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研究生：	吳書嫻 Wu, Shu-Hsien
論文名稱：	利用Clostridium acetobutylicum轉化微藻殘渣產製生質丁醇之研究 Study of utilizing oil-extracted microalgae residues for butanol production by Clostridium acetobutylicum
指導教授：	黃良銘 Whang, Liang-Ming
學位類別：	碩士 Master
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	106
中文關鍵詞：	ABE 發酵、微藻殘渣、生質丁酸、甲烷化發酵
外文關鍵詞：	ABE fermentation, oil-extracted microalgae residues, biological butyrate, methanogenesis
相關次數：	點閱：161 下載：1
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由於科技快速發展，人們對能源的需求大幅增長，使石化燃料被快速消耗，造成能源短缺與環境污染等相關問題，故開發新型替代能源與清潔永續的再生能源成為各國政府積極推行的首要政策。含油微藻之油脂取出後所剩之有機廢棄物尚含有相當比例的醣類，可為製造醇酮液體燃料之原料，如乙醇、丁醇、丙酮等，其中尤以生質丁醇(Biobutanol)逐漸受到重視。本研究的目的便是利用Clostridium acetobutylicum 的特性，回收微藻殘渣中有機物質作為ABE發酵的原料，並試驗不同的反應條件以提升丁醇產量。
本研究以經過2% 硫酸及2% 氫氧化鈉水解的微藻殘渣作為基質進行批次實驗。結果顯示，起始碳水化合物濃度大於30 g/L時即對菌種產生抑制現象，而使用 fed-batch 程序可有效去除基質的抑制效果，並使丁醇產量達8.83 g/L。在添加不同濃度丁酸的批次中，25 g/L 碳水化合物添加15 g/L 的丁酸有較佳的丁醇產率0.192 g-butanol/g-carbohydrogen。另外，先將殘渣提供產氫菌利用產製氫氣及丁酸，此丁酸再添加於ABE發酵當中；結果顯示，添加4.5 g/L 產氫出流之丁酸可使丁醇產率提升108%。而利用甲烷厭氧發酵回收微藻殘渣的批次實驗中，提供一階段反應程序20 g/L已水解殘渣有最好的產氣表現，氫氣及甲烷的產率分別是59 mL/g-OMR及243 mL/g-OMR。本研究中，一階段甲烷發酵有最佳的能源轉換效率10.43 kJ/g-OMR；ABE發酵添加4.5 g/L產氫出流之丁酸有最佳的能源轉換速率0.676 kJ/g-OMR/day。本研究發現Clostridium acetobutylicum可有效利用微藻殘渣，透過調整操作條件可進一步提升丁醇的產量及產率。

Since industrial resolution, fossil fuels supply most of the energy demand all over the world, which will be limited in the next few decades. The renewable energy becomes more and more important. During biodiesel extraction, the residues containing half of carbohydrate are usually remained which is the potential energy source that can be utilized. Therefore, this study has focused on the energy recovery from oil-extracted microalgae residues (OMR).
ABE fermentation carried out by Clostridium acetobutylicum was applied to treat the OMR. Bacteria could be inhibited when initial carbohydrate was higher than 30 g/L. Fed-batch process could eliminate the inhibitory effect by substrate and butanol production reached 8.83 g/L. Addition of 15 g/L butyrate to carbohydrate obtained the maximum butanol yield 0.192 g-butanol/g-carbohydrogen. On the other hand, OMR was fed to hydrogen producing bacteria to produce hydrogen and butyrate. This 4.5 g/L of biological butyrate was added to ABE fermentation and promoted the butanol yield about 108%. OMR was also fed to CH4 fermentation that one-stage process with 20 g/L pretreated OMR obtained the best performance. The yield of H2 and CH4 were 59 mL/g-OMR and 243 mL/g-OMR, respectively. The one-stage process of CH4 fermentation had the maximum energy generation 10.43 kJ/g-OMR. The ABE fermentation with 4.5 g/L biological butyrate addition obtained maximum energy generation rate 0.676 kJ/g-OMR/day. Results indicate that Clostridium acetobutylicum can utilize OMR and butanol can be enhance by appropriate operation.

Content
Abstract	I
摘要	III
誌謝	V
Content	VII
List of figures	X
List of tables	XII
Chapter 1	Introduction	1
Chapter 2	Literature Review	3
2.1	The trend of energy utilization and renewable energy	3
2.2	Biomass energy and its production technology	5
2.2.1	Overview of biomass energy	5
2.2.2	ABE fermentation	9
2.2.3	The advantages and of biobutanol	10
2.3	Butanol producing bacteria	11
2.3.1	Clostridium acetobutylicum	13
2.3.2	Clostridium beijerinckii	13
2.3.3	Clostridium saccharoperbutlacetonicum	14
2.4	The metabolic pathway of ABE fermentation	14
2.4.1	Acidogenesis pathway	16
2.4.2	Solventogenesis pathway	16
2.4.3	Fermentation phases and the corresponding cell cycle stages	18
2.4.4	Electron and energy flow	19
2.4.5	Redox balance and stoichiometric reactions in ABE fermentation	21
2.5	Different factors on ABE fermentation	23
2.5.1	The effect of pH	23
2.5.2	The utilization of lactate	25
2.5.3	The effect of acetate	26
2.5.4	The effect of butyrate	28
2.5.5	The pH buffer solutions and nutritional factors for ABE fermentation	31
2.6	Biohydrogen and methane production	31
2.6.1	Biohydrogen production and butyrate fermentation	31
2.6.2	Methane fermentation	32
2.7	Microalgae residues as substrate for ABE fermentation	33
2.8	Enhancement of performance in ABE fermentation	37
Chapter 3	Materials and Methods	41
3.1.1	Strain and growth conditions	41
3.1.2	Medium preparation	41
3.1.3	Oil-extracted microalgae residues pretreatment	42
3.1.4	Batched fermentation experiment	43
3.1.5	Analytical methods	45
Chapter 4	Results and Discussion	47
4.1	Fermentative biobutanol test under different initial OMR concentration	49
4.2	Fermentative biobutanol test carried out by fed-batch fermentation	54
4.3	Fermentative biobutanol test under addition of butyrate	61
4.4	Addition of butyrate from hydrogen fermentation effluent for ABE fermentation	66
4.4.1	Fermentative biobutyrate test with different hydrolysis methods	66
4.4.2	Fermentative biobutanol test with biological butyrate	68
4.5	Biofuel generation from OMR in other anaerobic fermentation	72
4.5.1	Methane fermentation by feeding pretreated and untreated OMR	72
4.5.2	Two-stage process by integrating hydrogen and methane fermentation	76
4.6	Comparison of overall performance between ABE fermentation and other anaerobic fermentation	81
Chapter 5	Conclusions and Suggestions	85
5.1	Conclusions	85
5.2	Suggestions	86
Chapter 6	Reference	87



