濾膜阻塞控制對於薄膜生物反應器(MBR)的操作與維護相當關鍵，生物膜則是造成濾膜阻塞的重要因素且可受到群體感應(quorum sensing, QS)系統的調控。Acyl homoserine lactones為革蘭氏陰性菌QS系統中普遍採用的一類訊息分子，易受到環境的pH值影響進而改變其化學結構。因此本研究擬探討利用此一AHLs的化學特性，藉由電化學法提升與濾膜相鄰的陰極表面的pH值，而在高pH值的環境中進一步將AHLs將會水解，阻斷AHLs在QS中的功能，達到降低革蘭氏陰性菌產生胞外聚合物的產量，最終並評估此法延緩濾膜阻塞的成效。
　　本研究中先以批次試驗觀察電化學法降低人工廢水中AHLs濃度的效果，結果顯示施加-1.8 V的DC電源能提高人工廢水的pH值至9.5，並且在X-gal平板法與冷光法的生物檢測中，五種的AHLs至少失去76%以上的活性，並且證明AHLs是因被水解而失去其QS功能。接著測試不同的電位、電極配置與有無攪拌等組合，分析電化學法水解AHLs的效率，結果顯示以分離陰陽電極且無攪拌的方式有最佳的AHLs處理效率，分別為65.2%與54.8%的去除率。最後將電化學法實際應用在實驗室規模的MBR，在以鐵金屬的陽極的試驗中，應用電化學的eMBR (electro-MBR)的濾膜阻塞速率反而增加，20天內濾膜阻塞5次，而對照組的MBR的濾膜則是尚未阻塞，推測是陰極與濾膜模組的距離接近，造成混凝劑與污泥容易附著在陰極上，並造成濾膜的阻塞加劇。而在將陽極改為鈦金屬後，eMBR為平均38.0天阻塞，MBR則是平均19.4天阻塞，試驗中並未產生使用鐵陽極時污泥附著的問題，結果顯示使用鈦陽極的電化學法具有延緩濾膜阻塞的效果。

Biofouling control is a crucial factor for operation and maintenance of membrane bioreactor (MBR). Bacteria utilize quorum sensing (QS) systems to regulate biofilm formation which is thought to be critical in causing biofouling. Acyl homoserine lactone (AHL), a QS signaling molecule frequently used by many Gram-negative bacteria, is pH-sensitive. Exploiting this chemical properties of AHLs, we utilized electrochemical approach to raise the pH value on the surface of cathode in the vicinity of filtration membrane. After AHLs hydrolysis occurring in high pH circumstance, interruption of QS systems would inhibit biofilm formation, therefore, the rate of biofouling can be mitigated.
  In this study, we first conducted experiments in batch tests to evaluate the effect of the electrochemical approach on removal rate of AHL in synthetic wastewater. The results show that pH of solution can be increased to 9.5 when applying -1.8 V DC to synthetic wastewater. At least 76% loss of QS activity of five types of AHLs were evidenced by X-gal plate and luminescence biodetection methods and the mechanism of AHLs deactivation was caused by AHLs hydrolysis. Different electrode configurations, electric potential from -1.2V to -2.0V, and with or without mixing were performed to estimate the efficiency of AHLs hydrolysis. Results indicate that isolated electrodes without stir is the best condition to treat AHLs, with the efficiency of 65.2% and 54.8%, respectively.
  Applying the electrochemical approach to a lab-scale MBR was first conducted with iron anode, but conversely increased the rate of fouling in eMBR (electro-MBR) by 5 times in 20-day compared to a control MBR. This is probably due to the aggregation of sludge and coagulants on the cathode and nearby membrane. After altering to titanium anode, the average time of fouling for eMBR and normal MBR were 38.0 days and 19.4 days, respectively. This result shows that eMBR utilizing titanium anode can alleviate biofilm formation and has potential to be applied to full-scale MBR for biofouling control.

