Screening of freshwater fungi for decolorizing multiple synthetic dyes

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

Environmental

Microbiology

Screening

of

freshwater

fungi

for

decolorizing

multiple

synthetic

dyes

Panpan

Yang

,

Wenxiao

Shi

_,

_Hongkai

_Wang

_,

_Hongmei

_Liu

a_{ZhejiangUniversity,BiotechnologyInstitute,StateKeyLaboratoryofRiceBiology,Hangzhou,Zhejiang,China}

b_{ShandongAgriculturalUniversity,CollegeofLifeScience,StateKeyLaboratoryofCropBiology,Tai’an,Shandong,China}

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t

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o

Received22May2015 Accepted6March2016 Availableonline4July2016 AssociateEditor:CynthiaCanêdoda Silva Keywords: Freshwaterfungi Biodegradation Syntheticdyes Decolorization

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Thebiodegradationofsyntheticdyesbyfungiisemergingasaneffectiveandpromising approach.Inthepresentstudy,freshwaterfungalstrainsisolatedfromsubmergedwoods werescreenedforthedecolorizationof7syntheticdyes.Subsequently,13isolateswith highdecolorizationcapabilitywereassessedinaliquidsystem;theybelongedto9different fungalspecies.Severalstrainsexhibitedahighlyeffectivedecolorizationofmultipletypes ofdyes.Newabsorbancepeaksappearedafterthetreatmentwith3fungalstrains,which suggeststhatabiotransformationprocessoccurredthroughfungalbiodegradation.These resultsshowedtheunexploitedandvaluablecapabilityoffreshwaterfungiforthetreatment ofdye-containingeffluents.Theabilityofcertainfungitodecolorizedyesisreportedhere forthefirsttime.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Syntheticdyesarewidelyusedforcoloringtheproductsof severalindustriessuchastextiles,leather,cosmetics,paper, printingmaterials,andplastics.Itisestimatedthat1–2%of dyeproductionislost,and5–10%isdischargedtothe environ-mentwhenthedyesareused.1,2_{Severaldyesandthe}

chemi-calsusedtoproducethemareoftentoxic,carcinogenicoreven explosive.3,4 _{Effluentsfromindustriesthatusevariousdyes}

areconsideredaspollutantsthatcancausesevere environ-mentalproblemsaswellasmedicalandestheticproblems.2

Thedecolorizationofthisindustrial wasteisachallenging taskbecausecertaindyesareresistanttodegradation.4

Physical and chemical decolorizing methods have been developedtoremovedyesfromwastewater;however,several

∗ _{Correspondingauthor.}

E-mail:[email protected](H.Wang).

ofthemhavedisadvantagessuchashighcostsand/orlimited applicability.2,5 _{Studiesonthecapabilityofmicroorganisms}

toperformdyedecolorizationhasreceivedincreasing atten-tionbecausetheuseofmicroorganismsisconsideredacost effectiveandenvironmentallyfriendlymethodforremoving organicdyesfromwastewaterbeforetheyaredischarged.6,7

Likewise,thecapabilitiesandmechanismsofdecolorization by bacteria have been studied.8,9 _{However, the}

applica-tion of bacteria was limited due to the narrow substrate range of various degrading bacteria.10 _{Moreover, to}

com-pletethedegradationofdyes,differentgroupsofbacteriaare requiredinanalternationprocessfromanaerobictoaerobic conditions.2,7

Researchonthefungaldecolorizationofdyewastewater hasbeen performedinrecentyears.11,12 _{Severalfungiwith}

thecapabilitytodecolorizeawide rangeofdyeshavebeen

http://dx.doi.org/10.1016/j.bjm.2016.06.010

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

reported.Forexample,thewhite-rotfungiandbrown-rotfungi arewell-studiedfungigroupswithdecolorizationabilities.12

Otherfungi,suchasAspergillusniger, Rhizopusarrhizus, and

R.oryzae,canalsodecolorizeand/orabsorbdiversedyesand possessexcellentcolorremovalcapabilities.13,14

