Enzymatic potential of heterotrophic bacteria from a neutral copper mine drainage

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Enzymatic

potential

of

heterotrophic

bacteria

from

a

neutral

copper

mine

drainage

Bruna

Zucoloto

da

Costa

_,

_Viviane

_Drumond

_Rodrigues

_,

_Valéria

_Maia

_de

_Oliveira

_,

Laura

Maria

Mariscal

Ottoboni

_,

_Anita

_Jocelyne

_Marsaioli

a_{UniversidadeEstadualdeCampinas,InstitutodeQuímica,Campinas,SP,Brazil}

b_{UniversidadeEstadualdeCampinas,CentrodeBiologiaMoleculareEngenhariaGenética,Campinas,SP,Brazil}

c_{UniversidadeEstadualdeCampinas,CentroPluridisciplinardePesquisasQuímicas,BiológicaseAgrícolas,Campinas,Brazil}

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r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25March2015 Accepted22February2016 Availableonline26July2016 AssociateEditor:CynthiaCanêdoda Silva

Keywords:

Copperminedrainage High-throughputscreening Hydrolases

Monooxygenases

a

b

s

t

r

a

c

t

Copperminedrainagesarerestrictedenvironmentsthathavebeenoverlookedassourcesof newbiocatalystsforbioremediationandorganicsyntheses.Therefore,thisstudyaimedto determinetheenzymaticactivities(esterase,epoxidehydrolaseandmonooxygenase)of56 heterotrophicbacteriaisolatedfromaneutralcopperminedrainage(SossegoMine,Canaã dosCarajás,Brazil).Hydrolaseandmonooxygenaseactivitiesweredetectedin75%and20% oftheevaluatedbacteria,respectively.Bacterialstrainswithgoodoxidativeperformance werealsoevaluatedforbiotransformationoforganicsulfides.Fourteenstrainswithgood enzymaticactivitywereidentifiedby16SrRNAgenesequencing,revealingthepresenceof threegenera:Bacillus,PseudomonasandStenotrophomonas.ThebacterialstrainsB.megaterium

(SO5-4andSO6-2)andPseudomonassp.(SO5-9)efficientlyoxidizedthreedifferentorganic sulfidestotheircorrespondingsulfoxides.Inconclusion,thisstudyrevealedthatneutral copperminedrainagesareapromisingsourceofbiocatalystsforesterhydrolysisand sul-fideoxidation/bioremediation.Furthermore,thisisanovelbiotechnologicaloverviewofthe heterotrophicbacteriafromacopperminedrainage,andthisreportmaysupportfurther microbiologicalmonitoringofthistypeofmineenvironment.

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

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

Introduction

Brazil is the world’s 15th largest producer of copper concentrate.1_{TheminingcompanyValeS.A.isresponsiblefor}

themajorityofcopperproduction,havingreached275,000mt

∗ _{Correspondingauthorat:ChemistryInstitute,UniversityofCampinas(UNICAMP),POB6154,13084-971Campinas,SP,Brazil.}

E-mail:anita@iqm.unicamp.br(A.J.Marsaioli).

1 _{Theseauthorscontributedequallytothiswork.}

ofcopperconcentrateinthefirstninemonthsof2013,2_and

operates atthe SossegoMine(CanaãdosCarajás,PA,since 2004) andattheSaloboMine (Marabá,PA,since2012).The processusedforcopperoreconcentrationinvolvescrushing andsubsequentsemi-autogenousgrinding(SAG),followedby

http://dx.doi.org/10.1016/j.bjm.2016.07.004

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

comminutioninaballmill.Thematerialisclassified accord-ingtotheparticlesizeand thenpassesthroughaflotation process.Thegeneratedwaste(aslurry)isdepositedinadam approximately5200minlength.

