以側向流免疫層析法快速偵測活性污泥中厭氧氨氧化菌(Anammox)總菌數與評估氨氮去除效能

Rapid Detection of Total Anaerobic Ammonium Oxidizing(Anammox) Bacteria in Activated Sludge and Evaluation of Ammonia Removal Efficiency Using the Lateral-flow Immunochromatographic Assay

鄭旭傑(Hsu-Chieh Cheng)

指導教授 ： 吳瑞璋 ； 黃郁慈

中原大學/工學院/化學工程研究所/碩士(2020年)

https://doi.org/10.6840/cycu202000165

摘要

台灣工業的蓬勃發展，工業廢水的排放量也相對增加，根據行政院環保署統計，化工、石化產業及科學園區佔氨氮廢水排放比例的80%，這些富含氨氮汙染物的廢水排進河川、湖泊後，會造成水體溶氧量下降、優養化以及危害水中生物。因此，如何使廢水達到放流標準則成了重要的課題。
相較於傳統脫氮程序，使用厭氧氨氧化菌(anaerobic ammonium oxidizing，Anammox)來進行脫氮，由於不須額外曝氣以及碳源，使成本下降不少。但在現今一直缺少一項能在短時間內得知Anammox含量的技術，因此，本研究欲利用薄膜試條對活性污泥裡的Anammox含量做快速偵測，從活性污泥裡萃取DNA，並將設計好的forward、reverse primer分別標定上biotin及digoxigenin，經由PCR擴增後，將PCR產物滴至薄膜試條上觀察結果。首先建立一條標準曲線，藉由DNA標準樣品的連續稀釋分別經qPCR以及上試條，欲從qPCR中獲得的Cq值與薄膜試條上訊號的結果建立關係，最後盲測其他活性污泥裡Anammox的含量分別為多少。結果顯示，薄膜試條的訊號雖然不如預期的靈敏，但大致上給出一個可供參考的正向趨勢。氨氮去除效能的部分，發現在氨氮濃度在500以及1000 ppm的活性污泥，薄膜試條上的訊號以及Cq值(1000 ppm:15.16，500 ppm:15.07)都是相當接近的，但水質監測的結果顯示氨氮去除量有明顯的差異，由於Anammox菌種分布十分複雜，推測是不同環境培養的汙泥樣品有著不同的菌相分布，也影響著各自的氨氮去除效率，日後若能找出菌相分布與環境條件的關係，相信薄膜試條訊號與氨氮去除效能會有更良好的相關性。

關鍵字

快速偵測 ； 厭氧氨氧化菌 ； 奈米金 ； 側向流免疫層析法

並列摘要

The vigorous development of Taiwan’s industry has led to a relatively large increase in the discharge of industrial wastewater. According to the statistics of the Environmental Protection Administration, Executive Yuan, the chemical, petrochemical industry and science industrial parks account for 80% of the ammonia nitrogen wastewater discharge. These wastewaters rich in ammonia nitrogen pollutants after being discharged into rivers, lake, it will cause a decrease in dissolved oxygen in the waterbody, eutrophication and harm to aquatic life. Therefore, how to make the wastewater reach the discharge standard has become an important issue.
Compared with the traditional denitrification process, anaerobic ammonium oxidizing (Anammox) is used for denitrification. Since no additional aeration and carbon source are required, the cost is reduced a lot. However, there is still a lack of a method that can know the content of Anammox in a short time. Therefore, this study wants to use a membrane-based lateral flow strip to quickly detect the content of Anammox in activated sludge. Extract DNA from activated sludge, and mark the designed forward and reverse primers with biotin and digoxigenin respectively. After PCR amplification, drop the PCR product onto the strip and observe the results. First create a standard curve, through serial dilution of the DNA standard sample. The relationship between the Cq value obtained from the qPCR and the signal result on the strip is established, and finally the blind test of the content of Anammox in other activated sludge. The results show that although the signal of the membrane-based strip is not as sensitive as expected, it generally gives a positive trend for reference. In the part of ammonia nitrogen removal efficiency, it was found that the activated sludge with ammonia nitrogen concentration of 500 and 1000 ppm, the signal on the strip and the Cq value (1000 ppm: 15.16, 500 ppm: 15.07) are quite close. But according to the results of quality of water, there is obvious difference in the amount of ammonia nitrogen removal. Because the community of Anammox is very complicated, it is presumed that the sludge samples cultured in different environments have different bacterial community, which also affects the ammonia nitrogen removal efficiency. If we can find the relationship between community of Anammox and the condition of environment, it is believed that signal of the membrane-based strip and ammonia nitrogen removal performance will have a better correlation.

並列關鍵字

rapid detection ； Anaerobic Ammonium Oxidizing ； gold nanoparticle ； lateral-flow immunochromatographic assay

參考文獻

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