Degradation of polynuclear aromatic hydrocarbons by two strains of Pseudomonas

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

Environmental

Microbiology

Degradation

of

polynuclear

aromatic

hydrocarbons

by

two

strains

of

Pseudomonas

Obinna

C. Nwinyi

a,d,∗

_,

_Oluseyi

_O.

_Ajayi

b

_,

_Olukayode

_O.

_Amund

c

a_Department_of_Biological_Sciences,_School_of_Natural_and_Applied_Sciences,_College_of_Science_and_Technology,_Covenant_University,

CanaanLand,Ota,OgunState,Nigeria

b_Department_of_Mechanical_Engineering,_College_of_Science_and_Technology,_Covenant_University,_Canaan_Land,_Ota,_Ogun_State,_Nigeria

c_Department_of_{Microbiology,}_University_of_Lagos,_Lagos,_Nigeria

d_Department_of_{Biotechnology}_and_Food_technology,_University_of_{Johannesburg,}_{Doornfontein,}_{Johannesburg,}_South_Africa

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23January2015 Accepted22December2015 Availableonline24April2016 AssociateEditor:CynthiaCanêdoda Silva

Keywords:

16SrRNAgeneanalysis Formerindustrialsites Soilmicrobialcommunities CompetentPAHdegraders

a

b

s

t

r

a

c

t

Thegoalofthisinvestigationwastoisolatecompetentpolynucleararomatichydrocarbons degradersthatcanutilizepolynucleararomatichydrocarbons offormerindustrialsites atMcDoelSwitchyardinBloomington,Indiana.Usingconventionalenrichmentmethod basedonsoilslurry,weisolated,screenedandpuriﬁedtwobacterialspeciesstrainsPB1and PB2.Applyingtheribotypingtechniqueusingthe16SrRNAgeneanalysis,thestrainswere assignedtothegenusPseudomonas(PseudomonasplecoglossicidastrainPB1andPseudomonas

sp.PB2).BothisolatesshowedpromisingmetaboliccapacityonpyrenesprayedMSagar platesduringthepreliminaryinvestigations.Usingtimecoursestudiesintheliquidcultures atcalculatedconcentrations123,64,97and94ppmfornaphthalene,chrysene,ﬂuroanthene andpyrene,P.plecoglossicidastrainPB1andPseudomonassp.PB2showedpartialutilization ofthepolynucleararomatichydrocarbons.Naphthalenewasdegradedbetween26%and 40%,chrysene14%and16%,ﬂuroanthene5%and7%;pyrene8%and13%byP.plecoglossicida

strainPB1andPseudomonassp.PB2respectively.Basedontheirgrowthproﬁle,wedeveloped amodelR2₌₁_to_predict_the_degradation_rate_of_slow_polynuclear_aromatic hydrocarbon-degraderswhereallthenecessaryparametersareconstant.Fromthisinvestigation,we conﬁrmthattheformerindustrialsitesoilmicrobialcommunitiesmaybeexploredforthe biorestorationoftheindustrialsite.

Introduction

Global industrialization is without consequences, particu-larly with the deposition of hydrophobic contaminants in

∗ _{Corresponding}_author.

E-mails:nwinyiobinna@gmail.com,obinna.nwinyi@covenantuniversity.edu.ng(O.C.Nwinyi).

sediments, surface soil and dump sites. Polynuclear aro-matichydrocarbons(PAHs),ahydrophobiccontaminanthave been listedasoneofpriority/toxicenvironmental contami-nants.Theyarechemicalcompoundscomprisingmainlyof carbon (C) and hydrogen (H); arranged in form of two or

http://dx.doi.org/10.1016/j.bjm.2016.04.026

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morearomaticringswithvariousstructuralconﬁgurations.1

PAHs can be classified as alternant (e.g., benzo[a]pyrene, benz]a]anthracene,chrysene,dibenz[a,h]anthracene)or non-alternant (e.g., fluoranthene, benzo[k]fluoranthene, ben-zol[j]fluoranthene, indeno[1,2,3-c,d]pyrene). The alternant PAHscomprisesofaromaticringswithsixcarbonatomswhile thenon-alternant onescontainaromaticrings withlessor morethansixcarbons.Thispeculiarityisbasedonthe elec-trondensity associatedwith the molecule.Alternant PAHs haveanequally distributedelectron density,whereas non-alternantPAHsbehavealmostasiftheywere twodifferent moleculesbecauseofanunevendistributionofelectron den-sityfromoneportionofthemoleculetoanother.PAHsbeing aderivativeofbenzene,andthermodynamicallystable,have twoormorefusedaromaticringsarrangedinlinear,angular, orclusteredstructures.2,3_PAHs_are_ubiquitous_{environmental}

pollutant,enteringtheenvironmentfromnaturaland anthro-pogenic sources such as natural ﬁres, volcanic eruptions, aluminumsmelting,cokeproduction,andcreosote preserva-tion.Theanthropogenic sourcestypicallyareasaresultof incompletecombustionoftheorganicsubstancesthatmake upsuchsubstances.4,5

ThehumanbodycouldbeexposedtoPAHsthrough inhala-tion,dermal contactand ingestion. Withinthe body,PAHs beinghighlylipid-soluble arequicklyabsorbed bythe fatty tissuessuchas thekidney,liver and gastro-intestinaltract ofmammals.Inhumans,thehighestmetabolizing capacity presentistheliver, thenthelungs, intestinalmucosa,skin and kidneys.Metabolism may alsotake place innasal tis-sues,mammaryglands,spleen,brainfollicles,erythrocytes, platelets,leukocytes,placentaanduterus.Intheliver,PAHs areadaptedbycytochromeP450intoepoxidesamajor inter-mediatesthatarereactiveandenzymaticallymetabolizedto dihydrodiolsandphenols.Thedihydrodiolsandphenolsreact againstDNAandproteinscausingmutagenicdamagetocells.6

Naphthalene has been noted to be a hazardous air pollutant.7 _When _experimental _organisms _are _exposed _to

naphthalene,itcausesdecreaseintheirhemoglobin concen-trationandinhibitstheiroxygenconsumption.Naphthalene isusedasrawchemicalforindustrialsynthasesofphthatic anhydride.8 _Naphthalene _at_high _{concentration} _may _cause

hemolyticanemiaand someother conditions, whereasthe tumorigenicpotentialispresentlyconsideredlow.9,10

Naph-thalenehas been often used as a modelPAH dueto high speedofutilizationbymicroorganismscomparedtootherPAH andtherelativelysimplestructureoftheintermediatesinthe catabolicpathways.Thusinformationonbacterial degrada-tionofnaphthalenehasbeenusedtounderstandandpredict pathwaysofmostPAHs.

