典型培養細菌的方式大多以培養盤或試管的方式進行，此種封閉隔離的環境，無法讓物質轉換傳遞，而顯著的限制細菌生長。另外，在培養環境中若累積過多欲收集之代謝產物，也會產生負向調控並降低最終產物之產量。現有商品化的生物反應器可藉由有效的物質轉換傳遞機轉以克服此問題，但缺點是此操作方法較複雜精細，且需要大量的培養液。本研究計畫欲發展一可連續補給養份並持續清除代謝廢物之微型細菌培養系統。由於光敏性水膠PEG-DA，具有良好的生物相容性、多孔性和選擇性滲透擴散特性等，且可利用光微影技術簡易方便地製造出微結構，故以其為建構微型生物反應器之材料。本研究首先探討不同濃度PEG-DA吸水率，結果顯示含較高濃度PEG-DA微結構，例如：100 % PEG-DA，其吸水率大約為10%。而較低濃度PEG-DA微結構，例如：20% 其吸水率最高可達210%。另外，我們也分析不同大小的分子在不同濃度PEG-DA的擴散速率。結果顯示50% PEG-DA微結構，可允許分子量小於1000 Da 的物質擴散通過，且隨著物質分子量越小其擴散通過此微結構速度越快。接著，我們設計和建構幾種不同的微結構，並以分離克雷白氏肺炎桿菌的代謝產物：乙醯甲基甲醇的效果優劣，做為評估後續設計是否適合微結構之參考。在H型、U型和圓型等設計當中，我們發現最適合細菌培養是U型微結構的設計。此U型PEG-DA微型生物反應器是由21個U型PEG-DA微培養槽的陣列所構成，每個培養槽長、寬和高分別為2 mm, 300 μm 和500 μm。我們實驗結果測得以2μl / min，為最適當之流速，可得到最高產量之乙醯甲基甲醇。另外，細菌可被包覆在微型生物反應器之生長空間，且此空間可允許乙醯甲基甲醇持續擴散至收集處長達24小時。在這段期間，克雷氏肺炎桿菌可於U型PEG-DA微型生物反應器內維持90%生長率長達24小時，表示此系統具適合長期細菌培養之效用。總結而言，此創新微型晶片式的生物反應器系統未來對於物質純化、菌種開發及新藥的篩選，將會有很大的幫助。

Microorganisms are typically grown in a culture dish or a tube, an isolated environment does not allow mass transfer and therefore significantly limits their growth. The accumulation of metabolites in the culture can also exert feedback regulation and reduce the final yields of the desired product. Current commercially available bioreactors can overcome these problems by providing efficient mass transfer mechanisms, although generally the instrumentation of the systems is sophisticate and the required medium volume is large. This study aims to develop a microscale bacterial culture system with a capability of continuous nutrient supply and waste removal. The photoresponsive hydrgel poly-ethylene glycol diacrylate (PEG-DA) was employed in fabricating the bioreactor because it is biocompatible, meshed and permselective, and can be made into microstructure conveniently through photopatterning techniques. This study first investigates the swelling ratio of PEG-DA of different percentages. The results showed that the swelling ratios of 100% and 20% PEG-DA were 10% and 210%, respectively. The diffusion rates of compounds of different sizes in the PEG-DA were also determined. The results revealed that PEG-DA at 50% concentration allows free diffusion of molecules smaller than 1000 Da. Subsequently, two growth chambers in H-, U-, and round shape were designed, fabricated, and tested for acetoin (3-hydroxy-2-butanone) production by Klebsiella pneumoniae. Among them, the U-shape micro-chamber array is the most suitable for bacterial cultures. The U-shape micro-bioreactor contains an array of 21 U-shape PEG-DA micro-chambers, each with a dimension of 300 μm × 2 mm × 500 μm (w × l × h). The optimal flow rate to achieve the highest yield of acetoin was determined to be 2 μl/min. The growth chamber can hold the bacteria inside and allows the acetoin diffuse out and 90% viability can be maintained for at least 24 h, indicating its suitability for long term bacteria growth. To sum up, this novel micro-scale chip-based bioreactor system will be useful in applications such as metabolite collection, drug toxicity and high-yield strain screening.

1.Hagedorn, S., and Kaphammer, B. (1994) Microbial biocatalysis in the generation of flavor and fragrance chemicals, Annu Rev Microbiol 48, 773-800.
2.Walsh, G. (2005) Therapeutic insulins and their large-scale manufacture, Appl Microbiol Biot 67, 151-159.
3.Graumann, K., and Premstaller, A. (2006) Manufacturing of recombinant therapeutic proteins in microbial systems, Biotechnol J 1, 164-186.
