Bioremediation and Sustainability (Research and Applications) || Bioleaching

6 Bioleaching

Leo G. Leduc1 and Garry D. Ferroni1-2

department of Biology, Laurentian University, Sudbury, Ontario, Canada P3E 2C6

2Medical Sciences Division, Northern Ontario School of Medicine, Sudbury, Ontario, Canada P3E2C6

* Corresponding Author ([email protected])

Abstract Bioleaching, the solubilization of base metals from ores using microorgan-isms, is an important mining process in the world today. It is particularly useful for the recovery of metals from low-grade ores that cannot be pro-cessed by conventional hydrometallurgical means. Many different acido-philic microorganisms or consortia are involved in bioleaching, including members of the Bacteria and Archaea. The bacterium Acidithiobacillus ferrooxidans dominates the process in pyrite-containing ore deposits because of its unique physiology. This chapter provides a brief overview of bioleaching with an emphasis on pyritic ores. The following topics are examined: a) the mechanisms of bioleaching, b) the strategies of bioleach-ing, c) the microorganisms of bioleaching, d) factors affecting bioleaching, and e) environmental considerations. In addition, specific supplementary information is provided on uranium bioleaching.

Keywords: bioleaching, biomining, bio-oxidation

Introduction

Bioleaching is generally defined as the solubilization of base met-als from ores using microorganisms. It has also been referred to as biomining, by many. Bio-oxidation, on the other hand, involves the release of the metal, which is not necessarily soluble. In other words, the bio-oxidation process facilitates the chemical extraction

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of the metal. It has been used extensively for the release of valuable metals such as gold from ores [1]. Both processes, bioleaching and bio-oxidation, lead to the generation of acidity which is referred to as acid mine drainage (AMD) and/or acid rock drainage (ARD). The latter phenomenon relates to acid production by natural pro-cesses as opposed to mining activities.

The use of bioleaching in the commercial recovery of metals was first reported in the early 1960s. Indeed, Mashbir [2] reported on the bioleaching of low-grade uranium by Western Nuclear Incorporated in Wyoming, USA in 1964. Many projects followed and bioleaching quickly became a process of choice for the extraction of base metals, particularly low-grade ores that cannot be processed by conven-tional hydrometallurgical means. The high frequency of use of bio-leaching by mining companies is due to (a) its small environmental footprint, (b) its use of low-grade ores, (c) its ease of implementa-tion, and (d) its low capital costs [3].

The microorganisms involved in bioleaching live in an acidic environment and are, therefore, acidophiles. Acidophiles have tra-ditionally been defined as microorganisms that grow optimally at a pH of less than 5.0. It is generally accepted that various microbes including Bacteria and Archaea are involved in bioleaching, but the bacterium Acidithiobacillus ferrooxidans dominates the process in pyrite-containing ore deposits because it can oxidize both iron and reduced sulfur compounds and it flourishes in low acidity envi-ronments. Not surprisingly, it has been described by Lundgren & Silver [4] as "the principal acid-generating microorganism affili-ated with mineral leaching".

In this chapter, the following specific aspects of bioleaching will be briefly discussed with an emphasis on pyritic ores: a) the mecha-nisms of bioleaching, b) the strategies of bioleaching, c) the micro-organisms of bioleaching, d) factors affecting bioleaching, and e) environmental considerations. Additional information will also be provided on uranium bioleaching. As a complement to this chapter, the reader is referred to the following excellent articles which deal either exclusively or in part with bioleaching: Cardenas et al. [5], Gadd & Raven [6], Johnson [7], Olson et al. [8], and Suzuki [9].

Mechanisms of Bioleaching

Pyrite (FeS2) is relatively stable under anoxic and dry conditions [10]. However, when water and oxygen come in contact with

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pyrite at circumneutral pH, a relatively slow chemical oxidation (equation 6.1) occurs which converts some of the pyrite into ferrous sulfate and sulfuric acid. Moreover, the Fe2+ iron is quickly oxidized to Fe3+ iron with the concomitant production of protons resulting in acid generation. As more and more acid is produced abiotically, the pH becomes low enough for an accelerated acidification by the sulfur-oxidizing bacteria through their metabolic activity. At low pH, the importance of iron-oxidizing bacteria becomes evident as abiotic iron oxidation, in an acidic environment, is extremely slow [10].

