Naoyuki Miyata

Structure of nanocrystalline phyllomanganates produced by freshwater fungi

Abstract The crystal structures of biogenic Mn oxides produced by three fungal strains isolated from stream pebbles were determined using chemical analyses, XANES and EXAFS spectroscopy, and powder X-ray diffraction. The fungi-mediated oxidation of aqueous Mn2+ produces layered Mn oxides analogous to vernadite, a natural nanostructured and turbostratic variety of birnessite. The crystallites have domain dimensions of ~10 nm in the layer plane (equivalent to ~35 MnO6 octahedra), and ~1.5-2.2 nm perpendicularly (equivalent to ~2-3 layers), on average. The layers have hexagonal symmetry and from 22 to 30% vacant octahedral sites. This proportion likely includes edge sites, given the extremely small lateral size of the layers. The layer charge deficit, resulting from the missing layer Mn4+ cations, is balanced mainly by interlayer Mn3+ cations in triple-corner sharing position above and/or below vacant layer octahedra. The high surface area, defective crystal structure, and mixed Mn valence confer to these bio-minerals an extremely high chemical reactivity. They serve in the environment as sorption substrate for trace elements and possess catalytic redox properties.

Microbial decolorization of melanoidin-containing wastewaters: Combined use of activated sludge and the fungus Coriolus hirsutus

A white rot fungus, Coriolus hirsutus, exhibited a strong ability to decolorize melanoidin in cultures not supplemented with nitrogenous nutrients. Addition of peptone to the cultures lowered the ability of the fungus to decolorize melanoidin, but that of inorganic nitrogens (Ns), ammonium and nitrate did not bring about any marked reduction in the ability. These results suggest an inhibitory effect of organic N on melanoidin decolorization. Therefore, for enhancing the decolorization of melanoidin in wastewaters by the fungus, activated sludge pretreatment of the wastewaters was expected to be effective, i.e., activated sludge is capable of converting available organic N into inorganic N. To confirm this, waste sludge heat treatment liquor (HTL), wastewater from a sewage treatment plant, was pretreated with activated sludge. In practice, pretreatment of HTL under appropriate conditions accelerated the fungal decolorization of HTL. In the pretreated HTL, the fungus was shown to produce a high level of manganese-independent peroxidase (MIP). Addition of Mn(II) to the pretreated HTL caused a further increase in the decolorization efficiency of the fungus and a marked increase in the manganese peroxidase (MnP) activity. Consequently, the increases in MIP and MnP activities were considered to play an important role in the enhanced ability of C. hirsutus to decolorize HTL.

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

ABSTRACT Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.

Sorption of Co(II), Ni(II), and Zn(II) on Biogenic Manganese Oxides Produced by a Mn-Oxidizing Fungus, Strain KR21-2

Abstract The characteristics of Co(II), Ni(II), and Zn(II) sorption on freshly produced biogenic Mn oxides by a Mn-oxidizing fungus, strain KR21-2, were investigated. The biogenic Mn oxides showed about 10-fold higher efficiencies for sorbing the metal ions than a synthetic Mn oxide (γ-MnO2) on the basis of unit weight and unit surface area. The order of sorption efficiency on the biogenic Mn oxides was Co(II) > Zn(II) > Ni(II), while that on the synthetic Mn oxide was Zn(II) > Co(II) > Ni(II). These sorption selectivities were confirmed by both sorption isotherms and competitive sorption experiments. Two-step extraction, using 10 mM CuSO4 solution for exchangeable sorbed ions and 10–20 mM hydroxylamine hydrochloride for ions bound to reducible Mn oxide phase, showed higher irreversibility of Co(II) and Ni(II) sorption on the biogenic Mn oxides while Zn(II) sorption was mostly reversible (Cu(II)-exchangeable). Sorptions of Co(II), Ni(II), and Zn(II) on the synthetic Mn oxide were, however, found to be mostly reversible. Higher irreversibility of Co(II) and Ni(II) sorption on the biogenic Mn oxides may partly explain higher accumulation of these metal ions in Mn oxide phases in natural environments. The results obtained in this study raise the possibility to applying the biogenic Mn oxide formation to treatment of water contaminated with toxic metal ions.

