Screening of Trichoderma isolates for their potential of biosorption of nickel and cadmium

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

Environmental

Microbiology

Screening

of

Trichoderma

isolates

for

their

potential

of

biosorption

of

nickel

and

cadmium

Nabakishor

Nongmaithem,

Ayon

Roy

,

Prateek

Madhab

Bhattacharya

a

r

t

i

c

l

e

i

n

f

o

Received16February2014 Accepted10August2015 Availableonline2March2016 AssociateEditor:CynthiaCanêdoda Silva Keywords: Heavymetals Biosorption Trichoderma MIC50 Fungistasis

a

b

s

t

r

a

c

t

FourteenTrichodermaisolateswereevaluatedfortheirtolerancetotwoheavymetals,nickel andcadmium.Threeisolates,MT-4,UBT-18,andIBT-I, showedhighlevelsofnickel tol-erance,whereasMT-4,UBT-18,andIBT-IIshowedbettertoleranceofcadmiumthanthe other isolates.Undernickelstress,biomassproductionincreaseduptoaNi concentra-tionof60ppminallstrainsbutthendecreasedastheconcentrationsofnickelwerefurther increased.Amongthenickel-tolerantisolates,UBT-18producedsignificantlyhigherbiomass uponexposuretonickel(upto150ppm);however,theminimumconcentrationofnickel requiredtoinhibit50%ofgrowth(MIC50)washighestinIBT-I.Amongthecadmium-tolerant isolates,IBT-IIshowedbothmaximumbiomassproductionandamaximumMIC50valuein cadmiumstress.AsthebiomassoftheTrichodermaisolatesincreased,ahigherpercentage ofnickelremovalwasobserveduptoaconcentrationof40ppm,followedbyanincrease inresidualnickelandadecreaseinbiomassproductionathighernickelconcentrationsin themedium.Theincreaseincadmiumconcentrationsresultedinadecreaseinbiomass productionandpositivelycorrelatedwithanincreaseinresidualcadmiumintheculture broth.NickelandcadmiumstressalsoinfluencedthesensitivityoftheTrichodermaisolates tosoilfungistasis.IsolatesIBT-IandUBT-18weremosttoleranttofungistasisundernickel andcadmiumstress,respectively.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Trichoderma species are imperfect filamentous fungi with teleomorphs and belong to the order Hypocreales in the Ascomycetedivision. Trichoderma spp.are amongthe most frequentlyisolated soilfungiand are well known fortheir

∗ _{Correspondingauthor.}

E-mail:ayonroy.plantpathology@gmail.com(A.Roy).

biocontrolabilityagainst awide rangeofplant pathogenic fungi,1_{inductionoflocalizedandsystemicdefenseresponses} inplants,2 _{andplantgrowthenhancement.}3,4 _Theyplayan important role in ecology bytaking part in decomposition ofplantresidues,aswellasinbiodegradationofman-made chemicals and bioaccumulation of high amounts of vari-ous metals from wastewater and soil.5,6 _{Metal-containing}

http://dx.doi.org/10.1016/j.bjm.2016.01.008

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

pollutantsareincreasinglyreleasedintosoilfromindustrial wastewater, aswell asfrom wastes derivedfrom chemical fertilizersand pesticidesusedinagriculture.7 _Some metal-containingpollutantsare notbiodegradable;theyenterthe food chainand leadto bioaccumulation.8 _{Minuteamounts} of metals, except those that are non-essential for biologi-calfunctions,suchasmercury, arsenic,leadandcadmium, influencevital metabolicprocesses and are requiredbyall formsof life. However, metalsmay be toxic at concentra-tions higher than nutritional requirements. Evidence has suggestedthatTrichodermaspp.exhibitconsiderabletolerance formetalsandaccumulatehighamountsofmetalsfrom pol-luted habitats.1,8 _{Therefore, metal-tolerantTrichoderma spp.} maybecomedominantorganismsinsomepolluted environ-mentsandmayplayanimportantroleineco-friendlymetal removaltechnology.7_{Asacomponentofsoilfungistasis,metal} ionsmay influencethe growth,sporulationand enzymatic activities ofTrichoderma.9,10 _{Thiscan cause changes inthe} quantitiesofextracellularenzymes andmetabolites,11,12 _as wellasinoverallbiocontrolactivitiesagainstplantpathogenic fungiand in plantgrowth-stimulating activities. Katayama andMatsumura13_{demonstratedadegradationpotentialofa} rhizosphere-competentTrichodermasp.forseveralsynthetic dyes,pentachlorophenol,endosulfan,and dichlorodiphenyl-trichloroethane(DDT).Thus,Trichodermaspp.haveacquired anexceptionalroleaspartofasustainableapproachto biore-mediationofherbicide/pesticide-ladensoils.

