Leaching Behavior of Chromite Tailings

Article (Bio)leaching Behavior of Chromite Tailings

Viviana Bolaños-Benítez 1,2,3, Eric D. van Hullebusch 2,3,*, Piet N.L. Lens 3,Cécile Quantin 4, Jack van de Vossenberg 3, Sankaran Subramanian 5 and Yann Sivry 1

1 Institut de Physique du Globe de Paris, Sorbonne Paris Cité, University Paris Diderot, UMR 7154, CNRS, F-75005 Paris, France; [email protected] (V.B.-B.); [email protected] (Y.S.) 2 Laboratoire Géomatériaux et Environnement (LGE), Université Paris-Est, EA 4508, UPEM, 77454 Marne-la-Vallée, France 3 Department of Environmental Engineering and Water Technology, IHE Delft Institute for Water Education, P.O. Box 3015, 2601 DA Delft, The Netherlands; [email protected] (P.N.L.L.); [email protected] (J.v.d.V.) 4 UMR 8148 GEOPS, University Paris Sud-CNRS-Université Paris Saclay, 91405 Orsay CEDEX, France; [email protected] 5 Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India; [email protected] * Correspondence: [email protected]; Tel.: +31-152-152-305

Received: 20 April 2018; Accepted: 14 June 2018; Published: 20 June 2018

Abstract: Chromite beneficiation operations in Sukinda valley (India) produce large amounts of tailings, which are stored in open air. In this study, bioleaching experiments were carried out in batch reactors with Acidithiobacillus thiooxidans or Pseudomonas putida in order to determine the potential leachability of metals contained in these tailings due to biological activity. Acidic and alkaline pH resulted from the incubation of tailings with A. thiooxidans and P. putida, respectively. Tailings were characterized by X-ray diffraction (XRD), X-ray fluorescence (XRF), and scanning electron microscopy (SEM), and chemical extraction of Cr(VI) with KH2PO4 was performed. Mineralogical investigations showed that tailings are mainly composed of chromite, hematite, lizardite, chlorite, and goethite, which are all known as Cr-bearing phases. During the leaching with A. thiooxidans and P. putida, total Cr was initially extracted as Cr(VI) due to the presence of phosphates in the medium, and subsequently decreased because of Cr(VI) adsorption and reduction to Cr(III). Reduction was associated with bacterial activity, but also with the presence of ferrous iron. Despite the occurrence of siderophores in the tailings after incubation with P. putida, under acidic conditions, Fe extracted remained higher. Extracted Ni, Mn, and Al concentrations also increased over time. Given the significant amount of chromite tailings produced every year, this study shows that tailings storage and leachability represent a potential source of chromium. However, our findings suggest that the presence of bacterial communities, as well as physicochemical processes, favor Cr(VI) reduction.

Keywords: tailings; bioleaching; chromite mine; Cr(VI) reduction

1. Introduction In India, there are 9900 mining leases, spread over an area of 7453 km2, covering 55 minerals other than fuel. The Sukinda valley in Jajpur District, Orissa, known for its chromite (FeCr2O4) ore deposits, produces nearly 8% of the chromite ore in India [1]. Chromite can be found in stratiform deposits, with concentrations of up to 50%. Because of the high chromite content, no beneficiation process is required and the material can be directly extracted and used for metallurgical purposes. However, when the chromite concentration is lower than 33%, beneficiation with grinding and gravity methods is applied to concentrate the chromite ore [2]. Both in the mining and in the beneficiation process, a variety of waste residues are produced, including waste rock, overburden, and tailings. The inconsistent feed

Minerals 2018, 8, 261; doi:10.3390/min8060261 www.mdpi.com/journal/minerals Minerals 2018, 8, 261 2 of 23 quality during the beneficiation process results in a loss of efficiency and an increase of wastes, as is the case for tailings. Approximately 50% (by weight) of the total feed is discarded as tailings which still contain significant amounts of chromite (13–15%) [2]. Several studies have been carried out in order to increase the chromite extraction yield and consequently decrease the tailings production [3,4]. A maximum of 83% recovery of chromite was reached with 23% Cr2O3 content. Despite the efforts that have been made to reduce chromite losses or reprocess stockpiled tailings, the ultrafine particle size makes chromite recovery and management difficult. Therefore, several tons of tailings per year are still produced and stored in open air conditions [5,6]. Chromium (Cr) has several oxidation states from −2 to 6, and the trivalent (Cr(III)) and hexavalent (Cr(VI)) states are primary oxidation states in nature. Hexavalent chromium is known to be highly mobile, soluble, and toxic, while trivalent chromium is not toxic and largely immobile in the environment [7]. Nevertheless, Cr(III) can be mobilized by forming complexes with dissolved organic carbon [8]. Because of its toxicity, Cr(VI) has been shown to have several negative environmental impacts [9,10]. In groundwater, the natural occurrence of Cr(VI) is linked to the hydrolysis of feldspar, some common mafic minerals such as Cr-bearing pyroxenes and chromite, together with calcite, which causes alkaline groundwater conditions. This, coupled with the absence of natural reducing agents like Fe(II), organic matter, or reducing organisms, may allow the oxidation of Cr(III) to Cr(VI) [9]. In rocks, chromite grains are locked either within the iron ore minerals (goethite/hematite) and silicates or the chromite with inclusions of silicate [6]. Silicates are more susceptible to weathering than chromite, which represents a source of chromite nanoparticles (containing mainly Cr(III)), that after dissolution, could be oxidized to Cr(VI) [10,11]. According to Beukes et al. [9], Cr(VI) containing wastes generated by chromite mining are classified by three groups: (i) recycled within the process, (ii) re-purposed or re-used in other applications, and (iii) considered hazardous. Because tailings are not recycled or re-used and due to their potential to release Cr(VI), they belong to the third, hazardous, group of waste generated by chromite mining. Consequently, all the efforts are focused on the understanding of Cr(VI) dynamics [7,12–18] and the prevention or at least mitigation [19–23] of Cr(VI) production. Tiwary et al. [24] studied the migration of metals leached from an overburden dump of chromite ore to groundwater. This study revealed that due to the poor permeability of the seepage, the migration of metals occurs at a low rate and could only take place in the first layers down to 10 m in 10 years. Godgul [25] performed leaching experiments of chromite according to pH and highlighted that there is no relation between the total Cr in the solid sample and in the leached solution, because of the distribution of Cr in different mineral phases besides chromite. It was also observed that the maximum of Cr leached is obtained at pH 8 and all the elements (Cr, Fe, Al, Mn, and Ni) are leached from the same mineral phase. A great variety of microorganisms have been identified in mine waste, and microbial processes are usually responsible for the environmental hazard created by mine wastes. Microorganisms can, in principle, influence metals leaching from mine waste in several ways. However, they can also be used to retard the adverse impact of mine wastes on the environment [26]. Given the fact that some microorganisms contribute to metal immobilization, several studies have focused on the use of microorganisms such as bacteria for metal recovery or Cr(VI) reduction from mining residues. Das et al. [27] studied the selection of Cr(VI) reducing bacteria from soils impacted by tailings. Bacillus amyloliquefaciens was isolated from chromite mine soil and exhibited Cr(VI) tolerance and a Cr(VI) reduction rate of 2.2 mg Cr(VI)/L/h. Allegretti et al. [28] investigated the presence of intermediary compounds, produced by Acidithiobacillus and Thiobacillus, that were responsible for Cr(VI) reduction. They identified several polythionates associated with sulfur particles between 0.45 and 3 µm. Wang et al. [29] evaluated the leachability of Cr(VI) from tannery sludge with A. thiooxidans. After five days in a bubble column bioreactor at 30 ◦C, 99.7% of Cr was leached out from the tannery sludge. Kumar and Nagendran [30] also observed the efficient removal of metals, including Cr (95%), in soils through bioleaching with A. thiooxidans. Cr in the organic and residual fractions was unaltered Minerals 2018, 8, 261 3 of 23 during the course of the experiment, while the Fe-Mn oxide bound chromium was transformed to the exchangeable and carbonate fraction. Metal solubilization mechanisms by A. thiooxidans can be direct Minerals 2018, 8, x FOR PEER REVIEW 2− 3 of 23 or indirect. In the direct mechanism, metal sulfides are directly oxidized to SO4 by the bacteria, while in the indirect mechanism, H SO , a strong leaching agent, is produced by A. thiooxidans [31]. exchangeable and carbonate fraction2 . Metal4 solubilization mechanisms by A. thiooxidans can be direct Acidithiobacillus species have also been used in both the aerobic and anaerobic reductive dissolution or indirect. In the direct mechanism, metal sulfides are directly oxidized to SO42− by the bacteria, while in of iron rich nickel laterite to remove target metals [32,33] and in laterite tailings [34] to recover heavy the indirect mechanism, H2SO4, a strong leaching agent, is produced by A. thiooxidans [31]. metals.Acidi Mostthiobacillus of these species studies have target also been industrial used in applications.both the aerobic However, and anaerobic neither reductive the leaching dissolution nor the bioleachingof iron rich behavior nickel laterite of chromite to remove tailings target stockpiled metals [32,33] in open and in air laterite in the tailings environment [34] to recover has been heavy studied so farmetals. and the Most effect of these of bacteria studies on target chromite industrial tailings applications. bioleaching However has not, neither yet been the leaching identified. nor the Givenbioleaching the increasing behavior of importance chromite tailings of chromite stockpiled ores in foropen Cr air world in theproduction environment and has thebeen huge studied amount of tailingsso far producedand the effect (5 × of 10bacteria5 ton/year—average on chromite tailings production bioleaching between has not 2016 yet been and 2017identified in all. the chromite mines inGiven Sukinda the increasing valley) [35 importance], it remains of chromite of key ores interest for Cr to world fill theproduction knowledge and the gap huge regarding amount the of tailings produced (5 × 105 ton/year—average production between 2016 and 2017 in all the chromite tailing discarded after the chromite beneficiation process. In the present study, the influence of pH mines in Sukinda valley) [35], it remains of key interest to fill the knowledge gap regarding the tailing on the bioleaching of chromite tailings was evaluated in batch experiments. Acidic and alkaline discarded after the chromite beneficiation process. In the present study, the influence of pH on the conditionsbioleaching were of obtained chromite tailings through was the evaluated incubation in batch of tailings experiments. with AcidicAcidithiobacillus and alkaline thiooxidans conditions and Pseudomonaswere obtained putida through, respectively. the incubation The bacterial of tailings influence with Acidithiobacillus on tailings leachability thiooxidans and was Pseudomonas evaluated and the potentialputida, respectively. release of Cr(VI)The bacterial from tailingsinfluence was on tailings estimated. leachability Fresh andwas oldevaluated tailings and were the potential compared to estimaterelease the of potential Cr(VI) from risk tailings of chromite was estimated. tailings toFresh the and environment. old tailings were compared to estimate the potential risk of chromite tailings to the environment. 2. Materials and Methods 2. Materials and Methods 2.1. Field Settings 2.1. Field Settings Superficial fresh chromite tailings and concentrate ore, as well as a core in the stockpiled old chromite tailings,Superficial were fresh collected chromite in tailings May 2017 and fromconcentrate materials ore, generatedas well as a in core a typical in the beneficiationstockpiled oldplant chromite tailings, were collected in May 2017 from materials generated in a typical beneficiation plant of Sukinda valley, Jajpur district, Odisha, India (Figure1). The Sukinda valley is located in the eastern of Sukinda valley, Jajpur district, Odisha, India (Figure 1). The Sukinda valley is located in the eastern part of the Indian peninsula, where the average annual precipitation is about 2400 mm. Most of the part of the Indian peninsula, where the average annual precipitation is about 2400 mm. Most of the rain occursrain occurs during during the the monsoon monsoon season season between between May and and October. October. It is It primarily is primarily drained drained by the by the DamsalaDamsal Nalaa N River.ala River The. The river river flows flows westward westward through the the central central valley valley to meet to meet the major the major river, river, Brahmani,Brahmani which, which flows flows at aat distance a distance of of 15 15 km kmdownstream downstream of of the the mining mining belt belt [36] [36. ].

FigureFigure 1. 1.Chromite Chromite mine mine inin Sukinda valley valley (Odisha, (Odisha, India). India).

Minerals 2018, 8, 261 4 of 23

Most of the mines are located in the central part of the valley and are operated by 12 different companies [37]. The Sukinda ultramafic complex is composed of alternate bands of chromite, dunite, peridotite, and orthopyroxenite, which are extensively lateralized [38,39]. The chromite bands in the Sukinda valley present various compositions. When the Cr2O3 content is higher than 48%, the ore is “high grade” and is directly marketable, while between 32% and 45%, the ore is “medium grade”, and finally, lower than 30% is “low grade” [37]. For the last case, the presence of gangue minerals such as goethite, serpentine, olivine, and talc has led to the utilization of lean ore after beneficiation. The beneficiation process majorly consists of comminution to physically liberate the minerals followed by physical separation to generate the concentrate of the chromite ore [4]. During the comminution process, large quantities of fines are generated. The fines are commonly de-slimed using hydro-cyclone and cyclone underflow processed using gravity concentrators like spirals and tables. As a result, chromite concentrated ore is obtained, and roughly 50% to 70% of the chromite losses are in the fine fractions (tailings), where the fraction below 75 µm contains about 9–20% Cr2O3 [2,6].

2.2. Chemical and Biological Leaching Experiments The effect of bacterial bioleaching activity of fresh tailings was tested at three pulp densities of 5, 10, and 30 g/L in batch reactors. Batch reactors were selected in the present work, because they have been systematically used to study the kinetics of chromium transformations under typical environmental conditions [13], in order to establish the leachability of metals from laterite tailings [40] and to study the leaching characteristics of chromite ore processing residue (CORP) [41–43]. Also, batch experiments have been used to establish the microbial effect on chromium mobility [44,45]. All the material was autoclaved at 121 ◦C for 20 min prior to use. The 500 mL reactors were closed using a cotton plug, both previously sterilized, and placed in an orbital shaker at 190 rpm and 30 ◦C. Samples were collected after 4 h, 1, 2, 5, 6, 8, 10, 12, 15, and 30 days. A total of 20 mL of leachate was collected and replaced by fresh sterile medium to maintain the solid liquid ratio. In the samples collected, dissolved oxygen, pH, and electrical conductivity were monitored using a WTW 3410 Set 2 multiparameter probe (Xylem Inc., Rye Brook, NY, USA). In the case of bio-leaching with P. putida, approximately 5 mL of sample was frozen at −20 ◦C to determine the protein content and siderophores concentration later. The rest of the sample was filtered through 0.45 µm pore size nitrocellulose syringe filters, for the determination of major and trace element concentrations in the leachate. Samples were ◦ acidified with 16 N HNO3 and stored at 4 C until their analysis. Biological and chemical leaching experiments were done in duplicate to verify reproducibility. For this reason, the results presented in this work are an average of the duplicates with its associated standard deviation.

2.2.1. Bio-Leaching with A. thiooxidans and P. putida The bioleaching experiments of fresh tailings with A. thiooxidans and P. putida were performed in solutions containing the same components of the growth media where bacteria were pre-grown. The initial pH of the growth media was set at 3.5 for A. thiooxidans and 7 for P. putida, with 1 N H2SO4 and 1 N NaOH, respectively. The batch reactors were inoculated with one percent (v/v) of pre-grown bacterial culture. The sterilized fresh tailing sample plus culture media were used as a control and distilled water plus sterilized tailing sample as a blank. In both cases, sodium azide (NaN3) was added to avoid bacterial growth.

Acidithiobacillus thiooxidans The gram-negative bacterial strain A. thiooxidans (DSM 9463) was grown in a medium containing 2 g ammonium sulfate ((NH4)2SO4), 0.25 g magnesium sulfate (MgSO4·7H2O), 0.1 g dipotassium hydrogen phosphate (K2HPO4), and 0.1 g potassium chloride (KCl) per liter, in addition to 11% (wt/v) of elemental sulfur that was previously autoclaved at 121 ◦C for 20 min. The medium pH was adjusted to 3.5 before sterilization, and the sterilized sulfur was added subsequently together with the inoculum. Minerals 2018, 8, 261 5 of 23

The culture was maintained at 30 ◦C. The volume of the culture was increased gradually from 5 mL to 500 mL in separated Erlenmeyers.

Pseudomonas putida The gram-negative bacterium P. putida (WCS 358) bacterial strain was kindly provided by Peter Bakker, University of Utrecht (The Netherlands). For siderophore production, iron free succinate medium (SM) consisting of (in g/L): K2HPO4 6.0, KH2PO4 3.0, MgSO4·7H2O 0.2, (NH4)2SO4 1.0, and succinic acid 4.0, pH 7.0 was used to inoculate P. putida at a concentration of 1% (v/v) inoculum.

2.2.2. Chemical Leaching In addition to the experiments done with A. thiooxidans and P. putida, chemical leaching of the fresh tailing sample with distilled water at pH 2 (adjusted with H2SO4) and distilled water at pH 9 (adjusted with NaOH) was carried out at a pulp density of 30 g/L. The leachability of chromite was also studied through the leaching of the concentrated ore coming from the beneﬁciation plant.

