Bioleaching is the extraction of metals from ores using the...

Introduction

Chapter 1

Bioleaching is the extraction of metals from ores using the principal

components of water, air and microorganisms, all of which are found

readily within the environment. The development of new effective

techniques, using these sources, to process mineral material is one of the

decisive factors of scientific and technological progress. Lover metal

content and a more complicated mineral composition of ores to be

processed coupled with growing environmental awareness have made

mining and recovery of values more expensive (Ehrlich 1988).

Our natural mineral wealth has been exploited considerably to a greater

extent during the past 50 years. With increase in industrialization along

with population growth, the demand of metals has increased and is likely

to go up further in years to come.

This has lead to irreversible impacts like depleting high-grade ores with

simultaneous generation of solid wastes and effluents containing metals. It

is thus important to tackle the problem for control of pollution and

recovery of metal values in a cost-effective method.

Biotechnology holds greater importance in mineral engineering for the

development of economically viable processes for utilization of wastes and

low grade ores through biochemical leaching methods and up gradation of

ores through bio beneficiation. In these processes the natural ability of

microorganisms belonging to various groups has been effectively utilized

(Mishra et al 2005, Mohapatra et al 2009).

Worldwide reserves of high-grade ores are diminishing at an alarming rate

due to the rapid increase in the demand for metals. However there exist

large stockpiles of low and lean grade ores yet to be mined. But the

recovery of metals from them using conventional techniques is very

expensive due to high energy and capital inputs required. Another major

Introduction

Chapter 1

problem is environmental costs due to high level of pollution from these

technologies. Environmental standards continue to stiffen, particularly

regarding toxic wastes, so costs for ensuring environmental protection will

continue to rise.

Biohydrotechnology is regarded as one of the most promising and the most

revolutionary solution to these problems, compared to pyrometallurgy or

chemical metallurgy. It dramatically reduces the capital costs with

opportunity to reduce environmental pollution. Biological processes are

carried under mild conditions, usually without adding toxic chemicals. The

products of biological processes wind up in aqueous solution, easy to

containment and treatment than gaseous waste e.g. sulfur dioxide. (Preston

D et al. 2004, Ehrlich 1988)

Nowadays bioleaching occupies an increasingly important place among

the available mining technologies. Today bioleaching is no longer a

promising technology but an actual economical alternative for treating

specific mineral ores. An important number of the current large-scale

bioleaching operations are located in developing countries. This situation is

determined by the fact that several developing countries have significant

mineral reserves and by the characteristics of bioleaching that makes this

technique especially suitable for developing countries because of its

simplicity and low capital cost requirement. (Acevedo 2002)

1.1 History of biomining

The origin of mining industry goes back with beginning of civilization. It

has been carried out since at least Neolithic time. The earliest use of

microbial processes for mining occurred long before it was clear that

microbes were responsible for the effects observed. At the Rio Tinto (Rd

River) mine in Seville, Spain, copper mine workings were rediscovered in

Introduction

Chapter 1

1556. Evidence suggests that the mine used water from the Rio Tinto water

containing a very high concentration of ferric ion owing to microbial

activity in the area. When the water from this river was irrigated into

copper containing deposits, the copper dissolved and later precipitated as

smaller deposits. (Acevedo 2002, Dave et al. 2012, Ehrlich1988)

The roots of hydrometallurgy may be traced back to the period of

alchemists.Although the people at that time likely believed this process to

be magic; we now know that it was the first recorded use of

biomineralization. Also probably such methods may have been used since

prehistoric times and probably the Greeks and Romans extracted copper

from mine water more than 2000 years ago (Bosecker, 1997). It is reported

by Rawlings (2004), pre-Romans recovered silver, and the Romans

recovered copper from a mineral deposit located in the Seville province of

southern Spain. This deposit was later on became the site of the Rio Tinto

mine. Leaching of copper was practiced in Norway in the 15th century, in

Germany in 16th century and in England in the 18th century. In early 19th

century, heap and dump leaching was practiced. In 1947, actual evidence of

microbial leaching was obtained through the pioneer workers Colmer and

Hinkel. They isolated pure culture of Acidithiobacillus ferooxidans from mine

water. This gram negative chemolithotroph could oxidize the sulfide part

of minerals to sulphuric acid and ferrous ion to ferric at a very low pH. The

industrial scale bioleaching of copper in heaps has had a chequered 400

year career. Johnson and Hallberg (2009) reported that during the 18th and

19th centuries, the practice of allowing underground shafts and adits at

these mines to flood, and then periodically releasing and capturing the

metal-rich waters to recover copper from the leached subterranean rocks

by adding scrap iron (cementation) was adapted. This was, essentially, an

ignorant application of ‘‘in situ’’ leaching, which later deliberately applied

Introduction

Chapter 1

for uranium biomining from worked-out mines in Canada in 1970s. Over

the past 20 years this technology has blossomed with annualized world

copper production from the process increasing from 0.2% to approximately

8-10%. Bioleaching of copper in heaps was first recorded at the Rio Tinto

mine in 17th century. The first modern industrial scale copper heap

bioleach, producing 14,000tpa, commenced in 1980 at Lo Aguirre in Chile.

The first stand-alone mine using copper bioleaching – solvent extraction –

electrowinning was the Girilambone Copper Operation (managed by

Straits Resources and commissioned 1993) in central NSW, Australia

(Acevedo 2002, Ehrlich 1988, Rossi 1990).

1.2 Mineral bioprocessing mechanisms.

Biomining is a combination of chemical and biological reactions. During

metal sulphide bioleaching, metal sulphides are oxidized to metal ions and

sulphate by aerobic, acidophilic Fe2+ iron and/or sulphur compound

oxidizing bacteria or Archaea (Schippers, 2007).

Bioleaching is a heterogeneous reaction that takes place at the interface

between a solid and liquid phase and some-times a gaseous phase. At the

boundary between the two phases, a diffusion layer is formed. The

dissolution of mineral ore takes place through the following stages: (1)

diffusion of reactant through the diffusion layer, (2) adsorption of the

reactant on the solid, (3) chemical reaction between the reactant and the

solid, (4) desorption of the product from the solid and (5) diffusion of the

product through the diffusion layer. Any of these stages (1) - (5) may be the

rate controlling step depending on its relative speed to the others (Baba et

al., 2012)

A generalized reaction can be used to express the biological oxidation of a

mineral sulphide involved in leaching:

Introduction

Chapter 1

MS + 2O2 MSO4, Where M is a bivalent metal (1)

There are two dominant mechanisms, which are considered to be involved

in bioleaching.

In the first mechanism, bacterial membrane directly interacts with the

sulphide surface using enzymatic mechanisms. This mechanism is referred

to as the direct mechanism. Cell attachment to suspended mineral particles

takes place within minutes or hours with cell preferentially occupying

irregularities of the surface structure (Boon2001).The second mechanism

involve oxidation of reduced metal mediated through ferric (Fe+3)

generated from the microbial oxidation of ferrous ions (Fe2+) compounds

present in the mineral. Ferric ion is an oxidizing agent and is chemically

reduced to ferrous ions. Ferrous ions can 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

.The following equations describe the direct and indirect mechanism for

the oxidation of metal sulphides. (Thore et al.2007, Ana et al. 2003, Ehrlich

1988, Schippers et al. 1999)

Metal sulphides can be directly oxidized by A. ferrooxidans to soluble

metals sulphates according to equation,

MS + 2O2 M+2 +SO4-2 (2.1)

Because the metal sulphides exist in an insoluble form and the metal

sulphate (MSO4) is usually water soluble, this reaction is able to transform

a solid phase to a liquid one to which further treatment can be provided to

recover the metal. Theoretically, the mechanism can be continued until all

the substrate (MS) is converted to product (MSO4). Examples of the direct

mechanism are as follows:

A. ferrooxidans

Microorganisms

Introduction

Chapter 1

Pyrite:

4FeS2+15O2+2H2O→2Fe2 (SO4)3+2H2SO4 (eq.2.2)

Chalcopyrite:

4CuFeS2 + 17O2 + 2H2SO4 → 4CuSO4 + 2Fe2 (SO4)3 + 2 H2O (eq.2.3)

Chalcocite:

2Cu2S+5O2+2H2SO4→4CuSO4+2H2O (eq.2.4)

Covellite:

CuS + 2O2 → CuSO4 (eq.2.5)

Sphalerite:

ZnS + 2O2 → ZnSO4 (eq.2.6)

Oxidation of sulphide minerals is also contributed by ferric ions generated

by microorganisms. Reaction (3.1) takes place under the action of

Acidithiobacillus, whereas reaction (3.2) occurs chemically without any

association of bacteria. The oxidation of elemental sulphur (according to

equation 3.3) also occurs by Acidithiobacillus.

