Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

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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

Characterization

of

dioxygenases

and

biosurfactants

produced

by

crude

oil

degrading

soil

bacteria

Santhakumar

Muthukamalam,

Sivalingam

Sivagangavathi,

Dharmapal

Dhrishya,

Sadras

Sudha

Rani

∗

DepartmentofBiochemistryandMolecularBiology,SchoolofLifeSciences,PondicherryUniversity,Puducherry,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12April2016

Accepted15February2017

Availableonline3June2017

AssociateEditor:LucySeldin

Keywords: Crudeoil Biodegradation Bacteria Catecholdioxygenase Biosurfactant

a

b

s

t

r

a

c

t

Roleofmicrobesinbioremediationofoilspillshasbecomeinevitableowingtotheireco

friendlynature.Thisstudyfocusedontheisolationandcharacterizationofbacterialstrains

withsuperioroildegradingpotentialfromcrude-oilcontaminatedsoil.Threesuch

bacte-rialstrainswereselectedandsubsequentlyidentiﬁedby16SrRNAgenesequenceanalysis

asCorynebacteriumaurimucosum,AcinetobacterbaumanniiandMicrobacterium

hydrocarbonoxy-dansrespectively.Thespeciﬁcactivityofcatechol1,2dioxygenase(C12O)andcatechol2,3

dioxygenase(C23O)wasdeterminedinthesethreestrainswhereintheactivityofC12O

wasmorethanthatofC23O.Amongthethreestrains,Microbacteriumhydrocarbonoxydans

exhibitedsuperiorcrudeoildegradingabilityasevidencedbyitssuperiorgrowthratein

crudeoilenrichedmediumandenhancedactivityofdioxygenases.Alsodegradationoftotal

petroleumhydrocarbon(TPH)incrudeoilwashigherwithMicrobacterium

hydrocarbonoxy-dans.Thethreestrainsalsoproducedbiosurfactantsofglycolipidnatureasindicateddby

biochemical,FTIRandGCMSanalysis.Theseﬁndingsemphasizethatsuchbacterialstrains

withsuperioroildegradingcapacitymayﬁndtheirpotentialapplicationinbioremediation

ofoilspillsandconservationofmarineandsoilecosystem.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Oilpollutionisaperpetualproblemthataffectsterrestrialas

wellasmarineecosystems.Oilspillagemayariseeither

acci-dentallyoroperationallyduringproduction,transportation,

storage,processingorwhenusedatseaoronland.1_Crude_oil

∗ _{Corresponding}_author_at:_Department_of_Biochemistry_and_Molecular_Biology,_School_of_Life_sciences,_Pondicherry_University,_Puducherry

605014,India.

E-mails:dr.ssrlab@gmail.com,sadrassudha@yahoo.com(S.SudhaRani).

isamultifariousmixtureofmanypetroleum hydrocarbons

ofwhichpolycyclicaromatichydrocarbons(PAH)constitutea

majorfraction2_which_invite_greater_attention_owing_to_their

toxic,mutagenic andcarcinogenic nature.3 _The_recovery_of

spilledcrudeoilcouldbeachievedbyadoptingphysicaland

chemicalmethodsbuttheycantakecareofonly10–15%of

oilspillage.Ontheotherhand,bioremediationbyexploiting

http://dx.doi.org/10.1016/j.bjm.2017.02.007

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complex microbial communities can act as a self-driven,

economicalandeco-friendlymethod.Highlyhazardousoily

materialscaneasilybemineralizedtoharmlessendproducts

by introducing suitable microbial strains.4 _Poly _aromatic

hydrocarbons (PAH) can act as carbon source for certain

microbial communitiesinoil-polluted environment.5 _Many

bacterial species including Pseudomonas aeruginosa, Bacillus

subtilis, Bacillus megaterium, Corynebacterium kutscheri, have

been reported to possess oil degrading potential.6,7 _The

biochemical process of crude oil degradation by microbes

involves the action of several enzymes including

oxyge-nases, dehydrogenases and hydroxylases that fragment

aromaticandaliphatichydrocarbons.Amajorconstraintin

bio-degradationofoilisitshydrophobicitybutbiosurfactants

producedbyoildegradingbacteriafacilitateuptakeof

hydro-carbonsbythebacterialcells.Hence,thosebacterialstrains

that possess the ability to produce biosurfactants along

with enhanced oil degrading capacity are widely

recom-mendedforuseinordertoachievefastdegradationofcrude

oil.8

In this study, among the 12 bacterial isolates from oil

contaminatedsoilsamples,threestrainswhichhadthe

iso-late designation of PA, PM and BM were selected owing

totheir superior oildegrading activity.These threestrains

weresubsequently identiﬁedby16SrRNAgenesequencing

asCorynebacteriumaurimucosum,Acinetobacterbaumanniiand

Microbacterium hydrocarbonoxydans respectively. With these

three strains, their ability to utilize crude oil as the sole

carbonsource,determinationofactivity ofenzymes –

Cat-echol1,2dioxygenase(C12O) andCatechol 2,3dioxygenase

(C23O) and characterization of biosurfactants produced by

themwere carriedout.Additionally,the extentof

degrada-tionofaliphaticandaromaticcompoundsoftotalpetroleum

hydrocarbon(TPH)incrudeoilbythesestrainswasalso

ana-lyzed.

