International Biodeterioration & Biodegradation 119 (2017) 118e146

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Review

A review of sulfide minerals microbially assisted leaching in stirred

tank reactors
Akrama Mahmoud a, *, Pierre Ce

zac a, Andrew F.A. Hoadley b, François Contamine a,
Patrick D'Hugues c
a
Laboratoire de Thermique Energ

etique et Proc

ed
es (EAD 1932), UPPA, ENSGTI, BP 7511, 64075 Pau Cedex, France
b
Department of Chemical Engineering, Building 35, Clayton Campus, Monash University, Victoria 3800, Australia
c
Bureau des Recherches G

eologiques et Mini
eres, 3 Avenue Claude Guillemin, BP 36009, 45060 Orl
eans Cedex 2, France

a r t i c l e i n f o a b s t r a c t

Article history: Compared to conventional extractive techniques, bacterial assisted leaching, also called “biomining” is an
Received 30 June 2016 eco-friendly technology that provides improved metal/solid separations. These separations are enhanced
Received in revised form by the synergistic activities of astonishingly diverse groups of microorganisms, which lead to an
16 September 2016
extraction process with low energy consumption, low capital investment and low impact on the envi-
Accepted 16 September 2016
Available online 8 October 2016
ronment. Recently, biomining has received great attention in a variety of niche areas, especially in the
mineral industries and solid industrial waste materials (e.g. galvanic sludge, sewage sludge, fly ash,
electronic waste, spent petrochemical catalysts, medical waste, spent batteries, waste slag) where the
Keywords:
Biomining
metals values are low, or where the presence of certain elements would lead to smelter damage, or
Bioleaching where environmental considerations favor biological treatments options. It allows the recovery of metal
Biooxidation from low-grade sulfide ores and concentrates that cannot be processed economically by conventional
Sulfide minerals techniques, as well as the production of concentrated metal salt solutions, which could be recycled.
Microorganisms Bacterial assisted leaching processes are based on the ability of certain microorganisms to solubilize/or
Gas transfer expose the metals contained in the ores and concentrates by direct oxidation, or through indirect
chemical oxidation instigated by the corrosive metabolic by-products generated by an electrochemical
option, or a combination of both of these. The valuable metals in solution can be recovered using con-
ventional hydrometallurgical techniques. If the material of interest constitutes part of or is in the pre-
treated residue then it can be further processed for metal recovery.
The majority of microorganisms involved in bacterial assisted leaching processes are chemo-
lithotrophs. Carbon dioxide (CO2) and oxygen (O2) are essential nutrients that are used by microor-
ganisms for their growth, maintenance, metabolite production, and survival.
This literature review aims to provide a fundamental understanding of the various mechanisms
involved in microbial leaching of sulfide minerals and provide a brief look at the various factors affecting
this process. Special attention is focused on the mass transfer rates in the gas phase and how they exert a
pivotal role in microbially assisted leaching of sulfide minerals. Also reviewed are the major parameters
that can affect gas phase mass transfer, with particular emphasis on how it is related to the efficiency of
bacterial assisted leaching.
© 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2. Microorganisms species involved in biomining at laboratory scale and industry pilot scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3. Fundamental bacterial assisted leaching mechanisms of sulfide minerals oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

* Corresponding author. Laboratoire de Thermique Energe
tique et Proce

de

s (EAD
1932), UPPA, ENSGTI, rue Jules Ferry, BP 7511, 64075 Pau, France.
E-mail address: amahmoud@gmx.fr (A. Mahmoud).

http://dx.doi.org/10.1016/j.ibiod.2016.09.015
0964-8305/© 2016 Elsevier Ltd. All rights reserved.
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146 119

3.1. Direct/contact mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

3.2. Indirect mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.3. “Contact”, “non-contact” and “cooperative” sub-mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.4. Thiosulfate and polysulfide pathways of the metal sulfide dissolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
3.5. Bacterial attachment to the metal sulfide surface and the role of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.6. Galvanic interaction effect on metal sulfide bacterial assisted leaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4. Bacterial assisted leaching technology in continuous stirred tank reactors (CSTRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5. Factors influencing bacterial assisted leaching of sulfide minerals in stirred tank reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.1. Gas-liquid mass transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
5.1.1. Oxygen availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
5.1.2. Carbon availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.1.3. Aeration and stirring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5.2. Acidity (pH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5.3. Solids concentration and particle size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5.4. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6. Conclusions and future developments aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

1. Introduction encompasses both mineral “bioleaching” and “biooxidation” pro-

cesses (Rawlings, 2002; Rohwerder et al., 2003; Olson et al., 2003;
The global trend towards urbanization and industrialization Ehrlich, 2004; Johnson, 2013, 2014). These terms are sometimes
supports the increasing demand for industrial metals. Moreover, used interchangeably, however, “bioleaching” should be used to
the overall global reserves of high-grade ores are close to depletion refer to the conversion of an insoluble metal into a soluble form (e.
(Anjum et al., 2012). In this context, low-grade, complex ores, old g. from a metal sulfide into a metal sulfate), thereby extracting the
waste deposits related to past mining activities, and other sec- target metal into an aqueous solution. By contrast, mineral “bio-
ondary sources have received much more attention in recent years oxidation”, refers to microbial decomposition of the mineral matrix
(Kutschke et al., 2014). For both environmental and economical that occludes or locks the target metal (typically gold and silver)
exploitation of such ores and resources, efficient technologies for and exposing this entrapped metal making it more accessible for
the recovery of metals need to be developed and in this area, chemical extraction. In these cases, the target metal is not solubi-
bacterial assisted leaching is a promising emerging technology. lized during the biological process and subsequently requires the
Sulfide minerals ores such as pyrite (FeS2), chalcopyrite use of conventional techniques (e.g. cyanide leaching) for recovery
(CuFeS2), sphalerite (ZnS), galena (PbS) and pentlandite (FeNi)9S8 of this metal (Rawlings, 2002, 2004; Johnson, 2013, 2014). How-
are the major sources for recovery of associated metal values (Olson ever, both “bioleaching” and “biooxidation” processes, used to
et al., 2003; Jerez, 2011; Schippers et al., 2014) such as gold recover metals from minerals ores and concentrates, were medi-
(Kaksonen et al., 2014), uranium (Abhilash et al., 2011; Abhilash and ated by using essentially the same principals and a similar consortia
Pandey, 2013), and silver, and other base metals (Ehrlich, 1997; of microorganisms (Rawlings and Johnson, 2007; Johnson, 2014).
Brierley and Brierley, 2001; Olson et al., 2003; Viera et al., 2007; During the last decades, bacterial assisted leaching has seen
Schippers et al., 2014) such as cobalt (D'Hugues et al., 1997; much development with two main types of processes (irrigation-
Morin and D'Hugues, 2007), copper (Watling, 2006; Domic, 2007; type (involving in situ, dump, heap and vat leaching) and stirred
Pradhan et al., 2008; Watling et al., 2009), nickel (Watling, 2008) tank-type processes) and both have achieved industrial
and zinc (Konishi et al., 1992; Rodríguez et al., 2003a,2003b; Shi commercial-scale (Rawlings, 2002). A variety of full scale bacterial
et al., 2006). assisted leaching installations are in operation today, significantly
Initially it was thought that the dissolution of metals from ores contributing to the amounts of metals mined worldwide. At pre-
was purely a chemical process, mediated by water and atmospheric sent, approximately 15e25% of the world's copper production, 5%
oxygen, until the discovery of oxidizing microorganisms which of gold and smaller percentages of cobalt, nickel, uranium and zinc
were proven to catalyze/accelerate the mineral dissolution process are currently recovered by bacterial assisted leaching processes
and, consequently, to solubilize/or expose and recover the target (Puhakka et al., 2007; Brierley, 2008; Johnson, 2013; Kutschke et al.,
metals (Rudolfs, 1922; Colmer and Hinkle, 1947; Colmer et al., 1950; 2014; Schippers et al., 2014).
Temple and Colmer, 1951; Corrans et al., 1972; Torma, 1977; In recent years, several researches have investigated the other
Brierley, 1978, 1982, 1990; Suzuki, 2001; Tributsch and Rojas- practical applications of bacterial assisted leaching in solid wastes
Chapana, 2007). The extraction of metals by means of microor- from many various industrial sectors such as electroplating, metal-
ganisms, commonly referred to as “biomining”, is an ecologically finishing, steel and non-ferrous processes, petrochemical, phar-
alternative to conventional pyrometallurgical and chemical met- maceutical, sewage sludge, fly ash from municipal incinerators, and
allurgy processes for metals recovery. The principal benefits of the electronic wastes (Krebs et al., 1997; Brandl and Faramarzi, 2006;
“biomining” process compared to conventional extractive tech- Cui and Zhang, 2008; Pathak et al., 2009, 2014; Guo et al., 2010;
nologies are their lower labor and energy requirements, lower Hoque and Philip, 2011; Lee and Pandey, 2012; Erüst et al., 2013;
capital investment and reduced harm to the environment (Torma Mishra and Rhee, 2014; Guezennec et al., 2015).
and Banhegyi, 1984; Gentina and Acevedo, 1985; Bruynesteyn, An important challenge of biohydrometallurgy will be to exploit
1989; Acevedo et al., 1993; Agate, 1996; Poulin and Lawrence, refractory resources such as complex polymetallic concentrates,
1996; Das et al., 1999; Clark et al., 2006; Johnson, 2013, 2014; low-grade ores and concentrates, and various oxides. The bio-
Vainshtein, 2014). “Biomining” is a generic term that leaching operation must be matched to the available resources that
120 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
are lower grade, more complex, polymineral and polymetallic.
Some opportunities have been identified such as zinc sulfides and
black shales (Gahan et al., 2012). Biohydrometallurgy can also be an
option used to complement conventional processing routes, to
reprocess residues and mineral processing wastes for example. It
offers an opportunity as an auxiliary process for on-site generation
of acid for adjunct operations, such as base metal oxide heap
leaching. One remaining important bottleneck and technical chal-
lenge for biomining is the leaching of chalcopyrite. Indeed the main
limitation of biohydrometallurgy applied to the treatment of Cu
ores is due to the recalcitrance of chalcopyrite to leaching (Stott
et al., 2000; Sandstro € m et al., 2005; Dixon et al., 2008; Gericke
et al., 2010; Nazari et al., 2011, 2012a, 2012b, 2012c; Dixon and
Tshilombo, 2014). Various theories have been proposed to ac-
count for this phenomenon, mostly involving passivation of the
mineral by ferric precipitates and/or sulfur. If part of the researches
suggests that the use of high temperature and thermophilic archae
can be used in bioleaching systems to overcome incomplete
extraction of copper from chalcopyrite, other recent experimental
studies demonstrated that high rate of chalcopyrite leaching can
also be achieved by using moderate thermophile consortia. Other
approaches include operating at controlled redox levels (Pinches
et al., 2001b; Third et al., 2002; Co
rdoba et al., 2008a, 2008b;
Gericke et al., 2010; Ahmadi et al., 2010; Lotfalian et al., 2015), as
well as the addition of catalysts such as silver and by fine grinding
of the concentrate (Rhodes et al., 1998; Cancho et al., 2007;
Ballester et al., 2007; Nazari et al., 2011, 2012a, 2012b, 2012c;
Abdollahi et al., 2015). Despite this significant progress, the
development of an efficient bioleaching process for the recovery of
the copper in chalcopyrite is still a challenge that will have appli-
cation not only in the treatment of low-grade ore, but also in the
processing of mine tailings generated worldwide by mining
activities.
Recently, materials other than sulfides have been identified for
potential new applications of bioleaching processes. For example,
bioleaching is now considered as a viable option for the treatment
of all kinds of industrial and urban wastes such as dust, slags,
sludges and “End of Life products” containing valuable metals (Lee
and Pandey, 2012). Oxidized ores such as laterites have also been
investigated as potential targets for the development of a new
process concept called FERREDOX based on reductive bioleaching.
Du Plessis et al., 2011; Hallberg et al., 2011; Johnson et al., 2013
demonstrated that acidophilic iron and sulfur-oxidizing bacte-
rium, such as At. ferrooxidans can promote reductive dissolution of
ferric iron minerals and be used to accelerate the extraction of
nickel, cobalt or copper from a limonitic laterite ore. About 80% of
the nickel present in a limonitic deposit was solubilized using At.
ferrooxidans at 30
C using this reductive bioleaching approach
(Hallberg et al., 2011). This metabolic potential is opening new in-
sights in the field of bioprocessing applied to mineral resources.
Another important new concept suitable for all applications for
any kind of resources is known as “resources efficiency”. It aims to
optimize metal recovery of base metals (Cu, Ni, Zn) and precious
metals (gold, silver), but also other valuable associated minerals
(Co, Platinum-Group Metals (PGM)) for both economic and envi-
ronmental reasons.
Metal sulfides are the favorite nutrient for these microorgan-
isms and are present in practically every mineral substrate of eco-
nomic interest. Although the oxidation of metal sulfides by
microorganisms is a complex process, it is believed to result from
three different but concurrent mechanisms. Two of these mecha-
nisms are the “direct” bacterial oxidation and the “indirect”
chemical oxidation which involves the Fe2þ/Fe3þ couple as an in-
termediate electron carrier and acceptor, as shown in Fig. 1a for
pyrite (represented as a model mineral for this type of reaction).
The third mechanism is termed galvanic corrosion, because of its
electrochemical dissolution process mediated by bacteria, as shown
in Fig. 1b (adapted from Holmes and Crundwell, 1995; Ballester
et al., 2007; Ahmadi et al., 2012; Li et al., 2013). There is still con-
troversy over the relative significance of the three mechanisms as
to which plays the dominant role in the biooxidation process.
Besides the chemical/bacterial oxidation reactions involved in
the metal sulfides biooxidation process, other sub-processes can
also occur and may play a determinant role. First of all oxygen and
carbon dioxide could transfer from the gas to the liquid phase.
Precipitate layers on the mineral surface may impede transfer. Mass
transfer resistance of reactants and products between the bulk
liquid and the reaction surface could reduce the reaction rate.
Finally, the attachment of bacteria to the mineral surface and
temperature changes may also be important.
In this literature review, the principles of the sulfide minerals
“biomining” process, including both the various mechanisms
involved and the various engineering factors that significantly
affect the process are presented. Looking more closely at the
mechanism, a special attention on the gas mass transfer rates and
how they exert a pivotal role in microbially assisted leaching of
sulfide mineral is investigated.
2. Microorganisms species involved in biomining at
laboratory scale and industry pilot scale
The major role of the microorganisms in the “biomining” pro-
cesses is to oxidize metal sulfide species such as ferrous iron (Fe2þ)
and sulfur compounds, in order to produce ferric iron (Fe3þ) and
protons. These oxidation products are the metal sulfide attacking
agents. In addition, proton production keeps the pH low and thus,
the Fe ions in solution (Gericke, 2012; Brierley and Brierley, 2013;
Schippers et al., 2014). As mentioned earlier, irrespective of
whether mineral “bioleaching” or “biooxidation” are employed and
irrespectively of the type of biomining techniques (e.g. heap, dump,
in situ, or tank reactor), the microorganisms are required to grow in
an essentially inorganic, aerobic, and either extremely or moder-
ately acidic environment. There is a large variety of microorganisms
which can multiply under these conditions. The majority of these
are prokaryotes and they comprise a large number of
phylogenetically-diverse species of bacteria and archaea.
Acidophilic leaching bacteria are widely distributed among the
Proteobacteria (Acidithiobacillus, Acidiphilium, Acidiferrobacter, Fer-
rovum); Nitrospira (Leptospirillum); Actinobacteria (Ferrimicrobium,
Acidimicrobium, Ferrithrix); and Firmicutes (Alicyclobacillus, Sulfo-
bacillus) (Johnson, 2012). Besides these, leaching archaea have been
known for many years and mostly belong to the Sulfolobales group
of extremely thermophilic, sulfur and iron(II) oxidizers including
genera such as Sulfolobus, Acidianus, Metallosphaera, and Sulfuri-
sphaera (Norris et al., 2000). Also, within the Thermoplasmales, two
iron(II)-oxidizing species, Ferroplasma acidiphilum (Golyshina et al.,
2000, Golyshina and Timmis, 2005) and Ferroplasma acidarmanus
(Edwards et al., 2000), have been identified (Johnson, 2008).
The acidophiles can be conveniently gathered into three broad
groups according to their optimum temperature for growth: mes-
ophilic (20e40
C), moderately thermophilic (40e60
C), and
extremely thermophilic (above 60
C) (Clark and Norris, 1996;
Norris et al., 2000; Brierley, 2001; Olson and Clark, 2004;
Franzmann et al., 2005; Norris, 2007; Johnson, 2009; Schippers
et al., 2014). In fact, a consortium of different microorganisms is
responsible for the oxidative reactions involved in the bacterial
assisted leaching processes (Hallberg and Johnson, 2001;
Schippers, 2007).
The first isolated and the most widely studied of all mineral-
degrading microorganisms in biomining applications are the iron
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
Fig. 1. (a) Schematic of reactions involved in pyrite (FeS2) bacterial assisted leaching. (b) Schematic diagram of chalcopyrite/pyrite galvanic cell interactions (Adapted from Ballester
et al., 2007; Ahmadi et al., 2012; Li et al., 2013).
and sulfur oxidizing acidophiles (Acidithiobacillus ferrooxidans)
which proliferate autotrophically by fixation of atmospheric CO2 as
their carbon source much like green plants, gaining the energy
required for microbial growth from the oxidation of ferrous iron or
reduced inorganic sulfur compounds (Schippers and Sand, 1999;
Rawlings and Johnson, 2007; Vera et al., 2013).
From a practical point of view, the predominant microorganisms
involved in biomining include sulfur-oxidizing bacteria (e.g. Acid-
ithiobacillus thiooxidans previously known as Thiobacillus thioox-
idans and Acidithiobacillus caldus), iron-and sulfur-oxidizing
bacteria (e.g. Acidithiobacillus ferrooxidans previously known as
Thiobacillus ferrooxidans or Sulbacillus spp.), and iron-oxidizing
bacteria (e.g. Leptospirillum ferrooxidans and Leptospirillum ferri-
philum) (Bruynesteyn, 1989; Ewart and Hughes, 1991; Bosecker,
1997; Kelly and Wood, 2000).
In heap processes, the challenges for the growth of microor-
ganisms are due to several factors including the mineralogical and
physical heterogeneity of the ore, which influences temperature,
pH and the concentrations of dissolved solutes. Other factors also
include variations in ore wetting efficiency by irrigation, and the
efficacy of heap aeration (Brierley and Brierley, 2013). In the
multistage stirred tank reactors, the principal problems encoun-
tered by the microorganisms are the buildup of potentially toxic
components, physical damage to cells as a result of vigorous
agitation and aeration, and stresses due to the solids loading in the
reactors (Watling, 2011). All of these factors have to be considered
in the design and implementation “biomining” technologies in
engineered systems (Brierley and Brierley, 2013).
Except A. ferrooxidans (which are usually found in heap bio-
leaching operation), all the others will be of great significance in
stirred tank bioreactors commissioned for the pretreatment of re-
fractory sulfidic gold concentrates or for the Kasese plant in
Uganda, where a cobalt-rich pyrite concentrate was bioleached
121
(Morin and D'Hugues, 2007). These microorganisms operate be-
tween 40
C and 50
C and at a pH below 2 (they are moderate
thermophiles and thermotolerant acidophiles). These microor-
ganisms can support high solid concentration (above 20%). Most of
them are obligate autotrophs (carbon dioxide is their carbon
source), except Sulfobacillus spp. that proved to be facultative
autotroph.
Other researches have investigated the synergistic effects by
mixing different kinds of chemoautotrophic and heterotrophic
bacteria and demonstrated that the mixed cultures could improve
the leaching rate of heavy metals (Battaglia-Brunet et al., 1994,
1998; Falco et al., 2003; Akcil et al., 2007; Zhu et al., 2011, 2014).
Zheng and Li (2016) studied the synergy of Rhizobium phaseoli and
Acidithiobacillus ferrooxidans in the bioleaching process of copper
and showed that additional Rhizobium phaseoli increased the
leaching rate from 22 to 29% and the cell number of Acidithiobacillus
ferrooxidans. Guezennec et al. (2015) pointed out that the lixiviant
solutions obtained from the bioleaching of sulfidic mining wastes
using KCC-BRGM acidophilic and moderate thermophilic (40
C)
microbial consortium showed a good performance for base metal
recovery (Cu, Ni, Zn, Pb, Sn, Ga) from printed circuit boards waste.
The predominant organisms in their culture were affiliated to the
genera Leptospirillum, Acidithiobacillus and Sulfobacillus which are
known for their tolerance to high concentrations of various metals
especially copper. Hong et al. (2016) adapted a mixed culture of
Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans
achieving more than 70% biooxidation of refractory gold concen-
trates containing a high concentration of arsenic (As) in a stirred
tank reactor. Feng et al. (2014) adapted a novel integration strategy
for enhancing chalcopyrite bioleaching by Acidithiobacillus sp. First,


