台南中石化安順廠過去曾產生燒鹼 (NaOH)、五氯酚 (pentachlorophenol) 及各項農藥等化學物質，但製造五氯酚的過程產生了相當多戴奧辛(dioxin)副產物且未妥善處理，造成工廠廠址附近環境汙染，由於戴奧辛化合物相當穩定且半衰期長，因此當地居民健康與生態飽受危害。本次實驗試圖利用泥漿相生物反應器 (slurry phase bioreactor) 進行生物整治，其中輔以微生物添加法 (bioaugmentation) 加強戴奧辛之降解。實驗所使用的菌株-Pseudomonas mendocina NSYSU 為現地場址所篩選具有降解戴奧辛之菌種。為了檢視泥漿相生物反應槽可否用於無動力混和裝置，因此增加了以加強曝氣反應槽做對比。同時，利用 PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) 技術分析菌劑在實驗過程中菌量變化的情況。結果顯示，戴奧辛濃度隨著當時取樣土壤粒徑大小而受到顯著影響，因此實驗末期則改以泥漿相的表層取樣，以方便統一粒徑標準。在無動力生物反應器整治下汙染物濃度數值仍然不穩定，但動力攪拌組則由原先的 21,557 I-TEQ/kg 下降至17710.1 I-TEQ/kg ，顯示動力均質化在戴奧辛汙染整治上的重要性。PCR-DGGE 分析結果顯示，添加之P.mendocina NSYSU菌種能保持穩定在生物反應器中，持續發揮降解戴奧辛之功效。由於實驗規模較大，大量培養菌液之培養基必須符合經濟效益，本研究亦針對多項常用工業培養基原料進行探討，結果發現以 1% 魚溶漿組合下表現效果最佳，細菌濃度可達3.2*109 CFU/ml。

Sodium hydroxide, pentachlorophenol (PCP), and various pesticides were the main products of the China Petrochemical Development Corporation (CPDC) An-Shun Plant in the past. During the manufacturing of pentachlorophenol, many dioxin-like compounds were released into the environments. Because of dioxin compounds are fairly stable and possess long half-life in the nature, the dioxin contamination usually caused severe damages to the health of human as well as to the survival of wild animal and plants. One dioxin-degrading bacterium Pseudomonas mendocina NSYSU was isolated from this dioxin-contaminated site previously. In this study, we used a slurry phase bioreactor in combination with the addition of P. mendocina NSYSU strain to enhance the degradation of dioxins. In order to compare the effect of aeration during the treatment, another slurry phase bioreactor without mixing aeration was studied as well. The fluctuation of bacterial communities and diversities were analyzed in each bioreactor by using PCR-DGGE technique. Results showed that the measure of dioxin concentration was severely affected by the soil particle size. Therefore, all of the following samples were taken from the surface of the bioreactors in which the soil particle sizes were more uniform. The degradation of dioxin in the non-mixing bioreactor was not stable. However, in the aeration mixing bioreactor, the dioxin concentration decreased from 21,557 I-TEQ / kg to 17,710.1 I-TEQ / kg. This result indicated that mixing aeration is useful during the treatment of dioxin contaminated soil in a slurry phase bioreactor. PCR-DGGE analysis revealed that the P. mendocina NSYSU strain was constantly presented in the bioreactor and functioned dioxin degradation. For scale-up treatment of a polluted land site in the future, the production of large amount bacterial inoculant is also an important consideration. Our studied show that 1% of fish soluble extract medium provided the best growth for P. mendocina NSYSU strain (3.2 x 109 cfu/ml) during large amount production.

論文審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
第一章 緒論 1
1.1 前言 1
1.2 戴奧辛概述 2
1.2.1 戴奧辛基本結構及其物理化學性質 2
1.2.2 戴奧辛之生物毒性與對人體影響 3
1.2.3 戴奧辛自然來源與衰減 5
1.3 常見土壤戴奧辛處理方式 8
1.4 降解戴奧辛之微生物與反應機制 9
1.4.1 降解戴奧辛相關微生物及其降解機制之文獻 9
1.4.1.1 好氧環境下進行氧化破環 9
1.4.1.2 厭氧環境下進行還原性脫氯 15
1.4.1.3 好氧下進行脫氯反應 20
1.4.1.4真菌之木質素降解 21
1.5 Pseudomonas mendocina NSYSU 25
1.5.1 Pseudomonas mendocina NSYSU生化測試結果 26
1.5.2 Pseudomonas mendocina NSYSU 於本實驗室相關研究 27
1.6 研究目的 31
第二章 材料與方法 32
2.1 實驗架構 32
2.2大量生產菌液之培養基研發 34
2.3 實驗過程 35
2.3.1 菌液培養與放大 35
2.3.2 取樣方法與菌液補充 36
2.4 以16S rDNA 分析技術監測模槽菌相變化 36
2.4.1 土壤genomic DNA萃取 36
2.4.2 聚合酶連鎖反應PCR 38
2.4.3 變性梯度膠體設備組裝 39
2.4.4.變性梯度膠體配置 39
2.4.5變性梯度膠體清洗及電泳條件 40
第三章 結果與討論 41
3.1 模槽培養試驗與分生技術分析 41
3.1.1 模槽生菌數計數 41
3.1.2 模槽菌相分析 41
3.1.3 小結 42
3.2 各槽槽體實驗結果 42
3.2.1 槽體A與槽體B降解實驗數據變化 42
3-2-1-1 槽體A之戴奧辛數據濃度變化 42
3-2-1-2 槽體B之戴奧辛數據濃度變化 43
3-2-2 環境因子對戴奧辛降解之影響 43
3.2.3 小結 44
3.3 實場菌種培養基之測試與研發 45
第四章 結論與建議 47
參考文獻 48
圖附錄 65
表附錄 70

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