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Y Kamagata

Publications and source records attributed to Y Kamagata.

At least 19 recordsLinked to original sources
PubMed2005

Comparison of nitrification performance and microbial community between submerged membrane bioreactor and conventional activated sludge system.

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of more than 260 days on treating synthetic ammonia-bearing inorganic wastewater without sludge purge under decreased hydraulic retention times (HRTs). Conversion of NH4(+)-N to NO3(-)-N was achieved with an efficiency of over 98% at an HRT > or = 10 h in the SMBR, while similar performance was obtained at an HRT > or = 20 h in the CAS. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA was used to monitor variations of community structures in the two systems. With the prolongation of operation, the number of DGGE bands in the SMBR gradually increased from the initial 11 bands to the final 22 bands, whereas that in the CAS varied in a range between 13 and 183 Sequence analysis indicates that Nitrosomonas sp. and Nitrospira sp. were the dominating nitrification species responsible for ammonia and nitrite oxidation, respectively. Heterotrophic bacteria like Pseudomonas sp. and Flavobacteria sp. existed in both of the systems although only inorganic wastewater was fed. Substantive accumulation of extracellular polymeric substances (EPS) in the SMBR was confirmed by scanning electron microscopy and EPS analysis.

Ammonia↗
PubMed2005

Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors.

We developed a rapid and simple method for rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems. The method relies on the sequence-specific scission of 16S rRNA with ribonuclease H (RNase H) and oligonucleotides that specifically hybridize with targeted rRNA molecules. RNAs from a complex community were first mixed with an oligonucleotide and were subsequently digested with RNase H to achieve sequence-dependent rRNA cleavage at the hybridization site. For the quantitative detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel-electrophoresis, which separated and quantified cleaved and intact rRNA fragments. This method enabled the quantitative detection of microbes in a complex microbial community by a relatively simple and fast experimental procedure. We then applied the cleavage method to actual anaerobic microbial communities such as digested sewage sludge and UASB sludges. The results demonstrated that the present method was fully applicable to anaerobic digestor ecosystems containing complex anaerobic microorganisms.

Bioreactors↗
PubMed2004

Molecular analysis of bacterial community based on 16S rDNA and functional genes in activated sludge enriched with 2,4-dichlorophenoxyacetic acid (2,4-D) under different cultural conditions.

Differential emergence and diversity of bacterial communities from activated sludge in response to varied cultural conditions using 2,4-dichlorophenoxyacetic acid (2,4-D) were investigated by coupling molecular analyses based on 16S rDNA with functional genes. We employed three different cultural conditions: (1) a culture sequentially fed a high concentration (300 mg/L) of 2,4-D (HS); (2) a culture continuously fed a low concentration (10 mg/L) of 2,4-D (LC); and (3) a serial batch culture in which 1% (v/v) of culture was transferred to a fresh medium containing a high concentration (300 mg/L) of 2,4-D (HB). The HS and LC bioreactors were operated for 3 months and HB was repeatedly transferred for 1 month. The 2,4-D was stably degraded under all the cultural conditions tested. PCR amplification and cloning-based analysis of functional genes using community DNAs from the cultures revealed five different oxygenase genes that may be involved in the initial step of 2,4-D degradation. All five gene-types were present in HS, while one of the five genes, type V (tftA) was not detected in LC. Quantitative PCR analysis showed that in HS, Ralstonia eutropha JMP 134 type-tfdA4 (type I) was the most abundant in copy number (2.0 +/- 0.1 x 10(7) copies/microg DNA) followed by RASC type-tfdA (type II) (1.8 +/- 1.0 x 10(6) copies/microg DNA), putative cadA-like gene (type IV) (2.6 +/- 0.8 x 10(5) copies/microg DNA), cadA gene (type III) (1.3 +/- 1.0 x 10(4) copies/microg DNA), and tftA gene (type V) (3.5 +/- 1.1 x 10(3) copies/microg DNA). Similar results were obtained in LC. In contrast, HB contained only type I and type III genes, and the type I gene was five orders of magnitude greater in copy number than the type III gene. Denaturing gel gradient electrophoresis (DGGE) analysis of PCR, amplified 16S rDNA fragments of bacterial communities in the three different cultures showed low similarity coefficient values (< or =0.35) when compared to the original activated sludge, suggesting that 2,4-D amendment caused a drastic change in the bacterial community. Particularly, HB showed only six bands (16-18 bands in the other cultures) and very low similarity coefficient values when compared to the other communities (0.10 to HS, 0.17 to LC, and 0.0 to original sludge). These results indicated that serial batch culturing (HB) resulted in a phylogenetically limited number of 2,4-D degrading bacteria carrying limited catabolic genes whereas more diverse 2,4-D degraders and catabolic genes were present in HS and LC. Therefore, the approach used for monitoring should be taken into account when one evaluates the population dynamics of contaminant-degrading bacteria at bioremediation sites.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2002

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges.

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Bacteria, Anaerobic↗
PubMed2001

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY((R)) FL-labeled probe or primer.