an additional energy substrate such as Fe2þ and S was added for
shorting the strain adaptive-growing phase (SAG phase). Then, a
three-stage pH-stat control strategy (1.3 (0e8 days)-1.0 (8e14
122 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
days)-0.7 (14e40 days)) was proposed for weakening jarosite
passivation. The mineral substrate inhibition was further decreased
by using fed-batch fermentation. They reported that the final
copper ion concentration and productivity reached 89.1 mg/L and
2.23 mg/L.d, respectively, which is about 1.5 times that of the un-
controlled batch bioleaching. Feng et al. (2016), based on their
biochemical parameters analysis, pointed out that the effects of free
cells on chalcopyrite bioleaching in different stages were diverse,
ranging from 32.8% to 64.3%. The bioleaching contribution of free
cells of A. ferrooxidans in the stationary stage (8e14th days) was
higher than those of A. thiooxidans, while the situation was grad-
ually reversed in the jarosite passivation inhibited stage (26-40th
days).
Analysis of pilot-scale polymetallic sulfide stirred tanks opera-
tion running at 40e45
C showed that if the primary tank was
usually dominated by L. ferriphilum, At. caldus and Sulfobacillus spp,
the iron-oxidizing heterotroph Ferroplasma acidiphilum became
increasingly dominant in the final tanks (Bryan et al., 2011). Such
‘heterotrophically-inclined’ iron oxidizers may become increas-
ingly dominant in final reactors due to the increased availability of
organic carbon as a result of the primary production in the initial
tanks (Okibe et al., 2003; Norris, 2007).
Several studies carried out at laboratory scale and pilot scale
have demonstrated that extreme thermophilic cultures containing
Sulfolobus-like organisms (in between 70
C and 80
C) could
successfully bioleach chalcopyrite concentrates, a mineral that is
notoriously difficult to bioleach at lower temperatures (Batty and
Rorke, 2006). So far, this type of high temperature bioleaching
has never ended up as an industrial reality, but it presents a high
potential.
A list of some of the most studied microorganisms involved in
sulfide minerals biomining and some of their physiological prop-
erties is summarized in Table 1. While the identities of many of the
primary iron-and sulfur-oxidizing chemolithotrophs, and hetero-
trophic (or mixotrophic) acidophiles, frequently found in com-
mercial biomining operations are known (e.g., Leptospirillum spp.
Acidithiobacillus spp. Sulfobacillus spp. in tanks, and Acidithiobacillus
ferrooxidans in heaps) it is probable that many (novel) species and
genera present have not yet been identified. One reason for this is
that the compositions of microbial bioleaching communities
appear to vary from site to site, and are determined by a number of
factors such as the mineralogical composition of the ore or
concentrate being processed, operating temperatures and pH
(Johnson et al., 2008; Kimura et al., 2011; Bonnefoy and Holmes,
2012; Martinez et al., 2015).
Rawlings 2005, Rawlings and Johnson 2007 and more recently
Vera et al., 2013 presented an in depth review of the microbial
communities of biomining systems. The most important trait of an
effective mineral oxidizing community is the ability to generate the
ferric iron and sulfuric acid lixiviant. However, each organism must
be adaptable and able to compete in the non-sterile biomining
environment.
The interested readers may refer to a number of other articles
and books (Brierley, 1982; Bruynesteyn, 1989; Rawlings and
Kusano, 1994; Rawlings, 2001; Hallberg and Johnson, 2001;
Hedrich et al., 2011; Ehrlich, 2004; Norris, 2007; Plumb et al.,
2007; Schippers et al., 2010; Brierley and Brierley, 2013; Vera
et al., 2013) for a comprehensive coverage of the microorganisms
and their fundamental characteristics involved in biomining.
3. Fundamental bacterial assisted leaching mechanisms of
sulfide minerals oxidation
Originally, a model with two different mechanisms involved
concurrently in the bacterial assisted leaching of sulfide minerals
has been proposed (Silverman and Ehrlich, 1964; Silverman, 1967),
as shown in Fig. 1a. Firstly, there was the “direct” mechanism in
which the bacterial membrane directly interacts with the sulfide
surface using enzymatic mechanisms. Thus, the “direct” mecha-
nism is observed only if the cells were adsorbed by the mineral
surface. Rossi 1990; Bosecker 1997; Ehrlich and Newman 2009
explained the “direct” mechanism by the direct electron transfer
from metal sulfide to the cell attached to the mineral surface.
Secondly, the oxidation of reduced metal through the “indirect”
mechanisms is mediated by ferric ions (Fe3þ) generated from the
microbial oxidation of ferrous ion (Fe2þ) compounds present in the
mineral. The ferric ion (Fe3þ) acts as an oxidizing agent and can
oxidize metal sulfides and is chemically reduced to a ferrous ion.
Ferrous ions can subsequently be microbially oxidized to ferric ions
again. In this case, iron has a role as electron carrier. It has been
proposed that no direct physical contact is needed for the oxidation
of iron (Brandl, 2001). The two mechanisms may, of course, occur
alongside each other and there may also be synergistic interaction
between them.
It is now generally accepted that the originally discussed “direct
leaching mechanism” (which assumes that the cells are able to
enzymatically oxidize the sulfur moiety at the metal sulfide sur-
face) does not really occur as such (Vera et al., 2013). Instead,
attached cells provide an efficient extracellular polymeric sub-
stances EPS-filled reaction compartment for indirect leaching with
iron(III) ions (Sand et al., 1995, 2001). Therefore, to improve the
understanding of these processes, the “indirect mechanism” re-
mains and now comprises two submechanisms, the “contact”
mechanism and the “non-contact” mechanism for bioleaching by
attached and planktonic cells, respectively (Sand et al., 2001;
Rawlings, 2002). In the course of the “non-contact” mechanism,
iron(II) ions are oxidized by planktonic bacteria. The resulting
iron(III) ions come into contact with a sulfide surface, where they
are reduced and re-enter the cycle. The contact mechanism takes
into account that the majority of cells attach to the surface of sulfide
minerals and, by means of iron(III) ions, which are complexed in
their slime/glycocalyx or extracellular polymeric substances (EPS),
they begin to degrade the sulfide minerals, e.g. pyrite (Fig. 2)
(Gehrke et al., 1998; Sand et al., 2001; Sand and Gehrke, 2006). This
means that the electrochemical reactions between iron(III) ions and
metal sulfides resulting in the dissolution of sulfide minerals take
place at the interface between the bacterial cell and the mineral
sulfide surface.
Likewise, in both “contact” and “non contact” leaching, the
bacteria contribute to mineral dissolution by the generation of the
oxidizing agent (the iron(III)) and by a subsequent oxidation of the
released sulfur compounds arising from the metal sulfide to sulfuric
acid (Vera et al., 2013).
A third term, “cooperative” leaching, was introduced to describe
the dissolution of sulfur colloids, sulfur intermediates, and mineral
fragments by planktonic cells (Sand et al., 2001; Tributsch, 2001;
Rawlings, 2002; Rohwerder et al., 2003; Vera et al., 2013). Thus,
in practice, multiple patterns of bacterial leaching coexist and
therefore the above-mentioned mechanisms for the oxidation of
sulfide minerals (e.g. pyrite mineral illustrated as a model mineral)
are given in the reactions and discussed in detail in the following
section.
3.1. Direct/contact mechanism
The direct mechanism involves bacteria being physically
attached to the sulfide mineral, resulting in preferential oxidation
along the crystal lattice of the mineral, thereby causing dissolution.
This process can be represented according to the following
simplified overall reaction (Torma, 1977, 1988):
Table 1
Some of the most studied acidophilic microorganisms involved in metal sulfide minerals “biomining” operations and their physiological characteristics (Schippers, 2007; Schippers et al., 2014).
Microorganism
Bacteria
Acidithiobacillus
ferrooxidans
Acidithiobacillus thiooxidans
Acidithiobacillus caldus
Acidimicrobium ferrooxidans
Ferrimicrobium acidiphilum
Leptospirillum ferriphilum
Leptospirillum ferrooxidans
Sulfobacillus
thermosulfidooxidans
Sulfobacillus acidophilus
Alicyclobacillus tolerans
Archaea
Ferroplasma acidiphilum
Sulfolobus metallicus
Metallosphaera sedula
Acidianus brierleyi
Acidianus infernus
A: Autotroph; F: Facultative autotroph and/or mixotroph; H: Heterotroph; M: Mesophile; MT: Moderate thermophile; T: Thermophile; N.D.: No data given; *: Optimum; þ: physiological characteristic present; : physiological
characteristic absent.
a
S. Tank: Stirred Tank.
b
G þ C (mol%): Guanine þ Cytosine content of genomic DNA.
c
MS: Metal sulfides other than pyrite.
S. Tanka Phylogenetic
affiliation/Morphology
(G þ C (mol%))b
þ 58e59/b-/g-Proteobacteria
(Rods)
þ 58e59/b-/g-Proteobacteria
(Rods)
þ 62e64/b-/g-Proteobacteria
(Rods)
þ 67e68.5/Actiobacteria
(Rods)
N.D. 67e68.5/Actiobacteria
(Curves rods)
þ 55e58/Nitrospira (Spiral,
non spore forming)
þ 52/Nitrospira (Spiral, non
spore forming)
þ 48e50/Firmicutes (Rods
forms spores)
þ 55e57/Firmicutes
N.D. 49/Firmicutes
þ 36.5/Euryarchaeota
(Irregular)
þ 38/Crenarchaeota (Irregular
cocci)
þ 45/Crenarchaeota (Irregular
cocci)
þ 31/Crenarchaeota (Irregular
cocci)
þ 31/Crenarchaeota
Growth Growth pH range Mode of Oxidation of metal References
temperature nutrition sulfides pyrite MSc
range (
C) Fe2þ S
M 10e37 (30e35)* 1.3e4.5 (1.8e2.5)* A þ þ þ þ Colmer and Hinkle 1947; Torma 1977; Olson 1994; Krebs et al.,
1997; Johnson 1998, 2014; Nemati et al., 1998; Fowler et al.,
1999, 2001; Kelly and Wood 2000; Brandl 2001; Rawlings 2002;
Olson and Clark 2004; Norris 2007; Schippers 2007; Zhang et al.,
2008; Schippers et al., 2010, 2014
M 10e37 (28e30)* 0.5e5.5 (2.0e3.0)* A  þ  þ Krebs et al., 1997; Johnson 1998, 2014; Bacelar-Nicolau and
Johnson 1999; Brandl 2001; Rawlings 2002; Olson and Clark
2004; Norris 2007; Schippers 2007; Schippers et al., 2010, 2014
A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
MT 32e52 (45)* 1.0e3.5 (2.0e2.5)* F  þ  þ Hallberg and Lindstro €m 1994; Johnson 1998, 2014 Brandl 2001;
Rawlings 2002, 2005; Olson and Clark 2004; Watling 2006;
Norris 2007; Schippers 2007; Schippers et al., 2010, 2014
MT <30e55 (45e50)* N.D. (2.0)* F þ N.D. þ  Clark and Norris 1996; Johnson 1998, 2014; Brandl 2001; Olson
and Clark 2004; Norris 2007; Schippers 2007; Schippers et al.,
2010, 2014
M <37 (35)* 1.3e4.8 (2e2.5)* H þ N.D. þ  Johnson 1998; Okibe et al., 2003; Olson and Clark 2004; Norris
2007; Schippers 2007; Schippers et al., 2010, 2014
M <45 (30e37)* N.D. (1.3e1.8)* A þ þ þ  Coram and Rawlings, 2002; Rawlings 2002; Baker and Banfield
2003; Okibe et al., 2003; Kinnunen and Puhakka 2005; Norris
2007; Schippers 2007; Zhang et al., 2008; Schippers et al., 2010,
2014
M N.D. (28e30)* 1.3e4 (1.5e3.0)* A þ þ þ  Boon and Heijnen 1998a, 1998b; Johnson 1998; Brandl 2001;
Rawlings 2002; Baker and Banfield 2003; Norris 2007;
Schippers 2007; Schippers et al., 2010, 2014
MT 20e60 (45e48)* 1.5e5.5 (2.0)* F þ þ þ þ Norris et al., 1996; Johnson 1998; Brandl 2001; Rawlings 2002;
Olson and Clark 2004; Schippers 2007; Schippers et al., 2010,
2014
M <30e55 (45e50)* N.D. (2.0)* F þ þ þ þ Norris et al., 1996; Johnson 1998; Brandl 2001; Rawlings 2002;
Schippers 2007; Schippers et al., 2010, 2014
M <20e55 (37e42)* 1.5e5 (2.5e2.7)* F þ þ þ þ Kovalenko and Malakhova 1983; Johnson 1998; Brandl 2001;
Rawlings 2002; Karavaiko et al., 2005; Schippers 2007;
Schippers et al., 2010, 2014
M 15e45 (35)* 1.3e2.2 (1.7)* F þ N.D. þ  Golyshina et al., 2000; Olson and Clark 2004; Golyshina and
Timmis 2005; Schippers 2007; Schippers et al., 2010, 2014;
Johnson 2014
T 50e75 (65)* 1.0e4.5 (2.0e3.0)* A þ þ þ þ Nemati et al., 2000; Brandl 2001; Rawlings 2002; Olson and
Clark 2004; Norris 2007; Schippers 2007; Schippers et al., 2010,
2014; Johnson 2014
T 50e80 (75)* 1.0e4.5 (2.0e3.0)* F þ þ þ þ Brandl 2001; Rawlings 2002; Olson and Clark 2004; Schippers
2007; Schippers et al., 2010, 2014; Johnson 2014
* *
T 45e75 (70) 1.0e6.0 (1.5e2.0) F þ þ þ þ Segerer et al., 1986; Brierley and Brierley 1973; Larsson et al.,
1990; Konishi et al., 1995, 1997; Brandl 2001; Rawlings 2002;
Olson and Clark 2004; Schippers 2007; Schippers et al., 2010,
2014; Johnson 2014
T 65e96 (90)* 1.0e5.5 (2.0)* A þ þ þ þ Segerer et al., 1986; Brandl 2001; Rawlings 2002; Schippers
2007; Schippers et al., 2010, 2014; Johnson 2014
123
124 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146

Fig. 2. (i) Schematic diagram illustrating the proposed (a) “non contact”, (b) “contact” and (c) cooperative bacterial assisted leaching of sulfide minerals (e.g. pyrite). (Adapted from
Sand et al., 1995; Rawlings et al., 1999; Tributsch, 2001; Rawlings, 2002, 2004). (ii): Model for the contact mechanism catalyzed by a cell of Aciditiobacillus ferooxidans, showing the
bacterial cell embedded in its extracellular polymeric substances (EPS) attached to pyrite via electrostatic interactions. (Rohwerder et al., 2003; Vera et al., 2013).

