Simultaneous Sequestration of Co²⁺ and Mn²⁺ by Fungal Manganese Oxide through Asbolane Formation

Abstract

Biogenic manganese oxides (BMOs) have attractive environmental applications owing to their metal sequestration and oxidizing abilities. Although Co readily accumulates into Mn oxide phases in natural environments, the Co²⁺ sequestration process that accompanies the enzymatic Mn(II) oxidation of exogenous Mn²⁺ remains unknown. Therefore, we prepared newly formed BMOs in a liquid culture of Acremonium strictum KR21-2 and conducted repeated sequestration experiments in a Mn²⁺/Co²⁺ binary solution at pH 7.0. The sequestration of Co²⁺ by newly formed BMOs (~1 mM Mn) readily progressed in parallel with the oxidation of exogenous Mn²⁺, with higher efficiencies than that in single Co²⁺ solutions when the initial Co²⁺ concentrations (0.16–0.8 mM) were comparable to or lower than the exogenous Mn²⁺ concentration (~0.8 mM). This demonstrates a synergetic effect on Co sequestration. Powder X-ray diffraction showed a typical pattern for asbolane only when newly formed BMOs were treated in Mn²⁺/Co²⁺ binary systems, implying that the enzymatic Mn(II) oxidation by newly formed BMOs favored asbolane formation. Cobalt K-edge X-ray absorption near-edge structure measurements showed that both Co(II) and Co(III) participated in the formation of the asbolane phase in the binary solutions, whereas most of the primary Co²⁺ was sequestered as Co(III) in the single Co²⁺ solutions, which partly explains the synergetic effects on Co sequestration efficiency in the binary solutions. The results presented here provide new insights into the mechanism of Co interaction with Mn oxide phases through asbolane formation by enzymatic Mn(II) oxidation under circumneutral pH conditions.

1. Introduction

Cobalt (Co) is an economically important element because of its use in various applications, such as pigments, superalloys, mechanically resistant materials, and numerous electronic devices [1,2,3,4]. Moreover, the demand for Co has notably increased, especially as a raw material for rechargeable electrical batteries, which accounts for more than half of current cobalt usage [1]. Considering the predicted shift from internal combustion engines to electric vehicles worldwide, Co demand is expected to reach nearly twice its current levels by 2030 [5]. Therefore, to minimize anthropogenic contamination by Co and the subsequent adverse health effects on living organisms [2,3,4,6], the development of a cost-effective Co recovery system requires urgent attention. Under circumneutral pH conditions, the oxidation of Co²⁺ to insoluble Co(III) through manganese oxides is one of the most effective ways of sequestrating Co²⁺ from the aqueous phase [7,8]. However, the oxidation of Co²⁺ by abiotic Mn oxide causes a concomitant reduction of structural Mn⁴⁺ to dissolved Mn²⁺, which subsequently contaminates water during the remediation process.

In natural environments, several microorganisms (fungi and bacteria) can oxidize Mn²⁺ to Mn(III, IV) enzymatically using O₂ as an electron acceptor. Subsequently, biogenic Mn oxides (BMOs) are precipitated continuously under aerobic conditions. This biological process can proceed much faster than abiotic Mn(II) oxidation (by O₂) under circumneutral pHs [9,10]. Thus, Mn oxide biomineralization may primarily control oxidative sequestration of Co. In fact, many researchers have demonstrated the oxidation of Co(II) to Co(III) and its subsequent sequestration by BMOs [11,12,13,14,15,16,17]. Among them, fungal BMOs produced by the Acremonium strictum strain KR21-2 maintain the activity of Mn(II)-oxidizing enzymes (multicopper oxidase) in the oxide phase [18], and improve the oxidation efficiency of Co(II) to Co(III) through the continuous reoxidation of reduced Mn²⁺ (and possibly Mn³⁺) that readily halt(s) the Co(II) oxidation reaction. Consequently, fungal BMOs continuously accumulate insoluble Co(III) with virtually no detectable Mn²⁺ loss, resulting in cumulative Co sequestration in amounts up to ~2.5 times (molar basis) higher than the initial amounts of Mn in the BMO phase [16]. Such continuous Co(III) accumulation mineralizes poorly crystalline heterogenite (β-CoOOH), which is the most common Co oxide mineral [16,19], and contributes to heterogenite mineralization in natural environments. The Mn(II)-oxidizing activity retained in BMOs is also a prerequisite for the efficient oxidation of As(III) to As(V) [20], Ce(III) to Ce(IV) [21], and Cr(III) to Cr(VI) [22].

