Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process

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

Environmental

Microbiology

Polycyclic

aromatic

hydrocarbons

degradation

by

marine-derived

basidiomycetes:

optimization

of

the

degradation

process

Gabriela

A.L.

Vieira

_,

_Mariana

_Juventina

_Magrini

_,

_Rafaella

_C.

_{Bonugli-Santos}

_,

Marili

V.N.

Rodrigues

_,

_Lara

_D.

_Sette

a_{UniversidadeEstadualPaulistaJúliodeMesquitaFilho(UNESP),InstitutodeBiociências,DepartamentodeBioquímicaeMicrobiologia,} RioClaro,SP,Brazil

b_{UniversidadeEstadualdeCampinas(UNICAMP),CentroPluridisciplinardePesquisasQuímicas,BiológicaseAgrícolas,Paulínia,SP,} Brazil

c_{UniversidadeFederaldaIntegrac¸ãoLatino-Americana(UNILA),InstitutoLatinoAmericanodeCiênciasdaVidaedaNatureza,Fozdo} Iguac¸u,PR,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5November2017 Accepted13April2018 Availableonline3May2018 AssociateEditor:VâniaMelo

Keywords: Marinebiotechnology PAHs Fungi Acutetoxicity Experimentaldesign

a

b

s

t

r

a

c

t

Pyrene and benzo[a]pyrene(BaP)are highmolecular weight polycyclicaromatic hydro-carbons(PAHs)recalcitranttomicrobialattack.Althoughstudiesrelatedtothemicrobial degradationofPAHshavebeencarriedoutinthelastdecades,littleisknownabout degra-dationoftheseenvironmentalpollutantsbyfungifrommarineorigin.Therefore,thisstudy aimedtoselectonePAHsdegraderamongthreemarine-derivedbasidiomycetefungiand tostudyitspyrenedetoxification/degradation.Marasmiellussp.CBMAI1062showedhigher levelsofpyreneandBaPdegradationandwassubjectedtostudiesrelatedtopyrene degrada-tionoptimizationusingexperimentaldesign,acutetoxicity,organiccarbonremoval(TOC), andmetaboliteevaluation.Theexperimentaldesignresultedinanefficientpyrene degrada-tion,reducingtheexperimenttimewhilethePAHconcentrationappliedintheassayswas increased.Theselectedfunguswasabletodegradealmost100%ofpyrene(0.08mgmL−1) after48hofincubationundersalinecondition,withoutgeneratingtoxiccompoundsand withaTOCreductionof17%.Intermediatemetabolitesofpyrenedegradationwere identi-fied,suggestingthatthefungusdegradedthecompoundviathecytochromeP450system andepoxidehydrolases.Theseresultshighlighttherelevanceofmarine-derivedfungiin thefieldofPAHbioremediation,addingvaluetothebluebiotechnology.

©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

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

∗ _{Correspondingauthorat:DepartamentodeBioquímicaeMicrobiologia–IB,UniversidadeEstadualPaulistaJúliodeMesquitaFilho}

(UNESP),Av.24A,1515,13506-900,RioClaro,SP,Brazil. E-mail:larasette@rc.unesp.br(L.D.Sette).

https://doi.org/10.1016/j.bjm.2018.04.007

1517-8382/©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are organic com-poundscomposedoftwoormorefusedbenzenerings.These compounds are formed during the combustion of organic molecules and their subsequent recombination1,2 _{and are}

widelydistributedintheenvironment.3,4

PAHs discharged from various activities, such as com-bustion of fossil fuels, shipping, use and disposal of petroleum products, agricultural burning, coal, and wood-preserving products are persistently hazardous pollutants tothe environment.5 _{Through atmospheric depositionand}

municipal as well as industrial waste water discharges, PAHsaredistributedinthemarineenvironmentandfinally enrichedinthesedimentduetotheirgreatabilitytobindto particulatematter.6

Lowmolecularweight(LMW)PAHs(composedby2–3 aro-maticrings)arepredominantinpetrogenicsourcesandcan beintroducedinto aquaticenvironmentsthrough oilspills, dischargefromtankeroperations,andmunicipalandurban runoff.Highmolecularweight(HMW)PAHs(composedby4–6 aromaticrings),suchaspyreneandBaP,aremostimportant inpyrogenicsourcesandarereleasedintotheenvironment mainlyintheformofexhaustandsolidresidues.7,8Bothof thesecompoundsareclassifiedbytheUSEnvironmental Pro-tectionAgency(EPA)asprioritypollutants9,10_andhavebeen

accumulatinginsedimentsduetotheirlimitedwater solubil-ityandhighaffinityforparticulatematter.11

Microbialdegradationcanbeconsideredasanattractive biotechnologicalalternativeforachievingpossible mineraliza-tionofthepollutant12_{anditstransformationintolesstoxic}

productswithgreatersolubilityinwater,whichcanthenbe degradedbytheactionofothermicroorganisms.13,14

Hydrocarbon degradation using fungi from marine ori-gin was reported by Ahearn and Meyers.15 _{However, their}

application in this field is still poorly studied. The use of marine-derivedfungiforthebioremediationofpollutedsaline environments2_{isfacilitatedbytheirtolerancetosaline}

con-ditions,settingthesemicroorganismsasimportantmicrobial resourcesforbiotechnologicalapplicationinthedegradation ofPAH polluted environments, such as ocean and marine sediments.16

In the field of environmental pollutants biodegradation studies related to toxicity ofmetabolites generated during theprocessisrequired.Forthatend,crustaceansandrotifers larvaeareappliedinecotoxicologicalassaysduetotheir eco-logicalrelevanceandtheireasymaintenanceunderlaboratory conditions.17–19_{Additionally,PAHsbiodegradationgenerallyis}

incomplete,resultinginintermediatecompounds. Determi-nationsofPAHs degradationmetabolic pathwaysenableto predictthedestinyofsuchcompoundsintheenvironment andelucidatetoxicityloss.20,21

Considering that PAHs are pollutants commonly found inthe marine environmentand that little isknown about theirdegradationbymarinemicroorganism,thepresentstudy aimed to screen (among the three marine-derived basid-iomycetes)thebestfungusabletodegradepyreneandBaPand evaluateditscapacitytodetoxifyanddegradepyreneunder salinecondition.

