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GLOBALJOURNALOFENVIRONMENTALSCIENCEANDTECHNOLOGYPseudomonasveronii2EsurfaceinteractionswithZn(II)andCd(II)

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Pseudomonas veronii 2E surface interactions with Zn(II) and Cd(II)

Nerina Méndezb, Silvana A.M. Ramíreza, Helena M. Cerettia, Anita Zaltsa, Roberto Candalc,d, Diana L. Vulloa,b,*

a Área Química, Instituto de Ciencias, Universidad Nacional de General Sarmiento,

J.M. Gutiérrez 1150, B1613GSX, Los Polvorines, Buenos Aires, Argentina b Área Microbiología, Depto. de Química Biológica, Facultad de Ciencias Exactas y

Naturales (UBA), Pab. II, Piso 4, Ciudad Universitaria, (1428) Buenos Aires, Argentina c INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales (UBA), Pabellón II,

Ciudad Universitaria, (1428) Buenos Aires, Argentina d ECyT-UNSAM, Campus Miguelete, 25 de mayo y Francia, (1650) San Martín,

Buenos Aires, Argentina

*Author for correspondence: Diana L. Vullo, email: dvullo@ungs.edu.ar Received 8 May 2010; Accepted 30 Jun 2010

Abstract

Biosorption can be implemented as an immobilization technique for metal removal from wastewaters using either living or non-living biomass. The aim of this work is to study the biosorption process for Zn(II) and mixtures Zn(II)-Cd(II) by Pseudomonas veronii 2E, a microorganism with proven adsorptive capacity for Cd(II). A 82% of total Zn(II) biosorption was achieved in 30 hrs at 32ºC and the isotherm results suited the Freundlich

model. Cd(II) and Zn(II) simultaneous biosoption showed that all the adsorption sites are available for Cd(II), but only a fraction of them are for Zn(II). The electrophoretic mobility (µ) experiments that were performed with

either Zn(II) or Cd(II)-Zn(II) mixtures supported these results. In addition, electrical properties of bacterial surface were studied by isoelectric point determination and µ variations examined at different growth states. Zn(II) and

Cd(II) biosorption capacity of Pseudomonas veronii 2E makes possible its application as an efficient biosorbent in wastewater treatments. Keywords: Pseudomonas veronii 2E; Electrophoretic mobility; Biosorption; Anodic Stripping Voltammetry 1. Introduction

The use of microorganisms for environmental restoration involves exploiting microbial potential for remediation of metal contamination. Industrial effluents from electroplating, electronics and metal cleaning facilities, tanneries and other activities, contain large quantities of metals like Cd, Zn, Cu, Cr, Pb and Ni, reason for the need of wastewater treatment before being discharged to the environment. Biotreatments mediated by microorganisms are simple and usually compatible with the development of inexpensive technologies without causing an environmental damage [1-4].

Biosorption is a survival strategy of microorganisms based on metal-binding capacity of functional groups present in external cellular structures like exopolysaccharides (EPS), cell walls or other biopolymers [5�7]. These microbial interactions decrease the amount of bioavailable metal in the microenvironment, reducing toxic effects on cells. Biosorption can be used as an immobilization technique for metal removal from wastewaters using either living or non-living biomass [8-11]. Biomass capabilities

to immobilize metals rely on the type of biomass, the solution�s chemical composition

and the physicochemical conditions. After loading, the metals can be stripped from the biological matrix and the system may be regenerated for further sorption-desorption cycles thus opening the possibility for metal recovery. Compared to synthetic ion-exchange resins, biosorption methods seem to be more effective in removing dissolved metals at low concentrations (below 2-10 mg/L). The higher specificity of biosorbents to target metals is particularly interesting when high concentrations of alkaline-earth metals are present in wastewater so resin would be overloaded by these metals. Finally, biological systems offer the potential of genetic modification for a further increase of the specificity towards certain metal ions [1, 12, 13].

