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The complex formation of Eu(III) and Cm(III) was studied via tetradentate, hexadentate, and octadentate coordinating ligands of the aminopolycarboxylate family, viz., nitrilotriacetate (NTA3−), ethylenediaminetetraacetate (EDTA4−), and ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetate (EGTA4−), respectively. Based on the complexones’ pKa values obtained from 1H nuclear magnetic resonance (NMR) spectroscopic pH titration, complex formation constants were determined by means of the parallel-factor-analysis-assisted evaluation of Eu(III) and Cm(III) time-resolved laser-induced fluorescence spectroscopy (TRLFS). This was complemented by isothermal titration calorimetry (ITC), providing the enthalpy and entropy of the complex formation. This allowed us to obtain genuine species along with their molecular structures and corresponding reliable thermodynamic data. The three investigated complexones formed 1:1 complexes with both Eu(III) and Cm(III). Besides the established Eu(III)–NTA 1:1 and 1:2 complexes, we observed, for the first time, the existence of a Eu(III)–NTA 2:2 complex of millimolar metal and ligand concentrations. Demonstrated for thermodynamic studies on Eu(III) and Cm(III) interaction with complexones, the utilized approach is commonly applicable to many other metal–ligand systems, even to high-affinity ligands.

Etidronic acid (1-Hydroxyethylidene-1,1-diphosphonic acid, HEDP, H4L) is a proposed decorporation agent for U(VI). This paper studied its complex formation with Eu(III), an inactive analog of trivalent actinides, over a wide pH range, at varying metal-to-ligand ratios (M:L) and total concentrations. Combining spectroscopic, spectrometric, and quantum chemical methods, five distinct Eu(III)−HEDP complexes were found, four of which were characterized. The readily soluble EuH2L+ and Eu(H2L)2− species with log β values of 23.7 ± 0.1 and 45.1 ± 0.9 are formed at acidic pH. At near-neutral pH, EuHL0s forms with a log β of ~23.6 and, additionally, a most probably polynuclear complex. The readily dissolved EuL− species with a log β of ~11.2 is formed at alkaline pH. A six-membered chelate ring is the key motif in all solution structures. The equilibrium between the Eu(III)–HEDP species is influenced by several parameters, i.e., pH, M:L, total Eu(III) and HEDP concentrations, and time. Overall, the present work sheds light on the very complex speciation in the HEDP–Eu(III) system and indicates that, for risk assessment of potential decorporation scenarios, side reactions of HEDP with trivalent actinides and lanthanides should also be taken into account.

Microbial U(VI) reduction influences the uranium mobility in contaminated subsurface environments and can affect the disposal of high-level radioactive waste by transform-ing the water-soluble U(VI) to less mobile U(IV). The reduction of U(VI) by the sulfate-reducing bacterium Desulfosporosinus hippei DSM 8344T, a close phylogenetic relative to naturally occurring microorganism present in clay rock and bentonite, was investigat-ed. D. hippei DSM 8344T showed a relatively fast removal of uranium from the superna-tants in artificial Opalinus Clay pore water. Combined speciation calculations and lumi-nescence spectroscopic investigations showed the dependence of U(VI) reduction on the initial U(VI) species. Scanning transmission electron microscopy coupled with ener-gy-dispersive X-ray spectroscopy showed uranium-containing aggregates on the cell surface and the formation of membrane vesicles. By combining different spectroscopic techniques, including UV/Vis spectroscopy, as well as uranium M4-edge X-ray absorp-tion near-edge structure (XANES) recorded in high-energy-resolution fluorescence-detection (HERFD) mode and extended X-ray absorption fine structure (EXAFS) analy-sis, the partial reduction of U(VI) could be verified, whereby the formed U(IV) product has an unknown structure. Furthermore, the U M4 HERFD-XANES showed the presence of U(V) during the process, suggesting a single-electron transfer mechanism for the microbial U(VI) reduction by sulfate reducers. These findings offer new insights into the U(VI) reduction by sulfate-reducing bacteria and contribute to a comprehensive safety concept for a repository for high-level radioactive waste.

The biological relevance of early lanthanides, such as neodymium, remained undiscovered until methylotrophic bacteria with lanthanide-dependent metabolism were identified. The respective strains incorporate these elements into the active site of their key metabolic enzyme methanol dehydrogenase (MDH). Growth studies with the strictly lanthanide-dependent thermoacidophile Methylacidiphilum fumariolicum SolV and the mesophilic Methylorubrum extorquens AM1 ΔmxaF mutant demonstrate that the trivalent actinides americium and curium support growth in the absence of the essential lanthanides. In fact, the bacteria make no distinction between lanthanide and actinide ions if they have the correct size and oxidation state. Time-resolved laser-induced fluorescence spectroscopy, liquid scintillation counting, and inductively coupled plasma mass spectrometry confirm the bacterial uptake. The interchangeability of f-block elements is supported by very similar enzymatic activities of recombinant methanol dehydrogenase reconstituted with different metal ions. Our combined in vivo and in vitro results firmly establish that actinides support growth of methylotrophic bacteria, suggesting a potential biological role for these radioactive elements. Importantly, bacteria capable of utilizing actinides will also be able to mobilize these elements in the environment. This may lead to applications for bioremediation and recycling/separation of lanthanides and actinides.

