Heavy metal tolerance traits of filamentous fungi isolated from gold and gemstone mining sites

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

Environmental

Microbiology

Heavy

metal

tolerance

traits

of

filamentous

fungi

isolated

from

gold

and

gemstone

mining

sites

Oluwatosin

Gbemisola

Oladipo

_,

_Olusegun

_Olufemi

_Awotoye

_,

_Akinyemi

_Olayinka

_,

Cornelius

Carlos

Bezuidenhout

_,

_Mark

_Steve

_Maboeta

a_{North-WestUniversity,UnitforEnvironmentalSciencesandManagement,Potchefstroom,SouthAfrica} b_{ObafemiAwolowoUniversity,InstituteofEcologyandEnvironmentalStudies,Ile-Ife,Nigeria}

c_{ObafemiAwolowoUniversity,DepartmentofSoilandLandResourcesManagement,Ile-Ife,Nigeria}

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r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6August2016 Accepted5June2017

Availableonline8August2017 AssociateEditor:ValeriaOliveira

Keywords:

Fungimetaltolerance Mycoremediation Heavymetal Minesite

Eco-friendlyclean-upstrategy

a

b

s

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t

Increased environmental pollutionhasnecessitated theneed foreco-friendly clean-up strategies.Filamentousfungalspeciesfromgoldandgemstoneminesitesoilswere iso-lated,identifiedandassessedfortheirtolerancetovariedheavymetalconcentrationsof cadmium(Cd),copper(Cu),lead(Pb),arsenic(As)andiron(Fe).Theidentitiesofthefungal strainsweredeterminedbasedontheinternaltranscribedspacer1and2(ITS1andITS2) regions.Myceliagrowthofthefungalstrainsweresubjectedtoarangeof(0–100Cd),(0–1000 Cu),(0–400Pb),(0–500As)and(0–800Fe)concentrations(mgkg−1_{)incorporatedintomalt}

extractagar(MEA)intriplicates.Fungalradialgrowthswererecordedeverythreedaysover a13-days’incubationperiod.FungalstrainswereidentifiedasFomitopsismeliae,Trichoderma ghanenseandRhizopusmicrosporus.AlltestfungalexhibitedtolerancetoCu,Pb,andFeat alltestconcentrations(400–1000mgkg−1),notdifferingsignificantly(p>0.05)fromthe con-trolsandwithtoleranceindex>1.T.ghanenseandR.microsporusdemonstratedexceptional capacityforCdandAsconcentrations,whileshowingnosignificant(p>0.05)difference comparedtothecontrolsandwithatoleranceindex>1at25mgkg−1Cdand125mgkg−1 As.Remarkably,thesefungalstrainsshowedtolerancetometalconcentrationsexceeding globallypermissiblelimitsforcontaminatedsoils.Itisenvisagedthatthismetaltolerance traitexhibitedbythesefungalstrainsmayindicatetheirpotentialsaseffectiveagentsfor bioremediativeclean-upofheavymetalpollutedenvironments.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mails:tosin1oladipo@gmail.com,26940582@nwu.ac.za(O.G.Oladipo).

https://doi.org/10.1016/j.bjm.2017.06.003

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Increased heavy metal contamination of soil and water environments1 _{has necessitated the need for clean-up}

strategies.Recently,diverseeco-friendlyremediationoptions have been explored for the restoration of contaminated environments. These remediation options, among others, include the use of plants (phytoremediation),2 _bacteria

(bacterial bioremediation)3 _{and fungi (mycoremediation).}4

The employability of these bio-resources (plants, bacte-ria and fungi) for effective bioremediation has been well reported.2–4

Atpresentoftheseoptions,mycoremediationstrategyhas received increasedattention inthe bioremediation of con-taminated/pollutedenvironmentsduetoitsreasonablylow costimplicationsand significant successoutcomes.5–8

Fila-mentousfungalspecieshavebeenidentifiedfortheirdistinct attributes(ability tothriveunder extremepH,temperature andnutrientvariabilityconditions,aswellastolerancetohigh metalconcentrations)9–11_{andhencetheireffective}

remedia-tiontraitsofcontaminatedsites.

