Acidovorax bellezanensis sp. nov., a novel bacterium from uranium mill tailings repository sites with selenium bioremediation capabilities.

A Gram-stain-negative bacterial strain designated Be4T, belonging to the genus Acidovorax, was isolated from mining porewaters sampled in uranium mill tailings repository sites, located in Bellezane, near Bessines-sur-Gartempe (Limousin, France). Cells were facultative anaerobic, rod-shaped, non-endospore-forming and motile with flagella. The mean cell size was 1.25-1.31 µm long and 0.70-0.73 µm wide. Colonies were light yellow, opaque, circular, convex with smooth margins, and 1-2 mm in diameter. Growth occurs at 4-37 °C and between pH 5.5-9.0. It differed from its phylogenetically related strains by phenotypic and physiological characteristics such as growth at 4 °C, presence of acid phosphatase, naphthol-AS-BI-phosphohydrolase and ß-glucosidase enzymatic activities, and fermentation of l-xylose and esculin. The major fatty acids were C16:0, C16:1 ω7c/C16:1 ω6c, C17:0 cyclo and C18:1 ω7c. Phylogenetic analysis based on 16S rRNA and 938 core genes, confirmed its placement within the genus Acidovorax as a novel species. Strain Be4T showed highest 16S rRNA sequence similarity to Acidovorax antarcticus (98.2 %), Acidovorax radicis (97.9 %), Acidovorax temperans (97.8 %) and Acidovorax facilis (97.7 %). The genome of strain Be4T is 5,041,667 bp size with a DNA G + C content of 65.15 %. By automatic annotation numerous sequences involved in the interaction with metals/metalloids including some genes related to Se uptake and selenite resistance were detected in its genome. The average nucleotide identity (ANI) values calculated from whole genome sequences between strain Be4T and the most closely related strains A. radicis and A. facilis were below the threshold value of 95 %. Thus, the data from the phylogenetic, physiological, biochemical, and genomic analyses clearly indicates that strain Be4T represents a novel species with the suggested name Acidovorax bellezanensis sp. nov. The type strain is Acidovorax bellezanensis Be4T (=DSM116209T = CECT30865T). This novel species, due to its unique isolation source, genomic analysis, and preliminary laboratory tests where it was able to reduce toxic Se(IV) to less harmful Se(0) in the form of nanoparticles, holds great potential for further investigation in bioremediation, particularly concerning Se.

Past, present and future trends in the remediation of heavy-metal contaminated soil - Remediation techniques applied in real soil-contamination events.

Most worldwide policy frameworks, including the United Nations Sustainable Development Goals, highlight soil as a key non-renewable natural resource which should be rigorously preserved to achieve long-term global sustainability. Although some soil is naturally enriched with heavy metals (HMs), a series of anthropogenic activities are known to contribute to their redistribution, which may entail potentially harmful environmental and/or human health effects if certain concentrations are exceeded. If this occurs, the implementation of rehabilitation strategies is highly recommended. Although there are many publications dealing with the elimination of HMs using different methodologies, most of those works have been done in laboratories and there are not many comprehensive reviews about the results obtained under field conditions. Throughout this review, we examine the different methodologies that have been used in real scenarios and, based on representative case studies, we present the evolution and outcomes of the remediation strategies applied in real soil-contamination events where legacies of past metal mining activities or mine spills have posed a serious threat for soil conservation. So far, the best efficiencies at field-scale have been reported when using combined strategies such as physical containment and assisted-phytoremediation. We have also introduced the emerging problem of the heavy metal contamination of agricultural soils and the different strategies implemented to tackle this problem. Although remediation techniques used in real scenarios have not changed much in the last decades, there are also encouraging facts for the advances in this field. Thus, a growing number of mining companies publicise in their webpages their soil remediation strategies and efforts; moreover, the number of scientific publications about innovative highly-efficient and environmental-friendly methods is also increasing. In any case, better cooperation between scientists and other soil-related stakeholders is still required to improve remediation performance.

Effect of different phosphate sources on uranium biomineralization by the Microbacterium sp. Be9 strain: A multidisciplinary approach study.

