Microbiomes in an acidic rock-water cave system.

Belowground ecosystems are accessible by mining, where a specific microbial community can be discovered. The biodiversity of a former alum mine rich in carbon, but with a low pH of 2.6-3.7, was evaluated by DNA- and cultivation-dependent methods using samples of the black slate rock material, secondary mineralization phases and seepage water. Pyrite oxidation within the low-grade metamorphic Silurian black slate established high concentrations of Fe and $\rm{SO}_4^{2-}$ forming the extreme conditions visible with acidophilic and Fe-oxidizing microorganisms. In addition, an unexpected predominance of fungi in this C-rich and acidic cave ecosystem, including high numbers of Mucoromycota and Mortierellomycota, was detected. Therefore, fungal cultures were obtained, mainly from the secondary mineral phases that are iron phosphates. Hence, the fungi might well have been involved in phosphate mobilization there. The rock material itself is rich in organic carbon that can be used by oxidase activity. The cultivation setup mimicked the cave conditions (low temperature, low pH, oxic conditions), with one oligotrophic and one medium rich in nutrients that allowed for isolation of different fungal (and eutrophic bacterial) groups. The acidic conditions prevented the occurrence of many basidiomycetes, while the isolated fungi could survive these adverse conditions.

Changes in element availability induced by sterilization in heavy metal contaminated substrates: A comprehensive study.

Microbiome analyses of soils and microcosm experiments depend on conditions that include sterilization in order to perform experimental manipulation of microbial communities. Still, they should represent conditions close to nature. When using metal contaminated soils, sterilization methods might alter metal availability. Here, four typical metal contaminated substrates were analyzed, representing different contamination histories and soil types. They included two very poor substrates, as they are often found at metal contaminated sites. The low contents in organic carbon and nitrogen as well as two substrates with slightly higher nutrient availability were used to perform a comprehesive study for element availability changes induced by sterilization. Autoclaving, dry heat or gamma raγ sterilization were applied and compared to a non-treated control. The sterile substrates were analyzed using sequential extraction to account for different associations of the elements. Metals forming specific (hydro)oxide layers were specifically analyzed since they in turn may also impact other metals or ions. In addition, (heavy) metals and (micro)nutrients were analyzed for changes in speciation. The effects of autoclaving (wet heat) was found acceptable, while γ-ray irradiation did show unexpected changes in metal associations, especially for one substrate. Dry heat changed metal availability to the highest degree.

Microbial communities in carbonate rocks-from soil via groundwater to rocks.

Microbial communities in soil, groundwater, and rock of two sites in limestone were investigated to determine community parameters differentiating habitats in two lithostratigraphic untis. Lower Muschelkalk and Middle Muschelkalk associated soils, groundwater, and rock samples showed different, but overlapping microbial communities linked to carbon fluxes. The microbial diversities in soil were highest, groundwater revealed overlapping taxa but lower diversity, and rock samples were predominantly characterized by endospore forming bacteria and few archaea. Physiological profiles could establish a differentiation between habitats (soil, groundwater, rock). From community analyses and physiological profiles, different element cycles in limestone could be identified for the three habitats. While in soil, nitrogen cycling was identified as specific determinant, in rock methanogenesis linked carbonate rock to atmospheric methane cycles. These patterns specifically allowed for delineation of lithostratigraphic connections to physiological parameters.

Metals other than uranium affected microbial community composition in a historical uranium-mining site.

To understand the links between the long-term impact of uranium and other metals on microbial community composition, ground- and surface water-influenced soils varying greatly in uranium and metal concentrations were investigated at the former uranium-mining district in Ronneburg, Germany. A soil-based 16S PhyloChip approach revealed 2358 bacterial and 35 archaeal operational taxonomic units (OTU) within diverse phylogenetic groups with higher OTU numbers than at other uranium-contaminated sites, e.g., at Oak Ridge. Iron- and sulfate-reducing bacteria (FeRB and SRB), which have the potential to attenuate uranium and other metals by the enzymatic and/or abiotic reduction of metal ions, were found at all sites. Although soil concentrations of solid-phase uranium were high, ranging from 5 to 1569 µg·g (dry weight) soil(-1), redundancy analysis (RDA) and forward selection indicated that neither total nor bio-available uranium concentrations contributed significantly to the observed OTU distribution. Instead, microbial community composition appeared to be influenced more by redox potential. Bacterial communities were also influenced by bio-available manganese and total cobalt and cadmium concentrations. Bio-available cadmium impacted FeRB distribution while bio-available manganese and copper as well as solid-phase zinc concentrations in the soil affected SRB composition. Archaeal communities were influenced by the bio-available lead as well as total zinc and cobalt concentrations. These results suggest that (i) microbial richness was not impacted by heavy metals and radionuclides and that (ii) redox potential and secondary metal contaminants had the strongest effect on microbial community composition, as opposed to uranium, the primary source of contamination.

