Life in heaps: a review of microbial responses to variable acidity in sulfide mineral bioleaching heaps for metal extraction

Accepted Manuscript Life in heaps: A review of microbial responses to variable acidity in sulfide mineral bioleaching heaps for metal extraction D.W. Shiers, D.M. Collinson, H.R. Watling PII:

S0923-2508(16)30047-X

DOI:

10.1016/j.resmic.2016.05.007

Reference:

RESMIC 3507

To appear in:

Research in Microbiology

Received Date: 1 March 2016 Revised Date:

24 May 2016

Accepted Date: 25 May 2016

Please cite this article as: D.W. Shiers, D.M Collinson, H.R Watling, Life in heaps: A review of microbial responses to variable acidity in sulfide mineral bioleaching heaps for metal extraction, Research in Microbiologoy (2016), doi: 10.1016/j.resmic.2016.05.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Life in heaps: A review of microbial responses to variable acidity in sulfide mineral

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bioleaching heaps for metal extraction.

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Shiers, D.W.*, Collinson, D.M. and Watling, H.R.

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CSIRO Mineral Resources, Australian Minerals Research Centre, PO Box 7229, Karawara,

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WA 6152, Australia.

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E-mail addresses: [email protected] (D.W. Shiers)* correspondence and reprints,

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[email protected] (D.M. Collinson), [email protected] (H.R. Watling)

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Abstract

Industrial heap leaching of low grade mineral sulfide ores is catalysed by the use of

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acidophilic microorganisms. These microorganisms obtain energy for growth from the

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oxidation of reduced inorganic or organic compounds, including soluble ferrous ion, reduced

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inorganic sulfur compounds (RISC) and acid-stable organic compounds. By-products of these

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oxidative processes, such as soluble ferric ion and sulfuric acid create favourable chemical

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conditions for leaching. This review is focused on the behaviour of common bioleaching

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microorganisms, their responses to changing pH in an industrial setting, and how both

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changes and microbial responses can impact the micro and macro environment.

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Keywords: acid stress; chemolithotrophs; acidophiles

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1.

Introduction Heap (dump and in situ) leaching technologies were developed for the processing of ores

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where metal values are too low or otherwise not suited to flotation concentration and higher-

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intensity pyrometallurgical processing. Thus, heap leaching is mainly applied to low-grade

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ores such as secondary copper sulfides, pyritic gold ores or nickel or nickel-copper sulfide

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ores [1–3]. The technology is well established and accepted by the mineral processing

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industry and has been practiced in such diverse climates as the South American Andes (Fig.

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1), near the Arctic Circle and in the tropics [1,4,5].

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The technology is simple and based on natural, microbially-assisted processes first

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described more than 2000 years ago and today termed acid rock drainage (ARD). Some

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recent accounts of modern heap practice and the perceived economic benefits have been

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published [1,6,7]. Briefly, heaps are dynamic systems in which the substrate (sulfide) content

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diminishes with time, acid is consumed by gangue minerals but periodically augmented to

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maintain the leaching environment, giving rise to variations in solution pH. Secondary

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minerals may precipitate, changing the nature of particle surfaces or releasing/consuming

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acid during their formation. Thus microorganisms are subject to fluctuating solution acidity

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and substrate availability over the course of mineral sulfide dissolution.

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Metals extraction relies upon heap microorganisms to accelerate otherwise slow reactions

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in which the metal-oxygen bonds in sulfide minerals are broken and the minerals dissolved to

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their components [6]. Very slow mineral sulfide dissolution can occur via the action of strong

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acid (e.g., for chalcopyrite, reaction 1). Faster dissolution is achieved through the action of

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ferric ion, an oxidant, in an acidic sulfate medium (e.g., for covellite, reaction 2).

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CuFeS2 + O2 + 4H+ → Cu2+ + Fe2+ + 2S0 + 2H2O

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CuS + 2Fe3+ → Cu2+ + 2Fe2+ + S0

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The main reactions through which acidophiles contribute to accelerated extraction are the

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oxidation of ferrous ion and sulfur, the products of sulfide dissolution (reactions 3 and 4).

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Note that reaction 3 is acid consuming and reaction 4 is acid generating and that through

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these two reactions both the required oxidant (ferric ion) and acid are replenished in the

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process water. Heterotrophs and mixotrophs contribute through the utilisation of organic

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compounds that may inhibit the activities of some chemolithotrophs (e.g., glucose, reaction

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5).

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4Fe2+ + 4H+ + O2 → 4Fe3+ + 2H2O

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2S0 + 3O2 + 2H2O → 2H2SO4

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C6H12O6 + 6O2 → 6CO2 + 6H2O

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Under anaerobic conditions ferric ion reduction can take place, coupled with sulfur or

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organic compound oxidation (reactions 6 and 7). Reactions 3–7 provide energy for

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microorganisms, dependent upon their metabolic capabilities.

