Effect of microorganisms on the plutonium oxidation states

Applied Radiation and Isotopes 70 (2012) 442–449

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Effect of microorganisms on the plutonium oxidation states a Benedikta Lukˇsiene_ a,n, Ruta , Vidmantas Remeikis a, ¯ Druteikiene_ a, Dalia Pecˇiulyte_ b, Dalis Baltrunas ¯ b Algimantas Paˇskevicˇius a b

Center for Physical Sciences and Technology, Savanoriu˛ ave 231, LT–02300 Vilnius, Lithuania Nature Research Centre, Akademijos street 2, LT–08412 Vilnius, Lithuania

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 May 2011 Received in revised form 18 October 2011 Accepted 3 November 2011 Available online 12 November 2011

Particular microbes from substrates at the low-level radioactive waste repository in the Ignalina NPP territory were exposed to 239Pu (IV) at low pH under aerobic conditions. Pu(III) and Pu(IV) were separated and quantitatively evaluated using the modified anion exchange method and alpha spectrometry. Tested bacteria Bacillus mycoides and Serratia marcescens were more effective in Pu reduction than Rhodococcus fascians. Fungi Paecillomyces lilacinus and Absidia spinosa var. spinosa as well as bacterium Rhodococcus fascians did not alter the plutonium oxidation state. & 2011 Elsevier Ltd. All rights reserved.

Keywords: Plutonium Oxidation state Modified anion exchange method Bacteria Fungi

1. Introduction Safe handling and disposal of low, intermediate and especially high level nuclear waste are the major problems worldwide. It is well known that the most dangerous radioactive waste produced in nuclear power plants is spent nuclear fuel and high level waste. This waste mainly consists of fission products and actinoids. Due to high toxicity and long half-life, 239Pu and 240Pu are the most important radionuclides among them. The stability of various oxidation states of plutonium has an effect on chemical processing operations, on radioactive waste storage and treatment, and the reactivity and mobility of plutonium in the environment (Lee et al., 2008; Nitsche et al., 1988). The radionuclide behavior is often considered only from a physico-chemical point of view, though the biogenic factor, microbial activity, can affect either directly or indirectly their speciation and change mobility (Ehrlich, 2006; Fomina and Gadd, 2007; Keith-Roach, 2002; Levinskaite_ et al., 2009; Pedersen, 2005). Interaction of microorganisms with redox sensitive plutonium is very often related to a change in the oxidation state, which can affect plutonium dissolution and then enhance its environmental mobility (Francis, 2001; Francis et al., 2007; Neu et al., 2005). Understanding the role microbes can play in controlling radionuclide solubility and transport predetermined our further investigations. Potential scenarios for the plutonium oxidation state

n

Corresponding author. Tel.: þ370 5 2644857; fax: þ370 5 2602317. E-mail address: bena@ar.fi.lt (B. Lukˇsiene_ ).

0969-8043/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2011.11.016

alteration with emphasis on plutonium mobility under appropriate conditions have been developed. The chemical state of actinides released to the environment, combined with the complexity and diversity of their environmental behavior, involves difficulties in modeling their behavior over the lifetime of their radioactivity (Choppin, 2005). Scenarios foreseen for our investigations is an important part of modeling the disposal system and also are useful for sensitivity analysis of performance. Consequently, the aim of this study was to investigate the capability of microorganisms selected from those detected in samples at the low-level repository in the Ignalina NPP territory for participation in plutonium redox reactions under aerobic conditions and to develop a radiochemical method to differentiate plutonium oxidation states.

2. Experimental 2.1. Isolation and identification of microorganisms Microorganisms were isolated from the wood and cardboard samples disposed at the low-level radioactive waste repository in the terrain of the Ignalina NPP. Small pieces of either wood or cardboard samples (1 g and three replicates each) were pulverized in the mortar. Then the pulverized sample was mixed with 10 ml solution of NaCl (0.7%) and serial dilutions were prepared. Fungi were isolated on Malt Extract Agar (MEA, Liofilchem, Italy) and Potato Dextrose Agar (PDA, Liofilchem, Italy), using dilutions of 10  2, 10  3 and 10  4, bacteria—on Nutrient Agar (NA,