Materials

and

methods

Samplingandisolationofcrudeoildegradingbacterial

strains

Crudeoilandsoilsamplesfromcrudeoilcontaminatedsites

were collected from Indian OilCorporation Limited (IOCL),

Chennai,India.Fortheisolationofcrudeoildegrading

bacte-ria,1gofsoilsamplewasinoculatedinto100mLofmineral

saltmedium(MSMpH6.8±0.2)supplementedwith1%crude

oil9 _and _incubated _at _room _temperature _for_1-week. _From

this,1mLofactiveinoculumwasagaintransferredtofresh

MSMcontaining1%crudeoilandwasusedfortheisolation

ofcrudeoildegrading bacteriabyserial dilution technique

followedbyspreadplatetechniqueusingnutrientagar(NA)

plates (pH 7.4±0.2). Thebacterial colonies were then

cul-turedonfreshMSMagarplatessupplementedwith1%(w/v)

crudeoilandthosebacterialcoloniesgrownwerepickedand

preservedinliquidnutrientbrothcontaining70%glycerolat

−80◦_C.

DeterminationofbacterialgrowthandactivityofC12O

andC23Oenzymes

TheabilityofPA, PMandBMtoutilizecrudeoilascarbon

source was assessed bymeasuring their growth ascolony

formingunits9_(log_CFU)_in_MSM_containing_different

concen-trations(1,2and3%) ofcrudeoil(incubatedat30◦Cinan

orbital shakerat200rpm)for13daysat24hintervals.The

enzymeactivityofC12OandC23Owerealsodeterminedinthe

cellfreeextractsofPA,PMandBMculturesat24hintervals

for13days.Forthispurpose,cellswereharvestedby

centrifu-gationat5000rpm(4◦C),washedtwicewith0.01Mphosphate

buffer(pH7.0),subjectedtosonication(15s)andthecellfree

extracts were collected by centrifugation at20,000rpmfor

25min(4◦C).TheactivityofC12Owasdeterminedby

detec-tingthelevelsofcis,cis-muconate10_and_C23O_based_on_the

formation of 2-hydroxymuconic semialdehyde11 _by_UV–Vis

spectrophotometer(Shimadzu,UV-Pharmaspec1700).Protein

concentrationwasdeterminedbytheBradfordmethod.12

PuriﬁcationofC12O

ThepartialpuriﬁcationofC12Ofromthecellfreeextractsof

PA,PMandBMwascarriedoutbyammoniumsulphate

pre-cipitation followedbyionexchangechromatography (DEAE

cellulose).Thecellfreeextractwastreatedwithcoldsaturated

(NH4)2SO4solution(pH7.5)togive40,60and80%saturation

andtheprecipitatesformedwerecollectedbycentrifugation

at10,000rpmfor20min(4◦C).Thepelletsweredissolvedin

Tris–HClbuffer50mM,(pH7.5)andtheresultingprotein

solu-tionsweredesaltedbydialysis.13_The_dialyzed_protein_sample

wasthenloadedontoaDEAEcellulosecolumn(10cm×2cm)

thathadbeenpre-equilibratedwith50mMTris–HCl(pH7.5).

Elutionofproteinwascarriedoutwith50mMTris–HCl(pH7.5)

buffercontainingNaClinalineargradientfrom0.1to0.5M.14

Thefractionscollectedwereanalyzedforenzymeactivityand

those fractions withhighestactivity were pooledand

sub-jected to SDS PAGE analysis15 _to _determine _the _molecular

weightandthepurityoftheC12O.Molecularweightmarker

(97.4–14.3kDa)(Merckbioscience)wasusedinthisanalysis.

Physicochemicalandbiochemicalcharacterizationof

biosurfactants

Tocharacterize thebiosurfactants producedbyPA,PM and

BM, thebacterialstrains were inoculatedindividuallyinto

MSMbrothcontaining1%crudeoilandincubatedat30◦Cfor

5daysinashaker(200rpm).Thecell freesupernatantwas

collectedbycentrifugationat10,000rpmfor20minandwas

subjectedtophysicochemicalandbiochemical

characteriza-tion.Theemulsiﬁcationindex(E24)16andtheoildisplacement

capacity17_of_the_cell_free_supernatant_was_determined.

Qual-itativeanalysistodetectglycolipidsand lipoproteinsinthe

cell free supernatants of PA, PM and BM was carried out

respectivelybyPhenolsulfuricacid18_and_Bradford_Method.12

Thesugarcontentwasquantitativelydeterminedbyorcinol

(3)

ExtractionandcharacterizationofbiosurfactantsbyFTIR

andGCMSanalysis

ThebiosurfactantsfromthecellfreesupernatantsofPA,PM

andBMwereextractedbytheacidprecipitationmethod20_and

theextractedbiosurfactantsweresubjectedtoTLC(silicagel

60F254)analysisusingChloroform:methanol:water(65:25:4,

v/v)solventsystem.Theseparatedbandswerevisualizedwith

iodinevapor.Theywere dissolvedinchloroform:methanol

(2:1,v/v) and subjected toFTIR analysis21 _(Thermo_Nicolet

6700)intherangeof4000–400cm−1.Theextracted

biosurfac-tantswerealsosubjectedtoGCMSanalysisforidentiﬁcation

ofcomponentfattyacids(GCMS-PerkinElmerClarus680)and

thespectrawerecomparedwiththeGCMSNIST(2008)library

compounds.