The Mn oxide phase resulting from exogenous Mn²⁺ oxidation by fungal BMOs exhibits various mineralogical properties when the associated cations coexist at concentrations high enough to be incorporated [23,24]. Fungal BMOs effectively oxidize exogenous Mn²⁺ to form further BMO phases, the crystal structures of which are typically c-axis-disordered birnessite when no associated cation coexists. In contrast, the presence of coexisting Zn²⁺ during Mn oxidation by fungal BMOs provides a mixed Zn/Mn oxide phase of woodruffite (ZnMn^IV₃O₇·2H₂O) [23]. Additionally, coexisting Ba²⁺ at a Ba²⁺/Mn²⁺ molar ratio > 1 results in well-laminated birnessite, where Ba²⁺ is irreversibly incorporated into the interlayer [24]. This incorporation process favors Ba²⁺ over Sr²⁺, Ca²⁺, and Mg²⁺, and the sequestration of these ions is completely reversible [24]. Such mineralogical variation in the resultant BMO phases is important not only for biogeochemical association in Mn oxide phases [25], but also for metal resource recovery and the remediation of water contaminated with toxic elements [26,27].

Although fungal BMOs effectively transform Co²⁺ into insoluble Co(III) following the reoxidation of reduced Mn(II) [16], the Co²⁺ sequestration process and subsequent mineralogical alteration of BMOs during the concomitant process of exogenous Mn²⁺ oxidation remain unclear. Therefore, the aim of this study was to examine the Co²⁺ sequestration process associated with enzymatic BMO formation and elucidate the mineralogical alterations linked to Co²⁺ sequestration. The repeated treatment experiments of enzymatically active BMOs in Co²⁺/Mn²⁺ binary solutions demonstrated a synergetic effect on their sequestration process through the formation of asbolane and, in contrast, in single Co²⁺ solutions, BMOs precipitated heterogenite (β-CoOOH). The contributions of Mn oxide biomineralization in natural environments to the formation processes of asbolane and heterogenite, which are the two major Co oxide ore minerals, can be inferred from these data. The results of this study also demonstrate the potential application of enzymatically active BMOs for simultaneous Co²⁺ and Mn²⁺ recovery in recycling processes and contaminated wastewater treatment.

2. Materials and Methods

A. strictum strain KR21-2, which enzymatically oxidizes Mn(II) to BMOs [28,29,30,31], was incubated at 25 °C in a HAY liquid medium (pH 7.0) supplemented with 1 mM MnSO₄, as described previously [16,18,32,33,34]. After 72 h of incubation, BMOs with fungal mycelia were harvested using a cell strainer (100 μm nylon mesh, Falcon 352360, Corning Inc., Corning, NY, USA) and washed with Milli-Q water [24]; these were denoted as “newly formed BMOs” and used for Co²⁺ sequestration experiments within 1 h of washing. To elucidate the effects of the enzymatic Mn(II)-oxidizing activity in the BMOs, we inactivated the associated Mn(II) oxidase(s) by heating the newly formed BMOs for 2 h in a water bath (Thermo Minder Mini-80, Taitec, Nagoya, Aichi, Japan) at 85 °C [18], followed by cooling the samples to room temperature, i.e., approximately 20 °C (denoted as “heated BMOs” hereafter).