Bacteria
MSðsÞ þ 2O2ðgÞ ƒƒƒ
ƒ! MSO4ðaqÞ (1) 4FeSO4 þ O2 þ 2H2 SO4 ƒƒƒ
Bacteria
ƒ! 2Fe2 ðSO4 Þ3 þ 2H2 O (3)

where MS is the metal sulfide. In the older scientific literature, it is believed that the bacteria
For the sake of clarity the “direct” oxidation of pyrite by acido- act like a catalyst to assist oxidation of sulfide to sulfate (Equation
philic bacteria is described below by the following two reactions: (2)) in the presence of water and oxygen, thereby releasing ferrous
ions and producing sulfuric acid. The acid produced further reacts
Bacteria with ferrous and dissolved oxygen to produce ferric sulfate
4FeS2 þ 14O2 þ 4H2 O ƒƒƒ
ƒ! 4FeSO4 þ 4H2 SO4 (2)
[Fe2(SO4)3] (Equation (3)) which is vital for the “indirect” oxidation
reaction.
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
Palencia et al. (1991) suggested that pyrite is directly attacked by
oxidizing bacteria, according to the following reaction:
Bacteria
4FeS2 þ 15O2 þ 2H2 O ƒƒƒ
ƒ! 2Fe2 ðSO4 Þ3 þ 2H2 SO4
Reaction (Equation (4)), which describes the “direct” bacterial
oxidation, is the sum of bacteria-catalyzed oxidation of sulfur and
iron by oxygen.
According to the new insights derived from recent research, this
“direct” mechanism (i.e. enzymatic attack) does not exist (Sand
et al., 2001). In their study, Sand et al. (2001) demonstrated that
the metals sulfides are degraded by a chemical attack of iron(III)
ions and/or protons agents on the crystal lattice of metal sulfide.
The primary iron(III) ions are supplied by the bacterial extracellular
polymeric substances (EPS), where they are complexed to glucur-
onic acid residues. Based on their model, in a strict sense, the
mechanism is thus an “indirect” one.
3.2. Indirect mechanism
Initially, the “indirect” mechanism assumes that bacterially
lixiviant generated (Feþ3) plays an important role during bacterial
assisted leaching of a range of minerals. The reduction potential of
ferric ion (Feþ3) in acidic solution (0.67 V at pH ¼ 1.5) makes it an
effective oxidizing agent for solubilizing a range of minerals. The
metal solubilization can be therefore described according to the
following reaction:
Chemical
ƒ! MSO4 þ 2FeðSO4 Þ þ So
MS þ Fe2 ðSO4 Þ3 ƒƒƒ
In the case of “indirect” oxidation, pyrite is chemically oxidized
by (Feþ3) ions in the form of the oxidant (Fe2(SO4)3) according to
reactions shown below:
Chemical
ƒ! 3FeðSO4 Þ þ 2So
FeS2 þ Fe2 ðSO4 Þ3 ƒƒƒ
To keep enough iron in solution this chemical oxidation must
occur in an acid environment. The ferrous iron arising in this re-
action can be (re)oxidized to ferric iron by the action of bacteria and
as such can take part in the oxidation process again. In “indirect”
leaching the bacteria do not need to be in contact with the mineral
surface. They only have a catalytic function because they accelerate
the (re)oxidation of ferrous iron which would otherwise take place
very slowly by the presence of free oxygen in the atmosphere. In
the presence of bacteria and in the range of pH 2e3, the oxidation
of ferrous iron is about 105-106 times faster than the chemical
oxidation (Lacey and Lawson, 1970; Ahonen and Tuovinen, 1989a,
1989b). Moreover, the sulfur produced simultaneously (Equation
(6)) may be oxidized to sulfuric acid in the presence of sulfur-
oxidizing bacteria (Equation (7)).
Bacteria
2So þ O2 þ 2H2 O ƒƒƒ
ƒ! 2H2 SO4
In the absence of this type of bacteria, this elemental sulfur
deposited on the surface of the particles may grow in proportion so
as to create a thick enough layer to inhibit the progress of the
leaching process.
However, the relative contributions by each of the mechanisms
of “direct” and “indirect” pathways to pyrite oxidation remain
disputed. Silverman 1967 argued that the two mechanisms of py-
rite oxidation operate concurrently. Larsson et al. (1990) attribute
enhanced mineral dissolution to the “direct” mechanism, while
others are of the opinion that the “indirect” mechanism is the
principal leaching mechanism (Wakao et al., 1982, 1983, 1984;
Fowler and Crundwell, 1998; Fowler et al., 1999, 2001). They
125
based their conclusion on their observation that cells of Thiobacillus
ferrooxidans adsorbed onto the pyrite surface did not proliferate
and their oxidizing activity was strongly inhibited.
Sand et al. (2001) showed that the “indirect” mechanism i.e., the
(4)
non-enzymatic metal sulfide oxidation by iron(III) ions combined
with the enzymatic (re)oxidation of the resulting iron(II) ions, is to
be considered as the only relevant mechanism. Herein, the bacteria
have the two functions to (i) regenerate the iron(III) ions and/or
protons, and to (ii) concentrate them at the interface mineral/water
or mineral/bacterial cell in order to enhance the degradation/
attack. The indirect mechanism is now divided into two leaching
sub-mechanisms: the “contact” and the “non contact” one (Sand
et al., 2001; Tributsch, 2001; Rawlings, 2002; Rohwerder et al.,
2003; Vera et al., 2013). Also, the more recent works performed
by Sand et al., 1995, 2001; Schippers et al., 1996, 1999; Schippers
and Sand 1999 highlighted the existence of two different reaction
mechanisms to describe the dissolution of metal sulfides: (i) the
thiosulfate chemical pathway for the oxidation of acid-insoluble
metal such as pyrite (FeS2) and (ii) the polysulfide chemical
pathway for acid-soluble metals sulfides such as chalcopyrite
(CuFeS2). All the above-mentioned terms (contact, non-contact,
cooperative, thiosulfate pathway, and polysulfide pathway) are
given in the reactions and discussed in detail in the following
sections.
3.3. “Contact”, “non-contact” and “cooperative” sub-mechanisms
Recently, new insights have been derived from more advanced
techniques for the analysis of degradation products occurring in the
(5) bacterial assisted leaching process and the analysis of extracellular
polymeric substance (EPS). Furthermore, as mentioned earlier, it is
now generally accepted that the originally discussed “direct”
mechanism of bacterial assisted leaching of metal sulfides does not
exist. Consequently, the “indirect” mechanism has been revised and
the terms “contact” leaching and “non-contact” leaching have been
(6) introduced by Sand et al., 2001; Tributsch 2001; Rawlings 2002 for
bacterial assisted leaching by attached and planktonic cells (freely
suspended or swimming cells), respectively. A third term, “coop-
erative” leaching, was also proposed to describe the dissolution of
sulfur colloids, sulfur intermediates, and mineral fragments by
planktonic cells. Thus, in practice, multiple patterns of bacterial
leaching coexist, including “indirect” leaching, “contact” leaching
and “cooperative” leaching, as shown in Fig. 2 (Sand et al., 2001;
Tributsch, 2001; Rawlings, 2002; Rohwerder et al., 2003; Vera
et al., 2013). In the “non contact” sub-mechanism (Fig. 2), plank-
tonic cells oxidize the ferrous ions (Fe2þ) in the bulk solution and
regenerate in this way the oxidizing agent which afterwards
chemically reacts with metal sulfides. This in turn corresponds to
the previously designated “indirect” mechanism. In the “contact”
sub-mechanism (Fig. 2), the majority of the bacterial cells attach to
the surface of metals sulfides with the aid of extracellular poly-
meric substances (EPS), and this results in the dissolution of metals
(7)
sulfides at the interface between the bacterial cell and the mineral
surface by an electrochemical reaction between ferric ions (Fe3þ)
and metals sulfides. The “cooperative” sub-mechanism (Fig. 2) is
established between the contacted and planktonic cells. It de-
scribes the dissolution of sulfur colloids, sulfur intermediates, and
mineral fragments by planktonic cells, releasing protons and ferric
iron which are further used in “non-contact” leaching (Rojas-
Chapana et al., 1996, 1998; Tributsch, 2001; Sand et al., 2001;
Rawlings, 2002; Vera et al., 2013). There have been many studies
to ascertain the influence and the importance of each bioleaching
sub-mechanism, but a consensus has yet to be reached (Fowler and
Crundwell, 1998; Sand et al., 1995, 2001; Vera et al., 2013). For this
purpose, a separation of the leaching bacteria from the solid
126 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146

Fig. 3. (a): Scheme diagram of the two metal sulfide bioleaching pathways, the thiosulfate (i) and (ii) polysulfide mechanism. The main reaction products that accumulate in the
absence of sulfur oxidizer are boxed, i.e., sulfuric acid in (i) and elemental sulfur in (ii). (Schippers et al., 1996; Schippers and Sand, 1999; Sand et al., 2001; Rohwerder et al., 2003;
Vera et al., 2013). (b): Schematic diagram of the bioleaching model (Blight et al. (2000)).

particles, performed by installing a partition system using different
kinds of semi-permeable membrane, was used to avoid the bacteria
attachment and to analyze the influence of the “contact” mecha-
nism on the bioleaching process. By comparing the results obtained
from “contact” and “non-contact” conditions in the case of printed
circuit boards bioleaching process using Acidithiobacillus ferroox-
idans, Silva et al. (2015) demonstrated that a reduction of about 25%
in the copper extraction was observed when the “contact” mech-
anism was disabled.
3.4. Thiosulfate and polysulfide pathways of the metal sulfide
dissolution
These new terms may be useful for description of the physical
status of cells involved in bacterial assisted leaching, but they do
not provide any knowledge about the underlying chemical mech-
anisms of biological metal sulfide dissolution. Thanks to the work of
Schippers and co-workers (Sand et al., 1995, 2001; Schippers et al.,
1996, 1999; Schippers and Sand, 1999), only two different reaction
mechanisms can describe the dissolution of metal sulfides: (i) the
thiosulfate chemical pathway for the oxidation of acid-insoluble
metal such as pyrite (FeS2) and (ii) the polysulfide chemical
pathway for acid-soluble metal sulfides such as chalcopyrite
(CuFeS2) (Fig. 3a). In the thiosulfate mechanism, solubilization is
through the oxidative attack of ferric iron on the acid-insoluble
metal sulfides, such as pyrite, molybdenite and tungstenite (FeS2,
MoS2, and WS2, respectively), with thiosulfate being the main in-
termediate and sulfate the main end-product. Studies on the
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
mineralogy and semiconducting properties of (FeS2, MoS2, and
WS2) showed that the valence bands in these sulfides are only
derived from orbitals of metal atoms, whereas the valence bands of
all other metal sulfides are derived from both metal and sulfur
orbitals. Consequently, the valence bands of these sulfides do not
contribute to the bonding metal and sulfur moiety of the metal
sulfide which explains the resistance of these metals sulfides
against a proton attack. This is the reason why such sulfides can
only be degraded via multistep electron transfers by an oxidizing
attack with (Fe3þ) (Crundwell, 1988; Sand et al., 2001; Rodríguez
et al., 2003a; Schippers et al., 2014).
Overall, the thiosulfate pathway using pyrite (FeS2) as an
example can be summarized by the following reactions (Schippers
and Sand, 1999):
FeS2 þ 6Fe3þ þ 3H2 O/S2 O2
3 þ 7Fe
2þ
þ 6Hþ
S2 O2
3 þ 8Fe
3þ
þ 5H2 O/2SO2
4 þ 8Fe
2þ
þ 10Hþ
In acidic environments and in the absence of bacteria and
without detectable amounts of Fe3þ, pyrite leaching rates are
dramatically reduced (Rohwerder et al., 1998; Schippers et al.,
1999). But also under these conditions the thiosulfate mecha-
nisms seems to be effective, although the further reactions of
thiosulfate may deviate from the above mentioned ones. However,
the investigation of pyrite in acidic media by Descostes et al. (2004)
has been a point of criticism in recent work (Druschel and Borda,
2006).
On the other hand, in the case of the polysulfide mechanism,
solubilization of the acid-soluble metal sulfides, such as sphalerite,
galena, aresenopyrite, chalcopyrite, orpiment, realgar and hauerite
(ZnS, PbS, FeAsS, CuFeS2, As2S3, As4S4 and MnS2, respectively), is
through a combination of ferric ion and proton attack, with
elemental sulfur as the main intermediate. This elemental sulfur is
chemically stable in natural environments, but it may be oxidized
to sulfuric acid by sulfur-oxidizing bacteria. Overall, the polysulfide
pathway (e.g. ZnS, CuFeS2, or PbS) can be described by the following
reactions (Schippers and Sand, 1999; Sand et al., 2001):
MS þ Fe3þ þ Hþ /M2þ þ 0:5H2 Sn þ Fe2þ ðn  2Þ
0:5H2 Sn þ Fe3þ /0:125S8 þ Fe2þ þ Hþ
þ attach to the whole mineral surface but the attachment occurs at
0:125S8 þ 1:5O2 þ H2 O/SO2
4 þ 2H
As mentioned earlier, all of these sulfides have valence bands
formed by atomic orbitals from metallic and sulfur atoms. This
explains why these sulfides can be attacked not only by (Fe3þ) but
also by protons, which can remove electrons from the valence band,
causing a cleavage of the bonds between the metal and the sulfur
moiety of the sulfide (Vaughan and Craig, 1978; Crundwell, 1988;
Sand et al., 2001; Rodríguez et al., 2003b; Schippers et al., 2014).
Consequently, these metal sulfides are more or less soluble in acid,
whereas FeS2, MoS2, and WS2 are insoluble.
In both pathways, the main role of leaching bacteria consists of
the regeneration of Fe3þ ions: the most important oxidants in acidic
biotopes. The scheme in Fig. 3a gives an overview of the two metal
sulfides oxidation pathways, including some knowledge about
bacterial and/or chemically catalyzed parts.
Another bioleaching model is that of Blight et al. (2000) who
considered the bioleaching of pyrite using Acidithiobacillus fer-
rooxidans. They assume the following hypotheses for their pro-
posed model of pyrite bioleaching: (i) planktonic cells become
sessile on a mineral sulfide surface and an oxide film develops,
127
incorporating cells, extra cellular polysaccharides, iron oxy-
hydroxides and jarosite, (ii) below the surface film, iron species act
as a mediator, the iron is oxidized at the level of cell attachment and
reduced at the oxide/sulfide interface, (iii) sulfur species produced
by the action of the mediator at the oxide/sulfide interface diffuses
through the surface layers and is either metabolized or passes into
the bulk solution, and (iv) the iron released from the pyrite, and
reduced mediator, diffuse to the bio-film and are oxidized to ferric
form. The ferric ion hydrolyses water that diffuses past the biofilm
producing solid Fe(OH)3 and Hþ ion. A schematic view of the model
of pyrite bioleaching of Blight et al. (2000) and their proposed
stoichiometry reactions is shown in Fig. 3b.
In a stirred tank bioreactor, an improved oxidation efficiency
was correlated with more attachment of the bacteria to the solid
substrate and an increase in the formation of iron precipitates
(8) (D'Hugues et al., 1997). Other authors have also reported a positive
influence of precipitates on the oxidation of ores, for example,
Southam and Beveridge (1993) showed that bacterial cells were
(9) cemented to mineral surfaces by iron precipitates. As confirmed by
Blight et al. (2000) this phenomenon could play an important role
in sulfide mineral oxidation by creating an acidic oxidative micro-
environment which would favor chemical and biological substrate
degradation.
To summarize, in spite of the vast volume of literature on bac-
terial assisted leaching of pyrite, there is a little agreement on the
mechanism of the dissolution. Therefore, it can be stated that the
conclusions from these previous studies remain controversial and
still under discussion, and thus further studies and new observation
are necessary to obtain a better understanding of how bacteria can
dissolve/or expose metal sulfides.
3.5. Bacterial attachment to the metal sulfide surface and the role of
EPS
The attachment of bacteria onto a number of minerals surfaces
and consequent oxidative corrosion has been investigated by many
researchers (Weiss, 1973; Murr and Berry, 1976; Bennett and
Tributsch, 1978; Hiltunen et al., 1981; Wakao et al., 1982, 1983;
Rodriguez-Leiva and Tributsch, 1988; Konishi et al., 1990; Mustin
(10) et al., 1992; Crundwell, 1996, 2003; Sand and Gehrke, 1999, 2001;
Brandl, 2001; Cabral and Ignatiadis, 2001). However the mecha-
nism of attachment, the nature and selectivity of such attachment
(11) of bacteria onto the mineral surfaces, and its reversal is still under
investigation. Initially it was postulated that the bacteria do not
(12)
sites of crystal imperfection or dislocation and fractures, and metal
solubilization is due to electrochemical interactions (Bennett and
Tributsch, 1978; Brierley, 1982; Rodriguez-Leiva and Tributsch,
1988). Murr and Berry (1976) attributed the attachment mecha-
nism to electrostatic adhesion between bacteria and the mineral
surface, while Weiss (1973) demonstrated that the attachment
between sulfur crystals and bacteria is via the adhesive pili, and
others speculate moistening agents as being responsible for pro-
moting contact between bacteria and sulfur (Hiltunen et al., 1981).
Konishi et al. (1990) indicated that bacteria may multiply on the
pyrite surface as well as in the liquid phase. Mustin et al. (1992)
indicated that the specific surface areas of pyrite before and after
bioleaching were 1.1 and 1.6 m2/g, respectively. Thus the bacterial
oxidation increased the porosity and specific surface in pyrite
grains.
Crundwell 1996 showed that the attachment of bacteria to the
pyrite surface can occur as a biofilm and suggested that the indi-
vidual bacterium does not need to be attached to the pyrite for it to
harness the mineral as an energy source.
Recently, Sampson et al. (2000a), (2000b) reported that the
128 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
attachment of bacteria onto a mineral substrate is due to the pro-
tein apo-rusticyanin or a similar type of binding protein and the
degree of adhesion depends on the cultures growth history and the
substrate medium they were grown in.
On the other hand, Vandevivere and Kirchman 1993; Rohwerder
et al., 2003 and more recently Mangold et al. (2008) observed a
production of microbial extracellular polymeric substances (EPS)
during bioleaching experiments. Gehrke et al. (1998) and more
recently Sand and Gehrke 2006 demonstrated that EPS play a role
in the attachment of bacteria to the sulfide surface and in the
concentration of iron(III) ions by complexation at the mineral sur-
face; two phenomena that would facilitate the oxidation of sulfide.
The main bacterial contribution to the oxidation system would be
maintaining the Fe ions in the oxidized state. They also showed that
the yield of EPS varied when growth conditions, such as the sub-
strate, were varied. Recent work also suggests that in addition to
EPS, a “quorum sensing” type AI-1 system is involved in the for-
mation of biofilm on pyrite by bioleaching bacteria (Farah et al.,

