Analysis of element yield, bacterial community structure and the impact of carbon sources for bioleaching rare earth elements from high grade monazite.

Rare earth element (REE) recovery from waste streams, mine tailings or recyclable components using bioleaching is gaining traction due to the shortage and security of REE supply as well as the environmental problems that occur from processing and refining. Four heterotrophic microbial species with known phosphate solubilizing capabilities were evaluated for their ability to leach REE from a high-grade monazite when provided with either galactose, fructose or maltose. Supplying fructose resulted in the greatest amount of REE leached from the ore due to the largest amount of organic acid produced. Gluconic acid was the dominant organic acid identified produced by the cultures, followed by acetic acid. The monazite proved difficult to leach with the different carbon sources, with preferential release of Ce over La, Nd and Pr.

Structure illumination microscopy imaging of lipid vesicles in live bacteria with naphthalimide-appended organometallic complexes.

There is a lack of molecular probes for imaging bacteria, in comparison to the array of such tools available for the imaging of mammalian cells. Here, organometallic molecular probes have been developed and assessed for bacterial imaging, designed to have the potential to support multiple imaging modalities. The chemical structure of the probes is designed around a metal-naphthalimide structure. The 4-amino-1,8-naphthalimide moiety, covalently appended through a pyridine ancillary ligand, acts as a luminescent probe for super-resolution microscopy. On the other hand, the metal centre, rhenium(i) or platinum(ii) in the current study, enables techniques such as nanoSIMS. While the rhenium(i) complex was not sufficiently stable to be used as a probe, the platinum(ii) analogue showed good chemical and biological stability. Structured illumination microscopy (SIM) imaging on live Bacillus cereus confirmed the suitability of the probe for super-resolution microscopy. NanoSIMS analysis was used to monitor the uptake of the platinum(ii) complex within the bacteria and demonstrate the potential of this chemical architecture to enable multimodal imaging. The successful combination of these two moieties introduces a platform that could lead to a versatile range of multi-functional probes for bacteria.

Examining the Osmotic Response of Acidihalobacter aeolianus after Exposure to Salt Stress.

Acidihalobacter aeolianus is an acidophilic, halo-tolerant organism isolated from a marine environment near a hydrothermal vent, an ecosystem whereby levels of salinity and total dissolved salts are constantly fluctuating creating ongoing cellular stresses. In order to survive these continuing changes, the synthesis of compatible solutes-also known as organic osmolytes-is suspected to occur, aiding in minimising the overall impact of environmental instability. Previous studies on A. aeolianus identified genes necessary for the accumulation of proline, betaine and ectoine, which are known to act as compatible solutes in other halophilic species. In this study, the impact of increasing the osmotic stress as well as the toxic ion effect was investigated by subjecting A. aeolianus to concentrations of NaCl and MgSO4 up to 1.27 M. Exposure to high concentrations of Cl- resulted in the increase of ectC expression in log-phase cells with a corresponding accumulation of ectoine at stationary phase. Osmotic stress via MgSO4 exposure did not trigger the same up-regulation of ectC or accumulation of ectoine, indicating the transcriptionally regulated response against osmotic stress was induced by chloride toxicity. These findings have highlighted how the adaptive properties of halo-tolerant organisms in acidic environments are likely to differ and are dependent on the initial stressor.

Unlocking Survival Mechanisms for Metal and Oxidative Stress in the Extremely Acidophilic, Halotolerant Acidihalobacter Genus.

