Abstract
It is shown for the first time that haloalkaliphilic bacteria, isolated from soda-lake environments were capable of reducing Tc(VII)O4− to the Tc(V), Tc(IV) and Tc(III) at pH 10 in carbonate medium, whereas no reduction took place without bacteria or in the presence of dead biomass. After 34 h of incubation, 55% remained as Tc(VII), 36% was found as Tc(IV) and 8% as Tc(V) and after 2 months 80% of the technetium was reduced. Technetium has a toxic effect on bacteria. Reduction of TcO4− was drastically decreased at concentration above 1.5 mM. The microbial reduction has been suggested as a potential mechanism for the removal of Tc from contaminated environments or waste streams.
1 Introduction
Pollution of the environment by toxic metals and radionuclides takes place as a result of increasing industrial activity. The problem of radioactive wastes arises from experimental explosions of nuclear weapons, wastes from nuclear fuel cycle reprocessing plants and the use of isotopes for medical purposes. Among long-lived radionuclides, technetium (99Tc) is notable for its long half-life (2.13×105 years), its extreme mobility in the environment and its tendency for bioconcentration. Three years after the discharge of fuel from a reactor, the contribution of 99Tc to the total β-radioactivity is only 0.1%, but reaches 30–50% after 300 years, which makes it an important radionuclide in the fuel waste management [1]. During reprocessing, Tc is solubilized from nuclear spent fuels and is present in all waste streams as the pertechnetate anion (TcVIIO4−), which is weakly sorbed by most soils and subsurface sediments [2]. The 1990 99Tc inventory at the famous Hanford Site [3] showed that 510±210 kg was shipped off-site, 1310±220 kg remained within Hanford Site tanks and 80±10 kg was released to the environment [3]. According to this review, the pH of low-level waste is around 13. Tc can form several reduced species, including Tc(VI), Tc(V), Tc(IV) and Tc(III), which interact strongly with rocks, minerals (pyrrhotite, antimonite) and organic ligands [4]. Therefore, it is important to identify Tc forms accurately to predict the long-term stability and environmental mobility of microbially reduced Tc following in situ bioremediation.
Recently, microbial reduction of technetium was studied very intensively. Sulfate-reducing bacteria Desulfovibrio desulfuricans[5,6], metal-reducing bacteria Shewanella putrefaciens[7] and Geobacter sulfurreducans[8] and Escherichia coli[9] were found to be capable of reducing Tc(VII) at neutral pH. Under acidic conditions, Thiobacillus ferrooxidans and Thiobacillus thiooxidans can also reduce Tc(VII) to low-valency forms [10]. However, there is no information about technetium reduction under alkaline conditions.
Earlier, a group of alkaliphilic halotolerant heterotrophic bacteria from alkaline environments in Siberia, Kenya, Buryatiya and Mongolia was shown to oxidize sulfur compounds to tetrathionate [11,12]. Currently, the collection of these bacteria includes about 20 strains, which according to 16S rRNA sequencing, belong to the genus Halomonas in γ-Proteobacteria [11–13]. The objective of the present research was to determine whether these haloalkaliphilic soda-lake isolates would reduce Tc(VII) under anaerobic haloalkaline conditions.
2 Materials and methods
2.1 Reagents
All reagents were of analytical grade and purchased from Sigma-Aldrich.
2.2 Organisms and culture conditions
Bacterial strains (Se1, Se 3, Se4, Se5, AGJ 3) were obtained from D. Y. Sorokin (Institute Microbiology RAS, Moscow, Russia) and were isolated from Mongolian and Kenyan soda lakes. Strain Mono was isolated from Mono Lake (California, USA) by N.N. Lyalikova. The nutrient medium contained: Na2CO3– 13 g, NaHCO3– 4 g, NaCl – 50 g, K2HPO4– 0.5 g, MgSO4– 0.1 g, yeast extract – 0.1 g, NH4Cl – 0.1 g, sodium acetate – 2.8 g, 2 ml of microelement's solution [14] per liter of distilled water. The final pH of the medium was 10. All cultivations were conducted under anaerobic static conditions at 30°C. Medium was dispensed (4.5–9-ml aliquots) into 17-ml serum bottles, sealed with butyl rubber stoppers, deaerated with N2 and autoclaved. Cells aerobically pre-grown on acetate were washed twice with the same medium and aliquots (0.5–1 ml) and electron donors were then transferred to serum bottles. The concentration of electron donors (acetate, lactate, formate, ethanol, methanol) was 10 mM each and the concentration of TcO4− was 0.25 mM. To compare the effect of increasing concentration of TcO4−, it was added to 5 ml suspension of cells in the medium at final concentration ranging from 0.25 mM to 2 mM.
