| Project/Area Number |
14550546
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Civil and environmental engineering
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| Research Institution | University of Shizuoka |
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Principal Investigator |
IWAHORI Keisuke University of Shizuoka, Institute for Environmental Sciences, Professor, 環境科学研究所, 教授 (40183199)
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| Co-Investigator(Kenkyū-buntansha) |
MIYATA Naoyuki University of Shizuoka, Institute for Environmental Sciences, Assistant Professor, 環境科学研究所, 助手 (20285191)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | mycolic acid-containing bacteria / scum / immunomagnetic separation / hybridization / PCR-DGGE method / fluorescence in situ hybridization / Wuhrmann process / Eikelboom TYPE1851 / Gordonia sp. / Mycobacterium sp. / ミコール酸含有菌 / オリゴヌクレオチドプローブ / プライマー / 16S rRNA / 変性剤濃度勾配 / 抗ミコール酸ポリクローナル抗体 / PCR / 16SrDNA / 遺伝子配列 / サイクルシーケンシング |
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| Research Abstract |
This research aimed at the applicability of immunochemical and molecular-biological methods to analyse the microbial flora in the scum of activated sludge. The obtained results can be summarized as follows;
Operational modes of the immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody from Gordonia amarae SCI were investigated, and it was shown that 1 hr-treatment of PBS solution with Tween 20 of 0.2% was effective for the microbial dispersion and that proper dillution ratio of the polyclonal antibody was about 50 times. Though variation of the recovery for each mycolata was dependent on the specificity of the antibody, mycolata was found to be effectively recovered by this IMS procedure optimized with SCI cells. From the similar investigation on the hybridization, 45 min-ultrasonication with 20W and 20KHz for the cell fragmentation and spin colum method for the extraction of RNA fraction were applicable to the detection and determination of the mycolata. In the DGGE analysis with PCR-amplified fraction from the scum sample, denaturing gradient gel concentration of 30〜60% was shown to be suitable. The analytical investigations in the scum samples of Wuhrmann process, using the above-mentioned various metods, were carried out. As the results, ten or over bands were detected, and Gordonia alkalivorans, G. nitida, G. amicalis and Mycobacterium mucogenicum in mycolic acid-containing bacteria were identified from BLAST homologous detection to the base sequences of several bands with the higher fluorescence intensity. Existence of Eikelboom TYPE1851 in filamentous bacteria was simultaneously shown by the fluorescence in situ hybridization.
Judging from these results, the immunochemical and molecular-biological methods were concluded to be applicable and useful to analyse the microbial flora in the scum of activated sludge.
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