Research Article Biodegradation Ability and Catabolic Genes of …downloads.hindawi.com/journals/bmri/2014/127674.pdf · 2019-07-31 · Research Article Biodegradation Ability and

Research ArticleBiodegradation Ability and Catabolic Genes ofPetroleum-Degrading Sphingomonas koreensis Strain ASU-06Isolated from Egyptian Oily Soil

Abd El-Latif Hesham12 Asmaa M M Mawad3 Yasser M Mostafa4 and Ahmed Shoreit3

1 Genetics Department Faculty of Agriculture Assiut University Assiut 71516 Egypt2 Biology Department Faculty of Science King Khalid University Abha 61321 Saudi Arabia3 Botany and Microbiology Department Faculty of Science Assiut University Assiut 71516 Egypt4 Egyptian Petroleum Research Institute Nasr City Cairo 11727 Egypt

Correspondence should be addressed to Abd El-Latif Hesham hesham egypt5yahoocomand Ahmed Shoreit ashoreit1968yahoocom

Received 21 February 2014 Revised 7 July 2014 Accepted 14 July 2014 Published 10 August 2014

Academic Editor Gyorgy Schneider

Copyright copy 2014 Abd El-Latif Hesham et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards In this study 15 PAHs-degrading bacteriawere isolated from Egyptian oily soil Among them one Gram-negative strain (ASU-06) was selected and biodegradation abilityand initial catabolic genes of petroleum compounds were investigated Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensisStrain ASU-06 degraded 100 99 98 and 927 of 100mgL naphthalene phenanthrene anthracene and pyrene within 15 daysrespectively When these PAHs present in a mixed form the enhancement phenomenon appeared particularly in the degradationof pyrene whereas the degradation rate was 986 within the period This is the first report showing the degradation of differentPAHs by this species PCR experiments with specific primers for catabolic genes alkB alkB1 nahAc C12O and C23O suggestedthat ASU-06 might possess genes for aliphatic and PAHs degradation while PAH-RHD120572GP gene was not detected Production ofbiosurfactants and increasing cell-surface hydrophobicity were investigated GCMS analysis of intermediatemetabolites of studiedPAHs concluded that this strain utilized these compounds via two main pathways and phthalate was the major constant productthat appeared in each day of the degradation period

1 Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a group ofhydrophobic organic compounds composed of two or morefused aromatic rings in their chemical structure [1] PAHs arereleased into the environment from the incomplete combus-tion of fossil fuels and organic matter the accidental spillingof processed hydrocarbons and oils run-off from asphaltpavements coal liquefaction and gasification and naturalgeological processes [2] Due to their toxic carcinogenicand mutagenic properties PAHs are of environmental andhuman concern and 16 PAHs have been listed by the USEnvironmental Protection Agency as priority contaminantsin ecosystems [3]

Microbial degradation is the most dominant and signifi-cant process for removing PAHs from the environmentManymicroorganisms capable of metabolizing PAHs were isolatedincluding bacteria [4] yeasts [5] fungi [6] and algae [7]Most of the bacteria isolated belong to genera PseudomonasBurkholderiaMycobacteriaRhodococcusAlcaligenesRalsto-nia and others [4 8]

Usually contaminated sites are polluted by a mixture ofPAHs Thus for an efficient remediation process it is impor-tant that the bacteria involved have a complete degradationpathway so that no potentially toxic degradation productsaccumulate [9] Genetic analyses of PAH catabolic pathwaysin several PAH-degrading bacteria revealed the presenceof a group of genes for complete degradation of aromatic

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 127674 10 pageshttpdxdoiorg1011552014127674

2 BioMed Research International

compounds [10 11] PCR amplification using genes specificprimers or degenerate primers has been used to detect andstudy the diversity of aromatic-dioxygenase genes in PAH-degrading bacterial isolates [12ndash14]

Although many bacteria capable of degrading PAHs havealready been isolated it is still important to screen strainsthat can completely and rapidly decompose PAHs under thecondition of Egyptian environment Therefore the aim ofthe present study was to isolate strains capable of degradinglow and high molecular weight PAHs A strain was obtainedand identified as S koreensis according to morphologicalcharacteristics and 16S rRNA gene sequence analysis and itsability to degrade naphthalene phenanthrene anthraceneand pyrene were studied The production of biosurfactantsand increasing cell-surface hydrophobicity the metabolitesduring the degradation process and the genetics of catabolicgenes in the isolated PAH-degrading bacterium were alsoinvestigated

2 Materials and Methods

21 Sample Collection and Chemicals Oil contaminated soilwas collected in sterilized polyethylene bags from Oil Refin-ery Company in Assiut Egypt and stored at 4∘C in thelaboratory Naphthalene phenanthrene anthracene pyrene(all le99 purity) and mineral basal medium with tracemetals were purchased from Sigma-Aldrich

22 Enrichment Isolation and Evaluation of PAHs-DegradingBacteria Soil enrichment technique was used for the iso-lation of PAH-degrading bacteria as described in [5]About 10 g oil contaminated soil sample was suspended in90mL mineral basal salt medium (MBS) containing (gL)10(NH

4)2SO4 08K

2HPO4 02KH

2PO4 02MgSO

4sdot7H2O

01CaCl2sdot2H2O 0005FeSO

4sdot7H2O and 1mL of trace ele-

ments pH 70 plusmn 02 The medium was supplemented withone of the following PAHs naphthalene phenanthreneanthracene or pyrene at concentration of 100mgL as asole source of carbon The flasks were incubated on anorbital shaker at 150 rpm at 30∘C After 7-day incubation analiquot of 10 enriched cultures was transferred into another250mL conical flask containing 90mL fresh autoclaved MBSmedium supplemented with previously mentioned PAHsThis step was repeated five times to attain well-adapted PAH-degrading enriched bacterial consortia Bacterial strains wereisolated from MBS agar plates coated with the same PAHs asthe sole carbon source Colonies with different morphologieswere individually selected All isolates were evaluated fortheir PAHs-degrading abilities One pure strain of bacteriawith a high PAHs degradation rate was designated as ASU-06 and selected for further study

23 Physiological and Biochemical Tests of Strain ASU-06Conventional physiological and biochemical characteristicswere determined using the procedures described by John andKrieg [15]

24 16S rRNA Gene Amplification and Sequence Determi-nation The genomic DNA was isolated from strain ASU-06 according to the method described by Hesham [16] andthe 16S rRNA gene was amplified Amplification was carriedout with universal primers 27F (5-AGAGTTTGATCCTGG-CTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) in a final volume of 50120583L containing 10mM tris-HCl (pH83) 50mM KCl 15mMMgCl

2 each dNTP at a concentra-

tion of 02mM 125 IU of Taq polymerase each primer at aconcentration of 02mM and 1 120583L of the DNA template PCRwas performed with the following program 5min denatura-tion at 95∘C followed by 36 cycles of 1min denaturation at94∘C 1min annealing at 55∘C 15min extension at 72∘C anda final extension step of 7min at 72∘C 5 120583L of the amplifiedmixture was then analyzed using 15 05 times TBE agarose gelelectrophoresis The gel was stained with ethidium bromidevisualized under UV light and photographed Product of thecorrect size was purified and sequenced in both directionsusing an ABI automated sequencer