Introduction: Industrial activities related with the uranium industry are known to generate hazardous waste which must be managed adequately. Amongst the remediation activities available, eco-friendly strategies based on microbial activity have been investigated in depth in the last decades and biomineralization-based methods, mediated by microbial enzymes (e.g., phosphatase), have been proposed as a promising approach. However, the presence of different forms of phosphates in these environments plays a complicated role which must be thoroughly unraveled to optimize results when applying this remediation process. Methods: In this study, we have looked at the effect of different phosphate sources on the uranium (U) biomineralization process mediated by Microbacterium sp. Be9, a bacterial strain previously isolated from U mill tailings. We applied a multidisciplinary approach (cell surface characterization, phosphatase activity, inorganic phosphate release, cell viability, microscopy, etc.). Results and Discussion: It was clear that the U removal ability and related U interaction mechanisms by the strain depend on the type of phosphate substrate. In the absence of exogenous phosphate substrate, the cells interact with U through U phosphate biomineralization with a 98% removal of U within the first 48 h. However, the U solubilization process was the main U interaction mechanism of the cells in the presence of inorganic phosphate, demonstrating the phosphate solubilizing potential of the strain. These findings show the biotechnological use of this strain in the bioremediation of U as a function of phosphate substrate: U biomineralization (in a phosphate free system) and indirectly through the solubilization of orthophosphate from phosphate (P) containing waste products needed for U precipitation.

Current Insight into Culture-Dependent and Culture-Independent Methods in Discovering Ascomycetous Taxa.

Culture techniques are vital in both traditional and modern fungal taxonomy. Establishing sexual-asexual links and synanamorphs, extracting DNA and secondary metabolites are mainly based on cultures. However, it is widely accepted that a large number of species are not sporulating in nature while others cannot be cultured. Recent ecological studies based on culture-independent methods revealed these unculturable taxa, i.e., dark taxa. Recent fungal diversity estimation studies suggested that environmental sequencing plays a vital role in discovering missing species. However, Sanger sequencing is still the main approach in determining DNA sequences in culturable species. In this paper, we summarize culture-based and culture-independent methods in the study of ascomycetous taxa. High-throughput sequencing of leaf endophytes, leaf litter fungi and fungi in aquatic environments is important to determine dark taxa. Nevertheless, currently, naming dark taxa is not recognized by the ICN, thus provisional naming of them is essential as suggested by several studies.

Uranium removal from complex mining waters by alginate beads doped with cells of Stenotrophomonas sp. Br8: Novel perspectives for metal bioremediation.

Uranium-containing effluents generated by nuclear energy industry must be efficiently remediated before release to the environment. Currently, numerous microbial-based strategies are being developed for this purpose. In particular, the bacterial strain Stenotrophomonas sp. Br8, isolated from U mill tailings porewaters, has been already shown to efficiently precipitate U(VI) as stable U phosphates mediated by phosphatase activity. However, the upscaling of this strategy should overcome some constraints regarding cell exposure to harsh environmental conditions. In the present study, the immobilization of Br8 biomass in an inorganic matrix was optimized to provide protection to the cells as well as to make the process more convenient for real-scale utilization. The use of biocompatible, highly porous alginate beads for Br8 cells immobilization resulted the best alternative when investigating by a multidisciplinary approach (High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM), Environmental Scanning Electron Microscopy (ESEM), Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance, etc.) several consolidated entrapment methods. This biomaterial was applied to complex real U mining porewaters (containing 47 mg/L U) in presence of an organic phosphate source (glycerol-2-phosphate) to produce reactive free orthophosphates through Br8 phosphatase activity. Uranium immobilization rates around 98 % were observed after one cycle of 72 h. In terms of U removal ability as a function of biomass, Br8-doped alginate beads were determined to remove up to 1199.5 mg U/g dry biomass over two treatment cycles. Additionally, optimized conditions for storing Br8-doped beads and for a correct application were assessed. Results for U accumulation kinetics and HAADF-STEM/ESEM analyses revealed that U removal by the immobilized cells is a biphasic process combining a first passive U sorption onto bead and/or cell surfaces and a second slow active biomineralization. This work provides new practical insights into the biological and physico-chemical parameters governing a high-efficient U bioremediation process based on the phosphatase activity of immobilized bacterial cells when applied to complex mining waters under laboratory conditions.

Ancient lineages of arbuscular mycorrhizal fungi provide little plant benefit.

