Europium(III) as luminescence probe for interactions of a sulfate-reducing microorganism with potentially toxic metals.

Microorganisms show a high affinity for trivalent actinides and lanthanides, which play an important role in the safe disposal of high-level radioactive waste as well as in the mining of various rare earth elements. The interaction of the lanthanide Eu(III) with the sulfate-reducing microorganism Desulfosporosinus hippei DSM 8344T, a representative of the genus Desulfosporosinus that naturally occurs in clay rock and bentonite, was investigated. Eu(III) is often used as a non-radioactive analogue for the trivalent actinides Pu(III), Am(III), and Cm(III), which contribute to a major part of the radiotoxicity of the nuclear waste. D. hippei DSM 8344T showed a weak interaction with Eu(III), most likely due to a complexation with lactate in artificial Opalinus Clay pore water. Hence, a low removal of the lanthanide from the supernatant was observed. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed a bioprecipitation of Eu(III) with phosphates potentially excreted from the cells. This demonstrates that the ongoing interaction mechanisms are more complex than a simple biosorption process. The bioprecipitation was also verified by luminescence spectroscopy, which showed that the formation of the Eu(III) phosphate compounds starts almost immediately after the addition of the cells. Moreover, chemical microscopy provided information on the local distribution of the different Eu(III) species in the formed cell aggregates. These results provide first insights into the interaction mechanisms of Eu(III) with sulfate-reducing bacteria and contribute to a comprehensive safety concept for a high-level radioactive waste repository, as well as to a better understanding of the fate of heavy metals (especially rare earth elements) in the environment.

Presence of uranium(V) during uranium(VI) reduction by Desulfosporosinus hippei DSM 8344T.

Microbial U(VI) reduction influences uranium mobility in contaminated subsurface environments and can affect the disposal of high-level radioactive waste by transforming 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 investigated. D. hippei DSM 8344T showed a relatively fast removal of uranium from the supernatants in artificial Opalinus Clay pore water, but no removal in 30 mM bicarbonate solution. Combined speciation calculations and luminescence spectroscopic investigations showed the dependence of U(VI) reduction on the initial U(VI) species. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed uranium-containing aggregates on the cell surface and some membrane vesicles. By combining different spectroscopic techniques, including UV/Vis spectroscopy, as well as uranium M4-edge X-ray absorption near-edge structure recorded in high-energy-resolution fluorescence-detection mode and extended X-ray absorption fine structure analysis, 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. These findings offer new insights into U(VI) reduction by sulfate-reducing bacteria and contribute to a comprehensive safety concept for a repository for high-level radioactive waste.

Microscopic and spectroscopic bioassociation study of uranium(VI) with an archaeal Halobacterium isolate.

The safe disposal of high-level radioactive waste in a deep geological repository is a huge social and technical challenge. So far, one of the less considered factors needed for a long-term risk assessment, is the impact of microorganisms occurring in the different host rocks. Even under the harsh conditions of salt formations different bacterial and archaeal species were found, e. g. Halobacterium sp. GP5 1-1, which has been isolated from a German rock salt sample. The interactions of this archaeon with uranium(VI), one of the radionuclides of major concern for the long-term storage of high-level radioactive waste, were investigated. Different spectroscopic techniques, as well as microscopy, were used to examine the occurring mechanisms on a molecular level leading to a more profound process understanding. Batch experiments with different uranium(VI) concentrations showed that the interaction is not only a simple, but a more complex combination of different processes. With the help of in situ attenuated total reflection Fourier-transform infrared spectroscopy the association of uranium(VI) onto carboxylate groups was verified. In addition, time-resolved laser-induced luminescence spectroscopy revealed the formation of phosphate and carboxylate species within the cell pellets as a function of the uranium(VI) concentration and incubation time. The association behavior differs from another very closely related halophilic archaeon, especially with regard to uranium(VI) concentrations. This clearly demonstrates the importance of studying the interactions of different, at first sight very similar, microorganisms with uranium(VI). This work provides new insights into the microbe-uranium(VI) interactions at highly saline conditions relevant to the long-term storage of radioactive waste in rock salt.

Molecular Binding of EuIII/CmIII by Stenotrophomonas bentonitica and Its Impact on the Safety of Future Geodisposal of Radioactive Waste.

Microbial communities occurring in reference materials for artificial barriers (e.g., bentonites) in future deep geological repositories of radioactive waste can influence the migration behavior of radionuclides such as curium (CmIII). This study investigates the molecular interactions between CmIII and its inactive analogue europium (EuIII) with the indigenous bentonite bacterium Stenotrophomonas bentonitica at environmentally relevant concentrations. Potentiometric studies showed a remarkably high concentration of phosphates at the bacterial cell wall compared to other bacteria, revealing the great potential of S. bentonitica for metal binding. Infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the role of phosphates and carboxylate groups from the cell envelope in the bioassociation of EuIII. Additionally, time-resolved laser-induced fluorescence spectroscopy (TRLFS) identified phosphoryl and carboxyl groups from bacterial envelopes, among other released complexing agents, to be involved in the EuIII and CmIII coordination. The ability of this bacterium to form a biofilm at the surface of bentonites allows them to immobilize trivalent lanthanide and actinides in the environment.