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24Fe3+ + C6H12O6 + 6H2O → 24Fe2+ + 6CO2 + 24H+

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6Fe3+ S0 + 4H2O → 6Fe2+ + 8H+ + SO42–

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The isolation and characterisation of acidophiles from acidic mine drainage and other

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acidic metalliferous environments have been the foundation for numerous studies on

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microbial behaviours. These may be laboratory or larger-scale studies to examine the impacts

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of various parameters, such as process water composition and acidity, mineral compositions

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and impurities, and other solution chemistry parameters (oxidation-reduction potential

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(ORP), ionic strength). In this review some effects of variable acidity on substrate utilisation,

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attachment and microbial populations are discussed in the context of heap leaching,

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including: (i) a summary of the acidophiles that have been isolated from, or putatively

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detected, in heaps and the mechanisms that can be employed to protect them against acid

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stress; (ii) substrate utilisation responses to variable pH and how this can be used to maintain

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microbial viability; (iii) the importance of microbial attachment to sulfide minerals and the

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impacts of pH; (iv) the effects of pH on microbial community compositions, and (v) the

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impact of pH on microbial activity during leaching. Knowledge of the responses of

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microorganisms to acid stress will assist in interpreting and/or managing some problems

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arising during metal production from changeable sulfide-heap environments.

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2.

Acidophiles in heaps and waste dumps

There is no formal definition of what constitutes an acidophile [8], but rather a general

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consensus that an acidophile has an optimal pH (pHOPT) for growth significantly lower than

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pH 7, while an acid-tolerant microorganism has a pHOPT for growth closer to neutral pH. A

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further subdivision into “moderate acidophile” with pHOPT for growth in the range pH 3–5,

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and “extreme acidophile” with pHOPT for growth of pH < 3 has been proposed [9].

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Acidophiles can be grouped according to their adaptation to the acid environment (pH

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minima and maxima for growth) and to the substrates from which they gain energy for

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metabolic processes, oxidation of ferrous ions or RISC, or utilisation of organic carbon

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compounds (Table 1). These simple groupings ignore other equally important categories,

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such as temperature (psychrophiles, mesophiles, moderate thermophiles, thermophiles or

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hyperthermophiles), or method of obtaining carbon (obligate autotrophs, facultative

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autotrophs and obligate heterotrophs), which are discussed comprehensively elsewhere [8].

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The acidophiles or acid-tolerant microorganisms that colonise natural and/or managed

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acid drainage environments, including bioleaching heaps, waste dumps or in situ metal

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extraction operations, exhibit some remarkable characteristics. Detailed studies of the

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mechanisms used by bioleaching acidophiles to counter the deleterious effects of strong acid

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are outside the scope of this review. However, arguably, the most relevant is the ability to

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maintain an approximately neutral intracellular acidity (approximate range pH 5–7) while

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growing in solutions in the range pH 1–3. Important mechanisms for maintaining cell pH

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homeostasis include generating a reversed membrane potential, buffering the cytoplasm with

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various molecules, pumping excess protons out of the cell, modifying the cell wall and/or

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increasing the rates of protein and DNA repair [27]. All require the synthesis of additional

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proteins or enhanced cellular repair rates, which will lead to an increased expenditure of

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energy on cell maintenance. If cells fail or are inefficient, then acidification of the cytoplasm

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can lead to cell death [27]. Details of the physiological mechanisms by which cell pH

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homeostasis is maintained have been published in some informative reviews [28–30].

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Effects of pH on cell growth

Descriptions of new species often include an estimate of the pH range for growth. Those

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data may be complete, presenting a pH range extending to both high and low pH extremes for

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growth (together with an estimated pHOPT), or partial, where the pH range is restricted to the

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acidity thought relevant to a particular study. Other data can be retrieved from fundamental

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studies on the effects of acidity on growth or other physiological functions (e.g., Plumb et al.

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[31]). The compilation presented (Fig. 2) comprises data for 66 acidophiles, many of which

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have been isolated from or detected in heap samples (pH 1–4) or acid mine drainage

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environments (pH 2–6).

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Despite the above limitations, and based on a general analysis of the available data (Fig.

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2), the first point of difference is that archaea are generally more acidophilic or acid tolerant

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than bacteria. Some specific examples of archaea that grow in extremely acidic environments

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include Acidianus sulfidivorans, capable of growth on chalcopyrite at pH 0.35 with pHOPT

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0.8–1.4 for growth on ferrous ion [24]. Acidiplasma cupricumulans (formerly Ferroplasma

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cupricumulans), is capable of growth at pH 0.4 but has a pHOPT 1–1.2 for ferrous ion

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oxidation [5]. Acidianus brierleyi, a well-known bioleaching microorganism that has not so

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far been detected in heaps, grew on sulfur in the range pH 0.5–2.5 with pHOPT 1 [31], while

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the closely-related, Acidianus infernus grew on sulfur in the range pH 1–5.5 with pHOPT 2

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[32].

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The second point of difference is the narrow pH range for ferrous ion oxidation exhibited

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by the microorganisms, which may or may not represent the full pH range for growth. The

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cause is undoubtedly chemical, directly controlled by the formation of insoluble iron(III)

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compounds [33]. The removal of ferric ions, through the precipitation of compounds such as

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poorly-crystalline schwertmannite (Fe8O8(OH)6SO4), jarosite ((H,K,Na)Fe3(OH)6(SO4)2) or

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other mixed iron(III) precipitates with variable stoichiometries, impacts directly on the

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continued ferric-ion oxidation of mineral sulfides (e.g., reaction 2) and on the consequent

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release of ferrous ions for the ongoing growth of iron-oxidising microorganisms (reaction 3).

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At the same time, the formation of insoluble iron(III) compounds on the surface of

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microorganisms may prevent electron transfer through outer membrane mediators [34].

