Ligninolytic fungus Polyporus sp. S133 mediated metabolic degradation of fluorene

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

Environmental

Microbiology

Ligninolytic

fungus

Polyporus

sp.

S133

mediated

metabolic

degradation

of

ﬂuorene

Zainab

Mat

Lazim

a,b

_,

_Tony

_Hadibarata

a,b,∗

a_Centre_for_{Environmental}_{Sustainability}_and_Water_Security_(IPASA),_Research_Institute_for_Sustainable_Environment_(RISE),

UniversitiTeknologiMalaysia,Skudai,Johor,Malaysia

b_Department_of_{Environmental}_Engineering,_Faculty_of_Civil_Engineering,_Universiti_Teknologi_Malaysia,_Johor,_Malaysia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11July2015

Accepted22December2015

Availableonline23April2016

AssociateEditor:CynthiaCanêdoda

Silva Keywords: Metabolites Ligninolyticenzymes Polyporussp.S133 Non-ionicsurfactants Solubilization

a

b

s

t

r

a

c

t

Thisstudyaimedtoinvestigatetheimpactofnonionicsurfactantsontheefﬁcacyofﬂuorine

degradationbyPolyporussp.S133inaliquidculture.Fluorenewasobservedtobedegraded

initsentiretybyPolyporussp.S133subsequenttoa23-dayincubationperiod.Thefastest

cellgrowthratewasobservedintheinitial7daysintheculturethatwassupplemented

withTween80.Thedegradationprocesswasprimarilymodulatedbytheactivityoftwo

ligninolyticenzymes,laccaseandMnP.Thehighestlaccaseactivitywasstimulatedbythe

additionofTween80(2443U/L)followedbymixedsurfactant(1766U/L)andBrij35(1655U/L).

UV–visspectroscopy,TLCanalysisandmassspectrumanalysisofsamplessubsequentto

thedegradationprocessintheculturemediumconﬁrmedthebiotransformationofﬂuorene.

Twometabolites,9-ﬂuorenol(max270,tR8.0minandm/z254)andprotocatechuicacid(max

260,tR11.3minandm/z370),wereidentiﬁedinthetreatedmedium.

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

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

Introduction

Fungalbiotransformationisconsidered asoneofthe most

importantmethodsforremovingpollutantfromthe

ecosys-tem. In case oforganic pollutants, the primary issue that

impedes their clearance is their characteristically low

sol-ubilityinwater. Theinherenthydrophobicnature ofthese

organic pollutants results intheir selective partitioning in

the soil matrix such that their bioavailability is seriously

∗ _{Corresponding}_author.

E-mail:hadibarata@utm.my(T.Hadibarata).

compromised. This in turn jeopardizes the success of the

bioremediation treatment. Thereis anurgentneed forthe

developmentofalternativetreatmentmodalitieswhichwill

help remove these pollutants from the environment by

makingthemmoreavailableforthedegradationby

microor-ganisms. Surfactant-mediated biodegradation is one such

promisingprocess.Thismethodhasthepotentialtoincrease

the solubility of hydrophobic compounds, such as PAHs,

thereby increasingtheirbioavailabilityand expeditingtheir

removal from the environment.1–3 _Previous _studies _have

http://dx.doi.org/10.1016/j.bjm.2016.04.015

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shownthatdependingonthesurfaceofthesubstanceand

thetypeofthesurfactantused,theprocessofsurfactant

sorp-tiontomicroorganismscanresultineitheranincreaseora

decreaseinadhesion.4

Micellesarethesolutionaggregatesformedbysurfactant

moleculesinwaterwhentheirconcentrationisabovethe

crit-icalmicelleconcentrationorCMC.Micelleshavedualnature:

their hydrophilic outer surface is exposed to the solvent

whereasthecoreiscomposedofhydrophobicmoieties.