Biodegradationofcrudeoilanddeterminationoftotal

petroleumhydrocarbon(TPH)

To ascertain the crude oil degradation capacity of PA, PM

and BM, the TPH content of residual crude oilwas

deter-mined after culturingthe bacteria in MSM with 1% crude

oil for 13 days while the uninoculated medium served as

control. 500mg of residual crude oil was taken from the

mediumandextractedconsecutivelywithhexane,benzene,

chloroform and methanol.22 Theextracts were evaporated,

pooledtogether and dissolved inn-pentane and separated

into soluble and insoluble fractions and the TPH content

was determined by the gravitational method as further

(asphaltene). The TPH fraction was loaded on to a silica

gel column (100–200mesh) to separate aliphatic and

aro-matic fractions byusing hexane and benzene respectively.

Thealiphatic and aromaticfractionsofTPHwere analyzed

by GCMS (PerkinElmer Clarus 680) and the spectra was

comparedwith the library compounds (GC-MSNIST 2008).

Thepercentageof crude oildegradation was calculated as

[(control-treated)/control]*100.22 _{Quantiﬁcation} _of

hydroxy-lated aromatic compounds as an indicator of degradation

of polycyclic aromatic hydrocarbon (PAH) was determined

asresorcinolequivalent(RE)22_where_Resorcinol_served_as_a

standard.

Identiﬁcationofcrudeoildegradingbacteriaby16SrRNA

genesequencing

Genomic DNA from the strains PA, PM and BM was

iso-lated by the phenol chloroform extraction method.23

Ampliﬁcation of 16S rRNA gene from genomic DNA

was performed using universal forward and reverse

primers fD1 (5-GAGTTTGATCCTGGCTCA-3) and rP2 (5

-ACGGCTACCTTGTTACGACTT-3) in DNA thermo cycler

(Eppendorf,Mastercyclergradient).TheampliﬁedPCR

prod-ucts were puriﬁed and sequencing24 _was _carried _out _by

EuroﬁnsGenomicsIndiaPvt Ltd,Bangalore.Thenucleotide

sequences obtained were compared with sequences of

GenBankusing nBLAST search.The phylogenetic tree was

constructedbyusingMEGA5softwareversion5.1.25

Results

In thisstudy, 12bacterial strainswere isolated from crude

oilcontaminatedsoilsamplesbyusingMSMwith1%crude

oil.From these12strains, 3potentstrainswiththeisolate

designationofPA,PMandBMwereselectedbasedontheir

improvedcrudeoildegradingcapacity.Thecapacityofthese

strainstousecrudeoilassolecarbonsourcewasdetermined

byanalyzing the activity ofcatechol dioxygenaseenzymes

(C12OandC23O),characterizingthebiosurfactantsproduced

bythemandbymeasuringtheresidualTPHcontent.Thethree

strainswerealsosubjectedtomolecularidentiﬁcationby16S

rRNAgenesequenceanalysis.

DeterminationofgrowthcurveandC12OandC23O

activity

PureculturesofPA,PMandBMwereallowedtogrowonMSM

containingdifferentconcentrationsofcrudeoil(1,2and3%

(v/v)for13daysandduringthisperiodgrowthrateofbacterial

strains,theirproteincontentandactivityofC12OandC23O

weredeterminedat24hintervals.Amongthethreestrains,

PA and PMshowed maximumgrowth in mediacontaining

1%crudeoilwhileBMdisplayedmaximumgrowthinmedia

containing 3%crude oil. Exponential growth was observed

betweendays2and10forPA,2and7forPMand2and8for

BM(Fig.1a,dandg).AsPAandPMshowedoptimumgrowth

inpresenceof1%crudeoilandBMwith3%oil,theywere

cul-turedinMSMwithappropriateconcentrationofcrudeoilin

ordertodeterminetheactivityofenzymes–C12OandC23O.

Concomitantincreaseinproteincontent(Fig.1b,eandh)was

alsoobservedinallthethreestrainsafterthe5thdayof

incu-bation andpeakproteinlevelswereobservedondays10,7

and8respectivelyforPA(0.991mgmL−1),PM(0.891mgmL−1)

andBM(1.49mgmL−1).Thistrendclearlypointedtothe

abil-ity ofthreebacterialstrainstoutilizecrudeoilasasource

ofcarbon and energy. Ascould beseen inFig. 1c,f and i,

activityofC12OaswellasC23Oincreasedwithdaysof

incu-bationandthemaximumactivitywasattainedonday8for

PA,day4forPMandday5forBM.Thisincreasingtrendin

enzymeactivitycouldbecorrelatedwellwiththe

exponen-tialgrowthshownbythethreebacterialstrainsbyutilizing

crudeoilascarbonsource(Fig.1a,dandg).Another

interest-ingobservationmadeinthisstudywasthespeciﬁcactivity

ofC12OwashigherthanthatofC23Oinallthethreestrains

(Fig.1c,f andi).ThisincreasedactivityofC12Osignify the

roleofortho-cleavagepathwayasthepredominantpathway

ofoildegradation.Amongthethreestrainsusedinthisstudy,

BMexhibitedincreasedactivityofbothC12OandC23Owith

maximumspeciﬁcactivityof1.75U/mg−1 and0.973U/mg−1

respectively.Thisﬁndingmaybecorrelatedwiththeability

ofBMtoutilizehigherconcentrationofcrudeoilandshow

maximumgrowthinMSMcontaining3%crudeoil.Theother

twostrainsPAandPMalsoexhibitedmoderatelevelsof

activ-ityofC12OandC23O.ThemaximumspeciﬁcactivityofC12O

was0.967and0.436Umg−1andthatofC23Owas0.537and

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a

PA PA PA PM BM PM BM BM PM PA PM BM 10 6 15 10 5 0 4 2 0 0 5 10 15 0 5 10 15 8 6 4 2 0 1.5 1.0 2.0 1.5 1.0 0.5 0.0 0.8 0.6 0.4 0.2 0.0 1.0 0.5 0.0 1.0 0.5 _2.0 1.5 1.0 0.5 0.0 0.4 0.3 0.2 0.1 0.0 0.8 0.6 0.4 0.2 0.0 01 2345 678 9 10 11 12 13 0 1 23 4 56 78 9 10 11 12 13 01 23 45 6789 10 11 12 13 0.0 0.0 0.0 0.5 1.0 1.5 0.1 0.2 0.3 0.4 0.5 0.5 1.0 1.5 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15