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Thirdly, it is hypothesised that, in dynamic heap environments where pH changes can

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occur on the micro and macro-scales, microorganisms that can activate alternative metabolic

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pathways would be favoured over those restricted to ferrous ion oxidation (e.g., the

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autotrophic chemolithotrophic Leptospirillum spp.). The genus Sulfobacillus, which is

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ubiquitous in mine-impacted environments [35], provides an example [36]. In comparative

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tests, using the methods described in [20,35,37], growth curves of S. thermosulfidooxidans on

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different substrates revealed pHOPT values that increased from pH 1.6 (ferrous ions), to pH

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2.4 (tetrathionate) to pH 3 (glucose); pH ranges broadened in the same order indicating that

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growth across the pH range 1–6 would be possible by activating different metabolic pathways

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[36]. The results are consistent with data for S. sibiricus grown on ferrous ion (range pH 1.1–

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2.6, pHOPT 2.0) compared with elemental sulfur (range pH 2.0–3.5, pHOPT 2.2–2.5) [38] and

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for S. thermotolerans grown on ferrous ion (range pH 1.2–2.4, pHOPT 2.0) compared with

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elemental sulfur (range pH 2.0–5, pHOPT 2.5) [39].

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Similar results were reported for Alicyclobacillus-like strain FP1, isolated from copper-

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rich process water (pH 1.3, 3–4 g Cu L–1) where growth on ferrous ion was constrained

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within the range pH 1–3 (pHOPT 1.8), but with glucose as substrate, the pH range for growth

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was extended to pH 7 (pHOPT 4–5) [36]. The results are consistent with data for related,

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ferrous ion-oxidising heterotrophic Alicyclobacillus spp. For A. tolerans, the reported range

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for growth on ferrous ion was pH 1.5–4 with pHOPT 2.5–2.7 [40] compared with the

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equivalent data for growth on glucose by ferrous ion-oxidising species A. aeris (range pH 2–

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6, pHOPT 3.5) [41] and A. ferrooxydans (range pH 2–6, pHOPT 3) [42].

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In another comparison, the effect of pH on the growth of Acidithiobacillus caldus was

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examined for two substrates, tetrathionate and glucose. Glucose utilisation rates were high in

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the range pH 1–5, above which they slowed (pHOPT 3.5) but the effect of pH on tetrathionate

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utilisation was more closely defined, with high rates in the range pH 1–4 (pHOPT 1.8) [36].

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Previously, for At caldus, different pH ranges and optima for growth on tetrathionate (pH 1–

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3.5; pHOPT 2.2), sulfide (pH 2–6, pHOPT 4.5) or sulfite (pH 2–4, pHOPT 3) were reported

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[43,44]. A similar response occurred with Alicyclobacillus disulfidooxidans, a species lacking

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ferrous ion-oxidising capability; growth on sulfur, glutamate or glucose occurred over a

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broad range (pH 0.5–6) with the reported pHOPT 1.5–2.5 “depending on the substrate” [45]. Thus it can be deduced that bioleaching organisms in general, but specifically

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Sulfobacillus, Alicyclobacillus and Acidithiobacillus spp., are equipped metabolically to

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maintain growth in heaps with leach liquors from pH 1–6. They achieve this by utilising

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ferrous ions and/or sulfur generated by the oxidation of mineral sulfides (reactions 1, 2) but,

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if the inorganic secondary reaction products are consumed or the pH rises, remain viable by

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utilising the variety of organic compounds present in their environments.

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4.

Effects of pH on attachment

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During heap leaching of low grade ores, the exposure of sulfide mineral surfaces can be

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limited (Fig. 3). In this image, few of the sulfide grains were exposed on the surface of the

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particle and no micro-cracks or voids were detected that might permit contact between

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‘trapped’ grains and the lixiviant containing microorganisms. Thus, microbial mechanisms

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for the detection of a sulfide surface (the substrate), movement towards it and establishment

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of a close-association with it are requirements of microorganisms passing through a heap in

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the bulk solution. Some biomining bacteria and archaea are equipped with flagella or other

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cell appendages that assist their movement [46–49]. In an innovative study using a 3-D

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tracking microscope, the speed of movement by Sulfolobus acidocaldarius at 80 °C was

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estimated to be 7–15 µm s-1 and was largely unchanged with increased pH [50]. Chemotactic

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responses have been described for Acidithiobacillus ferrooxidans and Leptospirillum

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ferrooxidans, two of the earliest identified and most studied ‘biomining’ microorganisms.

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Their active movement towards sources of ferrous ion, tetrathionate or thiosulfate was

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described and L. ferrooxidans was also attracted to sources of nickel and copper ions [51–53].

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Both At. ferrooxidans and At. thiooxidans exhibited chemotactic responses when provided

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with spherical particles of two poorly-soluble substrates, sulfur or pyrite [54]. Once microorganisms find themselves in the vicinity of sulfide minerals, initial

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adsorption is relatively rapid. For example, greater than 80% of At. ferrooxidans cells adsorb

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to a substrate in 10–60 min [55,56]. Generally, the rates of attachment vary between species

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and depend on the mineral substrate with a rank order: pyrite>chalcopyrite, sphalerite,

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galena>>quartz [55–58]. This initial adsorption may be reversible but, with time, adhered

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microorganisms produce extracellular polymeric substances (EPS) of heterogeneous

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composition that anchor the colonies in the form of biofilms on the sulfide surface. Biofilms

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provide a protective microenvironment against a possibly inhospitable bulk-solution

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composition or other dynamic forces such as solution flows or abrasion [59].

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Despite strong interest in microbial attachment to sulfide minerals, only `four studies

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describing the effects of changing solution pH on attachment or the development of biofilms

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were found. In the first, the focus was a comparison of the early-stage adsorption rates of At.

