Mineralization of Ni²⁺-Bearing Mn Oxide through Simultaneous Sequestration of Ni²⁺ and Mn²⁺ by Enzymatically Active Fungal Mn Oxides

Abstract

A fungus, Acremonium strictum KR21-2, produces biogenic manganese oxides (BMOs) that can oxidize exogenous Mn²⁺ ions to form different BMO phases. When other guest ions are present during the BMO formation, it can strongly affect the mineralogical characteristics of the resultant BMO phase. The impact of coexisting Ni²⁺ ions on the mineralogy of BMO phases formed through enzymatic Mn(II) oxidation and its sequestration ability is not yet fully understood. To better understand it, repeated sequestration experiments were conducted using BMOs in Ni²⁺/Mn²⁺ binary, single Ni², and single Mn²⁺ solution systems with a pH range of 6.0 to 7.5. It was observed that simultaneous sequestration of Ni²⁺ and Mn²⁺ was efficient, with irreversible Ni²⁺ incorporation at pH values above 7.0. The resultant BMO phases showed that Ni²⁺-bearing Mn oxides resembling feitknechitite (β-MnOOH) were developed through enzymatic Mn(II) oxidation. At pH values below 6.5, the turbostratic birnessite structure was maintained even in Ni²⁺/Mn²⁺ binary solutions, and subsequently, the Ni²⁺ sequestration efficiency was low. The pseudo-first-order rate constants of enzymatically inactivated BMOs for Mn²⁺ sequestration were two orders of magnitude lower than those of active BMOs, indicating the crucial role of the enzymes in precipitating Ni²⁺-bearing Mn oxide phases. These findings provide new insights into the mechanism of Ni²⁺ interaction with Mn oxide through microbial activity under circumneutral pH conditions.

1. Introduction

Nickel (Ni) is a highly significant element in numerous industrial applications [1]. It has been increasingly used in rechargeable electrical batteries for electric vehicles, along with other rare metals like Co, Mn, Li, etc. [2]. While a trace amount of Ni is essential for several biological processes, an excessive amount of it can be toxic to plants, fishes, and animals, including humans [3,4,5,6]. Therefore, efficient Ni remediation is necessary to maintain a clean environment worldwide [5], and bioremediation offers a cost-effective solution to toxic heavy metal contamination [7,8,9,10,11,12].

Biogenic nanoparticles (BNPs) are gaining attention for their potential in remediation and recovery of heavy metals from wastewaters [13,14]. This is because BNPs possess unique properties such as high specific surface area, catalytic activity, and desirable morphology. Among BNPs, biogenic Mn oxides (BMOs), which are produced by bacteria and fungi, are known for their strong sorption and oxidation capabilities for various inorganic elements in natural environments [15,16]. Biogenic Mn oxide formation through biologically mediated redox reactions is an interesting process for developing an efficient remediation system, particularly for heavy metals [17,18,19,20]. Enzymatic Mn(II) oxidation with O₂ (as an electron acceptor) by multicopper oxidase (MCO) is a key process in both bacterial and fungal BMO formation [21,22,23]. A fungal Mn(II)-oxidizer, Acremonium strictum KR21-2, secretes an MCO-type Mn(II)-oxidizing enzyme to precipitate BMOs from Mn(II) at ~mM levels [22,23]. This strain is considered a model Mn(II)-oxidizing microorganism due to its potential in sequestering various elements.

In a previous study, it was discovered that newly formed BMOs in the cultivation of A. strictum KR21-2 can effectively oxidize exogenous Mn²⁺ to form different BMO phases [24]. This is because the enzymatic Mn(II)-oxidizing activity is stably maintained in the primary BMO phase [24]. This process strongly affects the mineralogical characteristics of the resultant BMO phase when the BMO formation progresses under the coexistence of guest elements (ions) sequestered into the BMO phase [25,26]. The crystal structure of BMOs is typically turbostratic birnessite when no associated cation coexists [27]. However, coexisting Zn²⁺ and Co²⁺ during Mn oxidation by fungal BMOs provide mixed Zn/Mn and Co/Mn oxide phases of woodruffite (ZnMn^IV₃O₇·2H₂O) [25] and asbolane [26]. Such mineralogical variations in the resultant solid phases are important not only for biogeochemical association in Mn oxide phases but also for metal resource recovery and remediation of water contaminated with toxic elements.

