Microbial influence in Spanish bentonite slurry microcosms: Unveiling a-year long geochemical evolution and early-stage copper corrosion related to nuclear waste repositories.

The deep geological repository (DGR) concept consists of storing radioactive waste in metal canisters, surrounded by compacted bentonite, and placed deeply into a geological formation. Here, bentonite slurry microcosms with copper canisters, inoculated with bacterial consortium and amended with acetate, lactate and sulfate were set up to investigate their geochemical evolution over a year under anoxic conditions. The impact of microbial communities on the corrosion of the copper canisters in an early-stage (45 days) was also assessed. The amended bacterial consortium and electron donors/acceptor accelerated the microbial activity, while the heat-shocked process had a retarding effect. The microbial communities partially oxidize lactate to acetate, which is subsequently consumed when the lactate is depleted. Early-stage microbial communities showed that the bacterial consortium reduced microbial diversity with Pseudomonas and Stenotrophomonas dominating the community. However, sulfate-reducing bacteria such as Desulfocurvibacter, Anaerosolibacter, and Desulfosporosinus were enriched coupling oxidation of lactate/acetate with reduction of sulfates. The generated biogenic sulfides, which could mediate the conversion of copper oxides (possibly formed by trapped oxygen molecules on the bentonite or driven by the reduction of H2O) to copper sulfide (Cu2S), were identified by X-ray photoelectron spectroscopy (XPS). Overall, these findings shed light on the ideal geochemical conditions that would affect the stability of DGR barriers, emphasizing the impact of the SRB on the corrosion of the metal canisters, the gas generation, and the interaction with components of the bentonite.

Long-term tracking of the microbiology of uranium-amended water-saturated bentonite microcosms: A mechanistic characterization of U speciation.

Deep geological repositories (DGRs) stand out as one of the optimal options for managing high-level radioactive waste (HLW) such as uranium (U) in the near future. Here, we provide novel insights into microbial behavior in the DGR bentonite barrier, addressing potential worst-case scenarios such as waste leakage (e.g., U) and groundwater infiltration of electron rich donors in the bentonite. After a three-year anaerobic incubation, Illumina sequencing results revealed a bacterial diversity dominated by anaerobic and spore-forming microorganisms mainly from the phylum Firmicutes. Highly U tolerant and viable bacterial isolates from the genera Peribacillus, Bacillus, and some SRB such as Desulfovibrio and Desulfosporosinus, were enriched from U-amended bentonite. The results obtained by XPS and XRD showed that U was present as U(VI) and as U(IV) species. Regarding U(VI), we have identified biogenic U(VI) phosphates, U(UO2)·(PO4)2, located in the inner part of the bacterial cell membranes in addition to U(VI)-adsorbed to clays such as montmorillonite. Biogenic U(IV) species as uraninite may be produced as result of bacterial enzymatic U(VI) reduction. These findings suggest that under electron donor-rich water-saturation conditions, bentonite microbial community can control U speciation, immobilizing it, and thus enhancing future DGR safety if container rupture and waste leakage occurs.

Insights into the Impact of Physicochemical and Microbiological Parameters on the Safety Performance of Deep Geological Repositories.

Currently, the production of radioactive waste from nuclear industries is increasing, leading to the development of reliable containment strategies. The deep geological repository (DGR) concept has emerged as a suitable storage solution, involving the underground emplacement of nuclear waste within stable geological formations. Bentonite clay, known for its exceptional properties, serves as a critical artificial barrier in the DGR system. Recent studies have suggested the stability of bentonite within DGR relevant conditions, indicating its potential to enhance the long-term safety performance of the repository. On the other hand, due to its high resistance to corrosion, copper is one of the most studied reference materials for canisters. This review provides a comprehensive perspective on the influence of nuclear waste conditions on the characteristics and properties of DGR engineered barriers. This paper outlines how evolving physico-chemical parameters (e.g., temperature, radiation) in a nuclear repository may impact these barriers over the lifespan of a repository and emphasizes the significance of understanding the impact of microbial processes, especially in the event of radionuclide leakage (e.g., U, Se) or canister corrosion. Therefore, this review aims to address the long-term safety of future DGRs, which is critical given the complexity of such future systems.

