Pseudomonas aeruginosa immobilized multiwalled carbon nanotubes as biosorbent for heavy metal ions.

Pseudomonas aeruginosa immobilized multiwalled carbon nanotubesas biosorbent for heavy metal ionsMustafa Tuzen a ,Kadriye Ozlem Saygi a ,Canan Usta b ,Mustafa Soylakc,*a Gaziosmanpasa University,Faculty of Science and Arts,Chemistry Department,60250Tokat,Turkey bGaziosmanpasa University,Faculty of Science and Arts,Biology Department,60250Tokat,Turkey cErciyes University,Faculty of Science and Arts,Chemistry Department,38039Kayseri,TurkeyReceived 6February 2007;received in revised form 9April 2007;accepted 10April 2007Available online 29May 2007AbstractPseudomonas aeruginosa immobilized multiwalled carbon nanotubes has been used as biosorbent for the solid phase extraction of some heavy metal ions in environmental samples.Cobalt(II),cadmium(II),lead(II),manganese(II),chromium(III)and nickel(II)ions have been selected as analytes for the presented study,due to their important negative and positive roles in human life.In order to inves-tigate quantitative biosorption conditions of the analytes,the influences of pH of the aqueous solution,eluent type,eluent volume,sam-ples volume,etc.were examined.The effects of alkaline,earth alkaline and some transitions metals on the biosorption of analyte ions on P.aeruginosa immobilized multiwalled carbon nanotubes were also investigated.The presented biosorption procedure was applied to the determination of analytes in tomato leaves,bovine liver,boiled wheat,canned fish,black tea,lichen and natural water samples.Ó2007Elsevier Ltd.All rights reserved.Keywords:Pseudomonas aeruginosa ;Multiwalled carbon nanotubes;Biosorption;Preconcentration;Trace metal1.IntroductionHeavy metals are extremely persistent in the environment at trace level.They are nonbiodegradable and nonthermo-degradable and thus readily accumulate to toxic levels (Sharma et al.,2007).Toxic levels of heavy metals may orig-inate from several sources including air,soil and water (Evans and Miller,2006;Szentmihalyi et al.,2006;Kutlu et al.,2006;Gunsen,2004).The roles of heavy metal trace amounts in the human body are still under investigation (Gunsen,2004;Subrahmanyam et al.,in press;Praveen et al.,in press;Yaman and Ince,2006).In these studies,atomic absorption spectrometer is one of the main instru-ments due to its simplicity and its low cost.However there are two big problems for the analytical chemist which are low levels of the metal ions and positive or negative effectsof the matrix components (Dadfarnia et al.,2006;Quina´ia et al.,2006;Kiran et al.,in press ).The usage of separa-tion-enrichment procedures could solve these problems,prior to determination of analytes (Lemos et al.,in press;Hakim et al.,2007;Ramesh et al.,2007;Ghaedi et al.,2006).Liquid–liquid extraction,electroanalytical techniques,cloud point extraction,solid phase extraction based on sorption or biosorption,etc.have been used for that pur-pose (Haji Shabani et al.,2006;Pourreza and Elhami,2006;Youcef et al.,2006;Seki et al.,2006a,b;Martinez-Garcia et al.,2006;Hosseini and Sarab,2007).Traces heavy metal ions could be adsorbed on the higher organisms including mosses,bacteria,algae (Seki et al.,2006a,b;Mar-tinez-Garcia et al.,2006;Yan and Viraraghavan,2001;Barros et al.,2007;Pamukoglu and Kargi,2007;Karthike-yan et al.,2007).The uptake of metals by biomass can take place actively,by means of a metabolic activity dependent process (bioaccumulation)or by means of a passive and usually rapid (several minutes)metabolism-independentprocess called biosorption (Godlewska-_Zyłkiewicz,2004;0960-8524/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2007.04.013*Corresponding author.Fax:+903524374933.E-mail