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系統識別號 U0026-0812200911392917
論文名稱(中文) 單醣與聚醣類混合蛋白腖基質於厭氧產氫程序分解機制之比較研究
論文名稱(英文) Comparative Study on Fermentation Mechanism of Glucose or Starch with Peptone in Anaerobic Hydrogenation Process
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 93
學期 2
出版年 94
研究生(中文) 李學霖
學號 P5692122
學位類別 碩士
語文別 中文
口試日期 2005-06-03
論文頁數 126頁
口試委員 口試委員-李季眉
指導教授-鄭幸雄
口試委員-林秋裕
口試委員-曾怡禎
口試委員-黃良銘
關鍵字(中) 鹽份
澱粉水解酵素
CSTR
水力停留時間
尾端修飾限制片段長度多形性
廚餘
葡萄糖
澱粉
蛋白腖
關鍵字(英) Peptone
Starch
NaCl
Amylase
Glucose
T-RFLP
Food waste
Hydraulic retention time
CSTR
學科別分類
中文摘要 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。

英文摘要 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.

論文目次 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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系統識別號 U0026-0812200911555726
論文名稱(中文) 探討厭氧產氫純菌Clostridium在不同pH下之反應動力機制
論文名稱(英文) Metabolic study of Clostridium species for biological hydrogen production under different pH conditions.
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 94
學期 2
出版年 95
研究生(中文) 任維傑
學號 p5693101
學位類別 碩士
語文別 中文
口試日期 2006-06-22
論文頁數 117頁
口試委員 指導教授-黃良銘
口試委員-曾怡禎
口試委員-張嘉修
口試委員-鄭幸雄
關鍵字(中) pH
Clostridium
數學模式模擬
glucose
生物產氫
關鍵字(英) biological hydrogen production
glucose
model
Clostridium
pH
學科別分類
中文摘要   隨著文明的發展及人口的成長,人類對於新興乾淨能源的需求將更為迫切。而因應化石能源的短缺及環境污染的日益嚴重,開發新型替代能源為目前各國政府所積極推行,永續且潔淨的再生能源亦是關注焦點。再生能源中,氫氣具有高能量及不會造成二次污染的特性,且利用厭氧微生物產生氫氣除了是清潔能源的生產外亦具有資源再利用的優勢,極具發展潛力。

在厭氧生物產氫程序中,由於大量的酸生成將形成一個低pH的環境,而造成hydrogenase的活性下降或微生物代謝途徑的改變。因此利用clostridia為厭氧產氫程序的主要微生物時,pH的控制成為一個重要的控制因子。近來許多研究報告指出Clostridium屬為厭氧發酵產氫系統中之主要優勢菌種,本研究利用由厭氧發酵產氫系統中分離出之純菌Clostridium butyricum、Clostridium tyrobutyricum及Clostridium beijerinckii進行生物氫氣產能試驗(BHP test),以10,000 mg/L glucose和peptone(3:2 (w/w))作為基質,溫度控制於35℃,且在pH控制為7、6、5和不控制pH狀況下探討其產氫特性。

在不控制pH的狀況之下,Clostridium tyrobutyricum有最佳之產氫量,產氫yield約為1.8 mmol /mmol glucose;在有控制pH的狀況之下,三株純菌皆在pH=6時有最佳的產氫量,其中以Clostridium beijerinckii和Clostridium butyricum產氫yield為1.7 mmol/mmol glucose最佳。此外,並利用數學模式模擬建立Clostridium菌種於批次實驗中降解葡萄糖之動力模式。此模式可正確描述葡萄糖降解、生物生長、乙酸、丁酸、氫氣及其他產物之生成趨勢。

在大部分的pH狀況下,Clostridium菌種降解葡萄糖產生揮發酸,其中以乙酸和丁酸為主要產物,但隨著pH的改變,微生物代謝途徑有移轉的現象,在pH控制為7的批次實驗中,觀察到揮發酸產物種類較多,包括乳酸、甲酸、乙酸、丙酸及丁酸,然而對於產氫量亦有一定的影響。



英文摘要   Due to the development of civilization, increasing population, and shortage of petrochemical energy in the near future, there is an urgent need to develope sustainable energy. Hydrogen is considered a clean and efficient energy among renewable resources, producing water as its only by-product when it burns. Moreover, hydrogen can be produced from renewable raw materials such as organic wastes by anaerobic microorganism. Therefore, hydrogen is a potential clean energy substitute for fossil fuels.

In anaerobic biological hydrogen production, a large amount of volatile fatty acids creates a low pH environment which causes the decrease of hydrogenase activity or alteration of metabolic pathways. Therefore, when using clostridia as the main microorganism in anaerobic hydrogen production process, the control of pH will be an important factor in overall hydrogen fermentation. Recent reports pointed out that Clostridium species were the dominant microorganisms in anaerobic hydrogen fermentation processes. The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism and hydrogen production performance of various Clostridium species, including Clostridium butyricum, Clostridium tyrobutyricum and Clostridium beijerinckii isolated from hydrogen fermentation processes. The reactor batch experiments were operated at 35℃ with 10,000 mg/L glucose, peptone (3:2(w/w)) as substrate.The pH value were controlled at 7, 6, 5 and uncontrolled condition to investigate hydrogen production performance of Clostridium species.

Among the strains examined, Clostridium tyrobutyricum had the highest hydrogen production yield of 1.8 mmol/mmol glucose at uncontrolled pH condition;Clostridium butyricum and Clostridium beijerinckii had the highest hydrogen production yield of 1.7 mmol/mmol glucose when pH were controlled at 6. A kinetic model was developed to evaluate the metabolism of glucose fermentation of the three Clostridium species in the batch reactors. The model, in general, was able to accurately describe the profile of glucose degradation as well as the production of biomass, acetate, butyrate, hydrogen and other products observed in the batch tests.

Almost in all batch tests, Clostridium species degraded glucose to produce acetate and butyrate as the major volatile fatty acids. But the metabolism pathway shifted with the variation of pH. When pH was controlled at 7, many kinds of volatile fatty acid products were observed, including lactate, formate, acetate, propionate and butyrate. However, it would make some influences on hydrogen production performance.



論文目次 考試合格證明....................................I
中文摘要.......................................II
英文摘要.......................................IV
誌謝...........................................VI
目錄.........................................VIII
表目錄.........................................XI
圖目錄.......................................XIII

第一章 前言....................................1

第二章 文獻回顧................................3
2-1 厭氧產氫微生物............................3
2-1-1 產氫生物之種類............................3
2-1-2 厭氧發酵產氫微生物........................7
2-2 厭氧生物產氫之機制.......................10
2-2-1 碳水化合物厭氧發酵產氫機制...............10
2-2-2 蛋白質厭氧發酵產氫機制...................17
2-2-3 Clostridium屬的hydrogenase...............18
2-3 厭氧微生物產氫的影響因子.................22

第三章 實驗方法與設備.........................29
3-1 厭氧生物氫氣產能試驗.....................29
3-1-1 基質儲備液...............................29
3-1-2 厭氧純菌營養鹽之製備.....................29
3-1-3 植種純菌製備.............................30
3-1-4 連續監測之批次實驗.......................30
3-2 中溫厭氧生物氫反應槽.....................35
3-3 水質分析項目與使用儀器...................36
3-3-1 一般水質分析項目.........................36
3-3-2 碳水化合物...............................36
3-3-3 氣體組成.................................37
3-3-4 揮發酸組成...............................37
3-3-5 醇類分析.................................37
3-4 數學模式模擬.............................38

第四章 結果與討論.............................41
4-1 Clostridium beijerinckii.................41
4-1-1 Uncontrolled pH..........................41
4-1-2 Controlled pH at 7.......................41
4-1-3 Controlled pH at 6.......................44
4-1-4 Controlled pH at 5.......................44
4-1-5 Controlled pH at 4.......................47
4-1-6 各pH狀況下之比較.........................48
4-2 Clostridium tyrobutyricum................52
4-2-1 Uncontrolled pH..........................52
4-2-2 Controlled pH at 7.......................52
4-2-3 Controlled pH at 6.......................55
4-2-4 Controlled pH at 5.......................56
4-2-5 Controlled pH at 4.......................56
4-2-6 各pH狀況下之比較.........................58
4-3 Clostridium butyricum....................63
4-3-1 Uncontrolled pH..........................63
4-3-2 Controlled pH at 7.......................63
4-3-3 Controlled pH at 6.......................66
4-3-4 Controlled pH at 5.......................66
4-3-5 Controlled pH at 4.......................69
4-3-6 各pH狀況下之比較.........................69
4-4 綜合討論.................................73
4-4-1 比較三株純菌在不控制pH的狀況下發酵產物
的差異...................................73
4-4-2 比較三株純菌在有控制pH的狀況下發酵產物
的差異...................................75
4-4-3 本研究與文獻中產氫yield的比較............81
4-4-4 產氫菌ATP與氫氣、生物量之間的關係比較....83
4-4-5 探討基質濃度對產氫的影響.................84
4-5 數學模式模擬.............................89
4-5-1 C. beijerinckii產氫動力參數模擬結果......89
4-5-2 C. tyrobutyricum產氫動力參數模擬結果.....92
4-5-3 C. butyricum產氫動力參數模擬結果.........94
4-5-4 探討三株純菌在各pH狀況下qGmax和Y值
的差異...................................96

第五章 結論與建議.............................99
5-1 結論.....................................99
5-2 建議....................................101

