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研究生:張曉婷
研究生(外文):Shiao-Ting Chang
論文名稱:包埋具酚類分解性的惡臭假單胞菌於多孔性幾丁聚醣顆粒之研究
論文名稱(外文):Entrapment of Pseudomonas putida in chitosan gel beads for biodegradation of phenol
指導教授:林睿哲
指導教授(外文):Jui-Che Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
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:中文
論文頁數:69
中文關鍵詞:酚的生物分解固定化惡臭假單胞菌幾丁聚醣甲殼素
外文關鍵詞:Pseudomonas putidaChitinPhenol BiodegradationImmobilizationChitosan
相關次數:
  • 被引用被引用:6
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本研究以幾丁聚醣作為固定化擔體,利用物理性膠凝作用,針對惡臭假單胞菌進行包埋固定化,作為一代謝酚的生物觸媒,此一固定化擔體有兩項性質是本實驗所關心的議題:
1. 顆粒表層與剖面形態:利用電子顯微鏡觀察不同膠凝條件下形成的顆粒,尋求表層孔洞分布程度高且孔徑小於0.2μm,避免菌體逸漏,達成固定化目的,而顆粒的核心層勿過於緻密,方可提供菌體足夠的生長空間。
2. 顆粒內包埋的活菌數:已固化成型的幾丁聚醣顆粒不易再度溶解,菌體的定量更加困難,採間接探討包埋過程中流失的菌數,與經包埋溶液接觸後的失活情形,來反推顆粒內包埋的活菌量。

利用2%幾丁聚醣溶液在三聚磷酸鈉中所膠凝的顆粒,可耐受曝氣測試與震盪測試,可見幾丁聚醣顆粒強度的穩定性佳。該顆粒表面孔徑為1~6μm,應該會有菌體逸漏的問題。幾丁聚醣分子量愈大與去乙醯程度愈高,都有助於幾丁聚醣滴入膠凝劑時,可以膠凝成顆粒狀。膠凝顆粒隨幾丁聚醣濃度增加,其顆粒核心愈密實,將會對菌體的生長空間有所限制。菌體一經幾丁聚醣溶液接觸後,立即失活,可能是幾丁聚醣的抑菌性與偏酸的幾丁聚醣溶液所致。
In this study, chitosan is used as an immobilization matrix. Pseudomonas putida is entrapped by physical iontropic gelation with chitosan and the resulting beads are used as a biocatalyst for the biodegradation of phenol. Two important issues were investigated about these beads:
1. The surface and cross-section morphology of the beads: the structure of the beads formed by different iontropic gelation procedures were observed by scanning electron microscopy (SEM). To avoid bacteria leakage and attain an effective immobilization, the high surface porosity and small pore diameter that is less than 0.2μm are sought. In addition, the core of the beads shouldn’t be too dense, so as to provide enough growing space for bacteria.
2. The viable bacteria concentration inside the bead: the quantification of bacteria is difficult since the lysis for the chitosan bead was hard. Thus, the following experiments were performed. One is the concentration of the bacterial loss during entrapment. The other is the viable ratio of the bacteria after contact with the solution used for immobilization. Henceforth, the viable bacteria concentration entrapped inside the bead can be calculated.

Chitosan beads formed by using 2% chitosan solution gelation in 1% sodium tripolyphosphate solution is strong enough to bear the aeration and shaking tests. This indicates the stability of the chitosan beads were satisfactory. The surface pore diameter of the beads formed is ranging from 1 to 6 μm. This might result in the leakage of bacteria entrapped. High molecular weight and high degree of deacetylation of the chitosan can lead to a better bead formation in the gelation solution. In addition, the core of the beads will become denser with the increase of the chitosan concentration, and this will limit bacteria growth. Most of all, it was found that the bacteria is denatured after its contact with chitosan solution. This may be attributed to the antibacterial property associated with chitosan as well as the acidic environment of the chitosan solution.
中文摘要 I
ABSTRACT II
誌謝 IV
目錄 V
圖目錄 VIII
表目錄 X
第一章 前言 1
第二章 文獻回顧 2
2-1外來有機物的生物轉化作用 2
2-1-1 酚污染的危害 2
2-1-2 微生物程序:生物分解作用 2
2-1-3 惡臭假單胞菌(Pseudomonas Putida) 3
2-2 細胞固定化技術 4
2-2-1 細胞固定化的概念 4
2-2-2 細胞固定化方法 5
2-2-3 固定化技術的限制 9
2-3 幾丁聚醣CHITOSAN 10
2-3-1 來源 10
2-3-2 製備 11
2-3-3 物理與化學性質 11
2-3-4 對金屬離子的螯合作用 15
2-3-5 幾丁聚醣在膠體包埋的應用性 16
2-3-6 應用與市場 17
2-4 研究動機與目的 18
第三章 實驗內容 19
3-1 實驗藥品與儀器 19
3-1-1 藥品與溶液配製 19
3-1-2 儀器 21
3-2 儀器分析原理與試樣處理 23
3-2-1 元素分析儀 23
3-2-2 掃描式電子顯微鏡 23
3-2-3 冷凍乾燥機 24
3-3 實驗步驟 25
3-3-1 使用菌株 25
3-3-2 菌株保存方法 25
3-3-3 培養基 25
3-3-4 培養方法 26
3-3-4 細胞的生長曲線 27
3-3-5 幾丁聚醣溶液的配製 27
3-3-6 膠體包埋固定化 28
3-3-7 膠凝顆粒的表面交聯 29
3-3-7 顆粒強度測試 29
3-3-8 菌體的包埋固定化 30
3-3-9 細胞的起始包埋率 31
3-4 分析與鑑定 35
3-4-1 幾丁聚醣分子量的測定 35
3-4-2 定量細胞濃度 36
3-4-3 酚的測定 38
第四章 結果與討論 41
4-1 膠凝顆粒的表面交聯 41
4-2 幾丁聚醣顆粒強度測試 41
4-3 惡臭假單胞菌的生長特性(營養培養基) 42
4-4 細胞的生長曲線與酚代謝曲線(酚培養基) 42
4-5 蒸氣滅菌對幾丁聚醣的影響 43
4-6 幾丁聚醣顆粒的表面型態分析 44
4-6-1 幾丁聚糖濃度的影響 44
4-6-2 膠凝劑的影響 45
4-6-3 膠凝劑濃度的影響 46
4-6-4 膠凝時間的影響 48
4-7 菌體的起始包埋率 49
4-7-1 菌體流失分析(Cell Leakage Assay) 49
4-7-2 殘存活細胞比例Cell Viability Percentage 50
結論與未來展望 51
參考文獻 64
自述 69
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