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研究生:林俊亨
研究生(外文):Lin, Chun-Heng
論文名稱:鹽田鹽單胞菌利用廢甘油生產PHB之研究
論文名稱(外文):PHB Production by Halomonas salina with Biodiesel By-product Glycerin
指導教授:徐嘉澤徐嘉澤引用關係
指導教授(外文):Hsu,Chia-Tse
口試委員:黃清龍潘建亮徐嘉澤
口試委員(外文):Huang,Ching-LungPan,Chien-LiangHsu,Chia-Tse
口試日期:2011-06-20
學位類別:碩士
校院名稱:高苑科技大學
系所名稱:化工與生化工程研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:92
中文關鍵詞:鹽田鹽單胞菌廢甘油聚β-羥基丁酸酯
外文關鍵詞:Halomonas salinaPHBBiodiesel By-product Glycerin
相關次數:
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  • 下載下載:24
  • 收藏至我的研究室書目清單書目收藏:0
近年來由於石油蘊藏即將用罄的憂慮逐漸加劇,使用石油為原料的燃油與物資價格不斷攀升。塑膠製品也是非常依賴石油的產業。面對逐漸減少的原料來源,學術與產業界已積極投注許多心力於替代原料與製程改良。生質塑膠是其中一個很好的選項々首先,生質塑膠由生物製造直接或間接來自植物的固碳作用,不會增加新的二氧化碳到空氣中。其次,生質塑膠具有高的生物相容性,應用於生醫材料時較無發炎或排斥問題。其三,生質塑膠具有生物可分解性,在使用過拋棄後可在環境中較快被分解,不會造成廢棄物破壞生態問題。 PHB 是生質塑膠的一種,性質類似熱塑性塑膠。由微生物製造,也可被其分解。本研究以 Halomonas salina 菌種為主,研究此菌種使用廢甘油生產PHB能力。結果發現此菌種可在含甲醇之廢甘油2%以下生長沒有抑制現象,同時累積35%PHB。分析累積特性發現此菌種的野生菌株與標準菌株可在豐富培養基中持續累積PHB,其分子量介於571480Da到679998Da之間。發酵完成之菌體經0.4N之NaOH純化後PHB純度可以從41%提升到62.5%。本實驗也檢驗此菌種使用有機高分子製造PHB之能力。綜合本研究之結果與中度嗜鹽菌廣譜的鹽濃度耐受能力,此菌種很適合應用在汙水處理再生系統中生產 PHB 。
Recent years, people are concerned about the exhausting of petroleum reserves. Fuel and materials mad from petroleum were rising priced. Plastic industry is also mainly dependent on petroleum. Facing the challenge, a lot of alternative raw materials and process improvements were done by academic and industry. Bio-plastic is one of the resolutions. The bio-plastic is bio-compatible and biodegradable when discarded after use. It is produced indirectly from plant carbon fixation. Polyhydroxybutyrate ( PHB ) is one of the bio-plastic, similar in nature to thermoplastics, produced and decomposed by many microorganisms. In this report, the production of PHB by Halomonas salina with simple carbon source and biodiesel waste glycerol was studied. The bacteria were found to grown normally in 2% methanol containing waste glycerol, and accumulate PHB to 35%. The accumulation was found from log phase to late stationary phase in both standard and wild strain, without nutrition limit. The molecular weights of the accumulated PHB were ranging between 571480Da to 679998kDa. The purity of PHB can raise to 62.5% fallowed by 0.4N NaOH treatment after fermentation. The ability of using organic polymer as carbon source to produce PHB has been investigated also. On the basis of the results, Halomonas salina is an ideal species for PHB production in sewage treatment systems.
