本研究以微生物發酵生合成PHB，使用之菌株為Bacillus megaterium YU-1，在氮源限制的生長環境下，於不同溫度 (26、30及35℃)進行批次培養，探討溫度對生質量、PHB產量及營養源消耗之影響。實驗結果顯示，於30℃下不調控pH值之培養，生質量與PHB產量分別為6.27與4.26 g/L，PHB為菌體生質量的68%；於26℃下，生質量與PHB產量分別為7.72與3.54 g/L，PHB為菌體生質量的48%；於35℃下，生質量與PHB產量分別為5.74與2.29 g/L，PHB佔菌體生質量為41%，其中以30℃為最佳之培養條件。
在調控pH為5.5時，於不同溫度下進行批次培養，探討溫度對菌體生質量、PHB產量及營養源消耗之影響。實驗結果顯示，於26℃下培養，可得最高之生質量與PHB產量，分別為8.31與5.59 g/L；但在PHB佔菌體之百分比中，以培養於30℃下，PHB佔菌體生質量(74%)為最高，而生質量與PHB產量分別為6.33與4.71 g/L；於35℃中，由於培養溫度較高，菌體的生長週期較短，生質量與PHB無法持續累積，因此產量較其它培養溫度下為低。
實驗結果顯示，於不同溫度下調控pH 為5.5之培養，菌體生質量與PHB產量，比不調控pH值的條件下來得高，其中又以30℃ pH 5.5為最佳之生長環境。

In this study, Bacillus megaterium YU-1 was cultivated in a batch fermenter under a nitrogen-limiting condition to explore the effect of temperature (26, 30 and 35℃) on microbial growth, PHB production and consumption of nutrition sources. For the case without pH control, the biomass and PHB reached 7.72 and 3.54 g/L, respectively, and PHB/Biomass, 48% at 26℃; the biomass and PHB reached 6.27 and 4.26 g/L, and PHB/Biomass, 68%, at 30℃; the biomass and PHB reached 5.74 and 2.29 g/L, and PHB/Biomass 41% at 35℃. From the above, 30℃ seemed to be the best culture condition among the three to produce PHB.
For the case with pH control at 5.5, Bacillus megaterium YU-1 was also cultivated in a batch fermenter under various temperatures (26, 30 and 35℃). Experimental results showed that, at 26℃, the biomass and PHB reached 8.31 and 5.59 g/L, respectively, which were the highest among the three temperatures. But the PHB/Biomass ratio ever reached 74%, being the best during cultivation, at 30℃, with a biomass of 6.33 g/L and a PHB of 4.71 g/L. As the temperature was raised to 35℃, the generation time became shorter; however, the biomass and PHB did not continue to accrue. Therefore, a lower temperature seemed to be better for the microbe to produce PHB.
In summary, microbial growth and PHB production were better for the case with pH control at 5.5, and 30℃ was the best among the three temperatures to produce PHB.

