Bioprospecting for and the applications of halophilic acidophiles in bioleaching operations

Keywords: acid saline lakes, Acidihalobacter, acidophiles, bioleaching, bioprospecting, genetic engineering, halophiles, saline drains.

As the global transition to, and the goal of governments for a zero emissions energy system are embraced worldwide, the demand for critical minerals such as cobalt, copper, nickel, lithium and rare earth elements is outstripping current availability.¹ Compounding the lack of materials are issues of low-grade ores (uneconomical using current recovery processes), water consumption and downstream co-contamination of unwanted elements such as uranium during traditional extraction processes. Commercial success of mining operations often includes reclaiming and recycling mine wastes with some sites operating bioleaching heaps as an inexpensive alternative to the traditional pyro-metallurgical methods for the extraction of metals from low grade sulfide ores.

Even though bioleaching has been employed successfully for decades, lengthy leaching cycles and low recovery efficiencies have hindered large scale investment and adoption of the process. The demand for critical minerals is increasing and the resulting upwards trajectory of mineral commodity prices showing no signs of slowing.² As a result, investigations into optimising bioleaching practices for the reclamation of elements (re-processing) from fresh or abandoned tailings is gathering interest, as is the application of this technology to lower grade primary ores. To optimise bioleaching processes, numerous avenues are being explored to not only increase mineral yield from these practices, but also to decrease operating, environmental and maintenance costs of the complex systems, tackling different parameters of the procedures involved. Prospective strategies for advancing bioleaching operations include deliberate adaptation of organisms to extreme conditions, genetic engineering and bioprospecting.

The microorganisms currently applied to bioleaching of sulfide ores are consistently a combination of acidophilic bacteria and archaea whose ability to prosper in low pH conditions enables the dissolution of iron and inorganic sulfur by way of metabolism and acid generation, thus liberating recoverable minerals. Under mesophilic conditions, bioleaching populations are dominated by genera such as Acidithiobacillus, Acidiphilium, Acidiferrobacter and Leptospirillum,³ whereas increasing temperatures (up to 60°C) alters the taxa to favour Sulfobacillus, Acidimicrobium and Ferroplasma. Communities comprising multiple species are more adept at undertaking a variety of tasks than a single species alone,⁴ but all can be adversely affected by changes in numerous parameters including water potential, ore porosity and surface area undesirably resulting in decreased retrieval efficiencies.

These bioleaching microorganisms were originally isolated from some of the most inhospitable regions on earth⁵ so adaptive culturing of both pure and mixed populations to extreme conditions (high compound metal concentrations, acidity, salinity) has seen some successes. Sub-culturing under situations of continuous or accumulative ore concentrations has been adopted by many researchers as a way to enhance bioleaching performance and increase element recovery.⁶ However, it can be a difficult and lengthy process maintaining discrete population numbers overtime in these adapted cultures. An alternative approach to the adaptation of microbial populations is to utilise a naturally occurring microbial consortium. A consortium of microbes can have superior benefits over cultures of pure isolates as a broader array of multifaceted functions including inter-species biofilm formation occur that have demonstrated a greater rate of mineral solubilisation⁷ than when pure cultures were applied.

Advances in proteomic and metabolomic analysis of microbial systems has enabled researchers to understand how microorganisms can tolerate extreme conditions. With this information, genetic manipulation and modification of existing bioleaching microorganisms to improve on leaching efficiencies has shown promise when applied to the recovery of rare earth elements.⁸ The incorporation of metabolic pathways involved in the degradation of organic compounds from heterotrophs such as Acidiphilum or Sulfobacillus into autotrophic iron and sulfur oxidisers (Acidithiobacillus) could reduce issues of organic toxicity that organics often bring to bioleaching operations.⁹ Designing and manufacturing synthetic microbial consortia for bioleaching applications is an emerging area of research, however, their application for in situ bioleaching (in particular heap leaching) remains to be seen because of strong environmental release laws. As microbial diversity, complete with wide metabolic potential, is influenced not only by the variable environment but also with the interactions between competing microorganisms, the genetic analysis of organisms often conducted on pure cultures does not reflect real world situations and should be taken into consideration if designing a synthetic population.

