Determination of some trace elements by flame atomic absorption spectrometry after preconcentration and separation by Escherichia coli immobilized on multiwalled carbon nanotubes


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We have immobilized living and non-living Escherichia coli (E. coli) bacteria on multiwalled carbon nanotubes (MWCNT) and used such materials as a biosorbent for the separation and preconcentration of copper, cobalt, cadmium and nickel prior to their
  ORIGINAL PAPER  Determination of some trace elements by flame atomicabsorption spectrometry after preconcentrationand separation by  Escherichia coli   immobilizedon multiwalled carbon nanotubes Nihat Aydemir  &  Nilgun Tokman  & Alper Tunga Akarsubasi  &  Asli Baysal  & Süleyman Akman Received: 26 April 2011 /Accepted: 15 July 2011 /Published online: 26 July 2011 # Springer-Verlag 2011 Abstract  We have immobilized living and non-living  Escherichia coli  (  E. coli ) bacteria on multiwalled carbonnanotubes (MWCNT) and used such materials as a  biosorbent for the separation and preconcentration of copper, cobalt, cadmium and nickel prior to their determi-nation by flame atomic absorption spectrometry (FAAS).  E.coli  bacteria cells were mixed with MWCNTs in a 1:1 ratio,dried and placed at the tip of a 50-mL syringe. The ionswere retained on the sorbent and then eluted by drawingand ejecting back the sample (or standard solution) and aneluent, respectively. The effects of various experimental parameters on the sorption and elution were investigated.The analytes were quantitatively retained (at pH values of 7) and eluted (with 0.5 M nitric acid) with high precision,the RSD being <5%. The performances of the new sorbentswere compared using certified reference materials. Thesorbent modified with living  E. coli  has a higher adsorptioncapacity and displays somewhat better recoveries comparedto sorbent based on non-living  E. coli . Both sorbents weresuccessfully used for the separation and preconcentration of copper, cobalt, cadmium and nickel prior to their determi-nation by flame atomic absorption spectrometry. Keywords  Preconcentration.Separation.Flame atomicabsorption spectrometry.  Escherichia coli .Biosorbtion.Multiwalled carbon nanotubes Introduction If the concentration of a trace analyte is too low to bedetermined directly and/or interferences due to matrixcannot be eliminated, the use of a separation/enrichment  procedure is compulsory. For this purpose, various separa-tion/preconcentration methods such as liquid-liquid extrac-tion, solid phase extraction, coprecipitation, ionic resinshaave been extensively applied .Biosorption, which uses the ability of biological materialsto remove heavy metals from solutions, has receivedconsiderable attention in recent years because of someadvantages compared to traditional methods such as ease of availability, low cost etc. To separate, preconcentrate andremove of heavy metal ions in waste water samplescould be quantitatively made by biosorption using theorganisms on the immobilized materials includingmosses, bacteria, algae [1  –  5]. Silica gel, sepiolit, multiwalled carbon nanotubes etc. can be used as theimmobilized materials. Immobilized microorganizms havesome advantages compared to the free microorganism,namely; (i) long usage time, (ii) mechanic stability of   N. Aydemir  : A. Baysal :  S. Akman ( * )Faculty of Science & Letters, Department of Chemistry,Istanbul Technical University,34469 Maslak,Istanbul, Turkeye-mail:  N. TokmanScientific and Technical Research Council of Turkey (TUBITAK), National Metrology Institute,PK 54 41470,Gebze, Kocaeli, TurkeyA. T. AkarsubasiFaculty of Science & Letters, Department of Molecular Biologyand Genetics, Istanbul Technical University,34469 Maslak,Istanbul, TurkeyMicrochim Acta (2011) 175:185  –  191DOI 10.1007/s00604-011-0668-2  immobilized microorganism (iii) easiness for treatment,(iv) high metal binding capacity, (v) selectivity for heavymetals. The literature is full of papers on preconcentration/ separation of trace elements by using microorganism onthe immobilized materials and using atomic spectrometry[6  –  16]. In order to biosorption of various analytes,different kind of microorganism and immobilized mate-rials were used. For