1. Introduction

Ever-increasing world demand for iron ores has led to exploitation of even lean-grade ores. However, acceptable specifications on the quality of iron ore concentrates necessitate close control of impurities such as alumina, silica, alkali and phosphates. Besides the availability of lean grade ores, significant tonnages of iron ore fines and even high iron containing processed wastes accumulated in various mining sites around the world call for immediate action. Under the circumstances, it becomes necessary to develop efficient mineral processing techniques which areenvironment-friendly, cost-effective and energy-efficient. Use ofmicroorganisms to bring out microbially-induced flotation or flocculation of the desirable mineral constituents could prove to be a novel alternative to existing physico-chemical methods.

Several microorganisms which are present ubiquitously in many iron ore deposits act as reagents, collectors or modifiers, to bring about selective mineral separation. Microbially-induced beneficiation of iron ores is a new concept having potential industrial applications in iron ore mining industries, worldwide. In this paper, biotechnological aspects of environmentally benign iron ore beneficiation are discussed with respect to biomineralisation,miningmicroorganisms, surface chemical aspects of microbe-mineral interactions and role of mineral-specific bioreagents.

Hematite is the most abundant and important iron bearing mineral relevant to iron and steel industries. But hematite is generally associated with oxide gangue minerals such as quartz, clayey matter, alumina and calcite. Various physico-chemical methods such as froth flotation and electrostatic separation used to separate the gangue minerals from hematite are considered to be expensive and are not environment friendly. Utility of microorganisms in iron ore beneficiation was understood and developed only in the last decade^[1-4]. Smith and co workers have reported extensive studies on the relevance and promise of microorganisms in mineral bioprocessing[5-9].

Utility of Bacillus subtilis, Saccharomyces cerevisiae and Desulfovibrio desulfuricans in the beneficiation of hematite ores has been recently studied[10-12]. More detailed studies involving microbiological and surface chemical aspects are needed to understand basic mechanisms governing microbially induced iron ore beneficiation. Considering the above scenario, a system of hematite along with quartz, corundum, calcite and apatite was chosen to separate hematite from rest of the oxide gangue minerals.

In this paper, the role of bioreagents secreted by Paenibacillus polymyxa in altering the surface chemistry of some iron ore minerals, such as hematite, corundum, calcite and quartz is discussed. The consequences ofmicrobe–mineral interactions of relevance in mineral beneficiation are brought out in terms of iron ore flotation and selective flocculation. Paenibacillus polymyxa[13] is a Gram-positive neutrophilic, periflagellated heterotroph, occurring indigenously associated with several mineral deposits. It secretes exopolysaccharides, proteins and several organic acids such as acetic, formic and oxalic acids. The utility of corundum-adapted bacterial cells in the removal of alumina from iron ores is discussed. Another bacterium namely, Bacillus subtilis was used to demonstrate separation of hematite from calcite, silica, corundum and apatite. Bacillus subtilis is a Gram-positive neutrophilic periflagellated aerobic, catalase-positive capsulated bacterium commonly found in soil[14]. Saccharomyces cerevisiae are unicellular and chemoorganotrophic, eukaryotes classified in the kingdom of fungi. Cells and metabolic products of Desulfovibrio desulfuricans and Saccharomyces cerrevisiae were successfully used to separate quartz from hematite through environmentally benign microbially induced flotation and flocculation. Desulfovibrio desulfuricans is a Gram negative sulfate reducing, facultative anaerobe, occurring indigenously associated with iron ore deposits and is implicated in the biomineralization of several iron oxides. It is essential to understand the mechanisms and resulting consequences of microbe-mineral interactions before the utility of microorganism in the processing of minerals could be established. Since mineral beneficiation processes such as flotation and flocculation depend on the surface chemical properties of the minerals, any changes in the surface chemistry brought about by biotreatment would be of significance.

