臺灣博碩士論文加值系統

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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CHAPTR 3
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CHEPTER 4
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CHAPTER 6
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