Keywords

7.1 Microbiology and Biomining

Prior to the 1930s, it was assumed sulfide mineral oxidation (and by extension dump and in place leaching) was an entirely chemical process. Research into the phenomenon of acid rock drainage (ARD) from mine wastes led to the identification of the biological role in mineral oxidation. The first acidophilic bacterium, Acidithiobacillus thiooxidans, had been described around 1920 and Colmer and Hinkle (1947) confirmed the presence of this sulfur-oxidiser in mine water, as well as an iron-oxidising bacterium, later named as Thiobacillus (now Acidithiobacillus) ferrooxidans. An increasing number of studies during this period demonstrated the abundance of such bacteria in mine waters, yet while their dependence on sulfur and/or iron oxidation was proven, it was not until the 1950s that the extent and scale of their role in pyrite oxidation and ARD formation began to be understood.

Consequently, the earliest commercial biomining systems were not designed to specifically promote microbial activity. An early study of dump leaching (Bhappu et al. 1969) showed that dumps were not conducive to microbial activity. The authors used most probable number (MPN) analysis to show that mesophiles were only active at the dump surface and theorised that the heaps were oxygen-depleted lower down. Studies such as this led to heaps being engineered to better promote microbial activity. However, there is still a tendency to “black-box” the microbiological processes that are fundamental to successful biohydrometallurgical applications. Without understanding these highly complex interactions, it is difficult to identify and target key operational parameters needed to maximise the potential of biohydrometallurgy. One of the pioneers of this field, Giovanni Rossi, was among the first to stress the intricate and important interrelationships between multiple scientific disciplines involved in biohydrometallurgy, and the need for scientists with expertise in these areas to work together (Rossi 1990).

7.2 Microbial Ecology of Biological Mineral-Oxidising Systems

Mineral-oxidising microorganisms may be found, and are active, in a wide variety of places (Chap. 1). The consequences of their actions are either desirable—and to be encouraged and optimised—or undesirable. The principal role of the microbial component of biohydrometallurgical processes is simple: the modification of the reduction–oxidation (redox) potential to manipulate the oxidation state of sulfur and some of the metals (chiefly iron) in the mineral, thereby influencing their solubility. In oxidative leaching, this is mediated through the (re)generation of lixiviant solutions containing ferric iron and sulfuric acid.

Bioleaching systems may be laboratory scale, industrial installations, or natural or anthropogenic environments. They may be saturated, such as acid rock/acid mine drainage (ARD/AMD) and stirred-tank reactors or unsaturated such as waste rock dumps and tailings ponds, heap- and dump leaching operations, or small-scale simulation columns. The microbiology of these systems is very different, and each presents different levels of complexity in terms of sampling and analysis, and how to extrapolate the data.

Bioleaching environments are generally considered to be extreme, with elevated concentrations of dissolved metals and acidic pH (typically <2, but more variable in heaps and dumps). Such conditions present challenges to both classical and molecular microbiology methods (Chap. 6). Advances in both cultivation techniques and DNA (as well as RNA and protein) extraction and analysis have led to step-changes in the understanding of the major microorganisms involved in bioleaching. To fully understand the role of the bioleaching microbiota, it is important to know not only who is present, but where they are and the roles that they play—both in the leaching of the metals and in interacting with other members of the microbial consortium. To have confidence in the data produced from these often heterogeneous and dynamic systems, it is critically important to have a robust and reliable sampling and sample processing strategy. Further, the relevance of standardised metabolic tests needs to be tested and validated under the extreme environments of biomining.

The challenges of sampling, sample processing, test work, and data analysis vary, depending on the system under study. Microbial communities are in a constant state of flux, with the growth cycles of different species overlaid on one another. Therefore, “snapshots” of microbial community composition need to be interpreted with this in mind. Overarching this, is the community response to environmental conditions. For example, if a sample spends several days or longer in transit prior to DNA extraction and analysis, there is the possibility that the environmental signature (of the heap or reactor) has been lost or modified, and therefore proper stabilisation of the sample during transportation and storage is critical. The geographical and relative isolation of the system is important. For example, commercial operations are often in remote locations and may have limited sample processing and microbiological facilities on site. As such, the sample transit time and conditions may need to be taken into consideration when interpreting the relevance of results.

