Alicyclobacillus sp. SO9, a novel halophilic acidophilic iron-oxidizing bacterium isolated from a tailings-contaminated beach, and its effect on copper extraction from chalcopyrite in the presence of high chloride concentration.

Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

Prokaryotic communities in the historic silver mine Reiche Zeche.

The research and education mine "Reiche Zeche" in Freiberg (Saxony, Germany) represents one of the most famous mining facilities reminiscent to the century-long history of silver production in the Ore Mountains. The mine was set up at the end of the fourteenth century and became part of the "Bergakademie Freiberg" in 1919. Galena, pyrite, sphalerite, arsenopyrite, and chalcopyrite are the most common minerals found in the mine. As acid mine drainage is generated from the dissolution of sulfidic ores, the microbial habitats within the adits and galleries are characterized by low pH and high concentrations of metal(loid)s. The community composition was investigated at locations characterized by biofilm formation and iron-rich bottom pools. Amplicon libraries were sequenced on a MiSeq instrument. The taxonomic survey yielded an unexpected diversity of 25 bacterial phyla including ten genera of iron-oxidizing taxa. The community composition in the snottites and biofilms only slightly differed from the communities found in acidic bottom pools regarding the diversity of iron oxidizers, the key players in most investigated habitats. Sequences of the Candidate Phyla Radiation as, e.g., Dojkabacteria and Eremiobacterota were found in almost all samples. Archaea of the classes Thermoplasmata and Nitrososphaeria were detected in some biofilm communities.

Microbial diversity of the hypersaline and lithium-rich Salar de Uyuni, Bolivia.

Salar de Uyuni, situated in the Southwest of the Bolivian Altiplano, is the largest salt flat on Earth. Brines of this athalassohaline hypersaline environment are rich in lithium and boron. Due to the ever- increasing commodity demand, the industrial exploitation of brines for metal recovery from the world's biggest lithium reservoir is likely to increase substantially in the near future. Studies on the composition of halophilic microbial communities in brines of the salar have not been published yet. Here we report for the first time on the prokaryotic diversity of four brine habitats across the salar. The brine is characterized by salinity values between 132 and 177 PSU, slightly acidic to near-neutral pH and lithium and boron concentrations of up to 2.0 and 1.4g/L, respectively. Community analysis was performed after sequencing the V3-V4 region of the 16S rRNA genes employing the Illumina MiSeq technology. The mothur software package was used for sequence processing and data analysis. Metagenomic analysis revealed the occurrence of an exclusively archaeal community comprising 26 halobacterial genera including only recently identified genera like Halapricum, Halorubellus and Salinarchaeum. Despite the high diversity of the halobacteria-dominated community in sample P3 (Shannon-Weaver index H'=3.12 at 3% OTU cutoff) almost 40% of the Halobacteriaceae-assigned sequences could not be classified on the genus level under stringent filtering conditions. Even if the limited taxonomic resolution of the V3-V4 region for halobacteria is considered, it seems likely to discover new, hitherto undescribed genera of the family halobacteriaceae in this particular habitat of Salar de Uyuni in future.

Uranium contents in plants and mushrooms grown on a uranium-contaminated site near Ronneburg in Eastern Thuringia/Germany.

Uranium concentrations in cultivated (sunflower, sunchoke, potato) and native plants, plant compartment specimens, and mushrooms, grown on a test site within a uranium-contaminated area in Eastern Thuringia, were analyzed and compared. This test site belongs to the Friedrich-Schiller University Jena and is situated on the ground of a former but now removed uranium mine waste leaching heap. For determination of the U concentrations in the biomaterials, the saps of the samples were squeezed out by using an ultracentrifuge, after that, the uranium concentrations in the saps and the remaining residue were measured, using ICP-MS. The study further showed that uranium concentrations observed in plant compartment and mushroom fruiting bodies sap samples were always higher than their associated solid residue sample. Also, it was found that the detected uranium concentration in the root samples were always higher than were observed in their associated above ground biomass, e.g., in shoots, leaves, blossoms etc. The highest uranium concentration was measured with almost 40 ppb U in a fruiting body of a mushroom and in roots of butterbur. However, the detected uranium concentrations in plants and mushrooms collected in this study were always lower than in the associated surface and soil water of the test site, indicating that under the encountered natural conditions, none of the studied plant and mushroom species turned out to be a hyperaccumulator for uranium, which could have extracted uranium in sufficient amounts out of the uranium-contaminated soil. In addition, it was found that the detected uranium concentrations in the sap samples, despite being above the sensitivity limit, proved to be too low-in combination with the presence of fluorescence quenching substances, e.g., iron and manganese ions, and/or organic quenchers-to extract a useful fluorescence signal, which could have helped to identify the uranium speciation in plants.

Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils.

The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon's index (alpha diversity) and Sørensen's Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

Microbially assisted phytoremediation approaches for two multi-element contaminated sites.

Phytoremediation is an environmental friendly, cost-effective technology for a soft restoration of abandoned mine sites. The grasses Agrostis capillaris, Deschampsia flexuosa and Festuca rubra, and the annual herb Helianthus annuus were combined with microbial consortia in pot experiments on multi-metal polluted substrates collected at a former uranium mine near Ronneburg, Germany, and a historic copper mine in Kopparberg, Sweden, to test for phytoextraction versus phytostabilization abilities. Metal uptake into plant biomass was evaluated to identify optimal plant-microbe combinations for each substrate. Metal bioavailability was found to be plant species and element specific, and influenced by the applied bacterial consortia of 10 strains, each isolated from the same soil to which it was applied. H. annuus showed high extraction capacity for several metals on the German soil independent of inoculation. Our study could also show a significant enhancement of extraction for F. rubra and A. capillaris when combined with the bacterial consortium, although usually grasses are considered metal excluder species. On the Swedish mixed substrate, due to its toxicity, with 30 % bark compost, A. capillaris inoculated with the respective consortium was able to extract multi-metal contaminants.

Metallomics: lessons for metalliferous soil remediation.

The term metallomics has been established for the investigation of transcriptome, proteome, and metabolome changes induced by metals. The mechanisms allowing the organisms to cope with metals in the environment, metal resistance factors, will in turn change biogeochemical cycles of metals in soil, coupling the metal pool with the root system of plants. This makes microorganisms key players in introducing metals into food webs, as well as for bioremediation strategies. Research on physiological and metabolic responses of microorganisms on metal stress in soil is thus essential for the selection of optimized consortia applicable in bioremediation strategies such as bioaugmentation or microbially enhanced phytoextraction. The results of metallomics studies will help to develop applications including identification of biomarkers for ecotoxicological studies, bioleaching, in situ soil regeneration, and microbially assisted phytoremediation of contaminated land. This review will therefore focus on the molecular understanding of metal resistance in bacteria and fungi, as can be derived from metallomics studies.

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress.

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.

"Ni-struvite" - a new biomineral formed by a nickel resistant Streptomyces acidiscabies.

Biomineralization dependent on bacterial activity has been described for struvite which is formed in soils, guano, putrescent matter and sediments. A new biomineral containing nickel instead of magnesium, Ni(NH4)(PO4) . 6H2O, has been identified. It was formed by nickel resistant Streptomyces acidiscabies E13, and putatively named nickel struvite. The mineral formation is dependent on biological activity since non-viable bacterial cells are not capable to induce formation of Ni-struvite under identical conditions. Formation of Ni-struvite was observed on colony surfaces upon prolonged incubation of solid minimal or complex media containing elevated concentrations of 8-15mM NiCl2. The formation of magnesium containing crystals was not observed although Mg2+ is present in the medium. However, the process was not depending on desiccation since small crystals attached to the mycelial biomass of the bacteria were observed microscopically also in liquid cultures of nickel supplemented minimal and complex media after two weeks of incubation. The capacity to induce biomineralization of a nickel containing mineral is postulated to constitute a resistance factor, allowing the soil bacterium to withstand high nickel concentrations. The strain shows nickel resistance as an adaption to its habitat, since this bacterium was isolated from a former uranium mining site in Eastern Thuringia, Germany, where nickel concentrations of up to 2000ppm (translating to appr. 30mM) occur as a result of former mining activities.

