博碩士論文 108326019 詳細資訊



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姓名 林子晞(Tzu-Hsi Lin)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 沼液沼渣的施用促成農地土壤抗生素抗性基因增殖的可能性探討
(Potential for the facilitated proliferation of antibiotic resistance genes in arable soil resulting from biogas residues applied as fertilizer)
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摘要(中) 抗生素的濫用已使得臨床抗生素抗藥性的問題以驚人的速度惡化，但多數藥廠仍因毛利過低而不願挹注資源開發新型的抗生素，這樣的局面可能導致未來感染時無藥可醫的困境，也因此世界衛生組織將抗生素抗藥性問題視為全球最大的健康威脅之一。而除了醫療之外，抗生素也作為生長促進劑在畜牧業中被廣泛使用，但無法被動物腸道所吸收的殘餘抗生素會隨著禽畜糞排出體外，造就抗生素抗性基因(ARGs)進入環境的途徑，讓禽畜糞回歸農用成為潛在的環境ARGs污染源與暴露風險。雖然已有文獻證實禽畜糞經好氧工法製成堆肥能有效削減其ARGs相對豐度，不過此效益卻常常無法在中、常溫厭氧消化程序被複製，但現今國內響應政府沼液沼渣再利用的畜牧場其消化設施卻以中、常溫的操作最為普遍，此現象值得深究。考量國內目前對於沼液沼渣ARGs相對豐度之調查仍然有限，本研究採集了新竹至雲林共27間牛/豬畜牧場之沼液沼渣樣品，分析其中4類ARGs (tet, sul, bla, erm)以及intI1之相對豐度，並與傳統肥料(即市售堆肥)中的ARGs豐度進行比較(本研究群先前碩論之數據)，以探討國內沼液沼渣再利用行為對於環境中ARGs增生是否有較高之潛力。調查結果顯示，國內沼液沼渣中多數ARGs相對豐度顯著高於市售堆肥(p < .05)，符合本研究最初預期，而不同種類(牛/豬)沼液沼渣中ARGs整體相對豐度也有顯著差異(p < .05)。為了更進一步確認沼液沼渣中ARGs進入農地後於土壤環境中的宿命，本研究也透過實驗室規模的培養試驗，檢視添加沼液沼渣土壤於一個月期間的ARGs豐度變化，且同樣和本研究群先前碩論之市售堆肥的縮模試驗結果對比。結果顯示添加豬沼液沼渣土壤及添加市售堆肥土壤在試驗第0天、第30天時，其ARGs整體相對豐度無顯著差異，但兩實驗組別均顯著高於添加牛沼液沼渣土壤以及環境背景值(p < .05)；而不論添加何種肥料之土壤，在縮模試驗第30天之ARGs整體相對豐度均無顯著削減，顯示來自這些有機肥料之ARGs整體相對豐度有一定持久性，若沒有適當控管施肥頻率，很有可能造成ARGs於土壤環境的累積，進而增加農地工作者暴露ARGs的可能性。即便如此，由於縮模系統在設計與操作上的侷限性，本研究結果僅能代表農地表層土壤添加沼液沼渣後ARGs之豐度概況，後續仍有待現地的採樣、調查、分析、比較，方能確切瞭解沼液沼渣再利用對於農地環境抗藥性發展之影響。
摘要(英) Over the past seven years, the Taiwan EPA and Council of Agriculture have been vigorously encouraging farmers of the husbandry to treat livestock excretion through conventional (i.e., non-thermophilic) anaerobic digestion processes and use the treated biogas slurry/residues as a new kind of fertilizer in arable soils, echoing the current "circular economy" policy. However, previous studies have shown that compared to the thermophilic biochemical treatment including composting, digestion of livestock feces under ambient temperature and mesophilic conditions fails to substantially diminish the abundance of the antibiotic resistance genes (ARGs) inherent in manure. Given that (i) environmental antibiotic resistance is being shown to have a cyclical relationship to clinical antimicrobial resistance and (ii) according to the WHO, the rising level of antimicrobial resistance is positioned to endanger “the very core of modern medicine”, the new fertilizer practice is of concern. To probe whether this policy execution would result in facilitating antibiotic resistome proliferation in farmland and thus ultimately imposing risks to public health, the first step undoubtedly is to compare the abundance of ARGs harbored in the new (i.e., biogas residue) versus old (i.e., compost) fertilizer. Consequently, in this study we collected 27 biogas residue samples of swine and cattle farms from Hsinchu to Yunlin. We then analyzed ARGs of four common antibiotics (namely tetracyclines, sulfonamides, β-lactams, and macrolides) as well as intI1, in addition to characterizing the basic physicochemical