For repeated treatment experiments, the newly formed or heated BMOs (1 mM as Mn) were mixed with 0–1.7 mM CoCl₂, with or without 0.8 mM exogenous MnSO₄ under air-equilibrated (aerobic) conditions at 25 °C, on a reciprocal shaker at 105 strokes min^–1 (NR–10, Taitec, Nagoya, Aichi, Japan). The solution pH was maintained at 7.0 with 100 mM HEPES buffer (adjusted using NaOH), because the optimum pH for the enzymatic Mn(II) oxidation of A. strictum KR21-2 is 7.0 [29], at which the kinetics of abiotic (catalytic) Mn(II) oxidation are negligible [28,29]. This procedure was conducted in triplicate and the bathing solution was renewed every 24 h. In all sequestration experiments, the supernatants were sampled at 0, 2, 4, 6, 8, and 24 h for each treatment and separated by centrifugation at 12,000× g for 2 min. The dissolved metal concentrations of the supernatants were measured using an Avio 200 inductively-coupled plasma optical emission spectrometer (ICP-OES, PerkinElmer, Waltham, MA, USA). For the two-step extraction protocol in this study, 10 mM aqueous CuSO₄ and subsequently 50 mM hydroxylamine hydrochloride were used for speciation of the Co and Mn sequestered by the BMOs, as described previously [16,32,33,34]. This extraction sequence commonly fractionates adsorbed Mn(II) and oxidized Mn from BMOs [35,36,37]. The Co and Mn fractions dissolved in aqueous CuSO₄ and the subsequent hydroxylamine hydrochloride extracts were termed the exchangeable (reversible) and reducible (irreversible) fractions, respectively. The total metal ions extracted via this two-step extraction sequence are hereafter referred to as the “solid”. The metal concentrations in the extracts were determined using an ICP-OES after dilution with 1.0 M HNO₃. All sequestration and extraction experiments were conducted in triplicate or quadruplicate (n = 3 or 4), with data in the figures and tables presented as the mean ± standard deviation.

Column experiments were conducted using glass columns with a porous polymer bed support (1.0 cm i.d., 10 cm length, Eco-Column Chromatography Column, 7371012, Bio-Rad, Hercules, CA, USA). The newly formed or heated BMOs (0.4 mmol as Mn) were wet-packed with 10 mM HEPES buffer solution (pH 7.0). As the inflows, aerobic (air-equilibrated) mixed solutions of MnSO₄ at ~0.09 mM and CoCl₂ at ~0.09 mM in 10 mM HEPES (pH 7.0) were loaded into the columns using peristaltic pumps (MP-2000, EYELA, Tokyo, Japan) with average flow rates of 50–55 mL/h. The outflows were received using Teflon bottles, which were renewed at time intervals of 17–30 h and subsequently diluted with 1% HNO₃. The concentrations of dissolved Mn²⁺ and Co²⁺ in each fraction of the outflow and inflow were determined using ICP-OES measurements. After the column experiments, the resultant BMO phases were unpacked, washed with Milli-Q water, and lyophilized.

X-ray diffraction (XRD) measurements were performed on the BMOs using a MiniFlex 600 diffractometer (Rigaku Co., Akishima, Tokyo, Japan) with Cu Kα radiation at 15 mA and 40 kV. Lyophilized BMOs obtained from repeated treatment experiments were placed on a glass holder and scanned over a 2θ range of 5–55°, at 2.0° min^–1, using a 0.02° interval. XRD measurements of BMO samples from column experiments were also conducted using a Rint2500 diffractometer (Rigaku Co., Akishima, Tokyo, Japan) with CuKα radiation at 26 mA and 40 kV over a 2θ range of 5–70°, at 1.0° min^–1, with 0.02° intervals.

Cobalt K-edge X-ray absorption near-edge structure (XANES) spectra for the BMO samples were obtained at BL12C in the Photon Factory, KEK (Tsukuba, Japan), as described previously [15]. Lyophilized BMO samples were diluted and adequately mixed with boron nitride (BN). After homogenization, the mixed BMO–BN powders were pressed into disks of appropriate thickness for XANES measurements in transmission mode.