25 Sequence Alignment and Phylogenetic Analysis The 16SrRNA gene sequences of the isolate obtained in this studywere aligned and compared with the known 16S rRNAgene sequences in Genbank database using the BLASTsearch at the National Center for Biotechnology Informa-tion (httpwwwncbinlmnihgovBLAST) to determinethe closest available database sequences To determine thetaxonomic position of the isolate a phylogenetic tree wasconstructedwithMEGAversion 40 using a neighbor-joiningalgorithm in addition the Jukes-Cantor distance estimationmethod with bootstrap analyses for 100 replicates was per-formed [17]

26 PAHs Degradation Analysis by HPLC Pure strain wasresuspended in 50mL MBS supplemented with either naph-thalene phenanthrene anthracene or pyrene at concentra-tion of 100mgL for each or mixed of them PAHs (25mgL)for each to a turbidity of 02 at OD (600 nm) Abiotic controlswere prepared in the same way but without addition ofbacteria Samples and controls were prepared in triplicateFlasks were incubated at 30∘C with rotation at 150 rpm for 15days After every three days of incubation 25mL aliquot ofeach sample was extracted three times with equal volumes ofethyl acetate according to the method described byManoharet al [18] The residual PAHs were analyzed by HPLC asdescribed by Bishnoi et al [19]

27 Detection of PAH-Degrading Enzymes The initial dioxy-genase activity catalyzing the conversion of indole to beindigo was determined following the standard methoddescribed by a previous worker [20] while Catechol 23-diox-ygenase activity was determined according to Ornston andStanier [21]

28 Detection of Aliphatic and PAH-Degrading Genes byPCR The presence of six genes including monooxygenaseand dioxygenase genes in the isolated bacterial strain ASU-06 was detected based on PCR amplification The primers

BioMed Research International 3

Table 1 List of primers used for detection of mono- and dioxygenase genes

Primer Sequence (51015840 to 31015840) Expected size (bp) ReferencealkBFalkBR

51015840-AACTACMTCGARCAYTACGG-3101584051015840-TGAMGATGTGGTYRCTGTTCC-31015840 100 Powell et al [22]

AlkB1FAlkB1R

51015840-TACGGGCACTTCGCGATTGA-3101584051015840-CGCCCAGTTCGAMACGATGTG-31015840 550 Kloos et al [13]

nahAc FnahAc R

51015840-TGGCGATGAAGAACTTTTCC-3101584051015840-AACGTACGCTGAACCGAGTC-31015840 487 Laurie and Jones [14]

PAH-RHD-GPfPAH-RHGPr

51015840-CGG CGC CGA CAA YTT YGT NGG-3101584051015840-GGG GAA CAC GGT GCC RTG DAT RAA-31015840 292 Cebron et al [12]

C12OFC12OR

51015840-GCCAACGTCGACGTCTGGCAGCA-3101584051015840-CGCCTTCAAAGTTGATCTGCGTGGTTGGT-31015840 350 Sei et al [23]

C23OFC23OR

51015840-AAGAGGCATGGGGGCGCACCGGTTCGA-3101584051015840-TCACCAGCAAACACCTCGTTGCGGTTGCC-31015840 900 Sei et al [23]

for the detection of n-alkanes monooxygenase (alkB andalkB1) dioxygenase (nahAc) Catechol dioxygenase (C12Oand C23O) and PAH-ring hydroxylating dioxygenase (PAH-RHD120572) genes were listed in Table 1 PCR conditions wereinitial denaturation for 5min at 95∘C 35 cycles with 40 s at94∘C 40 s at 55∘C 60 s at 72∘C and final elongation for 7minat 72∘C for the four genes alkB alkB1 nahAc and PAH-RHD120572[12ndash14 22] However PCR was performed for Catechol 12-dioxygenase (C12O) and Catechol 23-dioxygenase (C23O)genes with initial denaturation for 5min at 95∘C 35 cycleswith 20 s at 94∘C 30 s at 63∘C and 45 s at 72∘C and finalelongation for 5min at 72∘C [23] All PCR products wereseparated in 15 agarose gel stainedwith ethidium bromidevisualized under UV light and photographed

29 Detection Cell-Surface Properties Production of extra-cellular biosurfactant by the culture was analyzed by moni-toring the ability of surfactant to stabilize 1-naphthaldehydein water emulsion as described earlier by Phale et al [24] Analiquot of 5mL of sample was centrifuged at 2000 rpm thepellet was discharge and the supernatant was used as a sourceof biosurfactant(s) The total assay mixture (5mL) contained200120583L supernatant 38mL phosphate buffer (50mM pH75) and 1mL of 1 of 1-naphthaldehyde in water emulsion(100 120583L naphthaldehyde in 10mL double distilled waterfollowed by 1min sonication) The mixture was vortexedfor 1min and incubated at room temperature for 5 h Theabsorbance due to stability of emulsion was measured at660 nm against control that was prepared by using 200120583Ldistilled H

2O instead of the supernatant One unit is defined

as the amount of biosurfactants required to obtain an increasein absorbance of 10OD unit Affinity of cells towards variousPAHs was measured by the method of Rosenberg et al [25]Cells were grown on PAHs harvested and washed twice withphosphate buffer (50mM pH 75) and resuspended in thesame buffer to obtain a cell suspension with a final OD of0300 at 600 nm The 6mL assay mixture contained 3mLcell suspension and 3mL test PAHs After 5min of prein-cubation the mixture was vortexed for 60 s and incubatedfor additional 15min at room temperature The OD of theaqueous phase was measured at 600 nm The cell-surface

hydrophobicity was expressed as percent cells transferred tohydrocarbon phase by measuring the OD of the aqueousphase before and after mixing

210 Identification of PAHs Metabolites Using GC-MS Anal-ysis After growth on PAHs contents of the flasks wereextracted with three equal volumes of ethyl acetate Theaqueous fraction after extraction was acidified with concen-trated HCl to pH 2 and extracted again with three equalvolumes of ethyl acetate The residual extracts were driedover anhydrous sodium sulfate and evaporated with rotatoryevaporator at 40∘C to 10mL [26] The samples were dried invacuum and stored atminus20∘Cuntil being usedThe extract wasthen dissolved in hexane and introduced for GC-MS analysisGC-MS analysis was performed using an HP 6890 gaschromatograph with an HP 5973 mass spectrometer systemThe column was a TR-5MS (5 phenyl polysilphenylenesiloxane) (30mtimes 025mMdiameter 025 uMfilm thickness)Helium was the carrier gas at 1mLmin constant flow Thecolumn temperaturewas held at 70∘C for 5minutes increasedat a rate of 4∘Cmin to 290∘C and held for 10 minutes Toremove any remaining compounds the analysis was finishedwith a ramp of 20∘Cmin to 320∘C held for 20 minutesThe mass spectrometer was operated in electron impact (EI)mode at 70 electrons volts (EV) in the full scan mode from85 to 450 mz over 65ndash85 minutes Injector and detectortemperatures were 270∘C and 280∘C respectively [5 17]

211 GenBank Accession Number The nucleotide sequencesof 16S rRNA gene of Sphingomonas strain ASU-06 reportedin this study have been deposited in the DDBJ EMBL andGenBank nucleotide sequence databases under AccessionNumber KC420523