Theaqueouswastesgeneratedfromminingactivitiesare known as mine drainages and are described as restricted environmentsthatoftenhaveahighmetalcontentandlow organic matter concentration. In addition, water drainage fromminewastesandabandonedminesareoftenacidicas aresultoftheextendedexposureofsulfidicmineralstowater andoxygen.However,thepHofcopperminedrainagemay drasticallydifferaccordingtothewaste chemical composi-tion,thetimeofexposureandthemicrobialcommunity.3

Most microbial studies focus on the mineral-oxidizing prokaryotesfromacidminedrainages(AMD),whichusually consistofacidophilic bacteriaand archaea. These microor-ganismsareabletooxidizeferrousironand/orreducedforms ofsulfurandacceleratetheoxidativedissolution ofsulfidic minerals,whichmaybeappliedtometaloreprocessingand concentratesinabiotechnologicalprocessknownas biomin-ingandbioleaching.4–6

However, the microbial community from copper mine drainages(acidicorneutral)hasbeenoverlookedasasource ofnewbiocatalystsforbioremediationandthesynthesisof organiccompounds.Tothebestofourknowledge,thereare no reports about the use ofheterotrophic microorganisms isolatedfrom theserestrictedenvironments asbiocatalysts appliedforbioremediationoforganicpollutantsorfor syn-theticorganicchemistry.

Fromabiotechnologicalpointofview,esterases,epoxide hydrolasesandmonooxygenasesaresomeofthebest-studied andmost-appliedenzymes.Thesebiocatalystsarecommonly usedfortheenantioselectiveproductionofvalue-added com-pounds, which are important to pharmaceutical and fine chemicalindustries.Inthiscontext,ourresearchgrouphas been workingon the detectionofenzymatic activity using fluorogenic probes and organic substrates of interest. Our focus hasbeen onnew biocatalystsfrom theAmazon and Atlantic rainforest,7 _{petroleum oil and formation water,}8

humanskin,9_{andfromBrazilianCultureCollections.}10,11

The present work aimed to investigate hydrolases and monooxygenases inheterotrophic bacteria isolated from a neutralcoppermine drainage, applying fluorescence-based high-throughput screening (HTS) assays and multibioreac-tions tomonitororganic sulfide oxidations. Therefore, this studyrepresentsanewbiotechnologicalapplicationofpoorly investigated,heterotrophicmicrobiota.

Materials

and

methods

Generalmethods

Reagents were purchased from Sigma–Aldrich (Steinheim, Germany). Solvents were distilled from technical solvents. FluorogenicprobesandproductsforHTSassays(2–16)were synthesized by our group.12 _{A free sample of}

2-methyl-4-propyl-1,3-oxathiane(19)fromGivaudan(Jaguaré,SP,Brazil) was provided by Naturaperfumery and cosmetic industry (Cajamar,SP,Brazil).Ethylphenylsulfide(18)andoxidation

productsofsubstrates18,19and20(sulfoxides18a,19aand 20a,respectively)weresynthesizedasdescribedbyPortoetal. (2002).13_{Allchemicalreactionsweremonitoredbysilicagel}

TLC (aluminum foil, 60 F254 Merck), and visualization was

obtainedusingUVorbysprayingwithp-anisaldehyde/sulfuric

acid followed by heating at approximately 120◦C. Flash columnchromatographywasperformedusingMerck (White-houseStation,NJ,USA)silicagel60(0.04–0.063mm,230–400 mesh).NMRspectrawererecordedonaVarianInova500(Palo Alto,CA,USA)for1_{H(499.88MHz)and}13_C(125.69MHz)

mea-surements.Chemicalshifts(ı)aregiveninppm,andcoupling constants(J)aregiveninHertz.