Metaltolerance/resistancehasbeen definedas the abil-ity of an organism to survive metal toxicity by means of oneormoremechanismsdevisedindirectresponsetothe metal(s)concerned.7,12 _{Metaltolerancebyfilamentousfungi}

has been associated with their sites of isolation, toxicity of the metal tested, its concentration in medium, and on the isolate’s competence.10 _{Contaminated sites are known}

as principal sources of metal-resistant species18–22 _with

indigenousfungalstrainsisolatedfromheavymetal contam-inatedsitesexhibitingnotabletoleranceforhighheavymetal concentrations.9,21,23–25

However, of more importance is the specific and non-specific heavy metal tolerance mechanisms adopted by fungal species. According to Vadkertiova and Slavikova13

the introduction of heavy metals into the environment has induced physiological and morphological adaptation strategies in the microbial community. Specifically, fungal speciesadoptoneormoremetaltolerancestrategieswhich includeextracellularmetalsequestrationand precipitation, suppressedinflux,enhancedmetalefflux,productionof intra-cellular/extracellular enzymes, metal bindingto cell walls, intracellularsequestrationandcomplexation.14–17

Severalmetal-tolerantfilamentousfungi(Rhizopus, Tricho-derma,Aspergillus,Penicillium,andFusarium)havebeenisolated frommultipleheavymetalcontaminatedsoils.7_Zafaretal.7

reportedthatRhizopussp.,isolatedfrommetal-contaminated agriculturalsoilstoleratedCdandCrconcentrations.In addi-tion, Volesky26 _{observed that the mycelium of a Rhizopus}

speciewasbiosorbenttowardsPb,Cd,CuandZn.Trichoderma

specieshavealsobeenknowntoexhibittolerancetoarange oftoxicants27–29_{andCu,Cd,AsandZnheavymetalsininvitro}

conditions.8,23,27,30–34

However,there is a dearthof knowledgeof the growth responseand heavymetal tolerance offilamentous fungal speciesisolatedfromgoldandgemstoneminingsites.This studywasthereforedesignedtoisolate,identifyandassess thegrowthresponseandtolerance/resistanceoffilamentous fungiisolatedfromgoldandgemstoneminingsitestovaried

concentrations of selected heavy metals associated with miningsites.

Materials

and

methods

Studysitesandsoilsampling

Mine sitesoilsusedinthisstudy wereobtainedfrom gem-stoneandgoldminingsitesinSouthwestern,Nigerianamely: Awo (7◦46 N, 4◦24 E) and Itagunmodi (7◦30 N, 4◦49 E) as described.1,4,35 _{From previous studies,}1,4,35 _soil

prelim-inary heavy metal analysis of the sites recorded elevated concentrations of 0.20–0.35mgkg−1 Cd; 3.68–48.60mgkg−1 Cu; 19.05–35.00mgkg−1 Pb; 20.45–34.80mgkg−1 As and 240.24–296.18mgkg−1Fe.

Isolationofsoilfungi

Isolationofsoilfungiwasperformedbyserialdilutionandthe spreadplatemethodusingmaltextractagar(MEA)medium andincubatedat30◦Cforfivedaysaspreviouslydescribed.4,35

Streptomycin(35mgmL−1)wasaddedasasupplement into themediumtoinhibitbacterialgrowth.Afterincubation, iso-latesofsinglesporesweresuccessivelysub-culturedonMEA to obtain pure isolates. Fungal species were characterized on the basisofphenotypical/macroscopicobservation (pig-mentation,shape,diameter,colonyappearanceandtexture) andmicroscopicexamination(septationofmycelium,shape, form,diameterandtextureofspore/conidia).Theculturaland morphological characteristic featuresof the fungal species werecomparedwiththosedescribed.36_{Fungalspecieswere}

thenselectedforgenotypic-basedidentification.

DNAextractionandPCRamplification

TheZRfungal/bacterialDNAkit(ZymoResearch,Irvine,CA, USA) was used to extract genomic DNA from pure 5-day oldfungalculturesaccordingtothemanufacturer’smanual. About 40mg(wet wt.) myceliumwas harvestedaseptically intotheZRBashingBeadTM _{lysistubeandlysedin750␮Lof}

lysisbufferbybeadbeating.Thelysatewasthencentrifuged at13,400rpmfor300stoobtainclearsupernatant.Further pro-tocols,whicharebinding,washstepsandelutionofDNAwere performedasinstructedbythemanufacturer.Thequalityand integrityoftheextractedDNAwereverifiedon1%agarosegel, whileDNAconcentrationandpuritywereverifiedusing Nan-oDropspectrophotometer(ND-1000,NanoDropTechnologies Inc.,Wilmington,DelawareUSA).