3 Results

31 Isolation Characterization and Evaluation of PAHs-Degrading Bacteria The 15 bacterial colonies were isolatedfrom Egyptian oily soil By visualization these strains werevaried in colony shape color of colony and levels of growth


Sphingomonas yunnanensis (JQ6602901)

Sphingomonas phyllosphaerae (JQ6602701)

Sphingomonas sanguinis (JQ6601321)

Sphingomonas pseudosanguinis (JQ66008

Sphingomonas hankookensis (JQ6601661)

ASU-06 (KC4205231)

Sphingomonas koreensis (AB6810551)

Sphingomonas pruni (NR 0263731)

Sphingomonas mali (NR 0263741)

Sphingomonas aerolata (HE6811491)

Sphingomonas aurantiaca (JN1679621)

100

100

100

100

98

84

72

67

0005

Figure 1 Phylogenetic analysis of 16S rRNA gene of isolate ASU-06 and other related Sphingomonas spp By neighbor-joining methodNumbers at the nodes indicate bootstrap support () based on 100 replicates The scale bar indicates 0005 nucleotide substitutions pernucleotide position GenBank accession numbers are given in parentheses

on solid MBS medium Among these isolates one Gram-negative strain ASU-06 was selected for further biodegrada-tion studies of PAHs owing to its highest growth on all usedPAHs as a sole source of carbon Strain ASU-06 showed anability to produce indigo (blue) colored colonies as a result ofdioxygenase activity in the presence of indole as a substrateIt was also recorded positive when Catechol was introducedas a substrate and formed yellowish or brown coloniesConventional physiological and biochemical characteristicswere determined for ASU-06 using the procedures describedby John and Krieg [15] the results concluded that ASU-06could be identified as Sphingomonas sp

32 Molecular Identification and Phylogenetic Analysis Using16S rRNA Gene Sequence The genomic DNA was extractedfrom the isolated bacterial strain ASU-06 and universalprimers 27F and 1492R were used for the amplification andsequencing of the 16S rRNA gene fragment The alignmentand comparison of the 16S rRNA gene sequence of thestrain ASU-06 to the published 16S rRNA gene sequencesin GenBank database by BLAST search were determinedResults show that the 16S rRNA gene sequence of the isolatedstrain was highly homologous to Sphingomonas koreensiswith 100 sequence similarity To confirm the position ofthe strain ASU-06 in phylogeny a number of sequencesrepresentative of some Sphingomonas species were selectedfrom Genbank database for construction of a phylogenetictree As shown in Figure 1 the phylogenetic tree indicatedthat strain ASU-06 and S koreensis shared one clusterTherefore the strain ASU-06 was identified as S koreensis

33 PAHs Biodegradation Analysis UsingHPLC Degradationof sole PAHs by S koreensis strainASU-06 in liquid cultures isshown in Figure 2 S koreensis could degrade the four PAHs

Table 2 Some PAHs catabolic genes detected in ASU-06 using spe-cific primers for each gene

Gene StrainS koreensis Expected band (bp)

AlkB + 100AlkB1 + 550NahAc + 487PAH-RHD120572 minus 292C12O + 350C23O + 900

naphthalene phenanthrene anthracene and pyrene at con-centration of 100mgL for each with different capabilitiesof 100 99 98 and 927 after 15 days respectively Whenthese PAHs present in a mixed form the enhancement phe-nomenon appeared (Figure 3) particularly in degradation ofpyrene whereas the degradation rate was 986 within 15days

34 Detection of Catabolic Genes by PCR The catabolicgenes can be used to trace the specific activities of bacterialstrain We targeted six key enzyme coding genes includingmonooxygenase and dioxygenase responsible for the miner-alization of aliphatic and PAHs compounds The presence ofsix genes in S koreensis strain ASU-06 was detected basedon PCR amplificationThe existence ofmonooxygenase (alkBand alkB1) dioxygenase (nahAc) and Catechol dioxygenase(C12O and C23O) genes was confirmed and the results areshown in Figure 4 and Table 2 The sizes of PCR products formonooxygenases alkB and alkB1 were approximately 100 bpand 550 bp respectively while for nahAc C12O and C23Othey were 487 350 and 900 bp respectively Finally no signalfor PAH-RHD120572 gene was detected


TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

12

Rem

aini

ng n

apht

hale

ne (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14(L

n O

DtO

D0

) at6

00

nm

(a)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

Rem

aini

ng an

thra

cene

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14

(Ln

OD

tOD0

) at6

00

nm

(b)

TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

Rem

aini

ng p

hena

nthr

ene (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214

(Ln

OD

tOD0

) at6

00

nm

minus20

GrowthBiodegradation

Biosurfactant

(c)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

07

Rem

aini

ng p

yren

e (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214


Biosurfactant

(Ln

OD

tOD0

) at6

00

nm

(d)

Figure 2 The relation between growth curve of PAHs degradation and activity of biosurfactants (Umg protien) of S koreensis growingon MBS supplemented with 100mgL of (a) naphthalene (b) anthracene (c) phenanthrene and (d) pyrene ldquoas single substraterdquo

TD0 2 4 6 8 10 12 14 16

00020406081012141618

PAH

rem

oval

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

5

10

15

20

25

GrowthNaphAnth

PhenPYRBiosurfactant

(Ln

OD

tOD0

) at6

00

nm

Figure 3 The relation between growth curve at OD 600 ofPAHs degradation and activity of biosurfactants (Umg protien) ofS koreensis growing on MBS supplemented with 100mgL of fourPAHs ldquoas mixed substraterdquo

35 Biosurfactant Production and Cell-Surface Hydrophobic-ity Production of extracellular biosurfactant by the cultureand affinity of cells towards various PAHs were investi-gated The emulsification activity was increased graduallyfrom lag to reach the maximum values at exponentialphase (Figures 2 and 3) The values of percentage of cell-surface hydrophobicity (CSH) toward naphthalene phenan-threne anthracene pyrene and mixed PAHs were 37 5763 37 and 65 respectively The increasing of cell-surfacehydrophobicity facilitated the direct contact between cellsand the substrate particles that will stimulate the growth oforganism

36 Detection of PAH Metabolic Products Produced by GC-MS Analysis All detected metabolites retention times andfragmentation patterns were summarized in (Table 3) It wasnoted that phthalate was the major constant product thatappeared in each day of the degradation period The othermetabolic products were different and changed every threedays During degradation of naphthalene and phenanthrene


(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp

Figure 4 PCR products based on primers specific for the catabolic genes from the left to the right AlkBAlkB1 nahAc C23O and C12O thatare detected in S koreensis with 100 bp DNA ladder (M)

the presence of cinnamate salicylate and phthalate indi-cates that this strain has two major pathways for naph-thalene degradation Detecting of 1-hydroxyl or dihydroxylPAHs derivatives is confirming the activity of nahAc geneAppearance of to ciscis-dihydroxyl muconate during bothphenanthrene and pyrene degradation confirms the activityofC12O enzyme and also it could be concluded that the orthopathway is major for these carbon sources degradations

The percentage of phthalate increased gradually from1789 to 5725 at the 12th day and this percentage wasdecreased due to consumption to form other metabolitesduring pyrene assimilation S koreensiswas tested to grow onphthalate as a sole source of energy on MBS agar mediumand growth was obtained after 24 hours According tothe detected metabolic products it could be supposed theexpected metabolic pathway of pyrene degradation as anexample of four rings PAHs by S koreensis