Enzymatic reactions were monitored by GC–MS using an Agilent 7890gaschromatograph (Santa Clara,CA, USA) coupledwithaHewlettPackard5975C-MSD(70eV) spectrom-eterequippedwithafusedsilicacapillarycolumn(HP-5MS, 30m×0.25mm i.d.×0.25␮m film thickness).GC–MS analy-seswereconductedusinga1mLmin−1Heflow,splitmode (20:1) and the following temperature program: initial tem-perature 50◦C, increasing at 10◦Cmin−1 to 200◦C and at 20◦Cmin−1 to 300◦C, remaining constant for 5min. Enan-tiomer discrimination was performed on an Agilent 6850 gas chromatograph (Santa Clara, CA, USA) coupledwith a flameionizationdetector(GC-FID)usingafusedsilica capil-larycolumnLipodex-E(Macherey-NagelInc.,Bethlehem,PA, USA)withchiralphase Octakis-(2,6-di-O-pentyl-3-O-butyryl)-␥-cyclodextrin(28m×0.25mmi.d.×0.25␮mfilmthickness). TheGC-FIDanalyseswereconductedusinga1mLmin−1H2

flow,splitmode(10:1)andthefollowingtemperatureprogram: initialtemperature50◦C,increasingat10◦Cmin−1to100◦C andat5◦Cmin−1to180◦C,remainingconstantfor5min.

Samplecollectionandisolationofheterotrophicbacteria

SamplesfromtheSossegocopperminedrainage,Canaãdos Carajás,StateofPará,Brazil,werecollectedinAugust2009. ThesampleswerenamedSO5,SO6andSO7;collectedin ster-iledisposableflasks;andstoredat4◦C.TheSossegomining operationbegan in2004.Thus,theseare 5-year-old copper minedrainagesamples.

Thebacteriawere isolatedaccordingtoEatonand Fran-son(2005).14_{Eachsample(1g)washomogenizedwithsterile}

water(9mL)andseriallydiluted.Onemilliliterofeachdilution wastransferredtoaPetridishandhomogenizedwith12mL ofPlateCountAgar(PCA).Theplateswereincubatedfor48h at35◦C.Coloniesexhibitingadifferentmorphologyandcolor werere-isolatedintrypticasesoyagar(TSA).Thebacteriawere storedat−70◦_{CinLBmediumcontainingglycerol.}15

Fluorescence-basedhigh-throughputscreeningassays

Bacteriawere inoculatedinto LBsolidmedium15 _and

incu-bated for 16h at 37◦C. After growth, cell suspensions (0.2mgmL−1)werepreparedinboratebuffer(20mmolL−1pH 7.4).

Assayswere performedinflat-bottompolypropylene 96-well microtiter plates, which were incubated at28◦C and 200rpm.Fluorescenceintensitiesweremeasuredusingaplate reader spectrophotometer (FlashScan 530 AnalyticJena) at 460nm(excitationwavelength:390nm).

Scheme1–Fluorescence-basedHTSassayfordetectionofesterases(EST,2–4),epoxidehydrolases(EH,5–6)and monooxygenases(BVMO,7–10).

Hydrolase screening was performed using fluorogenic probes 2–6 (Scheme 1, 100␮molL−1), NaIO4 (2mmolL−1),

BSA(2mgmL−1)andbacterialcellsuspension(0.1mgmL−1) andwasmonitoredupto24h.Monooxygenaseswere inves-tigated using probes 7–10 (Scheme 1, 100␮molL−1), BSA (2mgmL−1)andbacterialcellsuspension(0.1mgmL−1)and were monitored up to 72h. All enzymatic reactions were followed by positive controls, including hydrolyzed or oxi-dized fluorogenic products 11–16 instead of probes, and usingnegativecontrolsdevelopedintheabsenceofbacterial cells.

Substrateconversion(%) into productwascalculated by comparingthefluorescenceintensitiesofthereactionassay andpositivecontrol,consideringthelatteras100%.Negative controlswereusedtomonitorspontaneousprobehydrolysis oroxidation.Enzymaticactivity wasconsideredpositivefor conversionsgreaterthan5%.