Incubation period (days)

Incubation period (days) Incubation period (days) Incubation period (days) Incubation period (days) Incubation period (days) Incubation period (days) Incubation period (days) Control 1% 2% 3% Control 1% 2% 3% Control 1% 2% 3% log CFU mI –1 Protein concentration (mg mI –1 ) C12O Umg –1 C12O Umg –1 C12O Umg –1 C23O Umg –1 C23O Umg –1 C23O Umg –1 C23O Umg–1 C12O Umg–1

C23O Umg–1 _{C23O Umg}–1

C12O Umg–1 C12O Umg–1 Protein concentration (mg mI –1 ) Protein concentration (mg mI –1 ) log CFU mI –1 log CFU mI –1

d

g

e

h

f

i

b

c

Fig.1–Growthcurvesofthethreebacterialstrains–(a)PA,(d)PMand(g)BMinthepresenceofdifferentconcentrations ofcrudeoil(1%,2%and3%v/v),proteincontent(mgmL−1)in(b,eandh)andSpeciﬁcactivities(U=1␮molofthe product/min/mgprotein)ofC12O(Umg−1)andC23O(Umg−1)ofbacterialstrainsgrowninMSMwithcrudeoil (concentrationin%)–(c,fandi)PA-1%,PM-1%andBM-3%.

PartialpuriﬁcationofC12Oenzymeanddeterminationof

molecularmass

AstheactivityofC12Owasfoundtobemorethan C23Oin

PA,PMand BM,partialpuriﬁcationofC12Oalone was

car-riedweresubjected toammoniumsuphateprecipitation at

40%,60%and80%saturationsandtheprecipitatesobtained

werecheckedforenzymeactivityafterdesaltingthepelletsby

dialysis.Astheprecipitateobtainedat60%saturationshowed

higherC12Oactivity,thesamewasusedforfurther

puriﬁca-tionbyion-exchangechromatographyusingDEAEcellulose

column.Thecolumnwaselutedwith0.1–0.5MNaClgradient

andfractionsobtainedat0.3MNaClwerefoundtoexhibit

bet-terenzymeactivity.ThespeciﬁcactivityofC12OfromPA,PM

andBMindifferentpuriﬁcationstepsaredepictedinTable1.

Amongthethreestrains,BMshowedreasonablyhigher

spe-ciﬁcactivityforC12Owith6.37Umg−1followedbyPAandPM

withrespectivespeciﬁcactivitiesof5.64and1.8Umg−1

follow-ingpuriﬁcationbyionexchangechromatography.Theparially

puriﬁedfractionofC12Ofromion-exchangecolumnshowed

adiscreteproteinbandwithanapproximatemolecularmass

of39KDainSDS-PAGE(Fig.2).

Characterizationofbiosurfactants

Severalmicroorganismsthatutilizepetroleumhydrocarbons

astheircarbonsourceareknowntoproducebiosurfactants

Lane 1 Lane 2 66 KDa 43 KDa 29 KDa 20.1 KDa 14_3 KDa Lane 3 Lane 4

Fig.2–SDSPAGEanalysisofpartiallypuriﬁedenzyme C12OfrombacterialstrainsPA,PMandBM.Lane1is Proteinmolecularmarker,lane2,3and4are0.3MNaCl elutedfractionofC12OenzymefromBM,PAandPM obtainedbyionexchangechromatography.

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Table1–PurificationofC12OenzymefromPA(Corynebacteriumaurimucosum),PM(Acinetobacterbaumannii)andBM (Microbacteriumhydrocarbonoxydans). Bacterial strains PA PM BM Purification steps Bacterial celllysate (NH4)2SO4 precipitation (60%) DEAE cellulose (0.3MNaCl) Bacterial celllysate (NH4)2SO4 precipitation (60%) DEAE cellulose (0.3MNaCl) Bacterial celllysate (NH4)2SO4 precipitation (60%) DEAE cellulose (0.3MNaCl) Volume(mL) 10 5 3 10 5 3 10 5 3 Protein (mg/mL) 2.84 1.11 0.86 1.35 0.98 0.42 3.58 1.64 0.98 Activity(U) 28.12 21.7 14.56 4.12 3.25 2.32 65.87 30.01 18.55 Specificactivity (Umg−1) 0.99 3.91 5.64 0.31 0.66 1.84 1.84 3.66 6.37 Purification fold 1 3.94 5.7 1 2.17 6.03 1 1.98 3.73 Recovery(%) 100 38.59 15.52 100 39.44 16.89 100 22.45 8.44

of glycolipid or lipoprotein nature. In this study,

biosur-factants produced by PA, PM and BM were characterized

byphysicochemical,biochemical, FTIR and GCMS analysis.

