Microbial Treatment of Lateritic Ni-ore for Iron Beneficiation and Their Characterization

Th is study aims at studying effect of anaerobic dissimilatory iron (III) reducing bacterial consortium on different phases of iron present in lateritic n ickel ore. Such conversion in lateritic nickel ore are helpful in better recovery of sorbed metal values like Ni and Co by subsequent bioleaching or acid leaching. Here properties of thermally and microbially reduced lateritic nickel ore are compared vis-à-v is orig inal ore. An anaerobic d issimilatory iron (III) reducing bacterial consortium capable of using glucose as carbon source and laterit ic n ickel ore as terminal electron acceptor was used for microbial reduction of ore under anaerobic condition. Microbial reduction changes the initial light brown colour of the lateritic n ickel ore to dark brown. The change in colour is due to the conversion of goethite to magnetite, which is confirmed from the XRD pattern. The FTIR spectra and the UV-Visib le spectra support the presence both goethite and hematite. The study shows that changes in phases brought about by microb ial treatment are d ifferent than those by thermal treatment. The carrier mediated exchange interaction between Fe and Fe ions in lateritic n ickel o re sample treated with IRB consortium is responsible for higher ferromagnetic ordering. The thermal reduction of the same sample showed lowering of ferromagnetic ordering due to the decreasing percentage of Fe and Fe ions.


Introduction
In laterit ic nickel ore, n ickel is associated with iron phase where as cobalt is associated with manganese phase [1]. Natural Fe(III) o xides are high in surface area and are reactive [2]. Fe(III) o xides adsorb a wide range of metal cations and anions by complexat ion to surface hydroxyl groups [3]. Nickel lateritic ore of Sukinda, Orissa, contains 0.8% Ni and 0.049% Co which makes them the only nickel deposit in India. More focus had been given by IMMT Bhubaneswar on this material for extraction of nickel and cobalt [4][5][6][7]. However, thermal act ivation or thermal reduction of ore is required for better recovery of metal values [8]. Heating of ore resulted in conversion of goethite to hematite, which was responsible for releasing nickel fro m Fe(II) lattice and resulted in better recovery [7]. Th is type of reduction is not cost effective in terms of energy consumption and may not be employed for large scale operations. Hence, it is desirable to have an alternative ecofriendly and lo w cost method, which ma kes the subsequent metal extract ion more feasib le.
Ferric iron o xides are widespread in anoxic aqueous environ ment and have been reported to act aselectron sink during b iodegradation of various natural and xenobiot ic compounds [9][10][11].
Microbial Fe (III) reduction results in the generation of several important Fe(II)-containing minerals in sedimentary environments, including magnetite Fe(II)Fe(III) 2 O 4 , which is a magnetic mineral. Magnetite format ion during dissimilatory Fe(III) reduction is reported for different pure cultures of bacteria [12] as well as for Fe(III)-reducing enrichment consortium [13]. Microbial dissimilatory Fe(III) reduction plays an important role in the geochemical cycling of iron and organic matter in ano xic ecological system [14]. Iron-reducing microbes generally belong to the genera Shewanella, Geobacter, Geovibrio, Desulfobulbus etc [15][16][17]. If bacterial reduction method is successful, cost intensive method of ore roasting process can be eliminated. Such 'dissimilatory' processes have opened up new and fascinating areas of research with potentially exciting practical applications.
Applying a bio logical approach to address this problem, we investigated the use of naturally occurring iron reducing bacteria for the reduction of lateritic n ickel ore (iron phase) in a low cost ecofriendly way.

Lateritic Nickel Ore
Lateritic Nickel ore was procured fro m Su kinda mines of Orissa Mining Corporation (OMC), India. The sample was a low grade lateritic ore which contains about 0.8% Ni, 0.049%Co, 1.92% Cr, 0.32% Mn, and 50.2% Fe. For the experimental purpose the ore samples were heated at 600 ℃ for 5 hours to convert the goethite to hematite which is reported to aid the release of the nickel bound to goethite matrix. Thermal activation of the lateritic n ickel ore has significant influence on nickel and cobalt recovery [18]. The thermal activation changes the mineral structure and brings about mineral phase transformat ion by dehydroxylat ion of the goethite [19].

