臺灣博碩士論文加值系統

Ralstonia eutropha已證實可在好氧環境下累積大量PHB (poly-3-hydroxybutyrate)，由於PHB可作為生物可分解塑膠的原料，因此本研究主要為探討Ralstonia eutropha在無氧脫硝環境下之生長條件及PHB累積特性，並與好氧的異營生長做比較，期能利用Ralstonia eutropha去除水中之硝酸塩，而脫硝所產生之污泥，由於含有高濃度之PHB，可進一步作為再利用資源，因此具有研究之價值性。
本研究皆以批次方式進行試驗，首先探討Ralstonia eutropha在無硝酸鹽之供氫環境下之生長情形，發現在無氧狀態下，硝酸塩為Ralstonia eutropha之唯一電子接受者，且當碳源不足時，氫氣可提供能量予細胞增殖。而無論在好氧與無氧狀態中，Ralstonia eutropha在生長期即開始合成累積PHB，而在穩定期達到最高濃度，進入內呼吸期後，菌體內之PHB即隨著菌體分解而降低含量。
在無氧狀態下，分別以不同C/N(5、10、20、25及30)、氨氮濃度(100及200 mgNH4-N/L)及硝酸鹽濃度(292、397及485 mgNO3-N/L)探討Ralstonia eutropha對PHB合成累積之影響，結果顯示在C/N<20時，有機物對PHB合成累積影響較顯著，且氨氮濃度越高，對於微生物生長速率及PHB之合成累積都有明顯提升，而較高之硝酸塩濃度，亦會提高PHB之合成累積量。
在好氧狀態方面，亦探討不同C/N對Ralstonia eutropha合成累積PHB之影響，發現此因子影響情形與無氧狀態類似，惟好氧狀態因生長速率較快，菌液增殖濃度較高，其在各個階段之PHB之合成累積量，皆高於無氧狀態。

It was verified that Ralstonia eutropha could accumulate a large amount of PHB in cells under the aerobic condition, and PHB is the raw material of plastic which can be decomposed by bacteria. So the aim of this study is to find out the growth condition and characteristic of PHB accumulation by Ralstonia eutropha in anoxic state, and then compare it with living under the aerobic and heterotrophic condition. It’s expected that Ralstonia eutropha can remove the nitrate from the waste water and the sludge which possesses high concentrated PHB can be reused. According to the reason above it’s worthy to do this research.
All experiments proceeded in batch reactor. First of all, we provide hydrogen and discuss the growth of Ralstonia eutropha in anoxic condition without nitrate. And then we find out that nitrate is the only electronic acceptor of Ralstonia eutropha in anoxic condition. In that condition hydrogen can be the energy source of Ralstonia eutropha to promote the growth when the carbon lacks in the liquid. In both anoxic and aerobic conditions, Ralstonia eutropha begins to accumulate PHB in growth phase. Furthermore, reaching the highest content of PHB in cells is in the stationary phase. And then declining the PHB content in cells accompanys the coming of endogenous phase.
In anoxic condition, i also researched PHB accumulation by Ralstonia eutropha in different C/N(5.10.20.25 and 30)、ammonia concentration(100 and 200 mgNH4-N/L) and nitrate concentration(292、397 and 485 mgNO3-N/L). The result reveals the difference of PHB accumulation in cells is obvious under C/N<20. The higher concentration of ammonia will accelerate the growth of Ralstonia eutropha and promote the PHB accumulation in cells apparently. There is also influence of enhancing the PHB accumulation in cells as the concentration of nitrate is promoting.
In the aspect of aerobic condition, the different C/N against the influence of PHB accumulation by Ralstonia eutropha was performed. It was found that the condition of aerobic is similar to anoxic condition. The most important difference between the two conditions is that the PHB accumulation in aerobic condition is higher than that in anoxic condition. Maybe it is the faster growth and higher microorganism concentration that leads to the outcome.

