竹材を効率的に分解する真菌を探索するため，竹材の腐朽過程における自然界での真菌の遷移を分子生態学的に解析した．竹ペレットをメッシュバッグに詰めたトラップを石川県金沢市のモウソウチク林２地点林内地上に設置し，一ヶ月おきにサンプルの一部を回収した．回収したペレットから，全DNAを抽出するとともに，常法に従って糸状菌の分離を試みた．ペレットから抽出したDNAは，真菌の18S rDNAを標的としたプライマーを用いてPCR増幅をおこない，変性剤濃度勾配ゲル電気泳動（）法によって解析した．解析によるDNAバンドパターンの質的・量的変化から，腐朽竹材中の真菌の種数や量が遷移しているということが示された．この中で優占すると考えられたバンドの塩基配列を解読してデータベース検索をした．その結果，子のう菌フンタマカビ網のConiochaeta属菌，Carpoligna属菌，Kionochaeta属菌，Pyrenomyxa属菌などが優占種としてあげられた．また，担子菌としては，シビレタケ属（Psilocybe）の２種が見いだされた．なお，既往の研究によりConiochaeta属菌はリグノセルロース分解能を有すること，Psilocybe属菌は白色腐朽性であることが示されている．今回の解析の結果からは，子のう菌の数種が竹材の腐朽過程で優占し，竹材の分解に主要な役割を果たしていることが示唆された．すなわち，野外での竹材分解菌としては子のう菌類の腐朽菌に注目する必要がある．一方，分離培養法では，Carpoligna属菌など一部の優占種は得られなかった．今後，解析によって明らかとなった腐朽竹材における優占種と分離菌株との対応をすすめ，それらの菌の竹材分解能の比較実験をおこなう．

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Bacteria in the rhizosphere influence plant growth and interact with plant roots. Microscopy and culture method have been used for studies of microorganisms of the rhizosphere, but these methods are insufficient for evaluation because most rhizosphere bacteria are viable but non-culturable (VBNC). Bacteria in the rhizosphere of rice cultivated in Andosol lowland and upland fields were analyzed in this study using PCR- and FISH, in combination with modified pretreatments. Results show that the two methods with the pretreatments, more than conventional methods, provided a rapid and simple analysis of rhizosphere bacteria. The 16S rDNA band pattern of bacteria in the rhizosphere obtained using PCR- indicated different species composition of bacterial community in the two ecosystems and greater diversity of bacteria in the rhizosphere in upland field. Sequencing of major 16S rDNA bands identified Bacterium A35 and Clostridium bifermentans as dominant bacteria in the rhizosphere of rice in lowland fields and Klebsiella planticola and Bacillus fusiformis in upland fields. Furthermore, FISH observation indicated the predominance of gram-positive low GC bacteria in both rhizospheres and a higher proportion of Clostridium spp. in lowland fields, which is consistent with results of PCR- analysis. The results suggest that the bacteria in the rice rhizosphere can be changed depending on aerobic and anaerobic conditions of fields. It is expected to apply the PCR- and FISH to agricultural field experiments as reliable methods to evaluate the rhizosphere bacteria.

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PCRより得られた3種の供試純粋菌株の16SrDNA断片を鋳型DNAとして用いて,微生物群集の構成相の評価法としてのプロファイリング有効性について検討した。PCRのサイクル数が30回の条件の下,エチジウムブロマイド染色によるPCR-分析により,バンドを検出するための鋳型DNAのコピー数はPCR溶液(100μL)中に10⁴から10⁵オーダーであった。同一オーダーにおける2種および3種の微生物間のDNA断片の存在比に対する蛍光強度比の変動性を検討した。鋳型DNAの存在比と蛍光強度比の差を小さくするためには,100μLのPCR反応系において10⁵オーダーのコピー数が最適であった。異なるオーダーでの2種および3種の微生物間の存在比の変動性を検討した。一方の鋳型DNAが86%以上で存在すれば,その鋳型DNAに相当するバンドが高い蛍光強度を呈したバンドとして検出でき,鋳型DNAの存在比と蛍光強度比の差は小さくなるが,鋳型DNAの存在比を正確な数値として反映することは難しいことが明らかになった。

