Sphingomonas from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities

brazilian journal of microbiology47(2016)271–278

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

Environmental

Microbiology

Sphingomonas

from

petroleum-contaminated

soils

in

Shenfu,

China

and

their

PAHs

degradation

abilities

Lisha

Zhou

_,

_Hui

_Li

_,

_Ying

_Zhang

_,

_Siqin

_Han

_,

_Hui

_Xu

a_{InstituteofAppliedEcology,ChineseAcademyofSciences,Shenyang,China}

b_{StateKeyLaboratoryofForestandSoilEcology,InstituteofAppliedEcology,ChineseAcademyofSciences,Shenyang,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22October2012 Accepted17August2015 Availableonline2March2016 AssociateEditor:RodrigoCosta

Keywords: Sphingomonas

Polycyclicaromatichydrocarbons (PAHs)

Degradation Genus

a

b

s

t

r

a

c

t

MembersoftheSphingomonasgenusareoftenisolatedfrompetroleum-contaminatedsoils duetotheir uniqueabilitiestodegradepolycyclicaromatichydrocarbons(PAHs),which areimportantforinsitubioremediation.Inthisstudy,acombinedphenotypicand geno-typicapproachusingstreptomycin-containingmediumandSphingomonas-specificPCRwas developedtoisolate andidentifyculturable Sphingomonasstrainspresentin petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined,12soilsyieldedyellowstreptomycin-resistantcolonies.Thelargestnumberof yellowcolony-formingunits(CFUs)couldreach105_CFUsg−1_{soil.ThenumberofyellowCFUs} hadasignificant positivecorrelation(p<0.05)withtheratioofPAHstototalpetroleum hydrocarbons(TPH),indicatingthatSphingomonasmayplayakeyroleindegradingthePAH fractionofthepetroleumcontaminantsatthissite.Sixtyyellowcolonieswereselected ran-domlyandanalyzedbycolonyPCRusingSphingomonas-specificprimers,outofwhich48 isolateshadPCR-positivesignals.The48positiveampliconsgenerated8distinctrestriction fragmentlengthpolymorphism(RFLP)patterns,and7outof8phylotypeswereidentified asSphingomonasby16SrRNAgenesequencingoftherepresentativestrains.Withinthese 7Sphingomonasstrains,6strainswerecapableofusingfluoreneasthesolecarbonsource, while2strainswerephenanthrene-degradingSphingomonas.Tothebestofourknowledge, thisisthefirstreporttoevaluatetherelationshipbetweenPAHscontaminationlevelsand culturableSphingomonasinenvironmentalsamples.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.} E-mail:huili@iae.ac.cn(H.Li).

http://dx.doi.org/10.1016/j.bjm.2016.01.001

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

Introduction

Polycyclicaromatichydrocarbons(PAHs)areconsidered haz-ardous due to their toxic, genotoxic, mutagenic and/or carcinogenic properties.1 _{Microbial degradation is a major}

process inthesuccessfulremovaland eliminationofPAHs from contaminated soils.2 _{The use of native or}

indige-nous microbiota is of great interest for bioremediation of petroleum-pollutedsitesbecausetheyareoftenmoreuseful and beneficial than commercial inocula that can be out-competedbyindigenousmicroorganisms.3

Members of the genus Sphingomonas, which was pro-posed by Yabuuchi et al.,4 _{are frequently isolated from}

PAH-contaminatedsoils,suggestingthattheSphingomonasare probably key members ofnatural PAH-degrading microbial consortia.3,5,6_{Anumberofstudiesdemonstratedthat} Sphin-gomonaswasoneofthedominantpopulationsinthemicrobial communitiesofPAH-contaminatedsoilandwatersamples.7–9

TheShenfuirrigationzoneislocatedbetweenShenyang and Fushun city in Liaoning province and is the largest petroleumwastewaterirrigation zonein China.Thepaddy fieldsinthisregionhavebeenirrigatedbyoil-bearing waste-waterdischargedbythepetroleumandcoal-refiningindustry sincethe1940sduetoalackofirrigationwater.Duringthe 1980–1990s,partialpaddyfieldswereconvertedtouplandin theup-andmid-stream.Mineraloil(especiallyPAHs)wasthe dominantcontaminantintheShenfuirrigationzone.10 _Our

previousstudydemonstratedthatalargepercentageof Sphin-gomonasspecieswaspresentinthepetroleum-contaminated soilsoftheShenfuirrigationareausingculture-independent methods,11_{indicatingapotentiallinkbetweenSphingomonas}

and the biodegradation of petroleum compounds in the Shenfuirrigationzone.However,duetothelackofareliable systemforthespecificdetectionand isolationofculturable

Sphingomonas, the functional strains in the environmental sampleswereonlyfoundatrandom.

