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研究生： 陳宥丞
You-Cheng Chen
論文名稱： 不同肥料引入的抗生素抗性基因 在實際農業土壤中的宿命
Fate of antibiotic resistance genes introduced from different fertilizers in practical agricultural soil
指導教授： 林居慶
Chu-Ching Lin
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 環境工程研究所
Graduate Institute of Environmental Engineering
論文出版年： 2023
畢業學年度： 112
語文別： 中文
論文頁數： 118
中文關鍵詞： 沼液沼渣 、不同肥料 、抗生素抗藥性 、農地土壤
外文關鍵詞： biogas slurry and residue, different fertilizers, antibiotic resistant, agricultural fields
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    • 我國政府近年來積極推動透過厭氧消化程序，將畜牧場所產生之禽畜糞尿製成沼液沼渣後，當作肥分施加至農地，藉此達到土壤改質及增加作物產量等廢棄物資源化後的成效。由於現今畜牧場養殖方式多已改成集約飼養，業者為了促進禽畜快速生長，會將抗生素添加在飼料中等方式引入到禽畜體內，導致使用禽畜糞所成之新型態肥料在製程中若處理不當，容易增加抗生素耐/抗藥性(AR)，並因此造就抗生素抗性菌和抗生素抗性基因(ARGs)容易藉由「施肥」此途徑進入周遭環境，間接促使現地土壤AR風險的提高。有鑒於現階段探討「國內沼液沼渣回歸現地土壤所產生環境中AR的增長與變化」相關文獻仍然不多，本論文藉由農地現場土壤的採樣與檢測，調查不同肥料所引入或誘發之ARGs，在現地土壤隨作物生長的豐度變化，除深入了解實際農地土壤在施用沼液沼渣、雞糞堆肥、化學肥料及芝麻粕粉等肥分後的AR發展外，也基於實驗室過去土壤縮模試驗所得之結果，測試施肥後的農地場址最終所累積的ARG含量，依序會是沼液沼渣＞雞糞堆肥＞化學肥料～芝麻粕粉此假說。
    本研究所採集土壤分別包括施用沼液沼渣、化學肥料與芝麻粕粉的水稻田，以及施用雞糞堆肥的菜田，並根據過去實驗室針對沼液沼渣的檢測結果，挑選4類ARGs中豐度較大的tetM, tetO, su1l, blaTEM, ermB等ARG，以及特定的MGE (intI1)作為目標基因進行分析與比較。調查結果顯示，肥料的施用會造成現地土壤ARG豐度上升，且沼液沼渣土壤的目標ARG相對豐度確實顯著高於化學肥料土壤(p < .05)，而雞糞堆肥土壤僅有少數ARGs會低於化學肥料土壤。研究同時分析環境因子與ARGs之間的關聯，並將數據利用兩種不同衰減模型予以擬合，使現地土壤之ARG衰減速率程度得以量化。結果顯示，相對於其他參數，pH與總氮為更能影響ARG豐度之環境因子；而在衰減情況方面，沼液沼渣的澆灌雖會造成土壤ARGs的顯著上升，但其削減速率與其他肥料的土壤相比，並無統計上的差異。本研究所觀察到「整個採樣期間施用沼液沼渣的土壤ARGs豐度始終高於其他肥料」的結果，說明沼液沼渣此類新型肥料的使用，對於促成現地環境AR增長的風險值得關注。但為確保此現象並非僅限於本研究的短期調查，尚需未來持續追蹤，才可進一步的證實沼液沼渣對於促進一般環境以及公眾衛生的AR增長與累積的實質風險。

    In recent years, the government of our country has been actively promoting the utilization of anaerobic digestion processes to convert livestock and poultry waste into biogas slurry and residue, which is subsequently used as a nutrient-rich fertilizer for agricultural land. This approach aims to improve soil quality and increase crop yields through the recycling of organic waste resources. However, with the shift towards intensive livestock farming practices, antibiotics are often administered to animals to promote rapid growth, potentially leading to the emergence of antibiotic resistance in the resulting animal waste. Poor management of these new-generation fertilizers, if derived from antibiotic-exposed livestock, can contribute to increased levels of antibiotic resistance (AR), allowing antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) to enter the environment via the "fertilization" pathway, indirectly elevating the risk of AR in local soils. Despite the importance of understanding the impact of returning biogas slurry and residue to local soils on AR, there is currently limited research on this topic. This study addresses this gap by conducting soil sampling and testing in agricultural fields where different fertilizers, including biogas slurry and residue, chicken manure compost, chemical fertilizers, and sesame meal powder, were applied. Furthermore, based on laboratory results from previous soil microcosm experiments, the study investigates the long-term accumulation of ARGs in soil following fertilizer application, hypothesizing that the order of ARG accumulation will be biogas slurry and residue > chicken manure compost > chemical fertilizers ~ sesame meal powder.
    Soil samples were collected from rice fields where biogas slurry and residue, chemical fertilizers, and sesame meal powder were applied, as well as from vegetable fields treated with chicken manure compost. Four predominant ARGs (tetM, tetO, sul1, blaTEM, ermB) and a specific mobile genetic element (intI1) were selected for analysis and comparison. The research findings indicate that fertilizer application increases the abundance of ARGs in local soils, with biogas slurry and residue soil showing significantly higher relative ARG abundance compared to chemical fertilizer soil (p < 0.05). In contrast, only a few ARGs in chicken manure compost-treated soil were lower than in chemical fertilizer soil. The study also explores the relationship between environmental factors and ARGs and quantifies the decay rates of ARGs in local soils using two different decay models. Results indicate that, among the parameters studied, pH and total nitrogen are the most influential environmental factors affecting ARG abundance. However, the decay rates of ARGs in soil amended with biogas slurry and residue are not statistically different from those in soil amended with other fertilizers. The study observed that "the abundance of ARGs in soil amended with biogas slurry and residue remained consistently higher than in soils treated with other fertilizers throughout the sampling period." This highlights the potential risk associated with the use of anaerobic digestate-derived fertilizers in promoting environmental AR. To ensure that this phenomenon is not limited to the scope of this short-term investigation, continuous monitoring and further research are essential to substantiate the substantial risks associated with the promotion of AR in the general environment and public health through the application of anaerobic digestate.