Microbial responses to elevated temperature: Evaluating bentonite mineralogy and copper canister corrosion within the long-term stability of deep geological repositories of nuclear waste.

Deep Geological Repositories (DGRs) consist of radioactive waste contained in corrosion-resistant canisters, surrounded by compacted bentonite clay, and buried few hundred meters in a stable geological formation. The effects of bentonite microbial communities on the long-term stability of the repository should be assessed. This study explores the impact of harsh conditions (60 °C, highly-compacted bentonite, low water activity), and acetate:lactate:sulfate addition, on the evolution of microbial communities, and their effect on the bentonite mineralogy, and corrosion of copper material under anoxic conditions. No bentonite illitization was observed in the treatments, confirming its mineralogical stability as an effective barrier for future DGR. Anoxic incubation at 60 °C reduced the microbial diversity, with Pseudomonas as the dominant genus. Culture-dependent methods showed survival and viability at 60 °C of moderate-thermophilic aerobic bacterial isolates (e.g., Aeribacillus). Despite the low presence of sulfate-reducing bacteria in the bentonite blocks, we proved their survival at 30 °C but not at 60 °C. Copper disk's surface remained visually unaltered. However, in the acetate:lactate:sulfate-treated samples, sulfide/sulfate signals were detected, along with microbial-related compounds. These findings offer new insights into the impact of high temperatures (60 °C) on the biogeochemical processes at the compacted bentonite/Cu canister interface post-repository closure.

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.

Impact of compacted bentonite microbial community on the clay mineralogy and copper canister corrosion: a multidisciplinary approach in view of a safe Deep Geological Repository of nuclear wastes.

Deep Geological Repository (DGR) is the preferred option for the final disposal of high-level radioactive waste. Microorganisms could affect the safety of the DGR by altering the mineralogical properties of the compacted bentonite or inducing the corrosion of the metal canisters. In this work, the impact of physicochemical parameters (bentonite dry density, heat shock, electron donors/acceptors) on the microbial activity, stability of compacted bentonite and corrosion of copper (Cu) discs was investigated after one-year anoxic incubation at 30 ºC. No-illitization in the bentonite was detected confirming its structural stability over 1 year under the experimental conditions. The microbial diversity analysis based on 16 S rRNA gene Next Generation Sequencing showed slight changes between the treatments with an increase of aerobic bacteria belonging to Micrococcaceae and Nocardioides in heat-shock tyndallized bentonites. The survival of sulfate-reducing bacteria (the main source of Cu anoxic corrosion) was demonstrated by the most probable number method. The detection of CuxS precipitates on the surface of Cu metal in the bentonite/Cu metal samples amended with acetate/lactate and sulfate, indicated an early stage of Cu corrosion. Overall, the outputs of this study help to better understand the predominant biogeochemical processes at the bentonite/Cu canister interface upon DGR closure.

Impact of microbial processes on the safety of deep geological repositories for radioactive waste.

To date, the increasing production of radioactive waste due to the extensive use of nuclear power is becoming a global environmental concern for society. For this reason, many countries have been considering the use of deep geological repositories (DGRs) for the safe disposal of this waste in the near future. Several DGR designs have been chemically, physically, and geologically well characterized. However, less is known about the influence of microbial processes for the safety of these disposal systems. The existence of microorganisms in many materials selected for their use as barriers for DGRs, including clay, cementitious materials, or crystalline rocks (e.g., granites), has previously been reported. The role that microbial processes could play in the metal corrosion of canisters containing radioactive waste, the transformation of clay minerals, gas production, and the mobility of the radionuclides characteristic of such residues is well known. Among the radionuclides present in radioactive waste, selenium (Se), uranium (U), and curium (Cm) are of great interest. Se and Cm are common components of the spent nuclear fuel residues, mainly as 79Se isotope (half-life 3.27 × 105 years), 247Cm (half-life: 1.6 × 107 years) and 248Cm (half-life: 3.5 × 106 years) isotopes, respectively. This review presents an up-to-date overview about how microbes occurring in the surroundings of a DGR may influence their safety, with a particular focus on the radionuclide-microbial interactions. Consequently, this paper will provide an exhaustive understanding about the influence of microorganisms in the safety of planned radioactive waste repositories, which in turn might improve their implementation and efficiency.