properties of the samples. Results indeed show that the relative abundance of the most target ARG in biogas residue samples was significantly higher than that in compost samples (p < .05). Moreover, the sum of ARG relative abundance was more elevated in swine biogas residue samples than in cattle samples (p < .05). To confirm the soil environmental fate of ARGs from biogas residues after fertilizing, the soil microcosm test was performed to quantify the ARGs reduction ratios of biogas residue-applied soil, and the results were also compared with the compost-soil microcosm test. The results show that the sums of ARG relative abundance in swine biogas residue-applied soils and compost-applied soils were not significantly different on the zeroth day and the thirtieth day, but the levels of the summed ARGs of both groups were significantly higher than cattle biogas residue-applied soils and environmental background (p < .05). The abundances of total ARGs in soil microcosms were not significantly reduced during 30 days after applying biogas residues/composts, which suggested the ARGs in biogas residue/compost-applied soils were quite persistent. If the frequencies of fertilizing were not controlled well, the ARGs perhaps accumulated in arable soil and promote agriculture workers′ ARGs exposure. However, the results of the soil microcosm only represented the situation of topsoil due to the limitation of the experimental design. Future research on the abundance and fate of in situ ARGs in arable soils is warranted, to obtain a complete picture of the potential risk and impact.
關鍵字(中) ★ 沼液沼渣
★ 抗生素抗性基因
★ 市售堆肥
★ 土壤縮模試驗		 關鍵字(英) ★ biogas slurry/residue
★ antibiotic resistance genes
★ compost
★ soil microcosm
論文目次 摘要 i
Abstract iii
致謝 v
目錄 vii
圖目錄 x
表目錄 xii
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 畜牧業與抗生素抗性之關聯 2
1.1.3 前處理程序對禽畜糞肥中ARGs豐度之影響 4
1.1.4 評估ARGs環境宿命之模型 5
1.1.5 沼液沼渣再利用的潛在抗生素抗性發展風險 6
1.2 研究目的 7
第二章 研究方法 9
2.1 研究流程及架構 9
2.2 第I階段實驗：沼液沼渣概況調查 11
2.2.1 沼液沼渣樣品採集 11
2.2.2 沼液沼渣基本特性分析 13
2.3 第II階段實驗：土壤縮模試驗 15
2.3.1 試驗土壤樣品採集與保存 15
2.3.2 試驗土壤基本特性分析 15
2.3.3 土壤縮模試驗 19
2.4 分子生物實驗 22
2.4.1 DNA萃取 23
2.4.2 目標基因定量 23
2.5 數據分析 34
2.5.1 統計分析 34
2.5.2 目標基因削減率/衰減係數計算 35
2.6 研究設備與試劑 37
第三章 結果與討論 40
3.1 沼液沼渣基本特性 40
3.1.1 沼液沼渣基本水質特性 40
3.1.2 沼液沼渣抗生素濃度 41
3.2 沼液沼渣目標基因豐度 44
3.2.1 沼液沼渣中ARGs/MGE豐度概況 44
3.2.2 不同料源沼液沼渣以及市售堆肥中目標基因豐度比較 54
3.3 沼液沼渣ARGs/MGE豐度與環境參數之間的潛在關聯 61
3.4 沼液沼渣土壤縮模試驗 64
3.4.1 試驗土壤基本特性 64
3.4.2 沼液沼渣縮模試驗ARGs/MGE豐度概況 65
3.4.3 縮模試驗各實驗組於不同時間之目標基因豐度 69
3.4.4 縮模試驗各實驗組目標基因豐度削減情況 81
3.5 環境意義 88
3.5.1 國內畜牧場沼液沼渣所含ARGs/MGE背景豐度 88
3.5.2 沼液沼渣中ARGs/MGE土壤環境宿命 88
3.5.3 潛在影響沼液沼渣ARGs/MGE豐度的要素或相關因子 89
3.5.4 研究脈絡以及環境意義 91
第四章 結論與建議 93
4.1 結論 93
4.2 建議 94
參考文獻 95
附錄 111
附錄一 Real-time PCR偵測極限測試 111
附錄二 Real-time PCR檢量線 112
附錄三 Melting curve 115
附錄四 原始數據 117
附錄五 沼液沼渣目標基因相對豐度與環境因子之Spearman相關性 132
附錄六 網絡分析 134
附錄七 學位考試委員意見回覆表 136
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指導教授 林居慶(Chu-Ching Lin) 審核日期 2023-1-7
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