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Reductive dissolution of minerals and selective recovery of metals using acidophilic iron- and sulfate-reducing acidophiles
Hydrometallurgy 127-128 (2012) 172–177
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Reductive dissolution of minerals and selective recovery of metals using acidophilic iron- and sulfate-reducing acidophiles☆ D. Barrie Johnson ⁎ School of Biological Sciences, Bangor University, Deiniol Road, Bangor LL57 2UW, UK
a r t i c l e
i n f o
Available online 3 August 2012 Keywords: Iron reduction Reductive mineral dissolution Metal precipitation Sulfate reduction Sulfidogenesis
a b s t r a c t Most microbiological applications in biohydrometallurgy use the abilities of some acidophilic bacteria and archaea to catalyze oxidative transformations of metals (e.g. iron) and non-metals (e.g. sulfur), and thereby either to facilitate metal extraction and recovery (bioleaching and bio-oxidation), or to immobilize metals and metalloids (iron, arsenic etc.) in bioremediation of mine wastes. Many acidophiles, including species more well known as iron- and sulfur-oxidizers, can also catalyze reductive transformations of these elements in anoxic or micro-aerobic environments, though the biotechnological potential of the latter has, for the most part, been ignored. Three potential applications of iron- and sulfate-reduction mediated by acidophilic bacteria are described in this review. The first of these uses the chemolithotroph Acidithiobacillus ferrooxidans to accelerate the dissolution of ferric iron oxy-hydroxides by coupling the oxidation of elemental sulfur to iron reduction when, grown under anaerobic conditions. This is the key reaction in the “Ferredox” process for extracting nickel from lateritic ores. Secondly, a continuous flow ferrous iron-generating bioreactor is described in which the heterotrophic acidophile Acidiphilium SJH, immobilized on porous beads, is used to couple the oxidation of glycerol to the reduction of soluble ferric iron in feed liquors. Iron-reducing bioreactors have potential both in mineral bio-processing (e.g. indirect leaching of oxidized ores) and mine water remediation. A laboratory-scale (2 L) reactor was demonstrated to reduce between 90 and 99.9% of ferric iron at dilution rates of up to 0.87 h−1, with ~1 g of ferric iron being reduced h−1 at the highest flow rates. The heterotrophic iron-reducing system also has the advantage of not requiring anoxic conditions to operate efficiently. Thirdly, selective precipitation of copper and zinc from synthetic mine waters containing a variety of soluble metals has been demonstrated in anaerobic bioreactors, operated at pH 2.2–3.8, using novel consortia of acidophilic sulfate-reducing bacteria. Environmentally-benign technologies for remediating acid mine and process waters, using controlled biosulfidogenesis in acidic liquors to ameliorate pH and to recover dissolved metals, have many advantages over conventional remediation strategies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Although biohydrometallurgy has established a distinct niche in the metal/mineral industries, the full potential of biological processes to extract metals from primary ores and secondary materials, and to recover metals from process waters and remediate metal-contaminated waste waters, has not been exploited. In the case of mineral bioprocessing, for example, all current full-scale applications involve using microbial consortia to accelerate the oxidative dissolution of sulfides in order to enhance the extraction of base and precious metals that are either an integral part of (e.g. copper in chalcocite) or intimately associated with (e.g. gold in refractory ores) the minerals (Rawlings and Johnson, 2007). Biosorption (using living or dead microbial, or other, biomass to remove metals from contaminated waters) has been the subject of ☆ This paper was originally presented at the International Biohydrometallurgy Symposium (IBS), Changsha, China, 18-22 September 2011. ⁎ Tel.: +44 1248 382358; Fax: +44 1248 370731. E-mail address: [email protected]. 0304-386X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hydromet.2012.07.015
large amount of research activity and the subject of many publications, though few full-scale applications have so far emerged from this technology (Kotrba et al., 2011). Bioaccumulation of metals by higher plants from soils and mine wastes has also been demonstrated to have potential for remediating sites contaminated by metal mining (Krämer, 2010). Some acidophilic bacteria and archaea catalyze reductive transformations of iron and sulfur, including several species (such as Acidithiobacillus ferrooxidans) that are better known for their propensities for oxidative transformations. While this phenomenon has received relatively little attention in terms of either fundamental or applied research, data are emerging which highlight the potential of reductive bioprocesses, mediated by acidophilic microorganisms for extracting and recovering metals. Three examples of this are described in this review. 2. Recovery of metals from oxidized ores by bacterially-catalyzed reductive mineral dissolution Metals occur in oxidized, as well as in reduced (sulfidic) ores. This is the case with nickel laterites, which are estimated to account for ~72%
D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177
of nickel ore reserves in the lithosphere (Dalvi et al., 2004). These are typically composed of an upper limonite zone that contains mainly ferric iron oxides, such as goethite (αFeO·OH), and a lower saprolite zone that is dominated by magnesium silicates. Nickel and cobalt, originating from the weathering surface parent material, are incorporated into the lattice structure of the precipitated hydrated iron oxides, either by co-adsorption or by substituting for iron. In the case of goethite, the modified mineral has a typical composition of (Fe0.97, Ni0.03, Co0.003)O·OH. Nickel laterites are not amenable to ferric iron-catalyzed oxidative dissolution, as are sulfide ores. There have been previous reports of enhanced nickel extraction from laterites by dissolving dissolution in strong mineral (sulfuric) acid or using an iron-chelating organic acid (e.g. citric acid), both of which can be produced by microorganisms (e.g. Coto et al., 2008; Simate and Ndlovu, 2008). However, rates of nickel recovery using these approaches are generally slow and yields of extraction are too limited to compete with conventional processing technologies. A different approach for extracting nickel from a limonite ore, in which acidophilic bacteria are used to mediate the reductive dissolution of oxidized minerals (chiefly goethite) with which most of the nickel is intimately associated, has been described (Hallberg et al., 2011) which is the core reaction in the “Ferredox” process (du Plessis et al., 2011). The dissimilatory reduction of ferric iron can be mediated by several species of heterotrophic and mixotrophic acidophiles, and some species of autotrophic acidophiles (reviewed in Johnson and Hallberg, 2009). Pronk et al. (1991) were the first to report the reduction of soluble ferric iron by At. ferrooxidans, but prior to the report of Hallberg et al. (2011) it was unclear whether this bacterium could also accelerate the reductive dissolution of ferric iron minerals, though the reductive dissolution of a number of such minerals (including goethite) had been reported for the obligately heterotroph Acidiphilium sp. SJH (Bridge and Johnson, 2000). At. ferrooxidans is a facultative anaerobe, and can couple the oxidation of elemental sulfur or hydrogen to the reduction of ferric iron in the absence of oxygen. Using sulfur, as opposed to an organic electron donor for iron reduction, has a number of advantages. For example, although the reductive dissolution of minerals like goethite is invariably an acid-consuming reaction, less protons are used when sulfur is used as the electron donor (illustrated in Eqs. (1) and (2), which are normalized in terms of goethite reduced; the organic electron donor illustrated in Eq. (2) is glucose): 0
þ
2þ
24α FeO·OH þ 4S þ 40H →24Fe
þ
2−
þ 4SO4 þ 32H2 O 2þ
24α FeO·OH þ C6 H12 O6 þ 48H →24Fe
þ 6CO2 þ 42H2 O
ð1Þ
ð2Þ
Details of the protocols used to assess the potential of At. ferrooxidans to enhance nickel extraction from a representative limonitic ore are contained in the report by Hallberg et al. (2011). In brief, following preliminary screening experiments in anaerobic serum bottles, bioleaching experiments were carried out in 2.3 L (working volume) bioreactors (Electrolab, U.K.) fitted with temperature and pH control (maintained at 30 °C and pH 1.8, respectively) and sparged continuously with either air or oxygen-free nitrogen (OFN). At. ferrooxidans was first grown aerobically on excess sulfur (50 g in each reactor vessel) to obtain planktonic cell numbers of >5×108 mL−1, at which point 112.5 g of milled (b 1 mm) limonite ore (which contained 7% (by wt) Fe, 0.4% Ni, 0.27% Cr, 0.13% Mn and 0.04% Co, with quartz (76%) and goethite (10%) as the major mineral phases) was added, and the gas supply changed to OFN. The bioreactors were operated in anaerobic mode for 30 days, and samples withdrawn at regular intervals to determine concentrations of soluble metals and the volume of 1 M sulfuric acid required to maintain pH homeostasis were recorded. A control culture, also maintained at 30 °C and pH 1.8 but under aerobic conditions, was also set up. Nickel was effectively leached from the limonite ore in the anaerobic bioreactors, and this was paralleled by an increase in concentration of