2. Biomineralisation and Biogenesis of Iron Oxides

A prior knowledge of indigenous microorganisms associated with natural iron ore deposits will not only enable an understanding of iron oxide-bacteria cycle in nature, but also would facilitate the right choice of mining bacteria for biobeneficiation. The formation of various iron oxides under the earth’s crust are due to both biological and abiotic reaction mechanisms[15-19]. Involvement of various microorganisms in extracellular and intracellular iron oxide formation is a definite indication of biogenesis of iron ores. In natural sediments, iron oxide particles often occur in intimate association with bacterial cell envelopes and their exopolymers. Biogenesis of oxyhydroxides such as goethite, lepidocrocite and limonite as well as oxides such as magnetite and hematite is likely in natural environments. From iron-rich seepage neutrophilic iron-oxidizing bacteria could be isolated. Anaerobic microorganisms such as Sulphate Reducing Bacteria (SRB) present in anoxic mining environments facilitate formation of ferrihydrite and magnetite. Microbial iron oxidation and reduction are involved in natural bacteria-iron cycles following various mechanisms and conditions of varying pH, oxygen availability, temperature and ionic concentrations. For example, Gallionella spp and Leptothrix spp are invariably associated with biogenic iron oxides at neutral pH in oxygen rich zones. In acidic environments, Acidithiobacillus spp. brings about ferrous iron oxidation. Iron oxidizing archea such as Thermoplasmales could be identified in extreme acidic environments. Bioreagents such asexopolysaccharides and proteins are secreted by iron-bacteria during the process of iron biogenesis and conversion. Microorganisms capable of producing polyphosphate granules, sulfur granules and other intra-cellular and inter-cellular inorganic polymers have also been located in mining environments. Many iron-bacteria exhibit magnetotaxis and are implicated in the biosynthesis of magnetite[20]. An iron-reducing bacterium, such as Shewanella putrefaciens is capable of production of intracellular particles of iron-minerals[21]. With reference to banded iron formations, it is possible for iron oxidizing bacteria to produce and precipitate iron-rich sediments on a large scale. Different metabolic activities, redox reactions and internal biomineralisation processes are thus involved in biological iron oxide formation.

3. Microorganisms Useful in Iron ore Beneficiation

Diversity of microbial life in iron ore deposits can be established through molecular biology tools. It will be useful to isolate and identify microbes which are indigenously present in an iron ore deposit for a beneficiation process. For example, mine-isolated phosphorous solubilising microbes could be utilized for dephosphorization of iron ores. Mineral specific organic and inorganic biopolymers secreted by microbes inhabiting a mining environment can find use in selective mineral separation. Close association between bacteria and precipitated silica as well as iron and alumina-silicates could pave the way for development of processes to separate clay and alumina particles from iron oxides.

Bacteria with enhanced iron reducing properties include:

Acidithiobacillus thiooxidans, Bacillus circulans, Bacillus pumilus, Paenibacillus polymyxa, Pseudomonas spp, Bacillus acidocaldarius, Bacillus mesentericus, Shewanella putrefaciens and Sulfate Reducing Bacteria (SRB). Many of the above microorganisms could be utilized to reduce ferric iron (present in iron oxides) to metallic iron.

Many types of microorganisms including autotrophic and heterotrophic bacteria, fungi, yeast and algae which inhabit iron ore deposits find use in iron ore beneficiation[22-24] Morphological features of some microorganisms useful in iron ore beneficiation are illustrated in Figure 1. Possible use of microbial cells and bioreagents as flotation, flocculation and dispersion reagents in iron ore beneficiation need be understood. For example, yeast cells can be used to agglomerate iron oxide fines. Bacteria such as Mycobacterium phlei, Bacillus subtilis and Rhodococcus opacus have been used to demonstrate their utility in iron ore beneficiation[25-26].

Mycobacterium phlei and their surface active products have been shown to function as flotation collectors and flocculation agents for hematite[5-9]. Best flotation was found to be at acidic pH of 2.5. Hematite can be selectively flocculated from silicates.

Utility of bacteria, metabolites, and other bioreagents in iron ore beneficiation has been reported[27-31].

Pure strains of Paenibacillus polymyxa (NCIM 2539), Bacillus subtilis (NCIM 2655), Desulfovibrio desulfuricans (NCIM 2047) and Saccharomyces cerevisiae (NCIM 3309) used in our studies were procured from National Collection of Industrial Microorganisms, National ChemicalLaboratory, Pune, India.

