臺灣博碩士論文加值系統

本研究運用分子生物科技中的螢光原位雜交技術（Fluorescence in situ Hybridization；簡稱FISH）方法，針對同時污泥好氧消化與重金屬溶出程序（simultaneous sludge digestion and metal leaching；SSDML）系統中的異營菌及硫氧化菌以現有的寡核苷酸探針作螢光原位雜交，並針對Thiobacillus thioparus設計具有專一性的寡核苷酸探針，對所設計的探針進行專一性與最佳雜交嚴格度測試，並運用於監測SSDML中微生物族群菌相、數量及菌群間之相互關係，評估所設計的探針用於環境樣品之可行性。
用（RDP II）網站的 Probe Match program（Small Subunit）模擬所設計的探針，除對T. thioparus 16S rRNA 序列有互補外並未發現有任何菌種的序列互補，顯示此設計之探針(THIOPA 511)確實有其專一性。在以探針對純菌株作FISH專一性測試方面，測得之訊號值偏低，推測可能是目標菌種的細胞膜滲透性不良，導致探針無法有效的進行原位雜交反應。在探針最佳雜交嚴格度測試中所測得的Formamide以20 %為最佳雜交條件。
實驗過程中發現馴養槽中約有50∼60 % 菌無法被本實驗所使用探針雜交，以Lysozyme、Urea、HCl及SDS（sodium dodecyl sulfate）四種前處理在本研究中皆無法有效增強雜交反應。將一般常用的固定劑 4 ％ Paraformaldehyde（PFA）改變成沉澱固定劑（100 % EtOH）配合雜交時改變SDS濃度為0.1 % ，雜交時間1.5 hours時，所得目標訊號強度和背景值便可明顯辨識。
在添加硫粉的污泥馴養實驗的操作初期雖然系統中之pH值變化極大，但整體而言總菌數並未有相對的變化發生，且以DAPI（4’,6 - diamidino - 2 - phenylindole）染色測得之總菌數變化與傳統微生物有機體含量（VSS）測定值間大致相符。以FISH監測菌群變化，過程中T. thiooxidans 和 T. ferrooxidans由複雜的菌群結構中逐漸被篩選出，且在特定pH範圍下形成優勢菌種。
在不同之固體物濃度條件下進行 SSDML試驗，以固體物含量為0.5 %時，T. thiooxidans 和 T. ferrooxidans 有較佳的生長狀況，有較佳的重金屬溶出效率，但pH值下降速率太快使得此條件下無明顯污泥消化作用。在不同元素硫添加量條件下進行同時污泥好氧消化與重金屬溶出程序(SSDML)試驗，T. thiooxidans 和 T. ferrooxidans增長隨著元素硫添加量增加而增加。元素硫添加量為0.1 % 時，由於硫添加量不足使得pH值下降緩慢，直到第10天pH值才下降至5.0以下，異營菌對有機物的分解有足夠時間進行而達到污泥好氧消化目的；元素硫添加量為0.5 % 時，有較佳的重金屬溶出效率。

