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Chemosphere 70 (2008) 13291337 www.elsevier.com/locate/chemosphere

Removal of heavy metals and arsenic from contaminated soils using bioremediation and chelant extraction techniques
Katerina Vaxevanidou, Nymphodora Papassiopi *, Ioannis Paspaliaris
School of Mining and Metallurgical Engineering, National Technical University of Athens, GR 157 80 Zografos, Greece Received 14 August 2007; received in revised form 12 October 2007; accepted 15 October 2007 Available online 26 November 2007

Abstract A combined chemical and biological treatment scheme was evaluated in this study aiming at obtaining the simultaneous removal of metalloid arsenic and cationic heavy metals from contaminated soils. The treatment involved the use of the iron reducing microorganism Desulfuromonas palmitatis, whose activity was combined with the chelating strength of EDTA. Taking into consideration that soil iron oxides are the main scavengers of As, treatment with iron reducing microorganisms aimed at inducing the reductive dissolution of soil oxides and thus obtaining the release of the retained As. The main objective of using EDTA was the removal of metal contaminants, such as Pb and Zn, through the formation of soluble metal chelates. Experimental results however indicated that EDTA was also indispensable for the biological reduction of Fe(III) oxides. The bacterial activity was found to have a pronounced positive eect on the removal of arsenic, which increased from the value of 35% obtained during the pure chemical treatment up to 90% in the presence of D. palmitatis. In the case of Pb, the major part, i.e. approximately 85%, was removed from soil with purely chemical mechanisms, whereas the biological activity slightly improved the extraction, increasing the nal removal up to 90%. Co-treatment had negative eect only for Zn, whose removal was reduced from 80% under abiotic condition to approximately 50% in the presence of bacteria. 2007 Elsevier Ltd. All rights reserved.
Keywords: Soil remediation; Iron reducing bacteria; EDTA; Arsenic; Heavy metals

1. Introduction Contamination of soils with heavy metals and metalloids, such as lead, cadmium, arsenic, etc., represents a serious threat for the ecosystem and human health and requires the implementation of appropriate remedial measures. Multi-contaminated soils usually require more than one single treatment option so that all contaminants which may be present in the soil are eciently removed. A typical case is the simultaneous presence of cationic heavy metals and anionic metalloids, which cannot be treated with the same techniques due to their dierent chemical characteristics. For instance, cations, such as Pb, Zn or Cd, can be generally removed from the soil using either acids or chelating reagents, as it has been demonstrated by several
*

Corresponding author. Tel.: +30 210 7722298; fax: +30 210 7722168. E-mail address: papasiop@metal.ntua.gr (N. Papassiopi).

studies (Neale et al., 1997; Wasay et al., 1998; Papassiopi et al., 1999; Theodoratos et al., 2000; Tampouris et al., 2001; Sun et al., 2001; Manouchehri et al., 2006). Acidic environments favour desorption of metal cations as well as the dissolution of oxides or carbonates, which are common mineral phases found in contaminated soils. On the other hand, various chelating reagents, such as EDTA (ethylene diamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid) etc., can form strong soluble complexes with most divalent and trivalent metal contaminants, resulting thus in the recovery of these contaminants in the aqueous solution. Arsenic however, which occurs in the form of oxyanions, AsO4 3 or AsO3 3 , can not be mobilised with the same mechanisms. The techniques, which have been investigated for the removal of arsenic from soils take into consideration the strong association of As oxyanions with soil iron oxides and try to obtain the release of arsenic either through the selective desorption

0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.10.025

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K. Vaxevanidou et al. / Chemosphere 70 (2008) 13291337 Table 1 Typical reactions taking place during the treatment of soil with CaEDTA2 and D. palmitatis Typical reactions 8FeOOH CH3 COO 15H ! 8Fe2 2HCO 3 12H2 O ! FeEDTA2 + Ca2+ Fe2+ + CaEDTA2 ! PbEDTA2 + Ca2+ Pb + CaEDTA2 2 Zn + CaEDTA ! ZnEDTA2 + Ca2+ Ca2+ + HCO3 ! CaCO3 + H+ Pb, Zn: denote Pb and Zn species in soil.
D:palmitatis