4FeSO4+2H2SO4+O2 A. ferrooxidans 2Fe (SO4)3 + 2H2O (eq.3.1)

MS + 2Fe (SO4)3 MSO4+2FeSO4+S0 (eq.3.2)

2S0+3O2+2H2O Acidithiobacillus 4H++2SO4-2 (eq.3.3)

Therefore, metal dissolution occurs by a cyclic process between reaction 2.7

and 2.8 and the formation of H+ during the sulphur oxidation (equation

2.9) enhances the overall efficiency. Examples of chemical oxidation

processes are as follows:

Introduction

Chapter 1

Pyrite:

FeS2 + Fe2 (SO4)3 → 3FeSO4 + 2S° (3.4)

Chalcopyrite:

CuFeS2 + 2Fe2 (SO4)3 → CuSO4 + 5FeSO4 + 2S° (3.5)

Chalcocite:

Cu2S + 2Fe2 (SO4)3 → 2CuSO4 + 4FeSO4 + S° (3.6)

Covellite:

CuS + Fe2 (SO4)3 → CuSO4 + 2FeSO4 + S° (3.7)

Sphalerite:

ZnS + Fe2 (SO4)3 → ZnSO4 + 2FeSO4 + S° (3.8)

The model of direct and indirect metal leaching is still under discussion,

especially the hypothesis of the direct mechanism. New insights have been

derived from recent research, with more advanced techniques for the

analysis of degradation products occurring in the bioleaching process and

the analysis of extracellular polymeric substances. (Rohwerder and Sand,

2007, Bevilaqua 2007)

Recently, two indirect mechanisms have been proposed whereas no

evidence for a direct enzymatically mediated process has been found

(Schippers and Sand, 1999; Sand et al, 2001). Thiosulphate and

polysulphide have been found as intermediates during the oxidation of

galena, sphalerite, chalcopyrite, hauerite, orpiment, or realgar. One

mechanism is based on the oxidative attack of iron (III) ions on the acid-

insoluble metal sulphides FeS2, MoS2, and WS2 with thiosulphate as an

intermediate. The second mechanism deals with metal dissolution by the

attack of ferric ions and/or by protons in which case the main sulphur

intermediate is polysulphide and elemental sulphur. The following

Introduction

Chapter 1

equations summaries the two mechanisms (Schippers and Sand, 1999; Sand

et al, 2001):

Thiosulphate mechanism (for FeS2, MoS2, WS2):

FeS2 + 6Fe+3 +3H2O → S2O3-2 + 7Fe+2 + 6H+

S2O3-2 + 8Fe+3 +5H2O Bacteria 2SO2- +8 Fe+2 +10H+

Polysulphide mechanism (for ZnS, CuFeS2, PbS):

MS + Fe3+ + H+ → M2+ + 0.5 H2Sn + Fe2+ (n ≥ 2)

0.5H2Sn+Fe+3 Bacteria 0.125S8+Fe+2+H+ (2.7)

0.125 S8 + 1.5 O2 +H2O Bacteria SO4 -2 + 2H+

The main characteristic of these two mechanisms is the hypothesis that

ferric ions and/or protons are the only chemical agents involved in

dissolving a metal sulphide.

1.3 Bacterial leaching techniques

The two major techniques used in leaching are percolation and agitation

leaching. Percolation leaching involves the percolation of a lixiviant

through a static bed, whereas agitation leaching involves finer particle sizes

agitated in a lixiviant. Due to the large scale operations involved in

bacterial leaching, percolation leaching is preferred commercially.

The principal commercial methods are in situ, dump, heap and vat

leaching. In situ leaching involves pumping of solution and air under

pressure into a mine or into ore bodies made permeable by explosive

charging. The resulting metal-enriched solutions are recovered through

wells drilled below the ore body. Three types of ore bodies are generally

considered for in situ leaching surface deposits above the water table,

Introduction

Chapter 1

surface deposits below the water table and deep deposits below the water

table.

Dump leaching involves uncrushed waste rock which is piled up. These

dumps generally contain about 0.1-0.5% Cu, too low to recover profitably

by conventional procedures. Some of these dumps are huge, containing in

excess of 10 million tons of waste rock. Heap leaching requires the

preparation of the ore, primarily size reduction, so as to maximize mineral-

lixiviant interaction and the laying of an impermeable base to prevent

lixiviant loss and pollution of water bodies. Essentially, both dump and

heap leaching involve the application of lixiviant to the top of the dump or

heap surface and the recovery of the metal laden solution that seeps to the

bottom by gravity flow. The dilute sulphuric acid sprinkled on top

percolates down through the dump, lowering the pH and promoting the

growth of acidophilic microorganisms. The acid run-off is collected at the

bottom of the dump, from where it is pumped to a recovery station.

Copper is extracted from the acid run-off by cementation or solvent

extraction or electrowining. All the above processes are essentially

uncontrolled from a biological and engineering standpoint. Beside these

processes are slow in nature and require long periods to recover a portion

of the metal. Vat leaching as currently applied to oxide ores involves the

dissolution of crushed materials in a confined tank. More controls can be

brought in for enhanced recovery by the use of bioreactors, though

necessarily these involve higher costs. However for ore concentrates and

precious metals they are being considered actively. (Schippers et al. 1999,

Thore et al.2007)

Introduction

Chapter 1

1.4 Metals extracted by bioleaching

The conversion of an insoluble metal (usually a metal sulphide, e.g., CuS,

NiS, ZnS) into a soluble form (usually the metal sulphate, e.g., CuSO4,

NiSO4, ZnSO4) because of various activities of microorganisms is termed as

‘bioleaching’. Because these processes are oxidations, this process may also

be termed ‘biooxidation’. However, the term biooxidation is usually used

to refer to processes in which the recovery of a metal is enhanced by

microbial decomposition of the mineral, but the metal being recovered is

not solubilised. An example is the recovery of gold from arsenopyrite ores

where the gold remains in the mineral after biooxidation and is extracted

by cyanide in a subsequent step. Hence, the term bioleaching is clearly

inappropriate when referring to gold recovery (although arsenic, iron, and

sulphur are bioleached from the mineral). ‘Biomining’ is a general term

that may be used to refer to both processes (Bosecker, 1997; Ehrlich, 2001,

Ehrlich 2004; Olson et al., 2003; Rawlings, 1995, 2002; Rohwerder et al.,

2003).

1.4.1 Bioleaching of Copper:

Biological copper leaching is practiced in many countries including

Australia, Canada, Chile, Mexico, Peru, Russia and the U.S.A. Copper

recovery from bioleaching accounts for about 25% of the world copper

production. Following the initial isolation of Acidithiobacillus ferrooxidans

from coal mine water in 1947, studies quickly disclosed its presence in

copper-leaching operations. Acidithiobacillus ferrooxidans is also found in the

Malanjkhand Copper Mines in Madhya Pradesh, India. The physical

configurations of bioleaching operations world-wide for copper are mostly

uniform. Typically copper ore mined from open pits is segregated; higher

grade metal is concentrated to produce feed for smelting, while the lower

grade ore is subjected to leaching. The ore is piled on an impermeable

Introduction

Chapter 1

surface until a dump of suitable dimension forms. After the top is leveled,

leach solution is flooded or sprayed onto the dump. A copper dump

represents a complex and heterogeneous microbiological habitat. It

contains solids ranging in size from boulders to fine sand and includes

material of complex mineralogy. Bacterial colonization occurs in the top 1

meter or so. The temperature may reach 90oC in the interior of the dump

and supports a range of thermophilic microorganisms, which are often

anaerobic, or microaerophillic. In these regions, indirect leaching by ferric

sulphate also prevails. The exterior of the dump is at ambient temperature

and undergoes changes in temperature reflecting seasonal fluctuations.

Many different microorganisms have been isolated from copper dumps,

some of which have been studied in the laboratory. These include a variety

of mesophilic, aerobic iron and sulphur oxidizing microorganisms,

Thermophillic iron and sulphur oxidizing microorganisms, and anaerobic

sulphate reducing bacteria. Some are heterotrophic bacteria, which

indirectly affect metal solubilisation by affecting the growth and activity of

metal solubilising bacteria. Others are protozoa, which interact with and

prey on different types of bacteria. Leach solutions enriched with copper

exit at the base of the dump and are conveyed to a central recovery facility.

In most large-scale operations the leach solution, containing 0.5-2 g/l

copper is pumped into large cementation units containing iron scrapings

for cementation and then electrolysis. A typical large dump may have an

operating life of over 10 years (Harneit et al. 2006, Thore et al.2007, Ehrlich

1988, Schippers et al. 1999)

1.4.2 Bioleaching of Zinc

According to the International Zinc Association, the mining, smelting and

refining of zinc contributes US$ 18.5 billion to the world economy each

year (International Zinc Association, 2005). Over the last 25–30 years the

Introduction

Chapter 1

zinc industry has moved away from traditional pyrometallurgy to

hydrometallurgy. Nowadays, about 80% of the world’s total zinc is

produced through conventional hydrometallurgical methods (roast-leach-

electro winning) (Viera et al. 2007, Carranza 1997).

The potential benefits of commercial bioleaching of zinc minerals are

significant in the treatment of concentrate, which is difficult to process

using conventional technologies. In addition, zinc concentrate bioleaching,

compared to hydrometallurgy, has the advantage of not requiring roasting,

sulfuric acid plants and washing of the gaseous effluents (Viera et al. 2007).

The main zinc mineral is sphalerite, which is degraded by acidophilic

autotrophic bacteria through the polysulfide mechanism.