instance, Madrid and Camara wereinvestigated some biological substrates for metal pre-concentration and speciation [1]. This article summarizedthe metal preconcentration and speciation using biologicalorganisms such as algae, plant-derived materials, bacteria,yeast, fungi and erythrocytes. Although biosorption has been the subject of increasing research for a variety of reasons, including its potential application in the recoveryof metals from manufacturing processes, the treatment of contaminated water and the recovery of precious metals inmining operations, only recently has it been exploited for analytical measurement. Basic techniques and principlesare discussed and recent developments reviewed. Specialattention were given to immobilization procedures andsome biosorption mechanisms were considered. Besidesthis article, Bag et al. [6] has given the sample of this topicusing  Escherichia coli  immobilized on sepiolite. Theywere developed the method for the determination of Cu,Zn, Fe, Ni and Cd by flame atomic absorption spectro- photometry (FAAS) after preconcentrating on a columncontaining  Escherichia coli  immobilized on sepiolite andthe method was applied to the determination of tracemetals in alloys. Baytak and Turker investigated the use of the  A. tumefacients  immobilized on Amberlite XAD-4 as a new biosorbent in trace metal determination of Fe(III), Co(II), Mn(II) and Cr(III) by FAAS in water, food and alloysamples [9]. In another study, the adsorption properties of two different marine algae ( Ulva fasciata  (green algae)and  Sargassum sp.  (brown algae) were investigated for thedetermination of Cu (II) by ICP-OES [12]. Tuzen et al. were used  Pseudomonas aeruginosa  immobilized multi-walled carbon nanotubes as biosorbent for the solid phaseextraction of cobalt(II), cadmium(II), lead(II), manganese(II), chromium(III) and nickel(II) ions in environmentalsamples [13]. With the same methodology,  Algea  cells onsilica gel [2],  Sacchar. Cerevisiae  in flow injection [7], Saccha. cerevisiae  immobilized on sepiolite for Cr (III)[8],  Sacchar. Carlsbergensis  immobilized on AmberliteXAD-4 for the determination of Iron(III), Cobalt(II) andChromium(III) [10],  Penicillium digitatum  Loaded on pumice stone for preconcentration of Co(II), Fe(III) and Ni(II) [14], filamentous fungal biomass-loaded TiO2nanoparticles for the lead determination [15],  Penicilliumitalicum  –  loaded on Sepabeads SP 70 for the determinationof trace elements [16] were used as an biosorbent for various metals by FAAS. However, in all those and other similar studies, the microorganisms were non-living. Theliving microorganisms as sorbent for trace elements havealmost not been studied for analytical purposes.We desribed here the use of non-living or living  E. coli immobilized on multiwalled carbon nanotubes (MWCNT)as a biosorbent for the separation and preconcentration of trace copper, cobalt, cadmium and nickel and their analytical performances were compared. For this purpose,living or non-living  E. coli  bacteria cells were immobilizedon multiwalled carbon nanotubes and the sorbent  prepared was filled in a syringe-mountable filter whichwas succesfully applied in a previous paper as well[17]. The analyte elements were retained and then eluted by drawing and ejecting back the the sample and eluent,respectively. The capacities as well as effects of experimental parameters on recoveries of the analytes were investigated.The merits of the method was discussed. The comparisons of sorption properties of nonliving and living bacteria on thesorbent and their use in syringe mountable filter for enrichment/separation are novel. Experimental InstrumentalA Analytik Jena Vario 6 flame atomic absorptionspectrophotometer (FAAS) (Analytik Jena AG, Germany, equipped with an acetylene-air  burner was used for the determination of cadmium, cobalt,copper and nickel. Coded hollow cathode lamps were usedeach of the analyte as the spectral radiation sources. Thewavelength were adjusted 228. 