活性污泥脫硝基因及脫硝菌多樣性之研究

The Diversity of Nitrite Reductase Gene in Activated Sludge Systems

林茗儀(Ming-Yi Lin)

指導教授 ： 游勝傑

中原大學/工學院/土木工程研究所/碩士(2005年)

https://doi.org/10.6840/cycu200500327

摘要

都市污水處理中多以生物處理法來減少水中氮化物，而生物處理法是指污水處理廠之反應槽中，馴養出具有硝化及脫硝功能之菌株來降低水中氮化物；本研究針對在脫硝作用之關鍵步驟，為將亞硝酸鹽經由電子轉移還原成一氧化氮，然而脫硝菌中具有此項功能基因有nirS和nirK基因兩種，因此採用該兩種基因之引子對偵測內湖污水處理廠、SBMBR反應槽及其薄膜上之生物膜污泥，並應用兩種分離方法(純菌培養和基因轉殖)來探討其脫硝基因與脫硝菌之多樣性。
由實驗結果顯示，其內湖污水處理廠之污泥經由PCR偵測結果，推估具有nirS基因(+)之脫硝菌佔該廠之活性污泥比例約為22.5%；而具有nirK基因(+)之脫硝菌約佔該廠活性污泥比例約為12.5%，可知脫硝菌佔該廠之活性污泥比例甚高。
將由內湖污水處理廠採回之污泥經由SBMBR系統長時間馴養後，並分別採用純菌培養及基因轉殖兩菌種分離之方法，進而比較兩種結果；經由實驗結果發現利用純菌培養可分離之菌種類別較少，但其在類緣關係間之比較與使用基因轉殖分類方法，所分離之菌株之分佈情形相似其可分離出較多種類之菌株，但觀察其類緣圖會發現其彼此間分佈情形較為均勻。
考量浸置於MBR反應槽中之薄膜表面所增生之污泥亦具有脫硝作用，因此亦將其生物膜污泥採集並進行基因轉殖之分類，其結果發現該部分之污泥亦有具脫硝基因之菌株存在，但可被發現之種類較少；另外，在類緣關係分佈情形中發現其菌株種類與槽內污泥分離出之菌株均勻分布，此與操作模廠時進行排泥作用會將部分生物膜污泥同時排出，或抽出出流水時抽出部分污泥，結果顯示SBMBR槽中與薄膜上污泥之其脫硝基因呈現之親緣關係相近。
本研究並將設計該引子對之菌株一同研究比對，將其結果繪置類緣圖並觀察與所偵測之菌種間親緣關係，其在與脫硝菌之親緣圖中群集關係較遠，可發現其所能偵測範圍極廣；另外，為考量其與菌種間序列差異，未來可將測得之脫硝菌株一併與以之設計該引子對之菌株設計出較完整性適合本土性引子對。

關鍵字

都市污水 ； 脫硝作用

並列摘要

Municipal sewage treatment employs biological treatment to remove nitric compound, and biological treatment means to cultivate microscopic organisms with nitrification and denitrification in the sewage plant. This study is to investigate the key step of denitrification. The step is the reduction of nitrite to nitric oxide by electron transfer. The denitrifying bacteria include two fuctional gene( nirS and nirK genes). Therefore, this investigation would detect the denitrifying bacteria of active sludge from the Nei Hu sewage treatment plant、sequencing batch membrane bioreactor (SBMBR) and the biological membrane by using two primers.
Experimental results have shown the denitrify bacteria include nirS gene(+) is around 22.5% in active sludge of the Nei Hu sewage treatment plant. In addition, the denitrify bacteria include nirK gene (+)is around 12.5%. The percentage of the denitrify bacteria from the Nei Hu sewage treatment plant is very high.
The sludge collected from the Nei Hu sewage treatment plant have domesticated by SBMBR system for a long time. Then this investigation uased pure culture and gene cloning to separate the sludge and compared these two different methods. The results have shown that the bacteria from pure culture is fewer, but the phylogenic tree is similarity as other method.
Considering the membrane which dipped into the SBMBR may proliferate sludge with denitrification. Hence the sludge was employed to proceed by gene cloning. The experimental results showed that there were denitrifying bacteria existed in it, but the species are fewer. In addition, the distribution of phylogenic trees shows that the species of sludge from SBMBR and the biological membrane are well mixed.
This study would also compare the bacteria which designed the nirS and nirK primers with the denitrifying bacteria from detection. The results were drawn on phylogenic trees, and the two different clusters were made. The two cluster were observed which related farther. As a result of the relation between these two clusters was extremely wide, can be used simultaneously to design a more complete local primers with the denitrifying bacteria have known.

並列關鍵字

DGGE ； denitrification ； PCR ； nirK gene ； nirSgene ； RFLP

參考文獻

34.史慧萍(2000)，好氧脫硝菌之篩選及其特性之研究，碩士論文，國立台灣大學環境工程所。

1.Baek, S.H., Kim, K.H., Yin, C.R., Jeon,C.O., Im, W.T., Kim, K.K. and Lee,S.T.(2003) Isolation and characterization of bacteria capable of degrading phenol and reducing nitrate under low-oxygen conditions. Curr. Microbiol. 47 (6), 462-466.

4.Braker, G., Zhou J., Wu Liyou, Devol A. H., and Tiedje J. M. (2000) Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment communities. Appl. Environ. Microbiol.66:2096-2104.

5.de Boer, A.P., Reijnders, W.N., Kuenen, J.G., Stouthamer, A.H. and van Spanning,R.J.(1994). Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans. Antonie Van Leeuwenhoek 66 (1-3), 111-127 .

6.Diekmann, H.(1999) Thauera mechernichensis sp. nov., an aerobic denitrifier from a leachate treatment plant. Int. J.Syst. Bacteriol. 49(3):1045-1051 .