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研究生:林昀輝
研究生(外文):Yun-Huin Lin
論文名稱:微生物固定化與發酵技術之開發
論文名稱(外文):The technology developments in microorganisms immobilization abd fermentation
指導教授:陳國誠陳國誠引用關係
指導教授(外文):Kuo-Cheng Chen
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:111
中文關鍵詞:微生物固定化聚胺基甲酸酯聚乙烯醇聚丙烯醯胺批式培養
外文關鍵詞:microorganismsimmobilizationpolyurethanepolyvinyl alcholPolyacrylamideclavulanic acidStreptomyces clavuligerusfed-batch
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本研究以開發微生物固定化與發酵技術為主軸,探討不同的固定化技術的特性和其在不同領域的應用,並探討二次代謝產物clavulanic acid 的發酵生產,以做為固定化生產生產之基礎。內容主要包括四大部分,分別為1.利用本實驗是先前所開發成功的polyvinyl alcohol (PVA)固定化活性污泥,進行養豬場實際廢水的處理。2.改良利用polyacrylamide固定化微生物的程序。3.利用新型水性polyurethane開發微生物固定化技術。4.探討clavulanic acid生產的特性,做為固定化生產的基礎。
在第一部分利用一個PVA固定化菌體反應器,以連續曝氣、間歇曝氣和以氧化還原電位即時監控的間歇曝氣三種操作模式,探討其對養豬場實際廢水同時去除碳與氮的效率。結果顯示在間歇曝氣的操作中的COD和T-N的去除率均比在連續曝氣的操作下高;而且以ORP 控制曝氣可以使循環時間減少20%。
第二部分探討伴隨褐藻膠在水溶液中將Candida tropicalis包埋在PAA,研究中探討單體與交聯劑濃度對顆粒內基質擴散的影響,且將此固定化菌體在氣舉式反應器中處理含高濃度酚的人工廢水。在反應器的連續操作中,在酚的入流濃度範圍高達 5000 mg l-1時系統仍可保持95%的去除率,此時反應器最大去除速率為7.68 g l-1 d-1。
第三部分是利用陰離子型水性PU開發一種新型細胞固定化技術,研究中以酵母菌做為測試細胞,陰離子型水性PU顯現無毒性且可在溫和的條件下包埋細胞。所生成的膠體顆粒具有高機械強度,研究終將探討其成膠的機制,並將其應用在酒精生產程序,結果顯示本固定化方法具有很大的應用潛力。
本論文最後兩章則探討在Streptomyces clavuligerus中clavulanic acid的生化合成。在所使用的基質中,甘油是最重要的營養原,持續添加甘油可持續菌體代謝維持clavulanic acid的生成,而添加ornithine可促進clavulanic acid的生產,但是添加arginine則無效果。由結果可知甘油的提供是速率決定步驟而非氨基酸的添加。Ornithine主要扮演的角色是抑制其他使用甘油生成cephamycin的路徑, 因此,在甘油充足之下ornithine比arginine更能促進clavulanic acid的生產。
The objectives of this research are to gain more science and engineering insights regarding the development and application of cell immobilization and fermentation technique. Three kinds of material, polyvinyl alcohol (PVA), polyacrylamide (PAA) and anionic polyurethane (APU) will be investigated in this thesis. In addition, a system for the production of clavulanic acid, a -lactamase inhibitor, was established which is expected to be used in immobilization process.
A single stage PVA immobilized-cell reactor with three operation modes, i.e. continuous aeration, intermittent aeration (IA), and IA with real-time control using oxidation-reduction-potential (ORP), was used to investigate the efficiency of simultaneous removal of carbon and nitrogen from raw swine wastewater. The results revealed that the chemical oxygen demand (COD) removal efficiency and total-nitrogen (T-N) removal efficiency in the IA mode were higher than that in the continuous aeration mode. When the ORP control was adopted in the IA mode, the total cycle time could be reduced by about 20%.