(1) ZnS + H++Fe3+ Zn 2++0.5H2Sn + Fe2+ (n ≥ 2) (reaction 1)

(2) 0.5H2Sn +Fe3+ 0.125S8 + Fe2+ + H+ (reaction 2)

(3) 0.125S8+1.5O2+H2O SO4 2– + 2H+ (reaction 3)

(4) 2 Fe2++0.5O2 + 2H+ 2Fe3+ + H2O (reaction 4)

The ferrous iron produced in reactions 1 and 2 can be reoxidized to ferric

iron by iron-oxidizing microorganisms such as A. ferrooxidans,

Leptospirillum or Sulfobacillus. According to reaction (2), when zinc sulfide is

oxidized by ferric ions, a product layer of elemental sulfur is formed on the

surface of the mineral. The diffusion of ferrous ions across this sulfur layer

becomes the rate-limiting step when zinc sulfide is oxidized by ferric ions;

a product layer of elemental sulfur is formed on the surface of the mineral.

The diffusion of ferrous ions across this sulfur layer becomes the rate-

limiting step (Viera et al. 2007, Palencia et al 1990). Elemental sulfur is

relatively stable but it may be oxidized to sulfate by sulfur-oxidizing

microbes. In such a case, the surface reaction with ferric ions becomes the

Microorganisms

Introduction

Chapter 1

rate-limiting step. Significant slowing of the leaching process confirmed the

passivation of the sphalerite surface by a layer of elemental sulfur when

there was no sulfur oxidizing bacteria present. Even in the presence of

sulfur-oxidizing bacteria, if the rate of elemental sulfur oxidation (reaction

3) is inadequate, the diffusion through this layer may be the rate-limiting

step. To sum up, the role of the microorganisms in the solubilization of zinc

sulfides is to provide sulfuric acid (reaction 3) for a proton attack and to

keep the iron in the oxidized ferric state (reaction 4) for an oxidative attack

on the mineral (Rawlings, 2005).

Several experiments on the bioleaching of sphalerite have been carried out

using pure cultures or mixed cultures of iron-oxidizing and sulfur-

oxidizing bacteria. Comparisons between the different published results

are difficult because the experiments were carried out using different

culture conditions and temperatures. Bioleaching of sphalerite can be

improved by using mixed cultures of bacteria. In experiments using 20%

pulp density, zinc extraction as high as 84% was reached using a

consortium of acidophilic chemolithotrophic iron and sulfur-oxidizers (A.

ferrooxidans, L. ferrooxidans and A. thiooxidans) and heterotrophic organisms

(Tipre & Dave, 2004, Viera et al. 2007,Sand et al 2001).

1.4.3 Bioleaching of Lead

Galena (lead sulphide) is a main lead mineral commonly found together

with sphalerite. The microbial bioleaching of the insoluble lead sulphides

produces lead sulphate (anglesite), which also has a very low solubility.

Although lead is toxic for most microorganisms used in bioleaching, this

effect is reduced due to the low solubility of the product. In complex ores

and concentrates, the selective solubilisation of copper, nickel and zinc,

which often occurs with galena, could be the basis of the beneficiation of

lead ores where lead remains in the residue. A diffusion controlled indirect

Introduction

Chapter 1

mechanism was proposed for the bacterial oxidation of galena by a mixed

culture of iron- and sulphur-oxidizing bacteria. The product layer consisted

of lead sulphate and elemental sulphur. During bioleaching processes

galena was selectively oxidized to anglesite with partial passivation of

sphalerite .Some studies have been carried out to recover lead – using

chemical leaching – from the residues obtained in the bioleaching of

complex sulphides containing sphalerite and galena. In the latter case,

silver and gold were also recovered from the residues. Tipre & Dave in

2004 developed a consortium consisting of autotrophic and heterotrophic

microorganisms for bioleaching of metals from a copper-lead-zinc sulphide

concentrate. Results showed copper and zinc extraction levels above 80%;

in addition, bioleaching produced 83% galena oxidation from the

concentrate. (Martha E. L 2006, Thore et al.2007, Ehrlich 1988, Schippers et

al. 1999)

1.4.4 Bioliberation of Nikel

The nickel mineral most commonly mined is pentlandite (Fe,Ni)9S8). Pilot

plant tests have demonstrated the efficacy of mixed cultures of A.

ferrooxidans, A. thiooxidans and L. ferrooxidans in the bioleaching of nickel

from pentlandite in a complex sulfide concentrate. Billiton developed a

process named BioNIC™ to treat low-grade nickel ores based on the gold

bioleaching process (Clark et al., 2005). This process has been tested but it

has not been possible to identify an ore with a suitable concentration and

size to allow economic recovery of nickel (Rawlings et al., 2003). Nickel

could be recovered from lateritic minerals by bioleaching using

heterotrophic microorganisms. Studies on the bioleaching of copper, zinc

and nickel from a mining ore using organic acids produced by A. niger

have been published. The maximum metal dissolution obtained was 68%

for copper, 46% for zinc and 34% for nickel. (Viera et al. 2007)

Introduction

Chapter 1

1.4.5 Bioleaching of Uranium

Uranium leaching proceeds by the indirect mechanism as Acidithiobacillus

ferrooxidans does not directly interact with uranium minerals. The role of

Acidithiobacillus ferrooxidans in uranium leaching is the best example of the

indirect mechanism. Bacterial activity is limited to the oxidation of pyrite

and ferrous iron. The process involves periodic spraying or flooding of

worked-out stops and tunnels of underground mines with lixiviant.

Another method in use for uranium extraction is vat-leaching. Bioleaching

has also been used successfully to obtain uranium from waste gold ore.

(Abhilash et al. 2009 & 2011, Schippers et al. 1999)

1.4.6 Bioliberation of Gold

Iron- and sulphur- oxidizing acidophilic bacteria are able to oxidize certain

sulphidic ores containing encapsulated particles of elemental gold,

resulting in improved accessibility of gold to Complexation by leaching

agents such as cyanide. Bio-oxidation of gold ores is a less costly, less

polluting alternative to other oxidative pre-treatments such as roasting and

pressure oxidation. Recently bio-oxidation of gold ores has been

implemented as a commercial process, and is under study worldwide for

further application to refractory gold ores. Technology developed by K. A.

Natarajan and co-workers at the Indian Institute of Science is being applied

at the Hutti Gold Mines, Karnataka, India for extraction of gold. Bio-

oxidation involves treatment with Acidithiobacillus ferrooxidans to oxidize

the sulphur matrix prior to cyanide extraction. Commercial exploitation

has made use of heap leaching technology for refractory gold ores.

Refractory sulphidic gold ores contain mainly two types of sulfides: pyrite

and arsenopyrite. Since gold is usually finely disseminated in the sulfide

matrix, the objective of biooxidation of refractory gold ores is to break the

Introduction

Chapter 1

sulfide matrix by dissolution of pyrite and arsenopyrite (Mousavi et al.

2006, Thore et al.2007, Ehrlich1988, Schippers et al. 1999).

1.5 Microorganisms involved in bioleaching

Use of microbes in ore leaching offers advantages over the traditional

pyrometallurgical and hydrometallurgical processing. Microbial extraction

procedures are environment friendly. They require lesser amounts of

energy during roasting or smelting and do not produce sulphur dioxide or

other environmentally harmful gaseous emissions (Rawlings, 2002). It

reduces the capital costs dramatically (Devasia and Natarajan, 2004). Also,

microbiological leaching processes, makes it possible to recover metals

from low grade industrial wastes and mineral wastes, which can serve as

secondary raw materials (Siddiqui et al., 2009).

Microbial leaching is a procedure that refers to the natural ability of some

microorganisms to solubilise some mineral constituents of rock or ore. Two

main families of microorganisms are involved in biohydrometallurgy,

chemolithotrophs and heterotrophs. The predominant metal-sulphide

dissolving microorganisms include iron- and sulphur-oxidizing

chemolithrophic bacteria and archaea (Brierley, 1982; Rawlings, 2002,

2005).

There are three main groups of the Chemolithotrophic bacteria. The first of

which are mesophiles, which function near 30 ºC, such as the genera

Acidithiobacillus and Leptospirillum, the second are moderate thermophiles,

which function at elevated temperatures in the range of 40 ºC to 60 ºC, such

as the genus Sulfobacillus, the final are extreme thermophiles, which growth

in the temperature range of 60 ºC to 90 ºC, such as the genera Sulfolobus,

Acidanus, Metallosphaera and Sulfurococcus.

Introduction

Chapter 1

1.5.1 Mesophilic and moderately thermophilic bacteria

1.5.1.1 Acidithiobacillus

Acidithiobacillus is mainly known for its ability to oxidise elemental sulphur

and sulphur containing compounds, but the conditions required (e.g.,

temperature) may vary depending on the physiology of each species. The

genus Acidithiobacillus was proposed by Kelly and Wood (2000) after

reclassification of some species of the genus Thiobacillus. Bacteria of the

genera Acidithiobacillus are normally strict aerobes and are either obligate

or facultative chemolithotrophs or are mixotrophs. They grow in media of

pH values between 0.5 and 10. Some are acidophiles; others can grow at

neutral pH values. Acidithiobacillus are mesophiles having optimum

temperatures for growth at around 30 °C, however, they can grow and

oxidise inorganic substrates within a wide temperature range between 2 to

37 °C .Some Acidithiobacillus are moderately thermophilic bacteria such as

Acidithiobacillus caldus (sulphur-oxidiser). These bacteria oxidise sulphur

above 40 °C and have been used in bioleaching of gold from pyrite and

arsenopyrite (Thore et al.2007,Schippers et al. 1999, Ehrlich

1988).Chemolithotrophic bacteria including the genus Acidithiobacillus, can

oxidise a range of sulphur compounds (i.e. S2-, S°, S2O4, S2O3 2-, SO42-). Some

of the oxidation reactions are listed below.