第六章 參考文獻..............................103

自述..........................................117

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------------------------------------------------------------------------ 第 3 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200913394648
論文名稱(中文) 以生質能源程序探討廚餘厭氧氫醱酵之研究
論文名稱(英文) Anaerobic Bio-energy Process Study on Fermentative Hydrogen Production with Kitchen Waste
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 95
學期 2
出版年 96
研究生(中文) 林建勝
學號 p5694132
學位類別 碩士
語文別 中文
口試日期 2007-06-22
論文頁數 182頁
口試委員 口試委員-曾怡禎
口試委員-李季眉
口試委員-黃良銘
指導教授-鄭幸雄
口試委員-郭文健
口試委員-鄭幸雄
關鍵字(中) 生物產氫
厭氧醱酵
尾端修飾限制片段長度多形性
生質能源
廚餘
關鍵字(英) T-RFLP
kitchen waste
hydrogen production
anaerobic fermention
bio-energy
學科別分類
中文摘要   至 2006 年,台灣廚餘的回收率已可達三分之一,日回收量超過 1,500 噸。現行的廚餘回收再利用的方式以養豬(75%)及堆肥(22%)為主,但這兩種回收再利用的方式皆有衛生環境上的隱憂,例如傳染疾病以及二次污染之虞。在另一方面,由於台灣缺乏化石燃料(煤礦、石油、天然氣等)的來源,98% 以上的能源來源必需仰賴進口,更有迫切開發替代性能源之需求。基於環境與能源這兩者的共同考量,我們期望透過發展廚餘厭氧醱酵,利用此對環境較為友善的方法來達到有機廢棄物的減量,並且開發潔淨生質能源的技術平台。
  廚餘是一種高濃度的有機廢棄物,其總 COD 約 82-107 g/L,VSS/SS 為 90% 以上,且具有高濃度的固體物(約 40 g/L)。本研究針對台北及高雄兩市的廚餘分別進行 7 次及 27 次的採樣與特性分析,並估算廚餘的電子分布。油脂及固態的蛋白質佔廚餘電子分布的最大宗,兩者的和超過總電子數的一半,而總碳水化合物所佔的比例則相對較小,北高兩市分別為 7.7% 及 19.3%。揮發酸的分布兩市相去不大,均約為 10% 左右,而揮發酸中,又以乳酸的濃度最高,高雄市廚餘乳酸的濃度可達 10 g/L。
  本研究以一實驗室規模的 3 L 廚餘厭氧氫醱酵槽來進行其廚餘水解、酸化、產氫之程序研究,反應槽的形式定義為「間歇性進流完全攪拌反應槽」(Intermittent - Continuous Stirred Tank Reactor, I-CSTR)。廚餘基質在濃度及組成成分的變動造成其產氫速率上亦有明顯的起伏,在經過連續 300 天的長期操作,於第五試程發現有最高的平均體積產氫速率為 3.36±0.86 L H2/L-day,該試程亦操作在最高的體積荷負:100.5±24.8 g COD/L-day。最高的產氫 yield 及比產氫速率則皆發生在第四試程,分別為 96.4±37.9 mL H2/g VSSin 及 0.11±0.03 L H2/g VSS-day。
  經過 277 天的廚餘氫醱酵微生物馴養,於第四試程將 I-CSTR 反應槽的出流混合液進行不同食微比的廚餘生化產氫潛能批分次試驗,由實驗的結果得知,在初始食微比為 S0/X0 = 9.5 g COD/g VSS 有最高的比產氫速率(specific hydrogen production rate)478 mL H2/g VSS-day,相較於反應槽啟動之際初始植種的污泥活性(150 mL H2/g VSS-day,初始食微比為 10.8 g COD/g VSS),有 3.19 倍的大幅增加。
  以 I-CSTR 廚餘厭氧氫醱酵槽出流混合液進行 16S rRNA 基因選殖(clone library)及 DNA 定序(sequencing)實驗,在挑選的 154 個 clones 中發現 17 個 OTUs(operational taxonomic units)。62% 的菌比對最接近為 Butyrivibrio fibrisolvens(相似度為 92%),另有比對到兩株 Clostridium,分別為 Clostridium aminophilum(相似度為94%)及 Clostridum proteoclasticum(相似度為 91%),兩者均約佔 5%。在 I-CSTR 廚餘厭氧氫醱酵槽 12 次的尾端修飾限制片段長度多樣性(terminal restriction fragment length polymorphism, T-RFLP)圖譜分析中,發現反應槽內的微生物在不同操作條件下,其微生物菌相的組成有極大的變化。利用兩端引子修飾不同螢光染劑的改良式 T-RFLP,可應用於廚餘氫醱酵微生物探討並提高其對不同菌種的解析能力。
英文摘要   In Taiwan, almost one third of population contributed 1,500 tons per day of household kitchen waste that was reused till 2006. Kitchen waste in Taiwan was mainly reused for pig house feeding (75%) and composting (22%), but both of these two ways to treat the kitchen waste would cause some problems, such as infectious disease or secondary pollution. Anaerobic fermentation is one of the most environmentally friendly methods to treat the kitchen waste. In another hand, Taiwan was short of fossil fuel (coal, petroleum, natural gas etc.), and more than 98% of energy source was imported from other countries. Thus anaerobic fermentation might be the better method to treat the kitchen waste for both environment and energy consideration.
  Kitchen waste was a kind of organic waste that contained with highly nutritive composition (total COD was about 82-107 g/L and VSS/SS was great than 90%) and with about 40 g/L high concentration of suspended solid. The electron distribution of kitchen waste was calculated by characteristic analysis for Taipei City and Kaohsiung city (n=7 and 27 respectively). Lipid and solid protein were the most dominant items of all which contained more than 50% of electron while total carbohydrate only about 7.7 to 19.8%. Electron distribution of VFAs (volatile fatty acids) was about 10% which mainly contributed by lactic acid.
  A 3 L bench scale of bio-hydrogenation I-CSTR (Intermittent - Continuous Stirred Tank Reactor) was established to study kitchen waste treatment and bioenergy process with anaerobic fermentation. Large fluctuation of organic loading from kitchen waste attained to the high variation of biogas profile. Within 300 days of continuously long-term operation, the maximum average hydrogen production rate was observed in run 5 up to 3.36±0.86 L H2/L-day with the extremely high volumetric loading rate 100.5±24.8 g COD/L-day. The highest average hydrogen yield and specific hydrogen production rate was observed in run 4 with 96.4±37.9 mL H2/g VSSin and 0.11±0.03 L H2/g VSS-day, respectively.
  After 277-days enrichment of microbial existed in I-CSTR, the bio-activity (specific hydrogen production rate) measured by batch test was increased 3.19 folds with 478 mL H2/g VSS-day where S0/X0 = 9.5 g COD/g VSS than previous study when inoculum (150 mL H2/g VSS-day, 10.8 g COD/g VSS).
  As the result of clone library of 16S rRNA of the anaerobic microbes taken from I-CSRT bio-hydrogen reactor, there were 17 operational taxonomic units (OTUs) in 154 clones. 62% of clones could be identified as belonging to Butyrivibrio fibrisolvens (92% similarity) and both 5% of clones were Clostridium aminophilum (94% similarity) and Clostridum proteoclasticum (91% similarity). Significant population shift was observed through different runs by 12 T-RFLP analyses which total DNA extracted from mixed liquid of effluent in I-CSTR. Modified T-RFLP with both fluorescently labeled forward and reverse primers could improve the differential of bacteria existed in this reactor.
論文目次 考試合格證明 III
誌謝 V
中文摘要 IX
Abstract XI
目錄 XIII
表目錄 XV
圖目錄 XVIII

第一章 前言 1
第二章 文獻回顧 5
2-1. 潔淨再生能源之展望及全球能源使用趨勢 5
2-2. 台灣廚餘清運現況及回收再利用之方法比較 8
2-3. 國際氫能源與氫氣經濟 12
2-4. 厭氧生物產氫技術 17
2-4-1. 影響產氫因素之探討 17
2-4-2. 厭氧氫醱酵之微生物 22
2-4-3. 產氫酵素hydrogenase與厭氧氫醱酵 25
2-4-4. 有機物中主要成份與氫醱酵之探討 26
2-5. 生物氫醱酵反應槽設計 36
2-6. 分子生物技術應用於產氫微生物族群之探討 39
2-7. 廚餘厭氧醱酵之工程化案例 43
第三章 研究材料與方法 51
3-1. I-CSTR 中溫厭氧生物氫醱酵槽 51
3-2. 水質分析項目與使用儀器 53
3-2-1. 一般水質分析項目 53
3-2-2. 儀器分析 53
3-3. 生化氫氣產能試驗及生物活性量測 56
3-3-1. 生化氫氣產能試驗 56
3-3-2. 生物活性量測數據整理方式 56
3-4. 掃描式電子顯微鏡 Scanning Electron Microscope(SEM) 57
3-5. 分子生物檢測技術 58
3-5-1. 總DNA 萃取 58
3-5-2. 聚合酵素連鎖反應(Polymerase Chain Reaction, PCR) 61
3-5-3. 尾端修飾限制片段長度多形性(T-RFLP) 62
3-5-4. 16S rRNA基因選殖實驗(clone library) 64
第四章 結果與討論 67
4-1. 台灣廚餘之特性分析 67
4-2. 廚餘間歇進流完全攪拌氫醱酵槽試程功能之探討 74
4-2-1. 反應槽型式探討 74
4-2-2. 各試程操作參數與功能指標 78
4-2-3. 廚餘中基質成份之降解及代謝產物之生成 84
4-2-4. 廚餘中主要成份影響產氫速率之探討 93
4-2-5. 不同操作條件下間歇性進流之廚餘氫醱酵機制探討 100
4-3. 廚餘醱酵槽微生物之活性及反應動力與機制探討 106
4-3-1. 廚餘氫醱酵槽微生物分解廚餘之生化產氫潛能測試 106
4-3-2. 以飯盒模擬廚餘基質探討生物氫醱酵機制之批次試驗 120
4-4. 微生物族群結構之探討 133
4-4-1. 以掃描式電子顯微鏡觀察厭氧廚餘氫醱酵微生物之菌相 133
4-4-2. 16S rRNA基因選殖實驗(clone library) 137
4-4-3. 分子生物技術 T-RFLP 探討產氫菌族群變化 141
4-4-4. 以核酸序列定序結果改進 T-RFLP 於產氫菌族群探討之應用 146
4-5. 廚餘厭氧醱酵產氫與文獻之比較 150
第五章 結論與建議 153
5-1. 結論 153
5-2. 建議 155
第六章 參考文獻 157

