Recovery of Nickel and Cobalt from Laterite Tailings by Reductive

Apr 29, 2015 - Biomining of sulfidic ores has been applied for almost five decades. However, the bioprocessing of oxide ores such as laterites lags ...
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Recovery of Nickel and Cobalt from Laterite Tailings by Reductive Dissolution under Aerobic Conditions Using Acidithiobacillus Species J. Marrero,*,† O. Coto,‡ S. Goldmann,† T. Graupner,† and A. Schippers† †

Federal Institute for Geosciences and Natural Resources (BGR), Stilleweg 2, 30655 Hannover, Germany Laboratory of Metals, Department of Microbiology, University of Havana, Calle 25 e/J e I, Havana, Cuba



S Supporting Information *

ABSTRACT: Biomining of sulfidic ores has been applied for almost five decades. However, the bioprocessing of oxide ores such as laterites lags commercially behind. Recently, the Ferredox process was proposed to treat limonitic laterite ores by means of anaerobic reductive dissolution (AnRD), which was found to be more effective than aerobic bioleaching by fungi and other bacteria. We show here that the ferric iron reduction mediated by Acidithiobacillus thiooxidans can be applied to an aerobic reductive dissolution (AeRD) of nickel laterite tailings. AeRD using a consortium of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans extracted similar amounts of nickel (53−57%) and cobalt (55−60%) in only 7 days as AnRD using Acidithiobacillus ferrooxidans. The economic and environmental advantages of AeRD for processing of laterite tailings comprise no requirement for an anoxic atmosphere, 1.8-fold less acid consumption than for AnRD, as well as nickel and cobalt recovered in a ferrous-based pregnant leach solution (PLS), facilitating the subsequent metal recovery. In addition, an aerobic acid regeneration stage is proposed. Therefore, AeRD process development can be considered as environmentally friendly for treating laterites with low operational costs and as an attractive alternative to AnRD.



INTRODUCTION Ferric iron reduction coupled to microbial growth has been extensively studied at neutral pH and in neutrophilic iron reducing microorganisms.1 Nowadays, the increasing number of acidophilic bacteria isolated and characterized has demonstrated that reduction of iron might be particularly widespread among acidophiles as well. In fact, ferric iron reduction has potentially more significance in acidic environments because (i) the solubility of iron is higher at low pH and (ii) high concentrations of ferric iron are found in diverse anthropogenic and extremely acidic environments such as mines. Ironreducing acidophiles include facultative anaerobes or aerobes able to couple ferric iron reduction to growth only under microaerophilic conditions, which are the majority of the isolated strains.2 In contrast, very little attention has been given to ferric iron reduction by acidophilic bacteria under aerobic conditions. Acidithiobacillus (At.) species such as At. ferrooxidans and At. thiooxidans are important iron and/or sulfur oxidizing bacteria for acid mine/rock drainage formation and for biomining of sulfide ores at the industrial level.3 At. thiooxidans, which is an extremely acidophilic, chemolithotrophic, Gram-negative bacterium belonging to the recently proposed class Acidithiobacillia,4 has great value in biomining due to its distinct advantage to produce considerable amounts of acid via oxidation of sulfur compounds.5 Although iron reduction activity has been detected in At. thiooxidans for at least three decades,6 this microorganism is mostly known as a sulfur-oxidizer and its iron © 2015 American Chemical Society

reduction capacity under aerobic conditions tends to remain unexplored. In contrast to At. ferrooxidans, which can grow anaerobically using ferric iron as electron donor,7−9 the ferric iron reduction activity of At. thiooxidans is not associated with cell growth and is only detected in suspensions of cells grown aerobically.10 To date, it is still unknown whether ferric iron reduction is mediated abiotically by some of the reduced inorganic sulfur compounds (RISCs) produced during sulfur oxidation or directly by one of the enzymes belonging to the sulfur oxidizing pathways. Whereas biomining of sulfidic ores is applied for almost five decades,3,11,12 the bioprocessing of oxide ores such as laterites still remains at laboratory scale so far, despite more than 60% of the world’s nickel occurs in laterite ore.13 Fungi were proposed to bioleach nickel and cobalt from oxide ores in the late 1980s.14 However, the adsorption of metals by fungal biomass and the stability of metal−organic acid complexes can be mentioned as the most important reasons that makes this approach not attractive for recovery of metals from this type of ore.15,16 Chemolithotrophic bacteria grown under aerobic conditions or their products have been shown to be more efficient than fungi, promising to be an alternative to recover metals from Received: Revised: Accepted: Published: 6674

February April 27, April 29, April 29,

21, 2015 2015 2015 2015 DOI: 10.1021/acs.est.5b00944 Environ. Sci. Technol. 2015, 49, 6674−6682

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Environmental Science & Technology laterite, though this field is still relatively unexplored.17−20 Initial attempts to treat nickel laterite tailings using At. thiooxidans have shown its potential to recover nickel and cobalt from tailings.17,19,20 However, whether the mechanism of metal extraction by At. thiooxidans is merely acidolysis via sulfuric acid production from added sulfur needs to be clarified.17−21 Anaerobe reductive dissolution (AnRD) of ferric oxide ores has been well-studied in acidophiles.2 Only quite recently, the use of the iron- and sulfur-oxidizing bacterium At. ferrooxidans has been proposed to recover nickel by AnRD of low grade nickel laterite ores containing 7% of total iron at 5% pulp density to extract target metals (e.g., extraction of 70% nickel and 50% cobalt within 30 days).21,22 Although this process, which is the main stage of the Ferredox process, requires far less aggressive conditions (temperature and pressure) and consequently lower capital expenditure (capex) than the traditional CARON and PAL processes, the acid consumption remains still relatively high which adds to the operational costs (opex).21,23,24 The scheme of the Ferredox process includes one stage of reductive acid generation by AnRD22 and requires a supplement of nitrogen to ensure operation under anoxic conditions which could restrict the process to tank bioleaching under anaerobic conditions. Recently, the AnRD has been referred to as “biomining in reverse gear” since the dissimilatory reduction of ferric iron under anaerobic conditions is the opposite of the main reactions driven in conventional bioleaching of sulfide minerals by means of ferrous iron oxidation.25 The current paper describes the iron reduction ability of At. thiooxidans and a mixed culture of At. thiooxidans and At. ferrooxidans as well as the aerobic reductive dissolution (AeRD) of nickel laterite tailings containing 40% of total iron content using (i) a pure culture of At. thiooxidans and (ii) a mixed culture of At. thiooxidans with At. ferrooxidans in comparison to AnRD using At. ferrooxidans. Our results open the door to “biomining in reverse gear” under aerobic conditions to extract metals from oxidized ores with clear economic advantages since a subproduct of the metallurgy industry with a high content of iron at high pulp density can be treated and a significant reduction on acid consumption can be achieved. This study is the first to demonstrate that reductive bioleaching of laterite by acidolysis and iron reduction can be carried out by acidophilic bacteria (e.g., At. thiooxidans or mixed culture of At. thiooxidans and At. ferrooxidans) in the presence of oxygen.