FeS2 (pyrite) + 3.5Ü2 + H2O -> 2FeS04 + H2SO4 (6.1)

2FeS04 + 0.5O2 + H2SO4 -> Fe2(S04)3 + H 2 O (6.2)

Indeed, in the presence of iron-oxidizing microorgansims such as At. ferrooxidans, a rapid oxidation of the ferrous sulfate occurs to produce ferric sulfate and water (equation 6.2). Ferrous iron acts as an electron donor for the bacterium, and the energy generated by the transfer of electrons supports the various metabolic require-ments of the bacterium.

FeS2 + Fe2(S04)3 -> 3FeS04 + 2S (6.3)

The ferric ions produced act upon the pyrite to produce more ferrous ions for the iron-oxidizing bacterium (equation 6.3).

2S + 3 0 2 + 2 H 2 0 -> 2H2SO4 (6.4)

As seen in equation 6.4, the sulfur moiety is directly oxidizable to sulfuric acid. Thus, the bacteria establish a ferrous ion-ferric ion cycle that results in the production of large amounts of the oxidant for the bioleaching process [11].

Bioleaching microorganisms, including both Bacteria and Archaea, cause the solubilization of metals from ores by a direct mechanism, and in the case of pyrite-containing ores, at least, by an indirect mech-anism as well. Direct and indirect leaching are also referred to as contact and non-contact leaching by many. It is generally agreed that both mechanisms involve the oxidant Fe3+ that results in the extraction of the metal from the ore, the oxidant being produced by the metabolic activity of the microorganisms [7]. Both the direct and indirect modes of action occur in concert under in situ conditions. During contact leaching, the bacterium is attached by


its glycocalyx to the mineral substrate and the entrapped oxidant Fe3+medidates the dissolution of the ore "directly" whereas during non-contact leaching, the planktonic bacteria are free-swimming and the oxidant Fe3+ diffuses in the leach liquor to oxidize the ore "indirectly".

Strategies of Bioleaching

There are two basic strategies to bioleaching: irrigated systems and stirred tanks. In irrigated systems, the low-grade sulfidic ore, in the form of crushed rock, is piled into large dumps or heaps which are sprayed with an acidic liquor to favor the growth of acidophilic microorganisms. The microbial population is either naturally occur-ring in the ore dump or inoculated into the dump using a culture. As the acidic liquor percolates through the ore, the metal of interest within the ore is solubilized and the so-called pregnant leach solu-tion (PLS) is collected at the base of the dump or heap. The metal of value is subsequently recovered from the PLS.

In Elliot Lake, Ontario, Canada, a variant of the irrigated system referred to as in situ underground leaching was used for several years to recover uranium from the vast low-grade ore (less than 0.05% by weight) body there. For uranium bioleaching, the insol-uble tetravalent uranium is converted to the soluble hexavalent uranium species by microbially-produced ferric iron as shown in equation 6.5.

UO2 + Fe2(S04)3 -> U02(S04>2 + 2FeS04 (6.5)

Equation 6.5 is the key step in the uranium bioleaching process. The major differences between dump and heap leaching is that

the latter usually consists of finer particle size and often involves more engineering such as aeration and collection pipes. In addi-tion, heaps are usually much smaller than dumps. Overall, heap leaching extracts more metal from the ore than dump leaching, but it is considerably more expensive. Not surprisingly, therefore, heap leaching is usually used to extract copper, a base metal of consider-able value.

The use of stirred tank bioreactors is the most efficient bioleach-ing strategy for extracting metals from ore. However, it is the most expensive. Notwithstanding their high cost of construction and

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maintenance, such highly engineered systems are sometimes used for bioleaching because environmental factors such pH, tempera-ture, aeration, etc. can be controlled. Stirred tank bioreactors are usually restricted to the extraction of gold from ore.