Material

and

methods

Microorganisms

The marine derived fungal strains Tinctoporellus sp.CBMAI 1061,Marasmiellussp.CBMAI1062,andPeniophorasp.CBMAI 1063were isolated from the Braziliansponges Dragmacidon reticulatum andAmphimedon viridiscollected inthe townof SãoSebastião,SãoPauloState,Brazil.22_{Allofthemwere}

taxo-nomicallyidentifiedaspreviouslyreportedbyBonugli-Santos etal.23_{anddepositedintheBrazilianCollectionof}

Microor-ganisms from Environment and Industry –CBMAI (CPQBA, UNICAMP).

Fungalgrowthandinitialscreening

Thethreeisolateswerecultivatedin2%(m/v)maltextractagar (MA2)fortendaysat28◦C.ForinitialscreeningofPAHs degra-dation(pyreneandBaP),threefungalculturecylinders(0.5cm diameter) from the edgeof thecolony were transferredto 125mLErlenmeyerflaskscontaining50mLof2%maltextract broth(MB2)andculturedfor72hat28◦Cand120rpm(initial biomass).PyreneandBaPweredissolvedindimethylsulfoxide andwereaddedseparatelyattheconcentrationof0.04and 0.02mgmL−1,respectively.TheconcentrationofPAHsapplied inthisworkwerechosenbasedinpreviousstudyreportedby Passarini.9_{Theflaskswereincubated for7,14and 21days}

underthesameconditions.Nosalineconditionswereapplied fortheinitialscreening.

QuantitativeanalysesofPAHsdegradation

PAHs degradation were quantified using gas chromatography–mass spectrometry (GC–MS) analyses, as previously described byPassarini et al.9 _{Theextraction}

ofthePAHsand theirdegradationproductsinfermentative medium was accomplished using an Ultra-Turrax system. Ethylacetatewasusedassolventtorupturethefungalcellular wallsandremoveadsorbedcompounds.ToeachErlenmeyer flaskcontainingthefungusinfermentativemedium,50mL of ethylacetatewas added, and the funguswas subjected tocellulardisintegrationat14,000rpmfor1min.The mate-rial was then filtered, transferred to a 250mL separating funneland shakenvigorouslyfor1min. Theorganic phase was filtered by anhydrous sodium sulfate saturated with ethylacetatedirectlyintoa250mL,round-bottomflask;the aqueous phase was re-extracted with an additional 50mL of ethyl acetate. The organic phases were combined and evaporatedundervacuumat40–45◦Cuntilthevolumewas reduced to approximately 2mL. The contents of the flask were analyticallytransferred into a 10mL volumetricflask anddilutedtothemarkwithethylacetate.Analiquotof1mL ofthis solution was thendiluted in10mLofethylacetate containing 1mL of the internal standard solution prior to GC–MSanalysis.

FortheGC–MSanalyses,anAgilent(PaloAlto,CA)6890N GC that was equipped with anAgilent 7683B autosampler coupled to an Agilent 5975 mass-selective detector was used. Data acquisition and analyseswere performed using

the standard software supplied by the manufacturer. The compoundswereseparatedonafused-silicacapillarycolumn (HP-5MS,30m×0.25mm i.e., 0.25-␮m film thickness) (J&W Scientific,Cologne,Germany).TheGCtemperatureprogram wasasfollows:180◦C,5◦Cmin−1upto310◦C,heldfor10min. Thetemperaturesfortheinjectionportanddetectorwereset at290◦Cand300◦C,respectively.Thesplitlessinjectionmode wasusedwiththesplitoutletopenedafter3min,andhelium, withaflowrateof1.0mLmin−1,wasusedasthecarriergas. Theretentiontimesandcharacteristicmassfragmentswere recorded,and the chosendiagnostic massfragments were monitoredinthe selected ionmonitoring (SIM)mode. The characteristic ions that were used for quantification were as follows:benzo[a]pyrene (m/z252, 126, 250), pyrene (m/z 202,101,200)anddibutylphthalate(m/z149,205,223)asan internalstandard.Forquantification,thepeakarearatiosof the analytes tothe internal standard were calculated as a functionofthecompoundconcentration.

Metabolitesidentification

TheGC-MStechniquewasappliedformetabolitesdetection formedduringpyrenedegradation.Thebasidiomycete Maras-miellussp.wasincubated in50mLofliquid medium(MB2) for5and15daysinthepresenceofpyrene(0.04mgmL−1)at 28◦Cand120rpm.Theculturemediumcontainingthegrown myceliumwasextractedinUltra-Turraxhomogenizerusing 60mLofdichloromethaneastheextractoragent.Aftertwo extractions,organicphaseswerepooledanddriedinvacuum usingarotaryevaporatorinawaterbathat30◦C.Foreach cul-turetime,fivereplicasweregroupedinordertoobtainahigher concentrationoftheformedproducts.Thefinalresiduewas dissolvedin2mLofethylacetateand1␮Lwasinjectedintothe GC-MSsystem.Samplecomponentswereseparatedona HP-5MScapillarycolumn(30m×0.25mm×0.25␮m)followinga temperatureprogram:from60◦Cto280◦C,up5◦Cperminute, keptat280◦Cfor26min,andfrom280◦Cto310◦C,up5◦Cper minuteandheldat310◦Cfor5min.Theinjectortemperature was 250◦C (splitless injection) and the interface tempera-turewas300◦C.SampleswereanalyzedinFullScanmodein the40–600m/zrange.Peakswereidentifiedbycomparingthe massspectrumandthedataavailableinthelibrarysoftware NISTMSSEARCHversion2.0andindependentlythrough frag-mentationpatterninterpretation.Sampleswerealsoanalyzed intheSelectedIonMonitoringmode(SIM).Metaboliteswere identifiedbyoneidentificationionandatleastoneadditional confirmationionpercompound, asmassspectradescribed in literature.Control experiments, without PAH, were also analyzed.