Immobilized growing cells of Candida tropicalis were prepared by entrapment into polyacrylamide (PAA) gel beads. The formation of PAA gel beads was accomplished in aqueous phase associated with the gelling reaction of Ca-alginate. Effects of monomer concentration and crosslinker content on the diffusivity of phenol in gel beads were studied. The immobilized-cell beads were used to degrade phenol in an air-lift bioreactor undergoing continuous operation. A stable removal efficiency of more than 95 % was achieved even at an inlet phenol concentration as high as 5000 mg l-1. And, the maximum biodegradation rate of 7.68 g l-1 d-1 was reached.
A new cell immobilization method based on the gelation of anionic polyurethane (APU) has been developed. APU is non-toxic against living yeast cells and the preparation of gel-entrapped cells can easily be achieved under very mild conditions. The formed gel beads then posses high mechanical strength. The immobilization method was illustrated by an ethanol production process using bakers’ yeast (Saccharomyces cerevisiae). The results imply that the cells entrapped by this method are useful for a variety of purposes
The addition of glycerol, ornithine and arginine had significant effects on the biosynthesis of clavulanic acid in Streptomyces. clavuligerus. Glycerol at initial concentration of 10-20 g l-1 increases clavulanic acid production by Streptomyces clavuligerus in flask culture. Adding ornithine in batch culture could enhance clavulanic acid production but arginine showed little effect. In the fed-batch experiments, the results showed that glycerol (C3) supply rather than amino acid (C5) supply is rate-limiting. Feeding ornithine not only provided an enough supply of arginine for clavulanic acid production, but also inhibited the glycerol-utilizing cephamycin biosynthesis. The results reveal that ornithine rather than arginine could enhance effectively clavulanic acid production if the amount of C3-precursor was sufficient.
1 Introduction 1
2 Simultaneous Removal of Carbon and Nitrogen from Swine Wastewater Using Phosphorylated PVA-Immobilized microorganisms 11
3 Degradation of Phenol by PAA-Immobilized Candida tropicalis 36
4 A New Microbial Immobilization Method Using Anionic Polyurethane 60
5 Optimization of Glycerol Feeding for Clavulanic acid Production by Streptomyces clavuligerus with Glycerol Feeding 79
6 Enhancement of Clavulanic Acid Production in Streptomyces clavuligerus with Ornithine Feeding 91
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11. Sumino T, Nakamura H, Mori N, Kawaguchi Y, Immobilization of nitrifying bacteria in porous pellets of urethane gel for removal of ammonium nitrogen from wastewater. Appl. Microbiol. Biotechnol 1992; 36: 556-560.
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CHAPTER 2
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10. Tanaka K, Tada M, Harada S, Fujii Y, Mizuguchi T, Mori N, Emori H. Development of new nitrogen removal system using nitrifying bacteria immobilized in synthetic resin pellets. Water Sci Technol 1991; 23: 681-690.
11 Tramper J, De Man AWA. Operating performance of Nitrobacter agilia immobilized in carrageenan. Enzyme Microb Technol 1986; 8: 477-480.
12. Hashimoto S, Furukawa K. Immobilization of activated sludge by PVA-boric acid method. Biotechnol Bioeng 1987; 30: 52-59.
13. Chen KC, Lin YF. Immobilization of microorganism with phosphorylated polyvinyl alcohol (PVA) gel. Enzyme Microb Technol 1994; 16: 79-83.
14. Chen KC, Lin YF, Houng JY. Performance of a continuous stirred tank reactor with immobilized denitrifiers and methanogens. Water Environ Res 1997; 69: 233-239.
15. American Public Health Association; American Water Work Association; and Water Environment Federation. Standard Methods for the Examination of Water and Wastewater. 1985; 20th Ed. Washington, DC.
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17. Sumino T, Nakamura H, Mori N. Development of a high-efficiency wastewater treatment system using immobilized microorganisms. In: Industrial application of immobilized biocatalysts (Tanaka A, Tosa T, Kobayashi Y. Eds.). Maecel Dekker, New York, 1993, 377.
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20. Sumino T, Nakamura H, Mori N, Tada, M. Immobilization of nitrifying bacteria in porous pellets of urethane gel for removal of ammonium nitrogen from wastewater. Appl Microbiol Biotechnol 1992; 36: 556-560.