H2S + 2O2 H2SO4

2H2S + O2 2S°+ 2H2O

2S°+ 3O2 + 2H2O 2H2SO4

Na2S2O3 + 2O2 + H2O Na2SO4 + H2SO4

There are five main species of Acidithiobacillus, which are Acidithiobacillus

thioparus, Acidithiobacillus dentrificans, Acidithiobacillus thiooxidans,

Acidithiobacillus

Acidithiobacillus

Acidithiobacillus

Acidithiobacillus

Introduction

Chapter 1

Acidithiobacillus intermedius, and Acidithiobacillus ferrooxidans. The genus

Thiobacillus can be divided into two groups on the basis of pH values for

growth. The first of these are the species that can grow only in neutral pH

values. The two species that fit this type are A. thioparus and A. dentrificans

state that A. thioparus is responsible for the oxidation of sulphur (N-C-S- +

2O2 + 2H20 → SO4-2 +NH4 + CO2 + 220 kcal/moleO2) and T. dentrificans uses

the same kind of reaction as T. thioparus except that instead of O2, it uses

NO-3 as the terminal electron acceptor (5S + 6KNO3 + 2CaCO3 → 2CaSO4 +

3 K2SO4+ 2CO2 +N2).

The second type of Acidithiobacillus is the species that grow at lower pH

values, for example A. thiooxidans, A. intermedius, and A. ferrooxidans. A.

thiooxidans grows best in an acidic pH, can oxidize only sulphur and can be

used for removal of accumulating sulphur from minerals during indirect

leaching or in direct leaching of minerals in the absence of iron, e.g. ZnS +

2O2 → Zn2+ + SO42- A. intermedius is a facultative chemolithotroph with a

pH range of 3 to 7 and its growth is powered by S2O3 2- as an electron

donor A. ferrooxidans is the dominant organism in the biohydrometallurgy

field. A. ferrooxidans can use either ferrous ions or sulphur as an energy

source and has been studied extensively as the agent of bacterial leaching.

(Syed 2006, Nicolette et al. 2006, Ehrlich 1988, Thore et al.2007, Boon 2001)

1.5.1.2 Leptospirillum

Leptospirillum ferrooxidans (L. ferrooxidans) is a moderately thermophilic

ironoxidiser L. ferrooxidans can oxidize only ferrous ions, but can grow at

higher temperatures than the genus Thiobacillus, which have an optimum

temperature of around 30 °C, and also at stronger acidity levels. L.

ferrooxidans has a higher affinity for Fe2+ than A. ferrooxidans (apparent Km

0.25 mM Fe2+ versus 1.34 mM for A. ferrooxidans, where Km is the Michaelis

constant for reactant Fe2+) and a lower affinity for Fe3+, a competitive

Introduction

Chapter 1

inhibitor. These qualities make L. ferrooxidans suitable for mineral leaching

under conditions of high temperature, low pH, and high Fe3+/Fe2+ ratio.

However, A. ferrooxidans has a faster growth rate than L. ferrooxidans during

the initial stages of a mixed batch culture when the redox potential is low

and is likely to be the dominant iron-oxidizing bacterium in such a system

(Boon 2001). L. ferrooxidans also tolerates higher concentrations of uranium,

molybdenum and silver than A. ferrooxidans, but it is more sensitive to

copper and unable to oxidize sulphur or sulphur compounds by itself .This

can be done together with sulphur oxidizing acidophiles (e.g. A. caldus, A.

ferrooxidans, or A. thiooxidans). For example, the BIOX® plant uses a mixed

culture of Acidithiobacillus and Leptospirillum to oxidize sulphidic refractory

gold concentrate (Syed 2006, Nicolette et al. 2006, Ehrlich 1988, Thore et al.

2007, Bryn 2012, Van Aswegen et al 2007).

1.5.2 Thermophilic bacteria

The fact that higher temperature gives higher dissolution rate led to the

discovery of many thermophilic bacteria and Archaea (Brierley,

2008).Thermophilic iron-oxidizing bacteria can be divided into moderate

and extreme thermophiles. Temperature optimum for growth and metal

leaching are in the range between 65 and 85 °C for extreme thermophiles

and about 40 to 60 °C for moderate thermophiles. A variety of thermophilic

microorganisms, especially Sulfolobus species, have been enriched and

isolated from bioleaching environments. In general the bacteria grow

chemolithotrophically at the expense of iron. Some of the bacteria are

facultative autotrophs, which require the presence of small amounts of

yeast extract, cysteine, or glutathione for growth. A moderately

thermophilic bacterium, Sulfobacillus thermosulfidooxidans, is a gram

positive, nonmotile, spore forming, rod-shaped eubacterium. Its

temperature range for growth is 28-60 °C, with an optimum around 50 °C.

Introduction

Chapter 1

The bacteria can grow autotrophically on Fe (II), S°, or metal sulphide as

energy source. (Thore et al 2007).

Well-studied examples of the extreme thermophiles within acidophilic

iron-oxidizing bacteria are Sulfolobus acidocaldarius and Acidianus brierleyi

(Ehrlich 1988).Both are in the genera Archaebacteria. There are four of these

genera, which are Sulfolobus, Acidanus, Metallosphaera, and Sulfurococcus

(Schippers et al 1999, Ehrlich 1988) they are all aerobic, extremely

thermophilic and acidophilic bacteria oxidizing ferrous ions, elemental

sulphur and sulphide minerals. Their temperature range of growth is

between 55 and 90 °C, with an optimum in the range 70-75 °C. They grow

between pH 1 and 5, with optimum growth around pH 3.0. All the species

are facultatively chemolithotrophic and grow under autotrophic,

mixotrophic or heterotrophic conditions. The organisms grow more rapidly

in the presence of 0.01-0.02 % (w/v) yeast extract and grow

heterotrophically with yeast extract at high concentration (Schippers et al

1999, Ehrlich 1988, Thore et al. 2007).

1.5.3 Mesophilic and moderately thermophilic Archaea

In 1978, Brierley described that application of thermophiles could improve

metal sulphide biooxidation in at least two ways. First, by increased

reaction rates with increasing temperature and second, elevated

temperature increased the extent of metal extraction from certain minerals,

most notably bioleaching of copper from chalcopyrite (CuFeS2). There is

considerable interest today in applying thermophiles to bioleach

chalcopyrite in stirred tank reactors and in bioheap leaching. The discovery

of unusual, extremely thermophilic microorganisms in acidic, sulphidic hot

springs (Brierley, 1978) led to evaluations of these microorganisms for their

ability to oxidize “difficult” minerals such as chalcopyrite and molybdenite

Introduction

Chapter 1

(MoS2) (Olson et al., 2003). Now known as Archaea, these organisms have

expanded biomining options in terms of temperature and metal tolerance.

Three genera of extremely acidophilic euryarchaeotes are currently

recognized: Thermoplasma, Picrophilus and Ferroplasma. There are some

similarities between these Archaea, such as the absence of cell walls in

Thermoplasma and Ferroplasma spp. and the requirement of a complex

organic carbon source (such as yeast extract) to support growth. The metal

sulphide oxidizing species belong to the following genera of Sulfolobales,

Acidianus, Metallosphaera, Sulfolobus, Sulfurococcus, Sulfurisphaera and

Stygiolobus (Table 1.3). All metal sulphide oxidizing, mesophilic and

moderately thermophilic Archaea belong to the genus Ferroplasma. Species

of the genus Ferroplasma are acidophilic Archaea that oxidize Fe2+ ion,

pyrite and other metal sulphides. Cells lack a cell wall and are pleomorphic

(irregular cocci, varying from spherical to filamentous, forming duplex and

triplex forms). Ferroplasma acidiphilum was first described initially as an

obligate chemoautotroph, though it was noted that addition of yeast extract

was necessary for the growth of the original (and type) strain (Golyshina et

al., 2009, Golovacheva at al 1979, Golovacheva at al 1993). Dopson et al.

(2004) failed to detect uptake of 14CO2 by three strains of Ferroplasma

acidiphilum, or by the more acid-tolerant species ‘‘Ferroplasma acidarmanus.’’

Ferroplasma acidiphilum was isolated from a pilot plant bioreactor treating

arsenopyrite/pyrite in Kazakhstan. It oxidizes ferrous iron but not sulphur

and appears to be obligately aerobic. Ferroplasma acidarmanus, was isolated

from acid mine drainage (Rawlings, 2002, Bruneel 2008).

Introduction

Chapter 1

Table 1.1 Optimum and range of growth for pH and temperature of metal

sulfide oxidizing, acidophilic microorganisms (Schippers, 2007).