were grown in basal salts-trace element solution (BS-TE).26 The cultures were grown on 1% (wt/vol) elemental sulfur (S0) (sterilized by tyndalization) in a shake flask culture (30 °C; initial pH 2.5) before being used as inoculum in different experiments. Determination of Ferric Iron Reduction Mediated by Strains of At. thiooxidans. S0-grown cells of strains of At. thiooxidans (1%) were inoculated in 25 mL of BS-TE solution (pH 2.5) containing 1.2 g/L Fe2(SO4)3 and supplemented with 1% S0 in aerated 150 mL shake flasks at 120 rpm. A separate sterile control of fresh liquid medium supplemented with 1.2 g/L ferric iron sulfate was included. The concentration of ferric iron, ferrous iron, pH, and total cell amount was determined at regular time intervals in liquid samples during 25 days. Effect of Initial pH on the Ferric Iron Reduction Activity of Cell Suspension of At. thiooxidansT. Ferric iron reduction activity was determined by using cell suspensions of the strain grown in BS-TE solution at pH 2.5 containing 1% S0 to early stationary phase. Cells (10 mL) were harvested by centrifugation (10 000g, 15 min) when the pH of the culture medium dropped to a value of 0.9. Cells were then washed in parallel with basal salts solution at pH 0.9, 2.5, or 3.5 and suspended in 1 mL of basal salts solution at the respective pH. The protein concentration of the cell suspension was measured by the Bradford method.27 Ferric iron sulfate was added to give a final concentration of 1.2 g/L soluble ferric iron. At the start of the experiment, each tube contained 1 mL of cell suspension with a protein concentration of 0.5 mg/mL. The bacterial suspensions were incubated at 30 °C. The soluble ferrous iron concentration was measured at regular intervals during 6.5 h. Determination of Induction of Ferric Iron Reduction. Cultures grown to early stationary phase in BS-TE at pH 2.5 containing 1% S0 were diluted 1:20 into fresh medium containing either no (noninduced cells) or 1 mM ferric iron sulfate (induced cells). Incubation was continued with shaking at 30 °C. After 4 days, the cultures of noninduced cells and induced cells were separately diluted 1:20 in fresh medium containing a concentration of the 25 mM ferric iron sulfate, respectively. The soluble ferrous iron concentration and total cells were measured at regular intervals.28 Aerobic and Anaerobic Reductive Dissolution of Nickel Laterite Tailings. Four experiments were carried out using (i) At. thiooxidansT under aerobic conditions, (ii) a consortium of At. thiooxidansT and At. ferrooxidansT under aerobic conditions (Ae), (iii) a consortium of At. thiooxidansT and At. ferrooxidansT under aerobic followed by anaerobic conditions (Ae-An), and (iv) At. ferrooxidansT under anaerobic conditions (An). As controls for the bioleaching experiments described below, two not inoculated reactors were also run to account for chemical leaching, one operated under aerobic conditions and the second one under anaerobic conditions. Briefly, during the cell growth stage, all experiments were run in bioreactors (Electrolab, United Kingdom) containing 15 g of S0 and 1.5 L of BS-TE solution at 30 °C, aerated with sterile air at 1 L/min and stirred at 120 rpm. The initial and final pH values for the growth stage as well as experimental details are specified below for each individual experiment. The bioreactors were inoculated with 10% inoculum giving a final number of planktonic cells of 107 cells/mL. When the number of planktonic cells reached 109 cells/mL and the pH was decreased to 1.6 or 0.8 at the end of the growth stage, due to acid production during sulfur oxidation by the acidophiles, the bioleaching stage was started by adding 150 g of laterite



MATERIALS AND METHODS Laterite Tailings. The laterite tailings used in this study originated from a technical process involving a reduction roast and ammoniacal leach (CARON) applied by a nickel plant in Moa, Cuba. The pH of the lateritic residue was determined in water at a 1:2.5 ratio (tailings/water (w/v)). Chemical analysis of the laterite tailings used throughout this paper is given in Table 1. Bacteria and Growth Conditions. At. ferrooxidans DSM 14882T and the strains of At. thiooxidans DSM 14887T, DSM 622, DSM 504, and DSM 9463 were used in this study. Bacteria Table 1. Chemical Composition of Laterite Tailings metal concentration (ppm)

Ni 3711

Co 770

Fe 400667

Mn 5654

Al 25498

Cr 18020

6675

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operated under anaerobic conditions during the bioleaching experiment (anaerobic or An bioreactor). Analytical Techniques. Concentrations of soluble metals in the bioreactors were determined by ICP-OES. All liquid samples were filtered through 0.2 μm pore-sized polycarbonate filters. Ferrous iron concentrations were measured by a colorimetric assay using ferrozine.28 Total iron was quantified by adding an excess of ascorbic acid to reduce soluble ferric iron to ferrous iron. Ferric iron was determined from differences in total and ferrous iron concentrations. The elemental composition and mineralogical analyses of the solid residues were determined by X-ray fluorescence analysis as well as electron probe microanalysis (EPMA). Analytical conditions of EPMA applied in this study are summarized in Table S2, Supporting Information. Redox potentials were measured using a combined platinum−silver/silver chloride electrode coupled to a Knick Portamess meter and adjusted to be relative to a hydrogen reference electrode (EH values). Metal mass balance calculations were performed using (i) the amount of metal present in the not processed laterite tailings compared to (ii) that amount released into the process solutions plus that still present in the tailings residues.23,24,30 Total cells were stained on filters with SYBR Green I and counted under a fluorescence microscope using a previously described staining procedure.31 Numbers of living, cultivable cells of sulfur-oxidizing bacteria and iron-oxidizing bacteria were determined using the most probable number (MPN) technique.32 Numbers of bacteria were quantified by real-time PCR (Q-PCR).33

