近年來群體感應抑制法(quorum quenching,QQ)已被證明能有效控制薄膜生物反應器(membrane bioreactor, MBR)的生物性阻塞，其中最常使用的手段為添加可降解訊息分子的微生物，控制MBR內訊息分子的濃度於啟動群體感應(quorum sensing,QS)的閾值之下，進而延緩生物膜的形成達到抑制薄膜阻塞的目的。本實驗室先前於現地污水廠成功篩選出三隻具多種AHLs降解能力的潛力菌株，分別是Ochrobactrum anthropic A9、Bacillus cereus A12及Bacillus thuringiensis B11。雖然各菌株於不同AHLs的批次降解實驗中皆具有良好的QQ效果，但是否能在連續流MBR中達到QQ的效果仍須進一步的測試及觀察。
　　因此本研究的主要目的為：(1)評估固定化包埋A9、A12、B11菌株對MBR抑制濾膜阻塞之成效；(2)改良固定化包埋法實驗條件；(3)比較A9對不同污泥性質的MBR濾膜阻塞控制之成效。
　　本研究結果發現應用固定化技術包埋A9及A12兩隻菌株於連續流MBR中，平均可延緩17.5%及90.9%濾膜阻塞速度，而B11並未觀察到控制阻塞的成效。同時改良固定化的包埋條件，發現在121°C下溶解PVA及alginate，並以CaCl2-H3BO4及Na2SO4作為交聯劑反應2小時及8小時的條件下，可獲得較佳的水合膠體機械/化學強度。本實驗為首次探討應用QQ菌於含膨化污泥之MBR，並觀察其延緩濾膜阻塞成效之研究，實驗中共測試兩種不同性質之膨化污泥MBR (SVI >140)，結果顯示添加改良固定化條件的A9於MBR中，發現在含有較低MLSS (3947±25 mg/L)及較小污泥粒徑(70 μm)的MBR內，A9延緩濾膜阻塞能力可達113.5%。
    此研究成果對於非以沉澱特性篩選微生物而可能發生污泥沉澱性較差之MBR，提供利用群體感應抑制法控制濾膜生物性阻塞相關資訊與貢獻，唯未來若能針對膨化污泥內的菌群結構做進一步的分析，將可提供更多延緩濾膜阻塞機制之解釋與探討。

Recently, quorum quenching (QQ) has been proven as an effective approach for biofouling control in the membrane bioreactor. Among of QQ strategies, the most common approach is applying the bacteria which can degrade the signal molecules in quorum sensing (QS) systems. Addition of these QQ bacteria in MBR will control the concentration of QS signal molecules under the activation threshold. Therefore, applying QQ bacteria can control biofouling by inhibiting biofilm formation. Previously, three potential bacteria strains with variety AHLs degradation abilities was successfully isolated from the local wastewater treatment plant in our lab. They are Ochrobactrum anthropic A9、Bacillus cereus A12 and Bacillus thuringiensis B11. Although all strains have demonstrated the effective QQ ability in removing various AHLs in batch degradation experiments, their QQ efficacy in continuous flow MBR still needs further observation and evaluation.
　　Therefore, the main objectives of this study are: (1) evaluation of MBR membrane fouling control with immobilized A9、A12 and B11, (2) improve the experimental conditions of cell immobilization process, (3) applied A9 to two MBRs that containing different sludge properties and compare their anti-biofouling efficacy.
　　Results in this study showed immobilized A9 and A12 applied to the continuous MBR, can delay the membrane fouling rate by an average of 17.5% and 90.9%, respectively. However, immobilized B11 was found no apparent QQ efficacy. To improve the cell immobilized condition, it was found that under the temperature of dissolving PVA and alginate at 121°C, and cross-linking with CaCl2-H3BO4 and Na2SO4 for 2 and 8 hours, can achieve best mechanical/chemical strength of the hydrated colloid. To the best of our knowledge, this is the first study attempting to examine the biofouling control efficacy of applied QQ bacteria in MBR with sludge bulking. Two different sludge bulking MBR (SVI >140) were tested. The results showed that when A9 was applied to sludge bulking MBRs using improved immobilization conditions, the membrane fouling delay can reach 113.5% in one of the reactors with lower MLSS concentration (3947±25 mg/L) and smaller sludge particle size (70μm).
　　Unlike conventional activated sludge process that can select inhabiting microbes by settling ability through the secondary settling tank, MBR culture microorganisms does not based on settling properties and is inevitable to sludge bulking. This research contributes relevant information and contributions to control membrane biofouling with QQ strategy against this type of sludge. It would provide more detailed explanations and discussion on the mechanism of fouling delay, if microbial community of the system can be analyzed the in the future.