Figure 1. Some microorganisms useful in iron ore processing (a) Acidithiobacillus ferrooxidans (b) Bacillus subtilis (c) Saccharomyces cerevisiae (d) Paenibacillus polymyxa

The bacterial growth studies were carried using Bromfield medium, Luria broth medium, Baars medium and Yeast extract peptone dextrose medium respectively[32-35]. The structures of the cell walls of microorganisms play a significant role in bacterial adhesion to mineral substrates. In order to utilize the beneficial attributes of microbes through microbe-mineral interaction, surface chemical studies of both microbial cells and minerals along with their mutual attachment behavior becomes imperative.

Utility of various microorganisms in the separation of quartz, corundum, calcite and apatite from hematite is discussed below.

3.1. Bioseparation of Quartz from Hematite

Microbially–induced separation of quartz from hematite is demonstrated through the use of the following microorganisms, namely, Paenibacillus polymyxa, Bacillus subtilis, Desulfovibrio desulfuricans and Saccharomyces cerevisiae.

Among the microorganisms used, Paenibacillus polymyxa and Bacillus subtilis are aerobic neutrophilic bacteria, Desulfovibrio desulfuricans is an anaerobic sulphate reducing bacteria and Saccharomyces cerevisiae is an yeast (Figure 1).

Kinetics of bacterial and yeast cells adsorption was obtained by estimating their adsorption density onto quartz and hematite as a function of time. Adsorption behavior with respect to time was investigated in a 10^-3 M KNO₃ electrolyte solution at different pH values. In case of all the bacterial and yeast cells, adsorption was found to be significantly higher on hematite compared to that on quartz. Interaction with microbial cells, metabolites and proteins resulted in enhanced hydrophobicity for quartz unlike hematite which was rendered more hydrophilic after similar treatment.

Microbial metabolites contain exopolysaccharides and many proteins and their preferential adsorption on the minerals are responsible for the above observations. Predominant adsorption of polysaccharides would render the mineral more hydrophilic and of proteins confer enhanced hydrophobicity. Proteins were observed to be predominantly adsorbed onto quartz, while hematite exhibited enhanced affinity towards polysaccharides.

It has also been observed that when microbial cells were grown in the presence of minerals, amounts of protein and polysaccharide secretions differed depending on the mineral as illustrated in Table 1 below.

Table1. Production of bioproteins and polysaccharides in the presence of minerals [10-12, 37, 38] (C-control: Q-Quartz: H-Hematite) Microorganisms Protein (µg/µl) Polysaccharide (µg/µl) C Q H C Q H P.polymyxa 0.7 0.8 0.5 0.5 0.4 0.6 B.subtilis 0.7 1.0 0.5 0.02 0.01 0.05 S.cerevisiae 0.3 0.6 0.3 0.2 0.2 0.6 D.desulfuricans 0.3 0.5 0.2 0.2 0.2 0.7

Presence of quartz during growth of various bacterial and yeast cells promoted protein generation. On the other hand, enhanced biogeneration of exopolysaccharides could be observed when grown in presence of hematite.

Enhanced mineral affinity towards bacterial proteins and polysaccharides would also be reflected in the shifts in their measured zeta potentials as discussed below. In case of Paenibacillus polymyxa, Bacillus subtilis andSaccharomyces cerevisiae, IEP of quartz shifted from pH 2 to 3.6, 2.7and 3.2 respectively on interaction with cells and metabolites for an hour. Cell walls of Gram-positive bacteria, such as Paenibacillus polymyxa and Bacillus subtilis contain several groups of exopolymers such as carboxylic acids and amino-acid containing protein groups. Positive shifts in IEP can be attributed to enhanced presence of amino-group containing proteins secreted by the cells which were grown in the presence of quartz. On interaction of Paenibacillus polymyxa, Bacillus subtilis and Saccharomyces cerevisiae cells and metabolites with hematite, the IEP of hematite shifted from pH 6.2 to 3.0, 4.5 and 4.0 respectively. Such an observed negative shift in zeta potentials and IEP for hematite can be attributed to the presence of higher amounts of exopolysaccharides when grown in presence of hematite.

An effective method for establishing affinity of polysaccharides towards various minerals is through ruthenium red adsorption. Percent adsorption of ruthenium red onto bacterial and yeast cells before and after interaction or adaptation with minerals is given in Table 2. Higher the percent ruthenium red adsorption, higher the availability of polysaccharides on the cell surface and vice-versa.

