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研究生:張智聖
研究生(外文):Chih-Sheng Chang
論文名稱:抗生素抗性菌與抗性基因在污水處理程序中的動態變化
論文名稱(外文):Dynamics of antibiotic resistant bacteria and genes in the domestic wastewater treatment process
指導教授:林居慶
指導教授(外文):Chu-Ching Lin
學位類別:碩士
校院名稱:國立中央大學
系所名稱:環境工程研究所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:138
中文關鍵詞:都市污水處理廠抗生素抗性菌抗生素抗性基因消毒程序
外文關鍵詞:Domestic treatment plantantibiotic resistance bacteriaantibiotic resistance genedisinfection procedure
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近年來由於氣候變遷及人口快速上升,導致乾淨且可利用的水資源越來越稀少,因此污水回收再利用的需求及發展漸漸受到重視,未來也希望運用在用水量較多的農業灌溉,甚至是飲用水,但前提是再利用的水質需受到嚴格控管,這包括過往較常被忽略的生物性污染物,特別是抗生素抗性基因(antibiotic resistance genes, ARGs)與抗生素抗性菌(antibiotic resistance bacteria, ARB)。有鑒於污水處理廠已被認定為是原核生物獲取與交換抗性基因的潛在熱區,以及為了避免再生水中的抗生素抗藥性成為公共安全衛生和環境污染問題,本研究利用傳統細胞培養與qPCR基因定量等技術,並佐以參數彼此間相關性的統計分析,調查ARB與ARGs於污水處理單元的流佈,以期瞭解並掌握ARB與ARGs的動態變化、甚至預測其在污水處理程序過程的表現。調查結果顯示:(1)使用MBR處理後對於ARB (從5.2log – 6.2log CFU/mL降低至1.1log – 3.4log CFU/mL)、ARGs (從5.4log – 13.1log copy/L降低至0.6log – 8.2log copy/L)和class 1 integron (從11.5log – 12.9log copy/L降低至3.8log – 3.9log copy/L)具有顯著的絕對濃度去除效果;(2) ARB的絕對濃度經加氯或UV消毒後顯著下降(p < 0.05) (Acl廠:從4.3log – 6.1log CFU/mL降低至1.2log – 3.7log CFU/mL;Cuv廠:從3.0log – 4.6log CFU/mL降低至1.3log – 3.2log CFU/mL),且兩個處理廠的消毒處理前後變化率具顯著差異(p < 0.05);(3)有無加氯消毒或UV消毒對於ARGs和class 1 integron的絕對濃度前後變化率不具顯著差異(p > 0.05) (Acl廠:從5.5log – 11.2log copy/L降低至5.5log – 11.1log copy/L;Bmbr廠:從0.6log – 8.2log copy/L降低至1.6log – 5.6log copy/L;Cuv廠:從3.5log – 7.6log copy/L降低至3.4log – 8.5log copy/L);(4)經過MBR處理後對於ARGs和class 1 integron相對濃度有較好的處理效率;(5)經過加氯消毒和UV消毒對於ARGs的相對濃度的變化率不具顯著差異(p > 0.05),且加氯消毒對於整體ARGs和class 1 integron的相對濃度變化率呈現下降、UV消毒對於整體ARGs和class 1 integron呈現濃度上升;(6)冗餘分析顯示COD、總磷、總氮、總凱氏氮和pH與ARGs和class 1 integron具有相關係,與硝酸鹽氮、亞硝酸鹽氮、溶氧濃度和溫度的相關性較小。這些調查結果說明當處理廠使用MBR時可以有良好的 ARB和ARGs去除效果,但是對於目前常用的加氯和UV消毒方法則可能需要調整其操作參數或使用更有效的處理技術(如:臭氧、Fenton)以避免回收水中含有較多的 ARGs;此外,由相關性分析所得的結果表明ARGs在廢水處理程序的多寡似乎與一般的厭氧參數有較強的關聯性,對此值得後續加以追蹤與確認。
Due to climate change and population explosion, shortage of clean and safe water resources is becoming an increasingly serious problem worldwide. Development of reuse of domestic wastewater could be one of the solutions. Ultimately, it is aimed to use recycled wastewater in agricultural irrigation and even in drinking water. To meet this goal, the water quality of recycled wastewater has to be demanded absolutely safe at every level, both chemically and biologically. Given that wastewater treatment plants (WWTPs) have been considered hot spots for the proliferation of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), investigating the dynamics of ARB and ARG levels during the wastewater treatment process and their association with other generic water quality parameters will be one step closer to the goal of wastewater reuse. In this study, influents and effluents of each operating unit of three WWTPs were collected in the Fall and Spring. In addition to generic water quality parameters, the collected samples were analyzed for the abundance of ARB ARGs via cell incubation and qPCR methods. Results show that: (1) After MBR treatment, the absolute abundances of ARB (from 5.2log – 6.2log CFU/mL to 1.1log – 3.4log CFU/mL), ARGs (from 5.4log – 13.1log copy/L to 