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Changes in bacterial community structure were followed in two outdoor experimental ponds. To compare the changes in bacterial community, Microcystis was inoculated into one of the ponds but not to the other one. In both ponds, Chlorophyceae algae from the genera Cosmarium and Scenedesmus were dominant in the first and last months of the study. During the middle period of the study, cyanobacteria were dominant. Microcystis and Aphanizomenon dominated in one pond, and Planktothrix dominated in the other. To investigate bacterial phylogenetic abundance and these compositions, we used catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and denaturing gradient gel electrophoresis (). A significant relationship was observed between the number of α-proteobacterial operational taxonomic units (OTUs) and the abundance of Chlorophyceae algae (p < 0.001), although no significant relationship was identified between the abundances of the two groups. The sequencing analysis of bands detected microcystin-degrading bacteria belonging to the α-proteobacteria as one of the dominant bacterial phylogenetic groups when Microcystis was the dominant phytoplankton. To our knowledge, this is the first report demonstrating changes in the abundance and composition of bacterial groups during the wax and wane of dominant phytoplankton taxa, using two different molecular methods.

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本研究では,ウメ(Prunus mume)の衰弱樹(主に白紋羽病(Rosellinia necatrix))における有益細菌接種による樹勢回復効果についてPCR-法を用いてその細菌の生存を調査した.供試した有益細菌はナギナタガヤ(Vulpia myuros)およびバヒアグラス(Paspalum notatum Flugge)の茎葉および根から発見した白紋羽病菌を抑制する3種類の細菌であり,それらの混合液を草生ウメ園における衰弱樹の根圏に接種した.接種後の樹勢の回復性を葉色および根の拡がりで調査するとともに,接種細菌の生存をPCR-法で分析した.その結果,接種後,衰弱樹の葉色は黄から緑にもどり,幹近くにしかみられなかった衰弱樹の根は健全樹の広がりと同様までに回復した.また,衰弱樹周辺土壌に接種した有益細菌3種類の存在が見られた.これらの結果から,土壌潅注接種した有益細菌の生息を確認するための生物的土壌診断法として,PCR-法が有用であることが明らかとなった.

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Soil microbial community analysis is one of the most important elements in the environmental risk assessment of transgenic plants. Recent technical advances in this area now enable us to assess the impact of plant genotypes on soil microbial communities with rapid, simple and less biased molecular techniques than the previously used conventional microbiological methodologies. We review the use of these modern molecular techniques from the aspect of environmental assessments of transgenic plants.

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An analysis of microflora characteristic and water quality in swine liquid manure treated with a BMW processing method was examined. Microbial 16S rDNA banding patterns by PCR- analysis in the wastewater from the primary tank and the processing tanks 1 and 2 were almost similar. Pseudomonas sp. and Bacillus sp. were identified from each sample. These bacteria were estimated dominant species. In FISH analysis, the amount of Eubacteria was much in the first tank and decreased with the following three tanks. The most part of Eubacteria was bacillus, resulting in the same as the PCR- analysis. The concentration of BOD and COD_Mn in the processing tanks was lowered than that of the primary tank, which was confirmed to improve the water quality

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For the promotion of environment-friendly agriculture, use of organic fertilizers and green materials is increasingly attempted in rice farming. Although effects of organic fertilizers on soil bacteria in the rhizosphere can differ from those in non-rhizosphere soil, microbiological studies that specifically address the rice rhizosphere still limited. This study was undertaken to investigate the impact of organic fertilizers on soil bacteria communities through comparison of rhizosphere soils and bulk soils. Effects of soil types and seasonal change were also analyzed. Rice plants (Oryza sativa L. cv. Nipponbare) were cultivated in a lowland paddy field of Andosol soil. Applications of compost and rice bran in combination with chemical fertilizer were compared with control soil (chemical fertilizer only). Soil 16S rDNA extracted from rhizosphere soil collected using ultrasonic treatment of rice roots and from bulk soils were analyzed using PCR-denaturing gradient gel electrophoresis (). Principal component analysis based on PCR- profiles revealed clear differences in the community structures of soil bacteria between rhizosphere and bulk soils. Furthermore, rhizosphere bacterial community structures of compost and rice bran treatments were plainly different from that of control, and changed with the seasons. The organic fertilizers showed pronounced effects on bacterial communities until mid-summer, but small effects in autumn. Results of this study suggest that the rhizosphere microorganisms in paddy fields can be modified through organic fertilizer management. Moreover, effects of organic fertilizer application, soil type, and phenology on soil bacteria appear depending on interaction with the rice rhizosphere effects in paddy fields.