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Liofilchem, Italy), using dilutions of 10–5, 10–6 and 10–7. Isolation and enumeration of actinomycetes were performed by the plate technique using Starch–casein agar supplemented with Nystatin (50 mg ml  1) to avoid fungal contamination. The cultivable number of bacteria, fungi and actinomycetes, expressed as colony forming units (CFU) in 1 g of sample, was determined after 2–3, 7–14 and 7 days, respectively. Microorganisms were picked at random from culture plates and all strains were identified by morphological and biochemical tests. Standard procedures for the identification of fungi were followed (Domsch et al., 1980; Ellis, 1971; Gilman, 1966; Kiffer and Morelet, 1999; Watanabe, 2002). Bacteria and actinomycetes were isolated and characterized following Bergey’s manual (1974). Bacteria and fungi, isolated from the substrates, were treated under different conditions and only some strains were screened relying on their peculiarities: ability to reduce nitrate, H2S formation, organic acid production and resistance to low pH. Much attention was paid to bacteria and especially to facultative aerobes. Thioglycolate medium, standard medium, for the determination of oxygen relationships was used to confirm facultative aerobes. 2.2. Verification of microorganism properties to participate in reduction reactions 2.2.1. Nitrate reduction test for bacteria Bacteria were grown in the indole–nitrite medium (BBL Trypticase Nitrate Broth). This medium is used for the identification of microorganisms by means of the nitrate reduction. Due to the medium nutritive content, it supports the growth of aerobes, microaerobes and facultative and obligate anaerobes. The nitrite test was performed after 18–24 h of cultivation: three reagents (A, B and C) were used. Reagent A was sulfanilic acid (1 g) dissolved in 5 N acetic acid (125 mL); reagent B – N-(l-naphthyl) ethylenediamine (0.25 g) dissolved in 5 N the acetic; reagent C – a-Naphthol (1 g) dissolved in 5 N acetic acid (200 mL). To perform the test, 0.1–0.5 ml of reagent A and either reagent B or reagent C (as specified in the method) were added to the culture grown medium. A pink-to-red color develops after addition of reagents if nitrite is present, and indicates that nitrate reduction occurred. Since some organisms further reduce nitrite to ammonia, a small amount of zinc dust in the tubes showed no color. A colorless reaction indicates that nitrates have been completely reduced. 2.2.2. Hydrogen sulphide test To detect H2S production, the bacteria and actinomycetes were inoculated in the semi-liquid media (Peptone Iron Agar (PIA)), ¨ recommended by (Kuster and Williams, 1964). Lead acetate strips were inserted, and after the standard incubation period the formation of pigments and darkening of the strips were noted. The color and occurrence of these pigments and the reactions of the strips were then compared with those obtained when H2S detectors were included. Composition of the Peptone Iron Agar medium was the following (g/L): peptone, 15; proteose peptone, 5; ferric ammonium citrate, 0.5; dipotassium phosphate, 1; sodium thiosulphate, 0.08; agar, 15. The concentration of lead acetate was 0.2 g/L. The tubes were then incubated at 25 1C for 7 days. Positive control tubes were prepared for this experiment by passing chemically produced hydrogen sulphide into a tube of medium until the metal sulphide was formed. 2.2.3. Determination of organic acid production The isolates were inoculated into the agar medium containing 0.1% insoluble zinc oxide (ZnO). NA and Czapek’s Dox agar (CDA,

443

Liofilchem, Italy) were used for bacteria and fungi, respectively. The inoculated test organisms were incubated on these media at 25 and 30 1C for 48 h. Clear halos formed around the colonies show the presence of organic acids, which solubilize ZnO. 2.2.4. Resistance of microorganisms to low pH Bacteria and fungi were grown on the media, whose pH was adjusted to 2.5 with sterile HCl. Nutrient agar (NA) was used for bacteria, starch–casein agar (SCA) for actinomycetes and malt extract agar (MEA) for fungi. Cultures were checked every day and those, which were able to develop under test conditions, were chosen for the investigation of radiochemical oxidation states of plutonium. 2.3. Preparation of microorganisms for exposure to

239

Pu

Microorganism cells were collected by centrifugation at 3000 rpm for 5 min and washed thoroughly with 0.08 M NaCl solution. Microorganism cells (30 mg dry wt. basis) were suspended in 20 ml Na chloride solution of 0.08 M ionic strength. Pu(IV) solution of 9.2  10  12 M concentration was prepared using 239Pu stock solution purchased from Eckert & Ziegler Isotope Products. Prepared Pu(IV) solution was added to the microorganism cell suspension. At the beginning and at the end of the interaction time, solution pH was measured with WTW inoLab pH/Cond 720 using WTW Tetra Con 325 and Schott pHelectrode. Initial solution pH was kept in the range of 2.7–2.9 to reduce the influence of pH on Pu(IV) transformations because Pu(IV) hydrolysis even at pH o1 and the formation of polymeric/ colloidal species (Yun et al., 2007) are possible. One set of experiments included shaking of suspensions with microorganisms and Pu(IV) for 1 h at room temperature and the other set was arranged as static interaction of microorganisms with 239Pu (IV) for 48 h period. For each experiment, the mean value of three independent samples was reported. All reagents used were of analytical grade (Sigma Aldrich, Merck) and solutions were prepared in deionised water (TKA LAB MICRO system, conductivity 0.055 mS/cm; TOCo10 ppb). After the interaction time elapsed, suspensions were centrifuged at 3000 rpm for 5 min. Separated aliquots, both supernatant and microorganism biomass, were prepared for the plutonium oxidation state analysis. 2.4. Radiochemical oxidation state analysis At the initial stage of analysis the higher oxidation states (V and VI) of plutonium were separated from lower oxidation states (III and IV). 236Pu (III), 242Pu (IV) and 242Pu (V) were used as yield tracers. The tracer solutions of different oxidation states were prepared from 242Pu (IV) and 236Pu (IV) stock solutions in HNO3 referring to the methods reported (Lee et al., 2007, 2008; Nesmeyanova, 1968; Silver, 1967). Rare earth fluorides are very effective scavengers of actinides in oxidation state III and IV, while oxidized fraction remains in solution (Lovett and Nelson, 1981). The modified anion exchange method was employed to separate Pu (III) and Pu (IV). The separation of Pu(IV) from Pu(III) relies upon the differences in sorption of these Pu oxidation states onto the anion exchange resin in HNO3 medium. As it is known Pu(IV) in HNO3 medium forms anion complexes with NO3 and they show strong sorption on an anion exchange resin. Thus, from 8 mol L  1 HNO3 solution containing Pu(III) and Pu(IV) analytes only Pu(IV) is sorbed onto the anion exchange BiO RaD AG 1  8 resin column. Because of the absence of other transuranium elements and complex matrix ingredients in the tested solution the plutonium procedure could be simplified. The steps to remove Am, U and Th by