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ferrooxidans to several high-purity model minerals [60]. As a minor part of that study, the

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attachment of At. ferrooxidans to chalcopyrite surfaces was studied at pH 1.5, 2.0 and 5.6;

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there was little difference between pH 1.5–2.0, but the number of attached cells decreased by

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approximately 50% when the pH was increased to pH 5.6 [60]

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In the second study, the effects of physical factors (temperature, ionic strength, agitation

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and pH) and chemical factors (addition of hydrogen peroxide, ferric or cupric ions or sodium

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glucuronate) on biofilm formation of three ferrous ion-oxidising Acidithiobacillus strains (At.

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ferrooxidansT type strain, At. ferrivorans SS3 and At. ferrooxidans R1) were compared [61].

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A number of different techniques were employed to evaluate the attachment of

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Acidithiobacillus cells to pyrite grains, including epifluorescence microscopy and confocal

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laser scanning microscopy. Of general interest in the context of heap bioleaching, the

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research showed that solutions 0.435 M ionic strength caused At. ferrooxidansT attachment to

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halve, compared with the control (ionic strength 0.035 M). More specifically, in the context

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of the present review, it was shown that, after 8 days of incubation at 28 °C in medium of pH

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1.8, At. ferrooxidans R1 (29.6±4.5%) and At. ferrivorans SS3 (12.5±3.5%) colonised pyrite

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surfaces more efficiently, as a percentage coverage of the surface than At. ferrooxidansT

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(4.8±0.7%) [61]. After 8 days of incubation, there was no difference in attachment at pH 1.8

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or pH 3. However, in medium pH 1, the three strains exhibited significantly lower degrees of

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attachment at 8 days (At. ferrooxidans R1 (1.2±4.5%), At. ferrivorans SS3 (1.5±0.7%) and

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At. ferrooxidansT (2.6±0.7%).

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In the third study, the focus was the effect of increased acidity on the attachment of

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Sulfobacillus thermosulfidooxidans to pyrite surfaces [62]. Attached bacteria were obtained

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by immersing a pyrite coupon vertically in a bacterial suspension (pH 2.2) for 24 h. After

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positioning the coupon in the atomic force microscope cell and initiating solution flow over

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the surface, images were collected before and after the addition of acid. The rapid change

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from pH 2.2 to pH 1.0 in the cell, that is from a condition favourable to bacterial growth to an

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acid-stress condition, caused many bacteria to become dislodged from the surface. However,

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staged addition of acid, from pH 2.2 to pH 1.4 (favourable to bacterial growth) resulted in a

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surface with attached bacteria that became ‘blurred’ with time (Fig. 4a), an effect attributed to

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the eventual formation of a nearly coherent EPS layer on the pyrite surface. Subsequent

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addition of acid to pH 0.9 did not cause further marked changes in the images collected but

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there was a marked decrease in the thickness of the EPS layer (Fig. 4b), attributed to

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structural re-arrangements within the EPS network [62].

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In the fourth study [63], a mixed, moderately-thermophilic culture containing A. caldus, S.

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acidophilus and F. thermophilum was used to inoculate a chalcopyrite suspension and

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leached for 20 days without pH control; solutions were pH 2.1 (day 5), pH 1.6 (day 10), pH

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1.3 (day 15) and pH 0.9 (day 20). Attached microbial-population compositions were variable

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under conditions of uncontrolled pH. At caldus dominated compositions when the solutions

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were pH 2.1 (70%) and 1.6 (55%) but diminished as the pH became lower. S. acidophilus

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was also major component (40%) at solution pH 1.6 (near its pHOPT (Table 1)) and F.

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thermophilum was the major component (65%) when the solution was pH 1.3. Surprisingly,

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the species had equivalent proportions in the solution pH 0.9, an acidity that would be

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anticipated to favour F. thermophilum (pHOPT 1) [64].

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5.

Effects of pH on heap microbial communities

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The impact of pH on population structure has been the focus of a number of studies, some

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conducted using crushed ore in columns (simulated heaps) in which the recycled leach

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solution was initiated at a fixed pH. For example, changes in mesophilic populations in

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columns of polymetallic black-shale ore operated at 35 °C and, initially pH 1.2, 1.6 or 2.0,

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were examined after 102 days of leaching [65]. At pH 1.2, Thermogymnomonas,

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Leptospirillum and Ferrimicrobium spp. (native to the ore) dominated the population (Fig. 5).

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At pH 2.0, the dominant species were Leptospirillum, Ferrimicrobium and Acidithiobacillus

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spp., with a lesser Thermogymnomonas component in the population. Changes in physico-

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chemical solution parameters that occurred during 102 days of leaching at 35 °C were

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increases in total iron concentrations from an initial 1–2 to 13–16 g L–1 Fe, mainly being

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solubilised as ferrous ions from the oxidation of pyrite (reaction 8) but subsequently being

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oxidised by the dominant Leptospirillum and Ferrimicrobium spp. (reaction 3) [65].

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FeS2 + 7Fe2(SO4)3 + 8H2O → 15FeSO4 + 8H2SO4

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Leptospirillum ferriphilum can oxidise ferrous ions over a broad pH range (Table 1) and

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is selectively enriched at high ferric ion concentrations [66] and the growth of

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Ferrimicrobium acidiphilum (increase in biomass) was promoted strongly by the addition of

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ferrous ions in culture medium [22]. The presence of an heterotrophic Thermogymnomonas

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acidicola-like strain in the inoculum and all columns (both 35 and 50 °C) may be related to

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the organic content of the ore as the mixed inoculum used in the study was sourced from a

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coal mine and the species has broad ranges in both temperature and pH over which it grows

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[65]. At 50 °C, the key differences were the presence of Acidimicrobium spp. in the inoculum

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but not in subsequent column samples at either temperature (35 or 50 °C), and

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Metallosphaera spp. in the inoculum and in the 50 °C columns operated at pH 1.6 and 2.0.