2.3. Chemical Extraction In order to determine the chemically exchangeable pool of Cr(VI) in the fresh and stockpiled old tailings and chromite ore, chemical extraction was performed using distilled water and 0.1 M KH2PO4. The principle of the extraction with KH2PO4 is based on the fact that chromate ions can be desorbed by 3− reactions of the soil with other specifically desorbed anions, such as phosphates (PO4 ) and sulfates. A suspension of 1 g of soil was agitated with 25 mL of the reactant (distilled water or 0.1 M KH2PO4) for 1 h [46]. The supernatant was separated by centrifuging for 15 min at 2500 rpm, followed by filtration with a 0.22 µm pore size PES filter. Finally, the total Cr concentration in the supernatant was determined with ICP-AES. Cr(VI) was analyzed by the colorimetric technique (diphenylcarbazide (DPC) method) [47,48] and was measured with a Shimadzu UV-2550 spectrophotometer with a 1 cm quartz cell at λ of 540 nm.

2.4. Analytical Methods

2.4.1. Chromite Tailings Characterization The mineralogical composition of the fresh tailing sample and the concentrated ore was determined using X-ray diffraction (XRD) analysis on a PANalytical diffractometer and Cu Kα radiation (at 45 kV–40 mA) in the grazing incidence angle in the 5◦–70◦ 2θ range with a scan step of 0.013◦. In addition, total metal concentration was determined by X-ray ﬂuorescence (XRF), using an X ﬂuorescence PANalytical spectrometer (Malvern Panalytical, Malvern, UK) equipped with Energy Dispersive Minipal 4 (Rh X-ray tube 30 kV–9 W) at a resolution of 150 eV (Mn Kα). The fresh tailing samples were thin-coated with carbon prior observations with a Zeiss Auriga Scanning Electron Microscope (SEM) (Zeiss, Jena, Germany). The SEM was equipped with a Field emission Electron Gun (FEG) at 15 keV with an SE-Inlens detector.

2.4.2. Leachates Composition The concentration of major elements in filtered samples was determined using ICP-AES (ICAP 6200 Thermo Fisher, Thermo Fisher Scientific, Waltham, MA, USA), whereas HR-ICP-MS (Thermo Scientific Element II, Thermo Fisher Scientific, Waltham, MA, USA) was used for trace elements analysis. Detection limits were typically between 0.6 and 74 ng/L and the standard deviation associated with the measurements was smaller than 5%. Fe speciation in the leachate was measured with the ferrozine method [49]. Minerals 2018, 8, 261 6 of 23

2.4.3. Protein Determination In order to establish the growth of the bacterial community, the Lowry protein test [50,51] was performed in the supernatant collected in the leaching experiments. For the calibration curve, protein standards were prepared with bovine serum albuminat concentrations between 10 and 500 µg/mL. A total of 0.1 mL of 2 N NaOH was added to 0.1 mL of sample or standard. The mixture was hydrolysed at 100 ◦C for 10 min in a boiling water bath. After cooling the hydrolysate to room temperature, 1 mL of the complex-forming reagent was added and the solution was left for 10 min at room temperature. After that, 0.1 mL of Folin reagent was added to the mix using a vortex mixer. After 30 to 60 min, the absorbance was read at 750 nm for protein concentrations below 500 µg/mL [50].

2.4.4. Siderophores Assay Quantitative estimation of siderophores produced by P. putida was done by the CAS-shuttle assay [52]. This method is based on the high afﬁnity of siderophores for iron(III) [53]. The CAS reagent was prepared using: (1) 0.06 g of CAS in 50 mL of distilled water; (2) 0.0027 g of FeCl3·6H2O in 10 mL of 10 mM HCl; and (3) 0.073 g of HDTMA in 40 mL of distilled water. Solution (1) was mixed with 9 mL of solution (2) and after with solution (3). The CAS reagent was autoclaved and stored in a plastic bottle [54]. For the quantiﬁcation of the siderophores, 0.5 mL of supernatant (sample) was mixed with 0.5 mL of CAS reagent, and absorbance was measured at 630 nm. The siderophore content was calculated by using Equation (1), proposed by Sayyed et al. [55].

A − A % siderophore units = r s × 100 (1) Ar where Ar and As correspond to the absorbance of the reference and sample at 630 nm, respectively.

2.5. Geochemical Modeling The visual MINTEQ 3.1 equilibrium modelling program was used to identify the major mineral(s) controlling the chemistry of fresh tailings. The Saturation Index (SI), which is deﬁned as the logarithm of the ratio of the ion activity product and the solubility product (Ksp) (SI = lgIAP/Ksp), was calculated with the program. Positive SI values indicate that the solution is oversaturated and precipitation is possible, negatives values indicate that it will tend to dissolve, and zero shows equilibrium of the solution with a mineral phase. The input data for MINTEQ 3.1 included the pH, temperature, total 2− − − 3− cation (Ca, Mg, Na, K, Ni, Fe(II), Al, Cr(VI)), and anion (SO4 , NO3 , Cl , PO4 ) concentration. No adsorption parameters were included within the calculations.

3. Results

3.1. Tailings and Concentrated Ore Characterization A detailed chemical composition of the samples is given in Table1. X-ray Fluorescence analysis shows that in the fresh tailing sample, 18 wt % is chromium oxide and 56 wt % is iron oxides. For the concentrated ore sample, the proportion of Cr2O3 increases up to 33.17 wt %. The XRF composition is consistent with XRD results, which shows the presence of goethite (α-FeO(OH)), hematite (Fe2O3), gibbsite (Al(OH)3), chlorite ((Fe,Mg,Al)6(Si,Al)4O10(OH)8), and chromite (FeCr2O4), as the main crystalized mineral phases in the fresh tailing sample. Minerals 2018, 8, 261 7 of 23

Table 1. XRF results of the fresh tailing and chromite concentrated ore sample collected in the beneﬁciation plant (Sukinda valley) in May 2017.

Oxide Rate (%) Fresh Tailings Chromite Concentrated Ore

Al2O3 8.7 9.4 SiO2 13.4 3.7 Cr2O3 18.1 33.2 Fe2O3 56.7 52.0 Total 96.9 * 98.4 * * The 3.1% and 1.6% leftover for tailings and concentrated ores, respectively, correspond to traces of Mn, Mg, V, Ti, Ca, P, and S. Minerals 2018, 8, x FOR PEER REVIEW 7 of 23 Dwari et al. [6] found additional mineral phases in chromite tailings from Sukinda valley, includingDwari kaolinite et al. (Al[6] found2Si2O 5additional(OH)4), quartz mineral (SiO phases2), andin chromite magnesioferrite tailings from (Mg(Fe) Sukinda2O valley,4). The including concentrated ore samplekaolinite is (Al enriched2Si2O5(OH) in4), chromite quartz (SiO with2), and some magnesioferrite traces of lizardite, (Mg(Fe)2O hematite,4). The concentrated and goethite. ore sample is enrichedSEM-EDX in chromite images collected with some on traces the fresh of lizardite, tailing samplehematite are, and displayed goethite. in Figure2. As was previously shown bySEM the-EDX XRF image results,s collected the tailings on the are fresh dominated tailing sample by theare displayed presence in of Figure Fe, followed 2. As was by previously Cr, Al, and Si shown by the XRF results, the tailings are dominated by the presence of Fe, followed by Cr, Al, and oxides (Figure2a). In the fresh tailing sample, Cr is found as chromite (Figure2b). Si oxides (Figure 2a). In the fresh tailing sample, Cr is found as chromite (Figure 2b).

(a)

(b) (c) (d)

Figure 2. Chromite tailing (a) elementary map of EDS showing the dominance of Cr, Fe, Si, and Al. SEM Figure 2. Chromite tailing (a) elementary map of EDS showing the dominance of Cr, Fe, Si, and Al. images of (b) chromite (Cr 63 wt %), (c) iron oxide (Cr 9 wt %), and (d) aluminum oxide (Cr 6 wt %). SEM images of (b) chromite (Cr 63 wt %), (c) iron oxide (Cr 9 wt %), and (d) aluminum oxide (Cr 6 wt %). In Sukinda chromite tailings, Tripathy et al. [3] found two different types of chromite grains: the firstIn Sukinda one is rich chromite in Cr with tailings, a minimum Tripathy amount et al. of [ 3Fe,] found Al, and two Mg different; whereas typesthe second of chromite one is rich grains: in the ﬁrstFe, one Al is, and rich Mg in, along Cr with with a Cr. minimum The grain amountdisplay in of Figure Fe, Al, 2b corresponds and Mg; whereas to the second the second group, where one is rich in Fe,there Al, is and 63 wt Mg, % of along Cr2O with3, 18 wt Cr. % of The FeO, grain 9 wt display % of MnO, in Figure6 wt % 2ofb Al corresponds2O3, and 4 wt to% of the MgO. second Other group, Cr-bearing phases are goethite, FeO(OH), aluminum oxides, and other colloids with a positively where there is 63 wt % of Cr2O3, 18 wt % of FeO, 9 wt % of MnO, 6 wt % of Al2O3, and 4 wt % of charged surface where hexavalent Cr species, which are negatively charged (e.g., CrO42− and HCrO4−), MgO. Other Cr-bearing phases are goethite, FeO(OH), aluminum oxides, and other colloids with a can be adsorbed [56]. In the tailings, gangue minerals enriched in Fe oxy(hydroxides) (81 wt %) are positively charged surface where hexavalent Cr species, which are negatively charged (e.g., CrO 2− identified with the presence of Cr (Cr2O3 9 wt %), Si (SiO2 5 wt %), and Al (Al2O3 5 wt %) (Figure 2c). 4 − andAl HCrO oxide4 is), also can present be adsorbed in the fresh [56]. tailing In the sample, tailings, with gangue a lower minerals Cr content enriched (6 wt % in) (Figure Fe oxy(hydroxides) 2d). (81 wt %) are identified with the presence of Cr (Cr2O3 9 wt %), Si (SiO2 5 wt %), and Al (Al2O3 5 wt %) (Figure3.2.2 c).pH AlProfiles oxide is also present in the fresh tailing sample, with a lower Cr content (6 wt %) (Figure2d). The changes of pH observed in the biotic and abiotic leaching experiments in distilled water and growth medium with P. putida and A. thiooxidans are presented in Figure 3. The leaching of the fresh tailing sample with distilled water shows a stable pH close to 7, and is 0.2 units higher for the pulp density at 30 g/L compared to 5 and 10 g/L (Figure 3a).

MineralsMinerals 201 20188, ,8 8, ,x x FOR FOR PEER PEER REVIEW REVIEW 1313 of of 23 23 Minerals 2018, 8, x FOR PEER REVIEW 13 of 23 toto thethe AlAl mineral mineral phases, phases, suchsuch asas ggibbsiteibbsite andand chlorite, chlorite, inin thethe chromite chromite tailingstailings usedused inin the the present present to the Al mineral phases, such as gibbsite and chlorite, in the chromite tailings used in thework.work. present The The control control of of A. A. thiooxidans thiooxidans reveals reveals no no m mediumedium effect effect in in Al Al dissolution. dissolution. The The incubation incubation of of the the work. The control of A. thiooxidans reveals no medium effect in Al dissolution. The incubationfreshfresh of tailing tailingthe ssampleample withwith P.P. putidaputida showsshows notablynotably lessless AlAl alterationalteration comparedcompared withwith thethe incubationincubation fresh tailing sample with P. putida shows notably less Al alteration compared with the incubationwithwith A. A. thiooxidans thiooxidans . .In In addition, addition, Al Al does does not not dissol dissolveve continuously continuously; ;o onn the the contrary, contrary, two two phases phases w wereere with A. thiooxidans. In addition, Al does not dissolve continuously; on the contrary, two phasesidentified.identified. were AnAn initialinitial AlAl precipitation,precipitation, withwith anan incubationincubation periodperiod thatthat lastslasts approximatelyapproximately 1010 daysdays, , identified. An initial Al precipitation, with an incubation period that lasts approximatelywas 10 noted,days, followed by Al dissolution in the time remaining. In the first phase, 50% of Al precipitates for Minerals 2018, 8, 261 was noted, followed by Al dissolution in 8the of 23time remaining. In the first phase, 50% of Al precipitates for was noted, followed by Al dissolution in the time remaining. In the first phase, 50% of Al precipitatesthethe pulp pulp for density density atat 55 g/L,g/L, 64%64% forfor 1010 g/Lg/L, , andand 73% 73% forfor 3030 g/L.g/L. InIn thethe seconsecondd phase, phase, Al Al isis dissolved dissolved the pulp density at 5 g/L, 64% for 10 g/L, and 73% for 30 g/L. In the second phase, Al is againdissolvedagain ( (FFigureigure 8 8).) . again (Figure 8). 3.2. pH Proﬁles

The changes of pH observed in the biotic and abiotic leaching0.6 experiments0.6 in distilled water and (a) Blank 0.6 growth medium with P. putida and A. thiooxidans are presented in Figure(a)3. The leaching of the fresh Blank 0.50.5 (a) tailing sampleMineralsBlank 201 with8, 8, x distilledFOR PEER REVIEW water shows a stable pH close to 7, and is 0.2 units higher for8 of the 23 pulp 0.5 density at 30 g/L compared to 5 and 10 g/L (Figure3a). 0.40.4 0.4 0.30.3 10 0.3 (a) Al (mg/kg) Al Al (mg/kg) Al 0.20.2 8 Al (mg/kg) Al Distilled water (pH 7) (pH water Distilled 0.2 7) (pH water Distilled 0.10.1 Distilled water (pH 7) (pH water Distilled 0.1 6 0.00.0

pH value pH 2000 0.0 4 2000 Distilled water (pH 7) (pH water Distilled 1414 (b)(b) ControlControl (c)(c) 2000 (b) Control (c)(c) 14 (b) Control (c) 1212 2 Blank 15001500 12 1500 1010 10 10 8 (b) (c) 8 10001000 8 6 pH 2) pH 10008 6 Al (mg/kg) Al Al (mg/kg) Al 6 44 Al (mg/kg) Al 500500 6 A. thiooxidans2)(pH A. 4 500 thiooxidans2)(pH A. 22 A. thiooxidans2)(pH A. 2 value pH 4 00 00

0 thiooxidansA.( 0 5050 5050 (d)(d) ControlControl (e) 50 502 Control (d) Control (e)(e) (d) (d) Control (e) 4040 4040 10 40 40 (d) (e) 3030 3030 8 30 30 2020 2020 (pH 9) (pH Al (mg/kg) Al 6 (mg/kg) Al 20 20 P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. Al (mg/kg) Al

pH value pH 1010 1010

P. putida (pH 9)(pH putida P. 4

10 putida P. 10 00 00 2 Control 0 0 00 1010 2020 3030 4040 00 1010 2020 3030 4040 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 TimeTime40 (days) (days) TimeTime (days) (days) Time (days) Time (days) Time (days) Time (days) FigureFigure 8. 8. Al Al concentrations concentrations in in the the leachate leachate a ass a a function function of of time time during during (bio)leaching (bio)leaching experiments experiments of of Figure 3. pH evolution in leaching experiments of the fresh tailings sample with distilled water Figure 8. Al concentrations in the leachateFigure as a 3. functionpH evolution of time in during leaching (bio)leaching experiments experiments of the fresh tailings ofthe fresh sample tailing with sample distilled with water distilled (blank) water (a), incubated with A. thiooxidans (c) and P. putida (e). (blank) (a), and incubation with A. thiooxidans (c) and P. putidathe (e). freshControls tailing for A. sample thiooxidans with and distilled P. water (a), incubated with A. thiooxidans (c) and P. putida (e). the fresh tailing sample with distilled (watera), and (aputida incubation), incubated are display with withedA. in A. thiooxidans (b ,thiooxidansd), respectively.(c) ( andc) andThreeP. putida P. pulp putida( densitiese). Controls(e).Control Control werefor evaluated:ss for A.for thiooxidansA. A. thiooxidans thiooxidans5 g/L ( and and ),andP. 10 putida P. g/LP. putida putida are are are displayed displayed in in ( b(b,d,d),), respectively. respectively. Three Three pulp pulp densities densities were were Controls for A. thiooxidans and P. putida displayedare displayed( in) (, b inand,d (),b 30, respectively.d g/L), respectively. ( ). Three Three pulp pulp densities densities were were evaluated:evaluated: evaluated: 5 5 5 g/L g/L g/L ( ( ),),), 10 10 g/L g/L ( ( )),,) ,and and 30 30 g/L g/L ( ( ).). evaluated: 5 g/L ( ), 10 g/L ( ),and and 3030 g/Lg/L ( ).). For A. thiooxidans, in the control (growth medium3.4. 3.4.pH Chemical adjustedChemical to Extraction Extraction 3.5), pH is higher at a higher 3.4. Chemical Extraction ForpulpA. thiooxidansdensity. For, 5 in g/L the the control pH varies (growth from 3.8 medium to 4.3, forpH 10 g/L adjusted it varies to from 3.5), 4.1 pH to 5.2 is, higherand for 30 at g/L a higher from 5.3 to 6.0. When A. thiooxidans is inoculated, the pH isT consistentlyThehe chemicall chemicall lowyy (1.6exchangeable exchangeable in average) forpool pool all of of hexavalent hexavalent chromium chromium (E (ECr(VI)Cr(VI))) for for fresh fresh and and stockpiled stockpiled old old pulp density. For 5 g/L the pH varies from 3.8 to 4.3, for 10 g/L it varies from 4.1 to 5.2, and for 30 g/L The chemically exchangeable pool of hexavalentpulp densities. chromium The low pH (ECr(VI) is the) for result fresh of andthe sulfur stockpiledtailing tailingoxidation oldand and by chromite chromite A. thiooxidans ore, ore, in usingin mg mg dissolved of of Cr(VI) Cr(VI) per per kg kg of of tailing, tailing, is is presented presented in in Table Table 2. 2. For For the the chromite chromite from 5.3 to 6.0. When A. thiooxidans is inoculated, the pH is consistently low (1.6 in average) for all pulp tailing and chromite ore, in mg of Cr(VI) peroxygen kg ofas tailing,the electron is presented acceptor, and in theTable resulting 2. For H the2SOore ore4chromite production samples samples , ,[57]the the. total Intotal the Cr caseCr content content of P. putida in in the the, the solid solid ranges ranges between between 203 203 and and 236 236 g/kg. g/kg. For For the the chromite chromite ore ore 11, , densities.control The ( lowgrowth pH medium is the result pH adjusted of the to sulfur 7) has oxidation a pH close by toA. 7 and thiooxidans no differencesusing were dissolved observed oxygen ore samples, the total Cr content in the solid ranges between 203 and 236 g/kg. For the chromiteCr(VCr(V oreI)I) extracted extracted1, with with water water is is equivalent equivalent to to total total Cr Cr extracted extracted with with KH KH2PO2PO4,4 ,with with an an average average value value as the electronaccording acceptor, to the pulp and densities. the resulting However, H2SO in4 theproduction reactors where [57]. InP. theputida case is ofinoculatedP. putida, ,the the pH control Cr(VI) extracted with water is equivalent to total Cr extracted with KH2PO4, with an averageofof 3.4 3.4 value mg/kg, mg/kg, while while the the Cr(VI) Cr(VI) extracted extracted with with KH KH2PO2PO4 4(E (ECr(VI)Cr(VI))) remains remains lower lower (2.3 (2.3 mg/kg). mg/kg). In In the the case case (growthincreases medium significantly pH adjusted from to 7 7) to has9.2 in a the pH first close five to days 7 and, whatever no differences the pulp weredensity. observed Van Hullebusch according to of 3.4 mg/kg, while the Cr(VI) extracted with KH2PO4 (ECr(VI)) remains lower (2.3 mg/kg). In the case the pulpet densities. al. [58] explained However, the inpH the increase reactors in slag where leachingP. putida as ais result inoculated, of the initial the pHdissolution increases of significantlycations such as Ca2+ and Mg2+, followed by neutralization by H + from H2O. Potysz et al. [59] also reported a from 7 to 9.2 in the first five days, whatever the pulp density. Van Hullebusch et al. [58] explained + 2+ pH increase in Cu slags leaching due to H replacing Fe during fayalite dissolution. In batch 2+reactors 2+ the pH increaseinoculated in with slag P. leaching putida, dissolved as a result oxygen of the increases initial from dissolution 0 on the first of cations day to 4.7 such mg/L as Caon theand fifthMg , + followedday by, and neutralization remains constant by H for fromthe rest H 2ofO. the Potysz sampling etal. time [59 (Figure] also reportedS1). a pH increase in Cu slags + 2+ leaching due to H replacing Fe during fayalite dissolution. In batch reactors inoculated with P. putida, dissolved oxygen increases from 0 on the first day to 4.7 mg/L on the fifth day, and remains constant for the rest of the sampling time (Figure S1).