tailings. The pH increased due to addition of laterite tailings which is an alkaline material. Once the pH of the medium reached a value of 1.8 within the first 2 days, the pH was maintained automatically at this value during the bioleaching stage using 2.5 M H2SO4 and 1 M NaOH in reservoir flasks. The volumes of acid/base added to maintain the pH were recorded. Samples were removed from the reactors at regular intervals for metal concentration, pH, and redox potential analyses. In the experiments operated under anaerobic conditions, to change aerobic to anaerobic atmosphere, the cultures were deaerated with a stream of nitrogen (2 L/min) for 60 min and later nitrogen flow was provided by intermittently sparging the bioreactors daily (1 L/min for 30 min) as required. Moreover, carbon dioxide was provided by injecting 10% CO2 into the free headspace volume daily after each sparging with nitrogen. At the end of the experiment, gassing was stopped and the remaining solid residues were harvested and washed twice in deionized water at pH 1.8 before drying at 60 °C. Experiments were repeated twice independently. All reactors were generally operated as described above, and differences in operation conditions for specific experiments are given as follows. (i) Experiment with At. thiooxidansT. The bioreactor was inoculated in BS-TE solution at pH 1.5 as described above for the growth stage. Once the pH of the medium reached a value of 0.8 at the end of the growth stage, the bioleaching stage started with the addition of laterite tailings up to 10% pulp density in four steps (adding 2.5% pulp density each time at 0, 8, 24, and 48 h to complete a total of 10% pulp density). Sequential addition of the laterite tailings rather than one addition to the 10% pulp density was conducted to allow progressive adaptation of the culture and to minimize growth inhibition at the initial bioleaching stage. The bioreactor was aerated at 1 L/min during the whole experiment. (ii) and (iii) Experiment with a consortium of At. thiooxidansT and At. ferrooxidansT. Two bioreactors were inoculated with At. thiooxidans and At. ferrooxidans at a ratio of 3:1 in BS-TE solution at pH 1.5 as described above for the growth stage. In the bioleaching stage, laterite tailings were added in four steps (as described above), when the pH reached 0.8 at the end of the growth stage. These growth stage conditions for pH and ratio of the microorganisms were selected on the basis of the previous finding that the coculture of At. thiooxidans and At. ferrooxidans exhibits a low oxidation rate of ferrous iron and a high decrease in pH over time and that the two acidophiles could oxidize sulfur more efficiently at low pH in coculture than the respective pure cultures.29 After 3 days of aerated operations, the atmosphere was changed to an anoxic atmosphere in the first bioreactor (sequential aerobic-anaerobic or Ae-An bioreactor) as described above whereas the aeration was kept constant in the second bioreactor (aerobic or Ae bioreactor) at 1 L/min. Before the gas atmosphere change, fresh cells of At. ferrooxidans grown on S0 (∼1010 total cells) were additionally inoculated into each bioreactor. The subsequent inoculation with fresh cells of At. ferrooxidans was conducted to overcome the cell depletion due to the decrease of pH to 0.8 during the growth stage and for further comparison of the Ae and Ae-An bioreactors with the An bioreactor. (iv) Experiment with At. ferrooxidansT. The bioreactor was inoculated in BS-TE solution at initial pH of 1.8. Once pH decreased to 1.6, the bioleaching stage started with the addition of laterite tailings (10% w/v) in one step. The bioreactor was



RESULTS Reduction of Soluble Ferric Iron by At. thiooxidansT. Biological ferric iron reduction mediated by At. thiooxidansT was demonstrated under aerobic conditions. While the ferrous iron concentration increased, the ferric iron decreased during the growth of At. thiooxidansT (Figure S1, Supporting Information). All ferric iron added to the culture was converted to ferrous iron (Figure S1, Supporting Information), demonstrating that the variation in ferric iron concentration is due to ferric iron reduction rather than precipitation of iron. No changes were detected on concentrations of ferric iron and ferrous iron in the not inoculated control under the conditions tested (not shown). Iron reduction activity was also found to be mediated by the At. thiooxidans strains DSM 622, DSM 504, and DSM 9463 (not shown). The production of ferrous iron was linear over a period of at least 6.5 h at the three pH values evaluated (Figure S2, Supporting Information). Despite a decrease of the initial velocity and the specific rates of ferric iron reduction being observed over the decrease in pH of the incubation medium, there were no significant differences between the three pH values evaluated (Figure S2A, Table S1, Supporting Information). The curve of ferric iron reduction proceeded at zero order kinetics (Figure S2, Supporting Information). No significant differences in the iron reduction activity and growth were found between induced and noninduced cells of At. thiooxidans (data not shown), suggesting a mechanism of constitutive expression of the iron reduction mediated by At. thiooxidans. Aerobic Reductive Dissolution of Laterite Tailings by At. thiooxidansT. At. thiooxidansT was able to accelerate the reductive bioleaching of laterite tailings under aerobic conditions (Figure 1A). While the soluble iron concentration remained low, that of ferrous iron increased during the 6676

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Figure 1. Solubilization of iron (A), extraction of nickel (B) and cobalt (C), and variation of pH (D) in the aerobic bioreactor inoculated with At. thiooxidansT (closed diamond) and the not inoculated bioreactor (chemical control; open triangle). Ferrous iron: solid lines; ferric iron: dashed lines. Graphs show mean values for two independent experiments with an assay error less than 7.5%. The temperature was maintained at 30 °C. Once the pH of the medium reached a value of 1.8, the pH was maintained automatically at this value to the end of the experiment.

Figure 2. Iron in solution (A), acid consumption (B), variation of pH (C), and redox potential (D) in aerobic bioreactor (closed square), aerobic-anaerobic bioreactor (closed circle), anaerobic bioreactor (open square), and not inoculated bioreactor (open triangle) during the bioleaching phase of the experiment. Once the pH of the medium reached a value of 1.8, the pH was maintained automatically at this value to the end of the experiment. Ferrous iron: solid lines; ferric iron: dashed lines. Graphs show mean values for two independent experiments with an assay error less than 5%. After 3 days of operation, the aerobic condition was switched to anaerobic conditions in the AeAn bioreactor.

experimental time in the inoculated bioreactor. Nickel and cobalt were leached in 11 days (53% and 46%, respectively) (Figure 1B,C). The metal amounts solubilized in the inoculated reactor were up to almost 3 orders of magnitude higher than in the not inoculated reactor (Figure 1). The redox potential declined in the inoculated bioreactor during the iron dissolution. The number of living cells of At. thiooxidans increased from 9 × 109 to 2 × 1010 cell/mL at the end of the AeRD as determined by the MPN technique. It is noteworthy that the bioleaching of the laterite tailings used in this study requires an initial acidification step since this material is alkaline (pH 8.0). Despite the acid consumption levels being similar for both experiments, the ferrous iron amount released was far higher and much more acid was produced due to sulfur oxidation in the inoculated bioreactor than in the not inoculated reactor (Table 2).