of arsenic species from oxides surface or through the complete dissolution of iron oxides. Desorption of arsenic can be obtained by treating the soil with alkaline solutions or with solutions containing competitive oxyanions, like phosphates, which can replace arsenates on the surface coordination sites of oxides (Jackson and Miller, 2000; Alam et al., 2001; Jang et al., 2005). Acidic treatment, when applied for the remediation of arsenic contaminated soils, has as main target the dissolution of iron oxides (Tokunaga and Hakuta, 2001), in order to obtain indirect release of arsenic. Use of acids however is not appropriate for the treatment of calcareous soils due to their high acid buering capacity. Another alternative is the dissolution of Fe(III) oxides by reductive mechanisms, which can take place under less aggressive pH conditions, using either chemical or biological processes. Removal of As from contaminated soils applying anaerobic bioremediation techniques has been recently investigated by Chatain et al. (2005) and Ignatiadis and Battaglia-Brunet (2005). Both research teams showed that it is possible to obtain the mobilization of arsenic by stimulating the activity of indigenous microorganisms with addition of appropriate carbon and energy sources under anaerobic conditions. The degree of As mobilization was found to depend on the specic characteristics of contaminated soil samples, e.g. the crystallinity of iron oxides, the organic matter content, etc. (Ignatiadis and Battaglia-Brunet, 2005). The soil sample used in the current experimental work is heavily contaminated both with metal cations, mainly Pb and Zn, as well as with As oxyanions, due to past mining activities. Previous studies (Papassiopi et al., 1999; Theodoratos et al., 2000) have demonstrated that treatment of this soil with the strong chelator EDTA can be very ecient for the extraction of cationic metal contaminants, but has rather limited eciency in the removal of arsenic. It was also shown, that due to the calcareous matrix of the soil it is better to use the calcium salt of EDTA, Na2CaEDTA, instead of the commonly used sodium salts, in order to avoid the dissolution of calcite. In this work EDTA treatment is coupled with the use of the iron reducing microorganism Desulfuromonas palmitatis in order to enhance the reductive dissolution of iron oxides and obtain simultaneous release of arsenic in the aqueous solution. D. palmitatis is an anaerobic microorganism, which is able to use Fe(III) as terminal electron acceptor in its respiration process with simultaneous oxidation of a wide variety of organic compounds, such as acetate, fumarate, palmitate, stearate and succinate (Coates et al., 1995). The typical reactions, which are expected to take place during this treatment, are given in Table 1. The main contribution of D. palmitatis is the reductive dissolution of Fe(III)-oxides in order to obtain the release of the retained As (reaction (1) in Table 1). The addition of Na2CaEDTA has a double objective: (a) to maintain biogenic Fe(II) in solution (reaction (2) in Table 1) and (b) to remove the cationic metal contaminants, i.e. Pb, Zn, from soil (reactions (3) and (4) in Table 1). In the presence of carbonates, the

(1) (2) (3) (4) (5)

decomplexed calcium cations precipitate as CaCO3, as shown in reaction (5) of Table 1. Parameters examined during the experimental work include (a) the eect of NaHCO3, which was added to the slurries in order to facilitate precipitation of CaCO3 and increase the availability of EDTA for complexation with the target metals Pb, Zn and Fe(II) and (b) the liquid to solid (L/S) ratio, which was modied from the value of 20:1 to that of 2:1 ml g1. 2. Materials and methods 2.1. Soil material Selected properties of the contaminated soil material are presented in Table 2. It is a calcareous soil containing 39.5% of calcite equivalent and exhibiting alkaline pH of 8.65. It contains 4.8% total carbon, which is all inorganic. Chemical analysis indicated that the material contains high concentrations of contaminants, such as Pb and Zn, up to 19 600 and 24 000 mg kg1, respectively, As up to 950 mg kg1 and Cd 360 mg kg1. Speciation analysis showed that all soil As is in the pentavalent state, As(V). Examination of this soil material with scanning electron microscopy and microanalysis techniques revealed the presence of several discrete carbonate and arsenate minerals such as PbCO3, Pb3(CO3)2(OH)2, ZnCO3, Pb5(AsO4)3Cl, Zn2AsO4OH and FeAsO42H2O, as well as contaminants associated with Fe(III) oxy-hydroxides containing 25% PbO, 26% ZnO and 12% As2O5 (Xenidis et al., 2003).
Table 2 Selected properties of soil material pH CaCO3 equ. (%) Ctotal (%) Corg (%) 8.65 39.5 4.80 <0.1 mg kg1 Ca Mg Al Mn Fe Pb Zn Cd As 340600 8100 7300 1960 25000 19600 23900 357 946 mmol kg1 8494 333 270 36 448 95 365 3 13

