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Kaksonen, A.H., Boxall, N.J., Gumulya, Y., Khaleque, H.N., Morris, C., Bohu, T., Cheng, K.Y., Usher, K. and Lakaniemi, A-M (2018) Recent progress in biohydrometallurgy and microbial characterisation. Hydrometallurgy

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Recent progress in biohydrometallurgy and microbial characterisation

Anna H. Kaksonen, Naomi J. Boxall, Yosephine Gumulya, Himel Nahreen Khaleque, Christina Morris, Tsing Bohu, Ka Yu Cheng, Kayley Usher, Aino-Maija Lakaniemi

PII: S0304-386X(18)30180-4 DOI: doi:10.1016/j.hydromet.2018.06.018 Reference: HYDROM 4853 To appear in: Hydrometallurgy Received date: 26 February 2018 Revised date: 14 June 2018 Accepted date: 24 June 2018

Please cite this article as: Anna H. Kaksonen, Naomi J. Boxall, Yosephine Gumulya, Himel Nahreen Khaleque, Christina Morris, Tsing Bohu, Ka Yu Cheng, Kayley Usher, Aino-Maija Lakaniemi , Recent progress in biohydrometallurgy and microbial characterisation. Hydrom (2018), doi:10.1016/j.hydromet.2018.06.018

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Recent progress in biohydrometallurgy and microbial characterisation

Anna H Kaksonen1,2*, Naomi J Boxall1, Yosephine Gumulya1, Himel Nahreen Khaleque1, Christina

Morris1, Tsing Bohu3, Ka Yu Cheng1,4, Kayley Usher1, and Aino-Maija Lakaniemi1,5

1CSIRO Land and Water, 147 Underwood Avenue, Floreat WA 6014, Australia

2School of Pathology and Laboratory Medicine, and Oceans Institute, University of Western

Australia, Nedlands, Western Australia 6009, Australia

3CSIRO Mineral Resources, Kensington WA 6151, Australia

4School of Engineering and Information Technology, Murdoch University, Murdoch, Western

Australia 6150, Australia

5Tampere University of Technology, Faculty of Natural Sciences, Laboratory of Chemistry and

Bioengineering, P.O. Box 541, FI-33101 Tampere, Finland

*Corresponding author: [email protected]

Abstract. Since the discovery of microbiological metal dissolution, numerous biohydrometallurgical approaches have been developed to use microbially assisted aqueous extractive metallurgy for the recovery of metals fromACCEPTED ores, concentrates, and MANUSCRIPT recycled or residual materials. Biohydrometallurgy has helped to alleviate the challenges related to continually declining ore grades by transforming uneconomic ore resources to reserves. Engineering techniques used for biohydrometallurgy span from above ground reactor, vat, pond, heap and dump leaching to underground in situ leaching.

Traditionally biohydrometallurgy has been applied to the bioleaching of base metals and uranium from sulfides and biooxidation of sulfidic refractory gold ores and concentrates before cyanidation.

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More recently the interest in using bioleaching for oxide ore and waste processing, as well as extracting other commodities such as rare earth elements has been growing. Bioprospecting, adaptation, engineering and storing of microorganisms has increased the availability of suitable biocatalysts for biohydrometallurgical applications. Moreover, the advancement of microbial characterisation methods has increased the understanding of microbial communities and their capabilities in the processes. This paper reviews recent progress in biohydrometallurgy and microbial characterisation.

Keywords: bioleaching, biooxidation, biohydrometallurgy, characterization, microbiology

1. Introduction

Metals are used widely in modern society, and the demand for and the dependency on metals is further increasing due to global urbanisation, population growth and the consumption of portable electronic devices. As an example, global crude steel production has almost doubled from

850 to 1630 million tons between years 2000 and 2013, with the current production volumes varying between 1620-1670 million tons (World Steel Association, 2017). Similarly, global copper production has increased from 12.9 to 19.4 million tons from 2000 to 2016 (U.S. Department of the

Interior and U.S. Geological Survey, 2001; U.S. Geological Survey, 2017).

The productionACCEPTED of modern electronic devices MANUSCRIPT such as smartphones and tablets requires the use of over 60 different metals ranging from gold, silver, platinum and copper to rare earth elements (Rohrig, 2015). In contrast, exploitable primary metal resources are getting scarcer, ore grades are getting lower, and mineralogy of the exploitable ores is becoming increasingly complex

(Prior et al., 2012; Northey et al., 2014). As the ore grades get lower, the energy input and

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environmental challenges associated with traditional pyrometallurgical and hydrometallurgical mining processes increase (Norgate et al., 2007).

Biohydrometallurgy, a branch of hydrometallurgy, utilises the activity of microorganisms in aqueous extractive metallurgy for the recovery of metals from ores, concentrates, and recycled or residual materials. It is considered as a promising, environmentally benign option, especially for low-grade and complex ores. The cumulative number of publications listed in Scopus referring (in the title, abstract and/or keywords) to biomining, bioleaching, biooxidation or biosolubilisation, and copper, zinc, nickel, gold, cobalt, uranium or rare earth elements are shown in Figure 1A. The most widely studied commodity for biohydrometallurgy is copper, followed by zinc, gold, nickel cobalt and uranium. As the consumption and demand for rare earth elements have recently increased, research targeting the bioleaching of these has also been increasing (Reed et al.,

2016). Moreover, possibilities for extracting other commodities such as phosphorus (Mäkinen et al., 2016; Reed et al., 2016) and manganese are also being explored (Das et al., 2011).

Commercial-scale bioleaching and biooxidation have been mainly applied to sulfide ores.

The processes are based on the activity of acidophilic iron- and sulfur-oxidising microorganisms, which generate ferric iron and sulfuric acid containing lixiviants for sulfide mineral dissolution.

Commercial bioleaching and biooxidation applications have usually targeted copper, gold, uranium, nickel, cobalt and zinc (Morin and d’Hugues, 2007; Puhakka et al., 2007; Brierley and Brierley, 2013; SchippersACCEPTED et al., 2014). Based MANUSCRIPT on recent estimations 10-15% of copper (Roberto, 2017b) and approximately 5% of gold are recovered through bioleaching and biooxidation, respectively (Brierley and Brierley, 2013). The use of ferric iron (Fe(III)) reducing microorganisms has been proposed to expand bioleaching from sulfide to oxide ores, such as laterites (Johnson and du Plessis, 2015). Moreover, manganese (Mn(IV) reducing microorganisms have been explored for bioleaching manganese nodules (Lee et al., 2001). Also, biological processes are perceived suitable for the extraction of metals from various waste materials including metallurgical slags, 3

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electronic wastes and metal-laden wastewaters (Bryan et al., 2015; Erüst et al., 2013; Johnson,

2014; Kaksonen et al., 2017). With the expansion of bioleaching to new types of commodities, alternative biogenic lixiviants, such as organic acids or cyanide generated by heterotrophic and neutrophilic microorganisms are also being considered (Kaksonen et al., 2014c).

The objective of this review is to discuss recent progress in biohydrometallurgy and microbial characterisation, including various engineering techniques and application areas. Moreover, this paper reviews advances in bioprospecting, adaptation, engineering and storing of bioleaching microorganisms as well as the characterisation of the microorganisms through culture-based, molecular and microscopy approaches.

2. Engineering techniques in biohydrometallurgy

Some biohydrometallurgical engineering techniques have been developed to facilitate bioleaching and biooxidation. These include reactors, vats, lagoons, heaps, dumps, as well as in situ leaching (Figure 2). The cumulative number of publications listed in Scopus referring to these techniques, and words bioleaching, biooxidation, biomining or biosolubilisation is shown in Figure

1B. Publications related to reactors and tanks have been most abundant, followed by heap, in situ, dump applications. In-place and vat bioleaching have so far least numbers of publications.

Commercial-scale biohydrometallurgical applications have most commonly been based on reactors, heaps and dumps. However, there is a growing interest in using vats and in situ mining for low-grade ores. ACCEPTED MANUSCRIPT

2.1 Bioreactors

Bioreactors are typically used for high-value ores and concentrates, due to their relatively high capital and operating costs (Kinnunen, 2004). Bioreactors allow precise control of parameters such as aeration, temperature and pH to enable higher leaching efficiencies and metal recovery (Gahan 4

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et al., 2012). Moreover, continuous flow operation enables natural selection for microbial consortia that are proficient in catalysing dissolution of the sulfide minerals. Another advantage of bioreactors is the possibility to establish synergy between chemoautotrophic and heterotrophic microorganisms within a reactor, thus improving the overall leaching rate. Usually, mineral decomposition takes only days in stirred-tank reactors compared with months in heaps (Rawlings et al., 2003).

2.1.1 Technical bioreactor developments

Bioleaching and biooxidation have been explored at laboratory-scale using a wide range of bioreactor configurations, such as stirred tank reactors (STR), Pachuca airlift reactors (ALR), continuous bubble columns (CBC), and continuous revolving barrel bioreactors (Loi et al., 2006;

Mahmoud et al., 2017). Commercial-scale tank bioleaching and biooxidation are generally carried out using continuous stirred tank reactors (CSTR), where the slurry of finely ground ore or concentrate is aerated and agitated to provide good mass transfer (Mahmoud et al., 2017).

Typically, stirred-tank bioreactor processes involve three or more stages in series, whereby the first stage consists of several tanks configured in parallel to allow longer feed retention, and the subsequent stages are usually single tanks configured in series (Brierley, 1997). The reactors are usually designed with height to diameter ratio of approximately 1 to minimise static slurry pressure, and thereby low-pressure aeration blowers can be used to reduce energy consumption (Mahmoud et al., 2017).ACCEPTED Bioreactors often have MANUSCRIPT temperature control for cooling to compensate for heat generation, or for heating to allow bioleaching with thermophiles.

Technological developments in bioreactor mixing have included low energy impellers to minimise energy use, dispersion agitators to provide high gas utilisation, non-welded impellers to minimise corrosion, and corrugated reactor bottoms to reduce the power required for keeping solid particles in suspension (Outotec, 2018). Although not yet implemented for biohydrometallurgy, the

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swirl flow agitation technology developed by CSIRO could also hold potential for bioleaching and biooxidation applications. The technology is based on short shaft impellers, which create a tornado-like flow pattern inside mixing tanks to lift settling solids from the bottom. The advantages of the technology include low capital and operating costs, high mechanical reliability and reduced scale formation on reactor walls when compared to tanks with conventional impellers.

The technology has so far been used for chemical leaching, precipitation, de-silication and storage tank applications (Wu et al., 2014; Wu et al., 2016).

A new development in bioreactor technology is the combination of biotechnology with electrochemistry to facilitate the bioelectrochemical leaching and recovery of metals. The cathodes of bioelectrochemical systems (BES) can facilitate the reduction of oxidised compounds

(Jeon and Park, 2011), whereas the BES anodes can drive oxidation reactions (Xiao et al., 2008).