Thephysicochemical properties ofthe biosurfactants were

analyzed by determining the emulsiﬁcation index (E24) %

and oil displacement capacity ofthe culturesupernatants

of PA, PM and BM. The emulsiﬁcation index for PA, PM

and BM was 57, 45 and 69% respectively with BM

show-inghigher index.Thethree bacterialstrainsalsoexhibited

wellpronouncedoildisplacementpropertywithrespectiveoil

clearancezoneof16,14 and17mm.Thechemical natures

ofthe biosurfactants from culture supernatants ofPA, PM

and BM were identiﬁed qualitatively as glycolipids while

theygavenegativeresultforproteins.Further,quantitative

analysisforglucosecontent inthe culturesupernatantsof

PA, PM and BM showed 135, 120 and 187mg/L of glucose

respectively.

CharacterizationofbiosurfactantsbyFTIRandGCMS

analysis

ThebiosurfactantsextractedfromPA,PMandBMwas

sub-jectedtoFTIRanalysisandtheFTIRspectraarepresentedin

Fig.3.Thebroadbandat3543,3552and3238cm−1observed

respectivelyforPABMandPMshouldbeassignedtotheO–H

stretching vibration in the chemical structuresof the

bio-surfactants.The strong peaks observed at2981, 1469cm−1

for PA, 2960cm−1 for PM and 2870, 2773, 2698cm−1 from

BMcorrespondtotheC–H stretching vibrationsoftheCH2

and CH3 hydrocarbon chains. The characteristic peak

dis-playedat1745, 1742and 1769cm−1 respectivelyforPA,PM

andBMrelatestotheC Ostretchingvibrationsofthe

car-bonylgroups.Samewaythepeaksobservedat1242forPA,

1282forPMand1132,1088and1011cm−1forBMrepresent

C-Ostretchingbandsthatconﬁrmthepresenceofglycosidic

bondsformedbetweencarbon atomsandhydroxylgroups.

ThebiosurfactantsthatwereextractedfromPA,PMandBM

wasfurtheranalyzedbyGCMSanalysistodetect the

pres-enceoffattyacidsandthedataarepresentedinFig.4.The

GCMSspectraofPAPMandBMshowedfourmajorpeaksand

thecorresponding fatty acids/fatty acid estersare listed in

Table2.

a

PA PM BM 100 100 100 90 80 70 80 80 60 60 40 40 20 0 0 20 40 40 60 60 80 80 90 80 70 100 100 100 4000 4000 3000 3000 2000 2000 Wavenumbers (Cm–1₎ Wavenumbers (Cm–1) Wavenumbers (Cm–1₎ T ransmittance (%) T ransmittance (%) T ransmittance (%) 1000 1000 4000 3000 2000 1000

b

c

Fig.3–FTIRspectrumofbiosurfactantsproducedby bacterialstrainsPA(a),PM(b)andBM(c).

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a

b

c

d

e

f

B1-(16ES-0648) 2369 (14.648)

B1-(16ES-0648) 2708 (16.344)

B2-(16ES-0649) 1172 (8.661)

B2-(16ES-0649) 2384 (14.723)

B3-(16ES-0650) 2232 (13.963)

B3-(16ES-0650) 2830 (16.954)

Nist 149203: 1,2,4,-Benzenetricarboxylic acid, 4-dodecyl dimethyl ester

Nist 196965: Octadecanoic acid, 2-OXO-, Methyl ester

Nist 21781: Heptadecanoic acid, Heptadecyl ester

Nist 6636: Octadecanoic acid, 9,10-Epoxy-, Isopropyl ester

Nist 22299: Eicosane, 9-Cyclohexyl-R:791

R:703 R:816 R:840 R:458

Nist 143997: Tetradecanoic acid, 2-OXO-, Ethyl ester R:691

Fig.4–GCMSspectraofbiosurfactantsfromPA(aandb),PM(candd)andBM(eandf).

Table2–ListofcompoundsidentiﬁedinBiosurfactantsfromPA(Corynebacteriumaurimucosum),PM(Acinetobacter

baumannii)andBM(Microbacteriumhydrocarbonoxydans).

RT Compoundname(fromLibrarysearch) Molecularformula Molecularweight

StrainPA

4.954 1,6;3,4-Dianhydro-2-deoxy-beta-d-lyxo-hexopyranose C6H8O3 128

14.648 Tetradecanoicacid,2-oxo-,ethylester C16H30O3 270

16.334 1,2,4-Benzenetricarboxylicacid,4-dodecyldimethylester C23H34O6 406

20.155 Nonahexcontanoicacid C69H138O2 998

StrainPM

RT Compoundname(fromLibrarysearch) Molecularformula Molecularweight 8.661 Octadecanoicacid,2-oxo-,methylester C19H36O3 312

10.962 Oxalicacid,allylpentadecylester C20H36O4 340

12.182 Oxiraneoctanoicacid,3-octyl-,2-Ethylhexylester C26H50O3 410

14.723 Heptadecanoicacid,heptadecylester C34H68O2 508

StrainBM

RT Compoundname(fromLibrarysearch) Molecularformula Molecularweight 13.208 Oxalicacid,allyltetradecylester C19H34O4 326

