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系統識別號 U0026-0812200913533228
論文名稱(中文) 不同pH環境下厭氧產氫菌Clostridium競爭情形之研究
論文名稱(英文) Competition Between Clostridium Species for Biological Hydrogen Production – Effect of pH
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) 林佩瑩
研究生(英文) Pei-Ying Lin
電子信箱 p5694401@mail.ncku.edu.tw
學號 p5694401
學位類別 碩士
語文別 英文
論文頁數 77頁
口試委員 指導教授-黃良銘
口試委員-鄭幸雄
口試委員-黃良銘
口試委員-曾怡禎
口試委員-張嘉修
中文關鍵字 微生物競爭  數學模式模擬  生物產氫 
英文關鍵字 Clostridium  microbial competition  biological hydrogen production  mathematical model simulation 
學科別分類
中文摘要 工業革命後,由於工業技術及科技迅速發展,使人口大量增長,能源需求也隨之增加,目前化石燃料為人類主要的能源需求,然而地球上化石燃料含量有限,且其經燃燒後將會造成地球暖化及空氣污染,因此尋找乾淨的替代能源為目前各國所積極研究之目標。許多替代能源中,氫氣為一種乾淨、可再生的能源,燃燒僅產生水,且可以經由厭氧分解有機物過程取得氫氣,可達到廢棄物利用再生及產生能源的目的。
近年來生物產氫被廣泛地研究,且發現多種可以在厭氧情況下轉換碳氫化合物為氫氣的微生物,許多研究著重在混菌反應槽氫氣產率的提升,然而,基礎研究仍具有相當之重要性,其有助於提供複雜的混合系統基礎資訊。因此本實驗著重於簡單的基質及較單純之微生物厭氧發酵系統,利用前人研究結果迴歸之微生物動力參數,建立了數學模式模擬系統預測微生物競爭情形,並操做連續反應槽(CSTR)以了解不同pH下微生物的競爭情形。
本實驗藉由CSTR來探討三株產氫菌C. butyricum (CGS2), C. beijerinckii (L9), and C. tyrobutyricum (FYa102)的競爭情形。首先以批次進行單一純菌培養12小時,而後植種於2升反應槽以批次程序、恆溫35℃下培養12~15小時,於反應槽微生物濃度達一定量,啟動進流及出流,以連續進流方式進行生物產氫產能試驗(BHP test)。進流基質為葡萄糖(12,000 mg/L)及蛋白腖 (8,000 mg/L),葡萄糖為三株Clostridium 屬微生物生長限制因子,分別控制pH於6.0、5.0及4.5,水力停留時間分別為12小時及6小時。操作期間以T-RFLP偵測微生物族群的變化。並監測水質變化情形。
於pH為6、水力停留時間12及6小時的實驗中,C. butyricum為主要優勢菌種,pH為5、水力停留時間12的實驗中以C. beijerinckii為優勢菌種,但當其水力停留時間調整為6小時時卻以C. butyricum為優勢菌種,在pH為4.5、水力停留時間12小時的實驗中,當反應槽環境較趨近於還原態時,C. beijerinckii為優勢菌種,然而當環境趨於氧化態時,轉為C. tyrobutyricum為優勢菌種。pH為6且水力停留時間12及6小時、pH為5且水力停留時間6小時下實驗結果之優勢菌種與模式模擬結果相同,唯競爭趨勢及微生物濃度不一致,且試程結束時仍有兩株微生物共存於系統中。
英文摘要 The main energy requirements, fossil fuels, are finite resources and the combustion of these fossil fuels contributes global warming and air pollution. Hydrogen is considered as a clean and renewable energy alternative and can be produced in the acidogenesis process during anaerobic degradation of organics.
Recently, biological hydrogen production has been extensively studied and various bacteria have been found to convert carbohydrates to hydrogen in mixed culture. And much research has focused on strain improvement, using molecular biology to increase the product concentrations and product yields in the fermentation broth. In this study, a mathematical model simulation was set up to describe the microbial competition and the continuous reactor was conducted to investigate the competition under different pH control.