Themechanismoffungaldecolorizationmainlyinvolves twoaspects,biodegradationandbiosorption.15_The

biodegra-dationcapabilityoffungiisduetotheirextracellular, non-specificandnon-selectiveenzymesystem.6 _Fungalenzyme

productiondependsonnutrientlimitations,andtheir subse-quentdyedecolorizationabilityisachieveddependingonthe growthconditions.16Consideringthecomplexenvironmental factorsinvolvedinthedyewastewaterconditions,the screen-ingofmorefungiisnecessaryforuseindyedecolorization.

Aquaticfungiarethemaindecomposersofaquatic ecosys-temsandplaycrucialrolesinthecyclingofnutrients.17_In

addition, aunique characteristicoffungi istheirability to produceseveralnon-specificenzymes.18_{These non-specific}

extracellular and/or exoenzymes enable the aquatic fungi toattackstructurallydiverseorganiccompoundsthat corre-spondtodifferentpollutantclasses.19,20 _{Hence,thesefungi}

mayserveasanewresourcetotreatwastewater.However, littleresearchhasbeen performedonthe processof decol-orizing wastewater by freshwater fungi,10 _{and more fungi}

withpotentialforthebiodegradationofsyntheticdyesneed tobeexplored.Inthepresentstudy,severalfreshwaterfungi were isolated from streams in the Zhejiang Province, PR China, and their ability of decolorizing multiple synthetic dyeswasevaluated.

Materials

and

methods

Dyesandmedia

Sevendyeswereusedforscreeningthedecolorization abil-ity of the fungal isolates. All the dyes were dissolved in distilledwaterataconcentrationof10g/L;then,theywere filteredandsterilizedusinga0.22␮m-diameterbacteriafilter beforebeingtestedforuse.Allthedyeswerepurchasedfrom Sigma–Aldrich.Themolecularstructuresofthedyesarelisted

inTable1.

Wateragarmedium(WA;WA1.5%agar)wasusedforthe fungalisolation;potato-carrot-agarmedium(PCA;5%potato, 5%carrot,2%agar)wasusedfortheidentificationoffungal isolates;potato-dextrose-agarmedium(PDA;20%potato,2% dextrose,2%agar)wasusedtogrowthefungalculturesand fortheDNAextraction;andmalt-agarmedium(MEA;2%malt extract,1.5%agar)wasusedforscreeningthedecolorization byfungi.

TheliquidMEAmediumusedforthedecolorizationtest wasthesameasthatdescribedabovebutwithoutagar.The PDAmediumwasusedforcolonygrowth,andthegenomic DNAisolationwaspreparedfollowingthemethodof Wong-sawasetal.21

Fungalstrainisolationfromstreams

Submerged wood samples were randomly collected from streamsin the Zhejiang Province; then,theywere brought

Table1–Structuresofthesyntheticdyesusedinthe presentstudyandthewavelengthofmaximum absorption–max(nm).

Dye Structure max(nm)

AcidBlue40 620 ReactiveRed11 525 AcidBlue193 580 AcidBlue62 590 AcidBlue113 630 ReactiveBlue74 590 AcidRed73 500

backtolaboratoryand placedseparately insnaplock

plas-ticbagswithsterilemoistpapertowels,incubatedatroom

temperatureandexaminedperiodicallyduringathree-month

period.Singlesporeisolationswereobtainedusingthe

meth-odsdescribedbyChoietal.22,23

Dyedecolorizationonsolidmedia

ThesolidmediawerepreparedwithMEAmediumand the addition ofeach dyeto atotalconcentration of50mg/L.A myceliumplugderivedfromtheedgeoffungalstrainsgrown on WAmediumplates for4days at25◦C weretransferred to the center of a solid medium plate and inoculated at 25◦Cfor1week.Theformationofdecolorizedzonesunder or around the developing mycelia was monitored for dye decolorization. Alltheagar plateassays wereperformedin triplicate.

