以焦磷酸定序技術探討北勢溪細菌菌相組成

Abstract

北勢溪為台北集水區重要水源之一，此區域自水源發源地經坪林地區與坪林處理廠，最後流入翡翠水庫。我們針對人口密集區與污水廠前後採集水樣，藉由了解微生物群落的消長，顯示出此區域的汙染情況。 焦磷酸定序(pyrosequencing)為次世代高通量定序方法之一，近年來常用於研究環境之細菌群落變化。為了偵測水體真實存活的細胞，使用碘化丙啶(propidium monoazide, PMA)，其為一種DNA螯合染劑，在照光下能與死亡細胞之DNA結合，並抑制聚合酶鏈鎖反應(polymerase chain reaction, PCR)結果。 本研究以焦磷酸定序法分析台北水源集水區之北勢溪五個測站的細菌群落，並分為兩個部分。(1)針對所有細菌(16S rRNA gene之V1-V2超高變異區間)水體樣品並結合PMA方法偵測活/死細胞進行探討。(2)針對與擬桿菌門(Bacteroidetes)比較水體樣品與糞便樣品之異同。結果發現，水樣細菌群落中，Proteobacteria、Fusobacteria與Bacteroidetes各占整體水樣16.43-43.36%、0.84-19.77% 與 2.30-60.21%。PMA前處理與無PMA前處理細菌群落有小幅度差異，PMA前處理之樣品之物種豐富度大於無PMA前處理之樣品，推測原因為PMA的前處理會抑制一些優勢物種的增幅，導致被遮蔽的物種顯露出來。PMA前處理所偵測到之Enterobacteriaceae、Bacteroides、Arcobacter、Rickettsia等菌群可能與人口密集相關。 針對Bacteroidetes的部分，利用水樣與糞便樣品進行OTU交互分析，OTU交互分析是以水樣與糞便樣品以文氏圖做交集計算，藉由交集多寡表示河川污染情況。結果顯示，集水區河川經人口密集區後，水體中所測得之動物糞便交集比例從1.5%上升至18.59%；經污水廠後的坪林污水下游測站則下降至8.89%，其中以人類糞便之比例變化最顯著。經過資料庫比對後，發現這可能為Prevotella copri、Bacteroides plebeius等人類腸道存在之菌種。根據結果，藉由OTU交互分析可以了解河川之汙染程度。

Bei-Shih river is one of the raw water sources for Taipei metropolitan area. It flows through Ping-lin area, Ping-lin wastewater treatment plant, and enter the Fei-Tsui reservoir. Samples were collected at the upstream and downstream of the highly populated area and downstream of the Ping-lin wastewater treatment plant to investigate the bacterial community variation by pyrosequencing. The purpose of this study was to target the hypervariable regions V1-V2 of the 16S rRNA gene and investigate the bacterial communities from Bei-Shih river by combination of PMA treatment and pyrosequencing. Also we compared the Bacteroidales group with fecal sources and freshwater. In our results, the phyla Proteobacteria, Fusobacteria and Bacteroidetes in freshwater were 16.43-43.36%, 0.84-19.77% and 2.30-60.21%, respectively. The bacterial community profiles of PMA-treated sample and non-PMA-treated sample were slightly different. The PMA-treated samples had more OTUs than non-PMA-treated samples. The gourps of Enterobacteriaceae, Bacteroides, Arcobacter, Rickettsia, were found in PMA-treamted water samples, and the ratio of each group to total bacteria was positively correlated to total coliforms. The correlation of shared OTUs between Bacteroidales communities of water samples and fecal samples were calculated. The results showed that the percentage correlation in Wan-Tan was 1.5% and increased to 18.59% in highly populated area. The shared OTUs of Prevotella copri and Bacteroides plebeius species, which related to human intestine bacteria, can also be found in freshwater. As a result, the degree of contamination in a region can be analyzed by the percentage of total and shared OTUs.