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Both are thermophiles and can oxidise ferrous ions or utilise organic carbon, but the absence

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of Metallosphaera spp. in the pH 1.2 column is surprising, as Metallosphaera hakonensis has

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a range for growth of pH 1–4.5 (pHOPT 3) [65].

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Similarly, in an ambient-temperature column study on a pyrrhotite-rich, polymetallic

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black-schist ore, the effect of pH (1.5, 2.0, 2.5 or 3.0) on column populations was examined

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periodically [26]. Referring to the data for 111 days of leaching, differences were revealed

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between pH 1.5 (Fetotal 40 g L–1, ORP 550 mV), where the bacteria Leptospirillum and

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Acidithiobacillus spp. comprised 60% of the population, and pH 2.0 (a drop at 100 d from

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Fetotal 4 to 2 g L–1, ORP 620 mV), where bacterial numbers had decreased and archaea

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(Thermoplasma-like and Ferroplasma spp.) comprised more than 50% of the population. In

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contrast, microbial populations from the pH 2.5 and 3.0 columns were dominated by

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Sulfolobus and Thermoplasma-like spp. [26]. The absence of Ferroplasma at pH 2.5 and 3.0

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(both with approximate ORP 580 mV but Fetotal <20 mg L–1) is consistent with the reported

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range for growth of pH 1.3–2.2 (Table 1) and the near absence of soluble iron concentrations.

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Some data based primarily on the analysis of planktonic communities in the recycled

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leach solution from industrial heaps operated at different pH have been reported. Comparison

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of the community compositions for three copper heaps operated at pH 0.8 (Zijinshan, China),

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pH 1.3 (Escondida, Chile) and pH 2 (Dongxian, China), revealed that at Zijinshan [67] and

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Dongxian [68] mines, the communities were dominated by Acidithiobacillus and

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Leptospirillum spp. (together 80–90%) but that a more diverse population including archaea

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and bacteria had colonised the pH 1.3 Escondida heap [69]. From the dominance of

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Leptospirillum and Acidithiobacillus spp. at Zijinshan mine [67] it may be deduced that the

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species involved are Leptospirillum ferriphilum, Acidithiobacillus thiooxidans and/or

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Acidithiobacillus caldus, all of which are capable of growth at pH ≤1 (Table 1). For the

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Dongxian mine (pH 2) [68], iron-oxidising strains of Acidithiobacillus might be dominant,

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preferring to grow at the higher pH together with Leptospirillum spp. (Table 1). At

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Escondida, more comprehensive studies have been undertaken with the goal of linking

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changes in community composition to different stages in the leaching cycle [69]. Within the

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limits of the molecular method used to detect and estimate strains in microbial communities

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(DGGE band intensities), the authors examined possible links between dominant species in

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communities and a number of physico-chemical parameters in circulating solutions as a

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function of time. Data analysis revealed changes in community composition extending over a

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700 d monitoring period [69]: Acidithiobacillus / Sulfurisphaera-like spp. dominant in the

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period 0–250 d; Leptospirillum / Ferroplasma spp. in the period 250–350 d; and

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Ferroplasma / Sulfobacillus spp. in the period 350–700 d. Relevant to the present review of

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the effects of pH on bioleaching microorganisms, these shifts correlated approximately with

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increases from pH 1 to pH 1.1–1.2 and then to 1.3–1.5. However, at the same time, sulfate

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and ferric ion concentrations rose from approximately 20 to 150 g L–1 and from 0 to 4.5 g L–1,

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respectively, after 350 days. Subsequently, to the end of the leach, sulfate concentrations

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remained constant at approximately 150 g L–1 but ferric ion concentrations decreased to

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approximately 1 g L–1 [69]. The change to a Leptospirillum / Ferroplasma community could

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therefore have been promoted by increased sulfate and ferric ion concentrations (and

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concomitant high ORP). Subsequently, in the third period, Leptospirillum was apparently out-

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competed by Sulfobacillus spp. which, together with Ferroplasma, flourished in a period

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when ORP remained high but the overall iron concentration diminished strongly.

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In a study of a copper tailings impoundment (Shuimuchong, China) described as an

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“extreme and heterogeneous environment” [70], the relative abundances of microbial

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lineages in 90 samples, grouped according to pH (2–3, 3–4, 4–6, 6–7, ≥7), were screened.

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The authors noted that “shifts of microbial composition along the pH gradient were clearly

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evidenced and relative abundances of different lineages varied greatly across the tailing

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communities”. These authors [70] acquired a sufficiently large data set, to give weight to

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their conclusions regarding the relative abundances of Leptospirillum, Sulfobacillus and

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Ferroplasma being largely influenced by pH, and the variations in Acidithiobacillus being

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responses to ferric ion concentrations. The data are consistent with those obtained from the

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Escondida heap [69]. From the combined data of the studies summarised briefly, it can be

322

deduced that pH is only one of the factors controlling microbial community development in

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heaps.

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6.

