Applied Radiation and Isotopes 89 (2014) 85–94

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Behaviour of 99Tc in aqueous solutions in the presence of iron oxides and microorganisms Rūta Druteikienė a,n, Benedikta Lukšienė a, Dalia Pečiulytė b, Kęstutis Mažeika a, Arūnas Gudelis a, Dalis Baltrūnas a a b

Center for Physical Sciences and Technology, Savanorių ave. 231, LT-02300 Vilnius, Lithuania Nature Research Center, Akademijos str. 2, LT-08412 Vilnius, Lithuania

H I G H L I G H T S

 Abilities of wustite/magnetite and hematite to accumulate 99Tc(VII) were investigated.  The effect of microbial activity on the migration behaviour of technetium was tested. xxx Gradual sorption of [99Tc] TcO4 from aquatic solution onto wustite/magnetite (FeO/Fe3O4) under aerobic conditions was observed.  Specific microorganisms induced sorption of 99Tc(VII) by hematite.

art ic l e i nf o

a b s t r a c t

Article history: Received 23 April 2013 Received in revised form 13 January 2014 Accepted 18 February 2014 Available online 25 February 2014

A set of experiments was performed to determine the factors that influence TcO4 interaction with Febearing minerals and to explore the effect of microbial activity on the behaviour of Tc(VII) in solution, in the presence of iron oxides under oxidizing medium. Gradual sorption of TcO4 (aq) onto wustite/ magnetite was observed under alkaline conditions (pH 8–9). No pronounced effect of TcO4 (aq) interaction with hematite was observed in the investigating alkaline systems. At low pH values (2.7– 4.5), TcO4 retention on hematite increases, suggesting that the process is dependent on pH. Sorption of 99 Tc (VII) onto hematite at pH 7.6–8.0 was achieved because of the presence of specific microorganisms. & 2014 Elsevier Ltd. All rights reserved.

Keywords: Technetium Wustite/magnetite Hematite Microorganism

1. Introduction The long-lived radionuclides produced by nuclear accidents and in the operative fuel cycle represent a major concern to the public if they enter the environment (Liljenzin, 2002; Choppin, 2005; Jaisi et al., 2009; Buechele et al., 2012). To assess the potential of these radionuclides to cause harm to humans, their geochemical and biological behaviour must be evaluated. The fission product 99Tc is presently considered as a key mobile radionuclide for the disposal of spent fuel and high-level radioactive waste (Begg et al., 2007). Nuclear installations generate a relatively large amount of 99Tc (half-life  2.1  105 years), which originates from a 6% yield from the fission of 235U in nuclear power plants (Maset et al., 2006). Technetium has been introduced into the environment because of authorized and accidental discharges. For example, about 140 PBq of 99Tc was produced in nuclear power

n

Corresponding author. Tel.: þ 370 5 2661648; fax: þ 370 5 2602317. E-mail address: ruta@ar.fi.lt (R. Druteikienė).

http://dx.doi.org/10.1016/j.apradiso.2014.02.020 0969-8043 & 2014 Elsevier Ltd. All rights reserved.

plants worldwide from 1976 to 2006, and it was determined that until 2010, nearly 10 GBq of 99Tc was released to the environment (Shi et al., 2012). Because most of 99Tc remains in the nuclear fuel, it is one of the most important radionuclides with respect to the management of spent fuel and radioactive waste. Techetium-99 is relevant in geochemical systems of both intermediate and highlevel nuclear waste repositories; it is present in aquifers, the marine environment and rivers (Standring et al., 2002; KeithRoach et al., 2003; Frederickson et al., 2004; Burke et al., 2006; Shi et al., 2012). Therefore, 99Tc mobility is of major interest in environmental science, and is an important concern for the safe disposal of radioactive waste. Most of the studies investigating geochemical processes and their impact on the transformation and dynamics of technetium have demonstrated that the Tc behaviour is complex, depending on a series of factors such as electron transfer reactions (redox), biogenic factors, and microbial activity (Tagami and Uchida, 1996; Istok et al., 2004; Abdelouas et al., 2005; Peretyazhko et al., 2012). Technetium is a redox sensitive radionuclide and its migration xxxbehaviour primarily depends on its oxidation state, which in