Pyreneisahydrophobiccompounds and itspersistence withinecosystemsisduetolowwatersolubility,denseclouds ofp-electronson bothsidesofthering structures,making themresistanttonucleophilicattack.11

Soilcanactasasinkforcarbon.Itcouldreceive consid-erableamountofPAHsthatmaylikelyremainpersistentin theenvironmentduetolowsolubilityandsequestration in soiland sediments; to lackof versatile metabolic capacity ofmicroorganismstodegradethesecompound.11_According

to Regonne and co-workers, for effective cleanup of PAH-contaminatedsoils, cheaperand moreecologicallyfriendly

optionsareproposedoverchemicalandphysicalprocesses.12

Theseoptionswillinvolvegreaterunderstandingofthe pro-cessesinvolvedandfactorsthatlimitthedegradationofhigh molecularandlowmolecularweightPAHs.Bioremediationis aproﬁcientandsafemethodtocleanupPAHfrom contami-natedsites.Ithasbeenappliedtobothterrestrialandaquatic ecosystems;andmaypossiblyprovideapositionin biorestora-tion of contaminated soils. Microorganisms transform the PAHstoCO2andwaterthroughmetabolismorco-metabolism. TheyPAHsserveascarbonandenergysources,thusreducing theassociatedtoxicityandco-metabolicsubstratesofPAH.11

Sincethe1950seffortshavebeenmadetoselect microorgan-ismswithabilitytodegradePAHsfrompurecultures.13_Since

then,manybacteriastrainshavebeenisolatedfortheirability totransform,degradeandutilizePAHsasasourceofenergy and carbon.5,14–17_Hitherto, _it_has_been _recognized_that_few

bacteriahavebeenisolatedthatarecapableofutilizingPAHs withfour ormorearomatic ringsassole sourcesofcarbon andenergy.18,19_{Consequently,}_it_is_of_paramount_importance

toisolateandinvestigateversatiledegradersofPAHs.Inthis study,wereportfortheﬁrsttimetheisolationand charac-terization ofbacterial strains from aformer industrial site inBloomingtoninIndianausingconventionalenrichmentof soilslurry.ThebacteriaPseudomonasplecoglossicidastrainPB1

andPseudomonassp.PB2werescreenedfortheirgrowthand

degradationfluxesinnaphthalene,fluoranthene,pyreneand chrysene.Weproposedamathematicalmodeltoassistinthe simulation ofthe degradationratesofourbacterialspecies ontheselectedPAHs(naphthalene,fluoranthene,pyreneand chrysene).

Materials

and

methods

Chemicals

Thenaphthalene,fluoranthene,pyreneandchryseneof ana-lyticalgradeswerepurchasedfromSigmaAldrichCorp.(St. Louis,MO,USA).Sodiumbenzoate(99+%purity), 2,2,4,4,6,8,8-heptamethylnonane (HMN), and all other organic solvents wereobtainedfromFisherScientificCo.(Springfield,NJ,USA). Hexane, a high purity solvent for GC-chromatograph was obtainedfrom EMDChemicalsInc. Merck.ThePAH analyt-icalstandards were procuredfrom AccustandardInc. (New Haven,CT06513).Allotherchemicalsandreagentsusedwere ofreagentgradeorbetter.

Stocksolutionsandmedia

For the enrichment and degradation experiments, chlo-ride freeminimal salts (MS)mediumas described by20–23,5

were used. The medium consisted of (g) 0.5(NH4)2SO4, 0.1MgSO4·7H2O,0.076Ca(NO3)2·4H2Oand1.0mLeachoftrace metal and vitamin solutions per liter of 40mM phosphate buffer (pH7.25).Naphthalene stocksolutionwereprepared inHMN,anon-degradablecarriertoprovideaninitial concen-trationofca.123ppm.Theconcentrationrepresentsthetotal massinboththeaqueousandHMNphases,dividedbythe aqueousvolume.Theappropriatestocksolutionwasadded using agas-tightsyringe in250-␮Laliquots toprovidetest

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compoundconcentrationofca.100ppminthefinalmedium. Alongside,chrysene,fluorantheneandpyrenestocksolution wereprepareddifferentlybydissolvingtheweightedtest com-poundsinacetonerespectively.Fluoranthene,chryseneand pyrenewere addedfromthedifferent stocksolution ofthe testcompound intothe balchtubes usingaHamilton gas-tightsyringe in250-␮Laliquots, toprovide testcompound concentrationofca.97ppmforfluoranthene,ca.64ppmfor chrysene,andpyreneca.94ppminthefinalmedium.SolidMS mediumwasmadebytheadditionof1.8%Bacto-agar(Difco Laboratories,Detroit,MI,USA).

ThenaphthalenesolutionwasaddedwithHamiltongas tightsyringe250␮Laliquotsintothebalchtubestoprovide testcompoundconcentrationof100ppmintheﬁnalmedium. TheMSmediumwassupplementedwiththetestcompound, achievinganexperimentdependentconcentration.The cul-tureswereincubatedatroomtemperatureonashakertable toaidslowmasstransferofthetestcompoundintothe aque-ousphase. Initialinvestigationswere carried out usingMS medium supplementedwith HMN as the sole carbon and energysourcestodeterminethatHMNdidnotserveasgrowth substrate.

SoilsamplecollectionandenrichmentofPAHdegrading bacteria

Thesoilsampleswerecollectedfromformerindustrialsitesat theMcDoelswitchyardinBloomington,Indiana.Fordecades, the site had been contaminated with PAHs, other organic andinorganicpollutants.Soilsamplesweretakenfrom6to 11cmlayerofthecontaminatedsoilatthreelocations,with indicationsoflowtohighlevelofPAH-contaminationbased onpreliminaryenvironmentalaudit.Thesoilsampleswere placedinseparatesterilejarsandtransportedbacktothelab atambienttemperatures.Thesamplesweredarkin appear-ance.PAH-degrading bacteria were initially isolated bythe conventionalenrichmentmethods.Forthis,5.0gofthe dif-ferentsoilsampleswereweighedinto160mLserumbottles mixedwith30mLofsterileMSmedium.PAH-contaminated soilsmay have limited bioavailability due to sorption and strong hydrophobicity of PAH. Thus pyrene was added in theenrichmentbottlestoserveassupplementalcarbonand energysource. All the 160mL serum bottle bioreactor was set up in triplicates. Theserum bottles were crimp-sealed withteﬂon-coated, butyl rubberstoppersto preventlosses duetovolatilizationand/orsorption.These wereincubated horizontallyonanorbitalshakertable(LablineInstruments Inc.,MelrosePark,IL,USA)atambienttemperature.Air sparg-ingwasdoneweeklytore-aeratetheheadspaceandbiweekly periodictransfersweremadeusingabout15%inoculuminto newMSmediumsupplementedwiththetestPAHs.The pro-cedurewasrepeatedforsevensuccessivetimes.

