Sequestration of Oxyanions of V(V), Mo(VI), and W(VI) Enhanced through Enzymatic Formation of Fungal Manganese Oxides

: Biogenic Mn oxides (BMOs) have become captivating with regard to elemental sequestration, especially at circumneutral pH conditions. The interaction of BMOs with oxyanions, such as vanadate (V), molybdate (VI), and tungstate (VI), remains uncertain. This study examined the sequestration of V(V), Mo(VI), and W(VI) (up to ~1 mM) by BMOs formed by the Mn(II)-oxidizing fungus, Acremonium strictum KR21-2. When A. strictum KR21-2 was incubated in liquid cultures containing either Mo(VI) or W(VI) with soluble Mn 2+ , the oxyanions were sequestered in parallel with enzymatic Mn(II) oxidation with the maximum capacities of 8.8 mol% and 28.8 mol% (relative to solid Mn), respectively. More than 200 µ M V(V) showed an inhibitory effect on growth and Mn(II) oxidizing ability. Sequestration experiments using preformed primary BMOs that maintained the enzymatic Mn(II) oxidizing activity, with and without exogenous Mn 2+ , demonstrated the ongoing BMO deposition in the presence of absorbent oxyanions provided a higher sequestration capacity than the preformed BMOs. X-ray diffraction displayed a larger decline of the peak arising from (001) basal reﬂection of turbostratic birnessite with increasing sequestration capacity. The results presented herein increase our understanding of the role of ongoing BMO formation in sequestration processes for oxyanion species at circumneutral pH conditions.

Biogenic nanosized metal oxide particles, such as Fe and Mn oxides act as scavengers for various inorganic elements (ions) in natural environments.Consequently, the biogenic metal oxide formation (biomineralization) through biologically mediated redox reactions is one of the most interesting processes for developing an efficient element remediation system [13,14].Among the biomineralization processes frequently found in natural environments, biogenic Mn oxides (BMOs) formation processes by fungi and bacteria are captivating because the sequestration ability of the BMO is very high, and they subsequently determine the fate of a variety of elements [15][16][17][18].Both in bacterial and fungal Minerals 2022, 12, 1368 3 of 14 also conduct sequestration experiments for oxyanions using newly formed (preformed) BMOs under the condition where exogenous Mn 2+ was enzymatically oxidized and under that where it was not.The results demonstrate that the ongoing BMO formation readily facilitates the sequestration ability for coexisting V(V), Mo(VI), and W(VI) (normalized to oxide Mn).X-ray diffraction measurements show mineralogical alteration with a decline in the peak intensity of (001) basal reflection, when BMO is formed from exogenous Mn(II) with coexisting oxyanions, suggesting that the coexisting oxyanions prevent the sheet stacking of BMO, possibly due to complexation on the edge sites of BMO.

Sequestration of Oxyanions during the Cultivation of A. strictum KR21-2
All chemical reagents used in this study were of analytical grade and purchased from FUJIFILM Wako Chemical Co.(Osaka, Japan) except for the yeast extract (36802-16), purchased from Nacalai Tesque, Inc. (Kyoto, Japan).To evaluate the influence of coexisting V(V), Mo(VI), and W(VI) on the growth and Mn(II)-oxidizing ability, a conidium suspension (1 × 10 5 conidia mL −1 ) of A. strictum KR21-2 was incubated in HAY liquid medium (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) buffer adjusted at pH 7.0 with NaOH) [36,37] with MnSO 4 (1 mM) and either Na 3 VO 4 , Na 2 MoO 4 , or Na 2 WO 4 (up to 1 mM) at 25 • C for 72 h on a reciprocal shaker, set at 105 strokes min −1 (NR-10, Taitec, Nagoya, Aichi, Japan).After incubation for 72 h, dissolved Mn(II) and either V(V), Mo(VI), or W(VI) in the supernatant were measured by a Varian 730-ES inductively coupled plasma-atomic emission spectrometer (ICP-AES)(Agilent Inc., Santa Clara, CA, USA) to determine the Mn(II) oxidizing and oxyanion sequestration abilities.Growth was measured as a fungal mass weight as previously described [29,32,37].In some experiments, the supernatants were sampled at the appropriate times and used for analyzing dissolved species to measure the time courses of Mn(II) oxidation and sequestration of oxyanions during the cultivation [36].
Sequestration experiments for oxyanions were conducted to evaluate the effect of the enzymatic Mn(II) oxidation process by newly formed BMOs on the sequestration ability under three experiment procedures listed in Table 1, where a and b in a term "BMO a /ExMn b " denote the concentrations of primary BMO (mM as Mn) and exogenous Mn 2+ (mM), respectively, used in the sequestration experiments.The sequestration reactions were carried out aerobically for 24 h at 25 The newly formed BMO2 (1 mM as Mn) was reacted with exogenous Mn 2+ (1 mM) to form additional BMO phases in 20 mM HEPES at 7.0 for 24 h.This primary BMO contained 2 mM Mn.
V(V), Mo(VI), or W(VI) (up to ~1 mM) without exogenous Mn 2+ in 20 mM HEPES (at pH 7.0) for 24 h.All the sequestration and extraction experiments were conducted in triplicate (n = 3), and data in the figures and tables are shown as mean ± standard deviation.