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turn depends on redox conditions of the surrounding environment (Lieser and Bauscher, 1987; Lloyd and Macaskie, 1996; Wildung et al., 2000; Abdelouas et al., 2005; Begg et al., 2007; Zachara et al., 2007). Under oxic conditions, technetium forms the highly soluble pertechnetate anion, TcO4 , which weakly sorbs onto mineral surfaces (Lieser and Bauscher, 1987). However, the reduction and oxidation conditions of geochemical surroundings can bring about an alteration of the technetium oxidation state. In anoxic surroundings, the pertechnetate anion TcO4 is reduced to the lower valence form of Tc(IV), which through hydrolysis forms a sparingly soluble solid phase (e.g., TcO2 nH2O) which can be sorbed to mineral phases or organic fractions (Maes et al., 2004; Burke et al., 2005). A number of experiments have been conducted in order to assess the redox behaviour of technetium in natural sediments containing sedimentassociated Fe and Mn oxide, metal-reducing bacteria, organic cocontaminants, or dissolved iron and iron minerals (Frederickson et al., 2004; Maset et al., 2006; Peretyazhko et al., 2009). Reduction of TcO4 under iron- and nitrate-reducing conditions, as well as under sulphate-reducing conditions, was observed (Wharton et al., 2000; Istok et al., 2004; Burke et al., 2005; Abdelouas et al., 2005). The treatment of high-level radioactive waste and management of the nuclear waste repositories are relevant to the technetium chemistry. The main problem is the presence of different technetium species in heterogeneous waste (Lukens et al., 2002). Radiolysis of pertechnetate has been examined in the high-level nuclear waste tanks at the Savannah River and Hanford Sites (Lukens et al., 2001, 2002). The results show that the radiolysis of TcO4 in highly alkaline solution in the presence of Ō scavengers is – one more mechanism of reducing technetium by NO23 . Radiolysis  of TcO4 in alkaline solutions containing organic compounds, causes the formation of both insoluble radiolysis product TcO2 nH2O, and the soluble, lower-valent Tc complexes. Interaction between technetium and microorganisms plays an important role in the environmental fate of the radionuclide with a multitude of mechanisms affecting transformations of its oxidation state (Marshall et al., 2009; Mohapatra et al., 2010; Borsh et al., 2010). Studies of Tc behaviour in biologically active, organicrich soil show Tc(VII) reduction in the presence of anaerobic metal- and sulphate-reducing bacteria. However, nitrifying bacteria, growing under aerobic/anaerobic reducing conditions, do not affect the reduction of Tc(VII) to Tc(IV). Organic matter is a determining factor in the immobilization of Tc(IV) in soil because of its ability to form strong complex (Abdelouas et al., 2005). The fate of Tc(VII) introduced into organic-rich environments was studied using X-ray absorption fine structure spectroscopy (Maes et al., 2004). The authors reported the formation of small Tc(IV) polymer oxides, which might aggregate into larger units that could interact with dissolved/immobile humic substances, suggesting a potentially new model for metal-humate complexation in organicrich environments. A study of the effects of progressive anoxia on the solubility of Tc in sediments has demonstrated that the removal of Tc(VII) from water to estuarine sediments is conditioned by reduction of Tc(VII) to Tc(IV) during the development of microbial anoxia, and coincides with Fe(III) reduction in the presence of sulphate-reducing bacteria (Burke et al., 2005). The reduction of Tc(VII) by dissimilation in microorganisms under neutral, acidic and alkaline conditions was extensively studied (Lyalikova and Khizhnyak, 1996; Lloyd et al., 1997, 1999, 2000; Wildung et al., 2000). The biotransformation of redox sensitive radionuclides is the complex process, achieved by the direct enzymatic interaction at the metal/microorganism interface or by abiotic reaction of the radionuclide with microbial reduction products (Mohapatra et al., 2010; Law et al., 2010). In summary, different studies have reported that the potential either for migration or retention of 99Tc at contaminated sites depends on the geochemical behaviour of 99Tc determined by its

chemical speciation, the physical characteristics of the environment (mineralogy, pH, complexing agents, redox conditions), and on the microbial activity. However, most of the studies have been related to the investigation of technetium migration/retention in anaerobic reducing environments. The behaviour of technetium under aerobic conditions remains poorly characterized, regardless of a broad range of direct and indirect processes formed in the aerobic environment, where technetium can undergo chemically or biologically controlled redox changes. The goal of the present study was to investigate the behaviour of 99Tc(VII) in the heterogeneous water system containing welldefined amounts of hematite and magnetite with emphasis on transformation of the oxidation state through microbial mediated processes under aerobic conditions. A set of experiments was performed to determine the factors (mineralogy, pH, incubation time, microorganisms) that influenced TcO4 (aq) sorption onto iron oxides, and to explore the capability of microorganisms to participate in metal/radionuclide mobility processes, paying special attention to their involvement in redox processes.

2. Experimental 2.1. Materials and methods Batch experiments were conducted in the laboratory to examine the selective adsorption characteristics of 99Tc(VII) under ambient conditions. In each experiment, synthetic powdered iron oxides (hematite and wustite/magnetite) and/or microorganisms isolated from groundwater and waterlogged soils were used. The powders of synthetic iron oxides were purchased from AO REACHIM (Russia); since the compositions of iron oxides were not clear, Mössbauer spectroscopy was used for verification of the iron oxides. The particle sizes of iron oxides were determined by SEM Helios NanoLab 650 (FEI). The solutions and iron oxide (Fe2O3 and Fe3O4/FeO) powder suspensions were prepared in deionised water (TKA LAB MICRO system, conductivity 0.055 μS/cm; TOC o10 ppb). Technetium-99 was purchased from National Physical Laboratory (UK) in 0.1 mol dm  3 NH4OH aqueous solution. For the batch experiments TcO4 (3.5  10  6 mol dm  3) solutions were prepared by diluting aliquots of the stock solution. All batch experiments were performed in triplicate. All reagents used were of analytical grade. 2.1.1. Microorganisms 2.1.1.1. Isolation. Cultures of bacteria and fungi were isolated from samples of groundwater and waterlogged soil. For isolation of the microorganisms, groundwater samples were serially diluted by four orders of magnitude. Aliquots (0.1 mL) of the undiluted water and each dilution were spread onto appropriate media. Undiluted water samples were used for isolation of actinomycetes. The isolation of fungi and bacteria was performed using diluted and undiluted water samples. Ten grams of each soil sample were mixed with 95 mL of sterile (0.1 M, pH 7.0) phosphate buffer in 150 mL conical flasks and were shaken for 10 min at 150 rpm. Soil suspensions were decanted and serial dilutions by six orders of magnitude were prepared. Different plating media were used for the isolation of microorganisms. Samples for the fungi isolation were plated either onto the 2% malt extract agar (MEA, Liofilchem, Italy) or a potato dextrose agar (PDA, Liofilchem, Italy) in Petri dishes (9 cm diameter); bacteria were isolated on a nutrient agar (NA, Liofilchem, Italy) and actinomycetes on a Starch-casein agar (SCA, Liofilchem, Italy). Nystatin (50 μg mL  1) was added to avoid fungal contamination in the actinomycetes cultures; chloramphenicol

