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Role of bacterial interaction and bioreagents in iron ore flotation
Int. J. Miner. Process. 62 Ž2001. 143–157 www.elsevier.nlrlocaterijminpro
Role of bacterial interaction and bioreagents in iron ore flotation K.A. Natarajan ) , Namita Deo Department of Metallurgy, Indian Institute of Science, Bangalore, 560 012, India Received 15 August 1999; accepted 17 July 2000
Abstract Interaction between Paenibacillus polymyxa and iron ore minerals such as hematite, corundum, calcite, quartz and kaolinite brought about significant surface chemical changes on all the minerals. Quartz and kaolinite were rendered more hydrophobic, while the other three minerals became more hydrophilic after bacterial interaction. Predominance of bacterial polysaccharides on interacted hematite, corundum and calcite and of proteins on quartz and kaolinite was responsible for the surface chemical changes. The bacterial strains could be preadapted to different mineral substrates. Corundum-adapted strains were seen to secrete mineral-specific proteins which could be used to separate alumina from iron ores. The utility of bioprocessing in the beneficiation of iron ores for removal of silica and alumina is demonstrated. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Paenibacillus polymyxa; iron ore; beneficiation; surface chemistry
1. Introduction Bacillus polymyxa, recently renamed as Paenibacillus polymyxa ŽAsh et al., 1993., 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. They also generate levan forming the capsule of the organism from sucrose by the activity of a specific enzyme known as levan sucrase ŽMurphy, 1952.. The extracellular polysaccharide ŽECP. aids in biological uptake of metal ions necessary for metabolism and growth. Heavy metals are known to )
Corresponding author. Tel.: q91-80-3600-120; fax: q91-80-3600-472. E-mail address: [email protected] ŽK.A. Natarajan..
0301-7516r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 7 5 1 6 Ž 0 0 . 0 0 0 4 9 - 1
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bind to the cell walls through the ECP. Commercial use of this organism in the production of proteinases, amylases, butanediol and glycogens has been reported ŽReed, 1987.. The structure of the bacterial cell wall plays a significant role in bacterial adhesion to mineral substrates. Peptidoglycan, a prominent constituent of the cell wall, is covalently linked to other macromolecular constituents which include several types of polysaccharides and polyphosphate polymers known as teichoic acids ŽStanier et al., 1993.. The presence of different metal ions, such as Caqq and Mgqq as well as minerals such as iron, aluminium and calcium oxides during growth, influences the types and quantity of polysaccharides, proteins and enzymes secreted by the bacteria ŽReed, 1987.. Although the use of different microorganisms in ore leaching is well established, biological methods of mineral beneficiation are not even sufficiently understood. The utility of B. polymyxa in the biobeneficiation of bauxite and iron ore minerals has been recently reported ŽPhalguni Anand et al., 1996, Namita Deo and Natarajan, 1997a,b, 1998.. As different from bioleaching, biobeneficiation, by definition, refers to selective removal of undesirable mineral constituents from an ore through interaction with microorganisms, thus enriching it with respect to the desired valuable minerals. It is essential to understand the mechanisms and resulting consequences of microbe– mineral interactions before the utility of the 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 is of significance. In this paper, the role of bioreagents secreted by P. polymyxa in altering the surface chemistry of some iron ore minerals, such as hematite, corundum, calcite, quartz and kaolinite, is discussed. The consequences of microbe–mineral interactions of relevance in mineral beneficiation are brought out in terms of iron ore flotation and selective flocculation. The utility of corundum-adapted bacterial cells in the removal of alumina from iron ores is discussed. 2. Materials and methods 2.1. Bacterial strain and growth conditions A pure strain of P. polymyxa NCIM 2539 was used in all the studies. A 10% vrv of an active inoculum was added to Bromfield medium ŽPhalguni Anand et al., 1996. and incubated at 308C on a rotary shaker at 240 rpm. The bacterial growth pattern was studied by microscopic counting using a Petroff–Hausser counter under a phase contrast microscope and also by colony counting after plating. pH changes during bacterial growth was monitored and enough cell mass was generated by continuous growth for 8 h. The culture was filtered through filter paper ŽWhatman No. 1. and centrifuged at 27,000 = g for 15 min. The cell pellet was washed several times and resuspended in distilled water. In some tests, the bacterial strain was adapted to corundum by repeated subculturing upto 6 months in the Bromfield medium in the presence of 5% Žwrv. mineral. These adapted strains were used in some beneficiation tests and their performance compared with unadapted strains.
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A special medium containing 1% peptone, 1% starch, 0.005% neutral red dye and 2% agar was used for enrichment of bacterial cultures and purification of the strain. Isolated pure colonies were picked up and repropagated in the same medium, the process being repeated until pure strains were obtained and checked. Mineral adhered organisms were desorbed by agitation and centrifugation and counted by direct plating on solid medium. The bacterial growth pattern was studied by the dilution plate technique through enumeration of colony forming units. The viable cell counts were cross-checked microscopically. The cell counts by both these methods were found to be within "10%. A batch culture after growth in the Bromfield medium was centrifuged at 31,000 = g for 20 min to separate cells from the extracellular materials, followed by filtration of the supernatant through sterile filter membrane ŽMillipore, 0.45 mm pore size, the membrane made of surfactant-free cellulose nitrate and cellulose acetate.. Bacterial proteins present in the cell-free metabolite solution were precipitated by slow addition of ammonium sulphate to a saturation level of 65%. The precipitate was separated and the supernatant desalted for determination of its polysaccharide concentration. The proteinaceous precipitate was redissolved in minimum volume of 0.02 M Tris-hydrochloride buffer at pH 7 and dialized against the same buffer over 18 h. The precipitate formed during dialysis was removed by centrifugation and discarded. The supernatant was used for protein estimation. Total protein content was estimated by the Lowry method ŽLowry et al., 1951. and carbohydrates by the Phenol-sulphuric acid method ŽDubois et al., 1956.. 2.2. Minerals Hand-picked pure mineral samples of hematite, corundum, calcite, kaolinite and quartz used in this study were obtained from Alminrock Indscer Fabriks, Bangalore, India. The samples were subjected to dry grinding in a porcelain mill. The ground sample was then dry screened and the - 38 mm size fraction was used in adsorption, dissolution, flocculation and electrokinetic studies. A Ž75–110 mm. size fraction of the minerals was used in flotation tests. In some tests, two types of iron ore samples having the chemical composition shown in Table 1 was used. Surface areas of the pure mineral samples are also indicated in the table. 2.3. Electrokinetic measurements Electrophoretic measurements were made using Zeta-meter 3.0. Mineral samples before and after interaction with bacterial metabolite, bioproteins and exopolysaccharides were dispersed in 10y3 M KNO 3 solution for the purpose. Zeta potentials as a function of pH were determined for different minerals before and after interaction. 2.4. Adsorption studies Adsorption of bacterial proteins and exopolysaccharides onto different minerals was estimated at different pH values and concentrations. A 10y3 M KNO 3 solution was used
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Table 1 Characterization of mineral and ore samples
Iron ore A
Chemical composition Ž%.
Mineralogical analysis Ž%.
