EnvironmentAsiachemical synthetic dye are an important problem in the northeast of Thailand, especially in Chonabot district, Khon Kaen province (Mahachai et al., 2009), where wastewater

Removal of Lead from Wastewater Contaminated with Chemical Synthetic Dye by Aspergillus terreus

Lamyai Neeratanaphan a, b, Sirirat Dechmon a, Prasart Phonimdaeng c, Prodpran Khamon d and Somsak Intamat b, e

a Department of Environmental Science, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailandb Genetics and Environmental Toxicology (GET) Research Group, Khon Kaen University

c Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailandd Faculty of Science and Industrial Technology Prince of Songkla University, Suratthani Campus

e Thatphanom Crownprince Hospital

Abstract

Novel isolatedmicroorganismshavebeendemonstrated toefficiently remove lead fromwastewatercontaminatedwithchemical syntheticdye. In this study, thephysicalandchemicalparametersofwastewater samples (includingPbconcentrations)wereanalyzedbeforeandaftertreatmentwithmicroorganisms.ThehighestPbconcentrationdetectedinwastewaterwas0.788mg/l.InvestigationsofthePbtoleranceandremovalcapacitiesofmicroorganismstrainsisolatedfromthewastewatersedimentresultedintheselectionofthreefungalisolates(F102,F203andF302).Interestingly,isolateF203hadaPbtoleranceofupto100mg/l.UsingDNAbarcodingandmorphologicalcharacteristics,fungalisolateF203wasidentifiedasAspergillus terreus.WastewatercharacteristicsbeforetreatmentincludedagrayishblackcolorwithpH,TDS,BOD,CODandPbconcentrationshigherthantheThailandstandardvalues.WastewaterqualitiesaftertreatmentwithA. terreusshoweddefiniteimprovement;however,thevaluesofcertainparameterswerestillhigherthantheallowedvaluesbasedontheThailandstandard.TheonlyimprovementthatfellwithintheallowedstandardwasthePbconcentration. Next, A. terreuswasusedforPbadsorptioninwastewaterwithaninitialPbconcentrationof0.788mg/lattimepointscorrespondingto0,24,48,72,96,120,144and168hofincubation.TheresultsshowedthatA. terreuscouldadsorbandremovehigheramountsofPbfromwastewaterthantheotherfungalisolates.TimecourseadsorptionanalysisshowedtheremainingPbconcentrationsas0.788,0.213,0.162,0.117,0.100,0.066,0.042and0.032mg/l,respectively;thepercentageofPbremovalcouldbeestimatedas0,72.97,79.44,85.15,87.31,91.62,94.67and95.94%,respectively.Inconclusion,A. terreuspossessedtheabilitytoadsorbupto96%ofPbfromchemicalsyntheticdyewithin168h.Thus,A. terreus might besuitableforadaptationanduseinPbtreatment.

Keywords:leadremoval;fungi;wastewater;chemicalsyntheticdye;Aspergillus terreus

1. Introduction

Heavymetals released into the environment bytechnological activities pose significant threats to natural ecosystems and public health due to their toxicity, bioaccumulation, non-biodegradation and persistence in the food chain (Wang et al., 2006; Chojnacka,2010;Sousaet al.,2010;Mudhooet al.,2012).Thus,excessheavymetalsmustberemovedfromwastewater before beingdischarged into the aquaticenvironment.As a specificwastewater contaminant,chemical synthetic dye are an important problem in the northeast ofThailand, especially inChonabotdistrict,KhonKaenprovince(Mahachaiet al.,2009),wherewastewater from dyed silk productionwithhighlead(Pb)concentrationshasbeendischargedintotheaquaticenvironment (VoleskyandHolan,1995).Wastewatersfromdyeingoperationsarecharacterized by color caused by both organic and inorganic

compoundscontainingPb.Pbishighlytoxictohumans,causingdamage to thenervoussystem, reproductivesystem,andbloodcirculationsystem;additionally,Pbis also poisonous to other organisms (Cruz-Olivareset al., 2013).Therefore, it is necessary to eradicatetheproblem(Ahalyaet al.,2003).Thereareseveralmethodsforwastewatertreatmentthatrelyonphysical, chemical or biological processes. Each treatment regimenhascertainadvantagesandlimitations.Physicalandchemicalprocesseshavebeenshowntobethemosteffectiveprocesses.Nevertheless,inpractice,thelackofproperproceduresrenderstheprocessdifficult,withahighcostoftreatment.Hence,biologicalprocessesare a better alternative choice.These techniques arefeasibleforPbremovalduetotheirgoodperformance,convenientmethods and low cost compared to thephysical andchemicalmethods, although the abilityof organisms to removePb has not been optimized(Montazer-Rahmatiet al.,2011).Microorganismsare

The international journal published by the Thai Society of Higher Education Institutes on EnvironmentEnvironmentAsia

Genotoxicity Assessment of Mercuric Chloride in the Marine Fish Therapon jaruba

Nagarajan Nagarani, Arumugam Kuppusamy Kumaraguru, Velmurugan Janaki Deviand Chandrasekaran Archana Devi

Center for Marine and Coastal Studies, School of Energy, Environment and Natural Resources,Madurai Kamaraj University, Madurai-625021, India

Abstract

The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysisby Micronucleus (MN) test in fish erythrocytes as a biomarker for marine environmental contamination. Micronucleusfrequency baseline in erythrocytes was evaluated in and genotoxic potential of a common chemical was determinedin fish experimentally exposed in aquarium under controlled conditions. Fish (Therapon jaruba) were exposed for 96hrs to a single heavy metal (mercuric chloride). Chromosomal damage was determined as micronuclei frequency infish erythrocytes. Significant increase in MN frequency was observed in erythrocytes of fish exposed to mercuricchloride. Concentration of 0.25 ppm induced the highest MN frequency (2.95 micronucleated cells/1000 cells comparedto 1 MNcell/1000 cells in control animals). The study revealed that micronucleus test, as an index of cumulativeexposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions.

