群體感應抑制法（Quorum Quenching ,QQ）是近年來所發展之濾膜生物阻塞控制技術之一，群體感應抑制法之原理為利用微生物分解細胞間溝通所使用的訊息分子，使微生物無法感知其群體數量大小，進而影響其分泌胞外聚合物（extra-cellular polymeric substance, EPS）之生理表現，因此得以延長濾膜使用的時程。此方法與傳統清潔手段相比之優點為能事先預防生物膜之發展與成熟，在其形成前就具有抑制效果，是一種從原理上去控制阻塞發生的手段。目前環境工程應用上主要利用能分解訊息分子Acyl homoserine lactones (AHLs)的微生物，這些分解菌由於能製造AHL-acylase或AHL-lactonase等酵素，在添加於MBR後可降低環境中AHLs的濃度，進而控制生物膜之發展。
　　因此，本研究主要目的為建立AHL分解菌的群體感應抑制法，工作內容主要涵蓋四個部分：（1）AHL生物檢測方法之建立與比較；（2）AHL群體感應抑制菌(QQ菌)篩選方法測試及建立，希望從環境中篩選出更多可應用於控制MBR濾膜阻塞的QQ菌；（3）比較篩選到之QQ菌的AHLs降解能力差異；（4）應用固定化QQ菌於MBR，以評估其延緩透膜壓力的成效。
　　本研究於淡水水資源回收中心活性污泥池及淡江大學校園水體中，從總計185隻菌中篩選出4隻(A9、A12、B11及D3)具快速降解C6-HSL、C8-HSL、C10-HSL、C12-HSL、3-oxo-C6-HSL、3-oxo-C8-HSL、3-oxo-C10-HSL及3-oxo-C12-HSL 之廣效菌株。此4隻菌株經以包埋固定化成QQ beads後仍保有降解AHLs的能力，後續並實際應用於MBR中，目前尚未觀察到MBR顯著的透膜壓力變化，可能是初次添加之QQ菌數量太少，建議未來提高QQ菌量再持續觀察其群體感應抑制成效。

Quorum quenching (QQ) is one of the biologically strategies for controlling membrane fouling. QQ directly controls the Extracellular polymeric substances (EPS), which is an important polymer causing membrane fouling. The principle of the bacterial QQ is to degrade signal molecules (e.g. acyl homoserine lactones, AHLs), so that the microorganisms can’t communicate and develop mature biofilm. This method has specificity and preventiveness, and several successful applications have been reported in the membrane bioreactor (MBR) treating wastewater.
The main objectives of this study are: (1) to establish bioassy methods forthe detection of AHLs, (2) to develop QQ bacteria screening protocol in order to isolate QQ bacteria from local environments, (3) to compare the degradation efficiency of QQ bacteria against various AHLs, (4) to apply immobilized QQ bacteria in a MBR. Through this study, the goal is to mitigate biofouling in MBR in the future.
Activated sludge from Tamsui wastewater treatment plant and water samples from Tamkang University were collected as source for screening QQ bacteria. Among total of 185 bacteria screened, 4 isolates were capable of rapidly quenching various AHLs. After quorum quenching bacteria were entrapped in beads, the ability to removal AHLs was still observed. These QQ beads were further applied in MBR while no apparent transmembrane pressure difference was found. It was speculated that this result was due to low concentration of QQ bacteria applied in the reactor. Therefore, current results suggest to increase total amount of QQ bacteria in MBR and to evaluate the impact of QQ effect on membrane fouling control in the future.