XRD Measurements of BMOs after the Sequestration Experiments
XRD measurements were performed for the BMOs after the sequestration experiments.A Rint2500 diffractometer (Rigaku Co., Akishima, Tokyo, Japan) was operated with CuK α radiation at 26 mA and 40 kV.BMO samples treated under the experimental conditions listed in Table 1 were harvested by a 100 µm nylon mesh (FALCON Cell Strainer 352360, Corning Inc., Corning, NY, USA), washed three times with Milli-Q water, and frozen at −60 • C.After lyophilization using 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 5-70 • at 1.0 • min -1 using a 0.02 • step interval.

Sequestration of Oxyanions Concurrent with BMO Formation during the Incubation of A. strictum KR21-2
When the conidia suspension of A. strictum KR21-2 is incubated in HAY liquid culture media (20 mM HEPES at 7.0) with 1 mM MnSO 4 , the concentration of dissolved Mn(II) abruptly decreases at 36-45 h incubation due to the enzymatic formation of insoluble Mn oxide [19][20][21]36].When either ~20 µM V(V), Mo(VI), W(VI), or Cr(VI) coexisted during the incubation, A. strictum KR21-2 readily formed a BMO and subsequently dissolved Mn(II) (1 mM) completely (>99%) were converted to the solid phase Mn (Figure 1A-D).The solid phase Mn formed comprised ~20% exchangeable Mn and ~80% reducible Mn [28].The concentrations of V(V), Mo(VI), and W(VI) were decreased synchronously with Mn oxide formation (Figure 1A-C) while dissolved Cr(VI) concentration remained unchanged throughout incubation (Figure 1D).There were no significant changes in dissolved V(VI), Mo(VI), and W(VI) during incubation when MnSO4 was not supplemented (Figure 1A-C), indicating that the BMO formation mediated the sequestrations.Consequently, the sequestration on fungal hyphae (biomass) was negligible for all oxyanions used in this study.Sequestration efficiency was the highest for W(VI) at >99%, where the initial W(VI) at 22.0 ± 0.2 μM decreased to 0.06 ± 0.00 μM (Figure 1C).Sequestration efficiency of W(VI) at the initial concentration of 19.2 ± 1.0 μM was still high at 42% even when the MnSO4-supplementation was lower at 0.22 There were no significant changes in dissolved V(VI), Mo(VI), and W(VI) during incubation when MnSO 4 was not supplemented (Figure 1A-C), indicating that the BMO formation mediated the sequestrations.Consequently, the sequestration on fungal hyphae (biomass) was negligible for all oxyanions used in this study.Sequestration efficiency was the highest for W(VI) at >99%, where the initial W(VI) at 22.0 ± 0.2 µM decreased to 0.06 ± 0.00 µM (Figure 1C).Sequestration efficiency of W(VI) at the initial concentration of 19.2 ± 1.0 µM was still high at 42% even when the MnSO 4 -supplementation was lower at Minerals 2022, 12, 1368 6 of 14 0.22 ± 0.00 mM (Figure 1C), where the molar ratio of sequestered W relative to solid Mn was deduced at 0.037 (mol/mol).Sequestration efficiency for V(V) at the initial concentration of 23.0 ± 0.6 µM was 62% with the V/Mn molar ratio of 0.013 (mol/mol) when 1.07 ± 0.02 mM Mn 2+ was supplemented (Figure 1A).For Mo(VI), 42% of the initial Mo(VI) at 22.6 ± 0.3 µM was sequestered with 1.09 ± 0.00 mM Mn 2+ -supplementation (Figure 1B).Under the experimental condition, the sequestration affinity is W(VI) > V(V) > Mo(VI) >> Cr(VI).It should be noted that the HAY liquid culture medium contains 200 µM MgSO 4 , 50 µM CaCl 2 , 30 µM K 2 HPO 4 , and 0.2-1 mM MnSO 4 added as an exogenous Mn(II) supplement.Therefore, anionic species from such constituents in the liquid media should be potential competitors for sequestration on BMO.Thus, BMO favors W(VI), V(V), and Mo(VI) compared to SO 4 2− , Cl − , and HPO 4 2− (H 2 PO 4 − ).No significant sequestration of Cr(VI) by BMO (Figure 1D) was consistent with our previous study, which demonstrated a release of Cr(VI) due to the oxidation of Cr(III) being sorbed on BMOs [41].
To clear the influence of the coexisting oxyanions on the sequestration ability during the incubation of A. strictum KR21-2, we measured the sequestration efficiency of V(V), Mo(VI), and W(VI) concurrent with BMO formation with the initial concentrations of each oxyanion, up to ~1000 µM.The fungal growth (based on the fungal mass weight after 72 h of incubation) and the Mn oxidation ability (based on Mn conversion efficiency from aqueous to solid phase) were also monitored after 72 h of incubation.The increasing concentrations of V(V) slowed the growth rate of A. strictum KR21-2 with a relative fungal mass of only 10.7 ± 6.4% at 1.23 ± 0.02 mM V(V)(Figure 2A). A. strictum KR21-2 abruptly lost the Mn(II) oxidation ability (<12.1 ± 0.4%; Figure 2D) at the concentration of V(V) is above 239 ± 2 µM V(V).These results demonstrated that the adverse effect of V(V) on the growth and Mn oxidation ability of A. strictum KR21-2 is similar to heavy metal ions, such as Co 2+ , Ni 2+ , and Cd 2+ .