第一章	前言	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薄膜生物處理系統	4
2.2濾膜阻塞機制及控制方法	4
2.3群體感應(quorum sensing)系統	7
2.4群體感應抑制(quorum quenching)	8
2.4.1阻斷訊息分子的生成	8
2.4.2阻斷訊息分子的接收蛋白	9
2.4.3利用酵素降解訊息分子	9
2.5 螢光分析	10
第三章	實驗方法	11
3.1 薄膜生物反應器	11
3.1.1濾膜製備	11
3.1.2薄膜生物反應器	12
3.2 QQ Beads製備	14
3.2.1前置作業	14
3.2.2製作流程	15
3.3 AHL生物分析法	16
3.3.1冷光(Luminescence)生物偵測法	16
3.3.2 X-gal生物檢測法	17
3.3.3 AHLs唯一碳源測試	18
3.4實驗分析方法	19
3.4.1水中懸浮固體物	19
3.4.2胞外聚合物(EPS)、溶解性微生物產物(SMP)	19
3.4.3化學需氧量(COD)	19
3.4.4氨氮、硝酸鹽氮、亞硝酸鹽氮檢測方法	20
3.4.5 EEM螢光光譜	21
3.4.6 粒徑分析	21
第四章	實驗結果	22
4.1 AHLs抑制菌延緩濾膜阻塞之效能評估	22
4.1.1 MBR透膜壓力之變化	23
4.1.2 PVA-alginate固定化包埋QQ菌後之降解活性測試	30
4.1.3 生物膜形成與EPS、SMP濃度間的相關性	35
4.1.4操作期間對各項水質參數及污泥特性之影響	38
4.2 固定化技術改良及效能測試	43
4.2.1 AHLs抑制菌活性測試	43
4.2.2 QQ Beads物理吸附作用	46
4.2.3固定化包埋條件之優化	48
4.3 AHLs抑制菌控制濾膜阻塞與污泥特性之影響	51
4.3.1 污泥特性	51
4.3.2 MBR透膜壓力之變化	54
4.3.3 改良固定化包埋QQ菌之降解活性測試	57
4.3.4 EPS、SMP濃度變化及EEM分析結果	59
4.3.5 粒徑分析	67
4.3.6 水質參數及污泥特性之監測	69
第五章	結論與建議	75
5.1 結論	75
5.2 建議	76
附錄	77
參考文獻	82