It is noteworthy that the amount of polysaccharides present on hematite-grown cells is significantly higher than that on quartz- grown cells. This clearly confirms that bacterial or yeast cells adapted through growth on hematite will be more hydrophilic than those grown on quartz which is expected to be more hydrophobic due to enhanced secretion and adsorption of proteins.

Table 2. Percent adsorption of ruthenium red on bacterial and yeast cells grown in presence of minerals [10-12] Growth conditions Percent adsorption P.polymyxa B.subtilis S.cerevisiae D.desulfuricans In absence of minerals 30 70 32 42 In presence of quartz 09 60 24 28 In presence of hematite 80 72 99 79

SDS-PAGE is considered to be an important tool in protein characterization, as it is useful in identifying mineral specific protein bands and also in determining their molecular weights. Expressions of mineral-induced protein bands are indicated by arrows in Figure 2.

Figure 2. SDS-PAGE of proteins isolated from cell free metabolite of B.subtilis [10]

Different groups of proteins were secreted by mineral grown cells compared to a normal strain grown in the absence of any minerals. B. subtilis grown in presence of hematite secreted a conspicuous band of protein of 23.44 kDa which was not expressed in the control.

B. subtilis grown in the presence of quartz secreted four different types of specific proteins, such as 19.9, 33.5, 40.2 and 60.3 kDa and these proteins were not secreted in absence of mineral. Protein band of about 22.39 kDa was seen in case of the normal strain which was not present in mineral-grown strains. Bands formed by the proteins separated from mineral grown cell free metabolite were different and more in number than the bands formed by the metabolites derived from normal cell growth. Mineral-induced proteins were expressed in case of mineral-grown bacterial cells only[10].

Protein profiles of solution-grown and mineral-grown yeast cells were also similarly established. Protein bands of molecular weights 8.4 kDa, 14 kDa, 17.2 kDa, 18.8 kDa and 29 kDa have been identified as mineral specific proteins secreted by yeast cells grown in presence of quartz, since these bands were absent in case of solution and hematite-grown yeast cells. These observations further prove that mineral-specific stress proteins can be secreted by yeast cells when grown in the presence of quartz[36].

Protein profiles of unadapted and mineral-adapted D.desulfuricans cells were also studied[12]. Protein bands of molecular weights 105 kDa, 36.5 kDa and 25 kDa have been identified as mineral-specific proteins secreted by quartz adapted cells, since those bands were absent in case of proteins secreted by unadapted and hematite-adapted bacterial cells. The secretion of higher amounts ofmineral-specific proteins was observed in case of quartz-adapted bacterial cells, but not in case of unadapted and hematite-adapted cells.

Effect of surface chemical changes brought about on mineral surfaces due to bacterial interaction can now be examined with respect to selective separation. Prior interaction with bacterial cells or metabolic products renders hematite more hydrophilic while quartz surface was rendered more hydrophobic after similar treatment. Such microbially induced mineral surface chemical changes can bebeneficially used to bring about selective flotation/flocculation of minerals. Settling rates of hematite and quartz were studied before and after interaction with microbial cells and cell free metabolite as illustrated in Table 3. The settling rate of hematite was significantly increased after interaction with cells and cell free metabolites. Dispersion of quartz was facilitated after microbial interaction under similar conditions. Adsorption studies also showed that bacterial and yeast cells exhibited a higher affinity towards hematite compared to quartz

Table 3. Flocculation behavior of quartz and hematite at neutral pH after interaction with cells and cell free metabolites (CFM) [10-12, 37] Percent settled (3min) Minerals Control P. polymyxa B.subtilis S.cerevisiae (adapted) Cells CFM Cells CFM Cells CFM Quartz 60 20 15 10 21 28 24 Hematite 85 96 96 80 85 82 91

From Table 4 it can be observed that percent weight flotation for quartz was the highest on interaction with cells and cell free metabolites. Such an enhancement in hydrophobicity of quartz is due to mineral-induced proteins secreted by various microorganisms into the cell free metabolite during growth in the presence of quartz. Under all conditions of pretreatment whether interacted with cells or cell-free metabolites, hematite flotation was seriously impaired.

Apart from studying the flotation characteristics of individual minerals, selective separation of quartz from a binary mixture of quartz and hematite was also carried out after interaction with various microbial cells and cell free metabolites. Efficient separation of quartz from hematite could be obtained from hematite-quartz mixtures after microbial interaction.