附錄 169
自述 181
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系統識別號 U0026-0812200914131259
論文名稱(中文) 高溫廚餘厭氧氫醱酵程序控制與水解機制之研究
論文名稱(英文) Process Study and Hydrolysis Mechanism Study of Thermophilic Hydrogen Production with Starch-riched Kitchen Waste Fermentation
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 96
學期 2
出版年 97
研究生(中文) 王郁萱
學號 p5695413
學位類別 碩士
語文別 中文
口試日期 2008-06-06
論文頁數 130頁
口試委員 口試委員-曾怡禎
指導教授-鄭幸雄
口試委員-張嘉修
口試委員-黃介辰
口試委員-黃良銘
關鍵字(中) 基因選殖
尾端修飾限制片段長度多形性
厭氧氫醱酵
澱粉類廚餘
澱粉水解酶活性測試
關鍵字(英) Amylase activity assay
T-RFLP
Starch-riched kitchen waste
Clone library
Anaerobic hydrogen fermention
學科別分類
中文摘要 廚餘是一種高濃度的有機廢棄物,其總 COD 約 280-430 g/L,VSS/SS 為 95% 以上,具有高濃度的固體物(約 210 g/L),且固體物COD電子分佈佔33.3%。本研究針對台南市的高澱粉濃度廚餘分別進行 20 次的採樣與特性分析,並估算廚餘的電子分布。固態碳水化合物及溶解態碳水化合物佔廚餘電子分布的最大宗,兩者的和約為總電子數的一半,而總有機氮所佔的比例則相對較小,為 17.3%。揮發酸約為8% 左右,而揮發酸中,又以乳酸的濃度最高,為5 g/L。本研究以一實驗室規模的 3 L 廚餘厭氧氫醱酵槽來進行其高澱粉濃度廚餘水解、酸化、產氫之程序研究,反應槽的形式定義為「間歇性進流完全攪拌反應槽」(Intermittent - Continuous Stirred Tank Reactor, I-CSTR)。在經過連續 250 天的長期操作,於第3-2試程發現有最高的平均體積產氫速率為 2.2 L-H2/L/day,該試程操作在水力停留時間為八天,間歇性進流頻率為24小時一次。最高的產氫 yield 也在第3-2試程,為 2.1 mmole-H2/g-COD;揮發性固體物降解率,在第2試程有最佳表現為47%,此試程操作在水力停留時間四天,間歇性進流頻率為12小時一次。在澱粉水解酶的部分,則是在第1試程有最大活性11 U/mL,而在第3-2試程有最佳固體碳水化合物水解為45%。然而,由時間序列採樣分析實驗,得知各試程中水解酶是足夠將固態碳水化合物完全水解的,且最大理論還原糖生成量為176 g-glucose eq./L/hr;在試程2中可有最大一階懸浮性固體物降解常數,值為0.04 hr-1。由降解米飯的產氫潛能生物活性測試,得知試程2有較佳的比產氫速率為0.15 mmole-H2/g-VSS/hr。由酵素動力模式及水解酶最佳化測試,可得KM值為34 g/L,最大速率(Vmax)為3.6 U/mL,且於溫度為55oC及使用磷酸緩衝溶液在pH 5.5或醋酸緩衝液在pH 4.4有最佳活性。以 I-CSTR 廚餘厭氧氫醱酵槽出流混合液進行 16S rRNA 基因選殖及 DNA 定序實驗,在挑選的 148 個 clones 中發現 27 個 OTUs(operational taxonomic units)。42% 的菌比對最接近為 Thermoanaerobacterium thermosaccharolyticum(相似度為 98%),另有比對到Clostridium sp.(相似度為95%,約佔 24%)以及兩株乳酸菌,分別為Lactobacillus panis (相似度99%, 約佔16%)及Lactobacillus amylovorus(相似度99%, 約佔10%)。其中,Thermoanaerobacterium thermosaccharolytium及Lactobacillus amylovorus被報導為具有分泌澱粉水解酶的能力。在 I-CSTR 廚餘厭氧氫醱酵槽 14 次的尾端修飾限制片段長度多形性(terminal restriction fragment length polymorphism, T-RFLP)圖譜分析中,發現反應槽內的微生物在不同操作條件下,其微生物菌相的組成皆已主要的4 OTUs為主,其中TKW-HPB-2(Thermoanaerobacterium thermosaccharolytium)在三試程皆佔有30%以上,而TKW-HPB-1(Clostridium sp.)則會在試程穩定後呈為優勢菌。
英文摘要 Kitchen waste was a kind of waste that contains highly nutritive organic compounds (total COD was about 280-430 g/L and VSS/SS ratio was higher than 95%). The total solid fraction was about 200 g/L and occupied 33% of total COD. The electron distribution of Tainan City kitchen waste was calculated according to the analyses data (n=20). Solid and soluble carbohydrate were the most dominant items of all which occupied about 50% of electron while total protein only about 17.3%. Electron distribution of VFAs (volatile fatty acids) was about 8% which were mainly contributed by 5 g/L of lactic acid. A 3 L bench scale of bio-hydrogenation I-CSTR (Intermittent - Continuous Stirred Tank Reactor) was established for kitchen waste treatment and bio-energy recovering process by anaerobic operation. Within 250 days of continuously long-term operation, the maximum averaged hydrogen production rate of 2.2 L H2/L-day was observed in run 3-2 with 8-day hydraulic retention time (HRT) and 24-hour intermittent feeding period. The highest average hydrogen yield of 2.1 mmol-H2/g-COD was also observed in run 3-2. However, the best VSS removal of 47% was occurred in run 2 which was operated at 4-day HRT and 12-hour intermittent feeding period. According to the analyses of amylase and reducing sugar, the maximum average amylase activity was about 11 U/mL in run 1, but the maximum solid carbohydrate hydrolysis rate was about 45% in run 3. To clarify the starch hydrolysis mechanism, amylase activity of effluent and time series profile between each operated period were monitored, and the white rice batch test was also studied. According to the results of time series profile analyses, amylase activity was sufficient for starch hydrolysis and the maximum theoretical reducing sugar production of 176 g-glucose eq./L was calculated. According to the results, the maximum first order constant of VSS degradation of 0.04 hr-1 was occurred in run 2. At the white rice batch test, the hydrogen producing bacteria taken from run 2 achieved better hydrogen production rate of 264 mL-H2/hr than the other ones. According to the enzyme kinetic study, the KM was 17 g/L and the maximum amylase activity was 1.5 U/mL. The environmental factors effects of amylase in the supernatant were also studied. The best reacted temperature was found to be 55oC. With phosphate buffer, the best amylase activity achieved when pH was at 5.5; with acetate buffer, the best one was observed at pH = 4.4. As the results of 16S rDNA-based cloning screening of the anaerobic microbes taken from I-CSTR bio-hydrogen reactor, the 27 operational taxonomic units (OTUs) were found in 148 clones. Among these clones, 42% of clones could be identified as Thermoanaerobacterium thermosaccharolyticum (98% similarity) and 24% of clones were identified as Clostridium sp. (95% similarity). The other two dominant clones were identified as Lactobacillus panis (99% similarity, abundance of 16%) and Lactobacillus amylovorus (99% similarity, abundance of 10%). Four dominant OTUs were observed during different runs by 14 times of T-RFLP analyses. Thermoanaerobacterium thermosaccharolyticum always exists in the system and amounted to 30%. However, Clostridium sp. would become dominant when the system was steady operated.
論文目次 第一章 前言 1
第二章 文獻回顧 3
2-1 再生資源能源化及台灣氫經濟發展 3
2-2 台灣廚餘清運現況及回收在利用之方法比較 7
2-3 暗醱酵厭氧產氫因素探討 11
2-4 澱粉水解酶酵素動力及環境因子影響 16
2-4-1 澱粉結構的介紹 17
2-4-2 澱粉水解酵素種類簡介 18
2-4-3  澱粉水解酶活性分析方式 19
2-4-3 酵素動力學 23
2-4-4 環境因子於水解酵素之探討 24
2-4-5  水解與厭氧醱酵程序結合 26
2-5 全球高溫及中溫厭氧氫醱酵研究現況 30
2-5-1  中溫 30
2-5-2 高溫 32
第三章 材料與方法 37
3-1 I-CSTR 高溫厭氧生物氫醱酵槽 37
3-2 水質分析項目與使用儀器 40
3-2-1 水質分析 40
3-2-2 儀器分析 41
3-3 生化氫氣產能試驗及生物活性測量 42
3-4 掃描式電子顯微鏡Scanning Electron Microscope (SEM) 43
3-5 水解酵素分析 (Fazel-Madjlessi et al., 1980) 44
3-6 分子生物檢測技術 45
3-6-1 總DNA萃取 45
3-6-2 聚合酵素連鎖反應 46
3-6-3 尾端修飾限制片段長度多形性 47
3-6-4 16S rDNA 基因選殖實驗 49
第四章 結果與討論 51
4-1 台灣廚餘之生物有機化學成分 51
4-2 高濃度澱粉廚餘間歇進流完全攪拌氫醱酵槽功能之探討 56
4-2-1 各試程操作參數與功能指標 56
4-2-2 厭氧醱酵槽各試程澱粉水解酶活性探討 64
4-2-3 不同操作條件下間歇性進流之廚餘氫醱酵機制探討 67
4-3 澱粉類廚餘醱酵槽微生物之活性及反應動力與機制探討 74
4-4 澱粉類廚餘醱酵槽澱粉水解酶活性最佳化測試及反應動力探討 81
4-4-1 澱粉水解酶反應動力探討 81
4-4-2 澱粉水解酶最佳化測試 84
4-5 微生物族群結構之探討 87
4-5-1 以掃描式電子顯微鏡觀察厭氧廚餘氫醱酵微生物之菌相 87
4-5-2 16S rDNA基因選殖實驗 89
4-5-3 分子生物技術T-RFLP探討產氫菌族群變化 98
4-5-4 分子生物監測結果和試程中功能指標之綜合討論 103
第五章 結論與建議 107
5-1 結論 107
5-2 建議 109
第六章 參考文獻 111