Minerals 2018, 8, 261 Minerals 2018, 8, x FOR PEER REVIEW 9 of 23 13 of 23 MineralsMinerals 201 20188, ,8 8, ,x x FOR FOR PEER PEER REVIEW REVIEW 1313 of of 23 23 to the Al mineral phases, such as gibbsite and chlorite, in the chromite tailings used in the present toto thethe AlAl mineralmineral phases,phases, suchsuch3.3. asas Release ggibbsiteibbsite ofElements andand chlorite,chlorite, during inin Leaching thethe chromitechromite Experiments tailingstailings usedused inin thethework. presentpresent The control of A. thiooxidans reveals no medium effect in Al dissolution. The incubation of the Minerals 2018, 8, x FOR PEER REVIEW 9 of 23 work.work. TheThe controlcontrol ofof A.A. thiooxidansthiooxidans revealsreveals nono mmediumedium effecteffect inin AlAl dissolution.dissolution. TheThe incubationincubationfresh ofof tailing thethe sample with P. putida shows notably less Al alteration compared with the incubation The behavior of Cr, Fe, Ni, Mn, and Al during the bioleaching of the fresh tailing sample is freshfresh tailingtailing ssampleample withwith P.P. putidaputida showsshows notablynotably lessless AlAl alterationalteration comparedcompared withwith thethe incubationincubationwith A. thiooxidans . In addition, Al does not dissolve continuously; on the contrary, two phases were presented3.3. Release in Figures of Elements4–8 andduring the Leaching units areExperiments expressed in mg of element released per kg of tailings. withwith A.A. thiooxidansthiooxidans.. InIn addition,addition, AlAl doesdoes notnot dissoldissolveve continuouslycontinuously;; oonn thethe contrary,contrary, twotwo phasesphasesidentified. wwereere An initial Al precipitation, with an incubation period that lasts approximately 10 days, The use ofTheA. behavior thiooxidans of Cr,asa Fe, leaching Ni, Mn agent, and facilitatesAl during Fethe dissolution. bioleaching Atof athe pulp fresh density tailing of sample 5 g/L, is Fe identified.identified. AnAn initialinitial AlAl precipitation,precipitation, withwith anan incubationincubation periodperiod thatthat lastslasts approximatelyapproximatelywas 1010 daysnoted,days,, followed by Al dissolution in the time remaining. In the first phase, 50% of Al precipitates for extractedpresented from in the Figures fresh 4 tailings–8 and the sample units increases are express fromed in 421 mg to of 1234 element mg/kg, release andd for per 10 kg g/L, of tailings. the range The is waswas noted,noted, followedfollowed byby AlAl dissolutiondissolution inin thethe timetime remaining. remaining. InIn thethe firstfirst phase phase,, 50%50% ofof AlAl precipitates precipitatesthe pulp forfor density at 5 g/L, 64% for 10 g/L, and 73% for 30 g/L. In the second phase, Al is dissolved narrower,use of withA. thiooxidans values between as a leaching 510 and agent 940 mg/kgfacilitates (Figure Fe dissolution.4). For 30 g/L,At a nopulp significant density of fluctuations 5 g/L, Fe the pulp density at 5 g/L, 64% for 10 g/L, and 73% for 30 g/L. In the second phase, Al is againdissolved (Figure 8). the pulp density at 5 g/L, 64%were forextracted observed, 10 g/L ,from and with the73% an fresh averagefor 30tailings g/L. value sampleIn ofthe 190 secon increa (±21)dses phase, mg/kg from Al421 over is to dissolved time. 1234 Inmg/kg, the reactors and forwith 10 g/LA., thiooxidansthe range again (Figure 8). again (Figure 8). for theis narrower three pulp, with densities, values between the pH 510 is and always 940 mg/kg below (Figure 4 and 4 for). F theor 30 first g/L day,, no significant Fe2+ is the fluctuations dominant 2+ species;were however,observed, with after an that, average Fe(III) value species of 190are (±21 more) mg/kg abundant, over time. caused In the reactors by0.6 the with oxidation A. thiooxidans of Fe forby 2+ (a) Blank 0.60.6 A. thiooxidansthe three pulp[60]. dens Theities same, the Fe pH speciation is alwaysis below observed 4 and in for a the leaching first day test, Fe using is the fresh dominant tailings specie samples; (a)(a) however, afterBlankBlank that, Fe(III) species are more abundant, caused by the oxidation0.5 of Fe2+ by A. thiooxidans conducted with distilled water acidified with H2SO4 and without bacteria (Figure S2). Liu et al. [60] 0.50.5 performed[60]. The leaching same Fe experiments speciation is ofobserved chalcopyrite in a leaching with A. test ferrooxidans using fresh(pH tailings 2–2.5)0.4 sample and they conducted found total with Fe 0.40.4 concentrationsdistilled water up acidified to 0.6 g/L, with which H2SO were4 andpresent without as bacteria Fe2+ and (Figure Fe3+ S2)for. Liu the et first al. six[60] days, performed but afterwards, leaching experiments of chalcopyrite with A. ferrooxidans (pH 2–2.5) and they found0.3 total Fe concentrations up only Fe3+ was detected. In the control of A. thiooxidans, at the higher pH (6; pulp density 30 g/L) 0.30.3 2+ 3+ 3+ to 0.6 g/L, which were present as Fe and Fe for the first six days, but(mg/kg) Al afterwards, only Fe was the Fe extracted is lower, compared with the lower pH (4; pulp density0.2 5 g/L), where total iron Al (mg/kg) Al Distilled water (pH 7) (pH water Distilled Al (mg/kg) Al 0.20.2 detected. In the control of A. thiooxidans, at the higher pH (6; pulp density 30 g/L) the Fe extracted is reaches 7 mg/kg on the sixth day of leaching. This result suggests that the0.1 medium effect is negligible Distilled water (pH 7) (pH water Distilled

Distilled water (pH 7) (pH water Distilled lower, compared with the lower pH (4; pulp density 5 g/L), where total iron reaches 7 mg/kg on the 0.10.1 compared to the leaching produced by the presence of A. thiooxidans and more speciﬁcally by the sixth day of leaching. This result suggests that the medium effect is negligible0.0 compared to the leaching sulfuric acid produced. 0.0 produced by the presence of A. thiooxidans and more specifically by the sulfuric acid produced. 2000 0.0 14 (b) Control (c) 20002000 1414 (b)(b) 50ControlControl (c)(c) 2000 12 (a) Control (b) 1500 1212 10 40 15001500 1010 1500 8 AT1 - B 1000 30 88 10001000 6 1000 (mg/kg) Al 66 4

Al (mg/kg) Al 500 Al (mg/kg) Al 20 Fe (mg/kg) Fe 44 thiooxidans2)(pH A. 500500 500 2 A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans (pH 2) thiooxidans (pHA. 10 22 0 0 00 00 50 50 0 0 (d) Control (e) 5050 10 5050 50 (e) (d)(d) (d)(d) ControlControl(c) (e)(e) Control (d) 40 40 4040 8 4040 40 30 30 3030 6 3030 30 20 20 Al (mg/kg) Al 2020 4 20 20

20 9)(pH putida P. Fe (mg/kg) Fe Al (mg/kg) Al Al (mg/kg) Al 10

P. putida (pH9) putida P. 10 P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. 1010 2 1010 10 0 0 0 0 00 00 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 00 1010 2020 3030 4040 00 1010 2020 3030 4040 Time (days) Time (days) Time (days) Time (days) TimeTime (days) (days) TimeTime (days)(days) Figure 8. Al concentrations in the leachate as a function of time during (bio)leaching experiments of FigureFigure 8.8. AlAl concentrationsconcentrations inin thetheFigure leachateleachateFigure 4. Fe 4. aas concentrationsFe aa functionconcentrationfunction ofof in timetime in the the duringduring leachate leachate (bio)leaching(bio)leaching as as a functiona function experimentsexperiments of of time time during during ofof the (bio)leaching(bio)leaching fresh tailing experiments sample with ofof distilled water (a), incubated with A. thiooxidans (c) and P. putida (e). thethe freshfresh tailingtailing samplesample withwith distilleddistilledthe freshthe waterfreshwater tailing tailing ((aa), samples), incubatedincubated sample withs with withwithA. A. thiooxidans A.A. thiooxidans thiooxidansthiooxidans(b ()b and) ( (candc)) and withand with P.P.P. P. putida putida putida ((( ed(ed).).). Control ControlsControlss for for A. A. thiooxidans thiooxidans andandand P. putida are displayed in (b,d), respectively. Three pulp densities were ControlControlss for for A. A. thiooxidans thiooxidans and and P. P.P. putida putida P. putida are are displayed displayed are display in in (ed ( bba, ,d,cind),), respectively. (respectively.a,c), respectively. ThreeThree Three Three pulppulp pulp densitiesdensities pulpdensities densities werewere were evaluated:evaluated: were evaluated: 55 g/Lg/L (5 g/L (),), 1010 g/Lg/L), ( ), and 30 g/L ( ). evaluated:evaluated: 55 g/Lg/L (( ),), 1010 g/Lg/L ( ( 10),) ),g/L, andand ( 3030 g/Lg/L), and ( ( 30 g/L).).). ( ). 3.4. Chemical Extraction Because of its toxicity, mobility, and enrichment in chromite and other Cr bearing phases in 3.4.3.4. ChemicalChemical ExtractionExtraction Because of its toxicity, mobility, and enrichment in chromite and other Cr bearing phases in chromite tailings, the leaching behavior of chromium is of particularThe interest chemicall. Withy exchangeabledistilled water ,pool a of hexavalent chromium (ECr(VI)) for fresh and stockpiled old chromite tailings, the leaching behavior of chromium is of particular interest. With distilled water, TThehe chemicallchemicallyy exchangeableexchangeablemaximum poolpool ofof hexavalentofhexavalent chromium chromiumchromium is leached (E(E fromCr(VI)Cr(VI) ))the forfor fresh freshfresh tailing andand stockpiledstockpiled sampletailing in old oldtheand first chromite three daysore, infor mg all pulpof Cr(VI) per kg of tailing, is presented in Table 2. For the chromite a maximum of chromium is leached from the fresh tailing sample in the ﬁrst three days for all pulp tailingtailing andand chromitechromite ore,ore, inin mgmg ofdensitiesof Cr(VI)Cr(VI), perreachingper kgkg ofof values tailing,tailing, of is is94.8 presentedpresented (±1.41), 74.45 inin TableTable (±0.35 2.2. ) For,For and thethe 62.70 ore chromitechromite (samples±8.87) mg/kg, the total for pulp Cr content densities in ofthe solid ranges between 203 and 236 g/kg. For the chromite ore 1, densities, reaching values of 94.8 (±1.41), 74.45 (±0.35), and 62.70 (±8.87) mg/kg for pulp densities of 5, oreore samplessamples,, thethe totaltotal CrCr contentcontent 5,inin 10 thethe, and solidsolid 30 rangesg/L,ranges respectively. betweenbetween 203203 After andand the 236236 third g/kg.g/kg. day, ForFor the thethe chromium chromitechromiteCr(V extracted oreoreI) extracted 11,, slowly with increases water untilis equivalent day to total Cr extracted with KH2PO4, with an average value 10, and 30 g/L, respectively. After the third day, the chromium extracted slowly increases until day 34. Cr(VCr(VI)I) extractedextracted withwith waterwater isis equivalentequivalent34. When toA.to totalthiooxidanstotal CrCr extractedextracted is present withwith in theKHKH 2batch2POPO44,, withreactorwith anan, chromium averageaverageof 3.4 valuevalue ismg/kg, leach ed while from the the Cr(VI) fresh tailingextracted with KH2PO4 (ECr(VI)) remains lower (2.3 mg/kg). In the case When A. thiooxidans is present in the batch reactor, chromium is leached from the fresh tailing sample ofof 3.43.4 mg/kg,mg/kg, whilewhile thethe Cr(VI)Cr(VI) extractedextractedsample in withwith the first KHKH2 2hoursPOPO44 (E(E andCr(VI)Cr(VI) then)) remainsremains the curve lowerlower decreases (2.3(2.3 mg/kg).mg/kg).. The pulp InIn thethe density casecase at 5 g/L presents the highest Cr content (174–268 mg/kg), followed by 10 (159–236 mg/kg) and 30 g/L (130–191 mg/kg). In the control

MineralsMineralsMinerals 201201 20188,8, 8,8 ,8, x,x x FORFOR FOR PEERPEER PEER REVIEWREVIEW REVIEW 131313 ofof of 2323 23

tototo the thethe Al AlAl mineral mineralmineral phases, phases,phases, such suchsuch as asas g ggibbsiteibbsiteibbsite and andand chlorite, chlorite,chlorite, in inin the thethe chromite chromitechromite tailings tailingstailings used usedused in inin the thethe present presentpresent Mineralswork.work.work. 2018The TheThe, control8 controlcontrol, 261 of ofof A. A.A. thiooxidans thiooxidansthiooxidans reveals revealsreveals no nono m mmediumediumedium effect effecteffect in inin Al AlAl dissolution. dissolution.dissolution. The TheThe incubation incubationincubation10 of ofof of the the 23the freshfreshfresh tailing tailingtailing s samplesampleample with withwith P. P.P. putida putidaputida shows showsshows notably notablynotably less lessless Al AlAl alteration alterationalteration compared comparedcompared with withwith the thethe incubation incubationincubation withwithwithMinerals A. A.A. thiooxidans thiooxidansthiooxidans 2018, 8, x FOR.. . In In InPEER addition, addition,addition, REVIEW Al AlAl does doesdoes not notnot dissol dissoldissolveveve continuously continuouslycontinuously;; ; o oonnn the thethe contrary, contrary,contrary, two twotwo phases phasesphases10 of w w23wereere ere in the ﬁrst hours and then the curve decreases. The pulp density at 5 g/L presents the highest Cr identified.identified.identified. An AnAn initial initialinitial Al AlAl precipitation, precipitation,precipitation, with withwith an anan incubation incubationincubation period periodperiod that thatthat lasts lastslasts approximately approximatelyapproximately 10 1010 days daysdays,, , content (174–268 mg/kg), followed by 10 (159–236 mg/kg) and 30 g/L (130–191 mg/kg). In the control waswaswasof noted, noted,A.noted, thiooxidans followed followedfollowed, the by byby maximum Al AlAl dissolution dissolutiondissolution chromium in inin the thethe extracted time timetime remaining.remaining. remaining. for the 5 In In(259In the thethe mg/kg) first firstfirst phasephase phase and ,, 10, 50% 50%50% g/L of of of(245 Al AlAl precipitatesprecipitatesmg/kg) precipitates pulp for for for of A. thiooxidans, the maximum chromium extracted for the 5 (259 mg/kg) and 10 g/L (245 mg/kg) thethethedensity pulp pulppulp density densityisdensity similar at atat to 5 55 theg/L, g/L,g/L, batch 64% 64%64% incubated for forfor 10 1010 g/L g/Lg/L with,, , and andand A. 73% 73% 73%thiooxidans for forfor 30 3030 , g/L. g/L.forg/L. the In InIn samethe thethe secon seconsecon pulpd ddensitiesd phase, phase,phase, .Al Al AlFor is isis the dissolved dissolveddissolved pulp pulpdensity density at is30 similar g/L in tothe the control batch for incubated A. thiooxidans with,A. the thiooxidans chromium, for concentration the same pulp slightly densities. increases For in the againagainagain ( (F(FFigureigureigure 8 88)).). . pulpsol densityution from at 30 170 g/L to in202 the mg/kg control in the for firstA. thiooxidans two days and, the then chromium remains concentration stable (Figure slightly5). increases in solution from 170 to 202 mg/kg in the ﬁrst two days and then remains stable (Figure5). 0.60.60.6 (a) Blank 300(a)(a) BlankBlank 0.50.50.5 (a) 250 0.40.40.4 200 0.30.30.3 150 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al 0.20.20.2