Table 4. Flotation of quartz and hematite after microbial interactions at neutral pH [10-12, 37] Percent weight floated Minerals Control P. polymyxa B.subtilis Cells CFM Cells CFM Quartz 19 60 80 96 92 Hematite 7 8 10 4 11 Percent weight floated Minerals Control S.cerevisiae(adapted) D.desulfuricans(adapted) Cells CFM Cells CFM Quartz 19 95 94 78 85 Hematite 7 8 7 9 15

3.2. Role of Paenibacillus Polymyxa and Bacillus Subtilis in Hematite-Corundum Separation

The beneficial aspect of using corundum-adapted cells has been demonstrated with respect to efficient separation of alumina from hematite using Paenibacillus polymyxa[30]. Unadapted cells could not bring about hematite-alumina separation. Corundum-adapted cells were shown to secrete additional mineral specific proteins besides otheralumina-specific bio-surfactants unlike unadapted cells. Whilecorundum-adapted cells enhanced the flocculation of alumina particles, hematite-adapted cells selectively flocculated hematite.

Significant changes in the surface chemistry of iron oxides and associated minerals are brought about when cells and metabolic products of Paenibacillus polymyxa are interacted [27-31]. Cells of Paenibacillus polymyxa were grown in the presence of corundum with repeated subculturing for six months.

Polyacrylamide gel electrophoresis (PAGE) in the presence of an anionic detergent, namely, sodium dodecyl sulfate (SDS) by which proteins can be characterized in terms of molecular weight of the constituent peptides was carried out to reveal alumina-specific bioproteins generated due to bacterial growth and adaptation in the presence of the mineral.

Presence of new proteinaceous bands with positively charged NH₃⁺ functional groups could be readily identified on alumina-grown bacterial cell walls as well as in metabolic products. Bacterial cells grown and adapted in presence of alumina secreted mineral-specific proteins which were not present in unadapted bacterial cells. The new protein secreted during alumina adaptation is a cytoplasmic protein.The molecular weight of this alumina-specific protein which is present in both cytoplasm and the extracellular metabolite was found to be 31 kDa.

It has also been observed that when microbial cells were grown in the presence of corundum and hematite, amounts of protein and polysaccharide secretions also differeddepending on the mineral. Relatively higher amounts of proteins were generated by both types of bacterial cells when interacted with corundum.

Percent adsorption of ruthenium red onto bacterial cells before and after interaction or adaptation with minerals is given in Table 5.

Table 5. Percent adsorption of ruthenium red on bacterial cells grown in presence of minerals [10] Growth conditions Percent adsorption P.polymyxa B.subtilis In absence of minerals 30 70 In presence of corundum 50 60 In presence of hematite 80 75

Presence of exopolysaccharides confers enhanced hydrophilicity on the interacted mineral. The amount of polysaccharides present on the hematite-grown cells is significantly higher than that on corundum- grown cells.

This clearly confirms that bacterial cells adapted through growth on hematite will be more hydrophilic than those grown on corundum which is expected to be more hydrophobic due to enhanced secretion of proteins.

Corundum-adapted P.polymyxa cells and their metabolites as well as B.subtilis cells and cell free metabolite were used in the separation of corundum from hematite as shown in Table 6. Efficient corundum separation from hematite could be achieved using adapted cells. Settling rates of hematite and corundum was studied before and after interaction with Paenibacillus polymyxa and Bacillus subtilis cells and cell free metabolite as given in Table 6.

Table 6. Flotation and flocculation of corundum and hematite at neutral pH (3 min) [10, 37] Percent settled Minerals Control P. polymyxa B.subtilis Cells (adapted) CFM Cells CFM Corundum 60 16 15 20 60 Hematite 85 92 91 78 81 Percent floated Minerals Control P. polymyxa B.subtilis Cells (adapted) CFM Cells CFM Corundum 9 95 96 76 35 Hematite 6 8 10 4 12

The settling rate of hematite was significantly increased after interaction with cells. Dispersion of corundum was facilitated after interaction with cells. Hematite was almost completely flocculated with cells and cell free metabolite.

Adsorption studies showed that the bacterial cells exhibited a higher affinity towards hematite compared to corundum.