Biogeochemistry of manganese oxide coatings on pebble surfaces in the Kikukawa River System, Shizuoka, Japan

The biogeochemistry of Mn-oxide coatings formed over submerged pebble surfaces on the streambed of the Kikukawa river system has been investigated. Located in central Shizuoka, Japan, this system drains strongly acidified soils under tea plantations. Besides containing high amounts of Mn (up to 450 μg/cm2), the coatings are capable of scavenging and accumulating other elements including Ba, Zn, Ni, Co, W, Mo and Sb. When suspensions of the coating material were incubated with 0.2 mM Mn2+, the Mn(II) ion was microbially transformed into Mn-oxides. When the same suspensions were spread on agar plates containing acetate, yeast-extract, and 1.0 mM Mn2+ (AY agar medium) both Mn-oxidizing bacteria and fungi appeared, indicating the existence of a diversity of Mn-oxidizing microorganisms in the system. Plate counts using two agar culture media with varied nutrient levels indicated that the ability of these microorganisms to oxidize Mn(II) was strongly dependent on nutrient supply. The relatively nutrient-poor AY agar medium was more conducive to microbial growth than the K1 agar medium with a higher organic nutrient content. Concentrations of Mn dissolved in the stream waters did not correlate well with the amounts of solid Mn on submerged pebbles. Thus, factors other than dissolved Mn concentration (e.g., organic nutrient supply and pH) determined the ability of microorganisms to oxidize Mn in the streambeds. A survey of dissolved Mn in streams and water draining tea plantations combined with chemical analysis of Mn in the underlying soils indicate that the soils have been strongly acidified through excessive applications of N-fertilizers. As a result, Mn was leached from the soil column into the Kikukawa river system. Biogenic Mn-oxide coatings on streambeds can therefore serve as an indicator of soil acidification and metal leaching from soils of the corresponding watershed.

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

ABSTRACT The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-14C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state 14C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (Kt) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated 14C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (Kd) of 41 ± 21 nM (mean ± standard deviation). Given the low values of Kt and Kd, Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair.

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.

Production of Biogenic Manganese Oxides by Anamorphic Ascomycete Fungi Isolated from Streambed Pebbles

We characterized the production of biogenic Mn oxides by four anamorphic ascomycete fungi isolated from streambed pebbles with Mn oxide coatings. Based on the 18S rRNA gene sequences, one strain was related to members of the order Xylariales and the other three were within distinct lineages of the Pleosporales. These strains oxidized Mn(II) to deposit Mn oxides when their growth approached the stationary phase. The fungal Mn oxides showed X-ray diffraction patterns typical of poorly crystalline vernadite (δ -MnO2), and X-ray absorption near-edge structure spectroscopy confirmed that the Mn phases consisted predominantly of Mn(IV). Mn(II) oxidation in the four strains proceeded enzymatically. The Mn(II)-oxidizing proteins were inhibited by azide and o-phenanthroline, and the proteins also oxidized typical laccase substrates including 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), showing the role of laccase or a laccase-like metalloenzyme. The mineralogical traits of the biogenic Mn oxides, and the participation of laccase-like enzymes, are in accordance with our previous results obtained with one Hypocreales ascomycete. In conclusion, phylogenetically diverse ascomycetes may use this common enzymatic system to produce solid Mn phases similar to δ -MnO2.

Manganese-independent and -dependent decolorization of melanoidin by extracellular hydrogen peroxide and peroxidases from Coriolus hirsutus pellets☆

Abstract Coriolus hirsutus pellets growth in a melanoidin-containing medium produced extracellular H 2 O 2 up to 43 μM. However, nonenzymatic decolorization of melanoidin by H 2 O 2 , which has been previously reported, seemed very little in the fungal culture. The culture fluid contained two extracellular peroxidases, a manganese-independent peroxidase (MIP) and manganese peroxidase (MnP). Since both MIP and MnP showed melanoidin-decolorizing activities in the presence of H 2 O 2 , it was concluded that melanoidin decolorization in the C. hirsutus culture involved the production of extracellular H 2 O 2 and the peroxidases.

Formation of stable foam by the cells and culture supernatant of Gordonia (Nocardia) amarae

Gordonia amarae is the cause of foaming activated sludge. In this study, the mechanism of foam formation by G. amarae SC1 was investigated. A liquid culture of SC1 cells generated a stable foam when shaken reciprocally. This foam formation was dependent on the presence of both bacterial cells and culture supernatant. A high-molecular-weight fraction (Mw>10000) of the supernatant was capable of emulsifying n-hexadecane in addition to exhibiting foaming activity, indicating that it contains a surface-active substance(s). The bacterial cells showed a high affinity to hexadecane. This hydrophobic cell surface property might be involved in the attachment of cells to air bubbles to generate a stable foam. The results demonstrated the participation of cells and the extracellular biosurfactant in the formation and stabilization of foam in G. amarae SC1 culture.