Multibioreactionassays

BacteriaweregrowninLBliquidmediumfor16hat37◦Cand 200rpm.Cellswererecoveredbycentrifugationat4500×gfor 15min.Wetbacterialcells(0.25g)weremixedwithSørensen buffer16_{(5mL,100mmolL}−1_{,pH7.0)containing1.5␮Lofeach}

substrate (18–21) in Erlenmeyer flasks (25mL). The result-ingsolutionswereincubated at37◦Cand200rpm.After72 and120h,thereactionswereinterruptedbysaturationwith NaClandextractionwithethylacetate(2×2mL).Theorganic phaseswerecombined,driedoveranhydrousMgSO4,

deriva-tizedwithdiazomethane,andaninternalstandardwasadded (benzophenone;0.05mgmL−1).Allsampleswereanalyzedby GC–MS and chiral GC-FID. Substratestability and volatility underreactionconditionswere monitoredbyreaction con-trolsintheabsenceofbacterialcells.

Substrateconversions(%) were calculatedbycomparing thechromatographicpeakareasofthereactants,productsand internalstandards.

Identificationofbacteria

The bacterial strains displaying hydrolytic activity above 99% or monooxygenases capable of oxidizing organic sul-fur compoundswere identifiedbysequencing of16S rRNA. Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega), following the manufac-turer’s instructions. PCR amplification and sequencing of partial 16S rRNA genes were conducted as described by Miquelettoetal.(2011).17_{Thesequencesobtainedwere}

com-paredwithsequencesavailableatGenBank(http://www.ncbi.

nlm.nih.gov)andtheRibosomalDatabaseProject(http://www.

cme.msu.edu/RDP/html/index.html). Multiplealignmentsof

16S rRNA gene sequences were performed using ClustalW 1.6,18_{andevolutionaryanalyseswereconductedinthe}

Molec-ular Evolutionary Genetics Analysis(MEGA) 6.06 package,19

using the DNA substitution model Kimura 2-parameter.20

A discrete Gamma distribution was used to model evo-lutionary rate differences among sites (5 categories (+G, parameter=0.5288)).21 _{Thephylogenetic reconstructionwas}

performedusingtheMaximumLikelihoodmethodwith1000 bootstrappedreplicates.

Results

EnzymaticactivitydetectedbyHTS

The56 heterotrophicbacteriaisolatedfrom thethree sam-ples of a neutral copper mine drainage were evaluated by fluorescence-based HTS screening using probes 2–10

(Scheme1).

Assays performed in the presence of probes 2 and 3

(Table 1), which were used to detect esterases catalyzing

thehydrolysisofshortchainesters(acetateandpropionate, respectively),indicatedthat27bacteriawereabletohydrolyze probe2,fiveofwhichconvertedmorethan99%ofthe sub-strate (SO5-6, SO5-13, SO7-7, SO7-10 and SO7-15). Probe 3

Table1–Esteraseandmonooxygenaseactivitiesof heterotrophicbacteriaisolatedfromaneutralcopper minedrainagedetectedbyfluorescence-basedHTS assaysusingprobes2–4a_and7–10.a

StrainID Conversion(%) 2 3 4 7 8 9 10 SO5-1 – 29 – 5 7 – – SO5-2 – – – 9 – 12 – SO5-3 45 – – – – – – SO5-4 – – – – 16 – – SO5-5 – – – – 14 7 – SO5-6 >99 21 – – – – – SO5-7 – 10 5 – – 30 – SO5-8 63 – – – – – – SO5-9 – – – – 5 21 – SO5-10 – 17 – – – 29 – SO5-13 >99 11 – – – – – SO5-14 76 10 – – – – – SO5-16 64 – – – – – – SO5-17 70 – – – – – – SO5-18 90 8 – 8 – – – SO5-19 – 18 – – – – – SO5-21 76 – – – – – – SO6-2 – 56 – – 29 – – SO6-3 – 19 – – – – – SO6-4 – 15 – – – – – SO6-5 84 6 – – – – – SO6-6 33 – – – – – – SO6-7 89 – – – – – – SO6-8 69 – – – – – – SO6-9 57 – – – – – – SO6-12 14 >99 – 6 – – – SO6-14 – 17 – – – – – SO6-15 88 18 – – – – – SO6-16 – 15 – – – – – SO6-18 – 33 – – – – – SO6-19 – 9 – – – – – SO6-20 82 – – – – – – SO6-22 75 – – – – – – SO7-1 – 13 – – – – – SO7-2 – 27 11 7 – – – SO7-3 91 46 – – – – – SO7-6 25 12 – – – – – SO7-7 >99 – – – – – – SO7-8 19 11 – – – – – SO7-9 – 13 – – – – – SO7-10 >99 – – – – – – SO7-11 80 – – – – – – SO7-12 10 11 – – – – – SO7-13 – 31 – – – – – SO7-14 21 11 – – – – – SO7-15 >99 – – – – – – a _SeeScheme1.

