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Int J Miner Process 62 2001 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 corun dum 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 q2001 Elsevier Science B V All rights reserved Keywords Paenibacillus polymyxa iron ore beneficiation surface chemistry 1 Introduction Bacillus polymyxa recently renamed as Paenibacillus polymyxa Ash et al 1993 is a Gram positive neutrophilic periflagellated heterotroph occurring indigenously associ ated 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 Murphy 1952 The extracellular polysaccharide ECP aids in biological uptake of metal ions necessary for metabolism and growth Heavy metals are known to Corresponding author Tel q 91 80 3600 120 fax q 91 80 3600 472 E mail address kan metalrg iisc ernet in K A Natarajan 0301 7516r01r see front matter q2001 Elsevier Science B V All rights reserved PII S0301 7516 00 00049 1 K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157144 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 Reed 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 Stanier et al qqqq 1993 The presence of different metal ions such as Caand Mgas 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 Reed 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 Phalguni 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 Phalguni 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 Whatman 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 wrv mineral These adapted strains were used in some beneficiation tests and their performance compared with unadapted strains K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157145 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 Millipore 0 45 mm pore size the mem brane 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 proteina ceous 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 Lowry et al 1951and carbohydrates by the Phenol sulphuric acid method Dubois 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 75 110 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 exopolysaccha rides were dispersed in 10y3M KNOsolution for the purpose Zeta potentials as a 3 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 10y3M KNO solution was used 3 K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157146 Table 1 Characterization of mineral and ore samples Chemical composition Mineralogical analysis Iron ore AFe 44 6Hematite 45 50 Goethite 10 15 SiO 32 1Magnetite 3 5 2 Quartz 30 35 Iron ore BFe 51 4Hematite 73 5 SiO 1 73Quartz 1 7 2 Al 10 0Alumina 14 7 LOI 2 0Kaolinite 3 0 2 Surface area m rg Hematite1 00 Corundum0 70 Calcite0 80 Quartz1 577 Kaolinite16 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 7 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 Taggart 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 K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157147 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 interac tion 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 Namita 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 BSA and polysaccharide starch 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 IEP of quartz shifted from its initialbefore 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 K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157148 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 metabo lites and biopolysaccharides For example freshly prepared corundum exhibited an IEP corresponding to a pH of about 7 2 Fig 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 Figs 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 Namita Deo and Natarajan 1998 The bacterium possesses negatively charged groups phosphate 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 pKvalues of free carboxylate and phosphate a groups lie in the pH range of about 2 to 4 and the values of bound species may be quite different 4 from that of the free species due to charge density created by neighbour ing groupsAchouak 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 less 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 avail able on mineral surfaces thus exhibiting different physico chemical affinities Environ mental 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 y1 new surface species Bands due to amide I 1650 cm CONH amide II at about y1 y1 1550 cm primary alcoholic stretching 1050 cmand amide IV and C O bending y1 in proteins700 795 cmcould be seen on quartz and kaolinite surfaces after interaction with bacterial metabolites On the other hand bands due to carboxylate anion y1 y1 1630 cm C OH stretching1120 1140 cmand CHvibrations400 600 2 y1 cmcould be seen on hematite calcite and corundum surfaces after interaction with metabolite The following order with respect to the presence of bacterial protein and K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157149 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 Figs 1 and 2after 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 K A Natarajan Namita DeorInt J Miner Process 62 2001 143 157150 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 Figs 3 5 after similar pretreatments can be due to surface adsorption of excess polysaccharides To further substantiate that the b