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.

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.

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.

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.

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.

BACKGROUND: Diversity-generating retroelements (DGRs) are a unique family of retroelements that generate sequence diversity of DNA to benefit their hosts by introducing variations and accelerating the evolution of target proteins. They exist widely in bacteria, archaea, phage and plasmid. However, our understanding about DGRs in natural environments was still very limited.
RESULTS: We developed an efficient computational algorithm to identify DGRs, and applied it to characterize DGRs in more than 80,000 sequenced bacterial genomes as well as more than 4,000 human metagenome datasets. In total, we identified 948 non-redundant DGRs, which expanded the number of known DGRs in bacterial genomes and human microbiomes by about 55%, and provided a much more comprehensive reference for the study of DGRs. Phylogenetic analysis was done for identified DGRs. The putative target genes of DGRs were searched, and the functions of these target genes were investigated with a comprehensive alignment against the nr database.
CONCLUSIONS: DGR system is a powerful and universal mechanism to generate diversity. DGR evolution is closely associated with the living environment and their cassette structures. Furthermore, it may impact a wide range of functional processes in addition to receptor-binding. These results significantly improved our understanding about DGRs.

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

There are two RBMK-1500 type graphite moderated reactors at the Ignalina nuclear power plant in Lithuania, and they are under decommissioning now. The graphite cannot be disposed of in a near surface repository, because of large amounts of (14)C. Therefore, disposal of the graphite in a geological repository is a reasonable solution. This study presents evaluation of the (14)C transfer by the groundwater pathway into the geosphere from the irradiated graphite in a generic geological repository in crystalline rocks and demonstration of the role of the different components of the engineered barrier system by performing local sensitivity analysis. The speciation of the released (14)C into organic and inorganic compounds as well as the most recent information on (14)C source term was taken into account. Two alternatives were considered in the analysis: disposal of graphite in containers with encapsulant and without it. It was evaluated that the maximal fractional flux of inorganic (14)C into the geosphere can vary from 10(-11)y(-1) (for non-encapsulated graphite) to 10(-12)y(-1) (for encapsulated graphite) while of organic (14)C it was about 10(-3)y(-1) of its inventory. Such difference demonstrates that investigations on the (14)C inventory and chemical form in which it is released are especially important. The parameter with the highest influence on the maximal flux into the geosphere for inorganic (14)C transfer was the sorption coefficient in the backfill and for organic (14)C transfer - the backfill hydraulic conductivity.