Aquifer community structure in dependence of lithostratigraphy in groundwater reservoirs.

Groundwater microbiology with respect to different host rocks offers new possibilities to describe and map the habitat harboring approximately half of Earths' biomass. The Thuringian Basin (Germany) contains formations of the Permian (Zechstein) and Triassic (Muschelkalk and Buntsandstein) with outcrops and deeper regions at the border and central part. Hydro(geo)chemistry and bacterial community structure of 11 natural springs and 20 groundwater wells were analyzed to define typical patterns for each formation. Widespread were Gammaproteobacteria, while Bacilli were present in all wells. Halotolerant and halophilic taxa were present in Zechstein. The occurrence of specific taxa allowed a clear separation of communities from all three lithostratigraphic groups. These specific taxa could be used to follow fluid movement, e.g., from the underlying Zechstein or from nearby saline reservoirs into Buntsandstein aquifers. Thus, we developed a new tool to identify the lithostratigraphic origin of sources in mixed waters. This was verified with entry of surface water, as species not present in the underground Zechstein environments were isolated from the water samples. Thus, our tool shows a higher resolution as compared to hydrochemistry, which is prone to undergo fast dilution if water mixes with other aquifers. Furthermore, the bacteria well adapted to their respective environment showed geographic clustering allowing to differentiate regional aquifers.

Phytoremediation using microbially mediated metal accumulation in Sorghum bicolor.

Reclaiming land that has been anthropogenically contaminated with multiple heavy metal elements, e.g., during mining operations, is a growing challenge worldwide. The use of phytoremediation has been discussed with varying success. Here, we show that a careful examination of options of microbial determination of plant performance is a key element in providing a multielement remediation option for such landscapes. We used both (a) mycorrhiza with Rhizophagus irregularis and (b) bacterial amendments with Streptomyces acidiscabies E13 and Streptomyces tendae F4 to mediate plant-promoting and metal-accumulating properties to Sorghum bicolor. In pot experiments, the effects on plant growth and metal uptake were scored, and in a field trial at a former uranium leaching heap site near Ronneburg, Germany, we could show the efficacy under field conditions. Different metals could be extracted at the same time, with varying microbial inoculation and soil amendment scenarios possible when a certain metal is the focus of interest. Especially, manganese was extracted at very high levels which might be useful even for phytomining approaches.

Identification of Mn(II)-oxidizing bacteria from a low-pH contaminated former uranium mine.

Biological Mn oxidation is responsible for producing highly reactive and abundant Mn oxide phases in the environment that can mitigate metal contamination. However, little is known about Mn oxidation in low-pH environments, where metal contamination often is a problem as the result of mining activities. We isolated two Mn(II)-oxidizing bacteria (MOB) at pH 5.5 (Duganella isolate AB_14 and Albidiferax isolate TB-2) and nine strains at pH 7 from a former uranium mining site. Isolate TB-2 may contribute to Mn oxidation in the acidic Mn-rich subsoil, as a closely related clone represented 16% of the total community. All isolates oxidized Mn over a small pH range, and isolates from low-pH samples only oxidized Mn below pH 6. Two strains with different pH optima differed in their Fe requirements for Mn oxidation, suggesting that Mn oxidation by the strain found at neutral pH was linked to Fe oxidation. Isolates tolerated Ni, Cu, and Cd and produced Mn oxides with similarities to todorokite and birnessite, with the latter being present in subsurface layers where metal enrichment was associated with Mn oxides. This demonstrates that MOB can be involved in the formation of biogenic Mn oxides in both moderately acidic and neutral pH environments.

Origin of middle rare earth element enrichment in acid mine drainage-impacted areas.

