Biosorption of copper(II) and zinc(II) from aqueous solution by Pseudomonas putida CZ1

Biosorption of copper(II) and zinc(II) from aqueous solution by Pseudomonas putida CZ1
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  Colloids and Surfaces B: Biointerfaces 46 (2005) 101–107 Biosorption of copper(II) and zinc(II) from aqueous solution by Pseudomonas putida  CZ1 Xin Cai Chen, Yuan Peng Wang, Qi Lin, Ji Yan Shi ∗ , Wei Xiang Wu, Ying Xu Chen  Department of Environmental Engineering, Zhejiang University, Hangzhou 310029, China Received 31 July 2005; received in revised form 10 October 2005; accepted 12 October 2005 Abstract To study  Pseudomonas putida  CZ1, having high tolerance to copper and zinc on the removal of toxic metals from aqueous solutions, thebiosorption of Cu(II) and Zn(II) by living and nonliving  P. putida  CZ1 were studied as functions of reaction time, initial pH of the solutionand metal concentration. It was found that the optimum pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5,respectively, for Cu(II) removal. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration increased. Theadsorption data with respect to both metals provide an excellent fit to the Langmuir isotherm. The binding capacity of living cells is significantlyhigher than that of nonliving cells at tested conditions. It demonstrated that about 40–50% of the metals were actively taken up by  P. putida  CZ1,withtheremainderbeingpassivelyboundtothebacterium.Moreover,desorptionefficiencyofCu(II)andZn(II)bylivingcellswas72.5and45.6%under 0.1M HCl and it was 95.3 and 83.8% by nonliving cells, respectively. It may be due to Cu(II) and Zn(II) uptake by the living cells enhancedby intracellular accumulation.© 2005 Elsevier B.V. All rights reserved. Keywords: Pseudomonas putida  CZ1; Copper; Zinc; Biosorption; Bioremediation 1. Introduction Human activities, such as mining operations and the dis-chargeofindustrialwastes,haveresultedintheaccumulationof metals in the environment [1,2]. Some metals (e.g. Ca, Co, Cr, Cu,Zn,Fe,K,Mg,Mn,NaandNi)areessentialmicro-nutrientsfor most, if not all, living organisms. One of the most importantfunctions of micro-nutrients is their role in metalloenzymes.However, when the concentrations of beneficial metals in theenvironment are excessively high, for instance, copper or zinc,can become toxic to these microorganisms and human [3].Therefore, growing attention is being paid to remove heavymetals from industrial waste water to protect the environmentandhumanhealth.Althoughremovaloftoxicheavymetalsfromindustrial wastewater has been practised for several decades,the conventional physico-chemical removal methods, such aschemical precipitation, electro winning, membrane separation,evaporation or resin ionic exchange, are usually expensive and ∗ Corresponding author. Tel.: +86 571 869 71975; fax: +86 571 869 71898.  E-mail address: (J.Y. Shi). sometimes, not effective. Therefore, there is a need for somealternative technique, which is efficient and cost effective.Biosorption, based on living or nonliving microorganisms orplants, could be such an alternative method of treatment. It hasdistinct advantages over conventional methods of treatments:the process does not produce chemical sludge, hence no sec-ondary pollution, more efficient and easy to operate. High metalbinding capacities of several biological materials have alreadybeen identified in part. Among the biosorbents, there are marinealgae [4], bacteria [5], yeasts [6], fungi and waste mycelia from the fermentation [7] and food industry [8]. Further, the capacities of these microorganisms to accumulate an amplerange of metal species have also been described [9,10,4].Compared to other metals, copper, especially zinc sequestra-tion by bacteria and in relation to  Pseudomonas  sp. remainslittle explored. So far some studies have been conducted on P. aeruginosa  [11–14],  Pseudomonas putida  [15–17] and  