臺灣博碩士論文加值系統

氫氣被視為下一世代的能源，是目前最理想的清潔燃料之一，因此產氫之技術成為一大研究領域。近年環保意識抬頭，環境汙染問題也是一大課題。產氫菌發酵系統不僅可以處理農工業之廢水，還能夠產生氫氣作為能量來源，是許多科學家研究之議題，然而因發酵系統為複雜之微生物反應，為了解並發展該技術需耗費大量時間、人力及資源。本研究期望發展縮小化之培養系統，使技術可同步測試多組參數。本研究利用連續式產氫發酵系統作為實驗之菌源(初始菌種：Clostridium butyricum CGS5、Clostridium pasteurianum CH4、Klebsiella sp. HE1)，以不同方式去追求縮小之發酵系統。實驗中證明可將油品覆蓋在12孔盤中的在培養基上幫助阻絕空氣中之氧氣，而溶液中之溶氧可透過添加還原劑於培養基中來消除，也可依靠混菌中兼性菌的代謝消耗液體內溶氧，但無添加還原劑之系統需更長的培養時間系統才能逐漸趨向厭氧狀態。本研究也用PDMS建構密閉式縮小型發酵槽，並可在其中培養產氫之混菌，且不需覆蓋油品或於培養基中添加還原劑，但培養時間需拉長至10天以上。以12孔盤和PDMS縮小型發酵槽做比較，密閉式培養系統相較之下，其優點在於可蒐集氣體產物，但培養體積小所以氣體產量少，影響數據可信度，因此需要搭配微型氫氣感測器做即時偵測，方能有潛力取代實驗室規模之實驗。

Hydrogen has been regarded as the fuel of the next generation due to its usefulness as a compact energy source in fuel cells and batteries. It has been discovered that hydrogen can be generated through natural biological processes. However, high production cost is the major challenge for the commercialization of biohydrogen. Scientists try to solve the problem by conducting numerous lab-scale experiments, which are high-cost, labor-intensive, and space-taking. In this study, we aim to reduce the cost of experiments by developing miniature culture system which can provide valuable information for optimizing larger scale experiments.

In this study, a 7 ml fermenter made by PDMS was tested with semi-batch cultivations. HPLC and GC were applied to evaluate the metabolites of a mixed culture bacteria. The results show that the air-tight PDMS device can successfully cultivate mixed culture bacteria of Clostridium butyricum CGS5, Clostridium pasteurianum CH4, and Klebsiella sp. HE1. Metabolites including 1.8 g/l acetate and 1.43 g/l butyrate were measured to be the main products of the system after 10 days of cultivation. However, the amount of gas sample was small and hard to collect, which made the quantification easily compromised by leakage. Therefore, we try to quantify hydrogen in real time by palladium nanoparticles coated with temperature-sensitive luminophores, EuTTA. Unfortunately, the results show no obvious relationship between hydrogen partial pressure and luminescence strength.

摘要 I
Extended Abstract II
致謝 X
目錄 XI
表目錄 XIII
圖目錄 XIV
第一章 緒論 1
1-1 研究動機與目的 1
1-2 實驗架構 2
第二章 文獻回顧 3
2-1 傳統產氫技術 3
2-2 生物產氫技術 5
2-3生物產氫技術挑戰 11
2-4 增進產氫效率之策略 13
2-5 微小化培養系統之需求 16
第三章 實驗材料與方法 18
3-1 連續式產氫發酵系統 18
3-1-1 實驗材料 18
3-1-2 實驗方法 20
3-2 微流道包菌 24
3-2-1 實驗材料 24
3-2-2 實驗方法 27
3-3 12孔盤培養 34
3-3-1 實驗材料 34
3-3-2 實驗方法 35
3-4 縮小型發酵槽培養 36
3-4-1 實驗材料 36
3-4-2 實驗方法 36
3-5 微型氫氣感測器 39
3-5-1 實驗材料 39
3-5-2 實驗方法 41
第四章 結果與討論 43
4-1 連續式產氫發酵系統 43
4-2 固定化細菌培養 47
4-2-1 微流道裝置生成包菌粒子 47
4-2-2 微流道凹槽固定化 49
4-2-3 高分子膠體固定化 51
4-3 12孔盤培養 56
4-3-1 培養時間 56
4-3-2 彌封油品 60
4-3-3 還原劑影響 65
4-4 縮小型發酵槽培養 67
4-5 微型氫氣感測器 70
4-5-1 製造鈀奈米粒子之結果 70
4-5-2 鈀奈米粒子附加螢光染劑感測氫氣 75
第五章 結論 80
參考文獻 81

