臺灣博碩士論文加值系統

聚羥基烷酯類因性質近似熱塑型塑膠且同時具有生物相容與環境友善特性，為時下綠色材料開發的熱門選擇。目前製造方式多數以細菌醱酵為主，因此世人尋找適當的醱酵菌株，正方興未艾。吾人觀察聚羥基烷酯類累積細菌體內，多在碳源過量與特定營養因子；如氮源受限的不平衡生長時發生。考慮轉劣培養程序也會導致細菌暫時性不平衡的生長現象，本研究報告以糖蜜與蛋白水解物調製培養基，配合轉劣培養程序探討轉劣現象與細菌累積聚羥基烷酯類的關係，同時評估其可應用的方式。研究結果顯示聚羥基烷酯類累積確屬轉劣現象。此現象為本研究挑選的革蘭氏陽性桿菌與革蘭氏陰性假單胞菌所共有，不因這兩類細菌所需養分的碳氮比不同而受影響，應可作為一簡單、廣用的聚羥基烷酯類生產菌株篩選方法。研究中也挑選二環境篩選菌株以搖瓶方式進行轉劣培養生產測試。結果Bacillus sp. B1與Pseudomonas sp. H1以轉劣培養分別得到0.03 和0.07 g/L聚羥基烷酯類，經分析為聚羥基丁酯(PHB)。顯示轉劣培養程序也可作為大量生產聚羥基烷酯的批次醱酵程序。

Polyhydroxyalkanoates (PHAs), one of the biodegradable biosynthetic polymers were function as carbon and energy reservoirs in numerous bacteria. They were known to accumulate inside the bacteria usually when carbon source is excess and one else essential growth nutrient is limited. Shift-down, a temporary unbalanced bacteria culture method established by simple dilution culture procedure can equivalently induce the PHAs accumulation. A temporary unbalanced bacteria growth phenomenon established by shift-down culture procedure was applied to screening PHAs accumulating environment bacteria isolates. The culture method was applied to test PHAs accumulating in standard and environment bacteria isolates. Despite the typical limiting nutrient or C/N ratio, PHAs accumulation seems to be common in bacteria applied of the culture method. This result shown that the shift-down culture method is simple and broad spectrum in screening environment bacteria to find new industry available PHAs produced candidates. The products were characterized as PHB in recovery of Pseudomonas sp. and Bacillus sp.0.03 g/L and 0.07g/L respectively.

中文摘要 I
英文摘要 VI
誌謝 VII
目錄 VIII
圖目錄 XI
表目錄 XII
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微生物不平衡生長現象介紹 4
2.1.1 Shift-Up現象 4
2.1.2 Shift-Medium現象 5
2.1.3Shift-Down現象 5
2.2 C/N Ratio生長因子要素 6
2.3 PHB鑑別方法介紹 7
2.3.1分子生物法 7
2.3.2螢光分析法 7
2.3.2.1尼羅藍 7
2.3.2.2尼羅紅 7
2.4單一養分對PHB累積影響 8
2.5 PHA代謝途徑 8
2.6生產PHAs菌種介紹 11
2.7各種碳源累積PHAs 種類介紹 12
2.8 PHB搖瓶醱酵培養 12
第三章實驗方法 14
3.1藥品 14
3.2儀器設備 15
3.3實驗菌株 16
3.4染劑與培養基配製 16
3.5實驗步驟 16
3.5.1革蘭氏陽性與陰性環境菌株篩選 16
3.5.2蘇丹黑染色 18
3.5.3菌株B1與H1於不同碳氮比生長曲線及PHAs累積情形探討 18
3.5.4菌株B1與H1轉劣培養程序及PHAs累積情形 19
3.5.5不同培養基的轉劣培養程序下菌株B1與H1 PHAs累積情形 20
3.5.6轉劣培養程序使標準菌株與環境菌株累積PHAs的情形 20
3.5.7菌株B1與H1以轉劣培養程序生產PHAs 21
3.5.8分離純化PHAs 21
第四章結果與討論 22
4.1革蘭氏陽性與陰性環境菌株篩選 22
4.2菌株B1與H1不同碳氮比生長曲線特徵探討 22
4.3菌株B1與H1不同碳氮比PHAs累積情形 23
4.4菌株B1與H1於常態與轉劣培養程序及PHAs累積情形 24
4.5不同培養基的轉劣培養程序下菌株B1與H1 PHAs累積情形 26
4.6轉劣培養程序使標準菌株與環境菌株累積PHAs的情形 28
4.7菌株B1與H1以轉劣培養程序生產PHAs 28
第五章結論 29
參考文獻 30
附件一環境篩選菌株16S rRNA gene序列 49


 
圖目錄
圖一、PHA之代謝路徑 10
圖二、細菌在不同碳、氮源比例下的生長曲線特徵 39
圖三、細菌B1在不同碳、氮源比例下的PHAs累積情形 40
圖四、細菌H1在不同碳、氮源比例下的PHAs累積情形 41
圖五、細菌於常態與轉劣培養程序的生長曲線特徵 42
圖六、細菌於轉劣培養程序的PHAs 蘇丹黑染色 43
圖七、細菌B1在 LB培養基之Shift Down程序PHAs累積情形 44
圖八、細菌 B1在 NB培養基之Shift Down程序PHAs累積情形 45
圖九、細菌 H1在 LB培養基之Shift Down程序PHAs累積情形 46
圖十、細菌 H1在 NB培養基之Shift Down程序PHAs累積情形 47
圖十一、環境菌株轉劣培養程序的PHAs 蘇丹黑染色. 48

 
表目錄
表一、用於16S rRNA gene定序的廣用PCR引子 36
表二、不同碳氮比的蔗糖水解蛋白培養液 37
表三、實驗菌種列表 38

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