lez et al., 2013).
2005; Ruiz et al., 2008; Gonza
Sand and Gehrke (1999) noted that EPS produced by bioleaching
bacteria consist mainly of carbohydrates, uronic acids and lipids.
They reported that proteins were not detected. They also found that
EPS from sulfur-grown cells are composed mainly of lipids, whereas
EPS produced by cells grown on iron(II) sulfate or pyrite as sub-
strate produce a high amount of carbohydrates and glucuronic acid
(Sand and Gehrke, 2006). In general, carbohydrates are known to be
crucial in the formation and properties of biofilms (Wingender
et al., 1999; Michel et al., 2009).
Recently, Michel et al. (2009) pointed out that exopoly-
saccharides and probably all EPS in a continuous stirred tank
reactor system are mainly in a free state and that even if the
presence of exopolysaccharides can be related to bioleaching ac-
tivity, there is no direct link between measured exopolysaccharide
concentration and bioleaching efficiency. They stated that the role
of EPS is not only dedicated to bacterial attachment to pyrite for
bioleaching activity; it is possible that part of the EPS might play a
protective role against the stress conditions that characterise bio-
leaching media (low pH, presence of metals, stirring conditions,
complex medium among others).
3.6. Galvanic interaction effect on metal sulfide bacterial assisted
leaching
Galvanic interactions are caused by electrochemical interactions
as a result of the differing rest potentials of the conducting or semi-
conducting sulfide minerals, resulting in a change of the rate of the
anodic and cathodic half-reactions that occur at the surface of each
mineral (Berry et al., 1978; Mehta and Murr, 1983; Hutchins et al.,
1986; Torma, 1988; Holmes and Crundwell, 2000; Abraitis et al.,
2004; Ballester et al., 2007; You et al., 2007; Dixon et al., 2008;
Koleini et al., 2011; Li et al., 2013).
Table 2
The rest potential of some common sulfide minerals at 25
C and pH ¼ 4 (Vaughan and Craig, 1978; Mehta and
Murr, 1983; Holmes and Crundwell, 1995; Ballester et al., 2007; Chandra and Gerson, 2010; Li et al., 2013).
Mineral
Pyrite (FeS2)
Marcasite (FeS2)
Chalcopyrite (CuFeS2)
Sphalerite (ZnS)
Covellite (CuS)
Bornite (Cu5FeS4)
Galena (PbS)
Argentite (Ag2S)
Stibnite (Sb2S3)
The rest potential of a sulfide mineral may vary from sample to
sample, depending on the nature, type and amount of impurity, the
leaching medium, and the pH (Jyothi et al., 1989). The rest poten-
tials of some common sulfide minerals are listed in Table 2. The data
reported in Table 2 reveal that pyrite mineral has a relatively high
rest potential 0.66 V (vs. Standard Hydrogen Electrode (SHE))
making it the least reactive (the most electrochemically inert) of
the common sulfides minerals (Majima, 1969; Hiskey and
Wadsworth, 1975).
When two sulfide minerals of different rest potential are in
electrical contact with each other in acidic solution, the mineral
with the higher rest potential tends to act as the cathode in the
galvanic couple and is galvanically protected. Conversely, the
mineral with the lower rest potential, acting as an anode, will be
dissolved by the following reactions:
At the cathode side:
1
O þ 2Hþ þ 2e /H2 O (13)
2 2
At the anode side:
MSðsÞ /M2þ þ SoðsÞ þ 2e (14)
where MS is the metal sulfide and M2þ is the bivalent metal.
The galvanic reaction at the mixed potential can be expressed
as:
1
MSðsÞ þ O2 þ 2Hþ /M2þ þ SoðsÞ þ H2 O (15)
2
In the case of chalcopyrite/pyrite (CuFeS2/FeS2) galvanic cell, the
chalcopyrite with lower rest potential (0.56 V vs. SHE) dissolves
rapidly while the pyrite (0.66 V vs. SHE) remains practically unaf-
fected (Mehta and Murr, 1983; Holmes and Crundwell, 2000;
Koleini et al., 2011). Fig. 1b illustrates schematically this galvanic
interaction mechanism and describes the main reactions occurred
in the system i.e. anodic oxidation of chalcopyrite in contact with
pyrite as a result of galvanic effect (Adapted from Ballester et al.,
2007; Li et al., 2013; Tao and Dongwei, 2014).
The role of bacteria in such galvanic cell system is to oxidize
continuously the passivation layer of elemental sulfur formed at the
surface mineral due to oxidation of sulfide mineral (Munoz et al.,
1979; McMillan et al., 1982; Dutrizac, 1989), which otherwise
inhibit the dissolution process, to sulfate resulting in the exposure
of fresh mineral surface and the generation of acidic ferric-sulfate
lixiviant (Berry et al., 1978; Hutchins et al., 1986). Thus, the leach
rate of chalcopyrite is significantly increased. Mehta and Murr
(1983) demonstrated that the rate of chalcopyrite dissolution,
associated with pyrite at mixing ratio of 5:5 in the presence of
Thiobacillus ferrooxidans, was increased by a factor between of 2e15
times. They also showed that there was an increase in redox
Rest potential (V vs Standard hydrogen electrode (SHE))
0.66
0.63
0.56
0.46
0.45
0.42
0.40
0.28
0.12
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
potential from 338 mV to 580 mV and a decrease in pH from 2.30 to
1.56. Tshilombo (2004) reported that in the absence of pyrite, the
anodic dissolution of chalcopyrite is coupled to the reduction of
ferric and/or oxygen, which must occur exclusively on the chalco-
pyrite surface. While, when the chalcopyrite is in contact with
pyrite, the reduction of ferric and/or oxygen takes place on the
surfaces of both pyrite and chalcopyrite. Tshilombo (2004) also
noted that the rate of reduction of ferric is greater on the pyrite
surface than on the chalcopyrite surface. The galvanic effect would
be more significant at higher pyrite/chalcopyrite ratios, because
pyrite provides more cathodic area for the reduction process. It
could also be expected that increasing pulp density enhances the
contact between chalcopyrite and pyrite particles which may in-
crease the galvanic effect. On the other hand, at a constant pulp
density, more pyrite in the feed, lower copper presents in it, so the
toxicity effect of dissolved copper ions for bacteria diminishes
(Ahmadi et al., 2012).
Where chalcopyrite is in contact with pyrite, in the presence of
dissolved ferric ions (Fe3þ), the following cathodic reaction needs
to be considered:
Fe3þ þ e /Fe2þ
The galvanic reaction of chalcopyrite in the presence of pyrite
can be expressed by the overall equation (Torma, 1988):
CuFeS2ðsÞ þ O2 þ 4Hþ /Cu2þ þ Fe2þ þ 2SoðsÞ þ 2H2 O
The elemental sulfur and ferrous irons produced by Equation
(17) will be oxidized by bacteria.
The nature of the above-mentioned passive layer is not well
known, but it is believed that a compact sulfur layer (Munoz et al.,
1979; Dutrizac, 1989), a metal deficient chalcopyrite-like sulfide
layer (Warren et al., 1982), a polysulfide like CuxSy (Biegler and
Horn, 1985) or an iron-bearing precipitate like jarosite (Stott
et al., 2000) is responsible for passivity of chalcopyrite surface,
which can lead to slow and incomplete extraction of copper. Jar-
osite is formed under the conditions of high pH and high ferric iron
concentration, as might be expected from Equation (18) (Stott et al.,
2000):
3Fe3þ þ Xþ þ 2HSO
ð4Þ þ 6H2 O/XFe3 ðSO4 Þ2 ðOHÞ6 þ 8H
þ
where X ¼ K þ ; Naþ ; NH4þ or H3 Oþ .
One possible way to prevent the formation of this passive layer
is the possible control of pulp oxidation reduction potential (ORP)
during the bioleaching process. This can be achieved either elec-
trochemically by applying a potential from an external source
through a working electrode or chemically by the addition of
reducing and oxidizing agents, as reported by Ahmadi et al. (2010).
Pulp potential can be maintained also at the desired level by con-
trolling the oxygen mass transfer, by adjusting either impeller
speed or gas flow rate (see Gericke et al. (2008) amongst others).
Gericke et al. (2008), for example, showed that 97% of copper could
be bioleached from chalcopyrite concentrate at redox potential
level around 420 mV compared to 64% at around 600 mV, in pilote-
scale bioreactor using moderate thermophiles at 45
C.
The electrochemical reduction method has attracted more
attention, because it avoids contamination of the solution and
consumption of reducing/oxidizing agents (Fuentes-Aceituno et al.,
2008). Hiroyoshi et al., 1997, 2000, 2001; Pinches et al., 2001a;
Third et al., 2002; Sandstro € m et al., 2005; Co

rdoba et al., 2008a,
2008b; Gericke et al., 2010 and more recently Ahmadi et al., 2010,
2011 have reported that during the chemical leaching of
129
chalcopyrite in ferric sulfate media, both the rate and yield of
copper dissolution is at a maximum in a narrow range of ORP
around 400e450 mV (vs. Pt, Ag/AgCl), whereas at ORPs above this
range, the surface passivation of the mineral could occur. Moreover,
ferric iron is an inhibitor for bacterial growth and activity; hence
increasing ORP has a negative effect on the bacterial subsystem.
In this regard different studies have shown that applying
negative potentials to the pulp significantly enhances the activity
and growth of microorganisms and increases the dissolution rate of
chalcopyrite (Jyothi et al., 1989; Yelloji and Natarajan 1989;
Natarajan 1992a, 1992b; Nakasono et al., 1997). In their experi-
mental results, Jyothi et al. (1989); Yelloji and Natarajan (1989) and
Natarajan 1992a, 1992b showed that sphalerite could be selectively
bioleached in the presence of Thiobacillus ferrooxidans under an
applied potential of 500 mV (Saturated Calomel Electrode (SCE))
from mixed sulfides containing sphalerire, pyrite, and chalcopyrite.
They also observed that bacterial activity and growth were pro-
moted under electro-bioleaching conditions and the rate of chal-
copyrite leaching increased slightly (Conner, 2005). Also, the
formation of jarosite was significantly reduced by passing current
through the system. On the other hand, in their experimental re-
(16) sults to extract copper from chalcopyrite concentrates by electro-
chemical bioleaching at lower temperatures (50
C) using a mixed
culture of mesophilic bacteria at 35
C and a mixed culture of
moderately thermophilic bacteria at 50
C, operating at 10% (w/v)
pulp density in stirred bioreactor at lab-scale, Ahmadi et al. (2010)
(17) showed that, compared to chemical and bioleaching, the control of
solution ORP around 425 mV (vs. Pt, Ag/AgCl), by applying current
directly to the slurry, significantly increases both, the cell concen-
trations (near to 109 cells/ml) and copper recovery (up to 35%) in
both cultures. Under these conditions, the precipitation of iron oxy-
hydroxides on the surface of chalcopyrite is significantly reduced.
As iron oxy-hydroxides can act as a diffusion barrier which pre-
vents chalcopyrite dissolution, this leads to a higher electro-
chemical reduction of chalcopyrite and improved dissolution. In
another conventional and electrochemical bioleaching study,
Ahmadi et al., 2011 investigated the extraction of copper from
Sarcheshmeh chalcopyrite flotation concentrate at high pulp den-
sity (at 20% w/v) using a mixed culture of moderately thermophilic
bacteria. They showed that the final copper recovery of electro-
chemical bioleaching at 400e430 mV (77%) was higher by a factor
of 5.9, 3.9, 1.5 and 1.17 relative to the chemical leaching (control
(18) test).
Based on the galvanic interactions a new process known as
Galvanox™ is reported in the literature, where pyrite is used as a
catalyst for atmospheric leaching of chalcopyrite concentrates in
iron sulfate media (Dixon et al., 2008; Nazari et al., 2011, 2012a,
2012b, 2012c; Dixon and Tshilombo, 2014). In this process, pyrite
provides an alternative surface for ferric reduction. Providing a
larger surface area for the cathodic reaction on pyrite than for the
anodic reaction on chalcopyrite also increases the anodic dissolu-
tion rate (Tshilombo, 2004). However, not all pyrite samples have
the same catalytic effect on the Galvanox™ process. Nazari et al.,
2011 reported that pyrite samples from different sources could
influence the rate of chalcopyrite leaching differently. Some pyrite
samples accelerate the leaching rate significantly, while others have
less effect on the process. They stated that the effectiveness of
pyrite as catalysts for chalcopyrite leaching has a strong correlation
with the amount of silver associated with the pyrite samples.
Therefore, the Galvanox™ process was re-visited in order to
enhance the catalytic properties of pyrite to accelerate the rate of
copper extraction from chalcopyrite by pretreatment of natural
pyrite samples with extremely small amounts of silver ions by
soaking the ground pyrite in a dilute solution of soluble silver salt
such as silver nitrate before introducing the pyrite into the leaching
130 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
reactor. It was found that the addition of as little as 60 mg of Ag per
kg of copper was sufficient to ensure complete copper extraction
within 10 h of leaching (Nazari et al., 2011). Miller and Portillo
(1979) proposed the first mechanism to explain the silver cata-
lyzed leaching of chalcopyrite. Based on their model, the enhanced
rate of leaching is due to the formation of an intermediate Ag2S film
which forms on the chalcopyrite surface by an exchange reaction.
They have suggested that, under these conditions, the elemental
sulfur forms a non-protective reaction product on the Ag2S crys-
tallites, in contrast to uncatalyzed ferric sulfate leaching, in which a
dense elemental sulfur layer forms on the mineral surface and acts
as a diffusion barrier, thus delaying the oxidation of chalcopyrite by
ferric ions. The following reaction has been proposed for the silver-
catalyzed leaching of chalcopyrite:
CuFeS2 þ 4Agþ /2Ag2 Sðchalcopyrite surfaceÞ þ Cu2þ þ Fe2þ (19)
In the presence of Fe , the silver sulfide film is oxidized to Agþ
3þ