Fe 44.6
Hematite 45–50 Goethite 10–15 Magnetite 3–5 Quartz 30–35 Hematite 73.5 Quartz 1.7 Alumina 14.7 Kaolinite 3.0
SiO 2 32.1 Iron ore B
Fe 51.4 SiO 2 1.73 Al 10.0 LOI 2.0 Surface area, m2 rg
Hematite Corundum Calcite Quartz Kaolinite
1.00 0.70 0.80 1.577 16.925
as the base electrolyte for the purpose. In all adsorption tests, the time of equilibration was fixed at 15 min. After interaction, the residual reagent concentration in the supernatant was estimated. 2.5. Flotation tests One gram of the mineral sample was used for the flotation of the minerals to study the influence of bacterial pretreatment. The mineral sample was taken in 200 ml of deionised double distilled water and the pH was adjusted to the desired value Ž7–9.. The conditioning period was 5 min and the flotation was carried out for 5 min under a nitrogen flow rate of 40 mlrmin using a Hallimond tube. Flotation tests using actual iron ore samples were carried out using a Leaf and Knoll cell ŽTaggart, 1967. at 2.5% pulp density. Flotation tests with pure minerals and the iron ore samples were carried out under different conditions as detailed elsewhere to establish the influence of prior biotreatment. In some tests, an isododecyl oxypropyl amino propyl amine was also used as a collector reagent. 2.6. Settling and flocculation tests To study the settling rates of the different minerals, 5 g of each sample were dispersed in 100 ml of deionised double distilled water in a graduated cylinder. The weight settled as a function of time and pH was monitored in the presence and absence of the bacteria or metabolite.
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For selective flocculation studies, artificial binary and ternary mixtures of the minerals in equal proportions were placed in a 100-ml graduated cylinder and diluted with make-up water to near the 100 ml mark so that the subsequent addition of bioreagents would give a total pulp volume of 100 ml. At this point, the pH of the pulp was adjusted either with sodium hydroxide or nitric acid which was followed by the addition of reagents in three equal portions, mixing being performed between each addition by gently inverting the cylinder three times between each addition. After the final addition, the cylinder was inverted five times and set down. The settling of the mineral particles in the mixture began immediately. After 1 min, the mud line had reached the bottom. A total of 90 ml of the supernatant was decanted out. Another 90 ml of water, whose pH had been preadjusted to the same value, was added to the remaining solids. The cylinder was inverted five times, set down, and the settling and decantation procedure repeated. The desliming process was performed six times. The sink and float fractions were analyzed each time. Control experiments in the absence of bioreagents were also carried out under similar conditions for comparison purposes. 2.7. Fourier transform infrared spectroscopy Infrared absorption spectra were recorded on a Bruker Equinox-55 model Fourier Transform Infrared spectrophotometer using different minerals before and after interaction. The instrument was provided with a KBr beam splitter separator and DTGS as detector. The acquisition was through the transmission mode. After interaction with bioreagents, the mineral samples were thoroughly washed using deionized double distilled water and vacuum dried. The KBr pellet technique was used to record the spectra. The difference spectrum was obtained by subtracting the spectrum of unreacted minerals from that of interacted ones using a program available in the instrument.
3. Results and discussion 3.1. Electrokinetic and adsorption studies Microbe–mineral interactions result in significant surface chemical changes on mineral surfaces ŽNamita Deo and Natarajan, 1998.. The manner in which bioreagents bring about such changes on the electrokinetic properties of quartz, kaolinite, hematite, calcite and corundum is illustrated in Figs. 1–5, with reference to influence of bacterial metabolite, bacterial protein and polysaccharides. Influence of a standard protein ŽBSA. and polysaccharide Žstarch. on the electrokinetic properties of the above minerals was also studied for comparison purposes. For quartz, the measured zeta potentials were seen to be shifted in a more positive direction after interaction with bacterial metabolite, protein and BSA. For example, the isoelectric point ŽIEP. of quartz shifted from its initial Žbefore interaction. value of about 2.0 to 3.2, 3.6 and 4.6, respectively, after interaction with bacterial protein, metabolite and BSA. The effect of similar interaction
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with either bacterial metabolite or proteins on the electrokinetic behaviour of kaolinite was only marginal in that only slight positive shifts in measured zeta potentials were observed, especially in the alkaline pH ranges without any significant shift in IEP. Presence of bacterial polysaccharides or starch did not significantly alter the surface chemistry of quartz and kaolinite, in the concentration ranges used in this study. Unlike the case with quartz and kaolinite, the effect of bioreagents interaction on the surface chemical behaviour of hematite, corundum and calcite was different in that the measured zeta potentials were observed to shift towards more negative values, both with metabolites and biopolysaccharides. For example, freshly prepared corundum exhibited an IEP corresponding to a pH of about 7.2 ŽFig. 5., while after interaction with the metabolite and bacterial polysaccharide, the IEP of corundum shifted to a lower pH value of about 4.6 and 2.8, respectively. The observed negative shift in measured zeta potentials was more prominent at pH values lower than about 7. For hematite and calcite also ŽFigs. 3 and 4., the zeta potentials were observed to be significantly shifted towards more negative values after similar interaction. The influence of adsorption of bacterial cells on the electrokinetic behavior of the above five minerals has already been reported ŽNamita Deo and Natarajan, 1998.. The bacterium possesses negatively charged groups Žphosphate, carboxylate, etc.. on the outermost surface. The cell walls of Gram-positive bacteria are made up of many layers of murein and teichoic acids. The p K a values of free carboxylate and phosphate groups lie in the pH range of about 2 to 4 and the values of bound species may be quite different Ž) 4. from that of the free species due to charge density created by neighbouring groups ŽAchouak et al., 1994.. The negative charge observed on bacterial cells would enable electrostatic interaction with positively charged minerals such as hematite and corundum, especially at pH values lower than the mineral IEP values Žless than pH 7.. On the other hand, such electrostatic attraction is unlikely for the bacteria towards quartz and kaolinite which exhibit increasing negative surface charges similar to bacterial cells throughout the pH range of 2 to 13. Bacterial cells then may use alternative mechanisms for adhesion depending on the environmental conditions available on mineral surfaces, thus exhibiting different physico-chemical affinities. Environmental growth conditions influence the nature of bacterial metabolite products. FTIR spectra of the various mineral samples before and after interaction with bacterial metabolite, bacterial proteins and exopolysaccharides were also taken. Before bacterial interaction, quartz surfaces indicated the presence of silanol groups and vibrational spectra due to Si`O bonds. Surface hydroxyl groups, hydrogen bonded OH as well as metal to oxygen stretching vibrations were observed on fresh untreated hematite, corundum, calcite and kaolinite samples. However, difference spectra obtained through subtraction of biotreated mineral from unreacted mineral yielded evidence of new surface species. Bands due to amide I Ž1650 cmy1 ., CONH Žamide II. at about 1550 cmy1 , primary alcoholic stretching Ž1050 cmy1 . and amide IV and C-O bending in proteins Ž700–795 cmy1 . could be seen on quartz and kaolinite surfaces after interaction with bacterial metabolites. On the other hand, bands due to carboxylate anion Ž1630 cmy1 ., C–OH stretching Ž1120–1140 cmy1 . and CH 2 vibrations Ž400–600 cmy1 . could be seen on hematite, calcite and corundum surfaces after interaction with metabolite. The following order with respect to the presence of bacterial protein and
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Fig. 1. Measured zeta potentials as a function of pH for quartz before and after interaction with various reagents.
polysaccharide on different mineral surfaces after treatment with bacterial metabolites could be observed. Proteins: kaolinite) quartz ) corundum) hematite) calcite Polysaccharides: calcite) corundum) hematite) quartz ) kaolinite The changes in surface chemical behaviour of the minerals after interaction with bacterial metabolites could then attributed to the above surface species. Positive deviations in measured zeta potentials for quartz and kaolinite ŽFigs. 1 and 2. after interaction with bacterial metabolite and proteins can be attributed to the predominant
Fig. 2. Measured zeta potentials as a function of pH for kaolinite before and after interaction with various reagents.