was also hydrolyzed by 27 bacteria, and SO6-12 displayed outstandingperformance,convertingmorethan99% ofthe substrate.

The results for short-chain ester biohydrolysis can be dividedinto fourdistinctgroups:(a) 15bacteriathat exclu-sively catalyzed the hydrolysis of acetateesters (2), (b) 15 bacteriathatexclusivelyhydrolyzedpropionateesters(3),(c) 12bacteriathatwerenotabletodiscriminatebetween2and

Table2–Chromatographicconversion(%)of

multibioreactionassaysforsulfide(18–21)oxidation,a catalyzedbybacteriafromaneutralcoppermine drainage. StrainID Conversion(%) 18 19 20 21 SO5-1 3 1 – – SO5-2 1 – – – SO5-4 20 7 11 – SO5-5 7 – 1 – SO5-7 10 – 1 – SO5-9 22 7 30 – SO5-10 4 – – – SO6-2 12 5 9 – a _SeeScheme2.

3,and(d)14bacteriawithnoesteraseactivity,asdetermined usingthetestedfluorogenicprobes.

Theheterotrophicbacteriaevaluatedinthisworkclearly prefershort-chainestersasonlytwostrains(SO5-7andSO7-2) wereabletohydrolyzethemedium-chainester4(Table1).

Noneofthestrainswereabletohydrolyze5and6,which were specific for epoxide hydrolases, after a 24h reaction period.Activitytowardtheseprobeswasobservedonlyafter 72h(datanotshown);however,theconversionswerebelow 10%,andthesebiocatalystswerenotconsideredanyfurther.

Monooxygenasescreeningusingfluorogenicprobes7–10 indicatedthatamongthe56strainsunderevaluation,11were active(Table1).StrainsSO5-2,-18,SO6-12andSO7-2catalyzed theconversionofcyclicketone7into13.StrainsSO5-1,-4, -5andSO6-2catalyzedtheoxidationof8tolactone14, and strainsSO5-2,-5,-7,-9and-10oxidizedketone9toester15. Noneofthestrainsoxidizedprobe10.

Biocatalyticsulfideoxidationbymultibioreactionassays

Multibioreactionexperiments(Table2)revealedthatbacteria isolatedfromaneutralcopperminedrainagewerereadilyable tooxidizeorganicsulfurcompoundstotheirrespective sul-foxides(Scheme2).Alltestedstrainswereabletocatalyzethe oxidationofatleastoneofthesubstrates.Eightstrains oxi-dized18,andthreestrainsoxidized19and20;however,none ofthestrainsoxidized21.Thebestresultswereobservedfor strainsSO5-4,SO5-9andSO6-2,whichwereabletooxidize threeofthesulfidestested(18,19and20).

Theenantioselectivities ofthe sulfidebiooxidations cat-alyzed bySO5-4, SO5-9 and SO6-2 were assessedby chiral GC-FID,revealinglowenantiomericexcesses(from5to15%, datanotshown).Nonetheless,oxidationofcis-19washighly diastereoselective(over99%de),providingonlyaracemic mix-tureoftrans-trans-19a(2S,3S,4Sand2R,3R,4R,Scheme3).

Isolationandidentificationofheterotrophicbacteriafrom

mineenvironments

AlloftheevaluatedcopperminedrainagesampleshadapH between7and8.Thus,specificlowpHculturemediafor iso-lation of acidophilic bacteria were notused. Heterotrophic bacteria (56strains)were isolatedfrom theSossego copper

Scheme2–Substrates(18–21)usedfordetectionoforganic sulfidebiooxidationandtheirrespective,expected

products(18a–21a).