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(60 μg mL  1) was added to avoid bacterial growth in the fungi cultures. Fungi, bacteria and actinomycetes were incubated at 2572 1C, 3272 1C and 2872 1C, respectively, in the dark. After incubation, well separated colonies were counted (morphologically different colonies) and the percentage of occurrence was analysed to determine potential strains for further application in the investigation of 99Tc relationships with soil and water microorganisms. Dominant strains were maintained on nutrient agar slants at 4 1C. 2.1.1.2. Identification. Fungi from the primary cultures were subcultured onto a malt extract agar (MEA, Liofilchem, Italy) and a potato dextrose agar (PDA, Liofilchem, Italy), a corn meal agar (CMA, Liofilchem, Italy), and Czapek-Dox agar (CDA; Liofilchem, Italy) and were incubated for up to 21 days at 2571 1C. Morphologically different colonies of bacteria and actinobacteria were sub-cultured on the fresh NA and SCA plates, respectively. Bacteria were identified and attributed to the species level on the basis of their physiological characteristics, using the Gram staining reaction and other biochemical tests (Holt and Bergey, 1994). Morphology of the fungi was analysed both by observing colonies under a stereomicroscope (Nikon SMZ 745Te;  10 magnification) and by observing prepared slides under a light microscope (Motic B1 Series;  400 and  1000 magnification). The fungi were identified based on the morphological characteristics and taxonomic keys described by Ellis and Ellis (1997), Pitt (1985), Kiffer and Morelet (2000), Domsh et al. (2007). Velvety isolates of the actinomycetes were examined to determine their Gram-positive morphology and formation of long filamentous cells. Their microstructure was observed by the cover slip method; mycelium structure, colour, as well as arrangement of conidiophores and arthrospores on the mycelium, were observed microscopically (Motic B1 series) using an oil immersion (  100) objective. Visually determined structures were compared with those described by Claus and Berkeley (1986), to confirm species of the isolates. After identification, separate strains of the microbial species, sub-cultured on the nutrient media slants, were maintained in the Culture Collection of the Nature Research Centre (Vilnius, Lithuania). The Gram-positive actinobacterium (actinomycete) (Streptomycete coelicolor strain DPA-Tc3), three Gram-negative bacteria (Bacillus mycoides DPB-Tc2, Rhodococcus rubber DPB-Tc4, Pseudomonas

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aeruginosa DPK-TcX), and three fungi (Aspergillus niger Tiegh. DPFTc7, Penicillium simplicissimum (Oudem.) Thom DPF-Tc6, Spicaria sp. DPB-Tc) dominating in the groundwater as well as the Gramnegative bacterium Arthrobacter globiformis strain DPB-Tc11, grampositive bacterium Cellulomonas cellulans DPB-8, and fungus Fusarium oxysporum Schltdl. strain DPF-Tc3 dominating in the waterlogged soil were applied for the investigation of Tc behaviour in the presence of iron oxides. In addition to the dominance in natural habitats, specific microbial strains were chosen for further investigation based on their dependence on oxygen, their ability to reduce nitrate, form H2S, produce organic acid and to tolerate different pH values, as observed during the earlier investigation (Lukšienė et al., 2012). Chosen microbial strains were sub-cultured for 4–7 days at 25–32 1C in different media (depending on the microorganism): bacteria in a nutrient broth (NB; Liofilchem, Italy), actinomycete – in a starch casein medium (SC, without agar), and fungi – in malt extract broth (MEB; Liofilchem, Italy). After incubation by aerating on a shaker at 140 rpm, biomass of the microorganisms was separated by centrifugation (bacteria and actinomycetes) or by filtration (fungi), and then it was washed with deionised sterile water.

2.1.2. Iron oxides Powders of synthetic iron oxides (AO REACHIM, Russia) were used for 99Tc(VII) sorption experiments. Based on SEM analysis, particle sizes of wustite/magnetite were in the range of 2  10  6– 2  10  5 m and those of hematite ranged between 2  10  6 and 1  10  5 m. The SEM images of both minerals are presented in Fig. 1. The compositions of two powdered iron oxides were verified using Mössbauer spectroscopy. Mössbauer spectra of solid samples (Fig. 2) were measured in transmission geometry using 57Co (Rh) source and a Mössbauer spectrometer (Wissenschaftliche Elektronik GmbH) at room temperature. For the measurements, iron oxides powders were put into the plastic capsule (with thickness of E30 mg/cm2).The spectra were fitted using WinNormos software decomposing to sextets, doublet and singlet subspectra and determining hyperfine parameters: the hyperfine field B, the isomer shift δ, the quadrupole splitting Δ and a relative area. According to the hyperfine parameters the area of a spectrum is

Fig. 1. Scanning electron microscope images of hematite (Fe2O3) ((a) and (b)); and wustite/magnetite (FeO/Fe3O4) ((c) and (d)).

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2.3. Batch experiments 1.0

Fe1-xO

Relative Intensity

0.9

Fe3O4

1.0

0.9

α-Fe2O3

0.8 -10

-5

0

5

10

Velocity, mm/s Fig. 2. Mössbauer spectra of magnetite/wustite (Fe3O4/FeO) (a) and hematite (Fe2O3) (b).

attributed to Fe(II), Fe(III) species, that is to the specific compound (FeO, Fe2O3, etc.). Mössbauer spectra indicate the presence of wustite/magnetite (FeO/Fe3O4 in the first mineral powder (Fig. 2a) and hematite (Fe2O3) in the second mineral powder (Fig. 2b). For wustite, Fe(II) and Fe(III) components of Mössbauer spectra in the ratio of 82:18 are detected in agreement with wustite formula. Fe1  xO (Schweika et al.,1995; Park et al.,1999) with xE0.08. Two sextets which are attributed to tetrahedral and octahedral sublattices are characteristic of magnetite (Volenik et al., 1975). The ratio of areas of magnetite sextets shown in Fig. 1b is found to be 45:55. Because the tetrahedral sublattice of magnetite is occupied by Fe(III) ions and the octahedral sublattice is occupied by Fe(II)þFe(III) ions, the Fe(II) ions may be found either in magnetite or wustite. 2.2. Measurements 99 Tc was measured by a liquid scintillation counting (LSC) method. An ultra low-level LSC counter “Quantulus-1220” was used. The reference standard solution of technetium-99 in the form of ammonium pertechnetate in 0.1 M ammonium hydroxide (National Physical Laboratory, UK) was used for the counting window optimization, the quenching curve construction and the counting efficiency calibration. Beta-particles emitted by 99Tc were counted in the window [100–650] channels, while the counting efficiency for slightly quenched samples was around 90%. The quenching correction was applied to each sample using the external standard techniques (Hou et al., 2005; Hou 2009). For the measurement of 99Tc activity, 4 mL of the sample solution were mixed with 16 mL of the liquid scintillation cocktail, OptiPhase HiSafe 3 (Perkin Elmer, Inc.). The pH measurements were performed using a “WTW pHmeter pH 315i” with the combined glass/reference electrode calibrated against standard buffers pH4 and pH7 (HANNA pH standard buffer solutions, Sigma-Aldrich Chemie) with a measurement error of 70.01.