Experimentaldesignandoptimizationofpyrene degradation

In order to investigate the culture conditions effect on pyrene degradation process by Marasmiellus sp., and to obtainthe necessaryconditions forefficientdegradation,a seriesof experimentaldesignswere established.24 _Initially,

the Plackett-Burman design(Matrix with 12 assays) (PB12) was performed for the analyses of the following indepen-dent variables evaluation: temperature, pH, salinity and

initial concentrations of malt extract, glucose, peptone, yeast extract and PAH. The coded valuesare presentedin

Table 2. Salinity was adjustedwith artificial seawater that

waspreparedaccordingtoKesteretal.25_{Allofthedifferent}

variables were prepared on two levels, designated as −1 (lower) and+1(upper) (Table 2). Threecentralpoint assays were added tothe matrix inorder to determinethe stan-darderror.Inatotalof15trials,thefirstdesignallowedto evaluatetheeffectof8variablesonPAHdegradationafter7 daysofincubation.Initialbiomasswasobtainedat120rpm without the presence of pyrene, then a dimethylsulfoxide solution containing the pollutant was added in separately after72hofgrowth.Thedegradationquantificationwas per-formedaspreviouslydescribed.VariableseffectsonthePAH biodegradationresponseswereevaluatedusingtheprogram STATISTICA7.0.

Based on the results from PB12, a fractional factorial design24−1withthreecentralpointswaspurposedto eval-uate the effect offour variables on pyrene biodegradation

(Table 3): glucose, malt extract, peptone and PAHs after 4

days of incubation. Other growing conditions were 28◦C, pH 8, 35ppm salinity and the absence of yeast extract. The variables glucose and peptone were selected to carry out aCentralCompositeDesign22 _{(CCD)containing4axial}

points and three repetitions at the central point in order to improve pyrene degradation rates (concentration set at 0.08mgmL−1)after2daysofincubation(Table4).Themalt extractconcentrationwassetto25gL−1andothergrowth con-ditionswere maintainedasdescribed infractionalfactorial design24−1.

After culture conditions optimization for pyrene degra-dationthe metabolitesformedwereidentifiedasdescribed above.Additionally,acutetoxicityandthetotalorganiccarbon reduction(TOC)wereanalyzedasdescribedbelow.

Acutetoxicity

Artemia sp. (brine shrimp) dry eggs were purchased from a fish store. Artemia sp.larvae were obtained after hatch-ing fromdry eggsinartificial seawaterwithconstant light. Recentlyhatched(48h)larvaewereusedforthetests,which wereperformedintriplicate,usingtesttubescontaining10% (v/v) NaCl 30% and 4.5mL of samples, totalizing 5mL of testsolution.Afterpreparationofthetubes, 30larvaewere addedandwereexposedtothesamplesfor24hunderlight. After that period aspects as Artemia sp. motility and sur-vival were observed. The mortality rate was calculated to determinetoxicityandstatisticstests(meta-analysis– mul-tipleproportions–p-value<0.01)wererunusingthesoftware BioStat5.0.

TOCanalysis

The total organic carbon reduction (TOC) anal-ysis was performed with a total organic carbon analyzer, TOC-L Shimadzu, as previously described by Bonugli-Santos et al.26 _{TOC measurement is based on the}

combustionofthetotalcarbon(TC)bycatalyticoxidationat hightemperature.Thestandarddeviationislessthan2%in

Tinctoporellus sp. CBMAI 1061

B

A

Marasmiellus sp. CBMAI 1062 Peniophora sp. CBMAI 1063

Fig.1–Pyrene(A)andBaP(B)degradationbythethreemarine-derivedbasidiomycetesafter7,14and21daysofincubation at28◦Cand120rpm.Thevaluesaretriplicateaveragesandverticalbarsshowthestandarderror.

100 Deg radation (%) 0 0 8 BaP Time (days) Pyrene 7 6 5 4 3 2 1 10 20 30 40 50 60 70 80 90

Fig.2–PyreneandBaPdegradationbyMarasmiellussp. CBMAI1062after7daysofincubationat28◦Cand120rpm. Thevaluesaretriplicateaveragesandverticalbarsshow thestandarderror.

eachsampleanalyzedintriplicate.The%TOCreductionwas calculatedusingEq.(1):

% TOCreduction=



TOCinitial−TOC(t)

TOCinitial



×100 (1)

whereTOCinitialandTOC(t)representtheinitialTOCvalueand

theTOCvalueattime‘t’(h),respectively.Samplescontaining optimized conditions for pyrene degradation in 48h were analyzed. Abiotic controls (without microorganism) were includedduringtheexperiment.

Results

PAHsdegradationbymarine-derivedfungi

Thethreemarine-derivedbasidiomycetefungiwere ableto degradepyrene(Fig.1A)andBaP(Fig.1B)atdifferentrates.