The separation and recycling of lanthanides is an active area of research with a growing demand that calls for more environmentally friendly lanthanide sources. Likewise, the efficient and industrial separation of lanthanides from the minor actinides (Np, Am–Fm) is one of the key questions for closing the nuclear fuel cycle; reducing costs and increasing safety. With the advent of the field of lanthanide-dependent bacterial metabolism, bio-inspired applications are in reach. Here, we utilize the natural lanthanide chelator lanmodulin and the luminescent probes Eu3+ and Cm3+ to investigate the inter-metal competition behavior of all lanthanides (except Pm) and the major actinide plutonium as well as three minor actinides neptunium, americium and curium to lanmodulin. Using time-resolved laser-induced fluorescence spectroscopy we show that lanmodulin has the highest relative binding affinity to Nd3+ and Eu3+ among the lanthanide series. When equimolar mixtures of Cm3+ and Am3+ are added to lanmodulin, lanmodulin preferentially binds to Am3+ over Cm3+ whilst Nd3+ and Cm3+ bind with similar relative affinity. The results presented show that a natural lanthanide-binding protein can bind a major and various minor actinides with high relative affinity, paving the way to bio-inspired separation applications. In addition, an easy and versatile method was developed, using the fluorescence properties of only two elements, Eu and Cm, for inter-metal competition studies regarding lanthanides and selected actinides and their binding to biological molecules., In need of environmentally friendly methods for the separation and recycling of lanthanides and actinides, the binding of the protein lanmodulin to lanthanides and actinides was studied using time resolved laser induced fluorescence spectroscopy.

Lanmodulin (LanM), a naturally lanthanide (Ln)-binding protein with a remarkable selectivity for Lns over Ca(II ) and affinities in the picomolar range, is an attractive target to address challenges in Ln separation. Why LanM has such a high selectivity is currently not entirely understood; both specific amino acid sequences of the EF-Hand loops and cooperativity effects have been suggested. Here, we removed the effect of cooperativity and synthesised all four 12-amino acid EF-Hand loop peptides, and investigated their affinity for two Lns (Eu( III) and Tb(III)), the actinide Cm(III) and Ca(II). Using isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy (TRLFS) combined with parallel factor analysis, we show that the four short peptides behave very similarly, having affinities in the micromolar range for Eu(III) and Tb(III). Ca(II) was shown not to bind to the peptides, which was verified with circular dichroism spectroscopy. This technique also revealed an increase in structural organisation upon Eu(III) addition, which was supported by molecular dynamics simulations. Lastly, we put Eu(III) and Cm(III) in direct competition using TRLFS. Remarkably, a slightly higher affinity for Cm(III) was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring and therefore a reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.

Europium, as an easy-to-study analog of the trivalent actinides, is of particular importance for studying the behavior of lanthanides and actinides in the environment. Since different soil organisms can influence the migration behavior of these elements, a detailed knowledge of these interaction mechanisms is important. The aim of this study was to investigate the interaction of mycelia of selected wood-inhabiting (S. commune, P. ostreatus, L. tigrinus) and soil-inhabiting fungi (L. naucinus) with Eu(III). In addition to determining the Eu(III) complexes in the sorption solution, the formed Eu(III) fungal species were characterized using scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy, chemical microscopy in combination with the time-resolved laser-induced fluorescence spectroscopy. Our data show that S. commune exhibited significantly higher Eu(III) binding capacity in comparison to the other fungi. Depending on fungal strain, the metal was immobilized on the cell surface, in the cell membranes, and within the membranes of various organelles, or in the cytoplasm in some cases. During the bioassociation process two different Eu(III) fungal species were formed in all investigated fungal strain. The phosphate groups of organic ligands were identified as being important functional groups to bind Eu(III) and thus immobilize the metal in the fungal matrix. The information obtained contributes to a better understanding of the role of fungi in migration, removal or retention mechanisms of rare earth elements and trivalent actinides in the environment.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H₃PQQ to PQQ³⁻ as well as water adducts and in complex with lanthanides (Lns)) using NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Using TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu states: Eu in the active site of MDH, but also in solution as PQQ-bound Eu and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu in the active site.

A combination of biochemical preparation methods with microscopic, spectroscopic, and mass spectrometric analysis techniques as contemplating state of the art application, was used for direct visualization, localization, and chemical identification of europium in plants. This works illustrates the chemical journey of europium (Eu(III)) through winter rye (Secale cereale L.), providing insight into the possibilities of speciation for Rare Earth Elements (REE) and trivalent f-elements. Kinetic experiments of contaminated plants show a maximum europium concentration in Secale cereale L. after four days. Transport of the element through the vascular bundle was confirmed with Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray analysis (EDS). For chemical speciation, plants were grown in a liquid nutrition medium, whereby Eu(III) species distribution could be measured by mass spectrometry and luminescence measurements. Both techniques confirm the occurrence of Eu malate species in the nutrition medium, and further analysis of the plant was performed. Luminescence results indicate a change in Eu(III) species distribution from root tip to plant leaves. Microscopic analysis show at least three different Eu(III) species with potential binding to organic and inorganic phosphate groups and a Eu(III) protein complex. With plant root extraction, further europium species could be identified by using Electrospray Ionization Mass Spectrometry (ESI MS). Complexation with malate, citrate, a combined malate-citrate ligand, and aspartate was confirmed mostly in a 1:1 stoichiometry (Eu:ligand). The combination of the used analytical techniques opens new possibilities in direct species analysis, especially regarding to the understanding of rare earth elements (REE) uptake in plants. This work provides a contribution in better understanding of plant mechanisms of the f-elements and their species uptake.