3. Results and Discussion

3.1. Sequestration of Co²⁺ by Newly Formed and Heated BMOs with/without Exogenous Mn²⁺

The repeated treatment experiments of newly formed BMOs (1 mM as Mn) in binary solutions of 0.8 mM Co²⁺ and 0.8 mM Mn²⁺ revealed an almost complete sequestration of Mn²⁺, with efficiencies of > 99%, 98.5 ± 0.2%, and 87.3 ± 6.6% in the first, second, and third treatments, respectively, and a total sequestration efficiency of 95.0 ± 6.6% (Figure 1A and Table S1). The resultant solid-phase Mn consisted of > 94.5% reducible Mn (oxidized Mn) after treatment (Figure 2(Aa) and Table S1). Consequently, the Co²⁺ coexisting at 0.8 mM had minimal adverse effects on the Mn(II)-oxidizing ability of newly formed BMOs; however, the efficiencies were slightly lower than those in single Mn²⁺ solutions (total efficiency > 99%; Figure S1 and Table S1). These results are consistent with those of a previous study [16], which showed a high tolerance of newly formed BMOs in oxidizing Mn²⁺ to insoluble Mn against coexisting Co²⁺, and further indicated that newly formed BMOs are an efficient Mn(II)-oxidizing mediator. The sequestration of Co²⁺ progressed with a gradual decrease in the sequestration efficiency from the first to third treatment, i.e., 92.7 ± 1.0%, 71.6 ± 3.5%, and 48.4 ± 7.8%, respectively (total sequestration efficiency of 70.8 ± 4.0%; Figure 1A and Table S1). The solid-phase Co increased with repeated treatments, and the reducible Co dominated in the solid Co, with relative contents of 67.6 ± 0.2%, 73.8 ± 0.8%, and 78.5 ± 1.9%, implying irreversible Co sequestration on BMOs. The reducible Mn correlated well with reducible Co, exhibiting a Co/Mn molar ratio of 0.55 (Figure 2(Ba)), which demonstrates that Co incorporation coincided with Mn(II) oxidation throughout the repeated treatment experiments.

Newly formed BMOs completely lost their Mn(II)-oxidizing ability upon 2 h of heating at 85 °C because of the inactivation of the associated Mn(II)-oxidizing enzymes [18]. Abiotic (non-enzymatic) Mn(II) oxidation kinetics are very low at pH 7.0, even under aerobic conditions [9,10]. Therefore, in the Mn²⁺/Co²⁺ binary solutions, heated BMOs (1 mM as Mn) showed slight decreases in both dissolved Mn²⁺ and Co²⁺ (Figure 1B and Table S1), with total sequestration efficiencies of 11.1 ± 4.0% and 16.7 ± 2.8%, respectively, which are consistent with the slight increases in solid-phase Mn and Co deduced by the two-step extraction experiments (Figure 2(Ab) and Table S1). Surface passivation of the heated BMOs should be induced at the beginning of the treatment and subsequently cease further Co(II) oxidation, even though the excess BMOs (>86% as reducible Mn) remain unreacted [16].

The repeated treatments of newly formed BMOs (1 mM as Mn) in single Co²⁺ solutions showed Co sequestration efficiencies of 85.4 ± 0.8%, 47.7 ± 1.1%, and 24.4 ± 1.9%, respectively (total sequestration efficiency of 52.7 ± 0.7%; Figure 1C). Notably, these values were significantly lower than those in the Mn²⁺/Co²⁺ binary solutions (Figure 1A), indicating less cumulative Co sequestration (Figure 1E). Dissolved Mn was not detected in any of the treatments (Figure 1C). Consequently, reducible Co was preferentially accumulated (Figure 2(Bc)). In contrast, upon treatment with heated BMOs in the single Co²⁺ solution, dissolved Mn²⁺ was detected during the first treatment (Figure 1D), which exhibited slight Co sequestration ability (efficiency of 23.1 ± 2.8%; Figure 1D and Table S1), resulting in a negative correlation between Co and Mn in the reducible phase (Figure 2(Bd)).

In the single Co²⁺ solution, newly formed BMOs sequestered Co²⁺ into insoluble Co (Equation (1)) and subsequently re-oxidized the reduced Mn²⁺ through enzymatic Mn(II) oxidation (Equation (2)) as previously demonstrated [16]. The reoxidation of reduced Mn(II) (Equation (2)) maintained the oxidizing ability of newly formed BMOs for Co²⁺, whereas heated BMOs were susceptible to surface passivation because of a lack of reoxidation ability for reduced Mn(II) [16].

2Co²⁺ (aq) + Mn^IVO₂ (s) + 2H₂O (aq) → Mn²⁺ (aq) + 2Co^IIIOOH (s) + 2H⁺ (aq)

(1)

Mn²⁺ (aq) + 1/2O₂ (aq) + H₂O (aq) → Mn^IVO₂ (s) + 2H⁺ (aq)

(2)

