摘要

利用厭氧消化處理有機固體廢棄物，在近年來相當受到關注，這項廢棄物處理技術，能透過微生物生物代謝途徑，產生能源並作為電力和熱能使用。然而在都市體廢棄物中含有多元且複雜之毒性有害物質將影響都市固體廢棄物厭氧消化槽之操作效能與參數，因此研究都市固體廢棄物厭氧消化之影響因子，顯得格外重要。由於雙酚A (Bisphenol A, BPA)為環境荷爾蒙之一，使用範圍廣泛且全球釋放量已達46萬5千磅之多，且在垃圾掩埋場滲岀液中常被檢測出來，來源為塑膠垃圾之溶出，由此得知，BPA已成為目前都市固體廢棄物中主要污染物來源之一，因此本研究擬對BPA對都市固體廢棄物厭氧消化的影響進行探討。
研究分為三大主軸，探討含不同濃度BPA之有機廢棄物對厭氧消化之影響，與改變固體物停留時間(Solid retention time, SRT)，對BPA有機廢棄物對厭氧消化之影響，與利用PCR-DGGE鑑定厭氧消化系統中，菌相組成及親緣關係，並尋找可適應或降解BPA之菌群。厭氧反應槽工作體積為4公升，SRT分別為13.3天、20天、40天，BPA濃度分別為10 mg/L、100 mg/L、500 mg/L，進出量為厭氧反應槽工作體積/固體物停留時間(4公升/20天=0.2公升/天；4公升/13.3天=0.3公升/天；4公升/10天=0.1公升/天)，取出之消化基質作為參數分析使用。
研究結果顯示，在SRT20的操作條件，有機廢棄物厭氧消化槽受到BPA的影響，各濃度的反應槽全面產生抑制現象，在持續操作至第126天後，BPA 10 mg/L組和BPA 100 mg/L組，微生物已完全馴化為適應BPA之菌群，促進了沼氣產率，約1~ 2.46倍，而在BPA 500 mg/L組，呈現完全抑制。SRT調整為 13.3天後，BPA 10 mg/L組和BPA 100 mg/L組，產能有下降趨勢，約促進0.4~ 0.7倍之沼氣產率，當SRT調整為40天後，受到SRT13.3時有機物濃度累積的影響，微生物無法負荷，造成活性的衰退。BPA分析結果顯示，液相中BPA濃度在13.25~20.65 mg/L時，不會改變微生物菌群結構，而是適應了其生長環境，並有提升都市固體廢棄物厭氧消化槽的沼氣產量之潛力，然而BPA濃度在95.36 mg/L以上時，則不利於都市固體廢棄物進行厭氧消化。PCR-DGGE分析結果顯示，Clostridium sp.扮演了分解纖維素的角色，兼性厭氧菌如Sporobacterium olearium、Klebsiella pneumoniae和Leuconostoc mesenteroides，則可將環境中殘餘的氧氣消耗掉，以利於絕對厭氧菌的生長，產甲烷菌群以Methanosarcina sp.、Methanosarcina barkeri和Methanomicrobiales archaeon為主。