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2.2. Microorganisms and media The microorganism used for the biological treatment of soil, i.e. D. palmitatis (DSM 12391), was purchased from the German Collection of Microorganisms DSMZ (Deutschen Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). The initial growth was carried out in the medium suggested by DSMZ (Medium 837), which contained (mM): NaCl 267, NaHCO3 30, KCl 10, NH4Cl 19, NaH2PO4 H2O 0.72, MgSO4 7H2O 0.81, CH3COONa 3H2O 10, Na-fumarate 50, selenite-tungstate solution 1 ml l1, vitamin solution 10 ml l1, trace element solution 10 ml l1. In this medium fumarate (50 mM) is used as electron acceptor and acetate (10 mM) as electron donor. Incubations were carried out at 30 C under anaerobic conditions (N2-CO2 atmosphere of 80:20 ratio). It is noted that during growth the pH of the medium is buered close to the value of 7.0 by the presence of NaHCO3 in the aqueous solutions and the partial pressure of CO2 (20.3 kPa) in the headspace of serum bottles. Growth of bacteria was determined by measuring the corresponding total protein with Bradford assay (Bradford, 1976). 2.3. Experimental procedure during the treatment of soil Standard anaerobic techniques were used throughout. All the tests were carried out using 125 ml serum bottles sealed with butyl rubbers and aluminium crimps. For the preparation of experimental slurries, a predetermined amount of soil was placed in the serum bottles. The aqueous phase constituents were added from concentrated stock solutions and the volume was adjusted with deionised water to 72 and 80 ml for the biological and control tests, respectively. The slurries were bubbled for approximately 20 min with N2:CO2 (80:20) to remove dissolved oxygen and then sealed and autoclaved. After cooling, the slurries corresponding to the biological tests were inoculated with 8 ml from a freshly developed D. palmitatis culture containing a cell concentration equivalent to approximately 70 5 mg l1 proteins and the serum bottles were incubated horizontally on a shaking waterbath. During the tests, aliquots of 1 ml were withdrawn from the serum bottles using a syringe. The samples were ltered through a 0.2 lm RC-membrane lter. The ltrate was diluted to 10 ml with deionised water and analysed for Fe, As, Pb, Zn, Cd, and Ca with atomic adsorption spectrophotometry. The pH, the redox potential, Eh, and the concentration of EDTA anions (Wallace and Hinton, 1970) were determined only in the nal solutions. 2.4. Experimental conditions Two series of tests were conducted and the experimental conditions are summarised in Table 3. The rst series aimed at comparing the biological and the chemical treatment of soil using the iron reducing microorganism

Table 3 Summary of experimental conditions A. Comparison of chemical and biological treatment eect of Na2CaEDTA and NaHCO3 addition Standard conditions: Liquid/solid ratio: L/S = 20:1 (80 ml solution: 4 g soil) Temperature: 30 C pH = 7.0 (buered by the presence of NaHCO3 and pCO2 = 0.2 atm in the headspace) Slurries autoclaved before inoculation Variable conditions: Composition of aqueous solution: Basic mediuma (with Na-acetate 10 mM) Basic mediuma (with Na-acetate 10 mM) + Na2CaEDTA 100 mM Basic mediuma (with Na-acetate 10 mM) + Na2CaEDTA 100 mM + NaHCO3 100 mM Inoculated and non inoculated tests B. Eect of L/S ratio Standard conditions: Temperature: 30 C Composition of aqueous solution: Basic mediuma (with Na-acetate 20 mM) + Na2CaEDTA 100 mM + NaHCO3 100 mM pH = 7.0 (buered by the presence of NaHCO3 and pCO2 = 0.2 atm in the headspace) Slurries not autoclaved Variable conditions: L/S ratios: L/S = 20:1, 10:1, 5:1, 2:1 (80 ml solution: 4 g, 8 g, 16 g and 40 g soil)
a Constituents of basic medium as described in Section 2.2 without addition of Na-fumarate.