P.stutzeri  [18], but few of them investigated the characteristicsof living and nonliving cells of   P. putida  on the biosorption of Cu(II) and Zn(II) from aqueous solution. Moreover, the per-formance of any biosorbent also depends on biomass charac-teristics, physico-chemical properties of the target metals and 0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2005.10.003  102  X.C. Chen et al. / Colloids and Surfaces B: Biointerfaces 46 (2005) 101–107  the micro-environment of contact solution, i.e., the initial pH of solution, temperature and interaction with other ions, etc. [19].Therefore, it was considered that supplementary research in thisfield would be useful.Inthiswork,weaimedtostudythecharacteristicsof  P.putida CZ1 on its removal of toxic metals from aqueous solution. Theobjectives of the study are to compare the living and nonliv-ing cells of   P. putida  CZ1 in their removal capacity of Cu(II)and Zn(II) and to model the equilibria of the correspondingadsorption processes. For these purposes, the removal capacity,desorption efficiency of living and nonliving cells and variousfactors affecting the adsorption, such as react time, initial pH of the solution and metal concentration, were investigated with thebatch equilibration technique. 2. Materials and methods 2.1. Microorganism and its preparation for biosorption The bacterial strain used in the present study was  P. putida CZ1, isolated from metal contaminated soil of mining activi-ties as a copper and zinc tolerant strain described elsewhere. P. putida  CZ1 was grown and maintained on nutrient brothmedium, which contained 5.0g beef extract, 10.0g peptone and5.0gsodiumchlorideperliterwithaninitialpHof7.0–7.2.Cellsof   P. putida  CZ1 were inoculated into 250ml Erlenmeyer flaskscontaining 100ml of sterile medium and cultivated aerobicallyin an orbital rotary shaker (200rpm) at 30 ◦ C for 24h. Afterinoculation, cells were harvested by means of centrifugationat 13,000rpm for 5min and washed three times with deionizedwater.Livingcellswerepreparedbyresuspendingthecellpelletwith phosphate buffer solution (PBS) (pH 7.0). Cell concentra-tion in the suspension was determined by drying an aliquot ina pre-weighed aluminum foil container to a constant weight at60 ◦ C [20]. The remaining harvested cells which were freeze dried, autoclaved at 121 ◦ C for 30min, crushed in a blender andresuspended with deionized water, were defined as nonlivingcells. 2.2. Preparation of metal solutions Stock solutions (100mM) of Cu(II) and Zn(II) wereprepared by dissolving analytical grade CuSO 4 · 5H 2 O andZn(NO 3 ) 2 · 6H 2 O in distilled water. Before mixing with thebiosorbents, the stock solutions were diluted to required con-centration. 2.3. Metal biosorption experiments Biosorption experiments were conducted at 30 ◦ C in batchwith0.01gofthelivingornonlivingcellsina50mlplastictubecontaining 10ml of working solution volume. The tubes werethen shaked at 200rpm. Potassium nitrate (0.01M) was used asa supporting electrolyte for all experiments.Experiments for determining the kinetics of the process wereperformed at 63.5 and 65.3mg/l initial metal concentrations forCu(II) and Zn(II), respectively. Samples were taken at desiredintervals and were subsequently centrifuged at 15,000 × g  for5min. The heavy metal concentration in the resulting super-natant was determined. The impact of the solution pH on themetal biosorption was investigated in the same way except thatthe initial pH of the solutions was adjusted from 2.0 to 7.0 withthe addition of either 0.1M NaOH or 0.1M HCl. After 24hof incubation the metal concentration in supernatants was mea-sured. 