Bioelectrochemical leaching has been explored in laboratory scale for manganese oxides and chalcopyrite (Jeon and Park, 2011). Jeon and Park (2011) evaluated the bioelectrochemical leaching of manganese from manganese ore using Lactococcus (L.) lactis. When using graphite cathodes and neutral red as electron mediator with L. lactis, Jeon and Park (2011) achieved 4 times higher reduction of Mn(IV) to Mn(II) using electrochemical reduction conditions (2 V potential between anode and cathode) than control conditions (open circuit potential). The leaching yields in

30 days were 16 g L-1 and 3.8 g L-1 for electrochemical and control condition, respectively. Xiao et al. (2010) utilised chalcopyrite anodes and manganese oxides cathodes to evaluate the ACCEPTED MANUSCRIPT electrochemical and bioelectrochemical leaching of copper and manganese from the electrodes.

The presence of Acidithiobacillus (A.) thiooxidans increased the leaching of manganese from the cathode after 36 h from 26.1 % to 39.7 %, but the leaching of copper from the anode was similar in the presence (32.5 %) and absence (32.6 %) of bacteria. Ter Heijne et al. (2010) utilised microbial fuel cells (MFC) for combined electricity production and copper recovery. Organic compounds were oxidised at the MFC anode while copper was recovered from solution at the MFC cathode. The 6

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anode was made of graphite and cathode was a piece of graphite foil pressed on a mixed metal oxide coated titanium plate that functioned as a current collector (Ter Heijne et al., 2010).

Electrochemical processes can also be incorporated to bioleaching units to facilitate recovery/ or extraction of metal values from the produced leachate (Maes et al., 2017; Modin et al., 2012).

Recently Maes et al. (2017) reported a two-stage strategy to recover two model rare earth elements, namely neodymium (Nd) and lanthanum (La) from monazite ore. In their process, the metals were first leached from the ore using citric acid and spent fungal supernatant.

Subsequently, the metal-containing leachate was introduced to a middle chamber of a three- chamber electrochemical cell equipped with both anionic- (anode facing) and cationic- (cathode facing) ion exchange membranes to facilitate electrochemical extraction of the metals in a separate stream (as catholyte). With a fixed current density (at 40 A m-2 projected electrode surface area), the process effectively separated the two metals from the feed leachate. Also, the concentrations of both Nd and La in the catholyte increased (up to 154 mg Nd L-1 and 207 mg La L-1 were extracted in 6 days) (Maes et al., 2017). The process also enabled recovery of the lixiviant

(here the citrate) as well as separation of unwanted species (here radioactive element thorium and counter ion phosphate) from the product stream. Further research exploring the use of electrochemical processes for biohydrometallurgical application is warranted.

2.1.2 Bioreactor applications

Several CSTR-basedACCEPTED biohydrometallurgical MANUSCRIPTtechnologies have been commercialised, such as BIOX®, BioCOP®, BioNIC®, BioZINC®, HIOX®, BRISA, BACOX, and BacTech/Mintek reactors

(Kinnunen, 2004; Mahmoud, 2017). BIOX® process designed for the biooxidation of refractory sulfidic gold minerals is the first and most widely used commercial CSTR process. The value of the gold produced can offset the high capital and operational costs of bioreactor processing (Gahan et al., 2012; Schippers et al., 2014). The first commercial-scale BIOX® plant was established at the

Fairview mine, South Africa in 1988 (van Aswegen and Marais, 2001). Over the past 30 years after 7

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its first commission, 13 successful BIOX® plants have been commissioned worldwide with over 22 million ounces of gold already produced through this process (Outotec, 2017). A BIOX® plant typically consists of six reactors, where three are configured in parallel as the first stage and rest as secondary reactors operated in series. The process involves the continuous loading of a flotation concentrate slurry with a solid content typically around 20 % (w/w), although some recent studies found that it is possible to increase the solid loading up to 30 % without any notable impact on the process efficiency (Mahmoud et al., 2017). Typically, the reactor temperature is maintained at between 40 and 45oC, and the retention time in the reactors is 4 to 6 days. The operating pH is typically maintained at between 1.2 and 1.6, and dissolved oxygen concentration in the slurry is maintained at over 2 mg L-1 (van Aswegen et al., 2007).

Stirred-tank reactors have also been used for bioleaching of base metals such as cobalt, zinc, copper and nickel from their respective sulfides, and uranium from its oxides (Gahan et al., 2012).

One example is the stirred-tank bioleaching plant at Kasese, Uganda, where the processing circuit includes a primary stage with three parallel tanks and a secondary and a tertiary stage of one tank each. The process is maintained at 42oC, and the pH values in all tanks are maintained 1.4-1.5 and

1.5-1.7 in the primary and secondary/tertiary stage reactors, respectively (Mahmoud et al., 2017).

The plant is used for cobalt recovery from a cobaltiferous pyrite concentrate. Pyrite concentrate

(240 t) is processed daily, and about 1,100 t of cobalt are produced per year, corresponding to

1.25% of the world production of cobalt in 2010 (Schippers et al., 2014).

Most biohydrometallurgicalACCEPTED reactor processes MANUSCRIPT have been operated under mesophilic or moderately thermophilic conditions (40-50oC) (Rawlings et al., 2003). However, with refractory ores such as chalcopyrite (CuFeS2) and enargite (Cu3AsS4), which are resistive to leaching under such conditions, a higher temperature is required (Norris and Owen, 1993). A well-known example for this is the BioCOP® stirred-tank bioreactor technology developed by BHP Billiton and Codelco for the bioleaching of chalcopyrite concentrates under thermophilic condition (70-80oC) in Chile

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(Mahmoud et al. 2017; Rawlings et al., 2003; Havlik, 2014). The process targets an annual production of 20,000 tonnes copper cathodes from 77,200 tonnes of chalcopyrite concentrate

(Havlik, 2014).

A more recent example of using bioreactors for leaching metals from sulfide concentrates is the project jointly launched by Mondo Minerals and Mintek (Neale et al., 2017). Mondo Minerals is the second largest talc producer in the world and owns two talc mines in Finland (at Sotkamo and

Vuonos). A commercial plant has been constructed at Vuonos talc concentrator plant to process their talc production by-product, a high-grade nickel-cobalt-arsenic containing sulfide concentrate.

At full production, it is expected that the plant can treat 12,000 tonnes of concentrate and produce approximately 1,000 tonnes of nickel per annum. Mintek’s BacTech bioleaching technology was adopted for the process and consisted of seven stirred-tanks with an overall residence time of 7 days (the residence time in the three parallel primary reactors was 3 days) (Neale et al., 2017).

The process was operated at 45oC and was continuously fed with a 15 % solid content slurry, and the pH in the primary and secondary was approximately 1.7 and 1.6, respectively, with a redox potential of over +600 mV vs. Ag/AgCl in all tanks. The process was robust even in the presence of total soluble metal concentrations more than 60 g L-1. This process demonstrated the potential to unlock opportunities for processing other base metal sulfide resources that are not currently exploitable due to high arsenic content (Neale et al., 2017). In addition to ore and concentrate processing, bioreactors have also been utilised for the removal of excess iron and sulfate from

(bio)hydrometallurgicalACCEPTED solutions. Iron and sulfate MANUSCRIPT accumulation can cause precipitate formation

(Figures 3A and 3B) which negatively impacts process kinetics and downstream metal recovery

(Kaksonen et al., 2014a). As a solution to this challenge, Kaksonen et al. (2014b) proposed a two- stage bioreactor system that enabled continuous iron oxidation, and the removal of excess iron and sulfate. Co-precipitation losses of copper and nickel from pregnant leach liquor were low

(Kaksonen et al., 2014b). In the case of heap bioleaching, bioreactors for excess iron removal can

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be applied either to pregnant or barren leach liquor before or after metal recovery, respectively

(Figure 3C).

2.2 Vat and lagoon leaching

In vat leaching technology, also known as sand leaching, the ore is crushed and leached in a series of vats lined with acid-proof material, such as rubber. The vats are sometimes equipped with agitators. INNOVAT Ltd developed a continuous vat leaching process where part of the leach solution is pumped into a head tank, and the potential energy of the water in the head tank is used to create hydrostatic pressure upon water release to fluidise the ore (INNOVAT Mineral Processing

Solutions, 2018). Water is released through fluidisation pipes at the bottom of the vat and fluidises the ore as it percolates through the ore bed. The vat is fed via the ore slurry stream or dry ore by a conveyor. A French drain at the bottom of the vat allows the recovery of solution for processing.

The end of the vat is equipped with a slowly revolving wheel with inlet ports and compartments, which remove material from the vat. The slurry drains during the removal and liquid drains back into the vat while solids are discharged (Cope, 1999).

Vat leaching has been primarily superseded as reactor leaching has become a more economical and efficient method of processing ores and concentrates (Cope, 1999). However, vats are still used in commercial scale at Mantos Blancos mine in Chile for leaching copper oxide ore

(Watling, 2015), as less milling is required and hence operating costs are lower than for reactors. ACCEPTED MANUSCRIPT There is also increasing interest in using vat and lagoon type systems for biohydrometallurgical applications.

Vale has recently patented a process for the bioleaching of crushed sulfide ore in unaerated vats. In this process, iron oxidation occurs in a separate bioreactor, and the bioreactor effluent is mixed with recirculated leach liquor to adjust the redox potential of the vat influent for optimising leaching (du Plessis et al., 2015). The process is proposed for bioleaching copper from chalcopyrite 10

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and is suggested to be also suitable for extracting nickel, cobalt, manganese, gold and uranium

(du Plessis et al., 2015). So far the process has been evaluated at bench scale and is yet to be scaled up to commercial scale. BRGM has also recently patented a lagoon leaching method and facility, where the temperature of the ore suspension, bioleaching consortium and nutrient substrate are controlled by regulating gas flow and composition supplied to the lagoon. The gas is comprised of a mixture of oxygen and CO2. The ore is crushed and pre-conditioned with sulfuric acid and nutrient solution, and the lagoons are mixed with floating mixers (Guezennec et al.,

2015). The pond process was proposed to be suitable for various sulfide ores, such as pyrite, copper sulphides, galena, sphalerite as well as complex ores such as copper black shales

(Guezennec et al., 2015). The process has so far been explored in pilot-scale (few m3) and is yet to be implemented at commercial scale.

2.3 Dump and heap bioleaching

Dump and heap bioleaching is generally considered when the ore is low-grade, sulfides cannot be concentrated because of economic or mineralogical reasons, or the project is too small to support a high capital process (Brierley and Brierley, 2001).

2.3.1 Engineering dumps and heaps

Dumps and heaps are constructed of fractured and hauled rocks on engineered pads with a ACCEPTED MANUSCRIPT network of drainage lines and perforated air lines arranged within a rock layer at the bottom

(Brierley, 2008). Sometimes ore is agglomerated with acid in rotating drums to condition the ore.

The acidic solution is percolated through the dump or heap by sprinklers or drippers, and metal- rich leach liquor is collected at the base for metal recovery. Dump are typically used for processing ores with larger particle size compared to heaps, and hence capital costs associated with dump leaching are lower, which allows larger ore volumes to be processed. However, metal extraction is 11

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typically slow (years or decades) due to low oxygen concentrations, poor acid transport and issues associated with accessibility with large rock fragments (Brierley, 2008). In comparison, heap leaching requires more significant capital investment, as ore is crushed to smaller particle sizes (≤

19 mm). When compared to dump leaching, the heap leaching environment is more controlled, which in turn shortens the time taken for metal recovery from decades/years to months (Kinnunen,

2004; Rawlings, 2002; Rawlings et al., 2003; Riekkola-Vanhanen, 1999; Wiertz et al., 2001).