The Year-Long Development of Microorganisms in Uncompacted Bavarian Bentonite Slurries at 30 and 60 °C.

In the multibarrier concept for the deep geological disposal of high-level radioactive waste (HLW), bentonite is proposed as a potential barrier and buffer material for sealing the space between the steel canister containing the HLW and the surrounding host rock. In order to broaden the spectra of appropriate bentonites, we investigated the metabolic activity and diversity of naturally occurring microorganisms as well as their time-dependent evolution within the industrial B25 Bavarian bentonite under repository-relevant conditions. We conducted anaerobic microcosm experiments containing the B25 bentonite and a synthetic Opalinus Clay pore water solution, which were incubated for one year at 30 and 60 °C. Metabolic activity was only stimulated by the addition of lactate, acetate, or H2. The majority of lactate- and H2-containing microcosms at 30 °C were dominated by strictly anaerobic, sulfate-reducing, and spore-forming microorganisms. The subsequent generation of hydrogen sulfide led to the formation of iron-sulfur precipitations. Independent from the availability of substrates, thermophilic bacteria dominated microcosms that were incubated at 60 °C. However, in the respective microcosms, no significant metabolic activity occurred, and there was no change in the analyzed biogeochemical parameters. Our findings show that indigenous microorganisms of B25 bentonite evolve in a temperature- and substrate-dependent manner.

Association of Eu(III) and Cm(III) onto an extremely halophilic archaeon.

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.

Microbial Diversity in an Arid, Naturally Saline Environment.

The Arava Valley in is a rock desert within the Great African Rift valley. Soil from this area is covered with a salt crust. Here, we report microbial diversity from arid, naturally saline samples collected near Ein Yahav from the Arava Valley by culture-independent as well as culture-dependent analysis. High-throughput sequencing of the hypervariable region V4 of the 16S rRNA gene revealed that the microbial community consists of halophiles from the domain Bacteria as well as Archaea. Bacterial diversity was mainly represented by the genus Salinimicrobium of the order Flavobacteriales within the phylum Bacteroidetes, from the gammaproteobacterial orders Alteromonadales and Oceanospirillales as well as representatives from the order Bacillales of the phylum Firmicutes. Archaeal diversity was dominated by euryarchaeal Halobacteria from the orders Halobacteriales, Haloferacales, and Natrialbales. But more than 40% of the sequences affiliated with Archaea were assigned to unknown or unclassified archaea. Even if taxonomic resolution of the 16S rRNA gene V4 region for Archaea is limited, this study indicates the need of further and more detailed studies of Archaea. By using culture-dependent analysis, bacteria of the order Bacillales as well as archaea from all three halobacterial orders Halobacteriales, Haloferacales, and Natrialbales including potentially novel species from the genera Halorubrum and Haloparvum were isolated.

Impact of Haloarchaea on Speciation of Uranium-A Multispectroscopic Approach.

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.

Comparative analysis of uranium bioassociation with halophilic bacteria and archaea.

Rock salt represents a potential host rock formation for the final disposal of radioactive waste. The interactions between indigenous microorganisms and radionuclides, e.g. uranium, need to be investigated to better predict the influence of microorganisms on the safety assessment of the repository. Hence, the association process of uranium with two microorganisms isolated from rock salt was comparatively studied. Brachybacterium sp. G1, which was isolated from the German salt dome Gorleben, and Halobacterium noricense DSM15987T, were selected as examples of a moderately halophilic bacterium and an extremely halophilic archaeon, respectively. The microorganisms exhibited completely different association behaviors with uranium. While a pure biosorption process took place with Brachybacterium sp. G1 cells, a multistage association process occurred with the archaeon. In addition to batch experiments, in situ attenuated total reflection Fourier-transform infrared spectroscopy was applied to characterize the U(VI) interaction process. Biosorption was identified as the dominating process for Brachybacterium sp. G1 with this method. Carboxylic functionalities are the dominant interacting groups for the bacterium, whereas phosphoryl groups are also involved in U(VI) association by the archaeon H. noricense.

Plutonium interaction studies with the Mont Terri Opalinus Clay isolate Sporomusa sp. MT-2.99: changes in the plutonium speciation by solvent extractions.