Microorganisms of Bioleaching

The recent use of new molecular techniques such as the PCR-based amplification of 16S rRNA to detect microorganisms present in bio-leaching and AMD environments has considerably added to our understanding of the microbial diversity of such ecosystems [12]. Such studies are numerous and include bench-scale [13] and commercial stirred-tank [14] bioleaching operations, column biole-aching experiments [15-17], and heap bioleaching operations [18,19]. Indeed, such studies have revealed that the microbiota of bioleach-ing environments is diverse and members of the three domains of life, i.e. Bacteria, Archaea, and Eucarya, populate leaching sites. The consortium of iron- and sulfur-oxidizing acidophilic microorgan-isms that play a role in the leaching of metals from sulfidic ore has recently been categorized into three main groups: primary, second-ary, and tertiary microbes by Johnson & Hallberg [20]. In a nutshell, primary acidophiles are chemolithoautotrophs that can oxidize ferrous iron and, thus, generate ferric iron necessary for the oxida-tion of the mineral. Secondary acidophiles are sulfur-oxidizers that generate acidity required to maintain a thriving community of aci-dophilic microorganisms. Tertiary acidophiles are chemoorgano-trophs that scavenge the organic compounds that can accumulate in leaching environments. This last group of acidophiles is important as they maintain a suitable environment for those chemolithoauto-trophic microorganisms that are sensitive to organic compounds. The division between the three groups of bioleaching microorgan-isms is not clear cut as there is considerable overlap between the groups [7].

With respect to the primary acidophiles, the main partici-pants in mineral oxidation are the Gram-negative bacteria: Acidithiobacillus ferrooxidans [21], Acidithiobacillus ferrivorans [22], and Leptospirillum ferrooxidans [23] although Gram-positive bacte-ria such as Ferrimicrobium acidiphilum, Sulfobacillus acidophilus [24], and Acidimicrobium ferrooxidans [25] can play a role in the mineral oxidative process. Leaching primary acidophiles belonging to the

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domain Archaea are also known to participate, including Sulfolobus, Acidianus, Metallosphaera, and Sulfurisphaera [26]. Although some leaching bacteria have a restricted metabolism such as L. ferroox-idans which can only oxidize ferrous iron aerobically, others like At. ferrooxidans are very diverse metabolically. Indeed, At. ferrooxi-dans can oxidize ferrous iron and reduced sulfur compounds such as elemental sulfur and thiosulfate either aerobically or anaerobi-cally [27,28] and over a wide range of temperatures [11]. Although it is commonly characterized as a mesophilic bacterium, psychrotro-phic strains, growing in the temperature range 2 to 35°C, have been isolated [11]. Indeed, psychrotrophic strains have been isolated that show exponential growth on ferrous iron at temperatures near 2°C [29]. Its pH optimum for iron oxidation is around 2 and in the range 1.5 to 6. L. ferrooxidans is an acidophilic, obligate chemolitho-autotrophic bacterium which grows on the oxidation of ferrous iron, but is not able to use reduced-sulfur compounds [30]. It is often con-sidered a moderate thermophile as it is active at temperatures above 45°C with an optimum near 30°C. Although they grow more slowly than At. ferrooxidans on ferrous sulfate in batch cultures, they are still important members of bioleaching environments. Needless to say, the fact that the consortium of primary acidophiles is metaboli-cally diverse is important in biomining operations [31].

As mentioned earlier, the secondary acidophiles are important as they are involved in the acid-generation process. However, they are not directly involved in the solubilization of the base metal from the ore substrate although some can such as At. ferrooxidans. The Gram-negative bacteria At. thiooxidans and At. caldus are probably the best known secondary acidophiles as they are easily recovered from bioleaching environments and are active at very low pH values. At. thiooxidans derives its energy solely from the oxidation of reduced sulfur compounds such as elemental sulfur, thiosulfate, and tetra-thionate. It is a mesophile with an optimum growth temperature in the range 25 to 30°C. As mentioned above, it is active at very low pH and is probably the most acid-tolerant of the sulfur-oxidizing species, with a pH growth range of 0.5 to 4.0 [32]. At. caldus is simi-lar to At. thiooxidans, both phenotypically and genotypically, but some strains of At. caldus have been shown to grow mixotrophically using yeast extract or glucose [33]. At. caldus is a moderate thermo-phile with an optimum growth temperature of around 45°C and an optimum pH in the range 2 to 2.5 although it is known to be as tolerant as At. thiooxidans to acidity. At. caldus is often the dominant

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microbe of commercial bioleaching operations, particularly those involving the biooxidation of gold in stirred tanks and above 40°C [34,35]. Other secondary acidophiles include bacteria already men-tioned such as At. ferrooxidans and the Archaea Sulfolobus, Acidianus, Metallosphaera, and Sulfurisphaera. Interestingly, some acidophiles such as At. ferrooxidans can be considered both primary and second-ary acidophiles because they are directly involved in the solubiliza-tion of the metal through the ferrous-ferric cycle and they generate acidity through their oxidation of reduced sulfur compounds.