Morphologicalidentificationoffungalstrainswith

decolorizationcapability

Pure culturesoffungiwithdecolorization capabilitieswere incubated onPCA,WAand PDAmediaat25◦Cfor2weeks fortheiridentification.Themorphologicalcharactersoffungi were examined and recorded in detail under an Olympus BX-51 microscopeequippedwithan OlympusDP-50digital camerasystem.

Molecularidentificationofthefungalstrainstested

The total genomic DNA was extracted from the cultured myceliawithamodifiedCTABmethodprotocol.21_The

com-pleteinternaltranscribedspacers1and2andthe5.8SrDNA regions ofthe isolatestested were amplified byPCRusing ITS1andITS4primers.The18SrDNAregionofisolateJX-43 wasobtainedbyPCRusingtheNS1andNS4primersbecause thereisnoITSsequenceofthisspeciesinGenBank.ThePCR reactionprotocolandsequencingprocesswasperformedas describedbyWongsawasetal.21

Dyedecolorizationinliquidsystems

Thefungalstrainsthatshowedadecolorizedzoneonthesolid mediawere picked out forfurthertesting in liquidmedia. Theconcentrationofeachdyewassupplementedtoobtain 50mg/L in a previously autoclavedliquid medium. A plug derivedfromtheedgeoftheselectedfungalstrainsonPDA wastransferredtoa10mLliquidmediumthatcontainedthe respectivedyeandinoculatedat25◦Cfor1week.Then,the liquidmediumthatcontainedfungalmyceliawascentrifuged at5000rpmfor10min,andaliquotsofthesupernatantswere transferredintonewtubesanddilutedasnecessaryforthe absorbancereadings.Foreachdye,untreatedliquidmedium wasusedasacontrol.Theabsorbancespectrumofeachdye was examined using a U-2001 two-beam spectrophotome-ter usinga wavelengthrange from 200to 900nm, and the maximum absorbancewavelengthwas determined accord-ing to the single peak with the strongest absorption. The absorbancedeterminationsweremeasuredatthemaximum absorbancewavelengthonaGENios+microplatereader.The standardcurvesofeachdyewereperformedatthemaximum absorbance wavelength, and the dye concentration ranged from 0 to50mg/L. Thedyedecolorization of sampleswas

expressedasapercentage,asfollows:D=(AC−AA)/AC×100, whereDisthe decolorizationrate (%),and ACandAA are theconcentrationsmeasuredforthecontrolandthetreated sample,respectively.24

Thedyebiosorption offungal myceliawas evaluatedas follows:afterthecentrifugalprocessdescribedabove,the fun-galmyceliaineachcentrifugetubewereputonafilterpaper (Whatman)toremovetheadditionalliquid;then,theywere transferred toa new centrifuge tube that contained10mL ethanol and 10mLfresh liquid medium.These tubes were placedonarotaryshaker(160rpm)atroomtemperaturefor 20minandthencentrifugedat5000rpmfor10min.The con-trolforeachdyewasperformedbyadding10mLethanolinto 10mLliquidmediumthatcontained50mg/Lofeachdye.The dyebiosorptionratewasexpressedasapercentage,asfollows:

A=AM/AD×100,whereAisthedyeadsorptionrateofeach fungal mycelia(%),andAMand ADare theconcentrations measuredinthetreatmentandcontrol,respectively.

Theassayswereperformedintriplicate,theexperimental errorwascalculatedasthestandarddeviation(SD)and,inall thecases,theSDwasbelow3%.

Results

Freshwaterfungalstrainisolationandscreeningofthe

dyedecolorizationabilityonagarplates

Atotalof92 strainsoffreshwaterfungiwereisolated from decayedwoodysamplescollected fromstreamsinthe Zhe-jiangProvince,PRChina.Thedecolorizationcapabilityofthese fungal strains was assessedon dye-containing MEA plates with7typesofsyntheticdyesinoculatedat25◦Cfor1week.