Since the discovery of the biological relevance of lanthanides (Lns) for methylotrophic bacteria in the last decade, the field has seen a steady rise in discoveries of bacteria using Lns. The major role of lanthanides here is in the active sites of enzymes: methanol dehydrogenases. Additionally, lanthanide binding proteins have also been identified. One such protein is lanmodulin (LanM) and, with a remarkable selectivity for Lns over Ca(II) and affinities in the picomolar range, it makes an attractive target to address challenges in lanthanide separation. Why LanM has such a high selectivity is currently not entirely understood, both the specific amino acid sequences of the EF-hand loops, together with cooperativity effects have been suggested. Consequently, we decided to remove the effect of cooperativity by focusing on the amino acid level. Thus, we synthesized all four 12-amino acid EF-Hand loop peptides of LanM using solid phase peptide synthesis and investigated their affinity for Lns (Eu(III), Tb(III)), the actinide Cm(III) and Ca(II). Using isothermal titration calorimetry and time resolved laser fluorescence spectroscopy combined with parallel factor analysis, we show that in the absence of cooperativity the short EF-Hand loop peptides have all similar affinities for lanthanides and that these are all in the micromolar range. Furthermore, calcium was shown not to bind to the peptides which was verified with circular dichroism spectroscopy. This technique also revealed that the peptides undergo a change to a more ordered state when lanthanides are added. These experimental observations were further supported by molecular dynamics simulations. Lastly, we put Eu(III) and Cm(III) in direct competition using TRLFS. Remarkably, a slightly higher affinity for the actinide, as was also observed for LanM, was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring in the full protein and therefore reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.

Novel separation processes for end of life fluorescent lamp phosphors could greatly contribute towards a more sustainable rare-earth element market. Furthermore, surface-binding peptides bound to magnetic carriers are a promising biotechnological tool for selective particle separation processes. In this work, we investigate the magnetic carrier separation of the three most common rare-earth fluorescent lamp phosphors, facilitated by Dynabeads functionalized with previously identified selectively surface-binding peptides. We present an active ester-mediated coupling to chemically immobilize the peptides on amine and carboxylic acid coated beads. We report on the impact of the peptide functionalization on the surface properties of the beads, based on zeta potential investigations of variously functionalized beads and a Raman spectroscopic structural study of the investigated peptides. Fluorometrically, we show that the phosphor removal strongly depends on the medium and the surface coating of the beads. Furthermore, the Raman spectroscopic evidence of various simultaneously present disulfide bond conformations indicates an equilibrium of multiple peptide conformations and/or the presence of intermolecular disulfide bonds. Moreover, we found that carboxylic acid coated Dynabeads have a high affinity for the red phosphor Y2O3:Eu3+ and based on the determined isoelectric points we hypothesize that this is driven by electrostatic surface interactions. This work can contribute towards novel rare-earth phosphor separation processes and towards a better understanding of magnetic carrier separation processes facilitated by surface-binding peptides.

The interaction between Eu(III) ion and different pH buffers, popular in biology and biochemistry, viz. HEPES, PIPES, MES, MOPS, and TRIS, has been studied by solution nuclear magnetic resonance spectroscopy (NMR) and time-resolved laser-induced fluorescence spectroscopy (TRLFS) techniques. The Good’s buffers reveal non-negligible interaction with Eu(III) as determined from their complex stability constants, where the sites of interaction are the morpholine and piperazine nitrogen atoms, respectively. In contrast, TRIS buffer shows practically no affinity towards Eu(III). Therefore, when investigating lanthanides, TRIS buffer should be preferred over Good’s buffers. Graphical abstract

Chemical microscopy combines high-resolution emission spectra with Abbe-limited spatial resolution and is used for studies of inhomogeneous samples at the (sub-)micronscale. The spatial distinction of multiple Eu(III) coordination sites allows for a comprehensive understanding of environmental samples and highlights the applicability of Eu(III) as a molecular probe in medicine and biology.

Daucus carota suspension cells showed a high affinity towards Eu(III) and U(VI) based on a single-step bioassociation process with an equilibrium after 48-72 h. Cells responded with an increased metabolic activity towards heavy metal stress. Luminescence spectroscopy pointed to multiple species for both f-block elements in the culture media, providing initial hints of their interaction with cells and released metabolites. Using nuclear magnetic resonance spectroscopy, we could prove that malate, as an released metabolite in the culture medium, was found to complex with U. Luminescence spectroscopy also showed that Eu(III)-EDTA species are interacting with the cells. Furthermore, Eu(III) and U(VI) coordination is dominated by phosphate groups provided by the cells. We found that Ca ion channels of D. carota cells were involved in the uptake of U(VI), which led to a bioprecipitation of U(VI) in the vacuole of the cells, most probably as uranyl(VI) phosphates along with an intracellular sorption of U(VI) on biomembranes by lipid structures. Eu(III) could be found locally concentrated in the cell wall and in the cytoplasm with a co-localization with phosphorous and oxygen.