Taxonomic identification of isolates was between the internally transcribed spacer regions – 1 (ITS1) and 2 (ITS2). DNA amplification was done using primer sets ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3).37_{EachPCRreactioncontained}

12.5␮Lof2×DreamMastermix(ThermoScientific Technolo-gies,Waltham,MA,USA),50ngDNAtemplate,0.2Mofeach forward and reverse primers and nuclease-free waterto a finalvolumeof25␮L.PCRwasperformedinaC1000TM

ther-malcycler (Bio-Rad, Hercules,CA, USA)involvingan initial denaturationat95◦Cfor5min,29 cyclesofdenaturationat

95◦Cfor30s,annealingat55◦Cfor30sandextensionat72◦C for60s.Theamplificationprocesswasterminatedbyafinal extensionof72◦C for5min. ThePCRampliconswere then verifiedon1.5%agarosegelafterelectrophoresis.

Sequencingandphylogeneticreconstruction

Purified PCR amplicons were sequenced by using forward primerITS1andtheBigDyeterminatorv3.1cycle sequenc-ing kit (Applied Biosystems, Warrington, UK) on a 3130 Geneticanalyzer(AppliedBiosystems/Hitachi,Tokyo,Japan). Sequence electropherograms were inspected manually and edited with FinchTV (v. 1.4.0; http://www.geospiza.com/

Products/finchtv.shtml). For taxonomic assignment, edited

sequences were aligned with sequences on the UNITE ITS database (https://unite.ut.ee/index.php) while for, phyloge-netic reconstruction, the sequences, together with closely related sequences in the GenBank were selected. Multiple sequence alignment of the obtained sequences was done usingMUSCLE38 _{integrated inMEGA V. 6.0.}39 _Theresulting

multiplesequencealignmentsweretheneditedmanuallyand rectifiedforgapsusingDAMBEsoftware.40_Phylogeny

dendro-gramswereconstructedusingtheneighbour-joiningmethod oftheTamura–Neisubstitutionmodelandathousand boot-strapreplicationsinMEGA.

Heavymetaltoleranceassay

Isolated filamentous fungi were assessed for heavy metal toleranceatvaryingCd,Cu,Pb,AsandFeconcentrations. Fil-ter(0.25␮mporesize)sterilizedheavymetalsaltsofCdCl2,

CuSO4, PbSO4, AsSO4 and Fe2SO4 were incorporated into

sterileMEA.Mediaweresupplementedwith35mgmL−1 strep-tomycinandpHwasmaintainedat5.6bytheadditionof3M NaOH.Theexperimentwasconductedintriplicateswith con-troland fourothervariedtestconcentrations.Heavymetal concentrations(mgkg−1)were:(25,50,75and100)cadmium, (125,250,500and1000)copper,(100,200,300and400)lead, (125,250,375and500)arsenicand(200,400,600and800)iron. Thenon-amendedmediumservedasacontrol.

Testfungalstrainof8mmdiameterdisksfrom7-dayold purecultureeachwereindividuallyinoculatedintoan8mm wellasepticallyboredatthecentreofcontrolandtestMEA plates.Allplateswereincubatedat29±1◦Cfor13days, dur-ingwhichmycelialradialgrowthwasmonitoredandrecorded everythreedays.Heavymetaltolerancepotentialofthe fun-galspeciesinthetestmediumwascalculatedinrelationto thecontrolradialgrowths(Eq.(1)).Fungiheavymetal toler-ancewasratedthus:0.00–0.39(verylowtolerance),0.40–0.59 (lowtolerance),0.60–0.79(moderatetolerance),0.80–0.99(high tolerance)and1.00–>1.00(veryhightolerance),thehigherthe values,thehigherthefungaltolerancetotheheavymetal.