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washings with 8 mol L  1 HNO3 and 9–12 mol L  1 HCl could be omitted. Prior to loading the samples onto anion exchange column, 1 ml of saturated Al(NO3)3 was added to overcome interference of fluoride ion in anion exchange processing. The sample solution having passed through the column with plutonium (III) is loaded onto another BiO RaD AG 1  8 column but in this case valence adjustment to Pu(IV) using oxidation by nitrite is required (Lukˇsiene_ et al., 2006). For elution of plutonium sorbed onto the resin we used freshly prepared solution of 4 mol L  1 HCl with sodium sulphite. Electro-deposition was used to prepare thin layer plutonium sources for alpha spectrometric measurement. Plutonium was electrodeposited in 1 h under direct 0.6 A/cm2 current onto a stainless steel diskelectrode in sulphate medium. 2.5. Measurement of plutonium analytes The electrodeposited plutonium analytes were detected using conventional a-spectrometry. The resolution of the subsequent alpha spectra of the employed Octete-Plus (Ortec) spectrometer with detectors of 600 mm2square was 25–27 keV at 25% counting efficiency. The counting time, depending on activity, was from 2 to 4 days.

nitrate breakdown to the nitrite test. Our results agree with those of Otto and Glathe (1971) suggesting that Bacillus and Pseudomonas are facultative anaerobes. Bacillus genus, which dominated on the investigated substrates, as well as Pseudomonas sp. and Arthrobacter sp. showed the ability to reduce nitrate to nitrite under aerobic conditions (Table 1). Takaya et al. (2003) discovered two bacterial strains, aerobic denitrifies, which reduced nitrate (NO3) to N2. These two strains were Pseudomonas stutzeris and Pseudomonas sp. Results obtained by Shoun et al. (1998) suggested that many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide, however the reduction process is possible under anaerobic conditions. Perhaps, the aerobic conditions of our test were the reason that nitrate reduction test of the Streptomycetes was negative. Only three bacterial species (B. mycoides, S. marcescens and R. fascians) from ten bacteria presented in Table 1 resisted low pH conditions. Moreover, growth of those bacteria was inhibited by 12.3, 24.7 and 36.6% as compared with neutral pH. Two bacteria (B. subtilis and S. marcescens) showed a positive test in the medium with insoluble ZnO. All those peculiarities of the isolated bacteria helped us to select strains for the investigation of the bacterial participation in changes of the plutonium oxidation state. 3.2. Fungi selected for investigations

3. Results and discussion 3.1. Characteristics of bacterial isolates Different microorganisms were detected from wood and cardboard in the very low-activity INPP operational waste (ionizing radiation dose rate at the 10 cm distance from the surface does not exceed 0.6 mSv/h) disposed at the special dumping site in the territory of the Ignalina NPP (Lukauskas et al., 2006). Seven Gram-positive bacteria belonging to Arthrobacter, Bacillus and Rhodococcus genera, three Gram-positive actinomycetes from the genus Streptomyces and three Gram-negative bacteria belonging to Pseudomonas, Escherichia and Serratia genera were isolated (Table 1). Most of them are common soil bacteria. Only four morphotypes of bacteria were determined as facultative anaerobes, three of them showed a positive hydrogen sulphide test, almost all showed a positive catalase test, and seven species—positive

In addition to isolated bacteria, from both substrates (wood and cardboard) fungi belonging to the genera of Absidia, Chrysosporium, Paecilomyces, Penicillium and Trichoderma were isolated as well. Those fungi are common in soil, air, dust and they are good users of various substrates. Fungi from the genus Chrysosporium and one species of Paecilomyces genus – Paecilomyces lilacinus – dominated in all media plates used for fungus isolation. It can be stated that those fungi dominated in the fungus communities of investigated materials. After the dominance of fungus species in communities was determined, the growth of the dominant species was determined under low pH conditions and their ability to produce organic acids was detected as well. Two peculiarities – resistance to low pH and organic acid production – were analyzed to select fungus species for the investigation of the bacterial participation in alteration of the plutonium oxidation state. Species Chrysosporium merdarium and Penicillium chrysosporum were characterized as the best producers of organic acids (clearing zone 12.4 and 9.2, respectively), however, those species were very sensitive to pH 2.5