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Fig. 3. Measured zeta potentials as a function of pH for hematite before and after interaction with various reagents.
presence of amino groups from the proteins, while the reverse behaviour observed for hematite, calcite and corundum ŽFigs. 3–5. after similar pretreatments can be due to surface adsorption of excess polysaccharides. To further substantiate that the biotreated
Fig. 4. Measured zeta potentials as a function of pH for calcite before and after interaction with various reagents.
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Fig. 5. Measured zeta potentials as a function of pH for corundum before and after interaction with various reagents.
quartz and kaolinite surfaces indeed contained significant amounts of proteinaceous matter, they were treated with Proteinase-K after which the electrokinetic behaviour of the minerals was observed to shift to its original position. Zeta potential measurements Žnot shown in the figures. carried out with the above minerals after interaction with Bovine serum albumin Ža representative protein. and starch Ža representative polysaccharide. indicated similar electrokinetic behaviour further substantiating the above conclusions. 3.2. Influence of bioreagents on mineral surface hydrophobicity Microflotation results on the various mineral samples before and after interaction with, metabolite as well as with the separated bacterial proteins and polysaccharides are illustrated in Table 2. The changes in the surface chemistry of the interacted minerals with respect to surface hydrophobicity and hydrophilicity brought about by prior Table 2 Flotability of minerals under different biopretreatment conditions Ž1-h interaction period, natural pH. Mineral
Control
Metabolite
Bacterial protein Ž3 mgrg.
Bacterial polysaccharides Ž3 mgrg.
Quartz Ž75–110 mm. Kaolinite Ž - 38 mm. Corundum Ž75–110 mm. Hematite Ž75–110 mm. Calcite Ž75–110 mm.
4.0 38.0 5.0 4.0 8.0
80.0 80.0 6.0 4.0 7.0
100.0 94.0 7.0 4.0 8.0
4.0 36.7 1.0 0.8 1.3
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interaction with bioreagents are clearly evident. Interaction with bacterial metabolite and proteins resulted in enhanced hydrophobicity for quartz and kaolinite, unlike the case with hematite, calcite and corundum, which were rendered more hydrophilic after similar treatment, as reflected in the magnitudes of percent weight floated. Bacterial metabolites contain exopolysaccharides and many proteins and their preferential adhesion Žadsorption. on the minerals are responsible for the above observations. As indicated already, predominant adsorption of polysaccharides would render the mineral more hydrophilic and of proteins making them more hydrophobic. Results of flotation tests on various minerals after interaction with separated proteins and polysaccharides further substantiate the above conclusion; namely, interaction with proteins enhance flotability Žhydrophobicity. and with polysaccharides depresses the mineral Žhydrophilicity.. Also, proteins are predominantly adsorbed onto quartz and kaolinite, while hematite, calcite and corundum exhibited more affinity towards polysaccharide ŽFigs. 6 and 7.. To establish further the positive role of proteins in enhancing the flotability of certain minerals such as quartz and kaolinite, additional flotation tests were performed in the presence of different concentrations of a standard representative protein, namely, bovine seum albumin and the results indicated that significant enhancements in the flotability of quartz and kaolinite could be readily obtained Ž90–100%.. Interaction with proteins, on the other hand, did not significantly promote the flotability of calcite, corundum hematite. Adsorption densities of bacterial protein and polysaccharide onto different minerals as a function of time in the pH range 7–9 are illustrated in Figs. 6 and 7. Protein adsorption was the highest on kaolinite and quartz and the least on hematite and corundum. For example, after 45 min of equilibration, kaolinite adsorbed 3.7 mgrm2 ,
Fig. 6. Adsorption of bacterial protein on various minerals as a function of time.
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Fig. 7. Adsorption of bacterial polysaccharide on various minerals as a function of time.
while quartz, 3.5 mgrm2 of protein. During the same period, hematite and corundum exhibited about 0.4 and 2.0 mgrm2 of protein uptake. On the other hand, calcite, corundum and hematite adsorbed the highest amount of bacterial polysaccharide, namely, 15.0, 14.0 and 10.1 mgrm2 , respectively, within 45 min, compared to only about 0.2 and 1.0 mgrm2 uptake by kaolinite and quartz. As evident from the above results, interaction with bacterial metabolite and bioproteins promoted the flotation and dispersion of quartz and kaolinite, while such biotreatment resulted in the depression of hematite and corundum. Starch, a natural hydrophilic carbohydrate polymer, is extensively used in mineral processing operations for depression of iron oxides ŽBalajee and Iwasaki, 1969.. Dispersion of silica particles and selective flocculation followed by settling of iron oxides can be achieved by controlled additions of starch and other polysaccharides. Bacterial polysaccharides are essentially serving the same purpose. It has also been reported ŽMera and Beveridge, 1993. that silica and silicate containing minerals are chemically bound and modified through silicate-to-amine complexation in the peptidoglycan of Gram-positive bacteria. Binding of silicate ions to positively charged groups of proteinaceous compounds in proteins is suggested. The presence of the amino-groups has previously been found to negatively affect metal–wall interactions ŽBeveridge and Murray, 1980.. Similarly, exopolysaccharides containing COOy, OHyand PO43y groups can influence metal cation binding. Interaction and complex formation of iron and aluminium ion species with polysaccharides have been well documented in the literature ŽSubramanian, 1985.. The major mechanisms involved in such interactions are electrostatic, hydrogenbonding and chemical. Results from adsorption and electrokinetic studies reveal the existence of electrostatic and chemical interaction. Significant shifts in the IEP of hematite, corundum, calcite and quartz after interaction with bacterial metabolites is
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indicative of chemisorption. FTIR spectroscopic data gave further evidence of chemical interaction as well as possibilities of hydrogen-bonding of metabolic products on different minerals. Sorption and interaction of bacteria with clays have been well reported ŽStotzky, 1985.. The adsorption and binding of proteins and different amino acids to kaolinite has been reported ŽDashman and Stotzky, 1982.. Bacterial proteins are known to influence enhanced reactivity with clay minerals, the amount of such adsorption depends on the types of clay and with the cation saturating the clay ŽDashman and Stotzky, 1984.. 3.3. Flocculation and flotation of minerals through bacterial interaction Settling rates of various minerals corresponding to 1 min of interaction Ž- 38 mm. at different pH values in the presence of bacterial metabolite, bioproteins and exopolysaccharides are illustrated in Table 3. While the settling rates of hematite, calcite and corundum particles were increased after interaction with either bacterial metabolite or exopolysaccharides, those of quartz and kaolinite were seem to be significantly decreased after similar treatment Žwith metabolite or bioproteins.. Flocculation of hematite and corundum and dispersion quartz and kaolinite is facilitated by such interaction. Various polysaccharides such as starches and dextrans are used as depressants for iron oxides in iron ore flotation. Selective flocculation of iron oxide with dispersion of silica can be achieved by addition of the above polysaccharides. Biopolymers such as those containing exopolysaccharides can interlink the mineral particles through polymer bridging and flocculate the mineral fines selectively. Increased affinity of bacterial cells and excreted polysaccharides towards hematite, calcite and corundum bring about their selective flocculation facilitating their rapid settling in an aqueous medium. Results of selective flocculation tests carried out with mineral mixtures substantiate the above conclusion. Typical results of selective flocculation on 1:1 mineral mixtures are presented in Table 4. It could be readily seen that it is possible to separate efficiently silica and
Table 3 Settling of minerals under different biopretreatment conditions Ž5% pulp density, - 38 mm. Mineral
Quartz Kaolinite Corundum Hematite Calcite
pH
4–5 7.0 4–5 7.0 4–5 7.0 4–5 7.0 7.0
Percentage weight settled in 1 min Control
Metabolite
Bacterial proteins
Bacterial polysaccharides
58.0 45.0 60.0 50.0 85.0 82.0 85.0 82.0 45.0
16.0 – 17.0 – 92 – 96.0 – 93
– 19.0 – 20.0 – 78.0 – 77.0 45.0
– 45.0 – 50.0 – 96.0 – 95.0 91.0
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Table 4 Selective flocculation of mineral mixtures Ž1:1. to separate silica through interaction with bacterial metabolite and bacterial proteins Žy38 mm. Mineral combination
Number of desliming stages
Separation efficiency Ž%.
