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.

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.

Genetic mechanisms for Se(VI) reduction and synthesis of trigonal 1-D nanostructures in Stenotrophomonas bentonitica: Perspectives in eco-friendly nanomaterial production and bioremediation.

Selenate (Se(VI)) is one of the most soluble and toxic species of Se. Microbial Se(VI) reduction is an efficient tool for bioremediation strategies. However, this process is limited to a few microorganisms, and its molecular basis remains unknown. We present detailed Se(VI)-resistance mechanisms under 50 and 200 mM, in Stenotrophomonas bentonitica BII-R7, coupling enzymatic reduction of Se(VI) to formation of less toxic trigonal Se (t-Se). The results reveal a concentration-dependent response. Despite the lack of evidence of Se(VI)-reduction to Se(0) under 50 mM Se(VI), many genes were highly induced, indicating that Se(VI)-resistance could be based on intracellular reduction to Se(IV), mainly through molybdenum-dependent enzymes (e.g. respiratory nitrate reductase), and antioxidant activity by enzymes like glutathione peroxidase. Although exposure to 200 mM provoked a sharp drop in gene expression, a time-dependent process of reduction and formation of amorphous (a), monoclinic (m) and t-Se nanostructures was unravelled: a-Se nanospheres were initially synthesized intracellularly, which would transform into m-Se and finally into t-Se nanostructures during the following phases. This is the first work describing an intracellular Se(VI) reduction and biotransformation process to long-term stable and insoluble t-Se nanomaterials. These results expand the fundamental understanding of Se biogeochemical cycling, and the effectiveness of BII-R7 for bioremediation purposes.

Bacterial Diversity Evolution in Maya Plaster and Stone Following a Bio-Conservation Treatment.

To overcome the limitations of traditional conservation treatments used for protection and consolidation of stone and lime mortars and plasters, mostly based on polymers or alkoxysilanes, a novel treatment based on the activation of indigenous carbonatogenic bacteria has been recently proposed and applied both in the laboratory and in situ. Despite very positive results, little is known regarding its effect on the evolution of the indigenous bacterial communities, specially under hot and humid tropical conditions where proliferation of microorganisms is favored, as it is the case of the Maya area. Here, we studied changes in bacterial diversity of severely degraded tuff stone and lime plaster at the archeological Maya site of Copan (Honduras) after treatment with the patented sterile M-3P nutritional solution. High-throughput sequencing by Illumina MiSeq technology shows significant changes in the bacterial population of the treated stones, enhancing the development of Arthrobacter, Micrococcaceae, Nocardioides, Fictibacillus, and Streptomyces, and, in one case, Rubrobacter (carved stone blocks at Structure 18). In the lime plaster, Arthrobacter, Fictibacillus, Bacillus, Agrococcus, and Microbacterium dominated after treatment. Most of these detected genera have been shown to promote calcium carbonate biomineralization, thus implying that the novel bio-conservation treatment would be effective. Remarkably, the treatment induced the reduction or complete disappearance of deleterious acid-producing bacteria such as Marmoricola or the phylum Acidobacteria. The outcome of this study demonstrates that such a bio-conservation treatment can safely and effectively be applied on temples, sculptures and stuccos of the Maya area and, likely, in other hot and humid environments.

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.

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.

Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada.

BACKGROUND: Biomineralization processes have recently been applied in situ to protect and consolidate decayed ornamental stone of the Royal Chapel in Granada (Spain). While this promising method has demonstrated its efficacy regarding strengthening of the stone, little is known about its ecological sustainability. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report molecular monitoring of the stone-autochthonous microbiota before and at 5, 12 and 30 months after the bio-consolidation treatment (medium/long-term monitoring), employing the well-known molecular strategy of DGGE analyses. Before the bio-consolidation treatment, the bacterial diversity showed the exclusive dominance of Actinobacteria (100%), which decreased in the community (44.2%) after 5 months, and Gamma-proteobacteria (30.24%) and Chloroflexi (25.56%) appeared. After 12 months, Gamma-proteobacteria vanished from the community and Cyanobacteria (22.1%) appeared and remained dominant after thirty months, when the microbiota consisted of Actinobacteria (42.2%) and Cyanobacteria (57.8%) only. Fungal diversity showed that the Ascomycota phylum was dominant before treatment (100%), while, after five months, Basidiomycota (6.38%) appeared on the stone, and vanished again after twelve months. Thirty months after the treatment, the fungal population started to stabilize and Ascomycota dominated on the stone (83.33%) once again. Members of green algae (Chlorophyta, Viridiplantae) appeared on the stone at 5, 12 and 30 months after the treatment and accounted for 4.25%, 84.77% and 16.77%, respectively. CONCLUSIONS: The results clearly show that, although a temporary shift in the bacterial and fungal diversity was observed during the first five months, most probably promoted by the application of the bio-consolidation treatment, the microbiota tends to regain its initial stability in a few months. Thus, the treatment does not seem to have any negative side effects on the stone-autochthonous microbiota over that time. The molecular strategy employed here is suggested as an efficient monitoring tool to assess the impact on the stone-autochthonous microbiota of the application of biomineralization processes as a restoration/conservation procedure.