附錄 125
自述 129
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系統識別號 U0026-0812200914171144
論文名稱(中文) 蔬菜廚餘厭氧氫醱酵程序及流體化床醱酵槽改良設計之研究
論文名稱(英文) Anaerobic Bio-energy Process Study on Hydrogen Fermentation with Vegetable Kitchen Waste and Modification of AnFB Fermentor
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 96
學期 2
出版年 97
研究生(中文) 李澤坤
學號 p5695405
學位類別 碩士
語文別 中文
口試日期 2008-06-06
論文頁數 198頁
口試委員 指導教授-鄭幸雄
口試委員-曾怡禎
口試委員-張嘉修
口試委員-黃良銘
口試委員-黃介辰
關鍵字(中) 流體化床
纖維素
高溫厭氧氫醱酵
高溫廚餘堆肥
蔬菜廚餘
關鍵字(英) Thermosphilic anaerobic hydrogen fermentation
Cellulose
Vegetable kitchen waste
Anaerobic fluidized bed reactor
Kitchen waste compost
學科別分類
中文摘要 本研究團隊以工人挑選廚餘中蔬菜類的部分並進行11次的採樣特性分析,蔬菜廚餘主要由碳水化合物 (33%)、脂肪 (32%)、蛋白質 (23%)以及有機揮發酸 (8%)所組成,其中纖維素的重量約佔整體的10%左右。本研究所選用的植種源主要取自台南市城西里的高溫廚餘堆肥,在試程啟動前,進行一套蔬菜廚餘氫醱酵pH最適化試驗,結果顯示食微比S0/X0= 3 於pH 6.0的高溫環境下,有最佳的產氫速率0.57 mmol-H2/g-VSS/hr以及氫氣產率0.48 mmol-H2/g-COD。在反應器操作方面,使用20 L完全攪拌式反應槽以間歇性進流方式進行連續91天的操作,試程期間蔬菜廚餘的特性有很大的變異,使得產氫表現以及系統微生物生態有很大的變化。第二試程在體積荷負為28 g-COD/L/day下,有最高產氫速率約為1.0 L-H2/L/day以及氫氣產率1.7 mmol-H2/g-CODin;第一試程在體積負荷為19 g-COD/L/day下,有機固體物有較好的破壞率為45%,其中固體碳水化合物以及纖維素去除率分別為62、37%。藉由16S rRNA基因選殖與定序的方法建立系統微生物資料庫,發現系統的微生物生態以Thermoanaerobacterium thermosaccharolyticum為主約佔57%左右,在高溫厭氧環境下具有降解纖維素、澱粉等醣類物質進行產氫的能力。試程期間以微生物資料庫作為基礎進行「尾端修飾限制片段長度多形性」分析,達到快速監測系統微生物的變化資訊,試程期間微生物生態的變異很大,在Run 2操作期間發現進流基中有與系統微生物相似的Clostridium菌屬存在。在完全攪拌式反應槽操作經驗的基礎下,本研究亦以流體化床式反應槽進行改良設計,在槽內設置導流管造成流體在管內、外循環攪拌,藉由噴嘴(Jet)裝置混合迴流水和槽頂氣體作為攪拌動力來源。在廻流水置換率25次/小時、氣體迴流量為0.4 L/min時,延散程度可達D/uL =2.3,達到流體均勻分佈的效果,另外在槽頂設置污泥廻流系統將產生的浮渣返送至槽底。將前試程所馴養的污泥植入流體化床反應槽,在25天的試程操作期間受到浮渣堵塞問題導致氣體計量上的問題,造成產氫的功能被低估,在體積負荷為14 g-COD/L/day時其產氫速率以及氫氣產率分別為0.26 L-H2/L/day、0.8 mmol-H2/g-CODin。
英文摘要 The characterisitics analysis for wasted vegetable selected from kitchen waste collected from Taian City by local EPA. According to 11 times of analyses data, the complex composition of vegetable kitchen waste was found. It includes 33% of carbohydrates, 32% of oil and grease, 23% of protein, and 8% volatile fatty acids. The vegetable kitchen wastes which conent 10% of cellulose were considered as the hardly-degradable substrate because of its crystalline-like structure. The purpose of the optimal pH in the hydrogen fermentation fed with vegetable kitchen wasted and seed with kitchen waste compost was proceed at he batch test with pH control system. The maximum hydrogen production rate and hydrogen yield were found at pH = 6.0, and they were achieved to 0.57 mmol-H2/g-VSS/hr and 0.48 mmol-H2/g-COD, respectively. The continuous stirred tank reactor (CSTR) with 20 L working volume was established to study for anaerobic bio-H2 process fed with vegetable kitchen waste. In 91 days of operation, the maximum hydrogen production rate of 1.0 L-H2/L/day and the yield of 1.7 mmol-H2/g-CODin were observed with the volumetric loading rate (VLR) of 28 g-COD/L/day in Run 2. The higher VSS, carbohydrate, and cellulose removal were 45, 62, and 37%, respectively at VLR 19 g-COD/L/day in Run 1. According to the results of 16S rDNA clone library and sequence, the dominant species was Thermoanaerobacterium thermosaccharolyticum, which was considered as an anaerobic thermophilic hydrogen-prodcing bacteria degrading cellulose, starch and so on. Terminal restriction fragment length polymorphism, T-RFLP was established to monitor the microbial culturies in the period of process operation. The dynamics of microbial cultures change dramatically. It would find the similar dominant culture, Clostridium species, in vegetable kitchen waste to those in the reactor. For better mixing effieciency, we modified the fluidized bed reactor by quipping the draft tube and jet. According to residence time distribution analysis, the reactor type was closed to CSTR and tracer recovery was attended to 100%. When the D/uL ratio was on the level of 0.4 L/min, the dispersion effect was up to 2.3 (D/uL) at flow recirculation frequency 25 times per hour. In addition, we equipped the scum recirculation in the top of reactor for removal the cumalitive scum. At start-up bio-H2 ananerobic fluidized bed reactor (AnFB), the enriched slurry from above process was seeded as seeding source. Around 25 days operating period, it would find the hydrogen production rate and yield were 0.26 L-H2/L/day, 0.8 mmol-H2/g-CODin in the volumtirc loading rate 14 g-COD/L/day. Cause of block in the air pipe by scum, the hydrogen performace was discount by uncomplete gas collection.
論文目次 目錄
中文摘要 III
Abstract V
誌謝 VII
表目錄 XIII
圖目錄 XV
第一章 前言 1
第二章 文獻回顧 5
2-1. 全球能源發展趨勢與生物氫能未來之展望 5
2-2 台灣廚餘特性以及回收再利用之現況 7
2-3 纖維素結構及纖維素分解菌 11
2-3-1 纖維素的結構與特性 11
2-3-2 纖維素水解酵素的類型與作用機制 14
2-3-3 纖維素分解菌 16
2-4 厭氧生物產氫技術 25
2-4-1 厭氧醱酵產氫微生物 25
2-4-2 厭氧生物產氫機制探討 27
2-5 厭氧流體化床反應槽 37
2-6 流體停留時間分佈分析(Residence time distribution (RTD) analysis) 41
2-7 分子生物技術應用於產氫微生物族群之探討 45
2-8 台灣廚餘厭氧醱酵程序之發展現況 49
第三章 研究材料與方法 51
3-1 高溫厭氧生物氫醱酵槽 51
3-1-1 間歇性進流完全攪拌反應槽 (I-CSTR) 51
3-1-2 間歇進流厭氧流體化床 (AnFB) 53
3-2 pH自動控制之批次反應器 59
3-3 水質分析項目與使用儀器 61
3-3-1 一般水質分析項目 61
3-3-2 儀器分析 62
3-4 纖維素水解酵素活性分析 65
3-5 生化氫氣產能試驗及生物活性量測 67
3-5-1 生化氫氣產能試驗 67
3-5-2 生物活性量測數據整理方式 67
3-6 掃描式電子顯微鏡 Scanning Electron Microscope(SEM) 69
3-7 分子生物檢測技術 71
3-7-1 總DNA 萃取 71
3-7-2 聚合酵素連鎖反應(Polymerase Chain Reaction, PCR) 73
3-7-3 尾端修飾限制片段長度多形性(T-RFLP) 74
3-7-4 16S rDNA基因選殖實驗(clone library) 75
第四章 結果與討論 79
4-1 台南市蔬菜類廚餘特性分析 79
4-2 蔬菜類廚餘厭氧產氫影響因子篩選 87
4-2-1 多因子設計試驗 87
4-2-2 酸鹼環境(pH levels)的影響 95
4-3 蔬菜類廚餘間歇進流完全攪拌氫醱酵槽試程功能之探討 105
4-3-1 試程操作策略研擬 105
4-3-2 試程操作情形與功能指標之探討 107
4-3-3 試程期間反應物與代謝產物的轉變 112
4-3-4 間歇性進流之蔬菜氫醱酵機制探討 117
4-3-5 以掃描式電子顯微鏡觀察微生物菌相 121