Marasmiellussp.was abletodegrademorethan 90%ofthe initial amount of both pollutants (0.02 and 0.04mgmL−1 BaP and pyrene, respectively) after 7 days of incubation. After 21 days, Tinctoporellus sp. reached almost 50% of

degradation,and Peniophorasp.wasabletodegrade30%of bothPAHs.

Marasmiellus sp. reached high levels of pyrene and BaP degradationafter7daysofincubationandwasselectedand submittedtoadditionalPAHsdegradationstudiesthatwere carriedoutafter1,3,5,and7daysofincubation(Fig.2).After 24hofincubation,79.4%oftheBaPweredegraded,andatthe end ofthe 7thday97.2%ofBaPdegradation wasachieved. Pyrenewasdegradedataslowerrate,reaching18.5%ofinitial concentrationdegradationafter24hofincubation.However, after7daysofincubation92.8%ofpyrenewasdegraded.Since the results from pyrene degradation were moreconsistent indicatingadegradationcurve(Fig.2),thisenvironmental pol-lutantwas selectedandsubjected tothestudies relatedto the optimizationof degradation,including the analysesof metabolites,toxicity,andtotalorganiccarbonreduction(TOC).

PyrenedegradationanddetoxificationbyMarasmiellussp.

CBMAI1062

Analysesthroughthe SIMmode andFullScanenabled the detectionandidentificationoffourmetabolitesafterpyrene degradationbythefungusMarasmiellussp.(Table1). Metabo-liteIpresentedamolecular ion(M+)m/z218andfragment ionm/z189,resultinginthelossofa–CHOgroup.Itwasthe mostabundantmetaboliteidentifiedanditsstandardmass spectrumischaracteristicofthehydroxypyrenecompound. MetabolitesIIand IIIpresentedmolecular ion(M+)m/z236 andbasepeakm/z218.ThisprofilerepresentsH2Ounitloss

(M+−18).Fragmentionm/z189,m/z176andm/z94werealso

Table1–Monitoredionsduringpyrenedegradationby

Marasmiellussp.CBMAI1062throughtheSIMmodeand

FullScan.

Compound Monitoredions(m/z) Identification Confirmation

Hydroxypyrene 218 189

Dihydroxypyrene 234 205,176 Pyrenedihydrodiol 236 218,189,176,94

Table2–Plackett-Burman(matrix12)(PB12)experimentaldesignappliedtotheevaluationoftheeffectsof8factorsin

pyrenedegradationbyMarasmiellussp.CBMAI1062after7daysofincubation.

Assay Temperature (◦_C) pH Salinity (ppm) Glucose (g/50mL) Maltextract (g/50mL) Peptone (g/50mL) Yeastextract (g/50mL) Pyrene (mgmL−1₎ Pyrene degradation(%) 1 +1(28) −1(7) +1(35) −1(0.25) −1(0.25) −1(0) +1(0.1) +1(0.08) 45.9 2 +1(28) +1(9) −1(21) +1(0.75) −1(0.25) −1(0) −1(0) +1(0.08) 48.9 3 −1(22) +1(9) +1(35) −1(0.25) +1(0.75) −1(0) −1(0) −1(0.04) 91.0 4 +1(28) −1(7) +1(35) +1(0.75) −1(0.25) +1(0.1) −1(0) −1(0.04) 86.2 5 +1(28) +1(9) −1(21) +1(0.75) +1(0.75) −1(0) +1(0.1) −1(0.04) 96.7 6 +1(28) +1(9) +1(35) −1(0.25) +1(0.75) +1(0.1) −1(0) +1(0.08) 82.4 7 −1 (22) +1(9) +1(35) +1(0.75) −1 (0.25) +1(0.1) +1(0.1) −1(0.04) 93.7 8 −1 (22) −1 (7) +1(35) +1(0.75) +1(0.75) −1(0) +1(0.1) +1(0.08) 49.4 9 −1 (22) −1(7) −1(21) +1(0.75) +1(0.75) +1(0.1) −1(0) +1(0.08) 62.7 10 +1(28) −1(7) −1(21) −1(0.25) +1(0.75) +1(0.1) +1(0.1) −1(0.04) 99.2 11 −1(22) +1(9) −1(21) −1(0.25) −1(0.25) +1(0.1) +1(0.1) +1(0.08) 69.2 12 −1(22) −1(7) −1(21) −1(0.25) −1(0.25) −1(0) −1(0) −1(0.04) 52.8 13(C) 0(25) 0(8) 0(28) 0(0.5) 0(0.5) 0(0.05) 0(0.05) 0(0.06) 66.6 14(C) 0(25) 0(8) 0(28) 0(0.5) 0(0.5) 0(0.05) 0(0.05) 0(0.06) 67.3 15(C) 0(25) 0(8) 0(28) 0(0.5) 0(0.5) 0(0.05) 0(0.05) 0(0.06) 69.0 Inparenthesis,realvalues.

Fig.3–StandardizedParetochartshowingtheeffectsof8 factors(Plackett-Burmanmatrix12)inpyrenedegradation byMarasmiellussp.CBMAI1062,after7daysofincubation.

detected.Thepatternofthemassspectrumobtainedsuggests thatmetabolitesIIandIIIaredihydrodiol.Thetwoidentified peaksprobablycorrespondtodifferentringcleavageposition. ThemetaboliteIVpresentedamolecularion(M+)m/z234and fragmentionsm/z205(M+−COH)andm/z176(M+−2COH).The massspectrumpatternobtainedsuggeststhatthemetabolite IVisdihydroxypyrene.27_{Thehydroxylgroups’ positionwas}

notdetermined.Noneofthesemetabolitesweredetectedin theexperimentalcontrolwithoutPAH.