Thirteenfungalstrainsthatcausedeitherapartialorfull decolorization were screenedout of92 strains of freshwa-ter fungiisolatedfromthedecayedwoodsamples.Thedye

Fig.1–Decolorizationof7dyesbyfungalstrainC-1onMEAmediumthatcontained50mg/Lofdifferentdyesinoculated during7days.(A)ReactiveRed11;(B)AcidBlue40;(C)AcidBlue62;(D)AcidBlue113;(E)AcidBlue193;(F)AcidRed73;(G) ReactiveBlue74.

decolorizationresultsonagarplatesshowedthatthese fun-galstrainshadtheabilitytodecomposemorethan3typesof dyes.Likewise,differentfungihaddifferentdyedecolorization abilities.Forexample,strainC-1hadthecapabilityto decom-poseall7differenttypesofdyestestedintheseexperiments (Fig.1).

Identificationofthefungiwithpotentialfordye

decomposition

The identification of the 13 fungal strains was performed based on morphological characters and on the ITS rDNA sequence(Table2).Basedontherecordedmorphologicaldata (notshown)from3strains(DX-8,I-5andC-1),itappearsthat theybelongtothespeciesMyrotheciumverrucaria,withslightly differentculturalcharacteristics(Fig.2)anddifferentabilities todecomposedyes(Table3).TheITSregionsofstrainC-1were sequenced,andapreliminarysequencinganalysisconfirmed thatthetaxonbelongedtotheMyrotheciumspecies.The fun-galstrainI-6cwasidentifiedasColletotrichumdematium.The fungalstrainI-101wassimilartoCorynesporacassiicola,while strainsH-6andSy06possessedmorphologysimilartothatof

Dictyosporiumzhejiangensis.ThemorphologyofI-10andBD-42 wereconsistentwiththatofFusariumthapsinum.25_The

fun-galstrainJX-62hadthemorphologicalcharactersofAlternaria alternata.

Themorphologicalcharacteristics ofJ-1 indicate aclose resemblanceto Plectosporiumtabacinum. TheITS sequences furtherconfirmedsuchanaffinity.Strain JX-43wassimilar toAcrogenosporasphaerocephala;however,theITSsequenceof thisstraindidnotrevealhighsequencesimilaritiesbecause noITSsequencedataofthisspecieswerefoundinthe Gen-Bank database. Then, we sequenced the SSU sequence of strainJX-43,andablast searchresultindicatedthatit had 99%identitywithFarlowiellacarmichaeliana,theteleomorphof

A.sphaerocephala.TheD-1straincouldnotbeidentifiedbased onmorphologybecauseitwasunabletosporulate;however, theITSsequencesshowaclosesimilaritytoCeriporialacerate,

knownasthewhite-rotfungus.26

All7typesofdyeswere usedforthe decolorizationtest inliquidmedia.Thewavelengthsofmaximumabsorptionby

Fig.2–Colonialcharacterizationof3strainsofMyrothecium verrucariaincubatedonPDAfor5daysat25◦C.DX-8 producedabundantsporesontheculture,butI-5hadno sporesonthecolony;thesporulationabilityofC-1was betweenthatofDX-8andI-5.

eachdyearelistedinTable1.The13fungalstrainsthatwere significantlyactiveindyedecolorizationinsolidplateswere usedintheliquiddecolorizationsystemassay.Allthetested strainsshowedahighlyefficientdecolorizationofavariety

ofdyes(Table3).Thedecolorizingextentofthedyesbythe

differentstrainsexhibitedanindividualpatternbasedonthe dyetypeandthefungalstrains,eventhosethatbelongedto thesamefungalspecies.Eachstrainhadtheabilityof decol-orizemultipledyesand,atleast,theydecolorizedonedyewith highefficiency.Forexample,strainD-1coulddecolorizeallthe testeddyeswitharateofupto50%.StrainsJX-43andI-10were abletodecolorizetheReactiveRed11andAcidBlue113dyes almostcompletely;however,only34.1%and41.3% decoloriza-tionrateswereachievedwithReactiveBlue74,respectively.