Determinationofcarbon,hydrogenandnitrogen(CHN) ratioofthesoilsamples

Thecarbon,hydrogenandoxygenratiooftheobtainedsoil samplesfrom thespatialdistributionatMcDoelswitchyard were determined using the PerkinElmer 2400 series CHN analyzer. The sieved soil samples were thoroughly mixed

and oven-driedat85◦Cfor24h.Approximately6mgofthe oven-driedsoilwereweighedintotincapsules.Thecapsules werethenplacedintothePerkinElmer2400seriesCHN ana-lyzer for determination of carbon, hydrogen and nitrogen ratio.

Isolation,puriﬁcationandphylogeneticanalysis

Pureculturesfrompyrene-enrichedmediawereisolatedby directlyplatingaliquots(0.2mL)ofhighly-enrichedcultures ontoMSagar.Becausewewishedtopreventlossofcatabolic plasmids from capableisolates, we usedMS agar medium supplementedwithpyreneratherthannutrientagarto main-tainselectivepressure.Thepyrenewasaddedtothemedium using the spray plate technique as described by Kiyohara et al.24 _Immediately _after_{spread-plating}_the _0.2_mL_aliquot

of enrichmentculture, an ethereal solution of pyrene was uniformlysprayedontothe surfaceofthe agar. Theplates weresealed withparafilmfilmandincubatedfor1weekat 30◦Cindark.Pyrene-degradingmicroorganismswere identi-fiedbyclearedzonesaroundanindividualcolony.Theselected colonywaspurifiedbyrepetitivestreakingonMSagarsprayed withpyrene.Themostefficientbacterialisolateswereselected bycomparisonofgrowthrate,generationtime,andgeneration number.

Thepure culturewere routinely cultured and sustained on solid MSagar plates containing2.5mM pyrene. For 16S rRNAgeneanalysis,genomicDNAwasisolatedfromovernight cultures of isolates growing on 2.5mM benzoate using an UltraCleanMicrobialDNAIsolationkit(MoBioLaboratories, SolanaBeach,CA,USA).ThreeeubacterialPCRprimers; for-ward primer 8fm (AGAGTTTGATCMTGGTCAG) and reverse primers926r(CCGTCAATTCCTTTRAGTTT)and1387r (GGGCG-GWGTGTACAAGGC)wereusedtoamplifythe16SrRNAgene. The reaction mixtures were incubated at 95◦C for 2.5min andthencycled33timesthroughthefollowingtemperature proﬁle:95◦Cfor30s,48◦Cfor30s,and72◦Cfor1.5min, fol-lowedbyasingle10minincubationat72◦C.About 2␮Lof eachampliﬁcationmixturewasanalyzedbyagarosegel elec-trophoresis10.0␮gmL−1(w/v)ethidiumbromidetoestablish thatampliconswereoftheexpectedlength.ThePCR ampli-conswere subsequentlycleanedusingQIAquickNucleotide RemovalKitfromQiagenInc.(Turnberrylane,CA91355).For the16SrRNAsequencing,thePCRproductsweresequenced followinganABIBigDyeTerminatorCycleSequencing reac-tionusinganAppliedBiosystems3730automatedsequencing system(AppliedBiosystems,Inc.,FosterCity,CA,USA).The following settings were applied: denaturation for 3min at 94◦C,25 cycles at96◦C for10s, 50◦C for5s and 60◦C for 4min.Theresultantsequenceswereeditedandalignedusing Bio Edit software version 4.8.7 to check for reading errors and when possible, clarify ambiguities.25 _Sequences _were

subsequently compared withdeposited sequences in Gen-Bank database usingthe BLASTalgorithm availableatURL http://www.ncbi.nlm.nih.gov/BLAST/.26

Growthonthedifferentcarbonandenergysources

Investigationsofthepotentialsofthepureculturestogrowon naphthalene,ﬂuoranthene,pyreneandchrysenewerecarried

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out.ThetestswerecarriedoutinMSmediumsupplemented witheachPAHtestcompoundassolecarbonsource.These investigationswere conductedincrimp-sealedtubes(balch tubes) usually utilized for anaerobic studies. For issues of qualitycontrol/assurance,tubesutilizedforthisstudywere baked in muffle furnace at 500◦C to remove organic con-taminants.Growthanddegradationstudieswereperformed inbalchtubescontaining 10mLofMSmedium, thetested PAH, inoculum,and approximately 15mLair headspace to maintainaerobicconditions.Thedifferenttubes were sup-plemented with different PAHs respectively. Naphthalene was added from an HMN stock solution at a concentra-tionofca.123ppmasdescribed aboveand inoculatedwith 105_cells/mL_of_phosphate_buffer_(pH_7.25)_washed_cells pre-grown in 2.5mM benzoate. All the stock solutions were asepticallypreparedbeforeuse.Fluoranthene,chryseneand pyrene were added from the stocksolution into the balch tubesusingagas-tightsyringein250-␮Laliquots,toprovide test compound concentration ca. 97ppm for fluoranthene, ca.64ppmforchrysene,andpyreneca.94ppminthefinal medium.Balchtubeswerecrimp-sealedwithteflon-coated, butyl rubber stoppers to prevent losses due to volatiliza-tionorsorption.Thetubeswereincubatedhorizontallyona shakertableat(120rev/min)atambienttemperature. Epiflu-orescencemicroscopicexaminationwasutilizedtomonitor growthbycountingthecellsnumbersusingreplicatetubes. The cells were stained with acridine orange stain after fixation with 50␮L of glutraldehyde. The acridine orange stain bind tothe DNA of the cells and is usually used to determinethe totalbacteria cells present.Visual examina-tions in agreement with periodic GC analyses to measure the test compound disappearance was also done. In this study,growthwaspositivewhenthereisanincreasein tur-bidity greater than the killed or abiotic control that was used.For statistical estimate,atleast10 microscopicfields were randomlychosen and aminimum of1000cells were counted.Dataarepresentedasthemeancellnumbers±the SEM.