4.Carr, F. J., Chill, D., and Maida, N. (2002) The lactic acid bacteria: a literature survey, Crit Rev Microbiol 28, 281-370.
5.Ikeda, R., Yamamoto, T., and Funatsu, M. (1973) Chemical and Enzymatic Properties of Acid-Cellulase Produced by Aspergillus-Niger, Agr Biol Chem Tokyo 37, 1169-1175.
6.von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., and Habich, D. (2006) Antibacterial natural products in medicinal chemistry--exodus or revival?, Angew Chem Int Ed Engl 45, 5072-5129.
7.Martens, J. H., Barg, H., Warren, M. J., and Jahn, D. (2002) Microbial production of vitamin B12, Appl Microbiol Biotechnol 58, 275-285.
8.Macfarlane, G. T., and Cummings, J. H. (1999) Probiotics and prebiotics: can regulating the activities of intestinal bacteria benefit health?, West J Med 171, 187-191.
9.Paterson, R. R. (2006) Ganoderma - a therapeutic fungal biofactory, Phytochemistry 67, 1985-2001.
10.Holtzclaw, W. D., and Chapman, L. F. (1975) Degradative acetolactate synthase of Bacillus subtilis: purification and properties, J Bacteriol 121, 917-922.
11.Oppermann, F. B., Schmidt, B., and Steinbuchel, A. (1991) Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, J Bacteriol 173, 757-767.
12.Xiao, Z. J., and Xu, P. (2007) Acetoin metabolism in bacteria, Crit Rev Microbiol 33, 127-140.
13.Martin, I., Wendt, D., and Heberer, M. (2004) The role of bioreactors in tissue engineering, Trends Biotechnol 22, 80-86.
14.Szita, N., Boccazzi, P., Zhang, Z. Y., Boyle, P., Sinskey, A. J., and Jensen, K. F. (2005) Development of a multiplexed microbioreactor system for high-throughput bioprocessing, Lab Chip 5, 819-826.
15.Zhang, Z., Boccazzi, P., Choi, H. G., Perozziello, G., Sinskey, A. J., and Jensen, K. F. (2006) Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor, Lab Chip 6, 906-913.
16.Zhan, W., Seong, G. H., and Crooks, R. M. (2002) Hydrogel-based microreactors as a functional component of microfluidic systems, Analytical Chemistry 74, 4647-4652.
17.Becker, H., and Gartner, C. (2000) Polymer microfabrication methods for microfluidic analytical applications, Electrophoresis 21, 12-26.
18.Duffy, D. C., McDonald, J. C., Schueller, O. J. A., and Whitesides, G. M. (1998) Rapid prototyping of microfluidic systems in poly(dimethylsiloxane), Analytical Chemistry 70, 4974-4984.
19.McDonald, J. C., and Whitesides, G. M. (2002) Poly(dimethylsiloxane) as a material for fabricating microfluidic devices, Accounts Chem Res 35, 491-499.
20.McDonald, J. C., Duffy, D. C., Anderson, J. R., Chiu, D. T., Wu, H. K., Schueller, O. J. A., and Whitesides, G. M. (2000) Fabrication of microfluidic systems in poly(dimethylsiloxane), Electrophoresis 21, 27-40.
21.Wichterle, O., and Lim, D. (1960) Hydrophilic Gels for Biological Use, Nature 185, 117-118.
22.Hoffman, A. S. (2002) Hydrogels for biomedical applications, Adv Drug Deliv Rev 54, 3-12.
23.Tessmar, J. K., and Gopferich, A. M. (2007) Customized PEG-derived copolymers for tissue-engineering applications, Macromol Biosci 7, 23-39.
24.Nho, Y. C., Lim, Y. M., and Lee, Y. M. (2004) Preparation, properties and biological application of pH-sensitive poly(ethylene oxide) (PEO) hydrogels grafted with acrylic acid(AAc) using gamma-ray irradiation, Radiat Phys Chem 71, 239-242.
25.Moroni, L., De Wijn, J. R., and Van Blitterswijk, C. A. (2008) Integrating novel technologies to fabricate smart scaffolds, J Biomat Sci-Polym E 19, 543-572.
26.Zhu, J. M., Tang, C., Kottke-Marchant, K., and Marchant, R. E. (2009) Design and Synthesis of Biomimetic Hydrogel Scaffolds with Controlled Organization of Cyclic RGD Peptides, Bioconjugate Chem 20, 333-339.