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY((R)) FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY((R)) FL-modified cytosine at its 5'-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.

Boron Compounds↗
PubMed2001

Application of capillary electrophoresis to monitor populations of Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 in mixed culture.

A bacterial cell mixture ot Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 was analyzed by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE). Both pherograms showed two discrete peaks. The cells in the peaks were collected, Gram stained, and examined with a microscope. The cells of the two strains were well separated by OGE, and each OGE peak consisted almost entirely of cells of one strain (greater than 98% purity), whereas each CZE peak contained cells of both strains (about 90% purity). In the concentration range of 1.0 x 10(10) to 1.0 x 10(12) cells/mL, the area of CGE peaks was proportional to the amount of cells. The growth of the two strains in mixed culture was measured by OGE. The OGE quantification data were in good agreement with those obtained using fluorescence in situ hybridization. The CGE analyses were accomplished in 1 h, using a relatively uncomplicated procedure. Thus, OGE exhibited great advantages in accuracy, rapidity, and simplicity.

Actinomycetales↗
PubMed2001

Recent advances in methane fermentation technology.

In the past two decades, a number of biotechnologies for anaerobic (methanogenic) wastewater treatment have been created, and practical applications of these processes are now being extended to more recalcitrant wastewaters and to wastewaters at extreme temperatures. Our knowledge of methanogenic organic degradation associated with bioreactors is also accumulating at a rapid rate. The recent advancement of such fundamental understanding is attributed to modern molecular biology techniques applied to the study of microbial communities and to continuous challenges to the cultivation of many important but recalcitrant anaerobes in bioreactors.

Anaerobiosis↗
PubMed2001

In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I.

We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was cocultured with a hydrogen-utilizing methanogen, Methanothermobacter thermautotrophicus, suggesting that the strain is a sugar-fermenting bacterium, the growth of which is dependent on hydrogen consumers in the granules.

Animals↗
PubMed2001

Bacteria mediate methylation of iodine in marine and terrestrial environments.

Methyl iodide (CH(3)I) plays an important role in the natural iodine cycle and participates in atmospheric ozone destruction. However, the main source of this compound in nature is still unclear. Here we report that a wide variety of bacteria including terrestrial and marine bacteria are capable of methylating the environmental level of iodide (0.1 microM). Of the strains tested, Rhizobium sp. strain MRCD 19 was chosen for further analysis, and it was found that the cell extract catalyzed the methylation of iodide with S-adenosyl-L-methionine as the methyl donor. These results strongly indicate that bacteria contribute to iodine transfer from the terrestrial and marine ecosystems into the atmosphere.

Bacteria↗
PubMed2001

Involvement of formate as an interspecies electron carrier in a syntrophic acetate-oxidizing anaerobic microorganism in coculture with methanogens.

To determine whether formate is involved in interspecies electron transfer between substrate-oxidizing bacteria and hydrogenotrophic microorganisms under anaerobic conditions, a syntrophic acetate-oxidizing bacterium Thermacetogenium phaeum strain PB was cocultured either with a formate /H2-utilizing methanogen strain TM (designated as PB/TM coculture), or an H2-utilizing methanogen strain deltaH (designated as PB/deltaH coculture). Acetate oxidation and subsequent methanogenesis in PB/TM coculture were found to be significantly faster than in PB/deltaH coculture. Formate dehydrogenase and hydrogenase were both detected in strains PB and TM. H2 partial pressures in the PB/TM coculture were kept lower (20 to 40 Pa) than those of the PB/deltaH coculture (40 to 60 Pa) during the exponential growth phase. Formate was also detected in both PB/TM and PB/deltaH cocultures, and the concentration of formate was maintained at a lower level in the PB/TM coculture (5 to 9 microM) than in the PB/deltaH coculture. Thermodynamic calculations revealed that the concentrations of both H2 and formate severely affect the syntrophic oxidation of acetate. These results strongly indicate that not only H2 but also formate may be involved in interspecies electron transfer.

Journal Article↗
PubMed2001

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.

Algorithms↗
PubMed2000

Micropruina glycogenica gen. nov., sp. nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge.

A new Gram-positive non-spore-forming bacterium, strain Lg2T, was isolated from an activated sludge reactor showing enhanced biological phosphorus removal activity. The new isolate was a slowly growing organism and was capable of accumulating large amounts of intracellular glycogen from substrate taken up. Both oxidase and catalase were produced. The new isolate contained meso-diaminopimelic acid (DAP) in the cell wall. Complex fatty acid patterns with iso-C14:0, anteiso-C15:0, C16:0, iso-C16:0 and four other minor saturated or unsaturated straight-chain fatty acids were detected. The isolate contained a high genomic G+C content (70.5 mol%). Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate in the high G+C Gram-positive group with Microlunatus phosphovorus and Friedmanniella antarctica as the closest relatives (sequence similarities are 93 and 92 %, respectively). These three organisms shared common features in morphology, but strain Lg2T could be differentiated from the other species by its peptidoglycan type (meso-DAP), fatty acid composition, carbon source utilization profile and G+C content. On the basis of these findings, it is proposed that a new genus and species, Micropruina glycogenica, should be created for the new isolate; the type strain is strain Lg2T (= JCM 10248T).