8, 240.7, 324.8, 232.0 nm for Cd, Co, Cu and Ni, respectively. As well as spectral slit widthwere adjusted 0.5, 0.2, 0.5, 0.2 nm for Cd, Co, Cu and Ni,respectively.The pHof the samples wereadjusted by0.01mol L − 1  NH 3 or0.01mol L − 1 HNO 3  and controlled using WTW pH 340-A/ SET2 pH meter (WTW Wissenschaftlich-TechnischeWerkstätten GmbH, Germany, home.html). An Alpha 1  –  2 LD Plus Freeze Dryer (MartinChrist Gefriertrocknungsanlagen GmbH, Germany, http:// were used for bacteria modificationand grow-up procedure. A Beckman Coulter centrifugation(Beckman Coulter Inc., USA,, a Fisher Scientific FB15012 and a Forma Orbitalshaking apparatus (Thermo Fisher Scientific Inc., USA, were used for necessary proceses.ReagentsAll chemicals were of analytical grade (Merck, Germany,; Fluka, Switzerland, www. 186 N. Aydemir et al., while Certified Reference Materials(CRM), namely, Waste Water (CRM-TM), were bought fromHigh-Purity Standards (USA,, respectively.Stock solutions (1000 mg/L) of Cd, Co, Cu and Niwere prepared from Titrisol (Merck, Germany, and further diluted with distilled-deionized water daily.Tryptone, sodium chloride and yeast extract (Difco,Becton, Dickinson and Company, USA, were used for making media. C tube 100 (Cnt Co.Ltd., South Korea, wereused as multiwalled carbon nanotubes.Grown and modification conditions of bacteria cells Condition for growing of bacteria cells The broth medium was prepared by mixing 1 g of trypone,1 g NaCl, 1 g yeast extract dissolving in the 100 mldistilled-deionized water, and autoclaved at 120 °C for 20 min. The pH of the prepared liquid medium wasadjusted to 7.2  –  7.4, and 200  μ  L stock   E. coli  cells werecultivated in this liquid medium and incubated at 37 °Cshaking gently for 24 h. Prepared bacteria cells were storedat 4 °C to eliminate any contamination and to maintain their stabilization.  Immobilization of living E. coli cells on the MWCNT  After cultivation, bacteria cells were precipitated from themedia using centrifugation at 5000 rpm for 10 min. 0.1 g of grown  E. coli  bacteria cell isolates were mixed with 0.1 g of multiwalled carbon nanotubes in 20 mL distilled-deionizedwater at 200 rpm for 2 h. The mixture of the  E. coli  cellsand MWNCT was separated from liquid phase by centrifu-gation at 5000 rpm for 10 min. The modification product was dried and stabilized in a vacuum oven at 80  –  105 °C for 24 h and subsequently grinded to use as a sorbent.  Immobilization of non-living E. coli cells on the MWCNT  After cultivated  E. coli  cells were separated from the media using centrifugation at 5000 rpm for 10 min, the  E. coli  precipitate was washed three times with 0.1 mol L − 1 HCland then distilled-deionized water. The bacteria cells werenon-living after acid treatment, and subsequently dried in a freeze drier for 24 h. To prepare non-living  E. coli immobilized MWCNT, a 0.1 g portion of non-living  E.coli  cells was mixed with 0.1 g of MWCNT in 20 mLdistilled-deionized water at 200 rpm 2 h. The mixture wasseperated from liquid phase using centrifugation at 5000 rpm for 10 min. The product was dried and stabilizedin an vacuum oven at about 80  –  105 °C for 24 h. The product of   E. coli  modified MWCNT was grinded to beused as a biosorbent for the present work.General procedureAt first, two 0.45  μ   membrane filters were inserted in a screwed separable syringe mountable filter and mounted ona 50 mL of syringe. The filters were washed drawing andejecting back 0.1 mol L − 1 HNO 3 , distilled-deionized water,0.1 mol L − 1  NaOH, and then distilled-deionized water.Then the syringe mountable filter was opened and a 0.1 gliving or a 0.1 g non-living (dead)  E. coli  immobilized onthe MWCNT was placed between two 0.45  μ   membranefilters and then closed tightly.The living or non-living  E. coli. Coli  immobilizedMWCNT in a syringe mountable filter was conditioned by passing a solution at working pH prior to separation  –   preconcentration study. For enrichment procedure, up to250 mL of sample solution containing 0.025  μ  g mL − 1 of Cd 2+ , Co 2+ , Cu 2+ , Ni 2+ was passed through the syringesystem. After sorption, 10 mL of water was passed twicethrough the sorbent loaded with the analyte. The sorbedmetal ions on the filter mounted to the syringe were eluteddrawing and ejecting back 12.5 mL of 0.5 mol L − 1 HNO 3 .The flow rate of sample solution and eluate were around3 mL min − 1 . Since the nanoparticles always prevent thesamples and eluents to be drawn and ejected, it was no useof applying excessive force to provide higher