concentration was reached after 7 days in the bioreactors inoculated with the consortium. However, the soluble ferrous iron concentration further increased in the An bioreactor with the culture of At. ferrooxidans, reaching values of 11 g/L after 11 days. The redox potentials declined during iron dissolution (Figure 2D). It is therefore concluded that laterite tailings can be reductively dissolved under aerobic conditions using the mixed culture of At. thiooxidans and At. ferrooxidans as well as under anaerobic conditions using a pure culture of At. ferrooxidans. Interestingly, the AeRD of laterite tailings was faster by far using At. thiooxidans in consortium with At. ferrooxidans than using the pure culture of At. thiooxidans (Figures 1A and 2A). Acid Consumption. The one-step addition of nickel laterite tailings into the An bioreactor and into the not inoculated reactor stimulated the consumption of 346 and 137 mmol of sulfuric acid within the first day, respectively (Figure 2B). The highly rapid consumption of acid could be predominatly due to the alkaline pH of the laterite tailings. No acid was consumed within the first 2 days in the Ae and Ae-An bioreactors inoculated with the consortium of At. thiooxidans and At. ferrooxidans under aerobic conditions (Figure 2B). The use of the consortium of the two acidophiles and the addition of laterite tailings in multiple steps triggered the AeRD of laterite tailings with lower consumption of acid compared to the anaerobe process. Interestingly, the change to an anoxic atmosphere in the Ae-An bioreactor provoked a rapid increase in the acid consumption at day 3, likely due to growth inhibition of At. thiooxidans and the lower acid production of At. ferrooxidans under anaerobic conditions (Figures 2B and S3, Supporting Information, discussed below).

Table 2. Acid Consumption and Amount of Iron in Solution during Leaching of Laterite Tailings at 10% Pulp Density in the Bioreactor Inoculated with At. thiooxidansT in Comparison to the Not Inoculated Reactor (Chemical Control) bioreactor

acid consumed (mmole)

iron in solution (g/L) Fe(II)/ Fe(III)

inoculated not inoculated

307 387

3.4/1.3 1.6/3.8

Comparative Study of AeRD by a Consortium of At. thiooxidansT and At. ferrooxidansT and AnRD by At. ferrooxidansT. The kinetics of iron reductive dissolution of laterite tailings under aerobic, aerobic-anaerobic, and anaerobic conditions are shown in Figure 2A. The soluble ferric iron concentration remained low for all conditions tested. The concentration of ferrous iron in the three inoculated bioreactors was far higher than in the not inoculated control reactor (Figure 2A). The maximum of the soluble ferric iron 6677

DOI: 10.1021/acs.est.5b00944 Environ. Sci. Technol. 2015, 49, 6674−6682

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Extraction of Other Metals. The AeRD as well as the AeAnRD and AnRD allowed one to solubilize similar amounts of manganese, aluminum, and chromium within the first 6 days (Figure S4, Supporting Information). Although the percentage of extraction of chromium was low within the first 6 days (4− 6%), that for manganese and aluminum were higher (40−50% and 10−15%, respectively) (Figure S4, Supporting Information, Table 1). In contrast to the metals manganese, nickel, and cobalt, the dissolution of the metals iron, aluminum, and chromium was accelerated after 7 days in the An bioreactor but not in the Ae and Ae-An bioreactors (Figures 2 and S4, Supporting Information). It appears that these metals are strongly bound in the lateritic starting material (probably to aluminum silicates) and, consequently, are less available to any bioleaching process. This inference confirms the role of a wellestablished mineralogical characterization of the laterite for this type of study. The distribution of heavy metals into different chemical forms within the laterite plays an important role in the bioavailability and potential mobility of metals.21 Mass Balance of Metals before and after Reductive Dissolution and Analysis of the Biotreated Residues. Mass balance calculations indicated that between 95% and 97% of nickel and cobalt present in the untreated tailings was accounted for by that remaining in the residue after biotreatment and that detected in the PLS, confirming that nickel and cobalt had been efficiently leached from the tailings. The laterite tailings used in this work had 40.06 wt % of iron content with 0.37 wt % of nickel, 0.07 wt % of cobalt, 0.56 wt % of manganese, and 1.80 wt % of chromium. Mineralogical studies by EPMA showed that most of the nickel in the laterite tailings was associated with (i) unspecified iron oxyhydroxide phases, (ii) serpentine, (iii) serpentinized olivine, (iv) magnesium−iron pyroxene, and (v) magnesium−iron amphiboles. Accurate mineral determination was not possible in every case as the primary rock-forming minerals like olivine, pyroxene, and amphibole were already strongly altered, first during natural laterite formation (weathering) and second during the industrial treatment in the CARON process. These phases were also detected in the bioprocessed residues, suggesting that some nickel and cobalt remained in these mineral phases during the bioreductive processes, particularly in the Fe oxyhydroxide phases (Figures 3 and S5, Supporting Information). Serpentinized olivine, pyroxene, and amphibole were attacked during the bioprocessing of the laterite tailings and further altered into serpentine resulting in mobilization of nickel and cobalt (Figure S5B,C, Supporting Information). Chromite was relatively enriched during the biotreatment in Ae, Ae-An, and An bioreactors as it is a very stable mineral and is not significantly attacked (Figure S5D, Supporting Information). However, some additional chromium and aluminum started to be solubilized at the seventh day, most likely from the chromite during the anaerobic process (Figure S4, Supporting Information). Variation of Cell Numbers and Cell Attachment. The number of bacteria estimated by qPCR as well as the number of living iron-oxidizing and sulfur-oxidizing bacteria did not significantly change in the Ae bioreactor over the time of bioleaching operation. In contrast, a depletion of cell numbers over time was observed in the An as well as in the Ae-An bioreactor after day 3 of operation (Figure S3, Supporting Information). This observation corresponds to the previous finding that the population of At. ferrooxidans is drastically affected during reductive dissolution under anaerobic con-

Extraction of Nickel and Cobalt. The extraction of nickel and cobalt from laterite tailings in the Ae as well as in the AeAn bioreactor inoculated with the consortium of At. thiooxidans and At. ferrooxidans was as efficient as in the An bioreactor inoculated with the pure culture of At. ferrooxidans (Figure 3). The rate of solubilization of nickel and cobalt was clearly lower in the not inoculated control than in all inoculated bioreactors (Figure 3).

Figure 3. Changes in percentage of nickel (A) and cobalt (B) extracted in an aerobic bioreactor (closed square), aerobic-anaerobic bioreactor (closed circle), anaerobic bioreactor (open square), and not inoculated bioreactor (open triangle). Graphs show mean values for two independent experiments with an assay error less than 9%.

