Microbial adaptations and biogeochemical cycling of uranium in polymetallic tailings.

Microorganisms inhabiting uranium (U)-rich environments have specific physiological and biochemical coping mechanisms to deal with U toxicity, and thereby play a crucial role in the U biogeochemical cycling as well as associated heavy metals. We investigated the diversity and functional capabilities of indigenous bacterial communities inhabiting historic U- and Rare-Earth-Elements-rich polymetallic tailings from the Mount Painter Inlier, Northern Flinders Ranges, South Australia. Bacterial diversity profiling identified Actinobacteria as the predominant phylum in all samples. GeoChip analyses revealed the presence of diverse functional genes associated with biogenic element cycling, metal homeostasis/resistance, stress response, and secondary metabolism. The high abundance of metal-resistance and stress-tolerance genes indicates the adaptation of bacterial communities to the "harsh" environmental (metal-rich and semi-arid) conditions of the Northern Flinders Ranges. Additionally, a viable bacterial consortium was enriched from polymetallic tailings. Laboratory experiments demonstrated that the consortium scrubbed uranyl from solution by precipitating a uranyl phosphate biomineral (chernikovite), thus contributing to U biogeochemical cycling. These specialised microbial communities reflect the high specificity of the mineralogy/geochemistry, and biogeography of these U-rich settings. This study provides the fundamental knowledge to develop future applications in securing long-term stability of polymetallic mine waste, and for reprocessing this "waste" to further extract critical minerals.

From biomolecules to biogeochemistry: Exploring the interaction of an indigenous bacterium with gold.

Specialised microbial communities colonise the surface of gold particles in soils/sediments, and catalyse gold dissolution and re-precipitation, thereby contributing to the environmental mobility and toxicity of this 'inert' precious metal. We assessed the proteomic and physiological response of Serratia proteamaculans, the first metabolically active bacterium enriched and isolated directly from natural gold particles, when exposed to toxic levels of soluble Au3+ (10 µM). The results were compared to a metal-free blank, and to cultures exposed to similarly toxic levels of soluble Cu2+ (0.1 mM); Cu was chosen for comparison because it is closely associated with Au in nature due to similar geochemical properties. A total of 273 proteins were detected from the cells that experienced the oxidative effects of soluble Au, of which 139 (51%) were upregulated with either sole expression (31%) or had synthesis levels greater than the Au-free control (20%). The majority (54%) of upregulated proteins were functionally different from up-regulated proteins in the bacteria-copper treatment. These proteins were related to broad functions involving metabolism and biogenesis, followed by cellular process and signalling, indicating significant specificity for Au. This proteomic study revealed that the bacterium upregulates the synthesis of various proteins related to oxidative stress response (e.g., Monothiol-Glutaredoxin, Thiol Peroxidase, etc.) and cellular damage repair, which leads to the formation of metallic gold nanoparticles less toxic than ionic gold. Therefore, indigenous bacteria may mediate the toxicity of Au through two different yet simultaneous processes: i) repairing cellular components by replenishing damaged proteins and ii) neutralising reactive oxygen species (ROS) by up-regulating the synthesis of antioxidants. By connecting the fields of molecular bacteriology and environmental biogeochemistry, this study is the first step towards the development of biotechnologies based on indigenous bacteria applied to gold bio-recovery and bioremediation of contaminated environments.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Microbially influenced corrosion and rust tubercle formation on sheet piles in freshwater systems.

The extent of how complex natural microbial communities contribute to metal corrosion is still not fully resolved, especially not for freshwater environments. In order to elucidate the key processes, we investigated rust tubercles forming massively on sheet piles along the river Havel (Germany) applying a complementary set of techniques. In-situ microsensor profiling revealed steep gradients of O2 , redox potential and pH within the tubercle. Micro-computed tomography and scanning electron microscopy showed a multi-layered inner structure with chambers and channels and various organisms embedded in the mineral matrix. Using Mössbauer spectroscopy we identified typical corrosion products including electrically conductive iron (Fe) minerals. Determination of bacterial gene copy numbers and sequencing of 16S rRNA and 18S rRNA amplicons supported a densely populated tubercle matrix with a phylogenetically and metabolically diverse microbial community. Based on our results and previous models of physic(electro)chemical reactions, we propose here a comprehensive concept of tubercle formation highlighting the crucial reactions and microorganisms involved (such as phototrophs, fermenting bacteria, dissimilatory sulphate and Fe(III) reducers) in metal corrosion in freshwaters.