1.Asada, Y., ＆ Miyake, J. Photobiological hydrogen production. Journal of Bioscience and Bioengineering 88 (1):1-6. 1999
2.Basak, N., ＆ Das, D. The prospect of purple non-sulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art. World Journal of Microbiology and Biotechnology, 23(1), 31-42. 2007
3.Basak, N., ＆ Das, D. Photofermentative hydrogen production using purple non-sulfur bacteria Rhodobacter sphaeroides OU 001 in an annular photobioreactor: a case study. Biomass and Bioenergy, 33(6), 911-919. 2009
4.Beckers, L., Hiligsmann, S., Hamilton, C., Masset, J., ＆ Thonart, P. Fermentative hydrogen production by Clostridium butyricum CWBI1009 and Citrobacter freundii CWBI952 in pure and mixed cultures/Production d'hydrogène par Clostridium butyricum CWBI1009 et Citrobacter freundii CWBI952 en cultures pures et mixtes. Biotechnologie, Agronomie, Société et Environnement, 14, 541. 2010
5.Chaubey, R., Sahu, S., James, O. O., ＆ Maity, S. A review on development of industrial processes and emerging techniques for production of hydrogen from renewable and sustainable sources. Renewable and Sustainable Energy Reviews, 23, 443-462. 2013
6.Das, D., ＆ Veziroǧlu, T. N. Hydrogen production by biological processes: a survey of literature. International Journal of Hydrogen Energy, 26(1), 13-28. 2001
7.Das, D., Khanna, N., ＆ Veziroğlu, N. T. Recent developments in biological hydrogen production processes. Chemical Industry and Chemical Engineering Quarterly/CICEQ, 14(2), 57-67. 2008
8.Elsharnouby, O., Hafez, H., Nakhla, G., ＆ El Naggar, M. H. A critical literature review on biohydrogen production by pure cultures. International Journal of Hydrogen Energy, 38(12), 4945-4966. 2013
9.Fedorov, A. S., Tsygankov, A. A., Rao, K. K., ＆ Hall, D. O. Hydrogen photoproduction by Rhodobacter sphaeroides immobilised on polyurethane foam. Biotechnology letters, 20(11), 1007-1009. 1998
10.Ghosh, D., Sobro, I. F., ＆ Hallenbeck, P. C. Optimization of the hydrogen yield from single-stage photofermentation of glucose by Rhodobacter capsulatus JP91 using response surface methodology. Bioresource technology, 123, 199-206. 2012
11.Girbal, L., Croux, C., Vasconcelos, I. and Soucaille, P. Regulation of metabolic shift in Clostridium acetobutylicum ATCC 824. FEMS Microbiol Rev 17:287-297. 1995
12.Ginkel, S. V., Sung, S., ＆ Lay, J. J. Biohydrogen production as a function of pH and substrate concentration. Environmental science ＆ technology, 35(24), 4726-4730. 2001
13.Kensy, F. T. Online monitoring in continuously shaken microtiter plates for scalable upstream bioprocessing (Doctoral dissertation, Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen). 2010
14.Kumar Gupta, S., Kumari, S., Reddy, K., ＆ Bux, F. Trends in biohydrogen production: major challenges and state-of-the-art developments. Environmental technology, 34(13-14), 1653-1670. 2013
15.Levin, D. B., Pitt, L., ＆ Love, M. Biohydrogen production: prospects and limitations to practical application. International journal of hydrogen energy, 29(2), 173-185. 2004
16.Liu, Y., Yu, P., Song, X., ＆ Qu, Y. Hydrogen production from cellulose by co-culture of Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17. International journal of hydrogen energy, 33(12), 2927-2933. 2008
17.Lo, Y. C., Bai, M. D., Chen, W. M., ＆ Chang, J. S. Cellulosic hydrogen production with a sequencing bacterial hydrolysis and dark fermentation strategy. Bioresource technology, 99(17), 8299-8303. 2008
18.Lu, W., Wen, J., Chen, Y., Sun, B., Jia, X., Liu, M., ＆ Caiyin, Q. Synergistic effect of Candida maltosa HY-35 and Enterobacter aerogenes W-23 on hydrogen production. International journal of hydrogen energy, 32(8), 1059-1066. 2007
19.Masset, J., Calusinska, M., Hamilton, C., Hiligsmann, S., Joris, B., Wilmotte, A., ＆ Thonart, P. Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp. Biotechnology for biofuels, 5(1), 1. 2012
20.Maeda, K., Teramura, K., Lu, D., Takata, T., Saito, N., Inoue, Y., ＆ Domen, K. Photocatalyst releasing hydrogen from water. Nature, 440(7082), 295-295. 2006
