臺灣博碩士論文加值系統

本研究在描述單醣類基質（葡萄糖）混合蛋白腖與聚醣類（澱粉）混合蛋白腖基質之人工配置基質在批次實驗中產氫機之異同。本研究同時也探討到澱粉與蛋白腖連續流產氫之功能評估。最後利用廚餘產氫之可行性分析延伸本研究至應用性基質的產氫探討。

　　利用批次實驗對葡萄糖混合蛋白腖基質進行批次實驗之產氫機制探討。發現丁酸為主要之發酵副產物，產量為156 mg Hbu/ g CODa。在批次實驗中所得之氫氣產率為122.41 mL H2 /gVSS-hr。在利用蛋白腖為單一基質時，則發現有耗氫之現象，且主要之發酵副產物為乙酸。
利用尾端修飾限制片段長度多形性之方法對一進流葡萄糖與蛋白腖之連續產氫系統進行產氫菌微生物結構分析，發現在大部份試程中主要之產氫菌皆為Clostridium Cluster I、 Cluster II、Cluster III為最主要的菌群。此外，在部份試程中有發現Desulfotomaculum – like的菌群出現。
利用葡萄糖與蛋白腖基質探討產氫菌對鹽份的影響，發現在外加氯化鈉濃度為10 g/L時，有最大產氫量及產氫速率。

　　利用批次實驗對聚醣類混合蛋白腖基質進行探討，發現有耗氫現象之發生，在揮發酸表現方面，以丙酸為最大量之揮發酸，產量為99.37 mg HPr/ g CODa；氫氣產量為39 mL H2/g CODa，最大產氫速率為25mL H2 /gVSS-hr。在水解酵素的測定方面，產氫菌之澱粉水解酵素大部份是屬於Cell bound的型式，且與生長成正相關。藉由還原醣的分析結果，可以概略的描述產氫菌進行水解時之作用情形。
　　在本研究中亦操作一CSTR反應槽進行以估計在不同水力停留時間（HRT）下連續厭氧產氫發酵之功能評估。操作的結果發現產氫的結果跟水力停留時間有其密切的關係。在水力停留時間長於9小時下，即使澱粉與蛋白腖被降解的較完全，但整體系統觀察到並無淨產氫生成。在水力停留時間操作在3小時，觀測之產氫速率為435 mmol H2/L/day，在這試程中之產氫量明顯大於其他試程。以尾端修飾限制片段長度多形性方法分析不同試程中之菌群結構，發現以澱粉混合蛋白腖之反應槽內有相當繁複的生物多樣性，在水力停留時間為最短（3小時）之試程反而觀察到更多樣性之生物族群。此外，在大部份的試程中皆以Clostridium Cluster I、 Cluster II、Cluster III為最主要的菌群。使用掃描式電子顯微鏡做菌相觀察，可以發現在大部份試程中皆以似Clostridium菌屬之桿菌菌相為主。
　　
在本研究中也進行廚餘高溫發酵之可行性探討。在此取一廚餘發酵甲烷槽之污泥為植種來源，以破碎後之素食便當做為基質，進行高溫氫發酵批次實驗。發現可以得到良好的氫氣產量與氫氣產率。同時也藉由尾端修飾限制片段長度多形性的方法，發現在廚餘甲烷槽中也含有Clostridium Cluster I、 Cluster II、Cluster III等近似產氫菌的菌群。

This study is to describe the different hydrogen fermentation mechanism between monosaccharide (glucose) mixed with peptone and polysaccharide (starch) mixed with peptone, as artificial multiple substrates, in the batch test. This study also covered the performance evaluation of continuous hydrogen fermentation system feed with starch and peptone. Finally extended to the feasibility study of apply substrate-food waste.

The first part studied glucose and peptone fermentation mechanism by batch test. After fermentation, butyrate is the major by-product in the bulk solution, and the yield is 156 mg Hbu/ g CODa. The hydrogen producing rate is 122.41mL H2 /gVSS-hr. Use peptone as sole substrate in batch test, the hydrogen consuming phenomena is observed. The major by-product is acetate.
The molecular method, terminal restriction fragment length polymorpholysium (T-RFLP), was used to investigate the dynamic of microbial ecology of the biohydrogen fermentation process. The TRFLP results indicated that Clostridium clusters I and II presented in the fermentor at all HRT conditions. Desulfotomaculum – like can also be found in some of these operation periods.
The multiple substrate of glucose and peptone is also be used to study the effect of NaCl in hydrogen fermentation. The result shows it comes the maximum hydrogen yield and producing rate when the additional NaCl concentration is 10 g/L.

