Hydrometallurgy Volume 142 Issue 2014 (Doi 10.1016/j.hydromet.2013.11.008) Kaksonen, Anna H. Mudunuru, Bhavani Madhu Hackl, Ralph - The Role of Microorganisms in Gold Processing and Recovery-A Review

Hydrometallurgy 142 (2014) 7083
Contents lists available at ScienceDirect
Hydrometallurgy
journal homepage: www.elsevier.com/locate/hydromet
The role of microorganisms in gold processing and recoveryA review

Anna H. Kaksonen a,, Bhavani Madhu Mudunuru a, Ralph Hackl b,1
a
b
CSIRO Minerals Down Under Flagship, Underwood Avenue, Floreat, WA 6014, Australia
CSIRO Minerals Down Under Flagship, Waterford, WA 6152, Australia
a r t i c l e
i n f o
Article history:
Received 9 June 2013
Received in revised form 19 October 2013
Accepted 13 November 2013
Available online 4 December 2013
Keywords:
Gold
Biooxidation
Bioprocess
Leaching
Permeability
a b s t r a c t
With a projected steady decline of gold ore grade in mineral resources, mining applications enabling efcient
metal extraction from low-grade ores are of increasing interest to the minerals industry. Microbial processes
may provide one such solution since they can participate in the biogeochemical cycling of gold in many direct
and indirect ways. This review examines current literature on the role of microorganisms in gold processing
and recovery. The review covers aspects such as the biotechnical pre-treatment of gold ores and concentrates,
microbially catalysed permeability enhancement of ore bodies, gold solubilisation through biooxidation and
complexation with biogenic lixiviants, and microbially mediated gold recovery and loss from leach liquors.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Gold (Au) ore grades in Australia show long-term declining trends
over time (Mudd, 2009; Fig. 1). As the quality of gold deposits continues
to decrease, it is expected that processes which can economically extract gold from low grade ores will grow in importance to the minerals
industry. Biotechnology has the potential to transform uneconomic gold
reserves into resources. Bioprocessing can be attractive for: 1) low grade
gold ores that are too expensive to process using conventional processes
and 2) ores that contain impurities that foul conventional processing
equipment (e.g. arsenic in gold ore). Microorganisms can mediate
gold solubilisation by oxidising the sulphide matrix of refractory gold
ores making the gold more accessible to leaching by chemical lixiviants.
Microorganisms can also excrete ligands which are capable of stabilising
gold by forming gold-rich complexes and/or colloids (Reith et al.,
2007a). The solubilisation of gold can be facilitated by biologically
produced amino acids, cyanide and thiosulphate (Reith et al., 2007a).
Moreover, microorganisms can participate in the redox cycling of
iodine (Amachi, 2008), which is a potential alternative lixiviant
for gold leaching. Microorganisms can also decrease gold solubility by
consuming the ligands that have bound gold, or by biosorption, enzymatic reduction and precipitation, and by using gold as a micronutrient
(Fig. 2) (Reith et al., 2007a). Additionally, microorganisms can inuence
gold solubilisation indirectly by enhancing the permeability of
Corresponding author: Tel.: +61 8 9333 6253.

E-mail address: anna.kaksonen@csiro.au (A.H. Kaksonen).
1
Present address: Rio Tinto Technology and Innovation, 1 Research Avenue, Bundoora
VIC 3083, Australia
0304-386X/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.hydromet.2013.11.008
ore bodies (Brehm et al., 2005; Burford et al., 2003; Ehrlich, 1998;
Jongmans et al., 1997; Kumar and Kumar, 1999). Understanding the
possible activities of microorganisms is important, especially when
considering leaching applications, where the control of operational
conditions may be challenging. This literature review aims to identify
microbial processes which may be relevant or hold potential for the
processing and recovery of gold.
2. Biotechnical pre-treatment of refractory gold ores

2.1. Biooxidation of refractory sulphide ores
2.1.1. Principles of biooxidation
Many gold deposits are sulphidic in nature and contain gold in a
form that is inaccessible to lixiviants. Refractory gold ores often contain
nely disseminated gold particles encapsulated by a sulphide mineral
matrix containing arsenopyrite, pyrite and pyrrhotite (Bosecker,
1997). The inaccessibility of gold to lixiviant has been overcome by
biooxidising the sulphides contained in the ore, thereby liberating
gold particles from the sulphide matrix and rendering the gold amenable to dissolution using lixiviants (for example cyanidation) (Bosecker,
1997).
The oxidation of the sulphide matrix is based on the activity of acidophilic chemolithotrophic iron and sulphur-oxidising microorganisms
which obtain energy by oxidising ferrous iron (Fe2 +) to ferric iron
(Fe3+) or elemental sulphur (S0) or other reduced sulphur compounds
to sulphuric acid (H2SO4) (Sand et al., 1995):
2
4Fe
O2 4H 4Fe
2H2 O
A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083
Gold grade (g Au t-1)
60
50
40
30
20
10
1875
1925
1900
1950
2000
1975
2025
Year
Fig. 1. Gold ore grades over time in Australia (data from Mudd, 2009). 2014 CSIRO. All
Rights Reserved.
2Fe1x SAu 41xH 1xO2 21xFe

21xH2 O 2Au
0
1850
2S
71
3O2 2H2 O 4H 2SO4
Fe3 + and H+ ions attack the valence bonds of sulphide minerals

leading to the breakdown of sulphide matrix as shown below for pyrite
(FeS2) and pyrrhotite (Fe1 xS) as examples (Belzile et al., 2004; Morin,
1995; Nagpal et al., 1994).
3
FeS2 Au 2Fe
FeS2 Au 14Fe
3Fe
2S Au
2
8H2 O 15Fe
Fe1x SAu 22xFe
Fe1x SAu 82xFe
2SO4
33xFe
16H
Au
S Au
4H2 O 93xFe
SO4
5
2
8H
Au
2.1.2. Engineering applications

During the past 20 years bio-treatment of refractory gold ores has
been developed as an industrial application and applied commercially
in bioreactors and heaps. The development of the biooxidation technology has been well reviewed elsewhere (see e.g. Brierley, 2008;
Harvey and Bath, 2007; Ndlovu, 2008; Rawlings et al., 2003; van
Aswegen and Marais, 2001van Aswegen and van Niekerk, 2004; van
Aswegen et al., 2007) and hence will be only briey mentioned here.
The rst industrial scale plant was started at the Fairview Mine, South
Africa, in 1986 (Bosecker, 1997; Morin, 1995) (Figs. 34). Since then,
biooxidation operations have been commissioned in a number of
countries, such as Australia, Brazil, Ghana, Peru, China, Uganda, USA,
Kazakhstan, Uzbekistan and Russia (Table 1).
Oxidation of Fe2+ and reduced sulphur compounds
Pre-treatment
of ores and
concentrates
Carbon-adsorbable blanking agent

Bioreduction of Fe3+
Production of acids, bases or ligands
Physical forces
Oxidised ore or concentrate

Thiosulphate production
Gold
solubilisation
Biooxidation
and complexation
of gold
Organic acid production

Cyanide production
Iodide production
Gold complexes in solution

Biosorption
Recovery /
loss of gold
Reduction and
precipitation of gold
Microorganisms used in biooxidation processes include mesophilic

bacteria, such as iron- and sulphur-oxidising Acidithiobacillus
(At.) ferrooxidans, sulphur-oxidising At. thiooxidans, iron-oxidising
Leptospirillum (L.) ferrooxidans and L. ferriphilum, moderately thermophilic bacteria, such as iron- and sulphur-oxidising Sulfobacillus spp.
and sulphur-oxidising At. caldus and a variety of archaea including
mesophilic iron-oxidising Ferroplasma acidiphilum, moderately thermophilic iron-oxidising Acidiplasma cupricumulans, and thermophilic
Acidianus spp., Metallosphaera spp. and Thermoplasma-like species
(Bosecker, 1997; Brierley and Brierley, 2001; Golyshina et al., 2009;
Hawkes et al., 2006; Olson et al., 2003; Reith et al., 2007b; Schippers,
2007; van Hille et al., 2013).
In general biooxidation of the gold-containing sulphide ores is a pretreatment which can decrease the consumption of lixiviant for gold
solubilisation in subsequent parts of the operation and ultimately
increase gold yields. However, since it does not actually solubilise gold
biooxidation needs to be used in conjunction with other methods.
Primary gold ore or concentrate
Biooxidation
of sulphide minerals,
deactivation of
carboneous material
and permeability
enhancement
2S
Enzymatic reduction
Micronutrition
Ligand utilisation and loss
Concentrated gold
Fig. 2. Potential roles of microorganisms in gold processing and recovery (adapted from Reith et al., 2007a). 2014 CSIRO. All Rights Reserved.
72
Fig. 3. BIOX process for biooxidation of refractory gold ore concentrate at the Fairview Gold mine, South Africa. 2014 CSIRO. All Rights Reserved.
Biooxidation treatment in tank reactors is typically practised for high