Microorganisms involved in current biomining operations are predominantly acidophilic. Furthermore, they can be classified as those which can oxidise iron and/or sulfur, and whether they are capable of autotrophic or heterotrophic growth (obligate or facultative; Chap. 5). Further to this, their likelihood of dominating or thriving in a particular leaching environment is driven by their propensities for iron oxidation, e.g., their affinities for ferrous iron (or ferric iron in reductive bioprocessing) and sensitivities to ferric iron. Similarly, their ability to scavenge inorganic carbon, nitrogen, and sulfur or to grow rapidly when each is plentiful impacts the community, as does tolerance to potentially inhibiting solutes. Further to this, the location of the microorganisms in the reaction system and their planktonic or sessile nature, the mixing patterns and contacting in the system and the degree to which the system is open to flow together with the time period required for renewal of the liquid volume within it (retention time) impact culture dynamics. The dynamic conditions within closed or slowly renewed systems (such as heaps and dumps) lead to changing culture dominance in response to ability to thrive in the changing environment. Conversely, while conditions may be more stable in flow-through systems, small perturbations may lead to rapid changes in the microbial ecology due to wash out from the system of species and strains that thrive less well following perturbations.

Despite the sometimes, though misplaced, reported difficulties in cultivating acidophiles, biohydrometallurgy is somewhat unique in that, as far as is known, the majority of the microorganisms which regularly constitute greater than 95% of the total diversity in active bioleaching systems (i.e., heaps and tanks) have been cultivated in pure culture. Therefore, it is possible to work with them and determine important physiological features such as growth rates, substrate affinities, and inhibition constants. Further, their preferred carbon and energy sources and the metabolic by-products from each system can be determined. The interaction of these biokinetic traits and the interactive roles in the metabolic cycles are critical in determining the microbial community dynamics. Indeed, in an in silico age of bioinformatics, molecular biology, and high throughput sequencing, it is important not to overlook the essential in vitro work needed to determine the effects of environmental factors on microbial activity and limits.

Previously, measuring rates and amounts of, for example, ferrous iron oxidised or CO2 fixed, have been proposed as proxies for biomass production. However, it has been shown that there is no cardinal relationship between these measurements and biomass, but rather that operating conditions affect the relationships between biomass production and substrate utilisation. Indirect methods such as microcalorimetry and respirometry may be better measures of microbial activity and biomass production though they are generally carried out using small samples which may not be representative of the entire heap or waste material.

Several studies have suggested that some microorganisms have a greater propensity to attach to mineral and other surfaces than others, while elsewhere the microbial ecology of continuously operated commercial biooxidation tanks has found similar microbial communities in the planktonic (aqueous) phase and those attached to surfaces. These data suggest that the tracking of microbial ecology in the tank biooxidation system is sufficiently, although not absolutely, represented by the study of planktonic cells, thus simplifying routine monitoring approaches to provide lead (aspirational) and lag (current) indicators of the “health” of microbial communities in stirred tanks.

In biological mineral-oxidising systems, there are three primary microbial functions: the oxidation of iron, the oxidation of elemental sulfur, and sulfur oxy-anions, and the metabolism of organic carbon. In some cases, all three roles can be fulfilled by a single organism (e.g., Sulfobacillus spp.) but mostly several species work in concert and it is generally accepted that mixtures of organisms (consortia) perform optimally. While in biomining processes, nutrients (nitrogen, phosphorus, and potassium) are usually provided, in mine waste environments this is not the case, and there is even greater importance of other metabolic functions such as nitrogen cycling. In any case, the required ecological functions must be provided either by indigenous microorganisms or through inoculation. “Top-down” and “bottom-up” approaches have been proposed to determine the most effective bioleaching consortia for different applications (Rawlings and Johnson 2007) (Table 7.1).