2.3 Protein determination
Bacterial protein measurement was performed by Bradford assay [15].
2.4 Spectrophotometric determination of different Tc species in the cultural supernatants
UV-visible spectra of technetium were recorded in the digital form, using a Beckman DU-50 Series double-beam diode array UV-visible spectrophotometer in the range of 250–700 nm. Identification of bands was carried out according to [16] in carbonate-bicarbonate medium, where the band at 290 nm is characteristic for Tc(VII), the band at 512 nm for Tc(IV) and at 630 nm for Tc(III). When Tc(III) was recorded, experimental bottles had not been opened and placed directly to spectrophotometer, in all other cases the solution had been transferred to quartz cuvettes.
2.5 Chromatographic separation and identification of Tc
Samples (0.2 ml) were centrifuged with Sigma 3MK (Germany) at 5000×g for 10 min. Different Tc species were separated on Whatman 3MM chromatography paper with 0.3 N HCl as the mobile phase [17]. Chromatography paper was impregnated with 5–10-μl aliquots. After separation air dried chromatograms were exposed to phosphor storage screen for 16 h. Chromatograms were visualized and quantified with the PhosphorImager technique [18].
2.6 Extraction techniques for determination of Tc species
For comparison with chromatographic Tc determination, several extraction techniques were used to differentiate between Tc species in the cultural supernatant. To co-precipitate Tc(IV) and Tc(V), FeCl3 (final concentration 10 mg ml−1) and concentrated NH4OH (3 drops) were added to the sample (0.5 ml). Precipitate was washed three times and dissolved with concentrated HNO3 (0.5 ml). The amounts of Tc in the washes and the precipitate were determined. To extract Tc(VII), 1 ml of tetraphenylarsonium chloride in chloroform (0.05 M) was added to an equal volume of sample [19]. After 1 min of shaking, the phases were separated. The amounts of Tc in the organic and aqueous phase were determined. To extract the Tc(V) species, 4% (w/w) 8-hydroxyquinoline in chloroform [20] was added to the supernatant. After 5–10 min of shaking, the phases were separated. The amounts of Tc in the organic and aqueous phases were again determined. In all cases the radioactivity of each phase (organic, aqueous, precipitate, washes) was determined with a Beckman liquid scintillation analyzer and Insta Gel coctail, Packard Bioscience Company.
3 Results
3.1 Spectrophotometric analysis of cultural supernatant
Haloalkaliphilic bacteria were tested for the ability to reduce TcO4− under the alkaline anaerobic conditions. Under these conditions the carbonate–bicarbonate medium became progressively pink (after 23 h), then colorless with the time (10–12 days) as Tc(VII) was reduced by bacteria. Spectrophotometric analysis indicated the presence of Tc(IV) (well-defined band at 512 nm) and Tc(III) (very wide band with maximum at 628 nm) in the bacterial spent media in addition to Tc(VII) (well-defined band at 290 nm), which was added at the start of experiment (Fig. 1A–C). These band positions were in complete agreement with Tc(III), Tc(IV) and Tc(VII) in the bicarbonate solutions reported in [16] and shown in Fig. 1E. A small band was also detected at 418 nm. No reduced species of technetium were detected in the control solutions without bacteria or with dead biomass (Fig. 1A). As soon as colorless bacterial spent media was exposed to air, the pink color reappeared. At the same time, spectrophotometric data showed that the band at 628 nm had completely disappeared and the intensity of the band at 512 nm had slightly increased (Fig. 1D). If incubation of bacteria was continued, the pink color disappeared again.