21. Peddie CC, Mavinic DS, Jenkins CJ. Use of ORP for monitoring and control of aerobic sludge digestion. J Environ Eng 1990; 116: 461-471.
22. Wareham DG., Hall KJ, Mavinic DS. Real-time control of aerobic-anoxic sludge digestion using ORP. J Environ Eng 1993, 119, 120-136.
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26. Lo KV, Liao PH, Gao. Y.C. Anaerobic treatment of swine wastewater using hybrid UASB reactors. Biosource Technol 1994, 47: 153-157.
27. Cheng N, Lo KV, Yip KH. Swine wastewater treatment in a two stage sequencing batch reactor using real-time control. J Environ Sci Health 2000; B35: 379-398.
CHAPTR 3
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6. Shishido M, Kojima T, Araike YK, Toda M. Biological phenol degradation by immobilized activated sludge in gel bead with three-phase fluidized-bed bioreactor. Chem Eng Res Des 1995; 73: 719- 726.
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19. Rao BYK, Godbole SS, D’Souza SF. Preparation of lactose free milk by fermentation using immobilized Saccharomyces fragilis. Biotechol Lett 1988; 10: 427-430.
20. Doran PM, Baily JE. Effects of hydroxyurea on immobilized and suspended yeast fermentation rates and cell-cycle operation. Biotechnol Bioeng 1986; 28: 73-87.
21. Wu KYA, Wisecarver KD. Biological phenol degradation in a countercurrent three-phase fluidized bed using a novel cell immobilized technique. AIChE Symp Ser 1990; 86: 113-118.
22. Klein JA, Lee D. Biological treatment of aqueous wastes from coal conversion processes. Biotechnol Bioeng 1978; 8: 379-390.
CHEPTER 4
1. Fukushima S, Nagai T, Fujita K, Tanaka A, Fukui S. Hydrophillic urethane prepolymers: Convenient materials for enzyme entrapment. Biotechnol Bioeng 1978; 20: 1465-1469
2. Sumino T, Nakamura H, Mori N, Kawaguchi Y. Immobilization of nitrifying bacteria in porous pellets of urethane gel for removal of ammonium nitrogen from wastewater. Appl Microbiol Biotechnol 1992; 36: 556-560.
3. Sumino T, Nakamura H, Mori N, Kawaguchi Y, Tada, M. Immobilization of nitrifying bacteria in porous pellets of urethane for removal of ammonium nitrogen from waste-water. Appl. Microbiol. Biotechnol 1992; 36: 556-560.
4. Vorlop KD, Muscat A, Beyersdorf J. Entrapment of microbial cells within polyurethane hydrogel beads with the advantage of low toxicity. Biotech Techn 1992; 6: 483-488.
5. Leenen EJTM, Santos VAPD, Grolle KCF, Tramper J, Wijffels RH. Characteristics of and selection criteria for support materials for cell immobilization in wastewater treatment. Water Res 1996; 30: 2985-2996
6. Dieterich D, Keberle W, Witt H. Polyurethane ionomer, a new class of block polymers. Angew Chem Internat Edit 1970; 9: 40-50.
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9. Wada M, Kato J, Chibata IJ. Electron microscopic observation on immobilized growing yeast cells. Ferment Technol 1980; 58: 327-331.
10. Chen KC, Huang, CT. Effect of growth of Trichosporon cutaneum in calcium alginate gel beads upon bead structure and oxygen transfer characteristic. Enzyme Microb Technol 1988; 10: 284-292.
11. Wada M, Kato J, Chibata I. A new immobilization of microbial cells. Immobilized growing cells using carrageenan gel and their properties. Eur J Appl Microbiol Biotechnol 1979; 8: 241-247
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14. Pundle A, Prabhune A, SivaRaman H. Immobilization of Saccharomyves uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation. Appl Microbiol Biotechnol 1988; 29: 426-429
CHAPTER 5
1. Brown AG, Butterworth D, Cole M, Hanscomb G, Hood JD, Reading C, Rolinson GN. Naturally-occurring β-lactamase inhibitors with antibacterial activity. J Antibiot 1976; 29: 668-669.