The impact of coexisting Ni²⁺ on the mineralogy of BMO phases formed through enzymatic Mn(II) oxidation and its sequestration ability is still not fully understood. We hypothesized that the presence of Ni²⁺ affects the mineralogical properties of BMO as enzymatic BMO formations progress, which, in turn, has a significant effect on their sequestration ability. Therefore, in this study, we aimed to examine the Ni²⁺ sequestration process associated with enzymatic BMO formation and elucidate the mineralogical alterations linked to Ni²⁺ sequestration. Repeated-treatment experiments were conducted on newly formed (enzymatically active) and heated (enzymatically inactivated) BMOs in Ni²⁺/Mn²⁺ binary, single Mn²⁺, and single Ni²⁺ solutions under aerobic conditions with pH values ranging between 6.0 and 7.5. The results demonstrate that the enzymatic formation of Ni²⁺-bearing Mn oxides, most probably Ni(II)-substituted feitknechitite, can be attributed to the efficient simultaneous Ni²⁺ and Mn²⁺ sequestration at pH values above 7.0. This study suggests that the process of Mn oxide biomineralization plays a role in the formation of Mn oxides that irreversibly incorporate high levels of Ni²⁺ in natural environments. Additionally, this study demonstrates that enzymatically active BMOs have the potential to recover both Ni²⁺ and Mn²⁺ simultaneously in recycling processes and contaminated wastewater treatment.

2. Materials and Methods

2.1. Biogenic Manganese Oxide Production by Acremonium Strictum KR21-2

All the chemical reagents used in this study were of analytical grade and were purchased from FUJIFILM Wako Chemical Co. (Osaka, Japan), except for the yeast extract, which was acquired from Nacalai Tesque, Inc. (Kyoto, Japan). A. strictum KR21-2, a fungus that enzymatically oxidizes Mn(II) to BMOs, was incubated for 72 h at 25 °C in a HAY liquid medium (pH of 7.0 by 20 mM HEPES buffer) supplemented with 1 mM Mn(NO₃)₂. The isolation and phylogenetic position (based on 18S rRNA sequencing) of this strain have been described elsewhere [22]. BMOs with fungal mycelia were collected using a cell strainer (100 μm nylon mesh; 352360, Falcon, NY, USA) and washed with Milli-Q water [26]. These were referred to as “newly formed BMOs” and were used for sequestration experiments within 1 h of washing [26]. To study the effects of 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, followed by cooling of the samples to room temperature (approximately 20 °C) [24]. These were referred to as “heated BMOs” and used for further analysis.

2.2. Repeated-Treatment Experiments of Biogenic Manganese Oxides

For repeated-treatment experiments [26], the newly formed or heated BMOs (1 mM as Mn) were mixed with 0–4.6 mM Ni(NO₃)₂, with or without 0.9 mM exogenous Mn(NO₃)₂ under air-equilibrated (aerobic) conditions at 25 °C, on a reciprocal shaker at 105 strokes·min⁻¹ (NR–10, Taitec, Nagoya, Aichi, Japan). This procedure was repeated three times, and the solution was changed every 24 h. The solution pH was maintained either at 6.0, 6.5 (with 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer adjusted using NaOH), 7.0 or 7.5 (with 100 mM HEPES buffer adjusted using NaOH) [26]. In experiments where the pH was 7.5, the maximum concentration of Ni²⁺ was limited to 0.5 mM due to the solubility limitation of Ni²⁺ at 0.79 mM, which was determined by the solubility product (K_sp) of Ni(OH)₂ at 10^−16.1 (25 ± 2 °C) [28]. In all sequestration experiments, the supernatant was collected at 0, 2, 4, 6, and 24 h for each treatment and then separated through 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).

