臺灣博碩士論文加值系統

紫色不含硫光合作用細菌已經被成功地應用於高濃度有機廢水處理上，亦曾被利用於處理廢水或土壤中難分解之苯環物質；除了應用在污染物去除之外，紫色不含硫菌因具有產生氫氣的能力，近年來被許多學者將其應用於生物產氫之研究上；本實驗室於生物產氫的研究中，意外發現紫色不含硫菌具有在體內累積聚磷酸鹽的能力，其體內所累積之聚磷酸鹽濃度可達一般除磷菌的2 – 3倍以上。由上述內容可知，紫色不含硫菌於環工上具有相當多元之應用價值，因此至今仍為諸多環工學者進行研究之熱門對象，也將是本研究所針對的研究目標。
環境微生物學中，除了菌相之鑑定與生理特性研究外，菌種於環境中所佔有之數量也是相當重要之研究課題。早期以傳統方法進行菌量計算往往會發生低估的可能，即時定量聚合酶鏈鎖反應(Real-Time PCR)是分子生物技術應用於細胞定量上之一大突破，許多研究皆證實Real-Time PCR具有更高的靈敏度與準確性，近來已陸續應用於環境微生物的定量上，然而國內環工界仍鮮少看到相關之應用。因此，本研究之目的便以Real-Time PCR作為紫色不含硫光合作用細菌的定量工具，同時搭配不同的傳統定量方法進行比較，希望能強調出Real-Time PCR之定量優勢，建立出一套屬於紫色不含硫光合作用細菌的最佳定量方法。
研究中，首先以cloning的方式將已知濃度之研究目標序列(pufM gene)嵌入質體中，進行系列稀釋作為定量所需的標準品，由Real-Time PCR之螢光偵測，先建立出Real-Time PCR定量紫色不含硫菌之標準曲線。而樣本之準備上，許多學者曾提及，樣本DNA之萃取效率將影響到樣本以Real-Time PCR進行定量的準確性，為了決定DNA的最佳萃取方式，本實驗共挑選了六種不同之商業套組(編號：A – F)進行DNA萃取，以核酸偵測(A260 /A280)結果作為判斷依據，結果顯示A套組於相同菌液中萃取出之DNA濃度與純度皆最高，表示以此套組所萃取的DNA最能代表來自所有菌體，因此最適於用在樣本DNA之準備上。樣本DNA順利萃取後，便可直接以Real-Time PCR進行螢光偵測並換算出相對菌量，完成所有之定量程序。
本研究後續利用所建立之Real-Time PCR定量方法搭配其它定量方式，比較純種培養與環境樣本中紫色不含硫光合作用細菌的菌量。在純菌的定量上，實驗結果顯示平板計數法、DAPI染色與Real-Time PCR的定量結果相差不大，其中Real-Time PCR方法數值略高一些，判斷在純種培養的單純條件下，三種方法皆適於應用在菌株定量上，而Real-Time PCR則能克服平板計數法與DAPI染色於抹碟及濾膜轉貼時所造成部分菌體流失的缺點，使得定量結果更趨近實際菌量；此外，本研究針對多種環境水體與活性污泥進行定量比較，實驗結果顯示，Real-Time PCR的定量結果比平板計數高出101 – 104倍，本實驗證實傳統培養存在外來菌種競爭的問題，且以醋酸納為電子供給者之條件下，環境中部分紫色不含硫菌種亦可能無法利用其生長，因此平板計數法確實會造成嚴重低估的現象，而Real-Time PCR的結果可能與實際菌量較為接近。
除了準確性較高之外，以往傳統培養定量紫色不含硫菌的時間需求至少為5 – 7天，如今以Real-Time PCR操作則不需1小時，如此省時又準確之優勢條件，Real-Time PCR確實為紫色不含硫光合作用細菌之最佳定量方法。