4 Discussion

PAHs are serious pollutants and health hazards and theyoccur as complexmixtures including low and highmolecularweight therefore degradation of PAHs in the environmentis becoming more necessary and interesting Low molecularweight is more easily degraded while high molecular weightis more recalcitrant and requires specific microorganismsto perform the degradation Construction of consortia bymixing several known PAH degraders has failed to maximizecooperation among different species using synthetic consor-tia [27] Therefore the powerful way to obtain the promisingstrain that could utilize PAHs as the sole carbon source is theenrichment techniques [5] Since only a few reports on themicrobial metabolism of PAHs with four or more aromaticringswere published they toomainly focused on cometabolictransformations [28]Therefore in this study 15 isolates wereobtained fromEgyptian oily soil using an enriched techniqueFrom these isolates one Gram-negative strain ASU-06 waschosen based on its utilization of naphthalene phenanthreneanthracene and pyrene as a sole carbon source and the

ability for dioxygenase activities Mrozik et al [29] reportedthat most of PAHs degradable bacteria are Gram-negativeResults of conventional characteristics concluded that ASU-06 could be identified as Sphingomonas spThe alignment andcomparison of the 16S rRNAgene sequence of the strainASU-06 to the published 16S rRNA gene sequences in GenBankdatabase as well as phylogenetic analysis confirmed thetaxonomic position of the strain as Sphingomonas koreensis(Figure 1) Species of the genus Sphingomonas were isolatedfrom different samples and have shown degradation capa-bility toward different PAHs [30] Several PAH-degradingbacterial strains isolated frommangrove sediments belongingto the genera of Sphingomonas Mycobacterium Paracoccusand Rhodococcus showed different potential in degradingmixed PAHs [31] Results reported by Uyttebroek et al [32]demonstrated that Mycobacterium strains had higher ratein degrading phenanthrene than Sphingomonas strains Ourresults in Figure 2 showed the degradation rates of sole PAHby S koreensis strain ASU-06 in liquid cultures This bac-terium almost completely degraded 100mgL of naphthalenephenanthrene and anthracene while the degradation rate forpyrene was 927 during 15 days On the other hand whenthe 4 PAHs were simultaneously presented degradation ratewas enhanced from 927 to 986 for pyrene (Figure 3)Similar phenomenon has been observed by other researchersin different microorganisms Yuan et al [33] reported thatthe degradation efficiency using isolated bacteria is improvedwhen acenaphthene fluorene phenanthrene anthraceneand pyrene are present together compared to PAHs presentindividually In our previous work [5] using isolated yeastwe found that naphthalene was promoting the degradationof high molecular weight more than other components

Degradation of aliphatic andPAHswasmostly carried outby the mono- and dioxygenases produced by bacteriaThere-fore the presence of six key enzyme coding genes includingboth monooxygenase and dioxygenase in S koreensis strainASU-06 was investigated The existence of monooxygenase(alkB and alkB1) dioxygenase (nahAc) and Catechol dioxy-genase (C12O and C23O) genes was confirmed A possible


Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis

after15daysofincubatio

ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)

Phenanthrene-45-dicarbo

xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca

rboxy-benzalpyruvate

37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)


reason may be because these detected genes are highly con-served among different Gram-negative bacteria Howeverno signal for PAH-RHD120572 gene was detected since it isconserved among Gram-positive bacteria [12] The naph-thalene dioxygenase (nahAc) gene is of particular interestas an indicator for PAHs degradation because the enzymeencoded by this gene not only degrades naphthalene butalso mediates degradation of other PAHs compounds [3134] C12O and C23O dioxygenases play a key role in themetabolism of aromatic rings by the bacteria because they areresponsible for cleavage aromatic CndashC bond at ortho or metaposition [35] On the other hand the presence of catabolicgenes (alkB and alkB1) should be investigated because short-chain n-alkanes are directly toxic acting as solvents forcellular fats and membranes [36] Our results demonstratedthat the five genes were present in the S koreensis strainASU-06 suggesting that this bacterium played an importantrole in degradation of aliphatic and PAHs and could berecommended for petroleum compounds bioremediation inthe environment

Recent studies have suggested that microorganismsinvolved in the degradation of hydrophobic compounds likePAHs and alkanes possess special physiological properties[37] To investigate the effects of physiological propertieson PAHs degradation the emulsification activities and cell-surface hydrophobicity of S koreensis strain ASU-06 weredetected The emulsification activity was increased gradu-ally from lag to reach the maximum values at exponentialphase (Figures 2 and 3) Although both phenanthrene andanthracene consist of three rings the solubility of the phenan-threne 1290 120583gL is more than that of the anthracene 73 120583gL[1] However biosurfactant production by bacteria increasedtoward anthracene rather than phenanthrene [38 39] Thisfinding was assured by Pei et al [40] who demonstrated thatbiosurfactant (rhamnolipid) and cell-surface hydrophobicityproduction by Sphingomonas sp increased the solubilityof phenanthrene and subsequently enhanced the utiliza-tionuptake of carbon source The values of CSH towardnaphthalene phenanthrene anthracene pyrene and mixedPAHs were 37 57 63 37 and 65 respectively The increasein CSH facilitated the direct contact between cells and thesubstrate particles that will stimulate the growth of organismThe production of biosurfactants and increasing CSH aretwo possible strategies used by microorganisms to improvethe transportation of hydrophobic hydrocarbon substrates tocells [39]

GCMS analysis of intermediatemetabolites of two threeand four rings PAHs showed that phthalate was the majorconstant product that appeared in each day of degradationperiod The metabolic products were different and changedevery three days The percentage of phthalate increasedgradually from 1789 to 5725 at the 12th day and thispercentage was decreased due to consumption to form othermetabolites S koreensis was tested to grow on phthalate asa sole source of energy on MBS agar medium the heavygrowth was obtained after 24 hours Zhong et al [41] detectedonly two metabolites (monohydroxypyrene and pyrene diol)from pyrene degradation by mixed culture of Sphingomonas

and Mycobacterium while when Sphingomonas was used aspure culture for pyrene degradation no metabolites could bedetected

The accumulation of monohydroxypyrene at the firststep of degradation indicates that the strain added oneatom of oxygen via monooxygenase enzyme to form 1-hydyoxypyrene and then added another one of O

2via

pyrene dioxygenase to form either 45-dihydroxypyreneor 1-methoxy-2-hydroxypyrene Detection of phenanthrenedicarboxylic acid in the present study supports the presenceof an orthocleavage pathway of 12- and 45-dihydroxypyrene[42] Phenanthrene-45-dicarboxylic then converted to 1-hydroxy-2-naphthoic acid Two common metabolic path-ways for 1-hydroxy-2-naphthoic acid are (a) formation of 2-carboxy benzal pyruvate under the catalysis of 1-hydroxy-2-naphthoate dioxygenase [43] and (b) dihydroxynaphthaleneunder the catalysis of salicylate-1-hydroxylase [44] In thepresent study 2-carboxy benzal pyruvate was found butthere were no traces of dihydroxynaphthalene A numberof Gram-negative and Gram-positive bacteria in the generaof Pseudomonades Nocardioides and Mycobacterium areknown to have proteins or genes of 1-hydroxy-2-naphthoicacid dioxygenase [42 43] These results well agreed with theresults of Kim et al [42] and Seo et al [43] Phthalate tereph-thalate and 4-hydroxybenzoate were found to be degradedvia protocatechuate orthocleavage in Ralstonia jostii RHA1[45] The Sphingomonads were known to possess broadPAHs metabolic capabilities [46] and they were involved indegradation of various recalcitrant organic pollutants suchas biphenyl [47] and benzo[a]pyrene [28] Furthermore itseems likely that the degradation of individual PAH com-pounds by the isolated bacteria proceeds via independentpathways [10] From the metabolic pathway of S koreensisit could be concluded that this strain utilized 100mgL ofnaphthalene (two rings) phenanthrene (three rings) andpyrene (four rings) via two main pathways