According to these reaction schemes, oxidative Co sequestration on BMOs represses the oxidation of exogenous Mn²⁺ in Mn²⁺/Co²⁺ binary solutions because it concomitantly produces Mn²⁺ (Equation (1)). Notably, Co sequestration efficiencies in the Mn²⁺/Co²⁺ binary solutions were higher than those in the single Co²⁺ solutions when Co²⁺ loadings were comparable to (0.8 mM; Figure 1) or smaller than (0.16 mM and 0.4 mM) Mn²⁺ loadings (0.8 mM; Figures S2A and S3A). The repeated treatment of newly formed BMOs in 0.16 mM and 0.4 mM Co²⁺ with exogenous Mn²⁺ at 0.8 mM showed total sequestration efficiencies for Co²⁺ of > 99% and 82.5 ± 3.6%, respectively (Figure 3 and Table S1), which were significantly higher than those in the corresponding single Co²⁺ solutions (68.4 ± 3.6% and 51.0 ± 0.8%, respectively; Figure 3, Figures S2A and S3A, and Table S1). The efficient sequestration of Mn²⁺ was also maintained in the corresponding Mn²⁺/Co²⁺ binary solutions (total efficiencies > 99% and 95.9 ± 1.4%, respectively; Figure 3, Figures S2A and S3A, and Table S1). These results imply a synergetic effect of Mn²⁺ and Co²⁺ on their sequestration by newly formed BMOs under these experimental conditions. The cumulative amount of Co sequestration exhibited a good linear correlation with that of Mn (R² > 0.98) throughout the repeated treatment experiments, with Co/Mn molar ratios (deduced from the slopes of the Co vs. Mn plots) of 0.22, 0.44, and 0.69 with increasing Co²⁺ loadings of 0.16 mM, 0.4 mM, and 0.8 mM under a constant initial Mn²⁺ concentration of 0.8 mM (Figure 4A). As described above, Co and Mn in the reducible phases were also positively correlated with each other (R² > 0.99), with respective Co/Mn ratios of 0.17, 0.35, and 0.55 (Figure 4B).

In contrast, Co²⁺ loadings at 1.4 mM and 1.7 mM resulted in lower Co sequestration efficiencies in the Mn²⁺/Co²⁺ binary solutions than in the corresponding single Co²⁺ solutions (Figure S4 and Figure 5, respectively, and Table S1); the total Co sequestration efficiencies were 43.5 ± 3.2% and 33.7 ± 3.1% in the binary solutions and 49.6 ± 0.8% and 42.9 ± 3.1% in the single Co²⁺ solutions. Consequently, higher cumulative Co sequestrations were observed in the latter system (Figure 3). In addition, the total Mn sequestration was also lower (74.9 ± 3.0% and 70.9 ± 2.1% at Co²⁺ loadings of 1.4 mM and 1.7 mM; Figure 3 and Table S1) than that in the single Mn²⁺ system (Figure 3 and Figure S1), suggesting a repressive, rather than synergetic, effect in the binary systems at Co²⁺ loadings higher than those of Mn²⁺. Co²⁺ oxidation by structural Mn^IV produces Mn²⁺ that dissolves spontaneously, through the process denoted in Equation (1), and consequently interferes with the enzymatic oxidation of exogenous Mn²⁺ by newly formed BMOs. This may partly explain the repressive effect in Mn²⁺/Co²⁺ binary systems with higher Co²⁺ loadings (Figure 3), where oxidative Mn²⁺ sequestration was also much less efficient. Repressive Co and Mn sequestration may be responsible for the significant fluctuation in the relationship plots between the cumulative sequestration of Co and Mn (Figure 4A), although the correlation tended to be linear (R² > 0.93).