Table 7.1 Major described bioleaching microorganisms frequently detected in mine wastes, heap/dump bioleaching, and commercial/industrial pilot bioreactor systems (modified from Chap. 5)
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7.2.1 Heaps and Dumps

Mineral heaps, from engineered bioleaching heaps, to dumps and waste heaps of historical mine waste are much more difficult to study than well-mixed tank processes. In the laboratory and at pilot level, they are mimicked by small and large-scale studies, across a wide range of scales from a few grams of low-grade ore to tons of solid material. These are all typically unsaturated systems, with microorganisms found both free-swimming in flowing and interstitial liquid phases and attached (either electrostatically or within a biofilm) to the mineral surface. Brierley (2001) noted that both chemical and physical conditions changed radically within mineral heaps from their construction to the point at which they were decommissioned. Understanding the changes in chemical and biological parameters that occur provides immense scope for process optimisation.

Due to their economic importance, there are generally more studies covering the microbiology of heap and dump leaching operations than mine wastes and there are several comprehensive reviews available in the literature (e.g., Watling 2006). Increased process control (in heap leaching), and the usually greater concentrations of reactive minerals means that heap and dump leaching operations generate more extreme conditions. Traditionally, effluents draining heaps and other unsaturated ore beds have been analysed for both their chemistry and microbiology, and these data used to infer microbial ecology, activity, and physico-chemical conditions within the heap. However, increasingly, this is recognised to be an indirect approach which may identify trends, but not actual conditions, in the heap, and it may not be a valid approach when used as a sole index of microbial activity.

Sampling of large-scale heaps, dumps, and tailings dams is typically carried out by taking core samples, and data have been published of cores taken from active heaps and recently spent heap residues. However, there are disadvantages in adapting this as a routine approach. Coring is expensive and often not practical for production heaps, let alone mine wastes. Furthermore, coring disturbs the bed structure and may lead to channelling and non-ideal fluid flow. The size and location of samples must also be considered to address how representative they are. This leaves many questions on how best to analyse microbiology within heaps, including whether it is possible to interpret data from effluents draining heaps and dumps to infer what is happening within them.

The relative activities (for example, iron oxidation, sulfur oxidation, and gene expression) of the key microorganisms implicated in bioleaching in the different phases (planktonic or attached) is difficult to evaluate. Early work demonstrated that microbial attachment is not essential for bioleaching, since ferric iron produced by planktonic cells can oxidize sulfide minerals. While it might seem logical that attached cells might play a more important role in mineral dissolution owing to their spatial proximity, reduced diffusion barriers, and the ability to concentrate leach agents into the biofilm phase, this is not easily proven.

Heap and dump leaching systems are essentially unmixed batch processes (Chap. 2) and subject to temporal and spatial variation in conditions and microbial biodiversity as well as inherent variability in mineralogy. Heaps may be inoculated during agglomeration or through irrigation, either just once or continually, though indigenous microorganisms will still be present and active. Comprehensive studies using high throughput sequencing (HTS) methods may reveal the presence of hundreds of taxa, but typically at least 90% of the abundance (in terms of biomass) is made up of less than ten individual taxa at most, the majority of which are at least partially described. The potential roles of the remaining rare taxa are unclear. They may be remnants of the indigenous community that are just managing to survive as the conditions are not so extreme as to eliminate them, without necessarily contributing to ecological function in any meaningful way.

At the same time, heap and dumps contain many different microenvironments, within which these minor taxa may dominate, but which bulk sampling procedures homogenise. Furthermore, it is important not to conflate abundance with activity. For example, studies have shown that while Leptospirillum spp. may not numerically dominate a heap leaching system, they can account for the majority of the heap metatranscriptome, i.e., they are the most active component of the ecosystem in terms of gene expression.

Unlike bioreactors, in bioheaps there appears to be a significant difference between the microbial communities associated with the mineral (attached to the surface, or free-swimming in the interstitial liquid within and between agglomerates) and the flowing solution (leachate liquors; Fig. 7.1). Laboratory-based studies have indicated that the concentrations of cells and metals in the interstitial fluid may be orders of magnitude greater than in the flowing leachate, and that the microbial community structure is significantly different as well with acidithiobacilli more abundant in the interstitial phase and species such as Leptospirillum spp. and Acidiferrobacter spp. reported to be more abundant in the percolating liquors. Most of the biomass (over 90%) in a heap is thought to be associated with the ore (either attached or in the stagnant interstitial fluid) and that this biomass experiences solution physico-chemical conditions (pH, redox potentials, concentrations of soluble metals, etc.) that are very different to those inferred from the leachate chemistry. This may have implications in understanding microbial activity at the mineral surface and in developing ecological models of heap and dump bioleaching.