Introduction

Chapter 1

Table 1.2 Physiological properties of metal sulfide oxidizing, acidophilic

microorganisms (Schippers, 2007).

Introduction

Chapter 1

1.5.4 Heterotrophic microorganisms

A series of heterotrophic microorganisms (bacteria, fungi, and yeast) are

also part of microbial leaching communities. In the case of oxide, carbonate

and silicate ores, the use of Acidithiobacillus sp is limited because of its poor

ability to extract metal. For such ores, research is being carried out on the

use of heterotrophic bacteria and fungi. Of the heterotrophic bacteria, the

genus Bacillus is the most effective in metal solubilization whilst with

regard to fungi the genera Aspergillus and Penicillium are the most

important ones Some examples of the application of heterotrophic

microorganisms in microbial leaching are the removal of silica from bauxite

by using Bacillus mucilaginosus and Bacillus polymyxa, and the solubilization

of aluminum from alumino-silicates by Asperillus niger .The heterotrophic

microorganisms require organic molecules as energy sources. For example,

a medium of culture Bacillus sp. contains 0.5 % (w/v) sucrose, essential

salts, yeast extract as nitrogen source and CaCO3 .In addition the

microorganisms also require maintenance of a pH nearer neutrality and a

mesophilic temperature. The extraction of a mineral from an ore by

heterotrophic bacteria involves enzymatic reduction. Several heterotrophs

can contribute to metal extraction by the excretion of organic acids, for

example Bacillus megaterium excrete citrate, Pseudomonas putida excrete

citrate and gluconate, or Aspergillus niger excrete citrate, gluconate, oxalate,

malate, tartrate and succinate (Thore et al. 2007).

As may be expected, due to mineral-rich environments, the biomining

microbes are extremely tolerant to a wide range of metal ions with

considerable variation within and between species (Baker et al., 2007;

Dopson et al., 2001; Rawlings, 2005)

Introduction

Chapter 1

Table 1.3 Optimum and range of growth for pH and temperature of acidophilic heterotrophic microorganisms (Schippers, 2007).

Introduction

Chapter 1

1.6 Microbial community analysis

To control and optimize metal bioleaching, quick and reliable methods to

identify and quantify single species in complex bioleaching communities

are needed. Microbial communities can be analyzed using microscopic

techniques, cultivation techniques, immunological techniques and nucleic-

acid based molecular techniques (Harneit et al. 2006, Thore et al 2007).

1.6.1 Microscopic techniques

Total cell numbers can be determined by counting cells under a

fluorescence microscope after application of nucleic acid-staining

fluorochromes (e.g. SybrGreen, acridine orange, DAPI). However, these

fluorochromes bind unspecifically to nucleic acids and thus, do not provide

information on the viability of the cells. Potentially, a major part of the

counted cells could be dormant or even dead and yet retain stainable DNA

(Schippers, 2007).

1.6.2 Cultivation techniques

Using classical cultivation techniques, i.e., the most-probable-number

(MPN) cultivation method or the dual-layer agarose plate technique,

acidophilic autotrophic Fe (II) and sulfur oxidizing bioleaching

microorganisms have been enriched from bioleaching communities.

However, cultivation techniques allow only a subset of the whole microbial

community can be detected, though media have been designed to select

different groups on the basis of their physiologies. Also, cultivation

techniques are labor-intensive and results are only available after

incubation times of several days or even weeks which do not allow a

monitoring of bioleaching operations (Schippers, 2007).

Introduction

Chapter 1

1.6.3 Immunological techniques

Immunoassays with specific antibodies have been applied to enumerate A.

ferrooxidans and other bioleaching microorganisms. However, their

application is time-consuming and requires thorough knowledge of the

microbes occurring in the bioleaching operation (Schippers, 2007).

1.6.4 Nucleic-acid based molecular techniques

Nucleic-acid based molecular techniques have been extensively used to

identify and quantify microorganisms in the environmental sample since

last decade. They are based on the DNA extraction from a culture of, a

bioreactor or an environmental sample, followed by the amplification of

DNA by Polymerase Chain Reaction (PCR), and finally an analysis of the

DNA amplification products. In most of the cases, the 16S ribosomal RNA

gene (16S rDNA) of prokaryotes (Bacteria and Archaea) is targeted, but

also functional genes coding for key enzymes of particular metabolic

interest have been analyzed (e.g. the rus gene coding for rusticyanin in At.

ferrooxidans) (Thore et al 2007).

To study the biodiversity and to identify new species, PCR products can be

cloned and the 16S rRNA gene of the various clones in the clone library can

be sequenced. The similarities of the sequences can then be shown in a

phylogenetic tree, to address the phylogenetic affiliation of the

microorganisms in the sample. This approach has been chosen to analyze

the microbial communities in natural, acidic environments and bioleaching

operations (Thore et al 2007, Amouric 2011).

Alternatively to clone libraries, the PCR products can be separated in

denaturing gradient gel electrophoresis (DGGE) which allows a separation

of DNA fragments of the same length but different base-pair sequences.

Bands in the DGGE can be excised and the 16S rRNA gene sequenced to

Introduction

Chapter 1

address the phylogenetic affiliation of the organisms. This method has been

applied to bioleaching communities as well (Thore et al 2007).

The DNA fingerprinting techniques RFLP (restriction fragment length

polymorphism) and ARDREA (amplified ribosomal DNA restriction

enzyme analysis) have been applied to identify bioleaching organism.

These techniques involve the digestion of the PCR product with one or

more restriction enzymes to produce fragments of different sizes that are

resolved on appropriate gels. DNA fingerprinting techniques are less

labour intensive but allow only the identification of known organisms

(Thore et al 2007).

RAPD (randomly amplified polymorphic DNA) and rep-APD , SSCP

(single stranded conformation polymorphism, analysis of the PCR-

amplified 16S-23S rRNA gene intergenic spacer and the use of microbe-

specific PCR primers are also another PCR based techniques for the

identification of bioleaching organisms (Thore et al 2007).

A highly sensitive technique used in environmental microbiology to

quantify different phylogenetic groups and genera is real-time PCR

(quantitative PCR). The technique is based on the online fluorescence

detection of PCR products and allows the rapid detection and

quantification of gene sequences without the need for labor-intensive post-

PCR processing Real-time PCR has been applied to quantify Bacteria and

Archaea oxidizing metal sulfides in mine heaps .Protocols have been

developed to quantify single species in bioleaching communities(Thore et

al 2007).

RNA can be quantified after application of an additional reverse

transcription step (real-time RT-PCR), which allows quantification of gene

expression in environmental microbiology. Concerning bioleaching

organisms, gene expression has been studied in pure cultures of

Introduction

Chapter 1

Acidithiobacillus ferrooxidans. The expression of the rus gene coding for

rusticyanin, a protein involved in Fe (II) oxidation, and of several

additional genes important for Fe (II) and sulfur oxidation as well as for

CO2-fixation has been measured by real-time PCR. The rus gene in A.

ferrooxidans is particularly expressed during Fe (II) and metal sulfide

oxidation rather than during sulfur oxidation. Rusticyanin may not be

expressed in all strains classified as A. ferrooxidans. However, the rus gene

should be a good target to monitor the abundance and activity of A.

ferrooxidans in biomining (Carlos 2007).

FISH is also a powerful technique to quantify microbial cells in

environmental samples. The technique targets ribosomal RNA (rRNA) and

so is indicative of actively metabolizing bacteria. FISH has been

successfully applied to quantify acidophilic Fe(II) oxidizing Acidithiobacillus

Leptospirillum, Ferroplasma and other microorganisms in acid mine drainage

environments and in bioleaching operations . A drawback of the technique

is that a sufficient content of cellular ribosome is prerequisite for its

successful application. Recently, modified FISH protocols (CARD-FISH =

Catalyzed Reporter Deposition- Fluorescence in Situ Hybridization) have

been published which allow the detection of less active cells in

environmental samples as well. So far, these protocols have successfully

been applied to quantify Bacteria and Archaea oxidizing metal sulfides in

mine heaps (Schippers, 2007).

1.7 Metagenomics and comparative genomics

A. ferrooxidans was the first biomining microorganism to have its genome

almost entirely sequenced although the annotation of all its genes has not

yet been published. Initial studies on the isolation, cloning and sequencing

of mostly individual chromosomal genes and plasmids of A. ferrooxidans

and expression of some of them in Escherichia coli to generate functional

Introduction

Chapter 1

proteins have been reviewed.The genomic contexts of some of the

rhodanase-like genes and the determination of their expression at the

mRNA level by using macroarrays suggested their implication in sulfur

oxidation and metabolism, formation of Fe–S clusters or detoxification

mechanisms. Several of the putative rhodanase genes were successfully

isolated, cloned and over expressed in E. coli (Carlos 2007, Bruton 2000).

Study of “Pho regulon” induced during phosphate starvation and is

involved in metal resistance in A. ferrooxidans showed that heavy metals

stimulate polyphosphate hydrolysis and the metal–phosphate complexes

formed would be transported out of the cell as part of a possibly functional

heavy metal tolerance mechanism in this bacterium (Carlos 2007).

The regulation of the expression of the “rus operon” which encodes two

cytochromes c, a cytochrome oxidase and rusticyanin was also studied.