TransformationofPAHcompounds–naphthalene, ﬂuoranthene,pyreneandchryseneexperiments

ThedegradationstudiesofthePAHs–naphthalene, fluoran-thene,pyreneandchrysenewerelikewiseconductedinthe balchtubes.Thetubeswereinoculatedwiththedifferent bac-terialcultures,crimpsealed and incubated horizontallyon theshakertableatambienttemperature.After14days,the degradation reactions were stopped fornaphthalene while experimentswith fluoranthene, pyrene and chrysene were stopped after21 days. 5mLof hexane was added, vortex-ingfor1–2minandsubsequently,mixedcontinuouslyon a tube rotator for 12h to stop the degradation study. Beck-manGS-6seriescentrifugeat2190rpmfor20minwasused to separate the hexane fraction and the aqueous phase. Thehexaneextractswerecollectedforfurtheranalysis.The extracts were stored in target vials with a headspace of 1mLand crimpsealed usingan11mmTeflonrubber stop-per from National scientific and preserved at4◦C prior to analysis.

Analyticalmethods

Gaschromatographycoupledﬂameionizationdetectorand

statisticalanalysis

ThehexaneextractswereanalyzedonanHP5890SeriesIIgas chromatographyGC(HewlettPackardCo.,PaloAlto,CA,USA) fittedwithanHP3396seriesIIintegratorandequippedwith aflameionizationdetector(FID).Hexaneextracts(5␮L injec-tionvolume)wereinjectedusinga10-␮LHamiltongas-tight syringe through a 30m HP-5 megabore fused-silica capil-larycolumn(J&WScientific,Folsom,CA,USA; 0.32mmid, 0.25␮mfilmthickness).TheGCutilizedhelium(He) asthe carriergaswithalinearvelocityHeflowrate=∼3.33mL/min; H2=∼30mL/min;air=∼400mL/min.Theinjectorand detec-tor temperatures rangedbetween 180and 300◦C withtwo different rates. Theprogram cycle for naphthalenewas at aninitialtemperatureof50◦C;thiswasheldfor5minthen ramped at 30◦C/min to 180◦C for 2min, then ramped to 300◦Cat40◦C/minfor4min.Analytical standards ofPAHs were preparedinhexane. Typicalcoefficientsofcorrelation forstandardcurveswere0.98–0.99.Statisticaltestswas per-formedusingthePrism4.0computersoftwareprogram(Graph PadSoftware,SanDiego,CA,USA)andstatisticalpackagefor socialscientist(SPSS)15.0.

Results

IsolationandphylogeneticcharacterizationofthePAH degradingstrains

Tenmorphologicaldifferentmicrobialcolonieswereselected from the MS agar plates following initial enrichment on pyrene.UponscreeningindividualisolatesforgrowthonMS salicyclic acid, MS benzoate and we selected two isolates for furtherstudy.The colonymorphology ofsomeisolates observedundertheﬂuorescentmicroscopeshoweduniform bacillaryrods,producingnon-ﬂuorescentdiffusibleblue pig-ment.16SrRNAphylogeneticanalysesplacedourstrainsPB-1 andPB-2withinthegenusPseudomonas(Table1).Theclosest relativeofstrainPB-1had99%similarityasP.plecoglossicida

strain 2–3 (EU594553),27 _a _root_organism _isolated_from _root

ofsugarbeet.StrainPB-2had98%homologyasPseudomonas

sp.strainMTQ15(HQ143608),28_isolated_from_the_soil_around

therhizospheresurroundingtobaccoplantinChina.Wehave classiﬁedourisolatesasP.plecoglossicidastrainPB1,and

Pseu-domonassp.strainPB2(GenBankdatabaseaccessionnumbers

JN624752andJN624753issued). DeterminationofCHNratio

Theobtainedquantityofcarbon,nitrogenandhydrogenfrom thesoilsamplesfromMcDoelswitchyardfromdifferent loca-tionswithinthesiteisasshowninTable2.Itwasapparent thateachofthesoilsampleexaminedhadsimilartrendwhere nitrogenbeingthelowestvaluesandcarbonthehighest val-ues.

Degradationofnaphthalene

In this study,we observedthat the initialand ﬁnalvalues (datanotshown)obtainedfortheabioticandbioticcontrols

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Table1–Clonedfragmentsof16SrRNAgenomicDNAofPAHdegradingbacterialspecies.

Bacterialstrain Tentative identity Conﬁrmed identity Closest relative Bacterial subdivision %IDa_with closest relative Genbank accessionno. Length(nt)b PB-1 Pseudomonas putidastrain W30 Pseudomonas plecoglossicida strainPB1 Pseudomonas plecoglossicida strain2–3 (100%) EU594553 ␥-Proteobacteria 99 JN624752 841 PB-2 Pseudomonas sp.strain MTQ15 Pseudomonas sp.PB2 Pseudomonas sp.strain MTQ15(99%) HQ143608 ␥-Proteobacteria 98 JN624753 1299 a _ID_identity. b _nt_nucleotides.

Table2–DeterminationofmeanCHNratio(%w/w).

Soilsample Carbon Hydrogen Nitrogen

Sample1 7.778 0.285 0.178

Sample2 9.745 0.393 0.249

Sample3 14.363 0.816 0.459

had no signiﬁcant difference with the values obtained at the initial stage forthe test samples. The isolated strains PB-1 and PB-2 were evaluated on naphthalene to deter-mine its degradative potentialon the testcompound. The evaluation was done with strains PB-1 and PB-2 washed cells grown on MS-benzoate. We employed no other car-bonsourceotherthan theprovidednaphthalene.Following 14 daysofincubation, strainsPB-1 and PB-2 abilityto uti-lizenaphthalenewasappraisedbycomparing theGCpeak areas of the initial day time (0) and the ﬁnal time (t).

We established the growth of our strains by the intense increase in the turbidity of the test sample and consider-ablereductionintheconcentrationofnaphthalene.(Fig.1A) shows the values of the net reduction (percent reduction in total naphthalene content) in naphthalene concentra-tion. These were 26% and 40% respectively forstrain PB-1 and PB-2. Theinitialconcentrationofnaphthaleneattime zero was ca. 123ppmwhilethe ﬁnal concentrationranged between 65 and 91ppm. The mean biodegradation rate of naphthalenebystrain PB-1 was 0.095±0.004mgL−1h−1.

Naphthalene degradation

PB1 PB2 Killed control Abiotic control

20 40 60 80 100 120 140 PB1 PB2

Killed control Abiotic control

Conc. of napthalene in ppm

A

Growth curve of strains PB1 and PB2 on napthalene

1 2 3 0.1 1 10 100 PB1 PB2 Time in weeks No. of cells/ml x 10 7

B

Fig.1–(A)DegradationofnaphthalenebyMS-benzoategrowncellsofPB-1,PB-2,incubatedfor14days.Datarepresentthe meanandstandarddeviationoftriplicatedeterminationofinitialandﬁnalconcentrationrespectively.Theerrorbars (StandardDeviation)wereduetodifferentialresponseofcellsintriplicatetubes.(B)Naphthlane-dependentgrowthandcell numbersdistributionofstrainsPB-1,PB-2,innaphthaleneincubatedfor14days.Datarepresentthemeanofreplicates tubesforinitialtime(0)celldensityrepresentedas(1)andﬁnaltime(14days)representedas(3)respectively.Thex-axis

valuerangewaschosenassuchtoallowforevenspreadofthegrowthcurve.Thelargeerrorbars(StandardDeviation)were duetodifferentialresponseofcellsintriplicatetubes.