and S according to reaction:
Ag2 S þ 2Fe3þ /2Ag þ þ 2Fe2þ þ So (20)
Further information on these mechanisms can be found else-
where (Ballester et al., 2007; Nazari et al., 2011, 2012a, 2012b,
2012c; Abdollahi et al., 2015).
To the best of our knowledge and in spite of broad research, no
commercial process has been developed using silver as a catalyst
for copper extraction from chalcopyrite. One of the weaknesses of
this concept encountered is the low solubility of silver in bio-
leaching due the bioaccumulation of silver ions within the cell
membranes of the microorganisms. Also, silver ions are toxic for
most microorganisms. Its toxicity has been attributed to a
competing mechanism between silver and ferrous ions for active
sites on bacterial cell walls (Mun~ oz et al., 2007).
Other methods to improve chalcopyrite leaching have also been
investigated, including adding activated carbon (Nakazawa et al.,
1998; Liang et al., 2010; Ahmadi et al., 2013), iron powder
(Sanchez et al., 1996), or additions of chloride irons (Defreyne and
Cabral, 2009).
Therefore, electrochemical and the galvanic effects play a major
role in a multisulfide bacterial assisted leaching system and offer a
promising technology for recovery of metals from complex sulfide
ores like chalcopyrite.
4. Bacterial assisted leaching technology in continuous
stirred tank reactors (CSTRs)
The reactors which are used commonly at laboratory scale, in
batch and continuous modes for the bacterial assisted leaching
process, are the stirred tank reactor (STR), the airlift reactor (ALR) or
the so-called pneumatic reactor (Pachuca), and the continuous
bubble column (CBC) (Hoffmann et al., 1993; Bailey and Hansford,
1994; Haddadin, 1995; Van Weert et al., 1995; D'Hugues et al.,
1997; Garcia-Ochoa et al., 1997, 1999; Derksen et al., 2000; Witne
and Phillips, 2001; Acevedo and Gentina, 2007; Morin, 2007). At
commercial scale for processing of certain sulfide minerals, bacte-
rial assisted leaching is applied using two broad engineered pro-
cesses: dump or heap reactors, where ore or concentrate is
irrigated, and continuous stirred tank reactors (CSTRs) where the
finely ground mineral ore or concentrate is treated as an agitated/
aerated slurry. While CSTRs are mainly used for gold recovery via
biooxidation and for cobalt (1 operation), copper, nickel, and zinc
are extracted in large heap bioleaching operations (Kutschke et al.,
2014).
In the last decade, bacterial assisted leaching processes of
certain sulfide minerals for metals recovery, using stirred tank
technologies, have been developed rapidly and well established at
industrial scale (D'Hugues et al., 2008). Advantages of the CSTRs lie
in their ability to provide higher mass transfer, higher mixing and
suspension than the values from airlift and continuous bubble
column (Rossi, 2001; Acevedo and Gentina, 2007), and to offer
much more control operations (such as temperature, pH, aeration
among others), thus facilitating a much faster and efficient metal
recovery process (Rawlings and Johnson, 2007). Also, the contin-
uous flow mode of operation results in the continual selection for
the microorganisms and this is able to progress most efficiently in
tanks. In such conditions, the more efficient microorganisms will be
subjected to less cell washout and therefore dominate the microbial
population in the tank reactor. The mineral provides the energy
source and some nutrients for the microorganisms, and those or-
ganisms that are most efficient at degrading the sulfide mineral will
tend to dominate the process. This means that there will be
continual selection for microorganisms that either catalyze the
sulfide mineral dissolution or create the conditions favorable for
rapid dissolution of the sulfide minerals. Likewise, there is no need
for sterility. This “non-sterile” requirement is because the highly
acidophilic microorganisms create an environment that is not
suitable for the growth of other organisms (Rawlings and Johnson,
2007). Also, another important aspect is the adaptation of mixed
cultures of chemoautotrophic and heterotrophic bacteria using
their synergistic effects to improve the leaching rate of heavy
metals in CSTRs (Falco et al., 2003; Akcil et al., 2007; Zhu et al.,
2014; Hong et al., 2016; Zheng and Li, 2016).
However, these CSTRs are the more expensive to construct and
to operate, which makes their use restricted to high-value ores and
concentrates (Rawlings and Johnson, 2007). Indeed, the CSTRs may
represent a potential technical solution for recovery of metals from
complex sulfide ores with high amounts of calcium carbonates (e.g.
kupferschiefer). The carbonates dissolution in sulfuric environ-
ments leads to the precipitation of gypsum which interferes with
mineral contact and solution flow patterns in the heap biomining
processes. Clogging phenomena observed in such situation affects
both air and solution permeability and thus decrease leaching ef-
ficiency and metal recovery (Kutschke et al., 2014).
Generally, CSTRs technologies consist of a circuit of aerated
continuous flow tanks, arranged in series or in parallel or combi-
nation of the two, equipped with agitator impeller systems that
keep the finely ground minerals in suspension and ensure the
efficient transfer of oxygen and carbon dioxide required for the
sulfide oxidation as well as for microorganism growth. The CSTRs
are operated in continuous flow mode with the feed being added to
the first tank and overflowing from tank to tank. Microorganisms
are injected once at the beginning of the start-up of an operation
and a batch culture is maintained until a certain point as close as
possible to the middle of the exponential phase of the bacterial
growth when the feed of fresh substrate can begin. A continuous
flow of substrate and nutrients through the tanks is then ensured to
keep optimum growth of microorganisms required for the fastest
degradation of sulfide minerals (Morin, 2007). Retention time in
the whole system is set to allow for sufficiently complete microbial
mediated oxidation of the target sulfide minerals.
On the other hand, CSTRs technologies are generally designed
with height to diameter ratio of close to 1 in order to minimize the
static slurry pressure and thus enable the use of low pressure
blowers for aeration.
Fig. 4 illustrates current and historical, pilot and laboratory-
scale, and demonstration and industrial-scale stirred tank tech-
nology around the world used to process some pyritic gold and base
metal concentrates. The first bioreactor using the BIOX® technology
operated at 40e45
C, developed by Gencor for commercial
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146 131

Fig. 4. Main bacterial assisted leaching stirred tank reactors. ( )): Refractory gold sulfide, ( ): Base metal sulfide; (*): Mine depleted; (**): Under care and maintenance. (Adapted
from Brierley, 2008; Brierley and Brierley, 2013; Clark et al., 2006; Morin, 2007; Rawlings et al., 2003).

biooxidation of a refractory sulfide gold concentrate, was implanted
by Gold Fields in 1986 at the Fairview Mine in South Africa. BIOX®
plant typically consists of six equi-dimensional reactors configured
as three primary reactors operating in parallel followed by three
secondary reactors operating in series. The BIOX® process involves
the continuous feeding of the flotation concentrate slurry (20e30%
of solids) to a series of stirred reactors. The process utilizes a mixed
population of Acidithiobacillus ferrooxidans, Acidithiobacillus thio-
oxidans, Leptospirillum ferrooxidans, Acidithiobacillus caldus, Lep-
tospirillum ferriphilum and Ferroplasma acidiphilum to break down
the sulfide mineral matrix, thereby liberating the occluded gold for
subsequent cyanidation. The optimum temperature in the com-
mercial reactors is controlled between 40 and 45
C allowing
maximum sulfide oxidation rates with minimum cooling re-
quirements. The retention time in the biooxidation reactors is
typically 4e6 days depending on the oxidation rates achieved and
on both the sulfide sulfur content and mineralogical composition of
the concentrate. The typical operating pH range in the BIOX® pro-
cess is 1.2e1.6. Low-pressure air is injected into BIOX® reactors to
supply oxygen for the oxidation reactions. A dissolved oxygen
concentration of more than 2 mg/L is maintained at all times in the
slurry (Van Aswegen et al., 2007).
CSTRs technologies have advanced since its 1986 inception and
operating temperatures were increased to improve process per-
formance. In the 2000s, the Beaconsfield plant in Australia and
Laizhou plant in China using Mintek BacTech technology were
operated at moderately thermophilic temperatures (between 45
and 55
C). Another famous CSTRs technology is BioCop™ process.
The BioCop™ process has been developed by BHP Billiton bio-
technologies group based in Johannesburg, South Africa. The pro-
cess used thermophiles bacteria to leach chalcopyrite at 70e80
C
(Dreisinger, 2006). In 2000, Billiton and Codelco formed a joint
venture called Alliance Copper Limited (ACL) to commercialize the
BioCop™ technology in Chile. A 20,000 t of copper/year
132 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
demonstration plant has been built close to Chuquicamata Mine in
Chile (Batty and Rorke, 2006; Clark et al., 2006).
Another commercial stirred tank bioleaching plant is the Kasese
plant in Uganda (KCC) used for cobalt recovery from a cobaltiferous
pyrite concentrate. This process was developed by the Bureau de
Recherches Ge
ologiques et Minie res (BRGM), France (Morin, 2007;
Morin and D'Hugues, 2007). Pyrite concentrate is reclaimed from
the stockpiles at a nominal rate of 245 t/day using high-pressure
monitor guns and is then pumped to the process plant for the
production of cobalt, copper, nickel, and zinc. At Kasese about
1100 t of cobalt/year are produced, corresponding to 1.25% of the
world production of cobalt in 2010. The bioleaching circuit of the
KCC plant includes a primary stage with three tanks in parallel, and
a secondary and a tertiary stage of one tank each. Theoretically, this
configuration is optimized to ensure a safe retention time for the
bacterial growth in the primary stage and sufficient mineral bio-
oxidation in the later bioreactors. Air is supplied to the bioleach
tanks by five blowers, which can feed each tank up to the equiva-
lent value of 20 000 m3/h air in normal conditions.
The operating airflow rates in the tanks are in the range of
10,000e15,000 m3/h in the primary stage reactors and between
5000 and 10,000 m3/h in the secondary and tertiary stage reactors.
Aeration and mixing in the tanks are ensured by air injection
and a BROGIM system designed according to the size of the tanks, to
the oxygen transfer and mixing requirements. The nominal average
temperature in all reactors is maintained at 42
C. The pH is kept as
constant as possible in every tank by the continuous addition of
limestone slurry at a controlled rate. The nominal values of pH are
1.4e1.5 and 1.5e1.7 in the primary and secondary/tertiary tanks,
respectively. The retention time is about 6 days with the full circuit
running, and the cobalt dissolution yield in the bioreactor is
generally greater than 80%. Approximately 60% of the total pyrite is
oxidized in the primary bioleach stage and the rest of the oxidation
(20e30%) occurs in the following two stages. The pulp density of
the feed to the bioleach circuit is 20% (Morin and D'Hugues, 2007).
As shown in Fig. 4, the numbers of stirred tank reactor plants
built around the world and these in operation demonstrate the
practical use of this technology by the industry of bio-
hydrometallurgy. Not only are the numbers of industrial plants
increasing, but it seems that also the capacity of sulfide concentrate
processed in these technologies is increasing.
Fig. 5. Factors involved in stirred biomining bioreactor (Adapted from Norton et al., 2005; Batty and Rorke, 2006; Kundu and Kumar 2014).
However, due to the level of engineered and process control
involved in this type of technology, these are more expensive
capital and operations than irrigation type technologies, as
mentioned earlier. Another major constraint of CSTRs technologies
is the amount of solids to be treated. Generally, a maximum solids
loading of 20% (w/w) is used, since several factors such as oxygen
requirements, nutrient availability, shear stress induced by impel-
lers, could limit the bacterial assisted leaching efficiency (Ce
zac,
1997). However some recent papers show that it is possible to in-
crease the solids loading up to 30% (w/w) in bioleaching operation
with moderate thermophilic bacteria without any decrease in the
leaching efficiency (Guezennec et al., 2012, 2014).
The use of slurry bubble columns may provide an alternative to
overcome this limitation, as demonstrated at laboratory scale by
Garcia-Ochoa et al., 1997, 1999. Also, possible benefits of using a
bubble column instead of mechanically agitated reactor are to
reduce operating costs, particularly power costs, and decrease the
stress induced on the microorganisms by the propellers (Foucher
et al., 2003). However, a design of a large scale bacterial assisted
leaching slurry bubble column with high solids loading is not yet
possible. Although it is not sulfide minerals biomining process,
novel promising treatment possibilities are outlined for printed
circuit boards utilizing both flotation separation techniques and
acid bioleaching (Ma €kinen et al., 2015).
Finally, other enhancements have been developed around the
world, but these are not discussed in this paper. For more infor-
mation, the interested readers may refer to (Rawlings et al., 2003;
Clark et al., 2006; Rawlings and Johnson, 2007; Brierley, 2008;
Brierley and Brierley, 2013).
5. Factors influencing bacterial assisted leaching of sulfide
minerals in stirred tank reactors
The rate and efficiency of bacterial assisted leaching of metal
sulfides, like any other extraction process, depends greatly on a
number of different parameters, such as physicochemical, micro-
biological, and mineralogical factors, as shown in Fig. 5. Some of the
most important factors and their effects, reported in the scientific
literature, are summarized in Table 3.
Unfortunately, in spite of the vast volume of literature in this
field, results are sometimes conflicting and often the conditions
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146 133

Table 3
Main factors affecting bacterial assisted leaching of metal sulfides.