However, the distinction between dump and heap bioleaching is becoming vague with the many technical enhancements that have been applied to the practice of dump bioleaching such as aeration, pre-conditioning of the ore with an acidified ferric iron solution, and in some cases, crushing (Brierley, 2008). For example, the Escondida Mine dump bioleaching operation in Chile includes technical improvements to enhance microbial activity and copper recovery (Brierley,

2008).

Heap permeability can be managed through ore crush size, heap stacking, agglomeration, solution application management (Petersen, 2016), and the use of suitable filler materials, such as quartz (Hao et al., 2017). Various irrigation methods (sprinkler/dripper, continuous/intermittent, unsaturated/flooding), aeration regimes (continuous/intermittent), heap construction (single lift/multi-lift) and inoculation strategies (through agglomeration/irrigation, intermittent/continuous) can be used to optimise bioleaching efficiencies.

2.3.2 Dump and heapACCEPTED leach operations MANUSCRIPT

Heap bioleaching has been used worldwide to extract copper from secondary copper ores which contain chalcocite (Cu2S) and covellite (CuS) (Brierley, 2008), and more recently explored in pilot- and demonstration-scale for primary sulfide chalcopyrite (CuFeS2) (Panda et al., 2015). The majority of dump and heap bioleaching mines have been located in Chile. Some operations have also been established for example in Australia, USA, China, Peru, Myanmar and Finland (Brierley,

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2008; Panda et al., 2015; Puhakka et al., 2007). Some of the heap/dump leach operations have permanently ceased due either to ore depletion or the heap leach failing (Brierley, 2008).

Apart from copper leaching, heap leaching has also been applied to extract nickel, zinc and cobalt from complex black shale-hosted deposit at Talvivaara Mine (now known as Terrafame

Mine) in Finland since 2008 (Puhakka et al., 2007). Heap bioleaching of base metals has typically been conducted for whole ores. Moreover, Newmont Mining Corporation used whole ore heap biooxidation for refractory sulfidic gold ore at Carlin, Nevada 2000-2010 (Brierley, 2008; Logan et al., 2007; Roberto, 2017a). The operation used patented Newmont's BIOPRO technology to belt agglomerate ore with microbial inoculum. Stacking of heaps was conducted either using a front- end loader or radial stackers (Roberto, 2017a). The heat generated by the oxidation of pyrite and arsenopyrite sulfides initially favoured the mesophiles, which were succeeded by the moderate thermophiles when temperatures reached ~ 50 °C, and thermophiles at over 60 °C. The heap cooled when the sulfide minerals were depleted, and the ore was ready for cyanide leaching

(Brierley, 2008).

The heap bioleaching and biooxidation of fine materials have been facilitated by the development of the GEOCOAT® process which involves coating inert rocks with concentrates before heap construction (du Plessis et al., 2007; Olson et al., 2003). The GEOCOAT® process was implemented at the African Pioneer Mining’s Agnes mine in South Africa for the biooxidation of refractory gold ores (Harvey and Bath, 2007), and at Kumba Resources’ Rosh Pinah Mine in

Namibia for sphaleriteACCEPTED concentrates (Harvey et MANUSCRIPTal., 2002). The process has also been trialled for bioleaching chalcopyrite concentrates (Johansson et al., 1999; Petersen and Dixon, 2002). A patented thin-layer leaching method invented by Johnson (1977) has been applied at the Sociedad

Minera Pudahuel Lo Aguirre Plant in Chile for bioleaching mixed and secondary copper sulfide ores

(Bustos et al., 1993). The method includes mixing crushed ore (< inch with at least 25% > 4 mesh) with a small amount of acid, dehydrating and indurating the ore for two days. The hardened ore is

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then spread on a permeable substrate as a 0.5-1 m thick layer, and acidic liquor is percolated through the thin ore bed for metal extraction (Johnson, 1977). At the Sociedad Minera Pudahuel Lo

Aguirre Plant, copper recoveries of 75-85 % were achieved after 180-250 days leaching, depending on mineralogical composition, copper grade and acid consumption of the ore (Bustos et al., 1993).

2.3.3 Temperature control

Heap leaching with thermophiles (>55 °C) has been suggested to be the most comprehensively demonstrated technology for accessing the copper in chalcopyrite from marginal ores (du Plessis et al., 2007). As heat generation is mainly the result of microbial activity, enough sulfur must be available for sulfur oxidation (Brierley, 2008; du Plessis et al., 2007; Petersen and Dixon, 2002).

Addition of sulfur can be achieved by blending the marginal ore with pyrite or arsenopyrite (du

Plessis et al., 2007). BHP Billiton has successfully demonstrated high-temperature chalcopyrite heap bioleaching in a 7-ton pilot-scale column (Dew et al., 2011). National Iranian Copper

Industries Company and Mintek have also demonstrated the process in 20,000-ton demonstration scale heaps at the Sarcheshmeh Copper Complex in Iran (Gericke et al., 2009). However, to the best of our knowledge the process is yet to be applied in full scale (Ghorbani et al., 2016).

In cold climates and at high altitudes plastic sheeting may be applied to the surface of the heap to help conserve heat generated through the oxidation of reduced sulfur compounds (du

Plessis et al., 2007), while in arid climates plastic sheets can help decrease evaporative losses.

Covers on heap surfaceACCEPTED have been applied, e.g. MANUSCRIPT in Escondida mine in Chile (Watling, 2015).

Geobiotics developed a technology called GEOLEACHTM to maximise heat conservation through the control of irrigation and aeration rates in a heap (Harvey and Bath, 2007). Geobiotics developed this technology because although the oxidation of sulfides can release enough energy to raise temperatures, the lack of heat management prevented the expected temperature rise in heaps

(Harvey and Bath, 2007). A GEOCOATTM demonstration plant was constructed and commissioned

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at the QueMibrada Blanca copper mine in Chile in 2009 (Ghorbani et al., 2016). Murray et al.

(2017) have recently proposed the capture and use of solar thermal energy to raise the temperature in heaps and increase copper extraction rates. The authors simulated a heap bioleaching system, which included ponds and a solar thermal collector field using HeapSim and

TRNSYS. The maximum copper extraction was calculated to increase from 67 % to 85 % over a year with the installation of the solar thermal field (Murray et al., 2017).

2.3.4 Heap aeration strategies

Hollitt et al. (2009) patented a heap leaching process which combines the use of aerated and non-aerated heaps to optimise copper recovery. The aerated heap is aimed at oxidising pyrite and ferrous iron to generate ferric iron while recovering copper. The aerated heap also produces heat and acid, and allows evaporation to maintain the water balance. The non-aerated heap is operated to leach copper under conditions that minimise pyrite oxidation. This heap generates ferrous iron and consumes acid and sulfates. According to Hollitt et al. (2009), the proposed process achieves good leach rates for sulfidic copper-containing ores. Moreover, it does not consume excessive reagents due to uncontrolled reactions of other minerals present, such as iron-containing minerals

(e.g. pyrite) or acid or sulfate consuming minerals (e.g. amphibole, chlorite, biotite, muscovite and phlogopite). The process also allows the amount of iron, sulfuric acid, mineral acidity and neutral salts extracted into the leach liquor to be controlled and reduces the demand of acidifiers or the need to bleed an excessACCEPTED of these products from MANUSCRIPT the leach circuit for neutralisation (Hollitt et al.,

2009). To the best of our knowledge, the process has not yet been applied for commercial-scale bioleaching.

2.3.5 Heap modelling

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Mathematical models have the potential to determine the impact of changing parameters in a bioheap and evaluate if there are practices that could improve metal extraction (Watling, 2006).

For reviews on modelling, see Ritchie (1997), Dixon (2003), and Watling (2006). Heap bioleaching of sulfide ores has been described using many mathematical models, which incorporate sub- processes, such as chemistry, microbiology, and hydrodynamics (Watling, 2006). Optimally heap models would account for both micro- and macro-scale processes as well as their interactions

(Watling, 2006). The HeapSim model (Dixon and Petersen, 2003, Dixon and Petersen, 2004) includes both conceptual and mathematical descriptions of various processes that are relevant for heap leaching. Multidimensional models, such as the Phelps Dodge copper stockpile model

(Bennett et al., 2003) and the CSIRO Heap model (Leahy et al., 2007; Watling, 2006) have enabled the simulation of two phases (gas and liquid). While the Phelps Dodge model has helped to simulate conditions that may occur in separate lifts that are spatially separated, the CSIRO Heap

Model has been applied to study the conditions around individual solution drippers and air spargers (Watling, 2006).

Recently, data mining tools have been successfully used for improving the performance of industrial-scale bioleaching systems (Sota et al., 2013). Data mining is a statistical approach for generating new useful knowledge or information from sizeable multivariate data sets, which would otherwise be difficult to analyse manually (Witten and Frank, 2000). Using data obtained from the minerals analysis, Soto et al. (2013) adopted a "hierarchical clustering" data mining approach to demonstrate a relationshipACCEPTED between the microbial MANUSCRIPT activity and the dissolution rate of sulfide ores of an industrial bioleaching heap at Escondida mine in Chile (Soto et al., 2013). Demergasso et al.

(2017) developed a data mining based decision support system (DSS) that can provide operational recommendations based on historical data for optimising heap bioleaching processes. The DSS utilises information on ore mineralogy, metallurgical parameters, operational conditions,

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physicochemical parameters as well as microbiological data. The DSS has been validated using data collected at the Escondida mine (Demergasso et al., 2017).

2.4 In-place and in situ leaching

In-place and in situ leaching have recently gained increasing attention as viable options for metal recovery from deep-buried ore bodies (> 1 km below the land surface) and other low‐grade ore deposits. These are often considered inaccessible or uneconomical for conventional mining technologies relying on drilling, blasting, excavation and transporting the ore to the ground surface for processing (Johnson, 2015; Pakostova et al., 2017b). However, in situ leaching of metals is not a novel concept. For example, in situ recovery of uranium has been practiced since the early 1960s

(Mudd, 1998) and activity of microorganisms in underground stope leaching has been knowingly utilised in uranium recovery already in late 1970s (Tuovinen and Bhatti, 1999).

The concepts of in-place and in situ leaching of metals entail drilling wells, i.e. boreholes into the ore body for delivery of leaching solution and extraction of metal-rich leachate to the surface for metal harvesting (Johnson, 2015). Traditionally, the term in-place leaching has been used for applications where the ore body is fractured, e.g. through blasting whereas the term in situ leaching was used initially for metal recovery from unfractured ore bodies (Gallant and

Wadden, 1984). However, nowadays the term in situ leaching has also been used to refer to leaching of fractured ore bodies (Johnson, 2015). Hereafter the term in situ recovery (ISR) is used to refer to both in-placeACCEPTED and in situ leaching. Blasting,MANUSCRIPT crushing and grinding used in metal mining today has been estimated to consume 5-7 % of globally available energy and thus avoiding these process steps could significantly increase economic benefits and reduce environmental impacts of mining (Johnson, 2014; Johnson, 2015). As ISR enables the recovery of the target metals without excavation of the host rock, generation of tailings can be reduced or even prevented (Johnson,

2015; Haschke et al., 2016).