27.Seong, G. H., Zhan, W., and Crooks, R. M. (2002) Fabrication of microchambers defined by photopolymerized hydrogels and weirs within microfluidic systems: Application to DNA hybridization, Analytical Chemistry 74, 3372-3377.
28.Kim, S. H., Nair, S., and Moore, E. (2005) Reactive electrospinning of cross-linked poly(2-hydroxyethyl methacrylate) nanofibers and elastic properties of individual hydrogel nanofibers in aqueous solutions, Macromolecules 38, 3719-3723.
29.Jeong, B., Kim, S. W., and Bae, Y. H. (2002) Thermosensitive sol-gel reversible hydrogels, Adv Drug Deliver Rev 54, 37-51.
30.Lee, N. Y., Jung, Y. K., and Park, H. G. (2006) On-chip colorimetric biosensor based on polydiacetylene (PDA) embedded in photopolymerized poly(ethylene glycol) diacrylate (PEG-DA) hydrogel, Biochem Eng J 29, 103-108.
31.Pathak, C. P., Sawhney, A. S., and Hubbell, J. A. (1993) Rapid Photopolymerization of Immunoprotective Gels in Contact with Cells and Tissue (Vol 114, Pg 8311, 1992), J Am Chem Soc 115, 2548-2548.
32.Nguyen, K. T., and West, J. L. (2002) Photopolymerizable hydrogels for tissue engineering applications, Biomaterials 23, 4307-4314.
33.Revzin, A., Russell, R. J., Yadavalli, V. K., Koh, W. G., Deister, C., Hile, D. D., Mellott, M. B., and Pishko, M. V. (2001) Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography, Langmuir 17, 5440-5447.
34.Hahn, M. S., Taite, L. J., Moon, J. J., Rowland, M. C., Ruffino, K. A., and West, J. L. (2006) Photolithographic patterning of polyethylene glycol hydrogels, Biomaterials 27, 2519-2524.
35.Peppas, N. A., Hilt, J. Z., Khademhosseini, A., and Langer, R. (2006) Hydrogels in biology and medicine: From molecular principles to bionanotechnology, Adv Mater 18, 1345-1360.
36.Juni, E. (1952) Mechanisms of formation of acetoin by bacteria, J Biol Chem 195, 715-726.
37.Lopez, J. M., Thoms, B., and Rehbein, H. (1975) Acetoin degradation in Bacillus subtilis by direct oxidative cleavage, Eur J Biochem 57, 425-430.
38.Huang, M., Oppermann-Sanio, F. B., and Steinbuchel, A. (1999) Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway, J Bacteriol 181, 3837-3841.
39.Deng, W. L., Chang, H. Y., and Peng, H. L. (1994) Acetoin catabolic system of Klebsiella pneumoniae CG43: sequence, expression, and organization of the aco operon, J Bacteriol 176, 3527-3535.
40.Matsukawa, K., Fukui, S., Higashi, N., Niwa, M., and Inoue, H. (1999) Preparation and properties of organic-inorganic hybrid thin films containing polysilane segments from polysilane-methacrylate copolymers, Chem Lett, 1073-1074.
41.Krampitz, L. O. (1957) Preparation and Determination of Acetoin, Diacetyl, and Acetolactate, Method Enzymol 3, 277-283.
42.Levine, M. (1916) On the significance of the Voges-Proskauer reaction, Journal of Bacteriology 1, 153-164.
43.Eddy, B. P. (1961) The Voges-Proskauer reaction and its significance: a review, J Appl Bacteriol 24, 27-41.
44.Mcfeters, G. A., Singh, A., Byun, S., Callis, P. R., and Williams, S. (1991) Acridine-Orange Staining Reaction as an Index of Physiological-Activity in Escherichia-Coli, J Microbiol Meth 13, 87-97.
45.Gant, V. A., Warnes, G., Phillips, I., and Savidge, G. F. (1993) The Application of Flow-Cytometry to the Study of Bacterial Responses to Antibiotics, J Med Microbiol 39, 147-154.
46.Zuo, S. S., and Lundahl, P. (2000) A micro-Bradford membrane protein assay, Anal Biochem 284, 162-164.
47.Carballo, J., Martin, R., Bernardo, A., and Gonzalez, J. (1991) Purification, Characterization and Some Properties of Diacetyl(Acetoin) Reductase from Enterobacter-Aerogenes, Eur J Biochem 198, 327-332.
48.Kim, B. Y., Hong, L. Y., Chung, Y. M., Kim, D. P., and Lee, C. S. (2009) Solvent-Resistant PDMS Microfluidic Devices with Hybrid Inorganic/Organic Polymer Coatings, Adv Funct Mater 19, 3796-3803.