臺灣博碩士論文加值系統

本研究目的為探討 Wautersia eutropha 在自營脫硝狀態下，脫硝反應與PHB累積行為之相關性。該菌株已經證實在好氧環境及脫硝反應行無氧呼吸件下，均可大量累積PHB。
本研究以矽膠管反應槽與血清瓶進行批次特性試驗，結果顯示當Wautersia eutropha 進行脫硝反應時以氫氣作為能源、碳酸氫鹽及二氧化碳作為碳源。該菌株會先將硝酸鹽降解為亞硝酸鹽，之後再將亞硝酸鹽轉化為氮氣，為兩階段式的反應途徑。此外，Wautersia eutropha 在進行脫硝作用時，若菌體內有PHB累積，則會優先利用PHB作為碳源，造成反應初期細胞內PHB含量降低。批次試驗至亞硝酸鹽脫硝末期，已有少量的PHB合成；在脫硝反應結束後，該菌株才開始快速合成PHB。本研究最高之PHB累積量達污泥乾重之5.7 %，遠小於異營狀態下之累積量。脫硝反應結束後停止供氫，菌體不僅停止合成PHB，還會降解PHB作為菌株生存之能源及碳源；系統一旦恢復供氫，菌體即回復PHB之合成作用。
在初始C/N比為1時，可獲得良好的比脫硝速率（0.15 mg-N d-1 mg-1-biomass），此時最佳的比PHB合成速率為0.002 mg-PHB d-1 mg-1-biomass。改變不同基質濃度負荷（固定初始C/N比為1），在基質負荷濃度為300 mg/l時，有最佳之比脫硝速率。而改變基質負荷則對最終PHB累積量不會造成明顯的影響。
氨氮較硝酸鹽適合作為 Wautersia eutropha 細胞合成之氮源，添加氨氮則有助於提升菌體濃度，並提高脫硝速率及PHB合成速率。

This study is to investigate the relationship between denitrification and behaviors of PHB accumulation by Wautersia eutropha in autotrophic systems. This study concluded that Wautersia eutropha accumulate PHB by a significant amount under both aerobic and anoxic conditions.
This research based on batch experiments with membrane-feeding substrate bioreactor (MFSB) and serum bottles shows that Wautersia eutropha proceed two-stage denitrification which with nitrite as an intermediate by using hydrogen as a energy source and bicarbonate as a carbon source. It was also observed that Wautersia eutropha utilize PHB prior as a carbon source during denitrification reactions, which lowers the PHB level in cells. The largest accumulated amount of PHB in this study is 5.7 % of the dry weight of cells, however, much smaller than those with heterotrophic incubations. When the hydrogen supplied was stopped at the end of the denitrification reaction, Wauterrsia eutropha used PHB instead of accumulating it. As soon as the hydrogen returned to system, Wautersia eutropha immediately went back to accumulate PHB.
The system achieves highest nitrate removal and PHB accumulation at a C/N ratio of 1.0 and both nitrate and bicarbonate concentration are set to be 300 mg/l. In this given condition, the specific denitrification rate is 0.15 mg-N/day/mg-biomass, and the specific PHB accumulated rate is 0.002 mg-PHB/day/mg-biomass, also, the PHB accumulation shows no connections to nitrate and bicarbonate levels at this study.
The results of this study also suggest that ammonia, which raise the specific denitrification and the specific PHB accumulation, is a better nitrogen source than nitrate and nitrite for incubation of Wautersia eutropha.

