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Electrolyzed water is a novel disinfectant that is widely used in the food industry. We conducted an experimental model-based study to determine the effectiveness of neutral electrolyzed water (NEW) for the daily nozzle cleaning of artificially contaminated tankless and tank-type bidet toilet seats. The toilet seats were designed to automatically self-clean the spray nozzles using tap water or NEW after each use or at specified intervals. The numbers of Pseudomonas aeruginosa and Escherichia coli microorganisms in the spray water were measured twice or thrice per week. A Kruskal-Wallis test was used to compare the bacterial count in the spray water of various cleaning (NEW) and control (tap water) conditions. The number of bacteria was significantly lower in NEW conditions with and without periodic nozzle cleaning functions than in tap water conditions for both tank-type and tankless bidet toilet seats. Microorganisms were detected only on the surface area around the opening for ejecting spray water and not in the internal piping at the spray nozzle tip. These findings demonstrate that NEW has superior decontamination efficacy over tap water when used as a cleaning agent for the spray nozzles of warm-water bidet toilet seats.

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The biogenic magnetite nanoparticles presented here had a high capacity of adsorbing metal cations, which was approximately 30- to 40-fold greater than commercially available magnetite. These results suggest the potential application of microbial magnetite formation in the removal of toxic metal cations from water.

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KEY MESSAGE: Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance. Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F2:3 population derived from a cross between susceptible 'CS-PMR1' and resistant 'A192-18' cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym (A192-18) . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym (A192-18) locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3 cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym (A192-18) locus; using cucumber genomic and molecular marker resources and screening an F2 population of 2,429 plants, we narrowed down the zym (A192-18) locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes' coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym (A192-18) -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym (A192-18) locus. These results may be valuable for MAS for ZYMV resistance in cucumber.

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Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method.

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Diverse bacteria and fungi oxidize Mn(II) enzymatically and produce insoluble Mn(III, IV) oxides, and these organisms are considered to be the primal agents for the occurrence of natural Mn oxide phases in most environments. Biogenic Mn oxides have a high sorption capacity for metal cations and an ability to oxidize numerous inorganic and organic compounds, owing to their structural and redox features. Thus, the microbial process is of significance in both biogeochemical and biotechnological contexts. In this article we summarize the enzymatic Mn(II) oxidation and interactions of biogenic Mn oxides with toxic metal and metalloid ions. Although Mn oxide formation by fungi has not been fully characterized yet, recent researches with ascomycetes emphasize the similarity between the bacterial and fungal Mn(II) oxidation with respect to the involved catalyst (i.e., multicopper oxidase-type enzymes) and the reaction product [i.e., layer-type Mn(IV) oxides]. Laboratory cultures of bacterial and fungal Mn oxidizers are expected to provide fundamental knowledge in their potential use for remediation of environments and effluents contaminated with toxic metal(loid) ions.

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We investigated the production of manganese (Mn) oxides using repeated-batch bioreactors maintained over long periods under laboratory conditions. Freshwater epilithic biofilms were used as the initial inocula. The bioreactors yielded suspended solids that could remove 0.1 mM dissolved Mn(II) within a few days. Chemical titration, X-ray absorption near-edge structure spectroscopy, and X-ray diffraction analysis revealed that the Mn(II) had been converted to poorly crystallized layer-type Mn(IV) oxides, which were similar to known biogenic Mn oxides from pure bacterial cultures. Spherical or rod-shaped Mn microconcretions occurred in the suspended solids; transmission electron microscopy showed that these structures likely resulted from the microbial activity but not represent living cells. Instead, the presence of encapsulated, sheathed, and hyphal budding cells in the suspended solids indicated that a range of Mn-depositing bacteria contributed to the Mn oxide formation. To our knowledge, our data represent the first observation of production of such Mn oxides in a laboratory microcosm wherein a range of Mn-depositing bacteria coexist. The fact that sorption of trace Zn(II) and Ni(II) ions onto the suspended solids co-occurred with the removal of dissolved Mn(II) emphasizes the important role of Mn-oxidizing microorganisms in the fates of trace or contaminant metals in the aquatic environment.

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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.

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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 (gamma-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 10mM CuSO4 solution for exchangeable sorbed ions and 10-20mM 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.

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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-(14)C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state (14)C 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 (K(t)) of 26 +/- 3 nM (mean +/- standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated (14)C-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 (K(d)) of 41 +/- 21 nM (mean +/- standard deviation). Given the low values of K(t) and K(d), Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.

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The bacterial community structure of anaerobic enrichment cultures that are capable of degrading both cis-1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC) and isolation of the organism responsible for the degradation were investigated. Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rRNA gene from the cultures showed the possible predominance of Clostridium species. One isolate, designated strain DC1, was closely related to members of Clostridiaceae, based on 16S rRNA gene analysis, and the highest sequence similarity (98.9%) was obtained for Clostridium saccarobutylicum. In culture experiments, strain DC1 was shown to degrade cis-DCE and VC during the stationary phase of growth without accumulation of VC and/or ethene. The bacterial growth was not linked to the degradation of cis-DCE and VC. Stoichiometric analysis revealed that two moles of chloride ions as released from one mole of cis-DCE during the incubation period, indicating that cis-DCE was fully dechlorinated. The results appear consistent with the presence of a mechanism of oxidative dechlorination rather than respiratory reductive dechlorination; the latter is accompanied by transient formation of dechlorinated ethenes from cis-DCE and VC.