In this study of starch and peptone fermentation, the hydrogen consuming phenomena is observed in batch test. After fermentation, propionate is the major by-product in the bulk solution, and the yield is 99.37 mg HPr/ g CODa. The hydrogen producing rate is 25mL H2 /gVSS-hr. To detect the amylase activity, it is found that α-amylase characteristics of HPBs is more cell bound type and growth associated. By the data of reducing sugar analysis, the hydrolysis mechanism of HPBs can be generally described.
The performance of the anaerobic hydrogen fermentation process was evaluated at different hydraulic retention times (HRTs) using a continuous stirred tank reactor (CSTR) type fermentor fed with starch and peptone. The results of anaerobic fermentor operations indicated that hydrogen production performance were strongly dependent on HRTs. At HRTs longer than 9 hours, no net hydrogen production was observed in the fermentor although enormous amount of starch and peptone was consumed. At HRT of 3 hours, the CSTR had a hydrogen production rate of 435 mmol/L/d which was significantly higher than those observed at other HRT conditions. The TRFLP results indicated that Clostridium clusters I and II presented in the fermentor at all HRT conditions and were, presumably, responsible for hydrogen production from starch fermentation. The SEM observation, discovered the microbial morphology is rod-like shape and supposed to be Clostridium-like microorganism.

This study include the feasibility of food waste thermophilic hydrogen fermentation. The sludge from the food waste methanogenesis tank is taken as the inoculation, and the content of the vegetarian lunch box is mashed as the substrate. it gets great hydrogen yield and great hydrogen producing rate. By T-RFLP method, it can find Clostridium cluster I, cluster II, and cluster III, which is similarity to hydrogen producing bacterium.

摘要
Abstract
致謝

第一章　前言．．．．．．．．．．．．．．．．．．．．．．．．．．． 1

第二章　文獻回顧．．．．．．．．．．．．．．．．．．．．．．．．．． 3
　　2-1再生資源能源化之趨勢及重要性．．．．．．．．．．．．．．． 3
　　2-2厭氧消化機制及應用．．．．．．．．．．．．．．．．．．．． 7
　　2-3厭氧產氫機制及應用．．．．．．．．．．．．．．．．．．．． 8
2-3-1碳水化合物厭氧代謝發酵機制．．．．．．．．．．．．．． 9
3-3-2含氮物質厭氧發酵代謝途徑．．．．．．．．．．．．． 12
2-4.厭氧產氫微生物．．．．．．．．．．．．．．．．．．．．．． 16
　　　2-4-1產氫生物之種類．．．．．．．．．．．．．．．．．．．． 16
　　　2-4-2厭氧發酵產氫微生物．．．．．．．．．．．．．．．．．． 17
2-5分子生物方法應用於氫發酵程序微生物生態之研究．．． 18
　　2-6 厭氧產氫反應槽之設計．．．．．．．．．．．．．．．． 24
2-6-1發酵槽分類．．．．．．．．．．．．．．．．．．．． 24
　　　2-6-2 CSTR產氫槽的動力參數求解．．．．．．．．．．．．．． 24
　　　2-6-2中溫厭氧生物氫發酵槽雙基質動力模式．．．．．．．． 26
　　　2-6-3應用薄膜反應槽於生物產氫發酵．．．．．．．．．．． 29
　　　2-6-4 UASB產氫反應槽．．．．．．．．．．．．．．．．．．． 29
　　　2-6-5擔體誘發式顆粒污泥床．．．．．．．．．．．．． 30
2-6-6 各類厭氧產氫發酵槽之功能比較．．．．．．．．．． 31
　　2-7.澱粉水解酵素動力及環境因子影響．．．．．．．．．．． 33
2-7-1 澱粉結構．．．．．．．．．．．．．．．．．．．． 33
2-7-2 澱粉水解酵素．．．．．．．．．．．．．．．．．． 34
2-7-3酵素動力學．．．．．．．．．．．．．．．．．．．． 36
2-7-4 酵素可逆之抑制型態．．．．．．．．．．．．．．． 37
2-7-5環境因子於水解酵素之影響．．．．．．．．．．．．． 39
2-8 生物產氫程序之應用．．．．．．．．．．．．．．．．． 41
2-8-1厭氧發酵程序結合光合反應程序．．．．．．．．．．． 41
2-8-2一般厭氧氫發酵程序．．．．．．．．．．．．．．．． 42
2-8-3 薄膜程序組合與厭氧氫發酵程序．．．．．．．．．． 42
2-8-4水解與產氫程序結合．．．．．．．．．．．．．．．． 43