D. palmitatis and the chelating reagent Na2CaEDTA, when these two treatment options are separately applied on the soil or when they are combined in order to maximize the overall eectiveness of the treatment. The eect of NaHCO3 addition was also investigated during these initial tests. The tests were carried out using a L/S ratio of 20:1 ml g1 and the operation temperature was controlled at 30 C. The main parameter that was varied during these experiments was the composition of the aqueous solution. Two tests (one inoculated and one similar as abiotic control) were conducted with an aqueous solution containing only the constituents of the basic culture medium for D. palmitatis growth. The medium was prepared with 10 mM of Na-acetate as electron donor but without addition of fumurate in order to compel the microorganism to use the Fe(III) oxides of soil as electron acceptor. Two other tests were carried out under similar conditions but adding 100 mM of Na2CaEDTA in the aqueous solution. In the last two experiments the solution was spiked with both Na2CaEDTA 100 mM and NaHCO3 100 mM. The second series of tests aimed at investigating the eect of L/S ratio. Experiments were carried out using both Na2CaEDTA and NaHCO3 in the aqueous solution. The volume of the solution was kept constant, 80 ml, and the amount of soil was increased from 4 g up to 40 g to obtain four levels of L/S ratios, i.e. 20:1, 10:1, 5:1 and 2:1 ml g1.

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3. Results and discussion 3.1. Evaluation of chemical and biological treatment eect of Na2CaEDTA and NaHCO3 additions Results concerning the dissolution of iron and the extraction of contaminants are shown in Fig. 1. When the treatment was carried out using only the medium of D. palmitatis growth (dotted lines with diamonds in Fig. 1), either under abiotic or biological conditions, there was no detectable dissolution for none of the four elements examined. It is thus evident that the various constituents of

growth medium cannot mobilise chemically the contaminants. Moreover, as seen in Fig. 1e, inoculation with D. palmitatis had no apparent eect on Fe. Despite the absence of any increase on the levels of aqueous Fe during the biological treatment of soil, reduction of Fe(III) oxides and reprecipation of biogenic Fe(II) cannot be excluded. Precipitation of insoluble ferrous compounds, such as magnetite, green rust, siderite, vivianite or ferrous arsenates, during the biological reduction of ferric oxides, have been reported in several studies (Fredrikson et al., 1998; Cummings et al., 1999; Papassiopi et al., 2003). To examine this possibility, the solid residues of the biological tests were

80

(a) Fe, abiotic tests

80 60

(e) Fe, biological tests

Fe, % dissolution

60
Medium

40 20 0 100

Medium + CaEDTA Medium + CaEDTA + NaHCO3

40
Medium

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Med. + CaEDTA Med. + CaEDTA + NaHCO3

(b) As, abiotic tests

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(f) As, biological tests

As, % extraction

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(c) Pb, abiotic tests

(g) Pb, biological tests

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Pb, % extraction

80 60 40 20 0

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(d) Zn, abiotic tests

100 80 60 40 20 0

(h) Zn, biological tests

Zn, % extraction

80 60 40 20 0 0 50

t, days

100

150

50

t, days

100

150

Fig. 1. Comparison of abiotic (a)(d) and biological (e)(h) tests. Eect of Na2CaEDTA and NaHCO3 presence in the aqueous solution. (a), (e) dissolution of Fe; (b), (f) extraction of A; (c), (g) extraction of Pb, (d), (h) extraction of Zn.

