臺灣博碩士論文加值系統

飲用水與民眾健康息息相關，為了防止水媒疾病（water-born diseases）的傳播，消毒已成為飲用水處理中不可或缺的過程。加氯消毒因成本低、操作簡單、效果好，且是唯一有能力提供餘量的方法，因此為目前最普遍使用的消毒方式。臭氧因具有超強氧化力、反應速率快，且無副產物等優點，成為目前最具潛力的消毒方法之一。本研究的主旨便是評估臭氧與氯對水中微生物之殺菌效率。
本研究包括二部分：第一部分以培養方法評估水中臭氧與加氯消毒之劑量效應關係，探討各項參數（菌種、菌濃度、臭氧濃度、暴露時間）對殺菌效率之影響，並討論美國環境保護署水中臭氧與加氯消毒標準下，不同微生物的殺菌效率；第二部分為應用非培養方法（Non-culture Method）－螢光染色法搭配流式細胞儀（Flow Cytometry, FCM）評估水中臭氧與加氯消毒效率，且同時與傳統培養方法（Culture Method）比較。
培養方法結果顯示，對於臭氧與氯的抵抗力：枯草桿菌內孢子＞青黴菌孢子＞酵母菌＞大腸桿菌；對臭氧殺菌效率之影響：臭氧濃度＞菌種差異＞暴露時間＞菌濃度；對加氯消毒效率之影響：菌種差異＞餘氯濃度＞暴露時間＞菌濃度；在美國環境保護署水中臭氧與加氯消毒標準下，對於細胞型態微生物來說，足以達到良好的殺菌效果；但對於孢子型態微生物的殺菌效率卻不及99.9％。
非培養方法結果顯示，比起傳統培養方法，以螢光染劑搭配流式細胞儀評估水中臭氧與加氯消毒效率時，可在殺菌機制與過程上提供更多的資訊，例如微生物在殺菌過程中總濃度的變化、細胞膜的受損程度或變化等，為一快速又十分有用的評估方法。此外，研究結果顯示消毒過程中，當微生物失去在培養基上的生長能力時，仍能保有一定程度的活性，因此使用傳統培養方法確實會高估殺菌效率。

For preventing the spread of water-born diseases, disinfection has already become an essential part in drinking water treatment plant. Chlorine disinfection is the most popular disinfection method because of its cheap, simple, and efficient properties. On the other hand, Ozone is a very strong and fast-reacting oxidant without byproduct, as considered as a potential disinfection method. Our current study was to evaluate the inactivation efficiency of ozone and chlorine on microorganisms in water. By culture and non-culture（flow cytometry with fluorochrome, FCM/FL）methods, the influences of microorganism species, microorganism concentrations, ozone and chlorine concentrations, and exposure time on inactivation efficiencies of ozone and chlorine were evaluated.
By culture method, it was found that the resistant abilities of tested microorganisms to both ozone and chlorine were as follows：endospore of Bacillus subtilis＞Penicillium citrinum spores＞Candida famata＞Escherichia coli. For ozone disinfection, the importance of the evaluated parameters were ozone concentration > species > exposure time > microbial concentrations. For chlorine disinfection, the importance was species, chlorine concentration, exposure time and microbial concentrations. Under the USEPA standards, the suggested doses could not provide efficient disinfection for spore type microorganisms.
It is known that microorganism could be still viable when they lose their ability to grow on culture medium. In comparison with non-culture method, it was clearly demonstrated that inactivation efficiencies of ozone and chlorine on microorganisms were overestimated by culture method.