Keywords: genotoxicity; mercuric chloride; micronucleus

Available online at www.tshe.org/EAEnvironmentAsia 2 (2009) 50-54

1. Introduction

In India, about 200 tons of mercury and itscompounds are introduced into the environmentannually as effluents from industries (Saffi, 1981).Mercuric chloride has been used in agriculture as afungicide, in medicine as a topical antiseptic anddisinfectant, and in chemistry as an intermediate inthe production of other mercury compounds. Thecontamination of aquatic ecosystems by heavymetals and pesticides has gained increasing attentionin recent decades. Chronic exposure to andaccumulation of these chemicals in aquatic biotacan result in tissue burdens that produce adverseeffects not only in the directly exposed organisms,but also in human beings.

Fish provides a suitable model for monitoringaquatic genotoxicity and wastewater qualitybecause of its ability to metabolize xenobiotics andaccumulated pollutants. A micronucleus assay hasbeen used successfully in several species (De Flora,et al., 1993, Al-Sabti and Metcalfe, 1995). Themicronucleus (MN) test has been developedtogether with DNA-unwinding assays asperspective methods for mass monitoring ofclastogenicity and genotoxicity in fish and mussels(Dailianis et al., 2003).

The MN tests have been successfully used asa measure of genotoxic stress in fish, under both

laboratory and field conditions. In 2006 Soumendraet al., made an attempt to detect genetic biomarkersin two fish species, Labeo bata and Oreochromismossambica, by MN and binucleate (BN)erythrocytes in the gill and kidney erythrocytesexposed to thermal power plant discharge atTitagarh Thermal Power Plant, Kolkata, India.

The present study was conducted to determinethe acute genotoxicity of the heavy metal compoundHgCl2 in static systems. Mercuric chloride is toxic,solvable in water hence it can penetrate the aquaticanimals. Mutagenic studies with native fish speciesrepresent an important effort in determining thepotential effects of toxic agents. This study wascarried out to evaluate the use of the micronucleustest (MN) for the estimation of aquatic pollutionusing marine edible fish under lab conditions.

2. Materials and methods

2.1. Sample Collection

The fish species selected for the present studywas collected from Pudhumadam coast of Gulf ofMannar, Southeast Coast of India. Theraponjarbua belongs to the order Perciformes of thefamily Theraponidae. The fish species, Theraponjarbua (6-6.3 cm in length and 4-4.25 g in weight)was selected for the detection of genotoxic effect

Available online at www.tshe.org/EAEnvironmentAsia 8(2) (2015) 45-55

46

an accepted alternative, and there has been a great deal ofinterestintheiradoptionforwastewatertreatment.Manymicroorganismshavebeenexaminedfor theirbiosorption properties, and different types of biomasses have shown high levels ofmetal uptake (Merroun et al.,2001;Bermudezet al.,2012;Colinet al.,2012).Microorganisms possess different absorption anddegradation capabilities depending on several factors, suchasionexchange,adsorption/adsorption,enzymesystems, and control of genes involved in adsorption (Vijayaraghavanet al.,2004).Microbialspeciesthathave been applied forwastewater treatment includeBacillussp.(Nourbakhshet al.,2002),Streptomyces rimosus (Selatnia et al., 2004), Pseudomonas aeruginosa(Gabret al.,2008),Aspergillus flavus(AkarandTunali,2006),Botrytis cinerea(AkarandTunali,2005),Mucor rouxii (Yan andViraraghavan, 2003)and Neurospora crassa (Kiranet al.,2005).Several molecular techniqueshavebeenused to identify thespecies of isolated microorganisms and their genetic relationships.Forexample,molecularidentificationoffungithroughDNAbarcodinghasbecomeanintegratedandessentialpartoffungalresearchandhasprovidednewinsightsintothediversityofmanydifferentgroupsof fungi (Horton andBruns, 2001;Anderson and Cairney,2004;ChaseandFay,2009;Seifert,2009). In thiswork,westudiedwastewaterquality, thelevel of Pb contamination, and the bioremediation of Pb inwastewater usingmicroorganisms isolatedfromwastewatersedimentsandidentifiedthespeciesofmicroorganismsforfurtheruseinthetreatmentofwastewatercontaminatedwithchemicalsyntheticdye.

2. Materials and Methods

2.1. Sampling sites

WastewatercontaminatedwithchemicalsyntheticdyeisanimportantprobleminthenortheastofThailand.