第一章	研究緣起	1
1.1	研究緣起	1
1.2	研究目的	3
第二章	文獻回顧	4
2.1	MBR薄膜生物處理系統	4
2.1.1	MBR薄膜生物處理系統之原理及特性	4
2.1.2	MBR薄膜生物處理系統之優點及面臨問題	5
2.1.3	濾膜阻塞之問題	6
2.1.4	濾膜之阻塞控制方法	7
2.2	Quorum Sensing群體感應機制	12
2.2.1	QS訊息分子	12
2.2.2	AHL偵測分析方法	14
2.3 Quorum Quenching	16
2.3.1	群體感應抑制原理	16
2.3.2	群體感應抑制之應用	17
2.4	固定化方法於廢水中之應用	19
2.4.1	固定化包埋法	19
2.4.2	固定化方法之比較	20
2.4.3	海藻酸鈉	21
2.4.4 聚乙烯醇 (PVA)	22
2.4.5聚乙烯醇(PVA)–硫酸鈉固定化法	22
第三章	實驗方法	24
3.1	AHL生物分析法	26
3.1.1	X-gal bioassay檢測法	26
3.1.2	Luminescence Beta-glo生物檢測法	27
3.2	AHL降解菌篩菌方法	28
3.2.1	AHL- Quenching Strains 96孔盤篩選方法	28
3.2.2	Enrichment Culture篩選方法	30
3.3	批次降解測試實驗	32
3.4	AHL降解菌酵素分析	34
3.4.1	QQ酵素菌熱處理測試	34
3.4.2	酵素胞內胞外測試	36
3.5 降解菌菌種鑑定	39
3.5.1 16S rDNA鑑定	39
3.6 微生物固定化方法	41
3.6.1 PVA包埋法	41
3.7 MBR薄膜生物反應器	42
3.7.1 MBR配製圖	42
3.7.2	MBR操作參數	44
3.7.3	MBR反應器之人工廢水	45
第四章	實驗結果	46
4.1	AHL生物分析法	46
4.1.1	AHLs檢測法	47
4.1.2	X-gal bioassay檢測法	47
4.1.3	Luminescence Beta-glo生物檢測法	51
4.2	AHL降解菌篩菌	56
4.2.1	AHL Quenching Strains 96孔盤篩選法	56
4.2.2	Enrichment Culture篩選	61
4.2.3	降解能力之最後測試	63
4.3	批次降解測試實驗	65
4.3.1	AHLs濃度降解變化	66
4.3.2	菌懸液濃度吸光值(OD600 )變化	75
4.3.3	批次實驗pH變化	75
4.4	QQ酵素性降解分析	77
4.4.1 QQ酵素熱處理測試	77
4.4.2	酵素胞內胞外測試	78
4.5 降解菌菌種鑑定	80
4.5.1 16S rDNA鑑定	80
4.6	MBR薄膜生物反應器	82
4.6.1包埋固定化QQ beads	82
4.6.2菌株包埋固定於QQ beads後之降解能力	83
4.6.3 MBR之壓力變化	84
第五章	結論與建議	86
5.1	結論	86
5.2	建議	87
第六章	附錄	88
批次降解實驗濃度數值	88
批次降解實驗pH變化	91
Reference	92

