Sequestration and Oxidation of Cr(III) by Fungal Mn Oxides with Mn(II) Oxidizing Activity

: Biogenic manganese oxides (BMOs) have gained increasing attention for environmental application because of their sequestration and oxidizing abilities for various elements. Oxidation and sequestration of Cr(III) processes by BMOs, however, still remain unknown. We prepared BMOs in liquid cultures of Acremonium strictum strain KR21-2, and subsequently conducted single or repeated treatment experiments in Cr(NO 3 ) 3 at pH 6.0. Under aerobic conditions, newly formed BMOs exhibited a rapid production of Cr(VI) without a signiﬁcant release of Mn(II), demonstrating that newly formed BMO mediates a catalytic oxidation of Cr(III) with a self-regeneration step of reduced Mn. In anaerobic solution, newly formed BMOs showed a cessation of Cr(III) oxidation in the early stage of the reaction, and subsequently had a much smaller Cr(VI) production with signiﬁcant release of reduced Mn(II). Extraordinary sequestration of Cr(III) was observed during the repeated treatments under anaerobic conditions. Anaerobically sequestered Cr(III) was readily converted to Cr(VI) when the conditions became aerobic, which suggests that the surface passivation is responsible for the anaerobic cessation of Cr(VI) oxidation. The results presented herein increase our understanding of the roles of BMO in Cr(III) oxidation and sequestration processes in potential application of BMOs towards the remediation of Cr(III) / Cr(VI) in contaminated sites.


Introduction
Chromium (Cr) is widely used in various industries such as electroplating, pigment production, wood preservation, tanning of animal hides, etc. [1,2]. Concomitant anthropogenic emissions of Cr into the environment have increasingly induced a significant environmental concern because of the potential toxicity of Cr toward humans [3], fishes [4], and plants [5][6][7]. Therefore, development of cost-effective remediation systems for Cr is urgently needed [8,9].
In natural environments, Cr exists as trivalent (Cr [III]) and hexavalent (Cr[VI]) forms which have quite different physicochemical properties. Although Cr(III) is less toxic and nutritionally essential for some organisms, including humans, large amounts of Cr(III) can cause health problems such as lung

Cr(III) Oxidation Efficiency of Newly Formed BMOs with an Enzymatic Mn(II)-Oxidizing Activity
We conducted the treatment experiments of Cr(NO 3 ) 3 by newly formed BMO under aerobic condition to clear Cr(III) oxidation efficiency by newly formed BMO at pH 6.0 where the enzymatic Mn(II)-oxidizing activity is active to readily oxidized Mn 2+ to insoluble Mn oxide [54]. When newly formed BMOs (1 mM as Mn) were reacted with 0.1-0.5 mM Cr(NO 3 ) 3 in 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH at 6.0) under aerobic conditions, rapid production of Cr(VI) dissolved (Cr VI diss ) was observed within the first 4-h reaction (Figure 1), and then gradually increased to 79%-86% of the initial Cr(III) (Cr III Int ) at 24 h ( Figure 1E and Table S1). This indicates that they readily convert Cr(III) to Cr(VI) at pH 6.0, whereas chemically synthesized MnO 2 commonly ceases the Cr(III) oxidation reaction at pH > 5 [22,23]. At pH 6.0, HCr VI O 4 -(76%) and Cr VI O 4 2− (24%) coexists [55] according to the following acid dissociation equilibrium [56].
