碩士 / 國立中興大學 / 環境工程學系 / 90 / The objective of this study is to understand the bacterial community of SSDML（simultaneous sludge digestion and metal leaching）by applying Fluorescence in situ Hybridization (FISH) technique. Existing Oligonucleotide probes for monitoring heterotrophic bacteria and a new probe Thiopa511 specially designed for Thiobacillus were used. The specificity and chemical stringency for Thiop511 were tested before its experiments on monitoring the SSDML bacterial community、number of bacteria、and the future application on testing various environmental samples.
Using the Probe Match program（Small Subunit）in the RDPⅡ（Ribosomal Database project Ⅱ）to stimulate the probe specificity of Thiopa511, this probe was found to be specific for Thiobacillus thioparus. In the FISH probe specific using pure culture, the fluorescence signal were found to be weak. It was concluded that the cell membrane of these target bacteria could have poor permeability thus lead to ineffectiveness during the hybridization process. In addition, 20 % Formamide concentration was found to achieve optimal Thiopa511 hybridization results.
Only 50 to 60 % of the total bacteria in the reactor could be hybridized using the probes selected in the preliminary study. Four sample fixation methods (Lysozyme、Urea、HCl and Sodium dodecyl sulfate) were performed in attempt to achieve better hybridization result. The results were not satisfied. Finally, precipitate fixative of 100 % EtOH was selected to replace the common fixative of 4 % Paraformaldehyde. The final established procedure included 1.5 hours hybridization and 0.1 % SDS concentration which produced fluorescence signal strong enough to distinguish target cells from background fluorescence.
In the sulfur power addition experiment, pH values in the reactor decreased significantly. However, the total number of bacteria stayed constant using the traditional VSS measurement and DAPI staining method. Microbial community in the reactor changed from a complex composition to being dominant by T. thiooxidans and T. ferrooxidans in a particular pH range.
In different initial total solid concentration experiment, T. thiooxidans and T. ferrooxidans were found to have best growth rate at 0.5 % total solid concentration. In this condition, better heavy metal dissolution rate was achieved with fast decreasing of pH but with no noticeable sludge digestion. Growth rate of T. thiooxidans and T. ferrooxidans increased following higher sulfur addition concentration. Better heavy metal dissolution was achieved when increasing sulfur concentration form 0.1% to 0.5%。
| Identifer | oai:union.ndltd.org:TW/090NCHU0515020 |
| Date | January 2002 |
| Creators | 彭誌強 |
| Contributors | 洪俊雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 146 |
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