![Spectrophotometric studies of TcVIIO4− reduction. Spectra were recorded with strain Mono at cell protein concentration 0.035 mg ml−1. Different forms of technetium could be determined as Tc(VII) at 290 nm, Tc(IV) at 512 nm, Tc(III) at 628 nm according to [16]. A: Control experiment without bacteria; B: bacterial medium after 23 h of exposition; C: after 12 days of exposition; D: case C exposed to air; E: electrochemical reduction of TcO4− on the electrode (from [16]), where spectra for TcO4− (full line), Tc(IV) (dashed line), Tc(III) (dotted line) are shown.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/femsec/44/1/10.1016_S0168-6496%2803%2900018-7/1/m_FEM_109_f1.jpeg?Expires=1750034384&Signature=Sm9x-A7cU86621tikpnQcGVHhQ0VSsDvLxKv4kqtL6yBk9WBRoA9G7EZoaw5LDofxrYVFKUwBnAuFeGIKPhFJI~q5fH0SteQgSy-bctiPq6LhisjCqoiN5Sw75WN7aaoP0WSytaQQR~ES6D19-r9EojbU5uK1qGFWcB9tQcubjS6TjI1dyU1x4sARfd-2gn1RihvbyS8rHrHaWPaby1uAw3TTjI525istgxSOpD5Shx6iYyTGc3UP973SVpz08XfSfBZpkKvGvknhehaAx02PGIibu20ZKDCvoXMZ4v9rxI50srA4DQOdId5E0uq3uRqRD0Oamaj9NdfSAM65p1hzg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Spectrophotometric studies of TcVIIO4− reduction. Spectra were recorded with strain Mono at cell protein concentration 0.035 mg ml−1. Different forms of technetium could be determined as Tc(VII) at 290 nm, Tc(IV) at 512 nm, Tc(III) at 628 nm according to [16]. A: Control experiment without bacteria; B: bacterial medium after 23 h of exposition; C: after 12 days of exposition; D: case C exposed to air; E: electrochemical reduction of TcO4− on the electrode (from [16]), where spectra for TcO4− (full line), Tc(IV) (dashed line), Tc(III) (dotted line) are shown.
3.2 Tc(VII) reduction by haloalkaliphilic bacteria
All soda-lake isolates were able to reduce technetium and the profile of the resulting Tc species was very similar (Fig. 2). Reduced technetium species could be separated, identified and quantified, for example, using paper chromatography. The results clearly show that after 3 days of incubation under anaerobic alkaline conditions, an average of 62% of the 0.25 mM of pertechnetate had been reduced by haloalkaliphilic cultures (Se1, Se3, Se4, Se5, AGJ 3 and Mono) to Tc (IV) and Tc(V) species (Rf for Tc(IV)=0.9, for Tc(V)=0.0 and for Tc(VII)=0.7 [17]) (Fig. 2). The spot of Tc(IV) also includes that of Tc(III) because Tc(III) has been reoxidized at the starting line due to presence of oxygen. Thus, it is not possible to detect Tc(III) by paper chromatography. Note that pertechnetate ion was not reduced in the control experiments without bacteria or with dead biomass (Fig. 2). No technetium reduction was observed under the aerobic conditions. No Tc was precipitated from the medium over a 34-h period (bacterial spent medium was centrifuged at 10 000 rpm for 20 min, the technetium measurements were done before and after centrifugation). After 3 days of incubation, about 6% was co-precipitated from the medium with excess of carbonate. After 2 months, the amount of reduced technetium reached 80%; from that, 25% was precipitated and 55% remained in solution. To check the stability of Tc(V) and Tc(IV), cultural supernatant was exposed to air. Chromatography revealed that no changes in Tc species were observed over a 4-day period: paper chromatography showed the presence of only Tc(VII), Tc(V) and Tc(IV) in the bacterial spent medium.
![After 3 days of incubation, Tc(V) (Rf=0) and Tc(IV) (Rf=0.9) were detected in culture supernatants, in addition to Tc(VII) (Rf=0.7), which was added at the start of experiment. Technetium species were separated on Whatman 3MM cellulose paper with 0.3 N HCl as the mobile phase. Chromatograms were visualized with the PhosphorImager technique [17]. Lane 1 – control (no bacteria); lane 2 – dead (boiled) biomass; lanes 3–8 – bacterial reduction of Tc(VII) by strains Se1, Se3, Se4, Se5, AGJ 3, Mono, respectively.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/femsec/44/1/10.1016_S0168-6496%2803%2900018-7/1/m_FEM_109_f2.jpeg?Expires=1750034384&Signature=wCrukRY6oSOxckWINWf1ICgS0lhAtikj4bMTWMz9HbqvNh5Lnrpc5QTP6mjs0AaTbVIWdcPILFKQG6qLdKmYaqs-Hn84gPUEF7uYwnMHRgp2yGtGev7UPpMtpI949uy-~-DINWxzXLzyFV8joZnXm2UHKkIo~UO6SS4rkI2zsof-WU7N7sA5zGBmrTyxz5cd9uPavw1nVuWJDiP85pjy1tEg~1aGpwS~Us70abqbKkPZcQasUvJvBSife717KVmJ7jiY0IUXFNaK7aAjveOYxBOC7TI0~ByYrDdIc4ynmEpFUcXaHGZh54W46iLGot95qE0--wQO7hydZ9sBPzyjiQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
After 3 days of incubation, Tc(V) (Rf=0) and Tc(IV) (Rf=0.9) were detected in culture supernatants, in addition to Tc(VII) (Rf=0.7), which was added at the start of experiment. Technetium species were separated on Whatman 3MM cellulose paper with 0.3 N HCl as the mobile phase. Chromatograms were visualized with the PhosphorImager technique [17]. Lane 1 – control (no bacteria); lane 2 – dead (boiled) biomass; lanes 3–8 – bacterial reduction of Tc(VII) by strains Se1, Se3, Se4, Se5, AGJ 3, Mono, respectively.
