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研究生：	周威宇 Chou, Wei-Yu
論文名稱：	以固定化Clostridium acetobutylicum細胞進行高效率二階段連續式丁醇醱酵 Developing a highly efficient two-stage continuous butanol fermentation process using immobilized Clostridium acetobutylicum cells
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	126
中文關鍵詞：	生質丁醇、固定化細胞、兩階段醱酵策略、丁酸、Clostridium acetobutylicum
外文關鍵詞：	Biobutanol, immobilized cells, two-stage fermentation, butyrate, Clostridium acetobutylicum
相關次數：	點閱：194 下載：0
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過去本實驗室以Clostridium acetobutylicum固定化細胞進行連續式丁醇醱酵的結果顯示，當系統操作在低水利滯留時間的條件下，儘管有良好的丁醇生產速率，但葡萄糖利用率僅達50%。為了改善此缺點，本研究利用兩階段丁醇醱酵策略來提升葡萄糖利用率，並進一步提高丁醇生產速率。在兩階段醱酵策略中，將總量為60 g/l的葡萄糖分成兩部分進料，在第一階段進行丁酸醱酵，乃將其中30 g/l的葡萄糖用於丁醇前驅物-丁酸的生產；第二階段則進行丁醇醱酵，乃將剩下的30 g/l 葡萄糖加上前面得到的丁酸用於丁醇的生產。在實際系統啟動前，先利用合成的丁酸測試丁酸在批次及流續流的系統中對丁醇生產的影響。由實驗結果發現，在批次的反應中當添加的丁酸小於5 g/l時，有助於提升丁醇的產量及產率。而在流續流的系統中，以葡萄糖濃度60 g/l及丁酸濃度7 g/l為進料時，可得最佳的丁醇產量(8.1±0.5 g/l)、丁醇生產速率(1.36±0.09 g/l/h)及產率(0.62±0.03 mol butanol/mol glucose)。接著，進一步將進料葡萄糖濃度降至30 g/l而丁酸進料濃度仍維持7 g/l時，葡萄糖利用率從原本的53%上升至100%，且丁醇的生產並無明顯下降。此外，為了得到最佳操作條件，使用實驗設計法來探討連續式操作時最佳丁醇產率及葡萄糖利用率的條件。結果顯示，當進料濃度在30~33 g/l的葡萄糖及5~7 g/l 的丁酸時有最佳的丁醇生產速率(1.2 g/l)及葡萄糖利用率(95%)。最後，以相同菌株(Clostridium acetobutylicum)用於第一階段的丁酸醱酵，當pH 6.0時30 g/l的葡萄糖可生產約8 g/l的丁酸，將丁酸醱酵液中添加葡葡糖至30 g/l及些許的營養物質且丁酸濃度調整至7 g/l後，做為第二階段的進料，發現丁醇的生產與用合成丁酸作為進料時相當接近，結果證實此兩階段流續式的策略確實可成功地應用於丁醇的生產，並大大提升丁醇產量、丁醇產率、丁醇生產速率、產氫速率及葡萄糖利用率。

Previous studies in our laboratory showed that continuous butanol production with immobilized Clostridium acetobutylicum had great butanol productivity but suffered poor glucose utilization efficiency (ca. 53%) at a short HRT of 6 h. In this study, a two-stage fermentation strategy was applied for butanol production to improve glucose utilization efficiency and further increase butanol productivity. In this two-stage process, the carbon source (totally 60 g/l of glucose) was equally divided into two halves. The first half was used in the first stage for the generation of butyric acid via acidogenesis metabolism and the butyric acid was then used as the precursor for butanol production in the next stage, where the second half of carbon source (i.e., 30 g/l glucose) was used for butanol production via solventogenesis pathway. Prior to the use of the biologically-produced butyrate, a commercially-acquired (synthetic) butyrate was added in batch and continuous modes to investigate the effect of butyric acid on butanol production performance. The results indicated that in batch culture, the addition of 5 g/l butyric acid with a glucose concentration of 60 g/l resulted in an increase in butanol concentration and yield. In continuous culture with 6 h HRT, feeding glucose concentration of 60 g/l with the addition of 7 g/l butyric acid, the C. acetobutylicum culture could achieve the highest butanol concentration, productivity, and yield of 8.1±0.5 g/l, 1.36±0.09 g/l/h, and 0.62±0.03 mol butanol/mol glucose, respectively. When feeding concentration of glucose was decreased from 60 to 30 g/l at an optimal butyric acid supplement of 7 g/l, glucose utilization significantly increased from 53% to 100% without obvious changes in butanol production performance. Moreover, the stable operational period of continuous ABE fermentation with immobilized-cell system was further extended to 88 days without apparent degeneration. In addition, experimental design tool was applied to optimize the conditions for the continuous culture. The optimal conditions determined from experimental design analysis was: glucose concentration, 30-33 g/l; butyric acid, 5-7 g/l; HRT, 6 h. Under these optimal conditions, the immobilized C. acetobutylicum could obtain a butanol productivity and glucose utilization efficiency of 1.2 g/l/h and 95%, respectively. For the two-stage operation, 30 g/l glucose was fermented in the first stage using the same strain under acidogenic conditions, producing nearly 8 g/l butyrate in the culture broth. The effluent of the first-stage process was used to prepare the culture medium for butanol production in the second stage (solventogenic stage). The butanol fermentation in continuous culture using 30 g/l glucose supplemented with 7 g/l butyrate produced in the first stage showed similar performance to that obtained from using pure butyric acid. Therefore, the two-stage fermentation strategy could be successfully applied in butanol production to increase the butanol productivity and glucose utilization.