摘 要..................................................I
ABSTRACT...............................................II
總 目 錄............................................III
表 目 錄..............................................V
圖 目 錄.............................................VI
第一章 前 言...........................................1
1.1 研究緣起............................................1
1.2 研究目的............................................2
第二章 文獻回顧.........................................3
2.1 水中臭氧消毒........................................3
2.1.1 臭氧特性..........................................3
2.1.2 臭氧生成基本原理..................................3
2.1.3 臭氧消毒機制......................................5
2.1.4 影響臭氧消毒效率的因子............................6
2.2 加氯消毒...........................................11
2.2.1 氯的特性.........................................11
2.2.2 氯在水中的反應...................................11
2.2.3 加氯消毒機制.....................................12
2.2.4 影響加氯消毒效率的因子...........................12
2.3 微生物之分析.......................................14
2.3.1 培養方法（Culture Method）.......................14
2.3.2 非培養方法（Non–culture Methods）...............14
2.3.3 螢光顯微鏡（Epifluorescence Microscopy，EFM）....15
2.3.4 流式細胞儀（Flow Cytometry，FCM）................15
2.3.5 螢光染色法.......................................16
2.3.6 應用非培養方法於水中臭氧及加氯消毒之研究.........18
第三章 材料與方法......................................26
3.1驗儀器與材料........................................26
3.1.1 實驗儀器.........................................26
3.1.2 實驗材料.........................................27
3.1.3 實驗菌種之製備...................................28
3.2 實驗方法與步驟.....................................31
3.2.1 臭氧對水中微生物殺菌效率之評估...................31
3.2.2 氯對水中微生物殺菌效率之評估.....................33
3.2.3 應用螢光染色法搭配流式細胞儀於水中臭氧與加氯消毒效率評估...................................................35
3.3 統計分析...........................................39
第四章 結果與討論......................................45
4.1 臭氧對水中微生物殺菌效率之結果.....................45
4.1.1 臭氧對大腸桿菌殺菌效率之結果.....................45
4.1.2 臭氧對酵母菌殺菌效率之結果.......................47
4.1.3 臭氧對青黴菌孢子殺菌效率之結果...................49
4.1.4 臭氧對枯草桿菌內孢子殺菌效率之結果...............52
4.1.5 臭氧對水中微生物殺菌效率之綜合比較...............55
4.1.6 美國環保署水中臭氧消毒標準下之殺菌效率...........58
4.2 氯對水中微生物殺菌效率之結果.......................60
4.2.1 氯對大腸桿菌殺菌效率之結果.......................60
4.2.2 氯對酵母菌殺菌效率之結果.........................61
4.2.3 氯對青黴菌孢子殺菌效率之結果.....................64
4.2.4 氯對枯草桿菌內孢子殺菌效率之結果.................67
4.2.5 氯對水中微生物殺菌效率之綜合比較.................69
4.2.6 美國環保署加氯消毒標準下之殺菌效率...............71
4.3 應用螢光染劑搭配流式細胞儀於水中臭氧與加氯消毒效率評估之結果.................................................73
4.3.1臭氧殺菌效率評估之結果............................73
4.3.2氯對大腸桿菌殺菌效率評估之結果....................78
第五章 結論與建議....................................122
5.1 結論..............................................122
5.2建議...............................................123
參考文獻..............................................124

1.顧洋 (1995) 紫外線/臭氧氧化程序在廢水處理上之應用, 工業污染防治, 56: 248-261.
2.張鳴助 (1995) 臭氧產生器, 工業材料, 119: 98-100.
3.張禹斌 (1983) “飲水加氯消毒之餘氯曲線研究”, 國立陽明大學公共衛生研究所碩士論文
4.蔡清讚 (1988) “醫院廢水中微生物之消毒及懸浮固體中微生物之消毒動力學”, 國立台灣大學土木工程研究所博士論文
5.葉欣誠 (1992) “臭氧消毒的基本現象與機制研究”, 國立台灣大學環境工程研究所碩士論文
6.謝淑貞 (1993) “臭氧與次氯酸鈉對家庭污水消毒效率以及經驗模式之探討”, 國立台灣大學環境工程研究所碩士論文
7.陳育明 (1996) “控製污水消毒加氯量之可行性分析”, 淡江大學水資源及環境工程研究所碩士論文
8.白姍燕 (2000) “以臭氧氧化水中有機物之研究”, 國立台灣大學環境衛生研究所碩士論文
9.謝文慶 (2002)“常溫充填包裝水殺菌條件之探討” 國立中興大學食品科學研究所碩士論文
10.王玉純 (2002) “臭氧對生物氣膠殺菌效率之評估”, 國立台灣大學環境衛生研究所碩士論文
11.江婉嘉 (2004) “應用螢光染色法以螢光顯微鏡與流式細胞儀評估醫院污水處理廠廢水中微生物特性”, 國立台灣大學環境衛生研究所碩士論文
12.Abu-Shkara, F.; Neeman, I.; Sheinman, R.; Armon, R. (1998). The effect of fatty acid alteration in coliform bacteria on disinfection resistance and/or adaptation. Water Science and Technology. 38(12), 133-139.