Fig. 7.1
figure 1

Schematic representation of the four phases within the ore bed (modified after Govender et al. 2013)

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Bioleaching of full-scale run-of-mine secondary copper sulfide low-grade heaps operated at the Escondida Mine (Chile) has been monitored since 2006 (e.g., Demergasso et al. 2018). Monthly analysis of pregnant leach solutions (PLS) draining the heap using MPN counts of acidophilic iron- and sulfur-oxidisers, Q-PCR and activity tests were combined with daily measurement of PLS temperature, pH, redox potentials, and soluble metals. This allowed the development and implementation of data analysis tools which demonstrated that PLS analysis can indicate the stage in the life cycle of leaching and thereby expected dominant microbial community as well as prediction of metal release.

Such predictive studies do not seek to shed light on the complete microbial consortium or on its optimisation but provide key performance indicators. This approach has since been extended to include the initial mineralogy, expanded daily data collection on the PLS to include acidity and alkalinity as well as concentrations of soluble copper and iron, among others. The data analysis algorithms have been expanded to allow empirical pattern recognition to predict recoveries and also to determine rules for stacking heaps in order to maximise their performance.

7.2.1.1 Microbial Succession and Thermal Gradients

Initially heaps and dumps will be at ambient temperature. In general terms, studies tend to suggest that during these early stages sulfur-oxidising organisms such as Acidithiobacillus spp. are the most abundant. This may be due to the initial abundance of elemental sulfur and sulfur oxy-anions resulting from chemical dissolution of sulfide minerals and the relatively low redox potentials. Biological ferrous iron oxidation causes redox potentials to become more positive and concentration of total soluble iron increases, favouring iron-oxidisers such as Leptospirillum spp. and Ferroplasma spp. For example, Wakeman et al. (2008) monitored microbial community succession in leachates from laboratory-scale columns. The community was dominated initially by At. ferrooxidans, transitioning to At. ferrivorans (referred to as “Acidithiobacillus sp. NO37” at the time) and then L. ferriphilum, all of which were included in the inoculum. Indigenous taxa including Alicyclobacillus spp. were significant members of the leachate communities initially but their numbers declined as bioleaching progressed.

The oxidation of reduced forms of sulfur is exothermic and, depending on heap design and operation as well as mineralogy, the interior temperature typically increases, sometimes to above 60 °C. These changes in temperature cause temporal and spatial changes in microbial community structure. Over time, temperature in most parts of the heap will increase, with consequent shifts toward thermo-tolerant and moderately thermophilic microorganisms such as Sulfobacillus spp. (and some other iron-oxidising Gram-positive bacteria) and Ferroplasma spp. and eventually to more extremely thermophilic archaea such as Metallosphaera spp., Sulfolobus spp., and Thermoplasma spp.

While mesophilic bioleaching organisms are often indigenous to mined ore, the same is not considered true for thermophiles or, if they are present, they tend to be far fewer in number and therefore, it is desirable to add them to the heap in order to exploit elevated temperatures. This may be done during heap construction but would then need to remain viable and not washed out during temperature ramping, or they may be introduced in irrigating solutions.

Depending on the design and operating parameters, heaps will have spatial variation in temperature (Chaps. 2 and 10), leading also to variations in microbial communities. The implications of these heterogeneous communities on heap performance are important. In very general terms, iron- and sulfur-oxidising mesophiles have efficient CO2 fixation and assimilation pathways and are the primary producers in biomining systems. The acidithiobacilli are particularly good at scavenging CO2 at very low dissolved concentrations. As temperature increases above 50 °C, communities become dominated by organisms such as Sulfobacillus spp. and acidophilic archaea which are less efficient at fixing CO2. Some are considered to be obligate heterotrophs and most display better growth in laboratory cultures that are supplemented with organic carbon. Therefore, optimising microbial activity in these higher temperature phases requires a different approach to that at mesophilic temperatures.

