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研究生:陳建孝
研究生(外文):Scott Chen
論文名稱:蘇力菌之培養及其影像分析上之應用
論文名稱(外文):Application of Image Analysis on Cultivation of Bacillus thuringiensis
指導教授:吳文騰
指導教授(外文):Wen-Teng Wu
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:中文
論文頁數:129
中文關鍵詞:蘇力菌運動性運動強度影像分析
外文關鍵詞:Bacillus thuringiensismotilitymotile intensityimage analysis
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  • 被引用被引用:2
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中文摘要
細菌的運動性是一個重要的生理現象,對於菌體的生長以及培養環境的變化都會影響菌體運動性的表現。在本論文中,吾人利用影像分析技術量化菌體的運動性,並且定義了一個描述菌體運動性的參數,稱為「運動強度 (Motile intensity)」,其物理意義為單位菌體的動能表現。本文首先利用「運動強度」描述菌體的運動性在培養過程中的變化情形。再來吾人以蘇力菌與蘇力菌素作為研究對象,探討「運動強度」對於菌體的生理意義。最後吾人應用「運動強度」的特性,發展了一套具經濟價值的醱酵程序。
在第三章的研究裡,吾人選用兩種描述參數「運動強度」與「平均菌體游動速度」,以七種不同菌株作為觀察對象,發現以「運動強度」描述菌體的運動性會呈現出一致的結果。菌體的最大運動強度出現在菌體生長遲緩期的後期與指數生長期的初期之間,在指數生長期的中期,菌體的運動強度會逐漸減弱至一個穩定態。另外吾人也發現,菌體的運動強度與比基質消耗速率呈現出相同的變化趨勢。
在第四章的研究中,吾人在不同的運動強度時期對蘇力菌的進行環境因子(氧氣供應、溫度與酸鹼值)的脈衝變化。由實驗結果發現,當菌體的運動強度處於高峰時,蘇力菌素的生產所受到的傷害最為嚴重。此外,吾人也針對運動衰減的因素進行研究,吾人發現菌體TCA循壞的代謝流率逐漸下降是造成運動強度衰減的主要原因。另外吾人也發現,菌體的最高運動強度與蘇力菌素的最終產量呈現一個良好的線性關係。
在第五章中,吾人將菌體運動強度應用在醱酵程序上,分別對於最適化接種時間的決定、最適化培養基與環境因子的選擇以及合適的饋料批次醱酵策略的決定。由實驗結果顯示,當菌體運動強度處於高峰期,此時便是菌體的最適接種時間。此外,根據菌體運動強度的特性,吾人發展出一套最新的最適化培養基選擇方法,稱為”液滴式培養基選擇法”。透過這一個方法,僅需要一小滴的培養基與具有高運動強度的菌體,吾人即可以在短時間內決定最適化的培養基組成與培養的環境條件。在最後一節中,我們進行饋料批次醱酵策略的設計。吾人只要透過一組批次培養的運動強度表現,就可以設計出一套合適的饋料批次醱酵策略:首先,在運動強度表現的最高處進行饋料,並以最適化的培養基濃度作為控制饋料的設定點;最後,當運動強度降至穩定期後停止饋料,讓菌體自然成長至實驗結束。
本文利用蘇力菌的運動強度作為研究對象,以菌體生長與蘇力菌素的生產作為研究標的,討論菌體運動強度與這兩者的關係。吾人發現「運動強度」不只可以描述菌體的運動性,並且可以視為菌體的活力指標。