value otation concentrates (Brierley and Brierley, 2001). A number of
commercial bioreactor processes have been developed, such as BIOX,
BacTech, BACOX and BIONORD (Table 1) (Adamov et al., 2011;
Brierley, 2008; Brierley and Brierley, 2001). A typical BIOX tank
biooxidation plant consists of six bioreactors congured as three primary reactors operating in parallel followed by three secondary reactors
operating in series (van Aswegen et al., 2007). This design increases
the efciency of sulphide oxidation by reducing the short-circuiting
of sulphide particles. Typically the bioreactors operate with 1520%
slurry density (Olson et al., 2003). Pulp residence time in the bioreactors
is typically 46 days depending on the oxidation rate achieved,
sulphide-S content and mineralogical composition of the concentrate
(van Aswegen et al., 2007). Nutrients in the form of nitrogen, phosphorus and potassium salts are added to the primary reactors to promote
microbial growth (van Aswegen et al., 2007). The bioreactors are aerated
to maintain a dissolved oxygen concentration of N2 mg L1. As the oxidation of sulphide minerals is an exothermic process, the reactors are
cooled continuously. A minimum carbonate content of 2% in the otation
concentrate is usually required to ensure that sufcient carbon dioxide
is available to promote microbial growth. If no carbonate is present,

limestone or carbon dioxide enriched air can be added to the primary
reactors as a carbon source for microorganisms (Astudillo and Acevedo,
2009; van Aswegen et al., 2007). Moreover, the addition of organic
carbon sources has been proposed to promote the growth of mixotrophic
microorganisms (Muravyov and Bulaev, 2013). The oxidation of pyrite
produces acid, while the dissolution of carbonate minerals consumes
acid. The pH of the bioreactors is controlled with limestone and sulphuric
acid within the optimum range of 1.21.8 (van Aswegen et al., 2007).
Before cyanide leaching the oxidised concentrate is typically washed
in three-stage counter current decantation (CCD) circuit to remove
dissolved iron in order to promote gold recovery and reduce cyanide
consumption (van Aswegen et al., 2007). In some bioreactor operations
biooxidised concentrate is directly aerated and neutralised to precipitate
iron prior to cyanidation instead of solidliquid separation and washing
in counter-ow decantation thickeners (Adamov et al., 2011). Other
variations include a cyanide process before biooxidation, the use of
various temperatures and microbial communities in consecutive stages,
the use of high temperature (80 C) ferric leaching before biooxidation,
and the concentration of biomass from solutions and recycling it back
Nutrients
Primary
bioreactors
Thickened gold
concentrate
Secondary
reactors
Tertiary
reactors
Mixer
Air
Air
Air
Counter-flow
decantation
thickeners
Process
water
To tailings dam
Wash
water
Lime
neutralisation
Biooxidised
concentrate to
cyanide leaching
Fig. 4. A simplied ow sheet of the BIOX process for biooxidation of refractory gold ore concentrate at the Fairview gold mine, South Africa. 2014 CSIRO. All Rights Reserved.
73
Table 1
Examples of commercial biooxidation plants for gold recovery.
Mine
Country
Process
Design capacity (ore t d1)
Years in operation
References
Fairview
So Bento
Harbour Lights
Wiluna
Ashanti-Shansu
Youanmi
Tamboraque
Beaconseld
Laizhou
Olympiada
Suzdal
Fosterville
Bogoso
Jinfeng
Kokpatas
Au quarry mine
Agnes
South Africa
Brazil
Australia
Australia
Ghana
Australia
Peru
Australia
China
Russia
Kazakhsan
Australia
Ghana
China
Uzbekistan
USA
South Africa
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BacTech)
Reactor (BIOX)
Reactor (BACOX)
Reactor (BACOX)
Reactor (BIONORD)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Reactor (BIOX)
Heap (whole ore)
Heap (GEOCOAT)
55
380
40
158
960
120
60
68
100
1000
196
211
750
790
1069
10,400
50
1986present
1991present
19921994
1993present
1994present
19941998
19982003 (restarted in 2006)
2000present
2001present
2001present
2005present
2005present
2006present
2006present
2008present
2000present
20032006, 2009present
1, 2, 4, 11
1, 2, 4, 5
1, 2, 4
1, 2, 4
1, 2, 3, 4
1, 2, 4
1, 4, 11
2, 4, 12
2, 4, 12
13
11
11
11
11
11
2, 6, 7
8, 9, 10
References: 1) Brierley and Brierley, 2001, 2) Olson et al., 2003, 3) Rawlings, 2002, 4) Rawlings et al., 2003, 5) do Carmo et al., 2001, 6) Bhakta and Arthur, 2002, 7) Brierley, 2000, 8) Ndlovu,
2008, 9) Harvey and Bath, 2007, 10) GeoBiotics, 2010, 11) Brierley, 2008, 12) Gericke et al., 2009, 13) Sovmen et al., 2009.
to the bioreactors (Adamov et al., 2011; Fomchenko et al., 2010;