13.963 Octadecanoicacid,9,10-Epoxy-,Isopropylester C21H40O3 340

16.954 Eicosane,9-cyclohexyl- C26H52 364

17.354 Fumaricacid,Hexadecylpropagylester C23H38O4 378

DeterminationofTPH

Gravitational analysisofTPH from un-inoculated crude oil

indicatedthepresenceof0.282gofaliphatic,0.098gof

aro-matic and 0.04g of NSO compounds along with 0.059g of

asphaltenes.Ontheotherhand,drasticreductionintheTPH

contentwasobservedattheendof13daysincubationwithPA,

PMandBM.Thealiphatic,aromatic,NSOandasphaltene

frac-tionsofresidualTPHinpresenceofBMwasfoundrespectively

as 0.042,0.014, 0.024, 0.036gand 0.048,0.053g, 0.063gand

0.044ginpresenceofPAwhilewithPMitwas0.114,0.073,0.027

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a

100

C14

7.40

5.00 10.00 15.00 20.00 25.00

12.40 17.40

Retention time (min)

22.40 Ind 2,4-DTBP NA 27.40 C16 C18 C20 C22 C24 C26 C28 C30 100 100 100 100 100 100 100 % % % % % % % %

b

c

d

e

f

g

h

Fig.5–GCspectrumofaliphatichydrocarbons(a–control;b–PA;c–PM;d–BM)aromatichydrocarbon(e–control;f–PA; g–PM;h–BM)after13daysofincubationinMSMsupplementedwith1%crudeoil.(NA–Naphthalene;Ind–Indene;2, 4-DTBP–2,4-di-tert-butylphenol).

BMwasmorecompetentinutilizingcrudeoilanditbrought

about 74%total degradation ascomparedto un-inoculated

controlwhilePAandPMbroughtabout52and43%

degrada-tionrespectively.FurthertheGCchromatogramofaliphatic

andaromaticfractionofTPHfromun-inoculatedcontroland

ﬂasksinoculatedwithPA,PMandBMispresentedinFig.5.

ItwasobservedthatBMbroughtaboutsubstantial

degrada-tionofaliphaticcompoundswith81%ofC14,12%ofC16,27%

ofC18andmorethan85%ofC20–C30(Fig.5d)ascompared

toun-inoculatedcontrol(Fig.5a).Sameway,PAalsoshowed

enhanceddegradationwith87%ofC14,34%ofC16,68%of

C18 and more than 85% of C20–C30(Fig. 5b). WhereasPM

broughtaboutlessereffectwith3.5%degradationofC14,11%

ofC16,8%ofC18,64%ofC20,81%ofC22,and85%ofC24–C30

(Fig. 5c). Similareffects wereobservedwiththe percentage

degradationofaromaticcompounds-indeneandnaphthalene

inpresenceofBMwhichwas90and87%respectively(Fig.5h)

ascomparedtoun-inoculatedcontrol(Fig.5e)andinthe

pres-ence ofPAit was of42 and 52% (Fig.5f) whilewithPM it

was11and31%respectively(Fig.5g).Also,anincreaseinthe

formationofintermediate metabolite2,4-di-tert-butyl

phe-nol (2,4-DTBP) during the degradation ofnaphthalene was

observedmoreprominentlyinpresenceofBM(Fig.5h).The

intermediatesformedduringthedegradationPAHwere

deter-minedasresorcinol equivalent(RE)whichwasfoundtobe

higherwithBM(87.43␮gmL−1)followedbyPA(67.68␮gmL−1)

andPM(65.89␮gmL−1)ascomparedtoun-inoculatedcontrol

(1.76␮gmL−1).

Identiﬁcationofbacteria

Thethreebacterial isolates-PA,PMand BMwere subjected

tomorphologicalandmolecularidentiﬁcation.Morphological

analysisindicatedthatallthethreestrainswereaerobic,

non-pigmentedandnon-sporulatingbacteria.ByGram’sstaining

procedure, PAandBMwere foundtobegram-positiverods

while PM was gram-negativerod. Further, molecular

char-acterizationofPA,PMandBMwasperformedby16SrRNA

genesequenceanalysisandthenucleotidesequences(958bp,

1028bp and 803bp) of the PA, PM and BM obtained were

deposited in the National center for Biotechnology

infor-mation (NCBI Gen Bank) under the respective accession

numbersKT372715,KT372716andKT372717anddesignated

asCorynebacteriumaurimucosumMKS1,Acinetobacterbaumannii

MKS2 and Microbacterium hydrocarbonoxydans MKS3

(8)

Fig.6–PhylogeneticpositionofbacterialstrainsPA,PMandBMandrepresentativesofsomeotherrelatedtaxabasedon the16SrRNAgenesequences.Corynebacteriumsimulans81-0613(AF537603.1),AcinetobacterbaumanniiCanadaBC-5 (JN667900.1)andMicrobacteriumhydrocarbonoxydansXAS-1(JF751054.1)servedasexternalreferenceforPA,PMandBM respectively.Bar(1,0.2and2forPA,PMandBMrespectively)substitutionpernucleotideposition.Bootstrapvalues expressedaspercentagesof1000replications.

and shown in Fig. 6. The 16S rRNA gene sequence of

PA revealed 97% identity to C. aurimucosum ATCC 700975

(AY536426.1),PMshowed99%identitytoA.baumanniiIARI-V-4

(JN411371.1)andBMshowed99%identitytoM.

hydrocarbonoxy-dansKBN-1-4(JQ954857.1).