2.4. Adsorption isotherms Cu(II)andZn(II)biosorptionisothermswereobtainedatcon-stant pH and ionic strength. For each tube, the initial Cu(II) andZn(II) concentrations were varied from 5.7 to 286.2 and 6.5 to279.4mg/l,respectively,andthenfollowedthesameproceduresfor the experiment of pH effect.To test the fit of data, the Langmuir and Freundlich isothermmodelswereappliedtothisstudy.TheLangmuirisothermmodelis valid for monolayer sorption onto surface and finite numberof identical sites and given by Eq. (1). q eq  = Q max bC eq 1 + bC eq (1)where  Q max  is the maximum amount of the metal ion per unitweight of cell to form a complete monolayer on the surfaceboundathigh C  eq  (mg/g)and b theconstantrelatedtotheaffinityof the binding sites,  Q max  represent a practical limiting adsorp-tion capacity when the surface is fully covered with metal ionsandassistsinthecomparisonofadsorptionperformance,partic-ularly in cases where the sorbent did not reach its full saturationin experiments.  Q max  and  b  can be determined from the linearplot of   C  eq  /  q eq  versus  C  eq .The empirical Freundlich isotherm model based on a hetero-geneous surface is given below by Eq. (2). q eq  = K f  C 1 /n eq  (2)where  K  f   and  n  are Freundlich constants characteristic of thesystem. K  f   and n areindicatorsofadsorptioncapacityandinten-sity, respectively. The values of   K  f   and  n  were evaluated fromthe intercept and the slope, respectively, of the linear plot of ln q eq  versus ln C  eq  based on experimental data. The Freundlichisotherm is also more widely used but provides on informationon the monolayer adsorption capacity, in contrast to the Lang-muir model [21–25]. 2.5. Desorption of Cu(II) and Zn(II) DesorptionofCu(II)andZn(II)frompreviouslyloadedlivingand nonliving  P. putida  CZ1 was studied by using 0.1M HCl aseluent. For this purpose, 0.01g of previously loaded living andnonliving  P. putida  CZ1 was added to 10ml of eluent in a 50mlplastic tube. After 24h of shaking, supernatants of centrifugedsamples were analyzed for the Cu(II) and Zn(II) concentrations.All experiments were conducted in triplicate. Control exper-iments without biomass were carried out in order to determinethe degree of removal of copper and zinc from solution by the   X.C. Chen et al. / Colloids and Surfaces B: Biointerfaces 46 (2005) 101–107   103 plastic tube. Extraneous metal contamination was found to benegligible. 2.6. Analysis of Cu(II) and Zn(II) The concentrations of initial and final Cu(II) and Zn(II) inthe biosorption experiments were determined by using flameatomic absorption spectrophotometry (FAAS, Thermo ElementMKII-M6).The results are given as a unit of adsorbed and unadsorbedmetal concentrations per gram of living or nonliving biosorbentin solution at equilibrium and calculated by Eq. (3). q e  = ( C 0 − C eq ) V X (3)where  X   is the biosorbent concentration (g/l),  q e  the adsorbedmetalionquantitypergramofbiosorbentatequilibrium(mg/g), C  0  the initial metal concentration (mg/l),  C  eq  the metal con-centration at equilibrium (mg/l) and  V   is the working solutionvolume. 3. Results and discussion 3.1. Effect of initial pH on Cu(II) and Zn(II) biosorption Earlier studies on heavy metal biosorption have shown thatpH was the single most important parameter affecting thebiosorptionprocess[26,27].Inallcases,metalbiosorptionbythe cells increases with increasing pH reaching to a maximum andthen showed a rapid decline in biosorption (Fig. 1a and b). Liv-ing cells demonstrated the maximum biosorption of 23.9mg/gCu(II)atpH5.0whereasthatwas12.8mg/gatpH4.5fornonliv-ing cells. The effect of pH on the biosorption capacity of Zn(II)withlivingandnonlivingcellsisshowninFig.1b.ItindicatedthesametrendsasinthebiosorptionofCu(II),butbothtypeofcellsachieved its maximum at pH 5.0. The maximum biosorption of Zn(II)bylivingcellswas26.9mg/gwhilethatwas12.4mg/gfornonliving cells. The results demonstrated that copper and zincbiosorption by living and nonliving cells were affected by theinitial pH of solution and suggest that the adsorption of Cu(II)and Zn(II) to the cells of   P. putida  CZ1 is mainly due to ionicattraction. Therefore, at low pH values the cell surface becomesmore