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Here we report the synthesis of the 6-(6-methyl-1,2,4,5-tetrazine-3-yl)-2,2‘-bipyridine (MTB) ligand, that has been developed for lanthanide/actinide separation. A multi-method study of the complexation of MTB with trivalent actinide and lanthanide ions is presented. Single crystal X-ray diffraction measurements reveal the formation of [Ce(MTB)2(NO3)3], [Pr(MTB)NO3)3H2O], and [Ln(MTB)(NO3)3MeCN] (Ln = Nd, Sm, Eu, Gd). In addition, the complexation of Cm(III) with MTB in solution was studied by time-resolved laser fluorescence spectroscopy. The results show the formation of [Cm(MTB)1-3] 3+ complexes, which occurs in two different isomers. Quantum chemical calculations reveal an energy difference between these isomers of 12 kJ mol-1, clarifying the initial observations made by TRLFS. Furthermore, QTAIM analysis of the Cm(III) and Ln(III) complexes was performed, indicating a stronger covalent contribution in the Cm-N interaction compared to the respective Ln-N interaction. These findings align well with extraction data showing a preferred extraction of Am and Cm over lanthanides (e.g., max. SFAm/Eu = 8.3) at nitric acid concentrations < 0.1 mol L-1 HNO3. .

The complexation of Np(V) with malonate and succinate is studied by different spectroscopic techniques, namely attenuated total reflection FT-IR (ATR FT-IR) and extended X-ray absorption fine structure (EXAFS) spectroscopy, as well as by quantum chemistry to determine the speciation, thermodynamic data and structural information of the formed complexes. For complex stoichiometries and thermodynamic functions (log beta0n(T), Delta rH0n, Delta rS0n) near infrared absorption spectroscopy (Vis/NIR) is applied. The complexation reactions are investigated as a function of the total ligand concentration ([Mal2-]total, [Succ2-]total), ionic strength (Im = 0.5 – 4.0 mol kg-1 Na+(Cl-/ClO4-)) and temperature (T = 20 – 85 °C). Besides the solvated NpO2+ ion, the formation of two Np(V) species with the stoichiometry NpO2(L)n1-2n (n = 1, 2, L = Mal2-, Succ2-) is observed. With increasing temperature the molar fractions of both complex species increase and the application of the law of mass action yields the temperature dependent conditional stability constants log beta`n(T) at given ionic strengths. The log beta`n(T) are extrapolated to IUPAC reference state conditions (Im = 0) according to the specific ion interaction theory (SIT) revealing thermodynamic log 0n(T) values. For all formed complexes (NpO2(Mal)-: log 01(25 °C) = 3.36 ± 0.11, NpO2(Mal)23-: log 02(25 °C) = 3.95 ± 0.19, NpO2(Succ)-: log 01(25 °C) = 2.05 ± 0.45, NpO2(Succ)23-: log 02(25 °C) = 0.75 ± 1.22) the stability constants increase with increasing temperature confirming an endothermic complexation reaction. The temperature dependence of the thermodynamic stability constants is described by the integrated Van’t Hoff equation yielding the standard reaction enthalpies and entropies for the complexation reactions. In addition, the sum of the specific binary ion-ion interaction coefficients 0n(T) for the complexation reactions are obtained from SIT modelling as a function of the temperature. The structure of the

The complexation of Np(V) with malonate and succinate is studied by different spectroscopic techniques, namely attenuated total reflection FT-IR (ATR FT-IR) and extended X-ray absorption fine structure (EXAFS) spectroscopy, as well as by quantum chemistry to determine the speciation, thermodynamic data and structural information of the formed complexes. For complex stoichiometries and thermodynamic functions (log beta0n(T), Delta rH0n, Delta rS0n) near infrared absorption spectroscopy (Vis/NIR) is applied. The complexation reactions are investigated as a function of the total ligand concentration ([Mal2-]total, [Succ2-]total), ionic strength (Im = 0.5 – 4.0 mol kg-1 Na+(Cl-/ClO4-)) and temperature (T = 20 – 85 °C). Besides the solvated NpO2+ ion, the formation of two Np(V) species with the stoichiometry NpO2(L)n1-2n (n = 1, 2, L = Mal2-, Succ2-) is observed. With increasing temperature the molar fractions of both complex species increase and the application of the law of mass action yields the temperature dependent conditional stability constants log beta`n(T) at given ionic strengths. The log beta`n(T) are extrapolated to IUPAC reference state conditions (Im = 0) according to the specific ion interaction theory (SIT) revealing thermodynamic log 0n(T) values. For all formed complexes (NpO2(Mal)-: log 01(25 °C) = 3.36 ± 0.11, NpO2(Mal)23-: log 02(25 °C) = 3.95 ± 0.19, NpO2(Succ)-: log 01(25 °C) = 2.05 ± 0.45, NpO2(Succ)23-: log 02(25 °C) = 0.75 ± 1.22) the stability constants increase with increasing temperature confirming an endothermic complexation reaction. The temperature dependence of the thermodynamic stability constants is described by the integrated Van’t Hoff equation yielding the standard reaction enthalpies and entropies for the complexation reactions. In addition, the sum of the specific binary ion-ion interaction coefficients 0n(T) for the complexation reactions are obtained from SIT modelling as a function of the temperature. The structure of the

The complexation of NpO2+ with lactate in aqueous solution is studied as a function of the total ligand concentration ([Lac-]total), ionic strength (Im = 0.5 – 4.0 mol kg-1 Na+(Cl-/ClO4-)) and temperature (T = 20 – 85 °C) by Vis/NIR absorption spectroscopy. The formation of two NpO2+ lactate species with the stoichiometry NpO2(Lac)n1-n (n = 1, 2) is observed at the studied experimental conditions. The temperature dependent conditional stability constants log beta 'j(T) at different ionic strengths are calculated with the law of mass action. The conditional data are extrapolated to IUPAC reference state conditions (Im = 0) with the specific ion interaction theory (SIT). With increasing temperature up to 85 °C log beta 01(20 °C) = 1.92 ± 0.14 decreases by 0.12 and log beta 02(20 °C) = 2.10 ± 0.13 decreases by 0.17. The thermodynamic stability constants correlate linearly with the reciprocal temperature according to the integrated Van’t Hoff equation. Thus, linear regression analyses yield the standard reaction enthalpy delta rH0 and entropy delta rS0 for the complexation reactions. In addition, the sum of the SIT specific binary ion-ion interaction coefficients delta epsilon j,k(T) of the complexation reactions are determined by variation of the ionic strength. Structural parameters of the formed complex species and the coordination mode of lactate towards the NpO2+ ion are investigated as a function of pHc by extended x-ray absorption fine structure spectroscopy (EXAFS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FT IR). The results show, that the coordination mode of lactate changes from end-on (coordination via only the COO- group) to side-on (formation of chelate rings involving the OH-group) with increasing pHc. The experiments are supported by quantum chemical calculations.

