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研究生:詹軒昀
研究生(外文):Hsuan-Yun Chan
論文名稱:利用Bacillussp.相關菌株生產聚羥基丁酸(PHB)與聚羥基戊酸(PHV)共聚物及其性質探討
論文名稱(外文):Production and characterization of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Bacillus sp. strains
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:101
中文關鍵詞:聚羥基丁酸聚羥基戊酸澱粉
外文關鍵詞:PHAPHBPHBVBacillus sp.starch
相關次數:
  • 被引用被引用:1
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聚羥基烷酸(Polyhydroxyalkanoates, PHA)是許多細菌或真菌,如Ralstonia eutrophas、Alcaligenes latuss、Bacillus cereus、Pseudomonas pscudoflava,在過量碳源或缺少某些營養素(如氮、磷、硫)時,用以儲存能量的產物。本研究主要利用自酒精廢水中篩選出的數株本土菌株Bacillus sp. YW系列 ,探討是否具有累積PHA的能力。研究顯示,篩選出的Bacillus sp. YW系列菌株,皆可生產PHA。研究進一步證實:上述菌株皆具有代謝多種醣類與澱粉進行生長,進而合成PHB之能力。添加低濃度多種不同酸類時作為碳源時,會促進菌體的生長;在添加tryptone 情況,具有累積PHA產物的能力。初步研究顯示Bacillus sp. YW1與YW2在LBS培養基下可得菌體乾重與PHB 菌體含量達10 g/L與30%以上。
培養條件調整之後,使用LBSS培養基,在溫度30°C、轉速 200 rpm與起始酸鹼值pH=7時,可使Bacillus sp. YW1與YW2可誘導合成PHV,且PHV佔有菌體含量12%以上。TEM觀察也顯示菌體是具有累積PHA之能力。將培養時間36小時的純化的PHA由NMR與GC-MS鑑定,確認菌體生產的為PHBV。再由NMR的分析,可知兩菌株的PHBV中PHV含量最高可占有總量的莫耳分率約近30%。藉由GPC的分析,菌株YW1與YW2生產的PHA平均重量分子量集中在Mw=300,000-400,000之間,YW1的PDI為4.71與YW2的PDI為2.62;再藉由TGA分析,可確認菌株生產的PHBV有良好的熱穩定性。DSC分析顯示,純化之PHBV應為具有半結晶性質的高分子。
Polyhydroxylalkanoates (PHA) are biodegradable polyesters that are accumulated in various microorganisms as intracellular carbon and energy storage material under nutrient-limited conditions. This study aimed to screen better production strains in order to obtain more efficient production of Polyhydroxylalkanoates from Bacillus sp. YW1, Bacillus sp. YW2 and Bacillus sp. YW3. This studies shown that these strains could be improved to grow and further synthesize PHB using various carbon sources. This work also suggests that the bacterial growth could be improved which the medium contain various organic acids. On the other hand, these strains could accumulate PHA when adding tryptone in media. A semi-synthesized medium (LBS medium) was developed for this study which could be able to support the growth of this Bacillus sp. YW1 and Bacillus sp.YW2 with 10 g/L PHB production and PHB content over 30%, respectively.
Temperature, pH and agitation rate are common environmental factors that play a crucial role in cell growth and PHB production. The optimal culture condition 30℃, pH 7, 200 rpm was used as the standard experimental condition. From this work, we found that the strains Bacillus sp. YW1 and YW2 could be induced to accumulate PHV over 12 %. PHA in the cell were purified primarily via different methods. The chemical structure and molecular masses of the purified PHA product were confirmed by nuclear magnetic resonance (1H-NMR & 13C-NMR) spectrometer and Gas Chromatography-Mass spectrometry (GC-MS), respectively. The property of the PHAs produced by Bacillus sp. strains isolated in this study was in consistent with the standard purchased from Sigma Co., indicating that Bacillus sp. strains are PHBV-synthesizing strain. Further analysis indicated that the PHV mole% of YW1 and YW2 strains were around 30%. According to analysis of GPC, the average Molecule weight was Mw=300,000 to 400,000; the strains YW1’s PDI was 4.71 and YW2’s PDI was 2.62. Besides, the data of TGA illustrated that PHBV has stable heat property. Moreover, the PHBV may be half-crystal polymer using DSC.
