Carla M. Zammit

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 biofilms on gold grains-implications for geomicrobial transformations of gold.

The biogeochemical cycling of gold (Au), i.e. its solubilization, transport and re-precipitation, leading to the (trans)formation of Au grains and nuggets has been demonstrated under a range of environmental conditions. Biogenic (trans)formations of Au grains are driven by (geo)biochemical processes mediated by distinct biofilm consortia living on these grains. This review summarizes the current knowledge concerning the composition and functional capabilities of Au-grain communities, and identifies contributions of key-species involved in Au-cycling. To date, community data are available from grains collected at 10 sites in Australia, New Zealand and South America. The majority of detected operational taxonomic units detected belong to the α-, β- and γ-Proteobacteria and the Actinobacteria. A range of organisms appears to contribute predominantly to biofilm establishment and nutrient cycling, some affect the mobilization of Au via excretion of Au-complexing ligands, e.g. organic acids, thiosulfate and cyanide, while a range of resident Proteobacteria, especially Cupriavidus metallidurans and Delftia acidovorans, have developed Au-specific biochemical responses to deal with Au-toxicity and reductively precipitate mobile Au-complexes. This leads to the biomineralization of secondary Au and drives the environmental cycle of Au.

The characterization of salt tolerance in biomining microorganisms and the search for novel salt tolerant strains

In this study an acidic saline drain in the Western Australian wheat belt was sampled and enriched for salt tolerant chemolithotrophic microorganisms in acidic media containing up to 100 gL-1 NaCl. A mixed consortium was obtained which grows at pH 1.8 and oxidises iron (II) in the presence of up to 30 gL-1 NaCl. In comparative tests (growth rates and iron (II) oxidation rates) it was found that NaCl concentrations >3.5 gL-1 generally cause reduced growth and iron (II) oxidation rates in known biomining organisms. The results help to set a benchmark for NaCl tolerance in known biomining microorganisms and will lead to the generation of a consortium of microorganisms that can oxidise iron (II) effectively in saline process water.

GEOGENIC FACTORS AS DRIVERS OF MICROBIAL COMMUNITY DIVERSITY IN SOILS OVERLYING POLYMETALLIC DEPOSITS

ABSTRACT This study shows that the geogenic factors landform, lithology, and underlying mineral deposits (expressed by elevated metal concentrations in overlying soils) are key drivers of microbial community diversity in naturally metal-rich Australian soils with different land uses, i.e., agriculture versus natural bushland. One hundred sixty-eight soil samples were obtained from two metal-rich provinces in Australia, i.e., the Fifield Au-Pt field (New South Wales) and the Hillside Cu-Au-U rare-earth-element (REE) deposit (South Australia). Soils were analyzed using three-domain multiplex terminal-restriction-fragment-length-polymorphism (M-TRFLP) and PhyloChip microarrays. Geogenic factors were determined using field-mapping techniques and analyses of >50 geochemical parameters. At Fifield, microbial communities differed significantly with geogenic factors and equally with land use (P < 0.05). At Hillside, communities in surface soils (0.03- to 0.2-m depth) differed significantly with landform and land use (P < 0.05). Communities in deeper soils (>0.2 m) differed significantly with lithology and mineral deposit (P < 0.05). Across both sites, elevated metal contents in soils overlying mineral deposits were selective for a range of bacterial taxa, most importantly Acidobacteria, Bacilli, Betaproteobacteria, and Epsilonproteobacteria. In conclusion, long-term geogenic factors can be just as important as land use in determining soil microbial community diversity.

Adaptation to Extreme Acidity and Osmotic Stress

Environments that are either acidic or have high osmotic potentials are found across the globe in a range of natural and anthropogenic systems. The organisms capable of inhabiting these systems are diverse, including archaea, bacteria and eukaryotes. However, environments where extreme acidity is combined with osmotic stress deriving from elevated concentrations of sodium chloride are seemingly rare. Subsequently, there is a relatively small number of species which have been identified and shown to tolerate both of these stresses simultaneously, and as a result the mechanisms that permit life in these harsh conditions has not been extensively studied. Recent genomic and proteomic studies indicate that several strategies may be employed by acidophilic microorganisms to combat the combined effects of low pH and high osmotic stress, most notably the production of osmo-protectants and the maintenance of membrane integrity. This chapter focuses on iron- and sulfur-oxidising microorganisms, which are able to tolerate acidic conditions, the effect of osmotic stress induced by salinity on their survival, and mechanisms used to survive these stresses both independently and in combination.

Proteomic responses to gold(III)-toxicity in the bacterium Cupriavidus metallidurans CH34

The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(i/iii)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(iii)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(iii)-chloride, 50 μM Cu(ii)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(iii)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(iii) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration samples.

High resolution two‐dimensional electrophoresis of native proteins

Blue native PAGE (BN‐PAGE) is a powerful method to separate protein complexes while preserving their native state. However, the resolution of the method is limited as complexes with similar molecular masses cannot be resolved. Here we describe native 2DE using immobilized pH‐gradients in combination with BN‐PAGE to resolve protein complexes by their pI and molecular mass. This method enables electrophoretic separation of proteins between pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spots at large molecular weight were identified using MS to confirm potential protein complexes. Several protein complexes could be identified, most prominent GroEL in complex with GroES, parts of the ribosomal machinery and membrane transport system. In summary, this method enables easy high‐resolution electrophoretic separation of protein complexes.

A Whole-Cell Biosensor for the Detection of Gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g−1 or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).

Nucleic acid extraction from biomining microorganisms

Various methods of nucleic acid (NA) extraction were investigated with the aim of developing a quantitative method of NA extraction from five representative strains of biomining microorganisms. The process of removing cells from mineral surfaces, lysing microorganisms, precipitating NA and purifying RNA were analysed. The success of each method was examined spectrophotometrically, by agarose gel electrophoresis and PCR or quantitative real time PCR (qPCR). The most important step was shown to be cellular lysis, which principally impacted on the quantity of NA extracted from each strain. The quantity and quality of extracted NA was highly dependent on the method of NA precipitation. This study resulted in the development of a NA extraction method that reliably and reproducibly extracted NA from five strains of biomining microorganisms.