addresses:soylak@.tr ,msoylak@ (M.Soylak).Available online at Bioresource Technology 99(2008)1563–1570Godlewska-_Zyłkiewicz and Kozlowska,2005).This point is used by the researchers on the preconcentration–separation of the heavy metals at trace level in the environment.The system is based on biosorption of the heavy metals and desorption of these metals from the organisms.Biosorption of trace metals by microorganisms can be realized in batch and continuous modes(Godlewska-_Zyłkiewicz,2004;God-lewska-_Zyłkiewicz and Kozlowska,2005).An important part of the studies on biosorption is based on the immobili-zation of the organisms on the natural or synthetic poly-meric materials(Godlewska-_Zyłkiewicz,2003,2004; Godlewska-_Zyłkiewicz and Kozlowska,2005;Baytak and Turker,2004,2005a,b;Menega´rio et al.,2005).Microor-ganisms immobilized natural and synthetic adsorbents have been used for trace heavy metal separation and preconcen-tration from various media with successfully results(God-lewska-_Zyłkiewicz,2003,2004;Godlewska-_Zyłkiewicz and Kozlowska,2005;Baytak and Turker,2004,2005a,b; Menega´rio et al.,2005).Saccharomyces carlsbergensis, Aspergillus niger,Agrobacterium tumefacients,Saccharomy-ces cerevisiae,etc.were the microorganisms used,while Amberlite XAD resins,silica,sephiolite,Diaion resins, etc.were used as supports(Baytak and Turker,2004, 2005a,b;Menega´rio et al.,2005;Godlewska-_Zyłkiewicz, 2003).Some applications of microorganisms loaded adsor-bent for heavy metal preconcentrations are summarized in Table1.Pseudomonas aeruginosa is a gram-negative,aerobic rod belonging to the bacterial family Pseudomonadaceae. P.aeruginosa is pathogens of humans(Menegario et al., 2006).P.aeruginosa is often preliminarily identified by its pearlescent appearance and grape-like odor in vitro.Defin-itive clinical identification of Pseudomonadaceae aeruginosa often includes identifying the production of pyocyanin and fluorescein as well as its ability to grow at42°C.Pseudo-monadaceae aeruginosa is capable of growth in diesel and jet fuel,where it is known as a hydrocarbon utilizing micro-organism,causing microbial corrosion(Gelmi et al.,1994).Carbon nanotubes(CNTs)are one of the most com-monly used building blocks of nanotechnology.With one hundred times the tensile strength of steel,thermal conduc-tivity better than all but the purest diamond,and electrical conductivity similar to copper,but with the ability to carry much higher currents,they seem to be a very interesting material(Seki et al.,2006a,b).Carbon nanotubes(CNTs) have been proposed as a novel solid phase extractor for various inorganic and organic materials at trace levels (.;Zhou et al.,2006;Iijima,1991;Liang et al.,2004,2005).According to our literature survey,P.aeruginosa and multiwalled carbon nanotubes combination is not used on the biosorption of traces heavy metal ions.Possible usage of the P.aeruginosa immobilized multiwalled carbon nanotubes for biosorption of metals was investigated.The analytical conditions for the quantitative recoveries of the analytes including pH of solutions,sample volume,etc. were investigated.2.Experimental2.1.InstrumentA Perkin Elmer AAnalyst700atomic absorption spec-trometer with deuterium background corrector was used. All measurements were carried out in an air/acetylene flame.A10cm long slot-burner head,a lamp and an air–acetyleneflame were used.The operating parameters for working elements were set as recommended by the manu-facturer.SEM image was obtained on a LEO440scanning electron microscope(SEM).A pH meter,Sartorius pp-15Model glass-electrode was employed for measuring pH values in the aqueous phase. Milestone Ethos D closed vessel microwave system(maxi-mum pressure1450psi,maximum