黑曲霉的固定化

Removal of heavy metals from industrial wastewater by free and immobilized cells of Aspergillus nigerK.Tsekova a ,*,D.Todorova a ,S.Ganeva ba Stephan Angeloff Institute of Microbiology e Bulgarian Academy of Sciences,Acad.G.Bonchev,Str.,bl.26,1113So fia,Bulgaria bFaculty of Chemistry e So fia University,1164So fia,Bulgariaa r t i c l e i n f oArticle history:Received 24March 2010Received in revised form 5May 2010Accepted 9May 2010Available online 7June 2010Keywords:Biosorption Heavy metals Wastewater Immobilization Aspergillus nigera b s t r a c tAspergillus niger ,strain B 77,was immobilized by inclusion in two different polymers:polyvinyl e alcohol hydrogel (PVA)and Ca e alginate.The biomass/polymer matrices were formed into equal size unites of the cubes and spheres,and the resulting biomass/polymer matrices were used to remove heavy metals (Cu 2þ,Mn 2þ,Zn 2þ,Ni 2þ,Fe 3þ,Pb 2þ,Cd 2þ)from wastewater in shake flask experiments.Total biosorption capacities of the biosorbents were in the following order:free cells (33.3mg/g)<PVA e biomass (39.8mg/g)<Ca alginate e biomass (44.6mg/g).The metal removal ef ficiencies of the beads Ca alginate e biomass were 96.2%for Cd 2þ;90.0%for Pb 2þ;80.0%for Fe 3þ;72.8%for Cu 2þ;55.4%for Zn 2þ;54.4%for Ni 2þand 52.3%for Mn 2þ,while the removal ef ficiencies of cubes PVA e biomass for the same heavy metals ions were:95.0%;88.0%;80.0%;67.1%;58.5%;48.9%and 44.6%,respectively.The results obtained from these experiments,were compared with those using dispersed biomass as a sorbent.Promising results were obtained in the laboratory,as effective metal removals were observed.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionThe increase use of heavy metals in difference industrial activ-ities causes their existence in wastewater.The discharge of heavy metal ions in industrial ef fluent is of great concern because of their toxic effect on living species,even at very low concentrations.The detoxi fication of metal ions from industrial ef fluent using biosorption processes is an area of extensive research during the last years (Volesky and Holan,1995;Tripathi et al.,2007).Biosorption is an innovative and low cost effective method for the removal of toxic substances from wastewaters (Zouboulis et al.,2003;Silva et al.,2009;Chatterjee et al.,2010).Fungi are recognized for their superior ability to produce a large variety of extra cellular proteins,organic acids,enzymes and other metabolites,and their waste biomass may be used as effective biosorbent for removal,reduction and detoxi fication of industrial ef fluents ingredients (Gupta and Mukerji,2001;Christian et al.,2005).Various fungal species under the genus Aspergillus ,Penicil-lium and Rhizopus have been shown to be effective in biosorption of heavy metals from polluted ef fluents both as immobilized cells and in the mobilized state (Kapoor and Viraraghvan,1995;Leitão,2009;Tsekova et al.,2010a,b ).During the last decade many research works have been focused on the development of immobilized systems of microorganisms into polymeric matrices suitable for metal ions uptake applications (Zouboulis et al.,2003;Tsekova et al.,2008;Mata et al.,2009).Biosorption has been considered as a promising technology for the removal of low levels of toxic metals from industrials ef fluents and natural waters.In view of potential applications in remediation of heavy metals from aqueous solutions the immobilization of the biomass is generally necessary.Immobilized cells are usually easier to handle,require less complex separation systems,allow a high biomass density to be maintained and provide a greater opportu-nity for reuse and recovery.Despite the current interest in microbial detoxi fication of ef fluents,relatively little work has been concerned with charac-terization of metal uptake by filamentous fungi,particularly when the heavy metals present at different and low concentrations.The purpose of this study was to determine the ability of Aspergillus niger (free and immobilized biomass)to remove toxic metals from an industrial wastewater by batch system.2.Materials and methods 2.1.Sample collection siteWastewater samples were from copper production factory in Pirdop,Bulgaria.The samples were collected for a relative long*Corresponding author.Tel.:þ35929793167;fax:þ35928700109.E-mail addresses:ktsekova@microbio.bas.bg (K.Tsekova),sganeva@chem.uni-so fia.bg (S.Ganeva).Contents lists available at ScienceDirectInternational Biodeterioration &Biodegradationjou rn al homepage:/locate/ibiod0964-8305/$e