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Pyrene degraders on chrysene

PB1 PB2 Killed control Abiotic control

0 25 50 75 100 PB1 PB2

Conc in ppm

A

1 2 3 4 0.1 1 10 100 PB1 PB2 Time in weeks

Pyrene degraders on chrysene (growth curve)

No of cells/mL x 10

7

B

Fig.2–(A)DegradationofchrysenebyMS-benzoategrowncellsofPB-1andPB-2,incubatedfor21days.Datarepresentthe meanandstandarddeviationoftriplicatedeterminationofinitialandﬁnalconcentrationrespectively.Theerrorbars (StandardDeviation)wereduetodifferentialresponseofcellsintriplicatetubes.(B)Chrysenedependentgrowthandcell numbersdistributionofstrainsPB-1andPB-2,inchryseneincubatedfor21days.Datarepresentthemeanofreplicates tubesforinitialtime(0)celldensityrepresentedas(1)andﬁnaltime(21days)representedas(4)respectively.Thex-axis

valuerangewaschosenassuchtoallowforevenspreadofthegrowthcurve.Theerrorbars(StandardDeviation)weredue todifferentialresponseofcellsintriplicatetubes.

The mean PB-2 biodegradation rate for naphthalene was 0.131±0.005mgL−1h−1.

GrowthofPB-1andPB-2onnaphthalene

In this study, we defined growth as increase in the cell numberofatleastone-order-of-magnitudeandconcomitant disappearanceofthe parentcompoundwhen comparedto the biotic and abiotic control. We determined that growth observedwasnotfromHMNbysupplementingitastheonly carbon source.Itwasobserved thatno appreciablegrowth occurred for strains PB-1 and PB-2 in HMN. Consequently inall cases,cellnumbersincreasedbyasignificant orders-of-magnitudethanthebalchtubesoftheabioticandbiotic control.Thisevidentlydemonstratedgrowthonnaphthalene. (Fig.1B)showstheresultsofgrowthprofilesofstrainsPB-1,2. Cellnumberswerecountedafter7days.StrainsPB-1andPB-2, haddeclineinthecellnumberwhencomparedattime(0)after oneweek.Overthecourseofthisinvestigation,strainPB-2, exhibitedanincreaseinthecellnumbersthatoccurreduntil theendof14daysincubationperiod.Possiblytheobserved declineincellnumbersmaybeduetostressexperiencedby thecellsthatwereactiveatlogphasehavingbeenpre-grown infreshMS-benzoate.

Degradationofchrysene

ThestrainsPB-1 and PB-2consumed chryseneas asource of its energy and carbon sources. Evidently, the strains exhibited increases in their cell numbers. However, each of the strains had a different growth pattern as shown by the growth proﬁlegraph (Fig. 2B). The strain PB-2 was able to degrade more of the chrysene than strain PB-1.

The (mean and standard deviation values) of chrysene used in this investigation was ca. 64ppm. At the end of the 21 days incubation period, the strains degraded about 14–16%ofthechrysene.Theﬁnalconcentration(meanand standard deviation) assessedwas 54ppm. Strain PB-1 con-sumedthechryseneat14%atvolumebiodegradationrateof 0.017±0.011mgL−1h−1.StrainPB-2utilized16%ofchrysene atthevolumebiodegradationrateof0.021±0.009mgL−1h−1. ItwasevidentfromthegrowthproﬁlestudyinFig.2Bthat afterthesecondweek,therewasadeclineinthecell num-bers of PB-1 and PB-2, this may perhaps be due to dead end products or intolerance to the produced intermediate products.

Degradationofﬂuoranthene

TheabilitiesofstrainsPB-1andPB-2todegradefluoranthene was assessedusing washedbenzoate-growncells.The per-centagenet reductionsforfluorantheneare 5%and7%for strainsPB-1andPB-2respectively.Representingthe concen-trationinppmtheinitialconcentrationca.97ppmand the finalca.91ppmthusthissignifiesminimalutilizationof flu-oranthene(Fig. 3A). Themeanbiodegradation rateusedby strain PB-1 was 0.009±0.0001mgL−1h−1. Strain PB-2 con-sumed fluoranthene at the rate of 0.013±0.005mgL−1h−1. From thegrowth profileinFig.3B,thestrainsdidnotshow anylagphasepossiblyduetoendogenousutilizationofthe substrates.Thislasted forabout aweekperiod.Thereafter, therewassharpdeclineinthecellnumbersthatcontinuedto theendoftheinvestigation.Thismaysuggestthatthe orga-nismscouldnottoleratehighconcentrationoffluoranthene. Inaddition,therateofdeclineforstrainPB-1andPB-2were similar.

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PB1 PB2 Killed control Abiotic control 0 20 40 60 80 100 120 PB1 PB2

Conc of fluoranthene in ppm

A

Pyrene degraders on fluoranthene

1 2 3 4 0.1 1 10 100 PB-1 PB-2 (Time in weeks) (10 7 x No. of cells/ml)

B

Pyrene degraders on fluoranthene (growth curve)

Fig.3–(A)DegradationoffluoranthenebyMS-benzoategrowncellsofPB-1andPB-2,incubatedfor21days.Datarepresent themeanandstandarddeviationoftriplicatedeterminationofinitialandfinalconcentrationrespectively.Theerrorbars (StandardDeviation)wereduetodifferentialresponseofcellsintriplicatetubes.(B)Fluoranthenedependentgrowthand cellnumbersdistributionofstrainsPB-1andPB-2,inchryseneincubatedfor21days.Datarepresentthemeanofreplicates tubesforinitialtime(0)celldensityrepresentedas(1)andfinaltime(21days)representedas(4)respectively.Thex-axis

Degradationofpyrene

Thedegradationpotentialofthe MS-benzoatewashedcells ofstrainsPB-1 andPB-2 were evaluatedon pyrene, having exhibited optimum potential in degrading pyrene on MS-agar sprayedpyrene.Nonetheless, the organismsexhibited low consumption of pyrene in balch tubes supplemented with initial concentration of pyrene ca. 94ppm and the ﬁnalconcentration ca. 83ppm After21 daysofincubation, strainPB-1wereabletoconsumeabout8%ofpyreneatthe biodegradation rate of0.016±0.001mgL−1h−1 while strain PB-2 utilized 13% of pyrene at the biodegradation rate of

