臺灣博碩士論文加值系統

根據世界衛生組織(WHO)和聯合國環境規劃署(UNEP)調查報告顯示，農村地區微生物數量約為139 CFU cm-2，而都市地區微生物數量約為72110 CFU cm-2。其中致病菌株如大腸桿菌(Escherichia coli)、金黃色葡萄球菌(Staphylococcus aureus)、克雷伯氏肺炎桿菌(Klebsiella pneumoniae)等易引起感染性疾病(Peng, et al. 2008) ; 麴菌類真菌如黑麴菌(Aspergillus niger)與黃麴菌(Aspergillus flavus)等則易引起食品及環境污染(Zhang, et al. 2012)。傳統消毒技術在殺菌/抑菌過程產生有毒物與致癌性物質，如碘化物、苯酚衍生物、鄰苯二甲酸二丁酯(Dibutyl phthalate)、三鹵甲烷(Trihalomethanes)和鹵乙酸(Haloacetic acids)等(Richardson, et al. 2003)。因此發展新興抗(真)菌綠色材料為當今全球抗菌課題之當務之急。
本研究使用自製光觸媒摻氮二氧化鈦 (N-TiO2)、摻氮與電氣石二氧化鈦 (N-T-TiO2)、摻碳二氧化鈦 (C-TiO2) 與摻鈀與碳二氧化鈦 (Pd-C-TiO2) 於可見光照射下進行光催化失活，失活反應參數如: 光觸媒劑量、初始菌數密度及光強度。研究指標菌種為克雷伯氏肺炎桿菌與黑麴菌。研究數據以失活動力學模型 (Chick-Waston model、Modified Hom model、Light Chick-Waston model與Light Modified Hom model) 模擬光催化綠色材料對上述指標菌種之失活效率。實驗結果顯示四種光觸媒綠色材料，其劑量1.0 g L-1和0.5 %、光強度7.32 mW cm-2、初始細菌濃度105 CFU mL-1和105 spore # mL-1條件下皆能有效使克雷伯氏肺炎桿菌 (1440分鐘) 與黑麴菌 (168小時) 達99.999%抗菌率 ; 各別材料抗克雷伯氏肺炎桿菌/黑麴菌效率依序為Pd-C-TiO2 > C-TiO2 > N-T-TiO2 > N-TiO2。不同菌種於光催化綠色材料其耐光催化失活能力克雷伯氏肺炎桿菌 (Pd-C-TiO2, 210分鐘) < 黑麴菌(Pd-C-TiO2, 96小時)，根據前人文獻與本研究所拍攝的電顯圖得知黑麴菌的細胞壁比克雷伯氏肺炎桿菌厚，故在相同條件下失活效率比克雷伯氏肺炎桿菌低。Chick-Waston model、Modified Hom model、Light-Chick-Waston model和Light-Modified Hom model皆有符合實驗數據的潛力，且k值皆具有規律性，如使用Modified Hom model進行模擬，克雷伯氏肺炎桿菌與黑麴菌的模擬，顯示兩者光催化失活反應的三階段參數變化一致，皆是第一階段 (緩衝期) 失活速率常數 (k1) 值趨勢會往上升 ; 第二階段 (對數期) 失活速率常數 (k2) 值趨勢會往上升 ; 第三階段 (遲滯期) 失活速率常數 (k3) 值趨勢會往下降。透過SEM、TEM、TXM和AFM了解改質過的二氧化鈦對克雷伯氏肺炎桿菌和黑麴菌進行光催化失活反應過程中細胞表面與型態的變化，並藉由K＋、CoA、MDA、DNA和蛋白質的釋出，探討進行光催化失活反應的機制。
本研究結果顯示自製光觸媒二氧化鈦具高效抗菌能力，對於環境中指標菌種如克雷伯氏肺炎桿菌與黑麴菌均可達99.999%抗菌率且無傳統耗能、產生具毒性之副產物等缺點，未來可廣泛應用於抗(真)菌材料與環境抗菌技術層面等並具高發展潛力。

According to the investigation report from World Health Organization (WHO) and the United Nations Environment Program (UNEP), with the number of bacteria being ∼139 CFU cm-2 in the countryside and 72,110 CFU cm-2 in an urban environment(Peng, et al. 2008). Among them, some pathogenic strains such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae were caused infectious diseases ; some fungus such as Aspergillus niger and Aspergillus flavus are easy to caused food and environmental pollution (Zhang, et al. 2012). Due to traditional disinfection techniques produce toxic and carcinogenic substances in sterilization and antibacterial processes, such as iodide, derivatives of phenol, dibutyl phthalate, trihalomethanes and haloacetic acids. Therefore, development of new anti-bacteria (fungus) green materials is the top priority issue for today.
