**2. Mechanisms of bioleaching**

The two majorly known mechanism in bacterial leaching are direct mechanism (involves physical contact of the organism with the insoluble sulphide) or hypothesized enzymatic reaction taking place between an attached cell and the underlying mineral surface which is independent of indirect mechanisms and it is where reduced sulfur dissolution takes place [16], it is only the direct attack of the bacteria can lead to leaching. Check the following reactions.

$$2\text{FeS}\_2 + 7\text{O}\_2 + 2\text{H}\_2\text{O} \to 2\text{FeSO}\_4 + 2\text{H}\_2\text{SO}\_4.\tag{1}$$

Indirect (involves the ferric-ferrous cycle) or it is a mechanism of sulfide oxidation involves non-specific oxidation of surfaces by Fe3+ that is generated by microorganisms that oxidize iron or oxidation of mineral by ferric ions [16]. The attached cells of bacterial oxidize the surface using either of the two mechanisms [9, 11, 14]. The reaction below shows oxidation of iron.

$$\text{FeS}\_2 + \text{Fe}\_2\text{(SO}\_4\text{)}\_3 \rightarrow \text{3FeSO}\_4 + 2\text{S}^0. \tag{2}$$

Minerals are broken due to the attack to their constituents, that results energy production for the microorganism. This energy production or oxidation passes through intermediates reaction processes. Two mechanisms have been proposed for the oxidation, viz. thiosulphate mechanism and polysulfide mechanism. Thiosulfate mechanism includes acid-insoluble metal sulfides like pyrite (FeS2) and molybdenite (MoS2) and polysulfide mechanism includes acid-soluble metal sulfides like chalcopyrite (CuFeS2) or galena (PbS) [15]. In thiosulfate mechanism, the attack of ferric ion on acid insoluble metal sulfides brings about solubilization via thiosulfate as an intermediate and sulfates as end product. The breaking reaction shown below.

$$\text{FeS}\_2 + \text{6Fe}^{3+} + \text{3H}\_2\text{O} \rightarrow \text{S}\_2\text{O}\_3^{2-} + \text{7Fe}^{2+} + \text{6H}^\*. \tag{3}$$

$$\text{S}\_2\text{O}\_3^{2-} + 8\text{Fe}^{3+} + 5\text{H}\_2\text{O} \rightarrow 2\text{SO}\_4^{2-} + 8\text{Fe}^{2+} + 10\text{H}^\*. \tag{4}$$

In polysulfide mechanism, a combined attack of ferric ion and protons on acidsoluble metal sulfides causes the solubilization with sulfur as an intermediate in its elemental form which can be oxidized to sulfate by sulfur-oxidizing microbes that the reaction is shown below [17].

$$\text{MS} + \text{Fe}^{3+} + \text{H}^{+} \rightarrow \text{M}^{2+} + \text{0.5} \,\text{H}\_{2}\text{Sn} + \text{Fe}^{2+} \,(\text{n} \geq 2). \tag{5}$$

$$\mathbf{0.5H\_2Sn} + \mathbf{Fe^{3+}} \rightarrow \mathbf{0.125S\_8} + \mathbf{Fe^{2+}} + \mathbf{H^{\*}}.\tag{6}$$

0.125 S8 + 1.5O2 + H2O → SO4 2− + 2H+ the reaction show the production of sulfuric acid results hydrogen (proton) for attacking mineral.

Fe (II) is re-oxidized to Fe (III) by iron oxidizing organisms (chemotrophic bacteria), the role of microorganisms in solubilization.

2Fe2+ + 0.5O2 + 2H+ → 2Fe3+ + H2O this reaction keep iron in ferric state that oxidize mineral.

The process of chemical attack takes place on a substrate or the mineral surface where the bacteria forms a composite and attach itself as firm as possible in order to increases maturity that finally detached and dispersed into the solution.

