Naomi J. Boxall

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

ABSTRACT 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.

In a quest for engineering acidophiles for biomining applications: challenges and opportunities

Biomining with acidophilic microorganisms has been used at commercial scale for the extraction of metals from various sulfide ores. With metal demand and energy prices on the rise and the concurrent decline in quality and availability of mineral resources, there is an increasing interest in applying biomining technology, in particular for leaching metals from low grade minerals and wastes. However, bioprocessing is often hampered by the presence of inhibitory compounds that originate from complex ores. Synthetic biology could provide tools to improve the tolerance of biomining microbes to various stress factors that are present in biomining environments, which would ultimately increase bioleaching efficiency. This paper reviews the state-of-the-art tools to genetically modify acidophilic biomining microorganisms and the limitations of these tools. The first part of this review discusses resilience pathways that can be engineered in acidophiles to enhance their robustness and tolerance in harsh environments that prevail in bioleaching. The second part of the paper reviews the efforts that have been carried out towards engineering robust microorganisms and developing metabolic modelling tools. Novel synthetic biology tools have the potential to transform the biomining industry and facilitate the extraction of value from ores and wastes that cannot be processed with existing biomining microorganisms.

Multistage leaching of metals from spent lithium ion battery waste using electrochemically generated acidic lixiviant

Lithium ion battery (LIB) waste contains significant valuable resources that could be recovered and reused to manufacture new products. This study aimed to develop an alternative process for extracting metals from LIB waste using acidic solutions generated by electrolysis for leaching. Results showed that solutions generated by electrolysis of 0.5 M NaCl at 8 V with graphite or mixed metal oxide (MMO) electrodes were weakly acidic and leach yields obtained under single stage (batch) leaching were poor (<10%). This was due to the highly acid-consuming nature of the battery waste. Multistage leaching with the graphite electrolyte solution improved leach yields overall, but the electrodes corroded over time. Though yields obtained with both electrolyte leach solutions were low when compared to the 4 M HCl control, there still remains potential to optimise the conditions for the generation of the acidic anolyte solution and the solubilisation of valuable metals from the LIB waste. A preliminary value proposition indicated that the process has the potential to be economically feasible if leach yields can be improved, especially based on the value of recoverable cobalt and lithium.

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.

Characterisation of Oxalate-Degrading Microorganisms in Bioreactors Treating Bayer Liquor Organic Materials

When bauxite is digested during Bayer processing, associated organic compounds and humic acids are degraded to produce sodium salts of organic acids, including sodium oxalate. If not removed from the liquor stream, sodium oxalate co-precipitates with the aluminium hydroxide resulting in poor crystallization and alumina and soda loss. Aerobic bioremediation processes have been developed as an economic and environmentally sound option for oxalate removal. Little research has been directed at characterising the microbial communities and biological processes underpinning these processes. Analysis of samples from both a moving bed biofilm reactor and bioreactor effluent using PCR-DGGE of 16S rRNA genes showed microorganisms of the genus Halomonas dominated the process. Most Probable Number (MPN) analyses also showed Halomonas spp. to be numerically dominant in all bioreactor samples.

Application of indirect non-contact bioleaching for extracting metals from waste lithium-ion batteries

Applying biohydrometallurgy for metal extraction and recovery from mixed and polymetallic wastes such as electronic waste is limited due to microbial inhibition at low pulp densities and substrate (iron and sulfur) limitation. Here, we investigated the application of indirect non-contact bioleaching with biogenic ferric iron and sulfuric acid to extract metals from lithium-ion battery (LIB) waste. Results showed that although a single leach stage at ambient temperature only facilitated low leach yields (<10%), leach yields for all metals improved with multiple sequential leach stages (4 × 1 h). Biogenic ferric leaching augmented with 100 mM H2SO4 further enabled the highest leach yields (53.2% cobalt, 60.0% lithium, 48.7% nickel, 81.8% manganese, 74.4% copper). The proposed use of bioreagents is a viable and a more environmentally benign alternative to traditional mineral processing, which could be further improved by appropriate pre-treatment of the LIB waste.