HCrO 4 − (aq) → H + (aq) + CrO 4 − (aq) pK a2 = 6.5 (2) Thus, oxidation reactions of Cr(III) to Cr(VI) by MnO 2 can be expressed as Equations (3) and (4) However, throughout the treatments, the Mn(II) concentrations released (Mn II rel ) from the BMO phase were consistently lower than 0.005 mM ( Figure S1 and Table S1), which is two orders of magnitude less than the calculated values from Equations (3) and (4). In the case of the aerobic treatments, in 0.5 mM Cr III Int (i.e., the highest concentration in this experiment) the observed Cr VI diss and Mn II rel were 0.39 mM and <0.005 mM (Table S1), respectively, giving the Cr VI diss /Mn II rel ratio of >78. Newly formed BMOs effectively oxidize exogenous Mn 2+ through the enzymatic Mn(II)-oxidizing activity maintained in the BMO under aerobic conditions at pH 6.0 [27,45,54] as: 2Mn 2+ (aq) + O 2 (aq) + 2H 2 O(aq) → 2Mn IV O 2 (s) + 4H + (aq) (5) The observed Cr VI diss /Mn II rel ratio suggests the effective reoxidation of Mn 2+ (and intermediate Mn III ) that was produced by the redox reaction with Cr(III), through Mn(II)-oxidizing enzyme activity. Thus, the enzymatically active BMOs act as a catalytic agent for Cr(III) oxidation with the terminal electron acceptor of dissolved O 2 , by which homogeneous oxidation of Cr(III) does not proceed under the same conditions ( Figure S2A). Our previous results have demonstrated such catalytic oxidation processes of As(III) to As(V) [29] and of Co(II) to Co(III) [30] (as the terminal electron acceptor of dissolved O 2 ) using newly formed BMOs, which continuously mediates the oxidation reactions without release of reduced Mn. Enzymatically active BMOs also performed, through catalytic mediation, effective Ce(III) to Ce(IV) oxidation with a lower release of Mn(II), where the Ce oxidized/Mn released molar of >200 [54]. Repeated treatments of newly formed BMO in 0.1 mM Cr(NO3)3 (100 mM MES at pH 6.0) solution with renewal of the bathing solution every 24 h confirmed that the catalytic Cr(III) oxidation by newly formed BMO continued for least 18 days ( Figure 2). Following each treatment, the Cr VI diss concentration ranged from 0.097 ± 0.003 mM for the 5th treatment to 0.068 ± 0.002 mM for the last treatments, whereas Mn II rel was <0.008 mM throughout, resulting in Cr VI diss/Mn II rel molar ratios from 150 ± 9 (1st treatment) to 8.6 ± 0.8 (last treatment). Decrease in Cr VI diss/Mn II rel molar ratios suggests that the Mn(II) oxidizing activity in BMOs gradually weakened as the treatment was repeatedly conducted. Even in the last treatment, the Cr VI diss/Mn II rel molar ratios were still higher than was expected from the redox stoichiometry of Cr VI diss/Mn II rel at 0.67 (Equations (3) and (4)). In this repeated treatment, the oxidative conversion (Cr VI diss relative to Cr III Int) was higher than 66% ± 1% for each treatment with the cumulative conversion of 86% ± 1% for 18 days (Figure 2). When newly formed BMO (1 mM as Mn) was repeatedly treated in a higher concentration of Cr(NO3)3 at 0.5 mM (100 mM MES at pH 6.0), conversion of Cr III Int to Cr VI diss was high at 86% ± 2% and 90% ± 2% for the 1st and 2nd treatments, respectively, without significant release of Mn(II) (Mn II rel) ( Figure 3 and Table S2). This shows the ability of newly formed BMOs to oxidize Cr(III) in these Cr III Int concentration ranges, in which some Mn(II)-oxidizing bacteria commonly lost growth and/or Mn(II)-oxidizing abilities; Psedomonas putida MnB-1 growth is partly inhibited at 0.2 mM and totally inhibited at 0.5 mM Cr(III) [18]. Murry et al. [17], also reported Cr(III) and Cr(VI) toxicity on Psedomonas putida GB-1 growth at concentrations ≥ 0.01 mM. Preformation of fungal BMOs in the culture without any Cr(III)/Cr(VI) can prevent the inhibitory effect of Cr and subsequently make the microbially-mediated Cr(III) oxidation sufficient, even at a high Cr(III) concentration. In the 3rd treatment, however, a smaller conversion (19% ± 0%) was observed (Figure 3), which was likely due to inhibitory effects through the exposure of higher Cr(III) and/or Cr(VI) concentrations on enzymatic Mn(II) oxidation.   