Corundum–quartz
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
28.0 40.0 59.0 78.0 90.0 99.0 20.0 35.0 55.0 70.0 88.0 98.0 25.0 38.0 56.0 71.0 89.0 97.0
Hematite–quartz
Calcite–quartz
silicates from alumina, calcite and iron oxide through bioflocculation in the presence of bacterial metabolites or bioproteins. Generation of bacterial proteins and exopolysaccharides was also found to be influenced by the nature of the mineral species present during bacterial growth as illustrated in Table 5. Presence of kaolinite or quartz during bacterial growth promoted protein generation while that of calcite, corundum and hematite resulted in higher amounts of exopolysaccharides in the metabolic product. It has been found not possible to separate alumina from hematite efficiently using the Bromfield-grown cells or their metabolites since bacterial interaction brings about similar surface-chemical changes on both of them. Tests with hematite–corundum mixtures have shown that both the minerals settled down faster in an aqueous medium after bacterial interaction without any selectivity ŽNamita Deo and Natarajan, 1999.. However, it has been observed that through the use of corundum-adapted strains and Table 5 Generation of proteins and polysaccharides during bacterial growth in the presence of different minerals Substrate
Protein Žmgrl.
Polysaccharide Žmgrl.
Bromfield Corundum Hematite Kaolinite Quartz Calcite
70.9 52.6 50.1 73.7 79.1 42.8
480.0 500.0 520.0 460.0 450.0 530.0
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Table 6 Separation of corundum–hematite Ž1:1. mixture through selective flocculation using bacterial protein and protein-free metabolites separated from corundum-adapted strains ŽpH 7.0, y38 mm, 5% pulp density. Desliming stages
Total % iron removal Bacterial protein separated from metabolite
Metabolite after protein separation
1 2 3 4 5 6
55.0 60.0 70.0 85.0 98.0 99.0
30.0 40.0 50.0 65.0 70.1 85.2
metabolites of P. polymyxa, efficient separation of alumina from iron oxides can be achieved. Typical selective flocculation results are shown in Table 6. At pH 7.0, 99% separation could be obtained between hematite and corundum, using bacterial protein and 85.2% separation using protein-free metabolite. Unlike the case with unadapted cells, corundum particles could be effectively settled Žflocculated. with the selective flocculation of only corundum using the metabolic products from corundum-adapted strain Žhematite dispersed.. The differences in bacterial protein and polysaccharide adsorption, nature of interaction products and the resulting alterations in the surface chemical behaviour of the minerals could be taken advantage of in the beneficiation of ores containing the various mineral constituents. The minerals studied in this work constitute a typical iron ore system. Removal of silica and alumina from iron ores is a major problem facing the iron and steel industries, the world over. Systematic studies carried out with hematite– corundum–quartz and hematite–kaolinite–quartz mixtures after interaction with adapted strains of P. polymyxa showed that silica, alumina and alumino-silicates can be effectively removed. The utility of biobeneficiation is further demonstrated with respect to the beneficiation of iron ore samples for removal of silica and alumina, as illustrated in Table 7. 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. Table 7 Selective flocculation of iron ore samples by metabolites separated from unadapted and corundum-adapted cells Ž5-min interaction at 5% pulp density. Number of desliming stages
% SiO 2 removal Žunadapted.
% Al 2 O 3 removal Žadapted.
1 2 3 4 5 6
31.0 51.0 66.0 79.9 90.3 97.9
25.0 35.7 57.9 76.0 89.9 96.0
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4. Conclusions The following major conclusions can be drawn from the present work. Interaction of metabolic products of P. polymyxa with minerals such as hematite, corundum, calcite, kaolinite and quartz resulted in significant surface-chemical changes. Quartz and kaolinite were rendered more hydrophobic, while hematite, calcite and corundum more hydrophilic after biotreatment. Enhanced adsorption of bacterial proteins on quartz and kaolinite and of polysaccharides on hematite, calcite and corundum was observed. Through biopretreatment of the above minerals, it becomes possible to selectively separate silica and alumina from the iron oxide either through flotation or selective flocculation. Bioprocessing is very useful in the beneficiation of iron ores for removal of either silica or alumino-silicates. References Achouak, W., Thomas, F., Heulin, T., 1994. Physico-chemical surface properties of rhizobacteria and their adhesion to rice roots. Colloids Surf., B 3, 131–137. Ash, C., Priest, F.G., Collins, M.D., 1993. Molecular identification of rRNA group of 3 Bacilli using a PCR probe test. Antonie van Leeuwenhoek 64, 253–260. Balajee, S.R., Iwasaki, I., 1969. Adsorption mechanism of starches in flotation and flocculation of iron ores. Trans. Soc. Min. Eng. 244, 401–406. Beveridge, T.J., Murray, G.E., 1980. Sites of metal deposition in the cell wall of Bacillus subtilis. J. Bacteriol. 141, 876–887. Dashman, T., Stotzky, G., 1982. Adsorption and binding of amino acids on homoionic montmorillonite and kaolinite. Soil Biol. Biochem. 14, 447–456. Dashman, T., Stotzky, G., 1984. Adsorption and binding of peptides on homoionic montmorillonite and kaolinite. Soil Biol. Biochem. 16, 51–55. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28, 350–356. Lowry, O.H., Rosenborough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265–275. Mera, M.U., Beveridge, T.J., 1993. Mechanism of silicate binding to the bacterial cell wall in Bacillus subtilis. J. Bacteriol. 7, 1936–1945. Murphy, D., 1952. Structure of levan produced by Bacillus polymyxa. Can. J. Chem. 30, 872–878. Namita Deo, Natarajan, K.A., 1997a. Surface modification and biobeneficiation of some oxide minerals using Bacillus polymyxa. Miner. Metall. Process. 32–39, August. Namita Deo, Natarajan, K.A., 1997b. Interaction of Bacillus polymyxa with some oxide minerals with reference to mineral beneficiation and environmental control. Min. Eng. 10, 1339–1354. Namita Deo, Natarajan, K.A., 1998. Studies on interaction of Paenibacillus polymyxa with iron ore minerals in relation to beneficiation. Int. J. Miner. Process. 55, 41–60. Namita Deo, Natarajan, K.A., 1999. Role of corundum-adapted strains of Bacillus polymyxa in the separation of hematite and alumina. Miner. Metall. Process. Žin print.. Phalguni Anand, Modak, J.M., Natarajan, K.A., 1996. Biobeneficiation of bauxite using Bacillus polymyxa: calcium and iron removal. Int. J. Miner. Process. 48, 51–60. Reed, G., 1987. Industrial Microbiology. 4th edn. CBS Publishers, New Delhi. Stanier, Y.R., Ingraham, J.L., Wheelis, M.L., Painter, P.R., 1993. General Microbiology. 5th edn. Macmillan, London. Stotzky, G., 1985. Mechanisms of adhesion to clays with reference to soil systems. In: Savage, D.C., Fletcher, M. ŽEds.., Bacterial Adhesion. Plenum, New York, pp. 195–253. Subramanian, S., 1985. Doctoral thesis, Univ. of Mysore. Taggart, A.F., 1967. Hand Book of Mineral Dressing. Wiley, New York.