0.024±0.005mgL−1h−1. In the growth proﬁlein Fig. 4B), it showedthattheorganismshadalagperiod,thatresultedin thedeclineofcellnumbersfollowingtheassaycarriedoutthe ﬁrstweek.Afterthethirdweek,therewasanincreaseinthe cell numbers.Nevertheless,the multiplicationincell num-berswasnotasharplogarithmicgrowth.Itshowedagradual increase.Therearepossibilities,thattheorganismsmightbe synthesizingdifferentmetabolicpathwayhavingbeengrown inMS-benzoatebeforethisevaluation.Theslowrateofthe biodegradationsuggeststhatthesestrainscouldutilizepyrene minimallyinaliquidmediumwhencomparedwithgrowthon thesprayedsolidMS-agar.Inaddition,itisevidentthatthese

PB1 Abiotic control PB2 Killed control 0 25 50 75 100 PB1 PB2 Killed control Abiotic control Conc. of pyrene in ppm

Pyrene degraders on pyrene

A

1 2 3 4 0.1 1 10 100 PB-1 PB-2 (Time in weeks) (10 7 x No. cells/ml)

Pyrene degraders on pyrene (growth curve)

B

Fig.4–(A)DegradationofpyrenebyMS-benzoategrowncellsofPB-1andPB-2,incubatedfor21days.Datarepresentthe meanandstandarddeviationoftriplicatedeterminationofinitialandﬁnalconcentrationrespectively.Theerrorbars (StandardDeviation)wereduetodifferentialresponseofcellsintriplicatetubes.(B)Pyrenedependentgrowthandcell numbersdistributionofstrainsPB-1andPB-2,inchryseneincubatedfor21days.Datarepresentthemeanofreplicates tubesforinitialtime(0)celldensityrepresentedas(1)andﬁnaltime(21days)representedas(4)respectively.Thex-axis

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y =–0.9808x3_+1.4275_x2_{+14.443x–10.54} R2₌₁ Gr owth value Time in weeks

A

B

y=–3.3865x3₊_23.139x2_{–45.016x+27.519} R2₌₁ Gr owth value Time in weeks

Fig.5–(A)PredictivegrowthmodelforPB1inchrysene.(B)PredictivemodelforPB2inchrysene.

organismsmay requirelong adaptationperiodforeffective utilizationofpyrene.

Discussion

ManyPAHs havebeennotedtobetoxicandassuch inter-est in understanding the physicochemical processes and microbial degradation activitiesthat inﬂuencesthesePAHs in soil are important. Soils subjected to a constant con-taminationcan yielda natural selectionof autochthonous pollutant–biodegradingmicrobialpopulation.29 Microorgan-ismsobtainedfromsuchpollutedsoilareefﬁcientinusing hydrocarbonsascarbonandenergysources.Thustheartof applicationofmicroorganismsforthebioremediationof PAH-contaminatedenvironmentisanattractivetechnologyforthe reclamationandsustainabledevelopmentofpollutedsites.In thisstudy,wehaveisolatedandcharacterizedP.plecoglossicida

strainPB1,and Pseudomonassp.strain PB2withcapacityto growandpartiallydegradetheselectedPAHs–naphthalene, ﬂuoranthene,pyreneandchrysene.

Oftentimes,sitescontaminatedwithhydrocarboncould bedifﬁcultreclaiming,partlyduetotheimbalanceinthe car-bon:nitrogen:phosphorus(C:N:P)ratio,causedbyhighcarbon and low nitrogen levels might slowdown or even prevent biodegradation.ThepercentageofC:H:Nratioobtainedfrom thesoilanalyses(samples1,2,3)from theMcDoel switch-yard is as shown in Table 2. An imbalance in the C:N:P ratiohasbeenfoundtoreducethecapacityofmicrobesto formviablebiomassinusingPAHsascarbon/energysource. Van Hamme and co-workers30 _revealed _that _nitrogen _and

phosphoruscontentsgreatlyaffectmicrobialdegradationof hydrocarbon.Besidesorganic compounds ascarbon source (C), productive PAH-metabolizing microorganisms require

inorganicmacronutrients suchasnitrogenandphosphorus for biomass production. Thus the macronutrients require-mentsforC,NandPforbacterialgrowtharenotconstantbut varywithtype,carbonsourceutilizedandthe environmen-talhabitat.Inaddition,Verdeandco-workers,31_reported_that

theamountofNandPrequiredformetabolismofacertain amountofPAHsdependsonthebacterialspeciﬁcgrowthrate, theelementalcompositionofthebacterialcellsformedand themaximumcarbonconversionefﬁciency.Byitself,thePAH conversionrateandgrowthofPAH-degradingspeciesinasoil environmentverymuchreliesontheavailableconcentrations ofNandP.ThusfromtheobtainedresultsofC:H:Nratio,these variationsintheseessentialnutrientsmighthaveaccounted forthehighlevelsofPAHrecordedduringtheenvironmental audit.

The evolution of degradative abilities among bacterial strains may beasaresult oftheplasmids.Plasmidsallow bacterialpopulationstoaccessthehorizontalgenepoolfor adaptivetraitsthatmaybeusefulfortheirsurvivalunderlocal selectivepressuresimposedbyorganicpollutants.

On the assessment of our strains PB-1 and PB-2 for their degradation and growth studies on naphthalene, we believed that the HMN a highly branched alkane did not inducearomatic dioxygenase,thus functionedasintended, i.e., reductionin volatility and facilitated mass transferof naphthaleneintothemedium.Fornaphthalene,theamount utilizedbystrainsPB-1andPB-2were26%and40% respec-tively from initial concentrationca. 123ppm. These values seemedlowwhencomparedtootherbacterialstrainsknown todegradenaphthalene.Thismaysuggestthatprevious expo-sureofourstrainstopyreneduringenrichmentmayinﬂuence thetypeofplasmid/catabolicenzymesexpressedbythe bac-terialspecies.FromthegrowthproﬁleinFig.1B,therewasa declineinthecellnumberwhichsuggeststhattheorganisms

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y=1.3467x3_–8.7575x2_{+14.921x –3E–13} R2=1 Gr owth value

A

B

Time in weeks y=–1.4745x2_{+5.4031x+0.7841} R2_=0.5232 Gr owth value Time in weeks

Fig.6–(A)PredictivemodelPB1innaphthalene.(B)PredictivemodelPB2innaphthalene.