However, the microhabitat behavior of Trichoderma spp. upon exposuretometal-containingcompoundsmay differ, dependingonthetypeofthemetalandtheTrichodermaisolate, andverylittleinformationinthisregardisavailable.Hence, anattemptwasmadetoscreenTrichodermaisolatesfornickel andcadmiumtoleranceandidentifystrainsthatcan poten-tiallybeusedforbioremediationofsoilspollutedwiththese metals.

Materials

and

methods

IsolationofTrichodermaspp.fromsoil

Trichoderma spp. were isolated from soil on a modified Trichoderma-specific medium(TSM)14 _{using a dilution plate} method.15_{Soilsampleswerecollectedfromdifferent} sugar-canegrowingareasofManipurandfromteaindustryareasin northerndistrictsofWestBengal,whereindiscriminateuseof chemicalsandeffluentshascausedheavy-metalsoiltoxicity. Thesampleswereair-driedandgroundtopowderusinga mor-tarandapestle.Soilsuspensions(1mLof10–3_,10–4_,and10–5

dilutions)wereplatedinPetriplatescontaining20mLof mod-ifiedTSM.Thesuspensionsweredistributeduniformlyover themediumsurfacebyhorizontalshakingandincubatedat 28±1◦Cforsevendays.Greencoloniesoftheantagonist usu-allyappearedafterfourorfivedaysofincubation.Eachcolony wasobservedunderamicroscopeusinglactophenolcotton bluestainandidentifiedtothegenuslevelbasedonthe avail-abletaxonomicliterature.16_{Theshape,sizeandaggregation} of phialospores and phialides were used as main identifi-cationcriteria,alongwithculturalcharacteristics onpotato dextroseagar(PDA).ThecoloniesidentifiedasTrichodermaspp.

weretransferredontoPDAslantsandkeptat4◦Cforfurther use.TheisolatesfromManipurweredesignatedasMT,and the isolatesfrom the tea plantationareas were designated as IBT,followed byanumber. Oneisolate,namely,UBT-18, wasobtainedfromtheculturecollectionoftheDepartment ofPlantPathology,UttarBangaKrishiViswavidyalaya.

Selectionofnickel-andcadmium-tolerantisolatesof Trichodermaspp.

InvitrotoleranceofTrichoderma spp.todifferent concentra-tionsofnickelandcadmiumwasdeterminedbythepoisoned foodtechnique.15_{PDAmedium(100mL)waspreparedin} 250-mLconicalflasks,thenappropriatequantitiesofnickeland cadmiumstocksolutionswereaddedtomoltenPDAtogetthe requiredconcentrations(40,60,100,150,and200mg/L),and theresultingmediawerepouredintoPetriplatesafter gen-tleshaking.Thenon-amendedmediumservedasacontrol. Theplateswere inoculatedbyplacing6-mmmycelialdiscs of4-day-oldculturesoftheTrichodermaisolatesontheagar surfaceandincubatedat28±1◦Cfor2–3days.Isolates show-ingmaximumradialgrowthonthemedia,irrespectiveofthe metalconcentration,wereselectedforfurtherstudies.

BiomassproductionbyTrichodermaisolatesand

determinationofminimuminhibitoryconcentrationofthe metals

For biomasspreparation,selected Trichoderma isolateswere inoculated on PDAplates and incubated atroom tempera-ture(27±1◦C).Afterfivedays,asmallportion(0.5mm)from the fungalmasswascut,transferredinto a250-mLconical flaskcontaining50mLofpotatodextrose(PD)broth supple-mentedwithdifferentconcentrationsofametal(0,40,60,100, 150,and200ppm),andincubatedintriplicatesat27±1◦Cfor sevendays.Thebiomasswasharvestedbyfilteringthrough Whatmanno.1filterpaperandthenwashedthoroughlywith deionizedwatertoremovethegrowthmedium.Theharvested myceliawereoven-driedat60◦Cfor48handthedryweight wasmeasuredusingaSartoriusLA8200Sdigitalweight bal-ancewithanaccuracyof0.1mg.Theinhibitionofbiomass productionwascalculatedbasedonthedryweightusingthe followingformula: PI=





where PI is the percentage of inhibition; X is biomass in the control (0ppm) broth; and Y is biomass in the metal-containingbroth.