Microorganisms
Soil sediment fro m a wetland sites around Bhubaneswar, Orissa, India was used as source of iron reducing bacteria. After collecting, these were immediately transferred to anaerobic vials and taken to the laboratory fo r subsequent enrich ment of Fe(III) reducing bacteria.

Enrichment and Isolation of Fe(III)-Reducers
One gram of sediment was transferred to a vial containing 10 ml of anaerobic saline solution. These vials were thoroughly mixed, allo wed to settle for 10 minutes and then 5 ml of liquid was used to inoculate 100 ml selective alkaline anaerobic iron reducing med iu m in volu metric flasks. This med iu m contains Nickel ore 1.0 g/l; K 2 HPO 4 3.0 g/l; KH 2 PO 4 0.8 g/l; KCl 0.2 g/l; NH 4 Cl 1.0 g/l; Mg Cl 2 0.2 g/l; CaCL 2 0.1 g/l; Yeast extract 0.05 g/l; mixture of vitamins and trace minerals solution 1% (v/v) and 10 mM sodium acetate (C 2 H 3 O 2 Na). pH of the media was adjusted to 7.2 with the help of 1N NaOH. Liquid surface was covered with paraffin oil to make the system anaerobic. The flasks were incubated at 30 o C for several days under dark condition. The enrich ment procedure was repeated four times with the transfer of 10 ml of culture as inoculu m into the fresh medium. The enrich ment cultures after five successive transfers were used as inoculum for lateritic nickel ore reduction experiments.

Treatment of Lateritic Nickel Ore with IRB Consortium
Lateritic nickel ore was treated with well enriched Fe(III) reducing bacterial consortium in an anaerobic, alkaline medium at 2% pulp density and kept at 30℃ for 7 days under dark condition. Simu ltaneously control experiments without any bacterial solution were also run. After treatment, the treated laterit ic nickel ore was filtered and air dried. In a similar set of experiments the heated lateritic nickel ore was taken instead of the orig inal o re. The reduction of h ighly insoluble Fe(III) o xides resulted in dissolution of soluble Fe(II) ions in the growth mediu m. Th is soluble ferrous-ion was determined by titration [20].
Samples are designated as O1 for laterit ic ore and O2 for laterit ic nickel ore sample treated with IRB consortium. Similarly R1 for heated laterit ic nickel ore samp le and R2 for heated lateritic nickel ore samp le treated with IRB consortium.

Analysis
X-ray diffraction analysis was carried out by an X-ray powder diffracto meter (Ph ilips X'pert Pro, Panalyt ical) using Mo Kα (λ= 0.7107 Å) as X-ray source and a programmab le divergence slit. The voltage and current of the x-ray source were 40 kV and 20 mA, respectively. UV-visib le absorption spectra of all the samples were taken with a Varian Cary 100 spectrophotometer in the region 200 -800 nm to determine the mineral species through absorption band gap. Fourier transform infrared spectra were recorded on a FTIR system (spectrum GX model supplied by Perkin Elmer instrument, USA ). The spectra were recorded fro m 400 to 4000 cm -1 . The surface morphology was observed in a Field Emission Scanning Electron Microscope (Zeiss Supra 55, observation conditions V-20 kV, I-0.6 nA) after coating the surface with gold to reduce the charging effect. M icroscopic image of the IRB consortium was taken in Nikon 80i optical microscope. The room temperature magnetic hysteresis studies of samples were carried out using Vibrating Sample Magnetometer (VSM ) (Model: Lakeshore 7410) at an applied magnetic field of 2T.