The tertiary acidophiles are usually not direct participants in the solubilization of metals nor in the acidification process but instead contribute to the establishment of an environment suitable for the growth and proliferation of the other acidophiles, namely the chemolithotrophs. Indeed, most tertiary acidophiles are heterotro-phic oligotrophs that utilize organic compounds that become avail-able due to natural processes such as cell lysis, cell leakage, etc. It is assumed that chemolithotrophic microorganisms are sensitive to the organic compounds and that such substances can become toxic as they accumulate in the bioleaching environment. Thus, the ter-tiary acidophiles metabolize the organic waste products produced by the iron- and sulfur-oxidizers and the autotrophs thrive in a less toxic environment, resulting in an increase in the solubilization rates [33,36,37]. Common tertiary acidophiles are members of the genera Acidiphilium [38,39], Acidocella [40], Addomonas [41], Acidisphaera [42], and Acidobacterium [43]. The species have mostly been isolated from acidic mining environments or cultures of At. ferrooxidans. The most studied tertiary acidophiles are Acidiphilium acidophilum and A. cryptum. A. acidophilum is an aerobic, Gram-negative facultative chemolithotrophic bacterium that is often found as a contaminant of cultures of At. ferrooxidans [44]. A. acidophilum can use elemental sulfur or tetrathionate, as well as organic acids as an energy source, and its source of carbon is carbon dioxide. The bacterium is typi-cally mesophilic, growing in the range 25 to 35°C. Its pH growth range is similar to that of At. ferrooxidans, ranging from 1.5 to 5.5, with optimal growth at 2.5. A. cryptum is a Gram-negative bacte-rium that grows in the pH range 2.5 to 5.9. It is a mesophile with a growth temperature range between 30 and 40°C [39]. The bacte-rium is an obligate chemoorganotroph, meaning that it acquires its energy and carbon source from organic compounds [39]. In con-trast to A. acidophilum, this microorganism cannot use inorganic compounds such as sulfur as its energy source.


Various biotic interactions between the different microorganisms of bioleaching environments come into play and ultimately affect leaching. Synergistic interactions between microorganisms impor-tant to biomining were described by Bacelar-Nicolau & Johnson [45] and Paiment et al. [36]. In the case of the earlier study, Fm. aci-dophilus, an obligately heterotrophic iron-oxidizing acidophilic bacterium, oxidizes pyrite if provided with an organic source of carbon. At. thiooxidans, an obligately sulfur-oxidizing autotroph, cannot oxidize pyrite. However, together, the bacteria enhance the solubilization of pyrite because Fm. acidophilus can liberate enough reduced sulfur compounds from the ore, thus allowing for the pro-liferation of At. thiooxidans. In return, the cells of At. thiooxidans release organic compounds during growth in the medium that can be used by Fm. acidophilus further promoting growth and iron oxidation, allowing a cycle to become established. With respect to the study by Paiment et al. [36], mixed cultures of At. ferrooxi-dans and A. acidophilus were shown to leach significantly greater quantities of copper from low-grade ore than the pure strain alone presumably because the facultative chemolithotrophic bacterium A. acidophilus prevented the accumulation of toxic organic com-pounds in the shake flasks. Interestingly, the synergistic interaction between the two microbes could not be shown in the case of nickel solubilization.

Factors Affecting Bioleaching

Bioleaching is affected by both biotic and abiotic factors. With respect to biotic factors, both synergistic and antagonistic effects are possible. Synergistic biotic effects involving bioleaching acido-philic bacteria are beneficial and have been mentioned earlier. In contrast, antagonistic biotic factors adversely affect bioleaching. For example, some bioleaching operations can harbor significant populations of single-celled and multicellular eucaryotes, includ-ing species of algae, fungi, protozoa and rotifers [46]. Given that some protozoa and rotifers are well known as predators of bacteria, such eucaryotic microorganisms could have deleterious effects on the community of primary and secondary leaching acidophiles in bioleaching operations. Abiotic factors that affect the leaching pro-cess are numerous, including temperature, pH, nutrients, and toxic substances.