4-3-6 16S rRNA 基因選殖實驗 (clone library) 123
4-3-7 厭氧氫醱酵系統微生物族群變化之探討 128
4-4 高溫厭氧氫醱酵流體化床改良設計與運轉操作之探討 133
4-4-1 流體化床反應槽改良設計之探討 134
4-4-2 流體化床反應槽流力試驗 137
4-4-3 改良型與一般的流體化床流力比較之探討 140
4-4-4 高溫厭氧氫醱酵流體化床試程操作之探討 142
4-5 有機固體廢棄物醱酵產氫研究之比較 151
第五章 結論與建議 154
第六章 參考文獻 157
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系統識別號 U0026-0812200914203048
論文名稱(中文) Clostridium tyrobutyricum 在不同水力停留時間下之代謝表現與產氫行為之研究
論文名稱(英文) Metabolic study and hydrogen production of Clostridium tyrobutyricum under different hydraulic retention time
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 96
學期 2
出版年 97
研究生(中文) 劉怡君
學號 p5695418
學位類別 碩士
語文別 中文
口試日期 2008-07-02
論文頁數 107頁
口試委員 口試委員-曾怡禎
口試委員-鄭幸雄
指導教授-黃良銘
口試委員-張嘉修
關鍵字(中) 水力停留時間
生物產氫
代謝
CSTR
Clostridium tyrobutyricum
關鍵字(英) Clostridium tyrobutyricum
bio-hydrogen
CSTR
hydraulic retention time
metabolism
學科別分類
中文摘要 本論文將 Clostridium tyrobutyricum 純菌植入連續流攪拌反應器 (Continuous-flow stirred tank reactor, CSTR),以 20,000 mg/L 葡萄糖及水解蛋白質 (3:2(w/w)) 作為進流基質,溫度控制於 35℃,所有試程的 pH 皆在 5.9-6.1 間,將反應槽操作在不同水力停留時間 (HRT = 18, 12, 8, 6, 4, 3, 2, 1.5, 1 hr,共 9 個試程),並收集穩定狀態下的氣體及水質數據,本論文的目的為探討 C. tyrobutyricum 在不同水力停留時間下之代謝表現及產氫行為的變化。
槽體內微生物濃度在前 8 個試程約維持在 2700±400 mg/L,直到水力停留時間 1 小時,槽體內微生物出現被洗出 (wash out) 的現象,故判定完成本株菌所有試程。在較低水力停留時間的試程 (HRT = 3, 2, 1.5 hr),微生物出現顆粒化現象,這個現象使得較低水力停留時間的試程微生物濃度有上升的趨勢,且使得出流水葡萄糖濃度可以維持 300 mg/L 以下。
出流水有機氮濃度隨水力停留時間減少而上升,而氨氮濃度隨水力停留時間減少而上升,顯示微生物在較高水力停留時間的試程中,有充足的時間進行反應,有利於水解蛋白質的降解作用;當反應槽操作在較高水力停留時間,水解蛋白質可被利用於發酵作用及進行生物體合成,隨著水力停留時間下降,水解蛋白質的作用主要為生物質體合成,或仍以有機氮的型式存在,只有少部分被代謝至氨氮型式。
C. tyrobutyricum 的產氫速率、比產氫速率與氫氣產率都是在水力停留時間 4 小時的試程出現最大值,分別為 416.61 mmol H2/L/d、8.92 L H2/g-VSS/d 及 3.47 mmol H2/g-CODapplied,而本試程也是實際產氫量最接近理論產氫量的試程;產二氧化碳速率則是在水力停留時間 6 小時的試程出現最大值,約為 292 mmol CO2/L-day。
在水力停留時間較低的試程,產氫速率下降,但微生物濃度並沒有明顯的變化,推測產氫速率下降是由於微生物代謝路徑移轉的關係,在較高水力停留時間的試程,出流水有機酸組成以丁酸及乙酸為主,在較低水力停留時間的試程則轉為乳酸生成為主。文獻指出,LDH 會被高 NADH/NAD+ 而激活,誘使乳酸的生成 (Garrigues et al., 1997),此外,LDH 的活性會被 NAD+ 抑制 (Fitzgerald et al.,1992)。故推測在低水力停留時間的操作下,微生物的比生長速率 (specific growth rate, μ) 提高,所需要的能量較多,使得體內 NADH 被快速產生,另一方面,往丁酸生成的代謝減少,推測是因為 PTB 的活性不佳;以上兩個因素造成微生物細胞內的 NADH 和丙酮酸的累積,使得 LDH 的活性增加,開始進行產乳酸的代謝路徑。
使用 CellNetAnalyzer 做代謝路徑的速率計算後,在 pyruvate 節點分析方面,製造 acetyl-CoA 並伴隨氫氣產生的路徑,隨著水力停留時間的降低而降低;往製造乳酸的路徑,隨著水力停留時間的降低而有很明顯的上升趨勢,判斷應該是 LDH 在低水力停留時間被活化所導致。從 acetyl-CoA 節點分析可以看出,在水力停留時間較高時,C. tyrobutyricum 控制丁酸生成的酵素群 (PTB及BK) 比控制乙酸生成的酵素群 (PTA及AK) 活性還高;當水力停留時間較低時則相反。
英文摘要 The objective of this thesis was to figure out the metabolism of Clostridium tyrobutyricum and evaluate the production of hydrogen in a continuous-flow stirred tank reactor (CSTR) as a function of hydraulic retention time (HRT). The influent substrates were 20,000 mg/L glucose and peptone (3:2(w/w)), temperature was at 35℃, and pH was controlled under 6±0.1 by computer. The reactor was operated at different hydraulic retention time (HRT = 18, 12, 8, 6, 4, 3, 2, 1.5, 1 hr), and collected gas production and water quality data under steady state for the nine runs.
The concentrations of biomass were around 2700±400 mg/L at HRT = 18-1.5 hrs, but C. tyrobutyricum was washed out at HRT = 1hr. Bacterial flocculation was occurred at HRT = 3, 2, 1.5 hrs. Biomass flocculation increased biomass concentrations and made concentrations of effluent glucose stay under 300 mg/L at lower HRTs.
Effluent organic nitrogen concentrations increased but effluent ammonia concentration decreased with decreasing HRT, because microorganism had enough time for degrading peptone when HRT was high. While the reactor was controlled at higher HRTs, peptone was used for biomass synthesis and fermentation. On the other hand, peptone was used for biomass synthesis mainly at lower HRTs.
Maximum hydrogen production rate, maximum specific hydrogen production rate and maximum hydrogen yield were 416.61 mmol H2/L/d, 8.92 L H2/g-VSS/d, and 3.47 mmol H2/g-CODapplied occurred at HRT = 4 hr. Moreover, experimental hydrogen production rate of HRT = 4 hr was also closest to theoretical hydrogen production rate among all the nine runs. Maximum carbon dioxide production rate was 292 mmol CO2/L-day occurred at HRT = 6 hr.
In addition, hydrogen production rate decreased when HRTs were reduced lower than 4 hr, but biomass concentrations were stable, so decreased hydrogen production rates were attributed to metabolic pathway shifting. The compositions of effluent organic acids were mainly butyrate and acetate at higher HRTs, but metabolic pathway shifted to lactate production at lower HRTs. LDH will be catalyzed by high NADH/NAD+ condition (Garrigues et al., 1997), and inhibited by NAD+ (Fitzgerald et al., 1992). When the reactor was controlled at lower HRTs, specific growth rates of microorganism were increased, and then required energy of microorganism was high, so NADH was produced faster. In addition, effluent butyrate concentrations decreased, maybe because of lower PTB activity. These two reasons caused accumulation of NADH and pyruvate in microorganism, LDH was activated, and then lactate was produced at lower HRTs.
Finally, metabolic flux analysis was studied. After using CellNetAnalyzer calculated the rates of metabolic pathways for eight runs, nodal analysis was applied. For nodal analysis of pyruvate, the pathway for producing acetyl-CoA with hydrogen production decreased when HRT decreased. On the other hand, the pathway for lactate production increased when HRT decreased, maybe because of high LDH activity. For nodal analysis of acetyl-CoA, the activities of PTB and PK were higher than PTA and AK, and vice versa.
論文目次 考試合格證明I
中文摘要II
英文摘要IV
致謝VI
目錄VIII
圖目錄XI
表目錄XIV