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soluble (ferrous) iron. The correlation between nickel and iron release was relatively weak (r2 = 0.76) during the early stages of reductive dissolution, but was much stronger (r2 =0.99) in the later stages (Fig. 1), and was interpreted as being due to a more readily leached nickel being exchangeable with protons (i.e. acid dissolution), while the bulk of the nickel recovered was intimately associated with the goethite fraction. There was also a strong correlation (r2 =0.97) between consumption of sulfuric acid and iron released from the goethite fraction of the ore, as predicted by Eq. (1). Over 75% of the nickel was extracted within the 30 day experimental period. In contrast, much smaller amounts of iron (exclusively ferric) and nickel were solubilized when aerobic conditions were maintained in the bioreactor (i.e. oxidative acid leaching; Fig. 1). Other metals were also found to be solubilized from the ore. These included manganese and cobalt, which were highly correlated (r2 =0.93). As was the case with nickel and iron, far greater amounts of manganese and cobalt were leached under anaerobic (140 mg Mn and 24 mg Co) than under aerobic conditions (40 mg Mn and 8 mg Co). The major deportment of both of these metals in the ore was the oxyhydroxide mineral asbolane ((Ni,Co)xMn(O,OH)4·nH2O). The oxidation state of manganese in asbolane is +4 and, since Mn(IV) is highly insoluble in pH 1.8 liquors (and therefore would not be detected as a soluble metal if its valency remained unchanged), the implication is that asbolane was also subjected to reductive dissolution. To confirm this, abiotic controls containing limonite ore with and without added ferrous iron were incubated at 30 °C (aerobically) for 7 days, and concentrations of soluble manganese and cobalt were measured. These were found to be much greater (35.6+/− 0.3 mg L−1, and 6.3 +/−0.4 mg L−1, respectively) in the ferrous iron-containing suspensions than in those incubated without ferrous iron (7.4+/−1.1 and 2.0 +/− 0.3 mg L−1). It was therefore concluded that, in the anaerobic bioreactors, At. ferrooxidans was accelerating the reductive dissolution of asbolane indirectly via its generation of ferrous iron. Chromium was also leached effectively in the anaerobic bioreactors. The major mineral that contained this metal in the limonite ore was chromite (FeCr2O4) in which the oxidation state of iron is ferrous (+2) and that of chromium is +3 (i.e. both metals are reduced). Chromite dissolution was considered to have occurred by direct acid attack on the mineral, and, under reducing conditions, both metals released would have been maintained in their reduced states. This is particularly significant in terms of chromium, as oxidation to the +6 state (as would be anticipated under aerobic conditions in the presence of ferric iron, and also under alternative processing options such as high pressure acid leaching) is undesirable given the highly toxic nature of Cr(VI). By using an acidophilic bacterium to catalyze reductive dissolution of the various minerals in the limonite ore, it was possible to maintain low solution pH throughout and therefore retain the metals that were released in the solution. This, in turn, facilitates their recovery in downstream processing.
Fig. 1. Comparison of iron (●, ○) and nickel (■, □) solubilized from the milled nickel limonite ore under anaerobic (solid symbols) or aerobic (hollow symbols) conditions. Modified from Hallberg et al. (2011).
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Table 1 shows the comparative costs of the main reagent (elemental sulfur) and of the metal values obtained on the basis of typical commodity prices in 2011. Most (~90%) of the sulfur used in the reductive dissolution bio-processing of nickel laterites is in the sulfuric acid consumed in maintaining the required acidity of the leach liquor. The remaining ~10% is that used as an electron donor coupled to ferric iron reduction.
3. Construction and testing of a laboratory-scale, continuous flow fixed bed ferric iron-reduction bioreactor There have been a number of reports describing fixed bed bioreactors containing immobilized iron-oxidizing bacteria, where the objective is to oxidize ferrous iron present in process or waste waters to the oxidized (ferric) form. Most of these have been designed to mitigate acidic, iron-containing mine drainage streams (e.g. Rowe and Johnson, 2008), as oxidation of ferrous iron (which occurs very slowly as an abiotic reaction at pH b 3.5 and ambient temperatures) is a prerequisite to efficient removal of soluble iron from mine waters (the solubility product (log Ksp) of Fe(OH)3 (− 38.6) is significantly smaller than that of Fe(OH)2 (− 16.3)). Similar bioreactors can also be used to generate ferric iron as an oxidant for indirect mineral processing, though because of the larger concentrations of ferric iron and lower pH of the liquors required, Leptospirillum spp. (rather than At. ferrooxidans and “Ferrovum myxofaciens”) are more appropriate iron-oxidizing bacteria in this context (Kinnunen and Puhakka, 2004). In other situations, however, there would be perceived advantages in generating ferrous iron-rich acidic liquors, for example: (i) where bioremediation of mine waters uses a reductive rather than an oxidative route, involving both sulfate and iron reduction; (ii) for indirect reductive dissolution of oxidized ores and concentrates, using a system analogous to indirect oxidative dissolution of sulfide concentrates (Frias et al., 2008). A laboratory-scale iron reduction bioreactor was set up and operated as a continuous flow system over several months. A strain of Acidiphilium cryptum (SJH) was selected as the most appropriate for the design and modus operandi of the bioreactor, where the major criteria were rapid rates of iron reduction and simple engineering design and system operation. Acidiphilium spp. were the first acidophilic heterotrophs to be shown to catalyze the dissimilatory reduction of ferric iron and Acidiphilium SJH was subsequently shown to be a particularly adept iron-reducer, capable of using crystalline and amorphous ferric iron minerals as well as soluble ferric iron as electron acceptors (Bridge and Johnson, 2000). Other perceived advantages of Acidiphilium SJH are: (i) its tolerance of extremely low pH (pH minimum 1.8, an important trait, as reduction of soluble ferric iron is an acid-generating reaction); (ii) its ability to use a wide range of small molecular weight soluble electron donors; (iii) its ability to reduce ferric iron in the presence of oxygen, removing the necessity to operate the bioreactor under strictly anaerobic conditions; and (iv)
its inability (in contrast to At. ferrooxidans) to also oxidize ferrous iron, thereby avoiding the potential problem of iron cycling. Cells of Acidiphilium SJH, grown in 5 mM glycerol liquid medium (pH 2.5), were immobilized onto 1–2 mm diameter porous beads (manufactured from recycled glass by Poraver Dennert, GmbH, Germany) that had previously been acid-washed and heat-sterilized. The liquid medium was replaced several times and the inoculated beads were transferred to a 2.3 L bioreactor vessel, to give a packed bed bead volume of about 1.1 L. Additional liquid medium (~1 L) was added to the reactor vessel and two pH electrodes were inserted (one into the bead bed and the other extending only into the upper liquid phase (Fig. 2)). The bioreactor vessel was then coupled to a reservoir containing the feed liquor, which was added to the bioreactor vessel using a variable-speed peristaltic pump. Reduced liquor drained from the bioreactor vessel by gravity flow through a tube with its open end set at the bioreactor liquid surface. No attempts were made to exclude oxygen from this vessel (e.g. in the air-gap above the liquid in the vessel) or to de-oxygenate the feed liquor. A heating jacket was used to maintain the reactor vessel temperature at 30 °C. The standard feed liquor contained 10 mM ferric sulfate and 2 mM glycerol, pH adjusted to ~2.4 with sulfuric acid. Glycerol was selected as electron donor for the process (and also for the sulfidogenic bioreactors; Section 4) as it is both a suitable carbon and energy source for Acidiphilium SJH and a relatively low cost organic electron donor. The stoichiometry of glycerol oxidized to ferric iron reduced is 14:1 (Eq. (3)). Excess glycerol was provided to promote micro-aerobic conditions within the packed bed (i.e. some of the glycerol would be oxidized using molecular oxygen as electron acceptor) to generate the micro-aerobic conditions that are optimum for iron reduction by Acidiphilium SJH. 3þ
C3 H8 O3 þ 14Fe
2þ
þ 3H2 O→3CO2 þ 14Fe
þ 14H
þ
ð3Þ
The feed liquor was pumped into the bioreactor at variable rates (135 to 1950 mL h −1), corresponding to dilution rates of between 0.06 and 0.87 h−1 (the bioreactor volume was calculated as that of the combined packed bead bed and overlying liquor). pH values within the bead bed and in the surface liquor were recorded, and ferrous:ferric iron ratios in the effluent liquor were determined from redox potential measurements, using the Nernst equation. The Acidiphilium SJH bioreactor was highly efficient at reducing ferric iron to ferrous. At dilution rates of b 0.124 h−1, 99% of the
Pump
Feed liquor
pH electrodes
Table 1 Comparison of reagent (sulfur) cost and metal values in the reductive bio-processing of the nickel laterite ore. Amount consumed/ solubilized (kg/t ore) Reagent (sulfur) Acid consumption Reduction of goethitea Total S consumed Metal values Nickel Cobalt Chromium Total metal value
39 3.75 42.75
3.0 0.25 4.5
Price/kg (US $) 0.32 0.32 0.32
19 32 5.5
Estimated cost/ value (US $) 12.5 1.2 13.7
57 8 25 90
a Calculated on the basis that the stoichiometry of elemental sulfur oxidized to sulfate (a 6-electron reaction) to the reduction of ferric to ferrous iron (a single electron reaction) is 6:1.