Sequestrations of Oxyanions by Newly Formed BMOs with an Mn(II) Oxidizing Enzymatic Activity
The growth and subsequently Mn(II) oxidation ability are susceptible to inhibition by V(V) at >100 μM similar to heavy metal ions, such as Co 2+ , Ni 2+ , and Cd 2+ .Our previous studies demonstrated that when A. strictum KR21-2 is incubated in HAY liquid media  Our previous studies have shown the growth inhibition at ≥100 µM Co 2+ [37], Ni 2+ [29], and Cd 2+ [29] (Zn 2+ did not show the growth inhibition up to 1 mM [32]) and the complete loss of Mn(II) oxidizing ability at ≥100 µM Co 2+ [37], ≥50 µM Zn 2+ [32], ≥30 µM Ni 2+ [29], and ≥50 µM Cd 2+ [29].Therefore, it seems that the adverse effect of V(V) on the growth and Mn(II) oxidizing ability is somewhat smaller than that of such heavy metal ions.The threshold of V(V) at ~200 µM (~10,000 µg/L V) for Mn(II) oxidation obtained in this study (Figure 2D) is much higher than the dissolved concentration of dissolved V that is reported to be ~0.7 µg/L in river water and ~1.8 µg/L in seawater [8].Thus, the in-situ remediation by the BMO formation process through the incubation of the Mn(II) oxidizing fungi is still applicable for remediating wastewater contaminated with V(V) below ~100 µM (~5000 µg/L).
In contrast to V(V), even when Mo(VI) and W(VI) were added up to 1.26 ± 0.02 mM and 1.19 ± 0.00 mM, respectively, the growth rate (>89% of the fungal mass base) and Mn(II) oxidation efficiency (>94%) were readily maintained after 72 h of incubation (Figure 2B,C).Subsequently, sequestrations of Mo(VI) and W(VI) progressed concurrently with BMO formation, with the highest Mo(VI) and W(VI) at 0.088 ± 0.003 (mol/mol) and 0.288 ± 0.004 (mol/mol) as the molar ratios relative to solid Mn in the solid phases, respectively (Figure 2H,I).These results demonstrated the applicability of the fungal BMO formation process to recover those oxyanions from aqueous solutions.When 1 mM Mn 2+ was used as an additive for fungal BMO formation, quantitative recovery (>93.8 ± 0.5% at 121 ± 15 µM initial W(VI) concentration) was almost achieved (Figure 2F).