In this research, homemade N-TiO2, N-T-TiO2, C-TiO2 and Pd-C-TiO2 were carried out photocatalytic deactivated under visible light irradiation. The inactivation reaction parameters including photocatalyst dose, the bacteria initial concentration and light intensity. The bioindicators were Klebsiella pneumoniae and Aspergillus niger. The experimental data were used to simulate the deactivation efficiency of the above-mentioned indicators strain by using the (Chick-Waston model, Modified Hom model, Light Chick-Waston model and Light Modified Hom model). The results showed that when the four photocatalytic materials were 1.0 g L-1 and 0.5%, the light intensity was 7.32 mW cm-2, and the initial bacterial concentrations were 105 CFU mL-1 and 105 spore # mL-1 can effectively make Klebsiella pneumoniae (1440 minutes) and Aspergillus niger (168 hours) reach 99.999% of the antibacterial rate. The antibacterial efficiency of each material on Klebsiella pneumoniae / Aspergillus niger was Pd-C-TiO2> C-TiO2> N-T-TiO2> N-TiO2. Effects of different microbial species on anti-photocatalytic deactivation with these four kinds of photocatalytic materials, Klebsiella pneumoniae (Pd-C-TiO2, 210 min) was lower than that of Aspergillus niger (Pd-C-TiO2, 96 hours). According to the electron micrograph of this study and predecessors studies, it was found that the cell wall of Aspergillus niger was thicker than Klebsiella pneumoniae, so the efficiency of inactivation was lower than that of Klebsiella pneumoniae under the same conditions. Chick-Waston model, Modified Hom model, Light-Chick-Waston model, and Light-Modified Hom model showed the potential to fit the experimental data and k values are regular, if the use of Modified Hom model for Klebsiella pneumoniae and Aspergillus niger simulation operation, showing the two photocatalytic deactivation reaction of the three-phase parameter changes are consistent. The trend of the first stage (buffer period) inactivation rate constant (k1) tends to rise; the second stage (logarithmic period) inactivation rate constant (k2) tends to rise; the third stage (lag phase) inactivation rate Constant (k3) value tends to decline. In this research, SEM, TEM, TXM and AFM were used to investigate the changes of cell surface and morphology in the photocatalytic deactivation of Klebsiella pneumoniae and Aspergillus niger during photocatalytic deactivation of modified titanium dioxide, and the mechanism of photocatalytic deactivation were explored by the release of K+, CoA, MDA, DNA and protein.
The results of this research show that the homemade photocatalyst titanium dioxide has a high efficiency of antibacterial activity with the bioindicators such as Klebsiella pneumoniae and Aspergillus niger can reach 99.999% antibacterial rate and no traditional energy consumption, resulting in toxic byproducts and other shortcomings. In the future, these four materials can be widely used in anti-(fungi) and environmental anti-bacterial technology level and a high potential for development.

致謝 i
摘要 ii
Abstract iv
目錄 vi
圖目錄 xiii
表目錄 xxiv
第一章 前言 1
1.1 研究背景 1
1.2 研究目的 5