Muravyov and Bulaev, 2013; Zaulochnyi et al., 2011). The use of various
by-products and/or waste materials such as mesalime, electric arc
furnace dust and otation tailings for biopulp neutralisation have also
been proposed (Adamov et al., 2011; Gahan et al., 2010). Moreover,
biooxidation has been used to increase the recovery of gold from
otation tailings. In the study by Kondrat'eva et al. (2012) biooxidation
of otation tailings resulted in additional recovery of 2627% gold and
a decreased cyanide consumption, compared to direct cyanide leaching.
Heap biooxidation treatment is generally considered when the ore is
low-grade, economics cannot sustain the cost of making a concentrate,
the mineralogy is such that the refractory sulphides cannot be concentrated, or the project is too small to support a high capital process
(Brierley and Brierley, 2001). Heap biooxidation of refractory gold concentrates has been conducted at the Agnes Gold Mine in South Africa
since 2003 by the GEOCOAT process developed by GeoBiotics LLC
(Ndlovu, 2008). In the process, sulphide concentrate slurry is coated
onto a crushed and sized carrier rock concentrate (Figs. 5 and 6). The
coated material is stacked on an impervious pad for biooxidation
(Ndlovu, 2008). After biooxidation, the heap is washed, the concentrate
is separated from the carrier, washed and then the gold is dissolved
by cyanidation.
Biooxidation pretreatment of lower-value, refractory gold-bearing
whole ores can be conducted in heaps, similar to those used for secondary copper ores (Brierley, 2000). Newmont Mining showed the practicality of heap biooxidation treatment of whole ores with a demonstrationscale heap at a 3.8-million t year1 facility at Gold Quarry Mine (Bhakta
and Arthur, 2002; Brierley, 2000). The ore is crushed to approximately
12.7 mm size (Brierley, 2000) and stacked on pads with an airventilation system at the base to supply oxygen and carbon dioxide to
the microorganisms inoculated on to the ore (Olson et al., 2003). The oxidation rate for the ore is typically between 0.30% and 0.34% sulphide
sulphur oxidation/day. Therefore, an oxidation cycle of 100150 days
could result in 3050% sulphidesulphur oxidation. Column test work
on sulphide oxidation versus gold recovery indicated a diminishing
return after 60% sulphide oxidation (Bhakta and Arthur, 2002). After
100270 days of biooxidation, the ore is removed from the pretreatment
pads and cyanide leached in an oxide mill facility (Brierley, 2000; Olson
et al., 2003). Gold recovery ranges from 60% to 70% of the contained
value with an ore grade range of 1.7 to 4.1 g t1 (Brierley, 2000). Gold
can be recovered from cyanide solutions using adsorption of gold onto
activated carbon, which is then chemically stripped of gold. In a nal
step, gold is precipitated electrolytically or by chemical substitution
(Reith et al., 2007b). More recently, non-cyanide lixiviants have been
evaluated as alternatives for leaching gold from biooxidised ores. A
column study simulating heap leaching indicated that thiosulphate
leaching could result in similar recoveries as cyanide leaching from
biooxidised refractory gold ores (Gudkov et al., 2011).
Although not yet demonstrated in large-scale, in situ and in place
leaching methods could be attractive alternatives for low grade gold
ores that are too expensive to process using conventional open-pit or
underground mining and processing methods. With in situ leaching
the leach solution is injected into the subsurface ore body to extract
target metals. The pregnant (metal-bearing) leach solution is collected
from production wells for metal recovery (Bosecker, 1997). In situ
leaching does not usually require extensive mine infrastructure and
Fig. 5. GEOCOAT process for biooxidation of refractory gold concentrate at the Agnes gold mine, South Africa. 2014 CSIRO. All Rights Reserved.
74
Gold concentrate
Microorganisms
H2SO4
Coating of support
rock and stacking
Nutrients
Make-up
Water
Heap
biooxidation
Pond
Air
Water to
treatment
Support
rock
returned to
coating
Rinsing of heap
Washing of support
rock
Oxidised concentrate
to gold leaching
Fig. 6. A simplied ow sheet of the GEOCOAT process at the Agnes gold mine, South Africa. 2014 CSIRO. All Rights Reserved.
may reduce the visual impact of the mining operation. However, in situ
leaching does require relatively long contact times between ore
minerals and uid and well developed reservoir permeability. According
to Steven (2009) the porous medium should have a hydraulic conductivity of greater than 0.043 m d1 for successful in situ leach operation.
Extensive knowledge of the hydrology and geology of the area and careful control of leaching solutions is required to prevent contamination of
nearby groundwater (Kinnunen, 2004; Nurmi, 2009). In place leaching
is similar to in situ leaching but the ore body is fractured, for example
by blasting, to improve the permeability before leaching (Wadden
and Gallant, 1985). With a laboratory scale study, Kaksonen et al.
(2014) showed that submerged oxidation of pyrite is possible using ferric iron that is biologically generated either externally or using underground aeration in the ore body. The simulated underground aeration
and the presence of bioleaching microorganisms clearly enhanced the
oxidation of pyrite. Moreover, microorganisms oxidised sulphur intermediates and thereby decreased the accumulation of elemental sulphur.
The removal of pyrite and elemental sulphur is expected to enhance subsequent gold leaching with chemical lixiviants and decrease the lixiviant
consumption (Kaksonen et al., 2014).
2.2. Microbial pre-treatment of refractory carbonaceous ores
Some gold ores have a carbon content that inhibits gold recovery
using leaching or lixiviant processes and thus renders them refractory
(Brierley and Kulpa, 1993). These include refractory carbonaceous
and/or carbonaceous-sulphidic ores. The refractory carbon content is a
signicant source of preg-robbing, which refers to its ability to remove
or rob gold that has been leached out of the ore and held in pregnant
lixiviant solution. It is believed that the carbonaceous component that
participates in the preg-robbing comprises an activated carbon-type
material, long-chain hydrocarbons and organic acids, such as humic
acid. Microbial pre-treatment processes have been developed to deactivate the carbonaceous component of these ores to prevent binding
of the dissolved gold onto this component. Brierly and Kulpa (1993)
patented a treatment process where the ore is inoculated with a
microbial consortium of bacteria that comprises at least two species selected from the following: Pseudomonas (P.) maltophila, P. oryzihabitans,
P. putida, P. uorescens, P. stutzeri, Achromobacter spp., Arthrobacter spp.,
and Rhodococcus spp. The carbon-adsorbable blanking agent produced
by the bacterial consortium is used together with a relatively high
concentration (25250 kg per ton of ore) of the chelating agent, ethylene diamine tetraacetic acid (EDTA). The microbial deactivation of
carboneous material can be conducted before, after or contemporaneously with biooxidation of sulphidic minerals. Yen et al. (2009) patented
an alternative process that is based on the use of fungal agents and/or
culture media. Although any suitable fungi may be employed, the
preferred heterotrophic agents listed in the patent are white rot fungi,
such as Trametes spp., Phanerochaete spp., Phlebia spp., Cyathus spp.,
and Tyromyces spp. It is belived that some carbonaceous materials are
converted into carbon dioxide by some fungi while other fungi passivate
the preg-robbing capacity of carbonaceous materials (Yen et al., 2009).
2.3. Permeability enhancement
One of the critical factors for the success of low-grade gold ore in situ
and in place leaching is the permeability of the ore body. Microorganisms can contribute to the breakdown of rock forming minerals by
both biochemical mechanisms and physical (mechanical) forces such
as through the action of microscopic fungi that spread within cracks
and even through entire mineral bodies (Brehm et al., 2005; Jongmans
et al., 1997). Some microorganisms promote rock weathering by
mobilising mineral constituents with the inorganic and organic acids
or ligands that they excrete. Others promote rock weathering by
redox attack of mineral constituents such as Fe and Mn (Ehrlich,
1998). Biochemical breakdown of rock forming minerals can result in
microtopographic change of mineral surfaces producing: pitting and
etching of their surfaces, mineral displacement reactions, widening of
pores and mineral interphases, and even complete dissolution of mineral
grains (Brehm et al., 2005; Burford et al., 2003; Ehrlich, 1998; Kumar and
Kumar, 1999). Biological solutions have also been reported to permeate
microfractures and affect the wettability of ores and solution contact
with leachable gold due to surfactant properties (Thompson and

MacCulloch, 2004).
2.3.1. Removal of passivating S0- and Fe3+-layers
The oxidation of sulphide minerals is thought to be hindered by the
formation of passivating layers of elemental sulphur, polysulphides and
jarosite (Stott et al., 2000). Bioleaching can be enhanced by using
microorganisms capable of removing passivating layers from mineral
surfaces. By doing this microorganisms may also be able to enhance
the permeability of ore deposits. For example some acidophilic microorganisms, such as At. ferrooxidans are capable of oxidising sulphur under
anaerobic conditions using ferric iron as the terminal electron acceptor
(Pronk et al., 1992):
0
6Fe
4H2 O SO4
6Fe
8H
The generation of acid in this process decreases pH, which in turn

decreases the precipitation of ferric iron as passivating iron hydroxy
compounds (Nurmi, 2009). In addition to At. ferrooxidans, several
other acidophilic microorganisms can also reduce ferric iron, including
At. ferrivorans, Acidiferrobacter thiooxydans, Ferrimicrobium acidiphilum,
Acidimicrobium ferrooxidans, Ferrithrix thermotolerans, several Acidiphilum
spp., Acidocella spp., Acidobacterium spp., Alicyclobacillus spp., Sulfobacillus
spp., Acidiplasma spp. and Ferroplasma spp. (Johnson et al., 2012). Ferric
iron reducing microorganisms can catalyse reductive dissolution of iron
hydroxy compounds which contain ferric iron, such as jarosite, goethite,
or schwertmannite (Johnson and Hallberg, 2009). Reaction 9 shows
the reductive dissolution of schwertmannite with glucose as an electron
donor (Coupland and Johnson, 2008):
2
3Fe8 O8 OH6 SO4 C6 H12 O6 6H2 O 24Fe
2
3SO4
42OH
6CO2
9
Reductive dissolution of iron hydroxy compounds would require the

addition of a suitable electron donor for the microorganisms. Although
the addition of organic substrates into the subsurface may enhance
the reductive dissolution of iron hydroxy compounds, the organic
substrate may also be utilised by microorganisms, such as sulphate
reducers, which can decrease the solubility of gold.
2.3.2. Dissolution of silicate minerals
Some microorganisms, such as bacteria and fungi are able to accelerate dissolution of silicates and aluminosilicates. According to Ehrlich
(1996) their action on these minerals has been characterised as nonenzymatic. The dissolution mechanisms may involve production of
metabolic products such as organic acids that act as acidulants and/or ligands, alkalinity in the form of NH3, and capsular slime (acid polysaccharide) from bacteria (Ehrlich, 1996). Among the acids, 2ketogluconic acid formed by some bacteria, and citric and oxalic acids
formed by some fungi, have been reported to be effective in the dissolution of silicates (Duff et al., 1963; Ehrlich, 1996; Vandevivere et al.,
1994). The organic acids furnish protons that help in breaking SiO
and AlO bonds through protonation. Some of the acids may also act
as ligands that pull cations from the framework of the crystal lattice, facilitating subsequent breakage of framework bonds. Some bacterial
slimes (acid polysaccharide) have been reported to form complexes
with silicate leading to silicate dissolution (e.g. Ehrlich, 1996; Liu et al.,
2006; Malinovskaya et al., 1990).
Quartz comprises 20% of the volume of the exposed Earth's crust and
is one of the most resistant of rock forming minerals (Brehm et al., 2005;
White and Brantley, 1995). Its rate of dissolution is slow, approximately
1017 mol cm1 s1 at 40 C at near neutral pH in pure water, because
the activity required to break SiO bonds is high (Brehm et al., 2005).
As a component of rocks (e.g. granite, gneiss, sandstone), quartz crystals
and grains have a higher resistance to weathering processes than many
75
other common minerals such as feldspar and mica (Brehm et al., 2005).
At pH values lower than 3.5, quartz dissolution is minimal, although mechanical processes of grain diminution may still continue (Brehm et al.,
2005). Quartz solubility increases signicantly in alkaline conditions of
pH 9 and above (Brehm et al., 2005). A number of biological processes
can generate alkalinity or consume acidity and therefore have potential
for increasing pH and thus quartz dissolution. These include: photosynthesis (Brehm et al., 2005; Johnson, 2000; Robb and Robinson, 1995;
van Hille et al., 1999), denitrication (Johnson, 1995; Kalin et al.,
1991), hydrolysis of urea (Fujita et al., 2000), ammonication,
methanogenesis, and reduction of iron and sulphate (Johnson, 1995,
2000; Kalin et al., 1991; White et al., 1997) (Table 2).
Brehm et al. (2005) reported that natural biolms composed of
diatoms, heterotrophic bacteria and cyanobacteria can actively attack
quartz and glass. Microscopic analysis of the quartz crystal from a
tepui, a type of quartzitic tabular mountain found in the Guiana Highlands of South America, revealed that the associated biolms can create
a local shift in the pH from 3.4 (pH of the water on the tepui), to N 9
(necessary for quartz dissolution) (Brehm et al., 2005). The quartz
covered with biolm was partially perforated to a depth of more than
4 mm (Brehm et al., 2005). However, Brehm et al. (2005) estimated
that the resident microbial community had been affecting the mineral
surface considerably longer than 10 years.
A consortium of diatoms (eukaryotic algae) and heterotrophic
bacteria created depressions or pitted zones to window glass in a
9 months study (Brehm et al., 2005). The diatoms and their accompanying bacteria were embedded in large amounts of extracellular polysaccharides covering the surface of the glass. Brehm et al. (2005)
suggested that diatoms produce polysaccharides useful for bacterial
metabolism and survival, whereas bacteria with their leaching activity
provide silicon ions for diatom frustule construction. The use of photosynthetic diatoms and cyanobacteria would not be applicable in dark
subsurface in situ leaching environments due to the lack of sunlight.
Quartz often contains iron as an impurity (tyriakov et al., 2003).
tyriakov et al. (2003) studied the biodestruction and deferrisation of
quartz sands with Bacillus spp. The bioleaching experiments showed
that Bacillus spp. can solubilise iron, silica, and aluminium from quartz
sands and reduce the iron oxyhydroxides present as impurities.
3. Gold solubilisation through biooxidation and complexation
A number of chemical and biologically produced lixiviants have
been assessed for their ability to oxidise and complex gold as alternatives to chemical cyanide solubilisation.
3.1. Thiosulphate
One of the most potential alternatives to cyanide systems is the
leaching of gold with thiosulphate in the presence of co-ligands, such
as ammonia and oxidants such as Cu2 + (Aylmore and Muir, 2001;
Reith et al., 2007b; Wan and LeVier, 2003). The leaching reaction is
described as follows (Reith et al., 2007b):
Au 5S2 O3
CuS2 O3 3
CuNH3 4
4NH3
h
i
3
AuS2 O3 2
10
Au(I)thiosulphate complex is stable in mildly acidic to highly alkaline pH