The complexation of NpO2+ with lactate in aqueous solution is studied as a function of the total ligand concentration ([Lac-]total), ionic strength (Im = 0.5 – 4.0 mol kg-1 Na+(Cl-/ClO4-)) and temperature (T = 20 – 85 °C) by Vis/NIR absorption spectroscopy. The formation of two NpO2+ lactate species with the stoichiometry NpO2(Lac)n1-n (n = 1, 2) is observed at the studied experimental conditions. The temperature dependent conditional stability constants log beta 'j(T) at different ionic strengths are calculated with the law of mass action. The conditional data are extrapolated to IUPAC reference state conditions (Im = 0) with the specific ion interaction theory (SIT). With increasing temperature up to 85 °C log beta 01(20 °C) = 1.92 ± 0.14 decreases by 0.12 and log beta 02(20 °C) = 2.10 ± 0.13 decreases by 0.17. The thermodynamic stability constants correlate linearly with the reciprocal temperature according to the integrated Van’t Hoff equation. Thus, linear regression analyses yield the standard reaction enthalpy delta rH0 and entropy delta rS0 for the complexation reactions. In addition, the sum of the SIT specific binary ion-ion interaction coefficients delta epsilon j,k(T) of the complexation reactions are determined by variation of the ionic strength. Structural parameters of the formed complex species and the coordination mode of lactate towards the NpO2+ ion are investigated as a function of pHc by extended x-ray absorption fine structure spectroscopy (EXAFS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FT IR). The results show, that the coordination mode of lactate changes from end-on (coordination via only the COO- group) to side-on (formation of chelate rings involving the OH-group) with increasing pHc. The experiments are supported by quantum chemical calculations.

In the advent of the 2nd International Workshop on Advanced Techniques in Actinide Spectroscopy (ATAS 2014), an inter-laboratory round-robin test (RRT) was initiated. The main goal of this RRT was the comprehensive molecular analysis of a simple aqueous com-plexing system – U(VI) acetate – which was selected to be independently investigated by various spectroscopic (vibrational, luminescence, X-ray absorption, and nuclear magnetic resonance spectroscopy) and quantum chemical methods applied by leading laboratories in actinide or geochemical research. Ultimately, more than 40 scientists hosted at twenty insti-tutions in seven countries participated. The outcome of this RRT can be considered on two levels: First, conformities as well as discrepancies in the results of each spectroscopic technique and their sources are evaluated. The raw data from the experimental approaches were found to be generally consistent. In particular, for complex setups such as accelerator-based X-ray absorption spectroscopy, the agreement between the raw data was surprisingly high. By contrast, the data obtained using luminescence spectroscopy turned out to be strongly related to the chosen acquisition pa-rameters. Second, the potentials and limitations of coupling various spectroscopic and, in particular, theoretical approaches for the comprehensive study of actinide molecule com-plexes are assessed. The additional benefits of the combined approach with regard to the exploration of the aqueous speciation of the U(VI) acetate system are elaborated.

The necessity to reprocess spent nuclear fuel has arisen from increasing volumes of highly active and long-lived radionuclides associated with nuclear fission alongside increasing environmental impact concerns. In particular, one major challenge is the separation of the long-lived trivalent minor actinides (here, AmIII and CmIII) from the trivalent lanthanides in order to allow the future transmutation of the actinides into shorter lived radionuclides for the purpose of reducing the long-term radiotoxicity of the waste and the volume build up in storage. There are currently a number of different processes under development world-wide, all of which rely on the coordination chemistry and selective extraction of the minor actinides from the trivalent lanthanides using solvent partitioning. Of these, The TALSPEAK process (Trivalent Actinide Lanthanide Separation by Phosphorus reagent Extraction from Aqueous Komplexations) has showed great promise in separating the trivalent lanthanides from the minor actinides. The process uses DTPA (diethylenetriamine pentaacetic acid) as the chelating holdback reagent, HDEHP (bis-2-ethylhexyl phosphoric acid) as the extracting ligand and lactic acid as the buffer to prevent precipitation of DTPA that occurs at below pH 3.6. However, despite the considerable separation factors obtainable, and the fact the process exhibits promising radiolysis resistance, the process operates at sub optimal pH values. To overcome these limitations, we have developed a family of DTPA-amino acid conjugates that operate under TALSPEAK-like conditions over a much lower pH range of 1.5-2.5. Additionally, these ligands show comparable selectivity to the TALSPEAK process and are also relatively resistant to radiolysis. We will discuss the coordination chemistry of these ligands with the lanthanides and minor actinides using a combination of luminescence and NMR spectroscopies.

The necessity to reprocess spent nuclear fuel has arisen from increasing volumes of highly active and long-lived radionuclides associated with nuclear fission alongside increasing environmental impact concerns. In particular, one major challenge is the separation of the long-lived trivalent minor actinides (here, AmIII and CmIII) from the trivalent lanthanides in order to allow the future transmutation of the actinides into shorter lived radionuclides for the purpose of reducing the long-term radiotoxicity of the waste and the volume build up in storage. There are currently a number of different processes under development world-wide, all of which rely on the coordination chemistry and selective extraction of the minor actinides from the trivalent lanthanides using solvent partitioning. Of these, The TALSPEAK process (Trivalent Actinide Lanthanide Separation by Phosphorus reagent Extraction from Aqueous Komplexations) has showed great promise in separating the trivalent lanthanides from the minor actinides. The process uses DTPA (diethylenetriamine pentaacetic acid) as the chelating holdback reagent, HDEHP (bis-2-ethylhexyl phosphoric acid) as the extracting ligand and lactic acid as the buffer to prevent precipitation of DTPA that occurs at below pH 3.6. However, despite the considerable separation factors obtainable, and the fact the process exhibits promising radiolysis resistance, the process operates at sub optimal pH values. To overcome these limitations, we have developed a family of DTPA-amino acid conjugates that operate under TALSPEAK-like conditions over a much lower pH range of 1.5-2.5. Additionally, these ligands show comparable selectivity to the TALSPEAK process and are also relatively resistant to radiolysis. We will discuss the coordination chemistry of these ligands with the lanthanides and minor actinides using a combination of luminescence and NMR spectroscopies.