Therearemanyhumancommunitieslivingwithinthecatchmentareawherethewastewaterissituated,withsmallwaterwaysrunningfromhighelevationatthetopoftheplateautolowerareas.Thesewaterwayscombine and eventually join the reservoir.Wastewater and sediment sampleswere collected in theChonabot districtoftheKhonKaenprovinceofThailand(Fig.1).

2.2. Wastewater sampling and analysis

Wastewatersampleswerecollectedfromthesmallwaterways prior to their joining the reservoir in theChonnabot district,KhonKaen province,Thailand(Fig.1).This isnormalwaterways that receivebothwastewaterandrainwater.Wastewatersweredirectlydischarge fromdyed silk activitieswithout furthertreatment.Thephysical and chemical parameters ofthewastewatersamples(Table1)wereanalyzedbeforeandaftertreatmentwiththeeffectivemicroorganisms.Pb concentrations in thewastewater sampleswere determined using a modelAnalyst 300 atomic absorptionspectrometer(AAS)(Correiaet al.,2003).

2.3. Microorganism screening and species identification

Themicroorganismstrainswereisolatedfromthesedimentofwastewatercontaminatedwithchemicalsynthetic dye thatwasobserved to have thehighestPb concentration (0.788mg/l).Themedium usedfor isolationwasnutrient agar (NA) (0.5%peptone,0.5%NaCl,0.2%yeastextract,0.2%beefextractand1.5%agar,pH7.0,allw/v)containing1mg/lofPb(II)acetate.NAwas used for primary isolation only.Atotalof8fungalisolatesand25bacterialisolateswerecollectedaftertestingtodeterminetheirPbresistance.ThetestingwasconductedinNAsupplementedwithPb concentrationlevelsof10,100,500,1000,1200,1400,1600and1800mg/l.Threebacterial isolates (B206,B308andB405)andthreefungalisolates(F102,F203

Table 1.Methods used for analysis of physical and chemical parameters ofwastewater contaminatedwith chemical synthetic dye

Physical and chemical parameters AnalysismethodsColor ThecolorchartHTML-RGBTurbidity Turbiditymeter,model966,MettlerToledoTotalDissolvedSolid;TDS ECmeter,modelCH-8603,MettlerToledopH pHmeter,modelEcoScanpH5,EutechElectricalConductivity;EC ECmeter,modelCH-8603,MettlerToledoBiochemicalOxygenDemand;BOD AzideModificationmethod(APHA,2005)ChemicalOxygenDemand;COD AzideModificationmethod(APHA,2005)Pb concentrations AAS,modelAanalyst300,Perkin-Elmer(Correiaet al.,2003).

L. Neeratanaphan et al. / EnvironmentAsia 8(2) (2015) 45-55

47

andF302)exhibitedgrowthinthepresenceofhighPbconcentrations,althoughthethreefungiwereobservedtobemoretolerantofPbthanthebacteria.Themosteffective fungiwas identified by gene sequencing modifiedinaccordingtoGostincaret al. (2012)andspecific primers (50-AATGCAAAAACTGCATA-ACCAC-30 and 50-GGGATAGGCTTACCTTC-GAAG-30).Theacquiredsequenceswerecomparedin dendrogram construction using theMEGA5program (Tamura et al., 2011) and comparisons togeneticsimilarityvaluesattheCollaborativeUnitof Biological Science andBiotechnology ofMahidol University and Osaka University.Additionally, morphological characteristics were examined by scanning electronmicroscopy (SEM) using aLEO1450VP apparatus. Specimenswere prepared by fixation, dehydration and coatingwith a very thin layerofcarbon(Lymanet al.,1990).

2.4. Pb resistance and absorption/adsorption of three fungal isolates in culture media

ThehighestlevelsofPbresistanceswerefoundin3fungalisolates(F102,F203andF302).Theisolateswereculturedinpotatodextroseagar(PDA)usinga

cork borer to remove colonies onto newPDAagarplates,wheretheywereincubatedat30°Cfor7days.Then,asterilespreaderwasusedtoscrapethesporesofthefunguswithdeionizeddistilledwaterintoapetri dish.The sporeswere filtered and counted using a hemacytometer.A0.2ml spore suspension from the3fungalisolates(5×106spores/ml)(Chatterjeeet al.,2010)wasinoculatedintoa250mlErlenmeyerflask,incubatedat30°Conanorbitalshaker(120rpm)for72handwashedwithdeionizeddistilledwater(Tsekova et al., 2010).Three fungal isolates from the fungal preparation process were used as living cells by suspending 0.35 g dry cellweight (the dryweightof cellsobtainedbybakingat70°Cuntil a constantweightwas obtained) intomalt yeast extract broth(MYB)mixedwithPbconcentrationsof50,60,70,80,90,100,110and120mg/linavolumeof100ml. Triplicateexperimentswereincubatedonashakerat30°Cwithagitation(120rpm)(Majumdaret al.,2010).The sampleswere collected at 24, 48, 72, 96, 120,144and168h.Thesampleswerecentrifugedat5,000rpmat4°Cfor10min,andPbconcentrationsinthe supernatant were determined by AAS model AAnalyze 300 (Perkin-Elmer). Fungal growth aftertreatmentwithPb inMYBwasdeterminedforeach

Figure1.OverviewoftheChonabotdistrictarea,Thelocationofthesamplingcollectionsiteislabeledwithastar.