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subjected to an acidic treatment with 6 M HCl and the extract was analysed for Fe(II) with the phenanthroline method (Harvey et al., 1955). This treatment is reported to dissolve most ferrous iron compounds including the refractory mixed Fe(II)Fe(III) oxide, magnetite (Dong et al., 2000). The concentration of ferrous iron, Fe(II), was found to be below the detection limit of the assay in all solid residues. It was thus concluded that D. palmitatis were not able to reduce the Fe(III) oxides of soil during the experiment that was carried out without addition of EDTA. The addition of EDTA had very limited eect on the dissolution of iron in the abiotic test. As seen in Fig. 1a, only 1.4% of Fe was found in solution, which indicates that CaEDTA does not dissolve chemically the Fe(III) of soil oxides. It should be noticed that EDTA forms very strong complexes with trivalent iron, as indicated by the value of log stability constant, logKFe(III)-EDTA = 27.7, which is several orders of magnitude higher than that of CaEDTA, logKCaEDTA = 12.4 (at 25 C and I = 0; Gustafsson, 2003). However most common iron(III) oxides, such as goethite, have very low solubility at neutral pHs and cannot be dissolved by chelation with EDTA. Visual Minteq 2.2 software (Gustafsson, 2003) was used to compare the solubility of goethite in water with its solubility in the presence of CaEDTA2. Calculations indicated that at pH = 7 the solubility of FeOOH increases from 7.6 1013 M in water to 1.1 103 M in the presence of CaEDTA2 (0.1 M). The concentration of aqueous Fe measured in this experimental work was of the same order of magnitude with the calculated value, i.e. 0.3 mM. This amount of dissolved Fe(III) in the presence of EDTA corresponds to an important increase in solubility but represents only a small percentage of soil iron oxides, i.e. 1.4%. Comparison between Fig. 1a and e clearly illustrates that appreciable dissolution of iron up to 60% is obtained only when the soil is subjected to the biological treatment with D. palmitatis and more particularly when the activity of D. palmitatis is combined with the presence of EDTA. When the aqueous solution contains only the constituents of culture medium, the bacteria have no apparent eect on soil iron oxides. Dissolution of iron is observed only when the activity of D. palmitatis is supported by the presence of EDTA. It was not possible to determine directly the oxidation state of dissolved iron during the experiments that were carried out in the presence of EDTA, due to the strong interference of EDTA with the phenanthroline method. However, an indirect indication that iron dissolution took place via reductive mechanisms was provided by the redox potential values, which were determined in the nal solutions. More particularly, the Eh values were found to be 45 mV (at pH = 7.78) and 53 mV (at pH = 7.82) in the two experiments that were carried out in the presence of D. palmitatis and EDTA. This combination of Eh, pH values indicate the establishment of mild reducing conditions, which are typical in ecosystems with rich iron reducing microbial activity.

On the contrary, the aqueous solution resulting from the biological treatment without EDTA had a high redox potential, Eh 385 mV (at pH = 7.46), providing an additional evidence that D. palmitatis were not able to develop their reducing activity in the absence of EDTA. The role of chelating reagents during the microbial reduction of Fe(III)-oxides has been the subject of several studies (Urrutia et al., 1999; Roden and Urrutia, 2002; Nevin and Lovely, 2002. Urrutia et al. (1999) and Roden and Urrutia (2002) attribute the enhancing eect of chelating reagents to the complexation of biogenic Fe(II). The above researchers suggest that the ferrous iron, which is generated during the initial stages of bioreduction, inhibits the continuation of the microbial reductive activity by two main mechanisms, i.e. sorption on the surface of Fe(III)oxides and biosorption on the cell surfaces of the microorganisms. According to this mechanism, the inhibitory eect of Fe(II) is decreased when bioreduction takes place in the presence of Fe(II) complexants. By complexing Fe(II) as soon as it is produced, the surface of Fe(III) oxides, as well as the surface of bacteria cells, remain free and bioreduction can freely proceed. Nevin and Lovely (2002) propose a dierent mechanism. They suggest that Fe(III) chelators may alleviate the need for the iron reducing microorganisms to establish direct contact with the solid Fe(III) oxide, by dissolving through chelation a small amount of Fe(III). In this case the terminal electron acceptor in the microbial metabolic process is a soluble form of Fe(III), which is generally much more bioavailable compared to the Fe(III) on the surface of crystalline ferric oxides. In the case of our experiments, the positive role of EDTA in the bioreduction of Fe(III)-oxides is evident, but it is not possible to distinguish, which one of the above suggested mechanisms is preponderant. The release of As during the abiotic and biological treatment of soil is shown in Fig. 1b and f, respectively. Treatment of soil only with culture medium under abiotic conditions had no eect on As. The addition of Na2CaEDTA under abiotic conditions resulted in 40% As extraction. This fraction of As corresponds probably to Pb and Zn arsenate compounds, which can be dissolved chemically via the chelating action of EDTA. The biological treatment without EDTA caused only a slight mobilisation of As in the order of 3%. In the presence of Na2CaEDTA, the removal of As was more than 70% and the addition of NaHCO3 further improved the extraction up to 90%. The observed increase of As extraction in the biological tests is apparently related to the bacterial dissolution of Fe(III)-oxides. Lead extraction is only slightly aected by the presence of D. palmitatis. As shown in Fig. 1c and g, lead is dissolved up to 80% in 120 days and this extraction is mainly obtained through the chelating action of EDTA. The addition of NaHCO3 slightly increases Pb concentration in solution to 89% in the inoculated sample. It is worthwhile to mention that a high percentage of Pb, i.e. more than