Toleranceindex=Radialgrowth (mm) oftestfungusinheavymetalincorporatedmedium

Radialgrowth (mm) offungusinnon-incorporatedmedium (1)

Statisticalanalysis

Statisticalanalysisofdataobtainedwasdoneusingone-way analysisofvariance(ANOVA)at5%levelofsignificanceusing

the StatisticalPackageforSocialSciences (SPSS)version 23 (IBM,Armonk,NY,USA).Aposthoctestwasperformedusing theDuncan’sNewMultipleRangeTest.

Results

Fungiidentification

Threeindigenousfungalspeciesisolatedfromgoldand gem-stoneminingsiteswereidentified.TheITS-basedtaxonomic assignmentofthefungalstrainsconfirmedtheidentitiesof

Fomitopsismeliae,Trichodermaghanense(twoisolates)and Rhi-zopusmicrosporus(Table1).Theisolationsourcesofthespecies revealedthepresenceoftwogenera–Fomitopsisand Tricho-derma from Itagunmodi, the goldmining site and Rhizopus

from thegemstoneminesite.Theevolutionaryrelatedness oftheisolateswithsimilarGenBanksequencesfurther con-firmedtheidentitiesofthestrains(Fig.1).

Growthresponseoftestedfungalstrainsinheavy

metal-richmedia

Mycelia growth response of F. meliae, T. ghanense and R. microsporustovariedconcentrationsofcadmium,copper,lead, arsenicandirondifferedamongthespecies(Fig.2).

Onexposuretoallcadmiumandarsenicconcentrations,F. meliaeexhibitedinhibitedgrowth,withmyceliagrowths differ-ingsignificantly(p<0.05)comparedtothecontrol.Although, when exposed to varied Cu, Pb and Fe-enriched media, a divergent trait was displayed as F. meliae revealed no sta-tistical (p>0.05) difference in radial growth compared to the control. Withrespect to T. ghanense and R. microsporus

strains,nostatistical(p>0.05)differenceswereobtainedinthe radialgrowthofthestrainscomparedtotheircontrolsinCd (25–100mgkg−1),Cu(125–1000mgkg−1),Pb(100–400mgkg−1), As(125–500mgkg−1)andFe(200–800mgkg−1)enrichedmedia. Overall, agrowth response trendofFe=Cu=Pb>As=Cd was observed among the fungal strains to heavy metal concentrations. In terms oftheir responsein heavy metal rich-media, a trend showing T. ghanense>R.microsporus>F. meliaewasobserved.Generally,F.meliaetolerated400mgkg−1 Pb, 800mgkg−1 Fe and 1000mgkg−1 Cu concentrations,but revealed high inhibition to all Cd and As concentrations. On the other hand, T. ghanense and R. microsporus showed tolerance to elevated Cd (100mgkg−1), Pb (400mgkg−1), Fe (500mgkg−1),As(800mgkg−1)andCu(1000mgkg−1) concen-trations.Comparingtheresponseofthesefungalspeciesto heavy metallimitsforcontaminated soils,it wasobserved thatthefungalspeciesfarexceededthesetpermissiblelimits

(Table2)exceptF.meliaewhichwasintoleranttoallCdandAs

Table1–TaxonomicidentificationoffungalspecieswithsimilarityontheUNITEITSdatabase.

LAB-ID Sampleorigin(miningsite) Closestrelative Sequencesimilarity(%) Accessionnumber

FUG-07 Itagunmodi Trichodermaghanense 99.5 KT819140

FUG-08 Itagunmodi Trichodermaghanense 99.8 KT819141

FUG-09 Itagunmodi Fomitopsismeliae 97.5 KT819142

FUG-14 Awo Rhizopusmicrosporus 100 KT819147

ToleranceindexratingofF.meliae,T.ghanenseandR.

microsporustoCd,Cu,Pb,AsandFeconcentrations

Inascertainingthetoleranceofthetestfungalstrainstoheavy metalconcentrations(Fig.2),wefurtherevaluatedtheheavy metaltolerancelevelsofthefungalspecies.Thiswasobtained bycalculatingthetoleranceindexofthetestfungalspecies relativetotheircontrolsusingthemyceliaradialgrowthdata inheavymetalenrichedmedia(Eq.(1)).