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2.4.1 Engineering and exploring in situ recovery (ISR)

Successful ISR of target metals requires the presence of fractures or channels in the ore to enable efficient lixiviant flow and sufficient contact between the ore and the lixiviant. If the deposit is not naturally porous or permeable, the channels can be created by blasting (Sand et al.,

1993). It has also been suggested that channels could be generated, e.g. by hydraulic fracking, which is already used in the recovery of gas and oil from deep-buried shales but is known to include a risk of groundwater contamination (Johnson, 2015). Efficient control of leaching solutions is of utmost importance on both economic and environmental viewpoint, as the solutions contain the valuable metal products, which can cause significant groundwater contamination if unintentionally discharged to the surroundings. Site hydrogeology and geochemistry should be well known and preferably include impermeable geological barriers, such as clays or shales, above and below the deposit to ensure that lixiviant cannot leak to the surrounding aquifers (Mudd, 1998). In stope leaching, where a stope filled with broken ore is repeatedly flooded, drained and allowed to rest (Chien et al. 1990), losses of leaching solutions can be minimised by, e.g. resin-coated concrete bulkheads (Sand et al., 1993) or by building dams (Miller, 1986). Also, the bores need to be designed and constructed with durable materials and have perforations enabling water flow only on the zone of mining (Mudd, 1998). The extraction wells should typically have 0.5-5% higher extraction volumes compared to injection volumes, and the injection wells need to be surrounded by several extractionACCEPTED wells (Mudd, 1998; Hascke MANUSCRIPT et al., 2016). Typical injection and extraction well geometries used in ISR of uranium include, for example, 5-spot and 7-spot patterns, in which injection well in the middle is surrounded by four or six evenly distributed extraction wells, respectively (Mudd, 1998).

Underground activity of acidophilic microorganisms has been utilised in stope leaching of copper and zinc (Miller, 1986; Sand et al., 1993). Sand et al. (1993) reported 10 % and 78 %

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mobilisation efficiency for copper and zinc after 650 days, but due to liquor losses (e.g. via small cracks in the bulkheads) recovery efficiencies were lower, being 5.7 % and 52 %, respectively.

Most recently proposed microbially-mediated ISR approaches are based on indirect leaching using microorganisms that are cultivated in bioreactors above ground to produce lixiviants. The lixiviants are then injected to the ore body facilitating solubilisation of target metals or pre-treatment of the ore to make it more amenable for leaching with chemical lixiviants (Kaksonen et al., 2014d,

Haschke et al., 2016, Pakostova et al., 2017a; Pakostova et al., 2017b). For example, Haschke et al.

(2016) reported about an ongoing project, which uses microbially produced chelating agents (e.g. enzymes, carbonic acids, siderophores and phospholipids) for indirect recovery of rare earth elements form ion-adsorption clays.

Kaksonen et al. (2014d) explored a submerged biooxidation concept mimicking vat, in-place or in situ type environments for refractory gold ores. They demonstrated that a biologically generated acidic ferric iron solution could be used to oxidise crushed refractory gold ore in upflow columns with and without aeration. Similarly, the potential of indirect bioleaching in unaerated columns using an acidic microbiologically-generated ferric iron solution was demonstrated for polymetallic black schist sulfide ore containing Fe, Zn, Ni, Cu, Ni and Co as the principal base metals (Pakostova et al., 2017a), and saline, calcareous copper sulfide ore (Pakostova et al.,

2017b). However, the latter ore type required a water washing step to remove soluble salts and an acid leaching stage to remove calcareous minerals and other acid soluble salts before the bioleaching step to ACCEPTEDprevent the inhibition of theMANUSCRIPT bioleaching microorganisms by high pH and salt concentrations (Pakostova et al., 2017b). For the polymetallic black schist sulfide ore, metal recovery efficiencies were 75 % for Fe, 93 % for Mn, 75 % for Zn, 55 % for Cu, 79 % for Ni, and 88 % for Co after 10 days of acid leaching followed by 106 of indirect bioleaching (Pakostova et al.,

2017a). Approximately 13 % of Cu and further 39-59 % Cu was recovered from the saline, calcareous copper sulfide ore using sulfuric acid for 3 days and indirect bioleaching for 40 days,

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respectively (Pakostova et al., 2017b). However, it should be noted that column studies with crushed ore can give too optimistic results on leaching efficiencies, as the contact area between the ore and the lixiviant is significantly higher than anticipated for fractured ore bodies.

Demonstration of the three-stage in situ bioleaching process described by Pakostova et al. (2017b) using a real fractured underground ore block is currently ongoing in Rudna mine, Poland as a part of the European Union Horizon 2020 project “BIOMOre” (BIOMOre, 2017).

2.4.2 Commercial scale microbially catalysed ISR applications

Microbially catalysed ISR has been used in large scale especially for uranium recovery. At

Agnew Lake Mine in Ontario, Canada, uranium bioleaching was implemented to a virgin ore body in the late 1970s. The mine utilised surface heap leaching and underground stope leaching.

However, because of the loss of leach solution to the fractured ore body, the mine site was closed within a few years (Tuovinen and Bhatti, 1999). Underground stope bioleaching of uranium from a fractured ore was also practised at the Denison Mine in the Elliot Lake area, Ontario in the 1980s and early 1990s (Tuovinen and Bhatti, 1999). ISR of base metals using biogenic lixiviants has also been considered economically attractive since the 1980s when underground tests of microbial ISR of Cu and Zn from low‐grade ore reserves were carried out at the Prieska copper-zinc mine situated at 1500 m above sea level in the northern Cape Province of South Africa (Miller, 1986). However, the leaching was discontinued after 9 months due to the collapse of safety pillar (caused by other work in the mine area)ACCEPTED and low metal extraction MANUSCRIPT efficiency. To the best of our knowledge, there are currently no large-scale ISR operations that actively utilise microbial activity.

2.4.3 Factors impacting subsurface microbial activity

Although most proposed in situ bioleaching and biooxidation approaches rely on indirect leaching, microorganisms can be injected into the subsurface with the biologically-produced

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lixiviant. In the case of acidic ferric iron leaching of metal sulfide ores, in situ microbial activity could be useful, as it would decrease the accumulation passivation layers consisting of elemental sulfur, polysulfides and jarosite that could otherwise complicate lixiviant flow and reduce leaching efficiency (Stott et al., 2000). Kaksonen et al. (2014d) reported that the presence of bioleaching microorganisms and the boosting of their activity by underground aeration enhanced the oxidation of pyrite and decreased the accumulation of elemental sulfur. However, delivering oxygen to the ore body would be very challenging to very deep ore bodies (Johnson, 2014), and it is more likely that anaerobic biooxidation of elemental sulfur using ferric iron as an electron acceptor would occur (Zhang et al., 2017). Another vital factor characteristic to deep-buried ore bodies is high hydrostatic pressure (Johnson, 2015). Zhang et al. (2017) reported that mixed cultures of mesophilic and thermophilic acidophilic bioleaching microorganisms could oxidise reduced sulfur compounds and reduce soluble ferric iron at a pressure of 100 bar, although ferric iron reduction efficiency and cell numbers were lower than at atmospheric pressure. Further research on pressure tolerance of bioleaching microorganisms is still needed.

As bioleaching organisms may retain their activity in the subsurface, recent studies have also been conducted to delineate efficient methods for eliminating the introduced bioleaching organisms from the subsurface after completion of in situ bioleaching. Bomberg et al. (2017) showed that saline water (65 g L-1 Cl-) could be used to inactivate bioleaching microorganisms, whereas Ballerstedt et al. (2017) reported that low concentrations of formic acid added simultaneously with ACCEPTEDNaCl eliminates bioleaching MANUSCRIPT prokaryotes efficiently.

3 Wastes as resources through bioleaching

As the global population increases, the consumption of raw materials also increases, as does the generation of waste. Waste generated by industry and valuable wastes such as electronic waste (e-waste) are increasingly being targeted for urban mining. Mining metals from wastes

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supplements dwindling volumes and quality of primary mineral reserves as these wastes often contain metals at concentrations higher than found in nature (Ongondo et al., 2015; Sommer et al.,

2015). Recycling of these products is now crucial to secure the future supply of critical materials such as rare earth elements, cobalt, magnesium and platinum group metals (European

Commission, 2017), as well as strategic metals not yet considered critical, such as lithium (Swain,

2017).

In the same way that acidophilic bioleaching microorganisms have been used for the processing of base metal sulfides and oxides, refractory gold ores and pyrite-bearing coals, bioleaching is increasingly being considered for the recovery of metals from various metallurgical waste materials and by-products such as e-wastes and smelter slags (Bryan et al., 2015; Erüst et al., 2013; Kaksonen et al., 2017).

Near-to-full recovery of metals from metallurgical wastes can be achieved using pyrometallurgy or hydrometallurgy with strong acids and alkalis. Often these methods also result in secondary emissions such as dioxins and furans or contaminating effluents. Moreover, these traditional methods are energy intensive and sensitive to changes in feedstock, or require high concentrations of metals to be economically feasible (Pagnanelli et al., 2016; Vestola et al., 2012).

Also, pyrometallurgy is relatively unselective, and often minor or inexpensive metals are not recovered (Pagnanelli et al., 2016; Zeng et al., 2014).

Bioleaching has been investigated for complex, polymetallic metallurgical wastes, such as e- waste, as a more environmentallyACCEPTED benign process, MANUSCRIPT and the results have been promising, albeit variable. Inhibition of microbial consortia during e-waste bioleaching has been reported. Inhibition can occur due to high pulp densities (e.g. above 2 %), organic contaminants and acid consumption by the waste, and a lack of essential growth substrates such as ferrous iron and sulfur (Valix,

2016; Niu et al., 2014; Xin et al., 2012; Zeng et al., 2012). Leaching times are often longer, with

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lower pulp densities and leach yields than can be obtained by hydrometallurgy with strong acid or alkali solutions (e.g. Mishra et al., 2007; Niu et al., 2014; Zeng et al., 2012).

Recently, non-contact spent medium bioleaching has been proposed as another technique for the recovery of metals from spent battery and e-wastes (Hong and Valix, 2014; Natarajan and

Ting, 2015). In this method, biological reagents such as biogenic cyanide, ferric iron or acid are generated by microbial cultures in the absence of the waste to be processed, and the cells are removed from the solution which is then used to leach metals from the waste. Indirect, non- contact bioleaching allows the waste pulp density to be increased to a level used in more traditional leaching processes (e.g. > 10 %), and also avoids microbial inhibition, allowing maximum generation of biogenic reagents. Also, higher yields can be obtained in a shorter period when compared to direct bioleaching of these wastes, and there is the potential to harness low- cost substrates such as mine wastes to drive the production of these bioreagents (Natarajan and

Ting, 2015). Despite advances in this field, further research is required to optimise bioleaching and the use of biological reagents for the recovery of metals from e-waste.