摘要 ……………………………………………………………………….…Ⅰ
英摘 ……………………………………………………………………… Ⅱ
目錄 ………………………………………………………………………… Ⅳ
表目錄 …………………………………………………………………....... Ⅶ
圖目錄 …………………………………………………………………….…Ⅷ

第一章 緒論 ……………………………………………………...………… 1
1-1 研究源起 …………………………………………………………. 1
1-2 研究目的 ……………………………………………………….… 2
1-3 研究內容 …………………………………………………………. 3

第二章 文獻回顧 …………………………………………………………... 5
2-1 微生物脫硝作用與原理 …………………………………………… 5
2-1-1 脫硝作用（Denitrification）………………………………..… 5
2-1-2 脫硝作用原理與生化反應 ………………………………... 6
2-2 自營性脫硝作用 …………………………………………………… 9
2-3 影響脫硝反應的因子 …………………………………………….. 14
2-3-1 溶氧 ………………………………………………………. 14
2-3-2 pH …………………………………………………………… 16
2-3-3 溫度 ……………………………………….……………… 18
2-3-4 ORP ………………………………………………………… 19
2-3-5 能量來源 …………………………………………………. 20
2-3-6 其他氮氧化物 …………………………………………… 23
2-3-7 抑制劑 ………………………………………………….… 24
2-4 PHB簡介 ………………………………………………………..… 24
2-4-1 PHB的特性與代謝途徑 ………………………………… 24
2-4-2 細菌合成PHB …………………………………………… 28
2-4-3 PHB之發展與應用 ……………………………………… 30
2-5 Wautersia eutropha特性 ………………………………………...… 32
2-6 矽膠管反應槽之技術與應用 …………………………………… 35
2-6-1 薄膜生物反應槽之種類 ………………………………… 35
2-6-2 利用矽膠管作為氫氣自營性生物脫硝反應槽之供氣系… 39
2-7 分子生物技術於生物反應槽菌相鑑定之應用 ………………….. 41


第三章 材料與方法 ……………………………………………………… 46
3-1 研究內容 ………………………………………………………..… 46
3-2 研究方法 ………………………………………………………… 47
3-2-1 菌種來源 ………………………………………………… 47
3-2-2 菌種馴養 ……………………………………………….… 47
3-2-3 批次試驗 …………………………………………………. 49
3-3 分析方法 ………………………………………………………… 52
3-3-1 水質分析 ………………………………………………… 52
3-3-2 污泥分析（PHB之分析）………………………………… 54
3-3-3 菌相觀察 …………………………………………………. 56
3-3-4 反應槽微生物菌種分析 ………………………………… 57
3-4 研究設備 ………………………………………………………… 66


第四章 結果與討論 …………………………………………………….… 68
4-1 菌種馴養 ………………………………………………………… 68
4-2 Wautersia eutropha 脫硝及累積PHB之批次特性實驗 ………… 72
4-2-1 Wautersia eutropha 脫硝反應 ………………………….. 72
4-2-2 Wautersia eutropha 脫硝反應與PHB之累積相關性 ….… 74
4-2-3 Wautersia eutropha 脫硝反應之氫氣影響試驗 ………… 78
4-2-4 Wautersia eutropha PHB累積之氫氣影響試驗 ………… 81
4-3 C/N比對Wautersia eutropha 脫硝與PHB累積之影響 ………… 85
4-3-1 矽膠管反應槽批次試驗 ……………………………….… 85
4-3-1-1 固定碳量，不同C/N比之批次試驗 …………… 85
4-3-1-2 固定氮量，不同C/N比之批次試驗 ………....… 90
4-3-2 血清瓶批次試驗 ……………………………………….… 94
4-3-2-1 固定碳量，不同C/N比之批次試驗 …………… 95
4-3-2-2 固定氮量，不同C/N比之批次試驗 …………… 99
4-4 不同基質濃度負荷對脫硝與PHB累積之影響 ………………… 102
4-5 氨氮對 Wautersia eutropha 脫硝與PHB累積之影響 ……… 106
4-6 微生物菌相觀察 ………………………………………………… 108
4-7 以分子生物技術鑑定反應槽菌相 ……………………………… 110

第五章 結論與建議 ………………………………………………………116
5-1 結論 …………………………………………………………….…116
5-2 建議 …………………………………………………………….… 118

參考文獻 ………………………………………………………………..… 119
附錄1 ………………………………………………………………………. 125
附錄2 ……………………………………………………………………… 130
附錄3 ………………………………………………………………………. 143

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