see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.ibiod.2010.05.003International Biodeterioration &Biodegradation 64(2010)447e 451period of time(April e November2009)in high e grade plastic bottles of1.5l capacity and stored in a refrigerator at4 C.From all taken wastes a representative sample was prepared mixing150ml from each separate bottle.2.2.Determination of heavy metal concentrationsThe initial concentration of the metals in the wastewater and samples after biosorption treatment were determined using an Atomic Absorption Spectrophotometer Perkin Elmer Analyst400, air-acetyleneflame.Cadmium and lead after biosorption were determined using electrothermal atomic absorption spectrom-eter Zeeman Perkin Elmer3030,pyrolytically coated graphite tubes and optimized temperature program for modifier freeETAAS.2.3.Preparation of biosorbents2.3.1.Microorganisms,medium and cultivationThe mutant strain A.niger B77(A.niger),an industrial producer of glucoamylase(N 65/1980,National Bank of Industrial Micro-organisms and Cell Cultures,Bulgaria)was used in this study. Spores from established culture(6e7days old)incubated on potato glucose agar slants at30 C were used for the preparation of inocula.The liquid growth medium consisted of(g/L):glucose30 and corn steep liqueur45;pH adjusted to4.8.Cultivation of A.niger was carried out in500e ml Erlenmeyer flasks with75ml growth medium on a rotary shaker at30 C.After 48h of cultivation,the mycelium was harvested byfiltration from the medium,washed twice with distilled water and stored at4 C until use.The dry weight of the free fungal biomass(FC)was determined after drying at85 C for48h.2.3.2.Immobilizing materials and techniquesPoly(vinyl)alcohol(PVA)hydrogel was obtained as describe earlier(Tsekova et al.,2008).The PVA e hydrogel used for immo-bilization was cut into small pieces(4Â4Â4mm in size).They were washed with distilled water and than submerged in500-ml Erlenmeyerflasks containing75ml growth medium(previously autoclaved for20min at120 C).The carrier pieces were inoculated with10ml A.niger spores suspension(1Â106/ml)were cultivated as described above.After48h of incubation,the PVA e immobi-lized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a bio-sorbent.The dry weight of PVA e biomass was determined as described for FC.Method described by El e Naggar et al.(2006)was used to prepare Ca alginate beads the spore suspension(10ml,1Â106/ml) was mixed with Na e alginate prior to its stabilization.Beads of approximately4mm diameter were obtained by selecting an appropriate orifice size through which the polymer/spores mixed passed.The cultivation was carried out as described above.After 48h of incubation,the Ca alginate e immobilized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a biosorbent.The dry weight of Ca alginate e biomass was determined as described for FC.2.4.Batch biosorption studiesBatch biosorption experiments were carried out in500ml Erlenmeyerflasks,as follows:preweighed biosorbent samples (wet or immobilized biomass)with concentration varying from0.1 to0.5g/l(dry weight)were examined.Each sample was added to 100ml of real wastewater,containing heavy metal ions.Biosorption studies were performed at different initial pH from5.5to7.5using 0.1N NaOH.The mixtures were then agitated at120rpm on a rotary shaker up to30min at25 C.Then the content of theflasks was separated byfiltration using a Whatman N 1filter paper.2.5.Metal uptake(q)Uptake of metal ions was calculated from a metal mass balance yielding:q¼VðC iÀC fÞ(1) where:q is mg metal ions per g dry biosorbent;V is the reaction volume(l),C i and C f are the initial and residual metal concen-trations(mg/l),respectively,and m is the amount of dry bio-sorbent(g).The concentration of the metal ions in thefiltrates was deter-mined using atomic absorption spectrophotometer with an air/ acetyleneflame(model2380;Perkin Elmer,Uberlingen,Germany).Aliquots of wet biomass as well as of immobilized biomass, followed by drying for48h at85 C,were considered as dry biosorbent to calculate the uptake.The efficiency of heavy metal removal was calculated from the amount of metal ions adsorbed on the biosorbent and the amount of metal ions available in the wastewater,as the following equation:%removal¼mg heavy metal ions removedmg heavy metal ions availableÂ100(2)2.6.ReproducibilityAll the experiments were run in triplicates and controls were also run on same pattern without addition of biosorbent.The data shown are average from three separate experiments.Table1Characteristics of the biosorption process for removal of heavy metals from waste-water using