temperature300°C)was used.Digestion conditions for microwave system were applied as2min for250W,2min for0W,6min forTable1Comparative data from some recent studies on biosorption of heavy metals on microorganism immobilized on adsorbentsElements Media Adsorptioncapacity(mg gÀ1)PF DL(l g lÀ1)RSD(%)ReferenceFe3+,Co2+,Mn2+,Cr3+Agrobacterium tumefacients immobilizedon Amberlite XAD-41.21–1.7125 2.8–3.6<10Baytak andTurker(2005)Fe3+,Co2+,Cr3+Saccharomyces carlsbergensis immobilizedon Amberlite XAD-41.41– 2.8–7.4<5Baytak andTurker(2005)Mn2+Saccharomyces carlsbergensis immobilizedon Amberlite XAD-4––60<5Baytak andTurker(2004)Cr3+,Cr6+Saccharomyces cerevisiae immobilizedon controlled pore glass –120.45–1.5–Menega´rioet al.(2005)Pt2+,Pd2+Saccharomyces cerevisiae and Chlorellavulgaris immobilized on silica gel ––0.4–0.8<5Godlewska-_Zyłkiewicz(2003)Cu2+,Pb2+,Fe3+,Co2+Bacillus sphaericus loaded Diaion SP850 4.3–9.2500.20–0.75<5Tuzen et al.(2007) Cu2+,Pb2+,Zn2+,Fe3+,Ni2+,Co2+Aspergillus fumigatus immobilized onDiaion HP-2MG4.4–8.5500.30–0.72<7Soylak et al.(2006)Co2+,Cd2+,Pb2+,Mn2+,Cr3+,Ni2+Pseudomonas aeruginosa immobilizedon multiwalled carbon nanotubes5.25–6.23500.24–2.60<10This studyPF,preconcentration factor.1564M.Tuzen et al./Bioresource Technology99(2008)1563–1570250W,5min for400W,8min for550W,ventilation: 8min(Tuzen et al.,2004,2005).2.2.Reagents and solutionAll chemicals used in this work,were of analytical reagent grade and were used without further purification. Deionised water(Milli-Q Millipore18.2M X cmÀ1resistiv-ity)was used for all dilutions.All the plastic and glassware were cleaned by soaking in dilute HNO3(1+9)and were rinsed with distilled water prior to use.The element stan-dard solutions used for calibration were produced by dilut-ing a stock solution of1000mg lÀ1of the given element supplied by Sigma and Aldrich.Stock solutions of diverse elements were prepared from high purity compounds.The calibration standards were not submitted to the preconcen-tration procedure.Multiwalled carbon nanotube(Aldrich no.:636630)was purchased from Aldrich,Milwaukee,WI,USA.The BET surface area and density of nanotubes were600m2gÀ1 and2.1g mlÀ1,respectively.It has high purity.Standard reference materials(NIST SRM1573a Tomato leaves and NIST SRM1577b Bovine liver)were used in the experiment.Phosphate buffer solutionsðH2POÀ4=H3PO4Þwere pre-pared by mixing of appropriate volumes of0.1mol lÀ1 sodium dihydrogen phosphate and phosphoric acid solu-tions for pH2,and 3.Acetate buffer solutions (CH3COOÀ/CH3COOH)were prepared by mixing of appropriate volumes of0.1mol lÀ1acetic acid and 0.1mol lÀ1sodium acetate solutions for pH4.Phosphatebuffer solutionsðH2POÀ4=HPO2À4Þwere prepared by mixingof appropriate volumes of0.1mol lÀ1sodium dihydrogen-phosphate and0.1mol lÀ1sodium hydrogen phosphate for pH5,6and7.Ammonium buffer solutions were prepared by mixing of appropriate amounts of0.1mol lÀ1ammonia and0.1mol lÀ1ammonium chloride solutions for pH8–10.2.3.Preparation of biomassThe liquid medium was prepared by mixing2g of pep-tone,2g meat extract and1g mineral medium(10g CaCl2Æ2H2O,20g MgCl2Æ6H2O,1g MnCl2Æ4H2O)and was dissolved in the200ml distilled water,and sterilized at120°C for20min.To prepare a starter culture,the bac-terial strain,P.aeruginosa was grown in solid stock med-ium.It was inoculated into a10ml liquid nutrient medium.It was incubated at30°C for24h.The previously prepared200ml sterile liquid mediums were inoculated with2ml of the starter culture,and incubated in10vials at pH7.2–7.4.The bacterial cultures were kept in continu-ous shaking at30°C.The stationary phases of each200ml liquid bacterial cultures were detected by microscopic observations.After reaching stationary phases,16–24h of incubation