Phototrophic purple non-sulfur bacteria had been applied to treat waste water with high concentration of organic matters and degrade aromatic hydrocarbons, such as benzoate or 3-chlorobenzoate, in waste water or soil. Besides contaminants removal, purple non-sulfur bacteria was also used in the study of biological H2 production. Other researches had observed that purple non-sulfur bacteria would accumulate polyphosphate (poly-P) inside the cells, and the capability of poly-P accumulating was 2 to 3 times higher than PAOs. Therefore, purple non-sulfur bacteria was a valuable microbial community for the environmental engineering field.
Not only species and physiological characteristics but also microbial population is important for studying environmental microbiology. In the past researches, quantification of microorganisms by traditional methods always led to underestimate. A new molecular biotechnique, Real-Time PCR, was expected to overcome this disadvantage, which with faster, reliable, sensitive and convenient advantage on cell quantification, and has recently been applied to quantify environmental microorganisms. Nevertheless, quantification of purple non-sulfur bacteria by molecular biotechnologies has not been reported to date. For this reason, the primarily goal of this study is to develop a Real-Time PCR assay to quantify the purple non-sulfur bacteria which is based on enumerating the copy number of pufM gene.
At first, known quantities of pufM gene inserted plasmid DNA was used as standard DNA, a series of 10-fold diluted standard DNA analyzed by Real-Time PCR for obtaining cycle threshold (CT), and then a standard curve was generated from these data. Due to the effect of sample DNA preparation on quantitative accuracy, six different commercial DNA extraction kits were used to compare the extracted DNA quality based on measuring nucleic acid assay (A260/A280). One of the commercial kit was finally chosen for further experimental using.
After Real-Time PCR assay was established, purple non-sulfur bacteria within both pure culture and environmental samples were quantified by using Real-Time PCR and other quantitative methods. From the quantification results of pure culture Rhodopseudomonas palustris GN11, there is no obvious difference between Real-Time PCR, DAPI staining and plate count methods. Real-Time PCR assay overcame the disadvantages which would lead cells losing on DAPI staining and plate count, and obtained more accurate amount. Quantification results of environmental samples indicated that date from Real-Time PCR were 101 to 104-folds higher than that from plate count method. This experiment confirmed that microorganisms in environmental samples would compete to each other while using traditional culture; moreover, a portion of purple non-sulfur bacteria probably couldn’t grow on sodium acetate based medium. Using plate count method actually resulted in data underestimate; on the other hand, results from Real-Time PCR should more reliable.
Quantification of purple non-sulfur bacteria by using plate count method, cost at least 5 to 7 days, but less than 1 hour using Real-Time PCR method. Besides more reliable, quantification using Real-Time PCR method was faster. For this reason, Real-Time PCR method was really a proper method for the quantification of phototrophic purple non-sulfur bacteria.