第二章 文獻回顧 6
2.1 微生物種類 6
2.1.1 細菌-克雷伯氏肺炎桿菌 6
2.1.2 真菌-黑麴菌 8
2.2 光催化劑材料介紹 10
2.2.1 傳統二氧化鈦 10
2.2.2 可見光應答二氧化鈦 13
2.2.3 二氧化鈦的光催化活 19
2.3 光催化失活參數影響 22
2.3.1 光催化劑劑量 22
2.3.2 微生物初始濃度 30
2.3.3 光強度 34
2.3.4 不同微生物類型 42
2.4 光催化失活機制 50
2.5 動力學模擬 53
2.5.1 Empirical model 53
2.5.2 Mechanistic model 58
第三章 材料與方法 61
3.1 實驗流程與條件 61
3.2 實驗材料與設備 64
3.2.1 材料與藥品 64
3.2.1.1 菌株 64
3.2.1.2 培養基 65
3.2.1.3 抗菌材料 65
3.2.1.4 藥品 65
3.2.2 實驗設備與器材 67
3.2.2.1 實驗設備 67
3.2.2.2 實驗器材 69
3.3 實驗方法 70
3.3.1 可見光催化劑製備 70
3.3.1.1 摻氮二氧化鈦 (N-TiO2) 合成 70
3.3.1.2 摻氮/電氣石二氧化鈦 (N-T-TiO2) 合成 70
3.3.1.3 摻碳二氧化鈦 (C-TiO2) 合成 70
3.3.1.4 摻鈀/碳二氧化鈦 (Pd-C-TiO2) 合成 71
3.3.2 微生物培養 71
3.3.2.1 細菌-克雷伯氏肺炎桿菌 71
3.3.2.2 真菌-黑麴菌 72
3.3.3 光催化失活 72
3.3.3.1 細菌-克雷伯氏肺炎桿菌 72
3.3.3.2 真菌-黑麴菌 73
3.3.4 光催化失活動力模擬 74
3.3.4.1 Chick-Watson model 74
3.3.4.2 Modified Hom model 75
3.3.4.3 Novel kinetic model-light intensity effect 76
3.3.5 微生物失活特性 76
3.3.5.1 Scanning Electron Microscope (SEM) 76
3.3.5.2 Transmission Electron Microscope (TEM) 76
3.3.5.3 Transmission X-Ray Microscope (TXM) 77
3.3.5.4 Atimic Force Microscope (AFM) 77
3.3.5.5 Fluorescence Microscope (FM) 78
3.3.5.6 Flow Cytomter (FCM) 78
3.3.5.7 鉀離子 (K+) 測定 79
3.3.5.8 CoA測定 79
3.3.5.9 MDA測定 79
3.3.5.10 DNA測定 79
3.3.5.11 Protein測定 80
第四章 結果與討論 81
4.1光催化失活 81
4.1.1 細菌-克雷伯氏肺炎桿菌 81
4.1.1.1 光催化劑劑量效應 81
4.1.1.2 初始菌數密度效應 92
4.1.1.3 光照強度效應 101
4.1.2 真菌-黑麴菌 106
4.1.2.1 光催化劑劑量效應 106
4.1.2.2 初始菌數密度效應 116
4.1.2.3 光照強度效應 125
4.1.3 材料效益 130
4.1.4 菌種效益 134
4.2光失活動力學模式 136
4.2.1 Chick-Watson model 137
4.2.1.1 細菌-克雷伯氏肺炎桿菌 138
4.2.1.2 真菌-黑麴菌 152
4.2.2 Modified Hom model 166
4.2.2.1 細菌-克雷伯氏肺炎桿菌 167
4.2.2.2 真菌-黑麴菌 185
4.2.3 Novel kinetic model-light intensity effect 200
4.2.3.1 Light-Chick-Watson model 200
4.2.3.2 Light-Modified Hom model 211
4.3細胞表面型態變異 224
4.3.1 SEM 影像 224
4.3.1.1 細菌-克雷伯氏肺炎桿菌 224
4.3.1.2 真菌-黑麴菌 228
4.3.2 TEM 影像 233
4.3.2.1 細菌-克雷伯氏肺炎桿菌 233
4.3.2.2 真菌-黑麴菌 236
4.3.3 TXM 影像 239
4.3.3.1 細菌-克雷伯氏肺炎桿菌 239
4.3.3.2 真菌-黑麴菌 241
4.3.4 AFM 測定 243
4.3.4.1 細菌-克雷伯氏肺炎桿菌 243
4.3.4.2 真菌-黑麴菌 245
4.4 細胞膜完整性 248
4.4.1 FM分析 248
4.4.1.1 細菌-克雷伯氏肺炎桿菌 248
4.4.1.2 真菌-黑麴菌 250
4.4.2 FCM 分析 250
4.4.2.1 細菌-克雷伯氏肺炎桿菌 250
4.4.2.2 真菌-黑麴菌 251
4.5 細胞胞內物破損與釋出 252
4.5.1 陽離子 (K+) 釋出 252
4.5.1.1 細菌-克雷伯氏肺炎桿菌 252
4.5.1.2 真菌-黑麴菌 255
4.5.2 CoA釋出 256
4.5.2.1 細菌-克雷伯氏肺炎桿菌 256
4.5.2.2 真菌-黑麴菌 257
4.5.3 MDA釋出 258
4.5.3.1 細菌-克雷伯氏肺炎桿菌 258
4.5.3.2 真菌-黑麴菌 259
4.5.4 DNA破損與釋出 261
4.5.4.1 細菌-克雷伯氏肺炎桿菌 261
4.5.4.2 真菌-黑麴菌 262
4.5.5 Protein釋出 263
4.5.5.1 細菌-克雷伯氏肺炎桿菌 263
4.5.5.2 真菌-黑麴菌 264
第五章 結論和建議 265
參考文獻 272
附件 286
Appendix I Experimental Raw Data Figures 286
I -1 N-TiO2 for killed K. pneumoniae 286
I -1-1 Dosage Effect 286
I -1-2 Light intensity Effect 286
I -1-3 Bacterial concentration Effect 287
I -2 N-TiO2 for killed A. niger 287
I -2-1 Dosage Effect 287
I-2-2 Light intensity Effect 288
I-2-3 Fungus concentration Effect 288
I -3 N-T-TiO2 for killed K. pneumoniae 289
I-3-1 Dosage Effect 289
I-3-2 Light intensity Effect 289
I -3-3 Bacterial concentration Effect 290
I -4 N-T-TiO2 for killed A. niger 290
I-4-1 Dosage Effect 290
I-4-2 Light intensity Effect 291
I-4-3 Fungus concentration Effect 291
I -5 C-TiO2 for killed K. pneumoniae 292