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食品残さを原料にした発酵リキッド飼料を豚に給与することで，ふん便中のFirmicutesの存在比率が上昇した。試験区のみで存在比率が上昇した菌については，Eubacterium tortuosumの近縁種であることは分かったが，相同性が86％と低かったため菌名の特定は出来なかった。また，この菌叢変化が豚に与える影響については，増体成績が良好であったこと，下痢などの体調不良を起こさなかったことなどから，少なくとも豚にとって害になるような菌叢変化ではないと考えられた。
一方，スターターとして添加したLactobacillus plantarum A305は，胃酸耐性の不足から，豚腸内への定着が不十分であることが分かった。このため，試験区と対照区の間で，期待したほどのプロバイオティック効果が出なかったものと考えられた。しかし，菌叢の変化自体は起こっていたため，乳酸菌の死菌体もしくは乳酸発酵時の代謝物の影響によって菌叢が変化したものと考えられた。

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Yamagawa Bay, located in Ibusuki, Kagoshima Prefecture, Japan, is a geographically enclosed coastal marine inlet, and its deteriorating seabed sediments are under an anoxic, reductive, sulfide-rich condition. In order to gain insight into diversity of anoxygenic photosynthetic bacteria (AnPBs) and their ecophysiological roles in the sediments, three approaches were adopted: isolation of AnPBs, PCR- of 16S rDNA, and PCR- of pufM. Among the bacterial isolates, relatives of Rhodobacter sphaeroides were most dominant, possibly contributing to transforming organic pollutants in the sediments. Abundance of Chlorobium phaeobacteroides BS1 was suggested by 16S rDNA PCR-. It could reflect intensive stratification and resultant formation of the anoxic, sulfide-rich layer in addition to extreme low-light adaptation of this strain. Diverse purple non-sulfur or sulfur bacteria as well as aerobic anoxygenic photoheterotrophs were also detected by pufM PCR-, which could be associated with organic or inorganic sulfur cycling. The outcome of the present study highlights ecophysiologically important roles of AnPBs in the organically polluted marine sediments.

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A new genotyping method of Cryptosporidium, denaturing gradient gel electrophoresis () followed by DNA sequencing, was developed to genotype Cryptosporidium spp. in river water. The method could successfully discriminate nine species of Cryptosporidium: C. parvum, C. hominis, C. canis, C. meleagridis, C. felis, C. sp. strain 938, C. andersoni, C. serpentis and C. saurophilum. The sequential combination of the QProbe PCR method for quantification; and the method and DNA sequencing for genotyping, enabled the simultaneous quantification and genotyping of Cryptosporidium spp. in the same water sample. This method was applied to analyze Cryptosporidium in the Koyama River, a tributary of the Tone River, both for the total concentration of organisms, and for their genotype. Seven Cryptosporidium genotypes (C. andersoni, C. sp. strain 938, C. parvum, C. hominis, C. sp. PG1-26, C. sp. t03, C. sp. t04) were found in 11 positive samples (positive ratio=69%). A bovine specific species, C. andersoni, was found most frequently (7 samples). The genotypes infectious to human accounted for only 16% of the concentration of all genotypes. These results showed that this detection method could provide valuable information on Cryptosporidium in river water both in the quantity and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