Cr (mg/kg) Cr 100 Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled 0.10.10.1 50 Blank 0.00.00.0 200020002000 141414300(b)(b)(b) ControlControlControl (c)(c)(c) (b) (c) AT1 - B 121212 250 150015001500 101010 200 888 10001000 150 1000 666 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al

Cr (mg/kg) Cr 100 444 500500500 A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. 222 50 Control 000 0 000 505050300 505050 (d)(d)(d) Control (d)(d)(d) (d) ControlControlControl (e)(e)(e)(e) 404040250 404040 200 303030 303030 150 202020 202020 Al (mg/kg) Al Cr (mg/kg) Cr Al (mg/kg) Al Al (mg/kg) Al P. putida (pH 9) putida(pH P. 100 P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. 101010 50 101010 Control 000 0 000 0 10 20 30 40 0 10 20 30 40 000 101010 202020 303030 404040 000 101010 202020 303030 404040 Time (days) Time (days) TimeTimeTime (days)(days) (days) TimeTimeTime (days) (days)(days) FigureFigureFigureFigureFigure 5. 8. 8.8. Cr Al5. AlAl Cr concentrations concentrationsconcentrations concentrations in in inin the the thethe leachate leachateleachate a aasass s a a aa functionfunction function of ofof timetime timetime during during duringduring (bio)leaching (bio)leaching (bio)leaching(bio)leaching (bio)leaching experiments experiments experimentsexperiments experiments of of ofof ofthethethe thethe fresh freshfresh freshfresh tailing tailingtailing tailingtailing sample samplesample samplesample with with with with distilled distilled distilled water waterwaterwater ( (a( aa),),), incubatedincubated incubated incubatedincubated withwith with with A. A. A.A.A. thiooxidans thiooxidans thiooxidans thiooxidans ( c ( )(c( c)cand) ) and andand P. P. P.putidaP. putidaputida putidaputida (e (). ( e(e (e).e).).). ControlsControlControlControlControlsssfor s forfor for forA. A.A. A. thiooxidansA. thiooxidansthiooxidans thiooxidans thiooxidansand andand andandP. P.P. P. putida putidaputida putidaare are are arearedisplayed displayed displayed displaydisplayededin inin in( ( b( b(b,b,d,d,d,),d),),), ), respectively. respectively.respectively. respectively. Three Three ThreeThree Three pulp pulp pulppulp pulp densitiesdensities densitiesdensities densities werewere werewere were evaluated:evaluated:evaluated:evaluated:evaluated: 5 5 5 g/L5 g/L g/L5g/L g/L ( (( ( ),),),), ), 1010 10 1010 g/L g/L g/L g/Lg/L ( ( ( )),),)), ,, and andandand 30 3030 g/L g/Lg/L ((( ( ).).).).

3.4.3.4.3.4. ChemicalIn ChemicalChemical theIn the case Extractioncase ExtractionExtraction of theof the fresh fresh tailing tailing sample sample incubated incubated with with P.P.putida putida,, forfor all pulp densities densities,, the the maximum maximum amount of chromium extracted (217 mg/kg in average) is reached in the first hours and then the chromium amount of chromium extracted (217 mg/kg in average) is reached in the ﬁrst hours and then the concentrationTTThehehe chemicall chemicallchemicall slowlyyyy exchangeable exchangeableexchangeable decreases. Starting pool poolpool of offromof hexavalent hexavalenthexavalent day 10, the chromium chromiumchromium differences (E (E(ECr(VI) Cr(VI)betweenCr(VI))) ) for forfor fresh freshpulpfresh anddensities andand stockpiled stockpiledstockpiled become old old old chromium concentration slowly decreases. Starting from day 10, the differences between pulp densities tailingtailingtailingrelevant and andand with chromite chromitechromite higher ore, ore,ore, amounts in inin mg mgmg of of ofof Cr Cr(VI) Cr(VI)Cr(VI) extracted per perper kg kgforkg of ofaof pulptailing, tailing,tailing, density is isis presented presentedpresented of 5 g/L in(135 inin Table TableTable mg/kg) 2. 2.2. For .For ForIn the thethe controlchromite chromitechromite become relevant with higher amounts of Cr extracted for a pulp density of 5 g/L (135 mg/kg). In the oreoreorefor samples samplessamples P. putida,, , the thethe, chromium total totaltotal Cr CrCr content content contentdecreases in inin theby thethe 15% solid solidsolid at ranges rangesranges5 and 10 between betweenbetween g/L and 203 203203 20% and andand at 30236 236236 g/L g/kg. g/kg.g/kg., from For ForFor day the thethe 1 chromite chromitetochromite 34. ore oreore 1 11,, , control for P. putida, chromium decreases by 15% at 5 and 10 g/L and 20% at 30 g/L, from day 1 to 34. Cr(VCr(VCr(VI)I)I) extractedFor extractedextracted both A. with with withthiooxidans water waterwater and is isis equivalent equivalent equivalentP. putida, the to toto maximum total totaltotal Cr CrCr extracted extractedCrextracted concentration with withwith KH KH KHis observed22PO2POPO44,4, , with withwith at thean anan averagebeginning averageaverage value value valueof For both A. thiooxidans and P. putida, the maximum Cr concentration is observed at the beginning ofofof 3.4the 3.43.4 mg/kg, mg/kg,incubation,mg/kg, while whilewhile and the thethe is Cr(VI) Cr(VI)Cr(VI)influenced extracted extractedextracted by the with withwith presence KH KHKH22PO2POPO of44 4 (EKH (E(ECr(VI)Cr(VI)Cr(VI)2PO)) ) 4remains remains remainsin the growth lower lowerlower (2.3 (2.3medium.(2.3 mg/kg). mg/kg).mg/kg). Phosphates In InIn the thethe case casecase of the incubation, and is inﬂuenced by the presence of KH PO in the growth medium. Phosphates (PO43−) can promote the desorption of anions including chromate2 4 ions, even if they are tightly bound 3− (PO4 ) can promote the desorption of anions including chromate ions, even if they are tightly bound compared with other anions such as chloride, nitrate, and sulfate [61,62]. However, not only the compared with other anions such as chloride, nitrate, and sulfate [61,62]. However, not only the

MineralsMineralsMinerals 201201 20188,8, 8,8 ,8, x,x x FORFOR FOR PEERPEER PEER REVIEWREVIEW REVIEW 131313 ofof of 2323 23

tototo the thethe Al AlAl mineral mineralmineral phases, phases,phases, such suchsuch as asas g ggibbsiteibbsiteibbsite and andand chlorite, chlorite,chlorite, in inin the thethe chromite chromitechromite tailings tailingstailings used usedused in inin the thethe present presentpresent work.work.work. The TheThe control controlcontrol of ofof A. A.A. thiooxidans thiooxidansthiooxidans reveals revealsreveals no nono m mmediumediumedium effect effecteffect in inin Al AlAl dissolution. dissolution.dissolution. The TheThe incubation incubationincubation of ofof the thethe freshfreshfresh tailing tailingtailing s samplesampleample with withwith P. P.P. putida putidaputida shows showsshows notably notablynotably less lessless Al AlAl alteration alterationalteration compared comparedcompared with withwith the thethe incubation incubationincubation withwithMineralswith A. A.A. 201 thiooxidans thiooxidansthiooxidans8, 8, x FOR.. PEER. In InIn addition, addition,addition, REVIEW Al Al Al does doesdoes not notnot dissol dissoldissolveveve continuously continuouslycontinuously;; ; o oonnn the thethe contrary, contrary,contrary, two twotwo phases phasesphases11 w ww ofereereere 23 Minerals 2018, 8, 261 11 of 23 identified.identified.identified. An AnAn initial initialinitial Al AlAl precipitation, precipitation,precipitation, with withwith an anan incubation incubationincubation period periodperiod that thatthat lasts lastslasts approximately approximatelyapproximately 10 1010 days daysdays,, , waswaspresencewas noted, noted,noted, of followed followedfollowed phosphates by byby Al AlAl promotes dissolution dissolutiondissolution the in inin presence the thethe time timetime remaining.remaining. ofremaining. chromium In InIn the theinthe firstsolution, firstfirst phasephase phase because,, , 50% 50%50% of ofof Alalmost AlAl precipitatesprecipitates precipitates 150 mg/kg for forfor presencethetheofthe Cr pulp pulppulp is extractedof density densitydensity phosphates at atduringat 5 55 g/L, g/L, promotesg/L, the 64% 64%64% leaching for forfor the 10 1010 presence g/L withg/Lg/L,, , and anddistilledand of 73% 73% chromium73% forwater forfor 30 3030 afterg/L.in g/L.g/L. solution, In 34InIn the daysthethe secon becauseseconsecon (Figureddd phase, phase,phase, almost 5). Al AlAl 150 is isis mg/kg dissolved dissolveddissolved of Cragainagainagain is extracted ( (F(FFigureigureigure 8 during88)).). . the leaching with distilled water after 34 days (Figure5).

1.0 0.60.60.6 (a) Blank (a)(a)(a) BlankBlankBlank 0.8 0.50.50.5

0.40.40.60.4

0.30.30.40.3 Ni (mg/kg) Ni Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al 0.20.20.2 Distilled water (pH 7) (pH water Distilled 0.2 Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled 0.10.10.1 0.0 0.00.00.050 100 (b) Control 200020002000 (c) 141414 (b)(b)(b) ControlControlControl (c)(c)(c) 40 80 121212 150015001500 AT1 - B 10103010 60 8 88 100010001000 20 40

Ni (mg/kg) Ni 666 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al 444 A. thiooxidans2)(pH A. 10 50050050020 A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. 222 0 0 00 00 500 500 505050 (d) Control 505050 (e) (d)(d)(d) Control (d)(d)(d) ControlControlControl (e)(e)(e) 40 40 404040 404040 30 30 303030 303030 20 20 Ni (mg/kg) Ni 202020 202020 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al P. putida (pH 9) putida(pH P. P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. 10 10 101010 101010 0 0 000 00 0 10 20 30 40 0 0 10 20 30 40 000 101010 202020 303030 404040 000 101010 202020 303030 404040 Time (days) Time (days) TimeTimeTime (days)(days) (days) TimeTimeTime (days) (days)(days) FigureFigureFigureFigure 6. 8. 8.6.8. Ni Al AlNiAl concentrations concentrationsconcentrationsconcentrations in ininin the thethethe leachate leachateleachateleachate a aasass s a aa a function functionfunction function of ofofof time timetimetime during duringduringduring (bio)leaching (bio)leaching(bio)leaching(bio)leaching (bio)leaching experiments experimentsexperiments experiments of ofof ofthethethethe the fresh freshfreshfresh fresh tailing tailingtailingtailing tailing sample samplesamplesample sample with withwith with distilled distilleddistilled water waterwater ( ((a( aa),),),), incubated incubatedincubated incubatedincubated with with with A. A.A.A. thiooxidans thiooxidans thiooxidansthiooxidans ( ((c(c)c)) ) and andandand P. P.P. putida putida putidaputida ( ( e((e(e).e).).).). ControlsControlControlControlsssfor s for for forforA. A. A. A. thiooxidans thiooxidans thiooxidans thiooxidansthiooxidansand and and andP. P. P. P. putida putida putida putidaare are are aredisplayed displayed displayed displayedin in in inin( (b ( (b(b,bd,,d,d,),d),),),), respectively. respectively. respectively. respectively.respectively. Three Three Three Three pulp pulp pulp pulpdensities densities densities densitieswere were were were evaluated:evaluated:evaluated:evaluated: 5 5 5 g/L5 g/L g/Lg/L ( ((( ),),),),), 1010 1010 g/L g/L g/Lg/L ( ((( )),)),), ,, and andandand 30 303030 g/L g/Lg/Lg/L ( ( ( ( ).).).).

3.4.3.4.3.4. ChemicalIt ChemicalChemicalIt is is known known Extraction ExtractionExtraction that that lateritic lateritic chromite chromite overburden overburden contains contains nearly nearly 0.4%–0.9% 0.4%–0.9% Ni, Ni which, which is is entrapped entrapped withinwithin the the goethite goethite (FeOOH) (FeOOH) matrix matrix [63 [63]]. It. isIt alsois also known know thatn that Ni Ni and and Cr Cr are are elements elements characteristic characteristic of TTThehehe chemicall chemicallchemicallyyy exchangeable exchangeableexchangeable pool poolpool of ofof hexavalent hexavalenthexavalent chromium chromiumchromium (E (E(ECr(VI)Cr(VI)Cr(VI))) ) for forfor fresh freshfresh and andand stockpiled stockpiledstockpiled old oldold of ultramafic areas, and both of them have species with a certain grade of toxicity. For this reason, ultramaﬁctailingtailingtailing and andand areas, chromite chromitechromite and ore,both ore,ore, in in ofin mg mg themmg of ofof haveCr(VI) Cr(VI)Cr(VI) species per perper kg kgkg with of ofof tailing, tailing,tailing, a certain is isis presented presented gradepresented of toxicity. in inin Table TableTableFor 2. 2.2. For For thisFor the thethe reason, chromite chromitechromite the the leachability of Ni is also evaluated. Figure 6 presents the Ni behavior in the leachate over time. leachabilityoreoreore samples samplessamples of,, , the thethe Ni total total istotal also Cr CrCr evaluated. content contentcontent in inin Figurethe thethe solid solidsolid6 presents ranges rangesranges between between thebetween Ni behavior 203 203203 and andand in236 236236 the g/kg. g/kg.g/kg. leachate For ForFor the thethe over chromite chromitechromite time. ore oreore 1 11,, , TheThe leaching leaching ofof thethe fresh tailings sample sample with with distilled distilled water water showed showed that that NiNi in the in the leachate leachate was Cr(VCr(VCr(VI)I)I) extracted extractedextracted with withwith water waterwater is isis equivalent equivalentequivalent to toto total totaltotal Cr CrCr extracted extractedextracted with withwith KH KHKH22PO2POPO44,4, , with withwith an anan average averageaverage value valuevalue waslower lower than than 1 mg/kg 1 mg/kg for forall allpulp pulp densities. densities. In the In the incubation incubation with with A. A.thiooxidans thiooxidans, the, the extracted extracted Ni ofofof 3.4 3.43.4 mg/kg, mg/kg,mg/kg, while whilewhile the thethe Cr(VI) Cr(VI)Cr(VI) extracted extractedextracted with withwith KH KHKH22PO2POPO44 4 (E (E(ECr(VI)Cr(VI)Cr(VI))) ) remains remainsremains lower lowerlower (2.3 (2.3(2.3 mg/kg). mg/kg).mg/kg). In InIn the thethe case casecase Niconcentration concentration increased increased over over time. time. For For a pulp a pulp density density ofof 5 5g/L, g/L, the the Ni Ni extracted extracted goes goes from from 20 20 to to 65 65mg/kg, mg/kg, for for 10 10 g/L g/L is isbetween between 44 44 and and 56 56 mg/kg mg/kg,, and and for for 30 30 g/L g/L is isbetween between 14 14 and and 36 36 mg/kg. mg/kg. The The A.