7.2.2 Mine Wastes

The microbiology of acid mine/rock drainage waters are more amenable to study than mineral-rich bioleaching ecosystems, and has been the focus of a number of reviews (e.g., Nordstrom et al. 2015). These communities are often relatively simple and structurally different to those found within the waste dumps that generate the ARD. In contrast, solid mine wastes represent the most heterogeneous mineral leaching systems, with widely varying ages, mineralogical composition, sulfide mineral content, pore/interstitial water pH, and other geochemical parameters such as bioavailable and total metal concentrations. There have been few systematic studies of mine waste microbial ecology and it is difficult to form generalised conclusions from published data.

The most comprehensive studies of mine wastes have combined molecular biology and cultivation-based approaches to elucidate not only the detectable biodiversity but also its potential for mineral oxidation and thus metal dissolution. The biomolecular and cultivation methodologies that have been used successfully to elucidate the microbiology biomining and mine-impacted environments are described in Chap. 6. Among examples of the importance of this approach for elucidating the microbial biodiversity of biological mineral-oxidising systems was the pioneering work done at the former Richmond mine at Iron Mountain, California (e.g., Edwards et al. 2000). Such work demonstrated how combined molecular and classical microbiology can be used to study the links between geochemical and environmental factors and microbial ecology in the ARD and the subsurface, ultimately improving understanding of key factors influencing microbial community structure and function.

In the early days of molecular microbiology, the methods were less able to detect the full breadth of microbial diversity. Consequently, it was assumed that the majority of taxa in mine wastes and ARD could be readily cultivated. However, advances in high throughput sequencing methods have radically altered this perception. In most cases, and in contrast to active bioleaching systems, the commonly isolated bioleaching microorganisms from weathered mine wastes make up a relatively small fraction of the total number of taxa (often less than 5%). Despite this, enrichments from such wastes can usually oxidise pyrite, though some enrichment cultures are better than others. As soon as wastes are put into leaching conditions (e.g., shake flasks for enrichment cultures) diversity decreases rapidly (within a single subculture) and significantly, from thousands of taxa to less than ten (Sbaffi et al. 2017). Such cultures are usually dominated by well-documented bioleaching organisms such as Leptospirillum spp., iron-oxidising acidithiobacilli, Sulfobacillus spp. (and other iron- and/or sulfur-oxidising Firmicutes), with varying abundances of other mineral-oxidising organisms such as Acidiferrobacter, Thermoplasma spp., and Ferroplasma spp. and non-mineral-oxidising heterotrophs such as Acidiphilium spp. Enrichment cultures are completely unlike their source waste in terms of community composition and structure, more so where the waste is particularly aged. Therefore, cultivation-based methods can be misleading in terms of both the microbial abundance and diversity revealed. Nevertheless, they are essential in determining the potential for such communities to catalyse mineral oxidation, given the right conditions, and in the goal of bioprospecting for novel strains and consortia with potentially exploitable traits.

Work done by Axel Schippers and co-workers using combinations of classical (e.g., MPN) and molecular microbiology methods began to elucidate some general trends in the microbial succession in mine wastes (e.g., Korehi et al. 2014; Schippers et al. 2010). When wastes are first deposited, they have neutral pH or even slightly alkaline (especially flotation tailings). They are initially colonised by a wide variety of microorganisms, including pioneer sulfur-oxidising Proteobacteria such as Thiobacillus spp. Due to the activities of these prokaryotes, the environment becomes more acidic. Closest to active sulfide oxidation zones (oxidation fronts or areas rich in reactive sulfides), biodiversity is more limited and communities tend to be dominated by mineral-oxidising taxa such as Acidithiobacillus spp., Leptospirillum spp., Sulfobacillus spp., and iron-oxidising archaea. However, in deeper parts of the waste, particularly saturated zones, these microorganisms may be present but in low numbers, and mineral oxidation is limited by oxygen supply.

Historic, unmanaged wastes often can still produce ARD, even after hundreds of years. These wastes typically have greater microbial diversity than more recent waste dumps, with often thousands of taxa routinely detectable by HTS. Communities can vary from mostly bacterial to predominantly archaeal. The most common bacterial phyla reported in weathered wastes include Proteobacteria, Acidobacteria, Chloroflexi, Planctomycetes, and candidate division AD3, with the Euryarchaeota and Crenarchaeota the most frequent archaeal phyla (e.g., Sbaffi et al. 2017). While most of these organisms represent poorly characterised, deeply branching phylogenetic groups, there is a general notion that they represent systems in transition from purely lithotrophic to more “heterotrophically-inclined” microbial communities. While many of these taxa represent novel lineages, similar organisms can be found in aging volcanic deposits or environments impacted by mine wastes and ARD. Currently, it is difficult to speculate as to their role in these systems.