mayhadtoadapttothenewsubstrateandsynthesizenew pathwayforutilization ofnaphthalene.Itmay alsobethat naphthalenecausedaphysiologicalstressonstrainPB-1and PB-2.FromstudiesofFoghtandWestlake,32_a_plasmid_may

encodeacomplete degradativepathwayorpartial degrada-tivestep.Someother plasmidsmaycode forenzymesthat havespecificityforseveralsubstrates.Inaddition,Foghtand Westlakeintheir investigation reported ofgenes encoding the upper and lower pathways ofnaphthalene within the NAHplasmidsofseveralpseudomonads.Thenotedofbroad specificitiesthatallowedthehosttogrowonseveraltwoand three-ringPAHs,assolecarbonandenergysources.Inviewof this,weassessedthedegradativecapacityofstrainPB-1and PB-2onchrysene.From theobtainedvalues,it wasevident thattheycouldutilizechrysenepartiallyat13%and16%from theoriginalconcentrationofabout79ppm.Influoranthene, thebacterialstrains(PB-1andPB-2)utilized4%and7%from theinitialconcentrationof98ppmrespectively.Onpyrene,8% and13%wereutilizedfromtheinitialconcentrationof94ppm. Itisobviousfromthegrowthprofile,thatchryseneandpyrene werepartiallydegradedbythesebacterialstrains.However,a declineincellnumberswasevidentinthefluoranthenestudy. Itcanbededucedfromourobtainedresults,thatthestrains utilizednaphthalene,pyreneandchryseneatslowrate.The slowrateofutilizationofpyrenemaybethattheorganisms werenotablesynthesizeenoughbiosurfactantsthatwillaid masstransferofthesubstrate.

Pyrenea HMW PAH ofubiquitous distribution, environ-mentalpersistence, and potentially with deleteriouseffect

on humanhealth hasprompted the developmentofclean upofpyrenecontaminatedsoils.Conversely,mostbacterial species show limited ability to degradethese hydrophobic compounds.33,34 _From _this_study,_the _strains_PB-1_and _PB-2

maypossessbroadspectrumcatabolicplasmidshavingshown potentialsofutilizingtheHMWPAHs–pyreneandchrysene. Inaddition,thelimiteddegradationofchryseneandpyrene maybeexplainedbasedonthestereochemistryoftheir struc-tures.Chryseneandpyrenehavefourofsixmemberedrings. Theonlydifferenceisintheirarrangement,wherepyreneis fusedtogethertoformacrystallineballstructurewhile chry-seneisalmostlinearcombinationofallrings.Inthisstudy, strains PB-1andPB-2were abletoexhibit similarbehavior tochryseneand pyrenebut nottofluoranthenewiththree sixmembered ring and onefivememberedring. Itgoesto showthatthestereochemistryinfluencestherecalcitranceof mostaromatichydrocarbons.Boldrinandotherworkers35

iso-latedMycobacteriumstrainBB1fromaformercoalgasiﬁcation site.Theorganismhadanexponentialgrowthonsolidpyrene (0.04h−1)assolecarbonandenergysource.Kastnerand co-workers29_isolated_Gordona_sp_BP9_capable_of_using_pyrene_as

asolecarbonandenergysourcefromPAHcontaminatedsite usingtheplatescreeningtechnique.

For the predictive models based on the growth curve plots Figs. 5–8(A and B), it showed that a nonlinear rela-tionship of the polynomial type describes adequately the relationship between both the PB-1 and PB-2 with time. Thus, based on the results of the statistical nonlinear regression modeling presented by the predictive growth

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y=2.4317x3_–17.44x2_{+35.958x–12.38} R2₌₁ Gr owth value Time in weeks

A

B

y=0.0875x2–0.1525x+1.515 R2₌₁ Gr owth value Time in weeks

Fig.7–(A)PredictivemodelPB1inpyrene.(B)PredictivemodelPB-2inpyrene.

plots, the values of the measure of explanatory power of the model (R2₎ _is _indicative _that _the _models _predicts accurately changes in the growth pattern as the time in weeks changes. Hence, variability in the growth pat-terns of the PBs is associated with the length of time of action.

Inconclusion,P.plecoglossicidastrainPB1andPseudomonas

sp.PB2couldpartiallydegradepyrene,chrysene,naphthalene andﬂuorantheneatlowconcentrations.Thepredictivemodel (R2₎_revalidates_the_growth_proﬁle_of_our_bacterial_strains_PB1 and PB2.However,this modelmaybeappliedtodetermine degradationratesofslow-growingPAHdegraders.Asawhole,

y=5.1942x3_–41.372x2_{+97.148x–58.345} R2₌₁ Growth value Time in weeks

A

B

y=3.6345x3–29.114x2+68.985x–42.051 R2=1 Growth value Time in weeks

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theresultssuggestedthatP.plecoglossicidastrainPB1and

Pseu-domonas sp. PB2 would be an excellent candidates with a

potentialforfutureapplicationinthebioremediationof PAH-contaminatedsites.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

Theauthors would like toacknowledge the ﬁnancial sup-portofInstituteofInternationalEducation,administratorsof theFulbrightScholarshipwhichsupportedO.Nwinyiandthe SchoolofPublicandEnvironmentalAffairs Indiana Univer-sity,Bloomington,IN,USA,foradditionalsupport.Theauthors appreciatetheinputsofAssociateProfessorFlynnW.Picardal andAnThuyoftheSchoolofPublicandEnvironmentalAffairs IndianaUniversity,Bloomington,IN,USA.

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s

1.JacquesRJS,OkekeBC,BentoFM,PeralbaMCR,CamaroFAV. Improvedenrichmentandisolationofpolycyclicaromatic hydrocarbons(PAH)-degradingmicroorganismsinsoilusing AnthraceneasaModelPAH.CurrMicrobiol.2009;58:628–634. 2.CheungP-Y,KinkleBK.Mycobacteriumdiversityandpyrene

mineralizationinpetroleum-contaminatedsoils.Appl EnvironMicrobiol.2001;67:2222–2229.

3.PengRH,XiongAS,XueY,etal.Microbialbiodegradationof polyaromatichydrocarbons.FEMSMicrobiolRev.

2008;32:927–955.

4.Maliszewska-KordybachB.Sources,concentrations,fateand effectsofpolycyclicaromatichydrocarbons(PAHs)inthe environment.PartA:PAHsinair.PolJEnvironStud. 1999;8:131–136.

5.NwinyiOC,PicardalFW,ThuyA,AmundOO.Aerobic degradationofnaphthalene,ﬂuoranthene,pyreneand chryseneusingindigenousstrainsofbacteriaisolatedfrom anoldpollutedindustrialsite.CanJPureApplSci.

2013;7:2303–2314.