中文摘要 ....................................................................................... I
英文摘要 ....................................................................................... II
誌謝 ......................................................................................... III
第一章 緒論 ..................................................................................... 1
1.1前言 ........................................................................................ 1
1.2研究動機與目的 ............................................................................... 3
第二章 文獻回顧 ................................................................................. 4
2.1 生物可分解材料的種類 ......................................................................... 4
2.1.1 天然可分解性高分子 ......................................................................... 4
2.1.2 人工合成可分解性高分子 ..................................................................... 5
2.1.3 微生物合成可分解性高分子 ................................................................... 6
2.2 PHB 生產 ................................................................................... 7
2.2.1 PHB 化學合成 .............................................................................. 7
2.2.2 PHB 生物合成 .............................................................................. 9
2.2.2.1 細菌醱酵合成PHB ......................................................................... 9
2.2.2.2 基因轉殖細菌生產 PHB ................................................................... 11
2.2.2.3 活性汙泥合成 PHB ....................................................................... 11
2.2.3 基因轉殖植物 ............................................................................. 12
2.3 PHB 改質與應用 ............................................................................. 12
2.3.1 改質 .................................................................................... 12
2.3.2 應用 .................................................................................... 13
2.3.2.1 可生物降解的熱塑性材料 .................................................................. 13
2.3.2.2 醫學上的應用 ........................................................................... 13
2.3.2.3 壓電製品 ............................................................................... 14
2.4 PHB 細菌生產研究 ........................................................................... 15
2.5 PHB PEG lation 研究 ....................................................................... 15
2.5.1 聚 ( β- 羥基丁酸酯 ) / 聚乙二醇共聚物的製備 ................................................ 16
2.5.1.1熔融酯交換反應 .......................................................................... 16
2.5.1.2互穿網路技術 ............................................................................ 16
2.5.1.3接枝共聚 ................................................................................ 17
2.5.1.4生物合成 ................................................................................ 17
2.6清潔生產 .................................................................................... 18
2.7 PHB 高分子物性分析 ......................................................................... 19
2.7.1 熱重分析( Thermogravity Analysis 々 TGA ) .............................................. 19
2.7.2凝膠滲透層析儀 ( Gel Permeation Cromatography々GPC ) ...................................... 19
第三章 實驗方法 ................................................................................ 21
3.1藥品 ....................................................................................... 21
3.2實驗菌株 .................................................................................... 22
3.3儀器設備 .................................................................................... 23
3.4實驗步驟 .................................................................................... 24
3.4.1菌種培養與保存 ............................................................................ 24
3.4.2小量搖瓶培養 .............................................................................. 24
3.4.3不同碳、氮源培養生產PHB檢測................................................................. 25
3.4.4 不同鹽濃度生長與PHB累積特徵測定............................................................. 26
3.4.4.1 長時間不同鹽濃度生長與PHB累積特徵測定 .................................................... 27
3.4.5不同碳源濃度培養生產PHB檢測................................................................. 27
3.4.6 PHB三氯甲烷純化法 ........................................................................ 28
3.4.7 NaOH純化法 .............................................................................. 29
3.4.8 熱重分析 ................................................................................. 29
3.4.9分子量分析 ................................................................................ 30
3.4.10 API 20 NE測試之結果 ..................................................................... 30
3.4.11 PHB/PEG混合物製備 ....................................................................... 31
第四章 結果與討論 ............................................................................... 32
4.1鹽單胞菌於不同鹽濃度培養基中的生長與PHB累積特徵 ................................................. 32
4.1.1 鹽單胞菌 CG1與DSM55928T於不同鹽濃度培養基中的生長特徵 ....................................... 33
4.1.2鹽單胞菌 CG1與DSM5928T 於不同鹽濃度生長PHB累積情形 ........................................... 35
4.1.2.1鹽單胞菌 CG1於不同鹽濃度生長PHB累積情形 ................................................... 35
4.1.2.2鹽單胞菌DSM5928T 於不同鹽濃度生長PHB累積情形 .............................................. 36
4.1.2.3鹽單胞菌 CG1長時間生長PHB累積情形 ......................................................... 37
4.1.2.4鹽單胞菌 CG1於不同鹽濃度培養基中PHB分子量分析 .............................................. 37
4.1.3鹽單胞菌 CG1 於不同碳源濃度生長PHB累積情形 ................................................... 41
4.1.4鹽單胞菌 CG1使用甘油與廢甘油碳源生長PHB累積情形 .............................................. 42
4.1.5鹽單胞菌 CG1 利用PEG生產PHB-PEG產物TGA分析 ................................................. 44
4.1.6鹽單胞菌 CG1 PHB使用NaOH純化之情形 ......................................................... 45
第五章 結論 .................................................................................... 46
第六章 參考文獻 ................................................................................ 47
圖 表 ......................................................................................... 56
表一、培養基配方表 .............................................................................. 57
表二、已知PHB 生產菌株 .......................................................................... 58
表三、CG1與Ventosa 發表之Halomonas salina生化測試之情形 .......................................... 59
圖一、PHB 結構細菌生產代謝路徑圖 ................................................................. 60
圖二、各種碳源於細菌中代謝途徑與相關基因產物圖 ..................................................... 61
圖三、鹽單胞菌於不同鹽濃度生長特徵 ............................................................... 62
圖四、鹽單胞菌CG1於不同鹽濃度生長累積PHB特徵 ...................................................... 63
圖五、鹽單胞菌DSM5928T於不同鹽濃度生長累積PHB特徵 ................................................. 64
圖六、鹽單胞菌 CG1 長時間生長PHB累積情形 ......................................................... 65
圖七、鹽單胞菌 CG1 於不同鹽濃度培養基中累積PHB分子量 ............................................... 66
圖八、鹽單胞菌 CG1 於不同碳源濃度生長PHB累積情形 .................................................. 67
圖九、鹽單胞菌 CG1使用甘油與廢甘油碳源生長PHB累積情形 .............................................. 68
圖十、鹽單胞菌 CG1使用甘油及廢甘油生產PHB之TGA分析 ................................................ 69
圖十一、鹽單胞菌CG1利用PEG1000生產PHB-PEG產物NMR分析 ............................................. 70
圖十二、鹽單胞菌CG1利用PEG生產PHB-PEG產物DSC分析 ................................................. 71
圖十三、鹽單胞菌CG1利用PEG生產PHB-PEG產物TGA分析 ................................................. 72
圖十四、鹽單胞菌 CG1 PHB使用NaOH純化之情形 ....................................................... 73
附件 .......................................................................................... 74
全國性技職院校師生創業競賽 ....................................................................... 75
高苑科技大學校園創業競賽 ........................................................................ 77
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