Metal-containing waste streams are produced by the mining, energy production and recycling industries, and these wastes often pose a significant risk to the environment if not treated appropriately (Kaksonen et al., 2017; Vestola et al., 2012). These waste streams contain a significant amount of value, with slags from copper and steel smelting often containing nickel, zinc, copper and cobalt, along with other trace metals that could be recovered, but vary in composition, makingACCEPTED reclamation difficult by either MANUSCRIPT hydrometallurgy or pyrometallurgy. Bioleaching has been hypothesised as an alternative and lower cost approach to meet variability in acid demand and composition, particularly, if elemental sulfur, reduced inorganic sulfur compounds or pyrite are used as the substrates to drive biological sulfur oxidation and acid generation (Kaksonen et al., 2017; Vestola et al., 2010, Vestola et al., 2012). Other waste streams, such as municipal solid waste incineration fly ash, effluents from electro-plating, manufacturing, refining,

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petrochemical and pharmaceutical industries also represent a significant secondary resource for metals and bioleaching has shown relatively good metals extraction yields from these wastes (Lee and Pandey, 2012).

Bioremediation using naturally-occurring microbial consortia, natural attenuation or bio- augmentation is often a low-cost, passive option for the treatment of contaminated sites containing heavy metals from industrial activity (Brune and Bayer, 2012; Navarro et al., 2013).

Similar to leaching of minerals, iron and sulfur oxidising microorganisms can catalyse the solubilisation of metals from contaminated soils under aerobic conditions, and iron-reducing microorganisms can catalyse this process under anaerobic conditions. These processes have been proposed for the treatment of mine tailings, acid mine drainage sites, or for the recovery of metals from anaerobic sludges (e.g. Akcil et al., 2015; Diaz et al., 2015; Dixit et al., 2015; Fonti et al.,

2016).

Though the use of bioleaching for the recovery of value from secondary resources and for urban mining is gaining traction, additional techno-economic, feasibility and environmental impact assessments are also required to determine how these processes compare to traditional methods.

However, bioleaching from secondary resources can be considered a significant area of research for securing critical metals as we move towards a future of limited primary resources and strive towards a circular economy.

ACCEPTED MANUSCRIPT 4. Bioprospecting, adaptation, engineering and storing of bioleaching microorganisms

4.1 Bioprospecting novel biocatalysts

Bioprospecting is the systematic search for economically valuable genetic and biochemical resources from nature. The extreme physicochemical nature of bioleaching liquors is highly toxic to the vast majority of life forms (Johnson, 2014). Bioprospecting from extreme environments,

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therefore, allows for the discovery of unique microorganisms that can tolerate the various stresses such as low pH, high metal loads, extreme temperatures and high salinity often present in such environments (Dopson, 2016; Rea et al., 2015; Zammit and Watkin, 2016).

The natural and man-made environments for the bioprospecting of iron- and sulfur oxidising microorganisms to be used for biohydrometallurgical operations have been extensively reviewed

(Dopson 2016; Hedrich and Schippers, 2016; Johnson and Quatrini, 2016). Natural environments include geothermal sites such as active volcanoes, deep-sea hydrothermal vents as well as cave systems, acidic brine lakes, acid sulfate soils and naturally exposed sulfide ore deposits. Man- made environments include metals and coal mines with acid mine drainage, pit lakes, mine heaps and waste rock dumps. Both types of environments are a rich source of microorganisms that are potentially adapted to high concentrations of metals and salt, low pH and extremes in temperature, and are therefore ideal for biohydrometallurgical operations (Hedrich and Schippers,

2016).

As higher grade ore reserves diminish, extraction of metals from lower grade ores has become a focus of the mining industry. The presence of high levels of chloride in ores and process waters in regions such as Western Australia and Chile has led to the requirement to bioprospect iron- and sulfur oxidising microorganisms that are able to tolerate high levels of chloride stress as well as low pH (Zammit and Watkin, 2016; Quatrini et al., 2017). Chloride tolerant acidophilic microorganisms would potentially also allow the use of seawater at mines in regions of fresh water scarcity. ACCEPTED MANUSCRIPT

As discussed by Zammit and Watkin (2016), the few geographical locations that provide both low pH and high chloride concentrations include acidic brine lakes, salt lakes, volcanoes near seawater and bioleaching environments. Acidihalobacter prosperus DSM 5130 was the first halotolerant, iron- and sulfur-oxidising acidophile to be isolated from a shallow geothermally heated seafloor at the beach of Porto di Levante, Vulcano, Italy (Huber and Stetter, 1989). Two

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other isolates, Acidihalobacter prosperus DSM 14174 (strain V6) and Acidihalobacter ferrooxidans

DSM 14175 (strain V8) were also isolated from acidic hydrothermal vents on the shore of the

Aeolian Island of Vulcano, Italy (Simmons and Norris, 2002). The strains were found to tolerate up to 45 g/L chloride ion and leach pyrite at up to 30 g/L chloride concentrations (Khaleque et al.,

2018). Furthermore, Acidihalobacter prosperus strain F5 was the first halotolerant, iron- and sulfur oxidising acidophile to be isolated from a mixed culture obtained from a saline drain in the Yilgarn

Crater region of Western Australia (Khaleque et al., 2017; Zammit et al., 2009). Ac. prosperus strain

F5 has shown the ability to withstand 45 g/L chloride ion stress at low pH. Also, this strain can leach base metals under chloride stress from pentlandite (at up to 45 g/L chloride ion), pyrite (at up to 30 g/L chloride ion) and from the recalcitrant ore, chalcopyrite (at up to 18 g/L chloride ion)

(Khaleque et al., 2017). Bioprospecting, therefore, leads to the discovery of novel indigenous microorganisms for potential use in biohydrometallurgy.

4.2 Adaptive evolution of bioleaching microorganisms

Adaptive evolution is a standard approach to improve microbial phenotypes, growth characteristics and tolerances to environmental stress and growth inhibitors. Adaptive evolution experiments consist of serial cultivation of a microorganism under defined conditions for prolonged periods of times (e.g. weeks to years) which allows for the selection of improved phenotypes as the growth conditions are continually changed (Dragosits and Mattanovich, 2013;

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Rea et al. (2015) employed adaptive evolution to increase the salt tolerance of bioleaching microorganisms with the view of promoting iron and sulfur oxidation in leaching environments with low-grade minerals, where fresh, potable water was scarce for processing. Also, adaptation to sulfate has also been targeted by the mining industry to use high sulfate-containing solutions derived from oxide processes to irrigate sulfide heaps (Adaos, 2013). Pure isolates and mixed

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strains derived from environmental samples were serially sub-cultured at increasing concentrations of sea salts, NaCl or magnesium sulfate (Rea et al., 2015). Enrichment cultures of mesophilic acidophilic iron and sulfur oxidising microorganisms resistant to up to 70 g/L sea salts and 350 g/L MgSO4.7H2O were obtained, able to tolerate considerably higher salt concentrations than previously reported (Rea et al., 2015). The efficiency of chalcopyrite bioleaching using high- sulfate adapted cultures was subsequently tested at elevated sulfate concentrations at three different temperatures (30 oC, 45 oC and 60 oC). The adapted cultures bioleached chalcopyrite at high sulfate concentrations with similar efficiency as previously reported in the literature for non- adapted cultures in a low-sulfate background (Boxall et al., 2017).

Adaptive evolution has also been used to increase acid resistance of archaea

Metallosphaera sedula and Sulfolobus solfataricus, which resulted in strains that grew well at pH

0.9 and pH 0.8, respectively (Ai et al., 2016; McCarthy et al., 2016). Enargite (Cu3AsS4) bioleaching carried out at pH 1.20 with the acid-adapted M. sedula strain resulted in 23.8 % higher leaching efficiency than the non-adapted parental strain (Ai et al., 2016). Feng et al. (2015) isolated

Acidithiobacillus spp. strains with enhanced adsorption performance to ore particles from serial passages of cultures with increasing elution strength. Bioleaching with the adapted cultures resulted in 25.8-36.5% increase in copper solubilisation from chalcopyrite compared to non- adapted cultures (Feng et al., 2015).

Despite the simplicity, adaptive laboratory evolution can take a long time (2-3 years) to obtain the desired ACCEPTED phenotypic improvements. MANUSCRIPT Several mutagenic agents (e.g. UV radiation or alkylating agents) can be used to increase the frequency of mutations. Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacterial Haloferax mediterranei resulted in a strain that had its spontaneous mutation rate increased up to 500-fold after mutagen exposure (Nieto et al., 1992). For microorganisms with genetic information and tools available, synthetic biology may provide an even faster way to improve microbial function.

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4.3 Engineering bioleaching microorganisms

Synthetic biology, defined as the synthesis of complex biologically-inspired systems that display functions which do not exist in nature (Serrano, 2007), has transformed the way scientists engineer naturally-existing microorganisms. There is growing interest in using synthetic biology tools for designing and constructing robust bioleaching microorganisms (Dunbar, 2017). Due to the limited molecular tools currently available for bioleaching microorganisms, efforts to genetically modify them are still lagging behind other microorganisms. Several initial studies have been conducted on the construction of shuttle vectors for plasmid conjugation from Escherichia coli into

A. ferrooxidans (Peng et al., 1994a; Peng et al., 1994b) or A. caldus (Meng et al., 2013; Zhang et al., 2014). Arsenic resistance and rusticyanin genes were overexpressed using the constructed vectors, resulting in increased tolerance to arsenic or enhanced iron oxidation activity, respectively. The arsenic tolerance improvement was low (resistant up to 26 mM NaAsO 2 for A. ferrooxidans and up to 45 mM for A. caldus compared to <10 mM of original strains (Peng et al.,

1994a; Zhang et al., 2014). However, future synthetic biology efforts can open opportunities to improve bioleaching of arsenic-bearing ores further.

Genetic tools for sulfur oxidising Sulfolobus spp. are more developed than those for other bioleaching microorganisms, as Sulfolobus spp. represent model organisms for studying how archaea survive in extreme environments (Reed et al., 2013). Transformation efficiencies obtained with the establishedACCEPTED electrotransformation protocols MANUSCRIPT for Sulfolobus are slightly better than with

Acidithiobacillus spp. The efficiencies were 102 - 104 transformants per g DNA for transformation of pRN1 vectors in S. acidocaldarius (Berkner and Lipps, 2008), compared to 1 successful transformant of 30 for transformation in A. ferrooxidans (Peng et al., 1994b). The challenge of genetic modification of these archaea is dependent on other factors as well. The selection process is vital due to the limited stability of antibiotics under the growth conditions of Sulfolobus (e.g. pH

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3 and 80 oC). Also, the genetic stability is a challenge due to the high numbers of mobile genetic elements which can produce spontaneous mutations. Lastly, the presence of restriction/modification activity in the hosts (e.g. due to the presence/absence of restriction endonuclease) also requires consideration for successful genetic modification of this genus

(Berkner and Lipps, 2008).