13.Arana, I., Santorum, P., Muela, A. and Barcina, I. (1999). Chlorination and ozonation of waste-water: comparative analysis of efficacy throuth the effect on Escherichia coli membranes. Journal of Applied Microbiology. 86, 883- 888.
14.Barbeau, B., Boulos, L., Desjardins, R., Coallier, J., Prévost, M. (1999). Examining the use of aerobic spore-forming bacteria to assess the efficiency of chlorination. Wat. Res. 33(13), 2941-2948.
15.Boulos, L., Prevost, B., Goalier, J., Desjardins, R. (1999). Live / Dead® BacLightTM：application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. Journal of Microbiological Method. 37, 77-86.
16.Chiang, C.F., Tsai, C.T., Lin, S. T., Huo, C. P. and Lo, K. V. (2003).
Disinfection of hospital wastewater by continuous ozonization. Journal of Environmental Science and Health. 38(12), 2895-2908.
17.Chii, S. and Ernest, R. Blatchleyiii. (2001). Chlorination of pure bacterial cultures in aqueous solution. Wat. Res. 35(1), 244-254.
18.Christen, E. and Giese, A.C. (1954). Changes in absorption spectra of nucleic acids and their derivatives following exposure to ozone and ultraviolet radiations. Archives of Biochemistry and Biophysics. 50, 206-209
19.Cho, M., Chung, H.M., Yoon, J. (2003). Quantitative evaluation of the synergistic sequential inactivation of Bacillus subtilis spores with ozone followed by chlorine. Environmental Science & Technology. 37 (10), 2134 -2138.
20.Cho, M., Chung, H., Yoon, J. (2002). Effect of pH and importance of ozone initiated radical reactions in inactivating bacillus subtilis spore. Ozone-science & Engineering. 24 (2), 145-150.
21.Cortezzo, D.E., Koziol-Dube, K., Setlow, B. (2004). Treatment with oxidizing agents damages the inner membrane of spores of bacillus subtilis and sensitizes spores to subsequent stress. Journal of Applied Microbiology. 97(4), 838-852.
22.Diao, H.F., Li, X.Y., Gu, J.D., Shi, H.C., Xie, z.m. (2004). Electron microscopic investigation of the bactericidal action of electrochemical disinfection in comparison with chlorination, ozonation and fenton reaction. Process Biochemistry. 39, 1421-1426.
23.Driedger, A., Staub, E., Pinkernell, U., Marinas, B., Koster, W. and Gunten, U. V. (2001). Inactivation of bacillus subtilis spores and formation of bromate during ozonation. Wat. Res. 35(12), 2950-2960.
24.Finch, G. R., Smith, D. W. and Stiles, M. E. (1988). Dose-response of Escherichia coli in ozone demand-free pfosphate buffer. Wat. Res. 22, 1563-1570.
25.Finch, G. R., Yuen, W.C. and Uibel, B. J. (1992). Inactivation of Escherichia coli using ozone and ozone-hydrogen peroxide. Environ. Technol. 13, 571-578.
26.Gwy-Am, S. and Mark, D. Sobsey. (2003). Reduction of norwalk virus，poliovirus 1，and bacteriophage MS2 by ozone disinfection of water. Apply and Enviromental Microbiology. 3975-3978.
27.Hamelin, C., Sarhan, F. and Chung, Y. S. (1978). Induction of deoxyribonucleic acid degradation in Escherichia coli. by ozone. Experentia 34, 1578-1579.
28.Huertas, A., Barbeau, B., Desjardins, C. (2003). Evaluation of bacillus subtilis and coliphage MS2 as indicators of advanced water treatment efficiency. Water Science and Technology. 47 (3), 255-259.
29.Hunt, Nimrata, K., Mariñas, Benito, J. (1999). Inactivation of Escherichia coli with ozone: chemical and inactivation kinetics. Wat. Res. 33(11), 2633-2641.
30.Jyoti, k.k., Pandit, A.B. (2004). Ozone and cavitation for water disinfection. Biochemical Engineering Journal. 18, 9-19.
31.Kaneko, M. (1998). Chlorination of pathogenic E. coli O157. Wat. Sci. Tech. 38(12), 141-144.
32.Katezenelson, E., Kletter, B. and Shuval, H. I. (1974). Inactivation Kinetics of viruses and bacteria in water by sue of ozone. J. Am. Wat. Works Assoc. 67, 725-729.