第一章 前言 1

第二章 文獻回顧 5
2-1 生物產氫程序的種類與發展 5
2-2 厭氧發酵產氫微生物 7
2-2-1 Clostridium 與其 hydrogenase 8
2-3 厭氧微生物產氫之機制 13
2-3-1 碳水化合物厭氧發酵代謝機制 13
2-3-2 含氮物質厭氧發酵代謝途徑 22
2-3-3 複合基質厭氧發酵 27
2-4 厭氧氫發酵的環境影響因子 28
2-5 C. tyrobutyricum 相關研究 35
2-6 CellNetAnalyzer 原理 38

第三章 實驗設備與方法 39
3-1 厭氧生物氫氣產能試驗(Biochemical Hydrogen potential test, BHP test) 39
3-1-1 反應槽啟動前基質之製備 39
3-1-2 反應槽啟動前營養鹽之製備 39
3-1-3 植種純菌製備 41
3-2 Clostridium tyrobutyricum 厭氧產氫純菌連續流反應槽 42
3-2-1 反應槽內純菌之培養 42
3-2-2 純菌反應槽進出流的啟動 42
3-3 水質分析項目與使用儀器 44
3-3-1 一般水質分析項目 44
3-3-2 碳水化合物 44
3-3-3 氣體組成 45
3-3-4 有機酸組成 45
3-3-5 醇類分析 45

第四章 結果與討論 47
4-1 Clostridium tyrobutyricum 純菌氫發酵槽之啟動與試程 47
4-2 C. tyrobutyricum 於各 HRT 下的表現 48
4-2-1 水力停留時間 18 小時 48
4-2-2 水力停留時間 12 小時 48
4-2-3 水力停留時間 8 小時 51
4-2-4 水力停留時間 6 小時 51
4-2-5 水力停留時間 4 小時 54
4-2-6 水力停留時間 3 小時 54
4-2-7 水力停留時間 2 小時 57
4-2-8 水力停留時間 1.5 小時 57
4-2-9 水力停留時間 1 小時 58
4-3 C. tyrobutyricum 在不同水力停留時間下表現之比較 61
4-3-1 揮發性懸浮固體物 (VSS) 64
4-3-2 氣體生成表現 65
4-3-3 殘留葡萄糖濃度 65
4-3-4 水解蛋白質轉化情形 67
4-3-5 有機酸與醇類 69
4-4 稀釋率對於 C. tyrobutyricum 的表現之影響 72
4-5 有機負荷對於 C. tyrobutyricum 產氫行為之影響 79
4-6 C. tyrobutyricum 複合基質發酵產物與氫氣生成之關係 81
4-7 利用CellNetAnalyzer 分析 C. tyrobutyricum 代謝路徑之速率 83
4-7-1 C. tyrobutyricum 的代謝路徑 83
4-7-2 CellNetAnalyzer 分析結果 85
4-7-3 節點分析(nodal analysis) 88

