Unlocking the key role of bentonite fungal isolates in tellurium and selenium bioremediation and biorecovery: Implications in the safety of radioactive waste disposal.

Research on eco-friendly bioremediation strategies for mitigating the environmental impact of toxic metals has gained attention in the last years. Among all promising solutions, bentonite clays, to be used as artificial barriers to isolate radioactive wastes within the deep geological repository (DGR) concept, have emerged as effective reservoir of microorganisms with remarkable bioremediation potential. The present study aims to investigate the impact of bentonite fungi in the speciation and mobility of selenium (Se) and tellurium (Te), as natural analogues 79Se and 132Te present in radioactive waste, to screen for those strains with bioremediation potential within the context of DGR. For this purpose, a multidisciplinary approach combining microbiology, biochemistry, and microscopy was performed. Notably, Aspergillus sp. 3A demonstrated a high tolerance to Te(IV) and Se(IV), as evidenced by minimal inhibitory concentrations of >16 and >32 mM, respectively, along with high tolerance indexes. The high metalloid tolerance of Aspergillus sp. 3A is mediated by its capability to reduce these mobile and toxic elements to their elemental less soluble forms [Te(0) and Se(0)], forming nanostructures of various morphologies. Advanced electron microscopy techniques revealed intracellular Te(0) manifesting as amorphous needle-like nanoparticles and extracellular Te(0) forming substantial microspheres and irregular accumulations, characterized by a trigonal crystalline phase. Similarly, Se(0) exhibited a diverse array of morphologies, including hexagonal, irregular, and needle-shaped structures, accompanied by a monoclinic crystalline phase. The formation of less mobile Te(0) and Se(0) nanostructures through novel and environmentally friendly processes by Aspergillus sp. 3A suggests it would be an excellent candidate for bioremediation in contaminated environments, such as the vicinity of deep geological repositories. It moreover holds immense potential for the recovery and synthesis of Te and Se nanostructures for use in numerous biotechnological and biomedical applications.

Effect of different phosphate sources on uranium biomineralization by the Microbacterium sp. Be9 strain: A multidisciplinary approach study.

Introduction: Industrial activities related with the uranium industry are known to generate hazardous waste which must be managed adequately. Amongst the remediation activities available, eco-friendly strategies based on microbial activity have been investigated in depth in the last decades and biomineralization-based methods, mediated by microbial enzymes (e.g., phosphatase), have been proposed as a promising approach. However, the presence of different forms of phosphates in these environments plays a complicated role which must be thoroughly unraveled to optimize results when applying this remediation process. Methods: In this study, we have looked at the effect of different phosphate sources on the uranium (U) biomineralization process mediated by Microbacterium sp. Be9, a bacterial strain previously isolated from U mill tailings. We applied a multidisciplinary approach (cell surface characterization, phosphatase activity, inorganic phosphate release, cell viability, microscopy, etc.). Results and Discussion: It was clear that the U removal ability and related U interaction mechanisms by the strain depend on the type of phosphate substrate. In the absence of exogenous phosphate substrate, the cells interact with U through U phosphate biomineralization with a 98% removal of U within the first 48 h. However, the U solubilization process was the main U interaction mechanism of the cells in the presence of inorganic phosphate, demonstrating the phosphate solubilizing potential of the strain. These findings show the biotechnological use of this strain in the bioremediation of U as a function of phosphate substrate: U biomineralization (in a phosphate free system) and indirectly through the solubilization of orthophosphate from phosphate (P) containing waste products needed for U precipitation.

Uranium removal from complex mining waters by alginate beads doped with cells of Stenotrophomonas sp. Br8: Novel perspectives for metal bioremediation.