Upper liquid phase
Acidiphiliumcolonised porous beads
Gravity drain
Bioreactor vessel Fig. 2. Set up of the ferric iron-reducing packed bed bioreactor.
D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177
inflowing iron was reduced, while at higher dilution rates (up to 0.87 h−1) conversion rates remained >90% (Fig. 3a). Flow rates and weights of ferric iron reduced were highly correlated (r2 >0.99), with ~1 g of ferric iron being reduced h−1 at the highest flow rate tested (Fig. 3b). As predicted by Eq. (3), the pH of the reduced liquor was always lower than that of the feed, but generally did not fall to below pH 2.2, which is well within the range for growth and activity of Acidiphilium SJH. Similar values were found within the bead bed and the surface liquor, indicating that most of the iron reduction was occurring within the lower packed bed layer. 4. Selective precipitation of transition metals from mine drainage waters using acidophilic sulfate-reducing bacterial consortia Mine water pollution is a global issue. While prevention of acid mine drainage (AMD) formation and migration is always the best option, in many situations this is not possible, and streams draining abandoned mines and mine spoils require treatment to prevent damage to the wider environment. The most common approaches used to remediate AMD – active chemical treatment and passive wetland/compost reactors – both have major detractions including, in both cases, non-recovery of metals and the production of metal-rich wastes (sludges and spent composts) that require careful disposal and storage. Active biological remediation, utilizing bacteria either to oxidize and precipitate iron, or to reduce sulfate and precipitate metal sulfides, is an alternative option which has the important advantage of facilitating the recovery of metals from waste. Waters draining metal mines often contain a variety of transition metals, such as copper, zinc manganese and iron, as well as other metals and metalloids, such as aluminum and arsenic. Many of these are chalcophilic (react with hydrogen sulfide) to form solid sulfide phases that have different solubility products, a trait that can be used, in theory, to remove many transition metals and As selectively from mine waters.
175
The key reactant involved is S2−, the concentration of which can be changed by varying solution pH, as illustrated by Eq. (4): −
þ
2−
þ
H2 S↔HS þ H ↔ S þ 2 H ρK 1 ¼ 7 ρK 2 ¼ 12:
As solution pH becomes more acidic liquors, the concentration of S 2− becomes increasingly small, thereby limiting the insoluble sulfides that can form to those with increasingly small solubility products. Control of pH can therefore be used to remove metals (and As) in a selective manner from mine waters. Aluminum does not form a sulfide phase, though it precipitates as hydroxides (gibbsite and basaluminite) at ~ pH 5, again allowing pH control to be used either to precipitate this metal or to cause it to remain in the solution. Hydrogen sulfide is generated as a waste product by dissimilatory sulfate- and sulfur-reducing bacteria and archaea. The vast majority of known species of these prokaryotes are highly pH-sensitive and are inactive or killed in acidic liquors such as AMD. This problem is circumvented in the BioSulfide process (Bratty et al., 2006) by separating bacterial growth chambers (that contain neutrophilic sulfur-reducing bacteria, rather than sulfate-reducers) and vessels where sulfide pumped from the bioreactors is contacted, at pre-determined pH values, with acidic metal-laden liquors. An alternative approach is to promote bacterial sulfate reduction and metal precipitation within a single reactor vessel maintained at low (and variable) pH, thereby simplifying engineering design and reducing construction and operation costs. The core requirement of such a system is a sulfidogenic microbial population that is active at an extremely low pH; such a consortium (active between pH 2.2 and 4.5) has recently been described by Ňancucheo and Johnson (2012). The inoculum used for this system came from two sources — a pure culture of an acid-tolerant Desulfosporosinus sp. (M1) isolated from the volcanic Caribbean island of Montserrat (Kimura et al., 2006), and an undefined sulfidogenic microbial mat found at an abandoned copper mine in Spain (Rowe et al., 2007). Details of the protocol used for the enrichment of the acidophilic sulfate-reducing mixed culture, and the set-up and operation of the sulfidogenic bioreactors that utilized this consortium, are given in Ňancucheo and Johnson (2012). In brief, two anaerobic bioreactors were operated in parallel for about one year as continuous flow systems. The bacteria were immobilized on Poraver beads in reactor vessels that contained ~1 L of packed colonized beads above which was ~1.2 L of liquor, similar to the set up described in Section 3. The acidic test solutions were pumped into the reactor vessels at rates required to maintain pre-determined values, using a pH electrode placed in the liquor above the packed bed coupled to a peristaltic pump. Glycerol was added to the feed liquors to act as an electron donor for sulfate reduction, in a proton-consuming reaction (Eq. (5)): þ
2−
2þ
4C3 H8 O3 þ 12H þ 7SO4 þ Cu →12CO2 þ 6H2 S þ CuS þ 16H2 O
Fig. 3. Ferric iron reduction in a packed bed bioreactor: (a) effect of flow rates on the percentages of ferric iron reduced; (b) relationship between flow rates and the weight of ferric iron reduced.