The total percentages of nickel and cobalt extracted were similar for the three bioprocesses, reaching between 53−57% of nickel and 55−60% of cobalt extracted (Table 3). In all cases, 6 Table 3. Acid Consumption and Metal Extraction in Aerobic Compared to Anaerobic Reductive Dissolution of Laterite Tailings in Bioreactor Experiments acid consumption (mmol/kg tailings/% metal extracted)

% metal extracted bioreactora

Ni

Co

(mmol)

(mmol/kg tailings)

Ni

Co

An Ae Ae-An control

58 56 53 20

56 60 58 16

725 306 532 347

3624 2043 3548 2310

63 37 67 115

65 34 61 144

a

An: anaerobic; Ae: aerobic; Ae-An: aerobic-anaerobic.

days were sufficient to reach the maximum dissolution of nickel and cobalt (Figure 3). Although the ferric iron continued dissolving after 6 days, no further nickel and cobalt were extracted in the anaerobic bioreactor (Figure 2A, Figure 3). Interestingly, the acid consumption of the AeRD is almost lower by a factor of 2 than that of the AnRD; i.e., 37 compared to 63 and 34 compared to 65 mmol of H+ per kg of laterite tailings are required to extract 1% of nickel and cobalt, respectively (Table 3). Similar to the An bioreactor, nickel and cobalt in the Ae and Ae-An bioreactors were recovered in a ferrous-based pregnant leach solution (PLS) containing exclusively ferrous iron (Figures 2A, 3, and 4). At the end of the experiment, the PLS obtained in the aerated processes contained as much solubilized nickel and cobalt as in the anoxic process. In contrast, the Ae and Ae-An bioreactors allowed one to obtain a ferrous-based PLS with less iron content at 11 days (Figures 2A and 3). 6678

DOI: 10.1021/acs.est.5b00944 Environ. Sci. Technol. 2015, 49, 6674−6682

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Figure 4. Comparative scheme of the anaerobic and the novel proposed aerobic reductive dissolution of laterite tailings. The overall scheme is adapted from the Ferredox process.21,24 Dashed lines indicate modifications proposed for the aerobe reductive dissolution.

ditions.24 In the Ae-An bioreactor, the depletion of living sulfuroxidizing bacteria was greater than that of living iron-oxidizing bacteria after the third day, indicating that the changed atmosphere provoked a growth inhibition of At. thiooxidans. This fact corresponded to the increased acid consumption after this time in the bioreactor with the consortium (Figure 2B). It is worth mentioning that a fresh inoculum of At. ferrooxidans added to the Ae bioreactor at the third day had no apparent effect on the iron reduction of the coculture (Figures 2A, 3, and S3, Supporting Information). On the other hand, assuming that the growth rate of At. thiooxidans and the acid resistance are higher than for At. ferrooxidans using S0 as energy source, the marginal difference in numbers of living iron- and sulfur-oxidizing bacteria becomes explainable (Figure S3, Supporting Information). Interestingly, a high density of cells attached to particles of laterite tailings was observed in the Ae bioreactor only (Figure S6, Supporting Information). Although attached cells to laterite tailings were observed in the Ae-An bioreactor during the three first days (Figure S6, Supporting Information), a change in the atmosphere from aerobic to anaerobic conditions provoked a drastic decreased colonization of the laterite tailings (Figure S6, Supporting Information). In contrast, most of the cells in the An bioreactor were observed as planktonic cells over the experimental time (Figure S6, Supporting Information). It has been shown recently that some genes could be differentially expressed in response to the presence of different members in the consortia.34 The understanding of biofilm formation in a consortium is crucial to develop procedures to influence biomining/bioremediation processes.35,36

recently proposed AnRD.21−25 Therefore, reductive dissolution of laterite can be further classified into aerobic and anaerobic reductive dissolution (AeRD or AnRD, respectively) (Figure 4). The data, taken together, show that At. thiooxidans is capable of bioaccelerating the reductive dissolution of ferric iron oxides (e.g., laterite tailings) under aerobic conditions following acidolysis in combination with bioreduction of ferric iron. The iron reduction activity is a trait conserved in the species At. thiooxidans, being consistent with the few studies addressing ferric iron reduction in others strains of the species.6,10,41,42 Evidence indicates the presence of a nongrowth-associated iron reduction activity and the absence of anaerobic ferric iron respiration in At. thiooxidans.10 Whether iron reduction might be mediated directly by one of the enzymes or indirectly by any RISCs with high reducing power is a matter of future work in progress. The only iron-oxidizing acidophilic proteobacteria known to present dissimilatory ferric iron reduction activity are the species At. ferrooxidans, At. ferrivorans, At. ferridurans, and Acidiferrobacter thiooxydans, which can couple the oxidation of S0 to the reduction of ferric iron, as well as Gram-positive acidophilic iron-oxidizers.37 At. ferrooxidans and At. ferridurans also have the capability to use hydrogen as electron donor to reduce ferric iron under anaerobic conditions.9,38 Although iron-oxidation pathways have been more extensively studied in acidophilic bacteria, those for iron reduction are almost unknown. Attempts to identify specific genes or proteins involved in the ferric reduction pathways in the iron-oxidizing acidophilic bacterium At. ferrooxidans using proteomics and transcriptomics were not successful, and no iron ferric reductase has been found until today.39,40 The reductive dissolution of ferric oxides catalyzed by different acidophilic microorganisms has been observed to be restricted to microaerophilic or anaerobic conditions.2 An almost unexplored area is the application of acidophiles in bioleaching of oxide ores under aerobic conditions. Previous reports showed acidolysis as the unique mechanism of dissolution of laterite by At. thiooxidansT.17,19,20,43 Combining the results described here with those from our previous studies, it is possible to infer that At. thiooxidans is capable of bioaccelerating the reductive dissolution of ferric iron oxides



DISCUSSION Nickel laterites are becoming an increasingly important source of nickel and cobalt. The increasing global demand and the fluctuating price of these target metals on the commodities market and the future depletion of the sulfidic ores have motivated one to look for alternative strategies for the bioprocessing of laterite ores or tailings, the remains of laterite ore processing. The AeRD of laterite tailings for recovering nickel and cobalt described in these trials meets several advantages over bioleaching via organic acid production by fungi14−16 or the 6679