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65%, was dissolved during the preparation of the slurries, which involved sterilization at 120 C for 20 min. Dierent results were obtained for Zn (Fig. 1d and h) since biological treatment seems to hinder the dissolution compared to simple chemical treatment. In the control test 90% of Zn was extracted in the aqueous phase, while only 70% was detected in the corresponding test with D. palmitatis. Without the addition of NaHCO3, zinc extraction in the inoculated test was further less, i.e. 62%. Immobilization of zinc in highly insoluble mineral phases during the microbial reduction of Fe(III) oxides has been reported by Cooper et al. (2000). These researchers have found that during the reduction of goethite (a-FeOOH) and lepidocrocite (c-FeOOH) with the iron reducing microorganism Shewanella putrefaciencs, Zn(II), which has the same ionic radius as Fe(II), was incorporated in the structure of characteristic Fe(II) mineral phases, such as siderite (FeCO3), magnetite (Fe3O4), and a new unidentied mineral. Zincsubstituted siderite was soluble in 0.5 M HCl, but Znenriched magnetite as well as the new unidentied mineral was insoluble when subjected to this treatment. Our experiments were conducted in the presence of CaEDTA2, which can dissolve many ferrous compounds. Calculations that were carried out using VMinteq 2.2 software indicated that FeCO3 can be quantitatively dissolved according to reaction: FeCO3 + CaEDTA2 ! FeEDTA2 + CaCO3. In contrast with siderite, magnetite, Fe3O4, cannot be dissolved by CaEDTA. The solubility of this compound was calculated to be very low, i.e. 0.32 mM at pH = 7 in the presence of 0.1 M CaEDTA2. Based on the above discussion a potential explanation for the lower degree of Zn dissolution during the biological treatment of soil with the Fe(III) reducing bacteria could be the sequestration of Zn in insoluble ferrous compounds, similar to that reported by Cooper et al. (2000). It should be mentioned however that it was not possible to detect any magnetite or other known ferrous compound, when the residue was subjected to XRD analysis. Even if such compounds have been formed, their relative percentage in the soil material would be rather low to be detected by XRD techniques. 3.2. Eect of L/S ratio Experiments investigating the eect of L/S ratio were conducted without prior sterilization of soil slurries. It was thus possible to evaluate whether any indigenous microorganisms can be stimulated to perform the microbial reduction of Fe(III)-oxides, as observed in other experimental studies (Chatain et al., 2005; Ignatiadis and Battaglia-Brunet, 2005). During the control tests the soil was mixed with aqueous solutions containing all micro and macro nutrients that were also used for the experiments carried out in the presence of D. palmitatis. However none of the control tests gave any indication of native iron reducing microbial activity. In all the cases, iron dissolution was less than 3% and was mainly due to the chemical action of EDTA. It is reminded that this soil is very poor in