21.Melis, A., ＆ Happe, T. Hydrogen production. Green algae as a source of energy. Plant physiology, 127(3), 740-748. 2001
22.Melis A., Green alga hydrogen production: progress, challenges and prospects. International Journal of Hydrogen Energy, 27, 1217-1228. 2002
23.Miyake, J. The science of biohydrogen: An energetic view. In BioHydrogen. Zaborsky, OR Ed., Plenum Press: New York, 7-18. 1998
24.Momirlan, M., ＆ Veziroǧlu, T. Recent directions of world hydrogen production. Renewable and Sustainable Energy Reviews, 3(2), 219-231. 1999
25.Muhich, C. L., Evanko, B. W., Weston, K. C., Lichty, P., Liang, X., Martinek, J., ... ＆ Weimer, A. W. Efficient generation of H2 by splitting water with an isothermal redox cycle. Science, 341(6145), 540-542. 2013
26.Ni, M., Leung, D. Y., Leung, M. K., ＆ Sumathy, K. An overview of hydrogen production from biomass. Fuel processing technology, 87(5), 461-472. 2006
27.Pataki, D. E., Alig, R. J., Fung, A. S., Golubiewski, N. E., Kennedy, C. A., McPherson, E. G., ... ＆ Romero Lankao, P. Urban ecosystems and the North American carbon cycle. Global Change Biology, 12(11), 2092-2102. 2006
28.Pan, C., Zhang, S., Fan, Y., ＆ Hou, H. Bioconversion of corncob to hydrogen using anaerobic mixed microflora. International Journal of Hydrogen Energy, 35(7), 2663-2669. 2010
29.Sakaue, H., Huang, C. Y., ＆ Sullivan, J. P. Optical hydrogen sensing method using temperature-sensitive luminophore on porous palladium. Sensors and Actuators B: Chemical, 155(1), 372-374. 2011
30.Schopf, J. W. The fossil record: tracing the roots of the cyanobacterial lineage. In The ecology of cyanobacteria (pp. 13-35). Springer Netherlands. 2000
31.Sekoai, P. T., ＆ Kana, E. B. G. Fermentative biohydrogen modelling and optimization research in light of miniaturized parallel bioreactors. Biotechnology ＆ Biotechnological Equipment, 27(4), 3901-3908. 2013
32.Seppälä, J. J., Puhakka, J. A., Yli-Harja, O., Karp, M. T., ＆ Santala, V. Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures. international journal of hydrogen energy, 36(17), 10701-10708. 2011
33.Smith, G. D., Ewart, G. D., ＆ Tucker, W. Hydrogen production by cyanobacteria. International journal of hydrogen energy, 17(9), 695-698. 1992
34.Teranishi, T., ＆ Miyake, M. Size control of palladium nanoparticles and their crystal structures. Chemistry of Materials, 10(2), 594-600. 1998
35.Tsygankov, A. A., Fedorov, A. S., Laurinavichene, T. V., Gogotov, I. N., Rao, K. K., ＆ Hall, D. O. Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus. Applied Microbiology and Biotechnology, 49(1), 102-107. 1998
36.Yetis, M., Gündüz, U., Eroglu, I., Yücel, M., ＆ Türker, L. Photoproduction of hydrogen from sugar refinery wastewater by Rhodobacter sphaeroides OU 001. International Journal of Hydrogen Energy, 25(11), 1035-1041. 2000
37.Yoshida, A., Nishimura, T., Kawaguchi, H., Inui, M., ＆ Yukawa, H. Enhanced hydrogen production from glucose using ldh-and frd-inactivated Escherichia coli strains. Applied microbiology and biotechnology, 73(1), 67-72. 2006
38.Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S., ＆ Takasaki, Y. H2 production from starch by a mixed culture of Clostridium butyricum and Enterobacter aerogenes. Biotechnology Letters, 20(2), 143-147. 1998
39.Zhang, Z., Perozziello, G., Boccazzi, P., Sinskey, A. J., Geschke, O., ＆ Jensen, K. F. Microbioreactors for bioprocess development. Journal of the Association for Laboratory Automation, 12(3), 143-151. 2007
40.Zhang, Z., Szita, N., Boccazzi, P., Sinskey, A. J., ＆ Jensen, K. F. A well‐mixed, polymer‐based microbioreactor with integrated optical measurements. Biotechnology and bioengineering, 93(2), 286-296. 2006
41.Zhang, C., Yang, H., Yang, F., ＆ Ma, Y. Current progress on butyric acid production by fermentation. Current microbiology, 59(6), 656-663. 2009
42.Zhu, H., Suzuki, T., Tsygankov, A. A., Asada, Y., ＆ Miyake, J. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. International Journal of Hydrogen Energy, 24(4), 305-310. 1999