Vanbroekhoven et al.12 _{developed a streptomycin-based} Sphingomonas growth medium that, together with the yel-lowpigmentationofSphingomonas,facilitated thedetection andisolationofculturableSphingomonasfromsoils.However, thisphenotypicdetectionmethodisnotefficientoraccurate because other bacterialtaxa alsoproduce yellowpigments and are resistant to streptomycin. A genotypic detection methodthatcircumventsthemajordrawbacksinherentinthe phenotypicdetectionmethoddescribedabovehasbeen devel-opedforthedetectionofspecificgroups.The16SrRNAgene sequenceoffersamplepossibilitiestodistinguish microorgan-ismsofvariousgeneraandminimizefalse-positivereactions using genus-specific primers.13,14 _{PCR-based detection has}

proven to be useful for the identification of the genera

Clostridium,Pseudonocardia,Saccharopolyspora,Nocardiopsisand

Saccharothrixusinggenus-specificprimers.14–16_{Inourprevious}

study,aprimersettargetingtheSphingomonas16SrRNAgene wasdesigned17_{;thus,wehypothesizedthattheuseof}

genus-specificcolonyPCRcombinedwithphenotypicidentification wouldallowthespecificdetectionofSphingomonasisolates.

Thespecificaimofthisstudywastoinvestigatethe rela-tionshipbetweenindigenousculturableSphingomonasinsoils fromthe Shenfupetroleum-wastewaterirrigationzoneand

thecontaminationlevelbythedirectandspecificdetection ofculturable Sphingomonas from thesoils. Additionally, the PAH-degrading capability ofthe isolated Sphingomonas was preliminarilycharacterized.

Materials

and

methods

Soilsamples

Soil samples were collected from the Shenfu

wastewater irrigation zone (41◦5046–41◦4124N, 123◦4443–123◦2538E), which is the biggest petroleum wastewaterirrigationzoneinChina.Thetotallengthofthe irrigationchannelisapproximately70km,and15 sampling siteswithdifferentrelativegeographicalpositions,irrigation historiesandsoilmanagementtypeswereselectedfrom up-to down-stream. The15 sampling sites included 11 paddy soils(soilsA–K)and4uplandsoils(soilsL,M,NandO)that werereferredtoassoilsA–Ointhefollowingtext.The phys-icochemicalsoilcharacteristicsandthePAHconcentrations inthe soilwerereported inourpreviousstudy.11 _Typically,

soil samples collected from the up- and mid-stream sites accumulatedmorepetroleumcontaminantscomparedwith thosefromthedown-streamsites.

Selectiveisolationandenumerationofputative Sphingomonas

The indigenous putative culturable Sphingomonas were iso-latedandenumeratedusingapreviouslydescribedmethod.12

Thestreptomycin-resistantyellowcoloniesthatoccurredon the plates were counted basedon the formationofcolony formingunits(CFUs).

PCRandRFLPanalysisoftheSphingomonasisolates Theidentityoftheyellowcoloniesgrownonthestreptomycin plateswasconfirmedbygenus-specificcolonyPCR.ThePCR protocolwasusedwiththeSA429f/933rprimersetdeveloped inourpreviousstudy.17_{Theampliconswiththeexpectedsize}

werefurtheranalyzedbyRFLPanalysisusingthree endonucle-ases[HaeIII(GGCC),HinfI(GANTC)andMspI(CCGG)]at37◦C for2h.TheRFLPpatternswerecomparedmanually,and dis-tinguishableRFLPpatternswereconsideredtorepresentone phylotype.

PhylogeneticidentificationofSphingomonasisolates

Two representatives of each phylotype were selected for nearlyfull-length16SrRNAgenesequencingforphylogenetic identification.PCRamplificationofthe16SrRNAgeneswas performedwiththeforwardprimer27Fandthereverseprimer 1492R.18_{Theobtainedsequenceswereusedforphylogenetic}

analysisasdescribedpreviously.11

MeasurementofPAHdegradation

To determine the PAH-degrading capability of the iso-latedSphingomonasstrains,thepurestrainswereinoculated

brazilian journal of microbiology47(2016)271–278

273

into mineral salts medium(MSM) containing a singlePAH compoundasthesolecarbonand energysource.TheMSM at pH 7.0–7.2 contained 2g K2HPO4, 0.5g KH2PO4, 0.5g

NaCl, 0.5g NH4Cl, 0.2g MgSO4, 10mg CaCl2, and 1ml of

trace element solution per liter of water. The composi-tion of the trace element solution was 2gL−1 FeSO4·7H2O,

2gL−1MnSO4·H2O,0.5gL−1Na2MoSO4·2H2O,0.2gL−1H3BO4,

0.4gL−1 CuSO4·5H2O, 0.5gL−1 ZnSO4, 0.2gL−1 NH4VO3,

0.5gL−1 CoCl2·6H2O, and 0.2gL−1 NiCl2·6H2O. Eight PAHs,

including naphthalene (NA), acenaphtene (ACY), fluorene (FLO),phenanthrene (PHE),anthracene(ANT), fluoranthene (FLU),pyrene(PYR)andbenzo[a]pyrene(BaP),wereprepared ataconcentrationof5gL−1inacetoneandfilteredpriorto analysiswitha0.22-␮mTeflonsyringefilter.