    摘要i Abstract ii 致謝 iii 目錄 iv 圖目錄 vii 表目錄 ix 第一章 研究緣起與目的 1 1.1研究緣起 1 1.1.1 抗生素緣起與抗生素抗藥性議題 1 1.1.2 各國政府應對抗生素抗藥性問題之策略 2 1.1.3 抗生素抗藥性與畜牧業之連結 3 1.1.4 禽畜糞尿再製成沼液沼渣之優勢 4 1.1.5 我國臺中地區之當地沼液沼渣政策 4 1.1.6 沼液沼渣製作與現地環境之隱藏風險 5 1.1.7 目前對於土壤抗生素抗藥性調查結果 5 1.2 研究目的 7 第二章 研究方法 8 2.1 研究流程與架構 8 2.2 樣品採集與保存 11 2.2.1 各組土壤樣品的採集 11 2.3 施用沼液沼渣／化學肥料／雞糞堆肥／芝麻粕粉土壤基本特性分析 14 2.3.1 pH (NIEA S410.62C) 14 2.3.2 土壤含水量(water content) (AFS2901-1) 14 2.3.3 土壤有機質含量 14 2.3.4 土壤粒徑分析 14 2.3.5 土壤總氮分析─燃燒法 16 2.3.6 土壤總鉀分析 17 2.3.7 土壤重金屬分析─微波輔助王水消化法 17 2.4 分子生物檢測 18 2.4.1 DNA萃取 18 2.4.2 目標基因之標準品製備 18 2.4.3 選定之目標基因real-time PCR分析 22 2.5 第三代長讀長定序菌種分析 26 2.6 數據統計分析 27 2.6.1 各組別與時間差異比較 27 2.6.2 熱點分析(heatmap) 27 2.6.3 Spearman相關係數統計分析 27 2.6.4 冗餘分析(redundancy analysis, RDA) 27 2.6.5 目標基因相對豐度削減率與衰減係數計算 28 第三章 結果與討論 30 3.1 現地土壤各時間樣貌 30 3.1.1 各組現地土壤質地、pH、OM 32 3.2 現地土壤基因調查結果 40 3.2.1 各組別目標ARGs/MGE之絕對豐度 40 3.2.2 各組別目標ARGs/MGE之相對豐度 45 3.3各組目標ARGs/MGE之間關聯性 50 3.3.1 沼液沼渣組(B) ARGs/MGE豐度Spearman分析 50 3.3.2 化學肥料組(C) ARGs/MGE豐度Spearman分析 50 3.3.3 雞糞堆肥組(M) ARGs/MGE豐度Spearman分析 51 3.4 現地土壤ARGs/MGE豐度和環境因子間潛在關聯 55 3.4.1 各組目標ARGs/MGE豐度與環境因子之冗餘分析 55 3.4.2 現地土壤目標ARGs/MGE豐度與環境因子(含重金屬)Spearman分析 60 3.4.3 沼液沼渣組(B)目標ARGs/MGE豐度與環境因子Spearman分析 60 3.4.4 化學肥料組(C)目標ARGs/MGE豐度與環境因子Spearman分析 61 3.4.5 雞糞堆肥組(M)目標ARGs/MGE豐度與環境因子Spearman分析 61 3.5 各組目標ARGs/MGE相對豐度衰減情況 67 3.6 現地土壤菌種分析結果 72 3.6.1 Alpha Diversity分析 72 3.6.2 菌種豐富度差異分析 73 3.6.3 Beta Diversity 76 3.7 環境意義 78 第四章 結論與建議 81 4.1結論 81 4.2建議 82 參考文獻 84 附錄 94 附錄一 Real-time PCR 檢量線 94 附錄二 解離曲線(Melting curve) 96 附錄三 各組別基本特性數據 97 附錄四 各廠區時間ARGs/MGE以對數迴歸模型評估現地豐度衰退情形之詳細數據 98 附錄五 各廠區時間ARGs/MGE以擬一階反應動力模型評估現地豐度衰退情形詳細數據 100 附錄六 學位考試委員意見回覆表 102

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