I-5-1 Dosage Effect 292
I-5-2 Light intensity Effect 292
I-5-3 Bacterial concentration Effect 293
I -6 C-TiO2 for killed A. niger 293
I-6-1 Dosage Effect 293
I-6-2 Light intensity Effect 294
I-6-3 Fungus concentration Effect 294
I-7 Pd-C-TiO2 for killed K. pneumoniae 295
I-7-1 Dosage Effect 295
I-7-2 Light intensity Effect 295
I-7-3 Bacterial concentration Effect 296
I -8 Pd-C-TiO2 for killed A. niger 296
I-8-1 Dosage Effect 296
I-8-2 Light intensity Effect 297
I-8-3 Fungus concentration Effect 297
I -9 The other figures of K. pneumoniae 298
I -10 The other figures of A. niger 302
I -11 Viable bacteria remaining in the NA-agar plates of K. pneumoniae 303
I -12 Viable bacteria remaining in the PDA-agar plates of A. niger 304
Appendix II Experimental Raw Data Tables 305
II-1 N-TiO2 for killed K. pneumoniae 305
II -1-1 Dosage Effect 305
II -1-2 Light intensity Effect 306
II-1-3 Bacterial concentration Effect 308
II -2 N-TiO2 for killed A. niger 309
II -2-1 Dosage Effect 309
II-2-2 Light intensity Effect 311
II-2-3 Fungus concentration Effect 312
II -3 N-T-TiO2 for killed K. pneumoniae 314
II-3-1 Dosage Effect 314
II-3-2 Light intensity Effect 315
II -3-3 Bacterial concentration Effect 317
II -4 N-T-TiO2 for killed A. niger 318
II-4-1 Dosage Effect 318
II-4-2 Light intensity Effect 320
II-4-3 Fungus concentration Effect 321
II -5 C-TiO2 for killed K. pneumoniae 323
II-5-1 Dosage Effect 323
II-5-2 Light intensity Effect 324
II-5-3 Bacterial concentration Effect 326
II -6 C-TiO2 for killed A. niger 327
II-6-1 Dosage Effect 327
II-6-2 Light intensity Effect 329
II-6-3 Fungus concentration Effect 330
II-7 Pd-C-TiO2 for killed K. pneumoniae 332
II-7-1 Dosage Effect 332
II-7-2 Light intensity Effect 333
II-7-3 Bacterial concentration Effect 335
II -8 Pd-C-TiO2 for killed A. niger 336
II-8-1 Dosage Effect 336
II-8-2 Light intensity Effect 338
II-8-3 Fungus concentration Effect 339
Appendix III Experimental Detection Limit and Calibration Curve 341
III -1 Potassium ion 341
III -1-1 Potassium detection limit 341
III -1-2 The calibration curve of potassium working standard 342
III -1-3 The concentration of potassium working standard (ppm) and instrument reading value 342
III -1-4 Potassium concentration (ppm cell-1) in K. pneumoniae 342
III -1-5 Potassium concentration (ppm cell-1) in A. niger 343
III -2 CoA 344
III -2-1 The calibration curve of CoA working standard 344
III -2-2 The concentration of CoA working standard (ppm) and instrument reading value 344
III -2-3 CoA concentration (ppm) in K. pneumoniae 345
III -2-4 CoA concentration (ppm) in A. niger 345
III -3 MDA 346
III -3-1 The calibration curve of MDA working standard 346
III -3-2 The concentration of MDA working standard (ppm) and instrument reading value 346
III -3-3 MDA concentration (ppm) in K. pneumoniae 347
III -3-4 MDA concentration (ppm) in A. niger 347
III -4 Filter test report 348
III -5 Light intensity conversion chart 349
III -6 Light intensity test 350
III -7 Model change chart 351
Appendix V Experimental Detection Limit and Calibration Curve 353

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