periods,P.aeruginosa cell density was4.0–4.6at600nm,and at this time the bacterial cells were har-vested and separated from the media using centrifugation at7000rpm for15min.The isolated biomass was washed three times with0.1mol lÀ1HCl,and rinsed with distilled water and dried.Hundred milligram of dry and dead P.aeruginosa was mixed with250mg of multiwalled carbon nanotubes.The mixture was wetted with2ml of doubly distilled water and thoroughly mixed.After mixing,the paste was heated in an oven at about105°C for1h to dry the mixture.The wetting and drying step were repeated to maximize the con-tact between P.aeruginosa and multiwalled carbon nano-tubes,thereby improving the immobilization efficiency. Then,the product obtained used as biosorbent for the pres-ent work.SEM photograph of P.aeruginosa immobilized multiwalled carbon nanotube is given in Fig.1.The P.aeruginosa immobilized multiwalled carbon nano-tubes column was10cm long,and1cm in diameter.A small plug of glass wool was placed on the bottom of the column. The column wasfilled with250mg of biosorbent according to literature(Tuzen et al.,2007;Soylak et al.,2006).The bed depth of biosorbent in the column was approximately 3.0cm.The resin column was prepared by aspirating water slurry of P.aeruginosa-immobilized multiwalled carbon nanotubes into the glass column.It was conditioned by passing10–15ml of ammonia(0.1mol lÀ1)/ammonium (0.1mol lÀ1)buffer solution then it was used for separa-tion–preconcentration study.After each use,the column was washed by passing10–15ml of ammonia (0.1mol lÀ1)/ammonium(0.1mol lÀ1)buffer solution for regeneration of the biosorbent.Theflow rates of the solu-tions were controlled by using stopcock of the column. 2.4.Biosorption procedureThe biosorption procedure presented was tested with model solutions.40–50ml of solution containing5–20l g of Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions was added10ml of ammonia(0.1mol lÀ1)/ammonium (0.1mol lÀ1)buffer solution.The P.aeruginosa immobi-lized multiwalled carbon nanotubes column was precondi-tioned by passing ammonia(0.1mol lÀ1)/ammonium (0.1mol lÀ1)buffer solution.The buffered metal solution was passed the column at aflow rate of5ml minÀ1.The sample solution was permitted toflow through the column under gravity.After passing of this solution completely,the column was rinsed with twice10ml of water.The sorbed metal ions on the column were eluted with8–10ml portion of1M HNO3.The residue is diluted to10.0ml with1.0M HNO3.The eluent was analyzed for the determinations of metal concentrations byflame atomic absorption spectrometer.3.Results and discussion3.1.Effects of pHDue to pH is the one of the important factor for the retentions of traces metal ions on the biosorption of theM.Tuzen et al./Bioresource Technology99(2008)1563–15701565metal ions on microorganisms as other preconcentration works (Lemos et al.,in press;Suvardhan et al.,2006;Soy-lak,1998),the influences of pH of the aqueous solution on the retentions of the analyte ions on P.aeruginosa immobi-lized multiwalled carbon nanotubes resin were investigated.The recovery values were given in Fig.2.Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions were quantitatively (P 95%)recovered at the pH range of 8.5–9.5for the ana-lytes.A competition between hydroniumions and analytes at the acidic pH values were occurred (Baytak and Turker,2005a,b;Turker and Baytak,2004).The cell surface becomes more positively charged at low pH values which decrease the attraction between metal ions and the func-tional groups on biosorbent (Baytak and Turker,2005a,b;Turker and Baytak,2004).The all further worksfor biosorption were performed at pH 9.0by using ammo-nia/ammonium buffer solution.The