Table 1 Characteristics of bacterial isolates from the woody substrates exposed at the repository in the Ignalina NPP territory. Identification was done according Bergey’s manual (Buchanan and Gibbons, 1974). Information about Bacillus species is from Harmon (1982) and Parry et al. (1983). Isolate

a Bacillus mycoides strain DPKI-01 (Gram-positive) Bacillus cereus DPKI-02 (Gram-positive) Bacillus sp. (Cereus group – No 1) (Gram-positive) Bacillus sp. (Cereus group – No 2) (Gram-positive) Bacillus subtilis (DPKI-03) (Gram-positive) Serratia marcescens Strain DPKI-06 (Gram-negative) Rhodococcus fascians (¼ Rhodococcus luteus) (strain DPKI-17) (Gram-positive) Pseudomonas sp. (Gram-negative) Arthrobacter sp (Gram-positive) Escherichia coli (Gram-negative) Streptomycetes (Gram-positive): No.1 No.2 No.3 nn

not determined (n. d.). a

pH-2 resistant strains are presented in bold.

Characteristics Viability at pH Organic acid 2.5 production

Facultative anaerobe

Nitrate breakdown to nitrite

Hydrogen sulphide

Catalase

þ     þ þ

    þ þ 

þ þ  þ   

þ þ   þ þ þ

þ    þ   /þ

þ þ þ þ þ þ þ

  

 n. d. 

þ þ 

þþ  þ

  

þ n. d. þ

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

  

þ  þ

þ þ þ

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Table 2 Influence of low pH (2.5) on growth of fungi in MEA medium (A). Organic acid production test (B). Species [Frequency of occurrence, %]

Absidia spinosa var. spinosa Lendn [24.5%] Botryobasidium asperulum (D.P. Rogers) Boidin [24.6%] Chrysosporium merdarium (Ehrenb.) J.W. Carmich. [23.3%] Clonostachys rosea var. rosea (Link) Schrosers, Samuels, SAeifer & W. Gams[2.6%] Chaetomium globosum Kunze [3.6%] Paecilomyces lilacinus (Thom) Samson [40.6%] Penicillium chrysogenum Thom [84.3%] Scopulariopsis brevicaulis (Sacc.) bainier [2.3%] Trichoderma virens (J.H. Mill., Giddens & A. A. Foster) Arx [1.6%] Trichoderma harzianum Rifai [0.6%]

(growth inhibition 100 and 89.3%, respectively) (Table 2). Absidia spinosa var. spinosa and Paecilomyces lilacinus species were the most resistant to low pH, though their ability to produce organic acids was less expressed. Different strains of the species A. spinosa var. spinosa and P. lilacinus were investigated in detail (data not presented) and more favorable ones (strain DPKI-25 and DPKI-12) were selected for the investigation of their capability to participate in the plutonium redox reactions. 3.3. Application of the separation method to Pu oxidation state Different techniques concerning Pu oxidation state determination are developed. A quantitative determination of the oxidation states of aqueous Pu by use of spectrophotometric methods (Lee et al., 2008; Schramke et al., 1989), the X-ray Absorption Near Edge structure (XANES) (Bielewski et al., 2009; Ervin and Conradson, 2002) or coupling of capillary electrophoresis (CE) ¨ with resonance ionization mass spectrometry (RIMS) (Burger et al.,. 2007) is generally effective only at relatively high concentrations. Consequently, it should be admitted that some above mentioned physical-instrumental methods are rather favorable. They could be applied to solids and liquids, and they require no sample preparation; however their usage is complex. Among the speciation technique methods listed (Choppin, 2005; Ervin and Conradson, 2002) the radiochemical trace analysis and Electron Spin Resonance (ESR) are the most advantageous methods to determine plutonium oxidation states because of their sensitivity (10  8 10  12 M) and (10  5  10  12 M), respectively. In our investigations, after Pu(IV) interaction with microorganisms, co-precipitation and the modified anion exchange method have been successfully tested to separate Pu oxidation states. Based on model experiments the following scheme of radiochemical analysis (Fig. 1) was adapted for inventory of Pu oxidation states in aliquots after centrifugation, i.e., in the biomass and supernatant. The radioanalytical scheme developed for the Pu oxidation state identification includes two stages. At the first stage Nd fluoride precipitate differentiates between reduced plutonium that has been regarded as the sum of oxidation states (III) and (IV) and oxidized plutonium as the sum of (V) and (VI). The second step is intended to separate Pu (III) and Pu (IV) using the two-column anion exchange method. An amine group in the anion exchange resin acts as a reducing agent in the HCl medium, particularly when dealing with microquantities (Lee et al., 2008); therefore in our investigations to prevent the influence of this factor, by studying Pu (IV) possible reduction due to microbial activity, procedures were carried out in the HNO3 medium. Of plutonium oxidation states, only Pu(IV) forms anion complexes