Microorganisms used for the biohydrometallurgical extraction of metals from minerals must be able to survive high levels of metal and oxidative stress found in bioleaching environments. The Acidihalobacter genus consists of four species of halotolerant, iron-sulfur-oxidizing acidophiles that are unique in their ability to tolerate chloride and acid stress while simultaneously bioleaching minerals. This paper uses bioinformatic tools to predict the genes and mechanisms used by Acidihalobacter members in their defense against a wide range of metals and oxidative stress. Analysis revealed the presence of multiple conserved mechanisms of metal tolerance. Ac. yilgarnensis F5T, the only member of this genus that oxidizes the mineral chalcopyrite, contained a 39.9 Kb gene cluster consisting of 40 genes encoding mobile elements and an array of proteins with direct functions in copper resistance. The analysis also revealed multiple strategies that the Acidihalobacter members can use to tolerate high levels of oxidative stress. Three of the Acidihalobacter genomes were found to contain genes encoding catalases, which are not common to acidophilic microorganisms. Of particular interest was a rubrerythrin genomic cluster containing genes that have a polyphyletic origin of stress-related functions.

Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov.

The genus Acidihalobacter has three validated species, Acidihalobacter ferrooxydans, Acidihalobacter prosperus and Acidihalobacter aeolinanus, all of which were isolated from Vulcano island, Italy. They are obligately chemolithotrophic, aerobic, acidophilic and halophilic in nature and use either ferrous iron or reduced sulphur as electron donors. Recently, a novel strain was isolated from an acidic, saline drain in the Yilgarn region of Western Australia. Strain F5T has an absolute requirement for sodium chloride (>5 mM) and is osmophilic, growing in elevated concentrations (>1 M) of magnesium sulphate. A defining feature of its physiology is its ability to catalyse the oxidative dissolution of the most abundant copper mineral, chalcopyrite, suggesting a potential role in biomining. Originally categorized as a strain of A. prosperus, 16S rRNA gene phylogeny and multiprotein phylogenies derived from clusters of orthologous proteins (COGS) of ribosomal protein families and universal protein families unambiguously demonstrate that strain F5T forms a well-supported separate branch as a sister clade to A. prosperus and is clearly distinguishable from A. ferrooxydans DSM 14175T and A. aeolinanus DSM14174T. Results of comparisons between strain F5T and the other Acidihalobacter species, using genome-based average nucleotide identity, average amino acid identity, correlation indices of tetra-nucleotide signatures (Tetra) and genome-to-genome distance (digital DNA-DNA hybridization), support the contention that strain F5T represents a novel species of the genus Acidihalobacter. It is proposed that strain F5T should be formally reclassified as Acidihalobacter yilgarnenesis F5T (=DSM 105917T=JCM 32255T).

Uncovering the Mechanisms of Halotolerance in the Extremely Acidophilic Members of the Acidihalobacter Genus Through Comparative Genome Analysis.

There are few naturally occurring environments where both acid and salinity stress exist together, consequently, there has been little evolutionary pressure for microorganisms to develop systems that enable them to deal with both stresses simultaneously. Members of the genus Acidihalobacter are iron- and sulfur-oxidizing, halotolerant acidophiles that have developed the ability to tolerate acid and saline stress and, therefore, have the potential to bioleach ores with brackish or saline process waters under acidic conditions. The genus consists of four members, A. prosperus DSM 5130T, A. prosperus DSM 14174, A. prosperus F5 and "A. ferrooxidans" DSM 14175. An in depth genome comparison was undertaken in order to provide a more comprehensive description of the mechanisms of halotolerance used by the different members of this genus. Pangenome analysis identified 29, 3 and 9 protein families related to halotolerance in the core, dispensable and unique genomes, respectively. The genes for halotolerance showed Ka/Ks ratios between 0 and 0.2, confirming that they are conserved and stabilized. All the Acidihalobacter genomes contained similar genes for the synthesis and transport of ectoine, which was recently found to be the dominant osmoprotectant in A. prosperus DSM 14174 and A. prosperus DSM 5130T. Similarities also existed in genes encoding low affinity potassium pumps, however, A. prosperus DSM 14174 was also found to contain genes encoding high affinity potassium pumps. Furthermore, only A. prosperus DSM 5130T and "A. ferrooxidans" DSM 14175 contained genes allowing the uptake of taurine as an osmoprotectant. Variations were also seen in genes encoding proteins involved in the synthesis and/or transport of periplasmic glucans, sucrose, proline, and glycine betaine. This suggests that versatility exists in the Acidihalobacter genus in terms of the mechanisms they can use for halotolerance. This information is useful for developing hypotheses for the search for life on exoplanets and moons.