3

Figure 1. Overview of the Chonabot district area, The location of the sampling collection site is labeled with a star.


The microorganism strains were isolated from the sediment of wastewater contaminated with chemical synthetic dye that was observed to have the highest Pb concentration (0.788 mg/l). The medium used for isolation was nutrient agar (NA) (0.5% peptone, 0.5% NaCl, 0.2% yeast extract, 0.2% beef extract and 1.5% agar, pH 7.0, all w/v) containing 1 mg/l of Pb(II)acetate. NA was used for primary isolation only. A total of 8 fungal isolates and 25 bacterial isolates were collected after testing to determine their Pb resistance. The testing was conducted in NA supplemented with Pb concentration levels of 10, 100, 500, 1000, 1200, 1400, 1600 and 1800 mg/l. Three bacterial isolates (B206, B308 and B405) and three fungal isolates (F102, F203 and F302) exhibited growth in the presence of high Pb concentrations, although the three fungi were observed to be more tolerant of Pb than the bacteria. The most effective fungi was identified by gene sequencing modified in according to Gostincar et al. (2012) and specific primers (50-AATGCAAAAACTGCATAACCAC-30 and 50-GGGATAGGCTTACCTTCGAAG-30). The acquired sequences were compared in dendrogram construction using the MEGA5 program (Tamura et al., 2011) and comparisons to genetic similarity values at the Collaborative Unit of Biological Science and Biotechnology of Mahidol University and Osaka University. Additionally, morphological characteristics were examined by scanning electron microscopy (SEM) using a LEO 1450VP apparatus. Specimens were prepared by fixation, dehydration and coating with a very thin layer of carbon (Lyman et al., 1990).

2.4. Pb resistance and absorption/adsorption of three fungal isolates in culture media

The highest levels of Pb resistances were found in 3 fungal isolates (F102, F203 and F302). The isolates were cultured in potato dextrose agar (PDA) using a cork borer to remove colonies onto


48

Pbconcentrationat168h.

2.5. Testing of Pb absorption/adsorption in wastewater contaminated with chemical synthetic dye by fungi

Atotalof0.35gofeachfungalisolatewasmixedwith50mlofwastewatercontaminatedwithchemicalsyntheticdyeinanErlenmeyerflask.TheErlenmeyerflaskswere incubated in a shaker incubator at 30°Cwithagitation(120rpm)(Majumdaret al.,2010),andsampleswerecollectedat0,24,48,72,96,120,144and 168 h.The sampleswere centrifuged at 5,000rpm at 4°C for 10min, and the concentrations oflead in the culture supernatantswere determinedbyAAS.Allmeasurementswereperformedintriplicate. Additionally, fungal growth after treatment with wastewatercontaminatedwithchemicalsyntheticdyewasdeterminedasdescribedbelow.

2.6. Testing of Pb remediation and release of Aspergillus terreus (F203) 2.6.1. Pb absorption/adsorption of A. terreus in wastewater contaminated with chemical synthetic dye A total of 0.35g ofA. terreuswasmixedwith50ml ofwastewater contaminatedwith chemicalsyntheticdyeinanErlenmeyerflask.TheErlenmeyerflaskswereincubatedat30°Conanorbitalshaker(120rpm)for168h.Then,thesampleswerecentrifugedat10,000rpmat4°Cfor15min,andPbconcentrationsin the supernatantswere determined byAAS.All measurements were performed in triplicate. The physical andchemicalparametersof thewastewater contaminated with chemical synthetic dye after treatmentwithA. terreuswereanalyzedasdescribedabove.

2.6.2. Pb release of A. terreus into HCl

AfterPb absorption/adsorption,A. terreusweremixedwith50mlof0.1MHClinanErlenmeyerflask.TheErlenmeyerflaskswereincubatedonashakerat30°Cwithagitation(120rpm)for3h.Then,thesampleswerecentrifugedat10,000rpmat4°Cfor15minandPbconcentrationsinthesupernatantsweredeterminedbyAAS.Allmeasurementswereperformedintriplicate(Majumdaret al.,2010).

2.7. The growth characteristics of A. terreus

SporesuspensionsofA. terreus(5×106spores/ml)wereinoculatedinto100mlofpotatodextrosebroth(PDB)ina250mlErlenmeyerflask.Thesporecultureswereincubatedat30°Cinanorbitalshaker(120rpm),andsampleswerecollectedat0,24,48,72,96,120,144 and168h.At the endof the incubationperiodmyceliawere harvested by filtration,washedwith deionizedwater,driedbysoakingonblottingpaper,cutintosmallpieces,andusedasalivebiomassforstudy.Adeadbiomasswaspreparedbyautoclavingtheflasksat121°Cfor15minbeforeharvestingthemycelia.Themoisture content of the biomasswas determinedbydryingat70°Cuntilaconstantweightwasobtained(Tsekovaet al.,2010).

2.8. Statistical analysis

Thedatawereanalyzedusingdescriptivestatistic.