2.3.1. 99Tc(VII) sorption onto the iron minerals The sorption of TcO4 (aq) onto iron oxides in alkaline (pH 8–9) medium, in the presence of both hematite (Fe2O3) and wustite/ magnetite (FeO/Fe3O4), was tested under ambient conditions. Technetium sorption onto hematite was also tested under acidic (pH 2.7, 4.5) and nearly neutral (pH 6.5) conditions, once again under ambient conditions. The iron oxides Fe2O3 (size fraction 2  10  6–1  10  5 m) and FeO/Fe3O4 (size fraction 2  10  6– 2  10  5 m) powder suspensions were prepared by adding 2 g of iron oxide powder into 100 mL flasks containing 50 mL of deionised water (conductivity  0.055 μS/cm, TOC o10 ppb). For preconditioning, the samples were shaken continuously on a vertical shaker for 24 h. Then, 400 μL of technetium (TcO4 ¼3.5  10  6 M) as ammonium pertechnetate in 0.1 M ammonium hydroxide, prepared from stock solution (National Physical Laboratory, UK), was added to each flask with the pre-conditioned suspensions. The pH of the suspensions was adjusted to 2.7, 4.5, 6.5 and 8–9 with 0.1 M HNO3 or 0.1 M NaOH, respectively. The suspensions were thoroughly stirred and the pH value was monitored. Then, the flasks were corked up and allowed to stand at room temperature. At fixed intervals of 48 h, 4 mL aliquots of samples were taken from the solution and filtered through 0.45 μm filters before 99 Tc measurement. The tests continued until the residual 99Tc concentration in the aqueous phase became o 1% of the initial concentration. All sorption experiments were carried out within 48–456 h.

2.3.2. Microbe-mediated sorption experiments The sorption of 99Tc(VII) was investigated in the presence of Fe(III) associated with hematite and microorganisms isolated from groundwater and waterlogged soil. The stains of microorganisms used in these experiments were selected on the basis of their unique physiological properties, paying special attention to their ability to grow in acetate-Fe(III)-citrate medium. Gram-positive bacteria, streptomycete S. coelicolor and C. cellulans, were selected as hydrogen sulphide (H2S) and catalase positive strains. Gramnegative bacteria, B. mycoides, C. cellulans and P. aeruginosa, were selected because they were facultative anaerobes under experimental conditions, and they are resistant to low pH (2.5–3.5) (Lukšienė et al., 2012). Fungi, A. niger and P. simplicissimum, were selected as good organic acid-producers and catalase positive organisms, while fungus F. oxysporum was selected as acid- and heavy metal-tolerant microorganism. Fungus F. oxysporum showed the highest Fe(III)-reduction rate, when used as an electron acceptor, and bacterium C. cellulans showed the lowest rate. The duration of the experiments ranged from 72 to 144 h. For the investigations of microbe-mediated processes, a live biomass of the microorganisms, separated from the growing media, was washed thoroughly with deionised water and 0.08 M NaCl, and then microorganisms were suspended in 0.08 M NaCl solution. The 1.8  10  6 M Tc(VII) solution was prepared using 99Tc stock solution. Hematite powder (0.1 g) was also suspended in 0.08 M NaCl solution. The mineral suspensions were generated by adding 5 mL of 0.08 M NaCl solution to the hematite. The volume to mass ratio (V/ms) was 50 (mL/g) in the series of experiments. The pH of the suspensions was adjusted to 6.3. Then, each type of microorganism (1.0–1.7 g of biomass) was added, and the suspensions of hematite and microorganisms were pre-conditioned at 30 1C without shaking for 24 h prior to the addition of TcO4 . Thereafter, the pH was measured and Tc was added. The flasks were kept at room temperature (  24 1C) for 48, 72 and 168 h in contact with

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the air. Prior to Tc analysis, the suspensions were filtered through 0.45 μm filters to remove bacteria cells. To test the ability of the microorganisms alone to accumulate technetium (KD), the batch experiment with selected microorganisms were carried out in the absence of hematite. The microorganism biomass was separated from the growing media, washed with deionised water and then microorganisms were suspended in 0.08 M NaCl solution. An appropriate amount of TcO4 was added to obtain the 1.8  10  6 M 99Tc(VII) solution. The pH of the solutions were adjusted to 7. After 48 h, the solution was filtered and 99Tc was measured in an aliquot of solution by LSC.

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Fig. 4. TcO4 (aq) distribution between the solution and hematite at pH values of 6.3, 4.5 and 2.7, after 456 and 48 h (white and black bars, respectively).

2.4. KD calculation Distribution coefficients (KD) were calculated to describe the adsorption behaviour of Tc in the experimental systems in the presence of iron oxides and microorganisms. The KD is defined as K D ¼ ðAi –Ae Þ=Ae  V=m; where Ai is the initial activity of 99Tc in solution, and Ae is the residual activity of 99Tc in solution after the sorption; V is the volume of the solution and m is the mass of the solid (Vejsada, 2006).