The aim of this work is to study the biosorption process for Zn(II) and mixtures of Zn(II)-Cd(II) mediated by Pseudomonas veronii 2E, a microorganism with proven adsorptive capacity for Cd(II) and potential application as biosorbent in biological treatments for industrial wastewaters. Biosorption was explored through two different approaches of metal-bacterial

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surface interactions studies. One is related to the study of adsorbent-adsorbate behaviour, obtained by measuring remaining metal in the solution. The second approach is the analysis of cell surface charges together with the modifications promoted by these metals and is accomplished by measuring bacterial electrophoretic mobility. 2. Experimental 2.1. Microorganism and culture conditions

Pseudomonas veronii 2E was isolated from polluted surface waters and identified both by biochemical and molecular methods as previously described [14]. This microorganism demonstrated adsorption capacity towards Cd(II) and Zn(II) at pH=7.5 together with its ability to develop biofilms on different polymeric matrices [14]. Bacteria were grown in PYG broth (casein peptone 2.5 g/L, yeast extract 1.25 g/L, glucose 0.5 g/L) supplemented with 0.05 mM of Zn(II) or Cd(II) according to the performed study. Growth was monitored by turbidity, measuring absorbance at 600 nm (A600nm) and culture pH was monitored when necessary.

For biosorption experiments, 50 mL early stationary phase cultures (A600nm= 1.1-1.2) in PYG broth supplemented with metals were centrifuged (3,000 x g, 15 min.). Cells were then washed and resuspended in 5 mL of water (18 MΩcm, Millipore). The resulting suspension

contained approximately 2 to 3 g dry weight living biomass /L.

For electrophoretic mobility experiments 2 mL early stationary phase cultures (A600nm= 1.1-1.2) in PYG broth (supplemented with metals, when needed) were centrifuged (5,800 x g, 15 min.). After that, cells were washed and resuspended in the same volume of water (18 MΩcm, Millipore). A 1:10 dilution

was prepared from this initial suspension to obtain a final concentration of approximately 50 mg dry weight living biomass /L. 2.2. Biosorption experiments

A biosorption assay was designed for testing bacterial retention of Cd(II) and Zn(II) in non-growth conditions [14]. Summarizing, each 10 mL biosoption mixture containing 5 mL of the 2 to 3 g dry weight /L cellular suspension (section 2.1.), Zn(II) or Zn(II)-Cd(II) at different concentrations, 10 mM buffer HEPES (N-2-hydroxyethyl]piperazine-N'-2-ethanesulfonic acid], pKa= 7.5, Aldrich) pH=7.5 was incubated at 32ºC and 200 rpm. Suspensions were then

centrifuged (3,000 x g, 20 min.) and filtered through 0.45 µm pore diameter cellulose

membrane; Zn or Cd equilibrium concentrations in cell free supernatants were determined in duplicates. Metal decreases related to a cell free

control biosorption mixture were obtained. Water 18 Mcm (Millipore) was used in every case. 2.2.1. Zn(II) biosorption kinetics: time optimization

Zinc biosorption was tested at different incubation times of the biosorption mixture, with initial 0.5 mM Zn(II). The biosorption mixtures were incubated from 0 to 30 hours and then were treated as mentioned for zinc quantification in supernatants.

2.2.2. Zn(II) biosorption isotherm

Experimental conditions previously established (pH=7.5, HEPES, 32ºC, 200 rpm,

incubation time 24 hours) were applied in order to obtain the biosorption isotherm. Pseudomonas veronii 2E cells were exposed to Zn(II) in the range of 0.005 to 0.5 mM. Zn(II) equilibrium concentration was measured in each filtered supernatant after incubation. 2.2.3. Biosorption of Cd(II) and Zn(II) mixtures

The described cellular suspensions were supplemented with Cd(II) and Zn(II) in order to evaluate simultaneous biosorption of metals at pH=7.5. Two different initial total cadmium and zinc concentrations were used for metal retention by cells: 0.5 mM in Cd(II)+Zn(II) and 0.1 mM in Cd(II)+Zn(II), as described in Table 1.

After 24 hrs of incubation, Cd(II) and Zn(II) equilibrium concentrations were analyzed in filtered supernatants. 2.2.4. Analytical procedures

Cd and Zn were determined by Anodic Stripping Voltammetry (ASV) using an Autolab PGStat10 (EcoChemie) and a Metrohm 663 VA polarographic stand (hanging mercury drop electrode mode). Merck Certipur certified standard solutions of Zn (1000 mg/L) and Cd (1000 mg/L) were used. 2.3. Electrophoretic mobility (µ) experiments

Electrophoretic mobility was determined in bacterial suspensions by laser light scattering using a Brookhaven 90-plus. In all cases ionic strength was fixed with 10 mM KCl. Results were the mean value of six measurements.