ThefungusMarasmiellussp.wassubmittedtoexperimental designstudiesinordertoevaluatetheinfluenceofdifferent variablesinpyrenedegradationandtoimprovethe degrada-tionprocess.Inthiscontext,theinfluenceof8independent factorsonpyrenedegradationbyMarasmiellussp.was investi-gatedusingPB12andtheresultsareshowninTable2.Pyrene biodegradationrangesfrom45.9%to99.2%.ThepHand tem-peratureshowedpositiveeffectsonpyrenedegradation,while

thePAHconcentrationdemonstratedanegativeeffect(Fig.3). Salinitydidnotinterferesignificantlyinthepyrene degrada-tion(Fig.3).However,higherdegradationvalueswereobserved insalineandalkalineconditions(assays5and7,Table2).

ConsideringtheresultsobtainedinthePB12experiment somevariableswere chosenforthenext stage,afractional factorialdesign24−1:glucose,maltextract,peptoneandPAH. Sincehigheramountsofpyreneweredegradedathighrates, theincubationtimewasreducedfromseventofourdays.High levelofpyrenedegradation(98.6%)wasalsoobtainedafterfour daysofincubation(assay8,Table3),withthethreenutrients setattheirupperlevel.

Since the malt extract had a positive effect on pyrene degradation in the fractional factorial design 24−1 _{it was}

set at 25gL−1 (Fig. 4). The PAH concentration was set at 0.08mgmL−1,andincubationtimewasreducedtotwodays. CentralCompositeDesign22_{(CCD)wasusedtoevaluatethe}

2.45 -0.65 -0.3 (4)Pyrene (3)Peptone* (2)Malt extract* (1)Glucose 1.0

Effect Estimate - Pyrene Degradation

3.0 3.5 2.5 2.0 p<0.1 1.5 0.5 0.0 2.9

Fig.4–StandardizedParetochartshowingtheeffectsof4 factors(Fractionalfactorialdesign24−1)inpyrene

degradationbyMarasmiellussp.CBMAI1062,after4daysof incubation.

Table3–Fractionalfactorialdesign24−1_{appliedtotheevaluationoftheeffectsof4factorsinpyrenedegradationby} Marasmiellussp.CBMAI1062after4daysofincubation.

Assay Glucose (g/50mL) Maltextract (g/50mL) Peptone (g/50mL) Pyrene (mgmL−1) Pyrene degrad.(%) 1 −1(0) −1(0.75) −1(0.1) −1(0.04) 94.2 2 +1(0.26) −1(0.75) −1(0.1) +1(0.08) 92.2 3 −1(0) +1(1.25) −1(0.1) +1(0.08) 97.8 4 +1(0.26) +1(1.25) −1(0.1) −1(0.04) 98.0 5 −1 (0) −1 (0.75) +1(0.2) +1(0.08) 97.2 6 +1(0.26) −1(0.75) +1(0.2) −1(0.04) 97.7 7 −1(0) +1(1.25) +1(0.2) −1(0.04) 98.5 8 +1(0.26) +1(1.25) +1(0.2) +1(0.08) 98.6 9(C) 0(0.13) 0(1.0) 0(0.15) 0(0.06) 95.3 10(C) 0(0.13) 0(1.0) 0(0.15) 0(0.06) 96.2 11(C) 0(0.13) 0(1.0) 0(0.15) 0(0.06) 97.9

Inparenthesis,realvalues.

effectsoftwofactors,glucoseandpeptone.Highrateof degra-dation (98.2%) was obtained using this strategy (assay 10,

Table4).Nosignificantdifferenceswereobservedamongthe

assays(listedinTable4).Thus,itwasnotpossibletopropose amathematicalmodelandtogettheresponsesurface.

Assaysofthecentralpoints,whichhadthehighestpyrene degradationratewereanalyzedbyGC-MSforthemetabolites identification.Thesamefourmetabolitespreviouslyidentified whenthefunguswasculturedunderthenonoptimized condi-tion(maltextract–nonsalinecondition)after5and15daysof incubationwereformedwhenthefunguswereculturedunder theoptimizedcondition(maltextract,glucoseandpeptone– salinecondition)after48hofincubation.

AcutetoxicityusingArtemiasp.wascarriedoutafterthe growthofMarasmiellussp.underpyreneoptimized degrada-tion conditions. In the medium containing pyrene (abiotic control)thepercentageofsurvivorswas67.2%,andlow motil-ityforthelivingindividualswasobserved.Inthepresenceof

Marasmiellussp.andpyrene(bioassay)95%ofsurvivorswith activemotilityweredetected.Statisticaltests(meta-analysis– multipleproportions)revealedsignificantdifferencebetween bothconditions consideringp-value <0.01. Resultsindicate

Table4–Centralcompositedesign(22_{)appliedtothe}

evaluationoftheeffectsoftwofactorsinpyrene

degradationbyMarasmiellussp.CBMAI1062after48hof

incubation. Assay Glucose (g/50mL) Peptone (g/50mL) Pyrene degradation(%) 1 −1(0.0175) −1(0.165) 92.5 2 +1(0.1025) −1(0.165) 95.1 3 −1(0.0175) +1(0.235) 98.0 4 +1(0.1025) +1(0.235) 97.5 5 −1.41(0) 0(0.2) 96.9 6 +1.41(0.12) 0(0.2) 98.0 7 0(0.06) −1.41(0.15) 97.9 8 0(0.06) +1.41(0.25) 97.8 9 0(0.06) 0(0.2) 97.1 10 0(0.06) 0(0.2) 98.2 11 0(0.06) 0(0.2) 98.0

Inparenthesis,realvalues.

thattherewasalossoftoxicityafterpyrenebiodegradation byMarasmiellussp.