2.3. Two-Step Extraction Procedure for Biogenic Manganese Oxide

Aqueous 10 mM Cu(NO₃)₂ and 50 mM hydroxylamine hydrochloride were used for speciation of Ni and Mn sequestered by the BMOs [29,30]. The two-step extraction process separated adsorbed Mn(II) and oxidized Mn from BMOs. The Ni and Mn fractions dissolved in aqueous Cu(NO₃)₂ and hydroxylamine hydrochloride extracts were called exchangeable (reversible) and reducible (irreversible) fractions, respectively [25,26]. 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), and the data in the figures and tables are presented as the mean ± standard deviation.

2.4. X-ray Diffraction Analysis

XRD measurements were conducted on the BMOs after repeated-treatment experiments [26]. A Rint2500 diffractometer (Rigaku Co., Akishima, Tokyo, Japan) was used with CuKα radiation at 26 mA and 40 kV. BMO samples were collected using a 100 μm nylon mesh (FALCON Cell Strainer 352360, Corning Inc., Corning, NY, USA), washed thrice with Milli-Q water, and then frozen at −60 °C. After lyophilization with an EYELA Freeze Dryer FD-1000 (Tokyo Rikakikai Co., LTD., Tokyo, Japan), lyophilized BMO samples were placed on a glass holder and scanned over a 2θ range of 5–55° at 1.0° min⁻¹ using a 0.02° step interval. Mineralogical phase identification was conducted using the ICDD database (version PDF-2 Release 2020).

2.5. Single-Treatment Experiments of Enzymatically Inactivated Biogenic Manganese Oxides

To measure the kinetic rate for Ni²⁺ and Mn²⁺ sequestration by heated BMOs, heated BMOs were treated either in 0.5 mM Ni(NO₃)₂/0.9 mM Mn(NO₃)₂ binary or single 0.9 mM Mn(NO₃)₂ solutions for 10 days. Pseudo-first-order rate constants for Mn²⁺ sequestration in the binary systems were calculated from the concentration changes from 0 to 10 d and compared to those for newly formed BMOs deduced from the data from 0 to 6 h.

3. Results and Discussion

3.1. Simultaneous Sequestration of Mn²⁺ and Ni²⁺ by Newly Formed Biogenic Manganese Oxides

Repeated-treatment experiments were conducted in Ni²⁺/Mn²⁺ binary solutions containing 0.9 mM Ni(NO₃)₂ and 0.9 mM Mn(NO₃)₂ at pH 7.0 (100 mM HEPES). The experiments showed that newly formed BMOs (1 mM as Mn) can sequester both Mn²⁺ and Ni²⁺ (Figure 1A), as previously reported [31]. The sequestration efficiencies for Mn²⁺ were >99%, 98.9 ± 1.2%, and 84.5 ± 3.7% in the first, second, and third treatments, respectively, with a cumulative sequestration efficiency of 94.4 ± 1.6% (Figure 1A and Table S1). However, the presence of Ni²⁺ lowered the kinetic rate for Mn²⁺ sequestration, resulting in a lower Mn²⁺ sequestration efficiency in Ni²⁺/Mn²⁺ binary solutions (Figure 1A) compared to that in single Mn²⁺ solutions (Figure 1B and Table S2). Dissolved Ni²⁺ was more readily sequestered, with efficiencies of 47.5 ± 1.8%, 30.6 ± 2.0%, and 22.8 ± 1.0% (cumulative efficiency of 33.6 ± 1.5%), in Ni²⁺/Mn²⁺ binary solutions than in single Ni²⁺ solutions, with 23.2 ± 0.2%, −1.1 ± 0.6%, and 0.9 ± 0.8% (cumulative efficiency of 7.7 ± 0.5%; Figure 1C and Table S3). These results demonstrate that continuous Ni²⁺ sequestration by newly formed BMOs requires an exogenous Mn²⁺ supplement, as shown in Figure 1D. The fungal mycelia grown without Mn²⁺ supplementation had a significantly lower Ni²⁺ sequestration capacity at ~0.02 mM and pH 7.0, and, consequently, a negligible contribution to Ni²⁺ sequestration [31].