It is clear that microbial diversity increases with mine waste age and that older waste deposits tend to be dominated by microorganisms that have yet to be isolated and characterised. However, it is not age, but pH that is by far the most significant factor that controls microbial diversity in mine wastes. Diversity decreases with decreasing pH and neither mineralogy nor chemistry have an effect to the same extent. While there does seem to be a possible link with the total metal(loid) content this is not entirely independent of pH itself.

Sulfidic ARD-generating mine wastes may be blended with amendments and planted with grasses and other plants during site rehabilitation or phytostabilisation. Interestingly, this does not necessarily lead to the displacement of the lithotrophic, mineral-oxidising community (e.g., Hottenstein et al. 2019). Even where a system is no longer net acid-generating these organisms are still found, juxtaposed with heterotrophic (non-mineral-oxidising) communities. This suggests that litho-autotrophic niches may still exist even in apparently stabilised waste dumps, and that these may be reactivated rapidly should conditions change, for example, through re-exposure of sulfidic material to both oxygen and water.

7.2.3 Bioreactors

Stirred-tank reactors (STRs; Chap. 3) are well-mixed systems and in theory, allow representative samples to be taken at any point within the reactor or at the outlet of a continuous STR. Typically, direct counting (using phase contrast microscopy) of planktonic cells in liquid phase is possible in order to estimate biomass and to follow growth rates, although this can be complicated due to the presence of fine particles. However, it is much more difficult to quantify microorganisms attached to the mineral surface and to determine what proportion of the total biomass is either attached or planktonic. Early work in laboratory-scale tanks suggested that planktonic cells accounted for about 66% of the microbial population present in the tank.

Attachment studies performed on fine-grained concentrates, using both flow-through systems in which cells are contacted with fine-grained mineral on coated beads and also submerged slurry systems, demonstrated rapid attachment of L. ferriphilum, At. ferrooxidans, and Metallosphaera hakonensis. L. ferriphilum and At. ferrooxidans previously adapted to a copper sulfide concentrate showed rapid attachment levels (75–79%) in a flow-through system, with M. hakonensis showing 30–45% attachment to mineral concentrate at 65 °C. Conversely, attachment to low-grade ores and quartzite gangue material was much lower (~25%) as was contacting at temperatures not optimal for metabolic growth. Data from growing and metabolically active, well-agitated slurry reactor systems at scales of 1 litre to 500 m3 and at solids concentrations of 10–25% have suggested that enumerating planktonic cells could be a reasonable proxy for the bioreactor as a whole. Shear stress, and slurry densities of fine particles of 10–15% are also important factors in controlling microbial attachment.

Bioreactor systems, whether laboratory-, pilot-, or commercial-scale, are the most homogeneous bioleaching environments spatially, and, at least in the case of continuous stirred-tank reactors (CSTR) at steady state, temporally. The operating conditions are tightly controlled, designed to maximise mineral dissolution and often push the microbial consortia to their limits. As a result, the microbial ecology is relatively noncomplex and usually comprise between two and four dominant taxa. Where other taxa have been identified through cultivation or HTS approaches, they are far more limited in diversity and abundance than in heaps and dumps.