Sev-eralresearchersareactivelyconductingstudiesinanattempt

toinvestigatetheeffectofsurfactantsonmicrobial

transfor-mationofpollutants;allthesestudiesarebasedonsurfactant

utilization atconcentrationsabove CMC.Hydrocarbons are

known to be partitioned into the hydrophobiccore of the

micelles.5,6_But,_it_is_well_established_that_extent_of

solubili-zationisinﬂuencedbyamultitudeoffactorssuchastypeand

concentrationofthesurfactant,theinteractionbetweenthe

surfactantandsoil,thecontacttimebetweenthe

contami-nantandsoilaswellasthehydrophobicityofthecompound

inquestion.7,8

Fluorene is a three-ring PAH found ubiquitously in the

environment.9 _It _is_produced _as _a _result _of_an _incomplete

combustionof certain products. Fluorene leaches into the

groundwatereitherasaresultofdirectcontaminationfrom

pollutedsurfacewatersorindirectlythroughthesoil.Fluorene

is not considered as a genotoxic agent but the

carcino-genic aspectof its molecularstructure hasbeen popularly

used as an indicator for the evaluation ofPAH-containing

pollutants.10_White-rot_fungi_have_been_frequently_employed

forthedegradationofvariousorganicenvironmental

pollut-ants.Theirexceptionalcapacitytodegradeandbiotransform

organiccontaminantscanbeattributedtotheproductionof

ligninolyticenzymessuchasMnP,LiP,laccase,andversatile

peroxidase.11–13_The_ligninolytic_enzymes_are_responsible_for

thedepolymerizationofPAHs,whichresultsinthe

produc-tionofvarioustypesofphenol.Theyalsooxidizesubstrates

inthepresenceofasuitableco-substrate.14_Extensive_studies

havebeenundertakeninordertoinvestigatethepotentialof

white-rotfungustodegradePAHs.15,16 _However,_the_impact

ofsolubilizationofﬂuorenewithsingleandmixednonionic

surfactantsonthedegradationrateremainsunexplored.

Thisstudy attempts toﬁll the above mentioned lacuna

byevaluatingthebiodegradationofﬂuorenebythePolyporus

speciesinliquid culturesin the presenceoftwo synthetic

surfactants.Ourstudyalsoquantiﬁestheresultantbiomass

produced,the extracellular enzymes and the glucose

con-sumptioninvolvedintheprocess.Furthermore,inanattempt

tocomprehensivelystudythedegradationprocess,

metabo-litesformedduringﬂuorenedegradationhavebeenidentiﬁed

andcharacterizedusingavarietyofchromatographic

tech-niques.Twononionicsurfactants,Tween80andBrij35,were

chosen to study the inﬂuence of the surfactant structure

onthebiodegradationandbiotransformationofﬂuoreneby

thefungal biomass.Studies haveestablishedthat nonionic

surfactants are more effective on account of their higher

adsorptioncapacityinclayfractionsascomparedtoanionicor

cationicsurfactants.6_The_relationship_between_enzyme

activ-ity, glucose consumption, ﬂuorene utilization and biomass

productioninthepresenceofTween80andBrij35wasalso

established.Thedatageneratedbythisstudywillcontribute

Table1–Structureandphysical–chemicalcharactersof

ﬂuorene.

Molecularstructure

Molecularformula C13H10

Appearance Whitecrystal

Molecularweight 166.22 Density 1.202g/mL Meltingpoint 117◦C Boilingpoint 295◦C Aqueoussolubility (20–25◦C) 1.98mg/L Octanol–waterpartition coefﬁcient(logL/kg) 4.0155

towarddefininganewapproachforbiotransformationofPAHs byimprovingourunderstandingabouttheinfluenceof surfac-tantsintheprocess.Itishopedthattheresultsofourstudy willbeasignificantcontributiontothefieldofbiodegradation especiallysurfactantenhancedbiodegradation(Table1).

Materials

and

methods

Chemicalsandmicroorganisms

Fluorene and an internal standard,4-chlorobiphenyl, were

obtainedfromSigma–Aldrich(Milwaukee,WI).Bothwere

sup-pliedat>97%purity.NonionicsurfactantssuchasBrij35and

Tween80,andextractionsolventssuchasethylacetateand

dichloromethanewereobtainedfromAcrosChemicals.Malt

extract as well as other chemicals required forliquid

cul-turewereprocuredfromDifco(Detroit,USA).Allthesolutions

obtainedorpreparedutilizedanalyticalgradeandhigh-purity

reagents. Thefungus usedin the study was isolated from

Matsuyama,Japan.ThePolyporussp.S133wascultivatedin

a100-mLErlenmeyerﬂaskcontainingmineralsaltmedium

(MSM).ThecompositionofthegrowthmediumMSMisgiven

inTable2.ThesterilityofMSMwasensuredbyautoclaving

the growthmediumpriortouse.Fluorene (10mg/L),Tween

80andBrij35solutionsweresterilizedbyvacuumﬁltration

inordertopreventanyconversionofthecompounds.15,17_For

estimationofenzymeactivity,theculturewassupplemented

withthedesiredamountofﬂuoreneandincubatedforspeciﬁc

timeintervals.Allexperimentswereconductedintriplicate.