Using an entirely bioinformatic approach other researchers found

candidate genes potentially involved in several other functions such as

metal resistance, amino acid biosynthesis pathways and others .In addition,

a cluster of five genes proposed to be involved in the formation of

extracellular polysaccharide (EPS) precursors via the Leloir pathway have

been identified recently in A. ferrooxidans .A large set of genes required

for pilus formation (pilA-D, pilS and pilR) and exopolymeric substances

(EPS) for attachment and a quorum sensing loci which are associated with

biofilm formation in other microorganisms have been identified. Potential

pathways were identified for sulfation of extracellular metabolites that may

possibly be involved in cellular attachment to pyrite, sulfur and other solid

substrates.

Metabolic modeling provides an important preliminary step in

understanding the unusual physiology of this extremophile especially

given the severe difficulties involved in its genetic manipulation and

Introduction

Chapter 1

biochemical analysis. However, all these predictions will have to be

demonstrated experimentally (Corinne et al 2006, Blanca et al. 2008,

Schippers et al 1999).

The first DNA macroarrays used with A. ferrooxidans have been very

useful for the partial study of gene expression in this microorganism.

However, in the near future, the use of microarrays based on the entire

genome of A. ferrooxidans and other microorganisms will allow having a

nearly complete view of gene expression of the members of the microbial

community under several biomining conditions, helping to monitor their

physiological state and adjustment made during the bioleaching process

(Blanca et al. 2008, Ana et al. 2003, Sambrook et al 2001).

Metagenomics is the culture-independent genomic analysis of microbial

communities. In conventional shotgun sequencing of microbial isolates, all

shotgun fragments are derived from clones of the same genome. However,

if the genomes of an environmental microbial community are to be

analyzed (Fig.1.1), the ideal situation is to have a low diversity

environment. The extremely acidic conditions of the biofilm (pH about 0.5)

and relatively restricted energy source combine to select for a small

number of species in bioleaching environments. Furthermore, the

frequency of genomic rearrangements and gene insertions or deletions was

very low, making this community ideal for testing these new culture-

independent genomic approaches in the environment. (Carlos 2007,

Bonnefoy 2011).

Introduction

Chapter 1

Fig.1.1 Proteomic and metaproteomic analysis of biomining

microorganisms and their relationship with genomics and Metagenomics

(Carlos 2007)

Chapter 1

1.8 Factors affecting bacterial leaching

The rate and efficiency of bacterial leaching of mineral ores depends upon

a number of different factors. Brandl, (2001) has summarized these factors,

which can be seen in table 1.4.

Table 1.4 Factors affecting bacterial leaching

2001).

Physico-chemical as well as microbiological factors of the leaching

environment affects bioleaching rates and efficiencies. Moreover, the

properties of the mineral ores and the manner in which they are processed

are also significant since they also affect bioleac

The influence of different microbiological, mineralogical, physicochemical

and process parameters on the oxidation of mineral ores has been reviewed

by many researchers. Unfortunately whilst much has been published in

this field, results are sometimes conflicting and often the conditions used

Introduction

Factors affecting bacterial leaching



which can be seen in table 1.4.

Factors affecting bacterial leaching (Pradhan et al. 2008, Bra

chemical as well as microbiological factors of the leaching



are also significant since they also affect bioleaching rates and efficiencies.




d, results are sometimes conflicting and often the conditions used



(Pradhan et al. 2008, Brandl

chemical as well as microbiological factors of the leaching



hing rates and efficiencies.




d, results are sometimes conflicting and often the conditions used

Introduction

Chapter 1

are not described in much detail. Given the inherent variability of the

systems used it is difficult to get consistent results. Some of the main

parameters affecting bioleaching are 6discussed in more detail below.

1.9 Biomining techniques

There are two main types of processes for commercial-scale microbially

assisted metal recovery. These are irrigation-type and stirred tank–type

processes (Fig. 1.2). Irrigation processes involve the percolation of leaching

solutions through crushed ore or concentrates that have been stacked in

columns, heaps, or dumps. There are also several examples of the irrigation

of an ore body in situ, that is, without bringing the ore to the surface.

Stirred tank–type processes employ continuously operating, highly aerated

stirred tank reactors. One feature of both types of processes is that, unlike

most other commercial fermentation processes, neither is sterile, nor any

attempt is made to maintain the sterility of the inoculum (Rawlings, 2002).

Fig. 1.2 Major process options used in biomining (Johnson, 2010).

Introduction

Chapter 1

1.9 Bioleaching technology

Bioleach heap leaching is a sub-category of an irrigation based biomining

processes. Irrigation based processes can be categorised based on the type

of resources to be processed dump leaching, heap leaching and in situ

leaching. Heap leaching deals with the newly mined materials

(intermediate grade oxides and secondary sulphides deposited in the form

of a heap on an impervious natural surface or a synthetically prepared pad

leached with circulation, percolation, and irrigation of the leaching

medium (Pradhan et al., 2008). Primary sulphides like chalcopyrite are also

suitable for this type of leaching.

Bioleach heap technology is emerged as the predominant technology route

for the recovery of metals from low-grade ores. In terms of revenue

generated, it is the most significant industrial application of biomining

(Rawlings, 2002). The technology has been in use since the 1960’s for the

acid leaching of copper oxide minerals, and since the 1970’s for the cyanide

leaching of gold and silver.

The static bioleaching techniques are based on the principle of circulating

water and air through heaps of ore coarsely fragmented to activate the

growth of microorganisms that amplify the oxidation of the sulphidic

minerals (Morin et al., 2006). This process involves stacking crushed ore

into piles constructed on an impermeable layer fitted with a solution

drainage system, or arranged on a slope to facilitate drainage. In many

cases the ore is agglomerated through tumbling with acid and/or irrigation

solution prior to stacking.

1.9.1 Basic Heap Design and the Importance of Heat Generation

Heap bioleaching technology relies on very similar operational heap

configurations, and civil and geotechnical engineering as for conventional

Introduction

Chapter 1

acid heap leaching. The basic configuration comprises an impermeable

leaching pad upon which the material to be leached is stacked. The PLS is

collected via a system of drainage pipes contained in a 1–2-m inert

overburden material at the bottom of the heap. An aeration distribution

pipe system is used to supply air into the heap.The PLS typically reports to

intermediate ponds and eventually to conventional solvent extraction and

electrowinning, while the raffinate is added to the top of the heap via a

network of irrigation pipelines. The leach material can either be run-of-

mine or crushed and agglomerated ore. The microbial reactions described

previously occur at the ore–liquid interface and in the solution and result

in the release of copper from the mineral to the solution phase. The key

distinguishing process feature of marginal metal sulfide heaps is the

achievement of elevated heap temperature (above 55ºC) in order to

facilitate effective recovery. The attainable temperature is the net result of

heat-generation and heat-loss/heat-retention factors. Heat generation is

mainly the result of microbial oxidation of sulfur to sulfate (Petersen and

Dixon 2002). The two main factors that govern the overall capacity for heat

generation are therefore the available sulfur content of the ore and

microbial activity.

Introduction

Chapter 1

Fig. 1.3 Whole-ore biooxidation heap flow diagram (Thomas et al. 2007) 1.9.2 Physical, chemical and biological factors affecting bioheapleaching

Although heap leaching appears to be a very simple process in concept, the

sub processes taking place within the ore bed are rather complex and their

interactions not yet fully understood.

To unravel the processes underlying heap bioleaching it is useful to

distinguish between phenomena taking place at different scales within the

heap (Peterson and Dixon, 2007a). Beginning at the heap scale, we can

distinguish a number of transport effects. More specifically these include:

(1) Solution flow: In unsaturated, coarsely granular packed beds solution

generally flows along tortuous pathways but remains stagnant in pores

and crevices between particles. This strongly influences heap

performance in terms of reagent delivery and product removal from the

reaction sites within the ore particles.

Introduction

Chapter 1

(2) Heat flow: Heat of reaction is significant in sulphide leaching. It is

transported through the heap downward as sensible heat with the

flowing solution and upward as latent heat with the flow of humid air.

Depending on air and solution flow rates, heaps can assume certain

temperature profiles and judicious manipulation of these variables

allow a certain degree of control.

(3) Gas flow: Although gas flow is usually well distributed in aerated

heaps, ensuring ample supply of oxygen throughout, the supply of CO2

may be limited under certain conditions. In non-aerated heaps O2

availability may also be limited, and gas distribution patterns are

complex.

Fig.1.4 A heap indicating the major heap-scale transport effects (Jochen et al. 2007)

The next level, at the meso-scale, represents a cluster of particles within a

heap bed. Here, two processes contribute to the overall rate of leaching:

(1) Diffusion transport: Diffusion is the main mode of transport of

dissolved constituents from and to the moving solution into pore spaces

between particles, and into cracks and fissures within particles. The

effect of pore diffusion on overall kinetics is determined by the length

Introduction

Chapter 1

of the diffusion path, which can be significant for systems with poor

solution distribution (Peterson and Dixon, 2007b)

(2) Microbial population dynamics: This encompasses the complex

interactions of different types of microorganisms in the liquid phase

and on the mineral surface. It includes the growth behaviour of each

strain as a function of temperature and concentration of dissolved

constituents (acid, Fe2+ and Fe3+ iron, O2, CO2 as carbon source, etc.),

and any synergies between these and the concomitant iron and sulphur

oxidation reactions.