Recent advances in biomining and microbial characterisation

Since the discovery of bioleaching microorganisms and their role in metal extraction in the 1940s, a number of technical approaches have been developed to enhance microbially catalysed solubilisation of metals from ores, concentrates and waste materials. Biomining has enabled the transformation of uneconomic resources to reserves, and thus help to alleviate the challenges related to continually declining ore grades. The rapid advancement of microbial characterisation methods has vastly increased our understanding of microbial communities in biomining processes. The objective of this paper is to review the recent advances in biomining processes and microbial characterisation.

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.

Biosolubilisation of Metals and Metalloids

The solubilisation of metals and metalloids is catalysed by a variety of microorganisms in natural and engineered environments. Biosolubilisation has a number of undesired implications, such as the generation of acid mine drainage and the formation of acid sulfate soils, which have harmful environmental impacts. Biosolubilisation also contributes to the corrosion of man-made structures causing significant economic losses. On the other hand biosolubilisation has been harnessed by the mining industry to recover valuable metals and uranium from low-grade ores and concentrates in large scale. This allows the utilisation of ores the processing of which would not be economically feasible through traditional mining methods. Biosolubilisation holds also potential for the recovery of resources from waste and clean-up of metal contaminated environments. This chapter reviews the role that microorganisms have in the solubilisation of various metals and metalloids, the mechanisms through which biosolubilisation occurs and microbial groups mediating the solubilisation. The environmental implications and industrial applications of biosolubilisation are also discussed. Microorganisms can catalyse biosolubilisation through oxidative and reductive dissolution, mediated by the oxidation and reduction of ferrous and ferric iron, respectively. Moreover, biosolubilisation can be achieved through the production of biogenic acids, alkali and ligands, such as cyanide, thiosulfate, organic acids and iodide. Mechanisms contributing to microbially influenced corrosion of metallic iron and steel include differential aeration cells, galvanic cells, attack by microbial oxidants, acids, sulfides and other metabolites, cathodic depolarisation and direct microbial extraction of electrons from steel. A wide range of microorganisms are able to facilitate solubilisation reactions, including bacteria, archaea and eukaryotes. Bioleaching has been explored for recovering metals from e.g. a variety of sulfide ores, metallurgical waste, electronic scrap, sludge from municipal and industrial wastewater treatment, municipal solid waste incineration fly ash and contaminated sites. Large-scale biosolubilisation has been mainly used for copper-, cobalt-, nickel-, zinc-, uranium- and gold-containing sulfidic ores through oxidative bioleaching, whereas reductive bioleaching is yet to be implemented at industrial scale.

Characterisation of a Novel Genus of Oxalate-Degrading Beta-Proteobacteria Isolated from a Full-Scale Bioreactor Treating Bayer Liquor Organic Wastes

A novel industrial-scale bioreactor was implemented by Alcoa of Australia (Alcoa) at its Kwinana alumina refinery (Western Australia) for the degradation of oxalate, an organic byproduct of the Bayer alumina refining process. At the Kwinana refinery oxalate is removed from the Bayer Liquor via a separate side-stream as it increases the operating costs associated with the process and, at sufficiently high levels, may adversely affect the quality and yield of the final alumina product. The bioreactor process provides a more economic and environmentally friendly method for the treatment of removed oxalate compared with chemical conversion or storage of the solid by-product. In previous studies, the microbial community composition of the bioreactor was investigated and was found to be largely dominated by microorganisms of the α-, β- and γ-Proteobacteria subgroups. During the present study, two bacteria that had the ability to use oxalate as a sole source of carbon and energy were isolated from samples obtained from the bioreactor. Phylogenetic and physiological analyses indicated that the two isolates were probably strains of a novel species of a novel genus within the β-Proteobacteria subgroup. Isolation and characterisation of the microbial communities within the bioreactor system has the potential to improve process operation, which may have a positive impact on the biological oxalate destruction process and the footprint of alumina production.