3.3 Kinetics of technetium reduction by haloalkaliphilic bacteria
Although the physiological properties of strains showed some differences, the patterns of technetium (VII) reduction were about the same (see Section 3.2). For that reason, only one strain was used for kinetic studies. The kinetics of Tc(VII) reduction were monitored over a 34-h period with pre-grown cells of strain Mono (Fig. 3a) and with growing culture of the same strain during 2 months (Fig. 3b). Microbial reduction of pertechnetate could be described as a two-phase process: first, very fast, where about 40% of pertechnetate was reduced within 23 h and second, very slow over days and weeks, when technetium reduction continued at a very low rate (Table 1). After 34 h of incubation 55% remained as Tc(VII), 36% was found as Tc(IV) and 8% as Tc(V).
Kinetics of microbial reduction of technetium by strain Mono over period of 34 h (a) and 2 months (b). Quantification was performed using PhosphorImager and verified with extraction techniques. Values represent the mean of four replicas. (♦) – Control without bacteria, (△) – control with boiled bacteria, (▪) – Tc(VII) and (●) – Tc-reduced in the bacterial experiment, where cell protein concentration was 0.035 mg ml−1.
Correlation of mathematical equations to experimental data from Fig. 3
| Time | Mathematical equations |
| 0<x<23, | %(TcVII)=−1.8407x+100 (R2=0.98) |
| where x– hours | %(TcIV)=1.7908x (R2=0.98) |
| 1<x<60, | %(TcVII)=−8.1851 ln(x)+53.379 (R2=0.95) |
| where x– days | %(TcIV)=8.0995 ln(x)+46.55 (R2=0.93) |
| Time | Mathematical equations |
| 0<x<23, | %(TcVII)=−1.8407x+100 (R2=0.98) |
| where x– hours | %(TcIV)=1.7908x (R2=0.98) |
| 1<x<60, | %(TcVII)=−8.1851 ln(x)+53.379 (R2=0.95) |
| where x– days | %(TcIV)=8.0995 ln(x)+46.55 (R2=0.93) |
The experimental data were treated with Microsoft Exell’97.
Correlation of mathematical equations to experimental data from Fig. 3
| Time | Mathematical equations |
| 0<x<23, | %(TcVII)=−1.8407x+100 (R2=0.98) |
| where x– hours | %(TcIV)=1.7908x (R2=0.98) |
| 1<x<60, | %(TcVII)=−8.1851 ln(x)+53.379 (R2=0.95) |
| where x– days | %(TcIV)=8.0995 ln(x)+46.55 (R2=0.93) |
| Time | Mathematical equations |
| 0<x<23, | %(TcVII)=−1.8407x+100 (R2=0.98) |
| where x– hours | %(TcIV)=1.7908x (R2=0.98) |
| 1<x<60, | %(TcVII)=−8.1851 ln(x)+53.379 (R2=0.95) |
| where x– days | %(TcIV)=8.0995 ln(x)+46.55 (R2=0.93) |
The experimental data were treated with Microsoft Exell’97.
3.4 Tc(VII) reduction coupled to different electron donors
A pre-grown cell suspension of strain Mono was tested for the ability to reduce Tc(VII) with different electron donors. Acetate, formate, lactate, ethanol and methanol all supported Tc(VII) reduction (Table 2). If electron donor was absent there was no technetium reduction observed.