3.2. Mineralogical Composition of Solid Phases Formed in Mn²⁺/Co²⁺ Binary Systems

XRD measurements were conducted to clarify the mineralogical variations in BMOs after repeated treatment experiments. Newly formed BMOs were layered analogously to vernadite, a nanostructured and turbostratic variety of birnessite [38] which typically shows XRD peaks at ~7.4 and 2.4 Å (Figure 6), which are assigned to the 001 basal reflection of vernadite and the 11,20 diffraction band of the C-centered two-dimensional unit cell, respectively [38]. The repeated treatments in single Mn²⁺ solutions resulted in a similar XRD pattern to that of the original BMOs, indicating that turbostratic birnessite is primarily mineralized through exogenic Mn²⁺ following enzymatic Mn(II) oxidation by newly formed BMOs at pH 7.0. XRD peaks newly appeared around 4.8 Å (strong), 2.4 Å (medium), and 9.6 Å (weak) for newly formed BMOs repeatedly treated in the Mn²⁺/Co²⁺ binary systems, although the peaks were not well defined in the case of 0.16 mM Co²⁺. This XRD pattern closely resembles that of asbolane (empirical formula: (Ni, Co)_xMn⁴⁺(O, OH)₄·nH₂O; [39]), according to Joint Committee on Powder Diffraction Standards (JCPDS) no. 43-1459 (Figure 6) and other literature [39,40,41]. Asbolane is commonly poorly crystallized with a highly variable chemical composition, consisting of MnO₆ octahedral layers alternating with “island-like” Co and/or Ni (hydr)oxides [39,42]. As the crystal structure is very close to that of lithiophorite [40,43], their XRD peaks mostly overlap, making it difficult to distinguish these minerals using XRD patterns alone [39]. However, naturally occurring Co-rich asbolane can bear very high Co contents of up to 15–20 wt.% [39]. The Co contents of newly formed BMOs repeatedly treated in the 0.8 mM Co²⁺/0.8 mM Mn²⁺ binary solutions were 19 wt.% and 15 wt.% (lyophilized samples) before and after extracting ion-exchangeable Co with 10 mM CuSO₄ solutions, respectively. These high Co contents, along with the XRD pattern, indicate the mineralization of Co-rich asbolane during repeated treatments of newly formed BMOs in the Mn²⁺/Co²⁺ binary solutions at pH 7.0. Since heated (enzymatically inactive) BMOs showed much lower sequestration efficiencies for both Mn²⁺ and Co²⁺ under the same experimental conditions (Figure 1B), the enzymatic Mn(II) oxidation by newly formed BMOs is a prerequisite for asbolane formation and concomitant Mn²⁺ and Co²⁺ sequestrations at pH 7.0. In contrast, asbolane formation through abiotic Mn oxidation should require more alkaline conditions [40]. Sanchez-Espana and Yusta [40] investigated the Mn oxide precipitation process and its ability to sequester heavy metals in neutralized acidic pit lake waters. They found that Co- and Ni-bearing asbolane precipitation occurred when a slow aerobic oxidation experiment was conducted at pH 8.5–9.0 with La Zarza (Spain) lake water, which contained the highest dissolved Co (up to 11,000 μg/L) and Ni (up to 11,400 μg/L) relative to dissolved Mn (up to 647 mg/L). It appears that asbolane precipitation through abiotic processes strongly depends on the Co and/or Ni concentrations relative to Mn in the reactant solution [40].

In the enzymatic process at pH 7.0, the formation of well-defined asbolane also required Co loadings greater than 0.4 mM; an incomplete XRD pattern was obtained in the 0.8 mM Mn²⁺/0.16 mM Co²⁺ binary solutions, where a birnessite–asbolane intermediate may have formed (Figure 6). In the 0.8 mM Mn²⁺/0.16 mM Co²⁺ binary solutions, the Co/Mn molar ratio in the reducible phase was 0.17, which was probably insufficient to maintain the asbolane structure. Co K-edge XANES spectra showed peaks for the aqueous Co^IICl₂ solution and chemically synthesized β-Co^IIIOOH at 7708 eV and 7713 eV, respectively, which were used as reference materials [15] (Figure 7). According to the linear combination fit, newly formed BMOs repeatedly treated in the 0.8 mM Co²⁺/0.8 mM Mn²⁺ binary solutions (asbolane precipitation) contained 40% Co(II) and 60% Co(III) (Figure 7). After the extraction treatment in 10 mM CuSO₄ to remove the ion-exchangeable Co (sorbed Co), the resultant solid consisted of 29% Co(II) and 71% Co(III) (Figure 7). The dependence of enzymatic asbolane formation on the solution pH remains uncertain. Further study is needed to discern the role of Mn(II)-oxidizing enzymes on asbolane formation in more acidic or alkaline conditions.

