臺灣博碩士論文加值系統

石油儲量枯竭及全球暖化問題已造成人類對較環保且可再生替代能源之迫切需求，而氫氣(H2)即是一個熱門的選項。且由於具有高能源效率與潔淨環保的特性，生物產氫預期可對全球未來之能源結構產生實質性的貢獻。生物產氫特別適用於小規模且分散未集中式(decentralized)的能源生產系統、可與農業和工業活動或廢棄物管理緊密結合。
大型海藻可作為醱酵產氫之可持續性料源，因爲它含有豐富的碳水化合物與少許的木質素。大型海藻可以養殖在缺少營養的海洋水體中，故亦可解決土地使用需求的問題。大型海藻被認為是能減少全球二氧化碳排放即降低全球暖化最有效率的生物系統之一。
本研究的主要目標爲設計與評估一套利用大型海藻為梭狀芽孢桿菌的料源進行暗醱酵生物產氫的系統，並建立大型海藻最佳的糖化技術及醱酵產氫條件。綠色大型海藻（石蓴）的主要成分包含了50.3％的碳水化合物、25.7％的蛋白質、2.3％的脂質與21.7％的灰份。大型海藻經採集之後，使用酸水解預處理進行糖化，接著進行梭狀芽孢桿菌之暗醱酵產氫。大型海藻在使用4%硫酸於120oC高溫下進行40分鐘水解後，其水解的成分含有32.1%總糖、9.8%總氮、1.5%總磷、0.6克/公升羥甲基糠醛、0.2克/公升糠醛、0.2%乙酸與0.31%丙酸。爲提高水解效率，本研究採用不同的硫酸濃度（0-20%）進行水解以探討大型海藻水解的最佳狀況。本研究先以蔗糖為碳源進行最佳產氫菌株的篩選，測試菌種為Clostridium pasteurianum CH1、CH4、 CH5、CHN 以及Clostridium bytyricum CGS2、CGS5，接著並探討攪拌速度與培養基成分（PM、Endo與改良式Endo 培養基）對生物產氫的影響。研究結果顯示，CH4菌種和CGS5菌種於200rpm 攪拌速度和Endo培養基條件下培養，可得最高的氫氣累計產量與氫氣產率。在使用大型海藻水解液進行暗醱酵方面，本研究探討不同的總糖濃度(4-16 g/L)與pH控制(5.0-6.0)對產氫之影響。結果顯示在12 g/L總糖與pH 5.0的條件下， CGS5菌株產生了最高的氫氣累計產量(2339.77 ml/L)、生產速率(208.28 ml/L/h)與產率(1.53 莫耳氫氣/莫耳總糖)。此最佳的暗醱酵菌種及醱酵條件被繼續使用於以大型海藻水解液作為碳源之連續式醱酵產氫，並探討在不同的水力滯留時間(hydraulic retention time; HRT)對產氫的影響。當滯留時間從8小時逐漸縮短至4小時的時候，氫氣產生速率從429 ml/L/h大幅提升至至812 ml/L/h，但氫氣產率則從1.62降至0.98莫耳氫氣/莫耳總糖。

The depletion of petroleum reserves and issues of global warming have created a need for the development of environmentally friendly alternative energy, such as hydrogen (H2). Biohydrogen holds the promise for a substantial contribution, because it is a highly energy efficient, clean burning and pollution-free fuel source. Production of biohydrogen seems particularly suitable for relatively small-scale, decentralized systems, integrated with agricultural and industrial activities or waste processing facilities.
Macroalgae are alternative and sustainable feedstock for biohydrogen production because of their high levels of carbohydrates and low levels of lignin. Macroalgae can be cultivated in non-arable seawater with minimal nutritional requirements, and hence they can overcome issues regarding land use changes. Macroalgae can be considered as one of the most productive biological systems for carbon capture in order to mitigate global CO2 emissions and reduce global warming.
The primary objective of this study is to design a suitable bioprocess to assess the potential of using macroalgae as the fermentation substrate for Clostridium sp. for bioH2 production. The optimal saccharification method for the macroalgae biomass, the suitable strain for fermenting the macroalgal hydrolysate and optimal fermentation conditions were determined. The green macroalgae used in this study (Ulva sp.) is composed of 50.3% carbohydrates, 25.7% protein, 2.3% lipid, and 21.7% ash. After harvesting, the macroalgal biomass was subjected to saccharification using physicochemical (acid-hydrothermal) pretreatment, followed by dark fermentation with Clostridium sp.. The macroalgal biomass was hydrolyzed by mild acid-thermal pretreatment with 4% H2SO4 and 121oC for 40 minutes. The macroalgae hydrolysate consisted of 32.1% total reducing sugar (TRS), 9.8% total nitrogen (TN), 1.5% total phosphorus (TP), 0.6 g/L hydroxy-methyl-furaldehyde (HMF), 0.2g/L furaldehyde (Furfural), 0.2% acetic acid, and 0.31% propionic acid. In order to enhance hydrolysis efficiency, different H2SO4 concentrations (0-20%) were used for hydrolysis to identify the best hydrolysis conditions for the macroalgae. Preliminary tests were performed using synthetic carbon source (sucrose) to determine the suitable H2 producing bacterial strain (Clostridium pasteurianum CH1, CH4, CH5, CHN, and Clodtridium butyricum CGS2, CGS5), agitation rate, and medium composition (PM, Endo, and Modified Endo medium). C. pasteurianum CH4 and C. butyricum CGS5 strains showed better fermentation performance with 200 rpm agitation and Endo Medium, attaining the highest cumulative H2 production, maximum H2 productivity, and H2 yield. For the fermentation of macroalgae hydrolysate, different initial total reducing sugar (RS) concentrations (4-16 g/L) and pH control (5.0-6.0) were evaluated for better fermentation performance. Using an initial reducing sugar concentration of 12 g/L and pH 5.5, C. butyricum CGS5 achieved the highest cumulative H2 production (2340 ml/L), maximum H2 productivity (208.3 ml/L/h), and H2 yield (1.53 mole H2/mole RS). The optimal fermenting conditions mentioned above were further used for continuous H2 production using macroalgae hydrolysate as the carbon source under different hydraulic retention time (HRT). When HRT was gradually shortened from 8 to 4 h, the H2 production rate increased from 429 to 812 ml/L/h, whereas the H2 yield decreased from 1.62 to 0.98 mol H2/mol reducing sugar (RS).