Table2–Fungalstrainsusedforthedecolorizationassayafterthescreeningtestandtheirblastresultsobtainedfrom GenBank.

Strainnumber Speciesname Accessionnumberof thetestedisolates

Identity AccessionwithhighIdent intheGenBank

C-1 Myrotheciumverrucaria JQ801304 100% FJ235085

DX-8 Myrotheciumverrucaria – – –

I-5 Myrotheciumverrucaria – – –

I-6C Colletotrichumdematium JQ801303 100% AB046608

I-101 Corynesporacassiicola JQ801302 99% GU461298

Sy06 Dictyosporiumzhejiangensis KF931342 99% FJ456893

H-6 Dictyosporiumzhejiangensis – – –

J-1 Plectosporiumtabacinum – 99% KC845227

I-10 Fusariumthapsinum – – –

DB-42 Fusariumthapsinum KF813067 100% JQ363735 JX-62 Alternariaalternata KF813070 100% JN681160 Jx-43 Acrogenosporasphaerocephala KF836060 93% AY265337

Table3–Decolorizationrates(%)of11dyesafterthetreatmentwith13fungalstrainsfor7daysinliquidmedia.

Dye AcidBlue40 ReactiveRed11 AcidBlue193 AcidBlue62 AcidBlue113 ReactiveBlue74 AcidRed73

Myrotheciumverrucaria C-1 46.50±0.15 50.64±0.18 46.05±0.32 67.09±0.33 79.83±1.06 66.74±0.46 63.23±0.52 M.verrucariaDX-8 45.41±0.55 56.28±0.46 41.47±1.05 68.79±0.23 77.59±0.44 60.96±0.96 61.54±0.42 M.verrucariaI-5 48.57±0.32 60.38±0.34 41.46±0.56 67.65±0.24 79.51±0.32 65.53±0.73 58.00±0.44 Colletotrichumdematium I-6c 69.60±0.21 86.02±0.29 53.58±0.61 91.59±0.32 85.86±0.92 79.71±0.86 54.96±0.16 Corynesporacassiicola I-101 24.59±0.33 44.39±0.20 25.96±0.12 48.69±0.21 48.21±0.82 51.74±0.56 30.28±0.86 Dictyosporium zhejiangensisSy06 77.67±0.90 62.56±0.46 44.78±0.11 61.55±0.87 77.87±0.23 66.43±0.22 56.34±0.63 D.zhejiangensisH-6 75.53±0.24 64.02±0.24 46.00±1.12 58.83±0.45 77.41±0.72 64.04±0.22 55.23±0.57 Plectosporiumtabacinum J-1 76.44±1.23 95.96±0.87 31.64±0.79 50.16±0.56 65.09±0.65 56.95±2.22 67.12±0.33

FusariumthapsinumI-10 93.97±0.46 100±0.00 92.93±0.43 60.04±0.68 98.64±0.24 34.12±0.33 51.76±0.65

F.thapsinumDB-42 90.86±0.94 92.81±0.72 90.58±0.61 66.21±0.89 96.16±0.36 40.29±0.83 55.55±0.54 AlternariaalternataJx-62 49.16±1.22 86.79±0.23 45.71±0.35 51.25±0.34 91.11±0.46 46.28±0.74 66.43±0.22 Acrogenospora sphaerocephalaJX-43 89.84±0.61 99.5±0.21 78.00±0.88 69.97±0.67 100.00±0.00 41.30±0.56 61.98±0.45 CeriporialacerataD-1 64.86±0.34 67.12±0.62 82.37±0.22 70.98±0.41 87.38±0.12 69.64±0.18 76.16±0.72

Certainisolatesofthisstudyhadadecolorizationefficiency

higherthanthatofthewhite-rotfungusCeriporialacerata.The

obtainedresultsaresummarizedinTable3.