recovery values for Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions on the column filled with multi-walled carbon nanotubes without P.aeruginosa at pH range of 8–10were below 70%.The recoveries for analytes on the column filled 100mg of P.aeruginosa without mul-tiwalled carbon nanotubes at pH range of 8–10were below 70%.These points show that for the quantitative recoveries of Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions,it is necessary that the combination of P.aeruginosa and mul-tiwalled carbon nanotubes as biosorbent.3.2.Eluent type and its volumeThe elution of biosorbed metal ions from the P.aerugin-osa immobilized multiwalled carbon nanotubes was also studied by using HCl and HNO 3at various concentrations.The results are given in Table 2.Quantitative recoveries were obtained by using 1M HCl and 1M HNO 3for analytes.Effect of volume of 1M HNO 3as eluent was also exam-ined on the recoveries of analytes.The results are given inFig.1.SEM photograph of Pseudomonas aeruginosa immobilized multiwalled carbon nanotube.Table 2Effects of various eluents on the recoveries of analytes (N =3)Eluent Recovery (%)CoCd Pb Mn Cr Ni 0.5M HCl 90±285±390±270±286±380±31M HCl 97±395±296±396±296±296±30.5M HNO 395±290±385±287±390±390±21M HNO 399±395±397±396±296±397±31566M.Tuzen et al./Bioresource Technology 99(2008)1563–1570Table 3.All the analytes were quantitatively recovered from P.aeruginosa immobilized multiwalled carbon nano-tubes at 8–10ml of 1M nitric acid.3.3.Flow rates of sample and eluent solutionsThe effects of the sample and eluent flow rates on the retentions and recoveries of Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions on P.aeruginosa immobilized mul-tiwalled carbon nanotubes were also examined in the flow rate range of 2–10ml min À1under optimal conditions with model solutions containing analyte elements.All the ana-lyte ions were quantitatively retained and recovered in the sample and eluent flow range of 1–6ml min À1.After 6ml min À1,the recoveries were not quantitative due to insufficient contact between analytes and biosorbent.In the all-further works,5ml min À1was selected as sample and eluent flow rate.3.4.Effect of sample volumeThe influences of sample volume on the recoveries of analyte ions on P.aeruginosa immobilized multiwalled car-bon nanotubes were investigated in the sample volume range of 25–750ml.The results are depicted in Fig.3.Ana-lyte ions were quantitatively (P 95%)recovered till 500ml.After 500ml of sample volume,recovery values not quan-titative.The preconcentration factor was calculated as 50when eluent volume is 10ml.3.5.Matrix influencesThe influences of the some ions which are known as interferic ions in the AAS determination were investigated on P.aeruginosa immobilized multiwalled carbon nano-tubes.The results for this study are given in Table 4.The tolerance limit of foreign ions was taken as that value which caused an error of not more than ±5%in the absor-bance.The ions normally present in water do not interfere under the experimental conditions used.Also,some of the transition metals at mg l À1levels were not interfered on the recoveries of the analyte ions.This results show that the proposed preconcentration/separation method could be applied to the highly saline samples and the samples that contains some transition metals at the tolerable levels given in Table 4.The samples analyzed in the presented work contain alkaline and earth alkaline ions at mg l À1and tran-sition metals at l g l À1levels.3.6.Adsorption capacityIn order to study the adsorptive capacity of biosorbent,batch method was used.To 0.1g of sorbent was added 50ml of solution containing 1.0mg of metal ion at pH 9.0.After shaking for 1h,the mixture