The U(VI) bioassociation on Magnetospirillum magneticum AMB-1 cells was investigated using a multidisciplinary approach combining wet chemistry, microscopy, and spectroscopy methods to provide deeper insight into the interaction of U(VI) with bioligands of Gram-negative bacteria for a better molecular understanding. Our findings suggest that the cell wall plays a prominent role in the bioassociation of U(VI). In time-dependent bioassociation studies, up to 95 % of the initial U(VI) was removed from the suspension and probably bound on the cell wall within the first hours due to the high removal capacity of predominantly alive Magnetospirillum magneticum AMB-1 cells. PARAFAC analysis of TRLFS data highlights that peptidoglycan is the most important ligand involved, showing a stable immobilization of U(VI) over a wide pH range with the formation of three characteristic species. In addition, in-situ ATR FT-IR reveals the predominant strong binding to carboxylic functionalities. At higher pH polynuclear species seem to play an important role. This comprehensive molecular study may initiate in future new remediation strategies on effective immobilization of U(VI). In combination with the magnetic properties of the bacteria, a simple technical water purification process could be realized not only for U(VI), but probably also for other heavy metals.

Gallium (as Ga3+) is a Group IIIa metal and its recovery from wastewaters has become increasingly important for its reuse. The use of peptides for recycling offers a low-cost and environmentally-friendly option but the structural characteristics of peptides likely to bind Ga3+ are largely unknown. Multiple computational methods, coupled with experimental verification via NMR and Isothermal Calorimetry (ITC), were used to establish that Ga3+ binds with high affinity to peptide sequences and to elucidate the structural characteristics that contributed. It was demonstrated that peptide pre-organisation is key to Ga3+ binding and that a favourable binding position is necessarily governed by the size and shape of the electrostatic environment as much as individual electrostatic interactions with peptide residues themselves. Given favourable conditions, Ga3+ retrieved plausible binding positions involving both charged and uncharged residues that greatly increases the range of bonding possibilities with other peptide sequences and offers insights for binding other metals. The addition of pH buffer substantially improved the affinity of Ga3+ and a structural role for a buffer component was demonstrated.

Clay formations are potential host rocks for the long-term storage of high-level radioactive waste in a deep geological repository. Bentonites are supposed to serve as backfill material, not only for a final disposal site in clay formations but also in crystalline rock. For a long-term safety assessment, various aspects must be taken into account. Besides geological, geochemical and geophysical considerations, naturally occurring microorganisms also play a crucial part in the environment of such a repository. In the event of a worst-case scenario when water enters the disposal site, they can interact with the radionuclides and change for example the chemical speciation or the oxidation state (Lloyd et al., 2002). Desulfosporosinus spp. are an important representative of anaerobic, sulfate-reducing microorganisms, which are present in clay formations as well as in bentonites. Various studies have shown that they play a major role in the microbial communities of these surroundings (Bagnoud et al., 2016; Matschiavelli et al., 2019). A closely related microorganism to the isolated species is Desulfosporosinus hippei DSM 8344, which was originally found in permafrost soil (Vatsurina et al., 2008). This bacterium was used to investigate its interactions with uranium(VI) especially regarding the reduction to the less mobile uranium(IV). Time-dependent reduction experiments in artificial Opalinus Clay pore water (Wersin et al., 2011) (100 µM uranium(VI), pH 5.5) showed the removal of about 80 % of the uranium(VI) from the supernatants within 48 h. Corresponding UV/Vis measurements of the dissolved cell pellets exhibited an increasing proportion of uranium(IV) in the cell-bound uranium. Calculations with the inclusion of extinction coefficients led to a ratio of 39 % uranium(IV) after 1 week. Therefore, a combined sorption-reduction process is a possible interaction mechanism. Time-resolved laser-induced luminescence spectroscopy verified the presence of two uranium(VI) species in the supernatant. A comparison with reference spectra led to an assignment to a uranyl(VI) lactate and a uranyl(VI) carbonate complex. The species distribution showed a decrease of the proportion of the lactate species with time, whereas the proportion of the carbonate species remained almost constant. Uranium aggregates are formed on the cell surface during the process, as determined by transmission electron microscopy (TEM). Furthermore, uranium occurs inside and outside the cells as well as vesicles containing uranium. These findings help to close existing gaps in a comprehensive safeguard concept for a repository for high-level radioactive waste in clay rock. Moreover, this study provides new insights into the interactions of sulfate-reducing microorganisms with uranium(VI).