ð4Þ
ð5Þ
Eq. (5) shows the formation of copper sulfide, with a glycerol:copper molar ratio in the feed liquor of 4:1. By modifying this ratio (by either increasing the copper concentration or decreasing the glycerol concentration) it was possible to devise a situation where there is ultimately no production of free H2S and consequently no net consumption of protons (i.e. the reaction is pH-neutral). However, the bioreactor set-up requires a pH differential between the feed liquor and that of the liquor within the bioreactor vessel, and so some production of free H2S was an integral part of the process. Two acidic sulfidogenic bioreactors were operated with different feed liquors. In one case, this was an acidic (pH 2.5) solution containing 3 mM of both zinc and ferrous iron, and variable concentrations (0.5–30 mM) of aluminum, and was based on the composition of AMD draining an abandoned Zn mine (Cwm Rheidol) in mid-Wales. The second bioreactor was fed with a more acidic (pH 2.1–2.5) solution containing (for the first 320 days of operation) 1 mM each of copper, zinc and ferrous iron (later (for ~60 days) changed to 0.7 mM copper, 1 mM zinc and 5–10 mM
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D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177
ferrous iron) 3.3 mM aluminum and 0.2 mM manganese. This was a synthetic mine water based on the composition of AMD draining an abandoned copper mine (Mynydd Parys) in north-Wales. Samples were withdrawn from the bioreactors at regular intervals to measure concentrations of soluble metals, glycerol and acetic acid, numbers of bacteria (total cell counts) and to determine microbial diversity (from terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis of extracted DNA). Both bioreactors achieved the objectives of demonstrating sulfidogenesis and selective metal precipitation in acidic (pH 2.2 to 4.8) conditions. In the case of the bioreactor fed with synthetic Cwm Rheidol AMD, zinc was efficiently precipitated (>99%) as a sulfide inside the reactor while both aluminum and ferrous iron remained in solution (>99%) and were washed out of the reactor vessel (Fig. 4). Sulfidogenesis and selective precipitation of zinc continued when the aluminum concentration in the feed liquor was increased to >50-times that found typically in Cwm Rheidol AMD, though the presence of 30 mM Al was noted to have a small negative impact on the performance of the bioreactor. Selective precipitation of metal sulfides was also demonstrated in the second bioreactor. Throughout the test period, all of the copper present in the feed liquor was precipitated (confirmed as copper sulfide) within this bioreactor, but none of the ferrous iron. Zinc (sulfide) biogenesis was more variable. When the bioreactor pH was set at pH 3.6, >99% of the soluble zinc in the influent liquor was precipitated. By progressively lowering both the bioreactor pH and the concentration of the electron donor (glycerol) in the influent liquor, it was possible to retain increasing amounts of zinc in the solution. When the bioreactor was maintained at pH 2.4, the amount of zinc precipitated stayed reasonably stable at 47%+/−1 6% (23 sampling time points) as the glycerol concentration in the influent liquor was lowered from 4 to 1.5 mM. By decreasing the bioreactor pH still further (ultimately to pH 2.2) and the influent glycerol concentration to 0.7 mM, it was possible to precipitate only 8+/−2% (9 samples) of zinc within the bioreactor, while maintaining >99% removal of copper from the solution. Bacteria could be enumerated in the upper liquid phases of the bioreactors, but not on the bead surfaces. Numbers fluctuated between ~106 and 5×107 mL−1 and were primarily influenced by both flow rates and glycerol concentrations in the feed liquor. T-RFLP analysis showed that both bioreactors contained consortia of bacteria that comprised non-sulfidogenic acidophiles as well as sulfate-reducing bacteria. The latter included Desulfosporosinus sp. M1, a novel genus/ species (“Desulfobacillus acidavidus”. CL4) previously isolated from the same abandoned Spanish copper mine used for the inoculum, and a previously unknown Firmicute (strain CEB3) that was isolated from the
bioreactors and is currently being characterized. No archaeal genes were amplified from either bioreactor. Molecular analysis also showed that the compositions of the microbial consortia within the bioreactors changed as the operational parameters (in particular, pH) were moderated. Both systems were noted, however, to be very robust, with occasional problems encountered in their operation (e.g. feedback of copper sulfate from attached H2S-capture bottles) causing only short-term perturbations in bacterial activity and bioreactor performance. 5. Discussion and conclusions The experimental work described has demonstrated how reductive transformations of iron and sulfur by acidophilic bacteria can be used for both extracting metals from mineral ores and for selectively recovering in acidic liquors. The reductive dissolution of nickel limonites represents a radical development for “biomining”. This is, in essence, a reversal of the conventional approach (oxidative dissolution, using microbial-generated ferric iron) currently used for bio-processing metal ores. The demonstration of bacteria effectively catalyzing a process at 30 °C that is currently carried out at ~800 °C using pyrometallurgical processing further demonstrates the remarkable potential of microorganisms. Evidence for indirect bio-reduction of asbolane, as well as goethite, points to the reductive dissolution of minerals potentially being of generic application for processing oxidized ores. The acidophilic ferric iron-reducing bioreactor described demonstrates how simple, rapid and effective such a set-up system can be. A typical application of this technology would be for off-line bioleaching of oxidized ores where, as in the current “indirect” approach used for sulfides (Frias et al., 2008), the biological reaction (ferrous iron generation) and the abiotic reaction (ferrous iron reduction of susceptible oxidized minerals) would be carried out in separate vessels where conditions for both processes could be optimized. Lastly, the selective precipitation of metals from synthetic mine waters highlights how an environmentally-benign approach to those presently could be used to remediate AMD streams and to capture metals from process and waste waters. Selective precipitation as sulfides facilitates the recovery and recycling of metals, and this can be used to offset, at least in part, the cost of the treatment process. At a time where disposal of potentially useful and valuable materials, such as metals, in landfill is being increasingly viewed as undesirable and unacceptable, a radically new approach for remediating AMD from metal mines is timely. Acknowledgments The author would like to acknowledge the many students and research officers of the Bangor Acidophile Research Team (BART), and other colleagues who have been involved in helping devise and in carrying out the research described in this review. In particular, thanks are due to Dr. Chris du Plessis (Vale, Perth, Australia), Drs. Barry Grail, Ivan Ňancucheo and Kevin Hallberg and Ms. Gemma Bowsher (all BART personnel). References
Fig. 4. Selective precipitation of zinc from synthetic (pH 2.5) mine water containing 3 mM of both ferrous iron (▼) and zinc (♦), using a sulfidogenic bioreactor. pH values (●) within the bioreactor were progressively lowered (from 4.8 to 4.0) during the course of the trial. Aluminum concentrations in the influent liquor increased from 0.5 to 30 mM during the experiment, but no precipitation of aluminum was observed within the bioreactor. Modified from Ňancucheo and Johnson (2012).
Bratty, M., Lawrence, R.W., Kratochvil, D., Marchant, P.B., 2006. Applications of biological H2S production from elemental sulfur in the treatment of heavy metal pollution including acid rock drainage. Proceedings of the 7th International Symposium of Acid Rock Drainage (ICARD). St. Louis, MO, pp. 271–281. Bridge, T.A.M., Johnson, D.B., 2000. Reductive dissolution of ferric iron minerals by Acidiphilium SJH. Geomicrobiol. J. 17, 193–206. Coto, O., Galizia, F., Hernandez, I., Marrero, J., Donati, E., 2008. Cobalt and nickel recoveries from lateritic tailings by organic and inorganic bio-acids. Hydrometallurgy 94, 18–22. Dalvi, A.D., Bacon, W.G., Osborne, R.C., 2004. The past and the future of nickel laterites. PDAC 2004 International Convention, Trade Show & Investors Exchange, March 7–10, 2004. du Plessis, C.A., Slabbert, W., Hallberg, K.B., Johnson, D.B., 2011. Ferredox: a biohydrometallurgical processing concept for limonitic nickel laterites. Hydrometallurgy 109, 221–229. Frias, C., Carranza, F., Sanchez, F., Mazuelos, A., Frades, M., Romero, R., Diaz, G., Iglesias, N., 2008. New developments in indirect bioleaching of zinc and lead sulfide concentrates.