Repeated treatments of newly formed BMO in 0.1 mM Cr(NO 3 ) 3 (100 mM MES at pH 6.0) solution with renewal of the bathing solution every 24 h confirmed that the catalytic Cr(III) oxidation by newly formed BMO continued for least 18 days ( Figure 2). Following each treatment, the Cr VI diss concentration ranged from 0.097 ± 0.003 mM for the 5th treatment to 0.068 ± 0.002 mM for the last treatments, whereas Mn II rel was <0.008 mM throughout, resulting in Cr VI diss /Mn II rel molar ratios from 150 ± 9 (1st treatment) to 8.6 ± 0.8 (last treatment). Decrease in Cr VI diss /Mn II rel molar ratios suggests that the Mn(II) oxidizing activity in BMOs gradually weakened as the treatment was repeatedly conducted. Even in the last treatment, the Cr VI diss /Mn II rel molar ratios were still higher than was expected from the redox stoichiometry of Cr VI diss /Mn II rel at 0.67 (Equations (3) and (4)). In this repeated treatment, the oxidative conversion (Cr VI diss relative to Cr III Int ) was higher than 66% ± 1% for each treatment with the cumulative conversion of 86% ± 1% for 18 days (Figure 2). When newly formed BMO (1 mM as Mn) was repeatedly treated in a higher concentration of Cr(NO 3 ) 3 at 0.5 mM (100 mM MES at pH 6.0), conversion of Cr III Int to Cr VI diss was high at 86% ± 2% and 90% ± 2% for the 1st and 2nd treatments, respectively, without significant release of Mn(II) (Mn II rel ) ( Figure 3 and Table S2). This shows the ability of newly formed BMOs to oxidize Cr(III) in these Cr III Int concentration ranges, in which some Mn(II)-oxidizing bacteria commonly lost growth and/or Mn(II)-oxidizing abilities; Psedomonas putida MnB-1 growth is partly inhibited at 0.2 mM and totally inhibited at 0.5 mM Cr(III) [18]. Murry et al. [17], also reported Cr(III) and Cr(VI) toxicity on Psedomonas putida GB-1 growth at concentrations ≥0.01 mM. Preformation of fungal BMOs in the culture without any Cr(III)/Cr(VI) can prevent the inhibitory effect of Cr and subsequently make the microbially-mediated Cr(III) oxidation sufficient, even at a high Cr(III) concentration. In the 3rd treatment, however, a smaller conversion (19% ± 0%) was observed (Figure 3), which was likely due to inhibitory effects through the exposure of higher Cr(III) and/or Cr(VI) concentrations on enzymatic Mn(II) oxidation.

Cessation of Cr(III) Oxidation by BMOs under Anaerobic Condition
Under anaerobic condition, enzymatic Mn(II) oxidation is suppressed due to the lack of dissolved O 2 as the terminal electron acceptor [27]. Anaerobic treatments of newly formed BMO in Cr(NO 3 ) 3 were conducted to deduce the Cr(III) oxidation efficiency when the catalytic mediation for Cr(III) oxidation by BMOs was inactive. Single treatment of newly formed BMOs (1 mM as Mn) with anaerobic 0.1-0.5 mM Cr(NO 3 ) 3 (100 mM MES at pH 6.0) showed that Cr VI diss production ceased within the first 2 h of the treatment ( Figure 4) and accompanied significant release of Mn(II) (Mn II rel ) (0.02-0.03 mM), because BMOs failed to reoxidize Mn II rel ( Figure S1B). The anaerobic cessation of Cr(III) oxidation resulted in much lower Cr VI diss concentrations, ranging from 0.03 (0.1 mM Cr III Int ) to 0.05 mM (0.5 mM Cr III Int ) ( Figure 4E and Table S1), demonstrating an important role of enzymatic Mn(II) oxidation for continuous mediation of Cr(III) oxidation by BMOs. Cessation of Cr(III) oxidation has been found for chemically synthesized Mn oxides [22][23][24][25][26], even under aerobic condition at pH > 5. In fact, heated BMOs, in which the enzymatic Mn(II) oxidizing activities were inactivated [27], showed cessation of Cr(III) oxidation ( Figure 5) and concomitant Mn(II) release ( Figure S1C) even under aerobic conditions, in a similar manner as abiotic Mn oxide phases.