Factor Parameter's effect References

Physiochemical parameters
Temperature -Influence on microbial activity and community composition Torma 1977; Acevedo and Gentina 1989; Ahonen and Tuovinen 1989a, 1989b,
-Influence on the rate of chemical kinetics 1990, 1991, 1992, 1995; Haddadin et al., 1995; Bosecker 1997; Norris 1997; Dew
-Influence on the formation of precipitates et al., 1999; Brandl 2001; Rawlings et al., 2003; Ojumu et al., 2006; Batty and
-Influence on the solubility of gaseous compounds Rorke 2006; Watling 2006; Morin 2007; Watling et al., 2014
pH -Influence on the microbial growth Tuovinen and Kelly 1973; Torma 1977; Acevedo and Gentina 1989; Ahonen and
-Influence on the formation of precipitates Tuovinen 1989b, 1991, 1995; Collinet and Morin 1990; Battaglia-Brunet et al.,
-Influence on the sulfide minerals dissolution 1994; Haddadin et al., 1995; Bosecker 1997; Rawlings 1997; Brandl 2001;
Rawlings et al., 2003; Deveci et al., 2004; Ojumu et al., 2006; Watling 2006; Morin
2007; Watling et al., 2014; Ngoma et al., 2015
Redox potential -Indicator on the electrochemical state of the environment Ahonen and Tuovinen 1995; Pinches et al., 2001a, 2001b; Third et al., 2002; Olson
(oxidizing or reducing conditions) et al., 2003; Watling 2006; Co
rdoba et al., 2008a, 2008b; Gericke et al., 2010;
-Influence on the leaching rate and thus controlling redox Khoshkhoo et al., 2014
potential is necessary to obtain the fastest leaching rate
O2 content and -Influence on the microbial growth and activity Myerson 1981; Bailey and Hansford 1993a, 1993b; Haddadin et al., 1995; Van
availability -Limiting factor caused by its low solubility Weert et al., 1995; Bosecker 1997; D'Hugues et al., 1997; Brandl 2001; Witne and
-Electron acceptor in microbial and chemical oxidation Phillips, 2001; Rawlings 2002; De Kock et al., 2004; Norton et al., 2005; Ojumu
-An oxidizing agent and can directly oxidize metal sulfides et al., 2006; Watling 2006; Du Plessis et al., 2007; Morin 2007
during the chemical leaching
CO2 content -Carbon source for microbial growth Torma et al., 1972; Holuigue et al., 1987; Norris et al., 1989; Barron and Lueking
-Limiting factor caused by its low solubility 1990; Boon et al., 1992; Bailey and Hansford 1993b; Haddadin et al., 1993, 1995;
-Inhibitory effects at high concentration Nagpal et al., 1993; Bosecker 1997; Nemati et al., 1998; Brandl 2001; Rawlings
et al., 2003; De Kock et al., 2004; Ojumu et al., 2006; Morin 2007; D'Hugues et al.,
2008
Gas mass transfer -Limiting factor in bacterial assisted leaching Myerson 1981; Bailey and Hansford 1993a, 1993b, 1994; Jordan et al., 1995;
D'Hugues et al., 1997; Boon and Heijnen 1998a, 1998b; Savi
c et al., 1998; Veglio
et al., 1998; Garcia-Ochoa et al., 1997, 1999; Witne and Phillips 2001; Du Plessis
et al., 2007; Morin 2007
Nutrients -Essential for the metabolic and optimal microbial growth Torma 1977; Acevedo and Gentina 1989; Battaglia-Brunet et al., 1994; Haddadin
(e.g. N, K, P, et al., 1995; Bosecker 1997; D'Hugues et al., 1997; Go
mez et al., 1999; Brandl
Mg) 2001; Watling 2006
Fe3þ -Inhibitory effects at high concentration Torma 1977; Collinet and Morin, 1990; Kumar and Gandhi, 1990; Curutchet
concentration -“Chemical instability” at high concentration which favors et al., 1992; Haddadin et al., 1995; Nemati et al., 1998; Brandl 2001; Rawlings
precipitation as jarosites
rdoba et al., 2008a, 2008b
et al., 2003; Co
Fe2þ -Inhibitory effects at high concentration Nikolov et al. 1988; Battaglia-Brunet et al., 1994; Nemati et al., 1998
concentration
Heavy metals -High concentration cause an inhibition of microbial activity Tuovinen et al., 1971; Norris and Kelly 1978; Hutchins et al., 1986; Battaglia-
Brunet et al., 1994; Haddadin et al., 1995; Harvey and Crundwell 1996; Pooley
and Shrestha 1996; Bosecker 1997; Das et al., 1997; Ojumu et al., 2006
Aeration and -Providing adequate O2 and CO2 supplies Hackl et al., 1989; Toma et al., 1991; Bailey and Hansford 1993a, 1993b; Oolman
stirring rate -Minimizing the resistance to gas transfer 1993; Van Weert et al., 1995; Clark and Norris 1996; Ce
zac 1997; D'Hugues et al.,
-Produce uniform distribution of bubbles through the liquid phase 1997; Derksen et al., 2000; Witne and Phillips, 2001; Chong et al., 2002; Deveci
-Produce uniform distribution of solids suspension, -Maintain 2002; Harrison et al., 2003; Morin 2007; Morin and D'Hugues 2007; Sun et al.,
uniform temperature, pH and concentration profiles through the 2012
bioreactor
-Ensure the continuous replacement of the liquid in contact with
the bubble surface
-Maximize the residence time of bubbles within the liquid
-Prevent flooding of the impeller
Mineralogical parameters
Solids -Influence on the available surface area for oxidation Sakaguchi et al., 1976; Norris 1997; Beyer et al., 1986; Toma et al., 1991; Bailey
concentration -Influence on the specific energy consumption and Hansford 1993b; Battaglia Brunet et al., 1994; Haddadin et al., 1995; Van
-Inhibitory, mechanical stress, and toxic effects on the microbial Weert et al., 1995; Kandler and Ko €nig 1998; Derksen et al., 2000; Witne and
growth at high concentration Phillips 2001; Rikmanis et al., 2007
-Decrease the mass transfer at high solids concentration
Particles size -Influence on the available surface area for oxidation Torma et al., 1972; Torma 1977; Groudev 1986; Acevedo and Gentina 1989;
-High metal leaching efficiency with the smaller particle size Komnitsas and Pooley 1991; Asai et al., 1992; Ahonen and Tuovinen 1995;
-Influence on the cost of the process Haddadin et al., 1995; Devasia et al., 1996; Bosecker 1997; Garcia-Ochoa et al.,
1997, 1999; Nemati et al., 2000; Brandl 2001; Sissing and Harrison 2003; Acevedo
et al., 2004; Morin 2007
Mineralogical -Influence on the acid consumption and on the pH Acevedo and Gentina 1989; Bosecker 1997; Rawlings et al., 2003; Rawlings
type and -Influence on the dissolution pathway 2004; Watling 2006; Olubambi et al., 2008
composition -Inhibitory effects on microbial activity at high carbonate content
-Influence on the cost of the process
Presence of -Mineral with the lowest rest potential tends to act as an anode in Berry et al., 1978; Mehta and Murr 1983; Hutchins et al., 1986; Torma, 1988;
different metal galvanic cell system Holmes and Crundwell 2000; Abraitis et al., 2004; Ballester et al., 2007; You et al.,
sulfides 2007; Dixon et al., 2008; Koleini et al., 2011; Li et al., 2013

used are not described in much detail. It can be assumed that the difficult to get consistent results. A selected number of the main
variability of the results also depends of the variability of the parameters affecting bioleaching are discussed in more detail
mineral resources that are treated and of the microbial consortia below.
that are used. Given the inherent variability of the systems, it is
134 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
5.1. Gas-liquid mass transfer
According to Peterson (2010), the possible different reaction
pathways that occur in biomining of sulfide minerals, regardless of
the reaction environment, can be gathered into: (i) gas (O2, CO2)
absorption from the gas phase to the liquid phase, (ii) microbial
oxidation of ferrous iron/or sulfur species, (iii) oxidative dissolution
of the mineral, and/or (iv) diffusion in and out of particles' pores
(depending on the nature of the mineral phase and the reactor
configuration).
As most of the metal bacterial assisted leaching are aerobic and
chemolithotrophic in nature, an adequate supply of both oxygen
and carbon dioxide is a prerequisite for good growth, metabolic
function, and in general a high activity of leaching microorganisms.
The adequate supply of gaseous substrates (O2, CO2), required for
the microorganisms, can be achieved by efficient aeration, stirring,
or shaking. In general, during the aerobic bioprocess, the oxygen is
transferred from a rising gas bubble into a liquid phase and ulti-
mately to the site of oxidative biochemical reaction, as argued by
Garcia-Ochoa and Gomez 2009. According to Garcia-Ochoa and
Gomez 2009, the transport of oxygen from the air bubbles to the
site of the oxygen reaction can be represented by a number of steps
and resistances. Taking into account the low solubility of oxygen in
aqueous solutions, it is commonly accepted that the principal
resistance to gas mass transfer is on the liquid film at the gas-liquid
interface and the gas phase resistance can usually be neglected.
Consequently, the liquid film resistances around bubbles will con-
trol the overall transfer rate and thus the oxygen mass transfer rate
(OTR) can be given by the following equation:
 
OTR ¼ kL aO2 CO* 2  CLO (21)
2
Using a similar approach, the carbon dioxide mass transfer rate
(CTR) can be expressed as follows:
 
*
CTR ¼ kL aco2 CCO 2
 CL CO
(22)
2
where, KL, refers to the liquid phase mass transfer coefficient, a, the
gas-liquid interfacial area per unit liquid volume, C*, the oxygen or
carbon dioxide saturation concentration in the bulk liquid which is
in equilibrium with the bulk gas phase and, CL, the instantaneous
dissolved oxygen or carbon dioxide concentration in the bulk
liquid. Due to the difficulty of measuring KL and a separately, usu-
ally the product KLa is measured and this parameter, called volu-
metric mass transfer coefficient, characterizes the transport from
gas to liquid. The saturated gaseous substrate concentration, C*,
depends on the respective chemical composition in the gas that is
being used for sparging (e.g. enriched or normal compressed air) as
well as the operating temperature and pressure and the chemical
composition of the liquid phase.
As pointed out by many researchers, the lack of adequate gas
transfer is a rate limiting step in many of the bacterial assisted
leaching processes (Myerson, 1981; Liu et al., 1988; Bailey and
Hansford, 1993a, 1993b; 1994; Jordan et al., 1995; Morin, 2007).
The gas-liquid transfer rate depends on a number of parameters
such as the reactor design, geometry and size, gas flowrate, sparger
design, stirring speed, impeller design, particles shape and size,
pulp density and viscosity. Many of these parameters are interre-
lated. They can impact both the gas liquid transfer efficiency and
the oxidation efficiency. Some of these parameters are perceived to
be important such as the gas (O2, CO2) supply, stirring and aeration,
pH, temperature, and solids concentration (loading) and particle
size and these will be discussed in some detail here.
5.1.1. Oxygen availability
The supply of oxygen represents the largest consumer of power
in bacterial assisted leaching process (Van Aswegen et al., 2007).
The oxygen requirement is mainly driven by the chemical oxygen
demand for the oxidation of the sulfide mineral. On the other hand,
in bacterial assisted leaching carbon dioxide is an important sub-
strate for microbial metabolism. It is well documented that there is
a lower limit of oxygen concentration, referred to as the critical
dissolved oxygen concentration CCRIT, at which oxygen becomes a
limiting factor and consequently slowing down the microbial
growth rate and ultimately the metal leaching rate (Myerson, 1981;
Liu et al., 1988; Bailey and Hansford, 1994; Van Weert et al., 1995;
D'Hugues et al., 1997). The maximum possible driving force in a
particular system would then be the difference between C* and
CCRIT. This allows the definition of an oxygen transfer potential
(OTP) as follows:


OTP ¼ kL a C *  CCrit (23)
Myerson (1981) reported that the critical oxygen concentrations
for Acidithiobacillus ferrooxidans pyrite leaching at several temper-
atures (from 25 to 35
C) and at a pH of 2.5 were varied from 0.35 to
0.41 mg/L. Liu et al. (1988) observed that the growth of Acid-
ithiobacillus ferrooxidans on ferrous iron was limited at a dissolved
oxygen concentration of approximately 0.7 mg/L and the oxidation
process completely ceased at the concentrations below 0.28 mg/L.
At 20% solids of cobaltiferous pyrite bioleaching, D'Hugues et al.
(1997) and Ce
zac (1997) found that growth was limited at dis-
solved oxygen concentration below 1.2 mg/L.
The minimum value of the volumetric oxygen mass transfer
coefficient, (KLa)min, required to maintain the oxygen concentration
above this value can be calculated from the equation:
xc
ðkL aÞmin ¼ (24)
YO2 ðC *  CCrit Þ
where c is the bacteria density (g bacterial cells/L), x the specific
growth rate (h1), YO2 the oxygen yield coefficient (g bacterial cells/
g O2 consumed).
Myerson (1981) showed that, for the continuous growth of
Acidithiobacillus ferrooxidans on pyrite at a specific growth rate of
0.1 h1, a typical value of (c·x) is 5 103 (g bacterial cells/L. h) which
corresponds to a (KLa)min of 58.5 h1. Moreover, Myerson (1981)
claimed in his study that to prevent oxygen from limiting growth
and hence limiting the leaching rate, the reactor must be designed
to operate with a (KLa)min equal to or greater than 58.5 h1. How-
ever, the values of the volumetric oxygen mass transfer coefficient
determined by this method are of doubtful validity. Therefore, the
early experimental relationship quoted above (Equation (24)) in
light of current bioreactors designs is no longer used. A number of
correlations for the volumetric gas mass transfer coefficient in
stirred tanks has been developed by a number of different re-
searchers (see Section 5.1.3). Generally, it is admitted that the ox-
ygen mass transfer coefficient, (KLa), for an agitator/aeration system
must meet or exceed the required (KLa)min determined by the ox-
ygen demand of the process.
The oxygen required by the leaching reactions must first be
dissolved into the liquid medium from air in the atmosphere or
another source via gas liquid mass transfer. On the other hand, the
various microbial and chemical oxidation reactions involved in the
oxidation and dissolution of sulfide minerals impose an overall
oxygen demand in the solution phase. This demand can only be
satisfied if the rate of transfer from the gas phase equals or exceeds
the rate of demand within the solution phase. This demand can
only be satisfied if the rate of transfer from gas phase equals or
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
exceeds the rate of demand within the solution phase. The
increased demand for oxygen under these conditions is generally
facilitated by increased aeration rates, higher impeller agitation
rates, and improved agitator designs. In the case of mesophiles and
moderate thermophiles, sufficient oxygen may be supplied to the
reactors by simply sparging with air and by improving agitator
designs. Agitation speeds and power input cannot however be
indefinitely increased to improve mass transfer limitations as cell
shear stress damage to biomining microorganisms becomes a
limiting factor at high agitation speeds and power inputs in the
presence of high pulp densities (Chong et al., 2002; Du Plessis et al.,
2007). In the case of thermophilic biomining conditions, mass
transfer limitation due to the reduced solubility of oxygen in higher
temperature is, however, a significant process challenge that
cannot be overcome by simply increasing agitation speeds and
aeration rates. In fact, the oxygen solubility in thermophilic systems
is reduced to 1/3 of what could be expected at mesophile condi-
tions (Morin, 2007).
As mentioned earlier, the solubility of oxygen decreases with
increasing temperature. Also, the nutrient solution has a major
influence on the solubility of oxygen. For comparison, only 7 mg O2
can be dissolved per liter of pure water at 35
C compared to
8.32 mg O2 per liter of pure water at 25
C in an air/water mixture.
The corresponding average value of the solubility of O2 in the Sil-
verman and Lundgren nutrient medium 9 K is only 6.49 mg/L at
35
C (Liu et al., 1988; Witne and Phillips, 2001). One way to tackle
this limitation in oxygen solubility in water or nutrient medium
and consequently to overcome the mass transfer difficulties at
elevated temperature is by increasing the driving force (e.g. raising
the oxygen partial pressure according to Henry's gas low) by the
use of oxygen or by enriching the air sparged to the bioreactor. As
the oxygen concentration increases in the gas phase, the oxygen
proportion in the nutrient medium would increase. Consequently,
the highest oxygen partial pressures could be achieved when air is
enriched with pure oxygen. For comparison, 40.3 mg O2 can be
dissolved per liter of pure water at 25
C in a pure oxygen/water
mixture compared to 36.2 mg per liter of pure water at 35
C (Witne
and Phillips, 2001). In the thermophile situation like for the Bio-
Cop™ process which implies a temperature of 78
C, Du Plessis et al.
(2007) mentioned that the use of oxygen-enriched air (as high as
90%) could potentially result in the increase of dissolved oxygen
concentration as high as 15 mg/L. However the dissolved oxygen
concentration can be much higher with moderate thermophile or
mesophile. Using Henry's law, it can be assumed that the dissolved
oxygen concentration may increase up to 28 mg/L for an oxygen
enrichment of 90% at 40
C. Such elevated dissolved oxygen con-
centrations could have detrimental effects on microorganisms. It is
reported that the optimal dissolved oxygen concentration has been
determined to range between 1.5 and 4.1 mg/L for thermophilic
bioleaching performed at 70
C (De Kock et al., 2004; Norton et al.,
2005; Du Plessis et al., 2007). To date, however, there is no agree-
ment on the upper limit of oxygen at which oxygen becomes
inhibitory to the microorganisms involved in bacterial assisted
leaching.
Many researchers have demonstrated that the oxygen enrich-
ment of the process air may enable the oxygen demand to be
satisfied more easily and have therefore beneficial effects on the
process efficiency (Corrans, 1974; Hoffmann et al., 1993; Ce
zac,
1997; D'Hugues et al., 1997; Witne and Phillips, 2001; Morin and
D'Hugues, 2007). Corrans 1974 demonstrated that the leach rate
was improved with increasing oxygen content of the process air
from 20 to 100%. These findings were further supported by
Hoffmann et al., 1993 during bacterial assisted leaching of a re-
fractory gold ore.
Similar observations were reported in the works of D'Hugues
135
et al. (1997) and Ce
zac (1997). They observed that the dissolution
of cobaltiferous pyrite was improved with increasing air flowrate. It
should be highlighted however that there is an optimal air flowrate
for bacterial assisted leaching. Further increase of the air flowrate
will result in a decrease of dissolution rate which can be attributed
to the excessive mixing, high turbulence and attrition adversely
affecting microorganism growth and ultimately hampering the
bacterial assisted leaching process. Witne and Phillips 2001 re-
ported that the dissolution of the OK Tedi copper concentrate was
also improved with increasing oxygen enrichment up to 30% oxy-
gen content (9% pure oxygen þ 21% O2 in atmospheric air). They
showed that an increase between of 2.6e9% copper extraction was
obtained for 30% O2 enrichment with various cultures compared
with atmospheric air for the same conditions. They also observed
that the volumetric mass transfer KLa was increased by about
12e37% with oxygen enriched air compared with atmospheric air
for the same conditions. However, no further improvement in the
extraction of copper for any culture was observed with further in-
crease of O2 from 30% onwards. Norton et al. (2005) have also
pointed out the importance of maintaining an adequate supply of
oxygen and hence a sufficiently high dissolved oxygen concentra-
tion to sustain microorganism growth and mineral oxidation. The
desired range of dissolved oxygen concentration may be obtained
by controlling operational conditions (e.g. the oxygen content of
feed gas, the rate of supply of the feed gas to the slurry, and the rate
of feed of slurry to the reactor).
On the other hand, the oxygen requirement for the oxidation of
the sulfide mineral will vary depending on the mineralogical
composition. For example, it varies from 1.8 to 2.6 kg of oxygen per
kilogram sulfide oxidized in the commercial BIOX® process (Van
Aswegen et al. (2007)), while it is estimated to be on average
0.89 kg of oxygen per kilogram pyrite oxidized in the case of the
KCC bioleaching operation (D'Hugues et al., 1997; Ce
zac, 1997;
Morin and D'Hugues, 2007).
5.1.2. Carbon availability
In addition to the oxygen requirement, the adequate supply of
carbon dioxide is a prerequisite for microbial growth. Chemo-
lithotrophic microorganisms involved in bacterial assisted leaching
assimilate or fix carbon dioxide via the Calvin-Benson Cycle to meet
their carbon requirement for growth. In the Calvin-Benson Cycle, it
is well documented that the Ribulose-1, 5-bisphosphate
carboxylase-oxygenase (RuBisCO) is the key enzyme for carbon
dioxide fixation (Nagpal et al., 1993; Shively et al., 1998).
In a general form, the microbial carbon dioxide uptake rate
(CUR) can be expressed by the following equation (Acevedo and
Gentina, 2007):
xc
CUR ¼ (25)
YCO2
where c is the bacteria density (g bacterial cells/L), x the specific
growth rate (h1), YCO2 is the carbon dioxide yield coefficient (g
bacterial cells/g CO2 consumed).
Similarly the microbial oxygen uptake rate (OUR) can be written
as:
xc
OUR ¼ (26)
YO2
Using equations (21) (22) (25) and (26) leads to:
 