3. Results 3.1. Sorption of Tc(VII) onto Fe-bearing minerals Immobilization of TcO4 (aq) on wustite/magnetite (FeO/Fe3O4) and hematite (Fe2O3) as a function of contact time is shown in Fig. 3. Under the investigation conditions (non-complexing solution, aerobic medium, pH 8–9), in the presence of wustite/ magnetite, the gradual removal of TcO4 (aq) from the solution was observed. The results show that under alkaline conditions (pH 8–9) and a short exposure period (48 h), more than 75% of TcO4 (aq) was associated with FeO/Fe3O4 particles and removed from solution. During the period of 360 h, o1% of TcO4 (aq) remained in the solution. Under these conditions, no pronounced effect of Tc accumulation onto hematite (Fe2O3) was observed. Rather, within the first 48 h, 99% of TcO4 remained in the solution. After 144 h of exposure, a negligible decrease to 97% of TcO4 concentration in the solution was observed. The amount of TcO4 (aq) in the solution remained almost unaltered during all 360 h of exposure. The investigation of TcO4 (aq) distribution (Fig. 4) between the solution and hematite (Fe2O3) under acidic (pH 2.7 and 4.5) and nearly neutral (pH 6.3) conditions suggests that the pH of solution had a slight influence on technetium sorption. The experiment with the hematite suspension at pH 6.3 indicated no alteration of TcO4 (aq) behaviour in solution. The partitioning of TcO4 (aq) between the solution and the solid phase either during 48 or 456 h was not observed, as the majority (  98%) of 99Tc remained as TcO4 (aq). A distinct effect of the behaviour of TcO4 (aq) was

Fig. 3. Immobilization of TcO4 (aq) onto iron oxides as a function of contact time at pH of 8–9.

obtained in solution at pH 2.7 and 4.5 during 48 h. Approximately 78% and 65% of TcO4 (aq) remained in solution at pH 2.7 and 4.5, respectively; thereafter, its concentration in the solution remained constant for further 456 h. This work shows that the presence of structural Fe(II) as mineral wustite/magnetite can affect the removal of 99Tc(VII) from the aqueous solution (pH of 8–9) even in the presence of oxygen. Results of our batch-type experiment with TcO4 (aq) and wustite/ magnetite are different from the general assumption stating that under oxidizing conditions TcO4 (aq) is stable and consequently mobile (Lieser and Bauscher, 1987; Skomurski et al., 2010). It can be seen that sorption of technetium on wustite/magnetite is rapid (48 h) and leads to a complete removal of TcO4 from the solution. The results obtained here suggest that a different mechanism of Tc removal from solution, in the presence of structural Fe(II), can occur. The removal of TcO4 from the solution can be attributed to the formation of hydrous TcO2  nH2O because of the indirect reduction of TcO4 by abiotic transfer of electrons from reduced species of Fe(II) (Jaisi et al., 2009). In addition, accumulation of soluble technetium by Fe-bearing mineral under alkaline conditions could result from the precipitation of 99Tc, as the solid TcO2  nH2O onto the mineral surface or incorporation into iron oxides (Lieser and Bauscher, 1987; Skomurski et al., 2010). Based on these observations the mechanisms of interaction of TcO4 (aq) and wustite/magnetite under oxidizing conditions remain ambiguous, so further comprehensive structural analysis is necessary. 3.2. Tc(VII) behaviour under the influence of microorganisms For investigation of the microorganisms' ability to accumulate Tc(VII), microbial-mediated batch experiments were conducted. As the batch experiment with TcO4 (aq) and hematite showed that hematite had no influence on the sorption of Tc(VII) in the noncomplexing, nearly neutral (pH 6.3) medium, the microbialmediated effects on TcO4 (aq) sorption onto hematite were investigated. The combined effect of microorganisms and mineral substrates on the fate of TcO4 (aq) was investigated in the presence of structural Fe(III) (hematite), and different bacterial species isolated from groundwater and waterlogged soil. The tests were performed under ambient conditions. The duration of the experiments was 144 h. As mentioned above, after adding biomass of each type of microorganisms, the pH of suspensions (hematite/ microorganisms ) was adjusted to a value of 6.3, and suspensions were pre-conditioned at 30 1C without shaking for 24 h. The pH of the pre-conditioned suspensions was 6.5. After adding 99Tc, the pH of suspensions increased to  7.9 and the pH was not corrected. The pH of the solution was monitored every 48 h and it ranged between 7.6 and 8.0 throughout the experiment, depending on the microorganisms species (Table 1). Results of the combined effect of individual microorganisms and hematite on TcO4 (aq) removal using batch-type experiments showed a different microbial ability to influence Tc removal from the solution indicating that generic

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Table 1 The percentage of residual Tc(VII) in alkaline solution (pH  7.9) with hematite and microorganisms after 144 h. Surroundings

Microorganism (type/species)

Waterlogged soil

Gram-negative bacteria Gram-positive bacteria Fungus

Groundwater

Fungus Gram-negative bacteria Gram-positive bacteria Gram-positive actinobacteria Fungus Gram-positive bacteria Fungus

pH (initial/end)

Tc (%) (in solution)

Arthrobacter globiformis Cellulomonas cellulans Fusarium oxysporum

7.9/8.0 7.7/7.8 7.6/8.2

78.0 76.2 98.0 76.0 15.0 71.2

Aspergillus niger Pseudomonas aeruginosa Bacillus mycoides Streptomyces coelicolor Penicillium simplicissimum Rhodococcus ruber Spicaria sp.

7.9/7.9 7.8/8.0 7.9/7.8 7.9/8.0 7.6/7.8 7.7/8.0 7.9/7.9

82.0 76.6 49.0 74.0 46.0 73.7 29.0 72.3 24.0 72.0 18.0 71.4 98.0 76.0

6

1.8 1.6 1.4

4

KD (mL g-1)

KD (mL g-1)

5

3 2

1 0.8 0.6 0.4

1 0

1.2

0.2 0

A.g.

C.c.

F.o. A.n.

P.a. B.m.

S.c.

P.s.

R.r.

Spic.