 
圖目錄
圖2- 1 革蘭氏陰性菌 Pseudomonas aeruginosa生物膜形成的過程	6
圖2- 2 群體感應誘導物質的種類與結構	13
圖2- 3革蘭氏陰性菌常見之群體感應誘導物質的種類與結構	14
圖2- 4 AHL信息通報菌株Agrobacterium tumefaciens A136	15
圖3- 1 本研究主要實驗流程	25
圖3- 2 篩選污水降解菌的基本流程圖	29
圖3- 3 批次降解測試實驗基本流程	33
圖3- 4 16S rDNA鑑定的基本流程	39
圖3- 5本實驗中MBR之配製圖	42
圖3- 6 操作運行中之薄膜生物反應器	43
圖4-1 培養於含X-gal之LB培養基之A136	46
圖4-2  X-gal生物檢測法之檢量線 (1)	48
圖4-3  X-gal生物檢測法之檢量線 (2)	48
圖4-4  X-gal生物檢測法之檢量線 (3)	49
圖4-5 在X-gal bioassay分析法之偵測結果(1)	49
圖4-6 在X-gal bioassay分析法之偵測結果(2)	50
圖4-7 在Luminescence冷光儀分析法之檢量線(1)	51
圖4-8 在Luminescence冷光儀分析法之檢量線(2)	53
圖4-9 在Luminescence冷光儀分析法之檢量線(3)	54
圖4-10淡水水資源回收中心活性污泥池，第一次篩選結果	57
圖4-11淡水水資源回收中心活性污泥池，第二次篩選結果	58
圖4-12淡水水資源回收中心活性污泥池，第三次篩選結果	59
圖4- 13 淡江大學的校園水樣，第一次篩選結果	60
圖4- 14 淡江大學的校園水樣，第二次篩選結果	60
圖4- 15  Enrichment culture篩選菌株於2 mM C8-HSL降解情形	61
圖4- 16  Enrichment culture篩選菌株降解C8-HSL之結果	62
圖4-17 降解菌之降解能力最後測試結果	64
圖4-18 降解菌在C6-HSL中，批次降解測試之降解變化圖	66
圖4- 19 降解菌在C8-HSL中，批次降解測試之降解變化圖	67
圖4- 20 降解菌在C10-HSL中，批次降解測試之降解變化圖	68
圖4- 21 降解菌在C12-HSL中，批次降解測試之降解變化圖	69
圖4- 22 降解菌在oxo-C6-HSL中，批次降解測試之降解變化圖	70
圖4- 23 降解菌在oxo-C8-HSL中，批次降解測試之降解變化圖	71
圖4- 24 降解菌在oxo-C10-HSL中，批次降解測試之降解變化圖	72
圖4- 25 降解菌在oxo-C12-HSL中，批次降解測試之降解變化圖	73
圖4- 26 批次降解實驗起始及結束時之pH值變化(1)	76
圖4- 27 批次降解實驗起始及結束時之pH值變化(2)	76
圖4-28  C8-HSL之QQ菌酵素熱處理測試結果。	77
圖4- 29  AHLs 之QQ菌酵素類型評估	78
圖4- 30  PVA-alginate QQ beads	82
圖4- 31  QQ菌及以包埋法製成QQbeads後之降解能力測試	83
圖4- 32  MBR之透膜壓力變化	84

 
表目錄
表2- 1 比較不同固定化方法之原理、材料及特色	21
表3- 1  Luria-Bertani (LB)洋菜培養基成分	26
表3- 2  Enrichment medium ① 	31
表3- 3  Enrichment medium ② 	31
表3- 4  Enrichment medium ③ 	31
表3- 5  Enrichment medium ④ 	31
表3- 6 本實驗使用之PCR Primer	39
表3- 7本實驗PCR之反應溫度及時間	40
表3- 8  MBR之操作相關參數	44
表3- 9  MBR之人工廢水組成配比	45
表4- 1  4隻降解QQ菌於8種AHLs中的降解情形	74
表4- 2  AHLs批次降解實驗起始菌懸液吸光值(OD600)	75
表4- 3  AHLs 之QQ菌酵素類型推估	79
表4- 4  4隻降解菌之16S rDNA鑑定結果	80
表4- 5  PVA-alginate QQ beads隨機挑選20顆之平均直徑	82
表6- 1 批次降解實驗中C6-HSL詳細濃度數值	88
表6- 2 批次降解實驗中C8-HSL詳細濃度數值	88
表6- 3 批次降解實驗中C10-HSL詳細濃度數值	88
表6- 4 批次降解實驗中C12-HSL詳細濃度數值	89
表6- 5 批次降解實驗中oxo-C6-HSL詳細濃度數值	89
表6- 6 批次降解實驗中oxo-C8-HSL詳細濃度數值	89
表6- 7 批次降解實驗中oxo-C10-HSL詳細濃度數值	90
表6- 8 批次降解實驗中oxo-C12-HSL詳細濃度數值	90
表6- 9  AHLs批次降解實驗起始及結束之pH值變化	91

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