33.Kim, J.G., Yousef, A. E. and Dave, S. (1999). Application of Ozone for enhancing the microbiological safety and quality of foods: A Review, J. Food Prot. 62(90), 1071-1087.
34.Komanapalli, I. R., Lau, B. H. S. (1996). Ozone-induced damageescherichia coli K-12 springer-verlag. Howard, K.,Timothy, Inglis, J.J. (2003). The effect of free chlorine on burkholderia pseudomallei in potable water. Wat. Res. 37, 4425-4432.
35.Larson, M. A., Marinas, B.J. (2003). Inactivation of bacillus subtilis spores with ozone and monochloramine. Wat. Res. 37(4), 833-844.
36.Lund, V. (1996). Evaluation of E. coli as an indicatorfor the presence of campylo bacter jejuni and yersinia enter ocolitica in chlorinated and untreated Oligotrophic lake water. Wat. Res. 30(6), 1528-1534.
37.Matthew, A., Larson, Benito, J. Marinas. (2003). Inactivation of Bacillus subtilis spores with ozone and monochloramine, Wat. Res. 37, 833-844.
38.Progress in nuclear energy. 29, 175-182.Nimrata, K. Hunt, and benito, J., Marinas. (1997). Kinetics of Escherichia coli inactivation with ozone. Wat. Res. 31, (6), 1355-1362.
39.Ohgaki, S. (1990) Disinfection by ozone, J. Water and Waste. 32(4) 936-945.
40.Poonkhum, R., and Srisukonth, C. (2002). Effects of ozone treatment on cell growth and ultrastructural changes in bacteria. J. Gen. Appl. Microbid. 48, 193-199.
41.Radziminski, C., Ballantyne, L., Hodson, J., Driedger, A., Staub, E., Pinkernell, U. (2001). Inactivation of bacillus subtilis spores and formation of bromate during ozonation. Wat. Res. 35(12), 2950-2960.
42.Sartory, D.P. and Holmes, P. (1997). Chlorine sensitivity of enviromental，distribution system and biofilm coliforms. Wat. Sci. Tech. 35(11-12), 289 -292.
43.Scott, D. B. M. and Lesher, E. C. (1963). Effect of ozone on survival and permeability of Escherichia coli. Journal of Bacteriology 85, 567-576.
44.Scott, D. B. M. (1975). The effect of ozone on nucleic acids and their derivatives. In Aquatic Application of ozone, eds. W. J. Blogoslawski and R. G. Rice, p.1. International ozone institute, New York, NY.
45.Scott, L., Burnett, Larry, R. Beuchat. (2004). Comparison of methods for fluorescent detection of viable，dead，and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers. Food Microbiology. 74, 37-45.
46.Sherri, A. Tyrrell, Scott, R. Rippey, and William, D. Watkins. (1995). Inactivation of bacterial and viral indicators in secondary sewage effluents，using chlorine and ozone. Wat. Res. 29(11), 2483-2490.
47.Sobey, S., Sibille, Y., Mathieu, L., Paquin, J. L., and Block, J. C. (1997) Influence of water chlorination on the counting of bacteria with DAPI (4’6-diamindino-2-phenylindole). Applied and Environmental Microbiology 63, 1564-1569.
48.Son, H., Cho, M., Kim, J. (2005). Enhanced disinfection efficiency of mechanically mixed oxidants with free chlorine. Wat. Res. 39 (4), 721-727.
49.Thanomsub, B., Anupunpisit, V., Chanphetch, S. (2002). Effects of ozone treatment on cell growth and ultrastructural changes in bacteria. Journal of General and Applied Microbiology. 48(4), 193-199.
50.Viera, M.R., Guiamet, P.S., de Mele, M.F.L. Videla, H.A. (1999). Use of dissolved ozone for controlling planktonic and sessile bacteria in industrial cooling system. International Biodeterioration and Biodegradation. 44, 201-207.
51.Young, S., Setlow, P. (2004). Mechanisms of bacillus subtilis spore resistance to and killing by aqueous ozone. Journal of Applied Microbiology 96 (5), 1133-1142.
52.Young, S.B. and Setiow, P. (2004). Mechanisms of bacillus subtilis spore resistance to and killing by aqueous ozone. Journal of Applied Microbiology, 96, 1133-1142.