TcO4

Fig. 5. Distribution coefficient (KD) values for (aq) in aqueous solutions with hematite and biomass of various microorganisms: Arthrobacter globiformis (A.g.), Cellulomonas cellulans (C.c.), Fusarium oxysporum (F.o.), Aspergillus niger (A.n.), Pseudomonas aeruginosa (P.a.), Bacillus mycoides (B.m.), Streptomyces coelicolor (S.c.), Penicillium simplicissimum (P.s.), Rhodococcus rubber (R.r.), Spicaria sp (Spic.).

peculiarities of the microorganisms are very important in these processes. Although two of the strains – bacterium C. cellulans and fungus F. oxysporum – were obtained from the same waterlogged soil, the first of them did not influence the immobilisation of Tc (VII) in hematite, while the latter, on the contrary, increased Tc removal (up to 85%) from the experimental solution compared with that in the system without microorganisms (Table 1). Differences in the removal of TcO4 (aq) from solution by hematite, depending on the activity of groundwater-borne microorganisms, were observed as well. Addition of any microorganisms (P. simplicissimum, R. rubber or S. coelicolor) to the tested system induced the accumulation of Tc(VII) onto hematite that ranged from 71% to 82%. The presence of P. simplicissimum, R. rubber and S. coelicolor in the experimental solution resulted in the 24%, 18% and 29% remainder of 99Tc concentration in solution, respective. The determined effect of B. mycoides and A. niger on 99Tc(VII) removal from the hematite suspension, ca 54% and 18%, respectively, was lower. The removal of 99Tc(VII) from hematite suspension impacted by fungus Spicaria sp. was not observed. The presence of Spicaria sp. in the experimental system with hematite resulted in approximately 98% remainder of 99Tc concentration in solution. The values of the distribution coefficient (KD) of the batch experiment showed an apparent influence of microbial activity on 99Tc (VII) removal as well (Fig. 5). These results suggest that the investigated microorganisms play a major role in the immobilization of technetium by iron-bearing minerals because of the possible reduction either of 99Tc(VII) directly or of Fe(III) to Fe(II) via biotransformation processes under aerobic conditions. Additional bioaccumulation experiments designed to investigate the abilities of selected microorganisms to immobilize 99Tc (VII) on/in their cells. For these experiments, the biomass of three microorganisms (S. coelicolor, P. simplicissimum, Rhodococcus ruber)

F. o.

S. c.

P. s.

R. r.

Fig. 6. Distribution coefficient (KD) values for TcO4 (aq) in hematite-free aqueous solutions with biomass of various microorganisms: F. oxysporum (F.o.), S. coelicolor (S.c.), P. simplicissimum (P.s.), and R. rubrum (R.r.).

isolated from the groundwater, and of one organism isolated from the waterlogged soil (F. oxysporum) was tested. Microorganisms used for this test were screened depending on their influence on 99 Tc sorption onto hematite as obtained in the previous experiment. The KD values obtained from this experiment (Fig. 5) show maximal sorption of technetium when hematite was combined with Gram-positive bacteria (S. coelicolor and R. rubber) or fungi (P. simplicissimum and F. oxysporum) biomass. Calculated KD values for Tc(VII) sorption by bacteria and fungi are plotted in Fig. 6. The KD values show that chosen microorganisms are capable of accumulating TcO4 (aq) in their biomass and may alter subsequent environmental behaviour of the radionuclide, regardless of the other environmental components. By comparing the results of both types of experiments (Table 2), the KD values of 99Tc were clearly higher in the batch experiment with the hematite and microorganisms combined, thus, demonstrating the effect of biotransformation in the total Tc removal process. In the microbial-mediated experiment without hematite, the maximum value of the 99Tc distribution coefficient (KD  1.7) was determined in the presence of P. simplicissimum biomass (Fig. 6). The KD value of the combined experiment with microorganisms and hematite was three times higher than that in the batch experiment without hematite. The distribution coefficient (KD  0.7) for the F. oxysporum biomass incubated without hematite (Fig. 6) was eight times higher (KD  5.7) than that in the batch experiment with hematite (Fig. 5). A KD of 1 was observed in the system with the biomass of bacterium R. rubber, and this value was four times lower than in the cells incubated with hematite. Comparatively different KD values were observed in the bioaccumulation experiment with the biomasses of actinomycete, S. coelicolor and R. rubber. Calculated KD values were 0.8 and 1.0, respectively, and they were lower than those in the corresponding batch experiment with hematite (KD 2.5 and 4.6, respectively). These results indicate that the investigated microorganisms could

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91

Table 2 Summary of microorganism-mediated accumulation (KD) experiment (with/without hematite). Surroundings

Microorganism (type/species)

Waterlogged soil

Gram-negative bacteria Gram-positive bacteria Fungus

Groundwater

Fungus Gram-negative bacteria Gram-positive bacteria Gram-positive actinobacteria Fungus Gram-positive bacteria Fungus

a

KD (Tc/Fe2O3/biomass)

KD (Tc/biomass)

A. globiformis C. cellulans F. oxysporum

0.3 0.02 5.7

–a – 0.7

A. niger P. aeruginosa B. mycoides S. coelicolor P. simplicissimum Rhodococcus ruber Spicaria sp.

0.2 1.0 1.2 2.5 3.2 4.6 0.02

– – – 0.8 1.7 1.0 –

Not measured for these systems.

stimulate the processes of technetium removal from solution in the presence of Fe(III)-containing mineral surfaces.