kL aO2 CO* 2  CO2 YO2
CUR ¼   CTR (27)
*
kL aCO2 CCO  C YCO2
2
CO2
136 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
The value of the proportional factor in this equation will
determine whether the bacterial assisted leaching will be limited
by carbon dioxide.
Several investigations have reported increases in bacterial
assisted leaching rate by the supplementation of sparged ambient
air with carbon dioxide (Torma et al., 1972; Brierley, 1978; Holuigue
et al., 1987; Liu et al., 1988; Barron and Lueking, 1990; Haddadin
et al., 1993, 1995; Nagpal et al., 1993; Witne and Phillips, 2001;
De Kock et al., 2004). Torma et al. (1972) showed that leach rate
of zinc from a zinc sulfide concentrate using Acidithiobacillus fer-
rooxidans was increased by increasing the carbon dioxide concen-
tration in the gas phase, up to 0.23% (v/v). At concentration above
0.23% (v/v) of CO2, no further enhancement in extraction rate was
observed by their study. Also, they reported that no apparent in-
hibition was observed, even at concentrations as high as 7.92% (v/v)
of CO2. Holuigue et al. (1987) reported that an increase in the car-
bon dioxide concentration from that of atmospheric air (0.03% (v/
v)) to 0.07 and 0.10% (v/v) increased the growth rate of the bacteria
twofold. Based on the experiments carried out by Liu et al. (1988)
with air enriched with 1% (v/v) carbon dioxide, one could inter-
pret that for atmospheric air conditions (not enriched with carbon
dioxide) carbon dioxide limitation occurred prior to oxygen limi-
tation. This assumption was borne out by the findings of Mandl
1984 who encountered carbon dioxide limitation before oxygen
limitation during the growth of Acidithiobacillus ferrooxidans on the
Silverman and Lundgren 9 K medium when sparging with normal
air. It is also in line with the findings by Boon et al. (1992) who
showed that carbon dioxide limitation occurred before oxygen
limitation unless the process air was enriched with carbon dioxide.
Norris (1989) and Norris et al. (1989) both found that the rate of
pyrite dissolution of A. ferrooxidans was only slightly reduced by the
use of air without additional carbon dioxide. At moderate ther-
mophilic temperatures, both the growth rate on ferrous iron (Wood
and Kelly 1985) and the pyrite dissolution rate (Norris and Owen,
1993) were enhanced with CO2 supplementation.
Barron and Lueking 1990 found that the optimal concentration
of carbon dioxide for growth was found to be between 7 and 8% (v/
v). Also, they reported that the use of elevated carbon dioxide (8%
onwards) inhibited the Thiobacillus ferrooxidans growth. Haddadin
(1995) stated that there is a strong relation between the supplied
carbon dioxide concentration and the bacterial oxidation of ferrous
ions Fe2þ and pyrite using a mixed culture of iron and/or sulfur-
oxidizing bacteria.
Nagpal et al. (1993) reported that aqueous phase carbon dioxide
concentrations in excess of 10 mg/L were found to be inhibitory to
the growth of Thiobacillus ferrooxidans on pyrite-arsenopyrite ore
concentrate at 16% solids. They also noted that the optimal aqueous
phase concentration of carbon dioxide for growth was found to be
3e7 mg/L. Carbon dioxide below this level sharply reduced the
bacterial growth rate. The same authors reported that the mineral
oxidation rates were not directly linked to bacterial growth rates.
Boon and Heijnen 1998a, 1998b attributed the observed decrease in
the biooxidation rates at increasing solids density to the exhaustion
of carbon dioxide and to gas-liquid carbon dioxide transfer
limitations.
Witne and Phillips 2001 demonstrated that the dissolution of
OK Tedi copper concentrate was enhanced with increasing carbon
dioxide content up to an optimum of 10% (v/v) CO2 (9.97% þ 0.03%
already in ambient air) for all the various cultures studied in their
investigation. They also noticed that the Sulfolobus culture allowed
the highest copper extraction (96.1%), corresponding to a 7.6% in-
crease in the copper extraction compared with normal air for the
same conditions. More recently, De Kock et al. (2004) investigated
the effect of dissolved oxygen and carbon dioxide concentration on
microbial activity of Sulfolobus sp. (a typical thermophilic archea).
They found that the optimal inlet carbon dioxide concentration for
ferrous iron oxidation was predicted to be between 7 and 17% (v/v).
On the other hand, other investigations stated that the specific
microbial growth rate was independent of carbon dioxide con-
centration in the range of 0.03 to 8e10% (v/v) (Kelly and Jones,
1978).
D'Hugues et al. (2008) showed that carbon dioxide limitation
had a very significant negative effect on bacterial productivity and
exopolysaccharide production. The direct consequence of the
decrease of the biomass was a significant decrease on the bio-
leaching efficiency.
It is noteworthy that many sulfide minerals concentrates
contain carbonates which react quickly in the highly acidic bioleach
slurry, releasing CO2. In such cases, it may not be necessary to
supplement air with CO2. Some concentrates are highly acid pro-
ducing when leached, and require the addition of an alkaline re-
agent to prevent the pH falling too low. In such cases, the use of
limestone (CaCO3) is recommended as this supplements the CO2
supply as well as controlling pH (Du Plessis et al. (2007)).
Finally, most authors agree on the fact that the addition of ox-
ygen and carbon dioxide can enhance the bacterial assisted leach-
ing process. Also, there is general agreement on the significant
limitation in the extraction rate due to insufficient mass transfer.
However, in light of the scientific literature available it could be
argued that the maximum oxidation rate may require an optimum
concentration different from the one required for maximum
growth rate or cell yield and this warrants further investigations to
elucidate these discrepancies.
During the bioleaching process, the decrease in the ratio of CUR/
pyrite oxidation rate, combined with the constant ratio of OUR/
pyrite oxidation rate, confirmed that substrate attack can not be
directly related to bacterial growth metabolism. In conclusion, the
bacteria seemed to act, in the pyrite oxidation process, as an
enzymatic catalyser which was independent of their growth
metabolism (D'Hugues et al. (1997)).
5.1.3. Aeration and stirring
As mentioned earlier, CSTRs provide higher mass transfer,
higher mixing and suspension than other types of reactors such as
ALR and CBC reactors (Rossi, 2001; Acevedo and Gentina, 2007).
However, in CSTR technology, an efficient aeration and stirring
system is required to provide adequate oxygen and carbon dioxide
supplies to the microorganisms, to obtain a high degree of homo-
geneity of the gas/liquid/solids system, to prevent flooding of the
impeller, and to enhance the gas transfer rates from gas/air or from
other sources to the liquid phase resulting in maximizing the
volumetric gas mass transfer coefficient, KLa, which in turn pro-
vides an improved process efficiency.
A number of correlations for the volumetric gas mass transfer
coefficient, KLa, in stirred tanks containing slurries of different
materials, have been developed by a number of different re-
searchers (Oguz et al., 1987; Schumpe et al., 1987). Oguz et al.
(1987) in a three-phase system which relates KLa to the relative
slurry viscosity as follows:
kL a ¼ 6:6 104 ðmÞ0:39 ðQ Þ0:5 ðP=VÞ0:75 (28)
where m is the relative slurry viscosity; Q is the gas sparge rate (m3/
h), and (P/V) the power input per unit volume (kW/m3).
It is believed that solids concentration affects the gas mass
transfer coefficient either by modification to the interfacial turbu-
lence at low solids concentration or by a diffusion blocking effect.
On the other hand, the gas-liquid interfacial area per unit liquid
volume, a, is affected by an increase in the viscosity and by altered
bubble coalescence rates. Also, it is reported that the relative slurry
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146 137

Fig. 6. (a) Influence of pyrite solids concentration on the value of kLa calculated from the Oguz et al. (1987) correlation (Bailey and Hansford, 1993a, 1993b; 1994). (b) kLa as a
function of the solids concentration at various impeller speed for Rushton turbine and Lightnin A315 in 67 L laboratory tank test. (c) kLa as a function of the gas flowrate for pyrite at
solids concentration (10% (m/v)) and at speed impeller of 500 rpm in 67 L laboratory tank test. (d) Comparison of kLa values for A315 impeller and BROGIM® system designed by
Milton Roy Mixing Industries in 850 L operating tank volume (Bouquet and Morin, 2006).