中文摘要 I
Abstract III
目 錄 V
表 目 錄 IX
圖 目 錄 X

第一章　前言 1
第一節　研究緣起 1
第二節　研究目的 3
第二章　文獻回顧 4
第一節　光合營微生物 4
一、　光合營微生物之光合作用 4
(一)　有氧光合作用： 4
(二)　無氧光合作用： 6
二、　光合營微生物種類及特性 7
(一)　藻類 (algae) 8
(二)　藍綠細菌 (cyanobacteria) 9
(三)　紫色及綠色含硫細菌 (purple and green sulfur bacteria) 9
(四)　紫色不含硫細菌 (purple nonsulfur bacteria) 10
三、　紫色不含硫光合作用細菌 10
(一)　紫色不含硫光合作用細菌之生理特性 10
(二)　環境中紫色不含硫光合作用細菌種類與分布 14
(三)　紫色不含硫菌Rhodopseudomonas palustris生理特性及相關應用 16
第二節　光合營微生物之應用 17
一、　光合作用細菌在環工上之應用 17
二、　紫色不含硫光合作用細菌之產氫特性 19
三、　紫色不含硫光合作用細菌之生物除磷潛力 21
第三節　傳統生物方法與分子生物技術 22
一、　傳統分析方法 22
(一)　傳統菌種鑑定之方法 22
(二)　傳統微生物之定量方法 24
二、　環工上常見之分子生物技術 24
(一)　16S rDNA與功能性基因 25
(二)　聚合酶鏈鎖反應 27
(三)　基因選殖 28
(四)　螢光原位雜交法 29
三、　分子生物技術新突破-即時定量聚合酶鏈鎖反應 31
(一)　Real-Time PCR操作機制與原理 31
(二)　Real-Time PCR操作之優點與限制 36
(三)　Real-Time PCR之應用範圍及在環工上的應用 38
四、　傳統生物方法與分子生物技術之比較 39
第四節　傳統方法與分子生物技術應用於光合作用細菌之研究 42
一、　傳統篩選與定量 42
(一)　傳統培養方法 42
(二)　吸收光譜分析法 42
(三)　以IREM定量光合作用細菌 43
二、　分子生物技術之應用 43
(一)　紫色光合作用細菌之光合作用基因 43
(二)　pufM功能性基因之相關研究與應用 45
第五節　文獻閱讀心得及研究方向擬定 46
第三章　材料與方法 48
第一節　實驗架構 48
第二節　實驗設備 49
第三節　Rhodopseudomonas palustris GN11之菌種來源及培養條件 50
第四節　環境樣本來源 51
一、　環境水體來源 51
二、　活性污泥來源 52
第五節　分析方法與操作技術 52
一、　DNA萃取 52
二、　聚合酶鏈鎖反應 (PCR) 53
(一)　引子對 53
(二)　PCR操作條件 54
(三)　瓊脂膠糖凝膠電泳分析 54
三、　pufM 基因選殖 (Cloning) 55
(一)　Agarose膠體之DNA純化 56
(二)　接合反應 (Ligation reaction) 56
(三)　轉形作用 (Transformation) 56
(四)　藍/白菌落篩選 (blue/white screening) 57
(五)　PCR 57
四、　質體萃取 57
五、　即時定量聚合酶鏈鎖反應 (Real-Time PCR) 58
六、　平板計數法 (Plate count method) 59
七、　DAPI染色 59
(一)　Gelatin Slide製作 60
(二)　濾膜轉貼 60
(三)　DAPI染色 60
八、　螢光原位雜交法 (FISH) 61
第四章　結果與討論 63
第一節　Real-Time PCR定量方法之建立 63
一、　pufM gene目標序列標準品製備 63
二、　Real-Time PCR反應偵測標準曲線 64
(一)　以核酸定量法定義標準品濃度 64
(二)　以轉殖後勝任細胞抹碟計數法定義標準品濃度 68
(三)　核酸定量法與轉殖後勝任細胞抹碟計數法於純菌株定量結果之比較 69
三、　分析樣本的準備與紫色不含硫菌之定量 71
(一)　DNA萃取-商業化DNA萃取套組之比較 71
(二)　DNA萃取-樣本前處理對定量之影響 74
(三)　背景螢光對Real-Time PCR定量之影響 76
第二節　以不同方法定量純種培養之紫色不含硫光合作用細菌 79
一、　不同定量方法之比較 79
(一)　Real-Time PCR螢光定量 79
(二)　DAPI染色計數 80
(三)　平板計數法 81
(四)　Real-Time PCR、DAPI染色與平板計數法之比較 82
二、　其它定量方法 84
(一)　以O.D.660 (Optical Density)換算總光合菌數 84
(二)　螢光原位雜交法 87
第三節　以不同方法定量環境樣本之紫色不含硫光合作用細菌 91
一、　自然界水體中紫色不含硫光合作用細菌之定量 91
(一)　不同環境水體中紫色不含硫菌之定量結果 91
(二)　不同環境水體中紫色不含硫菌數量之探討 92
二、　活性污泥中紫色不含硫光合作用細菌之定量 95
(一)　不同活性污泥中紫色不含硫菌之定量結果 96
(二)　不同活性污泥中紫色不含硫菌數量之探討 97
三、　綜合比較 99
第五章　結論與建議 102
第一節　結論 102
第二節　建議 104
參考文獻 106

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陳欣微 (2005) 完全混合厭氧醱酵產氫系統在不同操作條件下之菌群結構分析， 國立中興大學，碩士論文，台中。

陳郁璇 (2004) 利用Real-Time PCR偵測油污中芳香烴開環酵素Catechol 2,3-Dioxygenase之研究，國立中興大學，碩士論文，台中。

黃俊霖 (2001) 以分子生物技術探討厭氧生物產氫程序之菌群結構，國立中央大學，碩士論文，桃園。

葉穎緻 (2004) 紫色不含硫光合作用細菌在不同培養條件下累積聚磷酸鹽之能力探討，國立中興大學，碩士論文，台中。

鄭景鴻 (2005) 生物除磷及非除磷系統中添加紫色不含硫光合作用細菌之生物除磷加強效果，國立中興大學，國立中興大學，台中。

簡競擇 (2002) 生物除磷系統污泥特性之研究，國立雲林科技大學，碩士論文，雲林。




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