In summary the present study reports the isolation iden-tification and characterization of a very efficient bacteriumspecies capable of degrading low and high molecular weight(LMW and HMW) PAHs as a sole source of carbon andenergy The bacterium strain ASU-06 was identified basedon 16S rRNA gene sequence phylogeny and comparisonof this gene sequence with sequence in 16S rRNA genedatabase available in GenBank as S koreensis This bacteriumalmost completely removed 100mgL of LMW-PAHs whilethe degradation rate for pyrene as a HMW-PAH was 927during 15 days When the 4 PAHs were simultaneouslypresent degradation rate was enhanced for pyrene from927 to 986 The existence of catabolic genes includingmonooxygenase (alkB and alkB1) dioxygenase (nahAc) andCatechol dioxygenase (C12O and C23O) genes responsiblefor aliphatic and polyaromatic hydrocarbons degradationwas confirmed while no signal for PAH-RHD120572GP genefor Gram-positive strains was detected GCMS analysis ofintermediate metabolites of LMW and HMW-PAHs con-cluded that this strain utilized 100mgL of naphthalene(two rings) phenanthrene (three rings) and pyrene (fourrings) via two main pathways and phthalate was the majorconstant product that appeared in each day of degradation


period Our results suggest that this bacterium played animportant role in degradation of aliphatic and polyaromatichydrocarbons and could be recommended for petroleumcompounds bioremediation in the environment

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] A K Haritash and C P Kaushik ldquoBiodegradation aspects ofPolycyclic Aromatic Hydrocarbons (PAHs) a reviewrdquo Journalof Hazardous Materials vol 169 no 1ndash3 pp 1ndash15 2009

[2] H Zhang A Kallimanis A I Koukkou and C DrainasldquoIsolation and characterization of novel bacteria degradingpolycyclic aromatic hydrocarbons from polluted Greek soilsrdquoApplied Microbiology and Biotechnology vol 65 no 1 pp 124ndash131 2004

[3] R Goldman L Enewold E Pellizzari et al ldquoSmoking increasescarcinogenic polycyclic aromatic hydrocarbons in human lungtissuerdquo Cancer Research vol 61 no 17 pp 6367ndash6371 2001

[4] L Martınkova B Uhnakova M Patek J Nesvera and VRen ldquoBiodegradation potential of the genus RhodococcusrdquoEnvironment International vol 35 pp 162ndash177 2009

[5] AHesham Z YWang Y Zhang J Zhang andM Yang ldquoIsola-tion and identification of a yeast strain capable of degrading fourand five ring aromatic hydrocarbonsrdquo Annals of Microbiologyvol 56 pp 109ndash112 2006

[6] A Arun P P Raja R Arthi M Ananthi K S Kumar and MEyini ldquoPolycyclic aromatic hydrocarbons (PAHs) biodegrada-tion by basidiomycetes fungi pseudomonas isolate and theircocultures comparative in vivo and in silico approachrdquo AppliedBiochemistry and Biotechnology vol 151 no 2-3 pp 132ndash1422008

[7] A P Lei Y S Wong and N F Tam ldquoPyrene-induced changesof glutathione-S-transferase activities in different microalgalspeciesrdquo Chemosphere vol 50 no 3 pp 293ndash300 2003

[8] Z H Liang J Li Y He et al ldquoAFB1 bio-degradation by a newStrainmdashStenotrophomonas sprdquo Agricultural Sciences in Chinavol 7 no 12 pp 1433ndash1437 2008

[9] J Ma L Xu and L Jia ldquoDegradation of polycyclic aromatichydrocarbons by Pseudomonas sp JM2 isolated from activesewage sludge of chemical plantrdquo Journal of EnvironmentalSciences vol 24 no 12 pp 2141ndash2148 2012

[10] J D vanHamme A Singh andO PWard ldquoRecent advances inpetroleum microbiologyrdquo Microbiology and Molecular BiologyReviews vol 67 no 4 pp 503ndash549 2003

[11] Z Wang J Li A E Hesham et al ldquoCo-variations of bacterialcomposition and catabolic genes related to PAH degradationin a produced water treatment system consisting of successiveanoxic and aerobic unitsrdquo Science of the Total Environment vol373 no 1 pp 356ndash362 2007

[12] A Cebron M Norini T Beguiristain and C Leyval ldquoReal-Time PCR quantification of PAH-ring hydroxylating dioxy-genase (PAH-RHD120572) genes from Gram positive and Gramnegative bacteria in soil and sediment samplesrdquo Journal ofMicrobiological Methods vol 73 no 2 pp 148ndash159 2008

[13] K Kloos J C Munch and M Schloter ldquoA new method for thedetection of alkane-monooxygenase homologous genes (alkB)

in soils based on PCR-hybridizationrdquo Journal of MicrobiologicalMethods vol 66 no 3 pp 486ndash496 2006

[14] A D Laurie and G Jones ldquoQuantification of phnAc andnahAc in contaminatedNewZealand soils by competitive PCRrdquoApplied andEnvironmentalMicrobiology vol 66 no 5 pp 1814ndash1817 2000

[15] G H John and N R Krieg Bergeyrsquos Manual of DeterminativeBacteriology Williams amp Wilkins Baltimore Md USA 9thedition 1994

[16] A Hesham ldquoNew safety and rapid method for extractionofgenomic DNA from bacteria and yeast strains suitable forPCR amplificationsrdquo Journal of Pure and Applied Microbiolgyvol 8 no 1 pp 383ndash388 2014

[17] A Hesham S Khan Y Tao D Li Y Zhang and M YangldquoBiodegradation of high molecular weight PAHs using isolatedyeast mixtures application of meta-genomic methods for com-munity structure analysesrdquo Environmental Science and PollutionResearch vol 19 no 8 pp 3568ndash3578 2012

[18] S Manohar C Kim and T B Karegoudar ldquoDegradationof Anthracene by a Pseudomonas strain NGK1rdquo Journal ofMicrobiology vol 37 no 2 pp 73ndash79 1999

[19] K Bishnoi R Kumar and N R Bishnoi ldquoBiodegradation ofpolycyclic aromatic hydrocarbons by white rot fungi Phane-rochaete chrysosporium in sterile and unsterile soilrdquo Journal ofScientific and Industrial Research vol 67 no 7 pp 538ndash5422008

[20] F Dagher E Deziel P Lirette G Paquette J Bisaillon and RVillemur ldquoComparative study of five polycyclic aromatic hydro-carbon degrading bacterial strains isolated from contaminatedsoilsrdquo Canadian Journal of Microbiology vol 43 no 4 pp 368ndash377 1997