Flotation behavior of hematite and corundum was also studied before and after interaction with Paenibacillus polymyxa and Bacillus subtilis cells and cell free metabolite as depicted in Table 6.

Corundum could be efficiently separated from hematite using Paenibacillus polymyxa pre adapted to corundum.

In case of B.subtilis, separation of corundum from hematite was efficient only on interaction with cells and not with cell free metabolites.Selective separation of corundum from a binary mixture of corundum and hematite was also carried out after interaction with adapted bacterial cells as well as with cell free metabolite. Interaction of mineral mixture with bacterial cells and cell free metabolite resulted in efficient separation of corundum from hematite.

3.3. Role of Bacillus Subtilis in Hematite-Calcite Separation

Role of B.subtilis in separation of calcite from hematite has been studied. Typical scanning electron micrographs depicting adhesion of Bacillus subtilis onto calcite and hematite are illustrated in Figure 3. As could be readily seen, profuse and significant adhesion of bacterial cells could be seen on hematite when compared to calcite.

Figure 3. Scanning electron micrographs of B. subtilis attached onto (a) calcite and (b) hematite

Amounts of extracellular bacterial proteins and cell surface polysaccharides secreted by mineral-grown bacterial cells were compared with that secreted by cells grown in the medium in the absence of any minerals as illustrated in Table 7.

Table 7. Amount of protein and polysaccharide present in cell free metabolite and percent adsorption of ruthenium red onto the bacterial cell surfaces [10] Growth conditions Protein (µg/µl) Polysaccharide,(µg/µl) Percent adsorption of ruthenium red No Minerals 0.7 0.02 70 Hematite 0.5 0.05 75 Calcite 0.8 0.04 80

Bacterial growth in the presence of hematite resulted in a decrease in protein generation with enhanced secretion of exopolysaccharides. In the case of calcite, both proteins and polysaccharides appear to exert competitive influence on their surfaces.

Surface affinity of bacterial extracellular proteins onto the above minerals was then established through adsorption density measurements as illustrated in Figure 4.Protein adsorption was found to be higher on calcite than on hematite.

Prior interaction with bacterial cells or metabolic products renders hematite more hydrophilic while that of calcite surfaces were rendered more hydrophobic after similar treatment.

Such microbially-induced mineral surface chemical changes can be beneficially used to bring about selective flotation/flocculation of minerals.

Selective separation of calcite from a binary mixture with hematite was carried out after interaction with bacterial cells. Interaction of mineral mixture with bacterial cells resulted in 74 per cent flotation recovery of calcite and 15 per cent for hematite.

Figure 4. Adsorption behavior of proteins secreted by B.subtilis cells onto calcite and hematite

3.4. Protein, Genomic DNA and RAPD-PCR Fingerprinting Profiles of Bacillus Subtilis

Figure 5. SDS-PAGE of intracellular proteins isolated from cell free metabolite of B.subtilis in absence and presence of minerals

Intracellular protein (IP) profile of mineral – grown B.subtilis cells was then established as illustrated in Figure 5. Different groups of intracellular proteins were secreted by mineral grown cells compared to a normal strain grown in the absence of any minerals. B.subtilis grown in presence of hematite and quartz secreted a conspicuous thick band of proteins of 14 to 25 kDa range which was not expressed in the control.

B. subtilis grown in presence of corundum and calcite did not secrete any major mineral specific protein bands.

Bands formed by the proteins separated from mineral grown cell free metabolite were different and more in number than the bands formed by the metabolites derived from normal cell growth. Mineral-induced proteins were expressed in case of mineral - grown bacterial cells only.

Genomic DNA of B.subtilis in presence and absence of minerals was studied and is depicted in Figure 6. Lambda-HindIII digest was used as Marker. In all the isolates the genomic DNA was found to be of 23,130 bp. RAPD-PCR analysis was carried out to look into the genomic alterations of bacterial genomic DNA grown in presence of minerals.

Figure 6. Agarose gel showing genomic DNA of B.subtilis in absence and presence of different minerals

Figure 7. RAPD amplification patterns of PCR products of B.subtilis in absence and presence of different minerals

RAPD-PCR fingerprinting was carried out for gaining better understanding of the different base pair bands present in the bacteria in absence and presence of minerals (Figure 7). In total, 12-15 bands were detected.