ThePAHdegradationexperimentwasperformedby moni-toringthedisappearanceofPAHinliveversuskilledcontrols. The experiment was conducted in triplicate in sterile dry 250mlTeflon-cappedflasks.Afterall ofthe acetoneinthe PAHstockswasallowedtoevaporate,27mlofMSMbrothand 3mlofinoculum(OD600=0.8–1.0)wereaddedtoeachflaskata

finalconcentrationof200␮gml−1ofNA,ANT,FLU,PHE,ANT, andFLU,100␮gml−1ofPYR,and50␮gml−1ofBaP.Triplicate flaskswere inoculated withlive bacteriaand 4% formalin-killedcontrols.Thecultureswereincubated inthe darkon arotary shaker(150rpm) at30◦C for7days, andthen the flaskcontents were extracted for30min with hexane (1:1, vol/vol).FlaskswithPAHsbutwithoutthepurebacterial cul-tureswerealsorunasacontrol. Theextractwas analyzed byhigh performanceliquidchromatography(HPLC)using a Waters2695apparatus(WatersInc.,USA)equippedwithan ultravioletdetectoranda250×4.6mm2_{InertsilODS-P(Dikma}

Inc.,USA)reversephaseC18column.TheHPLCanalysiswas performedusing methanol:water (90:10,v/v) asthe mobile phaseataflowrateof0.7mlmin−1.PAHpeakswereidentified andintegratedusingtheMasslynxsoftware(Waters Corpora-tion,Milford,MA).

Nucleotidesequenceaccessionnumbers

Thenucleotidesequences havebeendepositedinthe Gen-Bank database under the following accession numbers: JF716058toJF716065.

Statisticalanalysis

Analysis ofall data was performed by ANOVA using SPSS version16.0.Linearcorrelationcoefficientsweredetermined betweendifferentbiologicaland biochemicalparameters. P

valuesbelow0.05wereconsideredstatisticallysignificant.

Results

and

discussion

EnumerationofputativeSphingomonasbyplatingon streptomycinagar

Most Sphingomonas strains are characterized as yellow-pigmented,straight,rod-shapedbacteria.Additionally, resis-tancetostreptomycin(Sm)isconsideredacommonfeature ofSphingomonas.12_{Hence,yellowcoloniesonplates}

contain-ing Sm were counted as putative culturable Sphingomonas.

Previous real-time PCR analysis indicated that all 15 petroleum-contaminated soilsamplesgavepositive signals for Sphingomonas.11 _{However, in this study only 12 soils}

yielded yellow colonies on the selective LB plates with Sm. The number of yellow CFUs ranged from 0.6×103 _to

1.6×105_CFUsg−1_{soil(Table1).Providedthatalloftheyellow}

coloniesbelongedtotheSphingomonasgenus,thecountsofthe

Sphingomonasstrainscouldonlyreach105_CFUsg−1_soil,which

wasmuchlessthan theabundance(107_–108 _copiesg−1_soil)

obtained byour previous real-timePCR assay inthe same tested soils.11 _{This discrepancy suggested that uncultured} Sphingomonasaccountedforaratherlargeproportioninthe petroleum-contaminatedsoils,whichwasconsistentwiththe resultsofVanbroekhovenetal.12 _andLeysetal.19_Although

culturableSphingomonasrepresentonlyasmallpercentagein thepollutedsoils,itisneverthelessimportanttoobtainpure

Sphingomonasculturesforfunctionalandmechanical charac-terizationduetotheirpotentialrolesinbioremediation.

ThelowestamountofyellowCFUswasdetectedinsoilK withlight contamination,whilethe uplandsoilLwith the highestratioofPAHstototalpetroleumhydrocarbons(TPH) hadthehighestnumberofyellowCFUs.Asignificant corre-lation(P<0.05)wasobservedbetweenthenumberofyellow CFUsandtheratioofPAHstoTPH(Table1).Itmaybeargued thattheheavilyPAH-pollutedsoilsaretootoxicforbacteriato survive,butthisiscertainlynotthecaseforSphingomonas. Sph-ingomonasare well-adaptedtocontaminatedenvironments, especiallythosewithhighPAHtoTPHratios,indicatingthat membersofthegenusSphingomonasareprobablykey mem-bersinnaturalPAHdegradation.Indeed,manySphingomonas

strainshavebeenisolatedbasedontheirabilitytodegradea varietyofPAHs,suchasSphingomonassp.LB126andS. pauci-mobilisEPA505.5,20

WealsofoundthatSphingomonasseemedtopreferentially associate with the sand fraction in the soil because the number ofyellow CFUs had a significant (P<0.05) positive correlationwiththeproportionofthesandfraction.In con-trast, a significant (P<0.05) negative correlationwas found between the number of yellow CFUsand the clay fraction

(Table1).Theassociation ofthe bacteriawithpollutedsoil

particleswasshownpreviously.Uyttebroeketal.21_reported

thatMycobacteriummainlyaccumulatedinthePAH-enriched clay fraction, indicating that Mycobacterium might experi-enceadvantagesconnectedtosubstratesourceattachment. However,tothebestofourknowledgenoresearchhasbeen performedonthenumberofculturableSphingomonasin dif-ferentsoilfractionsincontaminatedsoils.Bastiaensetal.20

compared twodifferent procedures toisolatePAH-utilizing bacteriafromPAH-contaminatedsamplesandrevealedthat liquidenrichmentmainlyledtotheisolationofSphingomonas,

whilethemembranemethodexclusivelyledtotheselection of Mycobacterium. Sphingomonas was postulated to prefer the sand habitat, in which the Sphingomonas were loosely associatedwithsoilparticlesandthusobtainedmoreoxygen toperformdegradation.