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Bacteria that pass through a 0.2-µm pore-size filter (0.2-µm-filterable bacteria) have been reported from various aquatic environments. It has been recognized that some 0.2-µm-filterable bacteria are “transiently” small/thin cells, which are considered as starvation forms of heterotrophic bacteria, especially in oligotrophic environments. However, whether these “transiently 0.2-μm-filterable bacteria” have roles in organic matter processing in aquatic environments has not yet been well described. Here we found high potential of the transiently 0.2-µm-filterable bacteria with extracellular protease activity in their natural nutritional condition. We conducted microcosm experiments using 0.2-µm-filtered coastal seawater (FSW) without any nutritional amendment, and monitored changes of the cell numbers trapped on a 0.2-µm filter (not 0.2-µm-filterable cells anymore) and their community structure in the FSW microcosms, with extracellular protease activities as indicators of heterotrophic microbial activity. We observed a rapid increase in the 0.2-µm-trapped cell number in the FSW microcosms that originally included only 0.2-µm-filterable microbes. The regenerating 0.2-µm-trapped cells were typical marine bacteria (Alphaproteobacteria, Gammaproteobacteria, Flavobacteriia). Extracellular aminopeptidase activities increased with increasing numbers of 0.2-µm-trapped cells. These results suggest that transiently 0.2-µm-filterable-form bacteria in the original coastal seawater recovered their size and were producing extracellular proteases, which might catalyze organic matter processing in seawater. Since organic nutrients were not added, the size increase might be caused by the reduction in competition from larger bacteria and/or the absence of grazing pressure. Our results demonstrate the growth potential and extracellular protease activity of “transiently 0.2-μm-filterable” bacteria in seawater, usually obscured due to the coexistence of grazers and other bacteria.

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We obtained ancient bronze mirrors and carried out: scanning electron microscopy (SEM) of fractured altered layers; biologic microscopic observation of Gram-stained samples; and DNA analyses of samples removed from altered sites.
Fine particles about 2 μm in length were confirmed in the altered layer by SEM. Microorganisms of identical size were observed in the Gram-stained sample removed from the altered layer through a biologic microscope. Fine particles observed under SEM were considered to be microorganisms. Many fine particles were confirmed, particularly in the altered sites, by SEM. Certain types of microorganisms may have played a part in the alteration (deterioration) of the ancient bronze mirrors while the latter were buried in soil.
From the base sequences obtained by analysis, two types of microorganisms were present in the altered layer of the mirror. One was 94.7% homologous to the 16S rDNA of the uncultured bacterium (accession number: AY 053488). It was also highly homologous to the sequence derived from the 16S rDNA of the Xanthomonadaceae family (e.g., Stenotrophomonas, Xanthomona). That is, the sequence was derived from a strain belonging to the Xanthomonadaceae family. The other base sequence was 97.4% homologous to the 16S rDNA of the Bacteroidales order such as uncultured Bacteroidales bacterium (accession number: AY 859647). That is, the sequence was derived from a strain belonging to the Bacteroidales order.
Genes of microorganisms, presumed to belong to the Acetobacter and Gluconacetobacter genera and the Fe(III)-reducing bacterium, Shewanella algae, were detected from base sequence analysis by cloning.
Microbial activity around the mirrors was assumed to be high. The alteration containing corrosion mechanism of bronze mirrors appears complex, but several types of microbes that possibly altered the bronze mirrors were verified.

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Permeable reactive barriers (PRBs) column tests were performed to investigate the contribution of anaerobic microbial community in sheep manure on the removal of As from groundwater. The column was packed with zero valent iron (ZVI), sheep manure, compost, wood chips, glass beads and gravels. All materials were sterilized except for sheep manure that contains anaerobic bacteria. Decrease in sulfate concentration was observed at the maximum rate of 0.26 mmol dm⁻³ day⁻¹. In addition, the sulfur isotopic ratio of δ³⁴S increased from the influent (−4.3\\ extperthousand) to the effluent (0.2\\ extperthousand), suggesting that there was sulfate-reducing activity in the microbial community. Arsenate was more effectively immobilized on ZVI than arsenite. Bacterial community analysis using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-) on 16S rRNA sequences suggested that majorities of bacteria were several Clostridium species and one species of Proteobacteria, Geobacter metallireducens GS-15, independently of PRBs column heights. Some of them might have contributed to the removal of arsenic.

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In intertidal sediments, bacteria attach to sand grains in mixed-species biofilms and inhabit the surrounding porewater as free-living communities. The large densities, high species diversity, and complex spatial distribution of sediment-attached bacteria implicate inter-specific competition as a likely force in structuring sediment communities. Both sediment-attached and free-living bacteria secrete antibiotics as a common means of competition. To establish the frequency of antibiotic production, bacteria isolated from intertidal sediments and porewater were screened using a disc-diffusion assay. Among sediment-attached bacteria, 39% displayed the ability to produce antibiotics, whereas significantly fewer of the porewater-associated bacteria (23.5%) produced inhibitory compounds. Denaturing gradient gel electrophoresis (

) was used to identify a selection of isolated antibiotic-producing bacteria within whole-community environmental samples. Through sequencing a region of the 16S rRNA gene, the relative abundances of 4 antibiotic producers were established to be between 4.3–9.4% of the community profile. The high frequency of antibiotic-producing bacteria in sediments, and their significant quantitative contribution to the community composition, suggest that antibiosis likely plays a significant role in structuring benthic microbial communities.