Fig.1.5 A cluster of particles within a heap bed (left) and the microbial colonies inhabiting the moist pore space between particles (right) (Peterson and Dixon 2007) (3) Grain topology

As the rate of leaching is usually proportional the total mineral surface

available to leaching, a further degree of complexity is added by the

distribution of grains of different size and accessibility within the ore. This

aspect is referred to as “mineral topology” Fig.1.6 illustrates, in simplified

form, the reaction network and associated mass exchanges found in heap

bioleaching. For this reaction to proceed at any particular location in the

heap, oxygen must be transferred from the gas phase into the solution

phase. Associated with this is the transport of air at the heap scale as well

as the gas–liquid mass transfer kinetics at the solution–gas interface. Acid,

Introduction

Chapter 1

on the other hand, must be supplied with the solution fed to the heap and

then transported to reaction sites by solution flow and interparticle

diffusion. Given the reagent concentrations, microbial population

characteristics and prevailing temperature, the oxidation reactions will

proceed at a certain rate. The ferric iron generated will have to migrate by

diffusion to mineral grains, which may be located deep inside a particle far

from the location of the microbial oxidation. The mineral is now oxidized

at a rate again depending on prevailing concentrations and temperatures.

The released heat of reaction determines, in interaction with all global gas

and liquid flow phenomena, the local temperature.

Finally, the dissolved copper migrates through the particle and stagnant

solution into the flowing solution to be transported out of the heap.

(Peterson and Dixon 2007)

Fig.1.6 The mineral biooxidation reaction-transport network between gas, liquid and solid phases in heaps (Peterson and Dixon 2007)

Finally, the smallest scale at which sub-processes of heap bioleaching need

to be analyzed is that of the individual mineral grain. Here leaching is

governed by the electrochemical interactions between the mineral grains

and reagents in solution.

Introduction

Chapter 1

1.9.3 Microbiology of bioleach heap

A wide variety of microorganisms consisting mainly of bacteria and

Archaea are found in natural leaching environments such as bioheap. The

majority of known acidophilic microorganisms have been isolated from

such natural environments. Understanding the microbiology of a bioheap

is important for advancement in commercial bioheap applications. Such

knowledge will increase the applications to various types of ores as well as

to the diversity of mineral deposits that can be processed by bioheap

technology. It will also enable the better control of conditions to improve

upon the leaching rates, metal recoveries and cost of production. A limited

comprehension is available of what actually occurs in a full-scale

microbiologically operated bioheap, despite the commercial achievement

in the copper ore bioheap leaching (Pradhan et al., 2008). Although

oxidative dissolution of simple and complex sulphide ores and

concentrates may be mediated by pure cultures of iron-oxidizing

acidophiles, as has often been described in laboratory studies, axenic

cultures are never found in actual biomining operations. Consortia of

microorganisms with synergistic (and sometimes complimentary)

metabolic physiologies have been identified in all commercial scale

systems that have been examined (Johnson, 2010).

These have tended to show, as would be expected, that microbial diversity

is far greater in heaps and dumps, which are highly heterogeneous and

uncontrolled environments, than in stirred tanks where conditions are far

more homogeneous. Both operate essentially as ‘‘inorganic’’ systems in

that, while inorganic nutrients (ammonium and phosphate) are added to

stimulate microbial activity, organic carbon is not. This, together with the

primary energy sources available being the sulphide minerals themselves,

means that the dominant prokaryotes present are invariably chemo-

Introduction

Chapter 1

autotrophic iron and sulphur oxidizers. However, organic carbon derived

from living (as exudates) and dead (as lysates) primary producers can

accumulate in leach liquors, and can support the growth of mixotrophic

and heterotrophic acidophiles. Hence, it is possible, as noted by Johnson

and Hallberg (2009), to divide micro-organisms in biomining operations

into three: (i) ‘‘primary acidophiles,’’ iron-oxidizing prokaryotes that

generate ferric iron and are responsible for initiating mineral dissolution;

(ii) ‘‘secondary acidophiles,’’ sulphur-oxidizing acidophiles that generate

sulphuric acid from reduced sulphur produced during mineral dissolution

and help maintain pH conditions that are conducive for the biooxidation of

sulphide minerals; (iii) ‘‘tertiary acidophiles,’’ heterotrophic and/or

mixotrophic microorganisms that degrade soluble organic carbon wastes

originating from the autotrophs, thereby detoxifying the environment for

some of the more organic-sensitive primary and secondary prokaryotes.

As shown in Fig. 1.6, Iron-oxidizing prokaryotes, either attached to the

mineral surface (Group Ia) or free-swimming (Group Ib) have the primary

role in mineral dissolution in that they generate ferric iron, the chemical

oxidant that attacks and degrades the mineral. Sulphur-oxidizing

prokaryotes (Group II) have a secondary role whereby they oxidize

reduced sulphur produced during pyrite dissolution, producing sulphuric

acid which maintains the required acidic conditions.

Introduction

Chapter 1

Fig. 1.7 Schematic representation of the roles of and interactions between

different physiological groups of acidophilic prokaryotes during the

oxidative dissolution of a representative sulphide mineral (pyrite; FeS2)

(adapted from Johnson and Hallberg, 2009).

Both the primary and secondary micro-organisms are predominantly

autotrophs and leak dissolved organic compounds (DOC) into their

environment. This is utilized by tertiary (Group III) prokaryotes

(heterotrophic and facultatively autotrophic acidophiles); mineralization of

DOC helps maintain a suitable environment for the autotrophic members

of the consortium.

Okibe and Johnson, (2004) evidently demonstrated the importance of

microbial consortia and interactions for optimizing sulphide mineral

dissolution in laboratory studies. As recently reviewed by Kondrat et al.

(2012), the microbial analysis of commercial-scale bioprocessing operations

have shown, in all cases so far reported, that bacteria and/or Archaea that

fulfil these primary, secondary and tertiary roles are present.

Introduction

Chapter 1

1.9.4 Present of heapbioleaching

A substantial number of heap-leaching metal recovery processes are in

operation, some for many years (Rawlings et al., 2003). ‘Thin layer’

leaching, where crushed and acid-cured ore is stacked 2 to 3 meters high

and then rinsed, was first applied at the Lo Aguirre Copper Mine in Chile

in 1980, and is regarded as the first instance of heap bioleaching. A further

significant milestone in heap bioleaching was the introduction of forced

aeration for the heap bioleaching of secondary copper sulphide ores at the

Girilambone Copper Mine in Australia in 1993 (Gerick et al., 2009).

As estimated by Brierley (2008) heap bioleaching of copper accounts for

some 7% (about 106t/year) of the total global annual production of

approximately 1.7×107 t of copper. This does not include copper recovered

using dump bioleaching processes. It is estimated that if dump bioleaching

is included some 20-25% of the world’s copper production is attributable to

bioleaching. Examples of very large copper leaching operations are those

by Sociedad Contractual Minera El Abra and the Codelco Division

Radimiro Tomic in Chile producing 225,000 and 180,000 tons of Cu per

annum, respectively (Pradhan et al., 2008). An excellent example of a

current commercial bioleach application is the Quebrada Blanca operation

in northern Chile (Brierley and Brierley, 2001) located on the Alti Plano at

an elevation of 4400 m under the cold temperatures and low oxygen partial

pressure of high altitudes (Gerick et al., 2009).

GeoBiotics, LLC has developed and patented several technologies for

biooxidizing or bioleaching of sulphide ores and concentrates in an

engineered heap environment. The two principal technologies are the

GEOCOAT™ and GEOLEACH™ processes. The process entails coating

refractory sulphide gold concentrates onto a screened support rock or ore.

Biooxidation pre-treatment takes place in a stacked heap configuration. The

Introduction

Chapter 1

oxidized concentrate is removed from the support rock for gold extraction

by conventional metallurgical processes. If the support rock is also a

refractory ore, this can also be leached following biooxidation to recover

additional gold values. The process can be applied to the biooxidation of

refractory sulphide gold concentrates and to the bioleaching of copper,

nickel, cobalt, zinc, and polymetallic base metal concentrates (Pradhan et

al., 2008). The GEOCOAT process has also been tested for bioleaching

copper from chalcopyrite concentrate using thermophilic microorganisms.

The GEOLEACH technology is designed to maximize heat conservation

through careful control of aeration and irrigation rates. Both the processes

are simple, robust, and ideally suited to operation in remote locations.

Fig. 1.8 GeoBiotics technology portfolio

Many heap-leach processes have targeted the extraction of marginal ores

that are not suitable for the production of concentrates or smelting.