positively charged, reducing the attraction between metalions and functional groups on the cell wall. In contrast, higherpH results in facilitation of the metal biosorption, since the cellsurfaceismorenegativelycharged[4,16,28,29].Andcopperand zincwilltransformintohydroxidecomplexesathighpHvalues,however, it could not be considered the biosorption behavior of the cells.However, the inconsistency in literature regarding the influ-ence of pH on biosorption seems to indicate the way that the pHwould alter the adsorption of metal ions to cells varies with thetypeofadsorbents(cells)andadsorbates(metalions)[12,16,17]. 3.2. Effect of reaction time on the biosorption Fig. 2 shows the effect of reaction time on the biosorptionof Cu(II) and Zn(II) by biosorbent from aqueous solutions.The rate of copper biosorption by the nonliving cells was veryrapid, reaching almost 96% of the maximum adsorption capac-ity within 10min of contact time. However, it took a longerbiosorption time for Zn(II), which reached approximate 90%of the maximum biosorption capacity within 60min and fol-lowed by a nearly constant after 5h. Noticeably, there wasa slight decreasing of Cu(II) sorption by nonliving cells at120min and then remained nearly constant. It may be becausea small amount of Cu(II) was released back into the solu-tion. Rapid Cu(II) biosorption by nonliving cells of   P. putida CZ1 is in agreement with Cu(II) biosorption by lyophilized P. aeruginosa  cells [14] and  P. cepacia  [3], which were com-pleted within 10min of reaction time. Such rapid biosorptionprocess have been correlated with the characteristics of thebiomass, and its physico-chemical interactions with the metalion [30]. Microbial metal uptake by nonliving cells, which ismetabolism-independent passive binding process to cell walls(adsorption), and to other external surfaces, and is generallyconsidered as a rapid process, taking place within a few minutes[31]. The rapid metal sorption is also highly desirable for suc-cessful deployment of the biosorbents for practical applications[32]. Fig. 1. Effect of initial pH on biosorption capacity of (a) Cu(II) and (b) Zn(II) by living and nonliving  Pseudomonas putida  CZ1.  104  X.C. Chen et al. / Colloids and Surfaces B: Biointerfaces 46 (2005) 101–107  Fig. 2. Effect of reaction time on (a) Cu(II) and (b) Zn(II) by living and nonliving  Pseudomonas putida  CZ1. Italsocanbeseenthatmetalsbiosorptionbylivingcellscon-sisted of two phases: a primary rapid phase (within 10–30min)andasecondslowphase.Itindicatesthatlivingcellsmaybenotonly having surface sorption but also slower and metabolism-dependentactiveuptakeofmetals.Inallcases,themetaladsorp-tioncapacityforlivingcellsisapparentlyhigherthanthatofnon-living cells (Fig. 2), which is consistent with Cu(II) biosorptionby resting cells and inactivated cells of   Pseudomonas aerugi-nosa PU21[12].Thisresultmaybeattributedtotheintracellularaccumulation of metal ions occurring in living cells, resulting inthe enhancement in metal uptake capacity. The other possibilityisthattheautoclave-sterilizationstep,whichmaydestroyorlosesome of metal binding sites, resulting in the decrease in metaluptake capacity of the nonliving cells. 3.3. Effects of initial metal concentrations on thebiosorption capacities Biosorption capacity of Cu(II) and Zn(II) by living cellsrapidly increased when the initial metal concentration increasedup to 50mg/l and a slight increase thereafter as shown in Fig. 3.Nonliving cells indicated a gradual increase of biosorption of Cu(II) and Zn(II) up to the concentration of 100mg/l and fol-lowedbyaslightincrease.WhentheinitialCu(II)concentrationwas increased from 5.7 to 286.2mg/l, the biosorption capacityof living cells increased from 3.8 to 28.6mg/g, but only from2.0 to 14.4mg/g for nonliving cells. The biosorption of Zn(II)seemed to the same trends as indicated for Cu(II). When theinitial concentration increased from 6.5 to 