Enzymeassaysandbiomassdetermination

The enzymeanalysis of the funguswas conducted as per

the protocoldescribed earlier.18 _Laccase_activity _was

deter-minedbyABTSoxidationandassayedspectrophotometrically

at420nm.MnPactivitywasdeterminedbytheoxidationof

malonate anddimethoxyphenol inan MnSO4 solution and

assayedbymonitoringchangesinabsorbanceat270nm.The

enzymeproductionwasexpressedinUL−1.Biomass

produc-tion was estimatedbycentrifugingthe culturefollowedby

ﬁltrationthroughaWhatmanNo.1ﬁlterpaper.Thebiomass

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Table2–Concentrationsofmineralsaltmedia(MSM)constituents.

MSMconstituents Concentration(g/L) Traceelement Concentration(mg/L)

Maltextract 10 FeSO4·7H2O 12

Glucose 10 MnSO4·7H2O 3 KH2PO4 2 ZnSO4·7H2O 3 MgSO4·7H2O 1 CoSO4·7H2O 1 CaCl2·2H2O 0.5 (NH4)6Mo7O24·4H2O 1 Ammoniumtartrate 0.5 Traceelement 10mL Solubilizationtest

The solubilization test was performed as described previously.19,20 _Single _nonionic _surfactants _were _used _in

the following concentrations: 0.1, 0.5, 1, 2, 4mM. Mixed

surfactantsolutions(Tween80andBrij35)werepreparedat

sameconcentrationina1:1ratio.

Instrumentalanalysis

Agilent 5975E FID GC-MS (DB–1 capillary column; 0.25␮m

ID;0.25mmdiameter;30mlength)wasusedtoidentifyand

characterize the metabolites present in the incubated

cul-ture.Asdescribedinourpreviousstudy,trimethylchlorosilane

wasusedinthesilylationprocedureemployedfordetecting

samplescontaininghydroxylandcarboxylgroups.18_The_GC

temperaturewasmaintainedasfollows:initial70◦C(1min),

increasedatthe rateof18◦Cmin−1 till150◦C,increasedat

therateof28◦Cmin−1 till330◦C,heldat330◦Cfor10min.

Theinjector and interfacetemperatures were set at260◦C

withasplitlesstimeof2min.Theinjectionconcentrationwas

1␮Linthesplitlessconditionandﬂowrateoftheheliumwas

adjustedto1mL/min.TheEIionizationmodewasusedwith

electronenergyof1.3eVandamassrangeof50–500amu.The

massspectraofthesampleswerecomparedwithauthentic

standardcompoundsaswell asthedatabaselibrary (Wiley

275L).21

Results

and

discussion

Solubilizationofﬂuorene

Fig.1showsthechangeinthesolubilityofﬂuoreneasaffected

bytheadditionofsingleandmixednonionicsurfactants.It

wasobservedthatthesolubilityofﬂuorenewassigniﬁcantly

enhancedbyadditionofsurfactantsand thatthesolubility

ofthepollutantwas directlyproportional tothesurfactant

concentrations.Thehighestsolubilitynotedinthisstudywas

achievedbytheadditionofTween80(39.4mgL−1);the

addi-tion ofmixed solutions resultedin amoderate rise inthe

solubility,whereasthelowestsolubilityvalueswereobserved

uponadditionofBrij35(20.4gL−1).Itishypothesizedthatthe

additionofﬂuoreneincombination withthenonionic

sur-factantsolutionfacilitatesthe formationofmixedmicelles

whichinturnimprovesthecontactbetweenmicroorganisms

andsurfactant.