The minimum inhibitory concentration of each metal, causing50%ofgrowthinhibition(MIC50)oftheselected

Tri-chodermaisolates,wascalculatedfromthegrowthinhibition results.

Estimationofresidualmetalsinculturebroth

After harvesting the biomass ofeach isolategrown in the PDbrothamendedwithdifferentconcentrationsofnickelor cadmium(0,10,25,50and100ppm),theculturebrothwas assayed forresidualmetal, followingthe methoddescribed

byTandon.17_{Fivemillilitersoftheculturebrothwasplacedin} a100-mLcleanbeaker,followedbytheadditionof10mLofa triacidmixture(HNO3:H2SO4:HClO4,9:4:1,v/v/v),andthe

con-tentwasmixedbyswirlingandkeptovernight.Themixture wasdigestedinadigestionchamberat60◦Cfollowedby heat-ingat90◦CuntiltheproductionofredNO2fumesceased.The

contentwasfurtherevaporateduntilthevolumewasreduced toabout2–3mL.Thecompletionofdigestionwasconfirmed whentheliquidbecame colorless.Aftercoolingthebeaker, itscontentwastransferredquantitativelytoa50-mLcapacity volumetricflask,dilutedto50mLwithdistilledwater,andkept overnight.Onthenextday,itwasfilteredthroughWhatman no.44filterpaper.Thefiltrateswereanalyzedforcadmiumor nickelusingaPerkinElmerAnalyst200AAflame spectropho-tometer.Eachsamplewasanalyzedtwoorthreetimesata wavelengthof445nmfornickelor229nmforcadmium. Resid-ualmetalconcentrationswereexpressedin␮g/mLofculture broth.

ToleranceofTrichodermaisolatestosoilfungistasisunder heavymetalstress

Fungistatic effects of soil were studied using the soil cel-lophane agar disk method18 _{with a slight modification.In} thisexperiment,soilsampleswere amendedwithdifferent concentrationsofnickelorcadmiumtoadjustthemetal con-tamination levels to 50, 100 or 150ppm and kept for one monthforstabilization.Well-saturated,metal-contaminated soil(100g)wasfilledinaplasticcup,andtheuppersurface wassmoothedusingthumbpressure.Cellophanepaperwas cuttothediameterofthecupandboiledtoeliminate plas-ticizereffects. A single pieceof cellophane was placedon thesmoothsoilsurface,andadisc(1cmindiameter)of2% wateragar wasplacedonthecellophane paper.Theentire systemwasrefrigeratedfor24htoactivatetheagardisc.On thenextday,aconidialsuspension(103_{cfu/mL)oftheselected}

metal-tolerantTrichodermaisolatewasappliedtotheagardisc andincubatedat28±1◦Cfor20h.Aftertheincubation,the discwastransferredtoaglassslideandstainedwith0.1% lac-tophenolcottonbluetoexamineconidialgerminationundera lightmicroscopeat20×magnification.Thepercentageof ger-minatedconidiawasrecorded,andthepercentofgermination inhibitionwascalculated.

Statistical

analysis

Theexperimentswereconductedusingafactorial,completely randomizeddesignwiththreereplications, consideringthe isolatesasfactorAandthemetalconcentrationsasfactorB. Ananalysisofvariance(ANOVA)wasperformedforall param-eters using the INDOSTAT package. Comparison of means wasdonebyDuncan’smultiplerangetestatthep<0.05level ofsignificance.19 _{Theidentical lettersinthe resultsdenote} non-significantdifferencesamongthetreatmentswithineach isolate.

Results

and

discussion

Trichoderma spp. areubiquitous microorganismsdistributed in almost all types ofcroprhizosphere20,21 _{and haveeven} beenfoundinmetal-pollutedecosystems.22,23 _Inthisstudy, 14Trichodermaisolates,namely,MT-1,MT-4,MT-7,MT-8, MT-11,MT-13,MT-18,MT-21,MT-23,MT-24,MT-25,IBT-I,IBT-II,and UBT-18,wereidentifiedandselectedforfurtherstudy,based ontheirculturalvariabilityandgrowthrates.Cultural variabil-ityexistedamongtheisolateswithrespecttotheirmycelial growthpattern,colorofsporulation,andpigmentationofthe medium(Table1),indicatingthattheisolatesmightbeableto producesecondarymetabolites. Themaximumgrowth rate wasdemonstratedbyUBT-18,followedbyMT-13,MT-8,MT-4, andMT-23.

