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研究生:羅凱軍
研究生(外文):Lo, KaiJuen
論文名稱:篩選菌株Gluconacetobacter sp. Wu3M生產細菌纖維薄膜及動力學解析
論文名稱(外文):Study on Production of Bacterial Cellulose Membrane by Isolated Gluconacetobacter sp. Wu3M and Kinetic Model of Production of Bacterial Cellulose
指導教授:吳建一
指導教授(外文):Wu, JaneYii
口試委員:吳建一陳晉照顏裕鴻
口試日期:2012-07-10
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:182
中文關鍵詞:細菌纖維素Gluconacetobacter sp. Wu3M動力學純化抗菌薄膜
外文關鍵詞:Bacterial celluloseGluconacetobacter sp. Wu3MKinetic modelpurificationantibacterial film.
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由Gluconacetobacter sp.菌株所生產之細菌纖維素是一生物聚合物,具有獨特的結構、機械強度和化學穩定性。在本研究中,由椰子汁中篩選具有生產細菌纖維素之菌株,經16S rDNA鑑定基因序列後屬於Gluconacetobacter sp.,並且命名為Gluconacetobacter sp. Wu3M (NCBI JX088028)。為了探討培養因子對Gluconacetobacter sp. Wu3M菌株生產細菌纖維之影響,在本研究中探討4個變因,包含醋酸濃度、培養基表面積、培養器皿壁面粗糙度及接菌量。實驗結果顯示,添加0.25%之試藥級醋酸可增加細菌纖維薄膜產量。雖然增加大的表面積可以提高細菌纖維產量,但其產率 (g BC/L medium volume)部分卻會下降。在表面積22×30 cm2下培養5天可得1.64 g/L之細菌纖維薄膜。純化實驗結果顯示,二階段處理可以明顯改善細菌纖維素色度之去除。在動力學部分別以Logistic model以及Luedeking-Piret model模擬微生物生長,細菌纖維素生產和基質消耗。結果顯示,細菌纖維素的生產屬於生長相關。將細菌纖維速乾膜浸泡於奈米銀與benzalkonium chloride溶液中製備抗菌薄膜。分析此薄膜對Bacillus subtilis和Escherichia coli.菌株之抑制活性。實驗結果顯示,細菌纖維素抗菌薄膜可有效抑制Bacillus subtilis和Escherichia coli.菌株生長。
Bacterial cellulose production from Gluconacetobacter specie is a unique biopolymer in terms of its structure, mechanical strength and chemical stability. In this study, we isolated the strain with high production bacterial cellulose capability from coconut milk and identified according to 16S rDNA gene sequences as Gluconacetobacter sp., called Gluconacetobacter sp. Wu3M (NCBI JX088028). In order to study the effects of different culture parameters on Gluconacetobacter sp. Wu3M to determine which conditions provided optimum BC production, four factors were investigated, including acetic acid concentration, culture surface area, wall of tray, inoculate volume. The results of study showed that BC membrane product was increased by adding the 0.25% reagent grade acetic acid. On the other hand, although a large surface area was important for good BC product, the yield (g BC/L medium volume) was lower. The BC produced was 1.64 g/L at the surface area of 22×30 cm2 by a static cultivation during 5 days of cultivation. A simple model was proposed using the Logistic equation for growth, the Luedeking-Piret equation for BC production and substrate consumption. The kinetic model showed the BC production is growth-associated model. Preparation antibacterial film by dry BC film was immersed in a nano-silver solution and benzalkonium chloride solution. Detailed studies on the antibacterial activity of these antibacterial films were carried out for Bacillus subtilis and Escherichia coli. The result of study showed, that BC antibacterial films were obtained especially against B. subtilis and E. coli..
目錄

