Spider web biomonitoring: A cost-effective source apportionment approach for urban particulate matter.

Elevated levels of particulate matter (PM) in urban atmospheres are one of the major environmental challenges of the Anthropocene. To effectively lower those levels, identification and quantification of sources of PM is required. Biomonitoring methods are helpful tools to tackle this problem but have not been fully established yet. An example is the sampling and subsequent analysis of spider webs to whose adhesive surface dust particles can attach. For a methodical inspection, webs of orb-weaving spiders were sampled repeatedly from 2016 to 2018 at 22 locations in the city of Jena, Germany. Contents of Ag, Al, As, B, Ba, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Sb, Si, Sn, Sr, Th, Ti, V, Y, Zn and Zr were determined in the samples using inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES) after aqua regia digestion. Multivariate statistical methods were applied for a detailed evaluation. A combination of cluster analysis and principal component analysis allows for the clear identification of three main sources in the study area: brake wear from car traffic, abrasion of tram/train tracks and particles of geogenic origin. Quantitative source contributions reveal that high amounts of most of the metals are derived from a combination of brake wear and geogenic particles, the latter of which are likely resuspended by moving vehicles. This emphasizes the importance of non-exhaust particles connected to road traffic. Once a source identification has been performed for an area of interest, classification models can be applied to assess air quality for further samples from within the whole study area, offering a tool for air quality assessment. The general validity of this approach is demonstrated using samples from other locations.

Rare earth element sequestration by Aspergillus oryzae biomass.

The fungus Aspergillus oryzae could be shown to be a viable alternative for biosorption of valuable metals from solution. Fungal biomass can be obtained easily in high quantities as a waste of biofermentation processes, and used in a complex, multi-phase solution mimicking naturally occurring, mining-affected water samples. With test solution formulated after natural conditions, formation of secondary Al and Fe phases co-precipitating Ce was recorded in addition to specific biosorption of rare earth elements. Remarkably, the latter were removed from the solution despite the presence of high concentrations of interfering Fe and Al. The biomass was viable even after prolonged incubation in the metal solution, and minimal inhibitory concentrations for single metals were higher than those in the test solution. While precipitation/biosorption of Ce (maximal biosorption efficiency was 58.0 ± 22.3% after 6 h of incubation) coincided with the gross removal of Fe from the metal solution, Y (81.5 ± 11.3% efficiency, 24 h incubation) and Nd (87.4 ± 9.1% efficiency, 24 h incubation) were sequestered later, similarly to Ni and Zn. The biphasic binding pattern specific to single metals could be connected to dynamically changing pH and NH4+ concentrations, which were attributed to the physiological changes taking place in starving A. oryzae biomass. The metals were found extracellularly in minerals associated with the cell wall, and intracellularly precipitated in the vacuoles. The latter process was explained with intracellular metal detoxification resulting in metal resistance.

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.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.

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.

The dendroanalysis of oak trees as a method of biomonitoring past and recent contamination in an area influenced by uranium mining.

We reconstructed the contamination history of an area influenced by 40 years of uranium mining and subsequent remediation actions using dendroanalysis (i.e., the determination of the elemental content of tree rings). The uranium content in the tree rings of four individual oak trees (Quercus sp.) was determined by laser ablation with inductively coupled plasma mass spectrometry (LA-ICP-MS). This technique allows the investigation of trace metals in solid samples with a spatial resolution of 250 µm and a detection limit below 0.01 µg/g for uranium. The investigations show that in three of the four oaks sampled, there were temporally similar uranium concentrations. These were approximately 2 orders of magnitude higher (0.15 to 0.4 µg/g) than those from before the period of active mining (concentrations below 0.01 µg/g). After the mining was terminated and the area was restored, the uranium contents in the wood decreased by approximately 1 order of magnitude. The similar radial uranium distribution patterns of the three trees were confirmed by correlation analysis. In combination with the results of soil analyses, it was determined that there was a heterogeneous contamination in the forest investigated. This could be confirmed by pre-remediation soil uranium contents from literature. The uranium contents in the tree rings of the oaks investigated reflect the contamination history of the study area. This study demonstrates that the dendrochemical analysis of oak tree rings is a suitable technique for investigating past and recent uranium contamination in mining areas.

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.

Siderophore production by streptomycetes-stability and alteration of ferrihydroxamates in heavy metal-contaminated soil.

Heavy metal-contaminated soil derived from a former uranium mining site in Ronneburg, Germany, was used for sterile mesocosms inoculated with the extremely metal-resistant Streptomyces mirabilis P16B-1 or the sensitive control strain Streptomyces lividans TK24. The production and fate of bacterial hydroxamate siderophores in soil was analyzed, and the presence of ferrioxamines E, B, D, and G was shown. While total ferrioxamine concentrations decreased in water-treated controls after 30 days of incubation, the sustained production by the bacteria was seen. For the individual molecules, alteration between neutral and cationic forms and linearization of hydroxamates was observed for the first time. Mesocosms inoculated with biomass of either strain showed changes of siderophore contents compared with the non-treated control indicating for auto-alteration and consumption, respectively, depending on the vital bacteria present. Heat stability and structural consistency of siderophores obtained from sterile culture filtrate were shown. In addition, low recovery (32 %) from soil was shown, indicating adsorption to soil particles or soil organic matter. Fate and behavior of hydroxamate siderophores in metal-contaminated soils may affect soil properties as well as conditions for its inhabiting (micro)organisms.

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.

Growth of streptomycetes in soil and their impact on bioremediation.

The impact of the extremely heavy metal resistant actinomycete Streptomyces mirabilis P16B-1 on heavy metal mobilization/stabilization, phytoremediation and stress level of plants was analyzed in the presence and absence of Sorghum bicolor in sterile microcosms containing highly metal contaminated or control soil. For control, a metal sensitive S. lividans TK24 was used. The metal contents with respect to the mobile and specifically adsorbed fractions of the contaminated soil were considerably decreased by addition of both, living and dead biomass of the strains, with the heavy metal resistant S. mirabilis P16B-1 showing considerably higher impact. Both strains could grow in control soil, while only S. mirabilis P16B-1 formed new tip growth in the metal contaminated soil. A plant growth promoting effect was visible for S. mirabilis P16B-1 in contaminated soil enhancing the dry weight of inoculated Sorghum plants. Thus, metal resistant strains like S. mirabilis P16B-1 are able to enhance phytoremediation of heavy metal contaminated soils.

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.

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.