BiodegradationofﬂuorenebyPolyporussp.S133

Itisevidentfromthedataavailableintheliteraturethat

ﬂuo-reneislikelytobeabsorbedontothefungalbiomasssurface

priortobeingaccumulated,degraded,andmetabolizedbythe

fungus.Inordertoidentifytheimpactofsingleandmixed

sur-factantsonﬂuoreneremoval,thegrowthmediumofPolyporus

sp.wassupplementedwiththeadditionofTween80,Brij35,

andmixturesofTween80andBrij35.S133culturewas

under-takeninordertoestimatebothbiomassproductionaswell

asﬂuorenedegradation.Resultsindicatedthatboththecell

growthaswellasthedegradationofﬂuorenewasinﬂuenced

bythepresenceofsurfactants(Fig.2).AsisshowninFig.2,

thepresenceofTween80signiﬁcantlyenhancedtheremoval

ofﬂuoreneaswellasthebiomassproduction.Fluorenewas

foundtobedegradedinentiretybyPolyporussp.S133afteran

incubationperiodof23d.Thefastestgrowthratewas

expe-riencedintheinitial7days(0.6gL−1 perday)intheculture

thatwassupplementedwithTween80.Tween80increased

thesorptionextentandnodistinctionwasfoundbetween

sur-factantandmycelia.Fig.2suggeststhattheadditionofTween

80andmixedsurfactantsenhancedtheﬂuorenedegradation

andbiomassproductionbyPolyporussp.S133.Itwasobserved

afterthe23-dayincubationperiodthatthecellularproduction

andgenerationslowlydecreasedlargelybecausemostofthe

ﬂuorenehadalreadybeendegraded.Theresultsshowthatthe

maximumﬂuorenedegradationwasobservedfortheTween

80culture(0.4mgperday),which0.2and0.27mg/dcompared

towasmixedsurfactantandBrij35.

0 10 20 30 40 4 3 2 1 0 Concentration (mM) S olubilit y (mg /L ) Tween 80 Brij 35 Tween 80-Brij 35

Fig.1–Solubilizationofﬂuorenebysingleandmixed

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40

A

B

C

5 4 3 2 1 0 5 4 3 2 1 0 5 4 3 2 1 0 30 20 10 0 40 30 20 10 0 40 30 20 10 0 0 5 10 15 20 Incubation time (d) Biomass production (g/L)

Fluorene residue Biomass production

Fluorene residue

(mg/L)

25 30 35

Fig.2–Utilizationofﬂuoreneandbiomassproductionin

singleandmixedsurfactant:Tween80(A);Brij35(B);

Tween80–Brij35(C).

Dioxygenase,peroxidaseandlaccaseenzymesplaya

crit-ical role in the biotransformation of ﬂuorene by Polyporus

sp.S133.Enzymessuchas1,2-dioxygenase,2,3-dioxygenase,

MnP, LiP, and laccase were studied with the aim of

gain-ingadditionalinsightintothemechanismofdecolorization

(Table3).Itwasnotedthatthedegradationprocesswas

pri-marilydeterminedbytheactivityoftwoligninolyticenzymes:

Table3–EnzymedetectionofPolyporussp.S133at

initialdegradation(10d).

Enzyme Activity(U/L)

Manganeseperoxidase 45.4

Ligninperoxidase 1.3

Laccase 113.8

1,2-Dioxygenase 0.2

2,3-Dioxygenase 0.5

Valuesaremeansofthreeexperiments.

2500 Glucose Laccase MnP

A

B

C

2000 1500 1000 500 0 5 4 3 2 1 0 5 4 3 2 1 0 5 5 10 15 20 Incubation time (d) Glucose (g/L) Laccase (U/L); Mnp (U/L) 25 30 35 4 3 2 1 0 0 2500 2000 1500 1000 500 0 2500 2000 1500 1000 500 0

Fig.3–Effectofnon-ionicsurfactantonenzymeactivities andglucoseconsumption.Tween80(A);Brij35(B);Tween 80–Brij35(C).

laccaseand MnP.In order toassay the impactofnonionic detergents on the enzyme activity as well as on the ﬂuo-renedegradationprocess,activityoftheenzymewasplotted againstglucoseconsumptionfordifferentconcentrationsof thesurfactants(Fig.3).