封面內頁
簽名頁
中文摘要iii
英文摘要iv
誌謝v
目錄vi
圖目錄ix
表目錄xv

1.前言1
2.文獻回顧4
2.1 纖維素簡介4
2.2 細菌纖維簡介6
2.2.1 細菌纖維之發展6
2.2.3 細菌纖維與植物纖維之比較10
2.2.4 細菌纖維之生合成路徑與機制13
2.3 細菌纖維薄膜之處理16
2.4 以微生物發酵生產細菌纖維之研究21
2.4.1 細菌纖維之生產菌株21
2.4.2 碳源對細菌纖維生產之影響30
2.4.3 氮源對細菌纖維生產之影響32
2.4.4 有機酸對細菌纖維生產之影響32
2.4.5 培養條件對細菌纖維生產之影響33
2.4.6 反應器類型對細菌纖維生產之影響35
2.5 利用基因轉殖微生物生產細菌纖維39
2.6 細菌纖維之應用40
2.6.1 食品上之應用40
2.6.2 醫學領域之應用41
2.6.3 電子紙44
2.6.4 燃料電池薄膜45
2.6.5 其他45
3. 材料與方法48
3.1實驗器材48
3.1.1藥品48
3.1.2儀器設備50
3.2 菌種來源51
3.2 菌種來源與篩選、鑑定51
3.3 菌株培養55
3.3.1菌株活化與保存55
3.3.2 影響細菌纖維薄膜生產之因子探討55
3.4 分析方法56
3.4.1 纖維素乾重與厚度分析56
3.4.2 發酵液分析57
3.4.2.1 還原糖分析57
3.4.2.2 培養基高度分析57
3.4.2.3 有機酸分析-高效能液相層析(High Performance Liquid Chromatography, HPLC)58
3.5 純化細菌纖維薄膜之結構分析60
3.5.1 掃描式電子顯微鏡 (Scanning Electron Microscopy, SEM)分析60
3.6 細菌纖維薄膜內毒素分析60
3.7 抗菌纖維薄膜製備63
4. 細菌纖維薄膜生產動力學解析65
5. 結果討論72
5.1 可生產細菌纖維素之菌株篩選與鑑定72
5.2額外添加物對細菌纖維薄膜生產之影響73
5.2.1 食用acetic acid對細菌纖維薄膜生產之影響 73
5.2.2 不同acetic acid濃度對細菌纖維薄膜生產之影響79
5.3培養條件對細菌纖維薄膜生產之影響85
5.3.1不同培養基之液面積對細菌纖維薄膜生產之影響85
5.3.2 不同培養器材之壁面粗糙度對細菌纖維薄膜生產之影響92
5.3.3 不同接菌量對細菌纖維薄膜生產之影響98
5.4 細菌纖維薄膜生產動力學解析103
5.5 細菌纖維薄膜之內毒素分析128
5.6 以細菌纖維薄膜製備抗菌薄膜之研究131
6. 結論143
參考文獻145