0.6log – 8.2log copy/L) and class 1 integron (from 11.5log – 12.9log copy/L to 3.8log – 3.9log copy/L) are substantively reduced; (2) The absolute abundance of ARB is significantly decreased after chlorination or UV disinfection (p < 0.05) (plant Acl: from 4.3log – 6.1log CFU/mL to as low as 1.2log – 3.7log CFU/ml; plant Cuv: from 3.0log – 4.6log CFU/mL and reduced to 1.3log – 3.2log CFU/mL), and the removal rates between the two plants are significantly different (p < 0.05); (3) Neither chlorination nor UV disinfection results in a significant decrease in the absolute abundance of ARG and intI (p > 0.05) (plant Acl: 5.5log – 11.2log copy/L reduced to 5.5log – 11.1log copy/L, plant Bmbr: 0.6log – 8.2log copy/L reduced to 1.6log – 5.6log copy/L, plant Cuv: 3.5log – 7.6log copy/L reduced to 3.4log – 8.5log copy/L); (4) higher removal efficiency of ARGs and class 1 integron relative abundance is observed in MBR treatment; (5) chlorination and UV disinfection do not significantly affect relative abundances of ARGs; (6) RDA show that relative abundances of ARGs and class 1 integron correlate well with COD, total phosphorus, total nitrogen, total Kjeldahl nitrogen concentration and pH, and little with nitrate nitrogen, nitrite nitrogen, dissolved oxygen concentration and temperature. Together, these results indicate that WWTPs implementing MRB can have better ARB and ARGs removal efficiency, and current disinfection units that use chlorination and UV may need to adjust operating parameters or to adopt more effective technologies (e.g., ozonation, Fenton oxidation) to reduce the risk of higher levels of ARGs appearing in the recovered water. In addition, the strong association between ARGs and certain anaerobic parameters suggests that future investigation on this regard is warranted.
誌謝 i
摘要 ii
Abstract iv
圖目錄 ix
表目錄 x
一、前言 1
1.1 研究動機 1
1.2 研究目的 3
二、文獻回顧 4
2.1 抗生素抗性基因(ARGs) 與抗生素抗性菌(ARB)的傳播及影響 4
2.2 環境水體中ARGs與ARB的表現 6
2.3.1 污水處理廠中ARB和ARGs的表現 7
2.3.2 二級處理程序中ARB和ARGs濃度變化 9
2.3.3 消毒處理程序中ARB和ARGs濃度變化 10
2.4 水環境中抗生素的濃度 11
2.5 污水處理廠中抗生素的濃度 12
2.6 亞致死劑量與抗性表現 13
2.7 共選擇機制與污水廠中共選擇的表現 14
2.8 抗生素的使用現況 15
2.9 抗生素的作用機制 16
2.10 ARGs的作用機制 20
2.11 移動性基因元 23
三、研究方法 25
3.1 樣品採集 26
3.2 樣品前處理與保存 27
3.3 DNA萃取 29
3.4 目標基因標準品製備-基因擴增與純化 29
3.5 目標基因標準品製備-TA克隆與檢量線製備 30
3.6 目標基因定量 31
3.7 平板記數法 37
3.8 水質分析 37
3.9 統計分析 38
3.10 試劑與儀器 39
3.10.1 實驗試劑與分生試劑 39
3.10.2 實驗儀器 41
四、結果與討論 43
4.1 水質分析結果 43
4.2 ARB通過污水處理單元的絕對濃度變化 50
4.2.1 ARB通過沉澱池或MBR處理單元的絕對濃度變化 50
4.2.2 ARB通過消毒處理單元的絕對濃度變化 51
4.3 ARGs與intI1在污水處理單元的絕對濃度變化 55
4.3.1 ARGs通過污泥處理單元的絕對濃度變化 55
4.3.2 ARGs通過沉澱池和MBR的絕對濃度變化 56
4.3.3 ARGs通過消毒處理單元的絕對濃度變化 57
4.4 ARGs與intI1之間在污水處理單元的相關性 62
4.4.1 ARGs和intI1通過污泥處理單元的相對濃度變化 67
4.4.2 ARGs和intI1通過沉澱池或MBR處理單元的相對濃度變化 68
4.4.3 ARGs和intI1通過消毒處理單元的相對濃度變化 69
4.4.4 ARGs和ARB在污水處理單元的相對濃度變化 73
4.5 ARGs和intI1與水質參數的相關性 79
4.6 污水廠中ARB、ARGs與intI1在環境中的意義 81
五、結論與建議 83
5.1 結論 83
5.2建議 85
參考文獻 86
附錄 103
附錄1 目標基因之標準曲線 103
附錄2 目標基因的標準品之解離曲線(melting curve) 107
附錄3樣品的目標基因之解離曲線(melting curve) 112
附錄4 Bmbr廠內16S rRNA, ARGs和intI1的絕對濃度 121
附錄5 Cuv廠內16S rRNA, ARGs和intI1的絕對濃度 122
附錄6 總異營性菌和抗生素抗性菌與水質參數的冗餘分析 123
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鄧教義(2018) 重金屬生物有效性對於抗生素抗性基因在農地土壤的分佈與持續之影響,國立中央大學,桃園縣
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