Effects of pH on heap bioleaching efficiency

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The impact of pH on mineral sulfide dissolution efficiency is directly related to the ability

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of a given microbial consortium to withstand pH stress and maintain its catalytic functions,

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particularly ferrous ion oxidation. During industrial-scale heap bioleaching of crushed ore,

329

pH gradients develop on both the macro- and micro-scales. On the macro-scale, pH gradients

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develop as a result of gangue mineral acid consumption leading to high-pH process water

331

and, on occasion from process water mismanagement (operator error or equipment

332

malfunction) (high or low pH process water).

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A macro-scale consequence of inadvertently high pH conditions, is the removal of ferric

334

ion, the oxidant in sulfide dissolution (e.g., reactions 1 and 2), via the formation of insoluble

335

iron(III) compounds for example jarosite (reaction 9) [33]. Microbial oxidation of ferrous

336

ions is therefore constrained by low iron concentrations, but those microorganisms that can

337

activate another metabolic pathway, such as carbon utilisation may remain viable [36],

338

although not directly beneficial to metal extraction. In high-pH process water, the overall low

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acidity reduces the direct but slow acid dissolution of gangue or sulfide minerals and the lack

340

of the oxidant (ferric ion) causes a reduction in mineral sulfide dissolution rates [26].

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Restoration of the process water acidity allows a variety of chemical dissolution reactions to

342

proceed, delivering ferrous ions to solution through gangue mineral dissolution, and re-

343

stimulating microbial ferrous ion oxidation. On the micro-scale, microorganisms may exhibit

344

less efficient attachment to exposed mineral sulfide (substrate) grains (Fig. 3; [60]) and the

345

formation of insoluble iron(III) compounds on cell surfaces [62] may interfere with electron

346

transfer through outer membrane mediators [34].

347

3Fe3+ + 2SO42– + 6H2O + Na+ → NaFe3(SO4)2(OH)6 + 6H+

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9

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At some heap leach operations, it may be advantageous to operate at stronger acidity than

349

‘normal’ to achieve a specific goal. For example, during pilot-plant operation at the Zijinshan

350

mine, where the high pyrite content was the main source of acid and ferrous ions (reaction 8),

351

the pH of both pregnant leach solution (PLS) and raffinate decreased from 1.8 and 2.5 to 1.1

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and 1.2, respectively, in a 9 month period [71]. Subsequently, the commercial heaps were

353

operated at pH 0.8 by which time a stable microbial community had developed [67]. Without

354

effective microbial adaptation, the mineral sulfide dissolution rate would be effected by acid

355

dissolution reactions rather than by an oxidative process and would therefore be greatly

356

reduced [6].

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While heap operation at low pH may be advantageous to an operation, microbial recovery

358

from inadvertent low pH is generally poor. For example, when a mesophilic culture

359

dominated by Leptospirillum ferriphilum was unexpectedly exposed to a pH of 0.4, well

360

below its active range (pH 0.9–1.5), microbial ferrous ion-oxidation was not resumed in a 10-

361

day period [72]. Similarly, the addition of acid to raffinate prior to solution recycle resulted in

362

decreased iron(II)- and RISC-oxidising activity in a heap [73] and a sudden decrease from pH

363

1.5 to 1.0 suppressed ferrous ion- oxidation during the leaching of pyrite in laboratory tests

364

[74]. Mazuelos et al. [75] reported that ferrous ion oxidation by a mixed culture of At.

365

ferrooxidans and L. ferrooxidans was suppressed when the pH was below or above tolerance

366

limits, but that oxidation rates recovered rapidly when the high-pH stress condition was

367

acidified appropriately. Tupikina et al. [76] also noted that microorganisms did not recover

368

from adverse pH conditions during column bioleaching of chalcopyrite. In that column study,

369

the effect of acid stress on a mixed culture subjected to a temperature increase from 25–50 °C

370

and a change in pH from an initial pH 1.7 to (in different columns) pH 1.0, 1.2, 1.4, 1.5 or 1.7

371

was studied. In the columns operated at pH 1.2–1.7 microbial activity did not abate.

372

However, at pH 1.0, the EH dropped from approximately 890 mV to 650–700 mV (relative to

373

the standard hydrogen electrode) and remained low, an indication that ferrous ion-oxidation

374

had ceased. In the context of heap leaching, the delay in microbial recovery would be

375

accompanied by a delay in the resumption of mineral sulfide oxidation by ferric ions.

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On a micro-scale, sudden increases in acidity impact directly on microbial attachment

377

efficiency, as demonstrated for three Acidithiobacillus species [61], and cause changes in the

378

nature and thickness of the EPS layer covering the cells, possibly as a protective layer against

379

an inhospitable environment [62]. This result is important in the context of the microbial

380

colonisation of pyrite grains, as in the pre-treatment of refractory gold ores or concentrates to

381

release occluded gold grains [2]. The oxidation of pyrite directly releases sulfuric acid at the

382

grain surface during mineral dissolution (reaction 8) and may result in a micro-layer that is

383

strongly acidic, compared with the bulk solution. Another consequence of extreme acidity on

384

the micro scale would be the inhibition of ferrous ion and sulfur oxidation at the pyrite

385

surface, for example, the lower pH limits for microbial activity (Fig. 2; Table 1). As already

386

noted, these microbial oxidation activities are necessary for rapid mineral sulfide dissolution.

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Depending upon ore mineralogy, another consequence of heap operation at low pH can be

388

increased rates of gangue mineral dissolution, resulting in high ionic-strength process water.