2. Liras P, Rodriguez-Garcia A. Clavulanic acid, a β-lactamase inhibitor: biosynthesis and molecular genetics. Appl Microbiol Biotechnol 2000; 54: 467-475.
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4. Mayer AF, Deckwer WD. Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations. Appl Microbiol Biotechnol 1996; 45: 41-46.
5. Kirk S, Avignone-Rossa CA, Bushell ME. Growth limiting substrate affects antibiotic production and associated metabolic flues in Streptomyces clavuligerus. Biotechnol Lett 2000; 22: 1803-1809.
6. Romero J, Liras P, Martin JF. Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 1984; 20: 318-325.
7. Elson SW, Oliver RS. Studies on the biosynthesis of clavulanic acid. I. Incorporation of 13C-labelled precursors. J Antibiot 1978; 31: 586-592.
8. Foulstone M, Reading C. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob. Agents Chemother 1982; 22: 753-762.
CHAPTER 6
1. Brown AG, Butterworth D, Cole M, Hanscomb G, Hood JD, Reading C, Rolinson GN. Naturally occurring-lactamase inhibitors with antibacterial activity. J Antibiot 1976; 29: 668-669.
2. Aharonowitz Y, Demain AL. Catabolite regulation of cephalosporin production in Streptomyces clavuligerus. Antimicrob agents chemother 1978; 14: 159-164.
3. Reed G, Peppler HJ. Backers’ yeast production. In Yeast Technology. Westport: AVI, 1973. p. 53-102.
4. Queener S, Swartz R. Penicillins; biosynthetic and semisynthetic. In Economic Microbiology, vol. 3, Secondary products of Metabolism. Edited by Rose AH. London: Academic Press, 1979. 35-123
5. Bushell ME, Bell SL, Scott, MF, Spier RE, Wardel JN, Sanders PG. Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population. Biotechnol Bioeng 1994; 44: 1099-1106.
6. Ives PR, Bushell ME. Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology 1997; 143: 3573-3579.
7. Elson SW, Oliver RS. Studies on the biosynthesis of clavulanic acid. I. Incorporation of 13C-labelled precursors. J Antibiot 1978; 31: 586-592
8. Townsend CA, Ho MF. Biosynthesis of clavulanic acid: Origin of the C5 Unit. J Am Chem Soc 1985; 107: 1066-1068.
9. Baggaley KH, Brown AG, Schofield CJ. Chemistry and biosynthesis of clavulanic acid and other clavams. Nat Prod Rep 1997; 14: 309-333.
10. Foulstone M, Reading C. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob Agents Chemother 1982; 22: 753-762.
11. Romero J, Liras P, Martin JF. Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus. Appl Microbial Biotechnol 1984; 20: 318-325.
12. Mayer AF, Deckwer WD. Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations. Appl Microbial Biotechnol 1996; 45: 41-46.
13. Hu WS, Brana AF, Demain AL. Carbon source regulation of cephem antibiotic production by resting cell of Streptomyces clavuligerus and its reversal by protein synthesis inhibitors. Enzyme Microb Technol 1984; 6: 155-160.
14. Romero J, Liras P, Martin JF. Utilization of ornithine and arginine as specific precursors of clavulanic acid. Appl Environ Microbiol 1986; 52: 892-897.
15. Kirk S, Avignone-Rossa CA, Bushell ME. Growth limiting substrate affected antibiotics production and associated metabolic fluxes in S. clavuliderus. Biotechnol Lett 2000; 22: 1803-1809
16. Elson SM, Oliver RS. Studies on the biosynthesis of clavulanic acid. I Incorporation of 13C-labelled precursor. J Antibiot 1978; 31: 586-592.
17. Valentine BP, Bailey CR, Doherty A, Morris J, Elson SW, Baggaley KH, Nicholson NH. Evidence that arginine is a later metabolic intermediate than ornithine in the biosynthesis of clavulanic acid by Streptomyces clavuligerus. J Chem Soc Chem Commun 1993; 1210-1211.
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