Itwasobservedthatforallsurfactants,themaximum

lac-case production was seen on day 30 of the incubation; it

wasalsoseenthattheactivitydeclinedsigniﬁcantlyatday

15 forBrij 35 andatday 23 formixedsurfactants. Growth

mediumsupplementedwithTween80exhibitedthehighest

activity(2443UL−1)forlaccasefollowedbythemixed

surfac-tant (1766UL−1)and lastlybyBrij35 (1655UL−1). Asimilar

analysis of the MnP activity with varying combinations of

thesurfactantprovedthatMnPactivitydecreasedafter7–15

days ofincubation and the downward trend continued till

the endoftheexperimentalperiod.Theglucose

consump-tion wassigniﬁcantlyhigh inthe ﬁrst7daysofincubation

but was observedtodecrease slowlyafterthat tillthe end

oftheexperimentalperiod.Thehighestconsumptionof

glu-cosewasseeninthegrowthmediumsupplementedbyBrij

35 (3.8gL−1)after30daysofincubation. Fromthis resultit

canbeconcludedthatwhenthegrowthmediumis

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fungalgrowth.Thefunctionofglucoseintheculturewasto

inducetheenzymaticsystemofthefungus.Fromtheresults

obtainedabove,wetheorizethattheproductionofligninolytic

enzymessuchaslaccaseandperoxidasewassuppressedby

additionofﬂuoreneandsurfactant.Ourresultsareinaccord

withapreviousstudythatanalyzedPAHdegradationby

var-iouswhite-rotfungi.Theauthorsofthestudyreportedthat

thedegradationrateswerefoundtobeassociatedbothwith

theactivityofligninolyticenzymesaswellaswiththetypeof

surfactantused.Theauthorsalsoproposedthattheenzymes

thatplayanimportantroleinPAHsdegradation,namely

lac-caseandMnP,arealsoimplicatedinthemetabolismofsome

PAHs.Laccaseisalsoconsideredtobeanimportantenzyme

foroxidizingPAHsinliquidculture.15,16_The_function_of_MnP

intheﬂuoreneremovalprocessissimilartothatoflaccasein

theearlyweeksofincubationanditsustainstilltheendofthe

incubationperiod.

Itwasobservedthatthemicroorganismsgrewatafaster

ratewhentheliquidculturewassupplementedwithacarbon

sourcesuchasglucose.Glucosewassupplementedintothe

fungalculturetofulﬁllthefunctionofaco-metabolicsubstrate

inthedegradationprocess.Previousstudieshaveestablished

thatglucoseplaysanimportantroleinthedepolymerization

oflongchainfattyacidsinthebiodegradationprocess.Also,

severalreportsinliteratureclaimthatmanyorganicpollutants

were morequicklytransformed when glucose was

supple-mented into the culture as a co-substrate for growth.22,23

Otherresearchershaveprovedthatformostwhite-rotfungi,

simple carbon sources, such as glucose, provide nutrients

thatarecrucialfortheproductionofextracellularenzymes

andbiomassproduction.Althoughligninolyticenzymesare

producedduringthesecondarymetabolismandarestrongly

inﬂuenced by nutrient limitation, the actual pollutant

II

OH O HO HO COOH COOH COOH

I

Fig.4–Proposedpathwayofﬂuorenemetabolismby

Polyporussp.S133.Bracket-compoundswereunidentiﬁed

inourcultureextract.

degradation process occurs after the production of

enzymes.24

Fluorenemetabolites

The culture was acidiﬁed with HCl in order to stop the

fungal growth following which the sample was extracted

with ethyl acetate.TLC and UV absorbance ofthe sample

extractsrevealedthepresenceofthreeﬂuorinemetabolites

(Table4).GC-MSanalysesoftheethylacetate-extracted

sam-pleswere foundtohavetwomajorchromatographic peaks

(Table4).MetaboliteI(Rf0.35)exhibitedmaxat270nmwhich

corresponds to the 9-ﬂuorenol standard. The spectrum of

CompoundI(tRof8.0min)showmaximam/z254,M+_,_a

frag-mentionatm/z239(M+₋₁₅₎_representing_loss_of_methyl,_as

wellastheexpectedfragmentionatm/z165(M+₋₈₉₎_which

couldbeaccountedforbythesequentiallossofOSi(CH3)3,and

Table4–UVabsorbanceandmassspectralanalysisofthemetabolicproductproducedfromﬂuorenebyPolyporussp.