The two-step extraction procedure for the BMO phases revealed that solid Mn contained mostly reducible Mn in all solution systems (Figure 2). Newly formed BMO progressively increased the amounts of reducible Mn in the presence of exogenous Mn²⁺. (Figure 2). When heated (enzyme-inactivated) BMOs were used in repeated-treatment experiments, Mn sequestration was scarce (Table S4). This suggests that exogenously added Mn²⁺ was precipitated through an enzymatic oxidation process by newly formed BMOs. In Ni²⁺/Mn²⁺ binary solutions, the amount of irreversibly sorbed Ni²⁺ (reducible Mn) increased with the number of repeated treatments and accounted for 81.4 ± 0.2% in the solid Ni after the third treatment (Figure 2 and Table S1). In contrast, exchangeable Ni²⁺ dominated sequestered Ni for single Ni²⁺ solution systems (Figure 2 and Table S2). Hence, it is likely that Ni²⁺ is incorporated into the precipitate during ongoing Mn oxide formation.

XRD experiments were conducted on newly formed BMOs that were treated in the Ni²⁺/Mn²⁺ binary solutions, and it was found that there was a clear mineralogical alteration (Figure 3). The newly formed BMOs were primarily layered analogously to vernadite, which is a nanostructured and turbostratic variety of birnessite [27]. The XRD peaks of the primary BMOs at approximately 7.3 and 2.4 Å (Figure 3 “Before treatment”) were identified as the 001 basal reflection of vernadite and the 11,20 diffraction band of the C-centered two-dimensional unit cell, respectively [27]. Repeated treatments of the newly formed BMOs in single Mn²⁺ solutions (0.9 mM Mn²⁺, 24 h, three times) did not alter the XRD pattern (Figure 3), indicating that enzymatic oxidation of Mn²⁺ by the newly formed BMOs still favored the mineralization of turbostratic birnessite in single Mn²⁺ solutions.

In contrast, XRD measurements for the resultant BMOs treated repeatedly in Ni²⁺/Mn²⁺ binary solutions showed prominent peaks at approximately 4.6 Å, with the intensity increasing as the treatment times increased (Figure 3). This XRD pattern resembled that of feitknechitite (β-Mn^IIIOOH, JCPDS No. 16-364; Figure 3) [32,33,34]. Lefkowitz and Elzinga [34,35] demonstrated the reductive transformation of chemically synthesized birnessite into secondary feitknechitite as a metastable phase when birnessite is reacted with solutions containing Mn²⁺ under anaerobic conditions at pH > 7. When Ni²⁺ is present, the secondary feitknechitite thus formed readily incorporates dissolved Ni(II) at pH 7.5, and consequently significantly affects the speciation and solubility of Ni(II) [34], indicating that feitknechitite crystallization favors Ni(II) incorporation above pH ~7.0, possibly through isomorphic substitution. In our experiments, the XRD pattern of feitknechitite did not appear for the resultant BMOs in the single Mn²⁺ solution system at pH 7.0 (Figure 3), where enzymatic Mn(II) oxidation readily progressed (as seen in Figure 1B). Consequently, we can surmise that coexisting Ni²⁺ is a prerequisite for feitknechitite formation in our experimental conditions. Manganese oxides with a feitknechitite XRD pattern thus formed in the Ni²⁺/Mn²⁺ binary solution system are most probably a Ni(II)-substituted feitknechitite [34,36], which may be attributed to Ni²⁺ sequestration concurrent with enzymatic Mn(II) oxidation by newly formed BMOs.