目錄
摘要……………………………………………………... I
目錄……………………………………….……………... IV
圖目錄…………………………………………………. VIII
表目錄…………………………………………………..... X
第一章 序論………………………..………………….…. 1
1-1 前言……………………….……………………….…. 1
1-2 研究內容………………………..……………..………….….. 3
第二章 文獻回顧…………………..……………….…. 4
2-1自然界中之氮循環…………………………….…….…. 4
2-2硝化作用（nitrification）…………………..………………... 4
2-3脫硝作用（denitrification）……………….…….……….…... 5
2-3-1脫硝菌………………………….………..…………....….. 6
2-3-2生化反應……………………………………..……….….. 6
2-3-3影響脫硝作用之因素………………….………....…..… 10
2-4 PHAs(polyhydroxyalkanoates)之合成與代謝….….……...… 14
2-4-1 PHAs(polyhydroxyalkanoates)之代謝……….……….… 14
2-4-2 影響PHAs合成的環境因子…………………...........… 16
2-5利用16S rDNA之序列比對分析菌種之相關研究….......… 19
第三章 實驗設備與方法…………………………..……. 23
3-1 研究內容與流程……………………………..……..… 23
3-2 研究方法………………………...………………………..… 24
3-2-1 菌種來源.......................................................................... 24
3-2-2 細菌馴養.......................................................................... 24
3-3 Ralstonia eutropha在無硝酸鹽及供給氫氣的環境下之生
長批次試驗……………………………………………...…… 26
3-3-1 Ralstonia eutropha在異營無硝酸鹽及氧氣生長的批
次實驗…...………………………………...……….…… 26
3-3-2 氫氣對Ralstonia eutropha在異營脫硝的批次實驗..... 26
3-4 菌株在不同生長階段比較之批次實驗………………..…... 27
3-5 菌株在不同C/N值之批次實驗……………….…..….…… 29
3-6 菌株在脫硝環境中，相同C/N值，不同氮源與碳源負
荷下之批次試驗……..…………………………..………… 31
3-7 菌株在脫硝環境中，相同C/N，不同硝酸鹽影響之批
次試驗…………..……………………………......………… 33
3-8 電子顯微鏡觀察………………..…………………..….…… 35
3-9 實驗設備、水質分析及污泥分析………………..….…….. 35
3-9-1實驗設備…………………..…………………..….…….. 35
3-9-2 水質分析………………..…...……………..…….…….. 36
3-9-3 污泥分析（PHB分析）………………..…...….……... 37
第四章 結果與討論……………………….……..…..….……. 40
4-1 Ralstonia eutropha 在無硝酸鹽及供氫環境下之生長試驗
….…..…………………………………………..…..…...…… 40
4-1-1 無硝酸鹽狀態下之試驗…………………..…..……… 41
4-1-2 供氫氣對Ralstonia eutropha在異營脫硝環境下的
影響…………………………………………….……… 41
4-2 Ralstonia eutropha在異營脫硝生長環境下之PHB合成
試驗..…..………………………………………..…..….…… 47
4-3 在固定硝酸鹽及氨態氮濃度，不同C/N值對Ralstonia
eutropha生長的脫硝反應及PHB合成的影響….….…… 52
4-4 在C/N值固定，不同氨氮濃度條件下對Ralstonia
eutropha生長脫硝反應及PHB合成的影響…….….…… 58
4-5 不同硝酸鹽氮負荷對Ralstonia eutropha生長脫硝反應及 PHB合成的影響..…………….………..……..…..….…… 64
4-6 在無氧脫硝環境與好氧環境中，Ralstonia eutropha之生
長及PHB合成累積量的比較…..……..…………….…… 68
4-6-1 Ralstonia eutropha 在好氧及無氧狀態下之生長及
PHB合成累積量之關係….……………....…..….…… 69
4-6-2 有機物對於Ralstonia eutropha在好氧及無氧狀態
下之生長及PHB合成累積之影響..…………….……. 74
第五章 結論與建議..………………………………..….…… 79
5-1 結論..………………………………………………….…… 79
5-2 建議..……………………………………………….....…… 81
參考文獻..………………………………………......……...…… 82
附錄..…………………………………………………….....…… 87
1. SEM電子顯微鏡下之Ralstonia eutropha圖….……..…… 87
2. 菌種鑑定..………………………………….…….....……….… 88

圖目錄
圖2-1 氮循環示意圖………………………………….......……. 5
圖2-2 Paracoccus denitrification 之電子傳遞模式…….…..…. 8
圖2-3 NO2-還原成N2O之可能反應途徑……………………… 9
圖2-4 pH 對Thiobacillus denitrificans 脫氮作用的影響…… 12
圖2-5 溫度對Thiobacillus denitrificans 脫氮速率的影響...… 12
圖2-6 PHAs常見的單體結構……………….……….……….. 15
圖2-7 Ralstonia eutropha 中PHB合成與代謝途徑………… 16
圖2-8 Ralstonia eutropha 中PHB合成率與C/N的關係....… 18
圖2-9 原核生物Ribosome之構成及其16S rDNA結構示意
圖………………………………...……………………… 21
圖2-10 Polymerase chain reaction (PCR) for a specific region.… 22
圖3-1 實驗流程圖………………………………...…………… 23
圖3-2 細菌的馴養裝置...……………………………………… 25
圖4-1 在溶液中無硝酸鹽之狀態下，各種水質變化……...… 42
圖4-2 在氫氣影響下的生長及各種水質變化...…...……… 45,46
圖4-3 微生物在無氧段與厭氧段試驗中，MLSS、COD、
pH值、硝酸鹽、亞硝酸鹽及PHB濃度變化曲線
與PHB佔微生物乾菌中比例柱狀圖...………...… 49,50
圖4-4 在硝酸鹽及氨氮固定，不同C/N值下，MLSS、
COD、pH值、硝酸鹽及亞硝酸鹽濃度變化曲線與
PHB佔微生物乾菌中比例柱狀圖……......…….… 55,56
圖4-5 在硝酸鹽及C/N值固定，不同氨氮濃度下，MLSS
、COD、pH值、硝酸鹽、亞硝酸鹽及PHB濃度變
化曲線與PHB佔微生物乾菌中比例柱狀圖...…… 61,62
圖4-6 在氨氮及C/N值固定，不同硝酸塩濃度下，
MLSS、COD、pH、硝酸鹽、亞硝酸鹽、氨氮
及PHB佔微生物乾菌中比例變化圖...………..… 65,66
圖4-7 在氨氮及C/N值固定，好氧及無氧狀態下，
MLSS、pH、氨氮、硝酸鹽、亞硝酸鹽、COD
及PHB佔微生物乾菌中比例變化圖...………..… 71,72
圖4-8 好氧狀態下，氨氮固定，不同之C/N，
MLSS、pH、COD及PHB佔微生物乾菌中比例
圖............................................................................... 75,76

表目錄
表2-1 目前發現脫硝菌種的菌屬名...………………………. 7
表3-1 菌種活化液態培養基質組成...……………………… 24
表3-2 Trace element的組成...……………………………… 25
表3-3 批次試驗基質組成...…………….………………...… 28
表3-4 批次試驗培養基質組成...…………………………… 31
表3-5 批次試驗培養基質組成...…………………………… 32
表3-6 批次試驗培養基質組成...…………………………… 34
表3-7 好氧狀態與無氧狀態單位COD產生PHB之比較... 73

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