A

B

Growth inhibition, %

Zone diameter, mm

8.7 100 100 64.3 26.7 12.4 89.3 100 86.7 100

4.3 1.3 12.4 3.8 0 8.8 9.2 0 0.2 0

with NO3 in the HNO3 medium. An interference of other ions is not available in the model laboratory experiments though by an anion exchange method in a 8 M HNO3 solution, a trace level of U has often been detected in the final Pu fraction during routine analysis (Warwick et al., 2001). Procedures of the two-column anion exchange method for Pu (III) and Pu (IV) separation are elucidated in more detail in Table 3. This procedure allows quantification of Pu (III) and Pu (IV), however separation of Pu (V) from Pu (VI) is not possible. 236Pu and 242Pu of different oxidation state were used to monitor the chemical yield, which during investigation of Pu oxidation states was in the range of 58–74%. 3.4. Analysis of the results Under the investigation conditions, the obtained dynamics of Pu oxidation states is presented in Fig. 2. Reductive dissolution of Pu (IV) induced by bacterial activity was observed at a larger extent in a case of plutonium interaction with gram-positive bacterium Bacillus mycoides strain DPKI-01 and gram-negative bacterium Serratia marcescens strain DPKI-06. The effect of bacterium Bacillus mycoides and bacterium Serratia marcescens on dynamics in Pu oxidation states was different. After 1 h interaction with plutonium Pu (IV), Bacillus mycoides generated 9.4% of Pu(III) in supernatant and about 1% of Pu (III) was found in biomass (Fig. 2, a). The reduction process during 24-hour interaction evolved and 14.6% of Pu (III) was produced in supernatant but in biomass the amount of Pu (III) was about 1% as during 1-hour interaction period. Bacterium Serratia marcescens during 1-hour interaction with Pu (IV) showed a very slight effect on Pu (IV) reduction process, while after 24-hour interaction this bacterium induced 16.1% of reductively dissolved Pu (IV) in supernatant (Fig. 2c). Those two bacteria of all studied microorganisms demonstrated the highest capability to reduce Pu(IV) to Pu(III). We can assume that practically production of Pu (III) was higher because radiochemical procedures were performed in the HNO3 medium that referring to Qiao et al. (2009) oxidizes part of Pu (III) ions. Bacterium Rhodococcus fascians, strain DPKI-17 within analytical uncertainties did not show capability to participate in Pu (IV) reduction processes under tested conditions (Fig. 2, b). The enhanced amount of Pu(III) fraction (Fig. 2c) in the liquid phase of the system Na chloride solution-Pu(IV)-gram-negative bacterium Serratia marcescens suggests that organic acids that are extruded products of cell metabolism (Neu et al., 2005) can solubilize plutonium and then enhance its environmental mobility. The bacterial membranes, proteins or redox agents can produce strongly reducing electrochemical zones and generate molecular Pu(III/IV) species or oxide particles (Neu et al., 2005).

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Supernatant,Pu (III-VI)

Biomass, Pu (III-VI) Ashing at 500?C in a muffle furnace

Tracers

Pu,

Pu (Pu (V); Pu (IV))

Treatment with HNO Tracers

Pu,

Pu (Pu (V) ;Pu (IV)) Solution of Pu with Nd and F

Tracer Supernatant Pu (V,VI )

Pu (III)

Centrifugation

NdF HNO +boric acid Heating Dissolution with boiling H O

HNO solution Pu (III, IV) NH OH, pH 8.5

Oxidation state adjustment Anion exchange column. Anion exchange column I without oxidation state adjustment

Eluate Pu (V,VI)

Electrodeposition

Hot conc. HNO , evaporation, dissolution in 8M HNO

Effluent Pu (III)

Electrodeposition

Alpha spectrometry

Eluate Pu (IV)

Oxidation state adjustment Anion exchange column II

Alpha spectrometry

Alpha spectrometry

Electrodeposition

Eluate Pu (III)

Fig. 1. Flow chart of separation and determination of different Pu oxidation states.

Table 3 Compilation of a two-column method for separation of Pu oxidation states. I column BiO RaD AG 1  8 Loading Pu3 þ , Pu4 þ

Oxidizing-redusing compound Not used

II column BiO RaD AG 1  8 Loading Oxidizing compound Pu3 þ NaNO2

Effluent Pu3 þ

Eluate Fraction for Pu4 þ

Effluent No Pu

Eluate Fraction for Pu3 þ

Distribution of Pu(IV) between supernatant and the biomass of tested microorganisms according to the obtained averaged results is presented in Fig. 3. Plutonium (IV) that was bound to the biomass of microorganisms could interact with microorganism cellular and extracellular structures containing metal-binding groups (Neu et al., 2005). It has also been indicated that microorganisms adsorb metals into complexes with organic acids (Yoshida et al., 2005). This finding can be attributed to the Pu (III) and Pu (IV) adsorption on S. marcescens cell walls (Fig. 2c) and Pu (IV) adsorption onto