Uranium-containing effluents generated by nuclear energy industry must be efficiently remediated before release to the environment. Currently, numerous microbial-based strategies are being developed for this purpose. In particular, the bacterial strain Stenotrophomonas sp. Br8, isolated from U mill tailings porewaters, has been already shown to efficiently precipitate U(VI) as stable U phosphates mediated by phosphatase activity. However, the upscaling of this strategy should overcome some constraints regarding cell exposure to harsh environmental conditions. In the present study, the immobilization of Br8 biomass in an inorganic matrix was optimized to provide protection to the cells as well as to make the process more convenient for real-scale utilization. The use of biocompatible, highly porous alginate beads for Br8 cells immobilization resulted the best alternative when investigating by a multidisciplinary approach (High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM), Environmental Scanning Electron Microscopy (ESEM), Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance, etc.) several consolidated entrapment methods. This biomaterial was applied to complex real U mining porewaters (containing 47 mg/L U) in presence of an organic phosphate source (glycerol-2-phosphate) to produce reactive free orthophosphates through Br8 phosphatase activity. Uranium immobilization rates around 98 % were observed after one cycle of 72 h. In terms of U removal ability as a function of biomass, Br8-doped alginate beads were determined to remove up to 1199.5 mg U/g dry biomass over two treatment cycles. Additionally, optimized conditions for storing Br8-doped beads and for a correct application were assessed. Results for U accumulation kinetics and HAADF-STEM/ESEM analyses revealed that U removal by the immobilized cells is a biphasic process combining a first passive U sorption onto bead and/or cell surfaces and a second slow active biomineralization. This work provides new practical insights into the biological and physico-chemical parameters governing a high-efficient U bioremediation process based on the phosphatase activity of immobilized bacterial cells when applied to complex mining waters under laboratory conditions.

High-efficient microbial immobilization of solved U(VI) by the Stenotrophomonas strain Br8.

The environmental impact of uranium released during nuclear power production and related mining activity is an issue of great concern. Innovative environmental-friendly water remediation strategies, like those based on U biomineralization through phosphatase activity, are desirable. Here, we report the great U biomineralization potential of Stenotrophomonas sp. Br8 CECT 9810 over a wide range of physicochemical and biological conditions. Br8 cells exhibited high phosphatase activity which mediated the release of orthophosphate in the presence of glycerol-2-phosphate around pH 6.3. Mobile uranyl ions were bioprecipitated as needle-like fibrils at the cell surface and in the extracellular space, as observed by Scanning Transmission Electron Microscopy (STEM). Extended X-Ray Absorption Fine Structure (EXAFS) and X-Ray Diffraction (XRD) analyses showed the local structure of biogenic U precipitates to be similar to that of meta-autunite. In addition to the active U phosphate biomineralization process, the cells interact with this radionuclide through passive biosorption, removing up to 373 mg of U per g of bacterial dry biomass. The high U biomineralization capacity of the studied strain was also observed under different conditions of pH, temperature, etc. Results presented in this work will help to design efficient U bioremediation strategies for real polluted waters.

The Bioreduction of Selenite under Anaerobic and Alkaline Conditions Analogous to Those Expected for a Deep Geological Repository System.

The environmental conditions for the planned geological disposal of radioactive waste -including hyper-alkaline pH, radiation or anoxia-are expected to be extremely harsh for microbial activity. However, it is thought that microbial communities will develop in these repositories, and this would have implications for geodisposal integrity and the control of radionuclide migration through the surrounding environment. Nuclear waste contains radioactive isotopes of selenium (Se) such as 79Se, which has been identified as one of the main radionuclides in a geodisposal system. Here, we use the bacterial species Stenotrophomonas bentonitica, isolated from bentonites serving as an artificial barrier reference material in repositories, to study the reduction of selenite (SeIV) under simulated geodisposal conditions. This bacterium is able to reduce toxic SeIV anaerobically from a neutral to alkaline initial pH (up to pH 10), thereby producing elemental selenium (Se0) nanospheres and nanowires. A transformation process from amorphous Se (a-Se) nanospheres to trigonal Se (t-Se) nanowires, through the formation of monoclinic Se (m-Se) aggregates as an intermediate step, is proposed. The lesser solubility of Se0 and t-Se makes S. bentonitica a potential candidate to positively influence the security of a geodisposal system, most probably with lower efficiency rates than those obtained aerobically.