The reduction of TcVIIO4− by strain Mono after 28 h of incubation
| Substrate | Technetium speciation in the supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| Formate | 65.0±1 | 32±1 | 3±0.3 |
| Acetate | 67.0±0.5 | 28.9±0.5 | 4±0.5 |
| Lactate | 68.0±0.75 | 28±0.75 | 3±0.3 |
| Methanol | 69.0±0.2 | 29±0.2 | 2±0.2 |
| Ethanol | 68.8±0.2 | 28.8±0.2 | 2.4±0.1 |
| Control without bacteria | 100.0±2 | 0 | 0 |
| Control without electron donors | 100.0±1.25 | 0 | 0 |
| Substrate | Technetium speciation in the supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| Formate | 65.0±1 | 32±1 | 3±0.3 |
| Acetate | 67.0±0.5 | 28.9±0.5 | 4±0.5 |
| Lactate | 68.0±0.75 | 28±0.75 | 3±0.3 |
| Methanol | 69.0±0.2 | 29±0.2 | 2±0.2 |
| Ethanol | 68.8±0.2 | 28.8±0.2 | 2.4±0.1 |
| Control without bacteria | 100.0±2 | 0 | 0 |
| Control without electron donors | 100.0±1.25 | 0 | 0 |
Cell protein concentration was 0.035 mg ml−1. Electron donors were at 10 mM each and [TcO4−] at 0.25 mM. Quantification was performed by means of PhosphorImager and verified with extraction techniques. The data shown are mean values of duplicate measurements.
The reduction of TcVIIO4− by strain Mono after 28 h of incubation
| Substrate | Technetium speciation in the supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| Formate | 65.0±1 | 32±1 | 3±0.3 |
| Acetate | 67.0±0.5 | 28.9±0.5 | 4±0.5 |
| Lactate | 68.0±0.75 | 28±0.75 | 3±0.3 |
| Methanol | 69.0±0.2 | 29±0.2 | 2±0.2 |
| Ethanol | 68.8±0.2 | 28.8±0.2 | 2.4±0.1 |
| Control without bacteria | 100.0±2 | 0 | 0 |
| Control without electron donors | 100.0±1.25 | 0 | 0 |
| Substrate | Technetium speciation in the supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| Formate | 65.0±1 | 32±1 | 3±0.3 |
| Acetate | 67.0±0.5 | 28.9±0.5 | 4±0.5 |
| Lactate | 68.0±0.75 | 28±0.75 | 3±0.3 |
| Methanol | 69.0±0.2 | 29±0.2 | 2±0.2 |
| Ethanol | 68.8±0.2 | 28.8±0.2 | 2.4±0.1 |
| Control without bacteria | 100.0±2 | 0 | 0 |
| Control without electron donors | 100.0±1.25 | 0 | 0 |
Cell protein concentration was 0.035 mg ml−1. Electron donors were at 10 mM each and [TcO4−] at 0.25 mM. Quantification was performed by means of PhosphorImager and verified with extraction techniques. The data shown are mean values of duplicate measurements.
3.5 Tc toxicity for haloalkaliphilic bacteria
When the effect of various concentrations of TcO4− on reduction of Tc(VII) was examined, it was found that the bacteria were resistant to 2 mM of Tc(VII) but TcO4− had a toxic effect. Reduction of TcO4− was drastically decreased at concentrations above 1.5 mM Tc (Table 3).
Toxic effect of Tc on the reduction of pertechnetate ion by suspension of strain Mono
| Tc added to experiment (mM) | Tc determined in the cultural supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| 0.25 (100%) | 62.4±1 | 29.2±1 | 8.4±1 |
| 0.50 (100%) | 82.6±1.75 | 11.6±1.75 | 5.8±1 |
| 1.00 (100%) | 94.5±0.7 | 3.5±0.4 | 2.0±0.4 |
| 1.50 (100%) | 96.3±1 | 2.6±0.5 | 1.0±0.3 |
| 2.00 (100%) | 98.4±2 | 1.0±0.2 | 0.5±0.2 |
| Tc added to experiment (mM) | Tc determined in the cultural supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| 0.25 (100%) | 62.4±1 | 29.2±1 | 8.4±1 |
| 0.50 (100%) | 82.6±1.75 | 11.6±1.75 | 5.8±1 |
| 1.00 (100%) | 94.5±0.7 | 3.5±0.4 | 2.0±0.4 |
| 1.50 (100%) | 96.3±1 | 2.6±0.5 | 1.0±0.3 |
| 2.00 (100%) | 98.4±2 | 1.0±0.2 | 0.5±0.2 |
Incubation 28 h and cell protein concentration 0.035 mg ml−1. Quantification was performed by means of PhosphorImager. The data shown are mean values of duplicate measurements.