Studies relatedtothe organiccarbonconsumption(TOC analysis)werealsoperformedusingpyreneoptimized degra-dationconditions.Marasmiellussp.wasabletoreduce17%of TOCafter48hofincubation,indicatingthatthefungusused thePAHsascarbonsources.

Discussion

Data derivedfromthe initialscreeningrevealed the poten-tial of the three marine-derived basidiomycete fungi for PAHs degradation.However,results contradicteddata from literature,28 _{whereinitialdegradationrateswere higherfor}

thecompoundwithlowermolecularweight(lessring num-bers).ThetoxicityofBaPcouldresultinaninductionofBaP degradationbyMarasmiellussp.,asasurvivingstrategy. Addi-tionally,itisworthwhiletomentionthattheconcentrationof BaP(0.02mgmL−1)waslowerthanthepyreneconcentration (0.04mgmL−1).

Ina previousstudy reportedbyourresearchgroup9 _the

best resultofPAHsdegradationwas achievedbyAspergillus sclerotiorum CBMAI 849 that was able to degrade 99.7% of pyrene(0.07mgmL−1)and76.6%ofBaP(0.03mgmL−1)after 8 and 16 days of incubation, respectively. Hadibarata and Kristanti29 _{alsoevaluatedthe pyrenebiodegradationbythe}

basidiomycetefungusArmillariasp.F022isolatedfroma trop-icalrainforestinIndonesia.About63%oftheinitialamountof pyrene(0.005mgmL−1)wasbiodegradedafter30daysof incu-bation.Inanotherstudythemaximumpyrene(0.01mgmL−1) degradationrate(99%)wasachievedbyPleurotuspulmonarius

F043after30daysofincubation.5

Consideringtheresultsobtainedinthescreening experi-mentsthefungusMarasmiellussp.wasselectedandcultured for5and15daysaimingtodetectandidentifythemetabolites formedduringpyrenedegradation.Thepresenceof metabo-litesI,II,IIIandIVsuggestthatpyrenewasdegradedbythe cytochrome P450 system and epoxide hydrolases activity. Althoughthehydroxylgrouppositionwasnotdetermined,the 1-hydroxypyrenehasbeendescribedasametaboliteoffungal pyrene degradation.30,31 _{Themetabolitesformed bypyrene}

degradationproductsbyseveralfungi,including representa-tivesofthegenusMarasmiellus.30,32_Langeetal.32_reportedthat

theisolatedMarasmiellusramealisJK314wasabletodegrade 76.5%ofpyreneproducing1-hydroxypyrene,1-pyrenylsulfate, 1,6-hydroxypyrene, 1,6-pyrenequinone and trans-43-dihydro-4,5-dihydroxypyrene metabolites after 14 days ofincubation.

It is worth emphasizing that the initial biotransforma-tion of a compound could facilitate the attack by other organisms.33 _{In addition, the metabolites formed initially}

as phenols and dihydrodiol can be detoxified by conju-gation to other molecules. The conjugates produced by fungalstrainsincludesulfates,glycosides,glucuronidesand xilosides.34 _{Theseconjugatesare generallymoresoluble in}

waterthantheparentcompoundandcanbeexcretedfrom thebody.

Resultsfromstatisticalexperimentaldesignsshowedthat efficient pyrene degradation was reached by Marasmiellus

sp.ina shorterperiodoftime (48h), higherconcentration (0.08mgmL−1),andundersalineconditions.Statistical exper-imentaldesignscanbeconsideredasanefficientapproach todeterminethebestcultureconditionsinbiotechnological processes,35,36 _{savingcostandtimeduetothereductionin}

thetotalnumberofexperiments.37_{Consideringthat}

marine-derived fungi are adapted to the ocean conditions, the greatperformanceofthefungusduringpyrenedegradation undersalineconditionwasexpected.However,several stud-iesshowedthatbiodegradationisnegativelyaffectedbythe increasinginsaltconcentration.38–40

AfterpyrenedegradationbyMarasmiellussp.therewasa lossoftoxicity,highlightingthebiotechnologicalpotentialof thisfungustodegradeanddetoxifythisenvironmental pollu-tant.SimilarresultwasreportedbyMunusamyetal.41_where

representativesofthewhiterotfungusPycnoporussanguineus

wereabletodegradePAH withthe productionofnontoxic compoundsbasedontoxicityanalysisusingArtemiasp.

Marasmiellussp.alsoshowedcapabilitytousepyrene as carbon source. Considering the short period oftime (48h) appliedforpyrenedegradationbythefungus,resultfromTOC reductioncanbeconsideredverysatisfactory.AhigherTOC reductioncouldbeobtainedafteralongerincubationtime. According to Russoet al.42 _{mushroom compostsubmitted}

tocombinedozonationandaerobicBaPbiodegradation pre-sented from 33% to72% of TOCremovals after30 days of incubation(soilsamples).