Discussion

Roleofmicrobialactivityinthebiodegradationof

hydrocar-bonshasbeenwellrecognizedformorethanacentury.1_The

nativebacterialpopulationsinthecrudeoilcontaminatedsoil

sitespossessthecapacitytomineralizehydrocarbons.26_In_the

presentstudy,thegrowthcurvesofPA,PMandBMdepicted

thatthecellsadaptedwelltothemediacontainingcrudeoil

within2daysand exhibitedexponentialgrowth between7

and10days.ItwasinterestingtoobservethatBMdisplayed

maximumgrowthinmediasupplementedwith3%crudeoil

suggestingitssuperiorabilitytoutilizehigherconcentration

ofcrudeoilwhilePAandPMshowed maximumgrowth in

presenceof1%crudeoil.Similarobservationshavebeenmade

withBacillussp.S6andS35whichshowedexponentialgrowth

inMSMcontaining1%crudeoiluptoday7.27_In_yet_another

study,Pseudomonassp.BP10andRhodococcussp.NJ2exhibited

slowgrowthinitiallybetween0and20daysinMSM

contain-ing2%crudeoilandshowedexponentialgrowthbetween20

and25days.22_The_concomitant_increase_in_the_protein

con-tentofPA,PMandBMobservedinthisstudycorrelatedwell

with increasingcellcountand their abilitytoutilizecrude

oilasasourceofcarbonand energy.Also,moderate

activ-ity enzymes C12Oaswell as C23Oenzymes was observed

inallthethreestrainswhichcorrelatedwellwiththe

expo-nentialgrowthshownbyPA,PMandBMutilizingcrudeoil.

Anotherinterestingobservationmadeinthisstudywasthat

thespeciﬁcactivityofC12OwashigherthanthatofC23Oin

allthethreebacterialstrains.ThisincreasedactivityofC12O

signifytheroleofortho-cleavagepathwayasthepredominant

pathwayofoildegradation.Amongthethreestrainsusedin

this study,BMexhibited higheractivity ofC12Oaswell as

(9)

maximum growth in MSM with 3% crude oil. Similar

to our ﬁndings, increased speciﬁc activity for C12O than

C23O was exhibited by Rhodococcus pyridinivorans BP101

and Pseudomonas sp. NJ2 strains cultured on MSM with

crude oil.22 _In_a _different_study, _increased_activity _of_C12O

(1.03␮molmin−1mg−1) than C23O (0.37␮molmin−1mg−1)

wasobservedinStenotrophomonasmaltophiliaKB2culturedon

MSMcontainingphenolascarbonsource.28

PartialpuriﬁcationofC12Ofrom PA,PMand BMinthis

study byusing ammonium sulphate precipitation followed

byion-exchangechromatographyspeciﬁedthattheactivity

oftheenzymewasprominentlyhigherinbacterialstrainBM

followedbyPA.ThisincreasedactivityofC12Ocanbe

corre-latedwiththeincreasedgrowthratesshownbyBMin3%crude

oilcontaining MSM inthis study. Ourﬁndings on

increas-ing speciﬁc activity of C12Oat different puriﬁcation steps

drawaparalleltothe observationsmadeinarecentstudy

wherethespeciﬁcactivityofC12Ofromcrudecelllysateof

RhodococcusspNCIM2891increasedfrom0.78to1.83Umg−1

afterionexchangechromatography.14_Similarly,_the_activity

ofcrudeC12OfromAcinetobacterradioresistenswasshownto

increase from 1.63 to 6.3 and then to 24.5Umg−1

respec-tivelyafterpuriﬁcationbyionexchangeandgelpermeation

chromatography.29 _The _molecular _mass_of_parially _puriﬁed

fractionofC12Oenzymefrom all the threestrains PA,PM

andBMinthisstudywasfoundtobe∼39KDaandthis

cor-roboratewellwiththeobservationsmadeonC12OfromA.