(510), and moderately oxidising to reducing conditions (Eh 0.17
0.76 V) (Reith, 2003).
Thiosulphate heap leaching has already been used in the Carlin
Nevada operation of Newmont Mining. A combination of biooxidation
and chemical thiosulphate heap leaching is applied for carbonaceous/
high sulphide ores and a direct thiosulphate heap leaching process for
carbonaceous/low sulphide ores (Wan and LeVier, 2003). Until 2003 a
total of 1.24 million tonnes of low-grade refractory gold ores were
76
Table 2
Alkalinity producing microbially catalysed bioprocesses.
Process
Reaction
Comments
Reference(s)
Photosynthesis
Ammonication
Urea hydrolysis
Methanogenesis
Sulphate reduction
H2O + CO2 + light O2 + CH2O

Organic-N NH3
2
CO(NH2)2 + 2H2O 2NH+
4 + CO3
CH3COO + H2O CH4 + HCO
3
SO2
+ 2CH2O H2S + 2HCO
4
3
1, 2, 3, 4
3, 5, 6, 7
8
3, 5, 6, 7
3, 5, 6, 7
Denitrication
6NO
3 + 5CH3OH 5CO2 + 3N2 + 7H2O + 6OH
Ferric iron reduction with

organic electron donors
4Fe(OH)3 + CH2O 4Fe2+ + H2CO3 + 2H2O + 8OH
Requires sunlight would not work underground

Requires organic N
Requires aerobic conditions and an organic substrate
Requires anaerobic conditions
Requires anaerobic conditions, sulphate,
and an electron donor
Requires anaerobic conditions, nitrate or nitrite,
Requires anaerobic conditions, Fe3+ compounds,
5, 6
3, 5, 6, 7
References: 1) Robb and Robinson, 1995; 2) van Hille et al., 1999; 3) Johnson, 2000; 4) Brehm et al., 2005; 5) Kalin et al., 1991; 6) Johnson, 1995; 7) White et al., 1997; 8) Fujita et al., 2000.
successfully processed with ammonium thiosulphate at the Nevada

operation (Wan and LeVier, 2003).
Thiosulphate and ammonium are produced and excreted by bacteria
and actinomycetes during a number of metabolic reactions (Reith et al.,
2007b). Ammonium is commonly produced through the hydrolysis of
urea by a wide range of yeasts and bacteria, including many alkaliphilic
Bacillus spp. (Reith et al., 2007b; Schmidt Mumm and Reith, 2007).
Sulphate-reducing bacteria (SRB), which are common in anoxic
sulphate-containing soils, form thiosulphate under certain
environmental conditions, such as during reduction of sulphite with
H2 and formate (Fitz and Cypionka, 1990; Reith et al., 2007a, 2007b).
A common soil actinomycete, Streptomycetes fradiae, produces
thiosulphate when metabolising sulphur from cystine (Kunert and
Stransky, 1988; Reith et al., 2007a). Thiosulphate has also been
suggested to be the main intermediate product of the biooxidation
of acid-insoluble sulphides, such as pyrite (FeS2) and molybdenite
(MoS2) and is oxidised further to sulphate (Schippers and Sand, 1999):
3
FeS2 6Fe
S2 O3
3H2 O S2 O3
3
8Fe
5H2 O2SO4
7Fe
8Fe
6H
11
10H
12
Gold solubilisation via the thiosulphate mechanism is expected in

organic carbon matter-poor environments, for example in primary sulphide bearing deposits (Reith et al., 2007a). Reith and McPhail (2006)
studied the solubilisation of sub-microscopic gold in carbon-limited
quartz vein materials with arsenopyrite and pyrite from Tomakin Park
Gold Mine in New South Wales, Australia. Reith et al. (2007a) proposed
that the gold solubilisation was mediated by microbially produced
thiosulphate. A maximum of 550 ng gold per gram (dry weight quartz
vein material, particle size b200 m) was solubilised in a biologically
active agitated slurry after 35 days of incubation and the concentration decreased thereafter. In contrast, a sterile control system
showed a ten times lower concentration of solubilised gold (Reith
and McPhail, 2006; Reith et al., 2007a). The gold grade of the quartz
vein material was not reported, and hence % gold extraction cannot be
calculated.
3.2. Organic acids
Organic acids (e.g. humic and fulvic acids, amino acids and carboxylic acids) have been shown to promote the solubilisation of native gold in
some experiments, whereas in other studies under different conditions
native gold was not oxidised and the formation of gold colloids was promoted (Baker, 1978; Fetzer, 1934, 1946; Reith et al., 2007a; Wood,
1996). A number of studies have indicated that amino acids produced
by heterotrophic microorganisms, such as Bacillus (B.) subtilis, B. alvei,
B. megaterium, B. mesentericus, Serratia marcescens, P. uorescens and
P. liquefaciens, can enhance gold solubilisation by forming gold-amino
acid complexes (Korobushkina et al., 1974; Reith et al., 2007a). According to Korobushkina et al. (1974), aspartic acid, histidine, serine, alanine
and glycine played a substantial part in gold dissolution by cultures isolated from gold-bearing deposits. Amino acid production by the strains
was increased by mutagenic factors (ultraviolet rays and ethylenimine)
and the solubility of gold increased in the presence of an oxidising agent
(2 g L1 sodium peroxide) under alkaline conditions (pH 910). Dissolution of gold by puried amino acid fractions yielded solutions with up
to 1415 mg L1 of gold in 20 days. The maximum concentration was
35 mg L 1 with no more than 0.20.3 mg L1 in the control experiment. The stability of gold-amino acid complexes varies with their
redox potentials. The complex forming capacity of amino acids may be
ranked according to the redox potentials as follows: cysteine N histidine N asparagines N methionine N glycine, alanine, valine, phenylalanine (Korobushkina et al., 1983). Jingrong et al. (1992)
suggested that the nitrogen atom in the amino group (NH2) shows a
strong tendency of complexing gold, and the oxygen atom in the carboxyl group (COO) also contributes to complexation. Jingron et al.
(1992) also reported that the solubilisation of gold by amino acids depends on pH and temperature. Salt- and alkaline-soluble proteins and
also water- and alcohol-soluble proteins were less effective in dissolving gold with soluble concentrations reaching 2.23.3 mg L1
and 0.150.57 mg L 1, respectively in 20 days at pH 910
(Korobushkina et al., 1983).
In the Tomakin Park Gold Mine study with gold-containing soils rich
in organic matter, biologically active samples displayed up to 80 wt.%
gold solubilisation (original gold concentration 1453 ng g1 d.w. soil)
within 45 days of incubation under aerobic conditions, after which
gold was re-adsorbed to the solid soil fractions (Reith and McPhail,
2006). In the early stages of incubation the microbial community apparently produced an excess of amino acids (up to 64.2 M of free amino
acids measured within the rst 20 days of incubation), which formed
complexes with gold. However, in the later stages of the incubation
the microbial community metabolised these gold-complexing ligands
(free amino acid concentration decreased to around 8 M by day
50), and gold, which apparently became unstable in the solution,
was re-adsorbed to the solid soil fractions. During the experiment
the bacterial community structure changed from a carbohydrateand polymer-utilising community to a carboxylic- and amino-acid
utilising community concurrently with the change from gold
solubilisation to re-adsorption. The microbiota of gold-containing
soils was also capable of dissolving gold from added gold pellets,
resulting in concentrations higher than the original soil sample and
the presence of biolms on the surfaces of the gold pellets. In contrast, the microbiota from soils 100 m from the mineralisation,
which displayed only background gold values, did not mobilise
gold nor form biolms on added gold pellets indicating that the microbiota were different or acted differently in gold-containing soils
(Reith and McPhail, 2006).
The interaction of gold and organic matter involves mostly electron
donor elements, such as nitrogen, oxygen and sulphur, rather than
carbon (Reith et al., 2007a). Vlassopoulos et al. (1990) showed that
gold binds preferentially to organic sulphur under reducing conditions,
and that complexation with organic nitrogen and carbon is more
important in oxidising environments. Gold solubilisation via complexation by organic acids may occur in organic matter-rich top and rhizosphere soils, where plant exudates may directly lead to gold
solubilisation or provide nutrients for organic acid-excreting microorganisms (Reith et al., 2007a).
Some amino acids are also precursors for the microbial production of
other gold-complexing ligands, e.g. cystine is a precursor for thiosulphate
(Kunert and Stransky, 1988; Reith et al., 2007b) and glycine is a precursor
for cyanide (Fairbrother et al., 2009; Reith et al., 2007a, 2007b; Rodgers
and Knowles, 1978). Moreover, amino acids have been shown to
complex Cu2+ during thiosulphate leaching of gold-bearing pyrite. This
decreased thiosulphate consumption due to reduced interaction between thiosulphate and the copper complexes (Feng and van Deventer,
2011). As with biogenic thiosulphate production, the transient stability
of the amino acids may indicate that economic exploitation of amino
acids for gold solubilisation may be challenging.
Current industrial amino acid production is a multi-billion dollar
business, with annual production estimated to be millions of tons.
Amino acids are used in a number of applications such as food additives,
pharmaceuticals, cosmetics, polymer materials, biofuels and antibiotics
(Park and Lee, 2008). The development of efcient amino acid producing strains has traditionally involved multiple rounds of random mutation and selection. More recently approaches for strain development
have shifted to targeted engineering strategies which purposefully
modify genes and pathways towards enhanced production of desired
amino-acids (Park and Lee, 2008).
3.3. Biogenic cyanide