3.2. Mineralization of Ni(II)-Incorporated Manganese Oxide Phases in Ni²⁺/Mn²⁺ Binary Solution Systems

Repeated-treatment experiments were conducted on newly formed BMOs in Ni²⁺/Mn²⁺ binary solutions with different ratios to investigate the simultaneous sequestration of Ni²⁺ and Mn²⁺, and the resultant mineralogical alteration of BMO phases. The experiments involved binary solutions with 0.22–4.6 mM Ni(NO₃)₂ and 0.9 mM Mn(NO₃)₂. The results showed that the newly formed BMOs efficiently sequestered exogenous Mn²⁺ with cumulative efficiencies of over 94.4% (Figure 4B and Table S1). Most of the Mn²⁺ was precipitated as reducible (oxidized) Mn, with a percentage of more than 90.3% (Figure 4C and Table S1). Heated BMOs had less ability to precipitate Mn²⁺ (Figure 4B and Table S4), indicating that enzymatic Mn(II) oxidation was responsible for the efficient Mn sequestration by newly formed BMOs. In addition, dissolved Ni²⁺ was also sequestered concurrently with Mn(II) oxidation (Figure 4A). The amounts of Ni²⁺ sequestered increased as the initial Ni²⁺ concentration increased (Figure 4B). In solutions with only Ni²⁺, the cumulative Ni²⁺ sequestered remained near constant at ~0.2 mM (Figure 4B) throughout the initial Ni²⁺ concentrations, suggesting that the sorption of Ni²⁺ on preformed (primary) BMOs reached saturation and that continuous Mn(II) oxidation was closely linked with Ni²⁺ sequestration.

The level of Ni²⁺ sequestration irreversibility (extracted as reducible Ni) in the Ni²⁺/Mn²⁺ binary solution system was significantly higher (60.9–84.0%; Figure 4C and Table S1) than the single Ni²⁺ sorption on preformed BMOs (30.9–45.2%; Figure 4C and Table S3). The reducible Ni was found to have a linear correlation with reducible Mn throughout the repeated-treatment experiments (R² > 0.98; Figure 4D). By analyzing the slope of each linear correlation equation (Figure 4D), the molar ratios of reducible Ni relative to reducible Mn were plotted as a function of initial Ni²⁺ concentrations in Figure 4E. These ratios depended on the initial Ni²⁺ concentrations and tended to reach saturation at a Ni/Mn molar ratio of about 0.55 (Figure 4E). This indicates that irreversible Ni²⁺ incorporation occurred concurrently with precipitation of the Mn oxide phase.

XRD peaks at approximately 4.6 Å were observed in newly formed BMOs for all Ni²⁺/Mn²⁺ binary solution systems, which were tentatively assigned to Ni(II)-substituted feitknechitite (refer to Section 3.1; Figure 5). The intensity of the peaks increased with the increase in irreversible Ni²⁺ incorporation (Figure 4E and Figure 5), indicating the crystallographic development of the feitknechitite structure through a simultaneous Ni²⁺ and Mn²⁺ sequestration process. When the initial Ni²⁺ concentrations were below 0.7 mM, an additional XRD peak appeared at around 9.5 Å. This XRD pattern closely resembled that of asbolane (JPCDS 43-1459, Figure 5). Asbolane is thought to consist of MnO₆ octahedra layers alternating with a poorly structured “island” of Co and Ni oxides [32]. Such an asbolane-type structure would be formed as an intermediate between turbostratic birnessite (no Ni²⁺ addition) and Ni(II)-substituted feitknechitite (Ni²⁺ > 1.0 mM) structures when lower Ni²⁺ loadings (<0.7 mM Ni²⁺ relative to 0.9 mM Mn²⁺). The single Ni²⁺ solution systems (Figure 5), however, did not show any distinguishable XRD peaks even at the highest concentration of Ni²⁺ at 4.4 mM, and a broad hump at around 4.6 Å was observed. This result indicates that repeated treatments of the ordinary BMO phase (turbostratic birnessite) with single Ni²⁺ solutions cannot bring about direct rearrangement in a well-defined feitknechitite structure under the experimental conditions used in this study.

