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研究生:陳裕憲
研究生(外文):Yu Sian Chen
論文名稱:限氮條件下培養Bacillusmegaterium生合成PHB之研究
論文名稱(外文):Biosynthesis of PHB by Bacillus megaterium in a Nitrogen-limiting Condition
指導教授:涂瑞澤涂瑞澤引用關係余世宗余世宗引用關係
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:2007
畢業學年度:95
語文別:中文
論文頁數:96
中文關鍵詞:有機鈉鹽批次發酵
外文關鍵詞:Bacillus megateriumPHB(V)
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本研究於限氮條件下,進行搖瓶與批次發酵培養Bacillus megaterium。搖瓶實驗中葡萄糖(主要碳源)濃度為20 g/L,以NH4NO3為氮源,並限制其濃度於0.6 g/L,另外添加有機鈉鹽(乙酸鈉、丙酸鈉、丁酸鈉及戊酸鈉)為第二碳源,探討各種有機鈉鹽最佳之添加濃度。批次發酵實驗中葡萄糖(主要碳源)濃度為40 g/L,以NH4NO3為氮源,並限制其濃度於0.6 g/L,另外添加有機鈉鹽(丙酸鈉與戊酸鈉)為第二碳源,探討含奇數碳鏈之有機酸鹽對菌體生合成PHB(V)之影響。
搖瓶實驗結果顯示:添加乙酸鈉2 g/L時具最高HB產量,為1.86 g/L,佔菌體重57.2%;添加丙酸鈉0.5 g/L時具最高HB產量,為0.62 g/L,佔菌體重27.6%;添加丁酸鈉0.5 g/L時具最高HB產量,為1.77 g/L,佔菌體重39.18%;添加戊酸鈉1 g/L時,具最高HB產量,為0.77 g/L,佔菌體重31.0%。在搖瓶試驗中,第二碳源的添加均無HV的產生。
批次發酵實驗結果顯示:添加丙酸鈉3 g/L時,培養至12 h,具最高菌體生質量與HB產量,分別達3.96與0.72 g/L,之後,隨著培養時間的增加而菌體生殖量與HB產量均遞減;添加戊酸鈉3 g/L時,結果顯示:HB產量有明顯增加,從未添加的0.73 g/L增加至添加後的1.70 g/L,增加了133.0%;添加兩種有機鈉鹽亦皆無HV的產生。
In this study, Bacillus megaterium was cultivated either in a flask or in a batch fermenter under a nitrogen-limiting condition. For a flask culture, the concentration of glucose (as the primary carbon source) was initially set at 20.0 g/L, and NH4NO3 (as the nitrogen source) was limited to be 0.6 g/L. In addition, one of the organic acid salts (sodium acetate, sodium propionate, sodium butyrate, and sodium valerate) was used as a second carbon source. Exploration of the optimal concentration was performed in order to obtain the highest yield of PHB. For the cultivation in a batch fermenter, the concentration of glucose (as the primary carbon source) was initially set at 40.0 g/L, and NH4NO3 (as the nitrogen source) was limited to be 0.6 g/L. In addition, sodium propionate or sodium valerate was used as a second carbon source to explore the effect of organic salt with odd number of carbon chains on the biosynthesis of PHB(V).
Experimental results showed that the biosynthesis of HB (57.2% of the biomass) was maximized, if the concentration of sodium acetate in the medium was 2.0 g/L. Similarly, the biosynthesis of HB (27.6% of the biomass) was maximized at 0.5 g/L of sodium propionate. Adding 0.5 g/L sodium butyrate maximized the HB yield (39.18% of the biomass), and adding 1.0 g/L sodium valerate maximized the HB yield (31.0% of the biomass). For cultivation in a flask, no HV was detected even though an organic acid salt was added into the medium as a second carbon source.
For cultivation in a fermenter, adding 3.0 g/L sodium propionate maximized the production of biomass and HB, being 3.96 and 0.72 g/L, respectively, at 12 h, and thereafter, both biomass and HB production decreased with the culture time. Adding sodium valerate resulted in the increase of HB production from 0.73 g/L (without adding valerate) to 1.70 g/L (adding valerate 3.0 g/L). However, adding either sodium propionate or sodium valerate as a second carbon source did not make the strain to synthesize HV.
封面內頁
簽名頁
授權書......................................................iii
中文摘要....................................................iv
英文摘要....................................................v
誌謝.......................................................vii
目錄.......................................................viii
圖目錄.....................................................xii
表目錄.....................................................xiv
1. 緒論....................................................1
2. 文獻回顧................................................3
2.1 塑膠的介紹.............................................3
2.2 生物分解性塑膠發展概況...................................4
2.2.1 分解性塑膠的分類......................................6
2.2.2 生物可分解性塑膠簡介..................................6
2.2.3 生物可分解性塑膠應用..................................8
2.3 微生物的代謝產物........................................8
2.3.1 ㄧ次代謝物的生合成....................................10
2.3.2 二次代謝物的生合成....................................10
2.4 PHAs簡介..............................................10
2.4.1 PHAs的物理化學性質...................................12
2.4.2 PHB之萃取...........................................16
2.5 生合成PHA的方式........................................18
2.5.1 搖瓶培養............................................18
2.5.2 批次發酵培養.........................................18
3. 材料與方法.............................................21
3.1 培養基................................................21
3.2 實驗藥品..............................................21
3.3 儀器設備..............................................26
3.4 菌株培養..............................................27
3.4.1 篩菌................................................27
3.4.2 篩菌方法............................................28
3.4.3 活化................................................28
3.5 碳、氮源的影響.........................................29
3.5.1限制氮源.............................................29
3.5.2不同氮源.............................................29
3.5.3添加不同碳源的探討....................................29
3.5.4添加最適碳源濃度探討..................................30
3.6 添加有機鈉鹽..........................................30
3.6.1 添加有機鈉鹽的時機探討...............................30
3.6.2 添加不同濃度有機鈉鹽之探討...........................31
3.7 批次發酵槽培養.......................................31
3.7.1 預培養............................................31
3.7.2 發酵槽培養........................................31
3.8分析方法.............................................33
3.8.1 生質體............................................33
3.8.2 葡萄糖............................................34
3.8.3 氮源..............................................34
3.8.4 菌體中PHB / PHBV的分析.............................35
4. 結果與討論............................................38
4.1 篩菌結果.............................................38
4.1.1 菌株B. megaterium之生長曲線.........................38
4.1.2 起始pH值對菌株B. megaterium生長之影響................43
4.2 碳、氮源之影響........................................43
4.2.1 硫酸銨濃度對B. megaterium生質量之影響................43
4.2.2 氮源對B. megaterium生合成PHB之影響..................43
4.2.3 碳源對B. megaterium生合成PHB之影響..................47
4.2.4 碳源濃度對B. megaterium生合成PHB之影響..............47
4.3 添加有機鈉鹽時機探討..................................47
4.4 添加不同濃度之有機鈉鹽探討............................50
4.4.1 乙酸鈉...........................................50
4.4.2 丙酸鈉...........................................53
4.4.3 丁酸鈉...........................................53
4.4.4 戊酸鈉...........................................53
4.5 限氮條件下添加丙酸鈉之批次發酵培養....................57
4.6 限氮條件下添加戊酸鈉之批次發酵培養....................61
5. 結論................................................66
5.1 結論...............................................66
5.2 未來展望............................................67
參考文獻................................................68
附錄.............................72
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