6.SamantaSK,SinghOV,JainRK.Polycyclicaromatic

hydrocarbons:environmentalpollutionandbioremediation. TrendsBiotechnol.2002;20:243–248.

7.USEPACleanAirAct(42USC,7401–7626).Washington,DC: UnitedStatesEnvironmentalProtectionAgency.Availableat http://www.epa.gov/air/caa/2004;Accessed2014.

8.PreussR,AngererJR,DrexlerH.Naphthalene–an environmentalandoccupationaltoxicant.IntArchOccup EnvironHealth.2003;76:556–576.

9.Umweltbundesamt.Naphthalin/naphtholeandhuman biomonitoring.BundesgesundheitsblGesundheitsforsch Gesundheitsschutz.2007;50:1357–1364.

10.BogenKT,BesonJM,YostGS,MorrisJB,DahlAR,ClewellHJ. Naphthalenemetabolisminrelationtotargettissue anatomy,physiology,cytotoxicityandtumorigenic mechanismofaction.RegulToxicolPharmacol.2008;51:27–36. 11.JohnsenAR,WickLY,HarmsL.Principlesofmicrobial

PAH-degradationinsoil.EnvironPoll.2005;133:71–84. 12.RegonneRK,MartinF,MbawalaA,NgassoumMR,Jouanneau

Y.Identiﬁcationofsoilbacteriaabletodegrade

phenanthreneboundtoahydrophobicsorbentinsitu. EnvironPoll.2013;180:145–151.

13.CernigliaCE.Fungalmetabolismofpolycyclicaromatic hydrocarbons:past,presentandfutureapplicationsin bioremediation.JIndMicrobiolBiotechnol.1997;19:324–333. 14.WikadaJ,NojiriH,KasugaK,YoshidaT,HabeH,OmoriT. Moleculardetectionanddiversityofpolycyclicaromatic hydrocarbon-degradingbacteriaisolatedfrom

geographicallydiversesites.ApplMicrobiolBiotechnol. 2002;58:202–209.

15.MroczekA,Piotrowska-SegetS,LabuzekA.Bacterial degradationandbioremediationofpolycyclicaromatic hydrocarbons.PolishJEnvironStud.2003;12:15–25.

16.AndreoniV,CavalcaL,RaoMA,etal.Bacterialcommunities andenzymeactivitiesofPAHspollutedsoils.Chemosphere. 2004;57:401–412.

17.HickeyAM,GordonL,DobsonAD,KellyCT,DoyleEM.Effect ofsurfactantsonﬂuoranthenedegradationbyPseudomonas alcaligenesPA-10.ApplMicrobiolBiotechnol.2007;74:851–856. 18.KanalyRA,HarayamaS.Biodegradationof

high-molecular-weightpolycyclicaromatichydrocarbonsby bacteria.JBacteriol.2000;182:2059–2067.

19.DavidovaIA,GiegLM,DuncanKE,SuﬂitaJM.Anaerobic phenanthrenemineralizationbyacarboxylating sulfate-reducingbacterialenrichment.ISMEJ. 2007;1:436–442.

20.KimS,PicardalFW.Anovelbacteriumthatutilizes monochlorobiphenylsand4-chlorobenzoateasgrowth substrates.FEMSMicrobiolLett.2000;185:225–229.

21.NwinyiOC,NwodoCS,AmundOO.Biodegradationpotential oftwoRhodococcusstrainscapableofutilizinganilineas carbonsourceintropicalecosystem.ResJMicrobiol. 2008;3:99–104.

22.NwinyiOC.Degradationofaskarel(PCBblend)byindigenous aerobicbacteriaisolatesfromdumpsitesinOre,OndoState, Nigeria.AustJBasicApplSci.2010;4:3938–3948.

23.NwinyiOC.EnrichmentandIdentiﬁcationofAskareloil(PCB blend)degradingbacteriaenrichedfromlandﬁllsitesinEdo State,Nigeria.AgricBiolJNorthAm.2011;2:89–100.

24.KiyoharaH,NagaoK,YanaK.Rapidscreenforbacteria degradingwaterinsoluble,solidhydrocarbononagarplates. ApplEnvironMicrobiol.1982;43:454–457.

25.HallTA.BioEdit:auser-friendlybiologicalsequence alignmenteditorandanalysisprogramforwindows 95/98NT.NucleicAcidsSympSer.1999;41:95–98.

26.AltschulSF,MaddenTL,SchafferAA,etal.GappedBLAST andPSI-Blast:anewgenerationofproteindatabasesearch programs.NucleicAcidsRes.1997;25:3389–3402.

27.ShiY.StudiesonCommunitiesofEndophyticMicroorganismsand

DynamicAnalysisfromSugarBeetontheNorthSlopeofXinjiang

TianshanMountain;2008.www.ncbi.nlm.gov;Accessed

2014.

28.JinF,DingY,DingW,ReddyMS,FernandoWG,DuB.Genetic diversityandphylogenyofantagonisticbacteriaagainst PhytophthoranicotianaeisolatedfromTobaccoRhizosphere. IntJMolSci.2011;12:3055–3071.

29.KastnerM,Breuer-JammaliM,MahroB.Enumerationand characterizationofthesoilmicroﬂorafrom

hydrocarbon-contaminatedsoilsitesabletomineralize polycyclicaromatichydrocarbons.ApplMicrobiolBiotechnol. 1994;41:267–273.

30.VanHammeJD,SinghA,WardOP.Recentadvancesin petroleummicrobiology.MicrobiolMolBiolRev. 2003;67:503–549.

31.VerdeK,HeldalM,NorlandS,BratbakG.Elemental composition(C,N,P)andcellvolumeofexponentially growingandnutrient-limitedbacterioplankton.ApplEnviron Microbiol.2002;68:2965–2971.

(12)

32.FoghtJM,WestlakeDW.Transposonandspontaneous deletionmutantsofplasmid-bornegenesencoding polycyclicaromaticdegradationbyastrainofPseudomonas ﬂuorescens.Biodegradation.1996;7:353–366.

33.LundstedtS[M.Sc.dissertation]AnalysisofPAHsandTheir TransformationProductsinContaminatedSoilandRemedial Processes.Sweden:DepartmentofChemistry-Environmental Chemistry,UmeåUnversity;2003,45pp.

34.WilsonSC,JonesKC.Bioremediationofsoilcontaminated withpolynucleararomatichydrocarbon(PAHs)–areview. EnvironPoll.1993;81:229–249.

35.BoldrinB,TiehmA,FritzscheC.Degradationof phenanthrene,ﬂuorine,ﬂuorantheneandpyrenebya Mycobacteriumsp.ApplEnvironMicrobiol.1993;59:1927–1930.