For a fully comprehensive safety concept of a nuclear repository, it is necessary to investigate not only the geological, geochemical and geophysical properties but also the influence of naturally occurring microorganisms in the deep geological layers. Clay rocks are a possible host rock formation for the long-term storage of the highly radioactive waste, with bentonite to be used as backfill material. Various studies show that, among other sulfate-reducing microorganisms, Desulfosporosinus species are present in both clay rock and bentonite.[1,2] A phylogenetically close relative to the isolated species is Desulfosporosinus hippei DSM 8344, an anaerobic, spore-forming microorganism originally found in permafrost soils.[3] Therefore, this strain was selected to get a deeper insight into the uranium(VI) interactions with naturally occurring microorganisms from deep geological layers. A time-dependent experiment in artificial Opalinus Clay pore water[4] (100 µM uranium(VI), pH 5.5) showed the removal of about 80 % of the uranium(VI) from the supernatants within 48 h. Corresponding live/dead images of the cells taken by fluorescence microscopy exhibit the formation of cell agglomerates and an increasing number of dead cells within the incubation time. Further examination of the supernatants using time-resolved laser-induced fluorescence spectroscopic techniques revealed the presence of two uranium(VI) species, a lactate and a carbonate complex. The proportion of the carbonate species remained constant over the incubation period, whereas the lactate species decreased. The comparison of UV/Vis band positions of the dissolved cell pellets with reference spectra provides clear proof of a partially reduction of uranium(VI) to uranium(IV), although bands of uranium(VI) were also still observable. Therefore, it could be that the ongoing interaction mechanism is a combined sorption-reduction process. These findings are an important contribution to a safety concept for a nuclear repository in clay rock and provide new insights into the interactions of sulfate-reducing microorganisms with uranium(VI). References [1] Bagnoud et al. (2016) Nat. Commun 7, 1–10. [2] Matschiavelli et al. (2019) Environ. Sci. Technol. 53, 10514–10524. [3] Vatsurina et al. (2008) Int. J. Syst. Evol. Microbiol. 58, 1228–1232. [4] Wersin et al. (2011) Appl. Geochemistry 26, 931–953.

After the Chernobyl and Fukushima incidents it has become clear that fungi can take up and accumulate large quantities of radionuclides and heavy metals, but the underlying processes are not well understood yet. For this study, the molecular interactions of uranium(VI) with the white-rot fungi, Schizophyllum commune and Pleurotus ostreatus, and the soil-living fungus, Leucoagaricus naucinus, were investigated. First, the uranium concentration in the biomass was determined by time-dependent bioassociation experiments. To characterize the molecular interactions, uranium was localized in the biomass by transmission electron microscopy analysis. Second, the formed uranyl complexes in both biomass and supernatant were determined by fluorescence spectroscopy. Additionally, possible bioligands in the supernatant were identified. The results show that the discernible interactions between metals and fungi are similar, namely biosorption, accumulation, and subsequent crystallization. But at the same time, the underlying biochemical mechanisms are different and specific to the fungal species. In addition, Schizophyllum commune was found to be the only fungus that, under the chosen experimental conditions, released tryptophan and other indole derivatives in the presence of uranium(VI) as determined by nuclear magnetic resonance spectroscopy. These released substances most likely act as messenger molecules rather than serving the direct detoxification of uranium(VI).

For a safety assessment of nuclear waste repositories, it is necessary to consider accident scenarios through which radionuclides (RN) can enter the biosphere up to the food chain via groundwater and soil. It is necessary to generate detailed knowledge about the uptake pathways and the interaction of RN with plants to contribute to the molecular process understanding required for a reliable biogeochemical modeling. We investigated the uptake and immobilization (bioassociation) of U(VI) and Eu(III) as a non-radioactive analogue for trivalent actinides by two typical crop plant cell cultures as model systems: canola (Brassica napus) and carrot (Daucus carota). For both metals a time- and concentration-dependent bioassociation behavior was observed, which shows differences between the two plant cell types. U(VI) and Eu(III) were used as luminescence probes to explore their speciation in the two systems. Therefore, time-resolved laser-induced fluorescence spectroscopy (TRLFS) was performed under cryogenic conditions. For an investigation of possible uptake pathways of the metals, it must be considered that both U and Eu are non-essential heavy metals for plants. Therefore, it can be assumed that they have no specific uptake pathway into the plant cells. Possible uptake routes are the use of transport systems of essential micronutrients, whose homeostasis can be disturbed by U(VI) and Eu(III), but uptake via endocytosis and mechanosensitive ion channels is also possible. Experiments were performed to investigate whether the metals can be unspecifically taken up into the cells by blocking Ca(II) ion channels with GdCl3. The investigations are supplemented by transmission electron microscopy combined with energy-dispersive X-ray spectroscopy (TEM with EDX mapping), which contribute to an improved understanding of the processes taking place by localizing the metals in the plant cell.

M. Basile, et al., Chem. Commun., 2015, 51, 5306-5309, showed that a sodium ion is sandwiched by uranyl(vi) oxygen atoms of two 3 : 3 uranyl(vi)-citrate complex molecules in single-crystals. By means of NMR spectroscopy supported by DFT calculations we provide unambiguous evidence for this complex to persist in aqueous solution above a critical concentration of 3 mM uranyl citrate. Unprecedented Ca

Here we provide a proof of principle for an application-oriented concept for the peptide-based recovery of gallium in industrial wastewater, which was supported by biosorption studies with a real wastewater sample. We investigated the interaction of the gallium-binding peptides TMHHAAIAHPPH, NYLPHQSSSPSR, SQALSTSRQDLR, HTQHIQSDDHLA, and NDLQRHRLTAGP with gallium and arsenic through different experimental and computational approaches. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution.