3. Results and Discussion

3.1. Wastewater quality and Pb concentrations

ThewastewaterqualityandPbconcentrationsareshown inTable 2.Thewastewater before treatment withA. terreus was grayish black in appearance. Additionally, the turbidity, pH,BOD,CODandPb

Table2.Physicalandchemicalparametersofwastewatercontaminatedwithchemicalsyntheticdye

Physical and chemical parametersWastewaterquality

Measurementvalue StandardvalueColor grayish black -Turbidity(FormazinNephelometricUnits;FNU) 183.0 -TDS(mg/l) 6.88 <3.0pH 9.40 5.5-9.0EC(μS/cm) 14.81 -BOD(mg/l) 4,228 <60COD(mg/l) 7,048 <400Pb(mg/l) 0.788 <0.2Remark:Thailand’swastewaterqualitystandard(PollutionControlDepartmentofThailand,2001)


49

concentration were all higher than Thailand’s wastewaterquality standard level (PollutionControlDepartmentofThailand,2001).


Weidentifiedthreebacterialisolates(B206,B308andB405)thatcouldgrowinthepresenceof10mg/lofPbandthreefungalisolates(F102,F203andF302)that couldgrow in thepresence100mg/l ofPb.As fungal isolateswere previously found to bemore durable than thebacterial isolates (data not shown),thefungalisolateswereselectedforfurtherstudyoftheabsorption/adsorptionprocess.Importantly,fungal isolate F203 was the most effective at treating wastewatercontaminatedwithchemicalsyntheticdye. Colonies of fungal isolate F203 are shown inFig.2.Thecoloniescontainedyellowish-brownfibers surrounding cream-colored colonies that had the appearance of cotton on the nutrient agar. The morphologyoffungalisolateF203wasfurtherstudiedusingSEM (Fig. 3).Basedon a dendrogramof thegeneticrelationshipsoffungalisolateF203comparedwith other fungal sequences downloaded from the NationalCenterforBiotechnologyInformation(NCBI)database,fungalisolateF203wasfoundtopossess98%genesequencesimilaritywithA. terreus(Fig.4).

3.3. Pb resistance, absorption/adsorption and fungal growth after treatment with Pb in MYB

WeobservedthatthethreefungalisolatesF102,F203andF302couldgrowinMYBinthepresenceofPbconcentrationsof50,60,70,80,90and100mg/l,butnotwithPbconcentrationsof110and120mg/l.ThePbconcentrationsinMYBaftertreatmentwithF102,F203andF302areshowninFig.5.Thepercentages of Pb absorption/adsorption demonstrated that

fungal isolate F203 adsorbed/absorbed higher concentrations of Pb than fungal isolates F302 andF102.ThepercentagesofPbadsorbed/absorbedwere97.47,95.47,88.97,58.95,18.36and14.76%forF203,96.63,21.87,20.51,16.20,10.44and7.96%forF302,and 83.92, 11.87, 11.89, 9.60, 12.89 and 7.68% forF102,respectively.WhenfungalgrowthwascomparedwithdryweightaftertreatmentwithPbinMYB(Fig.6), fungal isolateF203 exhibitedbetter growth thanF302andF102.

3.4. Pb adsorption/absorption by fungal isolates in wastewater contaminated with chemical synthetic dye

Threefungalisolatesweretestedforadsorption/absorptioninwastewatercontaminatedwithchemicalsyntheticdyeatmultipletimepoints(0,24,48,72,96,120,144and168h).Theresultsshownthatallthreefungal isolates decreased the Pb concentrations inthecontaminatedwastewater.However,lessresidual Pbwas detected in thewastewater incubatedwith fungal isolate F203 comparedwith F302 andF102(Fig.7).

3.5. Fungal growth after treating Pb in wastewater contaminated with chemical synthetic dye

Fungal growthwas compared based on the dryweightsofthefungalisolatesfollowingtreatmentofwastewatercontaminatedwithchemicalsyntheticdyefor0,24,48,72,96,120,144and168h.Theresultsshowed that the dryweights of F102were 0.3516,0.2739, 0.2900, 0.2910, 0.2987, 0.3070, 0.3130 and0.3175g,F203were0.3551,0.3201,0.3508,0.3765,0.3824,0.3897,0.3935and0.4164g;F302were0.3539,0.3210, 0.3414, 0.3776, 0.3821, 0.3900, 0.3982 and0.4131g,respectively(Fig.8).Thus,fungal isolatesF203andF302hadbettergrowththanF102.

Figure2.ColoniesoffungalisolateF203onPDA Figure 3. SEM images showing surfacemorphologies offungalisolateF203

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Figure 2. Colonies of fungal isolate F203 on PDA Figure 3. SEM images showing surface morphologies of fungal isolate F203

Figure 4. Dendrogram of the genetic relationship of F203 compared with other bacteria from the NCBI database


Three fungal isolates were tested for adsorption/absorption in wastewater contaminated with chemical synthetic dye at multiple time points (0, 24, 48, 72, 96, 120, 144 and 168 h). The results shown that all three fungal isolates decreased the Pb concentrations in the contaminated wastewater. However, less residual Pb was detected in the wastewater incubated with fungal isolate F203 compared with F302 and F102 (Fig. 7).