Mineral characterization and composition of Fe-rich flocs from wetlands of Iceland: Implications for Fe, C and trace element export.

In freshwater wetlands, redox interfaces characterized by circumneutral pH, steep gradients in O2, and a continual supply of Fe(II) form ecological niches favorable to microaerophilic iron(II) oxidizing bacteria (FeOB) and the formation of flocs; associations of (a)biotic mineral phases, microorganisms, and (microbially-derived) organic matter. On the volcanic island of Iceland, wetlands are replenished with Fe-rich surface-, ground- and springwater. Combined with extensive drainage of lowland wetlands, which forms artificial redox gradients, accumulations of bright orange (a)biotically-derived Fe-rich flocs are common features of Icelandic wetlands. These loosely consolidated flocs are easily mobilized, and, considering the proximity of Iceland's lowland wetlands to the coast, are likely to contribute to the suspended sediment load transported to coastal waters. To date, however, little is known regarding (Fe) mineral and elemental composition of the flocs. In this study, flocs from wetlands (n = 16) across Iceland were analyzed using X-ray diffraction and spectroscopic techniques (X-ray absorption and 57Fe Mössbauer) combined with chemical extractions and (electron) microscopy to comprehensively characterize floc mineral, elemental, and structural composition. All flocs were rich in Fe (229-414 mg/g), and floc Fe minerals comprised primarily ferrihydrite and nano-crystalline lepidocrocite, with a single floc sample containing nano-crystalline goethite. Floc mineralogy also included Fe in clay minerals and appreciable poorly-crystalline aluminosilicates, most likely allophane and/or imogolite. Microscopy images revealed that floc (bio)organics largely comprised mineral encrusted microbially-derived components (i.e. sheaths, stalks, and EPS) indicative of common FeOB Leptothrix spp. and Gallionella spp. Trace element contents in the flocs were in the low µg/g range, however nearly all trace elements were extracted with hydroxylamine hydrochloride. This finding suggests that the (a)biotic reductive dissolution of floc Fe minerals, plausibly driven by exposure to the varied geochemical conditions of coastal waters following floc mobilization, could lead to the release of associated trace elements. Thus, the flocs should be considered vectors for transport of Fe, organic carbon, and trace elements from Icelandic wetlands to coastal waters.

A genomic perspective of metal-resistant bacteria from gold particles: Possible survival mechanisms during gold biogeochemical cycling.

A bacterial consortium was enriched from gold particles that 'experienced' ca. 80 years of biotransformation within waste-rock piles (Australia). This bacterial consortium was exposed to 10 µM AuCl3 to obtain Au-tolerant bacteria. From these isolates, Serratia sp. and Stenotrophomonas sp. were the most Au-tolerant and reduced soluble Au as pure gold nanoparticles, indicating that passive mineralisation is a mechanism for mediating the toxic effect of soluble Au produced during particle dissolution. Genome-wide analysis demonstrated that these isolates also possessed various genes that could provide cellular defence enabling survival under heavy-metal stressed condition by mediating the toxicity of heavy metals through active efflux/reduction. Diverse metal-resistant genes or genes clusters (cop, cus, czc, zntand ars) were detected, which could confer resistance to soluble Au. Comparative genome analysis revealed that the majority of detected heavy-metal resistant genes were similar (i.e. orthologous) to those genes of Cupriavidus metallidurans CH34. The detection of heavy-metal resistance, nutrient cycling and biofilm formation genes (pgaABCD, bsmAandhmpS) may have indirect yet important roles when dealing with soluble Au during particle dissolution. In conclusion, the physiological and genomic results suggest that bacteria living on gold particles would likely use various genes to ensure survival during Au-biogeochemical cycling.