Since plutonium could be released from nuclear waste disposal sites, the exploration of the complex interaction processes between plutonium and bacteria is necessary for an improved understanding of the fate of plutonium in the vicinity of such a nuclear waste disposal site. In this basic study, the interaction of plutonium with cells of the bacterium, Sporomusa sp. MT-2.99, isolated from Mont Terri Opalinus Clay, was investigated anaerobically (in 0.1 M NaClO4) with or without adding Na-pyruvate as an electron donor. The cells displayed a strong pH-dependent affinity for Pu. In the absence of Na-pyruvate, a strong enrichment of stable Pu(V) in the supernatants was discovered, whereas Pu(IV) polymers dominated the Pu oxidation state distribution on the biomass at pH 6.1. A pH-dependent enrichment of the lower Pu oxidation states (e.g., Pu(III) at pH 6.1 which is considered to be more mobile than Pu(IV) formed at pH 4) was observed in the presence of up to 10 mM Na-pyruvate. In all cases, the presence of bacterial cells enhanced removal of Pu from solution and accelerated Pu interaction reactions, e.g., biosorption and bioreduction.

Multistage bioassociation of uranium onto an extremely halophilic archaeon revealed by a unique combination of spectroscopic and microscopic techniques.

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 repository, were used for these investigations. The kinetics of U(VI) bioassociation with 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 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, as shown with scanning electron microscopy in combination with energy dispersive X-ray spectroscopy. Our results demonstrate for the first time that association of uranium with the extremely halophilic archaeon is a multistage process, beginning with sorption and followed by another process, probably biomineralization.

Study of the Interaction of Eu3+ with Microbiologically Induced Calcium Carbonate Precipitates using TRLFS.

The microbial induced biomineralization of calcium carbonate using the ureolytic bacterium Sporosarcina pasteurii in the presence of trivalent europium, a substitute for trivalent actinides, was investigated by time-resolved laser-induced fluorescence spectroscopy (TRLFS) and a variety of physicochemical techniques. Results showed that the bacterial-driven hydrolysis of urea provides favorable conditions for CaCO3 precipitation and Eu3+ uptake due to subsequent increases in NH4+ and pH in the local environment. Precipitate morphologies were characteristic of biogenically formed CaCO3 and consistent with the respective mineral phase compositions. The formation of vaterite with some calcite was observed after 1 day, calcite with some vaterite after 1 week, and pure calcite after 2 weeks. The presence of organic material associated with the mineral was also identified and quantified. TRLFS was used to track the interaction and speciation of Eu3+ as a molecular probe with the mineral as a function of time. Initially, Eu3+ is incorporated into the vaterite phase, while during CaCO3 phase transformation Eu3+ speciation changes resulting in several species incorporated in the calcite phase either substituting at the Ca2+ site or in a previously unidentified, low-symmetry site. Comparison of the biogenic precipitates to an abiotic sample shows mineral origin can affect Eu3+ speciation within the mineral.

Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1.

Uranium (as UO2(2+)), technetium (as TcO4(-)) and neptunium (as NpO2(+)) are highly mobile radionuclides that can be reduced enzymatically by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble species. The redox chemistry of Pu is more complicated, but the dominant oxidation state in most environments is highly insoluble Pu(IV), which can be reduced to Pu(III) which has a potentially increased solubility which could enhance migration of Pu in the environment. Recently it was shown that flavins (riboflavin and flavin mononucleotide (FMN)) secreted by Shewanella oneidensis MR-1 can act as electron shuttles, promoting anoxic growth coupled to the accelerated reduction of poorly-crystalline Fe(III) oxides. Here, we studied the role of riboflavin in mediating the reduction of radionuclides in cultures of Shewanella oneidensis MR-1. Our results demonstrate that the addition of 10 µM riboflavin enhances the reduction rate of Tc(VII) to Tc(IV), Pu(IV) to Pu(III) and to a lesser extent, Np(V) to Np(IV), but has no significant influence on the reduction rate of U(VI) by Shewanella oneidensis MR-1. Thus riboflavin can act as an extracellular electron shuttle to enhance rates of Tc(VII), Np(V) and Pu(IV) reduction, and may therefore play a role in controlling the oxidation state of key redox active actinides and fission products in natural and engineered environments. These results also suggest that the addition of riboflavin could be used to accelerate the bioremediation of radionuclide-contaminated environments.

Bacterial Diversity in Bentonites, Engineered Barrier for Deep Geological Disposal of Radioactive Wastes.

The long-term disposal of radioactive wastes in a deep geological repository is the accepted international solution for the treatment and management of these special residues. The microbial community of the selected host rocks and engineered barriers for the deep geological repository may affect the performance and the safety of the radioactive waste disposal. In this work, the bacterial population of bentonite formations of Almeria (Spain), selected as a reference material for bentonite-engineered barriers in the disposal of radioactive wastes, was studied. 16S ribosomal RNA (rRNA) gene-based approaches were used to study the bacterial community of the bentonite samples by traditional clone libraries and Illumina sequencing. Using both techniques, the bacterial diversity analysis revealed similar results, with phylotypes belonging to 14 different bacterial phyla: Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria, Nitrospirae, Verrucomicrobia and an unknown phylum. The dominant groups of the community were represented by Proteobacteria and Bacteroidetes. A high diversity was found in three of the studied samples. However, two samples were less diverse and dominated by Betaproteobacteria.