D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177 In: Young, C.A., Taylor, P.R., Anderson, C.G., Choi, Y. (Eds.), Hydrometallurgy 2008: Proceedings of the Sixth International Symposium. Society for Mining, Metallurgy and Exploration Inc., Eaglewood, Colorado, pp. 497–505. Hallberg, K.B., Grail, B.M., du Plessis, C., Johnson, D.B., 2011. Reductive dissolution of ferric iron minerals: a new approach for bioprocessing nickel laterites. Min. Eng. 24, 620–624. Johnson, D.B., Hallberg, K.B., 2009. Carbon, iron and sulfur metabolism in acidophilic micro-organisms. Adv. Microb. Physiol. 54, 202–256. Kimura, S., Hallberg, K.B., Johnson, D.B., 2006. Sulfidogenesis in low pH (3.8–4.2) media by a mixed population of acidophilic bacteria. Biodegradation 17, 57–65. Kinnunen, H.-M., Puhakka, J.A., 2004. High-rate ferric sulfate generation by a Leptospirillum ferriphilum-dominated biofilm and the role of jarosite in biomass retainment in a fluidized-bed reactor. Biotechnol. Bioeng. 85, 697–705. Kotrba, P., Mackova, M., Macek, T. (Eds.), 2011. Microbial Biosorption of Metals. Springer, New York. Krämer, U., 2010. Metal hyperaccumulation in plants. Annu. Rev. Plant Biol. 61, 517–534.
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Ňancucheo, I., Johnson, D.B., 2012. Selective removal of transition metals from acidic mine waters by novel consortia of acidophilic sulfidogenic bacteria. Microb. Biotechnol. 5, 34–44. Pronk, J.T., Liem, K., Bos, P., Kuenen, J.G., 1991. Energy transduction by anaerobic ferric iron reduction in Thiobacillus ferrooxidan. Appl. Environ. Microbiol. 57, 2063–2068. Rawlings, D.E., Johnson, D.B. (Eds.), 2007. Biomining. Springer-Verlag, Heidelberg. Rowe, O.F., Johnson, D.B., 2008. Comparison of ferric iron generation by different species of acidophilic bacteria immobilised in packed-bed reactors. Syst. Appl. Microbiol. 31, 68–77. Rowe, O.F., Sánchez-España, J., Hallberg, K.B., Johnson, D.B., 2007. Microbial communities and geochemical dynamics in an extremely acidic, metal-rich stream at an abandoned sulfide mine (Huelva, Spain) underpinned by two functional primary production systems. Environ. Microbiol. 9, 1761–1771. Simate, G.S., Ndlovu, S., 2008. Bacterial leaching of nickel laterites using chemoautotrophic microorganisms: identifying influential factors using statistical design of experiments. Int. J. Min. Proc. 88, 31–36.
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Hydrometallurgy journal homepage: www.elsevier.com/locate/hydromet
Reductive dissolution of minerals and selective recovery of metals using acidophilic iron- and sulfate-reducing acidophiles☆ D. Barrie Johnson ⁎ School of Biological Sciences, Bangor University, Deiniol Road, Bangor LL57 2UW, UK
a r t i c l e
i n f o
Available online 3 August 2012 Keywords: Iron reduction Reductive mineral dissolution Metal precipitation Sulfate reduction Sulfidogenesis
a b s t r a c t Most microbiological applications in biohydrometallurgy use the abilities of some acidophilic bacteria and archaea to catalyze oxidative transformations of metals (e.g. iron) and non-metals (e.g. sulfur), and thereby either to facilitate metal extraction and recovery (bioleaching and bio-oxidation), or to immobilize metals and metalloids (iron, arsenic etc.) in bioremediation of mine wastes. Many acidophiles, including species more well known as iron- and sulfur-oxidizers, can also catalyze reductive transformations of these elements in anoxic or micro-aerobic environments, though the biotechnological potential of the latter has, for the most part, been ignored. Three potential applications of iron- and sulfate-reduction mediated by acidophilic bacteria are described in this review. The first of these uses the chemolithotroph Acidithiobacillus ferrooxidans to accelerate the dissolution of ferric iron oxy-hydroxides by coupling the oxidation of elemental sulfur to iron reduction when, grown under anaerobic conditions. This is the key reaction in the “Ferredox” process for extracting nickel from lateritic ores. Secondly, a continuous flow ferrous iron-generating bioreactor is described in which the heterotrophic acidophile Acidiphilium SJH, immobilized on porous beads, is used to couple the oxidation of glycerol to the reduction of soluble ferric iron in feed liquors. Iron-reducing bioreactors have potential both in mineral bio-processing (e.g. indirect leaching of oxidized ores) and mine water remediation. A laboratory-scale (2 L) reactor was demonstrated to reduce between 90 and 99.9% of ferric iron at dilution rates of up to 0.87 h−1, with ~1 g of ferric iron being reduced h−1 at the highest flow rates. The heterotrophic iron-reducing system also has the advantage of not requiring anoxic conditions to operate efficiently. Thirdly, selective precipitation of copper and zinc from synthetic mine waters containing a variety of soluble metals has been demonstrated in anaerobic bioreactors, operated at pH 2.2–3.8, using novel consortia of acidophilic sulfate-reducing bacteria. Environmentally-benign technologies for remediating acid mine and process waters, using controlled biosulfidogenesis in acidic liquors to ameliorate pH and to recover dissolved metals, have many advantages over conventional remediation strategies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Although biohydrometallurgy has established a distinct niche in the metal/mineral industries, the full potential of biological processes to extract metals from primary ores and secondary materials, and to recover metals from process waters and remediate metal-contaminated waste waters, has not been exploited. In the case of mineral bioprocessing, for example, all current full-scale applications involve using microbial consortia to accelerate the oxidative dissolution of sulfides in order to enhance the extraction of base and precious metals that are either an integral part of (e.g. copper in chalcocite) or intimately associated with (e.g. gold in refractory ores) the minerals (Rawlings and Johnson, 2007). Biosorption (using living or dead microbial, or other, biomass to remove metals from contaminated waters) has been the subject of ☆ This paper was originally presented at the International Biohydrometallurgy Symposium (IBS), Changsha, China, 18-22 September 2011. ⁎ Tel.: +44 1248 382358; Fax: +44 1248 370731. E-mail address: [email protected]. 0304-386X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hydromet.2012.07.015
large amount of research activity and the subject of many publications, though few full-scale applications have so far emerged from this technology (Kotrba et al., 2011). Bioaccumulation of metals by higher plants from soils and mine wastes has also been demonstrated to have potential for remediating sites contaminated by metal mining (Krämer, 2010). Some acidophilic bacteria and archaea catalyze reductive transformations of iron and sulfur, including several species (such as Acidithiobacillus ferrooxidans) that are better known for their propensities for oxidative transformations. While this phenomenon has received relatively little attention in terms of either fundamental or applied research, data are emerging which highlight the potential of reductive bioprocesses, mediated by acidophilic microorganisms for extracting and recovering metals. Three examples of this are described in this review. 2. Recovery of metals from oxidized ores by bacterially-catalyzed reductive mineral dissolution Metals occur in oxidized, as well as in reduced (sulfidic) ores. This is the case with nickel laterites, which are estimated to account for ~72%
D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177
