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.

Bioconsolidation of Damaged Construction Calcarenites and Evaluation of the Improvement in Their Petrophysical and Mechanical Properties.

Bioconsolidation treatment using bacterial carbonatogenesis has been proposed as an environmentally friendly strategy for the efficient preservation of damaged stones, particularly suitable for carbonate stones. The study presented here deals with the evaluation of the performance of this treatment, applied to damaged carbonate stones in two historical buildings in Spain. The methodology applied in this research serves as a reference for future similar studies. Results showed significant improvement in the petrophysical and mechanical properties of the damaged stone following the treatment through the production of calcite and vaterite by the abundant carbonatogenic bacteria inhabiting the stone. These bacteria were able to effectively consolidate weathered areas if an adequate nutritional solution was employed, thereby augmenting the stone's resistance, as evidenced by the Drilling Resistance Measurement System (DRMS). FESEM images showed calcified bacteria and calcified exopolymeric substances (EPS) consolidating stone minerals without blocking their pores. In addition to consolidation, this biotreatment improves the stone's behavior against water absorption and increases the contact angle of water droplets without significant modifications in the pore size or diminishing vapor permeability. No color changes are observed. Overall, these results show that the application of the nutritional solution (M-3P) for in situ consolidation of different types of porous carbonate building stones is a highly effective conservation method, with no modification of the chemical composition of the treated materials.

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.

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.

Microbial interaction with and tolerance of radionuclides: underlying mechanisms and biotechnological applications.

Radionuclides (RNs) generated by nuclear and civil industries are released in natural ecosystems and may have a hazardous impact on human health and the environment. RN-polluted environments harbour different microbial species that become highly tolerant of these elements through mechanisms including biosorption, biotransformation, biomineralization and intracellular accumulation. Such microbial-RN interaction processes hold biotechnological potential for the design of bioremediation strategies to deal with several contamination problems. This paper, with its multidisciplinary approach, provides a state-of-the-art review of most research endeavours aimed to elucidate how microbes deal with radionuclides and how they tolerate ionizing radiations. In addition, the most recent findings related to new biotechnological applications of microbes in the bioremediation of radionuclides and in the long-term disposal of nuclear wastes are described and discussed.

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.

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.

Exploring bacterial community composition in Mediterranean deep-sea sediments and their role in heavy metal accumulation.

The role of microbial processes in bioaccumulation of major and trace elements has been broadly demonstrated. However, microbial communities from marine sediments have been poorly investigated to this regard. In marine environments, particularly under high anthropogenic pressure, heavy metal accumulation increases constantly, which may lead to significant environmental issues. A better knowledge of bacterial diversity and its capability to bioaccumulate metals is essential to face environmental quality assessment. The oligotrophic westernmost Mediterranean, which is highly sensitive to environmental changes and subjected to increasing anthropogenic pressure, was selected for this study. A sediment core spanning the last two millennia was sampled at two intervals, with ages corresponding to 140 (S1) and 1400 (S2) yr BP. High-throughput sequencing showed an abundance of Bacillus, Micrococcus, unclassified members of Planococcaceae, Anaerolineaceae, Planctomycetaceae, Microlunatus, and Microbacterium in both intervals, with slight differences in their abundance, along with newly detected ones in S2, i.e., Propionibacterium, Fictibacillus, Thalassobacillus, and Bacteroides. Canonical correspondence analysis (CCA) and co-occurrence patterns confirmed strong correlations among the taxa and the environmental parameters, suggesting either shared and preferred environmental conditions, or the performance of functions similar to or complementary to each other. These results were further confirmed using culture-dependent methods. The diversity of the culturable bacterial community revealed a predominance of Bacillus, and Micrococcus or Kocuria. The interaction of these bacterial communities with selected heavy metals (Cu, Cr, Zn and Pb) was also investigated, and their capacity of bioaccumulating metals within the cells and/or in the extracellular polymeric substances (EPS) is demonstrated. Interestingly, biomineralization of Pb resulted in the precipitation of Pb phosphates (pyromorphite). Our study supports that remnants of marine bacterial communities can survive in deep-sea sediments over thousands of years. This is extremely important in terms of bioremediation, in particular when considering possible environmentally friendly strategies to bioremediate inorganic contaminants.

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.

Barium bioaccumulation by bacterial biofilms and implications for Ba cycling and use of Ba proxies.

Ba proxies have been broadly used to reconstruct past oceanic export production. However, the precise mechanisms underlying barite precipitation in undersaturated seawater are not known. The link between bacterial production and particulate Ba in the ocean suggests that bacteria may play a role. Here we show that under experimental conditions marine bacterial biofilms, particularly extracellular polymeric substances (EPS), are capable of bioaccumulating Ba, providing adequate conditions for barite precipitation. An amorphous P-rich phase is formed at the initial stages of Ba bioaccumulation, which evolves into barite crystals. This supports that in high productivity regions where large amounts of organic matter are subjected to bacterial degradation, the abundant EPS would serve to bind the necessary Ba and form nucleation sites leading to barite precipitation. This also provides new insights into barite precipitation and opens an exciting field to explore the role of EPS in mineral precipitation in the ocean.

