臺灣博碩士論文加值系統

五氯酚因普遍使用造成環境中土壤、水、空氣的污染，而生物復育是經濟可行的處理方法。本研究目的是從可降解高濃度五氯酚之五氯酚馴化堆肥土菌群中，分離純化出本土性五氯酚分解菌株探討其最適降解條件後增殖培養加入混合菌群，以生物放大方式提高五氯酚降解，以做為污染場址的應用。以變性梯度膠體電泳法分析菌群結構得知樹皮堆肥土馴化菌群及豬糞堆肥土馴化菌群有類似的族群結構，也比污染土馴化菌群結構複雜；而菌液以五氯酚活化次數愈多，其樣本之五氯酚分解菌菌群越趨單純。由馴化堆肥土菌群中篩選出較高降解能力之五氯酚分解菌株，經Biolog菌種鑑定為Pseudomonas citronellolis NPUEM001，NPUEM001於五氯酚濃度為75 mg/L時最適降解環境酸鹼值為7.48~8.99，溫度為30~35℃，提高接菌密度可加強五氯酚降解，以菌數在2.8×106 cell/mL時最佳，其降解率可達51.41 %，而1.4×106 cell/mL~2.8
×106 cell/mL為有效接菌密度範圍；除五氯酚外再添加葡萄糖碳源不但能有效的增加菌數，且降解率可提高17.3 %，但添加檸檬酸對五氯酚的降解卻無明顯效果。增殖NPUEM001加入馴化樹皮堆肥土菌群中進行生物放大降解實驗，降解率可提高20.8 %；NPUEM001降解前後菌液以Microtox進行生物毒性試驗，結果顯示經NPUEM001降解後五氯酚之毒性並無明顯下降。本研究所提供之生物復育選用菌株及各項環境參數值，未來將以醱酵方式大量培養，並製作為生物製劑，可施用於五氯酚污染場址中。

The widespread use of the pentachlorophenol (PCP) has caused certain pollutions in the soil, water, and air. However, Bioremediation is an economical and feasible disposing way that helps to reduce the damage to the environment. This study isolated the PCP degradating bacteria from three different PCP degradation microbial consortia in compost-soil that we have been climated for 150 days, and to find the most suitable environmental parameter on degradation. Then we used bioaugmentation to add screened bcteria into microbial consortia for enhancing PCP degradation activity.
The diversity of the three microbial consortia analysis by denaturant gradient gel electrophoresis (DGGE) showed the same structure in mature hog manure and bark compost-soils and they were more diversity than the structure of contaminated-soil only. Enriched more times with PCP added, the diversity of PCP degradation microbial consortia became simpler. Pseudomonas citronellolis NPUED001 was isolated from bark compost soil and it showed 24.3 % PCP degradation ratio in the 75 mg/L concentration of PCP. The most suitable degradation pH for PCP degradation by NPUEM001 is 7.88~8.99 under the temperature of 30~35℃ with 75 mg/L initial concentration of PCP. The higher inoculum density of NPUEM001 will
increase the PCP degradation ratio. The most efficient degradation ability is 51.41 % when the inoculum density is 2.8×106 cell/mL in 75 mg/L PCP-concentration. The initial concentration needs to reach the rang within 1.4×106~2.8×106 cell/mL of inoculum density for the effectiveness. When added with glucose as the second carbon source the PCP degradation ratio was raised up 17.3 % with 75 mg/L initial concentration of PCP but there was no obvious increase for citric acid addition. The bioaugmentation by added proliferation NPUEM001 into PCP degradation microbial consortia can effectively increase PCP degradation ratio to 50.7 %. The toxicity analysis of culture medium by Microtox showed the toxicity of PCP didn''t descend after the degradation of NPUEM001. The Pseudomonas citronellolis NPUEM001 and microbial consortia from bark compost-soil could be the inoculum for PCP contaminated site in Taiwan in the future.