Cessation of Cr(III) Oxidation by BMOs under Anaerobic Condition
Under anaerobic condition, enzymatic Mn(II) oxidation is suppressed due to the lack of dissolved O2 as the terminal electron acceptor [27]. Anaerobic treatments of newly formed BMO in Cr(NO3)3 were conducted to deduce the Cr(III) oxidation efficiency when the catalytic mediation for Cr(III) oxidation by BMOs was inactive. Single treatment of newly formed BMOs (1 mM as Mn) with anaerobic 0.1-0.5 mM Cr(NO3)3 (100 mM MES at pH 6.0) showed that Cr VI diss production ceased within the first 2 h of the treatment ( Figure 4) and accompanied significant release of Mn(II) (Mn II rel) (0.02-0.03 mM), because BMOs failed to reoxidize Mn II rel ( Figure S1B). The anaerobic cessation of Cr(III) oxidation resulted in much lower Cr VI diss concentrations, ranging from 0.03 (0.1 mM Cr III Int) to 0.05 mM (0.5 mM Cr III Int) ( Figure 4E and Table S1), demonstrating an important role of enzymatic Mn(II) oxidation for continuous mediation of Cr(III) oxidation by BMOs. Cessation of Cr(III) oxidation has been found for chemically synthesized Mn oxides [22][23][24][25][26], even under aerobic condition at pH > 5. In fact, heated BMOs, in which the enzymatic Mn(II) oxidizing activities were inactivated [27], showed cessation of Cr(III) oxidation ( Figure 5) and concomitant Mn(II) release ( Figure S1C) even under aerobic conditions, in a similar manner as abiotic Mn oxide phases.     Interestingly, this first phase (~2 h) of the anaerobic reaction involved the sequestration process of total Cr concentration (Cr T diss) until Cr T diss became equal mostly to Cr VI diss (Figure 4). Cr(VI) does not have significant sorption affinity to either Mn oxide phase or fungal hyphae in BMOs in 100 mM MES at pH 6.0 ( Figure S2B,C). Therefore, Cr T diss sequestration in the anaerobic treatments was mostly attributable to sorption and/or precipitation of Cr(III) on BMOs.  [46][47][48][49]. These polynuclear Cr(III) species may eventually precipitate as Cr(OH)3. Fendorf et al. [22] proposed that Cr(OH)3 precipitate on Mn oxide surface may be a causative factor for cessation of Cr(III) oxidation by acting as a barrier to the charge transfer between Cr(III) and structural Mn(III)/Mn(V), and as a redox stable sink for Cr(III). In contrast, Landrot et al. [22,23] observed no Cr precipitation on Mn oxide surfaces reacted with Cr(III) for 1 h, at which time the Cr(III) oxidation had already ceased, suggesting inhibition mechanisms other than Cr(OH)3 precipitation were the cause for the cessation.
To check the reversibility of the cessation of Cr(III) oxidation by newly formed BMOs with respect to enzymatic Mn(II) activity, we conducted the single treatment of newly formed BMO (1 mM as Mn) in 0.5 mM Cr(NO3)3 (100 mM MES at pH 6.0) under anaerobic, and subsequently, aerobic conditions. During the initial anaerobic treatment, Cr VI diss production and significant Mn II rel concomitantly ceased within the first 2 h, when the majority of the Cr III Int was sequestered (Cr T seq) in the solid phase ( Figure 6). When the condition was switched to aerobic, Cr VI diss was rapidly produced and concomitantly Mn II rel disappeared from the solution phase ( Figure 6). The reversible production of Cr VI diss with respect to enzymatic Mn(II) oxidation activity may deny the hypothesis that Cr(OH)3 Interestingly, this first phase (~2 h) of the anaerobic reaction involved the sequestration process of total Cr concentration (Cr T diss ) until Cr T diss became equal mostly to Cr VI diss ( Figure 4). Cr(VI) does not have significant sorption affinity to either Mn oxide phase or fungal hyphae in BMOs in 100 mM MES at pH 6.0 ( Figure S2B,C). Therefore, Cr T diss sequestration in the anaerobic treatments was mostly attributable to sorption and/or precipitation of Cr(III) on BMOs. At circumneutral pH in aqueous solutions, Cr 3+ [46][47][48][49]. These polynuclear Cr(III) species may eventually precipitate as Cr(OH) 3 . Fendorf et al. [22] proposed that Cr(OH) 3 precipitate on Mn oxide surface may be a causative factor for cessation of Cr(III) oxidation by acting as a barrier to the charge transfer between Cr(III) and structural Mn(III)/Mn(V), and as a redox stable sink for Cr(III). In contrast, Landrot et al. [22,23] observed no Cr precipitation on Mn oxide surfaces reacted with Cr(III) for 1 h, at which time the Cr(III) oxidation had already ceased, suggesting inhibition mechanisms other than Cr(OH) 3 precipitation were the cause for the cessation.