free biomass as a sorbent.Performance of biosorbentof A.niger biomassHeavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.8 1.10.460.060.0060.0048.1 Equilibrium time min20151555520 Heavy metal uptake mg/g140.66 1.50.460.150.2516.3 Removal efficiency%6015.448.970889537.7 Free biomass(m)0.3g/l;pH e6.5;number of parallels:(n)3;relative standard deviation(RSD)3e8%.Table2Characteristics of the biosorption process for removal of heavy metals from waste-water:PVA e biomass m¼0.3g/l;pH e6.5.Performance of biosorbent Heavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.30.540.460.040.0060.0047.2 Equilibrium time min1510555515 Heavy metal uptake mg/g15.6 2.5 1.50.530.140.2519.3 Removal efficiency%67.158.548.980889544.6 Number of parallels:(n)3;relative standard deviation(RSD)3e8%.K.Tsekova et al./International Biodeterioration&Biodegradation64(2010)447e451 4483.Results and discussion3.1.Sorption of heavy metals from wastewater by freeand immobilized cells of A.niger3.1.1.Sorption of heavy metals from industrial wastewater by free biomass of A.niger (FC)A.niger biomass absorbed Fe 3þ,Pb 2þand Cd 2þions from industrial wastewater more rapidly than other ions (Table 1)within 15e 20min.Experiments indicated that sorption equilibrium reached much faster in case of industrial wastewater sample (up to 20min)in comparison to single ions solution (up to 30min)using same biosorbent (Tsekova et al.,2010a ).These results are impor-tant,as equilibrium time is one of the important parameters for selecting a wastewater treatment system.This may be due to the presence of co e metal ions in the industrial ef fluents as well as to the differences in the heavy metal ions concentrations (Muhammad et al.,2009;Chatterjee et al.,2010).The removal percentages order at equilibrium was:Cd 2þ(95%)>Pb 2þ(88%)>Fe 3þ(70%)>Cu 2þ(60%)>Ni 2þ(48.9%)>Mn 2þ(37.7%)>Zn 2þ(15.4%)3.1.2.Sorption of heavy metals by immobilized biomass of A.nigerThe effect of immobilization of A.niger on PVA e hydrogel as well as on Ca alginate on the removal of heavy metal ions by adsorption was investigated.The metal removal study,illustrated in Tables 2,3showed that their removals were affected by immobilization of A.niger in comparison to the removal ef ficiency by free biomass (Table 1).In general,the both immobilized biosorbents displayed higher bio-sorption capacities for Mn 2þand Cu 2þ,presented in the ef fluent at higher initial concentrations.There was however considerabledifference in total biosorption capability of the test fungal bio-sorbents.Immobilized on Ca alginate cells of A.niger showed highest total biosorption capacity of 71.6mg/g for all heavy metal ions (Table 3),followed by PVA immobilized cells(68.9mg/g,Table 2)and free cells(59.3mg/g j ,Table 1),respectively.Meanwhile,the superior biosorption potential of Ca alginate e immobilized cells (22.6mg/g and 17mg/g)over PVA e immobilized ones (19.3mg/g and 15.6mg/g)was observed in the case of Mn 2þand Cu 2þions,respectively.In general,removal ef ficiency of the test biosorbents,for available metal ions,was observed to follow the sequence in following mode:FC (59.3%)<PVA e biomass (68.9%)<Ca alginate e biomass (71.6%).The both immobilized biosorbents displayed high removal potential for Cd 2,Pb 2þ,Fe 3þ,in comparison to other heavy metal ions from the industrial ef fluent.At the same time the immobilized biomass in Ca alginate exhibited the highest biosorption potency toward Mn 2þand Cu 2þions,that ’s why it was chosen for the following investigations.3.1.3.Effect of pHFig.1shows the effect of pH on the biosorption of different metals by A.niger immobilized biomass.Removal ef ficiency was analyzed over a pH range 5.5e 7.5.The results show that the metal sorption was a function of pH,as the pH increased from 5.5to 6.5,adsorption capacity increased at first for all metals.Maximum adsorption occurs at pH 6.5for Fe 3þ,Cu 2þ,Pb 2þand Cd 2þ,and at pH 7.5for Zn 2þ,Ni2þand Mn 2þ.The maximum removal ef ficiencies (%)for the different metals by Ca alginate e biomass were 96.3%for Cd 2þ(at pH 7.5)>90%for Pb 2þ(at pH 7.5)>80%for Fe 3þ(at pH 6.5)>72.8%for Cu 2þ(at pH 6.5)>61.5%for Mn 2þ(at pH 7.5)>59.7%for Zn 2þ(at pH 7.5)>58.9%for Ni 2þ(at pH 7.5).After pH 7.5the ef ficiency of the metal removal process increases drastically due to the formation of metal hydroxides with their respective metal ions (Zouboulis et al.,2003).This is mostly due to the metal precipitation as hydrox-ides which depend on the pH and ion concentration,but not due to the biosorption (Al e Qodah,2006;AjayKumar et al.,2009).pH value is one