The interactions between glutathione disulfide, GSSG, the redox partner and dimer of the intracellular detoxification agent glutathione, GSH, and hexavalent uranium, U(VI), were extensively studied by solution NMR, complemented by time-resolved laser-induced fluorescence and IR spectroscopies. As expected for the hard Lewis acid U(VI), coordination facilitates by the ligands’ O-donor carboxyl groups. However, owing to the adjacent cationic α-amino group, the glutamyl-COO reveal monodentate binding, while the COO of the glycyl residues show bidentate coordination. The log K value for the reaction UO₂2+ + H₃GSSG– = [UO₂H₃GSSG]+ (pH 3, 0.1 M NaClO₄) was determined for the first time, being 4.81 ± 0.08; extrapolation to infinite dilution gave log K° = 5.24 ± 0.08. U(VI) and GSSG form precipitates in the whole pD range studied (2 – 8), showing least solubility for 4 < pD < 6.5. Thus, particularly GSSG, hereby representing also other peptides and small proteins, affects the mobility of U(VI), strongly depending on the speciation of either component.

The interactions of two extremely halophilic archaea with uranium were investigated at high ionic strength as a function of time, pH and uranium concentration. Halobacterium noricense DSM-15987 and Halobacterium sp. putatively noricense isolated from the Waste Isolation Pilot Plant (WIPP) repository were used for these investigations. The kinetics of U(VI) bioassociation of both strains showed an atypical multistage behavior, meaning that after an initial phase of U(VI) sorption, an unexpected interim period of U(VI) release was observed, followed by a slow reassociation of uranium with the cells. By applying in situ attenuated total reflection Fourier-transform infrared (ATR FT-IR) spectroscopy, the involvement of phosphoryl and carboxylate groups in U(VI) complexation during the first biosorption phase was shown. Differences in cell morphology and uranium localization become visible at different stages of the bioassociation process, by using scanning electron microscopy (SEM) in combination with energy dispersive X-ray (EDX) spectroscopy. Our results demonstrate for the first time that association of uranium on the extremely halophilic archaeon is a multistage process, beginning with a sorption which is followed by another process, probably biomineralization.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA–peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

A comprehensive molecular analysis of a simple aqueous complexing system. U(VI) acetate. selected to be independently investigated by various spectroscopic (vibrational, luminescence, X-ray absorption, and nuclear magnetic resonance spectroscopy) and quantum chemical methods was achieved by an international round-robin test (RRT). Twenty laboratories from six different countries with a focus on actinide or geochemical research participated and contributed to this scientific endeavor. The outcomes of this RRT were considered on two levels of complexity: first, within each technical discipline, conformities as well as discrepancies of the results and their sources were evaluated. The raw data from the different 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 high. By contrast, luminescence spectroscopic data turned out to be strongly related to the chosen acquisition parameters. Second, the potentials and limitations of coupling various spectroscopic and theoretical approaches for the comprehensive study of actinide molecular complexes were assessed. Previous spectroscopic data from the literature were revised and the benchmark data on the U(VI) acetate system provided an unambiguous molecular interpretation based on the correlation of spectroscopic and theoretical results. The multimethodologic approach and the conclusions drawn address not only important aspects of actinide spectroscopy but particularly general aspects of modern molecular analytical chemistry.

The complexation of the trivalent lanthanides Nd(III) and Eu(III) and of the actinide Am(III) with malate was studied using a multi−method approach. The combination of structural and thermodynamic studies was required for the interpretation of the stoichiometry and thermodynamic data (logβ0, ΔrH0m,2, ΔrS0m, ΔrG0m) of the lanthanide/actinide malate complexes leading to a profound molecular understanding of the system. The structure-sensitive methods vibrational spectroscopy and extended X–ray absorption fine structure spectroscopy complemented with quantum-mechanical ab–initio molecular dynamics calculations revealed a tridentate ring structure of the respective metal complexes. The metal is coordinated by two carboxylate groups and a hydroxyl group. UV–Vis, laser fluorescence and calorimetric studies consistently yielded two complex species having a 1:1 and a 1:2 (metal:malate) stoichiometry. Parallel factor analysis and iterative transformation factor analysis were applied to decompose experimental spectra into their single components and to determine stability constants. The 1:1 and 1:2 Nd(III) malate complexation constants determined by isothermal titration calorimetry were extrapolated to zero ionic strength using the specific ion interaction theory, yielding logβ10 and logβ20 of about 6 and 9, respectively. The respective complexation enthalpies ΔrH0m,1 and ΔrH0m,2 showed average values of 5 kJ·mol−1 which are typical for small organic molecules. The comparison of Nd(III) and Am(III) malate complexes showed that the malate binding motif, the speciation and the thermodynamics can be transferred from lanthanides(III) to actinides(III) supporting the 4f–/ 5f–element homology.

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An3+/Ln3+ ions. As will be demonstrated herein, our data confirm the higher affinity of Cm3+ and Eu3+ compared to Ca2+ to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu3+ in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu3+ and Cm3+ to carry one or two additional waters compared to Ca2+-CaM, while also causing the structure of Cm3+/Eu3+-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca2+-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.