The amounts of yellow CFUs in 4 paddy soils (B, C, D andH)withlowpHvalues(6.4–6.8)were muchhigherthan the amounts in the other 5 paddy soils (A, F, I, J and K) with relatively high pH values (7.3–8.1). Correlation analy-sisshowedasignificantdecrease(P<0.01)intheamountsof

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 271–278

Table1–Totalnumbersofyellowstreptomycin-resistantcoloniesin12soilsfromtheShenfuirrigationzoneandthecorrelationbetweenthenumberofyellowcolonies

andsoilproperties.

SoilNo.a _Management

type

No.ofyellow colonies

Particlesize(%) pH TotalPAHs TPH TotalPAHs/TPH HMWPAHs/TPH

Sand Silt Clay (mgkg−1) (mgkg−1) (%) (%)

AU _{Paddy 2.2×10}3_{±3.6 67 21 12 7.5 4.69±417.3 1169.3±78.8 0.40 0.28} BU _{Paddy 6.0×10}4_{±1.1 86 12 2 6.5 2.56±237.4 1266.3±31.2 0.20 0.09} CU _{Paddy 9.0×10}4_{±3.9 84 13 3 6.8 1.87±154.4 1207.7±75.9 0.15 0.12} DU _{Paddy 7.2×10}4_{±1.1 84 13 3 6.8 1.35±151.3 716.2±31.1 0.19 0.15} EM _{Paddy N.D.}b _{42 24 34 8.1 4.04±334.2 5243.6±78.4 0.08 0.07} FM _{Paddy 4.0×10}3_{±1.3 41 28 31 7.7 2.72±241.9 1878.4±77.9 0.14 0.07} GM _{Paddy N.D. 41 27 32 7.6 1.96±210.4 661.4±31.2 0.30 0.19} HD _{Paddy 3.6×10}4_{±0.7 44 44 12 6.4 1.39±201.9 666.2±62.8 0.21 0.18} ID _{Paddy 2.4×10}3_{±2.3 49 32 19 7.3 4.30±188.0 937.1±31.2 0.46 0.43} JD _{Paddy 8.8×10}3_{±0.7 41 39 20 7.3 0.791±68.1 497.0±46.9 0.16 0.08} KD _{Paddy 0.6×10}3_{±0.4 43 42 15 7.3 0.39±31.7 277.2±15.7 0.14 0.11} LU _{Upland 1.6×10}5_{±5.4 59 27 14 7.3 2.99±25.8 331.4±15.6 0.90 0.74} MM _{Upland 2.6×10}3_{±1.9 40 30 30 7.3 2.36±79.3 604.4±26.8 0.39 0.25} ND _{Upland N.D. 45 37 18 7.5 4.50±35.4 2489.9±72.8 0.18 0.13} OD _{Upland 1.6×10}4_{±5.2 52 22 16 7.4 0.61±102.3 220.8±31.2 0.27 0.17} Correlationcoefficientc _{0.598(P<0.05) −0.402(P>0.05) −0.567(P<0.05) −0.754(P<0.01)}d _{−0.112(P>0.05) −0.259(P>0.05) 0.554(P<0.05) 0.541(P>0.05)}

a _{Thelocationsofsoilsamples;Usoilslocatedup-stream;Msoilslocatedmid-stream;Dsoilslocateddown-stream.} b _{N.D.:notdetected.}

c _{Thecorrelationcoefficientrepresentedthelinearcorrelationbetweenthetotalnumberofyellowcoloniesandthesoilparameterlistedinthatcolumn.} d_{Theanalysiswasconductedwiththe9paddysoilsthatyieldedyellowcolonies.}

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Sphingomonas sp. LB126 (AF335501) Sphingopyxis granuli (AY563034) Sphingopyxis soli (AB675376) Sphingopyxis ginsengisoli (AB245343) Sphingopyxis witflariensis (AJ416410) Sphingopyxis panaciterrae (AB245353) Sphingopyxis taejonensis (AF131297)

4c

Sphingopyxis chilensis (AF367204) Sphingopyxis alaskensis (Z73631)

Sphingopyxis baekryungensis (AY608604)

Novosphingobium pentaromativorans (AF502400)

23i

Novosphingobium naphthalenivorans (AB177883) Novosphingobium tardaugens (AB070237) Novosphingobium stygium (AB025013)

19e

Novosphingobium aromaticivorans F199 (U20756) Novosphingobium hassiacum (AJ416411) Novosphingobium lentum (AJ303009)

Sphingomonas desiccabilis (AJ871435) Sphingomonas mucosissima (AM229669) Sphingomonas paucimobilis (AM237364) Sphingomonas abaci (AJ575817) Sphingomonas fennica (AJ009706) Sphingomonas haloaromaticamans (X94101)

