季節效應對沼液沼渣中抗生素抗性基因豐度之影響

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李杰穎(Chieh-Ying Lee) 查詢紙本館藏

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論文名稱

季節效應對沼液沼渣中抗生素抗性基因豐度之影響
(Seasonal effect on the abundance of antibiotic resistance genes harbored in biogas residues)

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摘要(中)

抗生素常用於動物作為生長促進劑，已知用在動物身上的抗生素表明並未完全利用，在抗生素抗性基因(ARGs)產生的傳播下抗生素抗藥性(Antimicrobial resistance, AMR)的問題隨之而來，倘若新的抗生素研發無法跟上現在情勢腳步，無藥可醫不會是未來式，恐怕成為了現在式，ARGs因此也被賦予新興污染物代名詞。
近年國內推行肥份資源再利用計畫，將厭氧消化後動物糞尿取代有機化肥，在消化設施操作條件中、常溫最為普遍。有鑑於抗生素、抗生素抗性基因在沼液沼渣中潛在性不得而知，過往多數文獻對於溫度的機制在堆肥中針對抗生素、抗生素抗性基因帶來的抗藥性進行滅活、降解有所結果，不同於堆肥，厭氧消化也可比擬。
本研究調查北至南17間含動物豬、牛沼液沼渣畜牧場，季節氣溫促成ARGs/MGE的變化在相對豐度中相比冬季，夏季總四環素類抗性基因富集(p <.01)；臨床常見的β-內酰胺類則不一致，似乎較能因氣溫而有削減趨勢，大多數都是冬季高於夏季(Σbla, p < .001; blaTEM, p < .01; blaSHV, p < .001)；intI1在spearman分析表明夏季促成水平基因轉移機率提高，相對豐度與MGE正相關的ARGs (sul1, p < 0.01; sul2, p < 0.01; tetO, p < 0.01)正相關性，MGE整合酶的高度豐度對其它抗性基因豐度的介導能使環境系統抗藥性的提高。若單一季節拆開來看動物之間相比幾乎無差異性，即使溫度差異性不大，實則這些ARGs/MGE的豐度大多都被季節氣溫給影響。季節上溫度差距不大，表現的趨勢如文獻所述溫度影響ARGs/MGE的差異性。
事實上新型態肥料–沼液沼渣作為取代過往有機化肥成為肥料中減碳、節省成本的先驅，符合Recycle (循環回收)、Reuse (物盡其用)、Reduce (減少使用)，不過目前似乎在探討沼液沼渣中ARGs/MGE的影響因子還是有些侷限性，為了後續在探討環境公衛所帶來的風險潛勢，沼液沼渣再利用計畫的施行必須未雨綢繆發展適當的處理配套方法。

摘要(英)

Antimicrobials are commonly used in animals as growth promoters. Antibiotics known to be used in animals have been shown to be underutilized, and the problem of Antimicrobial resistance (AMR) has ensued due to the spread of antibiotic resistance genes (ARGs) , if the research and development of new antibiotics cannot keep up with the current situation, no cure will not be the future, and it may become the present. Therefore, ARGs are also given a synonym for emerging pollutants.
In recent years, China has promoted the reuse of fertilizer resources, replacing organic fertilizers with animal manure after anaerobic digestion. Among the operating conditions of digestion facilities, normal temperature is the most common. In view of the fact that the potential of antibiotics and antibiotic resistance genes in biogas residues is unknown, most of the previous literatures have found that the mechanism of temperature inactivates and degrades the resistance brought about by antibiotics and antibiotic resistance genes in compost. , unlike composting, anaerobic digestion is also comparable.
In this study, 17 livestock farms containing animal pigs and bovine biogas residues were investigated from north to south. Seasonal temperature contributed to the change of ARGs/MGE in relative abundance compared with winter and summer total tetracycline resistance gene enrichment (p <.01 ); clinically common β-lactams are inconsistent, and seem to have a tendency to decrease due to temperature, most of which are higher in winter than in summer (Σbla, p < .001; blaTEM, p < .01; blaSHV, p < .001); intI1 in spearman analysis showed that the probability of horizontal gene transfer increased in summer, and the relative abundance of ARGs positively correlated with MGE (sul1, p < 0.01; sul2, p < 0.01; tetO, p < 0.01) was positively correlated, MGE The high abundance of integrase can mediate the abundance of other resistance genes, which can increase the resistance of environmental systems. If a single season is taken apart, there is almost no difference between animals. Even if the temperature difference is not large, the abundance of these ARGs/MGEs is mostly affected by the seasonal temperature. There is little difference in temperature between seasons, and the trend of the performance is as mentioned in the literature that temperature affects the difference of ARGs/MGE.
In fact, a new type of fertilizer – biogas residues is a pioneer in reducing carbon and saving costs in fertilizers as a substitute for organic fertilizers in the past. It seems that there are still some limitations in exploring the influence factors of ARGs/MGE in biogas residues. In order to discuss the risk potential brought by environmental sanitation in the future, the implementation of the biogas residues reuse plan must develop appropriate treatment in advance Supporting method.