Role of bacterial interaction and bioreagents in iron ore flotation K.A. Natarajan ) , Namita Deo Department of Metallurgy, Indian Institute of Science, Bangalore, 560 012, India Received 15 August 1999; accepted 17 July 2000
Abstract Interaction between Paenibacillus polymyxa and iron ore minerals such as hematite, corundum, calcite, quartz and kaolinite brought about significant surface chemical changes on all the minerals. Quartz and kaolinite were rendered more hydrophobic, while the other three minerals became more hydrophilic after bacterial interaction. Predominance of bacterial polysaccharides on interacted hematite, corundum and calcite and of proteins on quartz and kaolinite was responsible for the surface chemical changes. The bacterial strains could be preadapted to different mineral substrates. Corundum-adapted strains were seen to secrete mineral-specific proteins which could be used to separate alumina from iron ores. The utility of bioprocessing in the beneficiation of iron ores for removal of silica and alumina is demonstrated. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Paenibacillus polymyxa; iron ore; beneficiation; surface chemistry
1. Introduction Bacillus polymyxa, recently renamed as Paenibacillus polymyxa ŽAsh et al., 1993., 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. They also generate levan forming the capsule of the organism from sucrose by the activity of a specific enzyme known as levan sucrase ŽMurphy, 1952.. The extracellular polysaccharide ŽECP. aids in biological uptake of metal ions necessary for metabolism and growth. Heavy metals are known to )
Corresponding author. Tel.: q91-80-3600-120; fax: q91-80-3600-472. E-mail address: [email protected] ŽK.A. Natarajan..
0301-7516r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 7 5 1 6 Ž 0 0 . 0 0 0 4 9 - 1
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bind to the cell walls through the ECP. Commercial use of this organism in the production of proteinases, amylases, butanediol and glycogens has been reported ŽReed, 1987.. The structure of the bacterial cell wall plays a significant role in bacterial adhesion to mineral substrates. Peptidoglycan, a prominent constituent of the cell wall, is covalently linked to other macromolecular constituents which include several types of polysaccharides and polyphosphate polymers known as teichoic acids ŽStanier et al., 1993.. The presence of different metal ions, such as Caqq and Mgqq as well as minerals such as iron, aluminium and calcium oxides during growth, influences the types and quantity of polysaccharides, proteins and enzymes secreted by the bacteria ŽReed, 1987.. Although the use of different microorganisms in ore leaching is well established, biological methods of mineral beneficiation are not even sufficiently understood. The utility of B. polymyxa in the biobeneficiation of bauxite and iron ore minerals has been recently reported ŽPhalguni Anand et al., 1996, Namita Deo and Natarajan, 1997a,b, 1998.. As different from bioleaching, biobeneficiation, by definition, refers to selective removal of undesirable mineral constituents from an ore through interaction with microorganisms, thus enriching it with respect to the desired valuable minerals. It is essential to understand the mechanisms and resulting consequences of microbe– mineral interactions before the utility of the 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 is of significance. In this paper, the role of bioreagents secreted by P. polymyxa in altering the surface chemistry of some iron ore minerals, such as hematite, corundum, calcite, quartz and kaolinite, is discussed. The consequences of microbe–mineral interactions of relevance in mineral beneficiation are brought out in terms of iron ore flotation and selective flocculation. The utility of corundum-adapted bacterial cells in the removal of alumina from iron ores is discussed. 2. Materials and methods 2.1. Bacterial strain and growth conditions A pure strain of P. polymyxa NCIM 2539 was used in all the studies. A 10% vrv of an active inoculum was added to Bromfield medium ŽPhalguni Anand et al., 1996. and incubated at 308C on a rotary shaker at 240 rpm. The bacterial growth pattern was studied by microscopic counting using a Petroff–Hausser counter under a phase contrast microscope and also by colony counting after plating. pH changes during bacterial growth was monitored and enough cell mass was generated by continuous growth for 8 h. The culture was filtered through filter paper ŽWhatman No. 1. and centrifuged at 27,000 = g for 15 min. The cell pellet was washed several times and resuspended in distilled water. In some tests, the bacterial strain was adapted to corundum by repeated subculturing upto 6 months in the Bromfield medium in the presence of 5% Žwrv. mineral. These adapted strains were used in some beneficiation tests and their performance compared with unadapted strains.
