Frank Reith

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans

While the role of microorganisms as main drivers of metal mobility and mineral formation under Earth surface conditions is now widely accepted, the formation of secondary gold (Au) is commonly attributed to abiotic processes. Here we report that the biomineralization of Au nanoparticles in the metallophillic bacterium Cupriavidus metallidurans CH34 is the result of Au-regulated gene expression leading to the energy-dependent reductive precipitation of toxic Au(III)-complexes. C. metallidurans, which forms biofilms on Au grains, rapidly accumulates Au(III)-complexes from solution. Bulk and microbeam synchrotron X-ray analyses revealed that cellular Au accumulation is coupled to the formation of Au(I)-S complexes. This process promotes Au toxicity and C. metallidurans reacts by inducing oxidative stress and metal resistances gene clusters (including a Au-specific operon) to promote cellular defense. As a result, Au detoxification is mediated by a combination of efflux, reduction, and possibly methylation of Au-complexes, leading to the formation of Au(I)-C-compounds and nanoparticulate Au0. Similar particles were observed in bacterial biofilms on Au grains, suggesting that bacteria actively contribute to the formation of Au grains in surface environments. The recognition of specific genetic responses to Au opens the way for the development of bioexploration and bioprocessing tools.

The geomicrobiology of gold

Microorganisms capable of actively solubilizing and precipitating gold appear to play a larger role in the biogeochemical cycling of gold than previously believed. Recent research suggests that bacteria and archaea are involved in every step of the biogeochemical cycle of gold, from the formation of primary mineralization in hydrothermal and deep subsurface systems to its solubilization, dispersion and re-concentration as secondary gold under surface conditions. Enzymatically catalysed precipitation of gold has been observed in thermophilic and hyperthermophilic bacteria and archaea (for example, Thermotoga maritime, Pyrobaculum islandicum), and their activity led to the formation of gold- and silver-bearing sinters in New Zealands hot spring systems. Sulphate-reducing bacteria (SRB), for example, Desulfovibrio sp., may be involved in the formation of gold-bearing sulphide minerals in deep subsurface environments; over geological timescales this may contribute to the formation of economic deposits. Iron- and sulphur-oxidizing bacteria (for example, Acidothiobacillus ferrooxidans, A. thiooxidans) are known to breakdown gold-hosting sulphide minerals in zones of primary mineralization, and release associated gold in the process. These and other bacteria (for example, actinobacteria) produce thiosulphate, which is known to oxidize gold and form stable, transportable complexes. Other microbial processes, for example, excretion of amino acids and cyanide, may control gold solubilization in auriferous top- and rhizosphere soils. A number of bacteria and archaea are capable of actively catalysing the precipitation of toxic gold(I/III) complexes. Reductive precipitation of these complexes may improve survival rates of bacterial populations that are capable of (1) detoxifying the immediate cell environment by detecting, excreting and reducing gold complexes, possibly using P-type ATPase efflux pumps as well as membrane vesicles (for example, Salmonella enterica, Cupriavidus (Ralstonia) metallidurans, Plectonema boryanum); (2) gaining metabolic energy by utilizing gold-complexing ligands (for example, thiosulphate by A. ferrooxidans) or (3) using gold as metal centre in enzymes (Micrococcus luteus). C. metallidurans containing biofilms were detected on gold grains from two Australian sites, indicating that gold bioaccumulation may lead to gold biomineralization by forming secondary ‘bacterioform’ gold. Formation of secondary octahedral gold crystals from gold(III) chloride solution, was promoted by a cyanobacterium (P. boryanum) via an amorphous gold(I) sulphide intermediate. ‘Bacterioform’ gold and secondary gold crystals are common in quartz pebble conglomerates (QPC), where they are often associated with bituminous organic matter possibly derived from cyanobacteria. This may suggest that cyanobacteria have played a role in the formation of the Witwatersrand QPC, the worlds largest gold deposit.

Nanoparticle factories: Biofilms hold the key to gold dispersion and nugget formation

Biofilms living on gold (Au) grains play a key role in the biogeochemical cycle of Au by promoting the dispersion of Au via the formation of Au nanoparticles as well as the formation of secondary biomorphic Au. Gold grains from Queensland, Australia, are covered by a polymorphic, organic-inorganic layer that is up to 40 μm thick. It consists of a bacterial biofilm containing Au nanoparticles associated with extracellular polymeric substances as well as bacterioform Au. Focused ion beam (FIB) sectioning through the biofilm revealed that aggregates of nanoparticulate Au line open spaces beneath the active biofilm layer. These aggregates (bacterioform Au type 1) resulted from the reprecipitation of dissolved Au, and their internal growth structures provide direct evidence for coarsening of the Au grains. At the contact between the polymorphic layer and the primary Au, bacterioform Au type 2 is present. It consists of solid rounded forms into which crystal boundaries of underlying primary Au extend, and is the result of dealloying and Ag dissolution from the primary Au. This study demonstrates that (1) microbially driven dissolution, precipitation, and aggregation lead to the formation of bacterioform Au and contribute to the growth of Au grains under supergene conditions, and (2) the microbially driven mobilization of coarse Au into nanoparticles plays a key role in mediating the mobility of Au in surface environments, because the release of nanoparticulate Au upon biofilm disintegration greatly enhances environmental mobility compared to Au complexes only.