Sequestrations of Oxyanions by Newly Formed BMOs with an Mn(II) Oxidizing Enzymatic Activity
The growth and subsequently Mn(II) oxidation ability are susceptible to inhibition by V(V) at >100 µM similar to heavy metal ions, such as Co 2+ , Ni 2+ , and Cd 2+ .Our previous studies demonstrated that when A. strictum KR21-2 is incubated in HAY liquid media with Mn 2+ , newly formed BMOs maintain the enzymatic activity to oxidize Mn 2+ and readily convert soluble Mn 2+ to Mn(III/IV) oxide even when Co 2+ , Ni 2+ , or Cd 2+ coexist at the mM level [33].To investigate the ability of newly formed BMOs to sequester V(V) and to oxidize Mn 2+ , the sequestration experiments for V(V) were conducted using the newly formed BMO (~1 mM as Mn) with and without exogenous Mn 2+ (~1 mM as MnSO 4 ) in 20 mM HEPES at pH 7.0.When newly formed BMOs were reacted with mixed solutions of exogenous Mn 2+ (~1 mM) and V(V) (80, 400, or 800 µM), the concentrations of Mn 2+ disappeared within 4 h of the reactions (Figure 3A).At 24 h, the two-step extraction procedure displayed that the solid phase Mn comprised 82.9-83.6%reducible Mn (Figure 3B), which was the same as Mn in the primary BMO phase, indicating that newly formed BMO successfully oxidized exogenous Mn 2+ even when V(V) coexisted at 800 µM.The concentrations of dissolved V(V) decreased in parallel with soluble Mn 2+ concentrations within 4 h of the reaction.Subsequently, they reached constant values (Figure 3A).The amounts of V(V) sequestered were considerably higher than that without exogenous Mn 2+ (Figure 3C), indicating that the V(V) sequestration was facilitated by the ongoing formation of Mn oxide from soluble Mn 2+ .Interestingly, the sequestration amounts of V(V) normalized to reducible (oxidized) Mn tended to be higher in the case of exogenous Mn 2+ .The BMO phase formed from exogenous Mn 2+ with coexisting V(V) may possess mineralogical characteristics (and consequently the V(V) sequestration ability) different from that of the primary BMO phase (formed without the coexisting V(V) before the V(V) sequestration experiments).For example, Ba 2+ coexisting during enzymatic Mn(II) oxidation leads to a well-laminated birnessite structure of the resultant Mn oxide phase and consequently leads to the high irreversibility of Ba 2+ sequestration [34].Our previous studies also demonstrated that Zn 2+ and Co 2+ coexisting during enzymatic Mn(II) oxidation causes the formation of binary oxide minerals, such as woodruffite [32] and asbolane [35], respectively, subsequently increasing the sequestration efficiencies of these heavy metal ions through the enzymatic Mn(II) oxidation processes.
well-laminated birnessite structure of the resultant Mn oxide phase and consequently leads to the high irreversibility of Ba 2+ sequestration [34].Our previous studies also demonstrated that Zn 2+ and Co 2+ coexisting during enzymatic Mn(II) oxidation causes the formation of binary oxide minerals, such as woodruffite [32] and asbolane [35], respectively, subsequently increasing the sequestration efficiencies of these heavy metal ions through the enzymatic Mn(II) oxidation processes.

Figure 3.
(A) Sequestration of V(V) (0.08-0.8 mM) by newly formed primary BMO (~1 mM as Mn) with and without exogenous Mn 2+ .Exogenous Mn 2+ was readily oxidized within 4 h of the reaction.(B) Two-step extraction for the resultant solid phases at 24 h of the reaction.(C) The concentration of V(V) sequestered (mM) and (D) the amounts of V(V) sequestered normalized to reducible Mn (mol/mol) with and without exogenous Mn 2+ at 24 h of the reaction.The error bars represent the standard deviation (n = 3).