Table1–CulturalvariabilityoftheTrichodermaisolates. Isolate Growthrateat48h

(mm)

ColonyappearanceonPDA

MT-1 53.2 Lightgreen,granularsporulationinconcentricring;greenishwhitemyceliagrowthatthemargin. MT-4 71.7 Whitishsporulationwithgreenishtinge,submergedmyceliagrowth,olivegreenpigmentationof

medium.

MT-7 66.3 Profusemycelialgrowthwhiteincolor,scantygreensporulationintermingledwithinthemycelia. MT-8 71.8 Greenishwhitesporulationwithprofusemycelialgrowthatmargin;lightyellowpigmentationin

themedium.

MT-11 38.0 Lightgreencolonywithprofusewhitefluffymycelialgrowthatmargin. MT-13 72.8 Greenishwhitescantysporulation;lightyellowpigmentationinthemedium.

MT-18 66.3 Darkgreengranularsporulationinconcentricringwithscantymycelialgrowth;lightgreen sporulationattheedgeofthecolony

MT-21 64.8 Profuse,greenishwhitefluffymycelialgrowthwithlittlegranulargreensporulationatthecenter. MT-23 68.7 Grayishwhite,fluffymycelialgrowthprofuselyatmargin;darkgreensporulationinhighamount. MT-24 59.7 ColonycharactermoreorlesssimilartoMT-23.

MT-25 38.0 ColonycharactermoreorlesssimilartoMT-23andMT-24.

IBT-I 58.3 Whitishsubmergedmycelialgrowthwithgreenishtingeattheedgeofthecolony,littleyellow pigmentationofthemedium.

IBT-II 54.4 Greenishwhiteraisedcolonywithfluffymycelialgrowth,darkgreensporulationintermingled withinthemyceliaatthecenter.

Table2–GrowthrateoftheTrichodermaisolatesonnickelamendedPDAmedium.

Isolate GrowthrateofTrichodermaisolates(mm)at48h

Concentrationofnickel 0ppm 40ppm 60ppm 100ppm 150ppm 200ppm MT-1 33.3 38.3 36.6 35.6 34.3 7.0 MT-4 64.8 66.0 57.0 54.9 53.2 30.0 MT-7 36.3 35.3 33.6 31.6 27.0 0.0 MT-8 36.6 38.6 36.3 31.6 30.0 0.0 MT-11 38.0 37.3 35.0 27.3 26.3 0.0 MT-13 38.6 38.6 37.0 34.0 27.6 0.0 MT-18 41.2 42.1 40.0 37.7 33.0 28.2 MT-21 49.7 59.6 53.6 49.7 35.7 34.0 MT-23 29.3 36.3 34.3 31.3 23.0 20.1 MT-24 39.3 46.0 40.5 40.0 29.6 18.6 MT-25 38.0 38.0 37.3 36.6 29.3 0.0 IBT-I 44.4 55.2 53.1 50.1 48.0 41.4 IBT-II 49.8 55.6 55.0 51.2 26.3 17.3 UBT-18 68.7 70.8 60.4 57.9 44.5 20.3 SEM±1.23;LSD(p=0.05)3.50. PDA,potatodextroseagar.

TheeffectsofdifferentconcentrationsofNionmycelial growthofthe14Trichodermaisolatesweretested(Table2),and themaximumradialgrowthwasshownbyUBT-18(70.8mm) at a Ni concentration of 40ppm, followed by isolate MT-4 (66.0mm),withasignificantdifferencebetweenthetwo iso-lates.Sixotherisolates,namely,IBT-I,IBT-II,MT-1,MT-23,and MT-24,exhibitedsignificantlyhighermycelialgrowthatthe same Ni concentrationcompared withtheir corresponding non-amended cultures. Witha further increase ofNi con-centrationinthemediumto60ppm,onlyfourisolates,IBT-I, IBT-II,MT-21,andMT-23,showedsignificantlyhighermycelial growth versus their respective controls. Athigher concen-trationsofNi,from100to200ppm,therewasasignificant reductioninmycelialgrowth ofallisolates, althoughthree ofthem,viz.,MT-4,IBT-I, and UBT-18,consistentlyshowed highermycelialgrowth atconcentrationsofup to150ppm. Although MT-21 showed a higher level of tolerance to Ni toxicityat200ppm,itsgrowthwascomparativelylowat con-centrations from 60 to 150ppm compared with the other Trichodermaisolates.