摘要 I
ABSTRACT III
ACKNOWLEDGEMENT V
CONTENT VI
LIST OF TABLES IX
LIST OF FIGURES XII
CHAPTER 1 INTRODUCTION 1
1.1 Motivation and Purpose 1
1.2 Research Scheme 2
CHAPTER 2 LITERATURE REVIEW 5
2.1 Hydrogen 5
2.1.1 General description 5
2.1.2 Hydrogen producing bacteria 6
2.1.3 Metabolic pathways, co-products, and yield 8
2.1.4 Hydrogen Production 11
2.1.5 Renewable feedstock for hydrogen production 21
2.2 Macroalgae 23
2.2.1 General description and structure of macroalgae 24
2.2.2. Composition of green macroalgae 25
2.2.3 Macroalgae cultivation and harvesting methods 28
2.2.4 Macroalgae as feedstock for biohydrogen 29
2.3 Macroalgae pretreatment and hydrolysis 31
CHAPTER 3 MATERIALS AND METHODS 33
3.1 Chemicals and materials 33
3.2 Equipment 35
3.3 Bacterial strain and fermentation 36
3.3.1 Dark fermentative H2 producing bacterial strain 36
3.3.2 Medium composition 37
3.3.3 Fermentation condition 39
3.4 Feedstock (Green macroalgae Ulva sp.) 43
3.4.1 Biomass composition 43
3.4.2 Hydrolysate composition 44
3.5 Analytical methods 45
3.5.1 Determination of cell concentration 45
3.5.2 Determination of gas products by gas chromatography (GC) 46
3.5.3 Determination of reducing sugar and soluble metabolites concentration by high performance liquid chromatography (HPLC) 46
3.5.4 Determination of hydroxymethyl furfural (HMF) and furaldehyde (furfural) contents 47
3.5.5 Determination of ionic compound (total nitrogen and total phosphorus) 48
3.5.6 Determination of carbohydrate content 48
3.5.7 Determination of amino acid content 49
3.5.8 Determination of lipid content 49
3.5.9 Determination of ash content 50
3.5.10 Analysis of transient behavior by modified Gompertz equation 51
3.5.11 Measurement of biohydrogen productivity, yield, and reducing sugar consumption 52
CHAPTER 4 RESULTS AND DISCUSSION 53
4.1 Mesophilic dark fermentation with H2 producing bacteria using a synthetic carbon source (sucrose) 53
4.1.1 Screening of various H2 producing bacteria for effective H2 production 53
4.1.2 Effect of media composition on H2 production 63
4.1.3 Effect of agitation rate on H2 production 72
4.2 Macroalgae biomass and hydrolysate 81
4.2.1 Macroalgae biomass and hydrolysate composition 82
4.2.2Effect of acid (H2SO4) concentrations on macroalgae hydrolysate 86
4.3 Mesophilic dark fermentation with H2 producing bacteria using macroalgae hydrolysate 88
4.3.1 Screening of various H2 producing bacteria for effective H2 production 88
4.3.2 H2 production using non-hydrolyzed macroalgae powder 94
4.3.3 Effect of macroalgae hydrolysate concentration on bioH2 production 98
4.3.4 Effect of pH control on H2 production 105
4.3.5 BioH2 production via continuous fermentation 111
CHAPTER 5 CONCLUSIONS 118
REFERENCES 120
APPENDIX 128

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