封面內頁
簽名頁
授權書iii
中文摘要iv
英文摘要v
誌謝vi
目錄vii
圖目錄x
表目錄xii
1. 緒論1
2. 文獻回顧2
2.1 前言2
2.1.1 石化塑膠概述2
2.1.2 生物可分解性塑膠5
2.2 PHAs6
2.2.1 PHAs之介紹6
2.2.2 PHAs的結構與性質6
2.2.3 可生合成PHA之菌株9
2.2.4 PHAs的代謝過程14
2.3 Bacillus megaterium生合成PHAs之探討17
3. 材料與方法22
3.1 實驗材料22
3.1.1 菌株22
3.1.2 實驗藥品22
3.1.3 儀器設備24
3.2 培養條件與步驟26
3.2.1 培養基26
3.2.2 菌株活化26
3.2.3 繼代培養26
3.2.4 預培養30
3.2.5 批次發酵培養30
3.3 分析方法31
3.3.1 菌體之生質量31
3.3.2 葡萄糖之測定31
3.3.3 氮源之測定33
3.3.4 代謝酸之測定34
3.3.5 PHB含量測定34
4. 結果與討論37
4.1 不同溫度對不調控pH之批次發酵培養37
4.1.1 26℃之批次發酵培養37
4.1.2 30℃之批次發酵培養41
4.1.3 35℃之批次發酵培養49
4.1.4 不同培養溫度之比較55
4.2 在控制pH為5.5條件下探討批次發酵PHB之影響60
4.2.1 26℃之批次發酵培養60
4.2.2 30℃之批次發酵培養62
4.2.3 35℃之批次發酵培養68
4.2.4 不同培養溫度之比較73
4.3 不調控pH與pH 5.5下培養Bacillus megaterium YU-1之比較76
5. 結論82
5.1 結論82
5.2 展望83
參考文獻84
附錄89
圖目錄
圖2.1 Alcaligenes eutrophus 生產 PHA 的合成與降解代謝路徑17
圖3.1 實驗架構圖23
圖3.2 樣品分析流程圖32
圖4.1 26℃不調控pH之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮的變化39
圖4.2 26℃不調控pH之批次發酵培養Bacillus megaterium YU-1之代謝酸變化42
圖4.3 30℃不調控pH之批次發酵培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮的變化(硝酸銨：1.5 g/L)44
圖4.4 30℃不調控pH之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮的變化(硝酸銨：0.75 g/L)46
圖4.5 30℃不調控pH之批次發酵培養Bacillus megaterium YU-1之代謝酸變化50
圖4.6不同硝酸銨濃度批次發酵培養Bacillus megaterium YU-1之PHB與PHB含量之變化51
圖4.7 35℃不調控pH之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮的變化53
圖4.8 35℃不調控pH之批次發酵培養Bacillus megaterium YU-1之代謝酸變化56
圖.4.9 不同溫度批次發酵培養Bacillus megaterium YU-1之生質量與PHB變化57
圖4.10 不同溫度批次發酵培養Bacillus megaterium YU-1之PHB佔菌體生質量百分比的變化59
圖4.11 26℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮源的變化61
圖4.12 26℃ pH5.5之批次發酵培養Bacillus megaterium YU-1之代謝酸變化64
圖4.13 30℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮源的變化66
圖4.14 30℃ pH5.5之批次發酵培養Bacillus megaterium YU-1之代謝酸變化69
圖4.15 35℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB、葡萄糖及氮源的變化71
圖4.16 35℃ pH5.5之批次發酵培養Bacillus megaterium YU-1之代謝酸變化74
圖4.17 不同溫度批次發酵培養Bacillus megaterium YU-1之生質量與PHB變化75
圖4.18 不同溫度批次發酵培養Bacillus megaterium YU-1之PHB佔菌體生質量百分比的變化77
表目錄
表2.1 各國廢棄塑膠佔都市垃圾含量之比較4
表2.2 微生物生合成PHAs之限制營養源7
表2.3 經由Alcaligenes sp. A-04生合成的三共聚合物的熱特性10
表2.4 經由Alcaligenes sp. A-04生合成三共聚合物的機械特性11
表2.5 能生合成PHB的微生物12
表2.6 利用基因轉殖菌株生合成PHAs15
表2.7 不同碳源培養B. megaterium於MSM培養基中對細胞濃度及PHB累積之影響19
表2.8 不同氮源培養B. megaterium於MSM培養基中對細胞濃度及PHB累積之影響20
表2.9 在不同溫度下培養Bacillus sp. INT005, B. megaterium ATCC 11561, 及R. eutropha ATCC 17699 生合成PHA之比較21
表3.1 基礎培養基的組成27
表3.2 微量金屬元素的組成28
表3.3 限氮培養基的組成29
表4.1 26℃不調控pH之批次發酵培養Bacillus megaterium YU-1之生質量、PHB產率及葡萄糖消耗速率40
表4.2 30℃不調控pH之批次發酵培養Bacillus megaterium YU-1 之生質量、PHB產率及葡萄糖消耗率(硝酸銨：1.5 g/L)45
表4.3 30℃不調控pH之批次發酵培養Bacillus megaterium YU-1 之生質量、PHB產率及葡萄糖消耗率(硝酸銨：0.75 g/L)48
表4.4 35℃不調控pH之批次發酵培養Bacillus megaterium YU-1之生質量、PHB產率及葡萄糖消耗速率54
表4.5 26℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB產率及葡萄糖消耗速率63
表4.6 30℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB產率及葡萄糖消耗速率67
表4.7 35℃ pH5.5之批次發酵槽培養Bacillus megaterium YU-1之生質量、PHB產率及葡萄糖消耗速率72
表4.8 不同溫度下分別在不調控pH與pH 5.5之批次發酵培養Bacillus megaterium YU-1之比較79
表4.9 不同溫度下分別在不調控pH與pH 5.5之批次發酵培養Bacillus megaterium YU-1之代謝酸比較80

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