Theeffectsofnickelonradialgrowthofthe14Trichoderma isolatesare presentedinFigs. 1and 2. Itwas noticedthat thegrowthoftheTrichodermaisolateswassignificantly influ-encedbytheheavymetal;inparticular, at40ppmofNi in theamendedmediumthegrowthwasevenhigherthanthat observedinthenon-amendedmedium.Athigher concentra-tionsofthe heavymetal,from 60to200mg/L,therewas a significantreductioninradialmycelialgrowthofallthe Tri-chodermaisolates.IsolateMT-4showedthehighesttolerance tonickel.Takentogether,isolatesMT-4,IBT-I,andUBT-18were consideredtoberesistantsincetheirradialgrowthdecreased ataslowerratethanthatoftheotherisolates,whileisolates MT-7,MT-18, MT-11,MT-13,andMT-25were mostsensitive andnomycelialgrowthwasdetectedat200mg/L.Hence,three isolates,namely,MT-4,IBT-IandUBT-18,werefinallyscreened duetotheirhighNitolerance.

ScreeningoftheTrichodermaisolatesfortheircadmium tol-erancerevealedthattheisolatesvariedsignificantlyintheir

0 10 20 30 40 50 60 MT -1 MT -4 MT -7 MT -8 MT-11 MT-13 MT-18 MT-21 MT-23 MT-24 MT-25 IB T -I IB T -II UB T -1 8 Gr o wt h rate ( mm)

Fig.1–ToleranceofTrichodermaisolatesatdifferent concentrationofnickel.

levels oftolerance,irrespective ofthecadmium concentra-tions tested (Table 3). There was asignificant reductionin themycelialgrowthoftheTrichodermaisolatesuponexposure tocadmium(Fig.3).Atacadmiumconcentrationof40ppm, themaximummycelialgrowthwasshownbyIBT-II(73.4mm), whichwassignificantlyhigherthanthatofUBT-18(66.6mm), followedbyMT-4andMT-24(62.6and62.2mm,respectively). ThedegreeoftoleranceoftheTrichodermaisolates,irrespective

0.00 10.00 20.00 30.00 40.00 50.00 100 ppm 200 ppm 60 ppm 40 ppm 0 ppm Gr o wt h rate ( mm) 150 ppm

Fig.2–EffectofnickelconcentrationsonTrichoderma isolates.

Table3–GrowthrateoftheTrichodermaisolatesatdifferentconcentrationsofcadmiuminPDA.

Isolate GrowthrateofTrichodermaisolates(mm)at72h

Concentrationofcadmium 0ppm 40ppm 60ppm 100ppm 150ppm 200ppm MT-1 90.0 44.5 40.2 34.0 24.3 11.3 MT-4 90.0 62.6 58.3 56.2 48.0 44.7 MT-7 90.0 55.2 49.1 49.0 43.5 39.3 MT-8 90.0 43.6 39.7 36.4 28.9 25.5 MT-11 90.0 56.5 56.2 53.6 43.0 35.3 MT-13 90.0 40.2 34.4 33.9 25.9 24.3 MT-18 90.0 55.1 52.8 46.1 45.2 29.7 MT-21 90.0 51.9 42.1 42.4 38.8 32.9 MT-23 90.0 50.2 46.7 44.4 40.4 33.7 MT-24 90.0 62.2 58.1 50.6 42.8 32.4 MT-25 90.0 61.1 59.2 47.5 46.1 35.8 IBT-I 90.0 57.7 24.3 13.0 0.0 0.0 IBT-II 90.0 73.4 63.0 52.0 39.3 34.0 UBT-18 90.0 66.6 54.8 50.7 43.6 40.6 SEM±1.13;LSD(p=0.05)3.10. PDA,potatodextroseagar.

0 10 20 30 40 50 60 70 MT-1 MT-4 MT-7 MT-8 _{MT-11 MT-13 MT-18 MT-21 MT-23 MT-24 MT-25} IBT-I _IBT-II UBT-18 Gr o wt h rate ( mm)

Fig.3–ToleranceofTrichodermaisolatesatdifferent concentrationsofcadmium.

ofthecadmiumlevel,ispresentedinFig.3.Thedataindicated thatMT-4,UBT-18,andIBT-IIweretolerant,IBT-I,MT-7, MT-11,MT-18,MT-21,andMT-23weremoderatelytolerant,and MT-1,MT-8,andMT-13weresusceptibletocadmiumtoxicity. InFig.4,theeffectsofcadmiumconcentrationsonthe Tricho-dermaisolatesaredepicted,andatrendofadecreasinggrowth

0.00 20.00 40.00 60.00 80.00 100.00 100 ppm 40 ppm 0 ppm 150 ppm 200 ppm Gr o wt h rate ( mm) 60 ppm

Fig.4–EffectofcadmiumconcentrationonTrichoderma isolates.

rateoftheisolateswasobservedwithincreasing concentra-tionsofcadmium.