mycelium and spores of the fungi Absidia spinosa var. spinosa and P. lilacinus (Fig. 3) due to their peculiarities to produce organic acids. In general, Fig. 3 shows that Pu is bound to the all microorganisms (bacteria and fungi) to a large extent. The mechanism of plutonium binding to microorganisms (a simple adsorption on the surface or accumulation in the organism cells) was not investigated. Certainly, if the Pu was uptaken and integrated to the organism cells, it should form a complex with the components of the organism. However, at this stage of investigations the organic associated form of Pu was not analyzed. On the other hand, bacteria and fungi synthesize a wide variety of low-molecular weight iron ligands that are called siderophores, such as bacterioferritin known in Absidia spinosa (Carrano et al., 1996) and fusarinine—in Paecilomyces spp. (Van der Helm and Winkelmann, 1994). Certain bacteria (especially pathogenic) employ iron sources that occur in their hosts and do not use siderophores; S. marcescens is just such a microorganism (Braun and Killmann, 1999). Siderophores being natural chelating reagents (Yoshida et al., 2005) form strong complexes with tetravalent actinides and enhance their solubility, therefore such

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0.3

0.3 0.25

0.2

Pu, Bq

1h exp. 24h exp.

0.25

0.15

1h exp.

0.2

24h exp.

0.15 0.1 0.05

0.1

(V

VI

)

I) (II Bm Pu

Bm

) (IV

) u SP

(V

Oxidation state

Bm

Bm Pu

Bm Pu

VI (V

(II

VI

)

I) (II

)

)

Bm Pu (IV

VI u SP

SP

SP

u

u

(V

(II

(IV

)

I)

SP

0

u

u

(IV

)

I)

0

0.05

SP

Pu, Bq

447

Oxidation state 0.3 0.25

1h exp. 24h exp.

Pu, Bq

0.2 0.15 0.1 0.05

) VI (V Bm

Bm Pu

Pu

(II I)

) (IV

) VI Bm

u SP

SP

SP

u

u

(V

(II

(IV

)

I)

0

Oxidation state

1h

B. myc.

R. fasc.

S. mar.

P.lil.

BmPu(IV)

SPu (IV)

BmPu(IV)

SPu (IV)

BmPu(IV)

SPu (IV)

BmPu(IV)

SPu (IV)

24h

BmPu(IV)

0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

SPu (IV)

Pu, Bq

Fig. 2. Distribution of plutonium oxidation states in supernatant (S) and biomass (Bm) following interaction of Pu(IV) with gram-positive bacteria (a) Bacillus mycoides strain DPKI-01 and (b) Rhodococcus fascians, strain DPKI-17 and (c) gram-negative bacterium Serratia marcescens strain DPKI-06.

A. spin.

Fig. 3. Dynamics of Pu (IV) after its interaction with microorganisms under aerobic conditions. B. myc.—bacterium Bacillus mycoides, R. fasc.—bacterium Rhodococcus fascians, S. mar.—bacterium Serratia marcescens, P. lil.—fungus Paecilomyces lilacinnus and A. spin.—fungus Absidia spinosa var. spinosa.

complexes can be responsible for significant Pu (IV) amount in the supernatant (Fig. 3). The results from the evaluation of interaction of Pu (IV) with fungi Absidia spinosa var. spinosa and P. lilacinus are shown in Fig. 4. The detected Pu(III) activity in the system Na chloride solution-fungus P. lilacinus was negligible (Fig. 4a). Pu (III) amount induced by A. spinosa var. spinosa was below the minimum detectable activity (MDA), i.e., practically this fungus has no

effect on plutonium (IV) reduction under appropriate conditions used (Fig. 4b). Fungi P. lilacinus and A. spinosa var. spinosa showed high resistance to low pH (growth inhibition 12.4 and 8.7%, respectively) but their organic acid production (clearing zone 8.8 and 4.3, respectively) was rather low (Table 2). Consequently, the relative contribution of mentioned above parameters probably resulted in high Pu accumulation on the fungus A. spinosa var. spinosa mycelium and spores and distribution of Pu between supernatant and fungus Paecilomyces lilacinus biomass.

4. Conclusions Of microorganisms detected near the low-level radioactive waste repository in the Ignalina NPP territory, microorganisms, which demonstrated properties to participate in reduction reactions were selected for the investigations. The study of Pu oxidation states after Pu (IV) interaction with microorganisms under aerobic conditions at low pH (2.7–2.9) was performed using radiochemical analysis. Individual Pu oxidation states, Pu (IV), Pu (III) and oxidized fraction Pu (V, VI), were separated using a combination of the neodymium fluoride co-precipitation method and the improved two-column anion exchange separation. The highest effect in a change of Pu(IV) to Pu(III) at low pH under aerobic conditions was demonstrated by Gram-possitive bacterium Bacillus mycoides (strain DPKI-01) and Gram-negative bacterium Serratia marcescens (strain DPKI-06). The amount of

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0.3

0.6 24h exp.