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A special medium containing 1% peptone, 1% starch, 0.005% neutral red dye and 2% agar was used for enrichment of bacterial cultures and purification of the strain. Isolated pure colonies were picked up and repropagated in the same medium, the process being repeated until pure strains were obtained and checked. Mineral adhered organisms were desorbed by agitation and centrifugation and counted by direct plating on solid medium. The bacterial growth pattern was studied by the dilution plate technique through enumeration of colony forming units. The viable cell counts were cross-checked microscopically. The cell counts by both these methods were found to be within "10%. A batch culture after growth in the Bromfield medium was centrifuged at 31,000 = g for 20 min to separate cells from the extracellular materials, followed by filtration of the supernatant through sterile filter membrane ŽMillipore, 0.45 mm pore size, the membrane made of surfactant-free cellulose nitrate and cellulose acetate.. Bacterial proteins present in the cell-free metabolite solution were precipitated by slow addition of ammonium sulphate to a saturation level of 65%. The precipitate was separated and the supernatant desalted for determination of its polysaccharide concentration. The proteinaceous precipitate was redissolved in minimum volume of 0.02 M Tris-hydrochloride buffer at pH 7 and dialized against the same buffer over 18 h. The precipitate formed during dialysis was removed by centrifugation and discarded. The supernatant was used for protein estimation. Total protein content was estimated by the Lowry method ŽLowry et al., 1951. and carbohydrates by the Phenol-sulphuric acid method ŽDubois et al., 1956.. 2.2. Minerals Hand-picked pure mineral samples of hematite, corundum, calcite, kaolinite and quartz used in this study were obtained from Alminrock Indscer Fabriks, Bangalore, India. The samples were subjected to dry grinding in a porcelain mill. The ground sample was then dry screened and the - 38 mm size fraction was used in adsorption, dissolution, flocculation and electrokinetic studies. A Ž75–110 mm. size fraction of the minerals was used in flotation tests. In some tests, two types of iron ore samples having the chemical composition shown in Table 1 was used. Surface areas of the pure mineral samples are also indicated in the table. 2.3. Electrokinetic measurements Electrophoretic measurements were made using Zeta-meter 3.0. Mineral samples before and after interaction with bacterial metabolite, bioproteins and exopolysaccharides were dispersed in 10y3 M KNO 3 solution for the purpose. Zeta potentials as a function of pH were determined for different minerals before and after interaction. 2.4. Adsorption studies Adsorption of bacterial proteins and exopolysaccharides onto different minerals was estimated at different pH values and concentrations. A 10y3 M KNO 3 solution was used
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Table 1 Characterization of mineral and ore samples
Iron ore A
Chemical composition Ž%.
Mineralogical analysis Ž%.
Fe 44.6
Hematite 45–50 Goethite 10–15 Magnetite 3–5 Quartz 30–35 Hematite 73.5 Quartz 1.7 Alumina 14.7 Kaolinite 3.0
SiO 2 32.1 Iron ore B
Fe 51.4 SiO 2 1.73 Al 10.0 LOI 2.0 Surface area, m2 rg
Hematite Corundum Calcite Quartz Kaolinite
1.00 0.70 0.80 1.577 16.925
as the base electrolyte for the purpose. In all adsorption tests, the time of equilibration was fixed at 15 min. After interaction, the residual reagent concentration in the supernatant was estimated. 2.5. Flotation tests One gram of the mineral sample was used for the flotation of the minerals to study the influence of bacterial pretreatment. The mineral sample was taken in 200 ml of deionised double distilled water and the pH was adjusted to the desired value Ž7–9.. The conditioning period was 5 min and the flotation was carried out for 5 min under a nitrogen flow rate of 40 mlrmin using a Hallimond tube. Flotation tests using actual iron ore samples were carried out using a Leaf and Knoll cell ŽTaggart, 1967. at 2.5% pulp density. Flotation tests with pure minerals and the iron ore samples were carried out under different conditions as detailed elsewhere to establish the influence of prior biotreatment. In some tests, an isododecyl oxypropyl amino propyl amine was also used as a collector reagent. 2.6. Settling and flocculation tests To study the settling rates of the different minerals, 5 g of each sample were dispersed in 100 ml of deionised double distilled water in a graduated cylinder. The weight settled as a function of time and pH was monitored in the presence and absence of the bacteria or metabolite.
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For selective flocculation studies, artificial binary and ternary mixtures of the minerals in equal proportions were placed in a 100-ml graduated cylinder and diluted with make-up water to near the 100 ml mark so that the subsequent addition of bioreagents would give a total pulp volume of 100 ml. At this point, the pH of the pulp was adjusted either with sodium hydroxide or nitric acid which was followed by the addition of reagents in three equal portions, mixing being performed between each addition by gently inverting the cylinder three times between each addition. After the final addition, the cylinder was inverted five times and set down. The settling of the mineral particles in the mixture began immediately. After 1 min, the mud line had reached the bottom. A total of 90 ml of the supernatant was decanted out. Another 90 ml of water, whose pH had been preadjusted to the same value, was added to the remaining solids. The cylinder was inverted five times, set down, and the settling and decantation procedure repeated. The desliming process was performed six times. The sink and float fractions were analyzed each time. Control experiments in the absence of bioreagents were also carried out under similar conditions for comparison purposes. 2.7. Fourier transform infrared spectroscopy Infrared absorption spectra were recorded on a Bruker Equinox-55 model Fourier Transform Infrared spectrophotometer using different minerals before and after interaction. The instrument was provided with a KBr beam splitter separator and DTGS as detector. The acquisition was through the transmission mode. After interaction with bioreagents, the mineral samples were thoroughly washed using deionized double distilled water and vacuum dried. The KBr pellet technique was used to record the spectra. The difference spectrum was obtained by subtracting the spectrum of unreacted minerals from that of interacted ones using a program available in the instrument.
3. Results and discussion 3.1. Electrokinetic and adsorption studies Microbe–mineral interactions result in significant surface chemical changes on mineral surfaces ŽNamita Deo and Natarajan, 1998.. The manner in which bioreagents bring about such changes on the electrokinetic properties of quartz, kaolinite, hematite, calcite and corundum is illustrated in Figs. 1–5, with reference to influence of bacterial metabolite, bacterial protein and polysaccharides. Influence of a standard protein ŽBSA. and polysaccharide Žstarch. on the electrokinetic properties of the above minerals was also studied for comparison purposes. For quartz, the measured zeta potentials were seen to be shifted in a more positive direction after interaction with bacterial metabolite, protein and BSA. For example, the isoelectric point ŽIEP. of quartz shifted from its initial Žbefore interaction. value of about 2.0 to 3.2, 3.6 and 4.6, respectively, after interaction with bacterial protein, metabolite and BSA. The effect of similar interaction
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with either bacterial metabolite or proteins on the electrokinetic behaviour of kaolinite was only marginal in that only slight positive shifts in measured zeta potentials were observed, especially in the alkaline pH ranges without any significant shift in IEP. Presence of bacterial polysaccharides or starch did not significantly alter the surface chemistry of quartz and kaolinite, in the concentration ranges used in this study. Unlike the case with quartz and kaolinite, the effect of bioreagents interaction on the surface chemical behaviour of hematite, corundum and calcite was different in that the measured zeta potentials were observed to shift towards more negative values, both with metabolites and biopolysaccharides. For example, freshly prepared corundum exhibited an IEP corresponding to a pH of about 7.2 ŽFig. 5., while after interaction with the metabolite and bacterial polysaccharide, the IEP of corundum shifted to a lower pH value of about 4.6 and 2.8, respectively. The observed negative shift in measured zeta potentials was more prominent at pH values lower than about 7. For hematite and calcite also ŽFigs. 3 and 4., the zeta potentials were observed to be significantly shifted towards more negative values after similar interaction. The influence of adsorption of bacterial cells on the electrokinetic behavior of the above five minerals has already been reported ŽNamita Deo and Natarajan, 1998.. The bacterium possesses negatively charged groups Žphosphate, carboxylate, etc.. on the outermost surface. The cell walls of Gram-positive bacteria are made up of many layers of murein and teichoic acids. The p K a values of free carboxylate and phosphate groups lie in the pH range of about 2 to 4 and the values of bound species may be quite different Ž) 4. from that of the free species due to charge density created by neighbouring groups ŽAchouak et al., 1994.. The negative charge observed on bacterial cells would enable electrostatic interaction with positively charged minerals such as hematite and corundum, especially at pH values lower than the mineral IEP values Žless than pH 7.. On the other hand, such electrostatic attraction is unlikely for the bacteria towards quartz and kaolinite which exhibit increasing negative surface charges similar to bacterial cells throughout the pH range of 2 to 13. Bacterial cells then may use alternative mechanisms for adhesion depending on the environmental conditions available on mineral surfaces, thus exhibiting different physico-chemical affinities. Environmental growth conditions influence the nature of bacterial metabolite products. FTIR spectra of the various mineral samples before and after interaction with bacterial metabolite, bacterial proteins and exopolysaccharides were also taken. Before bacterial interaction, quartz surfaces indicated the presence of silanol groups and vibrational spectra due to Si`O bonds. Surface hydroxyl groups, hydrogen bonded OH as well as metal to oxygen stretching vibrations were observed on fresh untreated hematite, corundum, calcite and kaolinite samples. However, difference spectra obtained through subtraction of biotreated mineral from unreacted mineral yielded evidence of new surface species. Bands due to amide I Ž1650 cmy1 ., CONH Žamide II. at about 1550 cmy1 , primary alcoholic stretching Ž1050 cmy1 . and amide IV and C-O bending in proteins Ž700–795 cmy1 . could be seen on quartz and kaolinite surfaces after interaction with bacterial metabolites. On the other hand, bands due to carboxylate anion Ž1630 cmy1 ., C–OH stretching Ž1120–1140 cmy1 . and CH 2 vibrations Ž400–600 cmy1 . could be seen on hematite, calcite and corundum surfaces after interaction with metabolite. The following order with respect to the presence of bacterial protein and
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149
Fig. 1. Measured zeta potentials as a function of pH for quartz before and after interaction with various reagents.