2.3.1. Bacterial isoelectric point

Bacterial electrophoretic mobility was evaluated at different pH values between 2 and 9.8 to determine the isoelectric point. HCl or NaOH were added to 1 mL of the 50 mg dry weight/L bacterial suspension (section 2.1.) and,

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after pH stabilization, a final dilution to 10 mL with 10 mM KCl was made. 2.3.2. Electrophoretic mobility and bacterial growth

The electrophoretic mobility was monitored at each stage of a bacterial batch culture. 50 mL of PYG broth was inoculated with 5 mL of an overnight culture. Aliquots representing different physiological states were taken every two hours of incubation (32ºC and 200 rpm) and turbidity (A600nm) and pH were measured in each sample. After centrifugation (5,800 x g, 15 min.), cells were washed and resuspended in the same volume of water (18 MΩcm, Millipore), being then a 1:10 dilution

from these initial suspensions prepared. For electrophoretic mobility determinations, 1 mL of each suspension was added to the final mixture (10 mL final volume).

2.3.3. Metal biosorption and electrophoretic mobility

Mixtures containing 1 mL of the 50 mg dry weight /L bacterial suspension (section 2.1.) with Zn(II) (0 to 0.75 mM) or mixed metal solutions (Zn(II)+Cd(II) 0.5 mM or 0.1 mM total),10 mM buffer HEPES (pH=7.5 or 6.5) and KCl solution to a final volume of 10 mL were prepared. Two different pH values were used according to the adsorptive capacity previously tested: biosorption was maximal at pH=7.5, while at pH=6.5 biosorption was not observed neither for Cd(II) nor Zn(II) [14]. Electrophoretic mobility determinations were carried out at pH=7.5, when metal�microbe interactions take place, and at pH=6.5 as a non-interaction control. Each measurement was performed after incubating the suspensions for 3 hrs at 32ºC.

Achievement of equilibrium condition was verified by two independent µ

measurements of the same sample with a delay of at least one hour, obtaining identical results for both.

3. Results and Discussion 3.1. Biosorption experiments Zn(II) biosorption kinetics showed that a 20% of Zn(II) was retained immediately after contacting cells with zinc solution. Nearly 50% total zinc was removed in the first 5 hours and 82% of total zinc was retained up to 30 hrs exposure. In the case of Cd(II), maximal biosorption efficiency (76.8%) was reached in 5 hours and no further changes in Cd(II) concentration were observed through the next 30 hours with the same experimental conditions [14]. Zinc biosorption isotherms were constructed in two Zn concentration ranges: 0 - 0.5 mM and 0 - 0.1 mM. Results were analyzed using the Langmuir and Freundlich models. On one hand, Langmuir model assumes that adsorption occurs in a monolayer, that all adsorption sites are identical and that no changes in adsorption free energy are observed at every site. According to this, the quantity of adsorbed metal (millimol Zn/ g of biomass), q, is given by: q = qmax . Ceq /(Kd + Ceq) or Ceq /q = (Kd / qmax)+ (Ceq / qmax) where qmax refers to the total number of adsorption sites, Ceq is Zn(II) final equilibrium concentration in supernatants and Kd the equilibrium constant for the dissociation of the surface complex. q was calculated as follows: q = (Ci- Ceq) x Vt / mt

where Ci is the initial Zn(II) concentration, Vt the total volume of the biosorption mixture assayed and mt the total biosorbent mass as dry weight. Zn(II) equilibrium concentration was determined as previously described.

Freundlich model, on the other hand, uses an empirical equation: q = KF . Ceq

1/n where KF and n are Freundlich constants

characteristic of each system and Ceq refers to the definition mentioned above. KF and n are indicators of adsorption capacity and intensity

Table 1. Cadmium and zinc concentrations for biosorption experiments.