Genome-based classification of two halotolerant extreme acidophiles, Acidihalobacter prosperus V6 (=DSM 14174 =JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175 =JCM 32254) as two new species, Acidihalobacter aeolianus sp. nov. and Acidihalobacter ferrooxydans sp. nov., respectively.

Phylogenomic analysis of recently released high-quality draft genome sequences of the halotolerant acidophiles, Acidihalobacter prosperus V6 (=DSM 14174=JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175=JCM 32254), was undertaken in order to clarify their taxonomic relationship. Sequence based phylogenomic approaches included 16S rRNA gene phylogeny, multi-gene phylogeny from the concatenated alignment of nine selected housekeeping genes and multiprotein phylogeny using clusters of orthologous groups of proteins from ribosomal protein families as well as those from complete sets of markers based on concatenated alignments of universal protein families. Non-sequence based approaches for species circumscription were based on analyses of average nucleotide identity, which was further reinforced by the correlation indices of tetra-nucleotide signatures as well as genome-to-genome distance (digital DNA-DNA hybridization) calculations. The different approaches undertaken in this study for species tree reconstruction resulted in a tree that was phylogenetically congruent, revealing that both micro-organisms are members of separate species of the genus Acidihalobacter. In accordance, it is proposed that A. prosperus V6T (=DSM 14174 T=JCM 32253 T) be formally classified as Acidihalobacter aeolianus sp. nov., and 'Acidihalobacter ferrooxidans' V8T (=DSM 14175 T=JCM 32254 T) as Acidihalobacter ferrooxydans sp. nov., and that both represent the type strains of their respective species.

Role of microorganisms in bioleaching of rare earth elements from primary and secondary resources.

In an era of environmental degradation, and water, and mineral scarcity, enhancing microbial function in sustainable mining has become a prerequisite for the future of the green economy. In recent years, the extensive use of rare earth elements (REEs) in green and smart technologies has led to an increase in the focus on recovery and separation of REEs from ore matrices. However, the recovery of REEs using traditional methods is complex and energy intensive, leading to the requirement to develop processes which are more economically feasible and environmentally friendly. The use of phosphate solubilizing microorganisms for bioleaching of REEs provides a biotechnical approach for the recovery of REEs from primary and secondary sources. However, managing and understanding the microbial-mineral interactions in order to develop a successful method for bioleaching of REEs still remains a major challenge. This review focuses on the use of microbes for the bioleaching of REEs and highlights the importance of genomic studies in order to narrow down potential microorganisms for the optimal extraction of REEs.

Complete Genome Sequence of Stenotrophomonas maltophilia AB550, an Environmental Solar Radiation- and Multidrug-Resistant Strain Isolated in Western Australia.

We report here the complete genome sequence of Stenotrophomonas maltophilia AB550, a multidrug- and solar radiation-resistant strain isolated from the effluents of an urban wastewater treatment plant in Western Australia. The genome consists of a single 4.9-Mb chromosome.

Quantitative proteomics using SWATH-MS identifies mechanisms of chloride tolerance in the halophilic acidophile Acidihalobacter prosperus DSM 14174.