Evidence of microbial activity in a uranium roll-front deposit: Unlocking their potential role as bioenhancers of the ore genesis.

Uranium (U) roll-front deposits constitute a valuable source for an economical extraction by in situ recovery (ISR) mining. Such technology may induce changes in the subsurface microbiota, raising questions about the way their activities could build a functional ecosystem in such extreme environments (i.e.: oligotrophy and high SO4 concentration and salinity). Additionally, more information is needed to dissipate the doubts about the microbial role in the genesis of such U orebodies. A U roll-front deposit hosted in an aquifer driven system (in Zoovch Ovoo, Mongolia), intended for mining by acid ISR, was previously explored and showed to be governed by a complex bacterial diversity, linked to the redox zonation and the geochemical conditions. Here for the first time, transcriptional activities of microorganisms living in such U ore deposits are determined and their metabolic capabilities allocated in the three redox-inherited compartments, naturally defined by the roll-front system. Several genes encoding for crucial metabolic pathways demonstrated a strong biological role controlling the subsurface cycling of many elements including nitrate, sulfate, metals and radionuclides (e.g.: uranium), through oxidation-reduction reactions. Interestingly, the discovered transcriptional behaviour gives important insights into the good microbial adaptation to the geochemical conditions and their active contribution to the stabilization of the U ore deposits. Overall, evidences on the importance of these microbial metabolic activities in the aquifer system are discussed that may clarify the doubts on the microbial role in the genesis of low-temperature U roll-front deposits, along the Zoovch Ovoo mine.

Unlocking the bentonite microbial diversity and its implications in selenium bioreduction and biotransformation: Advances in deep geological repositories.

Selenium, 79Se, is one of the most critical radionuclides in radioactive waste disposed in future deep geological repositories (DGRs). Here, we investigate the impact of bentonite microbial communities on the allotropic transformation of Se(IV) bioreduction products under DGR relevant conditions. In addition, Se amendment-dependent shifts in the bentonite microbial populations are assessed. Microcosms of water-saturated bentonites were spiked with a bacterial consortium, treated with selenite and incubated anaerobically for six months. A combination of X-Ray Absorption Spectroscopy, Electron Microscopy, and Raman Spectroscopy was used to track the allotropic changes of the Se bioreduction products. Interestingly, the color of bentonite shifted from orange to black in the selenite-treated microcosms. In the orange layers, amorphous or monoclinic Se(0) were identified, whilst black precipitates consisted of stable trigonal Se(0) form. Illumina DNA sequencing indicated the distribution of strains with Se(IV) reducing and Se(0) allotropic biotransformation potential, like Pseudomonas, Stenotrophomonas, Desulfosporosinus, and unclassified-Desulfuromonadaceae. The archaea Methanosarcina decreased its abundance in the presence of Se(IV), probably caused by this oxyanion toxicity. These findings provide an understanding of the bentonite microbial strategies involved in the immobilization of Se(IV) by reduction processes, and prove their implication in the allotropic biotransformation from amorphous to trigonal Se(0) under DGR relevant conditions.

Deciphering indigenous bacteria in compacted bentonite through a novel and efficient DNA extraction method: Insights into biogeochemical processes within the Deep Geological Disposal of nuclear waste concept.

Compacted bentonites are one of the best sealing and backfilling clays considered for use in Deep Geological Repositories of radioactive wastes. However, an in-depth understanding of their behavior after placement in the repository is required, including if the activity of indigenous microorganisms affects safety conditions. Here we provide an optimized phenol:chloroform based protocol that facilitates higher DNA-yields when other methods failed. To demonstrate the efficiency of this method, DNA was extracted from acetate-treated bentonites compacted at 1.5 and 1.7 g/cm3 densities after 24 months anoxic incubation. Among the 16S rRNA gene sequences identified, those most similar to taxa mediating biogeochemical sulfur cycling included sulfur oxidizing (e.g., Thiobacillus, and Sulfurimonas) and sulfate reducing (e.g., Desulfuromonas and Desulfosporosinus) bacteria. In addition, iron-cycling populations included iron oxidizing (e.g., Thiobacillus and Rhodobacter) plus reducing taxa (e.g., Geobacillus). Genera described for their capacity to utilize acetate as a carbon source were also detected such as Delftia and Stenotrophomonas. Lastly, microscopic analyses revealed pores and cracks that could host nanobacteria or spores. This study highlights the potential role of microbial driven biogeochemical processes in compacted bentonites and the effect of high compaction on microbial diversity in Deep Geological Repositories.