Anaerobic activities of bacteria and fungi in moderately acidic conifer and deciduous leaf litter.

Abstract The litter layer of forest soils harbors high amounts of labile organic matter, and anaerobic decomposition processes can be initiated when oxygen is consumed more rapidly than it is supplied by diffusion. In this study, two adjacent moderately acidic forest sites, a spruce and a beech-oak forest, were selected to compare the anaerobic bacterial and fungal activities and populations of conifer and deciduous leaf litter. Most probable number (MPN) estimates of general heterotrophic aerobes and anaerobes from conifer litter equaled those from deciduous leaf litter. H(2), ethanol, formate, and lactate were initially produced with similar rates in both anoxic conifer and deciduous leaf litter microcosms. These products were rapidly consumed in deciduous leaf but not in conifer litter microcosms. Supplemental ethanol and H(2) were consumed only by deciduous leaf litter and yielded additional amounts of acetate in stoichiometries indicative of ethanol- or H(2)-dependent acetogenesis. The negligible turnover of primary fermentation products in conifer litter might be due to the low numbers of acetogens and secondary fermenters present in conifer litter compared to deciduous leaf litter. Fungi capable of anaerobic growth made up only 0.01-0.1% of the total anaerobic microorganisms cultured from conifer and deciduous leaf litter, respectively. Metabolic product profiles obtained from the highest anoxic, growth-positive MPN dilutions supplemented with antibacterial agents indicated that the dominant population of fungi, apparently mainly yeast-like cells, produced H(2), ethanol, acetate, and lactate both in conifer and deciduous leaf litter. Thus, despite acidic conditions, bacteria appear to dominate in the decomposition of carbon in anoxic microsites of both conifer and deciduous leaf litter.

Influence of geogenic factors on microbial communities in metallogenic Australian soils

Links between microbial community assemblages and geogenic factors were assessed in 187 soil samples collected from four metal-rich provinces across Australia. Field-fresh soils and soils incubated with soluble Au(III) complexes were analysed using three-domain multiplex-terminal restriction fragment length polymorphism, and phylogenetic (PhyloChip) and functional (GeoChip) microarrays. Geogenic factors of soils were determined using lithological-, geomorphological- and soil-mapping combined with analyses of 51 geochemical parameters. Microbial communities differed significantly between landforms, soil horizons, lithologies and also with the occurrence of underlying Au deposits. The strongest responses to these factors, and to amendment with soluble Au(III) complexes, was observed in bacterial communities. PhyloChip analyses revealed a greater abundance and diversity of Alphaproteobacteria (especially Sphingomonas spp.), and Firmicutes (Bacillus spp.) in Au-containing and Au(III)-amended soils. Analyses of potential function (GeoChip) revealed higher abundances of metal-resistance genes in metal-rich soils. For example, genes that hybridised with metal-resistance genes copA, chrA and czcA of a prevalent aurophillic bacterium, Cupriavidus metallidurans CH34, occurred only in auriferous soils. These data help establish key links between geogenic factors and the phylogeny and function within soil microbial communities. In particular, the landform, which is a crucial factor in determining soil geochemistry, strongly affected microbial community structures.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database

BackgroundMicrobial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The ‘Biomes of Australian Soil Environments’ (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function.FindingsBASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project’s data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the ‘Atlas of Living Australia’.ConclusionsDeveloped within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services.

Bacterial communities associated with a mineral weathering profile at a sulphidic mine tailings dump in arid Western Australia.

We investigated bacterial community assemblages and functions down a hill slope contaminated by tailings from a volcanogenic massive sulphide mine in arid Western Australia. Weathering of waste rock, high in S and Fe, had resulted in a varying elemental dispersal down a face of the tailings hill. Bacterial community assemblage, characterised by PCR-DGGE fingerprinting, was significantly associated with electrical conductivity (E.C.) (ρ = 0.664; P < 0.01). Analysis of mobile salts showed that E.C. values were driven by ionic S, Zn, Cl and Al. The bacterial community assemblage was directly characterised across an E.C. gradient using an oligonucleotide microarray (PhyloChip). The dominant taxa at the site were Proteobacteria, Actinobacteria and Firmicutes; however, 37 phyla were detected. The most responsive taxa to variation in E.C. was Acidobacteria (negative correlation). Patterns of heterotrophic processes (BioLog analysis) were also best explained by variation in E.C. (ρ = 0.53; P < 0.01), showing a link between primary mineral weathering by lithotrophic bacteria and abiotic processes, and secondary biogeochemical processes by heterotrophic taxa. These data significantly broaden our knowledge of the bacteria present in metallomorphic ecosystems, establish that mobile phase elements are key drivers of community structure, and that primary biogeochemical cycling is directly influencing other geochemical interactions in the samples.