279.4mg/l, biosorp-tion increased from 4.5 to 26.1mg/g for living cells and 2.4to 15.5mg/g for nonliving cells. It was also found that thebiosorption capacities of living cells to Cu(II) and Zn(II) weresignificantly higher than that of the nonliving cells at all metalconcentrations. The increase of biosorption capacity of biomasswith the increase of metal concentration could be attributed tohigher probability of interaction between metal ions and biosor-bents[33].Moreover,higherinitialmetalconcentrationprovides an increased driving force to overcome all mass transfer resis-tanceofmetalsbetweenaqueousandsolidphasesandacceleratethe probable collision between metal ion and sorbents whichresults in higher metal uptake. Only slight difference in Cu(II)and Zn(II) binding capacity of nonliving  P. putida  CZ1 mightbe due to the similar properties of ionic size, atomic weight orreduction potential between these two metals [34]. Fig. 3. Effect of initial concentration of (a) Cu(II) and (b) Zn(II) on their biosorption by living and nonliving  Pseudomonas putida  CZ1.   X.C. Chen et al. / Colloids and Surfaces B: Biointerfaces 46 (2005) 101–107   105Fig. 4. Scatchard plots for (a) Cu(II) and (b) Zn(II) adsorption by living and nonliving  Pseudomonas putida  CZ1. 3.4. Langmuir and Freundlich adsorption isotherms Theisothermrepresentstheequilibriumrelationshipbetweenthemetaluptakebythesorbentandthefinalmetalconcentrationin the aqueous phase, showing the sorption capacity of the sor-bent [35]. The pH value of 5.0 was chosen as the experimentalcondition for the determination of adsorption isotherms exceptfor biosorption of nonliving  P. putida  CZ1 to Cu(II), which was4.5.To evaluate and compare the adsorption capacities of Cu(II)and Zn(II) by  P. putida  CZ1, the adsorption isotherms were ana-lyzed and fitted using Scatchard equation. When the Scatchardplot showed a deviation from linearity, greater emphasis wasplaced on the analysis of the adsorption data in terms of the Freundlich model in order to construct the adsorptionisotherms of the ligands at particular concentration in solu-tions. Fig. 4 presents the adsorption characteristics assessedfrom the Scatchard plot. In the adsorptions of metals, Scatchardanalysis of the equilibrium binding data for Cu(II) and Zn(II)on the cells of   P. putida  CZ1 nearly gave rise to a linearplot, indicating that the Langmuir model could be applied[31,36].The linearized Langmuir and Freundlich adsorptionisotherms of each metal for living and nonliving  P. putida CZ1 were shown in Figs. 5 and 6. The adsorption constants,metalbindingconstantandcorrelationcoefficientsforthemetalsobtained from Langmuir, Freundlich isotherms and Scatchardanalysis are given in Table 1. The adsorption data with respecttobothmetalsprovideanexcellentfittotheLangmuirisotherm.IntheexperimentsofCu(II)biosorption,the Q max  valueoflivingcells of   P. putida  CZ1 was 29.9mg/g, compared to 15.8mg/g of nonliving cells. It was also found that the  Q max  value of Zn(II)biosorption by living cells was higher (27.4mg/g) than that of nonliving cells (17.7mg/g). Compared to  P. aeruginosa  and  P.cepacia , copper biosorption capacity value of   P. putida  CZ1was lower [3,14]. But its biosorption capacity of living cells can compare well with  P. syringae  (25.4mg/g) [37] and  P. aerugi-nosa  PU21 (23.0mg/g) [12]. Moreover, both Cu(II) and Zn(II) biosorption by the cells of   P. putida  CZ1 are invariably higherthan  P. putida  (6.6 and 6.9mg/g) [17]. It is known that  b  is theconstant related to the affinity of the binding sites, which allowsus to make a comparison of the affinity of the biomass towardsthe metal ions. As shown in Table 1, the affinity of living cells to Cu(II) and Zn(II) (0.087 and 0.078l/mg, respectively) was Fig. 5. Langmuir adsorption isotherms of (a) Cu(II) and (b) Zn(II) on living and nonliving  Pseudomonas putida  CZ1.
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