Initial Cd (II) and Zn(II) total concentration 0.5 mM 0.1 mM

Cd(II) mM Zn(II) mM Cd(II) mM Zn(II) mM

0 0.5 0 0.1 0.1 0.4 0.01 0.09

0.25 0.25 0.025 0.075

0.4 0.1 0.05 0.05

0.5 0 0.075 0.025

0.09 0.01

0.1 0

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respectively and can be calculated from the linear plot ln q vs. ln Ceq. In this empirical model all sites on the surface are not considered equal. Adsorption becomes progressively more diffas more and more adsorbate accumulates. It is assumed that once the surface is covered, additional adsorbed species can still be accommodated, leaving no possible prediction of maximal monolayer adsorption. In other words, the Freundlich model does not consider the existence of a monolayer and a maximal adsorption capacity as the Langmuir model does [15].

Results obtained with veronii 2E are shown in Figure 1 A. At low Zn(II) initial concentrations q increases steeply as Zn(II) concentrations increase; at initial concentrations higher than 0.25 mM, though, there is a marked diminution in the slope and q increases slightly with Zn(II) concentration. Figure 1.B shows an adsorption isotherm performed with Zn(II) initial concentrations lower than 0.1 mM. In the assayed concentration ranges, Zn(II) did not saturate the adsorption capacity of bacteria.

The analysis of the isotherms indicates that Zn(II) biosorption matches the Freundlich model as shown in Figure 1.C. The Freundlich parameters calculated from the fitting of the experimental data are KF = 0.714 and n = 1.3,

Figure 1. A: Zn(II) biosorption isotherm for Zn 0C. Linearization by Freundlich and Langmuir models.

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respectively and can be calculated from the . In this empirical model

all sites on the surface are not considered equal. Adsorption becomes progressively more difficult as more and more adsorbate accumulates. It is assumed that once the surface is covered, additional adsorbed species can still be accommodated, leaving no possible prediction of maximal monolayer adsorption. In other words,

ot consider the existence of a monolayer and a maximal adsorption capacity as the Langmuir model does

Pseudomonas 2E are shown in Figure 1 A. At low

Zn(II) initial concentrations q increases steeply as Zn(II) concentrations increase; at initial concentrations higher than 0.25 mM, though,

inution in the slope and q increases slightly with Zn(II) concentration. Figure 1.B shows an adsorption isotherm performed with Zn(II) initial concentrations lower than 0.1 mM. In the assayed concentration ranges, Zn(II) did not saturate the adsorption

The analysis of the isotherms indicates that Zn(II) biosorption matches the Freundlich model as shown in Figure 1.C. The Freundlich parameters calculated from the fitting of the

= 0.714 and n = 1.3,

that are similar to the parameters found for other microorganisms [8, 16, 17].

Very interestingly, these results are different than those found for Cd(II) biosorption on the same bacteria. Unlike Zn(II), Cd(II) adsorption is better described by a Langmuir model, as was recently reported [14]. These differences in the adsorption behaviour suggest the presence of different sites for Cd(II) and Zn(II) on the bacterial cell wall.

Results of biosorption of cadmium and zinc from mixed solutions and for two Zn(II) + Cd(II) total concentrations (0.1 and 0.5 mM) are shown in Figure 2, where the initial concentrations were chosen considering the Cd(II) and Zn(II) biosorption isotherms ([14] and Figure 2). When only Zn(II) (or Cd(II)) is present in the solution, and for metal concentration = 0.5 mM, Cd(II) biosorption is higher than Zn(II) biosorption (Figure 2.A). In addition, the total amount of metal adsorbed increases as Cd(II) does. Figure 2.B shows that when the total metal concentration is 0.1 the amount of metal cations adsorbed remains practically constant within experimental error regardless of the concentration of each metal.

These results suggest that the surface acts as if it has completed the capacity to adsorb Zn for initial concentrations between 0.25 and 0.4 mM. The constancy in the sum of q values for Cd(II) and Zn(II) and the decrease of q when

A: Zn(II) biosorption isotherm for Zn 0-0.5 mM. B: Zn(II) biosorption isotherm for Zn 0-Langmuir models.