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Temperature

The efficiency of the bioleaching process is very much dependent upon the temperature of the environment as microbial processes are highly temperature dependent. We have been studying the interac-tion of temperature and At. ferrooxidans, with a particular interest in the natural occurrence of psychrotrophic and possibly psychrophilic strains of the bacterium. Both of these have growth temperature ranges that extend to 0°C or below, and the former are distinguish-able from the latter in having maximum temperatures for growth equal to or greater than 21°C. Given that mesophilic microbes usu-ally have their lower temperature limits in the 8 to 15°C range and have very slow rates of growth at these temperatures, there is a need for psychrotrophic bioleachers for underground applications in Canadian environments, at least. At such geographical latitudes, underground temperatures over the course of a year in actively-leaching stopes are usually in the range of 10 to 25°C.

In an investigation of uranium mine-water samples from which enrichment cultures for At. ferrooxidans were developed at temper-atures of 6, 12, 18, and 25°C, there was an approximate doubling of the growth rate for each approximately 6°C increment, and psy-chrotrophic strains of At. ferrooxidans were shown to be isolatable from the environment [47]. Therefore, the indigenous strains of At. ferrooxidans were shown to be capable of growth throughout the critical temperature range (12 to 25°C) for the particular ura-nium mine. Moreover, these results indicated that the time inter-vals between the flooding of stopes, which tended to be constant over the yearly cycle, should be seasonally adjusted to compensate for the different rates of bacterial activity expected at the different temperatures.

Subsequently, 10 isolates of At. ferrooxidans from Canadian and other mines were thermally characterized for the temperature range 2 to 35°C and four of the isolates proved to be psychrotrophs and six were mesophiles [29]. In a related study of mine-water samples collected from two Ontario, Canada uranium mines for which the temperatures at the time of sampling were in the range 13 to 18°C, psychrotrophic iron oxidizers, most of which were At. ferrooxidans, were found to be numerically predominant [48]. Interestingly, a fol-low-up study employing samples collected at much lower in situ temperatures (0.5 to 5.0°C), did not yield psychrophilic representa-tives of At. ferrooxidans [49].


In an investigation that emphasized temperature and the per-sistence of strains of At. ferrooxidans in the environment as a factor in ARD, we noted the absence of thermoduric types of At. ferro-oxidans, that strains survive well at temperatures just below their minimum temperatures for growth (around 2°C), and that strains are very susceptible to a slow freeze to -15°C [50]. Furthermore, heat-shocked cultures were shown to acquire thermotolerance but were not protected against loss of viability due to freezing, nor were cold-shock cultures [51].

The results of these temperature studies indicate that both meso-philic and psychrotrophic representatives of At. ferrooxidans exist in nature and that temperature relationships are variable from strain to strain even within these two groups. The natural occur-rence of psychrotrophs is important as they are indispensable in bioleaching applications at lower temperatures. Their broad tem-perature ranges for growth ensure that they will function, albeit at very different rates, over the seasonally-based temperature cycle of northern latitudes. Moreover, the conversion of a mesophile into a psychrotroph would be extremely challenging as the basis for psychrotrophy is undoubtedly multigenic. The apparent absence of psychrophilic representatives of At. ferrooxidans is not of practi-cal importance, as psychrophiles would probably have restrictive growth temperature ranges and as they would probably not have higher growth rates than psychrotrophs at the critical temperatures.

Recently, we [52] reported on the adaptation of the cell mem-brane of psychrotrophic and mesophilic strains of At. ferrooxidans in response to low temperatures, as such bacteria play an impor-tant role in metal leaching and AMD production in colder mining environments. Specifically, we determined changes in membrane fluidity and fatty acid composition in response to low tempera-tures (5 and 15°C). Significant differences in membrane fluidity were found where the psychrotrophic strains had a significantly more rigid membrane at cold temperatures. Membrane remodeling was shown to occur in all strains with a common trend of increased unsaturated fatty acid component in response to lower growth tem-peratures. In psychrotrophic strains of At. ferrooxidans, decreases in 12:0 fatty acids distinguished the 5°C fatty acid profiles from those of the mesophilic strains that showed decreases in 16:0, 17:0, and cyclo-19:0 fatty acids. It was concluded that psychrotrophic strains employ distinctive modulation of cytoplasmic membrane fluidity with uncommon membrane phase changes as part of their adapta-tion to the extreme AMD environment in colder climates.