To check the reversibility of the cessation of Cr(III) oxidation by newly formed BMOs with respect to enzymatic Mn(II) activity, we conducted the single treatment of newly formed BMO (1 mM as Mn) in 0.5 mM Cr(NO 3 ) 3 (100 mM MES at pH 6.0) under anaerobic, and subsequently, aerobic conditions. During the initial anaerobic treatment, Cr VI diss production and significant Mn II rel concomitantly ceased within the first 2 h, when the majority of the Cr III Int was sequestered (Cr T seq ) in the solid phase ( Figure 6). When the condition was switched to aerobic, Cr VI diss was rapidly produced and concomitantly Mn II rel disappeared from the solution phase ( Figure 6). The reversible production of Cr VI diss with respect to enzymatic Mn(II) oxidation activity may deny the hypothesis that Cr(OH) 3 precipitation is responsible for anaerobic cessation of Cr(III) oxidation by BMOs. Our previous studies demonstrated the anaerobic cessation of oxidative reactions of As(III) to As(V) [29] and Co(II) to Co(III) [30] by newly formed BMOs, whereas aerobic conditions prevented the cessation of As(III)and Co(II)-oxidation reactions. We assumed the anaerobic cessation of Cr(III) oxidation was due to the surface passivation by the accumulation of reduced Mn(II)/Mn(III) species at the redox reactive sites on BMOs, resulting in insufficient electron transfer from the BMO phase to the reductant species including Cr(III).
Catalysts 2020, 10, x FOR PEER REVIEW 8 of 14 precipitation is responsible for anaerobic cessation of Cr(III) oxidation by BMOs. Our previous studies demonstrated the anaerobic cessation of oxidative reactions of As(III) to As(V) [29] and Co(II) to Co(III) [30] by newly formed BMOs, whereas aerobic conditions prevented the cessation of As(III)and Co(II)-oxidation reactions. We assumed the anaerobic cessation of Cr(III) oxidation was due to the surface passivation by the accumulation of reduced Mn(II)/Mn(III) species at the redox reactive sites on BMOs, resulting in insufficient electron transfer from the BMO phase to the reductant species including Cr(III).

Anaerobic Sequestration of Cr(III) by BMOs
When the newly formed BMOs (1 mM as Mn) were repeatedly treated in anaerobic 0.5 mM Cr(NO3)3 in 100 mM MES with the renewal of the bathing solutions every 24 h (Figure 7), the cumulative Cr VI diss concentration was 0.06 ± 0.00 mM (0.03 ± 0.00, 0.01 ± 0.00, and 0.01 ± 0.00 mM for the 1st, 2nd, and 3rd treatments, respectively), resulting in only 4% of cumulative Cr III Int oxidation ( Figure 7 and Table S2). In contrast to very low oxidation efficiency, the cumulative C T seq concentration was high at 1.18 mM (0.43 ± 0.01, 0.46 ± 0.01, and 0.14 ± 0.00 mM for the 1st, 2nd, and 3rd treatments, respectively), corresponding to 83% ± 1% of the cumulative Cr III Int (Figure 7 and Table  S2). The molar ratio of C T seq relative to solid Mn was abnormally high (~120 mol%), implying a sequestration mechanism specific to Cr other than the simple sorption process of common divalent heavy metal ions, Zn 2+ and Cd 2+ (their maximum sorption relative to solid Mn was 20-25 mol%; [39,41]) or trivalent La 3+ ions (~30 mol% [45]). Heated BMOs possessed similar Cr sequestration capacity ( Figure S3) even under aerobic conditions with the domination of Cr(III) in the solid phase, which was revealed by X-ray Absorption Near-Edge Structure (XANES) (Figure 8). The XANES spectrum for the reference compound of K2Cr VI 2O4 showed a sharp pre-edge and a broad peak at 5988 eV and around 6030 eV, respectively. The former peak is assigned at the 1s→3d-4p hybrid orbital transition and characteristic of Cr(VI) [57]. The XANES spectrum for Cr III (NO3)3 9H2O has a main peak around 6005 eV, along with a pre-edge peak (the 1s→3d-4p hybrid orbital transition of Cr(III)) at 5985 eV with a much smaller intensity (Figure 8) [57]. The linear combination fit showed that BMO had >95% Cr(III) content after treatment in 0.5 mM Cr(NO3)3 for 24 h. Consequently, it is likely that sorption as the polynuclear Cr(III) species would lead to abnormally high Cr sequestration capacity of BMO when enzymatically inactivated. The XANES spectrum also showed that domination of Cr(III) remained in newly formed BMOs at 24 h of the aerobic Cr(III) treatments where >80% of Cr III Int

Anaerobic Sequestration of Cr(III) by BMOs