The pH-dependent (pH 2 - 4) formation of aqueous Am(III) complexes with formate (Form) is studied by EXAFS, iterative target transformation factor analysis (ITFA) and by quantum chemical calculations [1]. According to thermodynamical calculations three complexes (aq. Am3+, AmForm2+, AmForm2+) coexist and change their fractions, so that EXAFS spectral mixtures occur. A maximal spectral change of only 9% is observed between the different Am LIII-edge EXAFS spectra, which rules out the direct use of conventional shell fit analysis for structural investigation prior to the decomposition of the spectra into the single spectral components by ITFA. By combining pH-speciation calculations with quantum chemistry, the pH-dependent number of coordinated water and formate molecules is calculated and used as constraint for a modified ITFA-approach. The decomposition results in the separate spectral contributions of the exchanged molecules, hence the signal of a coordinated water and a monodentate coordinated formate molecule for which the structural parameters are determined by shell-fitting and are compared with twelve complex structures gained by quantum chemical calculations. Methodologically consistent, the prevailing coordination numbers are 9, 9 and 8 for the aq. Am3+, AmForm2+ and AmForm2+ complexes, respectively [2]. Low concentrations of species with other coordination numbers and modes cannot be excluded, as energy differences obtained by our quantum chemical calculations are small [2]. This work shows the power of the proposed ITFA-framework in obtaining structural information for weak ligand systems like formate, where conventional EXAFS data analysis fails due to the lower spectroscopic resolution in analysing mixtures of metal species. References [1] Rossberg et al., Anal. Bioanal. Chem. 376, 631-638 (2003). [2] Fröhlich et al., Inorg. Chem. submitted (2017).

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE. The ThermAc project is developed with the aim of improving the scientific basis for assessing nuclear waste disposal scenarios at elevated temperature conditions. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. A clear focus of ThermAc is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. Tetravalent actinides and detailed investigations of redox processes are excluded from the current ThermAc work programme. ThermAc mainly addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Chemical analogs for the actinide elements will be used, especially in order to gain information on solid phase transformation processes. Ion-interactions are treated with the Specific Ion Interaction Theory (SIT), in agreement with the approach favored by the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level. Within the scope of ThermAc, a significant impact can be realized within a strong collaborative and integrated concept with the following strategic components: (1) Systematic use of estimation methods for thermodynamic data and model parameters. (2) Compr

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE. The ThermAc project is developed with the aim of improving the scientific basis for assessing nuclear waste disposal scenarios at elevated temperature conditions. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. A clear focus of ThermAc is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. Tetravalent actinides and detailed investigations of redox processes are excluded from the current ThermAc work programme. ThermAc mainly addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Chemical analogs for the actinide elements will be used, especially in order to gain information on solid phase transformation processes. Ion-interactions are treated with the Specific Ion Interaction Theory (SIT), in agreement with the approach favored by the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level. Within the scope of ThermAc, a significant impact can be realized within a strong collaborative and integrated concept with the following strategic components: (1) Systematic use of estimation methods for thermodynamic data and model parameters. (2) Compr

The ThermAc project aims at extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. Such conditions are expected when storing highly active heat producing waste in a repository system over a significant period of time after starting repository operation. If early canister failure occurs, radionuclides therefore may contact aquatic systems at higher temperatures. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. ThermAc approaches this challenge by evaluating the capabilities of a variety of estimation methods to obtain thermodynamic parameters (formation constants, enthalpic and entropic data) as f(T). This is done by both intercomparison between such methods and pointwise checks with experimental results. There, a clear focus is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. ThermAc addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Only for selected cases with specific relevance or scientific interest, higher temperatures up to 200°C or salt brine solutions are investigated. Chemical analogs help to gain information on solid phase transformation processes. Ion-ion-interaction processes are treated with the SIT, in agreement with the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level.

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE.

Partitioning and transmutation (P&T) is a strategy for reducing the long-term radiotoxicity and heat load of spent nuclear fuel by separating actinides from the used fuel and converting them into shorterlived or stable products. A key step in this process is the separation of the trivalent actinides from lanthanides, which can be achieved by liquid-liquid extraction using selective N-donor extracting ligands, such as alkylated bis-triazinyl pyridines (BTP). These have high separation factors (>100) for trivalent americium over europium. However, little is known about the molecular origin of their selectivity. The aliphatic side chains of BTP ligands influence the stability against radiation and hydrolysis, the solubility, but also the selectivity and extraction behaviour. While nPrBTP (1) has been thoroughly studied over the past years, only few data are available for its isomer iPrBTP (2). TRLFS studies showed that the stability constants for the Eu(III) complex are almost three orders of magnitude higher than for nPrBTP, while the increase for Cm(III) complexes is less pronounced. This result prompted us to investigate Ln(III) and the Am(III) complexes by NMR spectroscopy, which offers insight into the metal-ligand bond properties. For the NMR studies, iPrBTP with 15N isotope labelling of the nitrogen atoms in the pyrazole moiety was synthesized. We will show a comparison of the bonding properties in complexes of (1) and (2) and discuss the share of covalence in the bonding. Furthermore, the implications for the mechanism of complex formation with ligand (2) will be evaluated.