目錄
第一章	序論	1
1.1	研究緣起	1
1.2	研究之假設與目的	3
第二章	文獻回顧	4
2.1	薄膜生物反應器	4
2.1.1	薄膜生物器之原理	4
2.1.2	濾膜之阻塞與控制.	4
2.2	群體感應(quorum sensing)系統	6
2.2.1	群體感應之原理與分類	6
2.2.2	Acyl-homoserine Lactones	6
2.2.3	AHLs的pH相依性	7
2.3	群體感應抑制(quorum quenching)	9
2.3.1	酵素法	9
2.3.2	生物法	10
2.4	Electro-MBR	11
第三章	實驗方法與材料	12
3.1	電化學法批次試驗	12
3.1.1	人工廢水(Synthetic Wastewater)	12
3.1.2	實驗步驟	12
3.2	AHL生物分析法	15
3.2.1	X-gal瓊脂平板生物檢測法	15
3.2.2	冷光(luminescence)生物檢測法	16
3.3	薄膜生物反應器連續試驗	17
3.3.1	濾膜製備	17
3.3.2	薄膜生物反應器	18
3.4	胞外聚合物(EPS)分析	20
3.4.1	SMPp與EPSp	20
3.4.2	SMPc與EPSc	21
3.5	化學需氧量(COD)之檢測	21
3.6	凱氏氮與氨氮之檢測	21
3.7	硝酸鹽氮之檢測	23
3.8	水中懸浮固體物(MLSS)之檢測	23
3.9	統計分析	23
第四章	實驗結果	25
4.1	電化學法處理AHLs之初步試驗	25
4.1.1	電化學批次試驗法提升pH值之初步測試	25
4.1.2	批次試驗中人工廢水之pH值變化率	26
4.1.3	電化學法處理AHLs之評估	29
4.2	電化學法之批次試驗	35
4.3	電化學法在反應槽中之分析	41
4.3.1	電化學法的連續試驗	41
4.3.2	胞外聚合物(EPS)與溶解性微生物產物(SMP)之變化	48
4.3.3	處理效能比較	52
第五章	結果與建議	57
5.1	結論	57
5.2	建議	59
參考文獻	60
 
圖目錄
圖2  1 Homeserine lactone訊息分子的不同結構	7
圖2- 2 AHLs的水解變化	8
圖2- 3 C4-HSL的內脂環(lactone ring)的「開環」與「閉環」途徑	8
圖3- 1 電化學法在批次試驗中之配置示意圖	12
圖3  2 批次試驗的配置圖	13
圖3- 3 尚未已樹脂固定的濾膜模組	17
圖3- 4 濾膜模組固定於電極網上	17
圖3- 5 薄膜生物反應器系統示意圖	18
圖3- 6 實驗流程圖	24
圖4-1 電化學法提高人工廢水pH值的初步測試	25
圖4- 2 陰極電極片在通電過程中產生的氣泡	26
圖4- 3 以電化學法施加-1.8V電位於人工廢水之pH變化率	27
圖4- 5 以5 N NaOH及5 N HCl處理C8-HSL後，並以X-gal生物檢測法觀察C8-HSL之濃度變化	30
圖4- 6 以電化學法及5 N HCl處理C8-HSL後，並以X-gal生物檢測法觀察C8-HSL之濃度變化	32
圖4- 7 AHLs經鐵金屬陽極電化學法(-1.8V/20 min)及5 N HCl處理後，以冷光生物檢測法觀察其濃度變化	34
圖4- 8 AHLs經鈦金屬陽極電化學法(-1.8V/20 min)及5 N HCl處理後，以冷光生物檢測法觀察其濃度變化	34
圖4- 9 暴露電極(exposed electrodes)電化學法在不同電位與攪拌與否條件下處理C8-HSL，以冷光生物檢測法觀察C8-HSL之濃度變化	36
圖4- 10 分離電極(isolated electrodes)電化學法在不同電位與攪拌與否條件下處理C8-HSL，以冷光生物檢測法觀察C8-HSL之濃度變化	37
圖4- 11電化學法在不同電位與攪拌與否條件下處理C8-HSL，以5 N HCl酸回復的樣本，並以冷光生物檢測法檢測C8-HSL之濃度	39
圖4- 12 第一階段試驗之透膜壓力變化	43
圖4- 13 第二階段試驗之透膜壓力變化	44
圖4- 14 不鏽鋼陰極網的污泥附著情形	45
圖4- 15 受腐蝕的不鏽鋼陽極比較圖	46
圖4- 16 第三階段試驗之透膜壓力變化	47
圖4- 17 鈦金屬陰極網的污泥附著情形	48
圖4- 18 EPSp在反應槽中之時間序列變化	49
圖4- 19 EPSc在反應槽中之時間序列變化	50
圖4- 20 SMPp在反應槽中之時間序列變化	50
圖4- 21 SMPc在反應槽中之時間序列變化	51
圖4- 23 MBR與eMBR在第一階段至第三階段中 MLSS的時間序列變化	53
圖4- 24 MBR在第一階段至第三階段中COD的時間序列變化	54
圖4- 25 eMBR在第一階段至第三階段中COD的時間序列變化	54
圖4- 26 MBR在第三階段中硝酸鹽氮的時間序列變化	55
圖4- 27 eMBR在第三階段中硝酸鹽氮的時間序列變化	55
 
表目錄
表3  1 人工廢水成份(Weerasekara et al. 2014)	12
表3- 2 批次試驗的參數	14
表3- 3 Lysogeny broth (LB) agar培養基成份	15
表3- 4 薄膜反應器之操作參數	18
表3- 5 Lowry混和液成份	20
表3- 6 消化試劑	22
表3- 7 氫氧化鈉-硫代硫酸鈉試劑	22
表3- 8 氧化試劑	22