When the newly formed BMOs (1 mM as Mn) were repeatedly treated in anaerobic 0.5 mM Cr(NO 3 ) 3 in 100 mM MES with the renewal of the bathing solutions every 24 h (Figure 7), the cumulative Cr VI diss concentration was 0.06 ± 0.00 mM (0.03 ± 0.00, 0.01 ± 0.00, and 0.01 ± 0.00 mM for the 1st, 2nd, and 3rd treatments, respectively), resulting in only 4% of cumulative Cr III Int oxidation (Figure 7 and Table S2). In contrast to very low oxidation efficiency, the cumulative C T seq concentration was high at 1.18 mM (0.43 ± 0.01, 0.46 ± 0.01, and 0.14 ± 0.00 mM for the 1st, 2nd, and 3rd treatments, respectively), corresponding to 83% ± 1% of the cumulative Cr III Int (Figure 7 and Table S2). The molar ratio of C T seq relative to solid Mn was abnormally high (~120 mol%), implying a sequestration mechanism specific to Cr other than the simple sorption process of common divalent heavy metal ions, Zn 2+ and Cd 2+ (their maximum sorption relative to solid Mn was 20-25 mol%; [39,41]) or trivalent La 3+ ions (~30 mol% [45]). Heated BMOs possessed similar Cr sequestration capacity ( Figure S3) even under aerobic conditions with the domination of Cr(III) in the solid phase, which was revealed by X-ray Absorption Near-Edge Structure (XANES) (Figure 8). The XANES spectrum for the reference compound of K 2 Cr VI 2 O 4 showed a sharp pre-edge and a broad peak at 5988 eV and around 6030 eV, respectively. The former peak is assigned at the 1s→3d-4p hybrid orbital transition and characteristic of Cr(VI) [57]. The XANES spectrum for Cr III (NO 3 ) 3 9H 2 O has a main peak around 6005 eV, along with a pre-edge peak (the 1s→3d-4p hybrid orbital transition of Cr(III)) at 5985 eV with a much smaller intensity (Figure 8) [57]. The linear combination fit showed that BMO had >95% Cr(III) content after treatment in 0.5 mM Cr(NO 3 ) 3 for 24 h. Consequently, it is likely that sorption as the polynuclear Cr(III) species would lead to abnormally high Cr sequestration capacity of BMO when enzymatically inactivated. The XANES spectrum also showed that domination of Cr(III) remained in newly formed BMOs at 24 h of the aerobic Cr(III) treatments where >80% of Cr III Int was converted to Cr VI diss , strongly indicating a preferential sorption of Cr(III) over Cr(VI) on BMO. Once Cr(III) is converted to Cr(VI) on BMO surface, it is readily leached to the solution phase as demonstrated in Figure 6.
Catalysts 2020, 10, x FOR PEER REVIEW 9 of 14 was converted to Cr VI diss, strongly indicating a preferential sorption of Cr(III) over Cr(VI) on BMO. Once Cr(III) is converted to Cr(VI) on BMO surface, it is readily leached to the solution phase as demonstrated in Figure 6.  Cumulative Cr and Mn (mM)  Figure 6.  Cumulative Cr and Mn (mM)  Newly formed BMOs produced by A. strictum KR21-2 are layered analogously to vernadite, a natural nanostructured and turbostratic variety of birnessite [32]. Prior to 0.5 mM Cr(NO 3 ) 3 treatments, no apparent changes in X-ray Diffraction (XRD) patterns were observed between newly formed and heated BMOs (Figure 9), for which the XRD peaks at~7.3, 2.4, and 1.4 Å were assigned to (001), (11,20), and (31,02), respectively [39]. Following the treatment, heated BMO exhibited a decline in XRD intensity arising from (001) basal reflection (Figure 9), suggesting that the ordering of the vernadite (birnessite) sheet stacking was mostly disturbed by sorption of polynuclear Cr(III) species with a high proportion of Cr T seq (0.42 ± 0.00 mM) relative to solid Mn (1 mM as Mn). In contrast, newly formed BMOs maintained a (001) XRD intensity even after the treatments (Figure 9) where much less Cr T seq remained in the solid (0.06 ± 0.02 mM). High sorption affinity of BMO for polynuclear Cr(III) species should play an as important role in efficient Cr(III) oxidation in aerobic treatments because significant amounts of Cr sequestration (i.e., a decrease in Cr T diss in Figure 1) progressed concomitantly with Cr VI diss production. Newly formed BMOs produced by A. strictum KR21-2 are layered analogously to vernadite, a natural nanostructured and turbostratic variety of birnessite [32]. Prior to 0.5 mM Cr(NO3)3 treatments, no apparent changes in X-ray Diffraction (XRD) patterns were observed between newly formed and heated BMOs (Figure 9), for which the XRD peaks at ~7.3, 2.4, and 1.4 Å were assigned to (001), (11,20), and (31,02), respectively [39]. Following the treatment, heated BMO exhibited a decline in XRD intensity arising from (001) basal reflection (Figure 9), suggesting that the ordering of the vernadite (birnessite) sheet stacking was mostly disturbed by sorption of polynuclear Cr(III) species with a high proportion of Cr T seq (0.42 ± 0.00 mM) relative to solid Mn (1 mM as Mn). In contrast, newly formed BMOs maintained a (001) XRD intensity even after the treatments (Figure 9) where much less Cr T seq remained in the solid (0.06 ± 0.02 mM). High sorption affinity of BMO for polynuclear Cr(III) species should play an as important role in efficient Cr(III) oxidation in aerobic treatments because significant amounts of Cr sequestration (i.e., a decrease in Cr T diss in Figure 1) progressed concomitantly with Cr VI diss production.