Conclusions

Resultsfromthepresentstudyrevealedthepotentialofthe marine-derivedfungiforenvironmentalpollutants degrada-tion.In particular,the fungusMarasmiellussp. CBMAI1062 showedgreatabilitytodegradePAHsandcanbeconsidered anewmicrobial resource forbioremediation. Experimental designwassuccessfullyappliedtooptimizethePAH degrada-tionbyMarasmiellussp.,resultinginaveryhighrate(almost 100%)of pyrene degradation afteronly 48h of incubation. Intermediatemetabolitesgeneratedsuggestedthatthe fun-gususedthecytochromeP450systemandepoxidehydrolases andhighlight theimportanceofthisorganism intheearly

stagesofpyrenedegradation.Pyrenetoxicitywasaround28% reducedandtotalorganiccarbonwas17%consumedafterthe degradationprocessunderoptimizedcondition,showingthat thefunguswasabletodetoxifyandusethepollutantas car-bonsource.Additionally,sincethisfunguswasisolatedfrom themarineenvironmentandwasabletodegradePAHsunder salineconditions,biologicaladvantagescanbeprovidedfor itsuseinsalineenvironmentsand/orprocesses.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

GabrielaA.L.VieirawassupportedbyaPhDscholarshipfrom the São Paulo Research Foundation – FAPESP (2012/12622-3) and Mariana J. Magrini was supported by a Master’s Degree scholarshipfrom theCoordinationforthe Improve-ment ofHigher Education Personnel– CAPES.The authors thank FAPESP for its financial support (Grants 2010/50190-2,2013/19486-0and 2016/07957-7).LaraD.Settethanksthe National Council for Scientific and Technological Develop-ment(CNPq)fortheProductivityFellowship(304103/2013-6).

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e

s

1.HaritashAK,KaushikCP.Biodegradationaspectsof

PolycyclicAromaticHydrocarbons(PAHs):areview.JHazard

Mater.2009;169(1–3):1–15.

2.RaghukumarC,D’Souza-TicloD,VermaA.Treatmentof

coloredeffluentswithlignin-degradingenzymes:an

emergingroleofmarine-derivedfungi.CritRevMicrobiol.

2008;34(3–4):189–206.

3.ShuttleworthKL,CernigliaE.EnvironmentalaspectsofPAH

biodegradation.ApplBiochemBiotechnol.1995;54(1):291–302.

4.Abdel-ShafyHI,MansourMSM.Areviewonpolycyclic

aromatichydrocarbons:source,environmentalimpact,

effectonhumanhealthandremediation.EgyptJPet.

2016;25(1):107–123.

5.HadibarataT,TehZC.Optimizationofpyrenedegradationby

white-rotfungusPleurotuspulmonariusF043and

characterizationofitsmetabolites.BioprocessBiosystEng.

2014;37(8):1679–1684.

6.LangS-C,HursthouseA,MayerP,etal.Equilibriumpassive

samplingasatooltostudypolycyclicaromatic

hydrocarbonsinBalticSeasedimentpore-watersystems.

MarPollutBull.2015;101(1):296–303.

7.SouzaHML,TaniguchiS,BícegoMC,etal.Polycyclicaromatic

hydrocarbonsinsuperficialsedimentsoftheNegroRiverin

theAmazonRegionofBrazil.JBrazChemSoc.

2015;26:1438–1449.

8.SyedJH,IqbalM,ZhongG,etal.Polycyclicaromatic

hydrocarbons(PAHs)inChineseforestsoils:profile

composition,spatialvariationsandsourceapportionment.

SciRep.2017;7:2692.

9.PassariniMRZ,RodriguesMVN,daSilvaM,SetteLD.

Marine-derivedfilamentousfungiandtheirpotential

applicationforpolycyclicaromatichydrocarbon

bioremediation.MarPollutBull.2011;62(2):364–370.

10.ZhangS,YaoH,LuY,etal.Uptakeandtranslocationof

bymaizefromsoilirrigatedwithwastewater.SciRep.

2017;7(1):12165.

11.RothermichMM,HayesLA,LovleyDR.Anaerobic,

sulfate-dependentdegradationofpolycyclicaromatic

hydrocarbonsinpetroleum-contaminatedharborsediment.

EnvironSciTechnol.2002;36(22):4811–4817.

12.RaghukumarC,ShailajaM,ParameswaranP,SinghS.

Removalofpolycyclicaromatichydrocarbonsfromaqueous

mediabythemarinefungusNIOCC#312:involvementof

lignin-degradingenzymesandexopolysaccharides.IndianJ

MarSci.2006.

13.CernigliaCE.Fungalmetabolismofpolycyclicaromatic

hydrocarbons:past,presentandfutureapplicationsin

bioremediation.JIndMicrobiolBiotechnol.1997;19(5):324–333.

14.CernigliaCE,SutherlandJB.BioremediationofPolycyclic

AromaticHydrocarbonsbyLigninolyticandNon-Ligninolytic Fungi.vol.23;2001:136–187.

15.AhearnDG,MeyersSP.Theroleoffungiinthe

decompositionofhydrocarbonsinthemarineenvironment.

In:WaltersAH,Hueck-vanderPlasEH,eds.Biodeteriorationof

Materials.London:AppliedScience;1972:12–18.

16.Bonugli-SantosRC,VasconcelosRdosS,PassariniMRZ,etal. Marine-derivedfungi:diversityofenzymesand

biotechnologicalapplications.FrontMicrobiol.2015;6,

http://dx.doi.org/10.3389/fmicb.2015.00269.

17.GaraventaF,GambardellaC,DiFinoA,PittoreM,FaimaliM.

SwimmingspeedalterationofArtemiasp.andBrachionus

plicatilisasasub-lethalbehaviouralend-pointfor

ecotoxicologicalsurveys.Ecotoxicology.2010;19(3):512–519.

18.PiazzaV,FerioliA,GiaccoE,etal.Astandardizationof

Amphibalanus(Balanus)amphitrite(Crustacea,Cirripedia)

larvalbioassayforecotoxicologicalstudies.EcotoxicolEnviron

Saf.2012;79:134–138.

19.CostaE,PiazzaV,GambardellaC,etal.Ecotoxicological

effectsofsedimentsfromMarPiccolo,SouthItaly:toxicity

testingwithorganismsfromdifferenttrophiclevels.Environ

SciPollutRes.2016;23(13):12755–12769.