viscosity remains constant with increasing the solids concentration
up to some critical solids concentration value (Bailey and Hansford,
1993a, 1993b). From this critical value onwards, however, the
relative slurry viscosity increases drastically. This increase in the
relative viscosity will cause a decrease in KLa and consequently a
lowering of the gas mass transfer, as shown by Bailey and Hansford
1994 using Oguz et al. (1987) relationship (Equation (28)) (Fig. 6a).
The gas mass transfer coefficient also depends on the gas rate
and the power input per unit volume from stirring. For this pur-
pose, several investigations have been carried out to enhance the
gas mass transfer by improving the reactor and impeller designs,
and gas enrichment (Hoffmann et al., 1993; D'Hugues et al., 1997;
Rossi, 2001; Bouquet and Morin, 2006; Morin, 2007). The
maximum gas rate, which is a function of the stirring speed, and the
extent of gas enrichment is largely determined by cost factors and
power requirement. On the other hand, the power input per unit
volume is bounded by the fact that high shear stresses are detri-
mental to the viability of the bacterial cells (Toma et al., 1991; Bailey
and Hansford, 1993a, 1993b; Oolman, 1993; D'Hugues et al., 1997;
Chong et al., 2002; Harrison et al., 2003; Sun et al., 2012). It was
suggested that exposure of microorganisms to strong shear and
“turbulent” environments could result in the lysis of cells, the in-
hibition of biomass growth or product synthesis, the denaturation
of extracellular proteins, the change in morphology of the micro-
organisms, the thickening of the cell wall, and the impediment of
the attachment of bacteria to solid substrate among others (Toma
et al., 1991; Sun et al., 2012).
Evidence of mechanical destruction of the microorganisms has
been noticed by several researchers using a range of different
equipment design. In their experiments, Hackl et al., 1989 found
that low leach rates were caused by excessive shear stress. Impeller
tip speeds of 5.3 m/s were found to be detrimental to the acido-
philic iron and sulfur oxidizing mesophiles. They showed that
leaching was improved when the speed of the Rushton turbine was
reduced to 3.3 m/s. Also, it is reported that thermophilic Sulfolobus
spp. bacteria using in biomining process appear to be sensitive to
hydrodynamic conditions (Clark and Norris, 1996) and the presence
of solids (Le Roux and Wakerley, 1988; Nemati and Harrison, 2000).
This can be attributed to the fact that Sulfolobus lack a rigid
peptidoglycan cell wall component (Ko € nig, 1988), from which
bacteria derive their structural strength (Sissing and Harrison,
2003). D'Hugues et al., (1997) found that the excessive turbu-
lence, associated with high agitation intensity during the cobaltif-
erous pyrite bioleaching process, affected the efficiency of the
bioleaching by limiting the bacterial productivity and by reducing
the bacterial contact with the solid substrate. To describe this in-
hibition, the term turbohypobiosis was introduced (Toma et al.,
1991). Turbohypobiosis was characterized by a stress factor
expressing interaction between microbial cells and medium flow,
considering energy distribution and residence time of cells in local
turbulent zones. The numerical value of turbohypobiosis may be
used in process control and regulation of microbiological process.
138 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
Deveci (2002) showed, during the bioleaching process in CSTRs,
that hydrodynamic shear alone as a characteristic function of
impeller type and speed has a very limited effect on the bacterial
cells. Mechanical damage to bacterial cells occurs, to a more sig-
nificant extent via attrition with the solid particles which is pro-
moted by the intensity of agitation. Deveci (2002) also reported
that extent of loss in the viability of bacterial population increases
as a function of time with increasing the solids density and/or the
intensity of agitation. More recently, Sun et al. (2012) demonstrated
that the higher agitation intensity on the refractory gold ores bio-
mining process by A. ferrooxidans in CSTRs not only damaged the
suspended cells but also prevented the adsorption of cells onto the
mineral surface. They also reported that the oxygen uptake rate
(OUR), the Fe2þ oxidation activity and the rus gene expression of
A. ferrooxidans during the biomining process decreased with the
increase of the agitation intensity, which might be responsible for
the decrease of the biomining rates with the agitation intensity. On
the other hand, Chong et al. (2002) studied the effect of particle
shearing on the process of pyrite biooxidation by A. ferrooxidans. In
their work, an immobilized ore bioreactor concept was used for the
elimination of particle shearing, while a mechanically agitated
slurry bioreactor was used as a frictional system. They showed that
when the oxygen mass transfer was not the rate limiting step, the
rate of bioleaching in the friction less bioreactor was 2.5 times
higher than that in a bioreactor with particle shearing. Indeed, the
concentration of free suspended cells in the frictionless bioreactor
was much lower than that in frictional one. This confirms that
particle friction strongly reduces the microbial attachment to the
sulfide surface which in turn reduces the rate of bioleaching. Based
on the experimental results, they concluded that reduction of
particle friction during bioleaching of sulfide minerals can not only
increase significantly the bioleaching rate, but also increase the rate
of gas-liquid oxygen mass transfer. It reduces both the formation of
iron precipitates and energy consumption.
From the point of view of bioreactor design, it is necessary to
strike a balance between stirring speed and aeration rate to in-
crease gas mass transfer rate into the liquid without hindering
bacterial growth. The power input for the design of the impeller is
normally determined by either the oxygen mass transfer rate
required or the aeration rate to prevent flooding of the impeller. For
many years, the radial flow Rushton turbine was the most widely
used in these bioreactors, but lately the axial flow Lightnin A315
impeller has been shown to outperform the Rushton turbine as it
requires a lower power level for the same performance, allowing
solids suspension at reduced shear stresses in suspensions. It also
gives equivalent gas transfer rates, but has in theory a relatively
limited efficiency in air dispersion compared to the Rushton tur-
bine. Derksen et al. (2000) investigated the influence of impeller
type on volumetric gas mass transfer coefficient for pyrite at
various impeller speed, gas flowrate, and solids concentrations, as
shown in Fig. 6b, c. They concluded that, at the same flowrate and
impeller speed conditions, the Rushton turbine showed slightly
higher kLa values compared to the Lightnin A315. They also high-
lighted that the power input by A315 impeller is typically half the
power need to revolve the Rushton turbine at the same impeller
speed. Their results showed that increasing the solids concentra-
tion in the range of 1e12.7% of pyrite increases kLa. Indeed, the
impeller speed and kLa are positively correlated, as shown in
Fig. 6b.
Recently, Bouquet and Morin 2006 made a comparative study
between BROGIM®, an alternative agitation system developed by
Milton Roy Mixing, and A315 impeller, as shown in Fig. 6d. The
alternative agitation BROGIM® system consists of two different
impellers mounted on the same rotating shaft. The lower impeller
is a flat blade disk turbine to disperse air and one or two propellers
located above the turbine is used to ensure the pumping effect. This
system showed an increase ranging from 1.49 to 1.90 times in kLa
compared to the A315 impeller alone under the experimental
conditions. Fig. 6d also clearly showed that with the increasing of
gas flowrate, kLa increased, and as a consequence the extraction
yield is also increased.
5.2. Acidity (pH)
The pH range employed in most industrial biomining processes
is largely influenced by the type of bacterial culture being used, the
mineral type being treated and the nature of the bacterial oxidation
processing method adopted. Likewise, the optimal pH range is
variable from one system to the other, one microorganism to the
other, as shown in Table 1.
A low pH value is harmful to microorganisms metabolism and
can be very selective for acid-tolerant species making the biological
system fragile. At low pH, the concentration of ferric iron from iron
sulfides is high in solution, which can reduce bacterial growth of
species sensitive to this ion (Collinet and Morin, 1990). As discussed
previously, the addition of carbonate neutralizing agents like
limestone have a beneficial effect on CO2 availability. In contrast,
where limestone is not required or in lower amounts, less carbon
dioxide is available in situ for stimulating bacterial growth and less
gypsum is precipitated, which may have consequences on the
retention time in neutralization operations (Morin, 2007). Gahan
et al. (2009) reported that considerable savings in operational
costs could be obtained by using steel slag as the neutralizing agent
rather than limestone without negative impact on bioleaching ef-
ficiency. Another aspect is related to nutrients as some authors
think that excess calcium could result in less phosphate being
available in solution to satisfy the bacterial metabolism needs
(Barrett et al., 1993).
Ahonen and Tuovinen 1995 reported that with a complex sulfide
ore, the leaching rate of cobalt, copper, nickel and zinc each
responded differently to pH changes. The rates of solubilization of
cobalt and copper were highest at low pH, whereas nickel and zinc
showed practically no pH dependency. Deveci et al., 2004, 2008
studied the effect of pH in the range of 1.0e2.0 on the dissolution
rate of zinc from the ore by mesophilic A. ferrooxidans, moderately
thermophilic S. yellowstonensis and S. thermosulfidooxidans, and
extremely thermophilic A. brierleyi. The bioleaching activity of
mesophile A. ferrooxidans, as indicated by the dissolution rate of the
zinc, (Fig. 7a), was adversely affected by decreasing pH to 1.4. But,
above this pH, there seemed no significant difference in the disso-
lution rate and extent (91e93%) of zinc, with an optimum bio-
leaching performance having been recorded at pH 1.8. In contrast to
A. ferrooxidans, the extraction rate of zinc by the extremely ther-
mophilic A. brierleyi (at 70
C) was observed to increase with
increasing acidity, as shown in Fig. 7a. The extraction of zinc,
although being comparable at pH  1.4 (95-92%) over the bio-
leaching period, was recorded to decrease by about of 10% with a
step increase in pH between 1.6 and 2.0. In the case of the moder-
ately thermophilic S. yellowstonensis and S. thermosulfidooxidans, the
increase in the acidity to pH 1.2e1.4 led to a decrease in the oxidizing
activity of both strains, indicating the inhibitory effect of increased
acidity on the strains. They also reported that the salinity has a
suppressing effect on the mesophiles, while the thermophils
appeared to be halophilic. Recently, Ngoma et al., 2015, investigated
the effect of acid stress in the terms of acid concentration and
duration of exposure in submerged culture on mesophilic microor-
ganisms implicated in sulfide mineral bioleaching. In their study,
following acid stress, the cultures were returned to standard oper-
ating conditions in a batch stirred reactor and their performance
were assessed and compared to that of a control test, not subjected
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
Fig. 7. (a) Effect of pH on the initial (maximum) dissolution rate of zinc from the ore by
mesophilic, moderately thermophilic, and extremely thermophilic microorganisms
(Deveci et al. (2004)). (b) Copper dissolution data for mesophiles and thermophiles of
predominantly chalcopyrite concentrate. (c) Copper dissolution profile for a contin-
uous pilot and batch test results of the same predominantly chalcopyrite concentrate
(Batty and Rorke, 2006).
to acid stress. They reported that increasing acid stress resulted in an
increase in the lag period before the onset of microbial growth and
iron oxidation due to decreased viable cell numbers, specific meta-
bolic activity or both. Following adaptation, typical growth rates and
ferrous iron oxidation rates were observed under low stress condi-
tions while a reduction in the rate and extent of microbial growth
and ferrous iron oxidation persisted at extreme conditions. A
reduction in yield (microbial cells produced per kg iron oxidized)
was observed with increased acid concentration over comparative
times.
5.3. Solids concentration and particle size
As metal solubilization in bacterial assisted leaching of sulfide
minerals takes place through the surface area of the particles, the
139
pulp density and particle size determine the available surface area
concentration and have an influence on the overall rate of bio-
leaching of minerals. A large number of researches have stated that
these factors together with others such as mechanical effects,
metabolic stress caused by uncomfortable environmental condi-
tions and inhibitory concentrations of ferric ion and heavy metals
can also lead to a negative overall effect (Torma et al., 1972;
Groudev, 1986; Nemati and Harrison, 2000; Nemati et al., 2000;
Sissing and Harrison, 2003; Acevedo et al., 2004). Particle size is a
critical parameter in biomining process because of its influence on
the bioleaching rate and the costs associated with size reduction.
Small particles of 100 mm or less are desirable in stirred tank system
in order to facilitate a uniform slurry. Using different size fractions
of a pyrite sample, the best results were obtained with the smallest
particle size fraction of 42 mm (Torma, 1977). In their study for
pyrite bioleaching by acidophilic thermophile Sulfolobus metallicus,
Nemati et al. (2000) found that the bioleaching rate was almost
doubled when the particle size is reduced from a mean diameter of
202 mm to a mean diameter of 42.5 mm. They also reported that
particles size distribution of the mineral in this range did not in-
fluence the morphology and growth kinetics of the cells. They also
showed that decreasing the particles size to a mean diameter of
6.4 mm adversely influenced the activity of the cell. The presence of
fine particles apparently increase the particle-particle collisions
causing sever attrition on the cells. Such situations would damage
the structure of the cells resulting in their inability to oxidize pyrite.
Ahonen and Tuovinen 1995 stated that the leaching rates for metal
sulfide ores increased with a reduction in particle size and this
effect was enhanced at lower pH values. In this case, the copper and
nickel leaching rate approximately doubled when the particle
diameter was decreased from greater than 5 mm to less than 10 mm
(þ5, 10 mm) to (þ1.68, 5 mm). Devasia et al. 1996 proposed that
bacterial adhesion and hence bioleaching rates depend on the
particle size of the minerals and compared a range of sizes between
(þ38, 53 mm) and (þ106, 150 mm). In practice the optimum size
must to be determined for each kind of sulfide mineral to be
leached. This size will be dictated by the relative economics which
should be based on the two alternatives: the gains resulted in the
increase of the rate of metal extraction by decreasing the particle
size and the grinding costs (Torma, 1977).
The effects of the pulp densities have been studied in bio-
leaching with mesophilic bacteria for a long time (Torma et al.,
1972; Groudev, 1986). When working with thermophilic Archaea,
the negative effect of high solids concentration is likely to be larger
because of their weaker cell wall (Bailey and Hansford, 1993a,
1993b; Kandler and Ko €nig, 1998) that makes them susceptible to
mechanical damage and metabolic stress (Toma et al., 1991; Norris,
1997; Rikmanis et al., 2007).
Generally, the bioleaching rate is maximal at 20% solids for high
sulfide content materials in the case of mesophiles and 12.5% in the
case of thermophiles (Morin, 2007). High solids concentrations
have been found to influence the lag time, biooxidation rate, and
ultimate extent of oxidation. The factors proposed as potential
causes of these phenomena include oxygen and carbon dioxide
availability, low bacteria to solids ratio, mechanical damage or in-
hibition of bacteria, inhibition of bacterial attachment, and the
buildup of toxic leach products (Bailey and Hansford, 1993a, 1993b).
5.4. Temperature
Microbial species may be classified according to the tempera-
ture range at which they can operate most efficiently. Thus the
operating temperature of a bacterial oxidation process is crucial,
because each bacterial culture has a preferred or optimum tem-
perature for growth and oxidation and then the optimum
140 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
temperature range may be defined as that range in which the
oxidation of sulfide minerals is most rapid. As discussed earlier,
three temperature ranges are considered for present and future
industrial-scale units of bioleaching in stirred tanks: the meso-
philes typical range, which is 35e40
C, the moderate thermo-
philes, 45e55
C, and the extreme thermophiles, 60e85
C, as
shown in Table 1. However, these ranges have no strict limits, and
for example moderate thermophile microorganisms like Sulfoba-
cillus thermosulfidoxidans of which optimal growth temperature is
45e50
C, can occur at 35
C (Norris, 1997; Foucher et al., 2001). All
the existing bioleach units in stirred tanks are at low temperature,
near 40
C, one used moderate thermophiles, near 50
C, and
several projects are in progress to use extreme thermophile for the
recovery of copper from chalcopyrite (Rawlings et al., 2003; Batty
and Rorke, 2006). The use of thermophilic bacteria follows the
demonstration of the ability of Sulfolobus microorganisms to
dissolve copper from chalcopyrite up to 95% (Norris, 1997), whereas
this mineral is refractory to complete degradation when using
mesophilic bacteria. Fig. 7b shows the difference in copper disso-
lution of a predominantly chalcopyrite concentrate using thermo-
philes and mesophiles (Batty and Rorke, 2006). The data shows that
final dissolution was doubled from 45 to more than 90% through
the use of thermophiles microorganisms. For comparison, meso-
philes achieved only 40% oxidation in 10 days while thermophiles
achieved 80% dissolution in the same time period. On the other
hand, Fig. 7c shows the comparative data from the batch reactor
and inventory results from the continuous pilot operation on the
same chalcopyrite concentrate. The same extent of dissolution was
achieved with half the leach time in the continuous system as
opposed to batch leach test (Batty and Rorke, 2006).
Temperature becomes not only a constraint in the design of
installation but also a parameter for the selection of the most
efficient biological system for dealing with a mineral in the most
economical condition (Dew et al., 1999).
As sulfide oxidation is exothermic heat removal is an important
aspect of the design of the bioleach tanks. The higher the temper-
ature in the tanks, the lower the heat removal requirement which
may be important when water supply is an issue or energy for
cooling would be too expensive. The heat generated by agitation is
also an input to be accounted for in the design of the heat removal
system. Energy outputs include heat absorption by the incoming
feed and the energy contained in exiting slurry product, convective
transfer, and the latent heat of moisture in the air leaving the sys-
tem. The heat removal is generally ensured by transfer to water
circulating in internal reactor coils that can be more than a kilo-
meter long in one tank. This piece of equipment is particularly
critical as a leak of cooling water in a tank can reduce the bio-
leaching activity by dilution (Morin, 2007).
6. Conclusions and future developments aspects
The art of biomining has been reviewed with emphasis on en-
gineering factors associated with CSTR technologies and it has been
shown that there is a strong fundamental basis for this enhanced
biotechnological process. Also significant efforts have been devoted
in this paper to provide comprehensive information about the
biomining mechanisms and to understand the relationship be-
tween these mechanisms and the engineering parameters. It is
hoped that this will assist with the development of more robust
designs to improve the productivity and further help in establishing
biomining as a promising and economical route for metal
extraction.
Continuous stirred tank bioleaching is often used to treat re-
fractory gold bearing sulfides prior to cyanidation leaching. These
commercial successes are very encouraging but (i) remain limited
to very specific resources, (ii) use a relatively low number (density)
of microorganisms and (iii) do not implement diversified technical
options.
One remaining important bottleneck and technical challenge for
biomining is the leaching of chalcopyrite. Indeed the main limita-
tion of biohydrometallurgy applied to the treatment of Cu ores is
due to the recalcitrance of chalcopyrite to leaching. Various the-
ories have been proposed to account for this phenomenon, mostly
involving passivation of the mineral by ferric precipitates and/or
sulfur, yet there is still no full consensus on the true mechanistic
phenomenon. If part of the researches suggests that the use of high
temperature and thermophilic archae can be used in bioleaching
systems to overcome incomplete extraction of copper from chal-
copyrite, other recent experimental studies demonstrated that high
rates of chalcopyrite leaching can also be achieved by using mod-
erate thermophile consortia. Other approaches include operating at
controlled redox levels, as well as the addition of catalysts such as
silver and by fine grinding of the concentrate.
Recently, new targets which are not sulfides materials have been
also identified for potential new applications of bioleaching pro-
cesses. Bioleaching is now considered as a viable technical option
for the treatment of all kind of industrial and urban wastes such as
dust, slags, sludges and “End of Life products” containing valuable
metals. Oxidized ores such as laterites have been also scrutinized as
potential targets for the development of a new process concept
called FERREDOX based on the reductive bioleaching. It has been
demonstrated that acidophilic sulfur-oxidizing bacterium can
promote a reductive dissolution of ferric iron minerals and be used
to accelerate the extraction of nickel, cobalt or copper from a
limonitic laterite ore. This metabolic potential is opening new in-
sights in the field of bioprocessing applied to mineral resources.
Another important new aspect for all applications related to
metal production from any kind of resources is the “resources ef-
ficiency” concept. There is a need to optimize the metal recovery
not only for the base metals (Cu, Ni, Zn) and precious metals (gold,
silver), but also for all the valuable associated ones (Co, Platinum-
Group Metals (PGM), …) for both economic and environmental
reasons. In this context it can be anticipated that bio-
hydrometallurgy, that can be applied on site and be a very flexible
technology, will also be a very promising and reasonable option.
Another area for additional research in the field of bio-
hydrometallurgy deals with a better comprehension of the micro-
bial composition in both bioheap systems and stirred tank reactors.
The dynamics of the heterogeneous and complex microbial popu-
lation microflora change with time and conditions in such systems.
There is a need to both define and understand the potential in-
teractions among the components of the microflora, but also the
link in between the biological aspects and the engineering ones. In
terms of microbiology, it is clear that the potential of using ther-
mophilic cultures (from 60
C up to more than 80
C) in industrial
applications remains a scientific and technical challenge that would
open opportunities to widen mineral targets amenable to bio-
leaching. For biohydrometallurgy to commercially advance with
new applications, the microbiologist must work cooperatively with
the practitioners of the technology for mutual understanding of
operational limitations and practical constraints affecting the
microbiological component.
This understanding is particularly important when considering
CSTR technologies. Oxidizing conditions, mass transfer, aeration
and stirring, temperature, acidity (pH), availability of nutrients,
solids loading, pulp density, particles size, oxygen and carbon
availability are deemed of prime importance for controlling and
optimizing biomining processes. It may be necessary for these
parameters to be continuously monitored and adjusted in CSTRs to
achieve the maximum microorganism activity and the optimum
 A. Mahmoud et al. / International Biodeterioration & Biodegradation 119 (2017) 118e146
biomining performance. However, the ability to predict accurately
the bioleaching efficiency for different sulfide minerals is still
limited. The biomining in CSTRs reactors remain a slow process and
with limited solids concentration of the feed. A better management
of the heat transfer is also still an important complementary en-
gineering challenge.
Besides the optimization of operating conditions, research and
innovation actions are still required in order to develop new types
of bioreactors that would fill the gap in between heap/dump
leaching (100% solids concentration) and CSTR (25% solids con-
centration). The objective is to find a type of reactor where the key
operating parameters can be controlled, but do not require the
same level of capital costs and operating costs as the CSTR. Working
with higher solids load, optimizing the gas and heat transfer would
be of great benefit for opening new opportunities to biomining
applications.
Finally, further fundamental studies are required to gather data
to allow the prediction of the biomining performance so that sys-
tems can be designed with greater confidence. Further experi-
mental developments are also needed to improve the productivity
of the biomining processes and to develop reliable design and
scale-up procedures. The future for biomining is exciting with
many benefits from this innovative technology still to be properly
assessed and with great potential to improve the design methods to
maximize further the process efficiency.
Acknowledgements
The authors gratefully acknowledge the financial and support
received for this research from the Agence Nationale de la
Recherche (ANR-13-RMNP-0006-04) under the collaborative
bilateral research project Ecometals between French and German
partners (BMBF under the Grant ID 033RF001). We would like to
thank all authors of the papers given in the reference list for
obtaining a great deal of useful information on biomining pro-
cesses, and thus in helping to prepare this paper. The principal
author (Dr. Akrama Mahmoud) would like to thank Jean Michel
Sorbet and Olivier Gelade for their kind technical assistance and
help. Dr. Akrama Mahmoud also would like to thank Anne-

nae€lle Guezennec for her careful reading of the paper and
Gwe
helpful comments. Finally, I dedicate this paper to my parents for
their love and sacrifices.
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