Metal resistant bacteria on gold particles: Implications of how anthropogenic contaminants could affect natural gold biogeochemical cycling.

In Earth's near-surface environments, gold biogeochemical cycling involves gold dissolution and precipitation processes, which are partly attributed to bacteria. These biogeochemical processes as well as abrasion (via physical transport) are known to act upon gold particles, thereby resulting in particle transformation including the development of pure secondary gold and altered morphology, respectively. While previous studies have inferred gold biogeochemical cycling from gold particles obtained from natural environments, little is known about how metal contamination in an environment could impact this cycle. Therefore, this study aims to infer how potentially toxic metal contaminants could affect the structure and chemistry of gold particles and therefore the biogeochemical cycling of gold. In doing so, river sediments and gold particles from the De Kaap Valley, South Africa, were analysed using both microanalytical and molecular techniques. Of the metal contaminants detected in the sediment, mercury can chemically interact with gold particles thereby directly altering particle morphology and "erasing" textural evidence indicative of particle transformation. Other metal contaminants (including mercury) indirectly affect gold cycling by exerting a selective pressure on bacteria living on the surface of gold particles. Particles harbouring gold-tolerant bacteria with diverse metal resistant genes, such as Arthrobacter sp. and Pseudomonas sp., contained nearly two times more secondary gold relative to particles harbouring bacteria with less gold-tolerance. In conclusion, metal contaminants can have a direct or indirect effect on gold biogeochemical cycling in natural environments impacted by anthropogenic activity.

Functional capabilities of bacterial biofilms on gold particles.

Gold particles contain gold and other toxic, heavy metals, making them 'extreme' geochemical microenvironments. To date, the functional capabilities of bacterial biofilms to deal with these conditions have been inferred from taxonomic analyses. The aims of this study are to evaluate the functional capabilities of bacterial communities on gold particles from six key locations using GeoChip 5.0 and to link functional and taxonomic data. Biofilm communities displayed a wide range of functional capabilities, with up to 53 505 gene probes detected. The capability of bacterial communities to (re)cycle carbon, nitrogen, and sulphur were detected. The cycling of major nutrients is important for maintaining the biofilm community as well as enabling the biogeochemical cycling and mobilisation of heavy and noble metals. Additionally, a multitude of stress- and heavy metal resistance capabilities were also detected, most notably from the α/ß/γ-Proteobacteria and Actinobacteria. The multi-copper-oxidase gene copA, which is directly involved in gold resistance and biomineralisation, was the 15th most intense response and was detected in 246 genera. The Parker Road and Belle Brooke sites were consistently the most different from other sites, which may be a result of local physicochemical conditions (extreme nutrient poverty and sulphur-richness, respectively). In conclusion, biofilms on gold particles display wide-ranging metabolic and stress-related capabilities, which may enable them to survive in these niche environments and drive biotransformation of gold particles.

Addressing challenges in providing a reliable ecotoxicology data for graphene-oxide (GO) using an algae (Raphidocelis subcapitata), and the trophic transfer consequence of GO-algae aggregates.

The graphene oxide (GO) due to its exceptional structure, physicochemical and mechanical properties is a very attractive material for industry application. Even though, the unique properties of GO (e.g. structure, size, shape, etc.) make the risk assessment of this nanomaterial very challenging in comparison with conventional ecotoxicology studies required by regulators. Thus, there is a need for standardized characterization techniques and methodology to secure a high quality/reliable data on the ecotoxicology of GO, and to establish environmentally acceptable levels. Herein, authors address the crucial quality criteria when evaluating the ecotoxicology of GO using an algae (Raphidocelis subcapitata) and a shrimp (Paratya australiensis). This study provides a detail characterization and modification of the used GO, robust quantification and a suspension stability in different media for ecotoxicology studies. It was observed that under the same exposure conditions the behavior of GO and the estimated outcomes (IC50 values) in modified algae media differed in comparison to the referent media. Further to that, the adverse effects of GO on the algae cell structure and the potential uptake of GO by the algae cells were examined using the TEM with different staining techniques to avoid artefacts. Shrimps which were exposed to GO-algae aggregates via the food intake did not indicate stress or accumulation of GO. Our work presents an important insight to necessity of establishing a benchmark ecotoxicology assays for GO (e.g. characterization techniques, choice of media, etc.) and providing a reliable data to be used by regulators in risk assessment of two-dimensional (2D) nanomaterials.