圖目錄

Figure 2-1. The structure of cellulose.5
Figure 2-2. Chemical structure of Cellulose.6
Figure 2-3. Schematic representation of the components in higher-plant cellulose.7
Figure 2-4. Outline of intra- and inter-molecular hydrogen bonds among cellulose chains.8
Figure 2-5. Assembly of cellulose microfibrils by A. xylinum.9
Figure 2-6. Comparison of Plant Cellulose Fibrils and Bacterial Cellulose Fibrils.12
Figure 2-7. Biochemical pathway for cellulose synthesis by A. xylinum.16
Figure 2-8. Comparison of typical pulping processes for purification of bacterial cellulose.20
Figure 2-9. The membranes of untreated bacterial cellulose and purification treated bacterial cellulose.21
Figure 2-10. Cross-section of a static bacterial cellulose culture.38
Figure 2-11. Nata de coco prepared from coconut water as a traditional dessert in Philippines.41
Figure 2-12. Wound dressing prepared from Bacterial cellulose membrane.43
Figure 2-13. Hollow tube made from bacterial cellulose using a silicon tube as a mold (A) and formation model (B).44
Figure 3-1. The standard calibration curve of glucose.57
Figure 3-2. Chromatogram of the mixture of standard organic acid.58
Figure 3-3. The standard calibration curve of organic acids.59
Figure 3-4. The standard calibration curve of endotoxin.62
Figure 3-5. Preparation of antibacterial fiber membrane.64
Figure 5-1. Phylogenetic tree based on 16S rDNA sequence comparisons of strain Wu3M and selected bacteria.72
Figure 5-2. Time course of cell growth and organic acids production by Gluconacetobacter sp. Wu3M at various mediums in different containers.76
Figure 5-3. Effect of various mediums on bacterial cellulose produce, gluconic acid production, glucose utilization and yield (BC and gluconic acid) by Gluconacetobacter sp. Wu3M.77
Figure 5-4. The photograph of bacterial cellulose membrane product by Gluconacetobacter sp. Wu3M at different culture methods. 78
Figure 5-5. Time course of cell growth and organic acids production by Gluconacetobacter sp. Wu3M at different concentrations of acetic acid. 82
Figure 5-6. Effect of various acetic acid concentration on bacterial cellulose produce, gluconic acid production, glucose utilization and yield by Gluconacetobacter sp. Wu3M. 83
Figure 5-7. The photograph of bacterial cellulose membrane by Gluconacetobacter sp. Wu3M at different acetic acid concentrations.84
Figure 5-8. Time course of cell growth and organic acids production by Gluconacetobacter sp. Wu3M at different medium surface area.89
Figure 5-9. The photograph of bacterial cellulose membrane production by Gluconacetobacter sp. Wu3M at different medium surface area.90
Figure 5-10. Time course of cell growth and organic acids production by Gluconacetobacter sp. Wu3M on different rough of wall.95
Figure 5-11. Effect of various rough of wall on bacterial cellulose produce, gluconic acid production, glucose utilization and yield (BC and gluconic acid) by Gluconacetobacter sp. Wu3M. 96
Figure 5-12. The photograph of bacterial cellulose membrane by Gluconacetobacter sp. Wu3M at different rough of wall. 97
Figure 5-13. Time course of BC and organic acids production by Gluconacetobacter sp. Wu3M at different inoculated volume. 100
Figure 5-14. Effect of various inoculated volume on bacterial cellulose produce, gluconic acid production, glucose utilization and yield (BC and glucuronic acid) by Gluconacetobacter sp. Wu3M.101
Figure 5-15. The photograph of bacterial membranes production at different inoculated volume.102
Figure 5-16. Comparison of experimental data and kinetic model predicitions of the growth of Gluconacetobacter sp. Wu3M by using Eq. (4-5).106
Figure 5-17. Comparison of experimental data and kinetic model predicitions of the formation of bacterial cellulose membrane by using Eq. (4-10).107
Figure 5-18. The relationship of αμX and βX time and in fermentation.108
Figure 5-19. Comparison of experimental data and kinetic model predicitions of the substrate utilization with different carbon source by using Eq. (4-19), (4-25) and (4-28).109
Figure 5-20. Evalation of 1/YX/Smax using Eq. (4-19).110
Figure 5-21. Evalation of 1/YX/Smax and 1/ YP/S max using Eq. (4-25).111
Figure 5-22. Evalation of 1/YX/Ssmax and 1/ YP/S max using Eq. (4-28).112
Figure 5-23. Comparison of experimental data and kinetic model predicitions of the growth of Gluconacetobacter sp. Wu3M by using Eq.(4-2).118
Figure 5-24. Comparison of experimental data and kinetic model predicitions of the formation of bacterial cellulose membrane by using Eq.(4-7).119
Figure 5-25. The relationship of αμX and βX time and in fermentation.120
Figure 5-26. Comparison of experimental data and kinetic model predicitions of the substrate utilization with different initial glucose concentration by using Eq.(4-19),(4-25) and (4-28).121
Figure 5-27. Evalation of 1/YX/Smax using Eq. (4-19).122
Figure 5-28. Evalation of 1/YX/Smax and 1/ YP/S max using Eq. (4-25).123
Figure 5-29. Evalation of 1/YX/Smax and 1/ YP/S max using Eq. (4-28).124
Figure 5-30. The kinetic curve of endotoxin by bacterial cellulose and culture broth in this study.130
Figure 5-31. Effect of concentrations of benzalkonium chloride solution and nano silver on antimicrobial activity against Bacillus subtilis on agar plates.135
Figure 5-32. Effect of concentrations of benzalkonium chloride solution and nano silver on antimicrobial activity against Escherichia coli on agar plates.136
Figure 5-33. Comparisons of antibacterial activity between benzalkonium chlorlde-BC dry films and nano-BC dry films.137
Figure 5-34. SEM image of bacterial cellulose membranes with different antimicrobial.138
Figure 5-35. Growth curves of Escherichia coli (a) and Bacillus subtilis (b) in the cultures with different antimicrobial BC film.139
Figure 5-36. The energy dispersive spectrometer of nano-silver BC films.140

表目錄

Table 2-1. Iα/Iβ ratio of different cellulose sources.10
Table 2-2. Differences in structural/mechanical properties of plant cellulose (cotton linters) and bacterail cellulose (from Acetobacter xylinus).11
Table 2-3. Different strain producing bacterial cellulose.24
Table 2-4. Examples of applications of bacterial cellulose.47
Table 3-1. HS medium.54
Table 3-2. PCR agent.54
Table 3-3. PCR program.54
Table 5-1. The yield of BC and gluconic acid by Gluconacetobacter sp. Wu3M at different medium surface area in trays.91
Table 5-2. Effect of carbon source on true and appearance YP/X for Gluconacetobacter sp. Wu3M.113
Table 5-3. Effect of carbon source on true and appearance YP/S and YX/S for Gluconacetobacter sp. Wu3M.114
Table 5-4. Effect of carbon source on true and appearance Yp/s and Yx/s for Gluconacetobacter sp. Wu3M.115
Table 5-5. Effect of initial glucose concentration on true and appearance YP/X for Gluconacetobacter sp. Wu3M.125
Table 5-6. Effect of initial glucose concentration on true and appearance YP/S and YX/S for Gluconacetobacter sp. Wu3M.126
Table 5-7. Effect of carbon source on true and appearance Yp/s and Yx/s for Gluconacetobacter sp. Wu3M.127
Table 5-8. Relationship between the feed drug concentrations and the drug-uploading capacities of BC films.141
Table 5-9. Swelling ratio of bacterial cellulose dry films in difference concentration solutions.142

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