Abstract
The motility of bacteria is a mechanism in response to the environment change and the growth of the cells. In this, a parameter, called “motile intensity”, for determining the motility of a bacterium is defined. The motile intensity is defined as the mean specific kinetic energy of the cells. In addition, the relationships among the growth of B. thuringiensis, the production of thuringiensin, and the motile intensity of the cells are investigated. Finally, an economical process of fermentation based on the motile intensity is developed.
In chapter 3, the first stage of the experiments, 7 kinds of bacteria and use two parameters, “motile intensity” and “mean swimming speed”, to characterize the motility of bacteria are observed. The results reveal that the motile intensity is an appropriate parameter for describe the motility of the cells. A rapid increase in motile intensity during the lag phase, with a peak between the post-lag phase and the early exponential phase is also observed. In addition, it is found that the specific glucose consumption rate of the cells exhibits the same curve as the motile intensity on cultivation.
In chapter 4, the second stage of the experiments, it is found that the motile intensity can be an indicator for the activity of the cell growth. If the environmental variables change at a stage where the motile intensity is at high level, the cells will get more damage for the thuringiensin production. In addition, the motile intensity commonly decreases during cultivation. The reason is that B. thuringiensis produces some compounds to inhibit the flux of the TCA cycle on cultivation. Most energy is consumed to keep the growth of the cells. So there is no enough energy left for the motility of the cells. Moreover, it is found that the maximum motile intensity of the cells exhibits a matching with the productivity of thuringiensin in an almost linear way.
In chapter 5, the finally stage of the experiments, a new process of fermentation based on the motile intensity for cultivation is developed. This process includes the following factors: optimum inoculum time decision, droplet medium selection, and the design of a suitable fed-batch strategy. The time that the motile intensity of the cells is at the maximum magnitude on cultivation is the optimum inoculum time. In addition, a new medium design method, called “droplet medium selection”, is developed. An optimum medium composition and the environmental conditions can be found very quickly by means of the droplet medium selection. Finally, a simple strategy for fed-batch cultivation is proposed. The strategy consists of two steps: (i) initiate the feeding at the maximum magnitude of the motile intensity of B. thuringiensis with an optimum substrate concentration control; (ii) terminate the feeding at low motile intensity (or non-motile) of the cells.
In summary, the motility of the Bacillus thuringiensis during cultivation is investigated, and the “motile intensity” for describing the motility is defined. Besides, an appropriate fermentation process is developed. The motile intensity can not only describe the motility of the cells, but also be an indicator for determining the activity of the cells on cultivation.

目錄
第一章 緒論 1
1-1 前言 1
1-2 細菌的運動性(MOTILITY) 2
1-3 蘇力菌與蘇力菌素簡介 5
1-4 影像處理與影像分析 8
1-5 影像處理分析技術於細菌運動性研究的應用 11
1-6 本論文之章節結構 12
第二章 實驗材料與分析方法 13
2-1 菌種與培養基組成 13
2-2實驗設備 16
2-3 分析方法 18
2-3-1 菌體量與菌體運動性分析 20
2-3-2 蘇力菌素濃度分析 22
2-3-3 葡萄糖濃度及比基質消耗速率的測定 23
第三章 微生物運動性的特性 24
3-1 前言 24
3-2實驗材料與方法 25
3-3實驗結果與討論 25
3-3結論 38
第四章 微生物運動性與發酵系統的關係 40
4-1 蘇力菌不同時期的運動強度與生長活性的關係 40
4-1-1 前言 40
4-1-2實驗材料與方法 41
4-1-3實驗結果與討論 43
4-1-4結論 56
4-2 蘇力菌運動強度消長因素的探討 57
4-2-1前言 57
4-2-2實驗材料與方法 57
4-2-3實驗結果與討論 58
4-2-4結論 62
4-3 蘇力菌最大運動強度在醱酵系統的應用 68
4-3-1前言 68
4-3-2實驗材料與方法 68
4-3-3實驗結果與討論 70
4-3-4結論 73
第五章 微生物運動性的應用 74
5-1蘇力菌最適接種時間 74
5-1-1前言 74
5-1-2實驗材料與方法 74
5-1-3結果與討論 75
5-1-3 結論 80
5-2液滴式培養基選擇法 82
5-2-1前言 82
5-2-2實驗材料與方法 83
5-2-3實驗結果與討論 84
5-2-3結論 86
5-3 根據運動強度設計合適的醱酵程序 91
5-3-1 前言 91
5-3-2實驗材料與方法 92
5-3-3實驗結果與討論 93
5-3-4結論 96
第六章 結論與未來展望 105
6-1結論 105
6-2未來展望 107
參 考 文 獻 108
附錄A 114

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