Prominent band of 500bp was seen in both control and bacteria grown in presence of minerals. 1kb band was prominently seen only in control and calcite adapted DNA, where as the 1kb band was very light in bacteria grown in presence of hematite, corundum and quartz.

This shows the possibility of some genomic alterations in the bacterial genomic DNA in presence of hematite, corundum and quartz.

Based on the RAPD results, the RAPD-PCR method can be used as an investigational tool for mineral induced genomic alterations. Furthermore, the present results suggest that RAPD-PCR fingerprinting in conjugation with gene sequencing can be a powerful screening tool for searching for mineral induced specific genomic variations which are caused when bacteria is grown in presence of the minerals.

3.5. Biobeneficiation of Iron Ores for Removal of Silica and Alumina

Having studied the amenability of microbially – induced flotation and flocculation using pure mineral samples, the investigations were extended to demonstrate potential of biobeneficiation to real iron ore samples.

Removal of silica and alumina from two typical Indian iron ore deposits using biobeneficiation is demonstrated.

The Kudremukh iron ore deposits situated in Karnataka, India have high silica (mainly as quartz) content. The Bolani iron ores used by Steel Authority of India Limited

(SAIL) have high alumina content (present as alumina and clays).

High alumina: silica ratios (>1) in the processed ore burden (sinter) create problems in blast furnace smelting and therefore, there is an urgent need for reducing the alumina content (and thereby reducing the alumina: silica ratio in the blast furnace feed) in such high alumina iron ores.

Table 8. Bioremoval of silica from Kudremukh iron ore [37]

No satisfactory beneficiation process for the reduction of alumina in Indian iron ores has so far been developed.

Exploratory tests were initially carried out[29] to assess the amenability of biological processes for beneficiation of different ores. Typical results obtained under different experimental conditions with the Kudremukh iron ore samples are illustrated in Table 8.

It could be readily seen that either through flotation or selective flocculation after biotreatment (unadapted cells), significant removal of silica from the iron concentrate could be achieved. For alumina removal from the Bolani iron ore, hematite-adapted cells were used.

Typical results are given in Table 9. Either through flotation or selective flocculation after bacterial interaction, significant reduction in alumina content of the iron concentrate could be obtained.

Table 9. Bioremoval of alumina from Bolani iron ores using hematite-adapted cells [37]

Typical laboratory results of a statistically designed and optimized biobeneficiation process for an alumina-silica rich Indian ore are illustrated in Figure 8. Alumina and hematite adapted cells of Paenibacillus polymyxa were used.

Figure 8. Selective bioflocculation of iron ore slimes using alumina-adapted strains of P. polymyxa. (a) Silica separation, (b) Alumina separation [37]

The beneficial role of biotreatment in silica and alumina removal from iron ore is clearly evident. Desiliconisation can be achieved either by flotation or selective flocculation in the presence of bacteria or their metabolic products.

Three dimensional response surface curves indicate that through appropriate control of pulp density and cell biomass significant reduction in alumina and silica levels in the iron concentrate could be achieved.

Growth of bacterial cells in the presence of minerals also brings about morphological and other environmentally necessitated changes. Structural features of bacterial cells grown in the presence of silica and calcite were studied through transmission electron microscopy.

Calcite-grown bacterial cells were seen to be encased by a capsule essentially of polysaccharides.

On the other hand, silica-grown cells exhibited neither any slime layer nor a capsule, evidently due to its lower affinity towards bacterial polysaccharides.

Presence of silica during bacterial growth promotes enhanced generation of proteins while that of calcite facilitates increased secretion of exopolysaccharides. These observations lead to the possibility of generation of mineral-specific bioreagents.

3.6. Role of Saccharomyces Cerevisiae in Hematite-apatite Separation

The attachment of yeast cells onto hematite and apatite was first compared. As illustrated in Figure 9, profuse attachment of yeast cells could be seen on apatite unlike onto hematite surfaces at neutral pH range, while attachment onto apatite was lower than that on hematite as the pH increased.

Figure 9. Scanning electron micrographs of S. cerevisiae attached onto (a) apatite and (b) hematite

Amounts of extracellular bacterial proteins and cell surface polysaccharides secreted by both the mineral-grown yeast cells and cells grown in absence of minerals were studied.

Yeast growth in presence of hematite promoted enhanced protein secretion compared to apatite and in the presence of apatite resulted in a decrease in protein generation.