The XRD patterns of asbolane did not appear for the newly formed BMOs treated in single Co²⁺ solutions at all Co²⁺ loadings, where the broad diffraction humps around 4.4 Å and 2.4 Å (Figure S5) suggested the formation of poorly crystalline heterogenite, as proposed in previous research [16]; the XRD patterns of heterogenite (β-CoOOH; JCPDS no. 26-1107) became apparent after repeated treatment with eight renewals of the bathing solutions. This observation clearly indicates that the simultaneous oxidation of exogenous Mn²⁺ and Co sequestration is a prerequisite for asbolane precipitation. The Co contents in the newly formed BMOs repeatedly treated in single 0.8 mM Co²⁺ solutions were 24 wt.% and 17 wt.% (lyophilized samples) before and after extracting the ion-exchangeable Co with 10 mM CuSO₄ solutions, respectively, which were significantly higher than those obtained in the Mn²⁺/Co²⁺ binary solutions (19 wt.% and 15 wt.%, respectively). Co preferentially accumulated over Mn in the solid phase as heterogenite in single Co²⁺ solutions. A linear combination fit to the XANES spectra showed that the newly formed BMOs repeatedly treated in single 0.8 mM Co²⁺ solution contained 24% Co(II) and 76% Co(III) (Figure 7). Removing sorbed (ion-exchangeable) Co by treatment in 10 mM CuSO₄ resulted in the dominance of Co(III) (91%) over Co(II) (9%) (Figure 7), indicating that heterogeneous precipitation mostly requires the oxidative transformation of Co(II) to Co(III), as described in Equation (1). In contrast, the XANES spectra demonstrated that asbolane formation from Mn²⁺/Co²⁺ binary solutions involves direct Co(II) incorporation into its structure, where 29% of Co in the reducible solid phase was Co(II). Direct Co(II) incorporation into asbolane may partly contribute to synergetic sequestration in Mn²⁺/Co²⁺ binary systems when the Co²⁺ concentration is comparable to or less than that of exogenous Mn(II).

3.3. Simultaneous Sequestration of Co²⁺ and Mn²⁺ in the Column Experiments

The column packed with newly formed BMOs (0.40 mmol as Mn) simultaneously recovered Co²⁺ and Mn²⁺ with efficiencies of 95% and 100% at the beginning of the experiments (Figure 8Aa–c). These efficiencies gradually decreased, reaching 13% for Mn²⁺ and 2% for Co²⁺ at a total flow volume of 22,000 mL on day 17 (Figure 8Ac). The cumulative amounts of recovered Co and Mn were 0.59 mmol and 1.14 mmol (Figure 8Ad), respectively, which were 1.4 and 2.9 times the initial Mn content in BMOs (0.40 mmol as Mn). The Co/Mn molar ratio in the recovered fraction (cumulative) ranged from 0.84 (day 1) to 0.51 (day 17), and was close to the Co/Mn molar ratio (0.69) obtained from the repeated treatment experiments of 0.8 mM Co²⁺ and 0.8 mM Mn²⁺ (Figure 4A). The Co²⁺/Mn²⁺ molar ratio in the reactant solutions was most likely the main factor determining the sequestration ratio of Co/Mn on newly formed BMOs. Heated BMOs exhibited a much smaller Co²⁺ recovery, from 20% (day 1) to 6% (day 5, total flow volume of 5900 mL), with leaching of Mn²⁺ from the BMO phases because of reduction of structural Mn(IV) (Figure 8Be–g). This resulted in a higher Mn²⁺ concentration in the outflow than in the inflow (Figure 8Bf). The XRD patterns for asbolane were obtained for the newly formed BMOs unpacked on day 17 (Figure 8C), which demonstrated the simultaneous sequestration of Mn²⁺ and Co²⁺ through “enzymatic” Mn(II) oxidation on newly formed BMOs.

4. Conclusions

The results presented in this study demonstrate that enzymatically active BMOs readily and simultaneously sequester Mn²⁺ and Co²⁺ through the formation of asbolane in Mn²⁺/Co²⁺ binary solutions at pH 7.0. In a binary solution with appropriate Co²⁺/Mn²⁺ molar ratios, asbolane formation indicated a higher Co²⁺ sequestration efficiency than that of a single Co²⁺ solution, where the formation of heterogenite (β-CoOOH) was the main pathway for Co²⁺ sequestration. The continuous formation of heterogenite also requires enzymatic Mn(II)-oxidizing activity to maintain the reoxidation reaction of reduced Mn²⁺, which causes the cessation of Co²⁺ oxidation. Therefore, this study strongly infers biological (fungal and bacterial) contributions to the formation processes of asbolane and heterogenite, which are the two major Co oxide ore minerals, in natural environments. The fungal BMOs possessed a high tolerance of Mn(II)-oxidizing ability to coexisting Co²⁺ up to ~1 mM (~60 mg/L); consequently, the BMOs assessed in this study may be applicable to the simultaneous recovery of Mn²⁺ and Co²⁺ from industrial wastewaters or in recycling processes of rechargeable battery materials.