Biogeochemical gold cycling selects metal-resistant bacteria that promote gold particle transformation.

Bacteria catalyze the dissolution and re-precipitation of gold, thereby driving the biogeochemical cycle of gold. Dissolution of gold/silver and re-precipitation of gold transforms gold particles by increasing gold purity. While soluble gold complexes are highly cytotoxic, little is known about how gold cycling affects bacterial communities residing on gold particles. Micro-analysis of gold particles obtained from Western Australia revealed porous textures and aggregates of pure gold nanoparticles, attributable to gold dissolution and re-precipitation, respectively. By interpreting structure and chemistry of particles, the kinetics of gold biogeochemical cycling at the site was estimated to be 1.60 × 10-9 M year-1. Bacterial communities residing on particles were composed of Proteobacteria (42.5%), Bacteroidetes (20.1%), Acidobacteria (19.1%), Firmicutes (8.2%), Actinobacteria (3.7%) and Verrucomicrobia (3.6%). A bacterial enrichment culture obtained from particles contained a similar composition. Exposure of enrichments to increasing concentrations of soluble gold decreased community diversity and selected for metal-resistant bacteria. Lower gold concentrations, which corresponded well with the concentration from the kinetic rate, provided a selective pressure for the selection of metal-resistant organisms while retaining the overall diversity. In conclusion, biogeochemical gold cycling directly influences bacterial communities on gold particles, thereby contributing to a continuum of particle transformation.

Carbon Sequestration in Biogenic Magnesite and Other Magnesium Carbonate Minerals.

The stability and longevity of carbonate minerals make them an ideal sink for surplus atmospheric carbon dioxide. Biogenic magnesium carbonate mineral precipitation from the magnesium-rich tailings generated by many mining operations could offset net mining greenhouse gas emissions, while simultaneously giving value to mine waste products. In this investigation, cyanobacteria in a wetland bioreactor enabled the precipitation of magnesite (MgCO3), hydromagnesite [Mg5(CO3)4(OH)2·4H2O], and dypingite [Mg5(CO3)4(OH)2·5H2O] from a synthetic wastewater comparable in chemistry to what is produced by acid leaching of ultramafic mine tailings. These precipitates occurred as micrometer-scale mineral grains and microcrystalline carbonate coatings that entombed filamentous cyanobacteria. This provides the first laboratory demonstration of low temperature, biogenic magnesite precipitation for carbon sequestration purposes. These findings demonstrate the importance of extracellular polymeric substances in microbially enabled carbonate mineral nucleation. Fluid composition was monitored to determine carbon sequestration rates. The results demonstrate that up to 238 t of CO2 could be stored per hectare of wetland/year if this method of carbon dioxide sequestration was implemented at an ultramafic mine tailing storage facility. The abundance of tailings available for carbonation and the anticipated global implementation of carbon pricing make this method of mineral carbonation worth further investigation.

Microbial Diversity in Actively Forming Iron Oxides from Weathered Banded Iron Formation Systems.