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lar to the parameters found for other

Very interestingly, these results are different than those found for Cd(II) biosorption on the same bacteria. Unlike Zn(II), Cd(II) adsorption is better described by a Langmuir

s recently reported [14]. These differences in the adsorption behaviour suggest the presence of different sites for Cd(II) and

Results of biosorption of cadmium and zinc from mixed solutions and for two Zn(II) +

tal concentrations (0.1 and 0.5 mM) are shown in Figure 2, where the initial concentrations were chosen considering the Cd(II) and Zn(II) biosorption isotherms ([14] and Figure 2). When only Zn(II) (or Cd(II)) is present in the solution, and for metal

entration = 0.5 mM, Cd(II) biosorption is higher than Zn(II) biosorption (Figure 2.A). In addition, the total amount of metal adsorbed increases as Cd(II) does. Figure 2.B shows that when the total metal concentration is 0.1 mM,

s adsorbed remains practically constant within experimental error

the concentration of each metal. These results suggest that the surface

acts as if it has completed the capacity to adsorb Zn for initial concentrations between 0.25 and

M. The constancy in the sum of q values for Cd(II) and Zn(II) and the decrease of q when

-0.1 mM.

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only Zn(II) is present in solution, indicate that the number of adsorption sites available for Cd(II) is greater than those available for Zn(II).

The effect of the simultaneous presence of both already mentioned metals during biosorption was compared to the results of the adsorption from individual solutions of Zn(II) or Cd(II) from previous work [14]. Metal ions adsorbed from the mixture have similar q as they do when they adsorb from individual solutions of the same concentration. This result points out the possibility of an independent behaviour of the metals during adsorption under our experimental conditions. 3.2. Electrophoretic mobility experiments

The interface between the outer cell envelope and the extracellular environment plays an important role within bacterial physiology. The outer cell surface mediates exchange and adhesive processes, affects interactions with immunological factors and is involved in cell growth and division [18]. From a chemical point of view, the macromolecules of the cell surface contain carboxylate, phosphate and amino functional groups. Ionization of these groups depends on pH conferring electrostatic charge to the cell periphery. Electrostatic charge affects polarity and hydrophilicity, which are fundamental properties for cell functions [19, 20, 21]. The surface charge is also responsible for electrokinetic phenomena, such as electrophoresis. It is assumed that the liquid

adhering to the particles surface (in this case the bacteria) and the mobile liquid are separated by a shear plane. The electrokinetic charge is the charge on the shear plane and the electric potential at the plane is defined as the zeta (ζ) potential which reflects the potential difference between the plane of shear and the bulk phase, being able to be estimated from electrophoretic mobility measurements by the Smoluchowski equation: ζ = (η . µ) / (ε0 . ε)

η is the viscosity of the medium, µ the

electrophoretic mobility, ε0 the permittivity of vacuum and finally, ε the dielectric constant of

the medium. Electrophoretic mobility is an intensive

property which indicates the particles� ability to

move when an electrical field is applied. It strictly depends on the particle�s charge and the

environment�s ionic strength. Establishing

experimental conditions for the determination of bacteria electrophoretic mobility represent a challenge since ionic strength, pH regulation and cell concentration need to be carefully established. The ionic strength had to be fixed at 10 mM KCl in order to buffer any possible change in the total ionic concentration due to the different concentrations of metal salts used in this work. The same importance exhibited the pH regulation: buffered solutions were strictly required since bacterial surface charges rely on pH. Cell concentration should be taken into account before testing metal-cell interactions; it

Figure 2. Simultaneous biosorption of Cd(II) and Zn(II) for total initial concentration conditions: A. 0.5 mM and B. 0.1 mM.

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must be clearly over the detection limits of the Brookhaven zetameter, so as to reach reliable µ

measurements.

3.2.1. Bacterial isoelectric point (pI) pI is defined as the pH value at which µ

becomes 0. Results of pI determination are shown in Figure 3, where pI is clearly close to pH=2. At higher pH values, µ shifts to more

negative values, referring to a higher charge on cell surface. At pH>6, µ remains almost constant

(between -3.7 and -4 (µm/s)/(V/cm)), providing

enough evidence to predict that this pH range is optimal to determine the effect of metal adsorption on mobility since µ variation could be

clearly attributed to the interactions between the cell surface and the metal cation. 3.2.2. Electrophoretic mobility and bacterial

growth The different bacterial physiological

states observed in batch cultures were characterized by the cell surface electrical properties. Figure 4 shows µ of bacterial

suspensions during bacterial growth. Throughout the exponential phase of growth evolution, cell division remains constant and µ exhibits more

negative values than in initial phases. These results could be related to the cell wall composition and the smaller cell size observed in this growing stage. When cells reach the stationary phase of growth, µ shifts to more

positive values. In this stage, cell size increases and changes in cell wall structures, related to non-growth states, are produced. This result is consistent with the reported for Pseudomonas aeruginosa and Escherichia coli, that are also Gram negative bacteria [19, 20]. Thus, µ changes

Figure 3. Determination of the isoelectric point of Pseudomonas veronii 2E.