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Environmental Science & Technology (e.g., laterite tailings) under aerobic conditions following acidolysis in combination with bioreduction of ferric iron. The iron reduction activity by the coculture of At. thiooxidans and At. ferrooxidans has not been previously predicted. The growth stage (pH 1.5−0.8) and the first 2 days of the bioleaching stage (0.8−1.6) impose a physiological-genetic adaptation of At. ferrooxidans in the mixed culture with At. thiooxidans to oxidize elemental sulfur at low pH. It has been found that the iron oxidation pathway is inhibited and the production of ferrous iron is stimulated in pure culture of At. ferrooxidans strain RAM-6F grown at extremely low pH.44 Under these conditions, resting cells of At. ferrooxidans produce ferrous iron during oxidation of sulfur to maintain the proton balance at low pH.44 Therefore, a complemented action on ferric iron reduction mediated by active sulfur-oxidizing At. thiooxidans cells and resting cells of At. ferrooxidans is found in the consortium during the first 2 days of the bioleaching stage at pH values between 0.8 and 1.6. The overall action is the iron reduction activity of the consortium, which together with the laterite attack by protons, produced biologically by the acidophilic culture, accelerate the dissolution of laterite. It would be expected that iron oxidation pathways of At. ferroooxidans are activated at pH above 1.6.44 However, the ferric iron concentration remained low and somehow constant, and ferrous iron was further produced in the Ae bioreactor inoculated with the consortium once the pH reached 1.6 due to the addition of laterite in the bioleaching stage. It cannot be ruled out that the adaptation to pH values above 1.6 and a consequent activation of iron oxidation pathways requires more than 12 days since no iron oxidation was detected during the time of the experiment. Living cells of At. ferrooxidans were present during the bioleaching experiment as indicated by the MPN data for iron-oxidizers. The production of active metabolites or intermediates by the acidophilic consortium could be considered. At. thiooxidans could produce intermediates with an inhibitory effect on the iron-oxidation pathways of At. ferrooxidans, which is forced to oxidize sulfur and not ferrous iron. It could be possible that some of the RISCs produced by the sulfur oxidation pathways could reduce ferric iron chemically and consequently produce ferrous iron. Contrarily, intermediates produced by At. ferrooxidans could upregulate the direct or indirect mechanism of ferric iron reduction mediated by At. thiooxidans. It might be possible that such biochemical intermediates allow cell−cell communication and interaction between the two acidophiles in coculture, provoking an improved activity of iron reduction. Previous studies have demonstrated an improvement in sulfide ore bioleaching using cocultures instead of pure cultures.11,34 The AeRD of laterite tailings by the coculture of At. thiooxidans and At. ferrooxidans consumed two times less acid than the AnRD by At. ferrooxidans alone. Previous studies have shown that AnRD consumes high amounts of acid for the extraction of nickel from goethite (α-FeOOH), cobalt and manganese from asbolane ((Co,Ni)1−y(MnO2)2−x(OH)2−2y+2x × n(H2O)), or copper from laterite using At. ferrooxidans.21,23,24 In the recently proposed Ferredox process, of 140 mmol of sulfuric acid consumed in 20 days, 75 mmol was already consumed within the first 3 days of operation.21,22 Similarly, the reductive dissolution of low-grade limonitic nickel laterite at 5% pulp density using A. ferrooxidans NCIMB 11820 consumed thereto about 70 mmol of sulfuric acid during the first days of the process of the 140 mmol in total.23

The release of ferric iron from ferric iron oxides in acidic solution by acidolysis (eq 1) is a very slow reaction as confirmed by the results of the not inoculated reactors. It is documented that scavenging the ferric iron by chelating agents or by reduction of ferric iron to ferrous iron provokes disequilibrium between ferric iron in solution and in the solid phase causing an acceleration of the release of ferric iron into solution.45,46 It could be predicted that iron may be released from an ore matrix by protons and the associated nickel and cobalt could dissolve according to eqs 2 and 3, respectively, and remain in the aqueous phase complexed with sulfate.47 FeOOH(s) + 3H+ → Fe3 + + 2H 2O

(1)

NiOOH(s) + 3H+ → Ni 2 + + 2H 2O

(2)

CoOOH(s) + 3H+ → Co2 + + 2H 2O

(3)

Noteworthy, the ferrous-based PLS is one of the main advantages of reductive dissolution since it facilitates downstream recovery of the metals and avoids interference with target metals recovery methods. Thereby, it reduces the loss of metals during iron precipitation (Figure 4).22,48 The scheme of the Ferredox process includes one stage to produce a ferrous-based acid lixiviant by reductive acid generation.22 This stage is based on the previous prediction that the oxidation of sulfur coupled to the reduction of soluble ferric iron by At. ferrooxidans generates four times more protons (acidity) per mol of sulfur oxidized than the equivalent reaction in which oxygen is used as the electron acceptor in place of ferric iron (eqs 4 and 5, respectively).22 However, the prediction is unlikely since higher growth rates of At. ferrooxidans grown on sulfur are observed under aerobic conditions compared to anaerobic conditions.40 This finding was confirmed by an upregulation of central carbon pathways under aerobic conditions, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth.40 The biomass yield of At. thiooxidans and the proton release through the oxidation of S0 under aerobic conditions are considered to be higher than that of At. ferrooxidans, under either aerobic or anaerobic conditions. This results in a higher production of acid lixiviant via the aerobic biooxidation of sulfur by At. thiooxidans than via the anaerobic biooxidation of sulfur using ferric iron as electron acceptor by At. ferrooxidans.5 Therefore, we propose that the overall aerobic acid generation process using a sulfur-oxidizing consortium with iron reduction ability under aerobic conditions might be considered as highly efficient for the production of an acid lixiviant agent (Figure 4). S0 + 6Fe3 + + 4H 2O → 6Fe 2 + + SO4 2 − + 8H+

(4)

S0 + 1.5O2 + H 2O → SO4 2 − + 2H+

(5)

The release of a similar amount of nickel and cobalt than for AnRD in a ferrous-based PLS with less consumption of acid in the case of the AeRD together with the possibility to eliminate an anoxic atmosphere points to an economic advantage reducing capex required for the future application of AeRD in biomining of laterites (Figure 4). Although tank biooxidation is carried out under controlled and homogeneous conditions, some microorganisms inhabiting biomining environments or ores could be enriched and dominate biomining tanks or bioleaching operations since ores are not sterile and cannot be sterilized on a commercial 6680