 
圖目錄
圖 2 1 N-acyl homoserine lactone(AHL)結構圖。	7
圖 2 2 EEM區域分類圖	10
圖 3 1 以環氧樹脂固定之濾膜膜組	11
圖 3 2 薄膜生物反應器系統配置圖	13
圖 3 3 PVA-alginate beads製作流程圖	15
圖 4 1 添加BH4 Beads與未添加QQ Beads反應槽透膜壓力變化圖	24
圖 4 2 添加A9 Beads與未添加QQ Beads反應槽透膜壓力變化圖	25
圖 4 3 添加A12 Beads與未添加QQ Beads反應槽透膜壓力變化圖	26
圖 4 4 添加B11 Beads與未添加QQ Beads反應槽透膜壓力變化圖。	27
圖 4 5 以冷光生物偵測法分析BH4 beads於實驗前後對C8-HSL之降解能力。	31
圖 4 6 以冷光生物偵測法分析A9 beads於實驗前後對C8-HSL之降解能力。	32
圖 4 7 以冷光生物偵測法分析A12 beads於實驗前後對C8-HSL之降解能力。	33
圖 4 8 以冷光生物偵測法分析B11 beads於實驗前後對C8-HSL之降解能力。	33
圖 4 9 馴養期及添加A9、A12、B11、BH4固定化球體於反應槽運行期間，MLSS濃度變化圖。	39
圖 4 10 馴養期及添加A9、A12、B11、BH4固定化球體於反應槽運行期間，進流水、出流水的化學需氧量及去除效率變化圖。	40
圖 4 11 馴養期及添加A9、A12、B11、BH4固定化球體於反應槽運行期間，進流水總氮去除率。	41
圖 4 12 馴養期及添加A9、A12、B11、BH4固定化球體於反應槽運行期間，進流水氨氮去除效率。	41
圖 4 13 馴養期及添加A9、A12、B11、BH4固定化球體於反應槽運行期間，活性污泥pH變化圖。	42
圖 4 14 以X-gal生物偵測法觀察各菌株對C8-HSL降解情形。	44
圖 4 15 A9(左)、BH4(右)以C8-HSL作為唯一碳源塗盤培養。	45
圖 4 16 A12(左)、BH4(右)以C8-HSL作為唯一碳源塗盤培養。	45
圖 4 17 空珠及A9 beads降解1µM C8-HSL趨勢圖。	46
圖 4 18 空珠及A9 beads降解10µM C8-HSL趨勢圖。	47
圖 4 19 10×10顯微鏡下R1槽活性污泥	52
圖 4 20 10×10顯微鏡下R2槽活性污泥	53
圖 4 21 10×10顯微鏡下R2槽活性污泥上清液	53
圖 4 22 添加A9 Beads與Vacant beads於R1槽之透膜壓力變化圖。	54
圖 4 23 添加A9 Beads與Vacant beads於R2槽之透膜壓力變化圖	56
圖 4 24 以冷光生物偵測法分析在R1槽內A9 bead於實驗前後對C8-HSL之降解能力。	57
圖 4 25 以冷光生物偵測法分析在R1槽內VB於實驗前後對C8-HSL之降解能力。	57
圖 4 26 以冷光生物偵測法分析在R2槽內A9 bead於實驗前後對C8-HSL之降解能力。	58
圖 4 27 以冷光生物偵測法分析在R2槽內VB於實驗前後對C8-HSL之降解能力。	58
圖 4 28  R1槽EPS之EEM圖。	61
圖 4 29  R1槽SMP之EEM圖。	63
圖 4 30  R2槽EPS之EEM圖。	65
圖 4 31  R2槽SMP之EEM圖。	66
圖 4 32 R1槽A9及VB的粒徑大小。	67
圖 4 33 R2槽A9及VB的粒徑大小。	68
圖 4 34 R1槽MLSS濃度變化圖	69
圖 4 35 R2槽MLSS濃度變化圖	69
圖 4 36 R1槽COD濃度變化圖	70
圖 4 37 R2槽COD濃度變化圖	70
圖 4 38 R1槽氨氮去除率	71
圖 4 39 R1槽總氮去除率	71
圖 4 40 R2槽氨氮去除率	72
圖 4 41 R2槽總氮去除率	72
圖 4 42 R1槽pH變化圖	73
圖 4 43 R2槽pH變化圖	73
圖 4 44 R1槽溶氧變化圖	74
圖 4 45 R2槽溶氧變化圖	74

圖S 1 PVA-alginate Beads投入反應槽產生之泡沫。	77
圖S 2 A9 Beads使用前(左)、使用後(右)狀態。	77
圖S 3 80 °C混和溫度下PVA-alginat beads剖面照。	78
圖S 4 121 °C混和溫度下PVA-alginate bead剖面照。	78
圖S 5 121 °C混和溫度下PVA-alginate beads在0分鐘、15分鐘、30分鐘下的去泡測試。	78
圖S 6 第一劑(CaCl2 + H3BO4)及第二劑(Na2SO4)在不同浸泡時間下，曝氣一小時的去泡測試。	79
圖S 7 薄膜生物反應器R1及R2活性污泥上清液	79
圖S 8 R2A9第一次阻塞濾膜積垢外觀，(左)原始狀態、(右)純水洗滌後	80
圖S 9 R2VB第一次阻塞濾膜積垢外觀，(左)原始狀態、(右)純水洗滌後	80
圖S 10 優化固定化方法後，添加PVA-alginate beads於反應槽中產生之泡沫。	81
圖S 11 優化固定化方法後，A9 beads使用後球體狀態。	81

 
表目錄
表 3 1 薄膜反應器設計之參數	12
表 3 2 人工廢水成份	13
表 3 3 LB Broth培養基成分	17
表 4 1 BH4槽及Control槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較	36
表 4 2 A9槽及Control槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較	37
表 4 3 A12槽及Control槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較	37
表 4 4 B11槽及Control槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較。	37
表 4 5 不同混和溫度下PVA-alginate beads膨脹狀況。	48
表 4 6 不同交聯劑浸泡時間實驗設計	49
表 4 7 不同浸泡時間下，球體在不同時間點的直徑大小	50
表 4 8 不同浸泡時間下，球體在不同時間點的膨脹率	50
表 4 9 薄膜生物反應器R1及R2各項污泥性質指標	52
表 4 10 R1A9槽及R1VB槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較	59
表 4 11 R2A9槽及R2VB槽懸浮污泥內EPS、SMP protein與polysaccharide含量比較	60

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