Base Composition↗
PubMed2000

Burkholderia kururiensis sp. nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE.

A trichloroethylene (TCE)-degrading bacterium was isolated from an aquifer sample collected at a TCE-polluted site in Japan by enriching with phenol as sole carbon source. The isolate, designated strain KP23T, was a Gram-negative, oval-shaped micro-organism. A phylogenetic study based on 16S rRNA gene sequences indicated that strain KP23T should be placed in the genus Burkholderia. Cellular fatty acids of the strain were mainly composed of C16:0, cyclopropanic acid C17:0 and cyclopropanic acid C19:0. Strain KP23T also contained notable amounts of C13:1 and C17:1. The G + C content of total DNA was 64.8 mol%. Strain KP23T oxidized various sugars and sugar alcohols as sole carbon source such as galactose, glucose, mannose, maltose, glycerol, inositol and mannitol. Comparisons of its phenotypic and genotypic characteristics with other known species belonging to the genus Burkholderia suggested that strain KP23T represents a new species in the genus. The name Burkholderia kururiensis is proposed for this species, with strain KP23T as the type strain (= JCM 10599T).

Bacterial Typing Techniques↗
PubMed2000

Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate.

A new anaerobic, thermophilic, syntrophic, fatty-acid-oxidizing bacterium designated strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor. The cells were slightly curved rods and were weakly motile. Spore formation was not observed. The optimal temperature for growth was around 55 degrees C and growth occurred in the range 45 to 60 degrees C. The pH range for growth was 5.8-7.5, and the optimum pH was 6.5-7.0. Crotonate was the only substrate that allowed the strain to grow in pure culture. However, in co-culture with the thermophilic, hydrogenotrophic Methanobacterium thermoautotrophicum strain delta H, the isolate could syntrophically oxidize saturated fatty acids with 4-10 carbon atoms, including isobutyrate. During the degradation of isobutyrate by the co-culture, isobutyrate was isomerized to butyrate, which was then oxidized. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate or Fe(III) as electron acceptor. The DNA base composition was 51.0 mol%. 16S rDNA sequence analysis revealed that the strain belongs to the family Syntrophomonadaceae, but it was only distantly related to other known species of beta-oxidizing syntrophs. Hence, the name Syntrophothermus lipocalidus is proposed for TGB-C1T as a new species of a new genus.

Bacterial Typing Techniques↗
PubMed2000

Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium.

A novel anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium, strain PB(T), was isolated from a thermophilic (55 degrees C) anaerobic methanogenic reactor which had been treating kraft-pulp waste water. The bacterium oxidized acetate in co-culture with a thermophilic hydrogenotrophic methanogen. Strain PB(T), a gram-positive, spore-forming, rod-shaped bacterium grew optimally at 58 degrees C and pH 6.8. The bacterium grew acetogenically on several alcohols, methoxylated aromatics, pyruvate, glycine, cysteine, formate and hydrogen/CO2. Strain PB(T) also oxidized acetate with reduction of sulfate or thiosulfate as the electron acceptor. The bacterium contained MK-7 as the major quinone. The G+C content of the DNA was 53.5 mol%. Comparative 16S rDNA analysis indicated that strain PB(T) belongs to the Bacillus-Clostridium subphylum. However, it was distant from any known genera or micro-organism. The closest known relative was Thermoterrabacterium ferrireducens with 87.4% similarity. The name Thermacetogenium phaeum gen. nov., sp. nov. is proposed. The type strain is strain PBT (= DSM 12270T).

Acetates↗
PubMed2000

Methanocalculus pumilus sp. nov., a heavy-metal-tolerant methanogen isolated from a waste-disposal site.

A mesophilic hydrogenotrophic methanogen, strain MHT-1T, was isolated from the leachate of a sea-based site for solid waste disposal (the port of Osaka, Japan). Strain MHT-1T was found to be an irregular coccus and was able to use H2/CO2 and formate as energy sources. Acetate was required for growth. The optimum temperature and pH for growth were 35 degrees C and 6.5-7.5, respectively. Strain MHT-1T was resistant to high concentrations of several heavy metals such as CdCl2 and CuSO4. The G+C content of the DNA was 51.9 mol%. Analysis of the 16S rRNA gene revealed that the isolate was a member of the genus Methanocalculus but distinct from its nearest neighbour, Methanocalculus halotolerans, there being a sequence similarity of 98.9%. DNA-DNA hybridization analysis revealed 51% relatedness with the DNA of M. halotolerans strain SEBR 4845T. The optimum NaCl concentration was 1.0%, whereas the optimum in M. halotolerans was 5.0%. A new species, Methanocalculus pumilus, is proposed for strain MHT-1T. The type strain is MHT-1T (= DSM 12632T = JCM 10627T).

Base Composition↗
PubMed2000

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge.

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.

Base Composition↗