A.thiooxidans thiooxidans mediummedium composition composition (Figure (Figure 6bb)) induces NiNi leachingleaching fromfrom thethe fresh fresh tailing tailing sample; sample; however,however it, onlyit only corresponds corresponds to theto 20%the 20% of the of Ni the extracted Ni extracted under under acidic conditionsacidic conditions in the batch in the reactor batch incubatedreactor incubated with A. thiooxidans with A. thiooxidans. This indicates. This that indicates the growth that the of A. growth thiooxidans of A.promotes thiooxidans the promotes leaching ofthe Ni.leaching In the leachingof Ni. In experimentsthe leaching withexperimentsP. putida ,with Ni tends P. putida to decrease, Ni tends from to thedecrease ﬁrst day from (33 the mg/kg first forday

Minerals 2018, 8, x FOR PEER REVIEW 13 of 23 MineralsMinerals 201 20188, ,8 8, ,x x FOR FOR PEER PEER REVIEW REVIEW 1313 of of 23 23 to the Al mineral phases, such as gibbsite and chlorite, in the chromite tailings used in the present toto thethe AlAl mineralmineral phases,phases, suchsuch asas ggibbsiteibbsite andand chlorite,chlorite, inin thethe chromitechromite tailingstailings usedused inin thethework. presentpresent The control of A. thiooxidans reveals no medium effect in Al dissolution. The incubation of the Minerals 2018, 8, x FOR PEER REVIEW 12 of 23 work.work. TheThe controlcontrol ofof A.A. thiooxidansthiooxidansMinerals revealsreveals2018, 8, 261 nono mmediumedium effecteffect inin AlAl dissolution.dissolution. TheThe incubationincubationfresh ofof tailing thethe sample with P. putida12 of shows 23 notably less Al alteration compared with the incubation freshfresh tailingtailing ssampleample withwith P.P. putidaputida showsshows notablynotably lessless AlAl alterationalteration comparedcompared withwith thethe incubationincubationwith A. thiooxidans . In addition, Al does not dissolve continuously; on the contrary, two phases were (33 mg/kg for 5 g/L, 6 mg/kg for 10 g/L, and 3 mg/kg for 30 g/L) to the sixth day (20 mg/kg for 5 g/L, withwith A.A. thiooxidansthiooxidans.. InIn addition,addition, AlAl doesdoes notnot dissoldissolveve continuouslycontinuously;; oonn thethe contrary,contrary, twotwo phasesphasesidentified. wwereere An initial Al precipitation, with an incubation period that lasts approximately 10 days, 50.5 g/L, mg/kg 6 mg/kg for 10 for g/L 10 and g/L, 0.2 and mg/kg 3 mg/kg for 30 for g/L) 30 g/L), and toafter the this, sixth part day of (20 it mg/kgis re-solubilized for 5 g/L, and/or 0.5 mg/kg more identified.identified. AnAn initialinitial AlAl precipitation,precipitation, withwith anan incubationincubation periodperiod thatthat lastslasts approximatelyapproximatelywas 1010 daysnoted,days,, followed by Al dissolution in the time remaining. In the first phase, 50% of Al precipitates for forNi 10 is leached g/L and from 0.2 mg/kg the tailings for 30. The g/L), P. putida and after medium this, partcomposition of it is re-solubilized effect is negligible. and/or more Ni is waswas noted,noted, followedfollowed byby AlAl dissolutiondissolution inin thethe timetime remaining. remaining. InIn thethe firstfirst phase phase,, 50%50% ofof AlAl precipitates precipitatesthe pulp forfor density at 5 g/L, 64% for 10 g/L, and 73% for 30 g/L. In the second phase, Al is dissolved leachedAs from occurs the tailings.with other The elements,P. putida mediummanganese composition is release effectd from is negligible.the fresh tailing sample under the pulp density at 5 g/L, 64% for 10 g/L, and 73% for 30 g/L. In the second phase, Al is againdissolved (Figure 8). the pulp density at 5 g/L, 64%incubation forAs 10 occurs g/L with, and with A. 73% thiooxidans other for 30 elements, .g/L. At aIn pulp the manganese densitysecond ophase,fis 5 g/L, released Al Mn is increasesdissolved from the at fresh a rate tailing of 21 mg/kg sample/day under in the againagain ((FFigureigure 88)).. incubationfirst three withdays A.and thiooxidans after this,. continue At a pulps to density increas ofe 5at g/L, a rate Mn ten increases times lower at a rate(2 mg/kg/day of 21 mg/kg/day). For 10 g/ inL, theMn ﬁrst extract threeed days varies and from after 94 this,to 138 continues mg/kg. For to increasethe pulp atdensity a rate of ten 10 times g/L, in0.6 lower the first (2 mg/kg/day). three days, the For Mn (a) Blank 0.60.6 10extracted g/L, Mn incr extractedeases at varies a rate fromof 8 mg/kg/day 94 to 138 mg/kg. and in the For period the pulp remaining density, ofdecrease 10 g/L,s to in the1.6 mg/kg/day ﬁrst three . 0.5 (a)(a) days,For 3 the0 g Mn/L, the extracted BlankBlankrange increasesof variation at ais rate smaller of 8 mg/kg/day(25 to 56 mg/kg), and in and the periodit also remaining,increases over decreases time. The to 0.50.5 1.6manganese mg/kg/day. chemically For 30 g/L,extracted the range by the of medium variation without is smaller the (25incubation to 56 mg/kg), of0.4 A. thiooxidans and it also corresponds increases 0.40.4 over time. The manganese chemically extracted by the medium without the incubation of A. thiooxidans to 10% of the total amount extracted after incubation. For the incubation0.3 with P. putida, the Mn corresponds to 10% of the total amount extracted after incubation. For the incubation with 2+P. putida, 0.30.3 concentrations are considerably lower than for A. thiooxidans, due to the instability of Mn at a pH thehigher Mn concentrationsthan 7. The Mn are variation considerably, during lower incubation than for ofA. the thiooxidans fresh tailing, due sample(mg/kg) Al to0.2 the instabilitywith P. putida of Mn, is2+ higherat a Al (mg/kg) Al Distilled water (pH 7) (pH water Distilled Al (mg/kg) Al 0.20.2 pHforhigher the pulp than density 7. The at Mn5 g/L, variation, with values during betw incubationeen 0.6 and of 23 the mg/kg fresh (Figure tailing0.1 7 sample). with P. putida, is Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled 0.10.1 higher for the pulp density at 5 g/L, with values between 0.6 and 23 mg/kg (Figure7). 0.0 0.0 2000 0.0 300 300 14 (b) Control (c) (a) 20002000 Control (b) 1414 (b)(b) ControlControl (c)(c) 12 250 250 1500 1212 10 15001500 200 200 1010 8 1000 150 150 88 10001000 6 Al (mg/kg) Al 66 Mn (mg/kg) Mn 100 4 Al (mg/kg) Al 100 500 Al (mg/kg) Al 44 500500 thiooxidans2)(pH A. 2 A. thiooxidans (pH 2) thiooxidans(pH A. 50 50 A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. 22 0 0 0 0 50 50 00 00 (d) 1.0 50 (d) Control (e) 5050 (c) 5050 (d)(d) Control (d) (d)(d) ControlControl (e)(e) 40 40 0.8 40 4040 4040 30 30 0.6 30 3030 3030 20 20 0.4 20 (mg/kg) Al 2020 Mn (mg/kg) Mn 20

20 9)(pH putida P. Al (mg/kg) Al Al (mg/kg) Al

P. putida (pH9) putida P. 10 10 P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. 0.2 10 1010 1010 0 0 0.0 0 00 00 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 00 1010 2020 3030 4040 Time (days) Time (days) Time (days) Time (days) TimeTime (days) (days) TimeTime (days)(days) Figure 8. Al concentrations in the leachate as a function of time during (bio)leaching experiments of FigureFigure 8.8. AlAl concentrationsconcentrations inin thetheFigureFigure leachateleachate 7. 7.Mn Mn aass concentration a aconcentration functionfunction ofof time intime in the the duringduring leachate leachate (bio)leaching(bio)leaching as as a a function function experimentsexperiments of of time, time, during during ofof the (bio)leaching fresh(bio)leaching tailing experimentssample experiments withof distilledof water (a), incubated with A. thiooxidans (c) and P. putida (e). thethe freshfresh tailingtailing samplesample withwith distilleddistilledthethe fresh fresh waterwater tailing tailing ((aa), sample), sample incubatedincubated with with withwithA. A. thiooxidans thiooxidans A.A. thiooxidansthiooxidans( b(b)) and (and(cc)) withand andwith P.P. P. putidaputida putida ((de e().).d). Control). Controls Controlss for for for A.A. A.thiooxidans thiooxidans thiooxidans andand and P. putida are displayed in (b,d), respectively. Three pulp densities were ControlControlss for for A. A. thiooxidans thiooxidans and and P. P.P. putidaP. putida putida are are aredisplayed displayed display in ined ( ( b bain,,d,cd ),),( arespectively. respectively.,c), respectively. ThreeThree Three Three pulppulp pulp densitiesdensities pulpdensities densities werewere were evaluated:evaluated: were evaluated: 55 g/Lg/L ( 5 g/L),), 10(10 g/Lg/L ),( ), and 30 g/L ( ). evaluated:evaluated: 55 g/Lg/L (( ),), 1010 g/Lg/L ( (10 g/L),)),, and(and 3030) , g/L g/Land ( ( 30 g/L).).). ( ). 3.4. Chemical Extraction 3.4.3.4. ChemicalChemical ExtractionExtraction Aluminum is the fourth most abundant element present on the chromite tailings, and it remains Aluminum is the fourth most abundant element present on theT chromitehe chemicall tailings,y exchangeable and it remains pool of hexavalent chromium (ECr(VI)) for fresh and stockpiled old presentpresent in in the the concentrated concentrated ore ore after after the the beneficiation beneficiation process. process. Leaching Leaching of of the the fresh fresh tailings tailings sample sample TThehe chemicallchemicallyy exchangeableexchangeable poolpool ofof hexavalenthexavalent chromiumchromium (E(ECr(VI)Cr(VI))) forfor freshfresh andand stockpiledstockpiledtailing old oldand chromite ore, in mg of Cr(VI) per kg of tailing, is presented in Table 2. For the chromite with distilled water shows that Al in solution is released in the first hours during the first six days. tailingtailing andand chromitechromite ore,ore, inin mgwithmg ofof distilled Cr(VI)Cr(VI) perper water kgkg ofshowsof tailing,tailing, that isisAl presentedpresented in solution inin TableTable is released 2.2. ForFor in thethe the ore chromitechromite first samples hours , the during total theCr content first six in days. the solid ranges between 203 and 236 g/kg. For the chromite ore 1, However, after this period of time, Al starts to continuously increase within 40 days of incubation, oreore samplessamples,, thethe totaltotal CrCr contentcontentHowever, inin thethe solid aftersolid ranges thisranges period betweenbetween of time, 203203 andand Al starts 236236 g/kg.g/kg. to continuously ForFor thethe chromitechromite increaseCr(V oreoreI) extracted 11 within,, 40 with days water of incubation, is equivalent to total Cr extracted with KH2PO4, with an average value exceptexcept for for the the pulp pulp density density of of 30 30 g/L, g/L,where where the the concentration concentration remains remains constant. constant. Cr(VCr(VI)I) extractedextracted withwith waterwater isis equivalentequivalent toto totaltotal CrCr extractedextracted withwith KHKH22POPO44,, withwith anan averageaverageof 3.4 valuevalue mg/kg, while the Cr(VI) extracted with KH2PO4 (ECr(VI)) remains lower (2.3 mg/kg). In the case WhenWhen the the fresh fresh tailing tailing sample sample is is incubated incubated with withA. A. thiooxidans thiooxidans,, there there is is a a release release of of Al Al starting starting ofof 3.43.4 mg/kg,mg/kg, whilewhile thethe Cr(VI)Cr(VI) extractedextracted withwith KHKH22POPO44 (E(ECr(VI)Cr(VI))) remainsremains lowerlower (2.3(2.3 mg/kg).mg/kg). InIn thethe casecase fromfrom 302 302 to to 1784 1784 mg/kg, mg/kg, from from 725 725 to to 1826 1826 mg/kg, mg/kg, and and from from 239 239 to to 1274 1274 mg/kg, mg/kg, for for the the pulp pulp densities densities

of 5, 10, and 30 g/L, respectively. This is due to the production of H2SO4 by A. thiooxidans and the of 5, 10, and 30 g/L, respectively. This is due to the production of H2SO4 by A. thiooxidans and the

dissolutiondissolution of of amorphous amorphous mineral mineral phases phases containing containing Al. Al. Chromite Chromite tailings tailings of of a a beneﬁciation beneficiation plant plant in in India were characterized by Tripathy et al. [3] and they found kaolinite (Al2Si2O5(OH)4), in addition

Minerals 2018, 8, 261 13 of 23 Minerals 2018, 8, x FOR PEER REVIEW 13 of 23 MineralsMineralsMinerals 201201 20188,8, 8,8 ,8, x,x x FORFOR FOR PEERPEER PEER REVIEWREVIEW REVIEW 131313 ofof of 2323 23 Indiato the were Al mineral characterized phases, by such Tripathy as gibbsite et al. [ 3and] and chlorite, they found in the kaolinite chromite (Al tailings2Si2O5(OH) used4 ),in in the addition present tototo the thethe Al AlAl mineral mineralmineral phases, phases,phases, such suchsuch as asas g ggibbsiteibbsiteibbsite and andand chlorite, chlorite,chlorite, in inin the thethe chromite chromitechromite tailings tailingstailings used usedused in inin the thethe present presentpresent towork. the Al The mineral control phases, of A. thiooxidans such as gibbsite reveals and no m chlorite,edium effect in the in chromite Al dissolution. tailings The used incubation in the present of the work.work.work. The TheThe control controlcontrol of ofof A. A.A. thiooxidans thiooxidansthiooxidans reveals revealsreveals no nono m mmediumediumedium effect effecteffect in inin Al AlAl dissolution. dissolution.dissolution. The TheThe incubation incubationincubation of ofof the thethe work.fresh Thetailing control sample of A.with thiooxidans P. putida revealsshows notably no medium less effectAl alteration in Al dissolution. compared with The incubationthe incubation of freshfreshfresh tailing tailingtailing s samplesampleample with withwith P. P.P. putida putidaputida shows showsshows notably notablynotably less lessless Al AlAl alteration alterationalteration compared comparedcompared with withwith the thethe incubation incubationincubation thewith fresh A. tailingthiooxidans sample. In addition, with P. putida Al doesshows not notablydissolve less continuously Al alteration; on compared the contrary, with two the phases incubation were withwithwith A. A.A. thiooxidans thiooxidansthiooxidans.. . In InIn addition, addition,addition, Al AlAl does doesdoes not notnot dissol dissoldissolveveve continuously continuouslycontinuously;; ; o oonnn the thethe contrary, contrary,contrary, two twotwo phases phasesphases w wwereereere withidentified.A. thiooxidans An initial. In addition,Al precipitation, Al does notwith dissolve an incubation continuously; period onthat the lasts contrary, approximately two phases 10 were days, identified.identified.identified. An AnAn initial initialinitial Al AlAl precipitation, precipitation,precipitation, with withwith an anan incubation incubationincubation period periodperiod that thatthat lasts lastslasts approximately approximatelyapproximately 10 1010 days daysdays,, , identiﬁed.was noted, An followed initial Al by precipitation, Al dissolution with in the an time incubation remaining. period In the that first lasts phase approximately, 50% of Al precipitates 10 days, was for waswaswas noted, noted,noted, followed followedfollowed by byby Al AlAl dissolution dissolutiondissolution in inin the thethe time timetime remaining.remaining. remaining. In InIn the thethe first firstfirst phasephase phase,, , 50% 50%50% of ofof Al AlAl precipitatesprecipitates precipitates for forfor noted,the pulp followed density by at Al 5dissolution g/L, 64% for in 10 the g/L time, and remaining. 73% for In30 theg/L. ﬁrst In the phase, secon 50%d phase, of Al precipitates Al is dissolved for thethethe pulp pulppulp densitythe densitydensity pulp at atat density 5 55 g/L, g/L,g/L, at64% 64%64% 5 g/L, for forfor 10 10 64%10 g/L g/Lg/L for,, , and andand 10 g/L,73% 73%73% for andforfor 30 3030 73% g/L. g/L.g/L. for In InIn 30 the thethe g/L. secon seconsecon Ind thedd phase, phase,phase, second Al AlAl phase, is isis dissolved dissolveddissolved Al is dissolved again (Figureagain 8). (Figure 8). againagain ((FFigureigureagain 88 (Figure)).. 8).

