Genetical and physiological studies of photocatalytic disinfection of Escherichia coli.

• Other Authors: Gao, Minghui
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Escherichia coli > Environmental aspects; Water > Purification > Photocatalysis; Water > Purification > Disinfection; Titanium dioxide
• Online Access: http://library.cuhk.edu.hk/record=b5549496; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328272

水資源缺乏引起的一系列問題在世界上已建得到廣泛關注，因此，確保提供潔淨衛生的水在保護人類健康和環境方面起著重要作用。近來，光催化作為頗有前景的替代方法被廣泛應用殺菌除污。二氧化鈦是目前研究最多應用最廣的光催化劑。基於紫外光譜照射，催化劑表面產生活性氧化物種，這些物種具有強氧化性能殺滅細胞。 === 本文首次研究了母體菌種大腸桿菌BW25113和它的同源單基因缺陷變異體對光催化殺菌的靈敏度差異。母體菌種和變異菌種表現出不同的耐受性。基於此，能幫助發掘出重要的變種。通過生物化學方法，可以檢測出不同菌種的生理性特徵。結合其他方法，可以進一步揭示光催化殺菌的生理性機理。 === 首先，我們篩選出了兩種重要的變異體。一種是大腸桿菌JW1081，即脂肪酸變異體，該菌種缺乏脂肪酸合成調節的關鍵基因。一種是大腸桿菌JW3942，即乙酰輔酶A變異體，該菌種缺乏乙酰輔酶A合成調控得到關鍵激酶。我們發現脂肪酸變異體對光催化處理的耐受性稍低，而乙酰輔酶A變異體則耐受性較高。 同時發現，溫度可以調節細胞膜的不飽和酸和飽和酸的比例。因此，我們提出脂肪酸和乙酰輔酶A是光催化殺菌中的重要影響因子。 === 更進一步研究發掘了細胞內酶和光催化產生的活性氧物種間的關係。大腸桿菌JW3914，即過氧化氫酶變異體，是發現的另一個重要的變異體。通過對母體和變異體的淬滅劑實驗，主要的殺菌活性氧物種是光催化產生的雙氧水，而不是羥基自由基。細胞體內的過氧化氫酶誘導在母體菌體內發現，而未在變異體內檢測到。 === 本課題採用母體/單基因變異體的研究方法，為全面深刻理解光催化殺菌的深層機理提供一種全新的研究思路。 === Many problems associated with the lack of clean, fresh water worldwide are well known. Developing countries will particularly be affected by water availability problems and there will be further pressure on water demand resulting from economic development and population growth. Therefore, the provision of safe and clean water plays a key role in protecting human health and the environment. Recently, photocatalytic oxidation (PCO) has been widely accepted as a promising alternative method of water disinfection. Titanium dioxide (TiO₂) has been investigated extensively and is the most widely used photocatalyst. Upon the irradiation of UVA lamp, reactive charged and oxidative species are generated on TiO₂ surface and can inactive the bacterial cells. === In this study, the photocatalytic performances of a parental strain (E.coli BW25113) and its isogenic single-gene deletion mutant strains have been investigated for the first time. These bacterial strains exhibited different sensitivies towards photocalytic inactivation. Based on this, it can help reveal some important mechanism from the mutations. Biotic factors were confirmed by physiological biochemical measurement. === Firstly, we screened out the potential mutation fabF⁻ mutant (E. coli JW1081, carrying the mutation of fabF759(del)::kan) and coaA⁻ mutant (E. coli JW3942, carrying the mutation of coaA755(del)::kan). The isogenic fabF⁻ mutant is slightly more susceptible, and coaA⁻ mutant is less susceptible to photocatalytic inactivation. Through conditioning temperature, it adjusts the ratio of unsaturated to saturated fatty acid (FA) of cell membrane. We propose that FA profile and coenzyme A level significantly affect photocatalytic inactivation of bacteria. Moreover, we show photogenerated electrons (e⁻) can directly inactivate the cells of E. coli. === Furthermore, we report the relationship between the bacterial intracellular enzyme and the reactive charged and oxidative species (ROSs) generated during photocataltic inactivation. The katG⁻ mutant, E. coli JW3914, carrying the mutation of katG729(del)::kan is another important mutation. The parental and katG⁻ mutant strains reveal that photogenerated H₂O₂ but not OH[subscript free] is another important reactive oxygen species to inactivate bacteria. The inducible catalase (CAT) corresponding to H₂O₂can be detected in parental strain but not in katG⁻ mutant. === The research methodology using parental/single-gene deletion mutant strains is expected to shed light on fully understanding of the fundamental mechanism of photocatalytic inactivation of E. coli. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Gao, Minghui. === Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references (leaves 130-177). === Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Abstract also in Chinese. === Acknowledgements --- p.i === Abstract --- p.v === Table of contents --- p.ix === List of Figures --- p.xiii === List of Plates --- p.xvii === List of Tables --- p.xviii === List of Equations --- p.xix === Abbreviations --- p.xxi === Chapter 1. --- Introduction --- p.1 === Chapter 1.1 --- Water crisis --- p.1 === Chapter 1.2 --- Traditional disinfection methods --- p.3 === Chapter 1.2.1 --- Chlorination --- p.4 === Chapter 1.2.2 --- Ozonation --- p.6 === Chapter 1.2.3 --- Ultraviolet irradiation --- p.8 === Chapter 1.2.4 --- Multiple disinfectants --- p.10 === Chapter 1.3 --- Advanced oxidation process (AOPs) --- p.10 === Chapter 1.3.1 --- Hydrogen Peroxide/Ozone (H₂O₂/O₃) --- p.11 === Chapter 1.3.2 --- Ozone/Ultraviolet Irradiation (O₃/UV) --- p.12 === Chapter 1.3.3 --- Hydrogen Peroxide/ Ultraviolet Irradiation (H₂O₂/UV) --- p.12 === Chapter 1.3.4 --- Fenton's --- p.Reaction === Chapter 1.4 --- Solar photocatalytic disinfection (SPC-DIS) --- p.14 === Chapter 1.4.1 --- Photocatalyst-TiO₂ --- p.31 === Chapter 1.4.2 --- Irradiation sources --- p.35 === Chapter 1.4.3 --- TiO₂ photocatalytic process --- p.35 === Chapter 1.4.4 --- The role of photogenerated reactive charged and oxidative species (ROSs) --- p.38 === Chapter 1.5 --- Bacteria --- p.40 === Chapter 1.5.1 --- E. coli BW25113 --- p.40 === Chapter 1.5.2 --- E. coli Keio Collection --- p.41 === Chapter 1.5.3 --- Bacterial defense mechanism towards oxidative stresses --- p.44 === Chapter 1.6 --- Photocalytic applications --- p.53 === Chapter 1.7 --- Significance of the project --- p.55 === Chapter 2. --- Objectives --- p.58 === Chapter 3. --- Genetic studies of the roles of fatty acid and coenzyme A in photocatalytic inactivation of Escherichia coli --- p.61 === Chapter 3.1 --- Introduction --- p.61 === Chapter 3.2 --- Materials and methods --- p.65 === Chapter 3.2.1 --- Photocatalyst --- p.65 === Chapter 3.2.2 --- Bacterial nutrient --- p.66 === Chapter 3.2.3 --- Bacterial strains --- p.67 === Chapter 3.2.4 --- Photocatalytic inactivation --- p.69 === Chapter 3.2.5 --- Fatty acid profile --- p.72 === Chapter 3.2.6 --- Fluorescent measurement of bacterial coenzyme A content --- p.74 === Chapter 3.2.7 --- The role of photogenerated electrons (e⁻) to bacterial inactivation --- p.74 === Chapter 3.2.8 --- Transmission Electron Microscopic (TEM) --- p.75 === Chapter 3.2.9 --- Photoelectrochemical measurement --- p.77 === Chapter 3.3 --- Results --- p.77 === Chapter 3.3.1 --- Photocatalytic inactivation --- p.77 === Chapter 3.3.2 --- Effects of pre-incubation at different temperatures --- p.80 === Chapter 3.3.3 --- Fatty acid profile --- p.83 === Chapter 3.3.4 --- Fluorescent measurement of bacterial coenzyme A content --- p.84 === Chapter 3.3.5 --- The role of electron (e⁻) in photocataytic inactivation --- p.84 === Chapter 3.3.6 --- Transmission electron microscopy (TEM) --- p.89 === Chapter 3.3.7 --- Photocurrent measurement --- p.90 === Chapter 3.4 --- Discussion --- p.90 === Chapter 3.5 --- Conclusions --- p.96 === Chapter 4 --- Genetic and physiological studies of the role of Catalase and H₂O₂ in photocatalytic inactivation of E. coli --- p.98 === Chapter 4.1 --- Introduction --- p.98 === Chapter 4.2 --- Materials and methods --- p.101 === Chapter 4.2.1 --- Bacterial strains --- p.101 === Chapter 4.2.2 --- Photocatalytic performance --- p.102 === Chapter 4.2.3 --- Scavenger studies --- p.103 === Chapter 4.2.4 --- Effects of different pHs on photocatalytic inactivation --- p.104 === Chapter 4.2.5 --- Measurement of bacterial catalase activity and H₂O₂ --- p.104 === Chapter 4.2.6 --- Transmission electron microscopy (TEM) --- p.105 === Chapter 4.2.7 --- Atomic absorption spectrophotometer (AAS) --- p.105 === Chapter 4.3 --- Results and discussion --- p.106 === Chapter 4.3.1 --- Photocatalytic performance --- p.106 === Chapter 4.3.2 --- Scavenger studies --- p.108 === Chapter 4.3.3 --- Contribution of hydrogen peroxide (H₂O₂) --- p.111 === Chapter 4.3.4 --- Effects of different pHs on photocatalytic inactivation --- p.114 === Chapter 4.3.5 --- Bacterial catalase (CAT) activity --- p.116 === Chapter 4.3.6 --- Destruction model of bacterial cells --- p.118 === Chapter 4.4 --- Conclusions --- p.120 === Chapter 5. --- General conclusions --- p.122 === Chapter 6. --- Prospectives --- p.125 === Chapter 7. --- Appendix --- p.127 === Chapter 8. --- References --- p.130