Partitioning and transmutation (P&T) facilitates a reduction of the long-term radiotoxicity and heat load of spent nuclear fuel by separation of the actinides and subsequent conversion into shorter-lived or stable nuclides. A chemically demanding key step in this process is the separation of the trivalent actinides from fission lanthanides. This can be achieved by liquid-liquid extraction using highly selective extraction agents, such as nPrBTP (1) or C5-BPP (2). These have high separation factors (>100) for trivalent americium over europium. However, the molecular origin of their selectivity is largely unclear. NMR spectroscopy on paramagnetic samples allows a separation of the observed paramagnetic shift into a part due to transferred electron spin density (Fermi contact shift, FCS) and a part due to dipolar coupling of electron and nuclear spin (Pseudo contact shift, PCS). Evaluation of the FCS thus allows an assessment of the share of covalence in the metal-ligand bond of the N-donor complexes. Several methods that enable the separation of the shift contributions have been proposed in literature. So-called “model free methods” that do not require structural models of the complexes appear most promising. We will present and compare the results of different temperature-dependent and temperature-independent model-free methods for complexes of both ligands. The merits, but also the limitations of currently available methods will be discussed in detail. Furthermore, we will evaluate the applicability of temperature-dependent methods for shift separation of Am(III) complexes and give a qualitative assessment on covalence in the bonding of these complexes.

Bistriazinyl-pyridine type ligands are important extracting agents for separating trivalent actinide ions from trivalent lanthanides. The alkyl substituents on the lateral triazine rings have a significant effect on the stability of the ligand against hydrolysis and radiolysis. Furthermore they influence solubility, extraction behaviour and selectivity. TRLFS and extraction studies suggest differences in complexation and extraction behaviour of BTP ligands bearing iso-propyl or n-propyl substituents, respectively. As NMR studies allow insight into the metal-ligand bonding, we conducted NMR studies on a range of 15N-labelled nPrBTP and iPrBTP complexes. Our results show that no strong change in the metal-ligand bonding occurs, thus excluding electronic reasons for differences in complexation behaviour, extraction kinetics and selectivity. This supports mechanistic reasons for the observed differences.

Bistriazinyl-pyridine type ligands are important extracting agents for separating trivalent actinide ions from trivalent lanthanides. The alkyl substituents on the lateral triazine rings have a significant effect on the stability of the ligand against hydrolysis and radiolysis. Furthermore they influence solubility, extraction behaviour and selectivity. TRLFS and extraction studies suggest differences in complexation and extraction behaviour of BTP ligands bearing iso-propyl or n-propyl substituents, respectively. As NMR studies allow insight into the metal-ligand bonding, we conducted NMR studies on a range of 15N-labelled nPrBTP and iPrBTP complexes. Our results show that no strong change in the metal-ligand bonding occurs, thus excluding electronic reasons for differences in complexation behaviour, extraction kinetics and selectivity. This supports mechanistic reasons for the observed differences.

The ThermAc project aims at extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. Such conditions are expected when storing highly active heat producing waste in a repository system over a significant period of time after starting repository operation. If early canister failure occurs, radionuclides therefore may contact aquatic systems at higher temperatures. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. ThermAc approaches this challenge by evaluating the capabilities of a variety of estimation methods to obtain thermodynamic parameters (formation constants, enthalpic and entropic data) as f(T). This is done by both intercomparison between such methods and pointwise checks with experimental results. There, a clear focus is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. ThermAc addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Only for selected cases with specific relevance or scientific interest, higher temperatures up to 200°C or salt brine solutions are investigated. Chemical analogs help to gain information on solid phase transformation processes. Ion-ion-interaction processes are treated with the SIT, in agreement with the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level.

Partitioning and transmutation (P&T) facilitates a reduction of the long-term radiotoxicity and heat load of spent nuclear fuel by separation of the actinides and subsequent conversion into shorter-lived or stable nuclides. A chemically demanding key step in this process is the separation of the trivalent actinides from fission lanthanides. This can be achieved by liquid-liquid extraction using highly selective extraction agents, such as nPrBTP (1) or C5-BPP (2). These have high separation factors (>100) for trivalent americium over europium. However, the molecular origin of their selectivity is largely unclear. NMR spectroscopy on paramagnetic samples allows a separation of the observed paramagnetic shift into a part due to transferred electron spin density (Fermi contact shift, FCS) and a part due to dipolar coupling of electron and nuclear spin (Pseudo contact shift, PCS). Evaluation of the FCS thus allows an assessment of the share of covalence in the metal-ligand bond of the N-donor complexes. Several methods that enable the separation of the shift contributions have been proposed in literature. So-called “model free methods” that do not require structural models of the complexes appear most promising. We will present and compare the results of different temperature-dependent and temperature-independent model-free methods for complexes of both ligands. The merits, but also the limitations of currently available methods will be discussed in detail. Furthermore, we will evaluate the applicability of temperature-dependent methods for shift separation of Am(III) complexes and give a qualitative assessment on covalence in the bonding of these complexes.

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE. The ThermAc project is developed with the aim of improving the scientific basis for assessing nuclear waste disposal scenarios at elevated temperature conditions. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. A clear focus of ThermAc is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. Tetravalent actinides and detailed investigations of redox processes are excluded from the current ThermAc work programme. ThermAc mainly addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Chemical analogs for the actinide elements will be used, especially in order to gain information on solid phase transformation processes. Ion-interactions are treated with the Specific Ion Interaction Theory (SIT), in agreement with the approach favored by the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level. Within the scope of ThermAc, a significant impact can be realized within a strong collaborative and integrated concept with the following strategic components: (1) Systematic use of estimation methods for thermodynamic data and model parameters. (2) Compr

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE.

The ThermAc project is extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures. To this end, a systematic use of estimation methods, new experimental investigations and quantum-chemistry based information is used. ThermAc has started in March 2015 and is projected for three years, running until 28.02.2018. The project is funded by the German Federal Ministry for Education and Research (BMBF) and is coordinated by KIT-INE. The ThermAc project is developed with the aim of improving the scientific basis for assessing nuclear waste disposal scenarios at elevated temperature conditions. Adequate scientific tools must be available to assess the related chemical effects and their impact upon safety. A clear focus of ThermAc is on long-lived actinides in oxidation states III, V and VI, with selected fission products and important redox controlling matrix elements like Fe also receiving attention. Tetravalent actinides and detailed investigations of redox processes are excluded from the current ThermAc work programme. ThermAc mainly addresses the temperature range from ~5°C up to ~90°C, focusing on systems at low or intermediate ionic strength. Chemical analogs for the actinide elements will be used, especially in order to gain information on solid phase transformation processes. Ion-interactions are treated with the Specific Ion Interaction Theory (SIT), in agreement with the approach favored by the NEA-TDB project. Quantum chemical calculations are used to support the interpretation of experimental findings, and establish a fundamental understanding of chemical effects on a molecular level. Within the scope of ThermAc, a significant impact can be realized within a strong collaborative and integrated concept with the following strategic components: (1) Systematic use of estimation methods for thermodynamic data and model parameters. (2) Compr