Competition among three species of hydrogen-producing bacteria – C. butyricum (CGS2), C. beijerinckii (L9), and C. tyrobutyricum (FYa102) – was studied in the continuous stirred tank reactor (CSTR). Each organism was grown separately in anaerobic batch cultures in the chemostat growth medium for 12 hours. Each culture was inoculated into the 2-L reactor in batch mode for 12~50 hours. Then, the reactor was conducted under continuous condition at 35℃ by the biochemical hydrogen potential (BHP) test. Multiple substrates containing glucose (12,000 mg/L) and peptone (8,000 mg/L) were fed into the CSTR; the competition for glucose as a growth-limiting substrate between three strains of Clostridium species. To clarify the microbial competition behavior of the reactor in response to pH changes, three runs whose pHs were controlled at 6.0, 5.0, and 4.5 by using a pH-controller. The populations were estimated using T-RFLP (terminal restriction fragment length polymorphism)
A kinetic model was developed to evaluate the microbial competition. The kinetic parameters were estimated in the previous research.
In the experiment, at pH 6 with HRT of 12 and 6 hours, C. butyricum was the predominant bacteria. At pH 5 with HRT of 12 hours, C. beijerinckii was dominant. However, at pH 5 with HRT 12 hours, C. butyricum was predominant. The fermentor was conducted at pH of 4.5 with 12-hour-HRT, C. beijerinckii was the dominant bacteria when the condition was in more reduce state. However, when the condition was in more oxidation state, C. tyrobutyricum was the dominant bacteria.
論文目次 摘要 III
ABSTRACT V
誌 謝 VII
TABLE OF CONTENTS VIII
TABLE OF TABLES X
TABLE OF FIGURES XI
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 LITERATURE REVIEW 3
2.1 The trend of energy utilization and renewable energy source 3
2.2 Anaerobic digestion 7
2.3 Hydrogen-producing bacteria and metabolic pathway 10
2.3.1 Clostridium 13
2.3.2 Clostridium butyricum 15
2.3.3 Clostridium beijerinckii 15
2.3.4 Clostridium tyrobutyricum 15
2.4 Hydrogenase of Clostridium spp. 15
2.5 Effect of pH and fermentation products on hydrogen production 21
2.5.1 The pH effect 21
2.5.2 The ATP and NAD(P)H effect 22
2.5.3 Volatile fatty acid and alcohol effects 25
CHAPTER 3 MATERIALS AND METHODS 27
3.1 Biochemical Hydrogen Potential test (BHP test) 27
3.1.1 Defined media 27
3.1.2 Stock solution of substrate 28
3.1.3 Inoculums 28
3.2 Continuous-Flow Stirred Tank Reactor (CSTR) 29
3.3 Water quality analysis 30
3.3.1 Normal water quality 30
3.3.2 Instrument analysis 31
3.3 Microbial community analysis 32
3.3.1 DNA extraction 32
3.3.2 Polymerase chain reaction (PCR) 35
3.3.3 Terminal restriction fragment length polymorphism (T-RFLP) 36
3.4 Mathematical model simulation 37
CHAPTER 4 RESULTS AND DISCUSSION 39
4.1 Mathematical model simulation 39
4.1.1 Mathematical model simulation for pH 6 40
4.1.2 Mathematical model simulation for pH 5 43
4.1.3 Mathematical model simulation for pH 4.5 46
4.2 Reactor operation and microbial competition 49
4.2.1 The reactor was controlled at pH of 6 and HRT of 12 hours 49
4.2.2 The reactor was controlled at pH of 6 and HRT of 6 hours 54
4.2.3 The reactor was controlled at pH of 5 and HRT of 12 hours 54
4.2.4 The reactor was controlled at pH of 5 and HRT of 6 hours 57
4.2.3 The reactor was controlled at pH 4.5 and HRT of 12 hours 58
4.3 Discussion 62
4.3.1 Reactor performance 62
4.3.2 Microbial competition 65
CHAPTER 5 CONCLUSIONS AND SUGGESTIONS 70
5.1 Conclusions 70
5.2 Suggestions 71
CHAPTER 6 REFERENCES 72
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