polysaccharide on different mineral surfaces after treatment with bacterial metabolites could be observed. Proteins: kaolinite) quartz ) corundum) hematite) calcite Polysaccharides: calcite) corundum) hematite) quartz ) kaolinite The changes in surface chemical behaviour of the minerals after interaction with bacterial metabolites could then attributed to the above surface species. Positive deviations in measured zeta potentials for quartz and kaolinite ŽFigs. 1 and 2. after interaction with bacterial metabolite and proteins can be attributed to the predominant
Fig. 2. Measured zeta potentials as a function of pH for kaolinite before and after interaction with various reagents.
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Fig. 3. Measured zeta potentials as a function of pH for hematite before and after interaction with various reagents.
presence of amino groups from the proteins, while the reverse behaviour observed for hematite, calcite and corundum ŽFigs. 3–5. after similar pretreatments can be due to surface adsorption of excess polysaccharides. To further substantiate that the biotreated
Fig. 4. Measured zeta potentials as a function of pH for calcite before and after interaction with various reagents.
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Fig. 5. Measured zeta potentials as a function of pH for corundum before and after interaction with various reagents.
quartz and kaolinite surfaces indeed contained significant amounts of proteinaceous matter, they were treated with Proteinase-K after which the electrokinetic behaviour of the minerals was observed to shift to its original position. Zeta potential measurements Žnot shown in the figures. carried out with the above minerals after interaction with Bovine serum albumin Ža representative protein. and starch Ža representative polysaccharide. indicated similar electrokinetic behaviour further substantiating the above conclusions. 3.2. Influence of bioreagents on mineral surface hydrophobicity Microflotation results on the various mineral samples before and after interaction with, metabolite as well as with the separated bacterial proteins and polysaccharides are illustrated in Table 2. The changes in the surface chemistry of the interacted minerals with respect to surface hydrophobicity and hydrophilicity brought about by prior Table 2 Flotability of minerals under different biopretreatment conditions Ž1-h interaction period, natural pH. Mineral
Control
Metabolite
Bacterial protein Ž3 mgrg.
Bacterial polysaccharides Ž3 mgrg.
Quartz Ž75–110 mm. Kaolinite Ž - 38 mm. Corundum Ž75–110 mm. Hematite Ž75–110 mm. Calcite Ž75–110 mm.
4.0 38.0 5.0 4.0 8.0
80.0 80.0 6.0 4.0 7.0
100.0 94.0 7.0 4.0 8.0
4.0 36.7 1.0 0.8 1.3
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interaction with bioreagents are clearly evident. Interaction with bacterial metabolite and proteins resulted in enhanced hydrophobicity for quartz and kaolinite, unlike the case with hematite, calcite and corundum, which were rendered more hydrophilic after similar treatment, as reflected in the magnitudes of percent weight floated. Bacterial metabolites contain exopolysaccharides and many proteins and their preferential adhesion Žadsorption. on the minerals are responsible for the above observations. As indicated already, predominant adsorption of polysaccharides would render the mineral more hydrophilic and of proteins making them more hydrophobic. Results of flotation tests on various minerals after interaction with separated proteins and polysaccharides further substantiate the above conclusion; namely, interaction with proteins enhance flotability Žhydrophobicity. and with polysaccharides depresses the mineral Žhydrophilicity.. Also, proteins are predominantly adsorbed onto quartz and kaolinite, while hematite, calcite and corundum exhibited more affinity towards polysaccharide ŽFigs. 6 and 7.. To establish further the positive role of proteins in enhancing the flotability of certain minerals such as quartz and kaolinite, additional flotation tests were performed in the presence of different concentrations of a standard representative protein, namely, bovine seum albumin and the results indicated that significant enhancements in the flotability of quartz and kaolinite could be readily obtained Ž90–100%.. Interaction with proteins, on the other hand, did not significantly promote the flotability of calcite, corundum hematite. Adsorption densities of bacterial protein and polysaccharide onto different minerals as a function of time in the pH range 7–9 are illustrated in Figs. 6 and 7. Protein adsorption was the highest on kaolinite and quartz and the least on hematite and corundum. For example, after 45 min of equilibration, kaolinite adsorbed 3.7 mgrm2 ,
Fig. 6. Adsorption of bacterial protein on various minerals as a function of time.