Bioleaching processes at high temperatures (>45°C) has seen success with the application of various thermophilic microbial species (Sulfolobus, Metallosphaera, Acidianus)¹⁰ for the recovery of copper, uranium and gold. Owing to the higher running costs of these operations (stirred tank reactors), this process is often restricted to high value minerals. This, nevertheless, has fuelled the search for more thermophilic organisms capable of solubilising iron–sulfide rich ores in locales such as hydrothermal vents¹¹ (which is now an emerging deep-sea biotechnology industry), volcanic areas¹² and hot springs.¹³

For bioleaching operations to be economically and environmentally sustainable, water consumption must be tightly controlled as it is essential for ore processing and recovery. Implementation of untreated groundwater sources for bioleaching applications runs the risk of tapping into sources with high levels of total dissolved solids, and the use of sea water requires understanding how chloride ions can affect mixed microbial communities.¹⁴ The mesophilic acidophiles listed earlier all demonstrate extreme sensitivity to chloride. Their cell membrane is permeable to the chloride ion, which, on entry to the cell, results in the negation of the positive membrane potential.¹⁵ Consequently, proton entry follows resulting in acidification of the cytoplasm, disturbing the proton motive force and eventually cell death. Microorganisms capable of surviving conditions of low pH, high salt and utilising Fe/S for energy are few in number as environments where these stresses co-exist are extraordinary unique. Therefore, bioprospecting for microorganisms that thrive in these conditions rapidly narrows the number of locations across the globe in which they may be found.

Our initial explorations took us to the acidic saline drains in the Yilgarn Craton of Western Australia in 2008 where we isolated a potential candidate known as F5.¹⁶ F5 effectively released base metals from pyrite, pentlandite and chalcopyrite under leaching conditions with up to 30 g L⁻¹ of chloride ions present, and following genomic sequencing was named Acidihalobacter prosperus.¹⁷ This organism was then further re-classified as A. yilgarnensis due to genomic sequence comparisons with related species demonstrating a clear difference between it and an already existing A. prosperus.¹⁸ Acidihalobacter prosperus (originally named Thiobacillus prosperus) was isolated from a shallow geothermally heated seafloor on the Aeolian Islands, Vulcano, Italy.¹⁹ Subsequently a further two halotolerant acidophiles of this genus, A. prosperus V6 and A. ferrooxidans V8, were isolated from mixed shallow acidic marine pools, also on the Aeolian Islands. These isolates have subsequently been renamed A. prosperus, A. aeolianus and A. ferrooxydans,^20,21 all of which are capable of growth at low pH (1.8–2.0) as well as tolerating high salt and oxidising pyritic compounds. The Acidihalobacter species may in the future be applied to bioleaching operations where concentrations of salt in the water render the usual consortium unviable.

Even though the organisms that currently make up the Acidihalobacter genus were isolated in locations greater than 13 000 km apart, similar environmental pressures have resulted in the conservation of genes essential for survival. Genome exploration of these organisms has revealed that the low pH and high salt tolerances evolved separately²² with the halophilic organism gaining genes for acid tolerance through horizontal gene transfer. Armed with this information, in vitro modelling of genetic alterations made to bioleaching organisms (Acidithiobacillus ferrooxidans) can allow us to predict the success and applicability of organisms for mineral recovery in high salt, low pH conditions.

As the Yilgarn Craton of Western Australia is dotted with numerous acidic saline lakes, it provides an ideal opportunity for further prospecting²³ while utilising the information gained from molecular modelling to target specific environments with the greatest chance of hosting prokaryotes suitable for bioleaching in regions where saline water is of concern. Another hypersaline lake in Australia whose pH fluctuates with the seasons is Lake Tyrell (a shallow, salt-crusted depression) in Victoria, a location where numerous bioprospecting expeditions have been conducted^24,25 in attempt to isolate organisms for biotechnological applications. A global search of other hypersaline lakes with low pH (<4) for iron–sulfur-oxidising halophiles to either adapt to acidic conditions or genetically modify to contain acid resistance mechanisms could allow for the construction of a consortia suitable for bioleaching in regions with salt contaminated waters (Fig. 1).

Fig. 1.  Life cycle of bioprospecting for halophilic iron- and sulfur-oxidising acidophile for use in the application of saline water bioleaching.

By understanding the microbial diversity, proteomic, lipidomic and genetic contributions, adaptations and pressures in the unique high salt, low pH environments, advances in the efficiency of bioleaching process could be accomplished. Although a range of approaches such as adaption and genetic modification could be applied to answering this problem, bioprospecting is the approach most likely to provide a successful solution.

Data sharing is not applicable as no new data were generated or analysed during this study.

The authors declare that they have no conflicts of interest.

This research did not receive any specific funding.