An important reaction mediated by Acidithiobacillus Ferrooxidans is:

$$2\text{ FeSO}\_4 + \text{O}\_2 + 2\text{H}\_2\text{SO}\_4 \rightarrow 2\text{Fe}\_2\left(\text{SO}\_4\right)\_3 + 2\text{H}\_2\text{O}.\tag{7}$$

Strong oxidizing agent, ferric sulfate that basically used to dissolve metal sulfide minerals, and leaching brought about by ferric sulphate is termed indirect leaching due to the absence of both oxygen and viable bacterial. Check the following leaching mechanism of reaction on several minerals.

$$2\text{ CuFeS}\_2\text{ (chalcopryite)} + 2\text{Fe}\left(\text{SO}\_4\right)\_3 \rightarrow \text{CuSO}\_4 + \text{5FeSO}\_4 + 2\text{S.}\tag{8}$$

$$\text{FeS}\_2\text{(Pyrite)} + \text{Fe}\_2\text{(SO}\_4\text{)}\_3 \rightarrow \text{3FeSO}\_4 + 2\text{S}.\tag{9}$$

$$\text{UO}\_2 + \text{Fe}\_2\text{(SO}\_4\text{)}\_3 + 2\text{H}\_2\text{SO}\_4 \rightarrow \text{UO}\_2\text{(SO}\_4\text{)}\_{4-3} + 2\text{FeSO}\_4 + 4\text{H}^+.\tag{10}$$

$$\text{24FeS}\_2 + \text{15O}\_2 + \text{H}\_2\text{O}^{\text{Acidh}\\\text{tho}\\\text{cillus ferrooxidans}}\text{ 2Fe}\_2\text{(SO}\_4\text{)}\_3 + \text{2H}\_2\text{SO}\_4.\tag{11}$$

$$\text{4CuFeS}\_2 + \text{17O}\_2 + \text{2H}\_2\text{SO}\_4^{\text{Acidihisicilus forrooxidama}}\\\text{4CuSO4} + \text{2Fe}\_2\text{(SO}\_4\text{)}\_3 + \text{2H}\_2\text{O}.\tag{12}$$

$$2\text{Cu}\_2\text{S} + 5\text{O}\_2 + 2\text{H}\_2\text{SO}\_4^{\text{Acidhiba\"less\" lines ferrooxidans}}\ 4\text{CuSO}\_4 + 2\text{H}\_2\text{O}.\tag{13}$$

$$\text{CuS} + \text{2O}\_2^{\text{Acidh\"{o}l\"{o}l\"{o}c\"{o}l\"{o}s\"{o}c\"{o}l\"{o}s} \text{CuSO}\_4.\tag{14}$$

Acidithiobacillus Ferrooxidans can convert elemental sulfur generated by indirect leaching to sulfuric acid –.

*Bio Hydrometallurgical Technology, Application and Process Enhancement DOI: http://dx.doi.org/10.5772/intechopen.94206*

$$2\text{SO} + 3\text{O}\_2 + 2\text{H}\_2\text{O}^{\text{Acidhidrobaillus ferrooxidans}}2\text{H}\_2\text{SO}\_4.\tag{15}$$

This sulfuric acid maintains the pH value at levels, which is favorable to the growth of bacteria and also helps for effective leaching of oxide minerals:

$$\text{CuO(Tenorite)} + 2\text{H}\_2\text{SO}\_4 \rightarrow \text{CuSO}\_4 + \text{H}\_2\text{O}.\tag{16}$$

$$\text{UO}\_3 + \text{3H}\_2\text{SO}\_4 \rightarrow \text{UO}\_2\text{\textdegree(SO}\_4\text{)}\_3 + \text{H}\_2\text{O} + 4\text{H}^+.\tag{17}$$

Chemolithotrophic (uses carbon for the synthesis of new cell material) bacteria can be categorized based on response to temperature as mesophiles, moderate thermoacidophiles and extreme thermoacidophiles.