Abstract

Anaerobic digestion treatment for organic solid waste has been increasingly used in recent years. This treatment technology can generate energy through microbial metabolic pathway. Municipal solid waste (MSW) contains complicated substance with potential toxicity and harm that might affect the MSW anaerobic digestion. Thus, understanding the affecting factors and potential harmful substance for MSW anaerobic digestion is important.
Bisphenol A (BPA) is one of the widely used endocrine disrupting chemicals (EDC). The global release amount has already found to be four hundred and sixty five thousand pounds. It has been detected very often in leachate of sanitary landfill. Recently, BPA has been found at MSW that might contain used polycarbonate. Release of BPA from MSW might be detrimental to the human health and the ecological environment. Thus, reducing the potential harm of BPA via MSW anaerobic digestion is the objective of this study.
There are three major themes in this study, investigating the effect f anaerobic digestion from various concentrations of BPA containing organic waste, changing solid retention time (SRT) to understand its effect on anaerobic digestion from BPA containing organic waste, and making use of PCR-DGGE to identify microbial community and their relationship to the anaerobic digestion.
The working volume of anaerobic reactor was 4 liters, SRT were 13.3, 20 and 40 days respectively. BPA concentrations were 10, 100, and 500 mg/L respectively. The input and output amount of substrates were 0.3, 0.2 and 0.1 L/d respectively (4 L/20 d= 0.2 L/d; 4 L/13.3 d= 0.3 L/d; 4 L/10 d= 0.1 L/d). The digestate taken out was used for parameter analysis.
Results showed, under SRT20 operation condition, anaerobic digestion of organic waste has been affected by BPA. The reactors with different concentrations were found to be inhibited in the beginning. However, BPA 10 mg/L and BPA 100 mg/L added bioreactors were found to acclimate and adapt BPA levels and the biogas production was enhanced to be around 1 ~ 2.46 times. However, BPA 500 m/L added bioreactors were found to be inhibited completely.
As the SRT operated at 13.3 days, BPA 10 mg/L and BPA 50 mg/L added bioreactors were found to produce about 0.4 ~ 0.7 times of biogas accumulation compared to SRT20. As SRT was changed to be 40 days, the biogas production was decreased increasingly due to the potential shortage of substrate supply.
BPA analysis result showed, when BPA concentration was at 13.25~20.65 mg/L in liquid phase, it will not change microbial structure. However, it could adapt to its growing environment and has the potential to improve biogas production for MSW anaerobic digestion. As BPA concentration increased more than 95.36 mg/L, it was detrimental to MSW anaerobic digestion. PCR-DGGE analysis result showed that Clostridium sp. payed s a role in dissolving cellulose, facultative anaerobe such as Sporobacterium olearium, Klebsiella pneumonia and Leuconostoc mesenteroides could consume residual oxygen in environment that was beneficial for the growth of obligate anaerobic bacteria. Methanogenic bacteria were found to be the major species of Methanosarcina sp., Methanosarcina barkeri and Methanomicrobiales archaeon.

摘要 I
ABSTRACT III
誌謝 V
總目錄 VI
圖目錄 XI
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 4
2.1 有機廢棄物厭氧消化 4
2.1.1 厭氧消化機制 6
2.1.2 厭氧消化影響因子 7
2.2 環境荷爾蒙(Environmental hormones, EHs) 11
2.3雙酚A (Bisphenol A, BPA) 14
2.3.1雙酚A之環境流布 15
2.3.2雙酚A處理技術應用 17
2.4 分子生物技術應用 18
2.4.1 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 18
2.4.2 變性梯度明膠電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 19
第三章 實驗材料與方法 21
3.1 雙酚A有機廢棄物厭氧消化系統 21
3.1.1植種微生物馴養 23
3.1.2合成有機廢棄物基質 24
3.1.3雙酚A(Bisphenol A, BPA) 25
3.1.4分析項目 25
3.1.4.1產氣量 26
3.1.4.2酸鹼度( pH) 26
3.1.4.3 氧化還原電位 (ORP) 26
3.1.4.4 導電度(EC) 26
3.1.4.5鹼度揮發酸 26
3.1.4.6 化學需氧量(COD)分析 27
3.1.4.7總固體物(TS)/發揮性固體物(VS) 28
3.1.4.8金屬成分分析 28
3.1.4.9 BPA液相萃取程序 29
3.1.4.10 BPA固相萃取程序 30
3.1.4.11 HPLC-BPA之分析方法 30
3.1.5藥品及儀器設備 31
3.2 微生物分析鑑定 32
3.2.1污泥前處理 32
3.2.2 DNA萃取 33
3.2.3微生物PCR分析 34
3.2.4微生物DGGE分析 35
3.2.5 親緣分析(Phylogenetic analyses) 36
3.2.6 藥品及儀器設備 38
第四章 結果與討論 40
4.1有機廢棄物厭氧消化槽最佳化操作條件評估 40
4.1.1 不同有機負荷率對產氣效果之影響 40
4.1.2 不同SRT對產氣效果之影響 41
4.2 雙酚A對厭氧消化之影響 44
4.2.1 沼氣產量之促進和抑制 44
4.2.2 厭氧化學參數分析 47
4.2.2.1 酸鹼值 (pH) 47
4.2.2.2導電度 (Electric conductivity, EC) 49
4.2.2.3 氧化還原電位 (Oxidation-Reduction Potential, ORP) 49
4.2.2.4 化學需氧量 (Chemical Oxygen Demand, COD) 52
4.2.3 反應槽金屬濃度分析 59
4.2.4 反應槽中雙酚A分佈之情形及濃度分析結果 64
4.3 雙酚A對厭氧反應槽之菌相變化分析 66
4.3.1 DNA萃取 66
4.3.2聚合酶鏈鎖反應結果 66
4.3.3變性梯度明膠電泳結果 69
4.3.4 親緣樹分析結果 72
第五章 結論與建議 75
5.1 結論 75
5.2 建議 76
參考文獻 77
附錄 89
附錄-A 89
附錄-B 92

