臺灣博碩士論文加值系統

本研究是利用疏水性離子液體，對Clostridium acetobutylicum進行丁醇萃取式醱酵並探討其影響及可行性。Clostridium acetobutylicum在進行ABE醱酵時，當產物丁醇達到10 g/L時，其菌體會受到產物抑制效應的影響而失活，萃取式醱酵可以降低產物濃度改善此問題，而本實驗將探討一項新的製程，利用離子液體[BMIM][PF6]、[HMIM][PF6]及[OMIM][PF6]作為萃取劑，實驗其最佳萃取體積、接觸時間長短對菌體的毒性及大型醱酵槽萃取式醱酵，其中以離子液體[HMIM][PF6]作為萃取劑效果最佳，在醱酵進行至48 小時進行萃取，萃取時間5 分鐘，其可將丁醇由7.956 g/L萃取至7.017 g/L，且在醱酵進行至第84 小時可達最大產量9.895 g/L，產率為0.118 g/L/hr，雖然產量和產率皆稍低於控制組實驗之10.111 g/L和0.140 g/L/hr，但其單位基質對菌重之轉化率及單位菌重對丁醇之轉化率皆略優於控制組實驗，即是其萃取後，使菌體較不被受到產物抑制效應影響，其利用葡萄糖維持自身活性的比例下降、生產丁醇的比例上升，因此利用離子液體作為萃取劑之製程的可行性，值得深入研究探討。

In this research, we use hydrophobic ionic liquid to butanol
extractive fermentation of Clostridium acetobutylicum, then discuss its effect and feasibility. When the concentration of butanol get 10 g/L in ABE fermentation, the Clostridium acetobutylicum will be inhibited by the product, butanol, and lose activation. The extractive fermentation will improve this problem by reducing the concentration of butanol. The experiment will explore a new process using ionic liquid [BMIM][PF6], [HMIM][PF6] and [OMIM][PF6] as the extractants, test the optimum volume of extraction, the toxic of contact time, and extractive fermentation of large fermentor. The best extractant in this experiment is ionic liquid [HMIM][PF6], when the fermentation gets to 48 hours, we extract by [HMIM][PF6] in 5 minutes, the concentration of butanol extracted from 7.956 g/L to 7.017 g/L. After 84 hours, the maximum yield of butanol is 9.895 g/L, and the productivity is 0.118 g/L/hr. Though the yield and productivity are little less than controlled experiment,
10.111 g/L and 0.140 g/L/hr, the Yx/s and Yp/x are more better than controlled. Because the broth after extracted, the bacteria used glucose to maintain itself is less, more used to produce butanol. Therefore, the process feasibility of using ionic liquids as extractant, is worth in-depth study.

摘要i
Abstractii
誌謝iii
目錄iv
圖目錄vii
表目錄ix
第一章　緒論2
1-1　研究動機2
1-2　研究目的3
第二章　文獻回顧4
2-1　再生能源4
2-1-1　再生能源之介紹4
2-1-2　生質能源發展及應用4
2-1-3　生質丁醇的介紹6
2-2　丁醇生產方法8
2-2-1　菌種之介紹9
2-2-2　ABE 醱酵10
2-3　ABE醱酵製程改良介紹12
2-3-1　氣提法12
2-3-2　固定化13
2-3-3　薄膜分離法13
2-3-4　萃取法14
2-3-5　基因工程14
2-4　離子液體之介紹15
2-4-1　離子液體的定義15
2-4-2　離子液體的發展17
2-4-3　離子液體的特殊物理性質17
2-4-4　離子液體的應用18
2-4-5　離子液體的毒性研究19
第三章　材料與方法22
3-1　實驗規劃22
3-2　實驗材料與設備23
3-2-1　實驗菌株23
3-2-2　實驗藥品24
3-2-3　實驗儀器及設備25
3-3　實驗方法27
3-3-1　菌種活化及保存27
3-3-2　培養基組成27
3-3-3　離子液體製備31
3-3-4　最佳萃取比例35
3-3-5　離子液體接觸時間毒性測試36
3-3-6　萃取式醱酵37
3-4　分析方法38
3-4-1　細胞乾重對光學密度之檢量線38
3-4-2　葡萄糖殘量分析38
3-4-3　丁醇濃度分析40
第四章　實驗結果與討論43
4-1　比較各種疏水性離子液體對丁醇水溶液最佳萃取比例43
4-2　培養基與離子液體之接觸時間對菌體抑制影響48
4-2-1　與離子液體接觸時間之探討48
4-2-2　進行萃取時間點之探討52
4-3　以離子液體對C. acetobutylicum進行萃取式醱酵53
4-3-1　控制組實驗57
4-3-2　以離子液體進行萃取式醱酵57
第五章　結論與建議60
5-1　結論 60
5-2　建議 61
第六章　參考文獻63