Almost all land plants form symbiotic associations with arbuscular mycorrhizal fungi (AMF). Individual plants usually are colonized by a wide range of phylogenetically diverse AMF species. The impact that different AMF taxa have on plant growth is only partly understood. We screened 44 AMF isolates for their effect on growth promotion and nutrient uptake of leek plants (Allium porrum), including isolates that have not been tested previously. In particular, we aimed to test weather AMF lineages with an ancient evolutionary age differ from relatively recent lineages in their effects on leek plants. The AMF isolates that were tested covered 18 species from all five AMF orders, eight families, and 13 genera. The experiment was conducted in a greenhouse. A soil-sand mixture was used as substrate for the leek plants. Plant growth response to inoculation with AMF varied from - 19 to 232% and depended on isolate, species, and family identity. Species from the ancient families Archaeosporaceae and Paraglomeraceae tended to be less beneficial, in terms of stimulation plant growth and nutrient uptake, than species of Glomeraceae, Entrophosporaceae, and Diversisporaceae, which are considered phylogenetically more recent than those ancient families. Root colonization levels also depended on AMF family. This study indicates that plant benefit in the symbiosis between plants and AMF is linked to fungal identity and phylogeny and it shows that there are large differences in effectiveness of different AMF.

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.

Draft genome sequence data of Microbacterium sp. strain Be9 isolated from uranium-mill tailings porewaters.

Microbacterium are Gram-positive, nonspore-forming, rod-shaped bacteria inhabiting a wide range of environments including soil, water, dairy products, other living organisms, etc. Microbacterium sp. strain Be9, isolated from mill tailings porewaters in France, shows a remarkable behavior in presence of uranium under distinct conditions, which is the main reason for the interest in sequencing its genome. In this work, we describe the draft genome sequence of Be9, comprising 4,046,806 bp, with a G+C content of 68.10% and containing 3,947 protein-coding sequences. The preliminary genome annotation analysis identified some genes encoding for resistance to antibiotics and toxic compounds like heavy metals. This draft genome has been deposited at DDBJ/ENA/GenBank under the accession PRJNA590666.

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.

Stenotrophomonas bentonitica sp. nov., isolated from bentonite formations.

A Gram-stain negative, rod-shaped, aerobic bacterial strain, BII-R7T, was isolated during a study targeting the culture-dependent microbial diversity occurring in bentonite formations from southern Spain. Comparative 16S rRNA gene sequence analysis showed that BII-R7T represented a member of the genus Stenotrophomonas (class Gammaproteobacteria), and was related most closely to Stenotrophomonas rhizophila e-p10T (99.2 % sequence similarity), followed by Stenotrophomonas pavanii ICB 89T (98.5 %), Stenotrophomonas maltophilia IAM 12423T, Stenotrophomonas chelatiphaga LPM-5T and Stenotrophomonas tumulicola T5916-2-1bT (all 98.3 %). Pairwise sequence similarities to all other type strains of species of the genus Stenotrophomonas were below 98 %. Genome-based calculations (orthologous average nucleotide identity, original average nucleotide identity, genome-to-genome distance and DNA G+C percentage) indicated clearly that the isolate represents a novel species within this genus. Different phenotypic analyses, such as the detection of a quinone system composed of the major compound ubiquinone Q-8 and a fatty acid profile with iso-C15 : 0 and anteiso-C15 : 0 as major components, supported this finding at the same time as contributing to a comprehensive characterization of BII-R7T. Based on this polyphasic approach comprising phenotypic and genotypic/molecular characterization, BII-R7T can be differentiated clearly from its phylogenetic neighbours, establishing a novel species for which the name Stenotrophomonas bentonitica sp. nov. is proposed with BII-R7T as the type strain (=LMG 29893T=CECT 9180T=DSM 103927T).

Draft Genome Sequence of Stenotrophomonas bentonitica BII-R7T, a Selenite-Reducing Bacterium Isolated from Spanish Bentonites.

The Gram-negative bacterium Stenotrophomonas bentonitica BII-R7T was isolated from bentonite formations. Like other species within the genus Stenotrophomonas, strain BII-R7T possesses high tolerance to numerous heavy metals, suggesting potential for bioremediation purposes. The draft genome sequence reported here comprises 4.37 Mb with a G+C content of 66.5% and 3,796 predicted protein-coding sequences.

Screening of bacterial strains isolated from uranium mill tailings porewaters for bioremediation purposes.