Many soil bacteria (such as P. uorescens, P. aeruginosa, P. putida,
P. syringae and B. megaterium), fungi and plants can produce and excrete
cyanide (Reith et al., 2007a). Cyanide has no apparent function in
primary metabolism, is optimally produced during growth limitation
and may offer the producer, which is usually cyanide tolerant, a
selective advantage by inducing cyanide toxicity in other organisms
(Bakker and Schippers, 1987; Castric, 1975; Reith et al., 2007a). At
neutral pH, cyanide mainly occurs as volatile HCN because of its pKa
value of 9.3. However, in the presence of salts and metal ions, volatility
is reduced, and thus biologically produced cyanide may directly affect
gold solubilisation (Faramarzi and Brandl, 2006; Reith et al., 2007a).
With cyanide Au+ forms a strong dicyanoaurate complex which is stable over a wide range of redox and pH conditions (Reith et al., 2007a):
4Au 8CN O2 2H2 O4AuCN2 4OH
13
An in vitro study using the cyanogenic bacterium Chromobacterium

(Chr.) violaceum showed that biolms grown on gold-covered glass
slides were able to solubilise 100% of the gold within 17 days, with concentrations of gold and free cyanide in solution reaching 35 and
14.4 mg L 1, respectively (Campbell et al., 2001). When incubating
Chr. violaceum with biooxidised gold concentrate up to 0.34 mg L 1
gold and 9 mg L 1 CN was detected in solution within 10 days
(Campbell et al., 2001). In a more recent study Fairbrother et al.
(2009) examined the effect of cyanide production by Chr. violaceum
on ultra-at gold foil by incubating the bacteria and foil in peptone
meat extract for up to 56 days. Total concentrations of solubilised gold
increased throughout the experiment and after 56 days 74.3 g L1 of
gold was detected in solution and 51.3 g L1 reversibly or irreversibly
bound with cells. Fairbrother et al. (2009) noted that the lower concentrations when compared to those observed by Campbell et al. (2001)
may have been due to the low surface roughness of the ultra at gold
foil used in the more recent study. Shin et al. (2013) suggested ore
grinding and pre-growing Chr. violaceum for increasing gold recovery.
A patent has already been granted for the biohydrometallurgical
processing of gold-containing ores using cyanide producing
77
microorganisms, such as Chr. violaceum and Chlorella (Chl.) vulgaris

(Kleid et al., 1995; Krebs et al., 1997).
Cyanide producing microorganisms have also been applied to leach
gold from metal-containing waste materials (Brandl et al., 2008;
Faramarzi et al., 2004). P. plecoglossicida solubilised gold from shredded
printed circuit boards producing up to 500 mg L1 [Au(CN)
2 ] in 80 h,
corresponding to gold concentration of 442 mg L1 and a 69% dissolution of the gold added (Faramarzi and Brandl, 2006; Reith et al., 2007a).
Faramarzi et al. (2004) and Brandl et al. (2008) demonstrated gold
solubilisation from shredded printed circuit boards with Chr. violaceum
and measured dicyanoaurate concentrations corresponding to 14.9%
and 68.5% gold dissolution, respectively in 7 days, after a 3-day lag
phase. A two-step bioleaching process has been proposed to overcome
the toxic effects of electronic waste on bioleaching cultures (Brandl
et al., 2001; Mishra and Rhee, 2010; Pradhan and Kumar, 2012).
Pradhan and Kumar (2012) applied a two-step bioleaching process
to rst generate cyanide forming biomass in the absence of
electronic waste followed by the addition of electronic waste for
metal solubilisation. Chr. violaceum was capable of leaching 69% of
gold and mixture of Chr. violaceum and P. aeruginosa exhibited 73%
gold leaching at an electronic waste concentration of 1% w/v (Pradhan
and Kumar, 2012). When P. uorescence was applied for electronic
waste leaching, dicyanoaurate did not remain stable in solution with
prolonged incubation times, probably due to sorption onto biomass or
biodegradation because cyanides can serve as carbon or nitrogen source
(Brandl et al., 2008). Kita et al. (2006) showed that increased dissolved
oxygen concentration enhanced gold solubilisation by Chr. violaceum
from electronic waste whereas Chi et al. (2011) showed that increasing
pH from 8 to 11 increased gold leaching by the same species. Pham and
Ting (2009) reported that biooxidation pretreatment of electronic
waste with At. ferrooxidans removed most of the copper present in the
waste and signicantly increased the gold recovery in subsequent
leaching with Chr. violaceum.
Amino acids, such as glycine, can function as metabolic precursors
for microbial cyanide production (Fairbrother et al., 2009; Reith et al.,
2007a; Rodgers and Knowles, 1978). The highest concentration of free
glycine in solution in the Tomakin Park Gold Mine soil study was detected
after 20 days of incubation (Reith and McPhail, 2006), and a cyanide
concentration up to 0.36 mg L 1 (soil solution) was measured in
another Tomakin soil study suggesting that the solubilisation of gold,
as a dicyanoaurate complex, may occur in addition to gold solubilisation
with amino acids (Reith et al., 2007a).
Gold solubilisation via cyanide mechanisms may occur in rhizosphere soils with tops rich in organic matter, where plant exudates
may lead directly to gold solubilisation or provide nutrients for cyanide
excreting microorganisms (Bakker and Schippers, 1987; Reith et al.,
2007a). Microbial in situ cyanide generation has also been suggested
as a way to reduce cyanide transport and reduce the total quantity of cyanide required (Zammit et al., 2012). The major challenge with microbial generation of cyanide is the rate of production and cost (Zammit et al.,
2012). Industrial application of this process would require that it
be more cost effective than chemical production and use of cyanide,
for which an industrial base is already available. Moreover, using biologically produced cyanide for in situ leaching may be prohibitive due
to the environmental issues. It would likely to be more acceptable to
produce cyanide in bioreactors and then use the biogenic cyanide in
traditional processing methods, such as reactors or heaps, as suggested
by Zammit et al. (2011).
Pintain Systems Inc. demonstrated enhanced gold recovery during
biological detoxication of cyanide in heap leached spent ore at Cripple
Creek and Victor Mining Company Ironclad Mine near Victor, Colorado,
USA. Gold recovery was at least two times higher than that expected
from a normal heap rinse operation producing an additional 156 kg of
gold from ve million tons of spent ore (Beckman and Thompson,
2004; Thompson and MacCulloch, 2004; Thompson et al., 1998).
The mechanism of the biologically enhanced gold solubilisation was
78
not disclosed. According to Thompson and MacCulloch (2004) Pintail