In this study, the differential protein expression of the acidophilic halophile, Acidihalobacter prosperus DSM 14174 (strain V6) was studied with the aim of understanding its mechanisms of tolerance to high chloride ion stress in the presence of low pH, using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). In acidophiles, chloride stress results in both osmotic stress as well as acidification of the cytoplasm due to the ability of chloride to permeate the cell membrane and disrupt the reversed transmembrane potential which normally extrudes protons. The proteomic response of A. prosperus DSM 14174 to elevated chloride concentrations included the production of osmotic stress regulators that potentially induced the production of compatibles solutes, of which the most significant increase was in the synthesis of ectoine. Other responses directly related to the increased chloride and acid stress, included the increased synthesis of glutathione, changes in carbon flux, the increased production of amino acids, the decreased production of ribosomal proteins, the efflux of metals and protons, and the increase in proteins involved in DNA repair and membrane biosynthesis. Energy generation through iron oxidation and sulphur oxidation were decreased, and energy was probably obtained from the metabolism of glycogen stores. Overall, these studies have helped to create a model of tolerance to elevated chloride under acidic conditions by A. prosperus DSM 14174 that differs from the previous model developed for the type strain, A. prosperus DSM 5130T.

Syntrophic effect of indigenous and inoculated microorganisms in the leaching of rare earth elements from Western Australian monazite.

The unique physiochemical properties exhibited by rare earth elements (REEs) and their increasing application in high-tech industries has created a demand for secure supply lines with established recovery procedures that create minimal environmental damage. Bioleaching experiments conducted on a non-sterile monazite concentrate with a known phosphate solubilising microorganism (PSM) resulted in greater mobilisation of REEs into solution in comparison to experiments conducted on sterile monazite. By combining the native consortia with an introduced PSM, a syntrophic effect between the populations effectively leached a greater amount of REEs than either a single PSM or the indigenous population alone. With sterile monazite, Penicillium sp.CF1 inoculated experiments released a total REE concentration of 12.32 mg L-1 after incubation for 8 days, whereas on non-sterile ore, double the soluble REE concentration was recorded (23.7 mg L-1). Comparable effects were recorded with Enterobacter aerogenes, Pantoea agglomerans and Pseudomonas putida. Alterations in the microbial populations during bioleaching of the monazite ore were determined by diversity profiling and demonstrated noticeable changes in community inhabitants over 14 days. The presence of native Firmicutes on the monazite appears to greatly contribute to the increased leaching recorded when using non-sterile monazite for REE recovery.

Microbial Diversity and Mineralogical-Mechanical Properties of Calcitic Cave Speleothems in Natural and in Vitro Biomineralization Conditions.

Natural mineral formations are a window into important processes leading to carbon storage and mineralized carbonate structures formed through abiotic and biotic processes. In the current study, we made an attempt to undertake a comprehensive approach to characterize the mineralogical, mechanical, and microbial properties of different kinds of speleothems from karstic caves; with an aim to understand the bio-geo-chemical processes in speleothem structures and their impact on nanomechanical properties. We also investigated the biomineralization abilities of speleothem surface associated microbial communities in vitro. Mineralogical profiling using techniques such as X-ray powder Diffraction (XRD) and Tescan Integrated Mineral Analyzer (TIMA) demonstrated that calcite was the dominant mineral in the majority of speleothems with Energy Dispersive X-ray Analysis (EDS) indicating a few variations in the elemental components. Differing proportions of polymorphs of calcium carbonate such as aragonite and vaterite were also recorded. Significant variations in trace metal content were recorded through Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Scanning Electron Microscopy (SEM) analysis revealed differences in morphological features of the crystals which varied from triangular prismatic shapes to etched spiky forms. Microbial imprints and associations were seen in a few sections. Analysis of the associated microbial diversity showed significant differences between various speleothems at Phylum level; although Proteobacteria and Actinobacteria were found to be the predominant groups. Genus level microbial associations showed a relationship with the geochemistry, mineralogical composition, and metal content of the speleothems. The assessment of nanomechanical properties measured by Nanoindentation revealed that the speleothems with a dominance of calcite were stronger than the speleothems with mixed calcium carbonate polymorphs and silica content. The in vitro metabolic activity of the microbial communities associated with the surfaces of the speleothems resulted in calcium carbonate crystal precipitation. Firmicutes and Proteobacteria dominated these populations, in contrast to the populations seen in natural systems. The precipitation of calcium carbonate crystals in vitro indicated that microbial metabolic activity may also play an important role in the synthesis and dissociation of biominerals in the natural environment. Our study provides novel evidence of the close relationship between mineralogy, microbial ecology, geochemistry, and nanomechanical properties of natural formations.

Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite.

Successful process development for the bioleaching of mineral ores, particularly the refractory copper sulfide ore chalcopyrite, remains a challenge in regions where freshwater is scarce and source water contains high concentrations of chloride ion. In this study, a pure isolate of Acidihalobacter prosperus strain F5 was characterized for its ability to leach base metals from sulfide ores (pyrite, chalcopyrite and pentlandite) at increasing chloride ion concentrations. F5 successfully released base metals from ores including pyrite and pentlandite at up to 30gL-1 chloride ion and chalcopyrite up to 18gL-1 chloride ion. In order to understand the genetic mechanisms of tolerance to high acid, saline and heavy metal stress the genome of F5 was sequenced and analysed. As well as being the first strain of Ac. prosperus to be isolated from Australia it is also the first complete genome of the Ac. prosperus species to be sequenced. The F5 genome contains genes involved in the biosynthesis of compatible solutes and genes encoding monovalent cation/proton antiporters and heavy metal transporters which could explain its abilities to tolerate high salinity, acidity and heavy metal stress. Genome analysis also confirmed the presence of genes involved in copper tolerance. The study demonstrates the potential biotechnological applicability of Ac. prosperus strain F5 for saline water bioleaching of mineral ores.

Draft Genome Sequence of Acidihalobacter ferrooxidans DSM 14175 (Strain V8), a New Iron- and Sulfur-Oxidizing, Halotolerant, Acidophilic Species.

The use of halotolerant acidophiles for bioleaching provides a biotechnical approach for the extraction of metals from regions where high salinity exists in the ores and source water. Here, we describe the first draft genome of a new species of a halotolerant and iron- and sulfur-oxidizing acidophile, Acidihalobacter ferrooxidans DSM 14175 (strain V8).

Interactions of phosphate solubilising microorganisms with natural rare-earth phosphate minerals: a study utilizing Western Australian monazite.

Many microbial species are capable of solubilising insoluble forms of phosphate and are used in agriculture to improve plant growth. In this study, we apply the use of known phosphate solubilising microbes (PSM) to the release of rare-earth elements (REE) from the rare-earth phosphate mineral, monazite. Two sources of monazite were used, a weathered monazite and mineral sand monazite, both from Western Australia. When incubated with PSM, the REE were preferentially released into the leachate. Penicillum sp. released a total concentration of 12.32 mg L-1 rare-earth elements (Ce, La, Nd, and Pr) from the weathered monazite after 192 h with little release of thorium and iron into solution. However, cultivation on the mineral sands monazite resulted in the preferential release of Fe and Th. Analysis of the leachate detected the production of numerous low-molecular weight organic acids. Gluconic acid was produced by all microorganisms; however, other organic acids produced differed between microbes and the monazite source provided. Abiotic leaching with equivalent combinations of organic acids resulted in the lower release of REE implying that other microbial processes are playing a role in solubilisation of the monazite ore. This study demonstrates that microbial solubilisation of monazite is promising; however, the extent of the reaction is highly dependent on the monazite matrix structure and elemental composition.

Draft Genome Sequence of the Acidophilic, Halotolerant, and Iron/Sulfur-Oxidizing Acidihalobacter prosperus DSM 14174 (Strain V6).

The principal genomic features of Acidihalobacter prosperus DSM 14174 (strain V6) are presented here. This is a mesophilic, halotolerant, and iron/sulfur-oxidizing acidophile that was isolated from seawater at Vulcano, Italy. It has potential for use in biomining applications in regions where high salinity exists in the source water and ores.

Saline-water bioleaching of chalcopyrite with thermophilic, iron(II)- and sulfur-oxidizing microorganisms.