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Others have also investigated the importance of suboptimal temperatures on the bioleaching process [53-57]. At temperatures above 40°C, it is clear that thermophilic Archaea are more impor-tant than Bacteria with species such as Sulfolobus and Metallosphaera being the prominent types [26]. However, it is not clear how these Archaea contribute to commercial bioleaching operations [58].

p H

The pH of the leaching environment is important in the dissolu-tion of sulfidic ores. Indeed, both the ferric iron and the hydrogen ion concentrations are important determinants in the dissolution of acid-soluble sulfide minerals such as sphalerite and chalcopyrite [31]. As noted by Plumb et al. [59], a high concentration of protons is necessary for mineral sulfide dissolution. At low pH, the oxidation of ferrous iron and sulfur is microbially mediated and very little occurs abiotically [10,60]. The low pH is essential for iron cycling, i.e. ferrous iron used an electron donor during aerobic respiration and ferric iron used an electron acceptor during anaerobic respira-tion. The low pH is also necessary for reverse electron transport to take place. The large difference in pH between the cytosol and the environment of the autotrophic microorganisms produces a high transmembrane proton gradient which is used to power reverse electron flow and the subsequent synthesis of reduced nucleotides such as NADPH needed to reduce carbon dioxide to biomass. Therefore, a low pH bioleaching environment is mandatory for the activity of iron- and sulfur-oxidizing bacteria.

The pH of the PLS is dependent on several factors, including the type of the mineral sulfide and the gangue material. It is clear that the pH of the PLS will vary as a function of the balance between the acid-producing and acid-consuming components of the ore and by the anthropogenic addition of acid or base at the bioleaching site [59]. According to Plumb et al. [59], the pH of the bioleaching liquor of most heap leaching operations is the range 1.5 to 2.5.

Surprisingly, very few studies have been conducted on the effect of pH on the activities of iron- and sulfur-oxidizing bacteria involved in bioleaching. Yahya and Johnson [61] showed that pH had a pro-found effect on the leaching activities of Gram-positive bacteria and that their tolerance to extreme acidity (pH < 1) was high. In a later study, Plumb et al. [59] showed that the Archaea tested proliferated optimally over a lower pH range than most of the bacteria tested. The study also showed that microbial activity varied as a function


of pH and that this variation was important in strain selection as applied to bioleaching processes. We have also commented on the importance of strain selection in a study on temperature and resis-tance to heavy metals in different strains of At. ferrooxidans [32]. In a more recent study using moderately thermophilic bacteria for the solubilization of metals from a Pb/Zn slag, Guo et al. [62] showed that the solubilization of heavy metals from the slag was signifi-cantly influenced by pH and that the optimum pH was 1.5.

We [63] have recently published on the importance of cell mem-brane remodeling in strains of At. ferrooxidans exposed to different pHs. The effects of sub- and supra-optimal pHs in different strains isolated from AMD water around Sudbury, Ontario, Canada were tested. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of diphenylhexatriene, and fatty acid profiles were compared. pH 1.5 produced the most pronounced effect compared to the other pHs tested. The effect was manifested in signif-icant changes in overall membrane fluidity and phase characteristics through modulations in fatty acid composition. Greater acid tolerance appears in strains that have a more rigid membrane and that can also change their membrane fatty acid composition to maintain a func-tional membrane. We demonstrated that membrane physiology per-mits differentiating pH tolerance in strains of this extreme acidophile.

Recent studies by Cameron et al. [64-66] showed that bioleaching of a low-grade nickel sulfide ore at elevated pH (>3) is possible. Their attempts to bioleach a low-grade metamorphosed ultramafic-dominated nickel ore at low pH resulted in an unacceptable amount of solubilized magnesium and a significantly high consumption of sulfuric acid. However, at higher pH (>3), the nickel to magnesium ratio in the leachate was increased substantially with a concomitant reduction in sulfuric acid consumption.

Nutrients

Leaching chemolithotrophic microorganisms need certain nutrients to grow and proliferate. With respect to the major nutrients required, the leaching microbiota requires carbon, oxygen, nitrogen, and phos-phorus in addition to a few other micronutrients. With respect to the autotrophic bacteria such as the acidithiobacilli and leptospirilli, the carbon source is atmospheric carbon dioxide. These microorganisms fix the carbon dioxide using the Calvin-Benson cycle. On the other hand, the heterotrophic microorganisms cannot use carbon dioxide as a source of carbon and, therefore, feed off the waste products of

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the autotrophic microbial community. As mentioned earlier, there is some evidence that heterotrophic microorganisms may actually help the bioleaching process even though they are not directly involved in metal solubilization [36,67]. Some strains of At. ferrooxidans have been shown to grow on formate [68] although it is not clear whether this ability is actually useful in a bioleaching setting [69].