Mesophiles-grows at a temperature values ranges (28°C -37°C) where Thiobacillus Ferrooxidans is able use the inorganic substrate to draw energy by oxidizing Fe (II) to Fe (III) and sulfur to sulfide and sulfate. The other mesophiles is Leptospirilium Ferrooxidans that use Thiobacillus Ferrooxidans to effect the oxidization of sulfur to sulfate. Moderate thermoacidophie-temperature values ranges (40–50°C), Sulfobacillus Thermosulfidooxidans is common one, which oxidize both sulfur and iron from energy production. This category includes Archaea and Eubacteria, and most of gram-positive microorganisms are included here. Extreme thermoacidophiles-temperature ranges 60–80°C, genera Sulfolobus, Acidanus, Metallosphaera and Sulfurococcus are in this category, [11, 18, 19]. Thermal value some time extends above the limitation values, it is due to exothermal reaction which is above the maximum growth temperature of microorganism, some microorganism genus like Archaea withstand thermal values up to 90° [19, 20].

This category is formed by closely related species that can act together with a common name given Sulfolobus. Sulfolobusa Acidocaldarius, Sulfolobus Sofataricus, Sulfolobus Brierley, and Sulfolobus Ambioalous that can oxidize Fe (II) to Fe (III) and sulfur to sulfate. Aspergillus Niger and Penicillium Simplicissimum are both used to leach sulfide minerals like copper with mobilization rate of 65% and aluminum, nickel, lead and zinc by more than 95% mobilization rate. Thiobacillus and Leptospirillum are characterized by the oxidation of sulphide minerals in acidic environment and temperature values less than 35°C, with regard to area of application these two are mostly used in dump and tank leaching of metal from sulphide based mixed ores [20, 21]. The other group of genus Sulphobacillus used under the same areas of application but relatively higher temperature up to 60°C, the temperature reaches up to 90°C in case of genera Sulpholobus and Acidianus, Organotrophic microorganisms like yeast, fungi and algae which destruct sulphide mineral and aluminum silicate, facilitate bio sorption of metals that solubilize gold, these microorganism uses carbonate and silicate ore for the extraction of metals and selective gold extraction from ore floatation and metal solution.

#### **2.1 Autotrophic and heterotrophic leaching**

The two bacterial leaching namely autotrophic and heterotrophic leaching has their distinct characteristics while bioleaching process takes place, in case of autotrophic bioleaching (effective on sulfide minerals) there are two proposed mechanism of Acidithiobacillus Ferrooxidans action on sulfide minerals, first the mechanism, that the bacterial oxidize ferrous ion to ferric ion in which the bulk solution where the mineral is leached counted as indirect, this mechanism which is indirect oxidation of ferrous ions to ferric ions is exopolymeric process, both takes place on the layer where the mineral is leached. The second proposed mechanism, does not require ferrous or ferric ions, the bacteria directly oxidize the minerals by biological means having direct contact mechanism of reaction. Autotrophic leaching uses both Thiobacillus Ferooxidans and Thiobacillus Thiooxidans to leach sulfide mineral and studies shows combining the two bacterial results an increase in selectivity and rate of leaching efficiency while leaching of nickel sulfide. From the heterotrophic genus of bacteria Thiobacillus and Pseudomonas are those used to leach non-sulfide minerals and from the genus of fungi Penicillium and Aspergillus (heterotrophic fungi) are those used in leaching process, a study shows 55–60% leaching rate for nickel and cobalt, some other studies indicates that 95% and 92% leaching rate achieved while using pretreated Aspergillus Niger by ultrasound for 14 and 20 days respectively which increase its stability [4, 11, 20, 22] (**Table 1**).

Heterotrophic fungi Aspergillus and Penicillium species combined to leach low-grade nickel-cobalt oxide ores, low-grade laterite ores and spudumene (aluminosilicate), these aluminosilicate (spudumene) also leached by heterotrophic yeasts (*Rhodotorula rubra*), Aspergillus Niger used to leach zinc and nickel silicate [11]. Bacterial leaching can be generalized in three mechanism redoxolysis, acidolysis, complexolysis, and in case fungal leaching bioaccumulation is important mechanism. To solubilize rock phosphorous, Aspergillus Niger has been used in many occasions due to the production of organic acids with low molecular weight and phosphorous is basically essential micronutrients for the growth of bio organism, these microorganism convert insoluble phosphate to soluble, the two filamentous fungi used in phosphate leaching are Aspergillus Niger and some Penicillium, the metabolic fungal reaction produces organic acid that result the formation of acidolysis, complex and chelate [22].