周世凱，許梅娟，新能源－生物產丁醇，科學發展，433　:　26-31,　2009.

陳勁中，淺談生質丁醇及未來研發趨勢，石油通訊，716　:　20-23,　2011.

黃浩宸，探討可控式包埋Saccharomyces cerevisiae 對於乙醇醱酵之影響，國立中央大學化學工程與材料工程學系碩士論文，2011。

蔡文慶，生質乙醇的生產與發展現況，朝陽科技大學應用化學系碩士論文，2010。

Hall D.O., Biomass Energy in Industrialized Countries: A View of the Further. Forest Ecology and Management, 91: 17-45, 1997.

Chen M., Zhao J., Xia L., Enzymatic Hydrolysis of Maize Straw Polysaccharides for the Production of Reducing Sugars. Carbohydrate Polymers, 71: 411-415, 2008.

Chum H. L., Koch V. R., Miller L. L., Osteryong R. A., Electrochemical scrutiny of organometallic iron complexes and hexamethylbenzene in a room temperature molten salt. Journal of the American Chemical Society, 97:3264-3275, 1975.
Couling D. J., Bernot R. J., Docherty K. M., Dixona J. K., Maginn E. J., Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure–property relationship modeling. Green Chem, 8:82–90, 2006.

Cserjesi P., Belafi-Bako K., Application of Ionic Liquids in Membrane Separation Processes. Applications and Perspectives, 25:561-586, 2011.

Desai R.P., Papoutsakis E.T., Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum. Appl. Environ Microbiol, 65: 936-945, 1999.

Fannin Jr A. A., Floreani D. A., King L. A., Landers J. S., Piersma B. J., Stech D. J., Properties of 1,3-dialkylimidazolium chloride-aluminum chloride ionic liquids. 2. Phase transitions, densities, electrical conductivities, and viscosities. American Chemical Society, 88:2614-2621, 1984.

Friedl A., Qureshi N., Maddox I. S., Continuous Acetone-Butanol-Ethanol (ABE) Fermentation Using Immobilized Cells of Clostridium acetobutylicum in a Packed Bed Reactor and Integration with Product Removal by Pervaporation. Biotechnology and Bioengineering, 38:518-527, 1991.

Gapes J. R., The Economics of Acetone Butanol Fermentation, Theoretical and Market Consideration. J. Mol Microbiol Biotechnol, 2: 27-32, 2000.

Ha S. H., Mai N. L., Koo Y. M., Butanol recovery from aqueous solution into ionic liquids by liquid–liquid extraction. Process Biochemistry, 45:1899-1903, 2010.

Hurley F. H., Wier T. P., The Electrodeposition of Aluminum from Nonaqueous Solutions at Room Temperature. Journal of the Electrochemical Society, 98:207-212, 1951.

Jastorff B., Molter K., Behrend P., Bottin-Weber U., Filser J., Heimers A., Ondruschka B., Ranke J., Schaefer M., Schroder H., Stark A.; Stepnowski P., Stock F., Stormann R., Stolte S., Welz-Biermann U., Ziegert S., Thoming J., Progress in evaluation of risk potential of ionic liquids-basis for an eco-design of sustainable products. Green Chem, 7:362-372, 2005.

Jork Nolling, Gary Breton, Zeng Q., Gibson R., Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum. J. Bacteriol., 183(16):4823-4838, 2001.

Keskina S., Kayrak-Talaya D., Akman U., Hortacsu O., A review of ionic liquids towards supercritical fluid applications. The Journal of Supercritical Fluids, 43:150-180, 2007.