3.3. pH Dependence of Sequestration Efficiency in Ni²⁺/Mn²⁺ Binary Solution Systems

Repeated-treatment experiments were conducted to determine the pH dependence of the sequestration efficiency of newly formed BMOs and how it relates to the mineralogical characteristics of the resultant solid phases in Ni²⁺/Mn²⁺ binary, single Mn²⁺, and single Ni²⁺ solutions at pH 6.0–7.5. In 0.5 mM Ni²⁺ and 0.9 mM Mn²⁺ binary solutions, the sequestration affinities for Ni²⁺ showed a strong pH dependence. Cumulative sequestration efficiencies of 16.2 ± 1.7, 34.2 ± 0.8, 52.8 ± 2.4, and 80.1 ± 2.2% were observed at pH 6.0, 6.5, 7.0, and 7.5, respectively. In contrast, almost quantitative sequestrations were observed for Mn²⁺ at pH 6.5–7.5 (>94.6 ± 2.4%) with an efficiency of 77.1 ± 2.3% at pH 6.0 (Figure 6A). The resultant solid phase showed a more intense XRD peak at approximately 4.6 Å at pH 7.5 than pH 7.0 (Figure 6E), demonstrating that Ni(II)-substituted feitknechitite formation is the most probable causative factor for efficient Ni²⁺ sequestration. It is noteworthy that in Ni²⁺/Mn²⁺ binary solutions, even with higher Ni²⁺ loading (0.9 mM), the resultant solid phases displayed mainly the XRD pattern for turbostratic birnessite at pH 6.0 and 6.5 (Figure 6E), at which small amounts of Ni²⁺ were sequestered (Figure 6A,B, Table S5). These results demonstrate that the pH of the solution is a crucial factor controlling the mineralogical characteristics of the resultant solid phases. This, in turn, affects the simultaneous sequestration efficiencies in Ni²⁺/Mn²⁺ binary solution systems. These findings may be related to the results found by Lefkowitz and Elzinga [34]. They demonstrated that the transformation of birnessite into Ni(II)-substituted feitknechitite, and subsequent Ni²⁺ sequestration, occurs easily in anaerobic Ni²⁺/Mn²⁺ binary solutions through interfacial Mn(II)-Mn(IV) comproportionation [34]. However, this reaction only takes place at pH 7.5 and not at pH 6.5 [34]. Therefore, the crystal growth of Ni(II)-substituted feitknechitite strictly favors values above pH 7. The pH dependence of sequestration for single Mn²⁺ (77.2–>99% for 0.5 mM Mn²⁺; Table S2) and single Ni²⁺ (11.2–14.5% for 0.5 mM Ni²⁺; Table S6) solution systems was not pronounced (Figure 6C,D) and, subsequently, their XRD patterns were analogized to that of the primary BMO phase (Figure 6F).