Protection and consolidation of stone heritage by self-inoculation with indigenous carbonatogenic bacterial communities.

Enhanced salt weathering resulting from global warming and increasing environmental pollution is endangering the survival of stone monuments and artworks. To mitigate the effects of these deleterious processes, numerous conservation treatments have been applied that, however, show limited efficacy. Here we present a novel, environmentally friendly, bacterial self-inoculation approach for the conservation of stone, based on the isolation of an indigenous community of carbonatogenic bacteria from salt damaged stone, followed by their culture and re-application back onto the same stone. This method results in an effective consolidation and protection due to the formation of an abundant and exceptionally strong hybrid cement consisting of nanostructured bacterial CaCO3 and bacterially derived organics, and the passivating effect of bacterial exopolymeric substances (EPS) covering the substrate. The fact that the isolated and identified bacterial community is common to many stone artworks may enable worldwide application of this novel conservation methodology.Salt weathering enhanced by global warming and environmental pollution is increasingly threatening stone monuments and artworks. Here, the authors present a bacterial self-inoculation approach with indigenous carbonatogenic bacteria and find that this technique consolidates and protects salt damaged stone.

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.

Consolidation of archaeological gypsum plaster by bacterial biomineralization of calcium carbonate.

Gypsum plasterworks and decorative surfaces are easily degraded, especially when exposed to humidity, and thus they require protection and/or consolidation. However, the conservation of historical gypsum-based structural and decorative materials by conventional organic and inorganic consolidants shows limited efficacy. Here, a new method based on the bioconsolidation capacity of carbonatogenic bacteria inhabiting the material was assayed on historical gypsum plasters and compared with conventional consolidation treatments (ethyl silicate; methylacrylate-ethylmethacrylate copolymer and polyvinyl butyral). Conventional products do not reach in-depth consolidation, typically forming a thin impervious surface layer which blocks pores. In contrast, the bacterial treatment produces vaterite (CaCO3) biocement, which does not block pores and produces a good level of consolidation, both at the surface and in-depth, as shown by drilling resistance measurement system analyses. Transmission electron microscopy analyses show that bacterial vaterite cement formed via oriented aggregation of CaCO3 nanoparticles (∼20nm in size), resulting in mesocrystals which incorporate bacterial biopolymers. Such a biocomposite has superior mechanical properties, thus explaining the fact that drilling resistance of bioconsolidated gypsum plasters is within the range of inorganic calcite materials of equivalent porosity, despite the fact that the bacterial vaterite cement accounts for only a 0.02 solid volume fraction. Bacterial bioconsolidation is proposed for the effective consolidation of this type of material. The potential applications of bacterial calcium carbonate consolidation of gypsum biomaterials used as bone graft substitutes are discussed.

Stone-isolated carbonatogenic bacteria as inoculants in bioconsolidation treatments for historical limestone.

Stone consolidation treatments that use bacterial biomineralization are mainly based on two strategies: (1) the inoculation of a bacterial culture with proven carbonatogenic ability and/or (2) the application of a culture medium capable of activating those bacteria able to induce the formation of calcium carbonate, from amongst the bacterial community of the stone. While the second strategy has been demonstrated to be effective and, unlike first strategy, it does not introduce any exogenous microorganism into the stone, problems may arise when the bacterial community of the stone is altered, for instance by the use of biocides in the cleaning process. In this study we isolate bacteria that belong to the natural microbial community of the stone and which have proven biomineralization capabilities, with the aim of preparing an inoculum that may be used in stone consolidation treatments wherein the natural community of those stones is altered. With this aim, outdoor experiments were undertaken to activate and isolate bacteria that display high biomineralization capacity from altered calcarenite stone. Most of the bacteria precipitated calcium carbonate in the form of calcite. The selected bacteria were phylogenetically affiliated with members of Actinobacteria, Gamma-proteobacteria and Firmicutes. Furthermore, the capability of these selected carbonatogenic bacteria to consolidate altered calcarenite stone slabs was studied in in vitro experiments, both in the presence and the absence of Myxococcus xanthus, as a potential reinforcement for the bacterial biomineralization. Herein, Acinetobacter species, belonging to the microbial community of the stone, are proposed as powerful carbonatogenic bacteria that, inoculated under appropriate conditions, may be used as inoculum for calcareous stone conservation/consolidation in restoration interventions where the microbial community of the stone is altered.