In addition to geological, geochemical, and geophysical aspects, also, microbial aspects have to be taken into account when considering the final storage of high-level radioactive waste in a deep geological repository. Rock salt is a potential host rock formation for such a repository. One indigenous microorganism, that is, common in rock salt, is the halophilic archaeon Halobacterium noricense DSM15987T, which was used in our study to investigate its interactions with the trivalent actinide curium and its inactive analogue europium as a function of time and concentration. Time-resolved laser-induced fluorescence spectroscopy was applied to characterize formed species in the micromolar europium concentration range. An extended evaluation of the data with parallel factor analysis revealed the association of Eu(III) to a phosphate compound released by the cells (F2/F1 ratio, 2.50) and a solid phosphate species (F2/F1 ratio, 1.80). The association with an aqueous phosphate species and a solid phosphate species was proven with site-selective TRLFS. Experiments with Cm(III) in the nanomolar concentration range showed a time- and pCH+-dependent species distribution. These species were characterized by red-shifted emission maxima, 600–602 nm, in comparison to the free Cm(III) aqueous ion, 593.8 nm. After 24 h, 40% of the luminescence intensity was measured on the cells corresponding to 0.18 μg Cm(III)/gDBM. Our results demonstrate that Halobacterium noricense DSM15987T interacts with Eu(III) by the formation of phosphate species, whereas for Cm(III), a complexation with carboxylic functional groups was also observed.

Haloarchaea represent a predominant part of the microbial community in rock salt, which can serve as host rock for the disposal of high level radioactive waste. However, knowledge is missing about how Haloarchaea interact with radionuclides. Here, we used a combination of spectroscopic and microscopic methods to study the interactions of an extremely halophilic archaeon with uranium, one of the major radionuclides in high level radioactive waste, on a molecular level. The obtained results show that Halobacterium noricense DSM 15987T influences uranium speciation as a function of uranium concentration and incubation time. X-ray absorption spectroscopy reveals the formation of U(VI) phosphate minerals, such as meta-autunite, as the major species at a lower uranium concentration of 30 μM, while U(VI) is mostly associated with carboxylate groups of the cell wall and extracellular polymeric substances at a higher uranium concentration of 85 μM. For the first time, we identified uranium biomineralization in the presence of Halobacterium noricense DSM 15987T cells. These findings highlight the potential importance of Archaea in geochemical cycling of uranium and their role in biomineralization in hypersaline environments, offering new insights into the microbe-actinide interactions in highly saline conditions relevant to the disposal of high-level radioactive waste as well as bioremediation.

For the only water coordinated "free" uranyl(VI) aquo ion in perchlorate solution we identified and assigned several different excited states and showed that the

For the only water coordinated "free" uranyl (VI) aquo ion in perchlorate solution we identified and assigned several different excited states and showed that the (3)Delta state is the luminescent triplet state from transient absorption spectroscopy. With additional data from other spectroscopic methods (TRLFS, UV/vis) we generated a detailed Jablonski diagram and determined rate constants for several state transitions, like the inner conversion rate constant from the (3)Phi state to the (3)Delta state transition to be 0.35 ps(-1). In contrast to luminescence measurements, it was possible to observe the highly quenched uranyl(VI) ion in highly concentrated chloride solution by TAS and we were able to propose a dynamic quenching mechanism, where chloride complexation is followed by the charge transfer from the excited state uranyl(VI) to chloride. This proposed quenching route is supported by TD-DFT calculations.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalization via coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides., Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via coacervation. Here authors demonstrate that a coacervate microenvironment supports RNA catalysis whilst selectively sequestering RNA based on length.

This study of aqueous metal speciation is an advanced combination of theoretical and experimental methods. Continuous wave (CW) and time-resolved laser-induced fluorescence spectroscopy (TRLFS) data of uranyl(VI) hydrolysis were analyzed using parallel factor analysis (PARAFAC). Distribution patterns of five major species were thereby derived under a fixed uranyl concentration (10−5 M) over a wide pH range from 2 to 11. UV (180 nm to 370 nm) excitation spectra were extracted for individual species. Time-dependent density functional theory (TD-DFT) calculations revealed ligand excitation (water, hydroxo, oxo) in this region and ligand-to-metal charge transfer (LMCT) responsible for luminescence. Thus excitation in the UV region is extreme ligand sensitive and specific. Combining findings from PARAFAC and DFT the [UO2(H2O)5]2+ cation (aquo complex 1 : 0) and four hydroxo complexes (1 : 1, 3 : 5, 3 : 7 and 1 : 3) were identified. The methodological concept used here is applicable to luminescent metals in general and thus enables acquisition of refined structural and thermodynamical data of lanthanide and actinide complexation.

The formation equilibria of salicylatoborate, lactatoborate and 3-hydroxybutyratoborate were studied by means of (11)B NMR spectroscopy. The smaller the pKa of the respective organic acid, the higher is the formation constant of the organoborate. The complexation of Eu(III) with salicylatoborate and lactatoborate was investigated by means of TRLFS (time-resolved laser-induced fluorescence spectroscopy) and (11)B NMR spectroscopy, yielding complexation constants lg β₁₁⁰ = 2.6-3.2. A Eu(III)-3-hydroxybutyrate complex was characterized by TRLFS and (1)H NMR spectroscopy (lg β₁₁⁰ = 2.89). DFT calculations of the investigated Eu(III)-organoborates and inorganic Eu(III)-(poly)borates provided information about the Eu(III) coordination (most likely chelate). They support the hypothesis that the complexation of Eu(III) with organic as well as inorganic borate structures containing the binding site "B(OR)4(-)" (R = H, threefold coordinated boron center(s), organic moiety) is comparable.