Partitioning and transmutation (P&T) is a strategy for reducing the long-term radiotoxicity and heat load of spent nuclear fuel by separating actinides from the used fuel and converting them into shorterlived or stable products. A key step in this process is the separation of the trivalent actinides from lanthanides, which can be achieved by liquid-liquid extraction using selective N-donor extracting ligands, such as alkylated bis-triazinyl pyridines (BTP). These have high separation factors (>100) for trivalent americium over europium. However, little is known about the molecular origin of their selectivity. The aliphatic side chains of BTP ligands influence the stability against radiation and hydrolysis, the solubility, but also the selectivity and extraction behaviour. While nPrBTP (1) has been thoroughly studied over the past years, only few data are available for its isomer iPrBTP (2). TRLFS studies showed that the stability constants for the Eu(III) complex are almost three orders of magnitude higher than for nPrBTP, while the increase for Cm(III) complexes is less pronounced. This result prompted us to investigate Ln(III) and the Am(III) complexes by NMR spectroscopy, which offers insight into the metal-ligand bond properties. For the NMR studies, iPrBTP with 15N isotope labelling of the nitrogen atoms in the pyrazole moiety was synthesized. We will show a comparison of the bonding properties in complexes of (1) and (2) and discuss the share of covalence in the bonding. Furthermore, the implications for the mechanism of complex formation with ligand (2) will be evaluated.

NMR spectroscopy on paramagnetic compounds is a sensitive and versatile method for structural investigations of metal-organic complexes. Furthermore, separation of the overall observed paramagnetic chemical shift into parts due to covalently transferred electron spin density (Fermi contact shift, FCS) and distance- and angle-dependent dipolar electron-nucleus spin coupling (pseudo contact shift, PCS) yields insights into metal-ligand bonding. The evaluation of the pure FCS allows to determine the share of covalance in this bond. Covalence is thought to be the reason for some ligands’ selectivity for the selective complexation of actinide over lanthanide ions in potential partitioning processes.[1,2] Since the advent of chemical shift reagents in NMR spectroscopy in 1969, several methods for the separation of FCS and PCS have been developed.[3-6] Modell-free methods rely on calculated values like spin expectation values, geometrical constants and crystal field parameters. All these values are still unknown for actinide compounds. On the other hand, the application of methods requiring a structural modell of the complex is only possible for metal ions with a large magnetic anisotropy, like the heavy lanthanides. As Am(III) has a low magnetic anisotropy, only modell-free methods can be applied to separate the observed paramagnetic shift and to elucidate the bonding in Am(III)-N-donor complexes. Currently, we evaluate the applicability of several approaches for separation of FCS and PCS in lanthanide complexes and their transferability to actinide compounds. This includes methods based on calculated values as well as temperature-dependent methods. We will report on our studies on a complete set of 15N-labeled lanthanide nPr-BTP and C5-BPP complexes and discuss the applicability of the methods on actinide complexes. This work is supported by the German Federal Ministry of Education and Research (BMBF) under contract numbers 02NUK020A and 02NUK020D. 1. C. Adam, B. B. Beel

The ThermAc project aims at extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures.

The complexation of acetate with Am(III) is studied as a function of the pH (1–6) by extended X-ray absorption fine-structure (EXAFS) spectroscopy. The molecular structure of the Am(III)–acetate complexes (coordination numbers, oxygen and carbon distances) is determined from the raw k3-weighted Am LIIIedge EXAFS spectra. The results show a continuous shift of Am(III) speciation with increasing pH value towards the complexed species. Furthermore, it is verified that acetate coordinates in a bidentate coordination mode to Am(III) (Am—C distance: 2.82 0.03 A ° ). The EXAFS data are analyzed by iterative transformation factor analysis to further verify the chemical speciation, which is calculated on the basis of thermodynamic constants, and the used structural model. The experimental results are in very good agreement with the thermodynamic modelling.

The ThermAc project aims at extending the chemical understanding and available thermodynamic database for actinides, long-lived fission products and relevant matrix elements in aquatic systems at elevated temperatures.

NMR spectroscopy on paramagnetic compounds is a sensitive and versatile method for structural investigations of metal-organic complexes. Furthermore, separation of the overall observed paramagnetic chemical shift into parts due to covalently transferred electron spin density (Fermi contact shift, FCS) and distance- and angle-dependent dipolar electron-nucleus spin coupling (pseudo contact shift, PCS) yields insights into metal-ligand bonding. The evaluation of the pure FCS allows to determine the share of covalance in this bond. Covalence is thought to be the reason for some ligands’ selectivity for the selective complexation of actinide over lanthanide ions in potential partitioning processes.[1,2] Since the advent of chemical shift reagents in NMR spectroscopy in 1969, several methods for the separation of FCS and PCS have been developed.[3-6] Modell-free methods rely on calculated values like spin expectation values, geometrical constants and crystal field parameters. All these values are still unknown for actinide compounds. On the other hand, the application of methods requiring a structural modell of the complex is only possible for metal ions with a large magnetic anisotropy, like the heavy lanthanides. As Am(III) has a low magnetic anisotropy, only modell-free methods can be applied to separate the observed paramagnetic shift and to elucidate the bonding in Am(III)-N-donor complexes. Currently, we evaluate the applicability of several approaches for separation of FCS and PCS in lanthanide complexes and their transferability to actinide compounds. This includes methods based on calculated values as well as temperature-dependent methods. We will report on our studies on a complete set of 15N-labeled lanthanide nPr-BTP and C5-BPP complexes and discuss the applicability of the methods on actinide complexes. This work is supported by the German Federal Ministry of Education and Research (BMBF) under contract numbers 02NUK020A and 02NUK020D. 1. C. Adam, B. B. Beel

The complexation of Cm(III) and Eu(III) with n-C3H7-BTP in non-aqueous organic solution was studied with extended X-ray absorption spectroscopy. Bond lengths are the same in both complexes. Quantum chemical calculations performed at different levels support this finding. On the other hand, the Cm•(n-C3H7-BTP)3 complex is formed at much lower metal-to-ligand concentration ratio than the Eu•(n-C3H7-BTP)3 complex, as shown by time-resolved laser-induced fluorescence spectroscopy. This is in good agreement with n-C3H7-BTP’s high selectivity for trivalent actinides over lanthanides in liquid-liquid extraction.