389

For example, samples of heap process water from a copper heap contained sulfate (up to 200

390

g L–1), iron (16 g L–1) and aluminium (22 g L–1), contributing to an estimated ionic strength

391

approximately 7 M [77]. On a micro scale, this is important because high ionic strength is

392

another of the heap ‘conditions’ resulting in reduced bacterial attachment [61] and activity

393

[78].

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7.0

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Summary

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This review highlights the substantial impacts of solution pH on the growth and activity

397

of microorganisms in heap bioleaching environments. pH gradients can occur spatially and

398

temporally throughout heaps. The impacts of these gradients on substrate availability and

399

utilisation, microbial attachment to sparsely-distributed sulfide mineral surfaces, and to

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community compositions and their activity during heap leaching is considerable. The varied

401

pH tolerances of acidophiles are evident from the collation of 'biomining' bacteria and

402

archaea. They are both species- and substrate-dependent. In high pH environments where

403

ferrous ion concentrations are limited, the utilisation of organic substrates represents a

404

survival mechanism for mixotrophic or heterotrophic organisms until conditions that favour

405

ferrous ion and RISC utilisation are restored. Utilisation of selected organic and RISC

406

compounds allow for a drop in pH in the immediate micro-environment, facilitating more

407

acidic conditions where ferrous ion oxidation is favourable. The attachment of

408

microorganisms to sulfide minerals is strongly impacted, with sharp pH changes causing

409

rapid new-cell detachment from sulfide minerals. This allows cells to be removed from

410

stressful chemical environments. Conversely, favourable pH conditions promote attachment,

411

biofilm development and growth on sulfide surfaces. Community compositions, as expected,

412

also vary with pH. Changes are dependent upon the heap environment, initial microbial

413

community and microorganisms indigenous to ore samples. Overall, the microbial

414

communities are equipped to meet the challenges of a dynamic heap environment.

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The impact of pH on microorganisms in heap environments should not be neglected

416

during operation. Excursions to extremes of pH, particularly extreme low pH, has been

417

shown to cause an irreversible loss of activity in heap microorganisms. The resulting changes

418

in community composition can impact metal production if ferrous ion- or RISC-oxidising

419

microorganisms are unable to facilitate mineral dissolution at a rate conducive with the

420

economic recovery of one or more valuable commodities. Deliberate inoculation strategies to

421

maximise a community that can tolerate acid stress (such as having a high proportion of

422

mixotrophic organisms) could potentially assist in maintaining a viable environment for

423

microorganisms, and thus enhance heap productivity.

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Acknowledgements We thank the Australian Government for funding through CSIRO Mineral Resources.

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Conflict of interest None declared.

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Liu XY, Chen B, Wen J, Ruan R. Leptospirillum forms a minor portion of the population in

Zijinshan commercial non-aeration copper bioleaching heap identified by 16S rRNA clone

606 607

AC C

602

He Z, Xiao S., Xie, X, Hu Y. Microbial diversity in acid mineral bioleaching systems of Donxiang copper mine and Yinshan lead-zinc mine. Extremophiles 2008;12:225–234.

[69.]

Demergasso CS, Galleguillos P PA, Escudero G LV, Zepeda VJ, Castillo D, Casamayor EO.

610

Molecular characterization of microbial populations in a low-grade copper ore bioleaching

611

test heap. Hydrometallurgy 2005;80:241–253.

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ACCEPTED MANUSCRIPT 612

[70.]

Liu J, Hua, ZS, Chen LX, Kuang JL, Li SJ, Shu WS, Huang LN. Correlating microbial

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diversity patterns with geochemistry in an extreme and heterogeneous environment of mine

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tailings. Appl Environ Microbiol 2014;80:3677–3686. [71.]

616 617

Ruan R, Wen J, Chen J. Bacterial heap-leaching: Practice in Jijinshan copper mine. Hydrometallurgy 2006;83:77–82.

[72.]

RI PT

615

Kinnunen PHM, Puhakka JA. High-rate iron oxidation at below pH 1 and at elevated iron and

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copper concentrations by a Leptospirillum ferriphilum dominated biofilm. Process Biochem

619

2005;40:3536–3541. [73.]

Zepeda VJ, Cautivo D, Galleguillos PA, Salazar CM, Velásquez A, Pinilla C et al. Effect of

SC

620

increased acid concentration on the microbial population inhabiting an industrial heap

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[76.]

TE D

623

M AN U

621

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AC C

631

EP

628

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[78.]

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1

ACCEPTED MANUSCRIPT

Table 1. Acidophiles isolated from managed bioleach heaps and/or sulfidic mine-waste

2

dumps or putatively identified by molecular methods in samples from heaps or mine waste

3

dumps (compiled from [8, 10-26] and references therein). Inverted commas indicate that the

4

species description is not yet formally ratified.