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine.

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.

Structural Analysis of Uranyl Complexation by the EF-Hand Motif of Calmodulin: Effect of Phosphorylation.

Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-Oeq distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes.

Stenotrophomonas bentonitica sp. nov., isolated from bentonite formations.

A Gram-stain negative, rod-shaped, aerobic bacterial strain, BII-R7T, was isolated during a study targeting the culture-dependent microbial diversity occurring in bentonite formations from southern Spain. Comparative 16S rRNA gene sequence analysis showed that BII-R7T represented a member of the genus Stenotrophomonas (class Gammaproteobacteria), and was related most closely to Stenotrophomonas rhizophila e-p10T (99.2 % sequence similarity), followed by Stenotrophomonas pavanii ICB 89T (98.5 %), Stenotrophomonas maltophilia IAM 12423T, Stenotrophomonas chelatiphaga LPM-5T and Stenotrophomonas tumulicola T5916-2-1bT (all 98.3 %). Pairwise sequence similarities to all other type strains of species of the genus Stenotrophomonas were below 98 %. Genome-based calculations (orthologous average nucleotide identity, original average nucleotide identity, genome-to-genome distance and DNA G+C percentage) indicated clearly that the isolate represents a novel species within this genus. Different phenotypic analyses, such as the detection of a quinone system composed of the major compound ubiquinone Q-8 and a fatty acid profile with iso-C15 : 0 and anteiso-C15 : 0 as major components, supported this finding at the same time as contributing to a comprehensive characterization of BII-R7T. Based on this polyphasic approach comprising phenotypic and genotypic/molecular characterization, BII-R7T can be differentiated clearly from its phylogenetic neighbours, establishing a novel species for which the name Stenotrophomonas bentonitica sp. nov. is proposed with BII-R7T as the type strain (=LMG 29893T=CECT 9180T=DSM 103927T).

Interaction of U(VI) with Schizophyllum commune studied by microscopic and spectroscopic methods.

Biosorption of actinides like uranium by fungal cells can play an important role in the mobilization or immobilization of these elements in nature. Sorption experiments of U(VI) with Schizophyllum commune at different initial uranium concentrations and varying metal speciation showed high uranium sorption capacities in the pH range of 4­7. A combination of high angle annular dark-field and scanning transmission electron microscopy analysis (HAADF-STEM) showed that living mycelium cells accumulate uranium at the cell wall and intracellular. For the first time the fluorescence properties of uranium accumulates were investigated by means of time-resolved laser-induced fluorescence spectroscopy (TRLFS) beside the determination of corresponding structural parameters using X-ray absorption fine structure spectroscopy (EXAFS). While the oxidation state of uranium remained unchanged during sorption, uranium speciation changed significantly. Extra and intracellular phosphate groups are mainly responsible for uranium binding. TRLFS spectra clearly show differences between the emission properties of dissolved species in the initial mineral medium and of uranium species on fungi. The latter were proved to be organic and inorganic uranyl phosphates formed depending on the uranyl initial concentration and in some cases on pH.

Complexation of uranium (VI) by three eco-types of Acidithiobacillus ferrooxidans studied using time-resolved laser-induced fluorescence spectroscopy and infrared spectroscopy.

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was used to study the properties of uranium complexes (emission spectra and fluorescence lifetimes) formed by the cells of the three recently described eco-types of Acidithiobacillus ferrooxidans. The results demonstrated that these complexes have different lifetimes which increase in the same order as the capability of the strains to accumulate uranium. The complexes built by the cells of the eco-type II were the strongest, whereas, those of the eco-types I and III were significantly weaker. The emission spectra of all A. ferrooxidans complexes were almost identical to those of the uranyl organic phosphate compounds. The latter finding was confirmed by infrared spectroscopic analysis.