Results and Discussion
Dissimilatory iron reducing bacterial consortium was enriched in an anaerobic mineral salt mediu m containing lateritic n ickel ore co mposed of insoluble ferric iron in the form of goethite. The med iu m contained acetate ions as carbon source (electron donor) and ammoniu m chloride as nit rogen source for bacterial metabolis m. In this system, bacteria oxidizes carbon source and the released electron pass through a series of electron carrier mo lecules to generate ATP, the b iochemical energy needed for bacterial metabolis m. The ult imate electron acceptor or electron sink in absence of oxygen is the Fe(III) present in goethite. After accepting the electron, Fe(III) gets reduced to Fe(II) which results in phase change. Due to the phase changes, original light brown colour of the laterit ic nickel ore transform to dark bro wn. No such change was observed in the control experiment. Gram staining of IRB consortium demonstrates the presence of both gram positive and negative bacteria of different morphology. An optical microscopic image shows 6 morphologically different species (Figure 1). Scanning electron micrograph show long curve rod shaped bacteria in close association with ore particles (Figure 2). Bacteria of length up to 6 µm were observed. The bio-film format ions around the ore particles were evident. So me granular material could be seen around bacteria wh ich may be extracellu lar magnetite deposition (encircled in the Figure 2). Similar depositions had been reported by Hansel [21]  The anaerobic IRB consortium grew well in the media with lateritic n ickel ore as only electron acceptor and convert Fe(III) to Fe (II) as evident by release of ferrous ion in the med iu m during growth of consortia ( Figure 3). The ferrous ions formed remain adsorbed to the solid ore particles. These Fe(II) ions were detectable only after washing the ore material with 0.1 N HCl. In itially pH decreases to 6 during first 15 days of incubation but after that it remains constant at around 7.4 ( Figure 3). The X-ray diffract ion peak is shown in Figure. 4. The XRD pattern of O1 shows the Bragg reflection peaks of goethite as well as hematite. The intensity of hematite peaks increased as the ore was treated with bacterial consortia in anaerobic condition. A small percentage of magnetite peaks was observed in the O2. The bacteria acts as a reducing agent and converts the goethite to hematite and magnetite. Such peaks were lower in the intensity in O1. The intensity ratio of hematite to goethite is in increased ratio in comparison to O1. However the presence of hemat ite, magnetite rep resents that the material is an admixture o f two iron states i.e. Fe (II) and Fe (III). When the ore O1 is annealed at 600 °C, the goethite phase converts to protohematite with a s mall percentage of goethite leaving behind. It is already reported in the literature that at around 250 °C, goethite converts to protohematite and at or above 800 °C, it converts to hematite [22]. As our annealing temperature was 600 °C, there is ma ximu m possibility of conversion of goethite to protohematite, wh ich is clearly reflected in the XRD pattern of R1. But the bacteria treated annealed ore (R2) showed improved crystalline peaks of hematite and reduction in the intensity of goethite peaks. The small amount of goethite present in R2 couldn't be established within the instrumental limitation. This feature is further well supported from the UV-Vis ible and FTIR studies. In UV-Vis ible spectra three absorption bands were observed at the wavelength of 367, 483 and 678 n m for O1 and O2 ( Figure 5). The h igher wavelength (678 n m) and lower wavelength (367 n m) is attributed to the optical absorptions band of goethite where as the optical band gap at 483 n m is attributed to the absorption bands of goethite and hematite. In R1 and R2, the optical absorption band is observed at 529 nm which co mp letely reflects the presence of hematite only and a sharp absorption band is observed at 367 n m, attributed to goethite. Even in R2, presence of goethite was not observed in XRD due to instrumental limitation, but was confirmed fro m the UV-Visib le spectra. It is well known that the band gap of goethite is higher than the band gap of hematite, which is well reflected fro m the shifting of bands in the spectrum.  (Table 1), whereas the FTIR peak observed in R1 and R2 are attributed to the hemat ite. Only the peak 465 cm -1 is attributing to the goethite. So me peaks are attributed to the hydroxyl group modes of v ibration that may be due to the surface contamination of water during experiment done at open at mosphere. Our well studied XRD, UV-Visible and FTIR analysis predicts that O1 and O2 is a composites of both goethite and hematite whereas R1 is a hematite with small percentages of goethite. R2 is co mpletely a hematite. The magnetic hysteresis recorded for all the samples are shown in Figure 6. The M H loop recorded for the O1 shows a small loop with remnant magnetizat ion (M r ) of 0.083 emu/g and coerecivity (H c ) of 311 Oe. No saturation magnetization (M s ) has been observed even at the applied field of 20000 Oe, whereas the MH loop recorded for R1 shows lower remnant magnetization of 0.021 emu/g and coereciv ity of 293 Oe. This may be possible due to the transformat ion of goethite to hematite at 600 o C. While O2 shows higher saturation magnetization value of 1.5 emu/g with remnant magnetization value of 0.328 emu/g and coercivity of 235 Oe. This observation provides evidence that the bacteria acts as a reducing agent to convert goethite to hemat ite in the solution. It is well known that hemat ite is weakly ferro magnetic at room temperature. R2 shows lower magnetization value of 0.876 emu/g with remnant magnetization of 0.185 emu/g and coercivity of 266 Oe than that of O2. Here the bacteria only act as reducing agent for converting goethite to hemat ite. Hence the percentage of goethite becomes less in comparison of the hematite. The M s , M r and H c value for all the samples are given in Table 2.  Goethite and hematite have iron in 3+valence state, whereas valence state of iron in magnetite is in-between 2 + and 3 +. So a well admixture of Fe(II) and Fe(III) in a material results in a ferro magnetic coupling interaction between the iron ions, wh ich enhances the ferro magnetic ordering and can be explained on the basis of RKKY exchange interaction. As observed in O2, Fe exists in the multip le valence state of 2 + and 3+, hence there is a strong possibility of carrier mediated exchange interaction in between Fe 3+ and Fe 2+ ions via oxygen. Such type of exchange interaction is known as double exchange interaction [23,24] because the conduction electron transfers fro m one Fe ions to another Fe ions through oxygen. Such type of carrier med iated exchange interaction imp roves the ferro magnetic ordering in the system and hence enhances the magnetizat ion. However, only hematite exists in R2 with a very small percentage of magnetite. So the short range exchange interaction between Fe(III) and Fe(III) is dominating over the long range Fe(II) and Fe(III) exchange interaction, thus lowering the saturation magnetizat ion in R2 in co mparison to O2. Therefore, the competing exchange interaction between short range ordered anti-ferro magnetic coupling in between similar type of Fe ions and long range ordered FM coupling between Fe(II) and Fe(III) ions reduces the saturation magnetization value. This is clearly evidenced fro m the MH loop of Figure 6.

Conclusions
Lateritic n ickel ore when subjected to treatment with anaerobic dissimilatory iron (III) reducing bacterial consortium show phase changes in iron minerals. Co mparison of properties of thermally and microbially reduced lateritic o re vis a vis original o re show that changes in phases brought about by microbial reduction were d ifferent than those by thermal reduction. Magnetite formation was more pro minent in case of microbial treat ment of orig inal o re than roasted ore which is well pictured in the XRD pattern. Roasting leads to the formation of hematite. The carrier med iated exchange interaction between Fe(II) and Fe(III) ions in laterite nickel ore sample treated with IRB consortium is responsible for higher ferro magnetic ordering.IRB consortia treated ore showed higher saturation magnetization value of 1.5 emu/g with remnant magnetizat ion value of 0.328 emu/g and coercivity of 235 Oe. The thermal activation of the same sample shows lowering in ferro magnetic ordering due to the decreasing percentage of Fe(II) and Fe(III) ions. Thermally act ivated ore was less amenable to further phase changes by microbial treatment. Fro m th is study we conclude that naturally occurring iron reducing bacterial consortia could be for better recovery of metal values in a cost effective and eco-friendly way.