Sphingomonas wittichii (EU730907) Sphingobium aromaticiconvertens (AM181012)

Sphingobium yanoikuyae (D13728) Sphingomonas sp. B1 (X94099)

Sphingomonas paucimobilis EPA505 (U37341) Sphingobium japonicum (AF039168)

Sphingomonas sp. GY2B (DQ139343) 9b 9e 18h 26b 100 84 100 100 99 91 98 96 98 95 92 85 71 59 81 72 64 58 71 64 56 88 57 0.1 Cluster I Cluster II Cluster III Cluster IV Cluster V 85

Fig.1–Phylogenetictreebasedonthealignmentof16SrRNAsequencesofsevenisolatesandrelatedsequencesofthe genusSphingomonaswasconstructedusingtheneighbor-joiningalgorithm.Thescalebarrepresents0.1substitutionsper baseposition.Thenumbersatthenodesindicatethepercentagesofoccurrencein500bootstrappedtrees.Species harboringPAH-degradingisolatesareindicatedby“”.Theaccessionnumbersforselectedspecies,whichweresuggested byYabuuchietal.4,23_{forgenusSphingomonasandincludeSphingomonassensustricto,Novosphingobium,Sphingobium,and}

Sphingopyxis,atthistimearegiveninparentheses.

yellowstreptomycin-resistantCFUswiththeincreaseinsoil pH(theanalysiswasconductedwiththe9paddysoilsthat yieldedyellowcolonies) (Table1).Thus, ourdataindicated that the culturableSphingomonas preferred an acidic pH in petroleum-contaminatedsoils.Previousstudiesshowedthat

SphingomonascouldsurviveinPAH-pollutedsoilswithpH val-uesrangingfrom7.0to8.9,7,12,19,22_{butthesuitablesoilpHfor}

theremovalofPAHsbySphingomonasstrainsrangedbetween 6.0and7.0.6_{Thismayexplaintosomeextentwhymore}

cul-turableSphingomonasweredetectedinsoilswitharelatively lowpHvalueinthisstudy.Nevertheless,theeffectofsoilpH onculturableSphingomonasinpetroleum-contaminatedsoils hasnotbeenpreviouslyreported.Thisfindingraisesfurther interestintherelationshipbetweensoilpHandtheamounts ofculturableSphingomonas(especiallythePAH-degrading iso-lates)inpollutedsoils.

Phylogeneticidentificationofisolates

Sixtystreptomycin-resistantyellowcolonieswererandomly selected from the 12 petroleum-contaminated soils and

tested with Sphingomonas specific-PCR toexclude the

non-Sphingomonas strains. A Sphingomonas species type strain

Sphingomonaspaucimobilis(1.3773,ChinaGeneral Microbiologi-calCultureCollectionCenter)wasusedasthepositivecontrol. Simultaneously,12randomlychosennon-yellowpigmented colonies with different morphologies were tested together withtheyellowcolonies.Nopositivesignalsweredisplayed whenSphingomonasspecific-PCRwasperformedonthese non-yellowpigmentedcolonies(datanotshown).Atotalof48out ofthe60yellowcoloniesproducedampliconsoftheexpected size,indicatingthatthestreptomycinmediumtogetherwith theyellowpigmentwasonlyasemi-selectivemethodforthe identification ofSphingomonas species. The combination of thisphenotypicdetectionmethodwithSphingomonas genus-specific PCR allowed genotypic confirmation ofthe yellow pigmentedcolonies,therebyfacilitatingthespecificdetection andisolationofculturableSphingomonasfromsoilsamples.

The48ampliconscouldbedividedinto8different phylo-typesaccordingtotheRFLPanalysis(Fig.1).Sequenceanalysis showedthatnosequencedifferencesexistedbetweenthetwo representative strains from each phylotype; thus,onlyone

Table2–DegradationrateofeightPAHsbySphingomonasstrains.a PAH %Degradation 4c 23i 9b 18c 10c 19e 9e FLO 87.2±3.8 85.4±4.5 74.9±4.7 83.0±5.4 68.7±3.1 63.2±1.9 N.G. PHE 64.8±7.7 60.6±6.3 N.G. N.G. N.G. N.G. N.G. NA N.G. N.G. N.G. N.G. N.G. N.G. N.G. ACE N.G. N.G. N.G. N.G. N.G. N.G. N.G. ANT N.G. N.G. N.G. N.G. N.G. N.G. N.G. FLU N.G. N.G. N.G. N.G. N.G. N.G. N.G. PYR N.G. N.G. N.G. N.G. N.G. N.G. N.G. BaP N.G. N.G. N.G. N.G. N.G. N.G. N.G.