With respect to the requirements for oxygen, molecular oxygen is the most common electron acceptor because it is the most thermody-namically favorable oxidant in aerobic metabolism. However, under anaerobic conditions, ferric iron can be used by some microorgan-isms. Indeed, the redox potential of the ferrous-ferric couple is nearly as positive as the oxygen-water couple. Pronk et al. [27] reported that some strains of At. ferrooxidans use ferric iron as the terminal electron acceptor when grown anaerobically on reduced sulfur compounds.

Nitrogen and phosphorus are also required by the microorgan-isms involved in bioleaching. For At. ferrooxidans, at least, the pre-ferred form of nitrogen is the ammonium ion and at a relative low concentration (0.2 mM) [70]. High concentrations of inorganic and organic forms of nitrogen inhibit the activity of At. ferrooxidans [69]. Common garden fertilizer containing ammonium sulfate is rou-tinely added to bioleaching operations [71]. Several strains of At. ferrooxidans have been reported to show a capacity for nitrogen fixation. However, given the oxygen sensitivity of nitrogenase, the nitrogen fixation activity is probably limited to areas of low oxy-gen tension in bioleaching environments. Although some strains of L. ferrooxidans have also been shown to harbor the genes required for nitrogen fixation [72], the fact that it is a strict aerobe makes it unlikely that this bacterium can fix significant amounts of dinitro-gen in leaching environments. Interestingly, there is some evidence that in the case of underground in situ bioleaching, the addition of nitrogen is not necessary as there is enough residual nitrogen fol-lowing blasting operations [73]. Phosphorus is commonly added in the form of either H3P04 or KH2P04. The micronutrients are natural contaminants of the ore and are not usually supplemented [74].

Toxic Substances

Several studies have shown that leaching microorganisms can be affected by a range of organic substances and metal cations. With respect to the inhibitory effects of organic compounds, solvent extraction compounds [75], surfactants [75], metabolic products [76], and simple organic compounds [77] have been reported. In the


Frattini et al. study [77], the effects of naturally-occurring compounds including glucose, cellobiose, galacturonic acid, and citric acid on the growth of At. ferrooxidans were determined in the view of using these compounds in the control of AMD. Each of these compounds had an inhibitory effect and the sensitivity to these compounds was strain dependent. Obviously, an inhibition of the activity of the microor-ganisms involved in bioleaching leads to reduced metal recovery.

With respect to metals, it is understood that the microbes involved must be resistant to any metals in the leaching environment and to the metal being extracted. It is also understood that the concentra-tion of the extractable metal will increase several fold as the leach-ing progresses. It was not necessarily predictable that the degree of metal resistance in At. ferrooxidans varies as a function of the elec-tron donor being oxidized [78]. Ferrous iron-grown At. ferrooxidans was shown to be 2000 times more resistant to copper, nickel, and zinc, than thiosulfate-grown cells. Similar results were reported by Silver & Torma [79], which indicates that metal resistance for this bacterium is dependent on the source of energy.

For uranium bioleaching by At. ferrooxidans, the metals of primary interest with regard to resistance are ferric iron, uranium and thorium. Their expected in situ concentrations are not likely to be inhibitory to the bioleaching process because, as pointed out by McCready & Gould [73], native microbes have acquired a tolerance to their chemi-cal and environmental conditions. Indeed, for Denison Mines (Elliot Lake, Ontario, Canada) mine water samples, Fe3+ concentrations were less than 2000 ppm, and laboratory studies showed that the bacterial growth rate was unaffected at Fe3+ concentrations of up to 6000 ppm and that growth was possible at even higher concentrations (unpub-lished data). Furthermore, a range of tolerance levels to uranium as U308 has been reported. Duncan & Bruynesteyn [80] isolated strains of At. ferrooxidans from uranium mines and these strains were active at concentrations in excess of 12 g L"1. Tuovinen et al. [78] showed strains resident in a uranium mine to be resistant to approximately 0.7 g L"1. Subsequently, Tuovinen & Kelly [81] reported growth inhi-bition in the range of 0.056 to 0.25 g of U30g L"1, but that cultures resistant to 1.40 g L_1 could be developed by successive subcultur-ing in media containing increased uranium concentrations. Martin et al. [82] and Huber & Stetter [83] reported resistances to uranium of 4.0 mM and 0.4 mM, respectively. The strains of Martin et al. [82] that were uranium resistant contained a plasmid, and loss of the plasmid was asssociated with loss of resistance to uranium. Again for the Denison mine water samples, we observed that growth rate