6






50

Figure4.DendrogramofthegeneticrelationshipofF203comparedwithotherbacteriafromtheNCBIdatabase

6





Figure5.ThepercentageofPbadsorbed/absorbedaftertreatmentwithF102,F203andF302

7

Figure 5. The percentage of Pb adsorbed/absorbed after treatment with F102, F203 and F302

Figure 6. The dry weight of F102, F203 and F302 grown in the presence of Pb concentrations of 50, 60, 70, 80, 90 and 100 mg/l for 168 h in MYB


51

7

Figure 5. The percentage of Pb adsorbed/absorbed after treatment with F102, F203 and F302

Figure 6. The dry weight of F102, F203 and F302 grown in the presence of Pb concentrations of 50, 60, 70, 80, 90 and 100 mg/l for 168 h in MYB

Figure6.ThedryweightofF102,F203andF302growninthepresenceofPbconcentrationsof50,60,70,80,90and100mg/lfor168hinMYB

Figure7.ResidualPbinwastewatercontaminatedwithchemicalsyntheticdyeaftertreatmentwithF102,F203andF302at0,24,48,72,96,120,144and168h

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Figure 7. Residual Pb in wastewater contaminated with chemical synthetic dye after treatment with F102, F203 and F302 at 0, 24, 48, 72, 96, 120, 144 and 168 h


Fungal growth was compared based on the dry weights of the fungal isolates following treatment of wastewater contaminated with chemical synthetic dye for 0, 24, 48, 72, 96, 120, 144 and 168 h. The results showed that the dry weights of F102 were 0.3516, 0.2739, 0.2900, 0.2910, 0.2987, 0.3070, 0.3130 and 0.3175 g, F203 were 0.3551, 0.3201, 0.3508, 0.3765, 0.3824, 0.3897, 0.3935 and 0.4164 g; F302 were 0.3539, 0.3210, 0.3414, 0.3776, 0.3821, 0.3900, 0.3982 and 0.4131 g, respectively (Fig. 8). Thus, fungal isolates F203 and F302 had better growth than F102.

Figure 8. Dry weights of F102, F203 and F302 after treatment with Pb in wastewater contaminated with chemical synthetic dye after 0, 24, 48, 72, 96, 120, 144 and 168 h of incubation

3.6. The physical and chemical parameters of waste-water contaminated with chemical synthetic dye after treatment with A. terreus (F203)

Wedemonstrated thatA. terreus is effective for treatingwastewatercontaminatedwithchemicalsyn-theticdye.ThephysicalandchemicalparametersofwastewatersamplesaftertreatmentwithA. terreus are shown inTable3.Thecolorof thewastewateraftertreatmentwasbrown.Importantly,theTDS,pH,BOD

andCODwerehigherthantheThailandstandardvalues,whilethePbconcentrationwaslowerthanthestandard.

3.7. Pb absorption/adsorption and release by A. terreus

ThePbconcentrationinwastewatercontaminatedwith chemical synthetic dye before treatmentwith A. terreuswas 0.788mg/l. Following incubation ofA. terreuswithwastewater for 168h,we tested thePbreleaseinHClfor3h(Fig.9).Theresultsshowed


52

that A. terreusabsorbed0.756mg/l(96%)ofPb,withthetotalamountofPbdesorbedinHClequalto0.702mg/l(89%).

3.8. Growth characteristics of A. terreus

The growth characteristics ofA. terreus werecomparedusingthedryweightsmeasuredfollowing0,24,48,72,96,120,144and168hof incubation(Fig.10).TheresultsshowedthatA. terreuswaswelladapted to environmental conditions in the time frame of0-12h.Then,A. terreus exhibited rapid cell division ataconstantrateduringthe12-72hperiod.Thisphaserepresentedthemaximumgrowthrate,witheachcelldivisioncycletakinganequalamountoftime.Finally,thedryweightofA.terreusdidnotincreaseduringthe72-168htimeperiod.

Inthisstudy,leadwasremovedfromwastewater contaminated with chemical synthetic dye using microbialisolationfromwastewatersedimentwithaproblematicPbcontamination.Threebacterialisolates(B206,B308andB405)grewathighPbconcentrationsofupto10mg/l,andthreefungalisolates(F102,F203andF302)grewatPbhighconcentrationsofupto100mg/l.Theresultsofthisstudyindicatedthatfungicantolerate higher Pb concentration than bacteria, and nativefungicanabsorbPbinwastewatercontaminatedwithchemical syntheticdyedue to the largefibrousstructure that bacteria do not possess.The study offungi used for the absorption/adsorption process byKapoorandViraraghavan(1995)reportedthatcommon filamentousfungicanabsorbheavymetalsfromaque-oussolutions.Inthisstudy,theisolationandselectionofmicroorganisms tolerating Pb concentrations revealed

Figure8.DryweightsofF102,F203andF302aftertreatmentwithPbinwastewatercontaminatedwithchemicalsyntheticdyeafter0,24,48,72,96,120,144and168hofincubation

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Figure 7. Residual Pb in wastewater contaminated with chemical synthetic dye after treatment with F102, F203 and F302 at 0, 24, 48, 72, 96, 120, 144 and 168 h


Fungal growth was compared based on the dry weights of the fungal isolates following treatment of wastewater contaminated with chemical synthetic dye for 0, 24, 48, 72, 96, 120, 144 and 168 h. The results showed that the dry weights of F102 were 0.3516, 0.2739, 0.2900, 0.2910, 0.2987, 0.3070, 0.3130 and 0.3175 g, F203 were 0.3551, 0.3201, 0.3508, 0.3765, 0.3824, 0.3897, 0.3935 and 0.4164 g; F302 were 0.3539, 0.3210, 0.3414, 0.3776, 0.3821, 0.3900, 0.3982 and 0.4131 g, respectively (Fig. 8). Thus, fungal isolates F203 and F302 had better growth than F102.