Intheabsenceofarationalmethodforanapriori predic-tionofabiosorptionpotentialofamicroorganism,theonly method foridentifying and developingnewer and efficient biosorbentsissustainedscreeningofmicrobes.24_Variationsin metaltoleranceamongdifferentspeciesofagenusorwithin thesamespeciesmightbeduetothepresenceofoneormore resistancemechanismsexhibitedbydifferentfungi.25_Sarkar etal.26_{reportedTrichodermaharzianumtobemoderately} toler-anttoupto60ppmofNi,atthatconcentrationthelevelof inhibitionofmycelialgrowthwas33.3%.Afurtherincreasein theNiconcentrationreducedthegrowth,andtotalinhibition wasobservedat200mg/L.Limaetal.27 _alsoobserved influ-enceofcadmiumonradialgrowthofT.harzianum.Theresults ofthepresentinvestigationareinlinewiththeseearlier obser-vations.

When the effects of different nickel concentrations on biomassproductionofthethreepromisingTrichoderma iso-lates(inorderofranking)werestudied,themaximumbiomass weightwasrecordedforUBT-18(359mg),whichwas signifi-cantlyhigherthanthevaluesobtainedforMT-4(256.67mg) and IBT-I(225mg),ataNi concentrationof40ppm.Witha furtherincreaseofthenickel concentrationinthemedium to60ppm, biomassproductionbyallthe isolatesscreened wasinsignificantcomparedwiththeirrespectivecontrols.At higherconcentrationsofnickel,from100to200ppm,there wasasignificant reductioninbiomassofallthreeisolates. AlthoughisolateUBT-18showedsignificantlyhigherbiomass productionatNiconcentrationsofupto150ppm,IBT-Ishowed somewhathigherbiomassproductioncomparedtotheother twoisolatesat200ppm(Table4).

Effects ofdifferent cadmiumconcentrationsonbiomass productionwerestudiedusingthethreemosttolerant Tricho-dermaisolates.Themaximumbiomassvaluewasrecordedfor IBT-II(523.70mg),anditwassignificantlyhigherthanthe val-uesobtainedforUBT-18(147.03mg)andMT-4(147.01mg)ata Cdconcentrationof40ppm(Table5).Withafurtherincrease

Table4–BiomassproductionoftheTrichodermaisolatesunderdifferentconcentrationofnickel.

Isolates Biomassproduction(mg)

Concentration(ppm)

0ppm 40ppm 60ppm 100ppm 150ppm 200ppm

MT-4 240.67def _256.67cde _225.67efg _223.00defg _200.33fgh _165.33h

UBT-18 321.67ab _359.00a _323.33ab _303.33bc _269.33cd _166.67h

IBT-I 215.33efgh _225.00defg _210.33efgh _196.33fgh _181.67gh _177.33gh

*ValuesfollowedbydifferentlettersdiffersignificantlyaccordingtoDuncan’smultiplerangetestatp=0.05.

Table5–BiomassproductionoftheTrichodermaisolatesunderdifferentconcentrationofcadmium.

Isolate Biomassproduction(mg)

Concentration(ppm)

0ppm 40ppm 60ppm 100ppm 150ppm 200ppm

MT-4 240.34d _147.01e _120.34efg _110.34efg _100.36efgh _60.34h

UBT-18 269.70d _147.03e _131.70ef _123.70efg _86.70fgh _79.70gh

IBT-II 577.03a _523.70b _453.70c _440.37c _410.37c _240.36d

*ValuesfollowedbydifferentlettersdiffersignificantlyaccordingtoDuncan’smultiplerangetestatp=0.05.

inthecadmiumconcentration,biomassproductionwas sig-nificantly reduced, except that IBT-II showed insignificant variations in biomass production at cadmium concentra-tionsofupto150ppm.IsolateMT-4(165.33mg)andUBT-18 (166.67mg) exhibited insignificant variations between each otherinbiomassproductionuponexposuretocadmium tox-icity(upto200ppm).