ThetoleranceratingofF.meliaeto25–100mgkg−1cadmium and 125–500mgkg−1 arsenic concentrations were observed tobeverylow,withtoleranceindicesrangingbetween0.17

and 0.32 (Table 3). However,in Cu and Pb concentrations,

F.meliaeindicatedhightolerancerevealed bythe veryhigh

toleranceindexvaluesof1.02–1.32in125–500mgkg−1Cuand 0.96–1.28 in 100–300mgkg−1 Pb. At higher Pb (400mgkg−1) andCu(1000mgkg−1)concentrations,adecreasedtolerance wasindicatedbyF.meliaewithtoleranceindexvaluesof0.67 and0.78respectively.

ForT.ghanenseandR.microsporusinallCdconcentrations, an indication of high to very high tolerances of 0.80–1.13 anda0.84–1.01toleranceindexwereobtainedrespectively.In addition,inCuandPbconcentrations,thespeciesindicated veryhightolerancewithindexrangesof1.02–1.27.Inarsenic enriched-media,T.ghanenseindicatedmoderate(0.72)tohigh (0.98) tolerance whileR.microsporus indicated high to very hightoleranceat375and500mgkg−1and125and250mgkg−1 concentrations. Exceptionally, the three fungal species

Trichoderma ghanense; EU280100

Trichoderma ghanense; EF442075

Trichoderma sp.; JX982450

FUG-07; KT819140 FUG-08; KT819141

Trichoderma saturnisporum; KC884818

Trichoderma pseudokoningii; X93971

Trichoderma reesei; KP263685

Trichoderma longibrachiatum; JX173863

Hypocrea sp.; JQ411368

Hypocrea koningii; JX174420

Curvularia sp.; KF811434

Curvularia lunata; KR012881

Curvularia lunata; KP131964

Helotiaceae sp.; EF060565

Curvularia lunata; JX966625

Curvularia lunata; JX966624

Cochliobolus kusanoi; JQ818178

Cochliobolus sp.; KP143696

Curvularia kusanoi; KT819137

FUG-09; KT819142

Fomitopsis meliae; KT210092

Fomitopsis meliae; KC585351

Fomitopsis sp.; KP012890

Piptoporus betulinus; AY966448

Polyporales sp.; JQ312181

Fomitopsis palustris; KJ995920

Fomitopsis palustris; AB733120

Antrodia bondartsevae; JQ700276

Antrodia wangii; JQ700277

Rhizopus sexualis; AB113011

FUG-14; KT819147

Rhizopus microsporus var.rhizopodiformis; KM527226

Rhizopus microsporus; KR149629

Rhizopus azygosporus; JN943010

Rhizopus microsporus var.chinensis; JN206355

100 68 78 66 76 50 98 91 61 92 83 81 89 68 63 100 75 68 0.1 Ascom yc ota B as ido m yc ota Zy go m yc ota

Fig.1–Unrootedneighbour-joiningtreeoffungalspecies.Sequencesobtainedinthisstudyareindicatedshadedcircles (䊉).Neighbour-joiningtreewasconstructedinMega(Version6)byusingtheTamura–Neisubstitutionmodelanda thousandbootstrapreplications.Bootstrapvaluesbelow50arenotshown.

Table2–Tolerancecapabilityoffungalspeciestoheavymetalconcentrations(mgkg−1).

Heavymetals Fungi Highestmetalconcentration

(mgkg−1_{)toleratedinmedia}

a_{Worldpermissiblelimitin}

soils(mgkg−1₎ Cadmium 0.41 F.meliae b_NT d_{T.ghanense 100} R.micosporus 100 Copper 38.90 F.meliae 1000 d_{T.ghanense 1000} R.micosporus 1000 Lead 27.0 F.meliae 400 d_{T.ghanense 400} R.micosporus 400 Arsenic 20.0 F.meliae b_NT d_{T.ghanense 500} R.micosporus 500 Iron c F.meliae 800 d_{T.ghanense 800} R.micosporus 800

a _FAO41_{andKabata-Pendias.}42

b _{‘NT’–nottoleratedatanyconcentration(mgkg}−1_).

c _{Notavailable.Dependentondifferentsoilparentalconstituents.}

d _{Meanconcentrationsoftaxonomicallysimilarfungalidentitiesinthestudywasused.}

Table3–Toleranceindexlevelsoffungalstrainsinmetal-richmediaconcentrations.