第五章 結論與建議 91
5-1 結論 91
5-2 建議 93

第六章 參考文獻 95

自述 107
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系統識別號 U0026-0812200915310226
論文名稱(中文) 以厭氧流體化床進行廚餘過篩液及狼尾草之氫醱酵程序研究
論文名稱(英文) Unit Operation of Anaerobic Fluidized Bed Reactor for Hydrogen Fermentation with Kitchen Waste Sieved Liquid and Napiergrass
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 陳怡傑
學號 p5696405
學位類別 碩士
語文別 中文
口試日期 2009-07-14
論文頁數 164頁
口試委員 口試委員-李季眉
口試委員-林秋裕
口試委員-黃良銘
口試委員-張嘉修
指導教授-鄭幸雄
關鍵字(中) 乳酸
流體化床
纖維素
木聚醣
廚餘過篩液
狼尾草
厭氧氫醱酵
關鍵字(英) Anaerobic fluidized bed reactor
Lactate
Napiergrass
Kitchen waste sieved
Anaerobic hydrogen fermentation
學科別分類
中文摘要 台灣現行之廚餘回收每天可達約1,900噸,是來源豐富的有機廢棄物之一,其中廚餘之70%以上為水,其液體部分可夾代豐富之營養成分適合用於產氫。本研究主要產氫基質選用廚餘過篩液,是將廚餘滲出的液體部分經過1~2 mm篩孔過篩,可有效控制懸浮固體物大小及濃度在26 g-SS/L(VSS/SS=0.91),其目的是降低SS含量使流體化床能操作順利,而流體化床的優點為藉由流力控制使懸浮固體物(基質及菌體)有更長停留時間且濃度更高的在反應區中反應。而此廚餘過篩液也能提供高濃度COD約104 g/L及總醣濃度總26.5 g /L供微生物產氫利用,其中碳水化合物的電子含量最高,約佔總COD之30.3%(溶解性佔總COD之26.9%),其次為油脂佔23%、蛋白質佔22.7%,再來為乳酸佔14.9%及乙酸2.6%。另外其TVS約73 g /L,可知以溶解性有機物為多。從批次實驗也顯示,廚餘過篩液本身所夾帶之嗜酸性微生物可有效分解其內養分且具良好產氫能力。本研究還有額外添加狼尾草當輔助基質與廚餘過篩液混合,其目的有三個:(1) 發揮流體化床擔體功能;(2)直接提高基質之有機負荷;(3) 培養出分解纖維及半纖維素之產氫菌。實驗所用的風乾狼尾草渣每克約可提供1.1 g-COD,有機成分佔總重的88.5%(含水率7.2%),而總醣佔總重的51%,夾帶有總重7.5%的溶解性醣類、纖維素20%及半纖維素及其他醣類約佔23%。蛋白質佔8.5%。而本研究利用110 L厭氧流體化床(反應溫度55oC、pH=6、HRT=7.3 day及總迴流量35 L/min)以廚餘過篩液為主要基質和狼尾草為輔助基質在193天連續氫醱酵操作下,第三試程(廚餘過篩液+20 g/L狼尾草)在有機荷負為17.7 g-COD/L/day下,有最高產氫速率約為1.64 L-H2/L/day(比前面第一及第二試程提升約16.6%),但是氫氣產率3.74 mmol-H2/g-CODin為各試程中最低的(也就是狼尾草中大部分醣類難分解而拉低產率);第一試程(只進廚餘過篩液)在體積負荷為14.2 g-COD/L/day下,產氫速率約為1.41 L-H2/L/day,其中氫氣產率約4.0 mmol-H2/g-CODin為各試程中最高的。各試程溶解性醣類去除率均可達約92%。流體化床在各試程的操作中,出流主要代謝產物以丁酸產生最多(13~17 g/L) ,乙酸次之 (2.3~3.3 g/L) ,而丙酸(生成345 mg/L)及乙醇(生成400 mg/L)生成量相當少,顯示此槽內微生物與廚餘過篩液在於產氫代謝是相當旺盛的,不利於產氫的代謝途徑並不顯著。另外值得注意的是各試程進料廚餘過篩液中高濃度乳酸(10.7~14.5 g/L)有被顯著降解的現象,其中第二試程有最高乳酸降解率,平均98.3%。由本研究乳酸降解試驗可證實槽內微生物可利用乳酸加上少量乙酸共基質代謝生成氫氣及丁酸。故可將槽內主要產氫途徑分為:
(1)乳酸代謝產氫
Lactate + 0.4 HAc +0.7 H+ → 0.7 HBu + 0.6 H2 + CO2 + 0.4 H2O
(2)醣類代謝產氫
C6H12O6 + 2H2O → 4H2 + 2CO2 +2HAc
C6H12O6 → 2H2 + 2CO2 + HBu
可知乳酸代謝產氫時,醣類代謝產氫所產生的乙酸會被消耗掉,所觀測到的乙酸生成濃度會減少,而乳酸代謝產氫也會同時生成丁酸,會與醣類代謝產氫所產生的丁酸累加,所觀測到的丁酸濃度會大量增加。以乳酸降解率最好的第二試程(98.3%)及第三試程(94.9%)為例,實測值的丁酸產生量與乙酸產生量的莫耳比值HBu/HAc會相當高(Run 2: 5.19, Run 3: 3.64),但將乳酸代謝干擾去除所得的醣類代謝之HBu/HAc會接近1(Run 2: 1.38, Run 3: 0.87) ,這表示槽內微生物利用醣類的反應接近此式: 3C6H12O6 + 2H2O → 8H2 + 6CO2 + 2 HAc + 2 HBu,故此式也顯示槽內最大的醣類代謝之氫氣產率約2.67 mol-H2/mol-hexose。而由揮發酸產量所推的理論產氫量與實際只誤差4.7~12.2%,顯示由其他非產氫代謝(如蛋白質)所產的乙酸及丁酸很少,而其中所降解的廚餘過篩液中之乳酸所產的氫氣約佔總產氫量的14.8%(Run 2)~17.1%(Run 3),故對乳酸濃度相當高廚餘滲出液來說,本流體化床內的乳酸產氫菌群除了可大幅的增加其產氫潛能外,此乳酸代謝過程還會消耗酸度,這對連續流進料的產氫酸化系統之pH維持恆定操作有很大的幫助,可緩和醣類代謝產氫時的酸化程度而減少液鹼回饋量。另外槽內及廚餘中微生物經流體化床經100多天的狼尾草馴養,對纖維素(CMC)完全沒水解及產氫活性,但對對木聚醣(半纖維素)有良好水解產氫活性,其可能因狼尾草物料僅進行初步的物理破碎,纖維素的露出率有限,微生物較難接觸到(木質素及半纖維素包在纖維素外面)。故第三試程加最多狼尾草20 g/L有最好之產氫量,其多出的產氫量可能主要由狼尾草所夾帶的少量溶解性醣類(7.5%)及水解部分半纖維素所提供。在槽內微生物菌相探討方面,由SEM可觀測到廚餘及流體化床的微生物型態幾乎都以桿菌為主,另外由兩端帶螢光之T-RFLP前後端之分生檢測結果,與本研究團隊所做的廚餘產氫系統菌群clone之 T-RFLP資料庫做比對,本研究槽內主要之微生物可能為Clostridium sp.和Thermoanaerobacterium sp.(reverse: 307 bp & 317 bp),這兩屬中很多菌種對多種複合醣類都具有產氫能力,而且皆為桿菌型態並有內生孢子能力。另外,乳酸降解試驗中產氫最好的組別在T-RFLP的forward 495 bp波峰,跟批次植種比有明顯的提升(4.3%升到36.1%),故推測forward 495 bp位置可能為乳酸產氫菌,而在第二試程中乳酸去除率(98.3%)最好的時候,495 bp位置能成為主流波峰之一,最高可佔總族群的41.3%。然而在在Run 3中後期時,495 bp位置及其他較小的波峰位置幾乎快消失(只有最主流波峰不變),可能是因為Run 3之進料SS過高,到中後期常會有堵塞問題,故在清理時會增加氧氣進入反應槽中的機會,使部分厭氧微生物可能會受到影響。
英文摘要 This study used kitchen waste sieved liquid (KWSL) (KW was sieved through 1~2 mm pore size) as a substrate for biohydrogenation by anaerobic fluidized bed reactor (AnFB). And later, different weights of napiergrass dregs were added to co-ferment with KWSL in long hydraulic retention time (HRT) (7.3 days).In Taiwan, the recycling amount of KW was up to 1900 ton/day in average. The moisture of KW was more than 75%, which (KWSL) could carry abundant nutrient and suit for biohydrogenation as well as the operation of AnFB (less suspended solid).The characteristic of KWML as follows: Total COD : 104,000 ± 16,000 mg/L (69% soluble) , Total Carbohydrate: 26,500 ± 6,300 mg/L (85% soluble ) , Total Org-N: 2,560 ± 270 mg/L (70% soluble) , lipid: 8,300 ± 3900 mg/L , suspended solid: 26,000 ± 5330 mg/L( 91 % volatile) , and it contained acetate (2,500 ± 340 mg/L) and high concentration lactate ( 10,700~14,500 mg/L ) . Napiergrass (1.1 g-COD/g- napiergrass dregs) is composed by about 20% cellulose and other lignin cellulose which are difficult to decomposition, this study expected that adding the napiergrass dregs with KWSL could degrade the cellulose and act as a kind of support material in the AnFBR.
The AnFBR has a draft tube in reacting zone, and let solid has longer retention time to reaction by controlling recycle flow rate .Working volume of the AnFBR is 110 L. The seeding was from the sludge which used KW vegetable as substrate to produce hydrogen. Operation parameters of the AnFBR: temperature:55oC , pH:6 ,HRT:7.3 days and recirculation liquid flow rate: 35 L/min
Total three runs: Run 1 (70 days) inputted KWSL only (organic loading rate: 14.2 ± 2.4 g-COD/L/day), and the hydrogen production rate (HPR) was 1.41 ± 0.27 L-H2/L/day. Run 2 (73 days) inputted KWSL with 5 g- napiergrass dregs/L , and the HPR was 1.41 ± 0.28 L-H2/L-day. Run 3 (47 days) increased the napiergrass concentration to 20 g- napiergrass dregs /L, and had the best HPR,1.64 ± 0.28 L-H2/L-day(Total biogas: 399.6 ± 40.4 L/L/day),in this study.
In metabolites aspect, the main volatile fatty acids (VFA) in the effluent were butyrate (13,000~17,000 mg/L) and acetate (2,300~33,00 mg/L) production. Especially, the high concentration lactate in KWSL was degraded obviously (maximum removal : 98.3% for Run 2),and from the batch test of lactate degradation, it showed that lactate was degraded and hydrogen was produced, which the reaction was similar to “Lactate + 0.4 Acetate +0.7 H+ → 0.7 Butyrate + 0.6 H2 + CO2 + 0.4 H2O”. Regarding KWSL contained high concentration lactate has advantages of biohydrogenation and reducing the feed-back of alkali.
論文目次 中文摘要 I
ABSTRACT IV
誌謝 VI
目錄 VIII
表目錄 XI
圖目錄 XIII
第一章 前言 1
第二章 文獻回顧 4
2-1 全球能源發展趨勢及生質氫能之發展 4
2-2 台灣廚餘清運現況及回收再利用之現況 8
2-3 厭氧生物產氫技術 11
2-3-1 厭氧生物氫醱酵機制 11
2-3-2 產氫酵素hydrogenase 17
2-3-3 厭氧氫醱酵之微生物 19
2-3-4 影響厭氧醱酵產氫之因素探討 23
2-4 廚餘中主要有機成分之水解及氫醱酵之探討 27
2-4-1 碳水化合物類(澱粉、纖維素及半纖維素)之厭氧水解及產氫 28
2-4-2 蛋白質之厭氧水解和代謝及對產氫之影響 37
2-4-3 油脂之厭氧水解和代謝產氫機制 39
2-5 厭氧流體化床反應槽 42
2-6 國外生物產氫應用於實際廢水之研究現況 45
2-6-1 化學廢水產氫研究結果 45
2-6-2 棕櫚油廠廢水產氫研究結果 47
第三章 材料與方法 49
3-1 110 L高溫脈衝式進料厭氧流體化床 (AnFB) 49
3-2 水質分析項目與使用儀器 54
3-2-1 一般水質分析項目 54
3-2-2 儀器分析 55
3-3 生化氫氣產能試驗及生物活性量測 57
3-3-1 生化氫氣產能試驗(BHP test) 57
3-3-2 生物活性量測數據整理方式 58
3-4 pH自動控制之批次反應器 59
3-5 掃描式電子顯微鏡 Scanning Electron Microscope(SEM) 60
3-6 分子生物檢測技術 61
3-6-1 總DNA 萃取 61
3-6-2 聚合酵素連鎖反應(Polymerase Chain Reaction, PCR) 63
3-6-3 尾端修飾限制片段長度多形性(T-RFLP) 64
第四章 結果與討論 66
4-1 110 L厭氧流體化床之基質、操作及硬體之改良與試程操作策略 66
4-1-1 先前進料基質(蔬菜廚餘)遭遇之問題與改良(廚餘過篩液) 66
4-1-2 流體化床硬體之問題與改良(pump) 68
4-1-3 流體化床操作之問題與改良(迴流量) 69
4-1-4 利用廚餘過篩液及狼尾草產氫之流體化床試程操作策略 70
4-2 台南市廚餘過篩液及狼尾草特性分析 75
4-2-1 廚餘過篩液特性分析 75
4-2-2 狼尾草特性分析 84
4-3 廚餘過篩液及狼尾草之高溫厭氧氫醱酵流體化床運轉操作探討 86
4-3-1 流體化床植種與廚餘過篩液之生化氫氣產能試驗 86
4-3-2 流體化床迴流量的控制及槽中懸浮固體物分布情況 88
4-3-3 各試程操作參數與狀況及功能指標 91
4-4 流體化床內微生物之乳酸降解批次試驗 108
4-5 廚餘過篩液之氫醱酵機制探討 117
4-6 由進出流揮發酸(乳酸、乙酸及丁酸)變化探討產氫平衡 122
4-7 槽內微生物針對纖維素及半纖維素及狼尾草的利用能力 128
4-8 微生物族群結構之探討 135