The understanding of molecular mechanisms for iron and sulfur oxidation, acid and metal tolerance and carbon fixation and degradation in acidophilic bioleaching microorganisms is improving (Dopson et al., 2003; Levicán et al., 2008; Cárdenas et al., 2012; Castro et al., 2017;

Quatrini et al., 2017). However, considerable research is still required to characterise all species relevant to bioleaching environments fully. For this reason, the use of synthetic biology for engineering these species is still a frontier science, gaining momentum as the need for microorganisms that can tolerate ever extreme conditions continues to build. Despite the difficulty in establishing genetic tools for these acidophiles, in combination with genome-based models, it should be feasible soon to construct robust acidophilic bioleaching microorganisms that are resistant to high salt and metal concentrations and to improve bioleaching efficiencies.

4.4 Preservation of bioleaching microorganisms

Generally, acidophilic bioleaching microorganisms are maintained as live cultures, with continual subculturing, and storage at temperatures below optimal growth requirements, with slow release substrates toACCEPTED prolong viability (Johnson, MANUSCRIPT 1995). Despite the recognised need for novel microbial cultures for biotechnical applications, their efficient storage after discovery has often been neglected, and the development of methods for preservation of these unique microorganisms has not been widely undertaken (Heylen et al., 2012). Bioleaching microorganisms are an example of a microbial group which often exhibit poor viability following preservation. Moreover, standard methods of preservation often fail in the case of acidophilic thermophilic microorganisms due to

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differences in cell structure and overall sensitivity of these microorganisms to ultra-low temperatures and desiccation processes (Balfour-Cunningham et al., 2017; Spring, 2006). A summary of the standard preservation methods is shown in Table 1.

Most preservation studies for bioleaching microorganisms have been conducted on pure cultures or those with limited diversity, and the results obtained were variable for individual species. In each study, the method of preservation tested, the cryoprotectants used to protect the cells and the storage time were all variable, and no standard preservation methods exist for any of the bioleaching microorganisms, or mixed microbial consortia that may have functional relevance for minerals industry (Morgan et al., 2006).

Wu et al. (2008) tested liquid nitrogen cryopreservation with glycerin at -196 °C on pure cultures of A. thiooxidans, A. ferrooxidans and Acidianus brierleyi. In this study, glycerin was used as the cryoprotectant instead of the commonly used glycerol as autotrophic bioleaching bacteria are generally inhibited by the presence of organic compounds (Rawlings, 2005). After 7 days of storage, the preserved cells were revived, and their measured growth, as well as ferrous or sulfur oxidising activities, were shown to be comparable to those of the unpreserved cultures. However, the storage period was relatively short, and it is likely that longer storage times could impact the overall revival rate of preserved cultures.

In another study, Zeng et al. (2009) tested the preservation of A. caldus using four different methods. The methodsACCEPTED were live culture maintenance, MANUSCRIPT sterile sand tube preservation (concentrated cell sample mixed with sterile sand and stored in a glass tube at room temperature), cryopreservation with mannitol (at -20 °C and -72 °C) and freeze-drying. The study showed that the sterile sand tube method was the best preservation method for short-term storage of A. caldus (6 months) and freeze-drying was the best method for long-term preservation (< 15 months), with 32

% and 17 % cell viability, respectively. A high cell death rate was reported for cells frozen at -20 °C, which was attributed to the formation of ice crystals and damage to cell structure, but the viability 30

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of cells was improved when the temperature was further decreased to -72 °C (12 % viability after

12 months). However, after 15 months cells could not be revived from preserved cultures that were stored at -72 °C. In the bioleaching activity experiments, an apparent lag in the growth time was observed for preserved cells when compared to non-preserved cells. The authors concluded that preservation affects cell activity and prolongs the lag phase but does not affect the long-term ability for Cu bioleaching.

A study by Zeng et al. (2010) showed similar results when testing the preservation and bioleaching activity of a moderately thermophilic mixed culture enriched from several chalcopyrite mines in China. Cells were freeze-dried, frozen at -72 °C and revived after 3, 6, 9, 12 and 15 months. Cell viability was shown to be directly proportional to the preserved cell storage time, which indicated that even successfully preserved cultures required consistent and regular maintenance to ensure proper revival and recovery of activity. Also, the preserved cells also had an extended lag phase compared to unpreserved cells, but the overall cell density and activity of the cultures reached similar maxima. The bioleaching tests compared cells preserved for 15 months to unpreserved cells. Bioleaching experiments confirmed a loss or slowing of bioleaching efficiency, with preserved cells able to extract only 42 % of copper from the chalcopyrite after 20 days, compared to 75 % Cu extraction by the non-preserved cells. However, extraction efficiency after 80 days was similar.

Most recently, Balfour-Cunningham et al. (2017) evaluated the cell viability of preserved ACCEPTED MANUSCRIPT mesophilic, moderately thermophilic and thermophilic salt-tolerant, mixed microbial cultures capable of bioleaching chalcopyrite. The preservation methods tested were cryopreservation with glycerol (-80 °C), liquid drying and cold storage (4 °C). The viability and activity of preserved cells were compared to non-preserved live cultures after 5 weeks of storage, and cell viability was over

60 % for all methods tested. Overall, cryopreservation with 10 % glycerol resulted in the highest cell recovery across all temperatures tested. The study showed that live culture maintenance 31

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resulted in the highest copper leach yields from chalcopyrite across all temperatures. However, it was also shown that the various preservation methods impacted the overall leaching activity of the cultures differently, and this was dependent on growth temperature. After live culture maintenance, cold storage, liquid drying and cryopreservation resulted in the highest leaching efficiencies following revival for mesophiles, moderate thermophiles and thermophiles, respectively. The authors recommended assessing the microbial community composition before and after preservation to determine the impact of preservation methods on microbial species diversity and bioleaching capacity.

There is variability observed with cell revival and viability for bioleaching microorganisms, which is dependent on the method of preservation, reagents used and time stored. This variability demonstrates the difficulty surrounding the maintenance and storage of functionally relevant cultures for biohydrometallurgical applications. Each species behaves differently under different preservation conditions, and this means that preservation of mixed microbial cultures is particularly difficult. Similarly, treatment of the cells after preservation is also essential, as incorrect revival and persistence of cryoprotectants into the growth phase of the culture can result in inhibition, prolonged lag phase and poor extraction efficiencies when compared to non- preserved/live maintenance cultures (Balfour-Cunningham et al., 2017).

Preservation of microorganisms can safeguard the long-term accessibility of cultures and provide a "pool" of resourcesACCEPTED for various biotechnical MANUSCRIPT applications (Heylen et al., 2012). Preservation also ensures the reproducibility of experiments and the sustainability of research and technology development that relies on biological resources (Smith and Ryan, 2012). Method development for the preservation of unique cultures ensures that microorganisms are not lost as a result of contamination, equipment failures or accidental loss due to disasters. Moreover, it can minimise possible genetic changes through gene mutations or loss of plasmids (Heylen et al.,

2012). The preservation of cultures may also be necessary for protecting intellectual property that 32

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is based on specific microbial cultures (Heylen et al., 2012). Culture preservation can also decrease the time, energy and material costs associated with routine subculturing.

5. Microbial characterisation

Characterisation of microbial communities can assist in identification of the composition, dynamics and role of microorganisms in various biohydrometallurgical processes. Traditional culture-based methods are essential for discovering new bioleaching strains, determining the optimal conditions for growth and thus allowing process optimisation. Also, new molecular and microscopy techniques have greatly assisted the characterisation of uncultured bioleaching communities. This section reviews current and developing methods for the characterisation of these communities.

5.1 Culture-based methods

Even with advances in culture-independent, modern molecular methods for identification and characterisation of microbial diversity, culture-dependent methods are essential to define the roles that bioleaching microorganisms play in the environment and industrial mining processes. Culture- dependent methods are also essential in optimising the growth and activity of microorganisms for industrial use, and for the isolation and traditional characterisation of novel, pure strains of microorganisms. As ACCEPTEDbioleaching microorganisms MANUSCRIPT have unique growth characteristics, such as requiring acidity, ferrous iron or reduced sulfur compounds, and nutrient-limited conditions for growth, their cultivation is not straightforward, and specific methods have been developed targeting the culturing, characterisation and monitoring of acidophilic bioleaching microorganisms.

5.1.1 Enrichment and isolation methods

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Before the development of modern day molecular characterisation techniques, culture-based characterisation methods first identified the role of microorganisms involved in the cycling of iron and sulfur- in iron-rich and acidic environments (Colmer and Hinkle, 1947). Since then, both culture-dependent and independent characterisation techniques have advanced, and are more sensitive, allowing further delineation in the roles of closely related bioleaching microorganisms and highlighting the diversity and dynamics in these environments (Quatrini and Johnson, 2018).

Bioleaching microorganisms are typically enriched in liquid culture using ferrous iron, reduced sulfur compounds, sulfide ore or mineral concentrate as substrate. Isolation of dominant bioleaching cultures is usually achieved using dilution to extinction, whereby a sequence of dilutions is done until only one or a few types of microorganisms are present in the last tube showing growth. Sequential dilutions are repeated at least three times to purify any isolates obtained. This method can be laborious and time-consuming and is most useful for samples with relatively low microbial diversity (Connon and Giovanni, 2002; Stewart, 2012).

To isolate less dominant members of the community, traditional plating methods such as the streak-plate method or serial dilution plating would be more beneficial, as these methods allow colonies to grow individually and facilitate their isolation and purification. However, autotrophic acidophilic microorganisms are sensitive to organic carbon, and agar and agarose are generally hydrolysed into compounds such as pyruvate at low pH (Ueoka et al., 2016; Johnson, 1995). The development of a selective overlay plating method allowed the growth of previously uncultivable autotrophic microorganismACCEPTEDs (Johnson, 1995). In MANUSCRIPT this method, agar containing a heterotrophic acidophilic microorganism (Acidophilium cryptum strain SJH) is poured at the bottom of the plate and allowed to set. An inorganic, iron-containing layer is then poured on top of the agar. The heterotrophic microorganisms in the underlayer detoxify the solid medium and allow the autotrophic microorganisms to grow on the top layer. For this method, washing the agar with distilled water to remove impurities, or with dilute sulfuric acid to hydrolyse acid-labile polymer

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bonds was also shown to improve plating efficiencies (Johnson, 1995). Many isolates have been obtained from bioleaching and acid mine drainage sites and hot springs using this method (e.g.

Arroyo et al., 2015; Kim et al., 2014; Ueoka et al., 2016). However, there is a risk of contamination at the interface of both layers if aseptic technique is poor. Besides, this method is not suitable for isolating thermophilic microorganisms, and instead, inorganic silica-based solidifying agents can be used. However, sometimes the enrichment and isolation of acidophilic microorganisms on medium containing silica-based gelling agents can be variable (Johnson, 1995; Ueoka et al., 2016).