 
List of figures
Fig. 2.1 World population growth from 1950 to 2050	3
Fig. 2.2 The trend of energy consumption. (Source: BP Energy Outlook, 2015)	4
Fig. 2.3 Global New Investment in Renewable Power and Fuels, Developed and	5
Fig. 2.4 Total world energy consumption by source in 2010 (Network, 2012)	6
Fig. 2.5 Different types of fuels obtainable from biomass (Muradov et al., 2008)	8
Fig. 2.6 Metabolic pathways of Clostridium acetobutylicum.	15
Fig. 2.7 Cell cycle of Clostridium acetobutylicum and its corresponding fermentation	19
Fig. 2.8 In vivo expression pattern of enzymes involved in carbon and electron flow in C. acetobutylicum ATCC 284. (Girbal et al., 1998)	20
Fig. 2.9 Tentative scheme of the terminal reactions in the fermentation of hexoses to butyrate, H2 and CO2 at low external pH values. Acetate formation has been left out for simplification of the scheme. A butyrate carrier (broken arrows) is considered not to be active under these conditions. (Gottwald et al., 1985)	29
Fig. 3.1 Diagram of one-stage bioprocess of CH4 fermentation applied in this study	44
Fig. 4.1 The product concentration profiles of different initial concentration of substrate test.	52
Fig. 4.2 The product concentration profiles of different substrate in bottle BK1 to BK4.	56
Fig. 4.3 The carbohydrate and product concentration profiles of different substrate in	59
Fig. 4.4 The product concentration profiles of different butyrate addition in bottle A to F.	63
Fig. 4.5 The butanol production with different concentration of butyrate addition.	64
Fig. 4.6 The product concentration profiles of butyrate fermentation and ABE fermentation with butyrate addition.	69
Fig. 4.7 The cumulative gas production in butyrate fermentation and ABE fermentation	71
Fig. 4.8 Profiles of fermentative CH4 production batch test with 20 g/L pretreated OMR.	74
Fig. 4.9 Profiles of fermentative CH4 production batch test with 20 g/L pretreated OMR.	75
Fig. 4.10 Profiles of two-stage process of hydrogen and methane fermentation with 30 g/L	78
 
List of tables
Table 2.1.1 Main renewable energy sources and their usage forms (Demirbaş, 2006)	5
Table 2.2.1 Options for biomass energy with different biomass resource (Rittmann, 2008)	7
Table 2.3.1 Species for ABE fermentation in different studies	13
Table 2.4.1 Stoichiometric reactions for Clostridium acetobutylicum (water and proton	22
Table 2.5.1 Three different stable metabolic state under different pH and conditions (Girbal et al., 1998)	24
Table 2.5.2 Acetate and glucose as co-substrate and pH for ABE fermentation	27
Table 2.5.3 Butyrate and glucose as co-substrate and pH for ABE fermentation	30
Table 2.7.1 Butanol fermentation from organic wastes by Clostridium sp.	36
Table 2.8.1 Different fermentation processes and performances of ABE fermentation	39
Table 4.1 Basic analysis of oil-extracted microalgae residues slurry........................48
Table 4.2 Detectable sugars from oil-extracted microalgae residues........................48
Table 4.1.1 The condition of different initial concentration of substrate test	49
Table 4.1.2 The production and yield of butanol and acids in different initial substrate test	53
Table 4.2.1 The condition of different substrate addition fed-batch test	55
Table 4.2.2 The production and yield of butanol and acids in contrast set	57
Table 4.2.3 The maximum butanol production and maximum butanol yield in fed-batch test	60
Table 4.3.1 The condition of different initial concentration of butyrate addition test	61
Table 4.3.2 The production and yield of solvent under different butyrate addition test	65
Table 4.4.1 The concentration of COD, carbohydrate and the fermentation results with different hydrolysis methods	67
Table 4.4.2 The condition of butyrate fermentation and the ABE fermentation with butyrate addition	68
Table 4.4.3 The production and yield of solvent and acid with butyrate addition from different source	70
Table 4.5.1 Methane, hydrogen yield and COD removal of one-stage and two-stage process	80
Table 4.6.1 Performance of ABE, H2 and CH4 production as well as energy generation	82

                                    

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