關鍵字(中)

★ 沼液沼渣
★ 厭氧消化
★ 抗生素抗性基因
★ 季節性

關鍵字(英)

★ Biogas residues
★ Anaerobic digestion
★ Antibiotic resistance genes
★ Seasonal

論文目次

摘要 i
ABSTRACT iii
致謝 v
目錄 vii
表目錄 x
圖目錄 xi
第一章前言 1
1.1 研究緣起 1
1.1.1 抗生素抗藥性議題 1
1.1.2 環境抗藥性背景 1
1.1.3 畜牧業與ARGs之關係 2
1.1.4 前處理程序對於抗藥性之影響 4
1.1.5 沼液沼渣施用促成的抗藥性風險潛在性 4
1.2 研究目的 5
第二章研究方法 6
2.1 研究流程及架構 6
2.2 沼液沼渣樣品採集 8
2.2.1 沼液沼渣樣品採集 8
2.3 沼液沼渣基本特性分析 10
2.3.1 水質參數(pH/電導度/化學需氧量/鹽度/溶解性有機碳)分析 10
2.3.2 營養鹽(總氮/總磷)含量分析 11
2.3.3 重(類)金屬分析 11
2.4 沼液沼渣目標基因檢測 12
2.4.1 目標基因選定 12
2.4.2 預處理 13
2.4.3 DNA萃取 13
2.4.4 目標基因片段定量 14
2.5 數據分析與比較 21
2.5.1 統計分析 21
2.5.2 其他統計分析與可視化物件 21
2.6 研究設備與試劑 22
第三章結果與討論 24
3.1 沼液沼渣基本特性 24
3.1.1 基本水質特性分析 24
3.1.2 重(類)金屬分析 26
3.2 單一季節沼液沼渣目標基因豐度 28
3.2.1 冬季目標基因絕對/相對豐度 28
3.2.2 冬季目標基因絕對/相對豐度熱圖 32
3.2.3 夏季目標基因絕對/相對豐度 36
3.2.4 夏季目標基因絕對/相對豐度熱圖 40
3.3 不同季節沼液沼渣中目標基因豐度比較 44
3.3.1 兩季目標基因絕對豐度(豬/牛合併比較) 44
3.3.2 兩季目標基因絕對豐度(豬/牛分開比較) 46
3.3.3 兩季目標基因相對豐度(豬/牛合併比較) 48
3.3.4 兩季目標基因相對豐度(豬/牛分開比較) 50
3.3.5 三季目標基因絕對豐度(豬/牛合併比較) 52
3.3.6 三季目標基因絕對豐度(豬/牛分開比較) 54
3.3.7 三季目標基因相對豐度(豬/牛合併比較) 56
3.3.8 三季目標基因相對豐度(豬/牛分開比較) 58
3.4 沼液沼渣與目標基因ARGs/MGE、環境因子影響性 60
3.4.1 溫度與兩季目標基因絕對豐度Spearman相關性 60
3.4.2 溫度與兩季目標基因相對豐度Spearman相關性 62
3.4.3 溫度與三季目標基因絕對豐度Spearman相關性 64
3.4.4 溫度與三季目標基因相對豐度Spearman相關性 66
3.4.5 冬季豬/牛目標基因相對豐度NMDS分析 68
3.4.6 夏季豬/牛目標基因相對豐度NMDS分析 69
3.4.7 冬季、夏季/豬牛目標基因相對豐度NMDS分析 70
3.4.8 冬季目標基因相對豐度與環境因子冗餘分析 71
3.4.9 夏季目標基因相對豐度與環境因子冗餘分析 73
3.5 沼液沼渣目標基因豐度與環境潛在性 75
3.5.1 沼液沼渣目標ARGs/MGE 75
3.5.2 氣溫因子所帶來的差異性 76
3.5.3 沼液沼渣與環境潛在性 78
3.5.4 研究理念 79
第四章結論與建議 81
4.1 結論 81
4.2 建議 81
參考文獻 82
附錄 96
附錄一 Real-time PCR 檢量線 96
附錄二 Melting curve 101
附錄三原始數據 103
附錄四沼液沼渣不同厭氧消化過程中ARGs/MGE消長規律 112
附錄五不同季節沼液沼渣中16S rRNA基因絕對豐度 115
附錄六學位考試委員意見回覆表 116

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指導教授

林居慶(Chu-Ching Ling)

審核日期

2023-8-14

推文