[21] L N Ornston and R Y Stanier ldquoThe conversion of catechol andprotocatechuate to beta-ketoadipate by Pseudomonas putidardquoThe Journal of Biological Chemistry vol 241 no 16 pp 3776ndash3786 1966

[22] S M Powell S H Ferguson J P Bowman and I Snape ldquoUsingreal-time PCR to assess changes in the hydrocarbon-degradingmicrobial community in antarctic soil during bioremediationrdquoMicrobial Ecology vol 52 no 3 pp 523ndash532 2006

[23] K Sei K Asano N Tateishi K Mori M Ike and M FujitaldquoDesign of PCR primers and gene probes for the generaldetection of bacterial populations capable of degrading aro-matic compounds via catechol cleavage pathwaysrdquo Journal ofBioscience and Bioengineering vol 88 no 5 pp 542ndash550 1999

[24] P S Phale H S Savithri N A Rao and C S VaidyanathanldquoProduction of biosurfactant ldquoBiosur-Pmrdquo by Pseudomonasmaltophila CSV89 characterization and role in hydrocarbonuptakerdquo Archives of Microbiology vol 163 no 6 pp 424ndash4311995

[25] M Rosenberg D Gutnick and E Rosenberg ldquoAdherence ofbacteria to hydrocarbons a simple method for measuring cell-surface hydrophobicityrdquo FEMS Microbiology Letters vol 9 no1 pp 29ndash33 1980

[26] H Zeinali M B Khoulanjani A R Golparvar M Jafarpourand A H Shiranirad ldquoEffect of different planting time andnitrogen fertilizer rates on flower yield and its components inGerman chamomile (Matricaria recutita)rdquo Iran Journal of CropScience vol 10 pp 220ndash230 2008

[27] F M Ghazali R N Z A Rahman A B Salleh and M BasrildquoBiodegradation of hydrocarbons in soil by microbial consor-tiumrdquo International Biodeterioration and Biodegradation vol54 no 1 pp 61ndash67 2004


[28] R A Kanaly R Bartha K Watanabe and S Harayama ldquoRapidmineralization of benzo[a]pyrene by a microbial consortiumgrowing on diesel fuelrdquo Applied and Environmental Microbiol-ogy vol 66 no 10 pp 4205ndash4211 2000

[29] A Mrozik Z Piotrowska-Seget and S Łabuzek ldquoBacterialdegradation and bioremediation of polycyclic aromatic hydro-carbonsrdquo Polish Journal of Environmental Studies vol 12 no 1pp 15ndash25 2003

[30] A M Desai R L Autenrieth P Dimitriou-Christidis and TJ McDonald ldquoBiodegradation kinetics of select polycyclic aro-matic hydrocarbon (PAH)mixtures by Sphingomonas paucimo-bilis EPA505rdquo Biodegradation vol 19 no 2 pp 223ndash233 2008

[31] C Guo Z Dang Y Wong and N F Tam ldquoBiodegradationability and dioxgenase genes of PAH-degrading SphingomonasandMycobacterium strains isolated frommangrove sedimentsrdquoInternational Biodeterioration andBiodegradation vol 64 no 6pp 419ndash426 2010

[32] M Uyttebroek J Ortega-Calvo P Breugelmans and DSpringael ldquoComparison of mineralization of solid-sorbedphenanthrene by polycyclic aromatic hydrocarbon (PAH)-degradingMycobacterium spp and Sphingomonas spprdquoAppliedMicrobiology and Biotechnology vol 72 no 4 pp 829ndash8362006

[33] S Y Yuan S HWei and B V Chang ldquoBiodegradation of poly-cyclic aromatic hydrocarbons by a mixed culturerdquo Chemo-sphere vol 41 no 9 pp 1463ndash1468 2000

[34] X Lu T Zhang H Han-Ping Fang K M Y Leung and GZhang ldquoBiodegradation of naphthalene by enriched marinedenitrifying bacteriardquo International Biodeterioration and Bio-degradation vol 65 no 1 pp 204ndash211 2011

[35] J Liu J A Curry W B Rossow J R Key and X WangldquoComparison of surface radiative flux data sets over the ArcticOceanrdquo Journal of Geophysical Research vol 110 no 2 pp 1ndash132005

[36] J Sikkema J A M De Bont and B Poolman ldquoMechanisms ofmembrane toxicity of hydrocarbonsrdquo Microbiological Reviewsvol 59 no 2 pp 201ndash222 1995

[37] W F Guerin and S A Boyd ldquoBioavailability of naphthaleneassociated with natural and synthetic sorbentsrdquoWater Researchvol 31 no 6 pp 1504ndash1512 1997

[38] P Dimitriou-Christidis R L Autenrieth T J McDonald andA M Desai ldquoMeasurement of biodegradability parametersfor single unsubstituted and methylated polycyclic aromatichydrocarbons in liquid bacterial suspensionsrdquo Biotechnologyand Bioengineering vol 97 no 4 pp 922ndash932 2007

[39] Y Prabhu and P S Phale ldquoBiodegradation of phenanthrene byPseudomonas sp strain PP2 novel metabolic pathway role ofbiosurfactant and cell surface hydrophobicity in hydrocarbonassimilationrdquo Applied Microbiology and Biotechnology vol 61no 4 pp 342ndash351 2003

[40] X H Pei X H Zhan S M Wang Y S Lin and L X ZhouldquoEffects of a biosurfactant and a synthetic surfactant on phenan-threne degradation by a Sphingomonas strainrdquo Pedosphere vol20 no 6 pp 771ndash779 2010

[41] Y Zhong T Luan L Lin H Liu and N F Y Tam ldquoProductionof metabolites in the biodegradation of phenanthrene fluoran-thene and pyrene by the mixed culture of Mycobacterium spand Sphingomonas sprdquo Bioresource Technology vol 102 no 3pp 2965ndash2972 2011

[42] YM Kim I H Nam KMurugesan S Schmidt D E Crowleyand Y S Chang ldquoBiodegradation of diphenyl ether and trans-formation of selected brominated congeners by Sphingomonas

sp PH-07rdquo Applied Microbiology and Biotechnology vol 77 no1 pp 187ndash194 2007

[43] J Seo Y Keum and Q X Li ldquoMycobacterium aromativoransJS19b1119879 degrades phenanthrene throughC-12 C-34 andC-910dioxygenation pathwaysrdquo International Biodeterioration andBiodegradation vol 70 pp 96ndash103 2012

[44] N V Balashova A Stolz H J Knackmuss I A KoshelevaA V Naumov and A M Boronin ldquoPurification and charac-terization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene andphenanthrene-degrading bacterial strain Pseudomonas putidaBS202-P1rdquo Biodegradation vol 12 no 3 pp 179ndash188 2001

[45] M A Patrauchan C Florizone M Dosanjh W W Mohn JDavies and LD Eltis ldquoCatabolismof benzoate and phthalate inRhodococcus sp strain RHA1 redundancies and convergencerdquoJournal of Bacteriology vol 187 no 12 pp 4050ndash4063 2005

[46] A Stolz ldquoMolecular characteristics of xenobiotic-degradingsphingomonadsrdquo Applied Microbiology and Biotechnology vol81 no 5 pp 793ndash811 2009