The surface crust that caps highly weathered banded iron formations (BIFs) supports a unique ecosystem that is a post-mining restoration priority in iron ore areas. Geochemical evidence indicates that biological processes drive the dissolution of iron oxide minerals and contribute to the ongoing evolution of this duricrust. However, limited information is available on present-day biogeochemical processes in these systems, particularly those that contribute to the precipitation of iron oxides and, thus, the cementation and stabilization of duricrusts. Freshly formed iron precipitates in water bodies perched on cangas in Karijini National Park, Western Australia, were sampled for microscopic and molecular analyses to understand currently active microbial contributions to iron precipitation in these areas. Microscopy revealed sheaths and stalks associated with iron-oxidizing bacteria. The iron-oxidizing lineages Sphaerotilus, Sideroxydans, and Pedomicrobium were identified in various samples and Leptothrix was common in four out of five samples. The iron-reducing bacteria Anaeromyxobacter dehalogens and Geobacter lovleyi were identified in the same four samples, with various heterotrophs and diverse cyanobacteria. Given this arid, deeply weathered environment, the driver of contemporary iron cycling in Karijini National Park appears to be iron-reducing bacteria, which may exist in anaerobic niches through associations with aerobic heterotrophs. Overall oxidizing conditions and Leptothrix iron-oxidizers contribute to net iron oxide precipitation in our sampes, rather than a closed biogeochemical cycle, which would result in net iron oxide dissolution as has been suggested for canga caves in Brazil. Enhancements in microbial iron oxide dissolution and subsequent reprecipitation have potential as a surface-crust-ecosystem remediation strategy at mine sites.

Progressive biogeochemical transformation of placer gold particles drives compositional changes in associated biofilm communities.

Biofilms on placer gold (Au)-particle surfaces drive Au solubilization and re-concentration thereby progressively transforming the particles. Gold solubilization induces Au-toxicity; however, Au-detoxifying community members ameliorates Au-toxicity by precipitating soluble Au to metallic Au. We hypothesize that Au-dissolution and re-concentration (precipitation) place selective pressures on associated microbial communities, leading to compositional changes and subsequent Au-particle transformation. We analyzed Au-particles from eight United Kingdom sites using next generation sequencing, electron microscopy and micro-analyses. Gold particles contained biofilms composed of prokaryotic cells and extracellular polymeric substances intermixed with (bio)minerals. Across all sites communities were dominated by Proteobacteria (689, 97% Operational Taxonomic Units, 59.3% of total reads), with ß-Proteobacteria being the most abundant. A wide range of Au-morphotypes including nanoparticles, micro-crystals, sheet-like Au and secondary rims, indicated that dissolution and re-precipitation occurred, and from this transformation indices were calculated. Multivariate statistical analyses showed a significant relationship between the extent of Au-particle transformation and biofilm community composition, with putative metal-resistant Au-cycling taxa linked to progressive Au transformation. These included the genera Pseudomonas, Leptothrix and Acinetobacter. Additionally, putative exoelectrogenic genera Rhodoferax and Geobacter were highly abundant. In conclusion, biogeochemical Au-cycling and Au-particle transformation occurred at all sites and exerted a strong influence on biofilm community composition.

Waveguide evanescent field scattering microscopy: bacterial biofilms and their sterilization response via UV irradiation.

Waveguide Evanescent Field Scattering (WEFS) microscopy is introduced as a new and simple tool for label-free, high contrast imaging of bacteria and bacteria sensors. Bacterial microcolonies and single bacteria were discriminated both by their bright field images and by their evanescent scattering intensity. By comparing bright field images with WEFS images, the proportion of planktonic: sessile (i.e., "floating": attached) bacteria were measured. Bacteria were irradiated with UV light, which limited their biofilm forming capability. A quantitative decrease in attachment of individual, sessile bacteria and in attached, microcolony occupied areas was easily determined within the apparent biofilms with increasing UV dose. WEFS microscopy is an ideal tool for providing rapid quantitative data on biofilm formation.

Gold biomineralization by a metallophore from a gold-associated microbe.

Microorganisms produce and secrete secondary metabolites to assist in their survival. We report that the gold resident bacterium Delftia acidovorans produces a secondary metabolite that protects from soluble gold through the generation of solid gold forms. This finding is the first demonstration that a secreted metabolite can protect against toxic gold and cause gold biomineralization.