radioresistens29andRhodococcusrhodochrous.30

Several micro organisms including Pseudomonas sp.,

Rhodococcussp.thatutilizespetroleumhydrocarbonsastheir

carbon sourceareknown toproduce biosurfactantsof

gly-colipids or lipoprotein nature.22 _In _the _present _study, _the

biosurfactants of from PA, PM and BM were qualitatively

identiﬁedtobeglycolipidinnature.Thebiosurfactantsalso

exhibited substantial emulsiﬁcation index as well as oil

displacementability.Thesurfacetensionattainedbythe

bio-surfactantsdisplacestheoilandthediameteroftheclearance

zoneontheoilsurfacegivesanindirectindicationof

biosur-factantactivity.17_Among_the_three_strains_used_in_this_study,

BMdisplayedsuperiorbiosurfactantactivity(E24=69%)and

oildisplacementcapacity(17mm).Thisenhancedsurfactant

productioncanbecorrelated withthesuperiorgrowthrate

ofBMobservedinthisstudy byutilizing higher

concentra-tionofcrudeoil(3%).Similarobservationshavebeenmade

withdifferentbacterialspeciessuchasStreptomycesmatensis

PLS-131_and_P._aeruginosa32_which_produced_{biosurfactants}_of

glycolipidnature.Inadifferentstudy,thecrudeoildegrading

capacityofAcinetobactercalcoaceticuswascorrelatedwiththe

levelofbiosurfactantsproduced(E24=64%)andsuperioroil

displacementactivity(7.2cm).33_FTIR_has_been_widely_used_to

characterizethesurfacegroupsastheIRtransmission

spec-trapresentspeakscharacteristicofspeciﬁcchemicalbonds.34

InthepresentstudytheFTIRspectraofextracted

biosurfac-tantsfromPA,PMandBMindicatedthepresenceofglycosidic

bondsandtherebyconﬁrmingtheglycolipidnatureofthe

bio-surfactants.Similartoourobservationsearlierworkershave

conﬁrmedthe glycolipidnatureofbiosurfactants produced

byBacillus megaterium7 _by_FTIR _analysis. _Subsequent_GCMS

analysisoftheextractedbiosurfactantsfromPA,PMandBM

conﬁrmedthepresenceoffatty acids/fattyacidesters. The

presenceofdecanoicacidestersinthebiosurfactant

prepa-rations observed in this study can be correlated with the

predominanceofmethylestersofdecanoicacidsobservedin

thebiosurfactantsfromA.Niger,A.ﬂavus35_and_Bacillus_sp.36

TheresidualTPHcontentfollowingcrudeoildegradation

byPA,PMandBMwasanalyzedusinggravitationalmethod

whichindicatedthatBMutilizedTPHmoreeffectivelythan

PAand PM. Similarobservations havebeenmade in

previ-ousstudieswithvariousotherbacterialstrains.Itwasshown

that P.aeruginosaDQ8degraded79.3% ofTPHinMSMwith

10%ofcrudeoil.37_Similarly,_A._{calcoaceticus}_BS_and

Alcanivo-raxdieselolei PG-12wereshown todegrade82%and 71%of

1%(v/v)crude oilrespectivelyowingtotheir high

emulsiﬁ-cationactivityandbiosurfactantproduction.38_In_this_study,_it

wasobservedthatthedegradationofaliphaticcompoundsof

TPHwaseffectivelybroughtaboutbybothPAandBM(more

than80%ofC14and85%ofC20-C30compounds)morethan

PM.Similarobservationshavebeenmadeinpreviousstudies

whereRhodococcussp.Moj-3449degradedcompoundsinthe

rangeofC14-C1939_while_A._{calcoaceticus}_and_Alcaligenes

odor-anspreferredC17-C24compoundsofcrudeoil.38_Same_way,

thedegradationofaromaticcompoundsofTPHbyPA,PMand

BMwhenanalyzed,itwasobservedthatBMwasmore

com-petentindegradingpolyaromatichydrocarbons(morethan

85%)ascomparedtoPAandPMwhichcoulddegradeonlyto

theextentof40and10%respectively.Inanearlierreport,

Strep-tomycesspp.wasshowntodegradepolyaromatichydrocarbon

naphthalenetoproduce2,4-di-tert-butylphenol(2,4-DTBP)as

byproduct.40_As_the_crude_oil_is_a_mixture_of_{hydrocarbons,}_it_is

difﬁculttoidentifytheintermediatesformedduringthe

degra-dationofcrudeoil.Sincethearomaticcompoundsaremore

unmanageablethanalkanes,theactivityofdioxygenase41_and

the productionof hydroxylated aromatic compounds were

usedasatooltotracetheinitialpathwaysofoildegradation.

Inthisstudy,BMproducedmoreresorcinolequivalent

inter-mediatesthanPAandPMduringthe13daysincubationperiod

whichsuggestedthatBMpossesseshighercrudeoildegrading

potential than PA and PM. Similar toour ﬁndings,

consid-erableincreaseinresorcinol equivalentintermediateswere

observedwithPseudomonassp.(86.7␮gmL−1)andRhodococcus

sp.(82.5␮gmL−1)following25daysofincubationwithcrude

oil.22

Based on the molecular characterization by 16S rRNA

genesequenceanalysis,thethreebacterialstrains–PA,PM

andBMwereidentiﬁedrespectivelyasC.aurimucosumMKS1

(KT372715), A.baumanniiMKS2 (KT372716)and M.

hydrocar-bonoxydansMKS3(KT372717).

Conclusion

This study focused on three potent oil degrading bacteria

strainsPA, PMandBMthatwere isolatedfrom oilpolluted

soil.Thesestrainsdisplayedsubstantialabilitytoutilizecrude

oilastheircarbonsourceandamongthethree,BMexhibited

superior capacitytodegradecrude oil. Asthe dioxygenase

enzymes C12Oand C23Oare known toplaya vitalrole in

thebiodegradationofaromaticmolecules,increasedactivity

ofC12O than C23Othat wasobserved inthis study in PA,

(10)

predominant pathway of oil degradation by these

bacte-ria. Also, their ability to produce biosurfactants seems to

compliment their biodegradation potential. The residual

TPHcontentanalysisandGCMSanalysisalsoconﬁrmedthe

efﬁciencyofcrudeoildegradationbythebacterialstrains.The

bacterialstrainsPA,PMandBMwereidentiﬁedasC.

aurimu-cosumMKS1,A. baumanniiMKS2and M. hydrocarbonoxydans

MKS3respectivelybythe16SrRNAgenesequenceanalysis.

Thus,resultsfromthisstudyconvincinglyspecifythatsuch

bacteriawithpotentoildegradingcapacitymaybevibrantly

usedforclearingunforeseenoilspillages.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest

Acknowledgements

The authors duly acknowledge instrument support from

the Department of Biochemistry and Molecular Biology,

Pondicherry University,Indiaand DST-FIST, Government of

India. The ﬁrst author (Muthukamalam S) conveys special

thankstoUGC,NewDelhi,India,forﬁnancialsupportinthe

formofJuniorResearchFellowship(CSIR-UGC-JRF316979;Ref

no.21/12/2014(ii)EU-V).Theauthorsconveyspecialthanksto

IndianOilCorporationLimited(IOCL),Chennai,TamilNadu,

Indiaforprovidingsoilsamplesandcrudeoil.

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