of nickel ore reserves in the lithosphere (Dalvi et al., 2004). These are typically composed of an upper limonite zone that contains mainly ferric iron oxides, such as goethite (αFeO·OH), and a lower saprolite zone that is dominated by magnesium silicates. Nickel and cobalt, originating from the weathering surface parent material, are incorporated into the lattice structure of the precipitated hydrated iron oxides, either by co-adsorption or by substituting for iron. In the case of goethite, the modified mineral has a typical composition of (Fe0.97, Ni0.03, Co0.003)O·OH. Nickel laterites are not amenable to ferric iron-catalyzed oxidative dissolution, as are sulfide ores. There have been previous reports of enhanced nickel extraction from laterites by dissolving dissolution in strong mineral (sulfuric) acid or using an iron-chelating organic acid (e.g. citric acid), both of which can be produced by microorganisms (e.g. Coto et al., 2008; Simate and Ndlovu, 2008). However, rates of nickel recovery using these approaches are generally slow and yields of extraction are too limited to compete with conventional processing technologies. A different approach for extracting nickel from a limonite ore, in which acidophilic bacteria are used to mediate the reductive dissolution of oxidized minerals (chiefly goethite) with which most of the nickel is intimately associated, has been described (Hallberg et al., 2011) which is the core reaction in the “Ferredox” process (du Plessis et al., 2011). The dissimilatory reduction of ferric iron can be mediated by several species of heterotrophic and mixotrophic acidophiles, and some species of autotrophic acidophiles (reviewed in Johnson and Hallberg, 2009). Pronk et al. (1991) were the first to report the reduction of soluble ferric iron by At. ferrooxidans, but prior to the report of Hallberg et al. (2011) it was unclear whether this bacterium could also accelerate the reductive dissolution of ferric iron minerals, though the reductive dissolution of a number of such minerals (including goethite) had been reported for the obligately heterotroph Acidiphilium sp. SJH (Bridge and Johnson, 2000). At. ferrooxidans is a facultative anaerobe, and can couple the oxidation of elemental sulfur or hydrogen to the reduction of ferric iron in the absence of oxygen. Using sulfur, as opposed to an organic electron donor for iron reduction, has a number of advantages. For example, although the reductive dissolution of minerals like goethite is invariably an acid-consuming reaction, less protons are used when sulfur is used as the electron donor (illustrated in Eqs. (1) and (2), which are normalized in terms of goethite reduced; the organic electron donor illustrated in Eq. (2) is glucose): 0
þ
2þ
24α FeO·OH þ 4S þ 40H →24Fe
þ
2−
þ 4SO4 þ 32H2 O 2þ
24α FeO·OH þ C6 H12 O6 þ 48H →24Fe
þ 6CO2 þ 42H2 O
ð1Þ
ð2Þ
Details of the protocols used to assess the potential of At. ferrooxidans to enhance nickel extraction from a representative limonitic ore are contained in the report by Hallberg et al. (2011). In brief, following preliminary screening experiments in anaerobic serum bottles, bioleaching experiments were carried out in 2.3 L (working volume) bioreactors (Electrolab, U.K.) fitted with temperature and pH control (maintained at 30 °C and pH 1.8, respectively) and sparged continuously with either air or oxygen-free nitrogen (OFN). At. ferrooxidans was first grown aerobically on excess sulfur (50 g in each reactor vessel) to obtain planktonic cell numbers of >5×108 mL−1, at which point 112.5 g of milled (b 1 mm) limonite ore (which contained 7% (by wt) Fe, 0.4% Ni, 0.27% Cr, 0.13% Mn and 0.04% Co, with quartz (76%) and goethite (10%) as the major mineral phases) was added, and the gas supply changed to OFN. The bioreactors were operated in anaerobic mode for 30 days, and samples withdrawn at regular intervals to determine concentrations of soluble metals and the volume of 1 M sulfuric acid required to maintain pH homeostasis were recorded. A control culture, also maintained at 30 °C and pH 1.8 but under aerobic conditions, was also set up. Nickel was effectively leached from the limonite ore in the anaerobic bioreactors, and this was paralleled by an increase in concentration of
173
soluble (ferrous) iron. The correlation between nickel and iron release was relatively weak (r2 = 0.76) during the early stages of reductive dissolution, but was much stronger (r2 =0.99) in the later stages (Fig. 1), and was interpreted as being due to a more readily leached nickel being exchangeable with protons (i.e. acid dissolution), while the bulk of the nickel recovered was intimately associated with the goethite fraction. There was also a strong correlation (r2 =0.97) between consumption of sulfuric acid and iron released from the goethite fraction of the ore, as predicted by Eq. (1). Over 75% of the nickel was extracted within the 30 day experimental period. In contrast, much smaller amounts of iron (exclusively ferric) and nickel were solubilized when aerobic conditions were maintained in the bioreactor (i.e. oxidative acid leaching; Fig. 1). Other metals were also found to be solubilized from the ore. These included manganese and cobalt, which were highly correlated (r2 =0.93). As was the case with nickel and iron, far greater amounts of manganese and cobalt were leached under anaerobic (140 mg Mn and 24 mg Co) than under aerobic conditions (40 mg Mn and 8 mg Co). The major deportment of both of these metals in the ore was the oxyhydroxide mineral asbolane ((Ni,Co)xMn(O,OH)4·nH2O). The oxidation state of manganese in asbolane is +4 and, since Mn(IV) is highly insoluble in pH 1.8 liquors (and therefore would not be detected as a soluble metal if its valency remained unchanged), the implication is that asbolane was also subjected to reductive dissolution. To confirm this, abiotic controls containing limonite ore with and without added ferrous iron were incubated at 30 °C (aerobically) for 7 days, and concentrations of soluble manganese and cobalt were measured. These were found to be much greater (35.6+/− 0.3 mg L−1, and 6.3 +/−0.4 mg L−1, respectively) in the ferrous iron-containing suspensions than in those incubated without ferrous iron (7.4+/−1.1 and 2.0 +/− 0.3 mg L−1). It was therefore concluded that, in the anaerobic bioreactors, At. ferrooxidans was accelerating the reductive dissolution of asbolane indirectly via its generation of ferrous iron. Chromium was also leached effectively in the anaerobic bioreactors. The major mineral that contained this metal in the limonite ore was chromite (FeCr2O4) in which the oxidation state of iron is ferrous (+2) and that of chromium is +3 (i.e. both metals are reduced). Chromite dissolution was considered to have occurred by direct acid attack on the mineral, and, under reducing conditions, both metals released would have been maintained in their reduced states. This is particularly significant in terms of chromium, as oxidation to the +6 state (as would be anticipated under aerobic conditions in the presence of ferric iron, and also under alternative processing options such as high pressure acid leaching) is undesirable given the highly toxic nature of Cr(VI). By using an acidophilic bacterium to catalyze reductive dissolution of the various minerals in the limonite ore, it was possible to maintain low solution pH throughout and therefore retain the metals that were released in the solution. This, in turn, facilitates their recovery in downstream processing.
Fig. 1. Comparison of iron (●, ○) and nickel (■, □) solubilized from the milled nickel limonite ore under anaerobic (solid symbols) or aerobic (hollow symbols) conditions. Modified from Hallberg et al. (2011).
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Table 1 shows the comparative costs of the main reagent (elemental sulfur) and of the metal values obtained on the basis of typical commodity prices in 2011. Most (~90%) of the sulfur used in the reductive dissolution bio-processing of nickel laterites is in the sulfuric acid consumed in maintaining the required acidity of the leach liquor. The remaining ~10% is that used as an electron donor coupled to ferric iron reduction.