Lee S. Y., Park J. H.; Jang S. H., Nielsen L. K., Kim J., Jung K. S., Fermentative Butanol Production by Clostridia. Biotechnology and Bioengineering. 101: 209-228, 2008.

Macfarlane D. R., Forsyth M., Howlett P. C., Ionic Liquids in Electrochemical Devices and Processes : Managing Interfacial Electrochemistry. Accounts of Chemical Research, 40:1165-1173, 2007.

Mariano A. P., Qureshi N., Filho R. M., Ezeji T. C., Assessment of in situ butanol recovery by vacuum during acetone butanol ethanol (ABE) fermentation. Society of Chemical Industry, 87(3):334-340, 2012.

Olivier-Bourbigou H., Magna L., Morvan D., Ionic liquids and catalysis: Recent progress from knowledge to applications. Applied Catalysis A: General, 373:1-56, 2010.

Park S., Kazlauskas R. J., Biocatalysis in ionic liquids-advantages beyond green technology. Current Opinion in Biotechnology, 14:432-437, 2003.

Qureshi N., Blaschek H. P., Recovery of butanol from fermentation broth by gas stripping. Renewable Energy, 22 : 557–564, 2001.


Qureshi N., Li X. L., Hughes S., Saha B. C., Cottaa M. A., Butanol Production from Corn Fiber Xylan Using Clostridium Acetobutylicum. Biotechnol Prog., 22(3): 673-680, 2006.

Qureshi N., Saha B. C., Cotta M. A., Butanol Production from Wheat Straw Hydrolysate Using Clostridium Beijerinckii. Bioprocess & Biosyst Eng., 30(6): 419-427, 2007.

Raves M. L., Harel M., Pang Y. P., Silman I., Kozikowski A. P., Sussman J. L., Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A. Nat Struct Biol, 4(1):57–63, 1997.

Sajilata M. G., Singhal R. S., Kulkarni P. R., Resistant Starch - A review. Comprehensive Reviews in Food Science and Food Safety, 5:1-17, 2006.

Silvester D. S., Aldous L., Hardacre C., An electrochemical study of the oxidation of hydrogen at platinum electrodes in several room temperature ionic liquids. Journal of Physical Chemistry B, 111:5000-5007, 2007.

Stolte S., Arning J., Bottin-Weber U., Matzke M., Stock F., Thiele K., Uerdingen, M., Welz-Biermann U., Jastorff B., Ranke J., Anion effects on the cytotoxicity of ionic liquids. Green Chem., 8: 621-629, 2006.


Stepnowski P., Składanowski A. C., Ludwiczak A., Laczyńska E., Evaluating the cytotoxicity of ionic liquids using human cell line HeLa. Hum. Exp. Toxicol, 23:513-517, 2004.

Tashiro Y., Takeda K., Kobayashi Y., Sonomoto K., Ishizaki A., Yoshino S., High Butanol Production by Clostridium saccharoperbutylacetonicum N1-4 in Fed Batch Culture with pH-stat Continuous Butyric Acid and Glucose Feeding Method. J. Biosci Bioeng., 98(4): 263-268, 2004.

Wells A. S., Coombe V. T., On the Freshwater Ecotoxicity and Biodegradation Properties of Some Common Ionic Liquids. Org. Process Res. Dev., 10(4):794-798, 2006.

Welton T., Room-Temperature Ionic Liquids. Solvents for Synthesis and Catalysis. Chemical reviews, 99:2071-2084, 1999.

Wilkes J. S., A short history of ionic liquids - from molten salts to neoteric solvents. Green Chemistry, 4:73-80, 2002.

Wilkes J. S., Levisky J. A., Wilson R. A., Hussey C. L., Dialkylimidazolium chloroaluminate melts: a new class of room-temperature ionic liquids for electrochemistry, spectroscopy and synthesis. Inorganic Chemistry, 21:1263-1274, 1982.

Wilkes J. S., Zaworotko M. J., Air and water stable 1-ethyl-3-methylimidazolium based ionic liquids. Journal of the Chemical Society, Chemical Communications, 965-967, 1992.

Zhao D., Wang Y., Duan E., Oxidative Desulfurization of Fuel Oil by Pyridinium-Based Ionic Liquids. Molecules, 14:4351-4357, 2009.