摘要	I
Abstract	III
Acknowledgements	V
Contents	VIII
List of tables	XII
List of figures	XIV
Chapter 1	Introduction	1
1	Motivation and purpose	1
2	Research Scheme	4
Chapter 2	Literature review	6
1	Butyrate production	6
1.1	Chemical synthesis of butyric acid	7
1.2	Biological synthesis of butyric acid	7
1.2.1	Butyric acid producing strains	8
1.2.2	Metabolic Pathway and Regulation of microbial Strains which produce butyric acid as an end product	11
1.2.3	Metabolic Pathway and Regulation of bacterial Strains which produce butanol as an end product	16
2	Butanol production	19
2.1	Industrial importance of butanol as a fuel	19
2.2	Chemical synthesis of butanol	23
2.3	Biological synthesis of butanol	25
2.3.1	History	25
2.3.2	Microorganism	26
2.3.3	Metabolic pathways	27
2.3.3.1	Acid producing pathways	27
2.3.3.2	Solvent producing pathway	28
2.3.4	Challenges and solutions	33
3	Hydrogen production	34
3.1	Conventional hydrogen production methods	35
3.2	Biological hydrogen production	37
Chapter 3	Materials and methods	40
1	Chemical and materials	40
2	Equipment	42
3	Bacterial strains and cultivation conditions	43
4	Analytical methods	46
4.1	Measurement of gaseous products	46
4.2	Determination of the concentration of soluble metabolites by HPLC	47
4.3	Determination of the concentration of acetone, butanol and ethanol by GC	47
4.4	Analysis of transient behavior by modified Gompertz equation	47
4.5	Determination of Cell Concentration	50
5	Experimental methods	51
5.1	Immobilization of Clostridium acetobutylicum with Poly-vinyl-alcohol (PVA)	51
5.2	Effect of butyric acid supplement on batch butanol fermentation with PVA-immobilized cells of Clostridium acetobutylicum	51
5.3	Effect of butyric acid supplement on continuous butanol fermentation with PVA-immobilized cells of Clostridium acetobutylicum	52
5.4	Effect of pH control on butyric acid production	52
5.5	Effect of different concentrations of supplemented media components (CRM) in butyrate fermentation broth with cell removing in batch culture	52
5.6	Effect of different concentrations of supplemented media components (CRM) in butyrate fermentation broth with cell removing in continuous culture	53
5.7	Effect of different concentrations of supplemented media components (CRM) in butyrate fermentation broth without cell removing in continuous culture	53
5.8	Integration of the two stage continuous fermentation	53
Chapter 4	Results and discussion	54
1	Effect of butyric acid concentration on immobilized C. acetobutylicum ATCC 824	54
1.1	Effect of initial butyric acid concentration on butanol production performance in batch culture	54
1.2	Effect of butyric acid (commercially-acquired) concentration on butanol production performance in continuous culture	64
1.3	Analysis by experimental design tool	80
2	Effect of controlled-pH on the butyrate performance of C. acetobutylicum	94
3	Effect of fermented butyric acid on the butanol performance of C. acetobutylicum	100
3.1	Effect of different concentrations of supplemented CRM components in butyrate fermentation broth with cell removing in batch culture	100
3.2	Effect of different concentrations of supplemented CRM components in butyrate fermentation broth with cell removing in continuous culture	108
3.3	Effect of different concentrations of supplemented CRM components in butyrate fermentation broth without cell removing in continuous culture	109
Chapter 5	Conclusions	112
Reference	114


                                    

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