of the main factors in biosorption ef ficiency of different biosorbents.The different pH binding pro files for different metal ions are due to the nature of the chemical interactions of metal ions with the biosorbent.Solution pH in fluences surface metal binding sites of the biosorbents and the chemistry of the cell walls,as well as physicochemistry and hydrolysis of the metals.Table 3Characteristics of the biosorption process for removal of heavy metals from waste-water:Ca alginate e biomass m ¼0.3g/l;pH e 6.5.Performance of biosorbentHeavy metals in mixed solution,mg/l CuZnNiFePbCdMn Initial concentrationsmg/l 7 1.30.90.20.050.0813Equilibrium concentrations mg/l 1.90.580.410.040.0050.003 6.2Equilibrium time min 1510555515Heavy metal uptake mg/g 17 2.4 1.60.530.150.3022.6Removal ef ficiency%72.855.454.4809096.252.3Number of parallels:(n)3;relative standard deviation (RSD)3e 8%.102030405060708090100Cu Zn Ni Fe Pb Cd Mnr e m o v a l , %pH - 6.5pH - 7.5Fig.1.Effect of pH on metal ions removal from wastewater using Ca alginate e immobilized cells of Aspergillus niger as a biosorbent.(m 0.3g/l dry weight).Vertical bars show standard error of means of three replicates.K.Tsekova et al./International Biodeterioration &Biodegradation 64(2010)447e 4514493.1.4.Effect of adsorbent weight (g/l)The effect of adsorbent weight (g/l)on the adsorption ef ficiency of the best fungal biosorbent (Ca alginate e immobilized cells)is shown on Fig.2.Adsorption experiments were carried out at different biosorbent doses ranging from 0.1to 0.5g/l in mixed ions solution.It was observed as a general trend that there is an increase of the removal percentage with increase in adsorbent weight from 0.1to 0.3g/l.The maximum removal of the most heavy metal ions was attained at an adsorbent dose of 0.3g/l with no further signi ficant increase in the removal percentage at higher biosorbent concentration tested was observed.In the case of Fe 3þ,Mn 2þand Ni 2þmaximum removal was attained at 0.5g/l of adsorbent weight.Removal ef ficiency increases for Fe 3þfrom 80%till 90%,Mn 2þfrom 52.3%till 61.5%,and for Ni 2þfrom 58.9%till 79%when the sorbent mass increases from 0.3g/l to 0.5g/l.These results are in agreement with previously studies on many other adsorbents (Yu et al.,2001;Dakiky et al.,2002).As the biosorbent mass increases the number of available binding sites or surface area for the heavy metal ions also increased.However,the removal ef ficiency of Pb 2þand Cd 2þretained constant when the biosorbent weight increased.Accord-ing to the previous works,higher biosorbent dose could produce a “screening ”effect on the binding sites,thus resulting in lower heavy metal uptake (Yahaya et al.,2009;Tsekova et al.,2010a,b ).3.1.5.Secondary chemical treatment of the filtrate after biosorptionTo the filtrate obtained after biosorption 5ml 2%sodium diethyldithiocarbamate (NaDDTC)solution and 4g activated char-coal were added,mixed for 10min and filtered through Whatman N 1filter paper.In this second filtrate the concentration of all the examined heavy metals was below the detection limit of the measurement method.Such successful procedure for completely removing of heavy metals from ef fluents has been not reported in the literature yet.4.ConclusionThe ability of A.niger biomass to bind and remove heavy metals,i.e.Cu 2þ,Zn 2þ,Ni 2þ,Pb 2þ,Cd 2þ,Fe 3þ,Mn 2þfrom real wastewater was investigated.To overcome the separation problems of using freely suspended biomass form,as well as,mass loss after regen-eration of the biosorbent,the biomass was immobilized in the polymer matrixes (PVA and Ca alginate gels).Biosorption studies of Ca alginate e A.niger beads have been found to be effective in removing of relatively low concentrations of these seven heavy metals from wastewater.The process was mainly in fluenced by pH and biosorbent dose.At pH 6.5and biosorbent dose of 0.5g/l dry weight the removal ef ficiencies obtained for Ca alginate e biomass beads were:for Cd 2þe 96.2%;for Pb 2þe 90%;for Fe 3þe 90%;for Cu 2þe 73.5%;for Ni 2þe 70.9%for Zn 2þe 60.9%and Mn 2þe 61.5%.The results obtained showed that immobilized biomass of A.niger ,appears as a possible biosorbent to be used for treatment of metal e polluted industrial wastewaters.The secondary chemical treatment with NaDDTC-activated charcoal showed complete removal of all the studied heavy metals.AcknowledgementsThis work was supported by the National Science Fund at the Ministry of Education and Science of Republic of Bulgaria (Grant DOO2-185/2008)and Operative 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