3. Construction and testing of a laboratory-scale, continuous flow fixed bed ferric iron-reduction bioreactor There have been a number of reports describing fixed bed bioreactors containing immobilized iron-oxidizing bacteria, where the objective is to oxidize ferrous iron present in process or waste waters to the oxidized (ferric) form. Most of these have been designed to mitigate acidic, iron-containing mine drainage streams (e.g. Rowe and Johnson, 2008), as oxidation of ferrous iron (which occurs very slowly as an abiotic reaction at pH b 3.5 and ambient temperatures) is a prerequisite to efficient removal of soluble iron from mine waters (the solubility product (log Ksp) of Fe(OH)3 (− 38.6) is significantly smaller than that of Fe(OH)2 (− 16.3)). Similar bioreactors can also be used to generate ferric iron as an oxidant for indirect mineral processing, though because of the larger concentrations of ferric iron and lower pH of the liquors required, Leptospirillum spp. (rather than At. ferrooxidans and “Ferrovum myxofaciens”) are more appropriate iron-oxidizing bacteria in this context (Kinnunen and Puhakka, 2004). In other situations, however, there would be perceived advantages in generating ferrous iron-rich acidic liquors, for example: (i) where bioremediation of mine waters uses a reductive rather than an oxidative route, involving both sulfate and iron reduction; (ii) for indirect reductive dissolution of oxidized ores and concentrates, using a system analogous to indirect oxidative dissolution of sulfide concentrates (Frias et al., 2008). A laboratory-scale iron reduction bioreactor was set up and operated as a continuous flow system over several months. A strain of Acidiphilium cryptum (SJH) was selected as the most appropriate for the design and modus operandi of the bioreactor, where the major criteria were rapid rates of iron reduction and simple engineering design and system operation. Acidiphilium spp. were the first acidophilic heterotrophs to be shown to catalyze the dissimilatory reduction of ferric iron and Acidiphilium SJH was subsequently shown to be a particularly adept iron-reducer, capable of using crystalline and amorphous ferric iron minerals as well as soluble ferric iron as electron acceptors (Bridge and Johnson, 2000). Other perceived advantages of Acidiphilium SJH are: (i) its tolerance of extremely low pH (pH minimum 1.8, an important trait, as reduction of soluble ferric iron is an acid-generating reaction); (ii) its ability to use a wide range of small molecular weight soluble electron donors; (iii) its ability to reduce ferric iron in the presence of oxygen, removing the necessity to operate the bioreactor under strictly anaerobic conditions; and (iv)
its inability (in contrast to At. ferrooxidans) to also oxidize ferrous iron, thereby avoiding the potential problem of iron cycling. Cells of Acidiphilium SJH, grown in 5 mM glycerol liquid medium (pH 2.5), were immobilized onto 1–2 mm diameter porous beads (manufactured from recycled glass by Poraver Dennert, GmbH, Germany) that had previously been acid-washed and heat-sterilized. The liquid medium was replaced several times and the inoculated beads were transferred to a 2.3 L bioreactor vessel, to give a packed bed bead volume of about 1.1 L. Additional liquid medium (~1 L) was added to the reactor vessel and two pH electrodes were inserted (one into the bead bed and the other extending only into the upper liquid phase (Fig. 2)). The bioreactor vessel was then coupled to a reservoir containing the feed liquor, which was added to the bioreactor vessel using a variable-speed peristaltic pump. Reduced liquor drained from the bioreactor vessel by gravity flow through a tube with its open end set at the bioreactor liquid surface. No attempts were made to exclude oxygen from this vessel (e.g. in the air-gap above the liquid in the vessel) or to de-oxygenate the feed liquor. A heating jacket was used to maintain the reactor vessel temperature at 30 °C. The standard feed liquor contained 10 mM ferric sulfate and 2 mM glycerol, pH adjusted to ~2.4 with sulfuric acid. Glycerol was selected as electron donor for the process (and also for the sulfidogenic bioreactors; Section 4) as it is both a suitable carbon and energy source for Acidiphilium SJH and a relatively low cost organic electron donor. The stoichiometry of glycerol oxidized to ferric iron reduced is 14:1 (Eq. (3)). Excess glycerol was provided to promote micro-aerobic conditions within the packed bed (i.e. some of the glycerol would be oxidized using molecular oxygen as electron acceptor) to generate the micro-aerobic conditions that are optimum for iron reduction by Acidiphilium SJH. 3þ
C3 H8 O3 þ 14Fe
2þ
þ 3H2 O→3CO2 þ 14Fe
þ 14H
þ
ð3Þ
The feed liquor was pumped into the bioreactor at variable rates (135 to 1950 mL h −1), corresponding to dilution rates of between 0.06 and 0.87 h−1 (the bioreactor volume was calculated as that of the combined packed bead bed and overlying liquor). pH values within the bead bed and in the surface liquor were recorded, and ferrous:ferric iron ratios in the effluent liquor were determined from redox potential measurements, using the Nernst equation. The Acidiphilium SJH bioreactor was highly efficient at reducing ferric iron to ferrous. At dilution rates of b 0.124 h−1, 99% of the
Pump
Feed liquor
pH electrodes
Table 1 Comparison of reagent (sulfur) cost and metal values in the reductive bio-processing of the nickel laterite ore. Amount consumed/ solubilized (kg/t ore) Reagent (sulfur) Acid consumption Reduction of goethitea Total S consumed Metal values Nickel Cobalt Chromium Total metal value
39 3.75 42.75
3.0 0.25 4.5
Price/kg (US $) 0.32 0.32 0.32
19 32 5.5
Estimated cost/ value (US $) 12.5 1.2 13.7
57 8 25 90
a Calculated on the basis that the stoichiometry of elemental sulfur oxidized to sulfate (a 6-electron reaction) to the reduction of ferric to ferrous iron (a single electron reaction) is 6:1.
Upper liquid phase
Acidiphiliumcolonised porous beads
Gravity drain
Bioreactor vessel Fig. 2. Set up of the ferric iron-reducing packed bed bioreactor.
D.B. Johnson / Hydrometallurgy 127-128 (2012) 172–177
inflowing iron was reduced, while at higher dilution rates (up to 0.87 h−1) conversion rates remained >90% (Fig. 3a). Flow rates and weights of ferric iron reduced were highly correlated (r2 >0.99), with ~1 g of ferric iron being reduced h−1 at the highest flow rate tested (Fig. 3b). As predicted by Eq. (3), the pH of the reduced liquor was always lower than that of the feed, but generally did not fall to below pH 2.2, which is well within the range for growth and activity of Acidiphilium SJH. Similar values were found within the bead bed and the surface liquor, indicating that most of the iron reduction was occurring within the lower packed bed layer. 4. Selective precipitation of transition metals from mine drainage waters using acidophilic sulfate-reducing bacterial consortia Mine water pollution is a global issue. While prevention of acid mine drainage (AMD) formation and migration is always the best option, in many situations this is not possible, and streams draining abandoned mines and mine spoils require treatment to prevent damage to the wider environment. The most common approaches used to remediate AMD – active chemical treatment and passive wetland/compost reactors – both have major detractions including, in both cases, non-recovery of metals and the production of metal-rich wastes (sludges and spent composts) that require careful disposal and storage. Active biological remediation, utilizing bacteria either to oxidize and precipitate iron, or to reduce sulfate and precipitate metal sulfides, is an alternative option which has the important advantage of facilitating the recovery of metals from waste. Waters draining metal mines often contain a variety of transition metals, such as copper, zinc manganese and iron, as well as other metals and metalloids, such as aluminum and arsenic. Many of these are chalcophilic (react with hydrogen sulfide) to form solid sulfide phases that have different solubility products, a trait that can be used, in theory, to remove many transition metals and As selectively from mine waters.
175
The key reactant involved is S2−, the concentration of which can be changed by varying solution pH, as illustrated by Eq. (4): −
þ
2−
þ
H2 S↔HS þ H ↔ S þ 2 H ρK 1 ¼ 7 ρK 2 ¼ 12:
As solution pH becomes more acidic liquors, the concentration of S 2− becomes increasingly small, thereby limiting the insoluble sulfides that can form to those with increasingly small solubility products. Control of pH can therefore be used to remove metals (and As) in a selective manner from mine waters. Aluminum does not form a sulfide phase, though it precipitates as hydroxides (gibbsite and basaluminite) at ~ pH 5, again allowing pH control to be used either to precipitate this metal or to cause it to remain in the solution. Hydrogen sulfide is generated as a waste product by dissimilatory sulfate- and sulfur-reducing bacteria and archaea. The vast majority of known species of these prokaryotes are highly pH-sensitive and are inactive or killed in acidic liquors such as AMD. This problem is circumvented in the BioSulfide process (Bratty et al., 2006) by separating bacterial growth chambers (that contain neutrophilic sulfur-reducing bacteria, rather than sulfate-reducers) and vessels where sulfide pumped from the bioreactors is contacted, at pre-determined pH values, with acidic metal-laden liquors. An alternative approach is to promote bacterial sulfate reduction and metal precipitation within a single reactor vessel maintained at low (and variable) pH, thereby simplifying engineering design and reducing construction and operation costs. The core requirement of such a system is a sulfidogenic microbial population that is active at an extremely low pH; such a consortium (active between pH 2.2 and 4.5) has recently been described by Ňancucheo and Johnson (2012). The inoculum used for this system came from two sources — a pure culture of an acid-tolerant Desulfosporosinus sp. (M1) isolated from the volcanic Caribbean island of Montserrat (Kimura et al., 2006), and an undefined sulfidogenic microbial mat found at an abandoned copper mine in Spain (Rowe et al., 2007). Details of the protocol used for the enrichment of the acidophilic sulfate-reducing mixed culture, and the set-up and operation of the sulfidogenic bioreactors that utilized this consortium, are given in Ňancucheo and Johnson (2012). In brief, two anaerobic bioreactors were operated in parallel for about one year as continuous flow systems. The bacteria were immobilized on Poraver beads in reactor vessels that contained ~1 L of packed colonized beads above which was ~1.2 L of liquor, similar to the set up described in Section 3. The acidic test solutions were pumped into the reactor vessels at rates required to maintain pre-determined values, using a pH electrode placed in the liquor above the packed bed coupled to a peristaltic pump. Glycerol was added to the feed liquors to act as an electron donor for sulfate reduction, in a proton-consuming reaction (Eq. (5)): þ
2−
2þ
4C3 H8 O3 þ 12H þ 7SO4 þ Cu →12CO2 þ 6H2 S þ CuS þ 16H2 O
Fig. 3. Ferric iron reduction in a packed bed bioreactor: (a) effect of flow rates on the percentages of ferric iron reduced; (b) relationship between flow rates and the weight of ferric iron reduced.