The commonly observed enrichment of middle rare earth elements (MREE) in water sampled in acid mine drainage (AMD)-impacted areas was found to be the result of preferential release from the widespread mineral pyrite (FeS2). Three different mining-impacted sites in Europe were sampled for water, and various pyrite samples were used in batch experiments with diluted sulphuric acid simulating AMD-impacted water with high sulphate concentration and high acidity. All water samples independent on their origin from groundwater, creek water or lake water as well as on the surrounding rock types showed MREE enrichment. Also the pyrite samples showed MREE enrichment in the respective acidic leachate but not always in their total contents indicating a process-controlled release. It is discussed that most probably complexation to sulphite (SO3 (2-)) or another intermediate S-species during pyrite oxidation is the reason for the MREE enrichment in the normalized REE patterns.

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.

Heavy metal tolerance of Fe(III)-reducing microbial communities in contaminated creek bank soils.

Fe(III)-reducing soil enrichment cultures can tolerate 100 µM Cu and Cd, 150 µM Co, 600 µM Ni, and 2,500 µM Zn. Metal-tolerant cultures were dominated by Geobacter-related Deltaproteobacteria and Gram-positive Firmicutes spp. (Clostridia and Sedimentibacter). A Cd- and Cu-tolerant Fe(III)-reducing coculture of Desulfosporosinus and Desulfitobacterium indicated the importance of the Firmicutes for Fe(III) reduction in the presence of metals.

Microbial links between sulfate reduction and metal retention in uranium- and heavy metal-contaminated soil.

Sulfate-reducing bacteria (SRB) can affect metal mobility either directly by reductive transformation of metal ions, e.g., uranium, into their insoluble forms or indirectly by formation of metal sulfides. This study evaluated in situ and biostimulated activity of SRB in groundwater-influenced soils from a creek bank contaminated with heavy metals and radionuclides within the former uranium mining district of Ronneburg, Germany. In situ activity of SRB, measured by the (35)SO(4)(2-) radiotracer method, was restricted to reduced soil horizons with rates of < or =142 +/- 20 nmol cm(-3) day(-1). Concentrations of heavy metals were enriched in the solid phase of the reduced horizons, whereas pore water concentrations were low. X-ray absorption near-edge structure (XANES) measurements demonstrated that approximately 80% of uranium was present as reduced uranium but appeared to occur as a sorbed complex. Soil-based dsrAB clone libraries were dominated by sequences affiliated with members of the Desulfobacterales but also the Desulfovibrionales, Syntrophobacteraceae, and Clostridiales. [(13)C]acetate- and [(13)C]lactate-biostimulated soil microcosms were dominated by sulfate and Fe(III) reduction. These processes were associated with enrichment of SRB and Geobacteraceae; enriched SRB were closely related to organisms detected in soils by using the dsrAB marker. Concentrations of soluble nickel, cobalt, and occasionally zinc declined < or =100% during anoxic soil incubations. In contrast to results in other studies, soluble uranium increased in carbon-amended treatments, reaching < or =1,407 nM in solution. Our results suggest that (i) ongoing sulfate reduction in contaminated soil resulted in in situ metal attenuation and (ii) the fate of uranium mobility is not predictable and may lead to downstream contamination of adjacent ecosystems.

Hydroxamate siderophores produced by Streptomyces acidiscabies E13 bind nickel and promote growth in cowpea (Vigna unguiculata L.) under nickel stress.

The siderophore-producing ability of nickel-resistant Streptomyces acidiscabies E13 and the role of the elicited siderophores in promoting plant growth under iron and nickel stress are described. Siderophore assays indicated that S. acidiscabies E13 can produce siderophores. Electrospray ionization mass spectrometry (ESI-MS) revealed that the bacterium simultaneously produces 3 different hydroxamate siderophores. ESI-MS showed that in addition to iron, all 3 siderophores can bind nickel. In vitro plant growth tests were conducted with cowpea (Vigna unguiculata) in the presence and absence of the elicited siderophores. Culture filtrates containing hydroxamate siderophores significantly increased cowpea height and biomass, irrespective of the iron status of the plants, under nickel stress. The presence of reduced iron was found to be high in siderophore-containing treatments in the presence of nickel. Measurements of iron and nickel contents of cowpea roots and shoots indicated that the siderophore-mediated plant growth promotion reported here involves the simultaneous inhibition of nickel uptake and solubilization and supply of iron to plants. We conclude that hydroxamate siderophores contained in culture filtrates of S. acidiscabies E13 promoted cowpea growth under nickel contamination by binding iron and nickel, thus playing a dual role of sourcing iron for plant use and protecting against nickel toxicity.

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.