0.6 0.60.60.6 (a) Blank (a)(a)(a) BlankBlankBlank 0.5 0.50.50.5 0.4 0.40.40.4 0.3 0.30.30.3

Al (mg/kg) Al 0.2 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al 0.20.20.2 Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled Distilled water (pH 7) (pH water Distilled 0.1 0.10.10.1 0.0 0.00.00.0 2000 2000 14 (b) 20002000Control (c) 141414 (b)(b)(b) ControlControlControl (c)(c)(c) 12 1212 12 1500 1500 10 15001500 101010 8 8 1000 88 100010001000 6

666 (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al 4 500 444 500500500 A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. A. thiooxidans2)(pH A. 2 222 0 0 000 000 50 50 505050 (d) 5050Control50 (d)(d)(d) (d) Control Control (e) (d)(d)(d) ControlControlControl (e)(e)(e) 40 40 404040 404040 30 30 303030 303030 20 20 202020 (mg/kg) Al 202020 Al (mg/kg) Al Al (mg/kg) Al Al (mg/kg) Al P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. P. putida (pH 9)(pH putida P. 10 10 101010 101010 0 0 000 000 0 10 20 30 40 0 10 20 30 40 000 101010 202020 303030 404040 000 101010 202020 303030 404040 Time (days) Time (days) TimeTimeTime (days)(days) (days) TimeTimeTime (days) (days)(days) Figure 8. Al concentrations in the leachate as a function of time during (bio)leaching experiments of FigureFigureFigure 8. 8.8. Al AlFigureAl concentrations concentrationsconcentrations 8. Al concentrations in inin the thethe leachate leachateleachate in the a leachateaass s a aa function functionfunction as a functionof ofof time timetime during duringofduring time (bio)leaching (bio)leachingduring(bio)leaching (bio)leaching experiments experimentsexperiments experiments of ofof of the the fresh tailing sample with distilled water (a), incubated with A. thiooxidans (c) and P. putida (e). thethethe fresh freshfresh tailing tailingfreshtailing tailing sample samplesample sample with withwith withdistilled distilleddistilled distilled water waterwater water ( (a(aa),),), incubated (incubatedincubateda), incubated with withwith with A. A.A. thiooxidans A.thiooxidansthiooxidans thiooxidans ( (c(c)c) () andc andand) and P. P.P. P. putida putidaputida putida ( (e(e().e).).). Controls Controls for A. thiooxidans and P. putida are displayed in (b,d), respectively. Three pulp densities were ControlControlControlss s forfor forfor A.A. A. thiooxidansthiooxidans thiooxidans andand and P.P. P. putidaputida putida areare areare displayeddisplayed displayeddisplayed inin in (( b(bb,,d,d,d),),), respectively.respectively. respectively.respectively. Three Three Three pulp pulp densitiesdensities densities werewere were evaluated: evaluated: 5 g/L ( ), 10 g/L ( ), and 30 g/L ( ). evaluated:evaluated:evaluated:5 5 5 g/L5 g/L g/Lg/L ( (( ),),),), 1010 1010 g/L g/L g/Lg/L ( (( )),),), , and andand 30 3030 g/L g/Lg/L (( ( ).).).). 3.4. Chemical Extraction 3.4.3.4.3.4. Chemical ChemicalChemical3.4. Extraction ChemicalExtractionExtraction Extraction

The chemically exchangeable pool of hexavalent chromium (ECr(VI)) for fresh and stockpiled old TTThehehe chemicall chemicallchemicallThe chemicallyyyy exchangeable exchangeableexchangeable exchangeable pool poolpool of ofof hexavalent hexavalenthexavalent pool of hexavalent chromium chromiumchromium chromium (E (E(ECr(VI)Cr(VI)Cr(VI))) ) for forfor (E Cr(VI)fresh freshfresh) and and forand fresh stockpiled stockpiledstockpiled and stockpiled old oldold old tailing and chromite ore, in mg of Cr(VI) per kg of tailing, is presented in Table 2. For the chromite tailingtailingtailing and andandtailing chromite chromitechromite and chromite ore, ore,ore, in inin mg mgmg ore, of ofof in Cr(VI) Cr(VI)Cr(VI) mg of per perper Cr(VI) kg kgkg of of perof tailing, tailing,tailing, kg of tailing,is isis presented presentedpresented is presented in inin Table TableTable in 2. Table2.2. For ForFor2 the .thethe For chromite chromitechromite the chromite ore ore samples, the total Cr content in the solid ranges between 203 and 236 g/kg. For the chromite ore 1, oreoreore samples samplessamplessamples,,, , the thethe total totaltotal the Cr CrCr total content contentcontent Cr content in inin the thethe solid insolidsolid the ranges rangesranges solid rangesbetween betweenbetween between 203 203203 and andand 203 236 236236 and g/kg. g/kg.g/kg. 236 For ForFor g/kg. the thethe chromite chromitechromite For the chromiteore oreore 1 11,, , ore 1, Cr(VI) extracted with water is equivalent to total Cr extracted with KH2PO4, with an average value Cr(VCr(VCr(VI)I)I) extracted extractedextractedCr(VI) extracted with withwith water waterwater with is isis waterequivalent equivalentequivalent is equivalent to toto total totaltotal Cr toCrCr total extracted extractedextracted Cr extracted with withwith KH KHKH with22PO2POPO KH44,4, , with withwith2PO an4 anan, with average averageaverage an average value valuevalue value of of 3.4 mg/kg, while the Cr(VI) extracted with KH2PO4 (ECr(VI)) remains lower (2.3 mg/kg). In the case ofofof 3.4 3.43.4 mg/kg, mg/kg,mg/kg, while whilewhile the thethe Cr(VI) Cr(VI)Cr(VI) extracted extractedextracted with withwith KH KHKH22PO2POPO44 4 (E (E(ECr(VI)Cr(VI)Cr(VI))) ) remains remainsremains lower lowerlower (2.3 (2.3(2.3 mg/kg). mg/kg).mg/kg). In InIn the thethe case casecase

Minerals 2018, 8, 261 14 of 23

3.4 mg/kg, while the Cr(VI) extracted with KH2PO4 (ECr(VI)) remains lower (2.3 mg/kg). In the case of chromite ore 2, the Cr(VI) extracted with water and KH2PO4 has the same value (1.6 mg/kg) and corresponds to 50% of the total Cr extracted with KH2PO4.

Table 2. Results of chemical extraction for the tailings and chromite ore sample.

Total Cr Cr(VI) Water Cr(VI) KH PO Cr(Tot.) KH PO Sample 2 4 2 4 (g/kg) (mg/kg) (mg/kg) (mg/kg) Chromite ore 1 203 3.5 2.3 3.4 Chromite ore 2 236 1.6 1.6 3.1 Fresh tailing 96 63 223 240 7–10 cm 99 36 149 158 Old tailing 15–20 cm 105 19 85 89 >30 cm 101 3.7 31 31

In the fresh tailing sample, the total Cr content in the solid (96 mg/kg) is approximately 50% of the total Cr contained in the chromite ore sample. However, the amount of Cr(VI) extracted with KH2PO4 (ECr(VI)) (223 mg/kg) is 100 times higher compared with the chromite ore sample. In the fresh tailings sample, Cr(VI) extracted with KH2PO4 corresponds to 93% of the total chromium extracted with KH2PO4. Total chromium in the stockpiled old tailing sample does not change signiﬁcantly with the depth (102 ± 3 g/kg); however, Cr(VI) extracted with KH2PO4 (ECr(VI)) decreased from the top to the bottom. For the Cr(VI) extracted with KH2PO4 (ECr(VI)), the range of variation goes from 36 and 149 mg/kg at the surface to 3.7 and 31 mg/kg in the deepest layer for extraction with water and KH2PO4, respectively. Between 94 and 100% of extracted Cr with KH2PO4 is present as a Cr(VI).

4. Discussion

4.1. Cr Leachability and Interaction with Other Ions Among the main chromium bearing phases in chromite tailings, iron and aluminum oxides play an important role in Cr mobility and leachability [7]. In leaching experiments of soils derived from chromium ore processing residue (CORP), Weng et al. [42] found that Cr(VI) was not detected in the leachate (pH < 2.5), because of the adsorption of Cr(VI) onto soil surfaces or the reduction of Cr(VI) to insoluble Cr(III). Significant amounts of Cr(VI) were leached between pH 4.5–12. Accordingly, Liu et al. [64] reported an optimal pH between 3 and 4 for hexavalent chromium leaching of chromium slag. Besides the importance of pH in the presence of Cr(III) and/or Cr(VI) in the dissolved fraction, pH also impacts the dominance of oxyanions species. According to visual MINTEQ simulation results, Cr precipitates during the leaching experiments and the Cr remaining in solution is mainly present as Cr(VI) oxyanions (Table S1). For leaching with P. putida growth medium (control) at a neutral pH, 2− − CrO4 and HCrO4 are the main Cr forms in the leachate, accounting for about 86% of total dissolved forms. The dominance of those species remains over time (Table S2). When P. putida is inoculated, 2− the pH increases up to 9.6 and CrO4 represents 93% of the dissolved chromium. This occurs for all the pulp densities evaluated. In addition, visual MINTEQ simulation results revealed that at pH 3.5 − (control of A. thiooxidans), HCrO4 dominates (94%), and at pH 1.6, when A. thiooxidans is inoculated, − 2− HCrO4 decreases (83%), while CrO3SO4 represents 13% of dissolved chromium (Table S1). In the blank, when the fresh tailing sample was leached with distilled water (pH = 7), 66% of the Cr was 2− − present as a CrO4 and the 34% remaining as HCrO4 . The oxidizing conditions and the pH explain − 2− the presence of Cr(VI) as HCrO4 or CrO4 [14]. The leaching of Cr for chromite tailings can be explained by two main processes. The first main process for Cr leaching is associated with the presence of phosphate anions in solution, which can Minerals 2018, 8, 261 15 of 23 exchange with chromate and bi-chromate anions, promoting the presence of Cr(VI) [46] in solution. According to the chemical extraction performed with KH2PO4 (Table2), the chemically exchangeable pool of chromium in the fresh tailing sample is 223 mg of Cr(VI) per kg of tailing. Chemical extraction was also performed with concentrated ore and the value obtained is 100 times lower. Given that the concentrated ore is mainly composed of chromite, it can be concluded that chromite contained in the fresh tailings is not the main source of Cr(VI), at least in the short term. However, it is important to consider that in the chromite deposit, which is the raw material for the concentrated ore, serpentinization and magnesium (Mg) ion release during the deuteric alteration of ultramafic rocks (and associated laterization) creates alkaline pore water, which generates Cr(VI) formation [65]. Chromite and Cr-magnetite have also been proposed by Garnier et al. [66] as a source of Cr in ultramafic soils, in spite of r-spinel resistance to alteration. These findings are supported with XANES evidence for the oxidation of Cr(III) to Cr(VI) by Mn-oxides in a lateritic regolith in New Caledonia [67]. In other words, Cr(VI) is potentially produced in the chromite deposit, and if it is not removed during the chromite beneficiation process, it can be still present in the concentrated ore and tailings. However, given the Cr release during leaching experiments with concentrated ore, in the tailings, chromite does not seem to be the main source of Cr(VI). Consequently, Cr(VI) leached from the tailings is coming from other bearing phases, such as iron and aluminum oxides and silicates. The second main process for Cr leaching that could explain the presence of Cr in the leachate is related to redox processes. Manganese oxide containing minerals acts as a transporter of electrons between dissolved oxygen and Cr(III), and therefore, Mn indirectly oxidizes Cr(III) to Cr(VI) [7]. On the other hand, Cr(VI) can reduce to Cr(III), which is less toxic. In this process, iron(II), reduced sulfur, and organic matter are the chief sources of electrons [56]. Reduction of Cr(VI) by Fe(II) (aq) can be expressed by the following general reaction [68]:

Cr(VI) (aq) + 3Fe(II) (aq) → Cr(III) (aq) + 3Fe(III) (aq) (2)

Given the visual MINTEQ calculations, during the leaching with A. thiooxidans, under acidic 2+ conditions (pH = 1.6), ferrous iron is mainly present in solution as FeSO4 (aq) and as a free ion Fe . The presence of ferrous iron in the leachate could explain the decrease of Cr over time, because of its iron dominant role in the reduction of Cr(VI) to Cr(III), a process which is 100 times faster than a biotic process [7]. In the case of Mn, it is mainly present as Mn2+, which is a non-stable species, and it does not play a role in Cr(III) oxidation. At an acidic pH, Cr(III) precipitation is not expected to occur, as is the case at a neutral pH, so one can expect to see the precipitation of Cr(III) produced after Cr(VI) reduction in leaching with distilled water and P. putida [69]. In the batch experiments with P. putida, due to pH (6.6–9.6) and oxidizing conditions, Fe is present in solution at a really low concentration, forming FeHPO4. A low content of Fe indicates that no significant colloidal transport of particles occurred [70]. Other elements of interest leached from the fresh tailing sample are Ni and Al. Given visual MINTEQ results, in the presence of P. putida medium (control), Ni is complexed by phosphates, which are concentrated in the growth medium. In total, 77% is complexed as NiHPO4 (aq), 14% is present as 2+ Ni , and 5% as NiSO4 (aq). When incubating the fresh tailing sample with P. putida, NiHPO4 remains dominant until day 4, when the pH reaches a value of 8.9. However, the diversification of Ni species increases over time, and at the end of the leaching period, Ni complexed with ammonia becomes 2+ 2+ 2+ dominant. Those species include Ni(NH3)2 (31%), Ni(NH3)3 (18%), and NiNH3 (17%). When incubating the fresh tailing sample with A. thiooxidans at pH 2, Ni is distributed as 68% Ni2+ and 31% NiSO4 (aq) (Table S2). The main Ni species in solution, according to visual MINTEQ results, shows its strong affinity with sulfur, nitrogen, and phosphorus compounds. A negative significant correlation between Cr and Ni (R2 = 0.8) in the leachate is observed for all pulp densities during the leaching experiment with A. thiooxidans (pH = 2). For Al, the presence of KH2PO4 and K2HPO4 in the P. putida growth medium induces the + formation of AlHPO4 as the dominant (97%) species in solution. When P. putida was inoculated at Minerals 2018, 8, 261 16 of 23 the beginning of the leaching experiment, when the pH was lower than 8, Al was present as three − + main species, including Al(OH)3, Al(OH)4 , and AlHPO4 , representing 3.4%, 49.4%, and 46.5%, respectively. After three days, Al started precipitating due to the high pH, which varied from 8.9 to 9.6 between day 4 and 34. Visual MINTEQ simulation results predict the precipitation of Al as Gibbsite (Al(OH)3) and kaolinite (Al2Si2O5(OH)4). Under more acidic conditions (leaching with the A. thiooxidans medium), with pH = 4, during the + + whole experiment, Al distribution did not change. The main Al species are AlHPO4 (50%), AlSO4 2− (25%), and AlSO4 (22%). Precipitation of Variscite (AlPO4 ·2H2O) is expected according to the thermodynamical modeling results (Table S3). During incubation of the fresh tailings sample with + 2− A. thiooxidans, at pH 2, aluminum sulfate complexes (AlSO4 and AlSO4 ) became the dominant species throughout the leaching period, accounting for about 90% of the dissolved forms. Due to the acidic conditions, no precipitation occurs. In the leaching experiment with distilled water, Al is mainly − present as Al(OH)3(aq) and Al(OH)4 ; however, part of the aluminum hydroxide could precipitate as a Gibbsite. A significant negative correlation between Cr and Al (R2 = 0.8) in the leachate is observed for all pulp densities during the leaching experiment with A. thiooxidans (pH = 2).

4.2. Bacterial Effect on Chromite Tailings Leachability In the present work, two bacterial strains, A. thiooxidans and P. putida, were used for acidic and alkaline leaching conditions, respectively. In the previous section, the leaching of chromite tailings was discussed without including the possible bacterial effects. However, the differences in element concentrations observed over time (Figures4–8) between blanks, controls, and batch experiments inoculated with both strains, indicate that bacteria may play an important role in tailings leachability, other than as a simple pH controller. Despite Schindler et al. [11] finding no direct bacterial effect on the release of Cr from altered chromatites, they affirmed that the presence of bacteria in tailings, mine waste piles, and soils may control pH and Eh and thus indirectly control Cr redox chemistry. Under this premise, the role of bacteria on Fe and Cr mobility, as the most abundant compounds on tailings, was selected to discuss this point. Initially, the only role of A. thiooxidans on Fe leaching was considered to be associated with the production of sulfuric acid, through the oxidation of both elemental sulfur and sulfide. However, Marreno et al. [32] demonstrated that A. thiooxidans is able to mediate reductive dissolution of laterite overburden coupled to the acidolysis under aerobic conditions. This chemolithoautotrophic bacterium has also been used to leach iron from low grade cobalt laterite [71], tailings [40,72], and laterite overburden [32]. As shown in Figure4c, in leaching experiments with A. thiooxidans, Fe is permanently leached for all pulp densities, with higher Fe concentrations for 5 and 10 g/L. Considering that the increase in total iron concentration does not evidence a bacterial effect, batch experiments inoculated with A. thiooxidans were compared with batch experiment with distilled water acidified at pH 2 with 1 N H2SO4 for a pulp density of 30 g/L (Figure S3b). In the batch experiment with acidified distilled water (pH 2), for a pulp density of 30 g/L, Fe extracted reached a total of 479 mg/kg in 11 days, while at the same pulp density, the batch experiment inoculated with A. thiooxidans total Fe only reached 213 mg/kg. Nevertheless, when fresh chromite tailings leaching with A. thiooxidans was performed with a pulp density of 5 and 10 g/L, the total amount of Fe extracted was 834 and 1108 mg/kg, respectively. This could suggest an inhibitory effect of A. thiooxidans at a pulp density of 30 g/L due to the release of toxic elements in the leachate such as Cr (946 µg/L) and Ni (843 µg/L). Jang and Valix [73] reported a bacteriostatic effect of Ni and Cu on A. thiooxidans for the leaching of saprolitic Ni laterite ores. The amount of total chromium extracted in the batch experiment with A. thiooxidans decreases over time. It has been reported that A. thiooxidans, A. ferrooxidans, and Thiobacillus thioparus are capable of Cr(VI) reduction when they are growing on sulfur compounds due to the production of a series of sulfur compounds with a high reducing power [28,74]. According to Viera et al. [74], in A. thiooxidans cultures, reduced glutathione is a required intermediate for the oxidation of elemental sulfur. The Minerals 2018, 8, 261 17 of 23 polysulfide formed is then successively oxidized to different compounds like sulfite, thiosulfate, and finally sulfate. Sulfite and polythionates could be responsible for reductive reactions. In addition, reducing compounds associated with sulfur particles (less than 3 µm) could promote Cr reduction in A. thiooxidans cultures [75]. Steudel [76] found that colloidal sulfur in cultures of Acidithiobacillus would be present as long-chain polythionates, forming micelles of globules of up to a few µm. Later, Viera et al. [74] observed that at pH 2 and 4, A. thiooxidans cultures reached higher free bacterial populations but lower chromium reduction values. Thus, the higher the pH, the higher the amount of reducing compounds associated with the colloidal sulfur and cells. Taking into consideration that in 3− the present work the Cr(VI) leached out due to the presence of PO4 in the medium, the Cr decrease could be explained by the reduction of Cr(VI) to Cr(III) mediated by the presence of A. thiooxidans and the low pH. During the incubation with P. putida, the pH is above 8, where Fe does exist at very low concentrations in solution. Siderophores (Figure S4a) produced by P. putida during growth in low-iron conditions solubilize and bind iron, and transport it back into the microbial cell, usually through siderophore specific membrane receptors [52]. In spite of the presence of Fe in the leachate, the Fe extracted remains considerably low (0.73–76 mg/kg) compared with the incubation with A. thiooxidans. The cell growth of P. putida was monitored with the total protein content in the leachate (Figure S4b). In the first two days, the protein content doubles for all pulp densities with a lower protein content for a higher pulp density. P. putida has a high affinity for goethite. The bacterial adhesion increases with K+ concentration and pH (2–7) [77]. Given the fact that goethite is one of the main mineral phases on the tailings studied, adsorption of P. putida on the solids is highly possible, which represents a limitation of its effect in the liquid phase. Other mineral phases that are positively charged or where the electrostatic repulsion force between them and the bacteria is overcome, could adsorb bacteria as well [77]. The lower leachability of the metals (Fe, Cr, Ni, Mn, Al) in the presence of P. putida at 30 g/L, compared to 5 and 10 g/L, suggests an effect of the pulp density on the culture growth. To establish the role of P. putida in the leachability of fresh chromite tailings, a batch experiment with distilled water at pH 9 (adjusted with 1 N NaOH) was carried out (Figure S3). Total Fe concentration on the leachate of the distilled water at pH 9 reaches 1.4 mg/kg after 262 h of leaching, while in the batch experiment with P. putida, the Fe extracted is higher (3.5 mg/kg) for the same pulp density. For Cr, there is only a 9 mg/kg difference between the extraction with distilled water at pH 9 and the batch experiment with P. putida, being higher for the last one. Reduction of hexavalent chromium by Pseudomonas dechromaticans, isolated from industrial sewage, has been reported, as it uses the chromate or dichromate as a terminal acceptor during anaerobic respiration. Under aerobic conditions, P. fluorescens uses a variety of electron acceptors for chromate reduction [45]. Additionally, microbial metabolite extracellular polymeric substances (EPS) enhance Cr(VI) reduction efficiency and form organo-Cr(III) complexes to protect the cell and chromate reductase from inactivation [78]. 2− 2− 2− 2− 3− − Nevertheless, the presence of anions such as SO4 , SO3 , MoO4 , VO4 , PO4 , and NO3 affects cellular chromate sensitivity [79]. Given the Cr(VI) reduction mechanisms by Pseudomonas spp. and the decrease of Cr concentration over time during incubation of the fresh tailing sample with P. putida, the reduction of Cr(VI) to Cr(III) is likely to happen [44,45]. Besides, the Cr decrease in the leachate is accompanied by an increase in protein and siderophore content, which ratifies the role of P. putida in Cr(VI) reduction. Nonliving biomass can also reduce Cr(VI) to Cr(III) through two different mechanisms. In the first case, Cr(VI) is directly reduced to Cr(III) in the aqueous phase by contact with electron-donor groups of the biomass, and the second consists of three steps: (a) binding of anionic Cr(VI) to the positively charged groups on the biomass surface; (b) reduction of Cr(VI) to Cr(III) by adjacent electron-donor groups; and (c) release of the aqueous phase due to electronic repulsion [79]. In the present work, A. thiooxidans bacteria have been shown to have a potential impact on chromite tailings leachability. Under aerobic conditions, A. thiooxidans promote Fe reduction, and acidic conditions associate with the production of sulfuric acid, which promotes heavy metals dissolution. Minerals 2018, 8, 261 18 of 23