Development of heap-leaching technology has been largely engineering-

Introduction

Chapter 1

focused rather than microbiology-focused. The main advances have

therefore come from improved acidification methods, solution

management and heat containment. Much of the progress in heap leaching

can be attributed to research into the modelling of leach liquor distribution,

oxygen diffusion and heat management (Bouffard and Dixon, 2009;

Petersen and Dixon, 2002). BIO SHALE Project has been running since 2004

in Finland for the extraction of nickel from black shale (Talvivaara

deposits) using bioheap leaching process (Watling, 2008). Recently in 2011,

a high temperature bioheap project has been called off, on which Nicico

and Mintek were collaboratively working in Iran for treatment of

Sarcheshmeh copper ore (www.mintek.co.za, last accessed on May 23,

2012). It was operating at the Sarcheshmeh copper complex in southern

Iran producing some 170,000 tons of copper a year.

Table 1.5 Industrial heap bioleaching operations for secondary copper

ores and mixed oxide/sulphide ores (Brierley, 2008; Gerick et al., 2009;

Watling, 2006).

Industrial heap bioleach plant and

location/owner

Cathode

copper

production

(t/a)

Years of

operation

Lo Aguirre, Chile/Sociedad Minera

Pudahuel Ltda. 15000

1980-1996

(deposit

depletion)

Mount Gordon (formerly Gunpowder),

Australia/Western Metals Ltd. 33000 1991-Present

Introduction

Chapter 1


location/owner

Cathode

copper

production

(t/a)

Years of

operation

Mt. Leyshon, Australia/(formerly

Normandy Poseidon) 750

1992-1995

(stockpile

depleted)

Cerro Colorado, Chile/BHP Billiton 115000 1993-present

Girilambone, Australia/Straits Resources

Ltd & Nord Pacific Ltd. 14000

1993-2003 (ore

depleted)

Ivan-Zar, Chile/Compañía Minera Milpro 10000–

12000 1994-Present

Punta del Cobre, Chile/Sociedad Punta

del Cobre, S.A. 7000-8000 1994-Present

Quebrada Blanca, Chile/Teck Cominco

Ltd. 75000 1994-present

Andacollo Cobre, Chile/Aur Resources,

del Pacifico & ENAMI 21000 1996-present

Dos Amigos, Chile/CEMIN 10000 1996-present

Skouriotissa Copper Mine (Phoenix pit),

Cyprus/Hellenic Copper Mines 8000 1996-present

Zaldivar, Chile/Barrick Gold Corp. 150000 1998-present

Lomas Bayas, Chile/XSTRATA plc 60000 1998-present

Introduction

Chapter 1


location/owner

Cathode

copper

production

(t/a)

Years of

operation

Cerro Verde, Peru/FreeportMcMoran &

Buenaventura 54200 1997-present

Lince II, Chile 27000 1991-present

Monywa, Myanmar/Ivanhoe Mines Ltd,

Myanmar No.1 Mining Enterprise 40000 1998-present

Nifty Copper, Australia/Straits

ResourcesLtd. 16000 1998-present

Equatorial Tonopah, Nevada/Equatorial

Tonopah, Inc.

25000

(projected) 2000-2001 Failed

Morenci, Arizona/FreeportMcMoran 380000 2001-present

Lisbon Valley, Utah/Constellation Copper

Corporation

27000

(projected) 2006-present

Jinchuan Copper, China/Zijin Mining

Group Ltd. 10000 2006-present

Spence, Chile/BHP Billiton 200000 Commissioned

2007

Whim Creek and Mons Cupri,

Australia/Straits Resources 17000 2006-present

Escondida, Chile 200000 -

Introduction

Chapter 1


location/owner

Cathode

copper

production

(t/a)

Years of

operation

Toquepala, Peru 40000 -

S&K Copper, Monywa, Myanmar 40000 1999-present

Phoenix deposit, Cyprus 8000 1996-present

The HydroZinc™ process of Teck Cominco and the BioHeap™ process

developed by Pacific Ore Technology for the heap bioleaching of zinc and

nickel-copper ores, respectively, are reported to be in an advanced stage of

development. Large-scale trials have demonstrated that high recoveries of

nickel, copper and cobalt can be achieved using the BioHeap™ proprietary

bacteria and patented processes. The process is also applicable to other

sulphide ores such as zinc, polymetallic and refractory gold ores

(Chadwick, 2007).

In addition, a substantial amount of unpublished operational research has

been carried out by companies such as Newmont Mining, Phelps Dodge,

BHP Billiton, Mintek, POT-Titan and Rio Tinto (Gonzalez et al 2003).

1.9.5 Limitations of heapbioleaching

Bioleach heap technology of metal sulphides is a standalone technology

that is

• robust and proven under different climatic conditions for oxides and

secondary sulphides;

• flexible — heap engineering and management can accommodate site

peculiarities in remote localities; suited to small deposits;

Introduction

Chapter 1

• simple—a technology that can be communicated to non-scientific

personnel;

• low cost — stacking, irrigation, aeration, solution collection are all

basic infrastructure;

However, this technology to date is still suffering from low metal

extraction rates and low ultimate metal recoveries. The drawbacks listed

below may outweigh the lower capital and operating costs of heap

processes (Brierley, 2010).

• Good permeability and porosity is required to allow leaching solution

to percolate through the heap and to ensure adequate wettability and

reagent access to all reaction zones. However, the fine particles

generated either during crushing or by the biochemical activity during

the operation cause the plugging of heaps. This leads to very low

recovery of metals due to a lack of solution contact with particles and

shortfall of aeration for bacterial growth. In addition, the presence of

‘‘dead zones’’, may be due to the gangue mineral reactivity (i.e. silicate

polymers, gelatinous materials coming from reaction between silica and

sulphuric acid, that form an impervious layer), further decrease the

metal recovery (Palencia et al., 2002).

• Heap reactors are cheaper to construct and operate and are therefore

more suited to the treatment of lower grade ores than are stirred-tank

reactors. However, heap reactors are more difficult to aerate efficiently

and to manage. Moreover, the rates of oxygen and carbon dioxide

transfer that can be obtained are low, and extended periods of

operation are required in order to achieve sufficient conversions

(Pradhan et al., 2008).

• In heap leaching, pH gradients occur at both the micro and macro scale.

Ores are usually a mixture of mineral and gangue material.

Introduction

Chapter 1

Biooxidation of the mineral leads to acid production, whereas most

gangue materials like quartz, mica, chlorite, potassium and calcium-

feldspar are acid-consuming. In practice it is difficult to maintain the

solution pH within the range of 1.8–2.2. When the pH rise above 2.5,

ferric iron precipitation might occur, this coats the mineral surfaces and

reduces the rate of metal solubilisation.

• Similarly, the effective provision of nutrients is more complicated in

heap reactors. The most important added nutrient is ammonium but, in

areas in which the pH is too high, the addition of ammonium can result

in the formation of jarosite precipitate, which removes ferric iron from

solution and also coats mineral surfaces. Once the jarosite is formed, it

precipitates on the mineral surfaces and decreases metal oxidation

mediated by acidophilic microorganisms (Pradhan et al., 2008).

• For example, pyrrhotite, which may be present in complex ores,

releases substantial amounts of heat rather quickly and consumes acid

creating operating conditions that must be carefully managed in order

to effectively utilize microbial leaching.

• Heaps are usually irrigated with raffinate (recycled leach solution from

which the metal has been removed) and a gradual build up of

inhibitory ions, such as sulphates and aluminium, has to be avoided.

• Heap reactors are also more difficult to inoculate than are tank reactors.

Different microbes exhibit different mineral adsorption isotherms, and

this might cause uneven initial microbial species distribution within a

heap.

• When a heap is constructed, fine material is agglomerated to the coarse

particles using acid and a microbial inoculum can be added at this

stage. A disadvantage of this option is that if the levels of acid used

during agglomeration are too high, cell viability can be reduced.

Introduction

Chapter 1

• Even in carefully designed heap reactors, larger particle sizes, less

effective aeration and reduced process control make the process less

efficient. Due to this, the biomineralization process is extended to

months rather than days.

1.10 Biomining in India – at a glance

Besides a host of minor and atomic minerals, India produces 65 minerals,

which are well-scattered in small reserves. Biomineral processing holds

great potential for such small reserves. In India, in spite of cheap labour,

liberalised market, vast consumer base and strong foundation of

hydrometallurgy, there exists a wide gap between the existing potential

and the potentials to be exploited for economic metal growth. In India,

bioleaching holds immense potential. Zinc extraction from concentrates at

laboratory scale was demonstrated 32 years back at Karnataka Regional

Engineering College (KREC) by Dave and Natarajan (Dave et al., 1981,

1982). Manganese leaching was carried out at Agharkar Research Institute

by Agate et al. (1974) Extensive academic research is being carried out at

Department of Microbiology, Gujarat University since 30 years by Dave

and his students. Cu and Zn were extracted at pilot scale bioreactors from

Cu-Pb-Zn bulk concentrate at Department of Microbiology, Gujarat

University by Tipre and Dave (2004)( Tipre 1999). Considerable

metallurgical research is going on at Institute of Minerals and Materials

Technology (IMMT), (formerly Regional Research Laboratory),

Bhubneshwar. The National Metallurgical Laboratory (NML), Jamshedpur,

is also involved in active research on biomining of base metals from sea

nodules along with uranium bioleaching (Abhilash and Pandey, 2011;

Abhilash et. al., 2011; Mehta et al., 2010). The only demonstration

biooxidation plant for gold recovery had been erected at Hutti gold mine,

India in 2002.

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