The second group of bacterial genus is Leptospirillum, which is categorized in moderate thermophilic bacteria that can only oxidize ferrous ions; it is dominate iron oxidizer, which is referred as Leptospirillum Ferrooxidans (L. Ferrooxidans). Oxidation process takes place under strong acidity and temperature up to 30°C, L. Ferrooxidans has high affinity to Fe2+ and low affinity to Fe3+ which results a working condition of high Fe3+/Fe2+ ratio, when redox potential is low, L. Ferrooxidans has low growth rate at the initial stage of a mixed batch culture, a native strain of Leptospirillum Ferrooxidans used to leach zinc from low grade sulfide complex from La Silvita and La Resbalosa (Patagonia Argentina) [23]. The leach liquor itself has been a place where microorganism found, higher amount of


#### **Table 1.**

*Some of microorganism and leachable ore [4, 11].*

#### *Bio Hydrometallurgical Technology, Application and Process Enhancement DOI: http://dx.doi.org/10.5772/intechopen.94206*

Leptospirillum Ferriphilum were in a leach liquor, in a study conducted to leach the effect of pH on the bioleaching of a low-grade, black schist ore from Finland using Acidithiobacillus Ferrooxidans and Leptospirillum Ferrooxidans as extractant [24]. The bacteria can relatively resist high concentration of uranium, molybdenum, and silver, this is due to its affinity towards to ferrous ions or resistivity to refractory elements, but it cannot oxidize sulfur or any sulfur related compounds. By combing it with other sulfur- oxidizing acidophiles, sulfur-oxidizing process can be achieved; these are T. Caldus, T. Ferrooxidans, or T. Thiooxidans, to oxidize sulphidic gold concentrate a mixed culture of Thiobacillus and Leptospirillum has been used [11].

The third group thermophic bacteria mainly characterized by oxidation of iron to assure growth chemolithorophically, some are facultative autotrophs that require synergetic effect of other microorganism to like yeasts extract, cysteine, or glutathione. Among the microorganism in this group Sulfolobus species is the major one, these organism categorized as moderate thermophilic at an average values of temperature 40°C -60°C and the second group is extreme thermophilc at an average values of temperature 65–85°C. One of the moderate thermophilic gram positive bacteria, Sulfobacillus Thermosulfidooxidans is facultative autotrophs in which its growth stimulated by yeasts extract, where the presence of CO2, weight and volume ratio (w/v) are factor to facilitate and inhibit growth. From of extreme theremophilic Sulfolobus Acidocaldarius and Acidianus Brierleyi are those in genera Archaebacteria, among the other four genera Sulfolobus, Acidanus, Metallosphaera, and Sulfurococcus act aerobically and categorized in extreme thermophilic acidophilic bacterial which oxidizes ferrous and elemental sulfur and sulphide based minerals. These bacteria grows under all conditions (auto, mixo, heterotrphic) depending on the yeast extract ratio (w/v), found in facultative chemolithotrophic species act in acidic medium and temperature value can be up to 90°C [11].

All the major concepts of bioleaching have been discussed, so what are factors affecting rate of bioleaching and leaching efficiency, the major factors can be summarized as microbiological, mineralogical and physiochemistry factors. A physiochemistry factor includes temperature, pH, redox potential, oxygen content, carbon dioxide content which facilitate mineral oxidation required for cell growth, mass transfer, light, surface tension which mean that the topography of mineral surface that indicate the rate adsorption and crystal structure which has direct relation on the rate of reaction. Microbiological factors includes microbial diversity that is the distinct nature of micro organisms with regard to range of unicellular organisms, variety of microorganism found in an environment suitable for bioleaching, these includes bacteria, fungi, algae, flagellates, and those found in microbial biocenosis, the other microbiological factors are population diversity, metal tolerance, spatial distribution microorganism and adaptation ability of microorganism. The third major factor is the nature of mineral processed, characteristics like grain size which affect rate of dissolution, porosity related to rate of chemical attack and digestion rate, hydrophobicity is another physiochemistry factor to determine the rate leaching, hydrophobicity is differentiating whether the elements are water hating or loving while floatation takes place. Process is the other major factor affecting leaching efficiency, techniques where bioleaching process takes place (heap, dump, in situ) which we will be discussed below, pulp density is the variable which results variation on dissolution rate, a study shows that dissolution metal increases while pulp density increases but it is based on (w/v) ratio that is between 5 and 20%, the other factor is concentration of target mineral, this can inhibit the growth of microorganism, that cause a limitation of pulp density usage [25]. Stirring speed is also another factor affecting rate of dissolution and geometry of the heap during heap leaching process, the other major factor is the presence of fluoride released from the ore sample, which inhibits the process of bioleaching, and when the release decreases the rate of inhibition eventually reduced.