Figure 4. Pseudomonas veronii 2E electrophoretic mobility during growth.

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could be attributed to cell interaction with Cd(II) or Zn(II) when working with non-growing cells. 3.2.3. Metal biosorption and electrophoretic mobility

Figure 5 shows the bacterial electrophoretic mobility in presence of Zn(II) (0 to 0.5 mM) at pH=7.5, when biosorption is maximal, and at pH=6.5 as well, when no Zn-cell interactions are detected. At pH=6.5 no significant changes in µ were observed. At

pH=7.5, however, for low Zn(II) concentrations the mobility slightly shifts to more negative values but, at concentrations higher than 0.05 mM, µ sharply increases until a value close to -1.7 (m/s)(V/cm). These results suggest that the outer negative charge on bacterial surface decreases directly as a consequence of Zn(II)-cell interactions.

Bacterial electrophoretic mobility was tested in presence of Zn(II) and Cd(II) mixtures (pH=7.5) and for the same total metal concentrations used in biosorption experiments: 0.5 mM and 0.1 mM as shown in Figure 6. As Cd(II) concentration increases, µ shifts to more

negative values in the 0.5 mM experiment. When Zn(II) concentration is maximal (and Cd(II) is absent), µ is more positive than when Cd(II)

concentration is maximal (and Zn(II) is absent) (Figure 6.A). When the total metal concentration is fixed at 0.1 mM, µ shifts to more positive

values when Cd(II) is present, remaining unchanged for all tested solutions containing Cd(II) (c.a. -2.35 (m/s)(V/cm)) (Figure 6.B).

The differences observed in µ of

bacterial cells in the two extreme cases, when only Cd(II) or Zn(II) are adsorbed, can be explained by considering the shielding effect of ions on bacterial negative surface charges. This effect derives from the fact that Zn(II) cations are smaller than Cd(II) ions, being more efficient in shielding the bacteria surface charge. The higher the electrokinetic charge, the higher will be the mobility.

Cell electrophoretic mobility is the integrated response to surface modifications introduced by metal adsorption as can be clearly seen in the experiments with variable concentration of Cd(II) and Zn(II). In the case in which the total metal concentration is kept at 0.1 mM, only small variations in µ are observed:

mobility is modulated by surface contributions of each metal as total q remains practically unchanged regardless of the concentration of each metal in the solution (Figure 6.B). When the total metal concentration is kept at 0.5 mM, the Cd(II) concentration increases while Zn(II) concentration decreases, the shielding effect of the metal cations on the surface charge decreases and the mobility increases monotonically until a value, that typically corresponds to Cd(II)-saturated bacteria, is reached (Figure 6.A).

These results suggest the presence of different binding sites for Zn(II) and Cd(II) and reinforce the idea that all the adsorption sites are available for Cd(II) but only a fraction is available for Zn(II), as concluded from Figure 2.

Figure 5. Electrophoretic mobility of Pesudomonas veronii 2E in presence of different Zn(II) concentrations at optimal biosorption conditions (pH=7.5) and no biosorption conditions (pH=6.5).

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4. Conclusions

Results obtained from both methodologies applied to metal-Pseudomonas veronii 2E surface interaction studies suggest the presence of different interactions of Zn(II) and Cd(II) with the microbial cells. Any possibility of competitive effects between Cd(II)and Zn(II) remains to be explored.

The high adsorption specificity for cadmium and zinc together with the easy and low cost production of the adsorbent make the application of Pseudomonas veronii 2E as an efficient biosorbent in wastewater treatments possible. Acknowledgment

This work was supported by the Universidad Nacional de General Sarmiento and Agencia Nacional de Promoción Científica y

Tecnológica (ANPCyT), PICTO Nº36782. We are grateful to Miss Leticia Rossi

for the English language revision.

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Cite this article as: Diana L. Vullo et al.: Pseudomonas veronii 2E surface interactions with Zn(II) and Cd(II). Global J. Environ. Sci. Technol. 2011, 1: 3