Table3–Identificationofheterotrophicbacterialstrains isolatedfromaneutralcopperminedrainageby sequencingofthe16SrRNAgene.

StrainID Sample Identification GenBank ID SO5-1 SO5 Stenotrophomonasmaltophilia KC859435 SO5-2 SO5 Pseudomonassp. KC859436 SO5-4 SO5 Bacillusmegaterium KC859437 SO5-5 SO5 Pseudomonassp. KC859438 SO5-6 SO5 Bacillusthuringiensis KC859439 SO5-7 SO5 Bacillussp. KC859440 SO5-9 SO5 Pseudomonassp. KC859441 SO5-10 SO5 Pseudomonassp. KC859442 SO5-13 SO5 Bacillusthuringiensis KC859443 SO6-2 SO6 Bacillusmegaterium KC859444 SO6-12 SO6 Bacillusmegaterium KC859445 SO7-7 SO7 Bacillusthuringiensis KC859446 SO7-10 SO7 Bacilluscereus KC859447 SO7-15 SO7 Bacilluscereus KC859448

minedrainagesamples(18strainswereisolatedfromsample SO5,23fromsampleSO6and15fromsampleSO7)usingserial dilutionsandPCA.

Fourteenstrainswithoutstandinghydrolyticoroxidative activity(above99%and10%,respectively)wereidentifiedafter partialsequencingofthe16SrRNAgene(Table3,Fig.1).Nine strainsofBacilluswereidentifiedasB.thuringiensis,B. mega-terium,B.cereusandBacillussp.Otherstrainsrecoveredwere

StenotrophomonasmaltophiliaandPseudomonassp.

Discussion

The 56 heterotrophic bacterial strains isolated from this neutralcopperminedrainagewereevaluatedby fluorescence-based HTS enzymatic screening and displayed distinct enzymaticactivitytowardtheselectedprobes.Esterasesand monooxygenases were found in 75% and 20% of strains, respectively.Hydrolasesandoxidoreductasesarefrequently employed in industrial biocatalyzed processes,22 _{and the}

search fornovel sourcesof theseenzymes isessential for the developmentofnewprocesses.TheHTSresults, there-fore,indicatethattherestrictedenvironmentofacoppermine drainageisapromisingsourceofnewbiocatalysts.

Thebestoxidationresultswereobtainedusingstrains iso-latedfromsampleSO5(Table1),whichwerefurtherevaluated bymultibioreactionassaysassessingsulfidebiooxidation.

Reducedorganicsulfurcompounds,suchassulfides,were chosenastargetsbecauseoftheirpresenceinindustrial aque-ous wastes. These organic sulfides are often bad smelling andenvironmentallydamaging.Theyareclassifiedasairand waterpollutants, and theirbioremediationisanimportant ecologicalissue.23

Sulfur atom oxidation is the first bioremediation step, producingsulfoxides(monooxygenation)orsulfones (dioxy-genation),whicharelessvolatileandhavealessunpleasant odor.Furthermore,sulfoxidesareindustriallyrelevant com-poundsthathavepotentialasdrugsandareusuallyapplied asprecursorsofaromasandflavorsandusedinasymmetric organicsyntheses.24

Compound18isusuallyemployedtodetectmicrobial sul-fideoxidationand,inthisstudy,wasoxidizedbyallscreened bacteria.Sulfide19isacommon,fruityodorconstituentand wasoxidizedtosulfoxide19abybacteriaSO5-4,SO5-9 and SO6-2,whichresultedinachange fromitsoriginalodorto anewaromavaluedbyfragranceindustries.25_Furthermore,

oxidation ofrac-cis-19provided onlytherac-trans-trans-19a

(Scheme3),withover99%ofdiastereomericexcesstoward

rac-cis-cis-19a,whichischemicallyexpectedasbothenantiomers have all substituents at equatorial positions. Oxidation of sulfide20wasalsoaccomplishedbySO5-4,SO5-9and SO6-2,providingsulfoxiderac-20ainsteadofrac-20b(Scheme 2) becausethelonepairoftheexternalsulfuratomof20isnot partofthearomaticbenzothiazolesystem.