was filtered.10ml of the supernatant solution was diluted to 100ml and determined by flame atomic absorption spectrometry.This procedure was repeated for each analyte ions separately.The capacity of sorbent for analytes were found as:Co:6.06mg g À1,Cd: 6.18mg g À1,Pb: 6.07mg g À1,Mn:5.83mg g À1,Cr:6.23mg g À1and Ni:5.25mg g À1.The stability of multiwalled carbon nanotubes was excellent.On storing for six mounts its properties and sorp-tion capacity do not change significantly.P.aeruginosa immobilized multiwalled carbon nanotubes filled columns could be used at least 50cycles without any loss their adsorption capacities.The adsorption could be attributed to ionic attraction between analytes and the biosorbent (Baytak and Turker,2005a,b;Turker and Baytak,2004).3.7.Figure of meritsThe relative standard deviations for flame atomic absorption spectrometric determinations for analytes are between 1.0%and 9.0%.The detection limits,defined asTable 3Effects of volume of 1M HNO 3as eluent on the recoveries of analyte ions (N =3)Volume (ml)Recovery (%)Co Cd Pb Mn Cr Ni 550±255±260±165±260±275±2660±365±275±270±270±280±2785±280±390±380±383±385±3896±397±399±398±399±499±31097±398±3100±298±298±399±3Table 4Influences of the matrix ions on the recoveries of analytes (N =3)IonAdded asTolerance limit (mg l À1)Na +NaCl 20,000Cl ÀNaCl25,000NO À3,SO 2À4,PO 3À4KNO 3,Na 2SO 4,Na 3PO 43000K+KCl5000Ca 2+,Mg 2+,F ÀCaCl 2,MgCl 2,NaF1000Cu 2+,Zn 2+,Al 3+,Fe 3+CuSO 4,ZnSO 4,Al 2(SO 4)3,FeCl 325M.Tuzen et al./Bioresource Technology 99(2008)1563–15701567the concentration equivalent to three times the standard deviation(N=11)of the reagent blank were found as: Co:0.74l g lÀ1,Cd:0.24l g lÀ1,Pb: 2.60l g lÀ1,Mn: 0.43l g lÀ1,Cr:1.18l g lÀ1and Ni:1.30l g lÀ1.In order to estimate the accuracy of the presented bio-sorption procedure,different amounts of the investigated metal ions were spiked in a tap water from Tokat-Turkey and spring water from Tokat-Turkey.The resulting solu-tions were submitted to the presented procedure given in Experimental.The results were given in Table5.The recov-ery values for Co(II),Cd(II),Pb(II),Mn(II),Cr(III)and Ni(II)ions were generally in the range of97–102%.It shows that the presented solid phase extraction method can be applied for biosorption of analyte ions in the real samples which have high salt content.3.8.Analysis of the real samplesThe validation of the presented procedure is performed by the analysis of two certified reference materials.NIST SRM1573a Tomato leaves,NIST SRM1577b Bovine liver standard reference materials(250mg)were digested with 6ml of HNO3(65%),2ml of H2O2(30%)in microwave digestion system and diluted to50ml with deionized water (Tuzen et al.,2004,2005).A blank digest was carried out in the same way.Then the preconcentration procedure given above was applied to thefinal solutions.The results are given in Table6.The certified and observed values for cer-tified reference materials were in good agreement with the certified values of SRMs.Spring,snow,tap waters from Tokat city analyzed was filtered through a cellulose membranefilter(Millipore)of 0.45l m pore size.The pH of the samples was adjusted to 9.0with ammonia((0.1mol lÀ1)/ammonium(0.1mol lÀ1)) buffer solution.The sample was passed through the col-umn.The metal adsorbed on P.aeruginosa immobilized multiwalled carbon nanotubes column were eluted with 1mol lÀ1HNO3.The levels of analyte ions in the samples were determined byflame atomic absorption spectrometry. The results were given in Table7.Maximum acceptable concentration of total chromium in drinking water was 50l g lÀ1(WPCRT,1989).The guideline value of Ni and Mn in drinking water is20l g lÀ1and0.1mg lÀ1,respec-tively(WPCRT,1989).Maximum acceptable