目錄
中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 vii
表目錄 x
第一章、緒論 1
1.1 前言 1
1.2 可分解性塑膠 2
1.3 生物分解性材料:聚羥基烷酸(酯)(PHA) 5
1.3.1 PHA簡史 5
1.3.2 PHA的化學結構與性質 6
1.3.3 PHA的在微生物系統的生成與代謝途徑 7
1.3.4 PHA的分解 11
1.3.5 PHA的萃取與純化 13
1.4 研究目的 15
第二章、材料與方法 16
2.1 實驗材料 16
2.1.1實驗菌株 16
2.1.2 實驗藥品 16
2.1.3 培養基 19
2.1.4 實驗儀器 20
2.2 實驗方法 22
2.2.1菌種的保存 22
2.2.2 搖瓶醱酵實驗 22
2.2.3 測定菌體之生長狀況 22
2.2.4 各種碳源配方測試 23
2.2.5 發酵槽實驗 23
2.2.6 PHA的萃取與純化 24
2.2.7 PHA氣相層析(GC )分析 24
2.2.8 PHA液態核磁共振儀(NMR)分析 25
2.2.9 PHA氣相層析質譜儀(GC-MS)分析 26
2.2.10 PHA凝膠層析儀(GPC)鑑定 26
2.2.11 PHA熱重量分析儀 (TGA)鑑定 27
2.2.12 PHA熱微差掃描分析儀(DSC) 鑑定 27
2.2.13穿透式電子顯微鏡(TEM)對菌體之觀察 27
第三章、結果與討論 29
3.1 論文研究大綱, 29
3.2 菌種對主要碳源之測試 30
3.3 菌種主培養基之探討 32
3.3.1 搖瓶與生長曲線測試 32
3.3.2 LB與澱粉為培養基搖瓶生產PHB測試 35
3.3.3 LBS培養基添加丙酸鈉(sodium propionate)生產PHBV 37
3.4不同種類的酸與菌種間之影響 40
3.4.1 各種酸類之測試 40
3.4.2 LBS培養基添加不同濃度的sodium propionate之影響 42
3.4.3 LBS培養基添加琥珀酸鈉(sodium succinate)之影響 45
3.4.4 LBS培養基添加蘋果酸(Malic acid)之影響 45
3.4.5 LBS培養基添加不同酸之影響 48
3.5離子對菌種之探討 51
3.5.1 培養基添加離子配方 51
3.6 LBS主培養基配方成分調整之研究 55
3.6.1 LB培養基影響因子之探討 55
3.7 外在環境條件最佳化對菌種生產PHA的影響探討 57
3.7.1 搖瓶選擇影響之探討 57
3.7.2 溫度影響之探討 60
3.7.3 轉速影響之探討 62
3.7.4 酸鹼值影響之探討 65
3.8 奶類蛋白影響之探討 68
3.8.1奶類蛋白相關衍生物之影響 68
3.9 PHA 性質鑑定與探討 71
3.9.1 PHA的純化與回收 71
3.9.2 GC分析PHA產量 71
3.9.3 NMR光學圖譜性質鑑定 73
3.9.4 GC-MS鑑定 77
3.9.5 GPC分子量鑑定 80
3.9.6 TGA分析 80
3.9.7 DSC分析 82
3.10 菌體型態觀察 86
3.10.1 菌體TEM觀察 86
第四章、結論 89
第五章、附錄 93
附錄 (一) 定序比對 93
附錄 (二) 標準品檢量線 95
附錄 (三) 藥品成分組成 96
第六章、參考文獻 98
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