Bioleaching environments were thought to be relatively simple, with low diversity. However, as advances in molecular biology have been applied to the research area, it has become apparent that the diversity is much higher than once thought (Acosta et al., 2017). An excellent example of this is the further delineation of Acidithiobacillus genera into four separate species including A. ferrooxidans, A. ferrivorans, A. ferridurans and A. ferriphilus (Amouric et al., 2011). Further culture- based research enabled the isolation and characterisation of these new species and complemented the developments of culture-independent research (Falagán and Johnson, 2016;

Hallberg et al., 2010; Hedrich and Johnson 2013).

Mimicking the environment in the laboratory is a crucial way to start to characterise microbial communities and their growth and activity. However, as culture-independent methods for characterisation of microbial diversity become more sensitive, the ability to routinely isolate and enrich newly identified, unidentified and unknown and closely related microorganisms from complex environmentsACCEPTED is getting harder. More studiesMANUSCRIPT are targeting the uncultivable microorganisms in environmental niches, such as the human gut, seawater and hot and cold deserts. These studies use systematic high-throughput culture-based approaches, now dubbed as

"culturomics" (Cherif et al., 2015; Diop et al., 2016; Lagier et al., 2015). There is potential to apply culturomics-based assessment to enrich further, isolate and identify the diversity in bioleaching environments, which currently are less than fully characterised.

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5.1.2 Defining growth characteristics and parameters

In addition to the enrichment, isolation and characterisation of microorganisms, culture- based methods are also essential for the identification of optimal growth conditions of pure and mixed cultures, especially in regard to process developments for biohydrometallurgical applications. Most commonly for the characterisation of the activity of bioleaching microorganisms, parameters such as optimal pH, redox and temperature are routinely measured using small-scale, laboratory-based culturing methods. Specialised equipment such as a temperature gradient incubator (TGI) can be used to quickly and efficiently allow the determination optimal growth temperatures for pure and mixed microbial cultures, as well as modelling the theoretical maximum and minimum growth temperatures (Franzmann et al., 2005). Each culture is grown at a range of temperatures simultaneously, and growth is measured as a function of cell density (using a Thoma-ruled counting chamber, or changes to culture turbidity). Alternatively, microbial iron or sulfur oxidation activity can be monitored. The data generated from these analyses are used for a Ratkowsky equation, and optimum, minimum and maximum temperatures can be calculated (Franzmann et al., 2005).

Similar experiments varying the pH, salt concentration and other determining growth conditions can also be conducted to define the overall optimum growth and activity window for pure and mixed cultures of bioleaching microorganisms. In addition to characterising how microorganisms optimallyACCEPTED grow, these methods MANUSCRIPTcan also be used to determine how microbial activity is impacted by the addition of contaminants, or varying mineral compositions. This information can be used to inform the design of new biohydrometallurgical processes or optimise and troubleshoot existing processes.

5.1.3 Online monitoring of growth and activity

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In addition to taking and analysing samples for process conditions, microbial numbers and activity, it is possible to monitor process parameters and the growth and activity of bioleaching microorganism using online monitoring. Online monitoring of redox has been applied to evaluate the activity of iron oxidising microorganisms in laboratory scale (Watling et al., 2008). Moreover, redox control was proposed in the vat leaching process patented by Vale for optimising leaching

(du Plessis et al., 2015). Similarly, online pH monitoring and control is also beneficial for ensuring that the environment does not become too acidic or alkaline as a result of biogenic sulfuric acid production or acid consumption by gangue minerals, respectively. For example, Okibe and Johnson

(2004) used pH-controlled bioreactors for evaluating the biooxidation of pyrite by moderately thermophilic acidophiles. Dissolved oxygen (DO) concentration is known to be a limiting factor for iron and sulfur oxidation, and online monitoring of DO can allow quick adjustment of process conditions to ensure iron and sulfur oxidation, leaching and microbial growth rates are not compromised. Respirometry provides a useful tool to monitor oxygen and carbon dioxide consumption and indicates microbial activity and growth (du Plessis et al., 2001). Song et al.

(2011) used a laboratory-scale respirometer to study the microbial growth and activity during bioleaching of low-grade chalcopyrite. Petersen et al. (2010) monitored oxygen and carbon dioxide concentrations continuously in the feed and exit gas of sizeable isothermal column used for bioleaching copper sulfide ore. Oxygen consumption correlated well with iron and copper leaching, while oxidation rate declined in sections of the column that were depleted in CO2 (Petersen et al.,

2010). PseudoadiabaticACCEPTED columns equipped with MANUSCRIPTtemperature monitoring and control have facilitated the evaluation of heat generation during the bioleaching of sulfide ores (Shiers et al.

2017). Temperature probes buried in the demonstration scale bioheap at Talvivaara Mine

(nowadays called Terrafame Mine) showed that sulfide oxidation increased the temperature in some parts of the heap up to 90 °C (Halinen et al., 2012). Process control software, such as

LabVIEW can facilitate online monitoring and control of critical process parameters, such as pH,

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dissolved oxygen and redox potential (Cheng et al., 2017). HotHeapTM is Geobiotic’s process control management system designed to optimise heap bioleaching through the control of irrigation and aeration rates allowing heat conservation and maximising extent of sulfide oxidation and metal extraction (Harvey and Bath, 2007).

5.2 Molecular methods

The development of molecular DNA- and RNA-based methods and information technology have provided unprecedented insights into the phylogeny and function of bioleaching microorganisms. Polymerase chain reaction (PCR)-dependent molecular fingerprinting techniques have often been used for characterising bioleaching microbial strains and communities. Examples of fingerprinting methods applied to isolated strains include random amplification of polymorphic

DNA (RAPD), arbitrary primed PCR (AP-PCR) and repetitive element PCR (rep-PCR) (Wu et al.,

2013). Fingerprinting methods applied to microbial communities include denaturing gradient gel electrophoresis (DGGE) (Halinen et al., 2012), terminal restriction fragment length polymorphism

(T-RFLP) and capillary electrophoresis single-strand conformation polymorphism (CE-SSCP)

(Hedrich et al., 2016). These methods focus on randomly targeted or specific genes (i.e. ribosome

RNA genes) in a metagenome. The pattern of the randomly amplified genome and the polymorphism of the specific gene, which is deciphered by electrophoresis or gas chromatography- mass spectrometry, correspond to the structural and functional diversity of the microbial communities. For instance,ACCEPTED Tang et al. (2016) MANUSCRIPT applied DGGE to investigate the allochthonous microbiome in a manganese mine and found a Lysinibacillus sp. was efficient in removal of high

Mn (II) concentrations (Tang et al., 2016). Microbial community profiling using fingerprinting techniques remain useful in identifying species and monitoring dynamic changes in relatively simple communities encountered in biohydrometallurgical applications (Halinen et al., 2012).

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Quantitative polymerase chain reaction (QPCR) with species-specific primer sets has been successfully used to quantify selected bioleaching microorganisms. Primer sets are available for example for A. caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans and

Sulfobacillus benefaciens (Hedrich et al., 2016). Galleguillos et al. (2013) applied reverse transcription (RT)-QPCR to evaluate the temporal dynamics of genes involved in carbon and nitrogen metabolism of Leptospirillum ferriphilum in the Escondida mine, Chile and showed that the expression levels of the genes varied as the leaching progressed. Zhu et al. (2013) used QPCR to quantify sulfur-oxidising soxB genes during laboratory-scale chalcopyrite bioleaching with thermophilic archaea.

Next-generation sequencing methods provide enhanced resolution by identifying low abundance species, which although not numerous, can alter the biochemical environment and the behaviour of other species. Next-generation sequencing methods also provide valuable information on the relative abundance of various microbial groups and can be used to track shifts in community composition in response to changes in the environment.

Metagenomic analysis combines data on phylogenetic diversity with functional capabilities of microbial communities (Cárdenas et al., 2016). Acosta and colleagues applied a variety of primer pairs to target the prokaryotic 16S ribosome genes in metagenomic analyses of the functional bioleaching microbial communities in Escondida mine bioleaching heap and laboratory columns (Acosta et al., 2017). They identified A. thiooxidans, F. acidarmanus, Leptospirillum spp.,

Acidiphilium sp. JA12ACCEPTED-A1, Acidiphilium spp., A. MANUSCRIPTferrivorans, and Leptospirillum ferriphilum were the most representative microorganisms, providing a clear temporospatial description of the functional microbial consortia in the metal extraction process. It is still equivocal whether DNA can genuinely reflect the real-time situation of the active microbiome in natural and engineered environments.

Therefore, the use of transcriptomic approaches is attractive to tackle time-series questions in bioleaching environments. For instance, Galleguillos et al. (2013) monitored the expression

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dynamics of multiple functional genes involved in metabolic pathways of C and N of L. ferriphilum in a copper bioleaching process (Galleguillos et al., 2013). Marín et al. (2017) applied transcriptomics to study the expression of genes in the CO 2 pathway of A. thiooxidans at various

CO2 concentrations. The results indicated that A. thiooxidans requires CO2 enriched air to reach high growth rates (Marín et al., 2017).

Because of the high coverage, high-throughput DNA and RNA sequencing have become a paramount technology to deeply profile whole genomes of isolated strains and microbial communities (Cárdenas et al., 2016). Various high-throughput sequencing methods have been developed (Table 2) and thoroughly reviewed (Porter and Hajibabaei, 2018). Among them,

Nanopore Sequencing (i.e. MinION) is of particular interest, as it allows real-time DNA and RNA sequencing to carried out at remote locations, such as mine sites using a portable device. MinION technology is based on the change of current as DNA or RNA bases pass through the nanopore.

With technical development, whole genome sequencing has become a powerful tool to understand the metabolic potential of biomining microorganisms, paving a way from genome sequences to industry applications. In 2015, the genomes of 86 prokaryotic acidophiles had been sequenced, with the number increasing by the year. The genomes of acidophilic biomining microorganisms have been identified as a source of many alternative metabolic pathways for acid survival, metal tolerance and carbon fixation (Gumulya et al., 2018). These metabolic pathways increase the understanding of their function and potential alternate physiological responses to mining environmentsACCEPTED (Quatrini et al., 2006; Valdés MANUSCRIPT et al., 2008). In a recent review, Zhang et al. thoroughly summarised the achievements of whole genome surveys on the ability of iron‐ or sulfur‐oxidising autotrophic acidophiles to adapt to acidic environmental conditions (Zhang et al.,

2016).

Although metagenomic data derived from high-throughput sequencing offers vast opportunities to explore the phylogenetic diversity and functional capabilities of microbial

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communities, the understanding of the interactions among different species in microbial communities is still a challenge. Molecular ecological networks analyses (including differential equation-based networks, Bayesian networks, and relevance/co-expression networks), have been applied as a tool to construct ecological networks that illustrate the interactions among the species and genes of microbial communities (Deng et al., 2012). Bordron et al. (2015) reconstructed a global metabolic network of five bacterial strains involved in copper bioleaching

(Acidiphilium cryptum, A. ferrooxidans, A. thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans). They found that no individual bacterial strain could accomplish the whole copper leaching process (Bordron et al., 2015).

Collectively, the application of culture-independent molecular methods helps to elucidate the structures and interactions in microbial communities and their responses to in situ circumstances.