Microbes and metals: interactions in the environment.

Research on the behaviour of microorganisms in geogenic or anthropogenic metallomorphic environments is an integral part of geomicrobiology. The investigation of microbial impact on the fate of minerals and geologically significant compounds of mining areas can lead to an understanding of biogeochemical cycles. Metabolic processes of microorganisms are the cause for the dissolution of minerals, and especially pyrite oxidation results in the generation of acid mine drainage which, in turn, leads to heavy metal contamination as a result of mining activities. On the other hand, microbial metabolism can also contribute to the formation of certain ore deposits over geological time. The adaptation to heavy metal rich environments is resulting in microorgansims which show activities for biosorption, bioprecipitation, extracellular sequestration, transport mechanisms, and/or chelation. Such resistance mechanisms are the basis for the use of microorganisms in bioremediation approaches. As only a small part of the worldwide occurring prokaryotes has been described yet, the understanding of the role bacteria play in a geogenic and pedogenic context is very likely to change deeply as soon as more habitat relevant microbial functions can be described. Examples for the identification of microbial processes from case studies may help to advance this field. The strongly interdisciplinary field of bio-geo-interactions spanning from the microorganism to the mineral holds much promise for future developments in both basic research as well as applied sciences.

Biosorption of metal and salt tolerant microbial isolates from a former uranium mining area. Their impact on changes in rare earth element patterns in acid mine drainage.

The concentration of metals in microbial habitats influenced by mining operations can reach enormous values. Worldwide, much emphasis is placed on the research of resistance and biosorptive capacities of microorganisms suitable for bioremediation purposes. Using a collection of isolates from a former uranium mining area in Eastern Thuringia, Germany, this study presents three Gram-positive bacterial strains with distinct metal tolerances. These strains were identified as members of the genera Bacillus, Micrococcus and Streptomyces. Acid mine drainage (AMD) originating from the same mining area is characterized by high metal concentrations of a broad range of elements and a very low pH. AMD was analyzed and used as incubation solution. The sorption of rare earth elements (REE), aluminum, cobalt, copper, manganese, nickel, strontium, and uranium through selected strains was studied during a time course of four weeks. Biosorption was investigated after one hour, one week and four weeks by analyzing the concentrations of metals in supernatant and biomass. Additionally, dead biomass was investigated after four weeks of incubation. The maximum of metal removal was reached after one week. Up to 80% of both Al and Cu, and more than 60% of U was shown to be removed from the solution. High concentrations of metals could be bound to the biomass, as for example 2.2 mg/g U. The strains could survive four weeks of incubation. Distinct and different patterns of rare earth elements of the inoculated and non-inoculated AMD water were observed. Changes in REE patterns hint at different binding types of heavy metals regarding incubation time and metabolic activity of the cells.

Superoxide dismutases of heavy metal resistant streptomycetes.

Heavy metal tolerant and resistant strains of streptomycetes isolated from a former uranium mining site were screened for their superoxide dismutase expression. From the strains tolerating high concentrations of different heavy metals, one was selected for its tolerance of concentrations of heavy metals (Ni, Cu, Cd, Cr, Mn, Zn, Fe). This strain, Streptomyces acidiscabies E13, was chosen for the purpose of superoxide dismutase analysis. Gel electrophoresis and activity staining revealed only one each of a nickel (NiSOD) and an iron (FeZnSOD) containing superoxide dismutase as shown by differential enzymatic repression studies. The gene for nickel containing superoxide dismutase, sodN, was cloned and sequenced from this strain. The genomic sequence shows 92.7% nucleotide identity and 96.1% amino acid identity to sodN of S. coelicolor. Expression can be activated by nickel as well as other heavy metals and active enzyme is produced in media lacking nickel but containing copper, iron or zinc. Thus, the selected strain is well suited for further characterization of the enzyme encoded by sodN.