Species

pHOPT (range)

Fe(II) oxidation

‘Acidibacillus ferrooxidans’

2




‘Acidibacillus sulfooxidans’

2 3.5–4 (2.5–4.5)

Bacteria

Substrate/Activity RISC Organic carbon oxidation

Fe(III) reductiona





(C)











(C)











(C)

SC




M AN U

Acidibacter ferrireducens

RI PT

1

2.5





Y





(C)

2 (1.4–3)


Y








(C)

3–3.5 (1.5–6)











(C)

(2.5–6)




nr







3.2 (1.9–5.9)




nr





(C)

3.2–4 (1.9–5.6)











(C)

(2.5–6)













3 (2–4.5)











(C)

Acidithiobacillus albertensis

3.5–4 (2–4.5)










nr

Acidithiobacillus caldus

1.8–2.2 (1–4)








S




Acidithiobacillus ferridurans

2.1 (1.4–3)













Acidithiobacillus ferrivorans

2.5 (1.9–3.4)













Acidithiobacillus ferrooxidans

2.5 (1.3–4.5)











(S,H2)

Acidithiobacillus thiooxidans

2–3 (0.5–5.5)








S


(S)

Acidobacterium capsulatum

(3–6)













Alicyclobacillus-like strain FP1

1.8 (1.2–3.5)


Y





SY




Alicyclobacillus tolerans

2–2.7 (1.5–5)


S







nr

3–4 (2–6)










nr

Acidicaldus organivorans Acidimicrobium ferrooxidans Acidiphilium acidophilum Acidiphilium angustum

Acidiphilium multivorum Acidiphilium rubrum

AC C

EP

Aciditerrimonas ferrireducens

TE D

Acidiphilium cryptum

Alicyclobacillus acidocaldarius

2

ACCEPTED MANUSCRIPT











Ferrithrix thermotolerans

1.8













‘Ferrovum myxofaciens’

3 (2–4.8)













Leptospirillum ferriphilum

1.9 (0.5–3.5)













Leptospirillum ferrooxidans

1.5–1.7 (1.3–4)













Metallibacterium scheffleri

5.5 (2–6.5)













Sulfobacillus acidophilus

1.7–1.9 (1–3)


Y








(C,S)

Sulfobacillus benefaciens

1.5 (0.8–2)











(C)

Sulfobacillus thermosulfidooxidans

1.6–1.8 (1–3)


Y








(C,S)

Sulfobacillus thermotolerans

1.3–1.5 (1–3)


Y





S

nr

0.8–1.4 (0.35–3.0)


S




1–1.2 (0.4–1.6)


Y








(S)

1.7 (1.3–2.2)


Y










2 (1–4.5)










nr

2
















nr

Acidianus sulfidivorans Acidiplasma cupricumulans Ferroplasma spp. Ferroplasma acidiphilum Metallosphaera prunae

6

S

1–2 (0.5–4)


(C)

component tested as electron donor: (S) RISC; (C) organic carbon; Y required yeast extract;

EP

a

required sulfur; nr not reported

AC C

5

TE D

Sulfolobus metallicus Thermoplasma spp.; Thermoplasma acidophilum

SC

M AN U

Archaea

RI PT

1.7–1.8

Ferrimicrobium acidiphilum

1

RI PT

ACCEPTED MANUSCRIPT

1

Fig. 1. The run-of-mine dump bioleach at Escondida, Chile, is the largest ‘bioreactor’ in the

3

world, with a surface area 107 m2 and ore stacked in 18 m lifts to a planned final height of

4

125 m. (a) surface with irrigation lines and (b) covers to retain heat and moisture. Arrows

5

indicate the distribution and direction of irrigation lines, also evidenced by surface ponding.

6

(Images provided by D. Shiers, CSIRO Mineral Resources. Images were previously

7

published in open access article, [36])

8

NOT TO BE PRINTED IN COLOUR

M AN U

TE D

EP AC C

9

SC

2

2

ACCEPTED MANUSCRIPT A (F)




B (F)




A (S)




A (C)




B (C)


0

1

2

3

4

5

SC

pH

6

RI PT




B (S)

10

Fig. 2. Compilation of mean reported upper and lower pH limits (bars) and mean pHOPT for

12

growth (circles) for acidophilic or acid-tolerant Bacteria (B) and Archaea (A) grown on

13

substrates: F, ferrous ion; S, elemental sulfur or RISC, or C, organic carbon compounds.

M AN U

11

AC C

EP

TE D

14

3

ACCEPTED MANUSCRIPT

SC

RI PT

15

M AN U

16

Fig. 3. High-resolution image of a ‘slice’ through an ore particle approximately 3 mM wide

18

obtained using X-ray CT microtomography. Few of the sulfide grains (white) distributed

19

through the silicate mineral matrix (shades of grey) were exposed at the particle edges.

20

(Image from P. Austin, CSIRO Mineral Resources).

EP

22

AC C

21

TE D

17

4

RI PT

ACCEPTED MANUSCRIPT

23 24

Fig. 4. Responses of S. thermosulfidooxidans to an increase in acidity: a, pair of partially-

26

joined cells attached to pyrite and covered in EPS after solution acidification from pH 2.2 to

27

pH 1.4; b, change in EPS thickness for
controlled pH 1.4 and varied pH
before and 

28

after acidification at 39 h from pH 1.4 to pH 0.9. (Data from [61], reprinted with permission).

29

NOT TO BE PRINTED IN COLOUR

AC C

EP

TE D

30

M AN U

SC

25

5

ACCEPTED MANUSCRIPT 30 °C

50 °C

100%

Acidimicrobium

100%

80%

Acidithiobacillus

80%

60%

Ferrimicrobium

60%

40%

Leptospirillum

40%

20%

Metallosphaera

20%

INOC

1.2

1.6

2

Thermogymnomonas Ferroplasma

pH

31

0%

RI PT

0%

INOC

1.2

1.6

2

pH

Fig. 5. Nominal distributions of genera in columns of polymetallic ore operated for 102 days

33

of leaching at different pH at 35 °C or 50 °C. Columns inoculated with culture enriched from

34

a self-combusting coal mine. Species identified using denaturing-gradient gel electrophoresis

35

[64].

M AN U

SC

32

AC C

EP

TE D

36