Most bacteria and all archaea possess as outermost cell envelope so called surface-layers (S-layers). These S-layers are formed by self-assembling proteins having a number of interesting intrinsic properties, e.g. they mediate selective exchange of molecules and therefore function as molecular sieves. Furthermore, S-layers from bacterial isolates recovered from heavy metal contaminated environments have outstanding metal binding properties and are highly stable. Thus they selectively bind several metals with different affinity. For using S-layer proteins for metal bioremediation and recycling three aspects of the metal-interactions with S-layer proteins must be taken into account. First, S-layers possess different functionalities, e.g. carboxyl, phosphoryl groups, binding toxic metals and metalloids, like U and As, unspecifically depending on pH-value. Second, precious metals like Au and Pd are likewise unspecifically bound to functional groups, but presumably covalently, making the binding irreversible unless the S-layer protein is destroyed completely. Third, some metals are needed for native protein folding of the S-layer protein monomer, self-assembly, and the formation of highly-ordered lattices. These particular metals are specifically bound bivalent cations, e.g. Ca2+. Important is that these binding sites allow selective binding not only of Ca2+, but also of chemical-equal elements including the trivalent lanthanides (Eu3+, Tb3+), possessing comparable ionic radii. Thus the metal dependent binding behavior of the proteins not only contributes to the development of biohybrid materials for the separation, removal or recovery of strategic relevant metals regulated by pH and allowing a conceivably release, but also allows a biochemical and mechanistic understanding of the interaction of different metal ions with S-layer proteins .

The mechanism by which oxygen bound in UO22+ exchanges with that from water under strong alkaline conditions remains a subject of controversy. Two recent NMR studies independently revealed that the key intermediate species is a binuclear uranyl(VI) hydroxide, presumably of the stoichiometry [(UO2(OH)42−)(UO2(OH)53−)]. The presence of UO2(OH)53− in highly alkaline solution was postulated in earlier experimental studies, yet the species has been little characterized. Quantum-chemical calculations (DFT and MP2) show that hydrolysis of UO2(OH)42− yields UO3(OH)33− preferentially over UO2(OH)53−. X-ray absorption spectroscopy was used to study the uranium(VI) speciation in a highly alkaline solution supporting the existence of a species with three U−O bonds, as expected for UO3(OH)33−. Therefore, we explored the oxygen exchange pathway through the binuclear adduct [(UO2(OH)42−)(UO3(OH)33−)] by quantum-chemical calculations. Assuming that the rate-dominating step is proton transfer between the oxygen atoms, the activation Gibbs energy for the intramolecular proton transfer within [(UO2(OH)42−)(UO3(OH)33−)] at the B3LYP level was estimated to be 64.7 kJ mol−1. This value is in good agreement with the activation energy for “yl”−oxygen exchange in [(UO2(OH)42−)(UO2(OH)53−)] obtained from experiment by Szabó and Grenthe (Inorg. Chem. 2010, 49, 4928−4933), which is 60.8 ± 2.4 kJ mol−1. Both the presence of UO3(OH)33− and the scenario of an “yl”−oxygen exchange through a binuclear species in strong alkaline solution are supported by the present study.

The uranium(VI) sorption onto orthoclase and muscovite, representing the mineral groups of feldspars and micas as important components of the earth crust, was investigated in the presence and absence of Ca2+ under aerobic conditions. Batch experiments were accompanied by time-resolved laser-induced fluorescence spectroscopy (TRLFS) as well as in situ attenuated total reflection Fourier-transform infrared (ATR FT-IR) spectroscopy. The results indicate that the U(VI) sorption is reduced by Ca2+ at pH ⩾ 8 up to 30% due to the formation of the neutral aqueous Ca2UO2(CO3)3 (aq) complex. TRLFS measurements on the supernatant confirmed the predominance of this Ca2UO2(CO3)3 (aq) complex in accordance with thermodynamic calculations. Furthermore, TRLFS measurements on the mineral suspension as a function of pH (4–9) and Ca2+ revealed the existence of several species. Parallel factor analysis (PARAFAC) indicated the formation of three surface species totally. In the absence of Ca2+, the XO UO2+ and XO UO2CO3− surface complexes were formed, whereas the presence of Ca2+ leads to the formation of XO UO2+ and XO UO2OH as the formation of the aqueous Ca2UO2(CO3)3 (aq) complex reduces the free UO22+ concentration in the solution. In addition, ATR FT-IR spectroscopy confirmed an outer-sphere surface species in the absence of Ca2+. These experimental results were used for the assessment of surface complexation parameters to improve the basis for a mechanistic modeling of the sorption processes of U(VI) onto orthoclase and muscovite including the influence of Ca2+. Namely, log K XO UO 2 + = 1.69 and log K XO UO 2 CO 3 - = 8.96 were determined for sorption onto orthoclase, whereas log K XO UO 2 + = 0.41 and log K XO UO 2 CO 3 - = 8.71 best describe sorption onto muscovite in the absence of Ca2+.

In the presence of polyborates (detected by B-11-NMR) the formation of a weak Eu(III) borate complex (lg beta(11) similar to 2, estimated) was observed by time-resolved laser-induced fluorescence spectroscopy (TRLFS). This complex is a precursor for the formation of a solid Eu(III) borate species. The formation of this solid in solution was investigated by TRLFS as a function of the total boron concentration: the lower the total boron concentration, the slower is the solid formation. The solid Eu(III) borate was characterized by IR spectroscopy, powder XRD and solid-state TRLFS. The determination of the europium to boron ratio portends the existence of pentaborate units in the amorphous solid.