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Fig. 7. Adsorption of bacterial polysaccharide on various minerals as a function of time.
while quartz, 3.5 mgrm2 of protein. During the same period, hematite and corundum exhibited about 0.4 and 2.0 mgrm2 of protein uptake. On the other hand, calcite, corundum and hematite adsorbed the highest amount of bacterial polysaccharide, namely, 15.0, 14.0 and 10.1 mgrm2 , respectively, within 45 min, compared to only about 0.2 and 1.0 mgrm2 uptake by kaolinite and quartz. As evident from the above results, interaction with bacterial metabolite and bioproteins promoted the flotation and dispersion of quartz and kaolinite, while such biotreatment resulted in the depression of hematite and corundum. Starch, a natural hydrophilic carbohydrate polymer, is extensively used in mineral processing operations for depression of iron oxides ŽBalajee and Iwasaki, 1969.. Dispersion of silica particles and selective flocculation followed by settling of iron oxides can be achieved by controlled additions of starch and other polysaccharides. Bacterial polysaccharides are essentially serving the same purpose. It has also been reported ŽMera and Beveridge, 1993. that silica and silicate containing minerals are chemically bound and modified through silicate-to-amine complexation in the peptidoglycan of Gram-positive bacteria. Binding of silicate ions to positively charged groups of proteinaceous compounds in proteins is suggested. The presence of the amino-groups has previously been found to negatively affect metal–wall interactions ŽBeveridge and Murray, 1980.. Similarly, exopolysaccharides containing COOy, OHyand PO43y groups can influence metal cation binding. Interaction and complex formation of iron and aluminium ion species with polysaccharides have been well documented in the literature ŽSubramanian, 1985.. The major mechanisms involved in such interactions are electrostatic, hydrogenbonding and chemical. Results from adsorption and electrokinetic studies reveal the existence of electrostatic and chemical interaction. Significant shifts in the IEP of hematite, corundum, calcite and quartz after interaction with bacterial metabolites is
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indicative of chemisorption. FTIR spectroscopic data gave further evidence of chemical interaction as well as possibilities of hydrogen-bonding of metabolic products on different minerals. Sorption and interaction of bacteria with clays have been well reported ŽStotzky, 1985.. The adsorption and binding of proteins and different amino acids to kaolinite has been reported ŽDashman and Stotzky, 1982.. Bacterial proteins are known to influence enhanced reactivity with clay minerals, the amount of such adsorption depends on the types of clay and with the cation saturating the clay ŽDashman and Stotzky, 1984.. 3.3. Flocculation and flotation of minerals through bacterial interaction Settling rates of various minerals corresponding to 1 min of interaction Ž- 38 mm. at different pH values in the presence of bacterial metabolite, bioproteins and exopolysaccharides are illustrated in Table 3. While the settling rates of hematite, calcite and corundum particles were increased after interaction with either bacterial metabolite or exopolysaccharides, those of quartz and kaolinite were seem to be significantly decreased after similar treatment Žwith metabolite or bioproteins.. Flocculation of hematite and corundum and dispersion quartz and kaolinite is facilitated by such interaction. Various polysaccharides such as starches and dextrans are used as depressants for iron oxides in iron ore flotation. Selective flocculation of iron oxide with dispersion of silica can be achieved by addition of the above polysaccharides. Biopolymers such as those containing exopolysaccharides can interlink the mineral particles through polymer bridging and flocculate the mineral fines selectively. Increased affinity of bacterial cells and excreted polysaccharides towards hematite, calcite and corundum bring about their selective flocculation facilitating their rapid settling in an aqueous medium. Results of selective flocculation tests carried out with mineral mixtures substantiate the above conclusion. Typical results of selective flocculation on 1:1 mineral mixtures are presented in Table 4. It could be readily seen that it is possible to separate efficiently silica and
Table 3 Settling of minerals under different biopretreatment conditions Ž5% pulp density, - 38 mm. Mineral
Quartz Kaolinite Corundum Hematite Calcite
pH
4–5 7.0 4–5 7.0 4–5 7.0 4–5 7.0 7.0
Percentage weight settled in 1 min Control
Metabolite
Bacterial proteins
Bacterial polysaccharides
58.0 45.0 60.0 50.0 85.0 82.0 85.0 82.0 45.0
16.0 – 17.0 – 92 – 96.0 – 93
– 19.0 – 20.0 – 78.0 – 77.0 45.0
– 45.0 – 50.0 – 96.0 – 95.0 91.0
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Table 4 Selective flocculation of mineral mixtures Ž1:1. to separate silica through interaction with bacterial metabolite and bacterial proteins Žy38 mm. Mineral combination
Number of desliming stages
Separation efficiency Ž%.
Corundum–quartz
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
28.0 40.0 59.0 78.0 90.0 99.0 20.0 35.0 55.0 70.0 88.0 98.0 25.0 38.0 56.0 71.0 89.0 97.0
Hematite–quartz
Calcite–quartz
silicates from alumina, calcite and iron oxide through bioflocculation in the presence of bacterial metabolites or bioproteins. Generation of bacterial proteins and exopolysaccharides was also found to be influenced by the nature of the mineral species present during bacterial growth as illustrated in Table 5. Presence of kaolinite or quartz during bacterial growth promoted protein generation while that of calcite, corundum and hematite resulted in higher amounts of exopolysaccharides in the metabolic product. It has been found not possible to separate alumina from hematite efficiently using the Bromfield-grown cells or their metabolites since bacterial interaction brings about similar surface-chemical changes on both of them. Tests with hematite–corundum mixtures have shown that both the minerals settled down faster in an aqueous medium after bacterial interaction without any selectivity ŽNamita Deo and Natarajan, 1999.. However, it has been observed that through the use of corundum-adapted strains and Table 5 Generation of proteins and polysaccharides during bacterial growth in the presence of different minerals Substrate
Protein Žmgrl.
Polysaccharide Žmgrl.
Bromfield Corundum Hematite Kaolinite Quartz Calcite
70.9 52.6 50.1 73.7 79.1 42.8
480.0 500.0 520.0 460.0 450.0 530.0
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Table 6 Separation of corundum–hematite Ž1:1. mixture through selective flocculation using bacterial protein and protein-free metabolites separated from corundum-adapted strains ŽpH 7.0, y38 mm, 5% pulp density. Desliming stages
Total % iron removal Bacterial protein separated from metabolite
Metabolite after protein separation
1 2 3 4 5 6
55.0 60.0 70.0 85.0 98.0 99.0
30.0 40.0 50.0 65.0 70.1 85.2
metabolites of P. polymyxa, efficient separation of alumina from iron oxides can be achieved. Typical selective flocculation results are shown in Table 6. At pH 7.0, 99% separation could be obtained between hematite and corundum, using bacterial protein and 85.2% separation using protein-free metabolite. Unlike the case with unadapted cells, corundum particles could be effectively settled Žflocculated. with the selective flocculation of only corundum using the metabolic products from corundum-adapted strain Žhematite dispersed.. The differences in bacterial protein and polysaccharide adsorption, nature of interaction products and the resulting alterations in the surface chemical behaviour of the minerals could be taken advantage of in the beneficiation of ores containing the various mineral constituents. The minerals studied in this work constitute a typical iron ore system. Removal of silica and alumina from iron ores is a major problem facing the iron and steel industries, the world over. Systematic studies carried out with hematite– corundum–quartz and hematite–kaolinite–quartz mixtures after interaction with adapted strains of P. polymyxa showed that silica, alumina and alumino-silicates can be effectively removed. The utility of biobeneficiation is further demonstrated with respect to the beneficiation of iron ore samples for removal of silica and alumina, as illustrated in Table 7. 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. Table 7 Selective flocculation of iron ore samples by metabolites separated from unadapted and corundum-adapted cells Ž5-min interaction at 5% pulp density. Number of desliming stages
% SiO 2 removal Žunadapted.
% Al 2 O 3 removal Žadapted.
1 2 3 4 5 6
31.0 51.0 66.0 79.9 90.3 97.9
25.0 35.7 57.9 76.0 89.9 96.0
K.A. Natarajan, Namita Deo r Int. J. Miner. Process. 62 (2001) 143–157
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