flow rates. Weapplied the max flow rate (3 mL min − 1 ) permitted by thesorbent by forcing max force because lower rates could not  provide betnter improvement for recoveries. In addition, the best reproducible flow rate could be maitained applyingmax force (i.e. max flow rate) 0204060801001200 2 4 6 8 10 pH Cd Co Cu Ni    R  e   t  e  n   t   i  o  n ,   % Fig. 1  The effects of pH on the recoveries of analytes using living  E. coli  immobilized on MWCNTPreconcentration and separation of some elements by  Escherichia coli  187  For living  E. coli  studies, after the analytes werecollected by the living microorganisms, they were killedand then the analytes retained were eluted using HNO 3 . Inother words, the elution of analytes was performed fromnon-living micoorganisms. The eluate was aspirated to theflame a few times for instrumental precision. However, toinvestigate the reproducibility of the whole procedure, thewhole procedure with living microorganisms i.e. collectionof analytes on  E. coli  immobilized MWCNT, their kiling,elution and aspiration to the flame) was repeated indepen-dently at least three times. Of course, independent replicateswere performed for non-living organisms as well. Since thenanoparticles always prevent the samples and eluents to bedrawn and ejected, it was no use of applying excessiveforce to provide higher flow rates. We applied the max flowrate (~3 mL min − 1 ) permitted by the sorbent by forcing maxforce because lower rates could not provide better improve-mentforrecoveries.Inaddition,thebestreproducibleflowratecould be maitained applying max force (i.e. max flow rate).For living  E. coli  studies, After the analytes werecollected by the living microorganisms, they were killedand then the analytes retained were eluted using HNO 3 . Inother words,, the elution of analytes from non-livingmicoorganisms was performed The eluate was aspirated tothe flame a few times for instrumental precision. However,to investigate the reproducibility of the whole procedure,the whole procedure with living microorganisms i.e.collection of analytes on  E. coli  immobilized MWCNT,their kiling, elution and aspiration to the flame) wasrepeated independently at least three times. Of course,independent replicates were performed for non-livingorganisms as well. The results were given as the averagesof at least three independent replicate analyses.To investigate the effects of potentially foreign ions on therecoveries of analytes, a model solution was prepared bydissolving 100 mg of NaCl, Na  2 SO 4 , Na  2 CO 3 , KCl, NH 4 Cland MgCl 2  of each in 100 mL of water simultaneously. Results and discussion We described here the effects of experimental parameters onsorption of Cd, Co, Cu, Ni by living and non-living  E. coli immobilized MWCNT as well as those on the elution of analytes from the sorbent and optimized for quantitativeanalysis. Finaly, method validation was made using waste-water certified reference material.Effect of pHOne of the most important parameters for sorption of analyte elements is the pH of analyte solution. The effect of  pH on the retention of analytes are depicted in Figs. 1 and2. The effect of pH on th sorption yield was investigated bymeans of the analyte concentration remaining in thesolution passed from the filter. Quantitative retention(>95%) was obtained pH  ≥ 7.0 if sample was drawn andejected back with flow rates around 3 mL min − 1 . Therefore,the pH of all solutions was adjusted to 7.0  –  8.0 throughout this work. Since nanocarbon layer was retarded the passageof solution through the filter, the flow rate could not beadjusted above 3 mL min − 1 . If it was forced to flow faster  by means of plunger of the syringe, the solution leaked out of the syringe mountable filter. 0204060801001200 2 4 6 8 10 pH    R  e   t  e  n   t   i  o  n ,   % Cd Co Cu Ni Fig. 2  The effects of pH on the recoveries of analytes using non-living  E. coli  immobilized on MWCNTAnalyte Mixture of analytes Individual analytesLiving  E. coli  onMWCNT, mmol g − 1  Non-living  E. coli  onMWCNT, mmol g − 1 Living  E. coli  onMWCNT, mmol g − 1  Non-living  E. coli  onMWCNT, mmol g − 1 Cd 0.043 0.037 0.124 0.061Co 0.059 0.032 0.144 0.072Cu 0.065 0.029 0.129 0.066 Ni 0.071 0.034 0.151 0.062 Table 1  The loading capacitiesof living and non-living  E. coli  immobilized MWCNTfor the individual and mixtureof analytes188 N. Aydemir et al.  