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%。

中文摘要………………………………………………….……………………….…Ⅰ
英文摘要………………………………………………….……………………….…Ⅲ
目錄……………………………………………………….………….………………Ⅴ
表目錄………….………………………………………...….……….……...……..…X
圖目錄………………………………………………….………………………….... XI
第一章 前言…………………………………………………………………………..1
1-1 研究緣起…………………………………………………………………….1
1-2 研究目的 ………………………………………………..……….………….4
第二章 文獻回顧………………..……………………………………...…………….6
2-1 同時污泥好氧消化與重金屬溶出程序…………………………………….6
2-1-1 同時污泥好氧消化與重金屬溶出程序的發展 ……...…………….6
2-1-2 同時污泥好氧消化與重金屬溶出程序的原理 ………...………….8
2-1-3 重金屬溶出機制 ………………………………….……..……..….10
2-1-4 同時污泥好氧消化與重金屬溶出程序中微生物之種類及生理特性……………..……………………………………………..….......12
2-1-5 影響同時污泥好氧消化與重金屬溶出程序之因子….…..…..…..16
2-2 螢光原位雜交技術（FISH）………………………………….……....….19
2-2-1 螢光原位雜交法原理………………………………………..…….20
2-2-2 16S rRNA…………………………………………………..……….21
2-2-3 螢光原位雜交優點………………………………………..……….23
2-2-4 螢光原位雜交的步驟…………………………..………………….25
2-2-4-1 固定（Fixation）……………………….….…………………….26
2-2-4-2 雜交（Hybridization）………………….……………………….27
2-2-4-3 清洗……………………….……………..……………………….28
2-2-4-4 顯像（Visualization）……………………..……………………….31
2-2-5 FISH與DAPI染色………………...…..….……….……..……….32
2-2-6 螢光原位雜交法限制因子……………….……………………..…32
2-2-7 螢光原位雜交法於環境工程之應用….………….……………….35
2-3 寡核苷酸探針的選擇與設計……………….……………………………. 37
2-3-1 寡核苷酸探針的選擇……………………..……………………….37
2-3-2 寡核苷酸探針的設計與合成………………..…………………….38
2-3-3 寡核苷酸探針專一性與嚴格度測試………..…………………….40
2-3-3-1 寡核苷酸探針專一性測試………………………...…………….40
2-3-3-2 寡核苷酸探針最佳雜交嚴格度測試…...……………………….41
第三章 實驗方法與設備………………………………...………………………….42
3-1 實驗用藥品………………………………………..……………………….42
3-1-1 實驗用水………………..………………………………………….42
3-1-2 固定劑………………………………...…...……………………….42
3-1-3 PBS（Phosphate Buffered Saline）……...………..……………….43
3-1-4 Hybridization Buffer……………...……………..………………….43
3-1-5 Washing Buffer…………………………………..………………….43
3-1-6 DAPI染劑………………………………………………………….44
3-2 實驗架構 ………………………………….……………………………….44
3-2-1 污泥來源………………………..………………………………….45
3-2-2 污泥前處理…………………………..…………………………….46
3-2-3 樣品的保存………………………………..……………………….46
3-2-3 污泥馴養(樣品由交通大學林志高教授研究室提供) ……………46
3-2-4 污泥固體物含量(樣品由交通大學林志高教授研究室提供) ……47
3-2-5 元素硫添加量(樣品由交通大學林志高教授研究室提供) ……....48
3-3 寡核苷酸探針的選擇、設計與合成……………………………………….49
3-3-1 寡核苷酸探針的選擇………………………………..…………….49
3-3-2 寡核苷酸探針的設計……………………………..……………….51
3-3-3 親緣樹分析（polygenetic tree analysis）…………………..…….53
3-3-4 寡核苷酸探針的合成與標定…………………………..………….53
3-3-5 寡核苷酸探針專一性測試……………………..………………….54
3-3-6 寡核苷酸探針最佳雜交嚴格度測試………..……..……..……….54
3-4 實驗步驟與方法 ……………………………………………..……….…..56
3-4-1 改變細胞膜滲透性…………………………..…………………….56
3-4-2 固定劑的選擇………………………………..…………………….57
3-5 分析設備與方法……………………………………….………………….58
3-5-1 分析設備………………………………..………………………….58
3-5-2 分析方法…………………………..……………………………….58
3-5-2-1 螢光原位雜交法………………...……………………………….58
3-5-2-1-1 固定（Fixation）……………………………………………….59
3-5-2-1-2 雜交（Hybridization）……………….……………………….59
3-5-2-1-3 清洗（Washing）……..……….…………………..…………. 60
3-5-2-1-4 FISH與DAPI染色……..…………………………………….60
3-5-2-1-5 觀察…………………………………………………………….61
3-5-2-2 總菌數測定………………………………………...…………….62
第四章 結果與討論………………………………………...……………………….64
4-1寡核苷酸探針的設計……………………………………...……………….64
4-2 探針專一性與最佳雜交嚴格度測試………………………..…………….70
4-2-1 探針專一性測試………………………….…….………………….70
4-2-2 探針最佳雜交嚴格度測試…………….………………….……….71
4-3 改變細胞膜滲透性……………………………..………………………….73
4-4 固定劑的選擇………………………………….….……………………….74
4-5 馴養槽污泥菌群結構分析……………………..……….………...……….77
4-5-1 總菌數分析…………………………….…………….…………….77
4-5-2 自體螢光的影響……………………….……….………………….79
4-5-3 菌群結構分析……………………….……….…………………….80
4-6 不同污泥固體物含量菌群結構分析………………..…………………….89
4-6-1 總菌數分析………………………..……………………………….90
4-6-2 污泥好氧消化…………………….….…………………………….91
4-6-3 菌群結構分析……………………………….…….……………….92
4-7 不同元素硫添加量菌群結構分析……………………………………….105
4-7-1 總菌數分析……………………………………………………….106
4-7-2 污泥好氧消化…………………………………………………….107
4-7-3 菌群結構分析…………………………………………………….109
第五章 結論與建議…………………………………………….………………….121
5-1 結論……………………………………………………………………….122
5-2 建議……………………………………………………………………….124
參考文獻………………………………………...………………………………….126
附錄一………………………………………………………………………………141

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