has developed a culture collection of bacteria and fungi which have
demonstrated a variety of gold solubilisation mechanisms. With a
column study they showed how various microbial strains were able to
sequentially recover gold from ore originating from Buffalo Valley pit
near Battle Mountain, Nevada, USA. Beckman and Thompson (2004)
used the term BioLix for biologically-derived lixiviants for precious
metals (gold and silver) recovery. The BioLix process is claimed to be
based on the generation of organic lixiviants with non-pathogenic naturally occurring microorganisms. For BioLix applications microorganisms are adapted to the target ore and cultured in a proprietary
broth to increase cell numbers. Thereafter, a proprietary enzymatic
inducing agent is added to initiate the microbial production of the
lixiviants. The microbial solution is then applied to the ore for the
solubilisation and complexation of precious metals (Beckman and
Thompson, 2004).
3.4. Iodide
One promising inorganic lixiviant for gold is the iodide(I)
iodine(I2) system. The most likely electrochemical half-reactions
involved in the dissolution of gold are the following (Davis and Tran,
1991):
Anodic :
Au 2I
Cathodic : I3
AuI2
2e
3I
14
15
Yielding the overall reaction:
2Au I I3
2AuI2
16
Gold dissolution may also occur according to the following overall

reaction (Angelidis et al., 1993):
2Au 3I3
2AuI4
17
As with other halogens, the aqueous iodideiodine system consists
of several known species, I2, I, I

3 , IO and HIO existing in equilibrium
(Davis and Tran, 1991):
HIOH IO
18
I2 I
20
CH3I
CH2ClI
Sorption
I3
19
These equilibria are inuenced by temperature, pH, and concentration

of I and I2 in the solution (Davis and Tran, 1991).
Major pathways in the biogeochemical cycling of iodine are oxidation and reduction of inorganic iodine species, the volatilisation of
organic iodine compounds into the atmosphere, accumulation of iodine
in living organisms, and sorption of iodine by soil and sediments (Fig. 7).
Considerable geochemical evidence has indicated that these processes
are inuenced or controlled by microbial activities, although the precise
mechanisms involved are still unclear (Amachi, 2008). Thus microorganisms may help to regenerate the iodideiodine lixiviant by oxidising
I to I2, and on the other hand, contribute to the loss of the lixiviant
through volatilisation, accumulation and sorption. Some microorgan
isms can also reduce iodate (IO
3 ) to I (for a review, see Amachi, 2008).
Microbial IO
3 reduction to I is still poorly understood largely due
to the limited number of isolates available as well as the paucity of information about key enzymes involved in the reaction. The average total
concentration of dissolved iodine in seawater is 0.45 M, and the predominant chemical forms are I and IO
3 . Thermodynamically, the con
centration ratio between IO
3 and I in oxygenated seawater at pH 8.1
and pE 12.5 should be 3.2 1013, indicating that IO
3 is the more stable
form. However, in deep waters (Nakayama et al., 1989), anoxic basins
(Farrenkopf et al., 1997; Wong and Brewer, 1977) and pore waters of
marine sediments (Muramatsu et al., 2007) I is often highly enriched
at concentrations of several M to more than one mM (Amachi, 2008).
In addition to abiotic chemical reduction of IO
3 and microbial
remineralisation of organic iodine compounds, microbial reduction of
IO
3 is likely to be an important process to maintain reduced form of
iodine in these environments (Amachi, 2008).
Recently, an IO
3 -reducing Pseudomonas sp. strain SCT was isolated
from marine sediment slurry by Amachi et al. (2007a). The strain
reduced 200 M IO
3 to I within 12 h in an anaerobic culture containing 10 mM nitrate, but could not grow with 5 or 10 mM IO
3 as the sole
electron acceptor. However, it showed signicant growth when much
lower concentrations (2, 3, and 4 mM) of IO
3 were added as the
electron acceptor. The growth was nearly proportional to the IO
3
concentration in the medium. The strain used malate, glycerol, lactate,
CH2I2
Microbial
oxidation and
volatilisation
I2 H2 OH I HIO
I-(-1)
Microbial
volatilisation
Microbial
reduction
IO3-(+5)
I2 (0)
Abiotic
oxidation
Abiotic
oxidation
HIO (+1)
Accumulation
and sorption
Fig. 7. Contribution of bacteria (solid arrows) to the biogeochemical cycling of iodine (modied from Amachi, 2008). 2014 CSIRO. All Rights Reserved.
succinate, acetate, and citrate as electron donors. The strain did not
reduce IO
3 under aerobic conditions (Amachi et al., 2007a).
Direct microbial reduction of IO
3 has also been demonstrated
with anaerobic cell suspensions of the sulphate-reducing bacterium
Desulfovibrio (D.) desulfuricans and the dissimilatory Fe3 +-reducing
bacterium Shewanella (S.) putrefaciens which were able to reduce IO
3
at pH 7 in 10 mM 4-2-hydroxyethyl-1-piperazineethanesulphonic
acid (HEPES) buffer (Councell et al., 1997). D. desulfuricans was also
able to reduce 96% of an initial 100 M IO

at pH 7 in 30 mM
3 to I
NaHCO3 buffer, whereas S. putrefaciens was not. Both soluble ferrous
iron and sulphide, as well as iron monosulphide (FeS) were shown to
abiologically reduce IO
. The study indicated that ferric iron
3 to I
and/or sulphate-reducing bacteria are capable of mediating direct,
enzymatic, as well as abiotic reduction of IO
3 in natural anaerobic
environments (Councell et al., 1997).
The oxidation of I to IO
3 does not occur spontaneously in slightly
alkaline solutions like seawater, since the rst step in the process,
i.e. the oxidation of I to I2 is thermodynamically unfavourable at the
pH of seawater. However, once I2 is formed, the hydrolysis of I2 to form
HIO will occur rapidly. Moreover, HIO disproportionates spontaneously
to form IO
3 (Amachi, 2008). A number of bacteria have been shown to
oxidise I. In 1968 Gozlan reported the isolation of an I-oxidising bacterium from experimental seawater aquaria (Gozlan, 1968). The isolate,
later named as Pseudomonas iodooxidans, was a heterotrophic Gramnegative bacterium which oxidised I to I2 through an extracellular
peroxidise with hydrogen peroxide as an electron acceptor (Gozlan
and Margalith, 1973, 1974):
H2 O2 2I 2H I2 2H2 O
21
More recently, Fuse et al. (2003) and Amachi et al. (2005b) isolated I -oxidising bacteria from marine environmental samples.
The bacteria were afliated with the -subclass of Proteobacteria.
Some of the strains were most closely related to Roseovarius tolerans
(9498% 16S rRNA gene sequence similarity) and others were related
to Rhodothalassium salexigens (8991% 16S rRNA gene sequence
similarity). The I-oxidising reaction was mediated by an extracellular
oxidase that requires oxygen (Amachi et al., 2005b):
4I O2 4H 2I2 2H2 O
22
Although the oxidation of I by oxygen as an electron acceptor is energetically favourable (G0 = 56 kJ per reaction), the extracellular
nature of the enzyme implies that energy conservation by this reaction
is not possible (Amachi et al., 2005b). I-oxidising bacteria seem to
prefer I-rich environments (Amachi et al., 2005b). I may enhance
the competitive advantage of I-oxidising bacteria over competing
microorganisms by toxic iodine species (Amachi, 2008). I2 produced
by I-oxidising bacteria is a highly active oxidising agent, and has strong
bactericidal, fungicidal, and sporicidal activities (McDonnell and Russell,
1999).
Microbially mediated cycling of iodine to regenerate the iodide
iodine lixiviant may hold potential for gold leaching. However, the practical feasibility of the concept has not been demonstrated. On the other
hand microbial volatilisation, accumulation and sorption of iodine lead
to the loss of the lixiviant. All of the processes occur at around neutral
pH and are stimulated by the supplementation of organic compounds.
The IO
3 reduction is favoured by anoxic conditions whereas all the
other biologically catalysed processes are favoured by oxic conditions
(Table 3).
4. Gold recovery and/or loss through bioprocesses decreasing
gold solubility
In contrast to most other metals, gold is extremely rare, inert,
and unstable as a free ion in aqueous solutions under atmospheric
79
Table 3
Examples of bacteria participating in geochemical cycling of iodine. All of the listed
bacteria grow at near neutral pH and benet from supplementation with organic
compounds.
Process
Microorganisms involved
Optimal
conditions
(oxic/anoxic)
Reference(s)
Reduction
of IO
3 to I
Denitrifying bacteria:
e.g. Pseudomonas sp. strain SCT;
sulphate-reducing bacteria
e.g. Desulfovibrio desulfuricans;
Fe3+ reducing bacteria:
e.g. Shewanella putrefaciens
Pseudomonas iodooxidans,
-subclass of Proteobacteria,
e.g. strains related to Roseovarius sp.
and Rhodothalassium sp.
Variovorax sp.
Rhizobium sp.
Arenibacter sp.
Not reported
Anoxic
1, 2
Oxic
3, 4, 5, 6
Oxic
Oxic
Oxic
8
6, 9, 10
Oxidation
of I to I2
Volatilisation
Accumulation
Sorption
References: 1) Amachi et al., 2007a; 2) Councell et al., 1997; 3) Gozlan and Margalith,
1974; 4) Fuse et al., 2003; 5) Amachi et al., 2005b; 6) Amachi, 2008; 7) Amachi et al.,
2001; 8) Amachi et al., 2005a; 9) Koch et al., 1989; 10) Bird and Schwartz, 1996.
conditions (Reith et al., 2007a). Gold complexes can be highly toxic to