New absorbance peaks appearedafter the treatment of dyeswith the fungal strains. A new absorbancepeak was shown whenAcid Blue 62 was treatedwith sy06 and I-6C respectively,whichhasamaximumabsorbancewavelength at590nm(Fig.3).

In this study, the dye biosorption by the fungal strains wasevaluated(Table4).Differentstrainsexhibiteddifferent extentsof dyeabsorbance depending on the typeof dyes. StrainI-101hadthe abilityofadsorbing6outof7dyes.In addition,8outof13strainswereabletoadsorbAcidRed73, whereasthedyeAcidBlue193exhibitedanulladsorptionby

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 740 710 680 650 620 590 560 530 500 470 440 410 380 350 320 290 260 230 200 Absorbance (OD) Wavelength (nm) ck0 ck1 I-6c I-10

Fig.3–SpectralcurvesofAcidBlue62treatedwith4fungal strainsafter7days.Thenewabsorbancepeakappearedat 500nmwhenthedyewastreatedwiththeSy06andI-6c strains.CK0:MEbrothmedium.CK1:MEbrothmedium with50mg/LAcidBlue62.

allthefungalstrainsusedinthisstudy.StrainI-101hasthe highestrateofbiosorptionamongthetestedfungalstrains, andtheefficiencyofdyebiosorptionwasmorethan20%in5 typesofdyes.However,atleast4strains,C-1,DX-8,I-5,DB-42, didnotshowanybiosorptionability.

Discussion

Thepresenceofdyesinindustrialproductscurrentlyresults in wastewater pollution, and these pollutants need to be treatedbeforetheyaredischarged.27_Theabilityof

microor-ganismstoperformdyedegradationhasreceivedconsiderable attention.7,16 _{The effluents from the textile industry are}

extremely variable in composition; even asmall structural differencecanaffectthedecolorizationprocess.2,28_Thereis

stillaneedforstudiestodiscoverthepotentialoffungal iso-latesfortheimprovementofdyewastewaterbecausecertain isolatescannotbesuitableforavarietyofdyesin wastewa-terconditions.1_{Thefungalisolatesinthisresearchprovidea}

newavenueforapproachingthebiodegradationofdyesinthe future.

Thefungiused fordyebiodegradationwere mainly iso-latedfromforestecosystems,suchaswhite-rotandbrown-rot fungi.Althoughthesefungihavebeenshowntodecolorize dyesinliquid fermentations,theirenzymeproductionwas shown tobeunreliable.Thisoutcome ismainlyduetothe unfamiliarenvironmentofliquidfermentationsforthese for-est fungi.12 _{New isolates offungiusedto decolorizewater}

conditions will probably have the efficacy of bioremedia-tion ofdyesbecausethesefungihavetheabilitytosecrete extracellular non-specific enzymes that contribute to the carbon recyclinginliquid environments.Junghannsetal.10

showedthatcertainisolatesofaquaticfungihadahighand unexploited potential for the treatment of dye-containing wastewaters, even at high concentrations. In the present study,13isolatesoffreshwaterfungishowedahighefficiency forthedecolorizationofmultipledyes,whichshouldprovide advantagesforthetreatmentofeffluentswithcomplexdye

Table4–Biosorptionrates(%)of11dyesafterthetreatmentwith13fungalstrainsfor7daysinliquidmedia.

Dye AcidBlue40 ReactiveRed11 Acid Blue193

AcidBlue62 AcidBlue113 ReactiveBlue74 AcidRed73

Myrotheciumverrucaria C-1 0 0 0 0 0 0 0 M.verrucariaDX-8 0 0 0 0 0 0 0 M.verrucariaI-5 0 0 0 0 0 0 0 Colletotrichumdematium I-6c 8.58±0.93 0 0 26.50±0.64 1.40±0.32 2.36±0.35 14.03±0.58 Corynesporacassiicola I-101 20.10±0.36 29.01±1.41 0 30.10±0.73 26.17±0.85 20.87±0.88 1.12±0.12 Dictyosporium zhejiangensisSy06 0 0 0 0 0 0 32.0±0.99 D.zhejiangensisH-6 0 0 0 0 0 0 41.84±2.41 Plectosporiumtabacinum J-1 0 0 0 0 0 0 10.03±1.88