Uranium(VI) complex formation at vegetative cells and spores of Bacillus cereus and Bacillus sphaericus was studied using uranium LII-edge and LIII-edge extended x-ray absorption fine structure (EXAFS) spectroscopy. A comparison of the measured equatorial U-O distances and other EXAFS structural parameters of uranyl complexes formed at the Bacillus strains with those of the uranyl structure family indicates that the uranium is predominantly bound as uranyl phosphate.

Mobilization of heavy metals in the environment due to industrial activities is of serious concern due to the toxicity of these metals in humans and other forms of life. Uranium is an example of these metals, and is considered one of the most seriously threatening heavy metals mainly because of its high toxicity, not so much radioactivity. Activities associated with the nuclear industry, mining and wastewater treatment have brought excessive amounts of uranium into the environment. In uranium deposits a number of acidophilic chemolithoautotrophic ( Acidithiobacillus ferrooxidans, for instance) bacteria have been identified which are able to oxidise sulphide minerals, elemental sulfur, ferrous iron. Sequence analysis of the 16S rRNA genes of several reference strains and uranium mining waste pile isolates of A. ferrooxidans revealed specific signatures which distinguish three types within the species. These types differ in their capability to accumulate and tolerate uranium /1/. Uranium (VI) complexes formation at cell surfaces of A. ferrooxidans types was studied using uranium LIII-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. In all samples uranium is coordinated by two axial oxygen atoms (Oax) at distance of 1.77-1.78 Å. The average distance between uranium and the equatorial oxygen atoms (Oeq) is 2.35 Å. The coordination number for Oeq is 5-6. Using U-C and U-P phase and amplitude functions, the third and the fourth peak in the FT of A. ferrooxidans give a distance of 2.91 and 3.58 ± 0.02Å, respectively. The latter is the same U-P bond distance as for the organic uranyl phosphate (U(VI)-ATP complex). But we do not exclude the possibility of implication of sulfur because these 2 elements (P and S) are close each other in the periodic system of elements and EXAFS can not distinguish between them. In addition, the P (or S) is bonded in a monodentate mode to the uranyl ion. A bidentate bounding would give a atomic distance of approximately 3.2 Å

Mobilization of heavy metals in the environment due to industrial activities is of serious concern due to the toxicity of these metals in humans and other forms of life. Uranium is an example of these metals, and is considered one of the most seriously threatening heavy metals mainly because of its high toxicity, not so much radioactivity. Activities associated with the nuclear industry, mining and wastewater treatment have brought excessive amounts of uranium into the environment. In uranium deposits a number of acidophilic chemolithoautotrophic ( Acidithiobacillus ferrooxidans, for instance) bacteria have been identified which are able to oxidise sulphide minerals, elemental sulfur, ferrous iron. Sequence analysis of the 16S rRNA genes of several reference strains and uranium mining waste pile isolates of A. ferrooxidans revealed specific signatures which distinguish three types within the species. These types differ in their capability to accumulate and tolerate uranium /1/. Uranium (VI) complexes formation at cell surfaces of A. ferrooxidans types was studied using uranium LIII-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. In all samples uranium is coordinated by two axial oxygen atoms (Oax) at distance of 1.77-1.78 Å. The average distance between uranium and the equatorial oxygen atoms (Oeq) is 2.35 Å. The coordination number for Oeq is 5-6. Using U-C and U-P phase and amplitude functions, the third and the fourth peak in the FT of A. ferrooxidans give a distance of 2.91 and 3.58 ± 0.02Å, respectively. The latter is the same U-P bond distance as for the organic uranyl phosphate (U(VI)-ATP complex). But we do not exclude the possibility of implication of sulfur because these 2 elements (P and S) are close each other in the periodic system of elements and EXAFS can not distinguish between them. In addition, the P (or S) is bonded in a monodentate mode to the uranyl ion. A bidentate bounding would give a atomic distance of approximately 3.2 Å

Certain bacterial strains can selectively accumulate various metal ions from aqueous systems /1/. Recently, it was demonstrated that two Bacillus strains, B. cereus JG-A30 and B. sphaericus JG-A12, recovered from a uranium mining waste site in Germany are able to accumulate selectively a large variety of heavy metals from the drain waters of the waste site /2/. In particular, it was shown that these strains accumulate large amounts of uranium. The aim of this study is the analysis of the structure of uranium complexes formed at vegetative cells and spores of the above mentioned Bacillus strains using extended x-ray absorption fine structure (EXAFS) spectroscopy. Analytical procedures allow separating radial distribution functions from the EXAFS, which contain information on bond lengths and coordination numbers. However, frequently the EXAFS result is dominated mainly by the major coordination type in the sample. A comparison of the determined structural parameters with those of the uranium structure family allows to determine the type of complex formed in the

Gram-positive spore-forming bacterial isolates belonging to the species Bacillus cereus, B. sphaericus and B. megaterium and also Gram-negative chemolithoautotrophic isolates of the species Thiobacillus ferrooxidans and Leptospirillum ferrooxidans were cultured from soil and sediment samples of several uranium polluted waste piles in Saxony, Germany. The interactions of these natural bacterial isolates with uranium (in particular, sorption - desorption, accumulation, reduction, and tolerance) were studied. We have demonstrated that the natural Bacillus isolates are able to accumulate selectively and reversible a variety of toxic metals from drain waters of a uranium mining waste pile (Selenska-Pobell et al., 1999). By the use of extended X-ray absorption fine structure (EXAFS) analysis and time resolved laser fluorescence spectroscopy (TRLFS) it was demonstrated that the main chemical group involved in the interaction of the mentioned Bacillus strains with U(VI) is the phosphate group (Hennig et al., 1999; Panak et al., 2000). Our studies on the interactions of the recently described in our laboratory three ecological types (eco-types) of T. ferrooxidans (Selenska-Pobell et al., 1999) and two eco-types of L. ferrooxidans (Tzvetkova et al., 1999) with uranium (VI) by the use of EXAFS, TRLFS, and infrared spectroscopy (IRS) indicate that the nature and the strength of the uranium complexes built by these Gram-negative acidophilic chemolithoauthotrops differ significantly from those built on the cell surfaces of the Gram-positive Bacilli. The tolerance and the interactions of the natural T. ferrooxidans isolates with U were type-specific.