Sequestrations of Oxyanions through Enzymatic Mn(II) Oxidation
To evaluate the effect of the enzymatic BMO formation process on the sequestration of V(V), Mo(VI), and W(VI), we conducted sequestration experiments under the three conditions with various primary BMOs (0.5-2 mM as Mn) and exogenous Mn 2+ (0-1.5 mM) as listed in Table 1.In the cases of sequestration experiments with exogenous Mn 2+ , i.e.,

Sequestrations of Oxyanions through Enzymatic Mn(II) Oxidation
To evaluate the effect of the enzymatic BMO formation process on the sequestration of V(V), Mo(VI), and W(VI), we conducted sequestration experiments under the three conditions with various primary BMOs (0.5-2 mM as Mn) and exogenous Mn 2+ (0-1.5 mM) as listed in Table 1.In the cases of sequestration experiments with exogenous Mn 2+ , i.e., BMO 1.0 /ExMn 1.0 and BMO 0.5 /ExMn 1.5 , >98.6 ± 0.0% for V(V), >98.0 ± 0.1% for Mo(VI), and >94.6 ± 1.4% for W(VI) (Figure 4A-C) of exogenous Mn 2+ were converted to solid phase Mn due to Mn(II) oxidation by the primary BMOs.In all cases, the resultant BMO phases comprised 80%-90% reducible (oxidized) Mn (Figure 4A-C).These results confirmed that there were no significant differences in the total concentration of solid Mn (acting as absorbents) at the termination of the sequestration experiments.

Mineralogical Characteristics of BMOs Formed with Coexisting V(V), Mo(VI), and W(VI)
Newly formed (primary) BMOs enzymatically deposited by A. strictum KR21-2 are referred to as a natural nanostructured and turbostratic variety of birnessite [27] of which the XRD peaks at ~7.3, 2.4, and 1.4 Å were assigned to (001), (11,20), and (31,02), respectively [39].In all BMO 2.0 /ExMn 0.0 cases, the resultant BMO after sequestration experiments showed a typical XRD pattern similar to that of the primary BMO (Figure 6A-D), suggesting that there were no apparent mineralogical alterations through the sequestration of V(V), Mo(VI), and W(VI) on the "preformed" BMO.For BMO 1.0 /ExMn 1.0 with V(V), Mo(VI), and W(VI), the resultant BMOs displayed declines in XRD intensity arising from (001) basal reflection, while the corresponding XRD peaks disappeared almost entirely for BMO 0.5 /ExMn 1.5 (Figure 6A-C).In contrast, the control experiments using SO 4 2− (added as Na 2 SO 4 ) did not affect the XRD patterns (Figure 6D).Thus, the more significant declines with increases in sequestration capacity suggested the disturbance of the ordering of the birnessite sheet stacking through the sorption of the oxyanions during enzymatic BMO formation.Generally, BMOs are negatively charged due to the high density of the Mn IVvacancy in their structure [27].This structural vacancy is one of the main factors causing the high sorption capacity of BMOs for cationic species, such as heavy metal cations.From the mineralogical characteristics of BMOs, sequestration (sorption) of oxyanions could likely occur mainly at the edge sites through the surface complexation.For example, V(V) sorbes on a chemically synthesized birnessite by forming monodentate corning-sharing complexes [42] and by forming a bidentate mononuclear edge-sharing complex [43].Kashi-wabara et al. [11], using wavelength dispersive X-ray absorption fine structure spectroscopy, demonstrated that Mn oxide is the main host phase for negatively charged WO 4 2− through inner-sphere complex formation.Tanaka et al. [12] proposed the adsorption mechanism of Mo(VI) through the formation of disordered octahedral Mo(VI) species on Mn oxides.These studies [11,12] explain that such oxyanions are preferentially hosted by "negativelycharged" Mn oxide phases in nature.Such complexation and adsorption of the coexisting oxyanions on the birnessite sheet structure possibly resulted in the disturbance of the sheet stacking of the resultant BMOs.Further studies need to evaluate the atomic-level mechanisms of the sequestration processes during enzymatic BMO formation.
formation.Generally, BMOs are negatively charged due to the high density of the Mn -vacancy in their structure [27].This structural vacancy is one of the main factors causing the high sorption capacity of BMOs for cationic species, such as heavy metal cations.From the mineralogical characteristics of BMOs, sequestration (sorption) of oxyanions could likely occur mainly at the edge sites through the surface complexation.For example, V(V) sorbes on a chemically synthesized birnessite by forming monodentate corning-sharing complexes [42] and by forming a bidentate mononuclear edge-sharing complex [43].Kashiwabara et al. [11], using wavelength dispersive X-ray absorption fine structure spectroscopy, demonstrated that Mn oxide is the main host phase for negatively charged WO4 2− through inner-sphere complex formation.Tanaka et al. [12] proposed the adsorption mechanism of Mo(VI) through the formation of disordered octahedral Mo(VI) species on Mn oxides.These studies [11,12] explain that such oxyanions are preferentially hosted by "negatively-charged" Mn oxide phases in nature.Such complexation and adsorption of the coexisting oxyanions on the birnessite sheet structure possibly resulted in the disturbance of the sheet stacking of the resultant BMOs.Further studies need to evaluate the atomic-level mechanisms of the sequestration processes during enzymatic BMO formation.1).(D) Na2SO4 was used as the experimental control, where no apparent changes in the XRD patterns were observed.The error bars represent the standard deviation (n = 3).(See Table 1).The amounts of oxyanions sequestered (Vseq.,  1).(D) Na 2 SO 4 was used as the experimental control, where no apparent changes in the XRD patterns were observed.The error bars represent the standard deviation (n = 3).(See Table 1).The amounts of oxyanions sequestered (V seq., Mo seq., and W seq. ) were normalized relative to reducible Mn.The error bars represent the standard deviation (n = 3).