Biomineralization of gold in biofilms of Cupriavidus metallidurans.

Cupriavidus metallidurans, a bacterium capable of reductively precipitating toxic, aqueous gold(I/III)-complexes, dominates biofilm communities on gold (Au) grains from Australia. To examine the importance of C. metallidurans biofilms in secondary Au formation, we assessed the biomineralization potential of biofilms growing in quartz-sand-packed columns to periodic amendment with Au(I)-thiosulfate. In these experiments, >99 wt % of Au, was retained compared to <30 wt % in sterilized and abiotic controls. Biomineralization of Au occurred in the presence of viable biofilms via the formation of intra- and extra-cellular spherical nanoparticles, which aggregated into spheroidal and framboidal microparticles of up to 2 μm in diameter. Aggregates of Au formed around cells, eventually encapsulating and ultimately replacing them. These particles were morphologically analogous to Au-particles commonly observed on natural Au grains. Bacterial cells were connected via exopolymer or nanowires to μm-sized, extracellular Au-aggregates, which would intuitively improve the flow of electrons through the biofilm. This study demonstrates the importance of C. metallidurans biofilms for the detoxification of Au-complexes and demonstrates a central role for bacterial biomineralization in the formation of highly pure Au in surface environments.

Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34

Cupriavidus metallidurans is associated with gold grains and may be involved in their formation. Gold(III) complexes influence the transcriptome of C. metallidurans (F. Reith et al., Proc. Natl. Acad. Sci. U. S. A. 106:17757-17762, 2009), leading to the upregulation of genes involved in the detoxification of reactive oxygen species and metal ions. In a systematic study, the involvement of these systems in gold transformation was investigated. Treatment of C. metallidurans cells with Au(I) complexes, which occur in this organisms natural environment, led to the upregulation of genes similar to those observed for treatment with Au(III) complexes. The two indigenous plasmids of C. metallidurans, which harbor several transition metal resistance determinants, were not involved in resistance to Au(I/III) complexes nor in their transformation to metallic nanoparticles. Upregulation of a cupA-lacZ fusion by the MerR-type regulator CupR with increasing Au(III) concentrations indicated the presence of gold ions in the cytoplasm. A hypothesis stating that the Gig system detoxifies gold complexes by the uptake and reduction of Au(III) to Au(I) or Au(0) reminiscent to detoxification of Hg(II) was disproven. ZupT and other secondary uptake systems for transition metal cations influenced Au(III) resistance but not the upregulation of the cupA-lacZ fusion. The two copper-exporting P-type ATPases CupA and CopF were also not essential for gold resistance. The copABCD determinant on chromosome 2, which encodes periplasmic proteins involved in copper resistance, was required for full gold resistance in C. metallidurans. In conclusion, biomineralization of gold particles via the reduction of mobile Au(I/III) complexes in C. metallidurans appears to primarily occur in the periplasmic space via copper-handling systems.

Analysis of gold(I/III)-complexes by HPLC-ICP-MS demonstrates gold(III) stability in surface waters.

Understanding the form in which gold is transported in surface- and groundwaters underpins our understanding of gold dispersion and (bio)geochemical cycling. Yet, to date, there are no direct techniques capable of identifying the oxidation state and complexation of gold in natural waters. We present a reversed phase ion-pairing HPLC-ICP-MS method for the separation and determination of aqueous gold(III)-chloro-hydroxyl, gold(III)-bromo-hydroxyl, gold(I)-thiosulfate, and gold(I)-cyanide complexes. Detection limits for the gold species range from 0.05 to 0.30 μg L(-1). The [Au(CN)2](-) gold cyanide complex was detected in five of six waters from tailings and adjacent monitoring bores of working gold mines. Contrary to thermodynamic predictions, evidence was obtained for the existence of Au(III)-complexes in circumneutral, hypersaline waters of a natural lake overlying a gold deposit in Western Australia. This first direct evidence for the existence and stability of Au(III)-complexes in natural surface waters suggests that Au(III)-complexes may be important for the transport and biogeochemical cycling of gold in surface environments. Overall, these results show that near-μg L(-1) enrichments of Au in environmental waters result from metastable ligands (e.g., CN(-)) as well as kinetically controlled redox processes leading to the stability of highly soluble Au(III)-complexes.