The application of thermoacidophiles for chalcopyrite (CuFeS2) bioleaching in hot, acidic, saline solution was investigated as a possible process route for rapid Cu extraction. The study comprised a discussion of protective mechanisms employed for the survival and/or adaptation of thermoacidophiles to osmotic stress, a compilation of chloride tolerances for three genera of thermoacidophiles applied in bioleaching and an experimental study of the activities of three species in a saline bioleaching system. The data showed that the oxidation rates of iron(II) and reduced inorganic sulfur compounds (tetrathionate) were reduced in the presence of chloride levels well below chloride concentrations in seawater, limiting the applicability of these microorganisms in the bioleaching of CuFeS2 in saline water.

Multiple Osmotic Stress Responses in Acidihalobacter prosperus Result in Tolerance to Chloride Ions.

Extremely acidophilic microorganisms (pH optima for growth of ≤3) are utilized for the extraction of metals from sulfide minerals in the industrial biotechnology of "biomining." A long term goal for biomining has been development of microbial consortia able to withstand increased chloride concentrations for use in regions where freshwater is scarce. However, when challenged by elevated salt, acidophiles experience both osmotic stress and an acidification of the cytoplasm due to a collapse of the inside positive membrane potential, leading to an influx of protons. In this study, we tested the ability of the halotolerant acidophile Acidihalobacter prosperus to grow and catalyze sulfide mineral dissolution in elevated concentrations of salt and identified chloride tolerance mechanisms in Ac. prosperus as well as the chloride susceptible species, Acidithiobacillus ferrooxidans. Ac. prosperus had optimum iron oxidation at 20 g L-1 NaCl while At. ferrooxidans iron oxidation was inhibited in the presence of 6 g L-1 NaCl. The tolerance to chloride in Ac. prosperus was consistent with electron microscopy, determination of cell viability, and bioleaching capability. The Ac. prosperus proteomic response to elevated chloride concentrations included the production of osmotic stress regulators that potentially induced production of the compatible solute, ectoine uptake protein, and increased iron oxidation resulting in heightened electron flow to drive proton export by the F0F1 ATPase. In contrast, At. ferrooxidans responded to low levels of Cl- with a generalized stress response, decreased iron oxidation, and an increase in central carbon metabolism. One potential adaptation to high chloride in the Ac. prosperus Rus protein involved in ferrous iron oxidation was an increase in the negativity of the surface potential of Rus Form I (and Form II) that could help explain how it can be active under elevated chloride concentrations. These data have been used to create a model of chloride tolerance in the salt tolerant and susceptible species Ac. prosperus and At. ferrooxidans, respectively.

Bioleaching in brackish waters--effect of chloride ions on the acidophile population and proteomes of model species.

High concentrations of chloride ions inhibit the growth of acidophilic microorganisms used in biomining, a problem particularly relevant to Western Australian and Chilean biomining operations. Despite this, little is known about the mechanisms acidophiles adopt in order to tolerate high chloride ion concentrations. This study aimed to investigate the impact of increasing concentrations of chloride ions on the population dynamics of a mixed culture during pyrite bioleaching and apply proteomics to elucidate how two species from this mixed culture alter their proteomes under chloride stress. A mixture consisting of well-known biomining microorganisms and an enrichment culture obtained from an acidic saline drain were tested for their ability to bioleach pyrite in the presence of 0, 3.5, 7, and 20 g L(-1) NaCl. Microorganisms from the enrichment culture were found to out-compete the known biomining microorganisms, independent of the chloride ion concentration. The proteomes of the Gram-positive acidophile Acidimicrobium ferrooxidans and the Gram-negative acidophile Acidithiobacillus caldus grown in the presence or absence of chloride ions were investigated. Analysis of differential expression showed that acidophilic microorganisms adopted several changes in their proteomes in the presence of chloride ions, suggesting the following strategies to combat the NaCl stress: adaptation of the cell membrane, the accumulation of amino acids possibly as a form of osmoprotectant, and the expression of a YceI family protein involved in acid and osmotic-related stress.