Toxic effect of Tc on the reduction of pertechnetate ion by suspension of strain Mono
| Tc added to experiment (mM) | Tc determined in the cultural supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| 0.25 (100%) | 62.4±1 | 29.2±1 | 8.4±1 |
| 0.50 (100%) | 82.6±1.75 | 11.6±1.75 | 5.8±1 |
| 1.00 (100%) | 94.5±0.7 | 3.5±0.4 | 2.0±0.4 |
| 1.50 (100%) | 96.3±1 | 2.6±0.5 | 1.0±0.3 |
| 2.00 (100%) | 98.4±2 | 1.0±0.2 | 0.5±0.2 |
| Tc added to experiment (mM) | Tc determined in the cultural supernatant (%) | ||
| Tc(VII) | Tc(IV) | Tc(V) | |
| 0.25 (100%) | 62.4±1 | 29.2±1 | 8.4±1 |
| 0.50 (100%) | 82.6±1.75 | 11.6±1.75 | 5.8±1 |
| 1.00 (100%) | 94.5±0.7 | 3.5±0.4 | 2.0±0.4 |
| 1.50 (100%) | 96.3±1 | 2.6±0.5 | 1.0±0.3 |
| 2.00 (100%) | 98.4±2 | 1.0±0.2 | 0.5±0.2 |
Incubation 28 h and cell protein concentration 0.035 mg ml−1. Quantification was performed by means of PhosphorImager. The data shown are mean values of duplicate measurements.
4 Discussion
Six heterotrophic haloalkaliphilic strains isolated from soda-lake environments were tested for ability to reduce Tc(VII) under anaerobic alkaline conditions. After 2 months of cultivation, the amount of reduced technetium reached 80%. At neutral pH several species of Desulfovibrio incompletely reduced TcO4−; 10% were present at the end as Tc(VII) [6]. As expected, Tc(VI) was not detected in our experiments because it disproportionated as 3Tc(VI)→Tc(IV)+2Tc(VII) within a period of only 10–50 ms [21]. Our experiment showed Tc(V) in the reduction process, but the amount of that form did not reach more than 3–5% (chromatographic determination). This may be explained by disproportion of the Tc(V) to the Tc(VII) and Tc(IV).
It is necessary to note that spectrum shape depends on the structure of the element. According to specific properties of electron orbitals, d-elements always have wider bands in the spectrum compared to f-elements (actinides). Structure of the external electron orbital of technetium (4s24p64d65s1) causes its properties as transitional element of second row of d-group. Such structure of electron orbital gives valent state from 7+ to 1−, inclination to disproportionation and complexation. As seen from Fig. 1 spectra of technetium have no sharp maximum but form a wide band that is sufficiently clear for identification. It is known that the optical spectra of Tc(V)-complexes have multiple bands, with at least one band in the range of 330–450 nm [22]. Based on these results, it may be assumed that the detected band at 418 nm belongs to Tc(V). Unlike bacterial reduction of TcO4− at neutral and acidic conditions, the reduction process under alkaline conditions in our experiments continued up to Tc(III). After exposure to air, however, the pink color reappeared and chromatography and spectrophotometric analyses revealed only Tc(IV). According to Paquette [16] Tc(IV) can be reduced to Tc(III), however, Tc(III) is very sensitive to oxygen and immediately reoxidizes to Tc(IV) [16], which is why in our experiments this form of technetium was detected only transiently.
With regard to the structure of reduced technetium, we can suggest that in our experiments reduced technetium existed as a complex TcIVO(OH)3(CO3)−. According to the literature data several aqueous complexes exist: TcO(OH)2 (aq), TcO(OH)3−, Tc(OH)2CO3 and TcIVO(OH)3(CO3)−. Within the pH range from 8 to 11, the anionic complex TcIVO(OH)3(CO3)− is the dominant one [23].
In conclusion, our results may be important for the understanding of the emission of technetium-99 by adding a new dimension to the chemistry of Tc in the environment. Haloalkalophilic heterotrophic microorganisms could provide a potential perspective for the biotechnological treatment of low level radioactive waste. Our isolates were capable of using formate, acetate, lactate, methanol and ethanol as electron donors and pertechnetate, nitrate, selenite, selenate, tellurite, chromate and elemental sulfur as electron acceptors. This wide variety of substrates and resistance to high concentration of technetium make such bacteria attractive for biotechnological applications.
Acknowledgements
Financial and human support from the French Ministry of Education and Research, CNRS, Bordeaux Region and from PACE (Programme sur l’Aval du Cycle Electronucléaire) is greatfully acknowledged. Also, this work was supported in part by Grant 02-04-48196 from the Russian Foundation for Basic Research.
References
