We examined the Mn(II)-oxidizing ability of the biogenic Mn oxide (BMO) formed in cultures of a Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2. The newly formed BMO effectively sequestered dissolved Mn(II) mainly by oxidizing Mn(II) to insoluble Mn under air-equilibrated conditions, and this ability lasted for at least 8 days. Deaerating the BMOs, poisoning them with NaN₃, or heating them all readily weakened their Mn(II) oxidation ability, indicating the involvement of enzymatic Mn(II) oxidation. There was no Mn(II)-oxidizing ability observed for mycelia cultivated without Mn(II) or for residual mycelia after the BMO phase was dissolved, suggesting the need for the oxide phase. A sodium dodecyl sulphate-polyacrylamide gel electrophoresis assay demonstrated that the oxide phase embeds the Mn(II) oxidase and thereby maintains the enzymatic activity in BMOs. Freezing at−80°C preserved the Mn(II)-oxidizing ability in BMOs for at least 4 weeks, while lyophilization caused a complete loss of this ability. Based on these results, we propose that fungal Mn oxides supporting Mn(II) oxidase activity are an effective Mn(II)-sequestering material capable of oxidizing Mn(II) continuously from solutions containing no additional nutrients to maintain biological 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) 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(NO₃)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(III) 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.

Diverse ascomycete fungi oxidize manganese(II) [Mn(II)] and produce Mn(III, IV) oxides in terrestrial and freshwater environments. Although multicopper oxidase (MCO) is considered to be a key catalyst in mediating Mn(II) oxidation in ascomycetes, the responsible gene and its product have not been identified. In this study, a gene, named mco1, encoding Mn(II)-oxidizing MCO from Acremonium strictum strain KR21-2 was cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Based on the phylogenetic relationship, similarity of putative copper-binding motifs, and homology modeling, the gene product Mco1 was assigned to a bilirubin oxidase. Mature Mco1 was predicted to be composed of 565 amino acids with a molecular mass of 64.0 kDa. The recombinant enzyme oxidized Mn(II) to yield spherical Mn oxides, several micrometers in diameter. Zinc(II) ions added to the reaction mixture were incorporated by the Mn oxides at a Zn/Mn molar ratio of 0.36. The results suggested that Mco1 facilitates the growth of the micrometer-sized Mn oxides and affects metal sequestration through Mn(II) oxidation. This is the first report on heterologous expression and identification of the Mn(II) oxidase enzyme in Mn(II)-oxidizing ascomycetes. The cell-free, homogenous catalytic system with recombinant Mco1 could be useful for understanding Mn biomineralization by ascomycetes and the sequestration of metal ions in the environment