Besides leaching process microorganisms are used for bioremediation of mining sites, treatment of gangue, tailing, and mineral wastes from the industry, contamination of sediments due heavy metals and soil from toxicities, sewage sludge can cured by microorganism in which the process is called bioremediation [26].

## **3. Bacterial leaching techniques**

The successful bioleaching process is characterize by the intimacy of microorganisms to a mineral surface, strong attachment result high rate of oxidation and dissolution on a substrate (mineral surface), this is achieved by the rate success of bio film formation. In general leaching techniques are two – Percolation leaching – a solution infiltrate through a fixed mineral location, and agitation leaching - mineral bearing ore stirred by a solution but while working in large scale, percolation leaching is usually chosen [7]. The principal commercial methods are aerated stirred-tanks, in situ, dump, heap, vat, bench scale, tank, column, reactor leaching are among the many. It was dump bioleaching process taken as the first commercial bioleaching in 1950 used to leach copper from sulfide minerals, since then bioleaching bloomed by copper oxide heap leaching, industrial microbial leaching process applied for sulfidic gold and bioheap commercial leaching of copper ore (chalcocite and covellite). The high production of bio heap leaching of copper in 1980 established at Lo Aguirre mine in Chile processing 16,000 tones ore/per year at the inception [27], these wipe the way that led to Chile's industrial bio copper production in large scale especially from the year 1984 [28].

#### **3.1 Stirred-tank biooxidation**

Aerated, stirred-tank bioreactors, used in mineral concentrate feeds, involve a series of stages that can have lots of tanks connected in parallel depending on the retention of the concentrate [7] a study conducted to check Na-chloride can possibly enhances the chemical and bacterial leaching of chalcopyrite uses three bioreactors engaged with inoculum of the bacteria [29]. Other tanks needed for value adding purposes which are usually single tanks might be connected in series, since these tanks subject to chemical attack, air, heat and sulfide mineral, they should be relatively resistant to corrosion, chemical attack, and soon, in order to have these character tanks can be lined with rubber, galvanized, or other corrosion protection method like using sacrificial anode or using high grade material like stainless steel, aluminum or copper.

Temperature maintained at optimum level by cooling coil or some time tanks are equipped with water jackets depending on the required temperature by the bacteria, these values can be conditioned based of the mineral to be leached, and sometimes the chemical used to enhance the leaching process [29]. Several tanks can be continuously arranged, named as continuous stirred tank reactor (CSTR), as per the above it can be followed by a series of small equal sized reactors [16]. Example of bioleaching of sulfidic gold concentrates, that the discharge from the final stage is subjected to water washing and solid/liquid separation in thickeners. Even though there is less power consumption basically used for agitator and blower, it has linear relationship with the amount of sulfide -sulfure which is required to oxidize and recover the target metal from the parent ore, rate of recovery depends up on the metal grade also.

The main advantages of these tank over other conventional methods like pressure autoclave, roasting, smelting, calcination and soon are; it has low capital and operational cost, relatively less construction period, less complicated requiring less skilled man power and most importantly it is environmental friendly. In general

Australia, Chile, USA, Brazil and South Africa are among those countries involved in bio oxidation by stirred tank [7, 16].