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scale.49 Leptospirillum and different Acidithiobacillus species have been found to be selected in sulfidic ore bioheaps over At. ferrooxidans.48 In aerobic leaching of metal oxides by a reductive process, the enrichment of purely ferrous iron oxidizers (e.g., Leptospirillum spp.) could be detrimental for the AeRD since the iron oxidation reaction could increase the ferric iron concentration leading to a deceleration of laterite dissolution. Whether purely iron-oxidizers could be present and dominate reductive tank or operational processes under aerobic conditions is an important question to be addressed during AeRD process development. The Ae-An bioreactor described here could be an attractive alternative if iron-oxidizers are enriched during the aerobic process. The anaerobiosis excludes potentially interfering obligate aerobes (such as Leptospirillum), and only facultative anaerobe iron-oxidizers (such as At. ferrooxidans) could continue the ferric iron reduction through sulfur oxidation. Thus, when designing an aerobic reductive leaching process, important considerations are (i) a high cell density of the initial culture with reductive capacity (higher than 109 cells/mL) and (ii) a continuous monitoring of the iron reduction/oxidation rates by the quantification of the soluble ferrous/ferric iron concentration or the related redox potential, in order to switch between AeRD and Ae-AnRD by controlling the gas composition. For commercial bioleaching of laterite ores, operational costs are another important issue. Lower opex for AeRD in comparison to AnRD for less acid consumption and less nitrogen gassing are reasons to favor AeRD, even if an anoxic phase is required (Ae-AnRD) to prevent growth of ferrous iron-oxidizers. Further work in this respect is required for a process development. Although this is the first comprehensive study describing AeRD and Ae-AnRD applied to laterite tailings, the approach described could also be modified and optimized for other lateritic resources. Furthermore, it significantly expands the applicability of sulfur-oxidizing aerobic acidophiles with iron reduction ability to biomining of oxide minerals, a field which has just begun to be explored.



REFERENCES

(1) Lovley, D. R. Dissimilatory Fe(III)- and Mn(IV)-reducing prokaryotes. In The prokaryotes, 3rd ed.; Dworkin, M., et al., Eds.; Springer: New York, 2006; Vol. 2, pp 635−658. (2) Coupland, K.; Johnson, D. B. Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria. FEMS Microbiol. Lett. 2008, 279, 30−35. (3) Brierley, C. L.; Brierley, J. A. Progress in bioleaching: Part B: Applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 2013, 97, 7543−7552. (4) Kelly, W.; Donovan, K. Proposal for a new class within the phylum Proteobacteria, Acidithiobacillia classis nov., with the type order Acidithiobacillales, and emended description of the class Gammaproteobacteria. Int. J. Syst. Environ. Microbiol. 2013, 63 (8), 2901−2906. (5) Olson, G. J.; Brierley, J. A.; Brierley, C. L. Bioleaching review part B: Progress in bioleaching: Applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 2003, 63 (3), 249−257. (6) Brock, T. D.; Gustafson, J. Ferric iron reduction by sulfur- and iron-oxidizing bacteria. Appl. Environ. Microbiol. 1976, 32, 567−571. (7) Pronk, J. T.; De Bruyn, J. C.; Bos, P.; Kuenen, J. G. Anaerobic growth of Thiobacillus ferrooxidans. Appl. Environ. Microbiol. 1992, 58, 2227−2230. (8) Das, A.; Mishra, A. K.; Roy, P. Anaerobic growth on elemental sulfur using dissimilar iron reduction by autotrophic Thiobacillus ferrooxidans. FEMS Microbiol. Lett. 1992, 97, 167−172. (9) Ohmura, N.; Sasaki, K.; Matsumoto, N.; Saiki, H. Anaerobic respiration using Fe3+, S0 and H2 in the chemolithotrophic bacterium Acidithiobacillus ferrooxidans. J. Bacteriol. 2002, 184, 2081−2087. (10) Hallberg, K. B.; Thomson, H. E. C.; Boeselt, I.; Johnson, D. B. Aerobic and anaerobic sulfur metabolism by acidophilic bacteria. In Biohydrometallurgy: Fundamentals, Technology and Sustainable Development, Process Metallurgy 11A; Ciminelli, V. S. T., Garcia, O., Jr., Eds.; Elsevier Press: Amsterdam, 2001; pp 423−431. (11) Rawlings, D. E.; Coram, N. J.; Gardner, M. N.; Deane, S. M. Thiobacillus caldus and Leptospirillum ferrooxidans are widely distributed in continuous-flow biooxidation tanks used to treat a variety of metal-containing ores and concentrates. In Biohydrometallurgy and the environment toward the mining of the 21st century. Part A.; Amils, R., Ballester, A., Eds.; Elsevier Press: Amsterdam, 1999; pp 777−786. (12) Schippers, A.; Hedrich, S.; Vasters, J.; Drobe, M.; Sand, W.; Willscher, S. Biomining: Metal recovery from ores with microorganisms. In Geobiotechnology I; Schippers, A., Glombitza, F., Sand, W., Eds.; Advances in Biochemical Engineering/Biotechnology; Springer-Verlag: Berlin, 2014; pp 1−47. (13) Berger, V. I.; Singer, D. A.; Bliss, J. D.; Moring, B. C. Ni-Co laterite deposits of the world; Database and grade and tonnage models; U.S. Geological Survey Open-File, Report 2011-1058, 2011; http:// pubs.usgs.gov/of/2011/1058/. (14) Bosecker, K. Leaching of lateritic nickel ores with heterotrophic microorganisms. In Fundamental and Applied Biohydrometallurgy; Lawrence, R. W., Branion, R. M. R., Ebner, H. G., Eds.; Elsevier Press: Amsterdam, 1986; pp 367−382. (15) Franz, A.; Burgstaller, W.; Schinner, F. Leaching with P. simplicissimum: Influence of metals and buffers on proton extrusion and citric acid production. Appl. Environ. Microbiol. 1991, 57, 769− 774. (16) Valix, M.; Tang, J. Y.; Cheung, W. H. The effects of mineralogy on the biological leaching of nickel laterite ores. Miner. Eng. 2001, 14 (12), 1629−1635. (17) Coto, O.; Galizia, F.; González, E.; Hernández, I.; Marrero, J.; Donati, E. Cobalt and nickel recoveries from lateritic tailings by organic and inorganic bioacids. Adv. Mater. Res. 2007, 20−21, 107− 110. (18) Simate, G. S.; Ndlovu, S. Bacterial leaching of nickel laterites using chemoautotrophic microorganisms: identifying influential factors using statistical design of experiments. Int. J. Miner. Process. 2008, 88, 31−36.

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figures S1−S6 and Supplementary Tables S1 and S2 showing additional study details. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b00944.



Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; fax: +49 511 643 2304; tel.: +49 511 643 2219. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The work of J.M. was financially supported by the award of the Georg Foster Fellowship of Alexander von Humboldt Foundation, Germany. A.S. and O.C. acknowledge the financial support given to this project by the German BMBF (BioLat grant 01DN14021). The authors would like to thank Mario Vera for image processing and discussion on cell attachment and Sabrina Hedrich for her advice and helpful comments. 6681

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(40) Osorio, H.; Mangold, S.; Denis, Y.; Ñ ancucheo, I.; Esparza, M.; Johnson, D. B.; Bonnefoy, V.; Dopson, M.; Holmes, D. S. Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in the extremophile Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 2013, 79 (7), 2172−2181. (41) Kino, K.; Usami, S. Biological reduction of ferric iron by ironand sulphur-oxidizing bacteria. Agric. Biol. Chem. 1982, 46 (3), 803− 805. (42) Donati, E.; Pogliani, C.; Curutchet, G. Indirect bioleaching of covellite by T. thiooxidans with an oxidant agent. Biotechnol. Lett. 1995, 17, 1251−1256. (43) Coto, O.; Schippers, A. Influence of sulfur source on nickel and cobalt bioleaching from laterite tailings using Acidithiobacillus thiooxidans. In Transactions of Nonferrous Metals Society of China indexed by SCI-Expanded and EI; Qiu, G., Jiang, T., Qin, W., Liu, X., Yang, Y., Wang, H., Eds.; Central South Universty Press: Changsha, 2011; pp 579−583. (44) Sand, W. Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values. Biogeochemistry 1989, 7, 195−201. (45) Stumm, W.; Furrer, G. The dissolution of oxides and aluminium silicates; Examples of surface-coordination-controlled kinetics. In Aquatic Surface Chemistry; Stumm, W., Ed.; John Wiley & Sons: New York, 1987; pp 197−219. (46) Banwart, S.; Davies, S.; Stumm, W. The role of oxalate in accelerating the reductive dissolution of hematite (α-Fe2O3) by ascorbate. Colloids Surf. 1989, 39, 303−309. (47) Georgiou, D.; Papangelakis, V. G. Sulphuric acid pressure leaching of a limonitic laterite: Chemistry and kinetics. Hydrometallurgy 1998, 49 (1−2), 23−46. (48) Willis, B. Downstream processing options for nickel laterite heap leach liquors. In Proceedings of ALTA Nickel/Cobalt Conference, 2007. (49) Dopson, M.; Holmes, D. S. Metal resistance in acidophilic microorganisms and its significance for biotechnologies. Appl. Microbiol. Biotechnol. 2014, 98, 8133−8144.

(19) Coto, O.; Galizia, F.; Hernandez, I.; Marrero, J.; Donati, E. Cobalt and nickel recoveries from lateritic tailings by organic and inorganic bio-acids. Hydrometallurgy 2008, 94, 18−22. (20) 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. (21) Hallberg, K. B.; Grail, B. M.; du Plessis, C.; Johnson, D. B. Reductive dissolution of ferric iron minerals: A new approach for bioprocessing nickel laterites. Miner. Eng. 2011, 24, 620−624. (22) du Plessis, C. A.; Slabbert, W.; Hallberg, K. B.; Johnson, D. B. Ferredox: A biohydrometallurgical processing concept for limonitic nickel laterites. Hydrometallurgy 2011, 109, 221−229. (23) Johnson, D. B.; Grail, B. M.; Hallberg, K. B. A new direction for biomining: Extraction of metals by reductive dissolution of oxidized ores. Minerals 2013, 3, 49−58. (24) Ñ ancucheo, I.; Grail, B. M.; du Hilario, F.; Plessis, C.; Johnson, D. B. Extraction of copper from an oxidised (lateritic) ore using bacterially-catalysed reductive dissolution. Appl. Microbiol. Biotechnol. 2014, 98, 6297−6305. (25) Johnson, D. B.; du Plessis, C. A. Biomining in reverse gear: Using bacteria to extract metals from oxidised ores. Miner. Eng. 2014; http://dx.doi.org/10.1016/j.mineng.2014.09.024. (26) Wakeman, K.; Auvinen, H.; Johnson, D. B. Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore. Biotechnol. Bioeng. 2008, 101 (4), 739−750. (27) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (28) Lovley, D. R.; Phillips, E. J. P. Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl. Environ. Microbiol. 1987, 53, 1536−1540. (29) Liang, G.; Tang, J.; Liu, W.; Zhou, Q. Optimizing mixed culture of two acidophiles to improve copper recovery from printed circuit boards (PCBs). J. Hazar. Mater. 2013, 250−251, 238−245. (30) Habashi, F. Engineering aspects. In Textbook of Hydrometallurgy; Habashi, F., Ed. Metallurgie Extractive: Quebec, 1999; pp 163−164. (31) Schippers, A.; Bosecker, K. Bioleaching: Analysis of microbial communities dissolving metal sulfides. In Methods in Biotechnology, Vol. 18, Microbial Processes and Products; Barredo, J. L., Ed.; Humana Press Inc.: Totowa, NJ, 2005; pp 405−412. (32) Breuker, A.; Stadler, S.; Schippers, A. Microbial community analysis of deeply buried marine sediments of the New Jersey shallow shelf (IODP Expedition 313). FEMS Microbiol. Ecol. 2013, 85 (3), 579−592. (33) Kock, D.; Schippers, A. Quantitative microbial community analysis of three different sulfidic tailings dumps generating acid mine drainage. Appl. Environ. Microbiol. 2008, 74, 5211−5219. (34) Xu, Y.; Yin, H.; Jiang, H.; Liang, Y.; Guo, X.; Ma, L.; Xiao, Y.; Liu, X. Comparative study of nickel resistance of pure culture and coculture of Acidithiobacillus thiooxidans and Leptospirillum feriphilum. Arch. Microbiol. 2013, 195 (9), 637−646. (35) Vera, M.; Schippers, A.; Sand, W. Progress in bioleaching: Fundamentals and mechanisms of bacterial metal sulfide oxidation part A. Appl. Microbiol. Biotechnol. 2013, 97, 7529−7541. (36) Bellenberg, S.; Díaz, M.; Noel, N.; Sand, W.; Poetsch, A.; Guiliani, N.; Vera, M. Biofilm formation, communication and interactions of leaching bacteria during colonization of pyrite and sulfur surfaces. Res. Microbiol. 2014, 165 (9), 773−781. (37) Hedrich, S.; Schlömann, M.; Johnson, D. B. The iron-oxidizing proteobacteria. Microbiology 2011, 157, 1551−1564. (38) Hedrich, S.; Johnson, D. B. Acidithiobacillus ferridurans sp. nov., an acidophilic iron-, sulfur- and hydrogen-metabolizing chemolithotrophic gammaproteobacterium. Int. J. Syst. Evol. Microbiol. 2013, 63, 4018−4025. (39) Kucera, J.; Bouchal, P.; Cerna, H.; Potesil, D.; Janiczek, O.; Zdrahal, Z.; Mandl, M. Kinetics of anaerobic elemental sulfur oxidation by ferric iron in Acidithiobacillus ferrooxidans and protein identification by comparative 2-DE-MS/MS. Antonie van Leeuwenhoek 2012, 101, 561−573. 6682

DOI: 10.1021/acs.est.5b00944 Environ. Sci. Technol. 2015, 49, 6674−6682