中文摘要. ........................................ I
英文摘要.............................................. III
誌謝.................................................. Ⅴ
目錄............................................... .. VI
圖目錄.......................................... XII
表目錄................................................ XVIII
附錄目錄.............................................. XX
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 .......................................5
2.1 氯酚化合物........................................5
2.1.1 氯酚化合物之特性................................5
2.1.2 氯酚類化合物之毒性............................. 9
2.2 五氯酚............................................12
2.2.1 特性............................................13
2.2.2 用途............................................16
2.2.3 污染情況........................................17
2.2.4 相關法規標準....................................18
2.3 五氯酚之整治復育技術..............................19
2.3.1 生物復育........................................19
2.3.2 分解五氯酚的微生物及代謝........................22
2.3.3 五氯酚分解機制..................................24
2.3.4 菌群結構分析....................................25
2.3.5 影響五氯酚分解環境因子..........................27
2.3.6 五氯酚降解後毒性評估............................28
2.3.7 生物放大（Bioaugmentation）.....................29
2.4 BIOLOG菌種鑑定系統................................30
第三章 材料與方法 ....................................31
3.1 實驗材料......................................... 31
3.1.1 堆肥土菌源 .....................................31
3.1.2 培養基......................................... 33
3.1.3 微生物基因體去氧核醣核酸萃取實驗材料........... 34
3.1.4 聚合酶連鎖反應實驗材料（Polymerase Chain Reaction）35
3.1.5 變性梯度膠體電泳實驗材料（Denaturant Gradient Gel Electrophoresis）......................................35
3.1.5.1 鑄膠（gel casting）材料.......................35
3.1.6 五氯酚馴化混合菌及純菌降解實驗材料............. 36
3.1.7 生物毒性分析實驗（Microtox......................37
3.2 實驗方法..........................................38
3.2.1 聚合酶鏈反應－變性梯度膠片電泳實驗............. 38
3.2.1.1 微生物基因體去氧核醣核酸萃取實驗..............38
3.2.1.2 瓊脂膠電泳確認DNA品質實驗............. 39
3.2.1.3 聚合酶連鎖反應實驗（Polymerase Chain Reaction）41
3.2.1.4 瓊脂膠電泳確認PCR反應物品質實驗...............41
3.2.1.5 變性梯度膠體電泳實驗
（Denaturant Gradient Gel Electrophoresis）........ 43
3.2.2 篩選分離純化五氯酚分解菌株………….…………… 46
3.2.3 五氯酚馴化混合菌及篩選菌降解實驗..……………... 46
3.2.4 Biolog菌種鑑定系統…………………………………. 47
3.2.5 最適降解環境探討…..…………….………………….. 48
3.2.5.1 活化菌液製備………..………………………….. 48
3.2.5.2 濃度（concentration）………………………….….. 48
3.2.5.3 酸鹼值（pH）………………………….……..…. 48
3.2.5.4 基質（carbon substrate）……..…………………. 49
3.2.5.5 溫度（temperature）………………………….…. 50
3.2.5.6 接菌密度（inoculum density）…………………. 50
3.2.6 生物放大（bioaugmentation）降解實驗…..………… 51
3.2.7 五氯酚萃取回收率實驗………………………………. 51
3.2.8 分析方法………………………………………………. 52
3.2.8.1 五氯酚的濃度測定方法……………………….… 52
3.2.8.2 總菌數測定方法……………...………………….. 54
3.2.8.3 菌數計數…………..………………..…….…...…. 55
3.2.8.4 水中氯離子檢測方法－離子層析法………….… 55
3.2.8.5 生物質量測定法…………………..………...…… 56
3.2.8.6 五氯酚降解副產物分析…...………………...…... 56
3.2.8.7 生物毒性分析…………….....…………...………. 57
第四章 結果與討論 .................................................. 59
4.1 菌群結構分析…..........................…. 59
4.2 萃取回收率…………………………………………………. 63
4.2.1 液體中五氯酚萃取回收效率………………………..... 63
4.3 分離純化五氯酚分解菌株………………………….……… 65
4.4 以五氯酚降解力篩選菌株…………………………………. 69
4.5 Biolog菌種鑑定……………………..………………….….. 73
4.6 最適降解環境探討……………………….………………… 74
4.6.1 濃度………………………..……..……………………. 74
4.6.1.1 氯離子濃度與分解速率相關性…………….…… 81
4.6.2 酸鹼值………………………………………….……… 84
4.6.3 溫度…………………………….…..………………….. 87
4.6.3.1 不同溫度下之五氯酚降解情況…………..…...… 87
4.6.3.2 溫度對分解速率影響…….……………………… 94
4.6.4 接菌密度…….……………….….……..…... 97
4.6.5 添加不同基質…….………………….….….… 101
4.7 生物毒性測試…………..………….……………… 103
4.8 生物放大效應（Bioaugmentation）…………..……… 104
第五章 結論..................................... 109
參考文獻 ..............................................111
附錄...................................................121
作者簡介.................................... 126

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