The present work characterizes at different levels a number of bacterial strains isolated from porewaters sampled in the vicinity of two French uranium tailing repositories. The 16S rRNA gene from 33 bacterial isolates, corresponding to the different morphotypes recovered, was almost fully sequenced. The resulting sequences belonged to 13 bacterial genera comprised in the phyla Firmicutes, Actinobacteria and Proteobacteria. Further characterization at physiological level and metals/metalloid tolerance provided evidences for an appropriate selection of bacterial strains potentially useful for immobilization of uranium and other common contaminants. By using High Resolution Transmission Electron Microscope (HRTEM), this potential ability to immobilize uranium as U phosphate mineral phases was confirmed for the bacterial strains Br3 and Br5 corresponding to Arthrobacter sp. and Microbacterium oxydans, respectively. Scanning Transmission Electron Microscope- High-Angle Annular Dark-Field (STEM-HAADF) analysis showed U accumulates on the surface and within bacterial cytoplasm, in addition to the extracellular space. Energy Dispersive X-ray (EDX) element-distribution maps demonstrated the presence of U and P within these accumulates. These results indicate the potential of certain bacterial strains isolated from porewaters of U mill tailings for immobilizing uranium, likely as uranium phosphates. Some of these bacterial isolates might be considered as promising candidates in the design of uranium bioremediation strategies.

Effects of different arbuscular mycorrhizal fungal backgrounds and soils on olive plants growth and water relation properties under well-watered and drought conditions.

The adaptation capacity of olive trees to different environments is well recognized. However, the presence of microorganisms in the soil is also a key factor in the response of these trees to drought. The objective of the present study was to elucidate the effects of different arbuscular mycorrhizal (AM) fungi coming from diverse soils on olive plant growth and water relations. Olive plants were inoculated with native AM fungal populations from two contrasting environments, that is, semi-arid - Freila (FL) and humid - Grazalema (GZ) regions, and subjected to drought stress. Results showed that plants grew better on GZ soil inoculated with GZ fungi, indicating a preference of AM fungi for their corresponding soil. Furthermore, under these conditions, the highest AM fungal diversity was found. However, the highest root hydraulic conductivity (Lpr ) value was achieved by plants inoculated with GZ fungi and growing in FL soil under drought conditions. So, this AM inoculum also functioned in soils from different origins. Nine novel aquaporin genes were also cloned from olive roots. Diverse correlation and association values were found among different aquaporin expressions and abundances and Lpr , indicating how the interaction of different aquaporins may render diverse Lpr values.

Bacterial Diversity in Bentonites, Engineered Barrier for Deep Geological Disposal of Radioactive Wastes.

The long-term disposal of radioactive wastes in a deep geological repository is the accepted international solution for the treatment and management of these special residues. The microbial community of the selected host rocks and engineered barriers for the deep geological repository may affect the performance and the safety of the radioactive waste disposal. In this work, the bacterial population of bentonite formations of Almeria (Spain), selected as a reference material for bentonite-engineered barriers in the disposal of radioactive wastes, was studied. 16S ribosomal RNA (rRNA) gene-based approaches were used to study the bacterial community of the bentonite samples by traditional clone libraries and Illumina sequencing. Using both techniques, the bacterial diversity analysis revealed similar results, with phylotypes belonging to 14 different bacterial phyla: Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria, Nitrospirae, Verrucomicrobia and an unknown phylum. The dominant groups of the community were represented by Proteobacteria and Bacteroidetes. A high diversity was found in three of the studied samples. However, two samples were less diverse and dominated by Betaproteobacteria.

Temporal dynamics of arbuscular mycorrhizal fungi colonizing roots of representative shrub species in a semi-arid Mediterranean ecosystem.

Arbuscular mycorrhizal (AM) symbiosis plays an important role in improving plant fitness and soil quality, particularly in fragile and stressed environments, as those in certain areas of Mediterranean ecosystems. AM fungal communities are usually affected by dynamic factors such as the plant community structure and composition, which in turn are imposed by seasonality. For this reason, a one-year-round time-course trial was performed by sampling the root system of two representative shrubland species (Rosmarinus officinalis and Thymus zygis) within a typical Mediterranean ecosystem from the Southeast of Spain. The 18S rDNA gene, of the AM fungal community in roots, was subjected to PCR-SSCP, sequencing, and phylogenetic analysis. Forty-three different AM fungal sequence types were found which clustered in 16 phylotypes: 14 belonged to the Glomeraceae and two to the Diversisporaceae. Surprisingly, only two of these phylotypes were related with sequences of morphologically defined species: Glomus intraradices and Glomus constrictum. Significant differences were detected for the relative abundance of some phylotypes while no effects were found for the calculated diversity indices. These results may help to design efficient mycorrhizal-based revegetation programs for this type of ecosystems.