表4- 1 各種不同的電化學條件以電化學法處理前後的C8-HSL平均濃度與去除濃度 38
表4- 2薄膜生物反應器之操作參數 41
表4- 3 濾膜長度 41
表4- 4 薄膜生物反應器在連續試驗中，在不同的階段之操作的參數與電極的使用 42
表4- 5 MBR的相關係數表  52
表4- 6 eMBR的相關係數表 52

Bani-Melhem, K., and Elektorowicz, M. (2010). "Development of a novel submerged membrane electro-bioreactor (SMEBR): performance for fouling reduction." Environmental science & technology, 44(9), 3298-3304.
Bani-Melhem, K., and Elektorowicz, M. (2011). "Performance of the submerged membrane electro-bioreactor (SMEBR) with iron electrodes for wastewater treatment and fouling reduction." Journal of Membrane Science, 379(1-2), 434-439.
Bani-Melhem, K. Q. (2008). "Development of a novel submerged membrane electro-bioreactor for wastewater treatment." Concordia University.
Bokhove, M., Jimenez, P. N., Quax, W. J., and Dijkstra, B. W. (2010). "The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket." Proceedings of the National Academy of Sciences, 107(2), 686-691.
Byers, J. T., Lucas, C., Salmond, G. P., and Welch, M. (2002). "Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule." Journal of Bacteriology, 184(4), 1163-1171.
Chan, Y. Y., Bian, H. S., Tan, T. M. C., Mattmann, M. E., Geske, G. D., Igarashi, J., Hatano, T., Suga, H., Blackwell, H. E., and Chua, K. L. (2007). "Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump." Journal of bacteriology, 189(11), 4320-4324.
Close, T., Zaitlin, D., and Kado, C. (1984). "Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58." Plasmid, 12(2), 111-118.
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W., and Greenberg, E. P. (1998). "The involvement of cell-to-cell signals in the development of a bacterial biofilm." Science 280(5361), 295-298.
Dong, Y.-H., Wang, L.-H., Xu, J.-L., Zhang, H.-B., Zhang, X.-F., and Zhang, L.-H. (2001). "Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase." Nature, 411(6839), 813-817.
Dong, Y.-H., Xu, J.-L., Li, X.-Z., and Zhang, L.-H. (2000). "AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora." Proceedings of the National Academy of Sciences, 97(7), 3526-3531.
Donlan, R. M. (2002). "Biofilms: microbial life on surfaces." Emerging infectious diseases, 8(9), 881.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. t., and Smith, F. (1956). "Colorimetric method for determination of sugars and related substances." Analytical chemistry, 28(3), 350-356.