Figure 8. Dry weights of F102, F203 and F302 after treatment with Pb in wastewater contaminated with chemical synthetic dye after 0, 24, 48, 72, 96, 120, 144 and 168 h of incubation

Table3.PhysicalandchemicalparametersofwastewaterbeforeandaftertreatmentwithA. terreus

Physical and chemical parametersWastewaterquality

before after ThailandStandardColor grayish black brown -Turbidity(FormazinNephelometricUnits;FNU) 183.0 31.6 -TDS(mg/l) 6.88 5.96 <3.0pH 9.40 9.37 5.5-9.0EC(μS/cm) 14.81 11.86 -BOD(mg/l) 4,228 3,190 <60COD(mg/l) 7,048 5,413 <400Pb(mg/l) 0.788 0.032 <0.2Remark:Thailand’swastewaterqualitystandard(PollutionControlDepartmentofThailand,2001)


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threefungal isolates(F102,F203andF302).FungalisolateF203hadaPbtoleranceofupto100mg/l.TheDNAbarcodingtechniqueisfrequentlyusedforfungalidentification.Basedontheanalysisofallofthedata,including themorphological characteristics assessedbySEMandDNAsequencing,thisfungalisolatewasconcludedtobeAspergillus terreus.Thepercentagesofabsorption/adsorptionofPbshowedthatA. terreus couldadsorb/absorbPbwithmuchhigherefficiencythan the other two fungal isolates. Pb adsorption/ absorptionbydifferentfungalspeciesdependsoncellsizeandthepresenceofmorphologicalandfunctionalgroupsthatacttoabsorbPbintothefungalcellwalls.Thefungusabsorbsleadfromtheenvironmentthroughthewallof themycelium,whicheffectivelyabsorbs

dependingonthecompositionofitsfunctionalgroups,includingcarboxylate,phosphate,andaminogroupsinthecellwall.Furthermore,thefungalcellwallcontainsnegativeionsthatareexchangedwiththecationsofPb(Iskandaret al.,2011).A. terreus demonstrated better growthcharacteristicsonMYBcontainingPbcomparedtotheotherfungalisolatesinthisstudy.FungalbiomassalsocontributestoeffectivePbremovalcapability.Thepotentialofafungalbiomasstofunctionasasorbentis partly dependent on the cell-wall fraction of the biomass,whichplaysanimportantroleinthesorptionof heavymetals (Kapoor andViraraghavan, 1995).AfteremployingA. terreusinwastewatertreatment,thewastewaterqualitiesimproved.However,thevaluesofcertainparameterswerestillhigherthanthoseallowed

Figure9.Pbabsorption/adsorptionandreleasebyA. terreus

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3.6. The physical and chemical parameters of wastewater contaminated with chemical synthetic dye after treatment with A. terreus (F203)

We demonstrated that A. terreus is effective for treating wastewater contaminated with chemical synthetic dye. The physical and chemical parameters of wastewater samples after treatment with A. terreus are shown in Table 3. The color of the wastewater after treatment was brown. Importantly, the TDS, pH, BOD and COD were higher than the Thailand standard values, while the Pb concentration was lower than the standard.

Table 3. Physical and chemical parameters of wastewater before and after treatment with A. terreus

Physical and chemical parameters Wastewater quality before after Thailand Standard

Color grayish black brown - Turbidity (Formazin Nephelometric Units; FNU) 183.0 31.6 - TDS (mg/l) 6.88 5.96 <3.0 pH 9.40 9.37 5.5-9.0 EC (S/cm) 14.81 11.86 - BOD (mg/l) 4,228 3,190 < 60 COD (mg/l) 7,048 5,413 < 400 Pb (mg/l) 0.788 0.032 < 0.2

Remark: Thailand’s wastewater quality standard (Pollution Control Department of Thailand, 2001)

3.7. Pb absorption/adsorption and release by A. terreus

The Pb concentration in wastewater contaminated with chemical synthetic dye beforetreatment with A. terreus was 0.788 mg/l. Following incubation of A. terreus with wastewater for 168 h, we tested the Pb release in HCl for 3 h (Fig. 9). The results showed that A. terreus absorbed 0.756 mg/l (96%) of Pb, with the total amount of Pb desorbed in HCl equal to 0.702 mg/l (89%).

Figure 9. Pb absorption/adsorption and release by A. terreus

3.8. Growth characteristics of A. terreus

The growth characteristics of A. terreus were compared using the dry weights measured following 0, 24, 48, 72, 96, 120, 144 and 168 h of incubation (Fig. 10). The results showed that A.terreus was well adapted to environmental conditions in the time frame of 0-12 h. Then, A. terreusexhibited rapid cell division at a constant rate during the 12-72 h period. This phase represented the maximum growth rate, with each cell division cycle taking an equal amount of time. Finally, the dry weight of A. terreus did not increase during the 72-168 h time period.