FusariumthapsinumI-10 0 0 0 0 0 0 8.03±2.46

F.thapsinumDB-42 0 0 0 0 0 0 0 AlternariaalternataJx-62 2.22±0.44 0 0 0 0 0 24.44±1.53 Acrogenospora sphaerocephalaJX-43 0 0 0 0 0 0 12.03±0.66 CeriporialacerataD-1 0 0 0 4.78±0.88 0 0 0

compositions. Thedecolorizationabilities ofseveral fungal

species indecolorizing synthetic dyes, such as A.

sphaero-cephala, D.zhejiangensis, and P. tabacinum, have never been

reportedbefore.

Themechanisms offungal dyeremoval are categorized

intothefollowingthreegroups:bioaccumulation,

bioabsorp-tionandbiodegradation.7,16_{Bioaccumulationisreferredtothe}

accumulationofpollutantsbyactivelygrowingcellsthrough theirmetabolism.16_{Biosorptioncanoccurineitherlivingor}

deadbiomass; amino, carboxyl, thiol,lipid and phosphate groupsthatexistinthefungalcellwallsareresponsiblefor binding the dye molecules in the biosorption process.16,29

Themainmechanismofdecolorizationbyfungiis biodegra-dationbecause the fungi canproduce various non-specific extracellularandintracellular enzymesinvolvedinthe dye decolorizationprocess,suchaslaccase,manganese peroxi-dase(MnP),manganeseindependentperoxidase(MIP),lignin peroxidase(LiP),tyrosinase,and others.1,16,29_The

biodegra-dation ability of fungi through enzymatic activities was confirmedbyusingpurifiedenzymesfordecolorization.11,16

Moreover, there are several factors that can influence the rate of fungal dye biodegradation, such as environmental parameters,thedyemoleculestructures,andtherelative con-tributionsofenzymes involvedinthedecolorization,which may bedifferent for each fungus.1,16,30 _{Further research is}

neededtounderstandthe factorsthat influencethe decol-orizationratewiththestudiedfungi.

Studiesonthebio-decolorizationoftextiledyeeffluents byfungihavebeenreportedeveninthepresenceofsaltsand highdyeconcentrationusingthewhite-rotfungi,31_Aspergillus

fumigatusand Phanerochaetesordid.28,32 _{Inthepresent study,}

oneisolate,D-1,whichwasidentifiedasthewhite-rot fun-gusC.lacerate,26_{alsoshowedapowerfulabilitytodecolorize}

multipledyes.Certainfungalisolatesinthisresearch,suchas

C.dematiumstrainI-6c,F.thapsinumstrainDB-42,F.thapsinum

strainI-10,andA.sphaerocephalastrainJX-43,showedahigher potentialfordyedecolorization than theD-1strain, which indicates theirsuitabilityforthe treatmentofdye-polluted

water.Theassessmentoftheremovalabilitiesofmultipledyes bycertainfungalstrainsindicatesthatseveralenzymeswere involvedinthedecolorizationprocessalthoughtheenzymes contributingtodyedecolourisationremaintobedefined,for thedyebiosorptioncontributingtodecolorisationbymycelia was very low. Moreover,the high frequency appearanceof newabsorbancepeaksafterfungaltreatmentdemonstrated therolesoftheenzymesinvolvedinboththedecomposition and thebiotransformationofdyes.Thischaracterizationof fungirevealedthattheymaybeusefulforthetreatmentof dye-containingwastewaters.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisresearchwas supportedbythe MajorScience& Tech-nologyProjectoftheXinjiangUygurAutonomousRegion(no. 201130102)andtheNatural ScienceFoundationofthe Zhe-jiangProvince(LY15C140001).

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