Eddy, M., Burton, F., Tchobanoglous, G., and Tsuchihashi, R. (2013). Wastewater engineering: treatment and Resource recovery, McGraw-Hill Education: New York, NY, USA.
Flynn, P. B., Busetti, A., Wielogorska, E., Chevallier, O. P., Elliott, C. T., Laverty, G., Gorman, S. P., Graham, W. G., and Gilmore, B. F. (2016). "Non-thermal plasma exposure rapidly attenuates bacterial AHL-dependent quorum sensing and virulence." Scientific reports, 6, 26320.
Fuqua, C., and Winans, S. C. (1996). "Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes." Journal of Bacteriology, 178(2), 435-440.
Galloway, W. R., Hodgkinson, J. T., Bowden, S. D., Welch, M., and Spring, D. R. (2010). "Quorum sensing in Gram-negative bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways." Chemical reviews, 111(1), 28-67.
Hasan, S. W., Elektorowicz, M., and Oleszkiewicz, J. A. (2012). "Correlations between trans-membrane pressure (TMP) and sludge properties in submerged membrane electro-bioreactor (SMEBR) and conventional membrane bioreactor (MBR)." Bioresource technology, 120, 199-205.
Hu, H., He, J., Liu, J., Yu, H., Tang, J., and Zhang, J. (2016). "Role of N-acyl-homoserine lactone (AHL) based quorum sensing on biofilm formation on packing media in wastewater treatment process." Rsc Advances, 6(14), 11128-11139.
Huber, B., Riedel, K., Hentzer, M., Heydorn, A., Gotschlich, A., Givskov, M., Molin, S., and Eberl, L. (2001). "The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility." Microbiology, 147(9), 2517-2528.
Judd, S. (2008). "The status of membrane bioreactor technology." 26(2), 109-116.
Kim, J.-H., Choi, D.-C., Yeon, K.-M., Kim, S.-R., and Lee, C.-H. (2011). "Enzyme-immobilized nanofiltration membrane to mitigate biofouling based on quorum quenching." Environmental science & technology, 45(4), 1601-1607.
Kim, M., Lee, S., Park, H.-d., Choi, S.-i., and Hong, S. (2012). "Biofouling control by quorum sensing inhibition and its dependence on membrane surface." Water Science and Technology, 66(7), 1424-1430.
Lade, H., Paul, D., and Kweon, J. H. (2014). "Quorum quenching mediated approaches for control of membrane biofouling." International journal of biological sciences, 10(5), 550.
Latifi, A., Winson, M. K., Foglino, M., Bycroft, B. W., Stewart, G. S., Lazdunski, A., and Williams, P. (1995). "Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1." Molecular microbiology, 17(2), 333-343.
Leadbetter, J. R., and Greenberg, E. (2000). "Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus." Journal of bacteriology, 182(24), 6921-6926.
Lee, S., Park, S.-K., Kwon, H., Lee, S. H., Lee, K., Nahm, C. H., Jo, S. J., Oh, H.-S., Park, P.-K., and Choo, K.-H. (2016). "Crossing the border between laboratory and field: bacterial quorum quenching for anti-biofouling strategy in an MBR." Environmental science & technology, 50(4), 1788-1795.
Lin, Y. H., Xu, J. L., Hu, J., Wang, L. H., Ong, S. L., Leadbetter, J. R., and Zhang, L. H. (2003). "Acyl‐homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum‐quenching enzymes." Molecular microbiology, 47(3), 849-860.
Liu, D., Momb, J., Thomas, P. W., Moulin, A., Petsko, G. A., Fast, W., and Ringe, D. (2008). "Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures." Biochemistry, 47(29), 7706-7714.
Lynch, M. J., Swift, S., Kirke, D. F., Keevil, C. W., Dodd, C. E., and Williams, P. (2002). "The regulation of biofilm development by quorum sensing in Aeromonas hydrophila." Environmental microbiology, 4(1), 18-28.