At the time of early bioreactor development for bioleaching in the latter part of the twentieth century, the acidithiobacilli were the most widely studied bioleaching organisms and were assumed to be the most important in stirred tanks. Moreover, bioleaching cultures were routinely enriched from ARD and mine wastes, where these organisms often dominated. As a result, early reactors were designed to run at mesophilic temperatures of around 35 °C. An example of this is the development of the cobaltiferous pyrite bioleaching operation in Kasese, Uganda, which began during the early 1990s (Morin and d'Hugues 2007) where the original inoculum used in laboratory studies, derived from a gold-bearing arsenopyrite bioleaching CSTR, was dominated by Acidithiobacillus-like bacteria. This was in line with the early BIOX development work (Chap. 4), with early reactors thought to comprise similar microbial consortia. Such results are incongruent with subsequent work which showed that even at such temperatures Leptospirillum spp. would be expected to be the dominant iron-oxidisers due to their ability to sustain higher rates of iron oxidation in high redox (<840 mV) environments (Rawlings et al. 1999). While it is theoretically possible that Leptospirillum spp. were absent from the inocula as well as the (unsterilised) concentrates, it is generally accepted that the inferred microbial community composition of these early gold biooxidation systems were erroneous observations due to limitations of the techniques available at the time, and the dominant acidophiles in the full-scale commercial tanks at the Kasese plant were found to be L. ferriphilum, At. caldus, Sb. benefaciens, and a Ferroplasma-like archaeon. However, the stirred tanks used in the BIONORD process developed by Polyus JSC (Chap. 11) which are typically maintained between 35 °C and 40 °C are dominated by Ferroplasma acidiphilum, Acidiferrobacter thiooxidans, At. thiooxidans, and Acidiphilium multivorum while L. ferriphilum accounts for less than 7% of the total biomass.

The oxidation of sulfide minerals such as pyrite and arsenopyrite is exothermic, and much of the capital and operating expenditure of tank bioleaching is associated with cooling. Moreover, increasing temperature increases reaction rate, and during the optimisation of operating parameters it is desirable to push the operating temperature to the highest possible before system instability. As a result, both the BIOX and Kasese plants were ultimately run above 40 °C, and most commercial biooxidation systems operate between 40 and 45 °C. It has been frequently reported that such bioreactor communities comprise At. caldus and L. ferriphilum (usually as the dominant prokaryotes) with Sulfobacillus spp. (frequently Sb. thermosulfidooxidans) present in smaller numbers. Acidophilic archaea (mostly Ferroplasma spp.) have also been detected, and their numbers tend to increase in downstream tanks as organic carbon concentrations increase.

Although it is not proven why Sb. thermosulfidooxidans is the dominant Sulfobacillus spp. in certain situations and Sb. benefaciens in others, it appears to be linked with CO2 availability and concentrations of organic carbon, with Sb. benefaciens better able to scavenge CO2 than Sb. thermosulfidooxidans (Johnson et al. 2008), and also their different degrees of tolerance to transition metals (e.g., Sb. thermosulfidooxidans is more resistant to copper). It is also interesting to note in this context that the Mintek bioleaching culture, used in the Mondo bioleaching process (Chap. 12) operates at 45 °C and is dominated by L. ferriphilum and At. caldus despite 45 °C being outside the accepted temperature range of many (though not all) strains of L. ferriphilum.

Whether L. ferriphilum is the principal iron-oxidising organism in all tank bioleaching systems that operate at 40–45 °C has been questioned. Analysis of a long-term continuous bioleaching system processing a nickel–copper concentrate at 40 °C indicated that it was dominated by Sb. thermosulfidooxidans and another Sulfobacillus sp. with very low levels of L. ferriphilum, despite having been inoculated with the BRGM-Kasese culture (Bryan, unpublished data). Moreover, analysis of the microbial communities of several commercial BIOX® plants in different countries showed archaeal dominance by organisms such as the iron-oxidiser Ferroplasma acidiphilum and a Thermoplasma spp. (Smart et al. 2017).

For a long time, ~45 °C has been considered the upper limit for tank bioleaching before stepping up to 60–80 °C for archaeal processes (MesoTHERM®, BioCOP, etc.). However, the IBCCO (Iranian Babak Copper Company) commercial bioreactors in Iran processing copper concentrate operate between 45 and 60 °C and are dominated by Sulfobacillus spp. suggesting that they can fulfil the iron oxidation demand in this system (Manafi et al. 2021). One of the challenges associated with Sulfobacillus-dominated bioleaching systems is that they are relatively poor at assimilating CO2. At lower temperatures, primary producers such as Leptospirillum spp. and Acidithiobacillus spp. efficiently fix and assimilate CO2, providing sulfobacilli with organic carbon in the form of cellular debris and exudates. A novel facultatively autotrophic, acidophilic actinobacterium (“Acidithiomicrobium P2”) has shown some promise in co-culture with Sb. thermosulfidooxidans and At. caldus in the leaching of nickel concentrate in continuous laboratory-scale culture at 49 °C (Norris 2017). However, its sensitivity to copper may limit its role in bioleaching operations.