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and the maximum amount of Fe3+ produced were unaffected by U308 in the range 0 to 1.37 g L_1. At 2.7 g L_1, the generation time increased from an average of 22 h to approximately 30 h, and at concentrations of 5.5 and 11.0 g L_1, growth did not occur. As the effluents from actively-leaching stopes contained less than 1 g of U30g L'\ we concluded that the indigenous strains of At. ferrooxidans were acceptably tolerant to uranium. In a study by Leduc et al. [84], the inhibitory concentrations for ferrous iron oxidation by copper, nickel, uranium, and thorium, for 10 different strains of At. ferro-oxidans were reported. For uranium, the inhibitory concentrations, defined as those concentrations that showed a significant (p < 0.01) decrease in the percentage of ferrous iron oxidized when compared to controls, were in the range of 1.0 to 8.0 mM.

With respect to metal resistance, unlike the situation for tem-perature, the genetic improvement of naturally-occurring strains of At. ferrooxidans through the use of mutagens or genetic engineer-ing is possible and an area of extreme interest. It is also possible that as a microorganism becomes acclimated to a particular ore, its resistance to the metals present increases due to selective pressures. Li & Ke [85] studied the effects of magnesium, nickel, and copper on a nickel-adapted strain of At. ferrooxidans. In their study, a wild strain of At. ferrooxidans was adapted to resist up to 30 g L_1 nickel by serial sub-culturing over a period of 12 months. Different com-binations of the metals were tested and the resistance varied as a function of the combination of metal used and the corresponding concentration. Recently, we [86] have reported on the use of growth characteristics, fluorescence polarization, and fatty acid composi-tion to determine the role of membrane structure and function in response to copper and nickel exposure in At. ferrooxidans. We found that the membrane of different strains of At. ferrooxidans shows sig-nificantly different modes of membrane adaptation in response to copper and nickel. Indeed, membrane fatty acid composition and the resulting fluidity and phase characteristics seemed to play an important role in determining the differential susceptibility of indi-vidual strains to metal toxicity.

Environmental Considerations

The environmental impact of bioleaching is a function of the type of leaching process employed. If the ore leached remains underground, as was the case with the Elliot Lake uranium leaching operations,


the environmental consequences to the neighboring ecosystems are insignificant [73]. There is some concern, however, with respect to the increased radon gas levels in such underground leaching sites. McCready [87] estimated that a mining rate of 10,000 tonnes per day at the Denison Mines Inc. bioleaching sites was equivalent to 15 days of mining operations. Therefore, the increased radon ema-nation from such sites could represent a health hazard to miners. Additional ventilation of leaching sites may offset this concentra-tion effect. In addition, a thorough monitoring regime during active leaching periods would be helpful in determining the contribution to radon gas levels from the leaching sites.

In contrast to in situ underground leaching, ores leached from heaps on the surface represent a potentially serious hazard to neighboring ecosystems, particularly aquatic ones. The resulting acid rock drainage (ARD) from such heaps can cause direct envi-ronmental damage to the biota and the reduced pH increases the mobility of heavy metals, including long-lived radioisotopes such as thorium, radium and residual uranium as is the case with ura-nium bioleaching. ARD and heavy metal contamination tend to decrease species diversity and community biomass. The severity of this reduction is dependent on the dilution and buffering capaci-ties of the receiving waters. In other words, if the volume of the receiving waters or the buffering capacity of such waters is low, the magnitude of the environmental damage is substantial and signifi-cant. There is no doubt, therefore, that surface bioleaching of ores presents the most significant waste management problems.

Conclusions

It is evident that an interest in the application of bioleaching has spawned numerous scientific investigations and that there is an extensive literature dealing with the participating microorganisms, especially At. ferrooxidans. As some important questions remain unanswered, bioleaching and bio-oxidation should continue to be active areas of experimentation.

Acknowledgements

The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada through the

BlOLEACHING 257

receipt of operating grants, and the contributions of their graduate and undergraduate students.

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