20.CarmonaM,ZamarroMT,BlázquezB,etal.Anaerobic

CatabolismofAromaticCompounds:aGeneticandGenomic

View.MicrobiolMolBiolRevMMBR.2009;73(1):71–133.

21.GhosalD,GhoshS,DuttaTK,AhnY.Currentstateof

knowledgeinmicrobialdegradationofpolycyclicaromatic

hydrocarbons(PAHs):areview.FrontMicrobiol.2016;7:1369.

22.MenezesCBA,Bonugli-SantosRC,MiquelettoPB,etal.

Microbialdiversityassociatedwithalgae,ascidiansand

spongesfromthenorthcoastofSãoPaulostate,Brazil.

MicrobiolRes.2010;165(6):466–482.

23.Bonugli-santosRC,DurrantLR,SetteLD.Laccaseactivityand

putativelaccasegenesinmarine-derivedbasidiomycetes.

FungalBiol.2010;114(10):863–872.

24.RodriguesM,IemmaA.Planejamentodeexperimentose

otimizac¸ãodeprocessos.2a_{Campinas;2009.}

25.KesterDR,DuedallIW,ConnorsDN,PytkowiczRM.

Preparationofartificialseawater.LimnolOceanogr.

1967;12(1):176–179.

26.Bonugli-SantosRC,VieiraGAL,CollinsC,etal.Enhanced

textiledyedecolorizationbymarine-derivedbasidiomycete

Peniophorasp.CBMAI1063usingintegratedstatisticaldesign.

EnvironSciPollutRes.2016;23(9):8659–8668.

27.LambertM,KremerS,SternerO,AnkeH.Metabolismof

pyrenebythebasidiomyceteCrinipellisstipitariaand

identificationofpyrenequinonesandtheirhydroxylated

precursorsinstrainJK375.ApplEnvironMicrobiol.

1994;60(10):3597–3601.

28.WangC,SunH,LiJ,LiY,ZhangQ.Enzymeactivitiesduring

degradationofpolycyclicaromatichydrocarbonsbywhite

rotfungusPhanerochaetechrysosporiuminsoils.Chemosphere.

2009;77(6):733–738.

29.HadibarataT,KristantiRA.Biodegradationandmetabolite

transformationofpyrenebybasidiomycetesfungalisolate

Armillariasp.F022.BioprocessBiosystEng.2013;36(4):461–468.

30.LangeB,KremerS,SternerO,AnkeH.Pyrenemetabolismin

Crinipellisstipitaria:identificationof

trans-4,5-dihydro-4,5-dihydroxypyreneand1-pyrenylsulfate

instrainJK364.ApplEnvironMicrobiol.1994;60(10):3602–3607.

31.CernigliaCE,SutherlandJB.Degradationofpolycyclic

aromatichydrocarbonsbyfungi.In:TimmisKN,ed.

HandbookofHydrocarbonandLipidMicrobiology.Berlin,

Heidelberg:SpringerBerlinHeidelberg;2010:2079–2110.

32.LangeB,KremerS,AnkeH,SternerO.Metabolismofpyrene

bybasidiomycetousfungiofthegeneraCrinipellis,

Marasmius,andMarasmiellus.CanJMicrobiol.1996;42(11).

33.KeckJ,SimsRC,CooverM,ParkK,SymonsB.Evidencefor

cooxidationofpolynucleararomatichydrocarbonsinsoil.

WaterRes.1989;23(12):1467–1476.

34.SutherlandJB.Detoxificationofpolycyclicaromatic

hydrocarbonsbyfungi.JIndMicrobiol.1992;9(1):53–61.

35.PearceAR,RizzoDM,WatzinMC,DruschelGK.Unraveling

associationsbetweencyanobacteriabloomsandin-lake

environmentalconditionsinMissisquoiBay,Lake

Champlain,USA,usingamodifiedself-organizingmap.

EnvironSciTechnol.2013;47(24):14267–14274.

36.ZengT,ArnoldWA.Clusteringchlorinereactivityof

haloaceticacidprecursorsininlandlakes.EnvironSciTechnol.

2014;48(1):139–148.

37.ChauhanK,TrivediU,PatelKC.Statisticalscreeningof

mediumcomponentsbyPlackett–Burmandesignforlactic

acidproductionbyLactobacillussp.KCP01usingdatejuice.

BioresourTechnol.2007;98(1):98–103.

38.PiedadDíazM,GrigsonJWS,PeppiattJC,BurgessGJ.Isolation

andcharacterizationofnovelhydrocarbon-degrading

euryhalineconsortiafromcrudeoilandmangrove

sediments.MarBiotechnol.2000;2(6):522–532.

39.ValentínL,FeijooG,MoreiraMT,LemaJM.Biodegradationof

polycyclicaromatichydrocarbonsinforestandsaltmarsh

soilsbywhite-rotfungi.IntBiodeteriorBiodegrad.

2006;58(1):15–21.

40.Minai-TehraniD,MinouiS,HerfatmaneshA.Effectof

salinityonbiodegradationofpolycyclicaromatic

hydrocarbons(PAHs)ofheavycrudeoilinsoil.BullEnviron

ContamToxicol.2009;82(2):179–184.

41.MunusamyU,SabaratnamV,MuniandyS,AbdullahN,

PandeyA,JonesE.Biodegradationofpolycyclicaromatic

hydrocarbonsbylaccaseofPycnoporussanguineusand

toxicityevaluationoftreatedPAH.Biotechnology.

2008;7(4):669–677.

42.RussoL,RizzoL,BelgiornoV.Ozoneoxidationandaerobic

biodegradationwithspentmushroomcompostfor

detoxificationandbenzo(a)pyreneremovalfrom