Oh, H.-S., Kim, S.-R., Cheong, W.-S., Lee, C.-H., and Lee, J.-K. (2013). "Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant." Applied microbiology and biotechnology, 97(23), 10223-10231.
Oh, H.-S., Yeon, K.-M., Yang, C.-S., Kim, S.-R., Lee, C.-H., Park, S. Y., Han, J. Y., and Lee, J.-K. (2012). "Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane." Environmental science & technology, 46(9), 4877-4884.
Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H., and Greenberg, E. (1994). "Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes." Proceedings of the National Academy of Sciences, 91(1), 197-201.
Pearson, J. P., Passador, L., Iglewski, B. H., and Greenberg, E. (1995). "A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa." Proceedings of the National Academy of Sciences, 92(5), 1490-1494.
Pearson, J. P., Van Delden, C., and Iglewski, B. H. (1999). "Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals." Journal of bacteriology, 181(4), 1203-1210.
Peterson, G. L. (1977). "A simplification of the protein assay method of Lowry et al. which is more generally applicable." Analytical biochemistry, 83(2), 346-356.
Pontefract, R. (1991). "Bacterial adherence: its consequences in food processing." Canadian Institute of Food Science and Technology Journal, 24(3-4), 113-117.
Tafti, A. D., Mirzaii, S. M. S., Andalibi, M. R., and Vossoughi, M. (2015). "Optimized coupling of an intermittent DC electric field with a membrane bioreactor for enhanced effluent quality and hindered membrane fouling." Separation and Purification Technology, 152, 7-13.
Trussell, R. S., Merlo, R. P., Hermanowicz, S. W., and Jenkins, D. (2006). "The effect of organic loading on process performance and membrane fouling in a submerged membrane bioreactor treating municipal wastewater." Water research, 40(14), 2675-2683.
Vu, B., Chen, M., Crawford, R. J., and Ivanova, E. P. (2009). "Bacterial extracellular polysaccharides involved in biofilm formation." Molecules, 14(7), 2535-2554.
Weerasekara, N. A., Choo, K.-H., and Lee, C.-H. (2014). "Hybridization of physical cleaning and quorum quenching to minimize membrane biofouling and energy consumption in a membrane bioreactor." Water research, 67, 1-10.
Wei, V., Oleszkiewicz, J., and Elektorowicz, M. (2009). "Nutrient removal in an electrically enhanced membrane bioreactor." Water Science and Technology, 60(12), 3159-3163.
Winson, M. K., Camara, M., Latifi, A., Foglino, M., Chhabra, S. R., Daykin, M., Bally, M., Chapon, V., Salmond, G., and Bycroft, B. W. (1995). "Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa." Proceedings of the National Academy of Sciences, 92(20), 9427-9431.
Yates, E. A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S. R., Sockett, R. E., Goldner, M., Dessaux, Y., Cámara, M., and Smith, H. (2002). "N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa." Infection and immunity, 70(10), 5635-5646.
Yeon, K.-M., Cheong, W.-S., Oh, H.-S., Lee, W.-N., Hwang, B.-K., Lee, C.-H., Beyenal, H., and Lewandowski, Z. (2008). "Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment." Environmental science & technology, 43(2), 380-385.
Yeon, K.-M., Cheong, W.-S., Oh, H.-S., Lee, W.-N., Hwang, B.-K., Lee, C.-H., Beyenal, H., and Lewandowski, Z. (2009). "Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment." Environmental science & technology, 43(2), 380-385.
Zhang, W., Cao, B., Wang, D., Ma, T., Xia, H., and Yu, D. (2016). "Influence of wastewater sludge treatment using combined peroxyacetic acid oxidation and inorganic coagulants re-flocculation on characteristics of extracellular polymeric substances (EPS)." Water research, 88, 728-739.
朱巧芸 (2018). "Acyl Homeserine Lactones (AHLs) 抑制菌篩選及其在薄膜生物反應器控制生物阻塞之效能研究." 碩士學位論文.
范姜仁茂, 莊連春, 曾迪華, 廖述良, 游勝傑, and 梁德明 (2009). "薄膜生物反應器(MBR)於廢水處理之技術評析." 工業污染防治, 109, 49-96.