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Aspergillus oryzae IFO 30113 was used for the treatment of the cassava starch processing (CSP) wastewater. The observations on the fungal morphology showed that, in the shake flasks containing the CSP wastewater with the high concentration of suspended solids, the formation of pellets originated from the adherence of germinated spores to solid particles in medium. The attached solid particles were also digested during the fungal fermentation and resulted in the formation of the smooth and hollow pellets. The changes of the culture conditions such as inoculum size, initial pH of wastewater, inoculum type and nutrient elements affected on the fungal morphology, biomass accumulation and treatment efficiencies of A. oryzae IFO 30113. In the typical pH range (pH 4-5) of the CSP wastewater, the formation of smooth pellets was predominant and A. oryzae IFO 30113 was satisfiable for the production of fungal biomass and treatment efficiencies. The supplementation of nitrogen sources has shown an improvement in the fungal biomass accumulation and the treatment efficiency of A. oryzae IFO 30113 growing in the CSP wastewater. Especially, high biomass yields (up to 0.8 g/g-COD) were achieved in flasks supplied with peptone. With ammonium sulfate as nitrogen source, 87% total organic carbon (TOC), 91% COD and 94% starch were removed after 96-h incubation. The possibility of the pellet formation despite the presence of the high content of suspended solids would be of great advantage to perform the treatment process and the fungal biomass production on the airlift-type bioreactors by lowering medium viscosity and better mass exchange of oxygen and nutrients.

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In batch culture experiments we examined oxidation of As(III) and adsorption of As(III/V) by biogenic manganese oxide formed by a manganese oxide-depositing fungus, strain KR21-2. We expected to gain insight into the applicability of Mn-depositing microorganisms for biological treatment of As-contaminated waters. In cultures containing Mn2+ and As(V), the solid Mn phase was rich in bound Mn2+ (molar ratio, approximately 30%) and showed a transiently high accumulation of As(V) during the early stage of manganese oxide formation. As manganese oxide formation progressed, a large proportion of adsorbed As(V) was subsequently released. The high proportion of bound Mn2+ may suppress a charge repulsion between As(V) and the manganese oxide surface, which has structural negative charges, promoting complex formation. In cultures containing Mn2+ and As(III), As(III) started to be oxidized to As(V) after manganese oxide formation was mostly completed. In suspensions of the biogenic manganese oxides with dissolved Mn2+, As(III) oxidation rates decreased with increasing dissolved Mn2+. These results indicate that biogenic manganese oxide with a high proportion of bound Mn2+ oxidizes As(III) less effectively than with a low proportion of bound Mn2+. Coexisting Zn2+, Ni2+, and Co2+ also showed similar effects to different extents. The present study demonstrates characteristic features of oxidation and adsorption of As by biogenic manganese oxides and suggests possibilities of developing a microbial treatment system for water contaminated with As that is suited to the actual situation of contamination.

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A Mn-depositing fungus, Acremonium-like hyphomycete strain KR21-2, was isolated from a Mn deposit occurring on the wall of a storage bottle containing Mn(III, IV) oxide-coated streambed pebbles and stream water. 18S rRNA gene sequence analysis revealed that strain KR21-2 was phylogenetically related to members of the order Hypocreales within the class Ascomycetes. The spent culture medium at the stationary phase of fungal growth contained a 54-kDa protein capable of depositing Mn oxides. The enzymatic activity was inhibited by azide and o-phenanthroline. The Mn(II)-oxidizing protein possessed a laccase activity, as indicated by direct oxidation of p-phenylenediamine and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). These results are consistent with the role assumed for laccase-like multicopper oxidase, which is proposed to be involved in the Mn(II)-oxidizing factors from some bacteria. Unlike laccases of basidiomycete fungi, however, the protein of strain KR21-2 did not produce soluble Mn(III) species in the presence of either of the Mn chelators pyrophosphate and malonate. This is the first report on the possible involvement of laccase and/or multicopper oxidase in Mn oxide deposition by ascomycetes (including their anamorphs) ubiquitous in natural environments.

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An anaerobic, Fe(III)-reducing enrichment culture, which originated from a sediment sample collected at a landfill in Nanji-do, Seoul, Korea, was capable of degrading cis-1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC). Although it exhibited the ability under Fe(III)-reducing conditions, the chlorinated ethenes degradation was not linked to the Fe(III) reduction. During cis-DCE degradation, no VC, ethene, or ethane was detected through the experimental period. Also, this culture did not accumulate ethene and ethane during the VC degradation. It was unlikely that cis-DCE was reductively dechlorinated to VC and then the VC formed was dechlorinated fast enough. Because the kinetic data showed that the rate of cis-DCE degradation was 3.5 times higher than that of VC. Whereas glucose supported the culture growth and the degradation, formate, acetate, butyrate, propionate, lactate, pyruvate, and yeast extract did not. The results appeared consistent with the involvement of oxidative degradation mechanism rather than reductive dechlorination mechanism. The traits of the culture described here are unusual in the anaerobic degradation of chlorinated ethenes and may be useful for searching an effective organism and mechanism regarding anaerobic cis-DCE and VC degradation.

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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.

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