第三章　研究方法與設備．．．．．．．．．．．．．．． 45
3-1.中溫厭氧生物氫發酵槽．．．．．．．．．．．．．．． 45
3-1-1 2.5公升連續流攪拌反應器(Continuous-Flow Stirred Tank Reactor, CSTR, 2.5L)．．．．．．．．．．．．．．．．．．．
45
3-1-2 25公升連續流攪拌反應器(Continuous-Flow Stirred Tank Reactor, CSTR, 25L)．．．．．．．．．．．．．．．．．．．．
45
　　3-2 水質分析項目與使用儀器．．．．．．．．．．．．．．．．．．．． 46
　　　3-2-1 一般水質分析項目．．．．．．．．．．．．．．．．．．．　 46
　　　3-2-2 儀器分析．．．．．．．．．．．．．．．．．．．．．．． 48
3-3 生化氫氣產能試驗（Biochemical Hydrogen Potential test, BHP test）．．．．．．．．．．．．．．．．．．．．．．．．
51
3-4 生物活性量測數據整理方式．．．．．．．．．．．．．． 52
　　3-5 掃描式電子顯微鏡．．．．．．．．．．．．．．．．．．．． 53
3-6 分子生物檢測技術．．．．．．．．．．．．．．．．．．．．．． 54
3-6-1總DNA 萃取．．．．．．．．．．．．．．．．．．．． 54
3-6-2聚合酵素連鎖反應．．．．．．．．．．．．．．．．．． 57
3-6-3尾端修飾限制片段長度多形性(T-RFLP)．．．．．．． 58
3-7 水解酵素分析．．．．．．．．．．．．．．．．．．．．．．． 61
3-8 生物電化學方法．．．．．．．．．．．．．．．．．．．．．． 62

第四章 葡萄糖與蛋白腖基質產氫程序之研究．．．．． 65
　　4-1葡萄糖與蛋白腖基質產氫發酵機制之探討．．．．．．．．．．． 65
4-1-1 葡萄糖與蛋白腖複合基質於批次氫發酵機制探討．．．．．．．．．．．．．．．．．．．．．．．．．
65
4-1-2 蛋白腖單一基質於批次氫發酵機制探討．．．．．．． 67
　　4-2以T-RFLP探討葡萄糖混合蛋白腖基質產氫反應槽菌群結構的變動．．．．．．．．．．．．．．．．．．．．．．．
70
　　4-4氧氫發酵程序中NaCl之影響．．．．．．．．．．．．．．． 74

第五章　澱粉與蛋白腖基質產氫程序之研究．．．．．．．．． 79
　　5-1澱粉與葡萄糖混合蛋白腖基質產氫發酵機制之比較．．．．．．．．．．．．．．．．．．．．．．． 79
　　5-2α型水解酵素活性分析與水解機制探討．．．．．．．．．． 84
5-3中溫CSTR進流澱粉及蛋白腖之生物產氫發酵功能評估．．．．．．．．．．．．．．．．．．．．．．．．．．．．
88
5-3-1 不同進流濃度下之產氫發酵槽之功能比較．．．．．． 91
5-3-2 不同體積負荷下之產氫發酵槽之功能比較．．．．．． 95
5-3-3以T-RFLP探討澱粉混合蛋白腖基質產氫反應槽菌群結構的變動．．．．．．．．．．．．．．．．．．．．．．．
102
　 　5-3-4中溫厭氧生物氫發酵槽菌相觀察．．．．．．．．．． 104

第六章　廚餘產氫可行性研究．．．．．．．．．．．．．．．．． 107
　　6-1廚餘高溫厭氧產氫可行性分析．．．．．．．．．．．．． 107
　　6-2以T-RFLP分析廚餘消化槽中之產氫族群分布．．．．．．． 110
6-3 廚餘產氫之應用性及可行性討論．．．．．．．．．．．． 111

第七章　結論與建議．．．．．．．．．．．．．．．．．．．．．．．．． 113

參考文獻．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 117
自述．．．．．．．．．．．．．．．．．．．．．．．．．． 125

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