NA–naphthalene;ACE–acenaphtene;FLO–fluorene;PHE–phenanthrene;ANT–anthracene;FLU–fluoranthene;PYR–pyrene;BaP– benzo[a]pyrene;N.G.–nogrowth.

a _{Thepresenteddegradationratewascalculatedafter7daysofincubationonarotaryshakerat30}◦_{C,andtheinitialconcentrationoftheeight} PAHswas200␮gml−1ofNA,ANT,FLU,PHE,ANT,andFLU,100␮gml−1ofPYR,and50␮gml−1ofBaP.

strainwasselectedforfurtheranalysis.Theresultsshowed that7phylotypesmatchedsignificantly(99–100%similarities) withtheSphingomonas16SrRNAgenesequencesinGenBank. Ofthese7Sphingomonasphylotypes,tworepresentativestrains (9band9e)bothsharedsimilaritiesof99%withSphingomonas aromaticivorans(currentlynominatedasNovosphingobium aro-maticivorans);however,RFLPanalysesindicatedthattheywere differentstrains(Fig.1).Onephylotype(2outofthe48total streptomycin-resistantyellowcolonies)wasnotaffiliatedwith

Sphingomonas,butwithErythrobacter.Itisnotunexpectedthat

ErythrobacterwasdetectedtogetherwithSphingomonasusing the phenotypic and genotypic methods developed in this study becausestreptomycinresistanceand yellow pigmen-tationhavebeenreportedpreviouslyforErythrobacterstrains (i.e., E. litoralis).12 _{Additionally, bothSphingomonas and} Ery-throbacter belong to the family Sphingomonadaceae, and the sequences ofprimerset SA429f/933rshowed 100% similar-ity tothe 16S rRNAgene sequences of Erythrobacterlongus

(datanotshown). Thus,it canbe inferredthat the pheno-andphylo-methodsdevelopedinthepresentstudythataimed tospecificallyisolateSphingomonasmemberscouldinevitably select a minor fraction of other groups from Sphingomon-adaceae.

Thephylogenetictree(Fig.1)constructedbyaligningthe isolated Sphingomonassequences with sequences oftypical speciesofgenusSphingomonasshowedthatonly3 representa-tivestrains(23i,4cand19e)clusteredwithknowncultured

Sphingomonas sequences. Strain 19e was closely related to

NovosphingobiumaromaticivoransF199,whichcouldutilize fluo-reneandnaphthaleneasthesolecarbonsource24_;thisresult

suggestedthatstrain19emighthavethecapabilityforPAH degradation. The other 4 isolated strains (9b, 9e, 18h and 26b)werenotplacedingroupswithknownSphingomonasbut weregroupedinasinglecluster,indicatingthattheymight representnewmembersofthegenusSphingomonas.Further analysesareneededtodeterminetheirphysiologicaland bio-chemicalcharacteristics.

UtilizationofPAHsasasolesourceofcarbonandenergy SevenSphingomonasisolatesrepresenting7phylotypeswere assessedfortheirabilitytouse8differentindividualPAHsas

theirsolecarbonandenergysourceinliquidculture.Growth was considered positive when increases in turbidity were observed;positivegrowthresponseswereconfirmedby ana-lyzingthedisappearanceofthePAHwithHPLC.Increasesin turbidity and the disappearance offluorenewere observed insixofthe seventested strains(9b,26b,4c, 18h,19e and 23i). Measurements of the degradation rates showed that

Sphingomonas4ccoulddegrade87.2%ofthefluorenewithin 7 days at 30◦C when the initialconcentration of fluorene was100mgL−1.Significantvariation(P<0.01)inthefluorene degradationratewasobservedamongthe6strains(Table2). Additionally,strain4candstrain23iwerecapableofgrowing onphenanthrene(Table2).Underthesameincubation con-ditions,Sphingomonas4candSphingomonas23icouldremove 64.8%and60.6%ofphenanthreneintheliquidculture, respec-tively.Overall,Sphingomonasstrain4cshowedacapabilityto degradefluoreneandphenanthrenewitharelativelyhigh effi-ciency,indicatingthatitmightserveasapromisingfunctional strainforfieldstudiesofinsitubioremediation.

Theselected Sphingomonas isolatesall failedto growon themediasupplementedwiththeothersixPAHcompounds as thesole carbon sources(Table 2). Althoughonly6 PAH-degrading Sphingomonasstrainswere obtainedinthestudy, based on the fact that most of the bacteria were uncul-tured and the limitations of the isolation method it is reasonabletodeducethatthediversityofPAH-degrading Sph-ingomonasstrainsinpetroleum-contaminatedsoilsishigher thanobservedinthepresentdata.

The6PAH-degradingstrainsoriginatedfrom8soil sam-ples(A,C,D,H,I,K,LandO)withdifferentcontamination levels. Despite the fact that all of these soils contained a relatively high ratio of high molecular weight (HMW)

PAHs to TPH (Table 1), noneofthe strains isolated in this

studycouldutilizeHMW-PAHs.Theprobablereasonforthis result may beexplained bythreefacts. First,several stud-ies have indicated that Sphingomonas are more adaptedto the degradationofPAHswithrelativelyhigh bioavailability, suchasphenanthrene.5,25,26_{Second,theco-existenceof}

bac-teria/bacteriaorbacteria/fungiknowncataboliccooperation is ubiquitous in soil. Thus, we may speculate that Sphin-gomonasmaybeinvolvedinHMW-PAHdegradationbyliving on the metabolites of other bacteria or fungi rather than

brazilian journal of microbiology47(2016)271–278

277

ontheHMW-PAHitself.Inthepresentstudy,degradationof HMW-PAHswasperformedbypurestrainsbutnotbymixed cultures,which may providea reduced chanceofisolating HMW-PAH-degradingSphingomonasstrains.Last,ourprevious resultsuggestedthatthe uncultivable Sphingomonasstrains werethedominantonesinthesecontaminatedsoils11_;hence,

the uncultured Sphingomonas strains might play important rolesinthedegradationofHMW-PAHs.