Significant reductions in microbial biomass and soil respirationhavebeenfoundinmetal-contaminatedsoils com-pared to uncontaminated ones.28–30 _{Optimum biosorption} conditionsdependonpH,biomassofthemicroorganism, con-tact time and temperature. The Langmuir, Freundlich and Dubinin–Radushkevichmodel,whichdescribesthe biosorp-tionisothermofametalion,hasindicatedthatbiosorption ofcadmiumbyHylocomiumsplendensbiomassoccursthrough chemicalionexchange.31_{Themainfunctionalgroups} respon-sible for a biosorption process are hydroxyls, carbonyls, carboxyls,sulfonates,amides,imidazoles,phosphonates,and phosphodiestergroupsasestablishedbyPradhanetal.32_and Volesky.33_{SomeofthesegroupsarepresentinTrichodermasp.} biomassandmayinteractwiththemetalions.Ithasalsobeen reportedthatbindingofNi(II)tobiopolymersoccursmainlyin thepeptidoglycanlayerofthecellsurface.34

Theminimuminhibitoryconcentrationsofnickeland cad-mium requiredfor50% ofgrowth inhibition(MIC50)ofthe

isolates were computed, and the results are presented in

Table 6. The highestminimum inhibitory concentration of

nickelwascalculatedforIBT-I(1884.93ppm),followedbyMT-4 (638.90ppm),whereasthehighestMIC50ofcadmiumwas

cal-culatedforIBT-II(227.92ppm),followedbyMT-4(71.16ppm). Determinationofresidualnickelintheculturebrothafter harvestingmycelialbiomassfromthemetal-amendedmedia revealedtotalremovalofnickelbytheincreasedbiomassof theTrichodermaisolatesatNiconcentrationsofupto40ppm, followed by an increase in residual nickel and a decrease in biomass production at higher nickel concentrations in the medium.Amongtheisolates,IBT-Iwas mostpotentin biosorptionofnickel,followedbyUBT-18andMT-4(Fig.5).The resultsareinagreementwiththefindingsofSarkaretal.,26 who recorded90.2%removalofNifroma50ppm-amended culture broth by T. harzianum after seven days of growth, beyondthat,therewasnoincreaseinmetaluptake.Theuseof asolid-phaseextractionprocesstodeterminebiosorptionof heavymetalsshowedthat0.59␮gofnickelcouldberemoved byAspergillusfumigatusfromaliterofpollutedwater.35

The trend of cadmium biosorption was quite different, so thatthe increasingcadmium concentrationsresultedin decreasingbiomassproductionbyallofthetestisolatesand positivelycorrelatedwithincreasedresidualcadmiuminthe culturebroth(Fig.6).

Ithasbeensuggestedthat metaluptakebyT.harzianum ishighlypH-andtemperature-dependentandthemaximum metaluptaketakesplaceatpH4.36,37_{AtpHvaluesabove7,} metaluptakeisreducedasmetalsexistashydroxidecolloids

Table6–MinimuminhibitoryconcentrationsofnickelandcadmiumfortheTrichodermaisolates.

Nickel Cadmium

Isolate Linearequation R2 _{MIC50(ppm) Isolate Linearequation R}2 _MIC50(ppm)

MT-4 Y=46.77x−81.21 0.896 638.90 MT-4 Y=43.52x−30.61 0.886 71.16

UBT-18 Y=73.07x−132.4 0.834 313.49 UBT-18 Y=36.76x−14.66 0.934 57.41 IBT-I Y=32.22x−55.53 0.992 1884.93 IBT-II Y=56.18x−82.46 0.768 227.92

-2 0 2 4 6 8 10 0 50 100 150 200 250 300 350 400 120 100 80 60 40 20 0 Residual nickel in culture broth (µ g/ml) Biomass dry wt. (mg ) Nickel concentration (ppm)

Biomass (MT-4) Biomass (UBT-18) Biomass (IBT-I)

Ni Conc-(MT-4) Ni Conc-(UBT-18) Ni Conc-(IBT-I)

Fig.5–BiosorptionofnickelinamendedmediabyTrichodermaisolates.