However, the use of molecular methods is still hampered by the challenge in extracting good quality DNA, especially from highly saline samples (Boxall et al., 2017).

5.3 Microscopy methods

A range of powerful optical and advanced microscopy and spectroscopy methods are now available, facilitating the visualisation of microorganism-mineral interactions and the characterisation of minerals and elements in microbial cells and extracellular polymeric substances (EPS). In many cases, these techniques can be used on fresh microbial cells or small mineral samples withoutACCEPTED the requirement for preservationMANUSCRIPT or sectioning. However, due to the high vacuum involved, electron microscopy typically requires specimens to be dried, which can cause chemical alterations.

Annular dark-field imaging can be used to show the distribution of elements at high resolution within a microbial cell or small thin mineral sample and can be combined with electron energy-loss spectroscopy. For example, annular dark-field imaging performed on a transmission

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electron microscope (TEM) and combined with Perle’s blue staining allowed the visualisation of

Fe3+ in the EPS of chalcopyrite grown A. ferrooxidans, while no Fe3+ accumulation was detected in the EPS of sulfur grown cells (Usher et al., 2010). These techniques have also demonstrated the presence of Fe3+ and P in intracellular granules and the accumulation of silica on cell walls and

EPS, which may hinder diffusion and bioleaching performance (Usher et al., 2010).

Advanced TEM techniques including energy-filtered TEM (Figure 4), TEM energy dispersive X- ray spectroscopy in scanning mode and electron energy-loss spectroscopy are valuable techniques

(Egerton, 2009) that can be performed on unpreserved microbial specimens (Usher et al., 2010).

These techniques can identify and map elements and their oxidation states within individual microbial cells at very high resolution (Midgley and Dunin-Borkowski, 2009). Sample preparation is simple, requiring only cells washing and then drying onto a carbon coated TEM grid.

Scanning electron microscopy (SEM) provides a wider field of view on thicker specimens and can be used to image microbial cells on minerals. However biological samples should be critical point dried following preservation and coated to provide conductivity. Quantitative and qualitative spectroscopy techniques can be combined with SEM imaging to determine the elements present and map their spatial associations with microorganisms (Usher et al., 2015), including energy- dispersive X-ray spectroscopy and wavelength dispersive X-ray spectroscopy.

CryoSEM avoids the need for biological samples to be preserved and dried by snap freezing specimens in liquid nitrogen and processing them through specialised equipment so they can be imaged and analysedACCEPTED in a frozen state in a cryogenicMANUSCRIPT chamber. Because there is no processing through multiple liquids, more microbial cells remain attached to minerals, and chemistry is not altered. CryoSEM is particularly useful for imaging EPS, which is typically lost or reduced to filaments in standard SEM (Dohnalkova et al., 2011; Usher et al., 2014).

Secondary ion mass spectroscopy (SIMS) requires dried samples with a polished surface.

Nano SIMS is used to analyse and map the distribution of multiple elements or isotopes within

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cells and mineral surfaces simultaneously at sub-micron resolution (Figure 5). Because isotopes of elements can be distinguished, nanoSIMS can be used to observe the uptake of rare isotopes into

EPS or cells by various microbial species in a community (Zimmermann et al., 2015). Also, probes used to label specific species in a mixed community can reveal how these species interact with microorganisms (Li et al., 2008) and minerals. However, SIMS instruments are expensive to use and analyses are slow to perform.

Raman microspectroscopy and variations of this technique allow the direct analysis of small quantities of fresh, wet samples without chemical alterations caused by drying, exposure to oxygen and preservation (Usher et al., 2015). The molecules in minerals, cells and cell products can be analysed at different time points in the same experiment to detect changes in mineral chemistry. Biofilm growth and maturation can be quickly assessed together with metabolic activities (Keleştemur and Çulha, 2017). Raman techniques have also been applied to identify strains of bacteria based on their spectra (Jarvis and Goodacre, 2004). Recent advances combine automated control and confocal microscopy to allow the collection of high-resolution images showing mineral composition and distribution, in addition to spectra.

A range of other microscopy techniques is also available. Some, such as confocal microscopy can be used in conjunction with fluorescent labelling to determine species function and distribution, providing 3D images. Fluorescent in situ hybridisation (FISH) and the more sensitive catalysed reporter deposition-FISH (CARD-FISH) are used for quantifying specific microorganisms using fluorescently ACCEPTED labelled probes. Schippers MANUSCRIPT et al. (2006) compared FISH and CARD-FISH for quantifying the bacterial numbers during the biooxidation of mine tailings concentrate and reported that the cell numbers determined by CARD-FISH were slightly higher than those obtained by FISH. Echeverría-Vega and Demergasso (2015) applied CARD-FISH to evaluate the numbers of

Acidithiobacillus and Leptospirillum species cell on chalcocite and pyrite surfaces over time and reported that the species showed differences in their attachment and detachment patterns. Atomic

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force microscopy (AFM) requires a polished surface, however wet samples can be analysed, and the distribution of EPS on minerals imaged. Mangold et al. (2008) used a combination of AFM and epifluorescence microscopy (EFM) to visualise the biofilm of A. ferrooxidans cells that formed on pyrite surfaces under a range of experimental conditions. They successfully demonstrated the application of selective stains for various cell components such as DNA (4′,6-diamidino-2- phenylindol, DAPI) and EPS (FITC-labelled lectin ConA). Florian et al. (2010) used AFM and EFM to study the initial attachment of bioleaching microorganisms and reported that Leptospirillum spp. were dominant on pyrite surfaces in the early stages of biofilm formation. Analytical microscopy and spectroscopy technologies continue to develop and advance together with software, becoming more powerful and permitting non-destructive, high-resolution imaging and analysis of microbial cells and cell products with minerals in real time.

6. Conclusions

Recent technological progress has enabled biohydrometallurgy to be an attractive option to extract value from ores, concentrates and wastes. Novel engineering solutions pave the way for more efficient processing, whereas bioprospecting, adapting and engineering bioleaching microorganisms facilitate the extraction of an increasing number of commodities from complicated feedstocks. The advances in characterising bioleaching microorganisms have deepened our understanding of theACCEPTED identity, function and MANUSCRIPT interactions between microbial catalysts, and facilitated the optimisation of biohydrometallurgical processes.

Acknowledgements

Funding from CSIRO Land and Water, CSIRO Mineral Resources, CSIRO Synthetic Biology

Future Science Platform and CSIRO ResearchPlus program is gratefully acknowledged. Melanie

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Bruckberger, Ana Mesquita and Jason Wylie from CSIRO are thanked for their valuable comments on the manuscript. The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for

Microscopy, Characterisation & Analysis, the University of Western Australia, a facility funded by the University, State and Commonwealth Governments.

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Table 1. Summary of the standard preservation methods. Preservation Method Impacts for bioleaching microorganisms References technique Cryopreservation A concentrated cell suspension is mixed There is a risk of cryoinjury to the cells during the Heylen et al., 2012; with a cryoprotectant and stored at a freezing and thawing process. Cryoprotectants may be Pegg, 2007; Spring, freezing temperature, preferably below - toxic to the cells. 2006 139 °C or lower, however -80 °C can be This is a low cost and easy method that does not used. Success rate and long term require access to specialised equipment. The method preservation (>5 years) depend on storage has been sucessfully applied to a broad range of temperature and temperature stability. microorganisms, including thermophilic prokaryotes. Freeze drying Water is removed from frozen cell samples This process is not suitable for low cell density strains Heylen et al., 2012; by vacuum dessication using specialised or anaerobes. Training is required for the use of the Pegg, 2007; Spring, equipment. The drying process can be very costly and specialised equipment. 2006 damaging to cells, however if sucessful, cells can be stored for > 35 years with little deterioration. Liquid drying Liquid samples are vacuum dried without This method has been successfully applied to Balfour-Cunningham freezing. Activated charcoal is used as a microorganisms sensitive to freeze drying or freezing et al., 2017; Malik, carrier material. Viable cells have been and it is suitable for Gram-negative microorganisms. It 1990 revived after 1-2 years of storage. has also been recently applied to mixed cultures of bioleaching microorganisms. Liquid drying requires specialist equipment and training. Storage times are shorter compared to cryopreservation and freeze drying. Cold storage Cultures are stored at 4 °C. This method This is a short term storage solution that could be Balfour-Cunningham has not beenACCEPTED formally tested for long term used toMANUSCRIPT extend the period between subculturing. et al., 2017 storage.

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Table 2. Comparison of selected high-throughput sequencing methods available for profiling bioleaching microbial communities (Adapted from

Goodwin et al. 2016).

Platform Read length (bp) Through-put Reads (M) Run time (h) Errors (%) Cost per Gb ($) (Gb) Sequencing by ligation (SOLiD) 50-75 80-320 700 -1400 144-244 ≤0.1 70-130 Sequencing by synthesis (Illumina) 25-300 0.54-900 12 - 4000 4-144 0.1-<1 7-1000 Sequencing by synthesis (454) 600-1000 0.035-0.7 0.1-1 10-23 1 9500-40000 Sequencing by synthesis (Ion) 200-400 0.06-15 0.4-80 2-23 1 25-3500 Oxford Nanopore MK 1 Minion Up to 200 kb Up to 1.5 >0.1 Up to 48 ~12 750

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Figure 1. The cumulative number of publications listed in Scopus referring to the words bioleaching, biooxidation, biomining or biosolubilisation (including various spellings thereof) and

A) specific techniquesACCEPTED (reactor/tank, vat, heap, MANUSCRIPT dump, in place or in situ); or B) commodities

(copper, zinc, nickel, gold, cobalt, uranium or rare earth) in title, abstract and/or keywords.

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Figure 2. Engineering techniques for bioleaching and biooxidation (adapted from Kinnunen, 2004).

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Figure 3. A) Heap coloured with yellow precipitates; B) Close-up photo of ore particles coated with precipitates; C) Heap bioleaching flow sheet with options for excess iron and sulfate removal indicated with dashed boxes.

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A B

C D

Figure 4. Energy-filtered TEM image Sulfolobus metallicus grown on iron sulfate in solution, then washed and dried onto a carbon coated TEM grid. For images B to D, bright areas indicate element of interest. Arrows ACCEPTED indicate granules containing MANUSCRIPT iron and Sulfur. A. Unfiltered image. B: Carbon map. C. Iron map, D. Sulfur map. Images taken at the Centre for Microscopy, Characterisation and

Analysis at the University of Western Australia.

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Slime layer around microorganism

Microorganism Mineral

Figure 5. A 3D image created from a nanoSIMS surface plot of Fe57 incorporated into EPS from culture medium by Acidithiobacullus ferrooxidans growing on chalcopyrite. Image taken at the

Centre for Microscopy, Characterisation and Analysis at the University of Western Australia.

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Highlights:

 Biohydrometallurgy has been mostly used for sulfidic gold, base metal and uranium ores.  Interest in waste bioleaching and extracting other commodities is increasing.  In situ and vat bioleaching are gaining interest along with heaps and bioreactors.  Bioprospecting, adaptation, engineering increase the availability of novel strains.  Microbial characterisation increases the understanding on biocatalysts.

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