organic matter as seen in Table 2, a fact which justies the absence of any apparent indigenous microbial activity. This series of tests was conducted using constant volume of aqueous solution (80 ml), constant initial concentration of Na2CaEDTA in the aqueous solution (100 mM) and variable amount of soil. Modifying the quantity of soil that was mixed with the constant solution volume had as consequence a proportional change in the amount of EDTA which is available for the chelation of target metals. In this treatment scheme the target metals are Pb and Zn, as main cationic contaminants, as well as biogenic Fe(II), which must be maintained in solution to avoid secondary precipitation of low solubility ferrous compounds. It must be noted that the initial amount of Fe + Pb + Zn in the soil is 909 mmol kg1 and that of Pb and Zn 463 mmol kg1. Considering the constant initial concentration 100 mM of Na2CaEDTA, it can be calculated that the molar ratios of EDTA versus the sum of target metals, i.e. EDTA/ (Fe + Pb + Zn), are equal to 0.22, 0.55, 1.1 and 2.2, when the L/S ratio is 2, 5, 10 and 20 ml g1, respectively. The experimental results at variable L/S ratios are presented in Fig. 2. Dissolution of Fe (Fig. 2a) was observed only in the biological tests that were carried out with L/S ratio greater than 2 ml g1. Percentage of dissolved Fe was 62% at L/S = 20 ml g1, 47% at L/S = 10 ml g1 and 14% at L/S = 5 ml g1. At the lower L/S ratio 2 ml g1, there was no Fe dissolution either in the control or in the biological test. In the dense slurries, i.e. L/S = 2 ml g1, all the chelating capacity of EDTA is used for the extraction of Pb and Zn and there are no more available chelant anions to induce the reduction of Fe(III)-oxides. This is illustrated in Fig. 3a and b presenting the distribution of divalent metal cations, which form complexes with EDTA, in the nal solutions of the tests carried out at various L/S ratios. In the case L/S = 2 ml g1, the sum of Pb and Zn concentrations in the aqueous solution is 98 mM, which is very close to the total EDTA concentration of 100 mM. As seen in Fig. 2b, the removal of As has the maximum value, 92%, at the higher L/S = 20 ml g1 ratio and is slightly lower, i.e. 87%, at L/S 10 ml g1. Decrease of L/S to 5 ml g1 depresses arsenic extraction to 62%. Finally operation at the lower L/S ratio 2 ml g1 reduces further arsenic removal down to the value of 15%. Lead is the element whose extraction is less aected by the variation of L/S ratios (Fig. 2c). At L/S 20 and 10 ml g1 Pb removal is close to 90%, at L/S 5 ml g1 is 80% and only at the lower ratio L/S 2 ml g1 Pb extraction is reduced to 62%. This is due to the fact that EDTA ligand has a higher anity for Pb than for Zn or Fe(II), as suggested by the relative stability of the corresponding 1-1 metal chelates, whose log stability constants (logKS) are equal to 19.7, 18.0 and 16.0 for Pb-, Zn- and Fe(II)-EDTA, respectively (at 25 C and I = 0; Gustafsson, 2003). As a consequence, when the amount of EDTA per gram of soil decreases at the lower L/S ratios, Pb is bound preferentially to the available EDTA to the expense of other competing elements.

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(a) Fe

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A B A B L/S 2/1 ml g-1 " 5/1 ml g-1 " EDTA/(Fe+Pb+Zn) 0.22 mol mol-1 " 0.55 mol mol-1 " A B A B L/S 10/1 ml g-1 " 20/1 ml g-1 " EDTA/(Fe+Pb+Zn) 1.1 mol mol-1 " 2.2 mol mol-1 "

A: Abiotic (control), B: Biological (inoculated with D. palmitatis)

Fig. 2. The eect of L/S ratio on metals extraction. Tests carried out at 30 C.

120

120 100 80 60 40 20 0

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Other Ca Fe Zn Pb

L/S=2

L/S=5

L/S=10

L/S=20

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L/S=5

L/S=10

L/S=20

Fig. 3. Distribution of Pb, Zn, Fe and Ca in the nal solutions for the tests carried out at various liquid to solid ratios. (a) Abiotic tests, (b) Biological tests.