4. Discussion Many studies have shown that Fe(II), when in the sorbed or mineral state can be a strong reductant for 99Tc(VII) under anoxic/ reducing conditions (Cui and Eriksen, 1996; Peretyazhko et al., 2009; Jaisi et al., 2009). At present, it is known that abiotic reduction of soluble TcO4 by both Fe(II)aq and Fe(II)ads can bring about the formation of relatively insoluble, amorphous TcO2  nH2O at pH above 3 (Lieser and Bauscher, 1987). Furthermore, TcO4 undergoes stageby-stage one-, two- or three-electron reductions process leading to the formation of different aqueous species (e.g., TcO2 þ , TcO (OH) þ , TcO2(s), TcO4̄ ) in non-complexing aqueous solutions under reducing conditions (Heller-Grossman et al., 1981; Warwick et al., 2007). On the other hand, the structural incorporation of Tc species into iron oxides is possible (Peretyazhko et al., 2009; Skomurski et al., 2010; Um et al., 2012). The origin of Tc(IV) species is possible in oxidizing environments with high pH values in the presence of a γ radiation source and specific organic compounds. Lukens et al. (2002) showed that Tc can exist as some soluble Tc(IV) phase in Hanford tank wastes under these conditions. Our research shows that ferrous iron Fe(II) associated with iron oxide wustite/magnetite strongly affects the behaviour of 99Tc(VII) in solution under aerobic/oxidizing conditions. Various pathways for Tc removal from solution under the investigated conditions are possible. The removal of TcO4 from solution can be attributed to the formation of hydrous TcO2 because of the indirect reduction of Tc(VII) by abiotic transfer of electrons from reduced species of Fe(II). On the other hand, rapid accumulation of TcO4 by this Fe-bearing mineral could result from the precipitation of Tc onto the wustite/magnetite surface. Taking into consideration the noncomplexing medium of the experimental system, and accessibility of O2, we assumed that Fe(II) subsequently reduced pertechnetate (TcO4 ) to TcO2 þ which eventually co-precipitated with the Fe(OH)3(s) formed as a consequence of the oxidation of Fe(II) to Fe(III) under these experimental conditions. However, a conclusive evaluation of TcO4 (aq) interaction with the Fe(II)-bearing minerals could be done after further analysis of post-reaction solids. Experiments with TcO4 (aq) and the Fe(III)-bearing mineral hematite showed limited to no Tc removal from solution under aerobic/oxidizing conditions. This result is in agreement with literature data stating that the affinity of TcO4 for Fe-bearing minerals is weak under most conditions (Ambe et al., 1996; Liu and Fan, 2005). The quantum-mechanical method used by Skomurski et al. (2010) to evaluate mechanisms for possible structural incorporation of Tc into the hematite structure suggests that the incorporation of TcO4 in the hematite lattice is not

feasible because of the energetic inadequacy and structural distortion required of the coordination environment. An investigation of TcO4 (aq) interaction with hematite under the influence of microorganisms has shown that microorganisms play a major role in Fe(III) reactivity toward Tc(VII). The biotransformation of radionuclides achieved either by reduction of radionuclides by the microorganisms and their indirect abiotic reduction by various reactive forms of iron or other reductants likely produced by the microorganisms. Many microorganisms are able to enzymatically reduce radionuclides for use as the energy source (Mohapatra et al., 2010). As it is known, bacteria exhibit different metabolic types (Holt and Bergey, 1994). Bacterial metabolism is classified on the basis of three major criteria: the kind of energy used for growth, the source of carbon, and the electron donors used for growth (Xu, 2006). In addition, microbial abilities depend not only on the species but also on the strain and the habitat from which it was isolated. An obvious difference in the ability of bacterial strains, isolated from the waterlogged soil and the groundwater, to remove TcO4 (aq) from solution was determined. Water-borne bacteria were significantly (with statistical significance P o0.002) more effective in TcO4 (aq) binding with the biomass than soil-borne bacteria. However, the differences between two soil-borne bacteria, and among the four water-borne bacteria were also determined. These findings confirm that bacterial activity in the investigated systems depends on the environmental conditions, from which bacterium was isolated, as well as bacterium taxonomy. This research showed that in the presence of Gram-negative bacteria P. aeruginosa, approximately 51% of TcO4 (aq) was removed from the experimental solution and sorbed onto hematite. P. aeruginosa is classified as an aerobic organism and is considered by many researchers to be a facultative anaerobe, as it is well adapted to proliferate under conditions of partial or total oxygen depletion (Palmer et al., 2007). P. aeruginosa tends to form biofilms, which are complex bacterial communities that adhere to a variety of surfaces. Biofilms can lead to retention of dissolved heavy metals at sorption sites of the extra cellular polymeric substance, cell cytoplasm, cell walls and cell membranes (McLean et al., 1996; Pollmann et al., 2006). KrawczykBaersch et al. (2008) showed that the presence of aqueous 238U(VI) in the biofilm microenvironment could lead to an increase of respiratory activity in the most metabolically active biofilm zone, and that oxygen consumption may induce 238U reduction and lead to the removal of 238U as 238U(IV) solids from solution. In the present investigation a biofilm formation of glass flasks was observed throughout the incubation period of P. aeruginosa, B. mycoides and S. coelicolor cultures. Investigations into the ability of hematite to accumulate TcO4 (aq) in the presence of water-borne eubacteria showed that B. mycoides, R. rubber and actinobacteria, S. coelicolor induced  54%, 82%, 71% removal of TcO4 (aq) from solution, respectively. The results suggest that the biofilm formation may play a role in