3.4. Abiotic Mn(II) Oxidation in Ni²⁺/Mn²⁺ Binary Solution Systems

To clarify the contribution of enzymatic and abiotic (auto-catalytic) oxidation processes to Mn(II) sequestration on BMO surfaces, we measured the kinetics of abiotic Mn(II) oxidation in Ni²⁺/Mn²⁺ binary and single Mn²⁺ solution systems using heated (enzymatic-inactivated) BMOs at pH 6.5, 7.0, and 7.5. At pH 7.5, dissolved Mn²⁺ and Ni²⁺ concentrations gradually decreased with reaction times both in the binary and single systems (Figure 7A). The sequestration efficiencies were 78.6 ± 1.8% for 0.5 mM Ni²⁺ and 56.4 ± 1.8% for 0.9 mM Mn²⁺ after 10 days in the Ni²⁺/Mn²⁺ binary solution systems (Figure 7A). The pseudo-first-order rate constant for Mn²⁺ sequestration of (3.5 ± 0.2) × 10⁻³ h⁻¹ (0–10 d) was two orders of magnitude lower than that by newly formed BMOs of (4.0 ± 1.4) × 10⁻¹ h⁻¹ (0–6 h; Table S5). The sequestration efficiency for Mn²⁺ in the single Mn²⁺ solution was 48.5 ± 5.7%, which was slightly lower than that in the binary system. At pH 7.0, 21.9 ± 1.8% of 0.9 mM Mn²⁺ and 39.4 ± 1.2% of 0.5 mM Ni²⁺ were sequestered for 10 d (Figure 7B). During this reaction time, the pseudo-first-order rate constant for Mn²⁺ sequestration was deduced to be (1.0 ± 0.1) × 10⁻³ h⁻¹ (0–10 d), which was also two orders of magnitude lower than that by newly formed BMOs of (8.4 ± 3.4) × 10⁻¹ h⁻¹ (0–6 h; Figure 4A and Table S1). Only 5.4 ± 4.0% of 0.9 mM Mn²⁺ was sequestered in the single Mn²⁺ system (Figure 7B). Therefore, the presence of Ni²⁺ exhibited synergistic effects on Mn²⁺ sequestration both at pH 7.5 and 7.0. XRD patterns of the resultant solid phases at pH 7.0 and 7.5 displayed a peak at around 4.6 Å (Figure 7E), which can most likely be assigned to Ni(II)-substituted feitknechitite, because of the simultaneous Ni²⁺ sequestration. In contrast, such a peak was not observed in the single Mn²⁺ solution systems (Figure 7E), even at pH 7.5, where Mn²⁺ was significantly sequestered (Figure 7A), and consequently, ordinary feitknechitite was scarcely formed in single Mn²⁺ solutions even at pH 7.5, as was observed for newly formed BMOs in the single Mn²⁺ solution systems (Figure 6F). No significant sequestration for Ni²⁺ or Mn²⁺ was observed at pH 6.5 for the binary and single systems (Figure 7C), where the XRD patterns for turbostratic birnessite were maintained despite the presence of Ni²⁺ (Figure 7E). The kinetic rate for sequestrations by heated BMOs was estimated to be <~1 × 10⁻⁴ h⁻¹(0–10 d) and, subsequently, much lower than those by newly formed BMOs (9.6 ± 0.5 × 10⁻¹ h⁻¹) (0–6 h; Figure 7D). These results demonstrated that the enzymatic Mn(II)-oxidizing activity has a crucial role in controlling the precipitation kinetics of Ni²⁺-bearing Mn oxides and subsequently Ni²⁺ sequestration in environments under circumneutral pH conditions.

4. Conclusions

The results of this study indicate that fungal BMOs can effectively remove Ni²⁺ and Mn²⁺ from binary solution systems at pH 7.0–7.5. The newly formed BMOs, which retain Mn(II) oxidizing activity, significantly enhance the kinetic rates for Mn(II) oxidation and subsequent Ni²⁺ incorporation. X-ray diffraction measurements reveal that the sequestration efficiencies closely relate to the mineralogical characteristics of the resultant solid phases. The ongoing formation of Ni(II)-substituted feitknechitite most likely controls sequestration efficiency in binary solution systems at pH values above 7.0. The molar ratio of irreversibly sequestrated Ni²⁺ relative to reducible Mn depends on the initial Ni²⁺ concentration and reaches saturation at Ni/Mn of ~0.55. In contrast, pH conditions below 6.5 are not favorable for the formation of Ni²⁺-substituted feitknechitite, either through enzymatic or abiotic (auto-catalytic) Mn(II) oxidation. The pseudo-first-order rate constants of the enzymatically active BMOs are two orders of magnitude higher than those of the inactivated BMOs. Therefore, the enzymatic active BMO is considered a valuable tool to recover (remove) Ni²⁺ and Mn²⁺ swiftly from contaminated water. The findings of this study strongly infer biological (fungal and bacterial) contributions to the mineralization process of Ni²⁺-bearing Mn oxide phases through enzymatic formation in natural environments.