Currently there are no commercial high-temperature biomining operations, although the development of the MesoTherm® process and the pilot-scale BioCOP process are discussed in more detail in Chaps. 3 and 4, respectively. Generally, commercial interest in thermophile bioleaching has centred around three culture types: Sulfuracidifex (previously Sulfolobus) metallicus at 64–68 °C, Acidianus brierleyi at ~70 °C (used in pilot work by Mintek for example), and the ICHT culture at ~78 °C (used in the BioCOP and BRGM HIOX pilot plant; Norris et al. 2013). The ICHT culture comprises 4–5 novel Sulfolobus-, Stygiolobus-, Acidianus-, and Metallosphaera-like archaeal species (Table 7.2).

Table 7.2 Summary of typical dominant microbial ecology of biological mineral-oxidising systems
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7.3 Challenges and Future Directions

Generally, in unsaturated systems, pH seems to be the factor that correlates most closely with greater microbial diversity. As pH increases, so does microbial diversity. However, it is not clear whether this is a direct or indirect effect, as pH will cause other parameters to change also, such as the solubility of transition metals, CO2 solubility, and organic acid protonation, which complicates the interpretation of data. In saturated systems where pH and temperature are constant, biodiversity is much more restricted. These systems are much more sensitive to changes in feed materials and resulting fluctuations in soluble metal(loid) concentrations and redox potentials. It is possible that as these systems are operating at their biological limits, the lack of biodiversity affects their resilience; they lack a biological buffer which may allow the system to adapt to changes. The links between environmental parameters like pH, system biodiversity, function, and resilience require further study.

The monitoring of heap and dump leaching operations remains a challenge. On the one hand, it is probable that the majority of heap biomass is found in interstitial (stagnant) fluid, or attached to the mineral surface, and that the microbial community of the leachate is probably not representative of the microbial community as a whole. On the other hand, microbiological and chemical analyses of heap leachates can be used to indicate heap performance. Nevertheless, better understanding of the microbial community of a heap and the conditions that it operates in (pH, solute concentrations, redox, etc.) will improve the ability to model heap performance and understand operational limits.

Understanding differences between microbial performance and adaptation is crucial to successful biomining operations. However, the best-adapted culture is not necessarily the best performing. Investigations have been carried out on top-down, designed cultures for bioleaching (particularly tank leaching), but organisms that have the most desirable traits may not be able to colonise a system where an indigenous community is present as these organisms may be better adapted (even if they are not the most efficient from an operational point of view). In other words, during either a bottom-up or top-down approach, it is important allow the more effective microbial consortia (in terms of accelerating mineral oxidation and solubilising metals) sufficient opportunity to adapt to the system (geochemistry, mineralogy, operating conditions, etc.). On the other hand, it has been suggested that, if during primary succession most environmental niches are highly colonised, it could be difficult for subsequent organisms to displace these, even if they are better adapted. Other approaches used to attempt to effect ecological control include continuous inoculation with appropriate microorganisms (e.g., thermophiles for self-heating bioheaps).

Ecological engineering of mine wastes is desirable for many reasons. From an environmental perspective, colonising mine waste with microorganisms that do not accelerate the breakdown of minerals, leading to acid generation and solubilisation of metals, could provide a useful method to limit ARD formation. How to achieve such control is less evident. Sterilising, or otherwise inhibiting the indigenous (or best-adapted) community is likely to be prohibitively expensive for most operations. Alternatively, continuous application of readily accessible organic matter has been shown to sustain the dominance of heterotrophic populations that not only do not oxidise residual sulfide minerals but can also immobilise soluble metals and generate alkalinity (Johnson 2014).

Bioleaching systems are complex, in terms of bio- and geochemical processes and interactions, sampling, analysis, and interpretation. Understanding microbial diversity, its function, and response to environmental conditions are key in improving process design, and system modelling and prediction. Improvements in microbiological tools such as DNA (and RNA) extraction, sequencing and ecological analytical approaches are now being applied more routinely to these systems. It is essential to properly consider sampling and experimental design with microbiology in mind. Important to this is not just knowing which microorganisms are present in a system, but their locations, activities and responses to environmental conditions.