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this process. Eubacteria are present in most habitats and grow in soil, water, and also in radioactive waste (Fredrickson et al., 2004). Gram-positive B. mycoides is an obligate aerobe, meaning that its cells can undergo morphological changes when starved of nutrients. For instance, bacterial cells can migrate to each other and behave as aggregates (Kaiser, 2004), or form biofilms on surfaces (Donlan, 2002). In these circumstances, bacteria perform separate tasks of a multicellular community. Differences in the ability of tested fungi to participate in TcO4 (aq) immobilisation in the presence of hematite were determined. The bacterium, C. cellulans, and fungus Spicaria sp. practically had no effect on the sorption of TcO4 (aq) onto hematite under the aerobic experimental conditions, since approximately only 2% of 99 Tc was removed from the solution. Significantly lower amounts of TcO4 (aq) (from 15% to 29%; P o0.01) remained in solutions when the biomass of bacterium, R. rubber, actinobacterium, S. coelicolor, or each of two fungi (F. oxysporum, and P. simplicissimum) was added. TcO4 (aq) was removed from solution at similar rates, with concomitant sorption of 15% to 29% of TcO4 (aq) from solution in 144 h. Almost half (46–49%) of the initial TcO4 (aq) amount was removed from solution by both Gram-positive bacterium, B. mycoides, and Gram-negative bacterium, P. aeruginosa. These findings show a tenuous ability of C. cellulanas and Spicaria sp. to bind the TcO4 (aq) with their biomass. We suppose, that the fungi F. oxysporum and P. simplicissimum, as well as Gram-positive bacterium, R. rubber, and actinomycete, S. coelicolor, were able to stimulate interaction between TcO4 (aq) and hematite via reduction of TcO4 (aq) or its accumulation in the biomass of microorganisms. In comparison, only 12% of the TcO4 (aq) was removed from solution when A. niger was added. Gram-negative bacterium, A. globiformis, was slightly more effective; approximately 22% of TcO4 (aq) remained in biomass. These hematite/microorganism batch experiments show that the removal of TcO4 from solution was facilitated by the activity of microorganisms. One mechanisms is biological reduction of structural Fe(III) to Fe(II), which may occur under these experimental conditions. Then, the reduction of TcO4 (aq) by Fe(II) is expected. However, further comprehensive structural and chemical analyses of the reaction products are necessary to obtain the knowledge of Tc removal mechanisms. The ability of a great variety of common bacteria (Bacillaceae, Pseudomonadaceae), as well as anaerobic, nitrogen-fixing bacteria, to reduce structural Fe(III) to soluble Fe(II) has been observed (Balashova and Zavarzin, 1980; Johnson and McGinness, 1991; Lovley, 1991; Lloyd and Macaskie, 1996; Lloyd et al., 1997, 1999; Wildung et al., 2000; Martin, 2005). Biological reduction of iron by fungi was shown by several authors (Ottow and von Klopotek, 1969; Jalal et al., 1987; Leong and Winkelmann, 1998; De Luca and Wood, 2000; Kosman, 2003). Woolfolk and Whiteley (1962) showed that the cell-free extract of Gram-negative bacterium, Vellionella atypica, reduced U(VI) along with a variety of other metals. Although the reduction of Tc(VII) has been demonstrated for a range of organisms (Lloyd and Macaskie, 1996; Lloyd et al., 1997, 1999; Wildung et al., 2000), under anaerobic conditions, very scarce information about Tc(VII) reduction under aerobic conditions is available (Istok et al., 2004; Burke et al., 2005). In addition, Martin (2005) showed that the dissolution of hematite occurred by ligand-promoted, proton-promoted, reductive and synergistic pathways. Reductants also rapidly accelerate iron oxide dissolution; examples of reductants are ascorbic acid, oxalic acid, hydrogen sulphate, and phenols (Stone and Morgan, 1984; Stone, 1987a, 1987b; Martin, 2005). Fungus A. niger, which was used in this study, produces oxalic acid which could serve as Tc(VII) reductant, or citric acid which could be involved in the reactions with the hematite (Sayer and Gadd, 1997). Fe(II)-oxalato aqueous complex is a strong reductant, which rapidly reduces iron oxide

through reductive dissolution. Oxalate secretion by fungi offers many advantages for their growth and colonization of substrates. Gram-positive bacteria produce succinic acid as well (Song and Lee, 2006). Three of the four Gram-positive bacteria used in the present investigation could be assigned to a group of active metalreductants. Fungi Fusarium sp. (Foster, 1949) and P. simplicissimum (Gallmetzer et al., 2002) are known to produce and secrete succinic acid. P. simplicissimum and F. oxysporum were effective in the 99Tc reduction and sorption processes (Gallmetzer et al., 2002). R. ruber synthesises red pigment may also participate in the enzymatic reduction of Tc (Lloyd et al., 1997). Many fungi (Müller and Raumond, 1984; Gadd and White, 1990) and bacteria (Sayyed et al., 2005) secrete specific hydroxamate siderophores when short of iron. In the present investigation, two fungal isolates, F. oxysporum and P. simplicissimum, showed effectiveness in TcO4 (aq) removing from solution under aerobic conditions. Further investigation is needed to confirm if these fungi are direct TcO4 (aq) or Fe(III) reducers, or if they serve as intermediates between TcO4 (aq) and Fe(III) in the sorption process.

5. Conclusions The presence of structural Fe(II) associated with wustite/magnetite in non-complexing aqueous solution (pH 8–9) under aerobic conditions can affect the removal of TcO4 (aq) from solution, thus limiting its mobility in oxidizing environments. The results show that under alkaline conditions (pH 8–9), after a short exposure period (48 h), more than 75% of TcO4 (aq) was associated with FeO/Fe3O4 particles and removed from solution. Structural Fe(III) associated with hematite was not able to immobilize TcO4 (aq) under similar alkaline conditions. However, low pH (2.7–4.5) increases TcO4 (aq) retention on hematite, which suggests that the process is dependent on pH. Retention of TcO4 (aq) onto hematite is achieved because of the presence of specific microorganisms isolated from waterlogged soil and groundwater. B. mycoides, R. rubber, actinobacteria, S. coelicolor and fungi F. oxysporum and P. simplicissimum induced the removal of TcO4 (aq) from solution in the presence of hematite. This research showed that in the presence of Gram-negative bacteria P. aeruginosa, approximately 51% of TcO4 (aq) was removed from the experimental solution. The bacterium, C. cellulans, and fungus Spicaria sp. practically had no effect on sorption of TcO4 onto hematite under the aerobic experimental conditions. An obvious difference between the bacterial strains, isolated from the waterlogged soil and the groundwater, in their ability to influence TcO4 (aq) behaviour in solution was determined. Waterborne bacteria were more effective in TcO4 (aq) immobilization with the biomass than soil-borne bacteria. The differences between two soil-borne bacteria, and among four water-borne bacteria were also determined. These findings have confirmed that the bacterial activity in the investigated processes really depends on the environmental conditions as well as on the bacterium taxonomy. Thus, we cannot yet use a classical explanation of the results obtained, because a genetic assignation to the genus must be investigated in future.

Acknowledgments The research leading to these results has received funding from the European Atomic Energy Community Seventh Framework Program [FP7/2007–2013] under grant agreement No. 212287, Collaborative Project Recosy and from the Lithuanian Agency for Science, Innovation and Technology (Grant no. 31V-6).

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Behaviour of (99)Tc in aqueous solutions in the presence of iron oxides and microorganisms.

A set of experiments was performed to determine the factors that influence TcO4(-) interaction with Fe-bearing minerals and to explore the effect of m...
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