10

Figure 10. Growth characteristics of A. terreus

In this study, lead was removed from wastewater contaminated with chemical synthetic dye using microbial isolation from wastewater sediment with a problematic Pb contamination. Three bacterial isolates (B206, B308 and B405) grew at high Pb concentrations of up to 10 mg/l, and three fungal isolates (F102, F203 and F302) grew at Pb high concentrations of up to 100 mg/l. The results of this study indicated that fungi can tolerate higher Pb concentration than bacteria, and native fungi can absorb Pb in wastewater contaminated with chemical synthetic dye due to the large fibrous structure that bacteria do not possess. The study of fungi used for the absorption/adsorption process by Kapoor and Viraraghavan (1995) reported that common filamentous fungi can sorb heavy metals from aqueous solutions. In this study, the isolation and selection of microorganisms tolerating Pb concentrations revealed three fungal isolates (F102, F203 and F302). Fungal isolate F203 had a Pb tolerance of up to 100 mg/l. The DNA barcoding technique is frequently used for fungal identification. Based on the analysis of all of the data, including the morphological characteristics assessed by SEM and DNA sequencing, this fungal isolate was concluded to be Aspergillus terreus. The percentages of absorption/adsorption of Pb showed that A. terreus could adsorb/absorb Pb with much higher efficiency than the other two fungal isolates. Pb adsorption/absorption by different fungal species depends on cell size and the presence of morphological and functional groups that act to absorb Pb into the fungal cell walls. The fungus absorbs lead from the environment through the wall of the mycelium, which effectively absorbs depending on the composition of its functional groups, including carboxylate, phosphate, and amino groups in the cell wall. Furthermore, the fungal cell wall contains negative ions that are exchanged with the cations of Pb (Iskandar et al., 2011). A. terreus demonstrated better growth characteristics on MYB containing Pb compared to the other fungal isolates in this study. Fungal biomass also contributes to effective Pb removal capability. The potential of a fungal biomass to function as a sorbent is partly dependent on the cell-wall fraction of the biomass, which plays an important role in the sorption of heavy metals (Kapoor and Viraraghavan, 1995). After employing A. terreus in wastewater treatment, the wastewater qualities improved. However, the values of certain parameters were still higher than those allowed based on Thailand’s wastewater quality standard. The only improvement was the Pb concentration, which was within the allowed standard. A.terreus was found to be the most efficient in this respect and removed up to 96% of Pb from wastewater contaminated with chemical synthetic dye. Moreover, Pb desorption in HCl was up to 89%, indicating that the Pb was degraded by A. terreus. Because increasing amounts of HCl result in increased release of Pb, pH changes may dictate functional changes in the cell (Gupta et al., 2000). Thus, our results showed that A. terreus could adsorb and remove Pb from wastewater contaminated with chemical synthetic dye.

Figure10.GrowthcharacteristicsofA. terreus


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basedonThailand’swastewaterqualitystandard.Theonly improvementwas thePb concentration,whichwaswithintheallowedstandard.A. terreuswasfoundtobe themost efficient in this respect and removedupto96%ofPbfromwastewatercontaminatedwithchemicalsyntheticdye.Moreover,PbdesorptioninHClwasupto89%,indicatingthatthePbwasdegradedbyA. terreus.BecauseincreasingamountsofHClresultin increased release ofPb, pH changesmaydictatefunctional changes in the cell (Gupta et al., 2000).Thus,ourresultsshowedthatA. terreuscouldadsorband removePb fromwastewater contaminatedwithchemicalsyntheticdye.

4. Conclusions

Microorganisms isolated from the sedimentsamples inwastewater contaminatedwith chemicalsyntheticdyeweretestedfortheirPbremovalcapacity. Fungiwere found to bemore tolerant of Pb than bacteria.One of the fungal isolateswas identified by gene sequence analysis and morphological characteristics as Aspergillus terreus.AfteremployingA. terreusinwastewater,thequalitiesofthewastewaterwereimproved;however,theonlyimprovementthatfellwithintheallowedstandardwasthePbconcentration.A. terreushasthecapacitytoremovePbandshowedimprovedgrowthcomparedtothetwootherfungi.A. terreus adsorbedup to 96%ofPb fromwastewatercontaminatedwithchemicalsyntheticdyewithin168h, suggesting thatA. terreusmight be suitable for adaptationanduseinPbtreatment.A. terreus has a high efficiencyandcanbeusedforwastewaterPbtreatment due to its ability to tolerate a Pb concentration of 0.788mg/landashighas100mg/linMYB.Therefore, A. terreus is considered to be a good target for the absorption/adsorption of Pb contamination for wastewater treatment.These resultsmay be useful for research into the amelioration of hazardous Pb-contaminatedareasworldwide.

Acknowledgments

This research was funded by the Genetics and EnvironmentalToxicologyResearchGroup, Faculty of Science,KhonKaenUniversity.

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Received 18 December 2014Accepted 12 March 2015

Correspondence toAssistantProfessorDr.LamyaiNeeratanaphanDepartmentofEnvironmentalScience,FacultyofScience,KhonKaenUniversity,KhonKaen40002ThailandEmail:[email protected]


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EnvironmentAsiachemical synthetic dye are an important problem in the northeast of Thailand, especially in Chonabot district, Khon Kaen province (Mahachai et al., 2009), where wastewater