GeneticanalysisofseveralPAH-degradingpathwaysin Sph-ingomonasstrainsrevealedthepresenceofauniquegroupof genesforaromaticcompounddegradationthatweredistantly relatedtothoseinothergenera.24_{Additionally,thecatabolic}

genesfrom fluorene-utilizingSphingomonaswere unlikethe otherdegradativegenesinvolvedinthedegradationofother PAHcomponents.27 Theprotocatechuic acid catabolicgene clusters found in the fluorene-degrading Sphingomonas sp. strainLB126displayedastrongsimilaritywithgenesfound inthelignin-degradingSphingomonaspaucimobilisstrain SYK-6.24,27_{However,littleinformationisavailableconcerningthe}

identificationofthedioxygenasegenesinvolvedinthefirst stepofPAHdegradationinSphingomonassp.LB126orother

Sphingomonasstrains.28 _{Notably,the strains9band9e were}

bothidentifiedasSphingomonasaromaticivorans;however,only 9b(butnot9e)coulddegradefluorene.Itshouldbenotedthat notallSphingomonasstrainshavetheabilitytodegradePAHs. ThismayreflectanabilityofsomeSphingomonasstrains to moreeffectivelyrecruitPAH-degradinggenesasanadaptive responseinPAH-contaminatedsoils.29_{Moreover,thisfinding}

couldindicate that differentstrains, eveniftheybelong to thesamespecies,mightrepresentdifferentfunctionalgroups. Withthedevelopmentofcomparativegenomics,similarities anddifferences betweenthe genomesofstrains9band 9e couldbecomparedtorevealhowthedegradinggeneswere obtainedorlost.

Conflict

of

interest

Theauthorsdeclarethatthereisnoconflictsofinterest.

Acknowledgments

Thiswork was supported by the National Natural Science Foundation ofChina No.30800157 and the Strategic Prior-ity ResearchProgram of the Chinese Academyof Sciences XDB15010103.

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1.HaritashA,KaushikC.Biodegradationaspectsofpolycyclic

aromatichydrocarbons(PAHs):areview.JHazardMater.

2009;169(1–30):1–15.

2.SimsJL,SimsRC,MatthewsJE.Approachtobioremediation

ofcontaminatedsoil.HazardWasteHazardMater.

1990;7(2):117–149.

3.GuoCL,ZhouHW,WongYS,TamNFY.Isolationof

PAH-degradingbacteriafrommangrovesedimentsandtheir

biodegradationpotential.MarPollutBull.

2005;51(8–12):1054–1061.

4.YabuuchiE,YanoI,OyaizuH,HashimotoY,EzakiT,

YamamotoH.ProposalsofSphingomonaspaucimobilisgen.

nov.andcomb.nov.,Sphingomonasparapaucimobilissp.nov.,

Sphingomonasyanoikuyaesp.nov.,Sphingomonasadhaesivasp.

nov.,Sphingomonascapsulatacomb.nov.,andtwo

genospeciesofthegenusSphingomonas.MicrobiolImmunol.

1990;34(2):99–100.

5.BastiaensL,SpringaelD,WattiauP,HarmsH,VerachtertH,

DielsL.Isolationofadherentpolycyclicaromatic

hydrocarbon(PAH)-degradingbacteriausingPAH-sorbing

carriers.ApplEnvironMicrob.2000;66(5):1834–1843.

6.LeysNM,RyngaertA,BastiaensL,TopEM,VerstraeteW,

SpringaelD.CultureindependentdetectionofSphingomonas

sp.EPA505relatedstrainsinsoilscontaminatedwith

polycyclicaromatichydrocarbons(PAHs).MicrobEcol.

2005;49(3):443–450.

7.BritoEMS,GuyoneaudR,Go ˜ni-UrrizaM,etal.

Characterizationofhydrocarbonoclasticbacterial

communitiesfrommangrovesedimentsinGuanabaraBay.

BrazilResMicrobiol.2006;157(8):752–762.

8.SaulDJ,AislabieJM,BrownCE,HarrisL,FoghtJM.

Hydrocarboncontaminationchangesthebacterialdiversity

ofsoilfromaroundScottBase,Antarctica.FEMSMicrobiol

Ecol.2005;53(1):141–155.

9.ShokrollahzadehS,AzizmohseniF,GolmohammadF,

ShokouhiH,KhademhaghighatF.Biodegradationpotential

andbacterialdiversityofapetrochemicalwastewater

treatmentplantinIran.BioresourTechnol.

2008;99(14):6127–6133.

10.LiX,LiP,LinX,etal.Spatialdistributionandsourcesof

polycyclicaromatichydrocarbons(PAHs)insoilsfromtypical

oil-sewageirrigationarea,NortheastChina.EnvironMonit

Assess.2008;143(1):257–265.