concentra-tion of cobalt in drinking water was10l g lÀ1(WPCRT, 1989).The concentrations of analyte ions in Table7were generally lower than the values given in literature (WPCRT,1989).For the microwave digestion of boiled wheat,canned fish,black tea,lichen(Homalothecium sericeum),1.0g of sample was digested with6ml of concentrated HNO3 and2ml of concentrated H2O2in microwave system.After digestion the samples,the volume of the digested sample was made up to25.0ml with distilled water.Blanks wereTable5The results for tests of addition/recovery for trace metal determination in some real samples(sample volume:50ml,final volume:10ml(N=3))Element Added(l g lÀ1)Tap water Spring waterFound(l g lÀ1)Recovery Found(l g lÀ1)RecoveryCo–ND–ND–5 4.9±0.298 4.8±0.3961010.1±0.41019.7±0.5972019.6±0.79819.6±0.898 Cd–ND–ND–2.5 2.5±0.1100 2.4±0.2965 5.1±0.1102 4.9±0.398109.9±0.4999.7±0.597 Pb–ND–ND–109.9±0.3999.5±0.4952020.2±0.610119.6±0.7984040.4±0.910139.5±0.899 Mn–ND–ND–2.5 2.4±0.196 2.4±0.2965 4.8±0.296 4.9±0.398109.8±0.3989.9±0.599 Cr–ND–ND–1010.2±0.41029.8±0.5982020.1±0.810119.7±0.9994039.7±0.69939.2±0.798 Ni–ND–ND–5 4.9±0.298 4.8±0.196109.7±0.4979.5±0.5952019.5±0.79819.2±0.996 ND,not detected.Table6The results for reference standard materials after application of presented procedure(N=4)Element Concentration(l g gÀ1)bNIST SRM1573a tomatoleavesNIST SRM1577b bovine liver Certified value Our value Certified value Our value Co0.570.60±0.05(0.25)a0.30±0.02 Cd 1.52 1.47±0.100.50.48±0.04 Pb–BDL0.1290.132±0.010 Mn246240±1410.510.2±0.50 Cr 1.99 1.92±0.10–BDLNi 1.59 1.52±0.12–BDLBDL,below the detection limit.a The value in the parenthesis is not certified.b Uncertainty at95%confidence limit.Table7The application of the presented method in natural water samples for contents of analyte ions(N=3)Element Concentration(l g lÀ1)aTap water Spring water Snow water Co8.7±0.510.6±0.78.1±0.4 Cd BDL 4.4±0.3 2.4±0.1 Pb BDL 6.2±0.49.2±0.5 Mn 1.8±0.17.3±0.3 3.5±0.1 Cr 4.7±0.214.1±0.8 2.4±0.1Ni7.2±0.48.3±0.5BDL BDL,below detection limit.a Uncertainty at95%confidence limit.1568M.Tuzen et al./Bioresource Technology99(2008)1563–1570prepared in the same way as the sample,but omitting the sample.The preconcentration–separation procedure given above was applied to the samples.The results are given in Table8.The maximum cadmium and lead level permit-ted for cannedfishes is0.05mg kgÀ1and0.2mg kgÀ1 according to Turkish Food Codex(2002).There is no information about maximum cobalt,manganese,chro-mium and nickel levels infish samples in Turkish stan-dards.It is reported that maximum nickel levels in some food samples as0.2mg kgÀ1(Turkish Food Codex, 2002).It is reported that maximum permitted levels of cad-mium and lead in grains is0.2mg kgÀ1(Turkish Food Codex,2002).The levels of analytes in Table8were gener-ally lower than the values given in literature(Turkish Food Codex,2002).4.ConclusionThe presented procedure is based on the immobilization of P.aeruginosa on multiwalled carbon nanotubes and bio-sorption of heavy metal ions on this biosorbent.The proce-dure is simple,economic and fast.Also preparation of the P.aeruginosa immobilized multiwalled carbon nanotubes is simple.The reusability of P.aeruginosa immobilized multi-walled carbon nanotubes was as high as greater than50 cycles without any loss in its sorption behavior.The pre-sented system was also successful in preconcentrating ana-lytes from large sample volume(500ml).The matrix effects were reasonably tolerable.AcknowledgementsThe authors are grateful for thefinancial support of the Unit of the Scientific Research Projects of Gaziosmanpasa University and the Unit of the Scientific Research Projects of Erciyes 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