Repeated treatment experiments were also carried out with renewal of the bathing solutions (0.1 or 0.5 mM Cr(NO 3 ) 3 solution with 100 mM MES) every 24 h under aerobic and anaerobic conditions.

X-ray Absorption Near-Edge Structure (XANES) Spectroscopy
Chromium K-edge XANES spectra were collected at the beamline 12C at the Photon Factory, KEK (Tsukuba, Japan). Newly formed (enzymatically active) and heated (enzymatically inactive) BMOs were treated with 0.5 mM Cr(NO 3 ) 3 in 100 mM MES (pH = 6.0) for under aerobic condition (24 h), harvested and lyophilized. As references for Cr(VI) and Cr(III) species, aqueous solutions of K 2 Cr 2 O 7 and Cr(NO 3 ) 3 ·9H 2 O, were measured respectively. The storage ring was operated at 2.5 GeV with a typical beam current of 450 mA. The incident X-ray beam was obtained by monochromatization of the broad band synchrotron radiation from the storage ring with a Si (111) double-crystal monochromator. The beam size of the incident X-ray was focused into an area 1 × 1 mm 2 at the sample position. The spectra of the reference solutions and the BMO samples were collected in transmission mode using ionization chambers.

Powder X-ray Diffraction (XRD)
The XRD measurements BMOs were carried out after single treatment experiments with 0.5 mM Cr(NO 3 ) 3 in 100 mM MES (pH = 6.0) for newly formed (enzymatically active) and heated (enzymatically inactive) BMOs under aerobic condition (24 h). Treated BMOs were harvested, and lyophilized. A RINT-2500 diffractometer (Rigaku Corp., Tokyo, Japan) was used with CuKα radiation at 26 mA and 40 kV. The scanning range was from 5 • to 70 • of 2θ and scanning rate was 1.0 • min −1 with a step interval of 0.02 • . The diffractograms were smoothed by the 10-point moving average method to display broad peaks more clearly.

Conclusions
The results presented here demonstrate that under aerobic conditions, enzymatically active BMOs efficiently mediate oxidation of Cr(III) to Cr(VI) without releasing reduced Mn(II) at pH 6.0. Thus, enzymatically active BMOs may aerobically leach (remove) Cr from the Cr(III)-contaminated sites, through catalytic oxidation to more labile Cr(VI). In contrast, Cr(III) oxidation readily ceases under anaerobic conditions due to the surface passivation of BMOs, whereas sequestration of Cr(III) (most probably as polynuclear Cr(III) species) progresses with extraordinary sequestration capacity of Cr(III) (up to 120% mol of Cr relative to solid Mn). This shows the potential applicability of BMOs as an efficient sorbent for Cr(III) from wastewater at a circumneutral pH. Interestingly, anaerobically sequestered Cr(III) can subsequently be released by switching to aerobic conditions, through the catalytic (enzymatic) Mn oxidation cycles by BMOs. These findings provide supporting evidence for the potential applications of BMOs towards the recovery of Cr species from industrial wastewater (anaerobic Cr sequestration) and environmental remediation of Cr-contaminated sites (aerobic oxidative Cr leaching).