[47] A D Davison and D A Veal ldquoSynergistic mineralization ofbiphenyl by Alcaligenes faecalis type II BPSI-2 and Sphin-gomonas paucimobilis BPSI-3rdquo Letters in Applied Microbiologyvol 25 no 1 pp 58ndash62 1997

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


compounds [10 11] PCR amplification using genes specificprimers or degenerate primers has been used to detect andstudy the diversity of aromatic-dioxygenase genes in PAH-degrading bacterial isolates [12ndash14]

Although many bacteria capable of degrading PAHs havealready been isolated it is still important to screen strainsthat can completely and rapidly decompose PAHs under thecondition of Egyptian environment Therefore the aim ofthe present study was to isolate strains capable of degradinglow and high molecular weight PAHs A strain was obtainedand identified as S koreensis according to morphologicalcharacteristics and 16S rRNA gene sequence analysis and itsability to degrade naphthalene phenanthrene anthraceneand pyrene were studied The production of biosurfactantsand increasing cell-surface hydrophobicity the metabolitesduring the degradation process and the genetics of catabolicgenes in the isolated PAH-degrading bacterium were alsoinvestigated

2 Materials and Methods

21 Sample Collection and Chemicals Oil contaminated soilwas collected in sterilized polyethylene bags from Oil Refin-ery Company in Assiut Egypt and stored at 4∘C in thelaboratory Naphthalene phenanthrene anthracene pyrene(all le99 purity) and mineral basal medium with tracemetals were purchased from Sigma-Aldrich

22 Enrichment Isolation and Evaluation of PAHs-DegradingBacteria Soil enrichment technique was used for the iso-lation of PAH-degrading bacteria as described in [5]About 10 g oil contaminated soil sample was suspended in90mL mineral basal salt medium (MBS) containing (gL)10(NH

4)2SO4 08K

2HPO4 02KH

2PO4 02MgSO

4sdot7H2O

01CaCl2sdot2H2O 0005FeSO

4sdot7H2O and 1mL of trace ele-

ments pH 70 plusmn 02 The medium was supplemented withone of the following PAHs naphthalene phenanthreneanthracene or pyrene at concentration of 100mgL as asole source of carbon The flasks were incubated on anorbital shaker at 150 rpm at 30∘C After 7-day incubation analiquot of 10 enriched cultures was transferred into another250mL conical flask containing 90mL fresh autoclaved MBSmedium supplemented with previously mentioned PAHsThis step was repeated five times to attain well-adapted PAH-degrading enriched bacterial consortia Bacterial strains wereisolated from MBS agar plates coated with the same PAHs asthe sole carbon source Colonies with different morphologieswere individually selected All isolates were evaluated fortheir PAHs-degrading abilities One pure strain of bacteriawith a high PAHs degradation rate was designated as ASU-06 and selected for further study

23 Physiological and Biochemical Tests of Strain ASU-06Conventional physiological and biochemical characteristicswere determined using the procedures described by John andKrieg [15]

24 16S rRNA Gene Amplification and Sequence Determi-nation The genomic DNA was isolated from strain ASU-06 according to the method described by Hesham [16] andthe 16S rRNA gene was amplified Amplification was carriedout with universal primers 27F (5-AGAGTTTGATCCTGG-CTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) in a final volume of 50120583L containing 10mM tris-HCl (pH83) 50mM KCl 15mMMgCl

2 each dNTP at a concentra-

tion of 02mM 125 IU of Taq polymerase each primer at aconcentration of 02mM and 1 120583L of the DNA template PCRwas performed with the following program 5min denatura-tion at 95∘C followed by 36 cycles of 1min denaturation at94∘C 1min annealing at 55∘C 15min extension at 72∘C anda final extension step of 7min at 72∘C 5 120583L of the amplifiedmixture was then analyzed using 15 05 times TBE agarose gelelectrophoresis The gel was stained with ethidium bromidevisualized under UV light and photographed Product of thecorrect size was purified and sequenced in both directionsusing an ABI automated sequencer

25 Sequence Alignment and Phylogenetic Analysis The 16SrRNA gene sequences of the isolate obtained in this studywere aligned and compared with the known 16S rRNAgene sequences in Genbank database using the BLASTsearch at the National Center for Biotechnology Informa-tion (httpwwwncbinlmnihgovBLAST) to determinethe closest available database sequences To determine thetaxonomic position of the isolate a phylogenetic tree wasconstructedwithMEGAversion 40 using a neighbor-joiningalgorithm in addition the Jukes-Cantor distance estimationmethod with bootstrap analyses for 100 replicates was per-formed [17]

26 PAHs Degradation Analysis by HPLC Pure strain wasresuspended in 50mL MBS supplemented with either naph-thalene phenanthrene anthracene or pyrene at concentra-tion of 100mgL for each or mixed of them PAHs (25mgL)for each to a turbidity of 02 at OD (600 nm) Abiotic controlswere prepared in the same way but without addition ofbacteria Samples and controls were prepared in triplicateFlasks were incubated at 30∘C with rotation at 150 rpm for 15days After every three days of incubation 25mL aliquot ofeach sample was extracted three times with equal volumes ofethyl acetate according to the method described byManoharet al [18] The residual PAHs were analyzed by HPLC asdescribed by Bishnoi et al [19]

27 Detection of PAH-Degrading Enzymes The initial dioxy-genase activity catalyzing the conversion of indole to beindigo was determined following the standard methoddescribed by a previous worker [20] while Catechol 23-diox-ygenase activity was determined according to Ornston andStanier [21]

28 Detection of Aliphatic and PAH-Degrading Genes byPCR The presence of six genes including monooxygenaseand dioxygenase genes in the isolated bacterial strain ASU-06 was detected based on PCR amplification The primers





AlkB1FAlkB1R


nahAc FnahAc R




C12OFC12OR


C23OFC23OR









3 Results








ASU-06 (KC4205231)






100

100

100

100

98

84

72

67

0005











TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

12

Rem

aini

ng n

apht

hale

ne (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14(L

n O

DtO

D0

) at6

00

nm

(a)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

Rem

aini

ng an

thra

cene

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14

(Ln

OD

tOD0

) at6

00

nm

(b)

TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

Rem

aini

ng p

hena

nthr

ene (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214

(Ln

OD

tOD0

) at6

00

nm

minus20


Biosurfactant

(c)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

07

Rem

aini

ng p

yren

e (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214


Biosurfactant

(Ln

OD

tOD0

) at6

00

nm

(d)


TD0 2 4 6 8 10 12 14 16

00020406081012141618

PAH

rem

oval

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

5

10

15

20

25

GrowthNaphAnth


(Ln

OD

tOD0

) at6

00

nm





(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp




4 Discussion





Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





AlkB1FAlkB1R


nahAc FnahAc R




C12OFC12OR


C23OFC23OR









3 Results








ASU-06 (KC4205231)






100

100

100

100

98

84

72

67

0005











TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

12

Rem

aini

ng n

apht

hale

ne (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14(L

n O

DtO

D0

) at6

00

nm

(a)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

Rem

aini

ng an

thra

cene

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14

(Ln

OD

tOD0

) at6

00

nm

(b)

TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

Rem

aini

ng p

hena

nthr

ene (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214

(Ln

OD

tOD0

) at6

00

nm

minus20


Biosurfactant

(c)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

07

Rem

aini

ng p

yren

e (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214


Biosurfactant

(Ln

OD

tOD0

) at6

00

nm

(d)