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高静水圧は発酵の制御に有望な技術だと考えられている．高静水圧の効果を把握するため，キムチ発酵における微生物叢の挙動をPCR増幅したDNAの変性密度勾配ゲル電気泳動とゲルから抽出したDNAの塩基配列シーケンシングにより調べた．キムチからLactobacillus，LeuconostocおよびWeissella属を含む11種の乳酸菌を分離した．60日間のキムチ発酵過程では，pHは3.9まで二段階で低下した．乳酸菌濃度は15日目に最大になり，その後3.2×10⁸ cfu/mlに保たれた．乳酸菌の中ではL. sakeiが一定して認められ，L. plantarumが21日目以降に見られた．酵母の菌濃度は15日目に最大となり1.4×10⁸ cfu/mlに達し，27日目以降は検出限界以下へと減少した．酵母ではKazachstania servazziiが圧倒的に優占化していた．発酵21日目に200 MPa，60分の高圧処理をキムチに加えたところ，以降のpH低下が見られなかった．乳酸菌と酵母の菌濃度は急激に低下し，それぞれ8.3×10⁵ cfu/mlならびに検出限界以下となり，乳酸菌は緩やかに回復したが，酵母は速やかな回復を見せた．しかし，高圧処理の有無による微生物叢の明確な違いは観察されなかった．発酵開始時におけるL. sakeiとK. servazziiの添加も試みたが，明確な効果は見られなかった．

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The fixation of carbon dioxide is one of the most important subjects in environmental research. The general approach to removing carbon from the atmosphere is to grow plants that sequester carbon dioxide in their biomass by photosynthesis. Recently, biochar, which is produced by the pyrolysis of trees, grasses or crop residues, has been undergoing a renaissance as a method for sequestering carbon dioxide. In our research, we applied a new compost containing biochar. The composting of agricultural and food industrial waste biomass occurred by the degradation of organic compounds by aerobic microorganisms in nature. Biochar has porous structures so that the blending of biochar into compost creates an aerobic environment which leads to the activation of aerobic microorganisms and the degradation of organic compounds in compost. In this study, we made a biochar-blended compost using waste biomass in Aomori. The biochar-blended compost contained a high amount of microorganisms. The growth of some vegetables was accelerated by treatment with this compost compared to ordinary compost. These results indicated that our biochar-blended compost has potential to be used in methods for the recuperation of degraded soil and the formation of soil environment.

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Due to an increased eutrophication of rivers and lakes, enhanced biological phosphorus removal (EBPR) process is now taken notice of. But the strategy of controlling EBPR process has not been established yet, because it is sometimes observed that the sludge suddenly loses the phosphorus removal activity. This is mainly because the bacteria which accumulate polyphosphate in the EBPR sludge have not been isolated, and because there is not enough information about the whole bacterial community in EBPR sludge.
Nowadays, new methods for analyzing microbial community such as molecular methods become available. In this study, the microbial community of EBPR sludge were characterized using the PCRDGGE (Polymerase chain reaction-Denaturing gradient gel electrophoresis) method.
Laboratory-scale sequencing batch reactor (SBR) was used, and the polyphosphate accumulating organisms (PAOs) were enriched using anaerobic aerobic configuration. The simple substrate which contained only acetate as a carbon source was fed. The microbial community was monitored usingthe PCR- method for about 2 months after the seed sludge was inoculated. The community gradually changed and got simple about lmonth later. The major bands on the gel were excised and the DNA recovered from them was sequenced. As a result, the bacteria that continued to increase during enrichment and became dominant at last were closely related to one of the putative PAO group which Crocetti et al.(2000) and Hesselmann et al (1999) have proposed. This PAO group is closely related to Rhodocyclus group (α Proteobacteria), but has no photosynthetic activity.

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