On the other hand, P. putida plays an important role in iron dissolution thanks to the production of siderophores, but the inﬂuence on Cr leaching is negligible. The role of those types of bacteria could be seen not only as a potential pollution factor, but also as a remediation pathway, as well as the possible opportunity to recover leached metal from residues (tailings).

4.3. Does Chromite Tailing Represent an Environmental Risk? From previous sections, it is concluded that due to chemical and/or biological processes, chromite tailings can release elements such as Fe, Mn, Ni, and Cr. Even if it has to be considered that some of the batch experiments do not represent the field conditions, previous studies have shown that the exposure of tailings to air, oxidation, and climatic conditions favored the release of heavy metals which are often a threat to the environment [80,81]. Meck et al. [82] studied the impact of mine dumps, including chromite mines, on river water quality. Based on the fact that the effluents were not acidic, this study concluded that chromite dumps do not bring major risks to river water quality. However, water bodies in the surrounding chromite mine areas are known to be impacted, in particular due to the presence of Cr(VI), which is toxic and highly mobile [1,36,38,83]. The extent and degree of heavy metal release around the mines vary depending on the geochemical characteristics and the mineralization degree of tailings [65]. The environmental risk is directly related to the mineralogical composition of chromite tailings and its susceptibility to release toxic elements into the environment. Typically, chromite ore process tailings contain a heterogeneous mixture of iron and chromium oxides, silicates, and aluminum silicate minerals [6]. In the tailings studies, mineral phases included chromite, which is the main Cr mineral on earth. In order to rule out chromite as a source of dissolved Cr, a leaching experiment with concentrated ore (product after the beneficiation plant) at pH 2, and a pulp density of 30 g/L, was performed (Figure S5). The results show three fold less Cr in the leachate of the concentrated ore sample (47 mg/kg) compared with the leachate of the fresh tailing sample (147 mg/kg), suggesting that chromite is not the main source of Cr release from the tailings. Chromite nanoparticles can be released as a consequence of silicate weathering; however, the production of Cr(VI) first requires a dissolution of the nanoparticle [10]. Other mineral phases such as iron and aluminum oxides are able to adsorb chromium, especially Cr(VI) anions. In the ring of Fire (Canada), various Cr bearing minerals occur such as clinochlore, phlogopite, amphibole, and clinopyroxene, with traces of Cr occurring in orthopyroxene, olivine, serpentine, and carbonates. Preliminary leaching tests indicated that Cr(VI) can be generated from those minerals in the presence of birnessite (Mn-containing mineral) [9]. In the present study area, birnessite was not detected. Fresh and old tailings were compared in order to evaluate the potential release of Cr(VI) over time (Table2). Chemical extraction with KH 2PO4 showed that 93% of total Cr extracted with KH2PO4 is present as Cr(VI), with a total concentration of 223 mg/kg. In superficial tailings collected in a stockpile (old tailings), 94% of the total Cr extracted is present as Cr(VI), which has a concentration of 158 mg/kg. The proportion of Cr(VI) compared with total Cr does not change with the depth; however, the Cr(VI) concentration decreases from 158 mg/kg in the first 10 cm to 31 mg/kg at 30 cm. These results evidence that the chemically exchangeable pool of Cr(VI) is higher in the fresh tailing sample compared with the stockpiled old tailing sample, and in the stockpiled old tailing sample, the pool of Cr(VI) decreases with depth. Stockpiled chromite tailings are susceptible to storing CO2 within the structures of minerals, a process known as natural carbonation, which is enhanced in mine tailings by a dramatic increase in mineral surface area from crushing during ore processing [84]. During accelerated mineral carbonation, Mg-carbonate mineral and Fe-oxyhydroxide phases sequester transition metals. Hamilton et al. [85] demonstrated that upon precipitation, MgCO3·3H2O (a common product of mineral carbonation at Earth’s surface conditions) rapidly sequesters transition metals (Cr, Ni, Mn, Co, and Cu) in solution. The trace metal uptake appears to occur by substitution of Mg2+ in the crystal structure, and also by incorporation into minor, metal-rich phases, such as Fe-oxyhydroxides. Additionally, the natural content of organic matter (NOM) in mine tailings exposed over long periods to atmospheric conditions Minerals 2018, 8, 261 19 of 23 increases over time. The NOM can directly affect the extraction of metals, reducing the production of oxidizing agents required for metal dissolution [86]. The natural carbonation and the NOM enrichment in old tailings could explain the lower exchangeable pool of Cr(VI), compared with fresh tailings, where the content of organic matter is negligible [87]. Mg-carbonate and hydromagnesite are common weathering products of serpentine minerals, such as lizardite, which is present in the stockpiled old tailing sample in all depths. Further analyses have to be done to understand the possible role of new secondary phases, NOM, and the passive carbonation in Cr(VI) mobility on chromite tailings. A broad estimation of the exchangeable pool of total Cr and Cr(VI) release from chromite tailings (for an average mine) could be estimated based on the average production between 2016 and 2017 in all the chromite mines in Sukinda valley [35] (Equation (3)).

kg mg Cr(VI) kg Cr(VI) = E × Tailings produced × 10−6 (3) yr Cr(VI) kg tailings yr

5 A total of 37.28 × 10 tons/year with a feed grade of Cr2O3 (dry) is processed on the beneﬁciation plant, and 4.72 × 105 tons/year of concentrated ore is produced (~50%), together with 5 × 105 tons/year (tailings produced) of tailings containing ~15% of Cr2O3 [35]. If the chemically exchangeable pool of Cr(VI) (ECr(VI)) in fresh tailings is considered, potentially 111,500 kg of Cr(VI) could be released per year. If ECr(VI) in the surface of stockpiled old tailings is considered, the Cr(VI) production decreases to 74,500 kg per year. This estimation suggests that the production and storage of chromite tailings could represent a risk for the surrounding soils and especially the water bodies, which could be severely affected by the presence of Cr(VI). Given the results of the batch experiments of this study, we can conclude that chromite is not the main source of Cr(VI) for tailings; however, other Cr bearing phases more susceptible to alteration could represent an important source of Cr(VI) in the environment. However, batch experiments with A. thiooxidans and P. putida, under the conditions studied, showed that the reduction of Cr(VI) to Cr(III) is a dominant process that is mediated by Fe and bacteria. In addition, the absence of Mn containing minerals such as birnessite, limits the oxidation of Cr(III) to Cr(VI). These results suggest the important role of bacteria in reducing the toxic hexavalent chromium, as a natural mechanism in stockpiled tailings.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-163X/8/6/261/s1. Figure S1: Dissolved oxygen evolution throughout the incubation of the fresh tailing sample with P. putida at different pulp densities, Figure S2: Total Fe, Fe(II) and Fe(III) evolution throughout the leaching of the fresh tailing sample with acidified (H2SO4) MQ water for a pulp density of 30 g/L, Figure S3: Cr and Fe extracted from the fresh tailing sample, after leaching with MQ water at pH 7, pH 2 with H2SO4 and at pH 9 with NaOH, Figure S4: Siderophores content under incubation of the fresh tailing sample with P. putida and protein content in the leached, Figure S5: Cr and Fe extracted from the concentrated ores sample, after leaching with MQ water at pH 7 and pH 2 with H2SO4, Table S1: Species distribution obtained in visual MINTEQ simulation, for the control and the leaching of the tailing sample with A. thiooxidans for a pulp density of 5 g/L, Table S2: Species distribution obtained in visual MINTEQ simulation, for the blank, the control and the leaching of the tailing sample with P. putida. Results are presented for a pulp density of 5 g/L and three subsamples collected at 0, 5 and 34 days in the batch with P. putida, Table S3: Ion Activity Product (IAP) and saturation index (SI) of MNHPO4 and variscite, obtained in visual MINTEQ simulation, for the control and the leaching of the tailing sample with A. thiooxidans. Results are presented for two pulp densities (5 and 10 g/L) and three subsamples collected at 0, 5 and 34 days. Author Contributions: Conceptualization, J.v.d.V. and S.S.; Methodology, E.D.v.H.; Formal Analysis and Original Draft Preparation, V.B.-B.; Solid sample characterization, C.Q. Supervision, E.D.v.H., Y.S. and P.N.L.L. Funding: This research was funded by Erasmus Mundus Join Doctorate Program ETeCoS3 (Environmental Technologies for Contaminated Solids, Soils and Sediments) grant agreement FPA No 2010-0009. The field trip was funded by the CEFIPRA Joint Research Project: “Assessment of chromium release from Sukinda mining overburden: an isotopic, chemical, physical and biological study”. Acknowledgments: The authors thank the European Commission (EC) for providing financial support through the Erasmus Mundus Join Doctorate Program ETeCoS3 (Environmental Technologies for Contaminated Solids, Soils and Sediments, grant agreement FPA No 2010-0009). This work was carried out as part of the CEFIPRA Joint Research Project: “Assessment of chromium release from Sukinda mining overburden: an isotopic, chemical, physical and biological study”. We greatly thank Sophie Novak for XRF acquisition of the Paris Diderot Chemistry Department. Minerals 2018, 8, 261 20 of 23

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Dhakate, R.; Singh, V.S.; Hodlur, G.K. Impact assessment of chromite mining on groundwater through simulation modeling study in Sukinda chromite mining area, Orissa, India. J. Hazard. Mater. 2008, 160, 535–547. [CrossRef][PubMed] 2. Murthy, Y.R.; Tripathy, S.K.; Kumar, C.R. Chrome ore beneficiation challenges & opportunities—A review. Miner. Eng. 2011, 24, 375–380. [CrossRef] 3. Tripathy, S.K.; Murthy, Y.R.; Singh, V. Characterisation and separation studies of Indian chromite beneficiation plant tailing. Int. J. Miner. Process. 2013, 122, 47–53. [CrossRef] 4. Kumar, C.R.; Tripathy, S.; Rao, D.S. Characterisation and Pre-concentration of Chromite Values from Plant Tailings Using Floatex Density Separator. J. Miner. Mater. Charact. Eng. Raghu Kumar Sunil Tripathy 2009, 88, 367–378. [CrossRef] 5. Çiçek, T.; Cöcen, I.; Engin, V.T.; Cengizler, H.; ¸Sen,S. Technical and economical applicability study of centrifugal force gravity separator (MGS) to Kef chromite concentration plant. Trans. Inst. Min. Metall. Sect. C Miner. Process. Extr. Metall. 2008, 117, 248–255. [CrossRef] 6. Dwari, R.K.; Angadi, S.I.; Tripathy, S.K. Studies on flocculation characteristics of chromite’s ore process tailing: Effect of flocculants ionicity and molecular mass. Colloids Surfaces A Physicochem. Eng. Asp. 2018, 537, 467–477. [CrossRef] 7. Choppala, G.; Bolan, N.; Park, J.H. Chromium Contamination and Its Risk Management in Complex Environmental Settings; Elsevier: New York, NY, USA, 2013; Volume 120, ISBN 9780124076860. 8. Sueker, J.K. Chromium. In Environmental Forensics: Contaminant Specific Guide; Murphy, B., Ed.; Academic Press: Burlington, NJ, USA, 1964; pp. 82–93, ISBN 978-0-12-507751-4. 9. Beukes, J.P.; du Preez, S.P.; van Zyl, P.G.; Paktunc, D.; Fabritius, T.; Päätalo, M.; Cramer, M. Review of Cr(VI) environmental practices in the chromite mining and smelting industry—Relevance to development of the Ring of Fire, Canada. J. Clean. Prod. 2017, 165, 874–889. [CrossRef] 10. Schindler, M.; Berti, D.; Hochella, M.F. Previously unknown mineral-nanomineral relationships with important environmental consequences: The case of chromium release from dissolving silicate minerals. Am. Miner. 2017, 102, 2142–2145. [CrossRef] 11. Schindler, M.; Lussier, A.J.; Principe, E.; Mykytczuk, N. Dissolution mechanisms of chromitite: Understanding the release and fate of chromium in the environment. Am. Miner. 2018, 103, 271–283. [CrossRef] 12. Zachara, J.M.; Ainsworth, C.C.; Cowan, C.E.; Resch, C.T. Adsorption of Chromate by Subsurface Soil Horizons. Soil Sci. Soc. Am. J. 1989, 53, 418–428. [CrossRef] 13. Salem, F.Y.; Parkerton, T.F.; Lewis, R.V.; Huang, J.H.; Dickson, K.L. Kinetics of chromium transformations in the environment. Sci. Total Environ. 1989, 86, 25–41. [CrossRef] 14. Stanin, F. The transport and fate of Cr(VI) in the Environment. In Chromium (VI) Handbook; CRC Press: Boca Raton, FL, USA, 2004; pp. 161–204, ISBN 9788276555. 15. Osaki, S. The effect of organic matter and colloidal particles on the determination of chromium (VI) in natural waters. Talanta 1983, 30, 523–526. [CrossRef] 16. Thacher, R.; Hsu, L.; Ravindran, V.; Nealson, K.H. Modeling the transport and bioreduction of hexavalent chromium in aquifers: Influence of natural organic matter. Chem. Eng. Sci. 2015, 138, 552–565. [CrossRef] 17. Kaprara, E.; Kazakis, N.; Simeonidis, K.; Coles, S.; Zouboulis, A.I.; Samaras, P.; Mitrakas, M. Occurrence of Cr(VI) in drinking water of Greece and relation to the geological background. J. Hazard. Mater. 2015, 281, 2–11. [CrossRef][PubMed] 18. Mcclain, C.N.; Maher, K. Chromium fluxes and speciation in ultramafic catchments and global rivers. Chem. Geol. 2016, 426, 135–157. [CrossRef] 19. Shahid, M.; Shamshad, S.; Rafiq, M.; Khalid, S.; Bibi, I.; Niazi, N.K.; Dumat, C.; Rashid, M.I. Chromium speciation, bioavailability, uptake, toxicity and detoxification in soil-plant system: A review. Chemosphere 2017, 178, 513–533. [CrossRef][PubMed] Minerals 2018, 8, 261 21 of 23