microorganisms. Hence, microorganisms have many mechanisms
to deal with toxic gold complexes and are able to precipitate gold
intra- and extracellularly, and in products of their metabolism, such as
exopolysaccharide (EPS) and sulphide minerals (Reith et al., 2007a).
Some of these mechanisms may hold potential for gold recovery, others
may result in unwanted gold loss from pregnant leach solutions.
4.1. Biosorption and accumulation of gold
Many bacteria (e.g. P. maltophilia, B. subtilis, Escherichia coli), actinomycetes (e.g. Streptomyces albus, S. fradiae, Saccharopolyspora erythraea),
algae (e.g. Ascophyllum nodosum, Chl. vulgaris, Sargassum natans), yeast
(Candida utilis, Saccharomyces cervisiae) and fungi (e.g. Aspergillus
(A.) niger, Cladosporium cladosporioides, Fusarium oxysporum, Rhizopus
arrhizus) can contribute to passive sorption of gold from solution (Cui
and Zhang, 2008; Labeda, 1987; Reith et al., 2007a). Some groups of bacteria have an unusual capacity to sequester gold and bioconcentrate it to
very high levels. One such strain is Hyphomonas adhaerens MHS-3,
which accumulates gold in an EPS capsule. Gold was sequestered by
cultures which were able to form the capsule whereas mutants without
capsules were not able to do so (Quintero et al., 2001). Accumulation of
gold into the EPS also contributed to the higher viability of P. aeruginosa
subjected to 0.1 mM Au3+ chloride when grown as a biolm compared
to free planktonic cells (Karthikeyan and Beveridge, 2002). Kenney et al.
(2012) used non-metabolising cells of B. subtilis and P. putida and
achieved over 85% gold removal from a solution with initial gold concentration of 5 ppm at pH b 5 in 2 h. At increasing pH the adsorption
slowed down. Dementyev and Voiloshnikov (2011) reported adsorption activity of A. niger and A. orizae for dissolved gold to be as high as
that of industrial activated carbon, and for gold colloids it was 810
times higher than for activated carbon. In pilot plant test the gold
recovery with the fungal biomass was 9698%.
4.2. Enzymatic reductive precipitation
Some bacteria and archaea are able to precipitate gold by reducing
Au3+ to Au0 (He et al., 2007; Kashe et al., 2001). Some species, including Pyrobaculum islandicum, Pyrococcus furiosus, Shewanella algae and
D. vulgaris precipitate Au0 extracellularly, whereas others, such as
Geobacter ferrireducens, precipitate Au0 within the periplasmic space.
According to Kashe et al. (2001) the Au3+ reduction in dissimilatory
Fe3 + reducers appears to be an enzymatically catalysed reaction
80
which is dependent upon the presence of a specic electron donor,

hydrogen, since alternative electron donors, such as lactate did not promote Au3+ reduction. The Fe3+-reducing microorganisms that reduced
Au3 + did not appear to have a signicant capacity for adsorption
of Au3 + prior to reduction, because there was no loss of Au3 + from
solution in the absence of hydrogen or when cells were incubated at
temperatures that inhibited metabolism. This suggests that the Fe3+reducing microorganisms reduced Au3 + prior to, or simultaneously
with, the adsorption of gold onto the cell surface (Kashe et al., 2001).
This mechanism appears to be clearly different from the mechanism of
aerobic microorganisms which adsorb Au3 + on the cell surface, with
the subsequent reduction of the adsorbed Au3+ to Au0, although the
mechanisms of these interactions are also poorly understood (Kashe
et al., 2001; Savvaidis et al., 1998).
Enzymatically catalysed precipitation of gold has been suggested to
have led to the formation of gold-bearing biomineralisations in various
environments (Fairbrother et al., 2013; Reith et al., 2006; Reith et al.,
2007a). Fairbrother et al. (2013) used quartz sand packed columns to
assess the biomineralisation potential of Cupriavidus metallidurans,
which can reductively precipitate gold as nanoparticles (Reith et al.,
2009). While abiotic control columns retained only b30 wt.% of the
gold added as Au(I)-thiosulphate, the inoculated columns achieved
N99 wt.% gold removal. The synthesis of gold nanoparticles has also
been successfully demonstrated with a variety of other microorganisms
(Gwynne, 2013) including bacteria, such as E. coli (Du et al., 2007),
Marinobacter pelagius (Sharma et al., 2012) and Delftia acidovorans
(Johnston et al., 2013), fungi, such as Verticillium luteoalbum (Gericke
and Pinches, 2006), yeast, such as Yarrowia lipolytica (Agnihotri et al.,
2009), and actinomycete, such as Rhodococcus (Ahmad et al., 2003a)
and Thermonospora (Ahmad et al., 2003b). Both intracellular and
extracellular formation of nanoparticles has been reported (Gericke
and Pinches, 2006; Johnston et al., 2013). Gold nanoparticles can be
used in several applications such as optoelectronics, photonics, catalysis,
imaging technology and drug delivery (Sharma et al., 2012).
4.3. Micronutrition
Gold is generally believed to be non-essential for microbial nutrition
(Reith et al., 2007a). However, in the presence of gold, Micrococcus (M.)
luteus produces an gold-containing protein, which oxidises methane to
methanol. The protein has a Au+/Au3+ redox couple in its active centre
and presumably helps the bacterium to survive when usual sources of
carbon and energy are scarce (Levchenko et al., 2000, 2002). M. luteus
is a mesophilic, aerobic and heterotrophic bacterium, which grows optimally near neutral pH values (Wieser et al., 2002). Methanotrophs have
been detected on secondary gold grains from an Australian mine, suggesting an environmental association of methane-oxidising bacteria
with gold (Reith et al., 2007a). It is not known if other gold-containing
enzymes exist in any other microorganisms (Reith et al., 2007a).
4.4. Ligand utilisation and loss
Microorganisms can decrease gold solubility by consuming the
ligands that bind gold. The ability of microorganisms to destabilise
gold ligands will need to be controlled if biogenic or other alternative
lixiviants are to be used for gold leaching.
4.4.1. Biooxidation and reduction
The precipitation of gold from Au(I)-thiosulphate solutions has been
observed in the presence of thiosulphate-oxidising bacteria (Lengke
and Southam, 2005). Gold precipitated by Acidithiobacillus thiooxidans
was accumulated inside the bacterial cells as ne-grained colloids (5
10 nm in diameter) and in the bulk liquid as crystalline micrometerscale gold. While gold was deposited throughout the cell, it was concentrated along the cytoplasmic membrane, suggesting that gold
precipitation was likely enhanced via electron transport processes