11.ZhouLS,LiH,ZhangY,WangYF,HanSQ,XuH.Abundance

anddiversityofSphingomonasinShenfu

petroleum-wastewaterirrigationzone,China.EnvironSci

PollutR.2012;19(1):282–294.

12.VanbroekhovenK,RyngaertA,BastiaensL,etal.

Streptomycinasaselectiveagenttofacilitaterecoveryand

isolationofintroducedandindigenousSphingomonasfrom

environmentalsamples.EnvironMicrobiol.

2004;6(11):1123–1136.

13.LudwigW,SchleiferK.Bacterialphylogenybasedon16Sand

23SrRNAsequenceanalysis.FEMSMicrobiolRev.

1994;15(2–3):155–173.

14.MorónR,GonzálezI,GenilloudO.Newgenus-specific

primersforthePCRidentificationofmembersofthegenera

PseudonocardiaandSaccharopolyspora.IntJSystBacteriol.

1999;49(1):149–162.

15.SalazarO,GonzI,GenilloudO.Newgenus-specificprimers

forthePCRidentificationofnovelisolatesofthegenera

NocardiopsisandSaccharothrix.IntJSystEvolMicr.

2002;52(4):1411–1421.

16.VanDykeM,McCarthyA.Molecularbiologicaldetectionand

characterizationofClostridiumpopulationsinmunicipal

landfillsites.ApplEnvironMicrob.2002;68(4):2049–2053.

17.ZhouLS,LiH,ZhangY,HanSQ,XuH.Developmentof

genus-specificprimersforbetterunderstandingthe

diversityandpopulationstructureofSphingomonasinsoils.J

BasicMicrob.2014;54(8):880–888.

18.FurushitaM,ShibaT,MaedaT,etal.Similarityof

tetracyclineresistancegenesisolatedfromfishfarmbacteria

tothosefromclinicalisolates.ApplEnvironMicrob.

2003;69(9):5336–5342.

19.LeysNM,RyngaertA,BastiaensL,VerstraeteW,TopEM,

SpringaelD.Occurrenceandphylogeneticdiversityof

aromatichydrocarbons.ApplEnvironMicrob.

2004;70(4):1944–1955.

20.MuellerJG,ChapmanPJ,BlattmannBO,PritchardP.Isolation

andcharacterizationofafluoranthene-utilizingstrainof

Pseudomonaspaucimobilis.ApplEnvironMicrob.

1990;56(4):1079–1086.

21.UyttebroekM,BreugelmansP,JanssenM,etal.Distribution

oftheMycobacteriumcommunityandpolycyclicaromatic

hydrocarbons(PAHs)amongdifferentsizefractionsofa

long-termPAH-contaminatedsoil.EnvironMicrobiol.

2006;8(5):836–847.

22.YimMS,YauYCW,MatlowA,etal.Anovelselectivegrowth

medium-PCRassaytoisolateanddetectSphingomonasin

environmentalsamples.JMicrobiolMeth.2010;82(1):19–27.

23.YabuuchiE,KosakoY,FujiwaraN,etal.Emendationofthe

genusSphingomonasYabuuchietal.,1990andjunior

objectivesynonymyofthespeciesofthreegenera,

Sphingobium,NovosphingobiumandSphingopyxis,in

conjunctionwithBlastomonasursincola.IntJSystEvol

Microbiol.2002;52(5):1485–1496.

24.PinyakongO,HabeH,OmoriT.Theuniquearomaticcatabolic

genesinSphingomonadsdegradingpolycyclicaromatic

hydrocarbons(PAHs).JGenApplMicrobiol.2003;49(1):

1–19.

25.GrosserRJ,FriedrichM,WardDM,InskeepWP.Effectof

modelsorptivephasesonphenanthrenebiodegradation:

differentenrichmentconditionsinfluencebioavailability

andselectionofphenanthrene-degradingisolates.Appl

EnvironMicrob.2000;66(7):2695–2702.

26.UyttebroekM,SpodenA,Ortega-CalvoJJ,etal.Differential

responsesofeubacterial,Mycobacterium,andSphingomonas

communitiesinpolycyclicaromatichydrocarbon

(PAH)-contaminatedsoiltoartificiallyinducedchangesin

PAHprofile.JEnvironQual.2007;36(5):1403–1411.

27.WattiauP,BastiaensL,vanHerwijnenR,etal.Fluorene

degradationbySphingomonassp.LB126proceedsthrough

protocatechuicacid:ageneticanalysis.ResMicrobiol.

2001;152(10):861–872.

28.PinyakongO,HabeH,KouzumaA,NojiriH,YamaneH,

OmoriT.Isolationandcharacterizationofgenesencoding

polycyclicaromatichydrocarbondioxygenasefrom

acenaphtheneandacenaphthylenedegrading

Sphingomonassp.strainA4.FEMSMicrobiolLett.

2004;238(2):297–305.

29.MuellerJG,DevereuxR,SantavyD,LantzS,WillisS,Pritchard

P.Phylogeneticandphysiologicalcomparisonsof

PAH-degradingbacteriafromgeographicallydiversesoils.