20. Watts, M.P.; Coker, V.S.; Parry, S.A.; Pattrick, R.A.D.; Thomas, R.A.P.; Kalin, R.; Lloyd, J.R. Biogenic nano-magnetite and nano-zero valent iron treatment of alkaline Cr(VI) leachate and chromite ore processing residue. Appl. Geochem. 2015, 54, 27–42. [CrossRef][PubMed] 21. Tokunaga, T.K.; Wan, J.; Firestone, M.K.; Hazen, T.C.; Olson, K.R.; Herman, D.J.; Sutton, S.R.; Lanzirotti, A. In situ reduction of chromium(VI) in heavily contaminated soils through organic carbon amendment. J. Environ. Qual. 1999, 32, 1641–1649. [CrossRef] 22. Hamdan, S.S.; El-Naas, M.H. Characterization of the removal of Chromium(VI) from groundwater by electrocoagulation. J. Ind. Eng. Chem. 2014, 20, 2775–2781. [CrossRef] 23. Zaitseva, N.; Zaitsev, V.; Walcarius, A. Chromium(VI) removal via reduction–sorption on bi-functional silica adsorbents. J. Hazard. Mater. 2013, 250–251, 454–461. [CrossRef][PubMed] 24. Tiwary, R.K.; Dhakate, R.; Ananda Rao, V.; Singh, V.S. Assessment and prediction of contaminant migration in ground water from chromite waste dump. Environ. Geol. 2005, 48, 420–429. [CrossRef] 25. Godgul, G. Chromium, its Analytical Techniques, Geogenic Contamination and Anthropogenic Pollution. Ph.D. Thesis, Submitted to Indian Institute of Technology (IIT) Bombay, Maharashtra, India, 1994; 194p. 26. Ledin, M.; Pedersen, K. The environmental impact of mine wastes—Roles of microorganisms and their significance in treatment of mine wastes. Earth-Sci. Rev. 1996, 41, 67–108. [CrossRef] 27. Das, S.; Mishra, J.; Das, S.K.; Pandey, S.; Rao, D.S.; Chakraborty, A.; Sudarshan, M.; Das, N.; Thatoi, H. Investigation on mechanism of Cr(VI) reduction and removal by Bacillus amyloliquefaciens, a novel chromate tolerant bacterium isolated from chromite mine soil. Chemosphere 2014, 96, 112–121. [CrossRef][PubMed] 28. Allegretti, P.; Furlong, J.; Donati, E. The role of higher polythionates in the reduction of chromium(VI) by Acidithiobacillus and Thiobacillus cultures. J. Biotechnol. 2006, 122, 55–61. [CrossRef][PubMed] 29. Wang, Y.S.; Pan, Z.Y.; Lang, J.M.; Xu, J.M.; Zheng, Y.G. Bioleaching of chromium from tannery sludge by indigenous Acidithiobacillus thiooxidans. J. Hazard. Mater. 2007, 147, 319–324. [CrossRef][PubMed] 30. Kumar, N.; Nagendran, R. Fractionation behavior of heavy metals in soil during bioleaching with Acidithiobacillus thiooxidans. J. Hazard. Mater. 2009, 169, 1119–1126. [CrossRef][PubMed] 31. Nguyen, V.K.; Lee, M.H.; Park, H.J.; Lee, J.U. Bioleaching of arsenic and heavy metals from mine tailings by pure and mixed cultures of Acidithiobacillus spp. J. Ind. Eng. Chem. 2015, 21, 451–458. [CrossRef] 32. Marrero, J.; Coto, O.; Schippers, A. Anaerobic and aerobic reductive dissolutions of iron-rich nickel laterite overburden by Acidithiobacillus. Hydrometallurgy 2017, 168, 49–55. [CrossRef] 33. Hallberg, K.B.; Grail, B.M.; Plessis, C.A.D.; Johnson, D.B. Reductive dissolution of ferric iron minerals: A new approach for bio-processing nickel laterites. Miner. Eng. 2011, 24, 620–624. [CrossRef] 34. Marrero, J.; Coto, O.; Goldmann, S.; Graupner, T.; Schippers, A. Recovery of nickel and cobalt from laterite tailings by reductive dissolution under aerobic conditions using Acidithiobacillus species. Environ. Sci. Technol. 2015, 49, 6674–6682. [CrossRef][PubMed] 35. Indian Bureau of Mines, Ministry of Mines, Government of India. Indian Minerals Yearbook, Part III (Mineral Reviews), Chromite; Indian Bureau of Mines: Nagpur, India, 2017; pp. 6-1–6-19. 36. Paulukat, C.; Døssing, L.N.; Mondal, S.K.; Voegelin, A.R.; Frei, R. Oxidative release of chromium from Archean ultramafic rocks, its transport and environmental impact—A Cr isotope perspective on the Sukinda valley ore district (Orissa, India). Appl. Geochem. 2015, 59, 125–138. [CrossRef] 37. BRGM. Development of Application Techniques in Relation to Environmental Management of Mnes and Waste Recoveries; Phase D—Task 1 Regional environmental impact assessment of Sukinda valley chromite mines (Orissa—India); BRGM: Orissa, India, 1999; 133 p. 38. Equeenuddin, S.M.; Pattnaik, B.K. Assessment of heavy metal contamination in sediment at Sukinda ultramafic complex using HAADF-STEM analysis. Chemosphere 2017, 185, 309–320. [CrossRef][PubMed] 39. Das, S.; Patnaik, S.C.; Sahu, H.K.; Chakraborty, A.; Sudarshan, M.; Thatoi, H.N. Heavy metal contamination, physico-chemical and microbial evaluation of water samples collected from chromite mine environment of Sukinda, India. Trans. Nonferr. Met. Soc. China 2013, 23, 484–493. [CrossRef] 40. Coto, O.; Galizia, F.; Hernández, I.; Marrero, J.; Donati, E. Cobalt and nickel recoveries from laterite tailings by organic and inorganic bio-acids. Hydrometallurgy 2008, 94, 18–22. [CrossRef] 41. Deakin, D.; West, L.J.; Stewart, D.I.; Yardley, B.W.D. The Leaching Characteristics of Chromite ore Processing Residue. Environ. Geochem. Health 2001, 23, 201–206. [CrossRef] 42. Weng, C.H.; Huang, C.P.; Allen, H.E.; Cheng, A.H.D.; Sanders, P.F. Chromium leaching behavior in soil derived from chromite ore processing waste. Sci. Total Environ. 1994, 154, 71–86. [CrossRef] Minerals 2018, 8, 261 22 of 23

43. Tinjum, J.M.; Benson, C.H.; Edil, T.B. Mobilization of Cr(VI) from chromite ore processing residue through acid treatment. Sci. Total Environ. 2008, 391, 13–25. [CrossRef][PubMed] 44. Desjardin, V.; Bayard, R.; Huck, N.; Manceau, a.; Gourdon, R. Effect of microbial activity on the mobility of chromium in soils. Waste Manag. 2002, 22, 195–200. [CrossRef] 45. DeLeo, P.C.; Ehrlich, H.L. Reduction of hexavalent chromium by Pseudomonas fluorescens in batch and continuous cultures. Appl. Microbiol. Biotechnol. 1994, 40, 756–759. [CrossRef] 46. Garnier, J.; Quantin, C.; Martins, E.S.; Becquer, T. Solid speciation and availability of chromium in ultramafic soils from Niquelândia, Brazil. J. Geochem. Explor. 2006, 88, 206–209. [CrossRef] 47. Onchoke, K.K.; Sasu, S.A. Determination of Hexavalent Chromium (Cr(VI)) Concentrations via Ion Chromatography and UV-Vis Spectrophotometry in Samples Collected from Nacogdoches Wastewater Treatment Plant, East Texas (USA). Adv. Environ. Chem. 2016, 2016, 10. [CrossRef] 48. Swietlik,´ R. Speciation analysis of chromium in waters. Pol. J. Environ. Stud. 1998, 7, 257–266. 49. Viollier, E.; Inglett, P.W.; Hunter, K.; Roychoudhury, A.N.; Van Cappellen, P. The ferrozine method revisited: Fe(II)/Fe(III) determination in natural waters. Appl. Geochem. 2000, 15, 785–790. [CrossRef] 50. Waterborg, J.H.; Matthews, H.R. The lowry method for protein quantitation. Methods Mol. Biol. 1984, 1. [CrossRef] 51. Rodríguez-Vico, F.; Martínez-Cayuela, M.; García-Peregrín, E.; Ramírez, H. A procedure for eliminating interferences in the lowry method of protein determination. Anal. Biochem. 1989, 183, 275–278. [CrossRef] 52. Payne, S.M. Detection, isolation, and characterization of siderophores. Methods Enzymol. 1994, 235, 329–344. [CrossRef][PubMed] 53. Schwyn, B.; Neilands, J.B. Universal chemical assay for the detection and determination of siderophore. Anal. Biochem. 1987, 160, 47–56. [CrossRef] 54. Lynne, A.M.; Haarmann, D.; Louden, B.C. Use of Blue Agar CAS Assay for Siderophore Detection. J. Microbiol. Biol. Educ. 2011, 12, 51–53. [CrossRef] 55. Sayyed, R.Z.; Badgujar, M.D.; Sonawane, H.M.; Mhaske, M.M.; Chincholkar, S.B. Production of microbial iron chelators (siderophores) by fluorescent Pseudomonads. Indian J. Biotechnol. 2005, 4, 484–490. 56. Kota´s,J.; Stasicka, Z. Chromium occurrence in the environment and methods of its speciation. Environ. Pollut. 2000, 107, 263–283. [CrossRef] 57. Gleisner, M.; Herbert, R.B. Sulfide mineral oxidation in freshly processed tailings: Batch experiments. J. Geochem. Explor. 2002, 76, 139–153. [CrossRef] 58. Van Hullebusch, E.D.; Yin, N.; Seignez, N.; Gauthier, A.; Lens, P.N.L.; Avril, C.; Sivry, Y. Bio-alteration of metallurgical wastes by Pseudomonas aeruginosa in a semi flow-through reactor. J. Environ. Manag. 2015, 147, 297–305. [CrossRef][PubMed] 59. Potysz, A.; Lens, P.N.L.; Vossenberg, J. Van De; Rene, E.R.; Grybos, M.; Guibaud, G.; Kierczak, J.; van Hullebusch, E.D. Applied Geochemistry Comparison of Cu, Zn and Fe bioleaching from Cu-metallurgical slags in the presence of Pseudomonas fluorescens and Acidithiobacillus thiooxidans. Appl. Geochem. 2016, 68, 39–52. [CrossRef] 60. Liu, H.C.; Xia, J.L.; Nie, Z.Y. Relatedness of Cu and Fe speciation to chalcopyrite bioleaching by Acidithiobacillus ferrooxidans. Hydrometallurgy 2015, 156, 40–46. [CrossRef] 61. Becquer, T.; Quantin, C.; Sicot, M.; Boudot, J.P. Chromium availability in ultramafic soils from New Caledonia. Sci. Total Environ. 2003, 301, 251–261. [CrossRef] 62. Garnier, J.; Quantin, C.; Guimarães, E.M.; Vantelon, D.; Montargès-Pelletier, E.; Becquer, T. Cr(VI) genesis and dynamics in Ferralsols developed from ultramafic rocks: The case of Niquelândia, Brazil. Geoderma 2013, 193–194, 256–264. [CrossRef] 63. Biswas, S.; Bhattacharjee, K. Fungal assisted bioleaching process optimization and kinetics: Scenario for Ni and Co recovery from a lateritic chromite overburden. Sep. Purif. Technol. 2014, 135, 100–109. [CrossRef] 64. Liu, S.; Chen, L.; Gao, Y. Hexavalent Chromium Leaching Influenced Factors in the Weathering Chrome Slag. Procedia Environ. Sci. 2013, 18, 783–787. [CrossRef] 65. Godgul, G.; Sahu, K.C. Chromium Contamination from Chromite Mine. Environ. Geol. 1995, 25, 251–257. [CrossRef] 66. Garnier, J.; Quantin, C.; Guimaraes, E.; Becquer, T. Can chromite weathering be a source of Cr in soils? Miner. Mag. 2008, 72, 49–53. [CrossRef] Minerals 2018, 8, 261 23 of 23

67. Fandeur, D.; Juillot, F.; Morin, G.; Livi, L.; Cognigni, A.; Webb, S.M.; Ambrosi, J.P.; Fritsch, E.; Guyot, F.; Brown, G.E. XANES evidence for oxidation of Cr(III) to Cr(VI) by Mn-oxides in a lateritic regolith developed on serpentinized ultramaﬁc rocks of New Caledonia. Environ. Sci. Technol. 2009, 43, 7384–7390. [CrossRef] [PubMed] 68. Fendorf, S.E. Surface reactions of chromium in soils and waters. Geoderma 1995, 67, 55–71. [CrossRef] 69. Oze, C.; Fendorf, S.; Bird, D.K.; Coleman, R.G. Chromium Geochemistry of Serpentine Soils. Int. Geol. Rev. 2004, 46, 97–126. [CrossRef] 70. Raous, S.; Becquer, T.; Garnier, J.; Martins, É.D.S.; Echevarria, G.; Sterckeman, T. Mobility of metals in nickel mine spoil materials. Appl. Geochem. 2010, 25, 1746–1755. [CrossRef] 71. Simate, G.S.; Ndlovu, S. Acid mine drainage: Challenges and opportunities. J. Environ. Chem. Eng. 2014, 2, 1785–1803. [CrossRef] 72. Cabrera, G.; Gómez, J.M.; Hernández, I.; Coto, O.; Cantero, D. Different strategies for recovering metals from CARON process residue. J. Hazard. Mater. 2011, 189, 836–842. [CrossRef][PubMed] 73. Jang, H.C.; Valix, M. Overcoming the bacteriostatic effects of heavy metals on Acidithiobacillus thiooxidans for direct bioleaching of saprolitic Ni laterite ores. Hydrometallurgy 2017, 168, 21–25. [CrossRef] 74. Viera, M.; Curutchet, G.; Donati, E. A combined bacterial process for the reduction and immobilization of chromium. Int. Biodeterior. Biodegrad. 2003, 52, 31–34. [CrossRef] 75. Quintana, M.; Curutchet, G.; Donati, E. Factors affecting the chromium(VI) reduction by microbial action. Biochem. Eng. J. 2001, 9, 11–15. [CrossRef] 76. Steudel, R. On the nature of “elemental sulfur” (S◦) produced by sulfur-oxidizing bacteria. In Autotrophic Bacteria; Schlegel, H.G., Bowien, B., Eds.; Springer: Berlin, Germany, 1989; pp. 289–303. 77. Rong, X.; Chen, W.; Huang, Q.; Cai, P.; Liang, W. Pseudomonas putida adhesion to goethite: Studied by equilibrium adsorption, SEM, FTIR and ITC. Colloids Surf. B Biointerfaces 2010, 80, 79–85. [CrossRef] [PubMed] 78. Jin, R.; Liu, Y.; Liu, G.; Tian, T.; Qiao, S.; Zhou, J. Characterization of Product and Potential Mechanism of Cr(VI) Reduction by Anaerobic Activated Sludge in a Sequencing Batch Reactor. Sci. Rep. 2017, 7.[CrossRef] [PubMed] 79. Park, D.; Yun, Y.S.; Jong, M.P. Studies on hexavalent chromium biosorption by chemically-treated biomass of Ecklonia sp. Chemosphere 2005, 60, 1356–1364. [CrossRef][PubMed] 80. Smuda, J.; Dold, B.; Spangenberg, J.E.; Pfeifer, H.R. Geochemistry and stable isotope composition of fresh alkaline porphyry copper tailings: Implications on sources and mobility of elements during transport and early stages of deposition. Chem. Geol. 2008, 256, 62–76. [CrossRef] 81. Adabanija, M.A.; Oladunjoye, M.A. Geoenvironmental assessment of abandoned mines and quarries in South-western Nigeria. J. Geochem. Explor. 2014, 145, 148–168. [CrossRef] 82. Meck, M.; Love, D.; Mapani, B. Zimbabwean mine dumps and their impacts on river water quality—A reconnaissance study. Phys. Chem. Earth 2006, 31, 797–803. [CrossRef] 83. Pattnaik, B.K.; Equeenuddin, S.M. Potentially toxic metal contamination and enzyme activities in soil around chromite mines at Sukinda Ultramaﬁc Complex, India. J. Geochem. Explor. 2016, 168, 127–136. [CrossRef] 84. Borja, D.; Nguyen, K.; Silva, R.; Park, J.; Gupta, V.; Han, Y.; Lee, Y.; Kim, H. Experiences and Future Challenges of Bioleaching Research in South Korea. Minerals 2016, 6, 128. [CrossRef] 85. Hamilton, J.L.; Wilson, S.A.; Morgan, B.; Turvey, C.C.; Paterson, D.J.; MacRae, C.; McCutcheon, J.; Southam, G.

Nesquehonite sequesters transition metals and CO2 during accelerated carbon mineralisation. Int. J. Greenh. Gas Control 2016, 55, 73–81. [CrossRef] 86. Silva, R.A.; Borja, D.; Hwang, G.; Hong, G.; Gupta, V.; Bradford, S.A.; Zhang, Y.; Kim, H. Analysis of the effects of natural organic matter in zinc beneﬁciation. J. Clean. Prod. 2017, 168, 814–822. [CrossRef] 87. Santini, T.C.; Banning, N.C. Alkaline tailings as novel soil forming substrates: Reframing perspectives on mining and reﬁning wastes. Hydrometallurgy 2016, 164, 38–47. [CrossRef]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).