associated with energy generation (Lengke and Southam, 2005).
SRB, such as Desulfovibrio spp., typically oxidise organic compounds
using sulphate as the terminal electron acceptor. Many SRB can also use
alternative electron acceptors, such as thiosulphate. SRB have been
shown to reduce the thiosulphate from Au(I)-thiosulphate complexes
and thus precipitate gold (Lengke and Southam, 2006). Lengke and
Southam (2007) studied the role of SRB in the precipitation of elemental
gold using column experiments. Bacterially mediated gold precipitation
from the Au(I)-thiosulphate complex was more efcient (98.299.6%)
than the precipitation in corresponding abiotic controls (074.3%).
SRB reduce thiosulphate and other sulphur compounds to H2S, which
precipitates metals ions, such as Fe2 +, leading to the formation
of metal sulphides. Thus, SRB may also indirectly contribute to gold
precipitation by production of H2S which precipitates gold as sulphide,
and iron sulphides which may lead to reduction of Au+ to Au0
(Lengke and Southam, 2006). The destabilised gold may be incorporated
into the newly forming sulphide minerals (Reith et al., 2007a).
Similarly to the thiosulphate-ligand utilisation, microbes utilising
gold-complexing carboxylic acids (such as amino-acids) or cyanide
may lead to destabilisation of gold complexes and contribute to gold
precipitation (Reith et al., 2007a). High pH has been suggested as an
approach to control cyanide degrading microbial populations (FedelMoen et al., 2000).
4.4.2. Sorption and accumulation of iodine
Iodine is a biophilic element, and accumulates in various organisms.
To date, however, the mechanisms of iodine uptake by living organisms
have been poorly understood with the exceptions of brown algae and
the thyroid gland in mammals (Amachi, 2008). Amachi et al. (2005a)
isolated a marine bacterium Arenibacter sp. C-21, which can actively
transport and accumulate iodine. When grown in a liquid medium
containing 0.1 M I, 7989% of the I was removed from the medium,
and a corresponding amount of I was detected in the cells. When
the strain was cultured with 0.1 M I, the maximum I content was
220 3.6 pmol of I per mg of dry cells, and the maximum concentration factor for I was 5.5 103. In the presence of much higher concentrations of I (1 M to 1 mM), I content increased, but decreased
concentration factors for I were observed (Amachi et al., 2005a).
Iodine transport assays revealed that glucose and oxygen were necessary
for the uptake of iodine (Amachi et al., 2007b). IO
3 , which is the other
dominant species of iodine in terrestrial and marine environments, was
not transported (Amachi et al., 2005a).
In terrestrial environments, iodine is strongly adsorbed by soils
(Amachi, 2008). Although the sorption by soils is affected by various
physico-chemical parameters including soil type, pH, Eh, salinity, and
organic matter content, a number of studies have indicated that microorganisms are also involved in the process (Amachi, 2008). Muramatsu
and Yoshida (1999) showed that autoclaving soils signicantly reduced
the sorption of I and the sorption was recovered after adding a small
amount (110%) of fresh soil, suggesting the role of microorganisms in
sorption. Koch et al. (1989) observed increased I sorption in soils
supplemented with glucose and decreased sorption in soils treated
with the antiseptic, thymol. Fumigation, air-drying and gammairradiation have also been shown to decrease iodine sorption
(Bors and Martens, 1992). Iodine sorption in soils occurs more
easily under oxic than under anoxic conditions (Bird and Schwartz,
1996).
4.4.3. Volatilisation of iodine
Iodine is thought to volatilise in the form of organic iodine
compounds, such as methyl iodide (CH3I), diiodomethane (CH2I2),
chloroiodomethane (CH 2 ClI) (Amachi, 2008). A wide variety of
marine and terrestrial bacteria have been shown to be capable of
methylating I to form CH3 I. Aerobic bacteria such as Variovorax
sp. strain MRCD 30 and Rhizobium sp. strain MRCD 19 showed
considerable production of CH3I, whereas anaerobic Clostridium novyi

and methanogens (Methanobacterium formicicum, Methanoculleus
bourgensis, Methanosarcina mazei, and Methanospirillum hungatei) did
not produce CH3I (Amachi et al., 2001; Asakawa and Nagaoka, 2003).
By using resting cells of a terrestrial bacterium Rhizobium sp. strain
MRCD 19 and a marine bacterium Alteromonas macleodii strain IAM
12920, Amachi et al. (2001) measured CH3I production at I2 concentrations of 0.1 mM to 5 mM. These strains showed increased CH3I production in the presence of increased I2 concentrations, indicating that
bacterial CH3I production depends greatly on the surrounding iodine
concentrations. The greatest observed production rate with Rhizobium
sp. was approximately 107 fmol CH3I day1 1010 cells1. Heat treated
(80 C) or autoclaved cells of Rhizobium sp. strain MRCD 19 did not
show any CH3I production, suggesting that the methylation was mediated by live cells (Amachi et al., 2001). The addition of yeast extract
and glucose to soil slurries stimulated iodine volatilisation, whereas
autoclaving and addition of antibiotics (streptomycin and tetracycline,
specic inhibitors of prokaryotes) decreased the volatilisation
(Amachi et al., 2003). Along with biosorption and bioaccumulation,
the volatilisation of iodide can lead to the loss of the lixiviant if
iodideiodine system is used for gold leaching.
5. Conclusions
Microorganisms play many roles in the biogeochemical cycling
of gold and can be utilised in a number of ways for gold processing
and recovery. Biooxidation of refractory gold bearing sulphide ores
with acidophilic iron and sulphur-oxidising microorganisms has been
already commercially practised since the 1980s, rst in bioreactors
and subsequently as heap leaching operations. Although in situ or in
place biooxidation of gold ores has not yet been industrially practised,
the indirect oxidation of the ores with Fe3 + biologically regenerated
above ground may be feasible. However, research is still needed to demonstrate practical applicability and economic feasibility of the concept.
Microbial processes can also be used to deactivate carbonaceous materials that would bind solubilised gold from pregnant leach solutions.
Mechanisms for biological permeability enhancement have also been
identied. However, these processes may be slow and the feasibility of
in situ permeability enhancement of ore bodies is yet to be demonstrated
through research.
Microorganisms can promote gold solubilisation by the excretion of
ligands, such as thiosulphate, organic acids, cyanide and iodideiodine,
which oxidise and forms complexes with gold. As thiosulphate, organic
acids and cyanide are intermediates in microbial metabolism, their long
term stability in an underground environment may be problematic. These
limitations may hinder the utilisation of microbial gold solubilisation in an
industrial process. The microbial stability of chemically lixiviants, such
as thiosulphate and iodideiodine also needs to be considered in long
term industrial application.
Microorganisms can contribute to the recovery and/or loss of gold by
decreasing the solubility of gold through biosorption and accumulation, reductive precipitation, ligand utilisation and by using gold
as a micronutrient for enzymes. Bioprecipitation and biorecovery
of gold would need to become economically attractive to be able
to replace already proven recovery processes such as activated carbon adsorption. Future research efforts are needed to quantify the
extent of gold loss from alternative lixiviant systems by microbial
activities.
In summary, several microbial processes are relevant for gold
leaching and recovery. Some of these may be applicable for in situ or
in place leaching of low grade gold ores, which the industry will increasingly depend on into the future. Other microbial processes
need to be considered as potential risks for lixiviant stability and
gold recovery. The understanding of these processes will enhance
industrial applications of biotechnology and lixiviant use by the
minerals industry.
81
Acknowledgements
The support of the CSIRO Minerals Down Under National Research
Flagship, sponsors of MERIWA project M409 and ORICA is gratefully
acknowledged.
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Hydrometallurgy 142 (2014) 7083

Contents lists available at ScienceDirect

The role of microorganisms in gold processing and recoveryA review

Corresponding author: Tel.: +61 8 9333 6253.

2. Biotechnical pre-treatment of refractory gold ores

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

Gold grade (g Au t-1)

2Fe1x SAu 41xH 1xO2 21xFe

3O2 2H2 O 4H 2SO4

Fe3 + and H+ ions attack the valence bonds of sulphide minerals

FeS2 Au 2Fe

FeS2 Au 14Fe

Fe1x SAu 22xFe

Fe1x SAu 82xFe

2.1.2. Engineering applications

Oxidation of Fe2+ and reduced sulphur compounds

Carbon-adsorbable blanking agent

Oxidised ore or concentrate

Organic acid production

Gold complexes in solution

Microorganisms used in biooxidation processes include mesophilic

Primary gold ore or concentrate

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

Biooxidation treatment in tank reactors is typically practised for high

is available to promote microbial growth. If no carbonate is present,

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

Design capacity (ore t d1)

to the bioreactors (Adamov et al., 2011; Fomchenko et al., 2010;

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

with leachable gold due to surfactant properties (Thompson and

The generation of acid in this process decreases pH, which in turn

3Fe8 O8 OH6 SO4 C6 H12 O6 6H2 O 24Fe

Reductive dissolution of iron hydroxy compounds would require the

Au(I)thiosulphate complex is stable in mildly acidic to highly alkaline pH

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

H2O + CO2 + light O2 + CH2O

Ferric iron reduction with

4Fe(OH)3 + CH2O 4Fe2+ + H2CO3 + 2H2O + 8OH

Requires sunlight would not work underground

successfully processed with ammonium thiosulphate at the Nevada

Gold solubilisation via the thiosulphate mechanism is expected in

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

3.3. Biogenic cyanide

4Au 8CN O2 2H2 O4AuCN2 4OH

An in vitro study using the cyanogenic bacterium Chromobacterium

microorganisms, such as Chr. violaceum and Chlorella (Chl.) vulgaris

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

not disclosed. According to Thompson and MacCulloch (2004) Pintail

Yielding the overall reaction:

Gold dissolution may also occur according to the following overall

As with other halogens, the aqueous iodideiodine system consists

of several known species, I2, I, I

These equilibria are inuenced by temperature, pH, and concentration

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

able to reduce 96% of an initial 100 M IO

conditions (Reith et al., 2007a). Gold complexes can be highly toxic to

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

which is dependent upon the presence of a specic electron donor,

precipitation was likely enhanced via electron transport processes

A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083

considerable production of CH3I, whereas anaerobic Clostridium novyi