0.3

) VI

(II

Bm

(V

)

) VI

(IV

Bm Pu

u

(II

) (IV u SP

(V

VI

I) (II

Bm

Bm Pu

(IV

)

) VI

Bm Pu

(V u

u

(II

SP

SP

(IV u SP

Oxidation state

Bm Pu

0 (V

0 )

0.1 I)

0.05

I)

0.2

SP

0.1

1h exp.

0.4

I)

Pu, Bq

0.15

0.5

u

24h exp.

0.2

SP

1h exp.

)

Pu, Bq

0.25

Oxidation state

Fig. 4. Distribution of plutonium oxidation states in supernatant (S) and biomass (Bm) following interaction of Pu(IV) with fungi: (a) Paecilomyces lilacinus, (b) Absidia spinosa var. spinosa.

reduced plutonium (III) came up to 15%. Tested fungi Absidia spinosa var. spinosa (strain DPKI-25) and Paecilomyces lilacinus (strain DPKI-12) showed no pronounced influence on the redox process in the used experimental system under aerobic conditions. Hence, bioreduction shows great promise to control the solubility and mobility of redox sensitive radionuclides at contaminated environmental sites. Microorganisms can alter the solubility of plutonium by facilitating (a) intra- or extracellular accumulation, (b) direct or indirect reduction or oxidation, (c) production of organic acids, which enhance the soluble fraction of metal and (d) the alteration of pH level, all of which impact the solubility and mobility in the environment. The efficient method to determine the Pu oxidation states in the tested matrices enhances the probability to model the fate and transport of plutonium in the environmental systems. Therefore, the models used should consider the different chemical plutonium species as well as their biogeochemical behavior due to the microbial activity.

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). The authors express their appreciation to engineer R. Gvozdaite for technical assistance in alpha-spectrometric measurements.

References Buchanan, R.E., Gibbons, N.E., 1974. Bergey’s manual of Determinative Bacteriology. Williams and Wilkins Co., Baltimore. Bielewski, M., Eriksson, E.M., Himbert, E.J., Betti, E.M., Belloni, E.F., Falkenberg, E.G., 2009. Fast method of XANES data collection suitable for oxidation state mapping. J. Radioanal. Nucl. Chem. 282, 355–359. Braun, V., Killmann, H., 1999. Bacterium solutions to the iron-supply problem. TIBS 24, 104–108. ¨ Burger, S., Banik, N.L., Buda, R.A., Kratz, J.W., Kuczewski, B., Trautmann, N., 2007. Speciation of the oxidation states of plutonium in aqueous solutions by UV/Vis spectroscopy, CE-ICP-MS and CE-RIMS. Radiochim. Acta 95 (8), 433–438. ¨ Carrano, C.J., Bohnke, R., Matzanke, B.F., 1996. Fungal ferritins: the ferritin from mycelia of Absidia spinosa is a bacterioferritin. FEBS Lett. 390, 261–264. Choppin, G.R., 2005. Actinide Science: Fundamental and Environmental Aspects. J. Nucl. Radiochem. Sci. 6 (1), 1–5. Domsch, K.A., Gams, W., Andersson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London. Ellis, M.B., 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England.