表目錄
表 2-1 國際能源總署所列各國有機廢棄物厭氧消化廠數量 5
表 2-2 國際能源總署所列有機廢棄物厭氧消化廠類別與數量 5
表 2-3 微生物之營養需求 9
表 2-4 生物所需微量元素及其機能 10
表 2-5 世界各國雙酚A環境流布 16
表 3-1 雙酚A有機廢棄物厭氧消化系統操作條件 21
表 3-2 植種污泥基本特性分析 23
表 3-3 馴化後污泥基本特性分析 23
表 3-4 垃圾化學組成表 24
表 3-5 雙酚A基本物化特性 25
表 3-6各類厭氧數及水質分析項目方法 25
表 3-7HPLC-BPA之設定條件 31
表 3-8雙酚A有機廢棄物厭氧消化系統實驗藥品 31
表 3-9雙酚A有機廢棄物厭氧消化系統儀器設備 32
表 3-10 PCR檢測使用之引子對與其序列 34
表 3-11所選用之引子對PCR操作條件 35
表 3-12 變性梯度膠所使用之各化學物質 36
表 3-13 微生物分析鑑定實驗藥品 38
表 3-14 微生物分析鑑定實驗儀器設備 39
表 4-1四組厭氧反應槽在SRT 20天條件下COD去除效能 52
表 4-2四組厭氧反應槽在SRT 13.3天條件下COD去除效能 53
表 4-3四組厭氧反應槽在SRT 40天條件下COD去除效能 54
表 4-4四組厭氧反應槽VS/TS比較表 57
表 4-5 反應槽中微生物的作用特性 74
附表-1 DGGE亮帶定序後之基因序列 89

圖目錄
圖 2-1 厭氧消化程序的生物代謝途徑 6
圖 2-2 環境荷爾蒙對正常內分泌系統之影響 12
圖 2-3 正常荷爾蒙和部分仿雌激素之化學結構 13
圖 2-4 雙酚A化學結構 14
圖 2-5 聚合酵素鏈鎖反應之過程示意圖 19
圖 3-1 雙酚A有機廢棄物厭氧消化系統實際模廠 22
圖 3-2 雙酚A有機廢棄物厭氧消化系統示意圖 22
圖 3-3 合成有機廢棄物基質 24
圖 3-4 BPA液相萃取法萃取步驟流程圖 29
圖 3-5 BPA固相萃取法萃取步驟流程圖 30
圖 4-1 厭氧微生物馴養階段及不同TS濃度之沼氣產量趨勢圖 42
圖 4-2 不同SRT條件下厭氧微生物之沼氣產量趨勢圖 43
圖 4-3 不同SRT條件下雙酚A厭氧消化之沼氣產量趨勢圖 46
圖 4-4 四組厭氧反應槽之酸鹼值統計箱型圖 48
圖 4-5 四組厭氧反應槽之導電度統計箱型圖 50
圖 4-6 四組厭氧反應槽之氧化還原電位統計箱型圖 51
圖 4-7 SRT改變對不同濃度之雙酚A厭氧消化槽COD的變化 55
圖 4-8 SRT改變對不同濃度之雙酚A厭氧消化槽COD去除率趨勢圖 56
圖 4-9 四組厭氧反應槽之總固體物及揮發性固體物統計箱型圖 58
圖 4-10 四組厭氧反應槽之總固體物及揮發性固體物統計箱型圖 59
圖 4-11 控制組厭氧反應槽之金屬成分統計箱型圖 60
圖 4-12 BPA 10mg/L厭氧反應槽之金屬成分統計箱型圖 61
圖 4-13 BPA 100mg/L厭氧反應槽之金屬成分統計箱型圖 62
圖 4-14 BPA 500mg/L厭氧反應槽之金屬成分統計箱型圖 63
圖 4-15 各厭氧反應槽固相中雙分A濃度趨勢圖 65
圖4-16 各厭氧反應槽液相中雙分A濃度趨勢圖 65
圖4-17 PCR產物之瓊脂膠糖明膠電泳圖 67
圖4-18 Cycle降為26後PCR產物之瓊脂膠糖明膠電泳圖 68
圖4-19 古細菌PCR產物之瓊脂膠糖明膠電泳圖 69
圖4-20 厭氧反應槽之古細菌菌群結構DGGE分析圖 70
圖4-21 厭氧反應槽之細菌菌群結構DGGE分析圖 71
圖4-21 厭氧反應槽之親緣樹分析圖 73
附圖-1 HPLC-UV分析之BPA檢量線 92

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