At low pH values, there is a competition between H + ions and metal ions. The cell surface becomes more positively charged at low pH values which decrease theattraction between metal ions and the functional groups oncell wall. At high pH values, the cell surface becomes morenegatively charged, increasing the attraction until a maxi-mum is reached at around pH 7  –  8. This result is consistent other examples in the literature [9, 10, 13]. Capacities of sorbentsThe maximum loading capacities of living and non-living  E. coli  modified MWCNT were determined with respect toeach analyte in a solution containing all analytes as wellas in different solutions containing only one analyteindividually. The reason for this set of experiments wasto determine and compare the loading capacities of thetwo sorbents (living and non-living  E. coli  modifiedMWCNTs) with respect to each analyte as well as to seethe effect of competition among the analytes to be boundon the sorbent. In order to calculate the sorption capacity,we treated the sorbent with excess of analytes. If it iscompletely sorbed, we repeated the experiment withhigher amount of analyte until the sorbent did not retainall the analyte. When the sorbent began not to retain allthe analyte, from the remaining analyte we calculated thesorption capacity. As can be seen from Table 1, for all theanalytes, the loading capacities of the living  E. coli modified MWCNT were significantly more than thoseof the non-living  E. coli  modified MWCNT. However, it is likely that the amount of living  E. coli  cannot beexactly controlled because it still grows somewhat after immobilization causing higher capacities. However, theelevated sorption capacities of living bacteria weremuch above expectations due to their growing. There-fore, the another/real reason for this situation may bethat living  E. coli  immobilized on MWCNT sorbs moreanalyte than the died  E. coli . Nevertheless, we hesitateto make speculation and overemphasize on the reasonsfor higher loading capacities and low precision for living  E. coli .As expected, if there is only one analyte in the media,the loading capacities with respect to each analyte werenaturally higher than those found in a mixture of allanalytes simultaneously due to competition of analytes tooccupy active sites on the sorbent. On the other hand, whenthe four analytes were present at the same time, there wasno significant preference for the favor of an element. Inother words, the four elements shared the active sites on thesorbents.Effects of eluentsThe effects of different concentrations of HCl and HNO 3 on the elution of biosorbed metal ions from the living or non-living  E. coli  immobilized on MWCNT are depictedin Table 2. The analyte elements collected on the sorbent was quantitatively recovered with 0.5 mol L − 1 of HNO 3 applying a flow rate of 3 mL min − 1 . When the concenra-tion of HNO 3  was increased from 0.5 mol L − 1 to1.0 mol L − 1 , there was a drop in recovery. However, wedid not make a reasonable explanation and hesitated to Eluent, mol L − 1 Recovery*,%Living  E. coli  on MWCNT Non-living  E. coli  on MWCNTCd Co Cu Ni Cd Co Cu NiHCl, 0.5 mol L − 1 80±3 88±7 85±2 84±3 74±4 60±5 62±7 74±3HCl, 1.0 mol L − 1 85±4 95±6 86±6 98±3 89±5 73±4 76±2 90±2HNO 3,  0.5 mol L − 1 97±3 98±3 98±3 96±3 98±2 97±2 97±2 99±2HNO 3,  1.0 mol L − 1 88±3 98±3 91±4 89±4 85±3 87±3 84±2 88±3 Table 2  The effects of different acids at different concentrationson the elution (analyte:1  μ  g mL − 1 , sorbent: 0.1 g,sample and eluent volumes:25 mL, N:3) * Mean ± standard deviationElement Concentration, ng mL − 1 Recovery,%Certified value Found * Living Non-living Living Non-livingCd 25.0 24.6±2.0 24.5±1.5 98 98Co 25.0 24.2±2.9 24.1±2.5 97 96Cu 25.0 24.3±1.8 24.5±1.2 97 98 Ni 25.0 4.5±2.5 24.4±1.4 98 98 Table 3  Recoveries of Cd,Co, Cu, Ni in Waste-water (CRM-TM) using living andnon-living  E. coli  immobilizedMWCNT (sorbent: 0.1 g;Enrichment: 20 times; Number of repetitions: 3) * Mean ± standard deviationPreconcentration and separation of some elements by  Escherichia coli  189
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