Ehrlich, H.L., 2006. Geomicrobiology: relative roles of bacteria and fungi as geomicrobial agents. In: Gadd, G.M. (Ed.), Fungi in Biogeochemical Cycles. , Cambridge University press, Cambridge. Ervin, P.F., Conradson, S.D., 2002. Plutonium contamination valence states determination using X-RAY absorption fine structure permits concrete recycling. In: WM’02 Conference, February 24–28, 2002, Tuscon, AZ, USA, pp. 1–7. Fomina, M., Gadd, G.M., 2007. Transformation of metals and minerals by microorganisms. J. Basic Microbiol. 36 (4), 269–282. Francis, A.J., 2001. Microbial transformations of plutonium and implications for its mobility. In: Kudo, A. (Ed.), Plutonium in the Environment. , Elsevier Science Ltd., Co., UK, pp. 201–219. Francis, A.J., Dodge, C.J., Ohnuki, T., 2007. Microbial transformations of plutonium. J. Nucl. Radiochem. Sci. 8 (2), 121–126. Gilman, L.C., 1966. A Manual of Soil Fungi. Iowa State University Press, Ames, Iowa, USA. Harmon, S.M., 1982. New method for differentiation members of the Bacillus cereus group: collaborative study. J. Assoc.Off. Anal. Chem. 65, 1134–1139. Yun, J.-I., Cho, H.-R., Neck, V., Altmaier, M., Seibert, A., Marquardt, C.M., Walther, C., ¨ Fanghanel, Th., 2007. Investigation of the hydrolysis of plutonium (IV) by a combination of spectroscopy and redox potential measurements. Radiochim. Acta 95, 89–95. Keith-Roach, M.J., 2002. Interactions of microorganisms with radionuclides. Elsevier, Oxford, UK. Kiffer, E., Morelet, M., 1999. The Deuteromycetes. Mitosporic Fungi. Classification and generic keys. Science Publishers Inc, Enfield, USA. Lee, M.H., Park, Y.J., Kim, W.H., 2007. Absorption spectroscopic properties for Pu(III, IV and VI) in nitric and hydrochloric acid media. J. Radioanal. Nucl. Chem. 273 (2), 375–382. Lee, M.H., Kim, J.Y., Kim, W.H., Jung, E.C., Jee, K.Y., 2008. Investigation of the oxidation states of Pu isotopes in a hydrochloric acid solution. Appl. Radiat. Isot. 66, 1975–1979. ¨ Kuster, E., Williams, S.T., 1964. Production of hydrogen sulfite by Streptomyces and methods for its detection. Appl. Microbiol. 12 (1), 46–52. Levinskaite_ , L., Smirnov, A., Lukˇsiene_ , B., Druteikiene_ , R., Remeikis, V., Baltrunas, ¯ D.A., 2009. Pu(IV) and Fe(III) accumulation ability of heavy metal-tolerant soil fungi. Nukleonika 54 (4), 285–290. Lovett, M.B., Nelson, D.M., 1981. Determination of some oxidation states of plutonium in sea water and associated particulate matter. In: Techniques for Identifying Transuranic Speciation in Aquatic Environments, International Atomic Energy Agency, Vienna, pp. 166–184. Lukauskas, D., Plukiene_ , R., Plukis, A., Gudelis, A., Duˇskesas, G., Juodis, L., Druteikiene_ , R., Lujaniene_ , G., Lukˇsiene_ , B., Remeikis, V., 2006. Method to determine the nuclide inventory of low-level activity waste of the RBMK–1500 reactor. Lithuanian J. Phys. 46 (4), 497–503 M.B. Lukˇsiene_ , B., Druteikiene_ , R., Gvozdaite_ , R., Gudelis, A., 2006. Comparative analysis of 239,240Pu, 137Cs, 210Pb and 40K spatial distributions in top soil layer at the Baltic coast. J. Environ. Radioact. 87 (3), 305–314. Nesmeyanova A.N. (1968). Guide to the Practical Lessons in Radiochemistry, Moscow, p. 67 (in Russian). Neu, M.P., Icopini, G.A., Boukhalfa, H., 2005. Plutonium speciation affected by environmental bacteria. Radiochim. Acta 93 (11), 705–714. Nitsche, H., Lee, S.C., Gatti, R.C., 1988. Determination of plutonium oxidation states at trace levels pertinent to nuclear waste disposal. J. Radioanal. Nucl. Chem. 124 (1), 171–185. Otto, J.C.G., Glathe, H., 1971. Isolation and identification of iron-reducing bacteria from gley soils. Soil Biol. Biochem. 3 (1), 43–45. Parry, J.M., Turnbull, P.C.B., Gibson, J.R., 1983. A collour atlass of Bacillus species. Wolfe Medical Publication Ltd., London. Pedersen, K., 2005. Microorganisms and their influence on radionuclide migration in igneous rock environments. J. Nucl. Radiochem. Sci. 6 (1), 11–15.

B. Lukˇsiene_ et al. / Applied Radiation and Isotopes 70 (2012) 442–449

Qiao, J., Hou, X., Miro´, M., Roos, P., 2009. Determination of plutonium isotopes in waters and environmental solids: a review. Anal. Chim. Acta 652, 66–84. Schramke, J.A., Rai, D., Fulton, R.W., Choppin, G.R., 1989. Determination of aqueous plutonium oxidation states by solvent extraction. J. Radioanal. Nucl. Chem., Art. 130 (2), 333–346. Shoun, H., Kano, M., Baba, I., Takaya, N., Matsuo, M., 1998. Denitrification by actinomycetes and purification of dissimilatory nitrite reductuase and azurin from Streptomyces thioluteus. J. Bacteriol. 180 (17), 4413–4415. Silver, G.L., 1967. Determination of plutonium oxidation states in dilute nitric acid by complementary tristimulus colorimetry. Talanta 14 (6), 637–641. Takaya, N., Catalan-Sakairi, M.A.B., Sakaguchi, Y., Kato, I., Zhou, Z., Shoun, H., 2003. Aerobic denitrifying bacteria that produce low levels of nitrous oxide. Appl. Environ. Microbiol. 69, 3152–3157.

449

Van der Helm, G., Winkelmann, G., 1994. Hydroxamates and polycarboxylates as iron transport agents (siderophoeres) in fungi. In: Winkelman, G., Winge, D.R. (Eds.), Metal ions in fungi, 11. , Marcel Dekker, New York, pp. 39–98. Watanabe, T., 2002. Pictorial atlas of soil and seed fungi: morphologies of cultural fungi and key to species, second ed. CRC Press, Boca Raton. Warwick, P.E., Croudace, I.W., Oh, J.-S., 2001. Radiochemical determination of 241Am and Pu (a) in environmental materials. Anal. Chem. 73, 3410– 3416. Yoshida, T., Ozaki, T., Ohnuki, T., Francis, A.J., 2005. Adsorption of Th(IV) and Pu(IV) on the Surface of Pseudomonas fluorescens and Bacillus subtilis in the Presence of Desferrioxamine Siderophore. J. Nucl. Radiochem. Sci 6 (1), 77–80.