0 1 2 3 4 5 6 0 100 200 300 400 500 600 700 120 100 80 60 40 20 0 Residual cadmium in culture broth (µ g/ml ) Dry myceli al b iomass (mg ) Cadmium concentration (ppm)

Biomass (UBT-18) Biomass (IBT-II) Biomass (MT-4)

Cd Conc-(MT-4) Cd Conc-(UBT-18) Cd Conc-(IBT-II)

Fig.6–BiosorptionofcadmiuminamendedmediabyTrichodermaisolates.

andprecipitate atalkalinepHdue toosmoticchangesand ahydrolyzing effect,38,39 _{thusresultinginadecrease inthe} sorption rate.40 _{As biomass ofT. harzianum increased, the} pH of the medium was shown to become more acidic41_; however,belowpH4biomassproductionwasreduced and subsequentlytheresidualmetalconcentrationincreased.Low absorption ofheavy metals at low pH is attributedto the

competitionbetweenthehydrogenionandthemetalionat thesorptionsite.42

Theresultsobtainedwhilestudyinggerminationofconidia oftheTrichodermaisolatesundernickelandcadmiumstress conditionsrevealedthattheisolatesdifferedsignificantlyin their responses tofungistatic effects(Table 7). Under non-amended conditions,asignificantlylower germinationrate

Table7–EffectoffungistasisonconidialgerminationoftheTrichodermaisolatesundernickelandcadmiumstressed condition.

Germinationofconidia(%)

Nickel Cadmium

Isolate 0ppm 50ppm 100ppm 150ppm Isolate 0ppm 50ppm 100ppm 150ppm

MT-4 91.73ab _89.09c _81.65ef _79.99f _{MT-4 88.37}b _68.33cde _63.22ef _57.04f UBT-18 88.23c _85.14d _79.18f _75.81g _{UBT-18 96.10}a _73.70c _69.36cde _66.83de IBT-I 93.73a _89.67bc _83.00de _81.31ef _{IBT-II 97.30}a _73.53cd _70.83cd _50.59g ValuesfollowedbydifferentlettersdiffersignificantlyaccordingtoDuncan’smultiplerangetestatp=0.05.

79 80 81 82 83 84 85 86 87 88 IBT-I UBT-18 MT-4 Ger mi n a tion of con idia (% )

Fig.7–FungistasisofTrichodermaisolatesirrespectiveof nickelgradient.

wasshownbyUBT-18(88.23%)comparedtoIBT-IandMT-4 (93.73and91.73%,respectively).Thedifferentlevelsofnickel stressalsohadsignificanteffectsonsporegermination; how-ever,thevariationintheinteractioneffectwiththeTrichoderma isolateswasnon-significant.Irrespectiveofthenickel concen-tration,IBT-Iwasmostresistanttofungistaticeffects(86.93% conidial germination),followed by MT-4 and UBT-18 (85.61 and82.09%conidialgermination,respectively)(Fig.7).Inthe absenceofcadmium,IBT-IIexhibitedthehighestgermination rate(97.30%),whichsignificantlydifferedfromthatofMT-4 (88.37%).Withthe increase inthe cadmiumconcentration, germinationwassignificantlyaffected,particularlyinthecase ofIBT-II,indicatingthattheisolatewasverysensitivetothe fungistaticeffect.UBT-18 wasfoundtobemosttolerantto thefungistaticeffect,evenatahighercadmiumstresslevel (66.83%conidialgermination),anditsgerminationratewas significantlydifferentfromthatofMT-4(57.04%).Irrespective ofthecadmium concentration,UBT-18 showedthe highest resistancetothefungistaticeffect(76.50%conidial germina-tion),followedbyIBT-IIandMT-4(73.06and69.24%conidial germination,respectively)(Fig.8).Partialannulmentofsoil fungistasishasasignificantimpactonsurvivaland popula-tiondynamicsofTrichodermaandGliocladiuminsoil.43_Royand Pan44_{reportedthatgammairradiationsignificantlyincreased} phialosporeandchlamydosporegerminationratesinmutants ofT.harzianumandGliocladiumvirenscomparedtotheirwild types.

Inconclusion,thepresentinvestigationhighlightsthe sig-nificanceofTrichodermaspp.aspotentialmetalbiosorbents. Fourisolatesobtainedinthisstudy,viz.,MT-4,UBT-18,IBT-I, andIBT-II,canbeexploitedaspotentbioremediationagents innickel-andcadmium-pollutedagriculturalfields.

64 66 68 70 72 74 76 78 IBT-II UBT-18 MT-4 Ger mi n a tion of con idia (% )

Fig.8–FungistasisofTrichodermaisolatesirrespectiveof

cadmiumgradient.

Conflicts

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