Figure 1 .
Figure 1.Sequestration of (A) V(V), (B) Mo(VI), (C) W(VI) (~20 µ M) synchronized with biogenic Mn oxidation (~1 mM, and ~0.2 mM for W(VI)) during the incubation of Acremonium strictum KR21-2 in HAY liquid media (20 mM HEPES buffer at pH 7.0).No significant sequestration was observed when no Mn 2+ was supplemented.(D) Sequestration of Cr(VI) was negligible even when the biogenic Mn oxide was formed.Shadows indicate the timing of biogenic Mn oxide formation typically 34-46 h of the incubation.The error bars represent the standard deviation (n = 3).

Figure 1 .
Figure 1.Sequestration of (A) V(V), (B) Mo(VI), (C) W(VI) (~20 µM) synchronized with biogenic Mn oxidation (~1 mM, and ~0.2 mM for W(VI)) during the incubation of Acremonium strictum KR21-2 in HAY liquid media (20 mM HEPES buffer at pH 7.0).No significant sequestration was observed when no Mn 2+ was supplemented.(D) Sequestration of Cr(VI) was negligible even when the biogenic Mn oxide was formed.Shadows indicate the timing of biogenic Mn oxide formation typically 34-46 h of the incubation.The error bars represent the standard deviation (n = 3).

Figure 3 .
Figure 3.(A) Sequestration of V(V) (0.08-0.8 mM) by newly formed primary BMO (~1 mM as Mn) with and without exogenous Mn 2+ .Exogenous Mn 2+ was readily oxidized within 4 h of the reaction.(B) Two-step extraction for the resultant solid phases at 24 h of the reaction.(C) The concentration of V(V) sequestered (mM) and (D) the amounts of V(V) sequestered normalized to reducible Mn (mol/mol) with and without exogenous Mn 2+ at 24 h of the reaction.The error bars represent the standard deviation (n = 3).

Figure 4 .
Figure 4.The effects of (A,D,G) V(V), (B,E,H) Mo(VI), and (C,F,I) W(VI) on (A-C) the conversion efficiency of exogenous Mn 2+ , (D-F) the contents of reducible (oxidized) Mn in the solid phase, measured by the two-step extraction procedure and (G-I) sequestration of oxyanions (normalized to reducible Mn).V(V) (0.08-0.8 mM) by newly formed primary BMO (~1 mM as Mn) with and without exogenous Mn 2+ at 24 h of the reaction.Green, blue, and red symbols represent data obtained under the experimental conditions of BMO 2.0 /ExMn 0.0 , BMO 1.0 /ExMn 1.0 , and BMO 0.5 /ExMn 1.5 , respectively (See Table1).The error bars represent the standard deviation (n = 3).

Table 1 .
• C at 105 strokes min −1 .Experimental conditions for sequestration experiments using newly formed BMOs with and without exogenous Mn 2+ .