TD0 2 4 6 8 10 12 14 16

00020406081012141618

PAH

rem

oval

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

5

10

15

20

25

GrowthNaphAnth


(Ln

OD

tOD0

) at6

00

nm





(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp




4 Discussion





Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







ASU-06 (KC4205231)






100

100

100

100

98

84

72

67

0005











TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

12

Rem

aini

ng n

apht

hale

ne (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14(L

n O

DtO

D0

) at6

00

nm

(a)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

Rem

aini

ng an

thra

cene

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14

(Ln

OD

tOD0

) at6

00

nm

(b)

TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

Rem

aini

ng p

hena

nthr

ene (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214

(Ln

OD

tOD0

) at6

00

nm

minus20


Biosurfactant

(c)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

07

Rem

aini

ng p

yren

e (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214


Biosurfactant

(Ln

OD

tOD0

) at6

00

nm

(d)


TD0 2 4 6 8 10 12 14 16

00020406081012141618

PAH

rem

oval

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

5

10

15

20

25

GrowthNaphAnth


(Ln

OD

tOD0

) at6

00

nm





(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp




4 Discussion





Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

12

Rem

aini

ng n

apht

hale

ne (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14(L

n O

DtO

D0

) at6

00

nm

(a)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

Rem

aini

ng an

thra

cene

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

2

4

6

8

10

12

14

(Ln

OD

tOD0

) at6

00

nm

(b)

TD0 2 4 6 8 10 12 14 16

00

02

04

06

08

10

Rem

aini

ng p

hena

nthr

ene (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214

(Ln

OD

tOD0

) at6

00

nm

minus20


Biosurfactant

(c)

TD0 2 4 6 8 10 12 14 16

00

01

02

03

04

05

06

07

Rem

aini

ng p

yren

e (

)

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

02468

101214


Biosurfactant

(Ln

OD

tOD0

) at6

00

nm

(d)


TD0 2 4 6 8 10 12 14 16

00020406081012141618

PAH

rem

oval

()

0

20

40

60

80

100

120

Bios

urfa

ctan

t act

ivity

(Um

g pr

otei

n) (

)

0

5

10

15

20

25

GrowthNaphAnth


(Ln

OD

tOD0

) at6

00

nm





(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp




4 Discussion





Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(M)

C12O

(M)

C23O

(M)

NahAc AlkB1

(M)(M)

AlkB

100 bp

550 bp

900 bp

350 bp

487 bp




4 Discussion





Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3

Thec

ompo

unds

identifi

edthroug

hGC-

MSa

nalysis

andtheirm

assfragm

entatio

npatte

rnform

edby

Sphingom

onaskoreensis


ninMBS

containing

(100

mgL)

naph

thaleneph

enanthreneand

pyreneseparately

Num

ber

Metabolites

RT(m

in)

mz(

)1(cont)

Naphthalene

2556

128(M

+ 100)102

(10)

2(6

d)1-M

etho

xynaph

thalene

1737

197(M

+ 3)153

(65)128

(100)102(6)

3(15d

)1-H

ydroxy-2-naphtho

ate

2363

237(M

+ 18)188(100)141(39)128(12)107

(28)86(20)53(71)

4(15d

)2-Hydroxy

4-metho

xycinn

amate

5517

239(M

+ 10)178(100)161(60)133(30)55(17)

5(6

d)Salicylate

2845

179(M

+ 8)137

(100)122(12)97(20)57(40)

6(615d

)Ph

thalate

5184

279(M

+ 10)180(2)167(42)149

(100)71

(12)57(20)

7(15d

)Ph

thalate3

4-dihydrodiol

4724

267(M

+ 2)202

(100)174(3)149(36)137

(1)101(12)98

(5)

8(15)

Pyruvate

812

101(M

+ 5)89(70)73(5)71

(100)56

(53)

1(cont)

Phenanthrene

4459

178(M

+ 100)152

(6)76

(5)

21-H

ydroxyph

enanthrene

5371

197(M

+ 5)193

(100)178(12)127

(12)95(25)69(16)60(23)

3Ph

thalate

5897

279(M

+ 12)219(3)167(42)149

(100)57

(14)

4Dihydroxy-cis

cism

ucon

ates

emialdehyde

4821

219(M

+ 2)175

(10)167

(100)142(5)101(10)57

(12)

52-Hydroxy-4-m

etho

xycinn

amate

5543

239(M

+ 10)178(100)161(58)133(30)55(15)

1(cont)

Pyrene

3859

202(M

+ 100)174

(3)150(5)135(1)122(3)101(35)88

(10)74(4)62

(2)50

(2)28

(3)

2(12d

)1-H

ydroxypyrene

3974

3218(M

+ 100)189

(25)174

(2)163(5)150(2)139(2)109(15)95(13)81(4)63(3)

3(15d

)45-Dihydroxypyrene

43239

236(M

+ 100)200

(35)174

(5)150(3)118

(18)100

(36)74(5)51

(2)28

(2)

4(12)


xylate

4712

5254(M

+ 19)236(15)221

(100)202(10)167

(25)149

(40)128

(21)113

(38)96(35)40(100)

5(12d

)34-Dihydroxyph

enanthrene

38287

208(M

+ 100)193

(80)176

(15)164

(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

6(9

d)1-H

ydroxy-2-naphtho

ate

1971

188(M

+ 100)146

(5)119

(30)105

(55)91(73)78

(10)65(15)51(3)41(5)

7(15d

)Be

nzocou

marin

6858

224(M

+ 15)207(50)191

(10)163

(15)147

(13)120

(80)92(48)63(25)44(100)16

(83)

8(12d

)trans-2-Ca


37426

203(M

+ 100)189

(9)175(5)149(5)137(1)123(1)101(15)88

(10)75(3)40

(12)

9(alld)

Phthalate

4645

297(M

+ 10)167(30)149

(100)132(1)113(15)93(2)71

(25)57(35)43(22)

10(12d

)Ph

thalate3

4-dihydrodiol

37676

202(M

+ 100)182

(19)151(65)133(32)114

(1)101(20)88

(8)63

(12)

11(6

d)34-Dihydroxyph

thalate

397

211(M

+ 5)195

(60)167

(25)146

(10)132

(26)117

(31)105

(5)90

(17)76(60)63(20)40(100)

12(6

d)Ca

rboxy-ciscis-mucon

ate

40023

183(M

+ 15)168(25)155

(20)141

(10)124

(21)111(21)97(20)85(25)71(20)57

(55)40(100)

13(15d

)1-M

etho

xy2-hydroxypyrene

4832

247(M

+ 100)217

(50)201

(100)189(50)174

(10)150

(5)100(20)87(5)

14(6

d)1-M

etho

xyph

enanthrene

382

208(M

+ 100)193

(80)177

(5)164(30)150

(20)132

(32)118

(28)105

(39)79(60)51(30)

Retentiontim

es(RT)

andmassp

ercharge

ion(m

z)(

)abu

ndancyIncub

ationtim

eper

days

(d)







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2via







References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Biodegradation Ability and Catabolic Genes of …downloads.hindawi.com/journals/bmri/2014/127674.pdf · 2019-07-31 · Research Article Biodegradation Ability and