臺灣博碩士論文加值系統

中文摘要
由Pseudomonas aeruginosa 菌株所生產的生物界面活性劑－鼠李醣脂(rhamnolipid)共有六種不同的結構，其擁有類似的化學結構及表面活性，平均分子量為577。鼠李醣脂的臨界微胞濃度大約在50-65 mg/L，可以將水的表面張力從70 mN/m 降至30 mN/m。本研究使用由油污染土壤中篩選之本土菌株Pseudomonas aeruginosa EM1 來進行生物界面活性劑鼠李醣脂的醱酵生產。由NMR 及mass spectrometry 的分析可以確認EM1菌株生產之鼠李醣脂產物的化學結構為RL1 (MW=527) 與RL2(MW=673)，且其擁有相當好的表面活性，臨界微胞濃度為24.8 mg/L，而臨界乳化指數為250-300 mg/L。探討不同碳源及氮源對鼠李醣脂產量的實驗中，發現最佳的碳源為葡萄糖或甘油(濃度分別為40 g/L)分別可以達到7.5 g/L 和4.9 g/L 的產量，而使用最佳氮源硝酸鈉(4.25 g/L)則可得到8.6 g/L 的產量。此外，碳氮源的最佳比值在使用不同的碳源時也會呈現不同的趨勢，當使用葡萄糖及甘油為碳源時，最佳碳氮比分別為26及52。為了更進一步的找出碳源及氮源的最佳濃度，本研究使用反應曲面法設計出二十組實驗來找尋葡萄糖、甘油及硝酸鈉的最佳濃度，最後得到當葡萄糖、甘油及硝酸鈉的濃度分別在30.5、18.1 和4.9 g/L 時可得最佳鼠李醣脂的產量為12.6 g/L。在醱酵槽的實驗中，發現消泡劑的添加會抑制鼠李醣脂的生產，而改良式的生物反應器可以控制醱酵中嚴重的發泡現象且得到較高的鼠李醣脂生產速率。
摘要： 鼠李醣脂，生物界面活性劑，Pseudomonas aeruginosa，生物反應器設計，培養基最適化，反應曲面法

Abstract
Rhamnolipid is a glycolipid biosurfactant produced by Pseudomonas aeruginosa strains. There are six types of rhamnolipids possessing similar chemical structure and surface activity with an average molecular weight (MW) of 577. Rhamnolipid can reduce surface tension of water from 72 mN/m to 30 mN/m with a critical micelle concentration of 50-65 mg/L. In this study, a newly isolated indigenous bacterial strain Pseudomonas aeruginosa EM1 originating from oil-contaminated site in southern Taiwan was used to develop effective technology for rhamnolipid production. Rhamnolipid produced by Pseudomonas aeruginosa EM1 has been characterized by NMR and mass spectrometry to confirm the chemical structure of the rhamnolipid product. The results indicated that the purified product was a mixture of RL1 (MW=527) and RL2 (MW=673). The critical micelle concentration and the critical emulsion index of the purified rhamnolipid were 24.81 mg/L and 250-300 mg/L, respectively. Different carbon and nitrogen sources were
examined for their effects on rhamnolipid production. The results show that the better rhamnolipid production 7.5 g/L and 4.9 g/L were obtained by using glucose or glycerol (both at 40 g/L) as the carbon substrate. Meanwhile, the
best nitrogen source for rhamnolipid production is sodium nitrate, giving a rhamnolipid yield of 8.6 g/L. Furthermore, the effect of carbon to nitrogen (C/N) ratio on rhamnolipid production was also studied. An optimal C/N ratio of 26 and 52 was obtained for glucose- and glycerol-based culture respectively by using sodium nitrate as the nitrogen source. In order to optimize the composition of fermentation, response surface methodology (RSM) was used to design 20 experiments to explore the best concentration of three critical components in the medium (i.e., glucose, glycerol, and NaNO3). The results of RSM analysis gave an optimal concentration of 30.5, 18.1, and 4.9 g/L for glucose, glycerol, and NaNO3, respectively, predicting a maximum RL yield of 12.6 g/L. In addition, fermentor studies showed that rhamnolipid production was inhibited by the addition of anti-foam agent. A modified bioreactor with foam collector and circulator was a better way to control the severe foaming, resulting a higher rhamnolipid production rate.
Keywords: Bioreactor design, biosurfactant, medium optimization, Pseudomonas aeruginosa, response surface methodology, rhamnolipid

Contents
Abstract (Chinese) I
Abstract (English) III
Acknowledgement V
Contents VII
List of Figures X
List of Tables XIII
Chapter 1 Introduction 1
1-1 Background 1
1-2 Motivation and purpose 1
Chapter 2 Literature review 3
2-1 Surfactants 3
2-2 Biosurfactants 6
2-3 Rhamnolipid 11
2-3-1 Rhamnolipid structure and properties 11
2-3-2 Biosynthesis function of rhamnolipid 11
2-3-3 Determination of rhamnolipid concentration 14
2-3-4 Rhamnolipid purification 15
2-4 Rhamnolipid production 16
2-4-1 Effect of medium compositions and culture conditions 16
2-4-2 Bioreactor designs 17
2-5 Response surface methodology 19
Chapter 3 Materials and methods 21
3-1 Chemicals and materials 21
3-2 Equipment 23
3-3 Medium composition 25
3-4 Flask fermentation 26
3-4-1 Bacterial strain and preparation of seed culture 26
3-4-2 Procedures for exploring the effect of carbon sources 26
3-4-3 Procedures for exploring the effect of nitrogen sources 26
3-4-4 Procedures for exploring the optimal carbon to nitrogen ratio 27
3-4-5 Experimental design for optimizing concentration of carbon and nitrogen sources 27
3-4-6 Procedures for exploring the effect of yeast extract
concentration on rhamnolipid production 28
3-5 Fermentor experiments 28
3-5-1 Bioreactor with addition of anti-foam 28
3-5-2 Bioreactor with foam collector 29
3-6 Analytical methods and rhamnolipid purification 29
3-6-1 Determination of cell concentration 29
3-6-2 Determination of Rhamnolipid concentration 30
3-6-3 Surface tension assay 30
3-6-4 Determination of emulsification index 30
3-6-5 Rhamnolipid purification 31
3-7 Definition of terms in data analysis 31
Chapter 4 Results and discussion 34
4-1 Analysis of rhamnolipid structure 34
4-2 Characterization of surface activity of the purified rhamnolipid 39
4-3 The Effects of carbon and nitrogen source on rhamnolipid production 42

4-4 Effect of carbon to nitrogen (C/N) ratio on rhamnolipid production 48
4-5 Improving rhamnolipid production of by RSM experimental design 51
4-6 The effect of yeast extract on rhamnolipid production 59
4-7 Fermentor production of rhamnolipid 61
4-7-1 Batch rhamnolipid production in fermentor 61
4-7-2 The effect of anti-foam on rhamnolipid production (flask tests) 62
4-7-3 The modified bioreactor with foam collector and circulator 67
Chapter 5 Conclusions 70
References 71
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