The genus Bacillus consists of more than 70 species of Gram-positive, aerobic or facultatively anaerobic spore-forming rod-shaped bacteria. Because of the high resistance of their spores, Bacilli were found in a large variety of natural habitats. For our accumulation studies with U(VI), we used vegetative cells and spores of three Bacillus isolates (JG-A 30, JG-A 12, JG-A 22, classified as Bacillus cereus, Bacillus sphaericus, Bacillus megaterium) from a uranium mining waste pile (Johanngeorgenstadt, Saxony) and their corresponding reference strains. Sorption studies at pH 5.0 have shown that in the concentration range examined (11 – 214 mg/L) Bacilli accumulate high amounts of uranium. Information on the binding strength and the reversibility were obtained from extraction studies with different extractants. With 0.01 M EDTA solution the uranium bound to the biomass was released almost quantitatively. The characterization of the bacterial-UO22+-complexes by time-resolved laser fluorescence spectroscopy (TRLFS) has proved the formation of inner-sphere complexes with the biomass. At pH 5 a ternary complex of uranyl with the biomass and hydroxide in solution was detected. After cell fractionation, we examined the spectroscopic properties of the U(VI)-complexes with the isolated cell walls and the isolated surface-layer protein fraction of the bacteria. Spectroscopic studies of these complexes have shown that with intact cells (vegetative cells or spores) the complexation of U(VI) is stronger than with isolated cell wall components. Therefore, the good complexation properties, the easy release of the bound uranium by EDTA-extraction, and the high resistance against harsh environmental conditions makes these bacteria applicable for bioremediation purposes.

Gram-positive spore-forming bacterial isolates belonging to the species Bacillus cereus, B. sphaericus and B. megaterium and also Gram-negative chemolithoautotrophic isolates of the species Thiobacillus ferrooxidans and Leptospirillum ferrooxidans were cultured from soil and sediment samples of several uranium polluted waste piles in Saxony, Germany. The interactions of these natural bacterial isolates with uranium (in particular, sorption - desorption, accumulation, reduction, and tolerance) were studied. We have demonstrated that the natural Bacillus isolates are able to accumulate selectively and reversible a variety of toxic metals from drain waters of a uranium mining waste pile (Selenska-Pobell et al., 1999). By the use of extended X-ray absorption fine structure (EXAFS) analysis and time resolved laser fluorescence spectroscopy (TRLFS) it was demonstrated that the main chemical group involved in the interaction of the mentioned Bacillus strains with U(VI) is the phosphate group (Hennig et al., 1999; Panak et al., 2000). Our studies on the interactions of the recently described in our laboratory three ecological types (eco-types) of T. ferrooxidans (Selenska-Pobell et al., 1999) and two eco-types of L. ferrooxidans (Tzvetkova et al., 1999) with uranium (VI) by the use of EXAFS, TRLFS, and infrared spectroscopy (IRS) indicate that the nature and the strength of the uranium complexes built by these Gram-negative acidophilic chemolithoauthotrops differ significantly from those built on the cell surfaces of the Gram-positive Bacilli. The tolerance and the interactions of the natural T. ferrooxidans isolates with U were type-specific.

Certain bacterial strains can selectively accumulate various metal ions from aqueous systems /1/. Recently, it was demonstrated that two Bacillus strains, B. cereus JG-A30 and B. sphaericus JG-A12, recovered from a uranium mining waste site in Germany are able to accumulate selectively a large variety of heavy metals from the drain waters of the waste site /2/. In particular, it was shown that these strains accumulate large amounts of uranium. The aim of this study is the analysis of the structure of uranium complexes formed at vegetative cells and spores of the above mentioned Bacillus strains using extended x-ray absorption fine structure (EXAFS) spectroscopy. Analytical procedures allow separating radial distribution functions from the EXAFS, which contain information on bond lengths and coordination numbers. However, frequently the EXAFS result is dominated mainly by the major coordination type in the sample. A comparison of the determined structural parameters with those of the uranium structure family allows to determine the type of complex formed in the

Bacterial diversity in subsurface soil samples drawn from different depths of several uranium waste piles was studied using the 16S rDNA retrieval. Extremely high diversity was found in all samples investigated. In particular, the presence of several dominant 16S rDNA groups related to the genara $\I Thiobacillus, Bacillus, Pseudomonas, Desulfovibrio $$END was demonstrated. One of these 16S rDNA groups was affiliated to the species $\IT. ferrooxidans$$END. Interestingly, this group includes three 16S rDNA types which possess slightly different sequences. Strains of $\IT. ferrooxidans$$END corresponding to two of the mentioned three 16S rDNA types were recovered from two soil samples polluted in different extend with heavy metals. The two groups of isolates have different genomic organization. In addition, the members of the group recovered from the more polluted sample are tollerant to higher concentrations of uranyl ions which are lethal for the isolates of the second group. The expression of at least three genes of the U-tolerant strains is influenced by by the presence of uranyl ions in the nutrient medium. The capability of the U-mine isolates to interact with U(VI) was studied, and it was found that they accumulate significantly higher amounts of U(VI) in comparison to several reference $\IT. ferrooxidans$$END strains recovered from other environments. Using time-resolved laser fluorescence spectroscopy it was shown that the complexes build by one of the U-waste isolates with U(VI) are much stronger than those build by the reference strains. This is the first report which demonstrates a microdiversity in closely related natural isolates of $\IT. ferrooxidans$$END. We suggest that the microdiversity obsrved reflects the genetic adaptation of the strains studied to the different heavy metal concentrations in their natural environments.