The extraction of zinc during the abiotic tests varies between 75 and 80% at the L/S ratios 20, 10 and 5 ml g1 (Fig. 2d) and is reduced to 40% at the lowest L/S ratio 2 ml g1. In the presence of D. palmitatis, the extraction of Zn is adversely aected by the microbial generation of

Fe(II). This eect was observed in the previous experimental tests and is also apparent in this series. The pH and redox potential were measured in the aqueous solutions after the completion of the tests. The pH values were found to increase from the value of 7.94 to 8.29,

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8.35 and 8.80 as the L/S ratio was reduced from 20 ml g1 to 10, 5 and 2 ml g1, respectively. It should be reminded however that the treatment was carried out under a N2:CO2 (80:20) atmosphere, which contributes in maintaining the pH close to neutral values. When the serum bottles were opened, there was no more CO2 atmosphere and solution pHs were shifted to more alkaline values, buered mainly by the CaCO3 content of treated soils. The redox potentials, Eh, were close to zero, i.e. 12 mV, 7 mV and 6 mV, in the nal solutions of the biological tests that were carried out at L/S 20, 10 and 5 ml g1, in accordance with the observed reductive dissolution of Fe. On the contrary, the redox potential of the nal solution at L/S 2 ml g1 was oxidic 212 mV, conrming the absence of any appreciable iron reducing microbial activity in this test. The results of theses tests indicate that the optimum dosage of EDTA for this type of combined biological and chemical treatment is an amount of the chelant stoichiometrically equivalent with the sum of the three target metals Fe, Pb and Zn. Applying this dosage As, Pb and Zn extraction was 87, 90 and 50%, respectively. Doubling the dose of EDTA had no eect on Pb and Zn extraction and increased only marginally the removal of As from 87 to 92%. Several other studies investigating the eect of EDTA dosage on the removal of contaminants result in similar conclusions. Di Palma and Mecozzi (2007) have studied the removal of Cu, Pb and Zn from contaminated sediments and found that increasing the dose of EDTA does not improve the removal of contaminants but results in the increased dissolution of competing cations, such as Ca and Fe, from sediment major components. Similarly, Finzgar and Lestan (2007) pointed out that above a certain level higher dosages of EDTA do not result in appreciable gain in Pb and Zn removal. Another important aspect in this type of combined treatment is the fate of EDTA and its eventual biodegradation by the microorganisms which are used in this specic remediation scheme. Although not easily biodegradable, EDTA has been reported to decompose under the action of several pure or mixed microbial populations (Thomas et al., 1998). To evaluate this eect, EDTA concentration was measured in all nal solutions after completion of the tests. The concentration was found to range between 90 and 80 mM, indicating a loss of 1020 mM. This loss cannot be attributed exclusively to the action of D. palmitatis, because it has been also observed in the abiotic tests. Probable reasons for this loss may be adsorption of EDTA on the surface of soil particles or partial photocatalytic decomposition during the execution of the tests, due to the known photosensitivity of iron chelates (Lambert et al., 1963). 4. Conclusions A treatment scheme combining the action of the iron reducing microorganism D. palmitatis with the chelating strength of EDTA was investigated in the framework of this study. This treatment resulted in high extraction rates

both for the cationic metal contaminants and for the metalloid arsenic. More specically, the removal of As was greatly enhanced by the bacterial activity with a nal extraction up to 90% whereas only 35% was obtained when pure chemical treatment was applied. The major part of Pb, approximately 85%, was removed from soil with purely chemical mechanisms, i.e. via the chelating action of EDTA. However, the biological activity was found to improve by 10% the extraction of Pb, increasing the nal removal up to 95%. The removal of Zn was limited to 50% in the presence of bacteria, due probably to the sequestration of Zn in low solubility compounds with biogenic Fe(II). Under abiotic conditions the extraction of Zn was almost 80%. A sequential treatment, consisting of a pure chemical step followed by the addition of bacteria at a second stage could be possibly applied to avoid this eect. Acknowledgments This work received the nancial support of the European Commission in the framework of METALBIOREDUCTION project (Contract No. EVK1-CT-199900033). Part of the research was conducted with nancial support by the European Social Fund (75%) and National Resources (25%) (EPEAEK II) PYTHAGORAS. References
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