Heavymetals Fungi Concentrations(mgkg−1)

Cadmium 25 50 75 100 F.meliae 0.17 0.17 0.17 0.17 a_{T.ghanense 1.13 0.85 0.96 0.80} R.micosporus 1.01 0.99 0.99 0.84 Copper 125 250 500 1000 F.meliae 1.32 1.12 1.02 0.78 a_{T.ghanense 1.25 1.27 1.27 1.27} R.micosporus 1.02 1.02 1.02 1.02 Lead 100 200 300 400 F.meliae 1.27 1.28 0.96 0.67 a_{T.ghanense 1.20 1.25 1.25 1.27} R.micosporus 1.02 1.02 1.02 1.02 Arsenic 125 250 375 500 F.meliae 0.32 0.21 0.17 0.17 a_{T.ghanense 0.98 0.91 0.72 0.87} R.micosporus 1.02 1.02 0.99 0.86 Iron 200 400 600 800 F.meliae 1.16 1.32 1.38 1.45 a_{T.ghanense 1.25 1.27 1.27 1.25} R.micosporus 1.02 1.02 1.02 1.02

Toleranceindexratingvaluesindicate: 0.00–0.39–verylowmetaltolerance. 0.40–0.59–lowmetaltolerance. 0.60–0.79–moderatemetaltolerance. 0.80–0.99–highmetaltolerance. 1.00–>1.00–veryhighmetaltolerance.

Fig.2–Effectofvariedconcentrationsof(A)Cd,(B)Cu,(C)Pb,(D)Asand(E)Feonfungiradialgrowth(mm)over13days incubationperiod.Key–F.meliae(Fomitopsismeliae);T.ghanense(Trichodermaghanense)andR.microsporus(Rhizopus microsporus).Meansof3replicates(±SE).Barsofthesamefungalspeciewithdifferentlettersaresignificantlydifferent (p<0.05)accordingtoDuncan’sNewMultipleRangeTest.

indicatedremarkablyhightolerances(toleranceindexranges of1.02–1.45)inallvariedFeenriched-mediaconcentrations, withF.meliaedemonstratingthe highesttoleranceindexof 1.45at800mgkg−1.

Whenassessingthetoleranceindexofthefungalspecies,

R. microsporus exhibited high to very high tolerance in all fiveheavymetalconcentrationstestedcloselyfollowedbyT.

ghanense whichrevealed high toveryhigh toleranceinCd, Cu,PbandFeconcentrationsexceptarsenic.F.meliae,onthe other hand,indicatedhightoveryhigh toleranceinCu,Pb andFeconcentrationexposuresbutverylowtoleranceinCd andAsconcentrations.Onthewhole,thetolerancelevelsof thespeciestotheheavymetalsshowedadecreasingtrendof Fe>Cu>Pb>As>Cd.

Discussion

TheoccurrenceofF.meliae,T.ghanenseandR.microsporuson heavymetalcontaminatedgoldandgemstoneminingsites wasconfirmedinthisstudy.Thepresenceoffungalspecies invariouscontaminated/pollutedsiteswithelevated heavy metalconcentrationshasbeenwelldocumented.Specifically, Zafaretal.7_{andFazlietal.}43_{reportedtheoccurrenceoffungal}

strainsinsoilswithelevatedCd,Cu,AsandZn concentra-tions.Inaddition,Anandetal.9_{andKarcprzakandMalina}29

confirmedthepresenceoffungiinheavymetalpollutedsoils. Irametal.,12_{Iskandaretal.,}21_{andLópezandVázquez}23_also

affirmedtheoccurrenceoffungal speciesinsewagesludge waterplants,heavymetalcontaminatedfreshwater ecosys-tem and sewage and industrial waste waters respectively. Furthermore,Moetal.,44_{Srivastavaetal.}45_andBabuetal.46

confirmedtheexistenceoffungalstrainsinPbandAspolluted sitesandminetailingssoils.