S133.

Metabolites Retentiontime(min) m/zoffragmentions(%

relativeabundance)

Possiblestructure UVabsorbance

I 8.0 73(62),104(6),139(12),152 (19),165(100),178(17),193 (5),221(5),223(6),239(82), 254(92,M+_),₂₅₅₍₃₁₎ 9-Fluorenol-BSTFA (conﬁrmedwitha withastandard) 0 0.2 0.4 0.6 0.8 1 220 260 300 Wavelength (nm) Absorbance II 11.3 73(100),74(12),165(17), 181(14),193(98),194(28), 223(15),311(33),355(54), 370(73,M+_),₃₇₁₍₂₉₎ Protochathecuic acid-TMSderivative (conﬁrmedwitha standard) 0 0.2 0.4 0.6 0.8 1 220 260 300 Wavelength (nm) Absorbance

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at73,178,and254.Furthermetabolitecharacterizationwas

donebygrowingPolyporussp.S133ina9-ﬂurenolacid

cul-ture.Intheculture,weidentiﬁedmetaboliteII(tRof11.3min),

whose spectra was found to have a main ion at m/z 370

(M+_),_a_fragment_ion_at_m/z₃₅₅_(M+₋₁₅₎_representing_loss_of

methylalongwithexpectedfragmentionsatm/z181[M+_−89;

OSi(CH3)3], 73[(CH3)3Si], 165and 193.Basedon the mass

spectraldataobtainedabove,weidentiﬁedcompoundII as

protocatechuicacid.

Polyporussp.S133isawhite-rotfungusthathasthe

capac-itytoeliminateandtransformmanytypesofPAHs.Inthis

study,weestablishedthatthefungussucceededinproducing

theligninolyticenzymeimportantforthedegradationof

ﬂu-orene.Twomajorenzymes,laccaseandMnP,werepresumed

tobevitalforthetransformationoftheﬂuorene.Duringthe

laccase-mediatedoxidationofPAHs,quinoneisformedduring

theinitialstepofdegradationwhile9-ﬂuorenol-1-carboxylic

acid is formed at a later stage. In the next step the

car-boxylicacid compound undergoes a ring cleavage to form

9-ﬂuorenolandprotocatechuicacidwhichisﬁnallyconverted

totricarboxylicacid.Unfortunately,ﬂuorenequinoneand

9-ﬂuorenol-1-carboxylicacid wasnotidentiﬁed inanyofthe

extractsobtainedinthisstudy(Fig.4).Itisknownthatcertain

metabolitesproducedasaby-productofPAHsmetabolismare

hazardousinnature.Forthisreasonfragmentarymetabolism

ofﬂuorenehas thepotential topose a gravethreat tothe

ecosystembecausethecharacteristicofintermediate

metabo-litesmay actuallybemoreharmfulthanthatoftheparent

compound.25–28

Conclusions

Fromtheresultspresentedaboveitcanbeconﬁdently

con-cludedthat ﬂuorene,athree-ringPAH, wasdegradedinits

entirety by a fungusPolyporus sp.S133 followinga 23-day

incubationintheliquidcultureformat.Thepositive

relation-shipbetweenbiomass,enzymeanddegradationestablished

thatthe ﬂuorenebioavailability was signiﬁcantlyincreased

bysurfactantmediatedbioaccumulationenhancement

espe-ciallythatbyTween80.Nonionicsurfactantswereobserved

toincreasethesorptionrateofﬂuoreneonthebiomassand

thusimprovetheremovalprocess.Twoimportantligninolytic

enzymes,laccaseand MnP,werefoundtoplayasigniﬁcant

role in the complex process of ﬂuorene transformation by

Polyporussp.S133.Toconclude,ourstudymakesapowerful

argumentinsupportoftheutilityandimportanceofTween

80inthePAHs-pollutedsoilbioremediationprocess.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

A part of this research was ﬁnancially supported by

a Fundamental Research Grant Scheme (FRGS) of

Min-istryofHighEducationMalaysia(R.J130000.7809.4F465)and

Research University Grant ofUniversiti Teknologi Malaysia

(Q.J130000.2522.10H17).

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