These resultsare promisingand indicate that biooxida-tion/bioremediationoforganicsulfidescanbeperformedby bacteriaisolatedfromtherestrictedenvironmentofneutral copperminedrainages.

Mostisolatedbacteriawithoutstandingenzymatic activi-tieswereidentifiedasbelongingtotheBacillusgenus,which isconsistentwiththewidespreaddistributionofBacillusspp.,

Fig.1–Phylogeneticanalysisbasedonpartial16rRNAgenesequencesoftheidentifiedbacterialstrainsandrelated microorganisms.TheevolutionaryhistorywasinferredusingtheMaximumLikelihoodmethodbasedontheKimura 2-parametermodel.ThenumbersatnodesindicatebootstrappercentvaluesfromtheMaximumLikelihoodanalysis,based on1000resampleddatasets.Onlysignificantbootstrapvaluesgreaterthan65%areshownneareachnode.Barsrepresent sequencedivergence,andthesequenceaccessionnumbersareindicatedinparenthesis.Nostocpunctiformewasusedasthe outgroup.

includinginminingenvironments.26–28_{Bacillusspecieshave}

displayedbroadcatabolicabilitiesandarethemajor indus-trialhydrolaseproducers,responsibleforapproximately50% ofthetotalenzymemarketproduction.29

The other genera recovered were Stenotrophomonas and

Pseudomonas,identified,respectively,asS.maltophiliaand Pseu-domonassp.BothStenotrophomonasandPseudomonashavebeen previouslyisolatedfromheavymetal-contaminatedsoilsand other environmentalminingsamples.30–33 _{Bacterial species}

fromtheStenotrophomonasandPseudomonasgeneraarewell knownfortheirmetabolicdiversity,multidrugresistanceand ability to degrade a wide range of compounds, including pollutants.34–36

Inthis work,theBacillus strains (SO5-6,SO5-13, SO6-12, SO7-7,SO7-10andSO7-15)displayedgoodhydrolaseactivity

(Tables1and3).Thesestrains,aswellasthestrains

identi-fiedasPseudomonas,presentedamoderatelevelofactivityfor monooxygenases(Tables1and3).

Thebestbiocatalyticperformanceforsulfideoxidationwas accomplished by Pseudomonas sp. (SO5-9), followed bytwo

Bacillusmegateriumstrains(SO5-4andSO6-2)(Tables2and3). Mahmood et al. (2009)37 _{isolated a strain of Pseudomonas}

stutzeri that was characterizedas a chemolithoautotrophic sulfide-oxidizingandnitrite-reducingstrainfromthesludge ofananoxicsulfide-oxidizingreactor.Leeetal.(2012)38 identi-fiedaPseudomonassp.strainisolatedfromasludgereactorthat couldsuccessfully convertsulfidetoelementarysulfurand

thusdemonstratedthatsulfideoxidationcouldbeappliedto electricitygenerationfromsulfide-containingindustrial efflu-ent.Therefore,ourresultsindicatetheminestrains’potential applicationasbiocatalystsinorganicsynthesesand bioreme-diation, corroboratingprevious datathat demonstrated the useofPseudomonasandBacillusinindustry.

In conclusion, this study provides a novel overview of the heterotrophic bacteria from a copper mine drainage, whichmightbeusefulforfutureinvestigationsofindustrial processes,enzymeexpressionandbioremediationof contam-inatedenvironments.Furthermore,theobtainedresultswill supportfurthermonitoringofthemicrobiologicalprocesses occurringinthistypeofrestrictedenvironment.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

We thankPablodos SantosPina forthe samples from the Sossegomine.WealsothankCompanhiaAmbientaldoEstado deSãoPaulo(CETESB)fortheisolationofthebacterialstrains, especiallyAnaPaulaG.ChristandNancydeCastroStoppe.

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