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.

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.

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.

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.

Predicting the long-term durability of hemp-lime renders in inland and coastal areas using Mediterranean, Tropical and Semi-arid climatic simulations.

Hemp-based composites are eco-friendly building materials as they improve energy efficiency in buildings and entail low waste production and pollutant emissions during their manufacturing process. Nevertheless, the organic nature of hemp enhances the bio-receptivity of the material, with likely negative consequences for its long-term performance in the building. The main purpose of this study was to study the response at macro- and micro-scale of hemp-lime renders subjected to weathering simulations in an environmental cabinet (one year was condensed in twelve days), so as to predict their long-term durability in coastal and inland areas with Mediterranean, Tropical and Semi-arid climates, also in relation with the lime type used. The simulated climatic conditions caused almost unnoticeable mass, volume and colour changes in hemp-lime renders. No efflorescence or physical breakdown was detected in samples subjected to NaCl, because the salt mainly precipitates on the surface of samples and is washed away by the rain. Although there was no visible microbial colonisation, alkaliphilic fungi (mainly Penicillium and Aspergillus) and bacteria (mainly Bacillus and Micrococcus) were isolated in all samples. Microbial growth and diversification were higher under Tropical climate, due to heavier rainfall. The influence of the bacterial activity on the hardening of samples has also been discussed here and related with the formation and stabilisation of vaterite in hemp-lime mixes. This study has demonstrated that hemp-lime renders show good durability towards a wide range of environmental conditions and factors. However, it might be useful to take some specific preventive and maintenance measures to reduce the bio-receptivity of this material, thus ensuring a longer durability on site.

The influence of the type of lime on the hygric behaviour and bio-receptivity of hemp lime composites used for rendering applications in sustainable new construction and repair works.

The benefits of using sustainable building materials are linked not only to the adoption of manufacturing processes that entail reduced pollution, CO2 emissions and energy consumption, but also to the onset of improved performance in the building. In particular, hemp-lime composite shows low shrinkage and high thermal and acoustic insulating properties. However, this material also shows a great ability to absorb water, an aspect that can turn out to be negative for the long-term durability of the building. For this reason, the hygric properties of hemp-based composites need to be studied to ensure the correct use of this material in construction and repair works. The water absorption, drying and transpirability of hemp composites made with aerial (in the form of dry powder and putty) and hydraulic limes were investigated here and related to the microbial growth induced by the water movements within the material. Results show that hemp-natural hydraulic lime mixes exhibit the highest transpirability and drying rate, the lowest water absorption by immersion and capillary uptake and the least intense microbial attack and chromatic change. A microscopical study of the hemp shives also related their great ability to absorb water to the near-irreversible swelling of their structure under dry-wet conditions.

Contribution of the microbial communities detected on an oil painting on canvas to its biodeterioration.

In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, "mock paintings," simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare "mock paintings." Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.

An approach to the study of the fungal deterioration of a classical art material: Mastic varnish

Mastic, one of the best natural varnishes, is frequently used as protective and finishing layer or as component of oleo-resinous media in paintings, both in the past and currently. However, this resin is affected by complex deterioration processes which can change its characteristics and thus the visual aspect of works of art. The alteration processes caused by radiation have been widely studied, but there is a lack of information on the biodeterioration of this natural product. In this paper, fungi from collections as well as from oil paintings of the Fine Arts Museum of Granada (Spain) were inoculated onto slides covered with mastic. The samples, after an incubation period of 15 days, were analysed by gas chromatography-mass spectrometry (GC-MS) to identify the chemical changes undergone, and a visual monitoring of the samples was performed to determine the formation of mycelia onto solidified resins. Major changes were detected in Chrysonilia sitophila, Phoma herbarum, and P. chrysogenum, showing evidence of alteration processes caused or favoured by these microorgamisms.

Biodeterioration patterns found in dammar resin used as art material

Since the middle of the XIX century, when dammar became popular in Occident, this natural resin is one of the most used in art painting techniques as final protective coating (varnish) as well as a component of pictorial media. The present work is the first approach to the study of the microbiological biodeterioration of this artistic material, which can seriously affect the appearance and integrity of works of art when bad conservation conditions -especially high humidity levels- take place. 12 microorganisms, fungi and bacteria, came from collection and from oil paintings affected by biodeterioration patterns, were inoculated on test specimens prepared with varnish dammar. These were incubated and analyzed by GC-MS to determine both the microbiological capacity of growth and chemical alteration on the resin. Some of the studied microorganisms have shown patterns of deterioration similar to those found in works dedicated to natural or accelerated photochemical ageing of triterpenoid varnishes.

Polyphasic approach to bacterial dynamics during the ripening of Spanish farmhouse cheese, using culture-dependent and -independent methods.

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.

Study of the microbiodegradation of terpenoid resin-based varnishes from easel painting using pyrolysis-gas chromatography-mass spectrometry and gas chromatography-mass spectrometry.

The alterations produced by microbiological attack on terpenoid resin-based varnishes from panel and canvas paintings have been evaluated using pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) and gas chromatography-mass spectrometry (GC-MS). The proposed methods include the on-line derivatisation of drying oils and diterpenoid resins using hexamethyldisilazane during pyrolysis and the application of methyl chloroformate as a derivatisation reagent for triterpenoid resins in GC-MS. Two types of specimens, consisting of model oil medium prepared from linseed oil and model spirit varnishes prepared from colophony and mastic resins dissolved in turpentine, have been used as reference materials. For a series of specimens upon which different genera of bacteria and fungi were inoculated and encouraged to grow, analyses indicated that no mechanisms that commonly occur during the attack of enzymes on drying oils and terpenoid biodegraders were observed to occur in the oil medium and varnishes studied. Thus, the degradation pathways observed in the performed trials usually occur as consequence of natural ageing. Specific trials consisting of the application of biocides to uninoculated colophony varnish resulted in the identification of processes that produce undesirable degradation of the varnish due to interactions between the biocide and the varnish components. Finally, the studied biocides--Biotin, New-Des and Nipagine--generally exhibited good inhibiting effects on the microorganisms studied, although some interesting differences were found between them regarding the application method and type of biocide.