Of more importance is the marked tolerance dis-played by these fungal species to heavy metals. Fungal species tolerate metals6,15,47 _{and thrive at elevated metal}

concentrations.9,24,48 _{In particular, indigenous filamentous}

fungi isolated from contaminated sites have shown toler-ance to heavy metals.12,18,19,49 This exceptional trait may be attributed to the isolates’ tolerance strategies to ele-vatedheavymetalcontaminations. Fominaetal.,14 _Turnau

etal.,15 _Gadd16 _{andValaand Sutariya}17 _{reportedthatthese}

tolerancemechanisms include metalbinding tocell walls, production of intracellular/extracellular enzymes, intracel-lular sequestration, extracellular metal sequestration and precipitation,suppressedinflux,enhancedmetalefflux,and complexation.

RemarkableheavymetaltolerancewasdemonstratedbyR. microsporusandT.ghanensespecies.TrichodermaandRhizopus

species have been widely reported for their notable toler-ancetovariousheavymetalsatvariedconcentrations.7,23,31,50

SomestrainsofRhizopusandTrichodermarevealedhigh resis-tance toa range ofheavy metals, such asCd,11,23,26,44,50,51

Cu,21,26,46_Pb26,52_andAs.17,45_{ValaandSutariya}17_reportedthat

Rhizopusspecies were highly tolerant to25 and 50mgkg−1 As concentrations, which confirms the findings of this study.Inaddition,strainsofTrichodermatoleratedCdat100 and 125mgkg−153,54 and Cu at 300mgkg−1,23 _500mgkg−1_,9

800mgkg−121 _{and 1000mgkg}−145 _{concentrations.}

Further-more, a strain of Trichoderma was found to tolerate Pb concentrationsof1000mgkg−1inmedium.21

Allthreefungalspeciesdemonstratedextraordinary pref-erence for Fe at all concentrations as observed in their toleranceindexvalues.Thismaybeascribedtothefactthat ironservesasamicronutrientandiscrucialinmanymetabolic processes.55_{Inaddition,Kosman,}56_Philpott57_andJohnson58

foundthatfungalspecieshaveahighaffinityandcapacityto takeupFeinvariousformsandvariety.AznarandDellagi55

andNeilands59_{statedthatmostfungalstrainssynthesizeand}

secretesiderophores(smallorganiccompoundsthatbind fer-ricFewithhighaffinityandspecificity)whichtheyutilizeto extractFe from their environment. Furthermore,according toKosman56_{microorganismsincludingfungibasicallydeploy}

threemainstrategiestoincreaseironsolubilitybyacidifying

theenvironment,reducingferricirontoamoresolubleferrous formandsecretingsolubleiron-chelatingmolecules.

Overall,T.ghanenseandR.microsporusexhibitedhigher tol-eranceinCd,Cu,PbandAs-enrichedmediacomparedtoF. meliaewhich specificallydisplayedsensitivitytoall Cdand As concentrations. Studies confirm that differing levels of metalresistancehavebeendemonstratedbydifferentfungal speciesisolatedfromthesamesourceofmetal-contaminated sites.12,60–64_{Thismaybeascribedtovariationsinthetolerance}

mechanismutilizedbythefungalspecies7_whichis

individu-allydependent.65_{Inaddition,theevidentsensitivitytoallCd}

andAsconcentrationsdisplayedbyF.meliaemaybeattributed totheknowntoxicityoftheseheavymetalsasreported.66–68

Conclusion

Indigenous filamentousfungal speciesfrom goldand gem-stone mine sites exhibited remarkable tolerance in heavy metal-richmedia.ExposuresofF. meliae,T.ghanense andR. microsporustoelevatedCu,PbandFelevelsrevealedhigh tol-erancewithindexvalues>1.Furthermore,T.ghanenseandR. microsporusdemonstratedextraordinarytoleranceforAsand Cdconcentrationswithatoleranceindex>1at25mgkg−1Cd and50mgkg−1As.Theseexceptionaltraitsdisplayedbythese fungalspeciestoelevatedheavymetallevelsmayindicatethe bioremediativepotentialsinherentintheindigenous filamen-tousfungalspecies.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisstudyacknowledgestheNorth-WestUniversity, Potchef-stroom,SouthAfricaforthefinancialsupportandappreciates Mr.ObinnaEzeokoliforassistingwiththemolecularaspect oftheworkandMr.AdesanyaAdebowaleforthestatistical analysis.

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