The amount of polysaccharides present on the hematite-grown cells was less than that on apatite- grown cells.

This clearly confirms that yeast cells adapted through growth on apatite will be more hydrophilic than those grown on hematite which is expected to be more hydrophobic due to enhanced secretion of proteins.

Zeta-potentials were also measured to assess the changes in the surface chemical characteristics of the minerals before after interaction with the cells and cell free metabolite of Saccharomyces cerevisiae. The IEP of hematite and apatite alone were found to be at pH 6.2 and 4.9 respectively. On interaction with the yeast cells the IEP of hematite shifted from pH 6.2 to 2.0. For apatite the IEP shifted from pH 4.9 to 2.0.

The surface charge of hematite interacted with yeast was found to be more negative when compared to apatite interacted with yeast cells. On interaction with the cell free metabolite the IEP of hematite and apatite was found to be at about pH 2.1 to 3.0.

On interaction with cell free metabolite the surface of both apatite and hematite became more negative when compared to that after interaction with cells.

Flocculation behavior of hematite and apatite before and after interaction with yeast cells was studied. At pH 3.0-4.0 range the flocculation behavior of both the minerals in absence and presence of yeast was found to be similar. At pH 6.0-7.0 range apatite interacted with yeast cells was found to settle around 85% in 5 min and hematite settled only 25% in 5 min. The settling rate of apatite was significantly increased after interaction with cells. Hematite was found to settle 98% at pH 9.0-10.0 range in 5 min and apatite showed only around 25% of settlement. Selective separation of hematite and apatite was possible at both neutral pH range and 9.0-10.0 pH range through flocculation.

Selective separation of apatite from a binary mixture with hematite was carried out after interaction with bacterial cells. Interaction of mineral mixture with bacterial cells resulted in 80 per cent dispersion of hematite and 28 per cent settlement of apatite.

3.7. Environmental Aspects

Biobeneficiation processes are more environment-friendly than physico-chemical alternatives. For example, many of the microorganisms reported to be useful in iron ore beneficiation are also implicated in bioremediation through degradation of organic reagents. Efficient biodegradation of various amines and oleate collectors used in iron ore flotation could be brought about by Paenibacillus polymyxa[28]. Bacteria such as Bacillus spp, Pseudomonas spp and Sulphate Reducing Bacteria (SRB) could also be used to degrade residual collector concentrations present in mill process effluents before environmental disposal. Similarly, the above organisms were also found to be useful in biological stripping of adsorbed reagents from mineral surfaces subsequent to flotation. Such biological surface removal of hydrophobic reagents from mineral particle surfaces would be very useful and beneficial in subsequent processing of a flotation concentrate. A case in point is pelletization of iron ore fines after beneficiation through flotation. Metabolic activity of iron-reducing bacteria could enhance the natural or engineered remediation of waste tailing disposal sites. Iron ore fines and slimes present in tailing dams can be efficiently agglomerated and stabilized using native microorganisms from an environmental angle. Also, such bioprocess would pave the way for recovery of valuable minerals from wastes and for reharvesting and recirculation of process water.

4. Summary

Many microorganisms such as Paenibacillus polymyxa, Bacillus subtilis, Saccharomyces cerevisiae and Desulfovibrio desulfuricans can be efficiently used for removal of silica, alumina and apatite from iron ores through microbially-induced flotation or selective flocculation. When microorganisms were grown in the presence of hematite, corundum, calcite and quartz mineral-specific bioproteins and exopolysaccharides were generated. Bacteria such as Paenibacillus polymyxa can be used for biodegradation of flotation collectors such as amines and oleates and also to strip residual collector reagents from floated concentrates. Microbially-induced iron ore beneficiation is thus environment-friendly and cost-effective. However, large scale tests using real ores need be carried out using real ore systems to establish techno-economic feasibility of the biobeneficiation process for commercial applications.

ACKNOWLEDGEMENTS

The authors are thankful to the Department of Science and Technology (DST) and Council of Scientific & Industrial Research (CSIR), Government of India, New Delhi for financial support, Indian Institute of Science and Bangalore University for the infrastructure. Thanks are due to the National Academy of Sciences (India) for Platinum Jubilee Senior Scientist Fellowship to the corresponding author (K.A. Natarajan).