4-8-1 以掃描式電子顯微鏡觀察厭氧流體化床內微生物菌相 135
4-8-2 分子生物技術T-RFLP探討各試程微生物族群變化 140
第五章 結論與建議 147
5-1 結論 147
5-2 建議 150
第六章 參考文獻 152
自述 163
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系統識別號 U0026-0812200915375083
論文名稱(中文) 利用生質酒精發酵殘渣產氫程序之研究
論文名稱(英文) Evaluation of Bioenergy Recovery Processes Treating Organic Residues from Ethanol Fermentation
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 莊崇柏
學號 P5696417
學位類別 碩士
語文別 中文
口試日期 2009-07-14
論文頁數 83頁
口試委員 口試委員-陳錫添
口試委員-張嘉修
口試委員-鄭幸雄
指導教授-黃良銘
關鍵字(中) 甲烷化發酵
兩階段式生質能源程序
酒精發酵後廢液
厭氧氫發酵
關鍵字(英) anaerobic hydrogen fermentation
alcohol fermentation residue
two-stage bioenergy process
methanogenesis
學科別分類
中文摘要 隨著人類對於能源的需求逐漸迫切,伴隨著嚴重的污染及化石燃料快速地消耗,開發新興且乾淨的再生能源成為各國努力並積極發展之重點。一般現有技術對能源作物進行酒精發酵之過程中,預期會有20~25%之殘渣無法完全利用,若可將20~25%之殘渣進行生質能再回收可解決生質能源工業化時的環保問題,同時能有效地回收寶貴的能源。本研究針對木薯之酒精發酵殘渣進行特性分析,酒精發酵後廢液含有大量的有機物質,包括碳水化合物、有機酸類、甲醇及乙醇等,COD高達64,000 mg/L,且富含營養成分(C/N為13)。在各成份之電子分佈方面,碳水化合物占總COD值的37 %,此部分可利用產氫發酵回收能源;另外,有機酸約佔12 %,甲醇及乙醇佔約42 %,加上氫發酵後產生的揮發酸類(Volatile fatty acids, VFAs),此部分可利用甲烷化回收剩餘之能源,故可利用兩階段式生質能源程序處理此生質酒精發酵後廢水。
經由生化產氫潛能測試(BHP test)探討pH與基質負荷(S0/X0) 對產氫程序之影響可知此股廢液具有產氫之潛能,其最佳操作之pH值為6.0而最佳之操作負荷為3.0 mg COD/mg VSS,最高產氫速率約為26 mL-H2/hr,轉化效率達0.7~1.3 mmole H2/g COD。且由額外添加乙醇之前導測試可知,當額外添加乙醇,將使氫氣產量降低(當酒精濃度達0.1 %以上,產氫速率以降低一倍),在此批次試驗中,產氫量皆不多,可見酒精對產氫之抑制。
本研究以CSTR厭氧氫發酵槽經過連續操作四個試程後,於第三試程有最高之反應速率約為0.77 mmol-H2/g-VSS/hr,此時之體積負荷為59 kg-COD/m3-day;第一試程在體積負荷25.2 kg-COD/m3-day有最好的氫氣產率,約為1.32 mmole-H2/g-COD,因此往後若欲實廠工程化,則須考量以產氫速率或是轉化效率做為操作之標的,以符合經濟效益。由於試程二沒有產氣效果不佳,且在試程後期量測不到氣體產生,因此將試程二之微生物取出,模擬試程一(S0/X0 = 6)、試程二產氣(S0/X0 = 12)與不產氣階段之食微比進行批次實驗,在此批次實驗中,S0/X0 = 12 g COD/g VSS產氫量及速率皆高於S0/X0 = 6組約5倍左右,且於有機酸的分析上,S0/X0 = 6組僅有乳酸消耗的現象,但S0/X0 = 12組則是消耗乳酸與乙酸且產生丁酸後,才有明顯產氣,故推測是因為代謝路徑不同,所以在氫氣產量上才有巨大之差異。
高食微比之批次實驗中,僅S0/X0 = 12, X0 = 2,000 mg/L此組有產氣之行為,而此組之產氣行為與上一批次實驗相同,同樣在分解麥芽糖時沒有氣體產生,而在分解乳酸及乙酸時才有產氣之行為;在微生物生長速率方面,當食微比越高比生長速率越快。在不同pH值的批次試驗中可發現,在馴養一段時間後,pH = 6有最佳之產氫效率,比產氫速率可達13.6 mmol-H2/g-VSS/hr,顯示在pH = 6馴養一段時間後,確實可培養出較適應此環境之微生物。
將厭氧氫發酵產生之揮發酸,及未去除之COD以甲烷化發酵處理,甲烷產率可達345 mL CH4/gCOD,比產甲烷速率最高為9.24 mL CH4/gVSS-hr,顯示甲烷發酵槽可有效率地將酒精發酵廢液中剩餘COD做生質能回收。將氫發酵槽及甲烷發酵槽之代謝物質進行電子平衡可知,經由兩階段生質能源程序後,可將2 % 的COD轉化成氫氣,67 % 轉換成甲烷;可利用總COD的91 % 左右;顯示兩階段之生質能源程序,可有效的進行生質能源的回收再利用。
英文摘要 In recent years, developing novel renewable “green energy” becomes a trend in many countries for the fast consumption of fossil fuels and the pollution after using them. Recent technologies can only convert 75-80% of the energy crop into energy by the alcohol fermentation process, about 20-25% of wasted as residue. The aim of this study was to re-utilized this fermentation residue to produce energy products. The characteristics of the tapioca alcohol fermentation residue were studied. Large amount of organics containing carbohydrates, organic acids, methanol and ethanol were found in the residue. The COD was as high as 64,000 mg/L, with C/N ratio of 13. For the electron distribution, carbohydrates comprised 37% of the total COD, and this portion can be fermented to hydrogen. On the other hand, the remaining organic acids (12%) and alcohols (42%), together with the volatile fatty acids produced in hydrogen fermentation, can further be utilized by methanogens to produce methane as energy product. Therefore, a two-stage bioreactor with hydrogen fermentation and methanogenesis was established in this study to treat this tapioca alcohol fermentation residue.
Effects of pH and substrate loading (S0/X0) on hydrogen production with this residue wastewater were evaluated from the biochemical hydrogen potential (BHP) test. The optimum pH and loading was 6.0 and 3.0 mg COD/mg VSS respectively. The highest hydrogen production rate in this test was 26 mL-H2/hr, with the conversation efficiency of 0.7-1.3 mmol H2/g COD. The effect of ethanol concentration was also study. When ethanol concentration was up to 0.1%, the hydrogen production rate was one fold lower compared to the blank. This shows that ethanol has certain inhibition effect on hydrogen production.
There were four runs in the CSTR anaerobic hydrogen fermentation tank. The highest specific hydrogen production rate, 0.77 mmol H2/g VSS/hr was obtained in run 3. The volumetric loading was 59 kg COD/m3-day. The highest hydrogen production, 1.32 mmol H2/g COD, was observed during run 1, with the volumetric loading of 25.2 kg COD/m3-day. During run 2, the hydrogen production efficiency was not good, with no gas production observed. Therefore, batch tests with different substrate loading (S0/X0=6 of run 1 and S0/X0=12 of run 2) were conducted using the sludge in run 2. Results showed that both hydrogen production and production rate of the S0/X0=12 group was five times higher than the S0/X0=6 group. For the organic acid analysis, only lactate consumption was observed in the S0/X0=6 group. For the S0/X0=12 group, hydrogen production was observed after the consumption of lactate and acetate and the production of butyrate. This showed that the differences in hydrogen production may be due to different metabolic pathways.
To study the hydrogen production under high F/M ratio conditions (S0/X0=12 and 24), batch tests was conducted to investigate the metabolic characteristics of the microorganisms. Sludge in run 4, which was acclimated under high F/M ratio for a period, was used in this test. In this test, hydrogen production was only observed in the group with S0/X0=12 and X0=2,000. Similar to previous test, no gas was produced during maltose degradation. Hydrogen began to produce when lactate and acetate was degraded. Higher microbial growth rate was obtained with higher F/M ratio. In the batch with different pH values, the highest hydrogen production rate, 13.6 mmol H2/g VSS/hr was observed under pH=6.
The fatty acids produced in the anaerobic hydrogen fermentation process, together with the residue COD was utilized by methanogens. The methane production was up to 345 mL CH4/g COD, and the highest specific methane production rate was 9.24 mL CH4/g VSS-hr. This indicated that the alcohol fermentation residue can effectively be converted to energy through methanogensis. The electron balance of the two-stage reactor showed that 2% of COD was converted to hydrogen and 67% to methane. Total 91% of COD was utilized in the process. This showed that this two-stage bioenergy process can effectively recover the energy from alcohol fermentation residue.
論文目次 考試合格證明 II
致謝 III
摘要 V
Abstract VII
目錄 IX
表目錄 XI
圖目錄 XIII
第一章 前言 1
第二章 文獻回顧 3
2-1 潔淨再生能源之展望及全球能源使用趨勢 3
2-2 生物產氫程序的種類與發展 5
2-3 厭氧發酵產氫微生物 7
2-3-1 Clostridium 與其 hydrogenase 8
2-3-2 Entrobacter 11
2-4 厭氧微生物產氫之機制 12
2-4-1 碳水化合物厭氧發酵代謝機制 13
2-4-2 含氮物質厭氧發酵代謝途徑 20
2-4-3 複合基質厭氧發酵 22
2-5 厭氧氫發酵的環境影響因子 23
第三章 材料與方法 29
3-1 兩階段式 CSTR 厭氧生物氫發酵槽 29
3-2 酒精發酵後廢液之來源 30
3-3 水質分析項目與使用儀器 30
3-3-1 一般水質分析項目 30
3-3-2 儀器分析 31
3-4 生化氫氣產能試驗及生物活性量測 32
3-4-1 生化氫氣產能試驗 32
3-4-2 生物活性量測數據整理方式 32
第四章 結果與討論 33
4-1 生質酒精發酵廢液特性分析 33
4-2 生質能源程序最佳化因子探討及水解產氫菌效能評估 38
4-2-1 生質能源程序最佳化因子探討 38
4-2-2 操作發酵殘渣有機物產氫程序 44
4-2-3 酒糟發酵殘渣厭氧氫發酵與文獻之比較 51
4-2-4 總結 53
4-3 酒糟發酵槽微生物之活性測試及反應動力機制探討 54
4-3-1 不同食微比對產氫程序之影響 54
4-3-2 高食微比對產氫程序之影響 62
4-3-3 不同pH下對產氫程序之影響 65
4-4 甲烷化程序與操作兩階段式發酵殘渣有機物 70
4-4-1 產氫後殘渣之甲烷化程序 70
4-4-2 兩階段式生質能源程序之效能評估 73
第五章 結論與建議 75
5-1 結論 75
5-2 建議 76
第六章 參考文獻 77
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