Bacterial diversity in soil samples drawn from different depths of several uranium waste piles was studied using 16S rDNA retrieval, RISA and rep-APD. Extremely high diversity was found in all samples investigated. Several dominant 16S rDNA groups related to the genera Thiobacillus, Bacillus, Pseudomonas, and Desulfovibrio were demonstrated. One of these groups was affiliated to the species T. ferrooxidans. This group includes three 16S rDNA types with slightly different sequences. Strains of T. ferrooxidans corresponding to two of the mentioned three 16S rDNA types were recovered from two samples of the waste polluted in different extend with heavy metals. The interactions of the natural T. ferrooxidans strains with U(VI) were group-specific. Microdiversity at the level of 16S- and IGS-rDNA was found also among the group of the uranium reducing Desulfovibrio waste isolates. The latter were affiliated to Desulfovibrio vulgaris subspecies oxamicus. These natural isolates are able to reduce more effectively higher amounts of U(VI) than the reference Desulfovibrio strains. In contrast to the reference strains, the waste isolates reduce U(VI) to U(IV) independently of the pH range of the medium. This is the first report demonstrating microdiversity of closely related natural isolates of T. ferrooxidans and D. vulgaris subsp. oxamicus. We suggest that the microdiversity observed reflects the genetic adaptation of the strains studied to the different heavy metal concentrations in their natural environments.

In the natural bioleaching systems autochtonic microorganisms are involved in the solubilization of metals from solid minerals. The widest spreaded mesophilic group of bioleaching microorganisms are the chemolithoautotrophic bacteria Thiobacillus ferrooxidans, Thiobacillus thiooxidans, and Leptospirillum ferrooxidans. From soil samples drawn from two particular sites of a former uranium mine in Saxony, Germany, which were polluted in different extend with heavy metals, several Thiobacillus strains were cultured. Using ARDREA the strains were classified in two uranium-waste-site-specific 16S rDNA subgroups of the species Thiobacillus ferrooxidans. Moreover, RAPD and PFGE analyses have demonstrated that the natural T. ferrooxidans isolates possess also group-specific genomic organization. The strains from the more polluted sample were tolerant to higher concentrations of uranyl ions which were lethal for the isolates of the second group. In these strains the expression of at least three genes was influenced by the presence of uranyl ions. The uranium binding capability of the uranium mine isolates was strain-specific and higher than those of the reference T. ferrooxidans strains recovered from other environments.

In the natural bioleaching systems autochtonic microorganisms are involved in the solubilization of metals from solid minerals. The widest spreaded mesophilic group of bioleaching microorganisms are the chemolithoautotrophic bacteria Thiobacillus ferrooxidans, Thiobacillus thiooxidans, and Leptospirillum ferrooxidans. From soil samples drawn from two particular sites of a former uranium mine in Saxony, Germany, which were polluted in different extend with heavy metals, several Thiobacillus strains were cultured. Using ARDREA the strains were classified in two uranium-waste-site-specific 16S rDNA subgroups of the species Thiobacillus ferrooxidans. Moreover, RAPD and PFGE analyses have demonstrated that the natural T. ferrooxidans isolates possess also group-specific genomic organization. The strains from the more polluted sample were tolerant to higher concentrations of uranyl ions which were lethal for the isolates of the second group. In these strains the expression of at least three genes was influenced by the presence of uranyl ions. The uranium binding capability of the uranium mine isolates was strain-specific and higher than those of the reference T. ferrooxidans strains recovered from other environments.

Bacterial diversity in subsurface soil samples drawn from different depths of several uranium waste piles was studied using the 16S rDNA retrieval. Extremely high diversity was found in all samples investigated. In particular, the presence of several dominant 16S rDNA groups related to the genara $\I Thiobacillus, Bacillus, Pseudomonas, Desulfovibrio $$END was demonstrated. One of these 16S rDNA groups was affiliated to the species $\IT. ferrooxidans$$END. Interestingly, this group includes three 16S rDNA types which possess slightly different sequences. Strains of $\IT. ferrooxidans$$END corresponding to two of the mentioned three 16S rDNA types were recovered from two soil samples polluted in different extend with heavy metals. The two groups of isolates have different genomic organization. In addition, the members of the group recovered from the more polluted sample are tollerant to higher concentrations of uranyl ions which are lethal for the isolates of the second group. The expression of at least three genes of the U-tolerant strains is influenced by by the presence of uranyl ions in the nutrient medium. The capability of the U-mine isolates to interact with U(VI) was studied, and it was found that they accumulate significantly higher amounts of U(VI) in comparison to several reference $\IT. ferrooxidans$$END strains recovered from other environments. Using time-resolved laser fluorescence spectroscopy it was shown that the complexes build by one of the U-waste isolates with U(VI) are much stronger than those build by the reference strains. This is the first report which demonstrates a microdiversity in closely related natural isolates of $\IT. ferrooxidans$$END. We suggest that the microdiversity obsrved reflects the genetic adaptation of the strains studied to the different heavy metal concentrations in their natural environments.

Bacterial diversity in soil samples drawn from different depths of several uranium waste piles was studied using 16S rDNA retrieval, RISA and rep-APD. Extremely high diversity was found in all samples investigated. Several dominant 16S rDNA groups related to the genera Thiobacillus, Bacillus, Pseudomonas, and Desulfovibrio were demonstrated. One of these groups was affiliated to the species T. ferrooxidans. This group includes three 16S rDNA types with slightly different sequences. Strains of T. ferrooxidans corresponding to two of the mentioned three 16S rDNA types were recovered from two samples of the waste polluted in different extend with heavy metals. The interactions of the natural T. ferrooxidans strains with U(VI) were group-specific. Microdiversity at the level of 16S- and IGS-rDNA was found also among the group of the uranium reducing Desulfovibrio waste isolates. The latter were affiliated to Desulfovibrio vulgaris subspecies oxamicus. These natural isolates are able to reduce more effectively higher amounts of U(VI) than the reference Desulfovibrio strains. In contrast to the reference strains, the waste isolates reduce U(VI) to U(IV) independently of the pH range of the medium. This is the first report demonstrating microdiversity of closely related natural isolates of T. ferrooxidans and D. vulgaris subsp. oxamicus. We suggest that the microdiversity observed reflects the genetic adaptation of the strains studied to the different heavy metal concentrations in their natural environments.