments.22,53,54 Amicrobial diversitystudy conductedwithinjectionwater samplesinplatformsoftheCamposBasin(Brazil)reported that 24% ofthe total 16S rRNA clones were related tothe

Achromobacter genus.55 _{The Pseudomonas, Bacillus and}

Acinetobacter spp. are commonly described as

inhabi-tants of petroleum-associated environments, including reservoirs3,22,42,56–58 _{and their role is often linked to the} hydrocarbon degradation process,including theproduction ofbiosurfactants.59,60_{Similarly,thegeneraKocuriaand}

Paeni-bacillushavealreadybeenrelatedtooilreservoirs61_andalso

tohydrocarbon degradation.62,63 _{ThePaenibacillus spp.have} been isolatedfrom Iranian oilwells61 _{andthe Kocuria from} Chineseoilfields.64

TheLeuconostocisabacterialgroupcommonlydescribed

livinginfreshplantsandplaysanimportantroleinseveral industrial and food fermentationprocesses.65 _Nonetheless, the presence of the genus Leuconostoc has already been reported in an oil field environment.21 _{Members of the} genus Weissella are usuallyisolated from food and vegeta-blesandhavebeeninvolvedwithfermentativeprocessesof foodproducts.66_{Recently,Silvaandco-workers}16_havefound

Kocuria,Bacillus,Weissella,Achromobacter,Acinetobacterand

Leu-conostocintwodifferentpetroleumsamples(PotiguarBasin,

RN)fromBrazilianreservoirs.

The genus Thermicanus was first described as a group encompassing thermophilic, fermentative microaerophilic bacteria living in soils.67 _{These bacteria were co-isolated} togetherwithathermophilicacetogen,Moorellathermoacetica,

from oxic soilobtained from Egypt,and these twospecies wereshowntogrowcommensallyonoligosaccharidesviathe interspeciestransferofH2andformateandlactate.67These

datasuggestthatThermicanusmightbeindirectlyinvolvedin thecomplexsyntrophicdegradationofhydrocarbonsin oil-associatedenvironments.

Theresultsobtainedusingthe differentsupport materi-als,polyurethanefoams,shaleandarenite,revealedthatall ofthemledtoanincreaseofthemicrobial biomass,when comparedtotheenrichmentwithoutanyphysicalsupport. However, it was expected that the use of shale and aren-itewouldallowtherecoveryofamorediversifiedmicrobiota fromoilsamples,consideringthattheyparticipateinthe com-positionofthereservoirrockand areanaturalsupportfor biofilmformationinsuchenvironments.Infact,polyurethane foamsyieldedthehighestmicrobialdiversity.Thiscouldbe explainedbythe porous natureofthe polyurethanefoams thatallowsanintensebiofilmformationandEPSproduction, whichinturnallowsmicroorganismsthatdonothaveaffinity withthematerialsurfacetogetattachedtothefirstbiofilm colonizers,thusincreasingthemicrobialdiversity.

Despitethediversityfoundintheanaerobicenrichments, members relatedtothe sulfatereductionmetabolismwere notobserved.Thisfactcouldbeexplainedbytheshortperiod ofincubation,60days,whichcould leadtoaninitial selec-tion offacultativeanaerobicheterotrophicbacteria,capable ofdegradingfasterthenutrientsource,becomingmore abun-dantthansulfate-reducingmembers.

Conclusion

This work describes the use of three different physical supports–shale,areniteandpolyurethanefoams–for effi-cientbiomassrecoveryinpetroleumenrichmentsinsulfate

reducing conditions. Molecular techniques and SEM were used as tools to assess the microbial diversity as a func-tion ofthephysicalsupport employedin theenrichments. ResultsrevealedPetrotogaasthemostabundantgenusinthe enrichmentsusingshaleandareniteasphysicalsupport.On the other hand,the enrichmentusing polyurethanefoams was morediverse and allowedthe identification of 12 dif-ferentbacterialgenera.Finally,thecombineddatagathered inthis work demonstrated the usefulnessofphysical sup-portsfortheenrichmentoflowabundancemicroorganisms foundinparticularenvironments,suchasdeepoilreservoirs, enablingsubsequentmicrobiological,physiological,genomic ormetagenomicanalyses.

Conflict

of

interest

Theauthorsdeclarethatthereisnoconflictofinterest.

Acknowledgement

TheauthorsthankEspac¸odaEscrita–CoordenadoriaGeralda Universidade–UNICAMP–forthelanguageservicesprovided.

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