博碩士論文 109326024 詳細資訊



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姓名 陳柏廷(Bo-Ting Chen)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 施用農廢所製生物炭對於澆灌沼液沼渣農地所含抗生素抗性基因豐度之影響
(Influence of employing agronomic waste-derived biochars on the abundance of antibiotic resistance genes in arable soils irrigated with biogas slurries/residues)
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摘要(中) 抗生素抗藥性已被世界衛生組織認定為全球最大健康威脅之一，原因在於除了醫療濫用外，抗生素也常被畜牧業者添加至飼料中作為預防牲畜染病及促進生長用，但殘餘的抗生素與抗生素抗性菌可隨糞尿排至環境中，進而造成抗生素抗性基因(antibiotic resistant genes, ARGs)在環境與臨床間的散播。近年來政府為了改善畜牧糞尿污染問題，積極輔導畜牧業者以厭氧消化方式處理畜牧糞尿，將其轉為沼液沼渣並回歸農用，達到循環經濟的效果。然而已有文獻指出，台灣畜牧場最常見的中、常溫厭氧消化程序無法有效地削減糞尿中的ARGs，且本研究群先前碩論也觀察到縮模系統中的土壤在澆灌沼液沼渣後，所引入的ARGs具有一定的持久性，暗示著農地ARGs的累積潛勢不可輕忽。對此國外雖有文獻嘗試使用生物炭處理糞肥澆灌農田所產生的ARGs污染問題，並觀察到ARGs因而豐度降低，但現階段針對將生物炭應用於澆灌沼液沼渣後的土壤，其內所含的ARG豐度變化的調查依然不多。有鑑於此，本研究收集廢棄菇包(M)、稻殼(R)，以不同溫度(300 ℃、600 ℃)燒製成四種生物炭(M3、M6、R3、R6)並比較其性質後，以2% (w/w)比例與土壤混合，加上未添加生物炭的空白組(CK)共5組，進行小白菜(Brassica rapa chinensis)種植的盆栽縮模方式，模擬農地環境在30天內根際與周圍土壤中4類ARG (tetM, sul1, ermB, blaTEM)和屬於移動性遺傳元件(MGE)的第一型整合子intI1，以及16S rRNA gene的豐度變化。結果顯示：(1)單純種植小白菜的CK組在30天內就可顯著降低土壤中的ARGs/MGE豐度，但在第30天有與第20天顯著差異的上升趨勢；(2)整體而言，添加生物炭組第30天ARGs/MGE豐度數值持續下降且常顯著低於CK組，但sul1及intI1豐度經處理後雖有顯著降低，但仍保有一定豐度，未來需特別注意；(3)菇包生物炭組第0天ARGs/MGE豐度即顯著低於CK組，稻殼生物炭組則僅略微下降但與CK組無顯著差異，兩種材料第0天時都是300 ℃組豐度較低，這可能為原料及奈米級生物炭所造成的差異。第30天時各生物炭組在不同基因有不同的狀況，但以相對豐度來看則是M3組常顯著低於各組；(4)使用兩種模型評估目標基因隨時間的衰退狀況時，除tetM與ermB外，其餘ARGs及總ARGs在兩種模型中都有相似的趨勢，且菇包生物炭組若比較衰退係數時在各基因中常為最慢的組別；(5)生物炭的添加顯著改變土壤微生物群落結構，造成各組菌相不同，尤其M3組與各組差異最大。而沼液沼渣中相關菌群相對豐度在30天後大幅下降；(6) Spearman相關分析結果顯示所有目標ARGs/MGE彼此間皆為顯著正相關，但與16S rRNA gene呈現顯著負相關，代表隨菌數越多則含ARGs的細菌可能因其體內帶有ARGs反而造成額外的適存度代價致使其傾向丟棄ARGs。最後，ARGs/MGE與環境因子之間的相關性分析因環境因子檢測項目較少，無法得知影響ARGs/MGE的環境因子整體面貌，且RDA分析解釋度較低，未來需進行更全面的分析以了解環境因子對ARGs/MGE影響能力。
摘要(英) The problem of antibiotic resistance has been recognized by the WHO as one of the greatest global public health threats. In addition to medical misuse, antibiotics are often added to animal feed by livestock farmers to both prevent diseases and promote growth of animals. The residual antibiotics and antibiotic resistant bacteria are inevitably excreted to the environment through feces and urine, leading to the problem of antibiotic resistant genes (ARGs) spread in the environment and ultimately the clinic. In recent years, in order to achieving a circular economy effect, our government has actively advocated using anaerobic digestion to treat livestock excrement, converting it into biogas digestate for agricultural use. However, existing literature has pointed out that the commonly used anaerobic digestion processes under ambient-air conditions in Taiwanese livestock farms cannot substantially diminish ARG levels from manure. Previous studies in our research group have also observed the persistence of ARGs in soil after the application of biogas digestate, emphasizing the potential of ARG accumulation in farmland. To address this issue, studies by others have attempted to use biochar to treat ARGs contamination from manure irrigation in farmland and have observed a decrease in ARGs abundance. However, there is still limited research on the use of biochar to treat ARGs contamination in soil receiving biogas digestate. Therefore, in this study, waste mushroom grow bag (M) and rice husk (R) were collected and pyrolyzed at different temperatures (300 ℃ and 600 ℃) to obtain four types of biochar samples (designated as M3, M6, R3, R6). After comparing their properties, the biochar was mixed with soil at a ratio of 2% (w/w). A control group (CK) without biochar addition was also included, resulting in a total of five groups. Pak choi (Brassica rapa chinensis) was planted in pots to simulate the agricultural environment, and the changes in the abundance of four types of ARGs (tetM, sul1, ermB, blaTEM), the class 1 integron gene intI1 (MGE), and the 16S rRNA gene were examined in the bulk and rhizosphere soil over a period of 30 days. The results showed that: (1) The CK group with only pak choi planting significantly reduced the abundance of ARGs/ MGE in the soil within 30 days, but there was a substantive increase on day 30 compared to day 20; (2) The biochar-added groups continuously decreased the abundance of ARGs/MGE on day 30 and generally had significantly lower values than the CK group. However, the abundance of sul1 and intI1, although significantly reduced after treatment, still maintained a certain level, indicating the need for special attention in the future; (3) The mushroom grow bag biochar group showed significantly lower ARGs/MGE abundance than the CK group on day 0, while the rice husk biochar group only showed a slight decrease without significant differences compared to the CK group. Both materials had lower abundance in the 300 ℃ group on day 0. On day 30, the biochar-added groups exhibited different conditions for different genes, but the M3 group consistently had significantly lower relative abundance than the other groups; (4) The assessment of soil ARGs/MGE decay using two models showed similar trends for sul1, intI1, blaTEM, and total ARGs. In the rice husk biochar group, the decay coefficients for sul1, intI1, and total ARGs were higher than those of the mushroom grow bag biochar group, while for blaTEM, the 300 ℃ biochar group had higher decay coefficients than the 600 ℃ biochar group, and the R6 group showed an increase. The mushroom grow bag biochar group consistently had the slowest decay coefficients among the different genes; (5) The addition of biochar significantly altered the microbial community structure in the soil, resulting in different bacterial compositions among the groups, with the M3 group showing the greatest difference compared to the other groups. The microbial communities from the biogas slurry/residue were suppressed by the original microorganisms in the soil, resulting in a significant decrease in the relative abundance of related microbial communities after 30 days; (6) All target ARGs/MGE showed significant positive correlations with each other and significant negative correlations with the 16S rRNA gene. This suggests that as the microbial population increases, bacteria carrying ARGs may incur additional fitness costs, leading to a tendency to discard ARGs. The correlation analysis between ARGs/MGE and environmental factors was limited due to the lack of comprehensive environmental factor measurements, and the redundancy analysis (RDA) had low explanatory power. Future research should conduct more comprehensive analyses to understand the overall impact of environmental factors on ARGs/MGE.
關鍵字(中) ★ 沼液沼渣
★ 農廢衍生生物炭
★ 抗生素抗性基因
★ 盆栽試驗		 關鍵字(英) ★ biogas digestate
★ agricultural wastes-derived biochar
★ antibiotic resistant genes
★ pot tests
論文目次 摘要 I
Abstract III
致謝 V
目錄 VI
圖目錄 VIII
表目錄 X
第一章 研究緣起與目的 1
1.1 研究緣起 1
1.1.1 抗生素抗藥性 1
1.1.2 畜牧業對抗生素抗藥性散播之影響 2
1.1.3 沼液沼渣再利用潛在風險 3
1.1.4 抗生素抗藥性基因控制對策 4
1.1.5 影響生物炭性質因素 6
1.2 研究目的 7
第二章 研究方法 9
2.1 實驗流程 9
2.2 生物炭製備方法 10
2.3 生物炭特性分析 11
2.3.1 生物炭含水率測定 11
2.3.2 生物炭pH測定 11
2.3.3 元素分析 11
2.3.4 重金屬分析—微波消化法 11
2.3.5 氮氣吸脫附測試 12
2.3.6 掃描式電子顯微鏡(SEM) 12
2.3.7 傅立葉轉換紅外線光譜儀(FTIR) 12
2.4 盆栽縮模土壤物化分析 13
2.4.1 土壤含水率測定 13
2.4.2 土壤最大保水力測定 13
2.4.2 土壤有機質測定 13
2.4.2 土壤pH測定 14
2.4.3 土壤電導度測定 14
2.4.3 土壤質地分析—比重計法 14
2.5 盆栽縮模試驗架設 17
2.6 分子生物檢測 19
2.6.1 DNA萃取 19
2.6.1 目標基因標準品製備 19
2.6.2 各基因real-time PCR分析 23
2.7 第三代長讀長定序 27
2.8 數據分析 28
2.8.1 不同處理組別比較 28
2.8.2 其餘統計分析 28
2.8.3 目標基因衰退係數模型 29
第三章 結果與討論 30
3.1 沼液沼渣性質 30
3.2 生物炭性質 33
3.3 盆栽縮模試驗 40
3.3.1 盆栽縮模土壤基本性質 40
3.3.2 盆栽植物生長概述 43
3.3.3 盆栽縮模試驗ARGs/MGE豐度概況 46
3.3.4 盆栽縮模試驗ARGs/MGE豐度與環境因子間的潛在關聯 49
3.3.5 盆栽縮模試驗各組別於不同時間目標基因之豐度 54
3.3.6 目標基因變化率 61
3.3.7 菌相分析 71
3.4 環境意義 88
第四章 結論與建議 92
4.1 結論 92
4.2 建議 93
參考文獻 95
附錄 109
附錄一 Real-time PCR檢量線 109
附錄二 Melting curve 110
附錄三 衰退係數數據表 111
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指導教授 林居慶(Chu-Ching Lin) 審核日期 2023-6-19
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