ð4Þ
ð5Þ
Eq. (5) shows the formation of copper sulfide, with a glycerol:copper molar ratio in the feed liquor of 4:1. By modifying this ratio (by either increasing the copper concentration or decreasing the glycerol concentration) it was possible to devise a situation where there is ultimately no production of free H2S and consequently no net consumption of protons (i.e. the reaction is pH-neutral). However, the bioreactor set-up requires a pH differential between the feed liquor and that of the liquor within the bioreactor vessel, and so some production of free H2S was an integral part of the process. Two acidic sulfidogenic bioreactors were operated with different feed liquors. In one case, this was an acidic (pH 2.5) solution containing 3 mM of both zinc and ferrous iron, and variable concentrations (0.5–30 mM) of aluminum, and was based on the composition of AMD draining an abandoned Zn mine (Cwm Rheidol) in mid-Wales. The second bioreactor was fed with a more acidic (pH 2.1–2.5) solution containing (for the first 320 days of operation) 1 mM each of copper, zinc and ferrous iron (later (for ~60 days) changed to 0.7 mM copper, 1 mM zinc and 5–10 mM
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ferrous iron) 3.3 mM aluminum and 0.2 mM manganese. This was a synthetic mine water based on the composition of AMD draining an abandoned copper mine (Mynydd Parys) in north-Wales. Samples were withdrawn from the bioreactors at regular intervals to measure concentrations of soluble metals, glycerol and acetic acid, numbers of bacteria (total cell counts) and to determine microbial diversity (from terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis of extracted DNA). Both bioreactors achieved the objectives of demonstrating sulfidogenesis and selective metal precipitation in acidic (pH 2.2 to 4.8) conditions. In the case of the bioreactor fed with synthetic Cwm Rheidol AMD, zinc was efficiently precipitated (>99%) as a sulfide inside the reactor while both aluminum and ferrous iron remained in solution (>99%) and were washed out of the reactor vessel (Fig. 4). Sulfidogenesis and selective precipitation of zinc continued when the aluminum concentration in the feed liquor was increased to >50-times that found typically in Cwm Rheidol AMD, though the presence of 30 mM Al was noted to have a small negative impact on the performance of the bioreactor. Selective precipitation of metal sulfides was also demonstrated in the second bioreactor. Throughout the test period, all of the copper present in the feed liquor was precipitated (confirmed as copper sulfide) within this bioreactor, but none of the ferrous iron. Zinc (sulfide) biogenesis was more variable. When the bioreactor pH was set at pH 3.6, >99% of the soluble zinc in the influent liquor was precipitated. By progressively lowering both the bioreactor pH and the concentration of the electron donor (glycerol) in the influent liquor, it was possible to retain increasing amounts of zinc in the solution. When the bioreactor was maintained at pH 2.4, the amount of zinc precipitated stayed reasonably stable at 47%+/−1 6% (23 sampling time points) as the glycerol concentration in the influent liquor was lowered from 4 to 1.5 mM. By decreasing the bioreactor pH still further (ultimately to pH 2.2) and the influent glycerol concentration to 0.7 mM, it was possible to precipitate only 8+/−2% (9 samples) of zinc within the bioreactor, while maintaining >99% removal of copper from the solution. Bacteria could be enumerated in the upper liquid phases of the bioreactors, but not on the bead surfaces. Numbers fluctuated between ~106 and 5×107 mL−1 and were primarily influenced by both flow rates and glycerol concentrations in the feed liquor. T-RFLP analysis showed that both bioreactors contained consortia of bacteria that comprised non-sulfidogenic acidophiles as well as sulfate-reducing bacteria. The latter included Desulfosporosinus sp. M1, a novel genus/ species (“Desulfobacillus acidavidus”. CL4) previously isolated from the same abandoned Spanish copper mine used for the inoculum, and a previously unknown Firmicute (strain CEB3) that was isolated from the
bioreactors and is currently being characterized. No archaeal genes were amplified from either bioreactor. Molecular analysis also showed that the compositions of the microbial consortia within the bioreactors changed as the operational parameters (in particular, pH) were moderated. Both systems were noted, however, to be very robust, with occasional problems encountered in their operation (e.g. feedback of copper sulfate from attached H2S-capture bottles) causing only short-term perturbations in bacterial activity and bioreactor performance. 5. Discussion and conclusions The experimental work described has demonstrated how reductive transformations of iron and sulfur by acidophilic bacteria can be used for both extracting metals from mineral ores and for selectively recovering in acidic liquors. The reductive dissolution of nickel limonites represents a radical development for “biomining”. This is, in essence, a reversal of the conventional approach (oxidative dissolution, using microbial-generated ferric iron) currently used for bio-processing metal ores. The demonstration of bacteria effectively catalyzing a process at 30 °C that is currently carried out at ~800 °C using pyrometallurgical processing further demonstrates the remarkable potential of microorganisms. Evidence for indirect bio-reduction of asbolane, as well as goethite, points to the reductive dissolution of minerals potentially being of generic application for processing oxidized ores. The acidophilic ferric iron-reducing bioreactor described demonstrates how simple, rapid and effective such a set-up system can be. A typical application of this technology would be for off-line bioleaching of oxidized ores where, as in the current “indirect” approach used for sulfides (Frias et al., 2008), the biological reaction (ferrous iron generation) and the abiotic reaction (ferrous iron reduction of susceptible oxidized minerals) would be carried out in separate vessels where conditions for both processes could be optimized. Lastly, the selective precipitation of metals from synthetic mine waters highlights how an environmentally-benign approach to those presently could be used to remediate AMD streams and to capture metals from process and waste waters. Selective precipitation as sulfides facilitates the recovery and recycling of metals, and this can be used to offset, at least in part, the cost of the treatment process. At a time where disposal of potentially useful and valuable materials, such as metals, in landfill is being increasingly viewed as undesirable and unacceptable, a radically new approach for remediating AMD from metal mines is timely. Acknowledgments The author would like to acknowledge the many students and research officers of the Bangor Acidophile Research Team (BART), and other colleagues who have been involved in helping devise and in carrying out the research described in this review. In particular, thanks are due to Dr. Chris du Plessis (Vale, Perth, Australia), Drs. Barry Grail, Ivan Ňancucheo and Kevin Hallberg and Ms. Gemma Bowsher (all BART personnel). References
Fig. 4. Selective precipitation of zinc from synthetic (pH 2.5) mine water containing 3 mM of both ferrous iron (▼) and zinc (♦), using a sulfidogenic bioreactor. pH values (●) within the bioreactor were progressively lowered (from 4.8 to 4.0) during the course of the trial. Aluminum concentrations in the influent liquor increased from 0.5 to 30 mM during the experiment, but no precipitation of aluminum was observed within the bioreactor. Modified from Ňancucheo and Johnson (2012).
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