Profiling native aquifer bacteria in a uranium roll-front deposit and their role in biogeochemical cycle dynamics: Insights regarding in situ recovery mining.

A uranium-mineralized sandy aquifer, planned for mining by means of uranium in situ recovery (U ISR), harbors a reservoir of bacterial life that may influence the biogeochemical cycles surrounding uranium roll-front deposits. Since microorganisms play an important role at all stages of U ISR, a better knowledge of the resident bacteria before any ISR actuations is essential to face environmental quality assessment. The focus here was on the characterization of bacteria residing in an aquifer surrounding a uranium roll-front deposit that forms part of an ISR facility project at Zoovch Ovoo (Mongolia). Water samples were collected following the natural redox zonation inherited in the native aquifer, including the mineralized orebody, as well as compartments located both upstream (oxidized waters) and downstream (reduced waters) of this area. An imposed chemical zonation for all sensitive redox elements through the roll-front system was observed. In addition, high-throughput sequencing data showed that the bacterial community structure was shaped by the redox gradient and oxygen availability. Several interesting bacteria were identified, including sulphate-reducing (e.g. Desulfovibrio, Nitrospira), iron-reducing (e.g. Gallionella, Sideroxydans), iron-oxidizing (e.g. Rhodobacter, Albidiferax, Ferribacterium), and nitrate-reducing bacteria (e.g. Pseudomonas, Aquabacterium), which may also be involved in metal reduction (e.g. Desulfovibrio, Ferribacterium, Pseudomonas, Albidiferax, Caulobacter, Zooglea). Canonical correspondence analysis (CCA) and co-occurrence patterns confirmed strong correlations among the bacterial genera, suggesting either shared/preferred environmental conditions or the performance of similar/complementary functions. As a whole, the bacterial community residing in each aquifer compartment would appear to define an ecologically functional ecosystem, containing suitable microorganisms (e.g. acidophilic bacteria) prone to promote the remediation of the acidified aquifer by natural attenuation. Assessing the composition and structure of the aquifer's native bacteria is a prerequisite for understanding natural attenuation and predicting the role of bacterial input in improving ISR efficiency.

Shifts in bentonite bacterial community and mineralogy in response to uranium and glycerol-2-phosphate exposure.

The multi-barrier deep geological repository system is currently considered as one of the safest option for the disposal of high-level radioactive wastes. Indigenous microorganisms of bentonites may affect the structure and stability of these clays through Fe-containing minerals biotransformation and radionuclides mobilization. The present work aimed to investigate the behavior of bentonite and its bacterial community in the case of a uranium leakage from the waste containers. Hence, bentonite microcosms were amended with uranyl nitrate (U) and glycerol-2-phosphate (G2P) and incubated aerobically for 6 months. Next generation 16S rRNA gene sequencing revealed that the bacterial populations of all treated microcosms were dominated by Actinobacteria and Proteobacteria, accounting for >50% of the community. Additionally, G2P and nitrate had a remarkable effect on the bacterial diversity of bentonites by the enrichment of bacteria involved in the nitrogen and carbon biogeochemical cycles (e.g. Azotobacter). A significant presence of sulfate-reducing bacteria such as Desulfonauticus and Desulfomicrobium were detected in the U-treated microcosms. The actinobacteria Amycolatopsis was enriched in G2P­uranium amended bentonites. High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy analyses showed the capacity of Amycolatopsis and a bentonite consortium formed by Bradyrhizobium-Rhizobium and Pseudomonas to precipitate U as U phosphate mineral phases, probably due to the phosphatase activity. The different amendments did not affect the mineralogy of the bentonite pointing to a high structural stability. These results would help to predict the impact of microbial processes on the biogeochemical cycles of elements (N and U) within the bentonite barrier under repository relevant conditions and to determine the changes in the microbial community induced by a uranium release.