探討生物炭改質對於降低澆灌沼液沼渣土壤所含抗生素抗性基因豐度之效應

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劉丞軒(Cheng-Xuan Liu) 查詢紙本館藏

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論文名稱

探討生物炭改質對於降低澆灌沼液沼渣土壤所含抗生素抗性基因豐度之效應
(Exploring the effect of biochar modification on reducing the abundance of antibiotic resistance genes contained in soil irrigated with biogas slurries and residues)

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摘要(中)

近年來國內環保署所大力推動的畜牧糞尿資源化政策，此舉雖有效改善承受水體的污染狀況以及實現農廢農用的訴求，但從抗生素抗藥性的角度來看，沼渣沼液作為農地肥分對於環境依舊有公共衛生的風險。基於當前政策的限制下，採取適當的管末處理以降低經沼液沼渣施肥後農地抗生素抗藥性的傳播，或許為一可行方案。本研究提出以生物炭應用於經沼液沼渣施肥後土壤中，可能影響重金屬生物有效性及土壤微生物的群落變化導致ARGs在土壤生態系統中的傳播有所減緩的假說，進一步以多種統計分析判斷不同生物炭對於經沼液沼渣澆灌後土壤中的抗生素抗藥性影響程度及影響機制。生物炭特性分析結果顯示以高錳酸鉀改質後生物炭會存在錳官能基並被確實固定在材料上，證實了錳改質生物炭 (M300、M600)的製備。而以不同生物炭吸附水相中Cu(II)、Zn(II)的實驗結果則表明錳改質生物炭 (M300、M600)對兩種重金屬的吸附能力遠高於常規生物炭 (B300、B600)，且低溫煅燒而成生物炭吸附能力更強，M300對Cu(II)及Zn(II)的最高吸附容量達到136.986 μmol/g 及133.333 μmol/g，同時發現所有生物炭對於Cu(II)、Zn(II)的吸附模式皆屬於化學吸附。本研究也以不同生物炭進行為期一個月的縮模試驗後，在土壤環境參數分析結果中發現經生物炭處理後的土壤樣品中的TOC及TN顯著上升，但生物可利用性銅含量則顯著下降，與控制組C2相比，加入M300生物炭後可從23%下降至13%，其次在各目標基因檢測中，檢測了1種管家基因 (16S rRNA gene)，3種不同的抗生素抗性基因(ermB、sul1、tetM)，還有1種流動性基因元素 (intI1)，結果發現高溫煅燒之生物炭 (B600、M600)使土壤樣品中的16S rRNA基因豐度隨著培養時間的增加顯著減少 (p < .05)，顯示B600、M600生物炭對於降低經沼液沼渣澆灌後土壤中的抗生素抗藥性機制，應屬於減少縮模試驗土壤系統中的菌數進而減少其ARGs/MGE豐度；而在相對豐度的統計結果中，M300生物炭對於土壤中抗生素抗藥性的減輕更為顯著 (p < .05)，發現其減輕機制可能為M300生物炭固定了土壤中的Cu(II)，使土壤系統中的可被微生物利用的Cu(II)轉換為更不易被利用的相態。相關性分析與冗餘分析中則證實生物可利用性銅含量與ARGs/MGE的強烈正相關 (p < .05)，進一步表明沼液沼渣中的重金屬Cu(II)對土壤中的ARGs/MGE豐度具有一定程度上的貢獻。可惜的是並未在次世代定序分析中發現生物炭對於縮模試驗土壤中在以門 (Phylum) 為層級的細菌群落組成所造成的變化，但細究對於可能帶有ARGs的Genus菌屬 (Clostridium、Nocardioides) 的豐度下降趨勢，則可以看到B300及B600組別受到細菌群落的影響可能更多。但根據上述結果而言，生物炭，尤其是改質生物炭應可透過不同機制減輕以沼液沼渣施肥後土壤中的抗生素抗藥性。

摘要(英)

In recent years, the domestic Environmental Protection Agency has vigorously promoted the policy of resource utilization of livestock manure. Although this measure has effectively improved its pollution of water bodies and fulfilled the demands of agricultural waste utilization, the commonly used mesophilic and thermophilic anaerobic digestion processes adopted by domestic livestock farmers are not sufficient to effectively reduce the total amount of antibiotic resistance genes (ARGs) in livestock manure. Therefore, the application of biogas slurries and residues as fertilizer in agricultural land may pose risks of promoting the development and dissemination of antibiotic resistance in the environment. Given the limitations of current policies, implementing appropriate post-treatment measures to reduce the spread of antibiotic resistance after the application of biogas slurries and residues as fertilizer on farmland may be a feasible solution. This study proposes a hypothesis that the application of biochar in the soil after the application of biogas slurries and residues as fertilizer may affect the bioavailability of heavy metals and the community changes of soil microorganisms, resulting in a reduction in the spread of ARGs in the soil ecosystem. Furthermore, multiple statistical analyses are conducted to determine the degree and mechanisms of the impact of different biochars on antibiotic resistance in the soil after the application of biogas slurries and residues as irrigation. The experimental results of biochar characterization analysis show that biochar modified with potassium permanganate possesses manganese functional groups that are effectively fixed on the material, confirming the preparation of manganese-modified biochar (M300, M600). The experimental results of different biochar adsorbing Cu(II) and Zn(II) from the aqueous phase indicate that manganese-modified biochar (M300, M600) has a much higher adsorption capacity for both heavy metals compared to conventional biochar (B300, B600), and biochar produced by low-temperature calcination exhibits even stronger adsorption capacity. The maximum adsorption capacities for Cu(II) and Zn(II) reached 136.986 and 133.333 μmol/g, respectively. It is also found that all biochars exhibit chemical adsorption modes for Cu(II) and Zn(II). Subsequently, after conducting a one-month simulated test using different biochars, the analysis of soil environmental parameters reveals a significant increase in total organic carbon (TOC) and total nitrogen (TN) in soil samples treated with biochar. The bioavailable copper (bioCu) content significantly decreased, dropping from 23% to 13% when M300 biochar was added compared to the control group C2. One stewardship gene (16S rRNA gene), three different antibiotic resistance genes (ermB, sul1, tetM), and one mobile genetic element (intI1) were tested, and the results indicated that high-temperature biochars (B600, M600) significantly reduced the abundance of 16S rRNA gene in soil samples with increasing incubation time (p < .05). The results indicate that B600 and M600 biochars likely reduce antibiotic resistance mechanisms in soil after biogas slurry irrigation by decreasing bacterial counts and subsequently reducing ARGs/MGE abundance in the shrinkage test soil
iv
system. In terms of relative abundance, the statistical results indicate that M300 biochar has a more significant effect on reducing antibiotic resistance in the soil (p < .05), and its mechanism may be related to the fixation of Cu(II) in the soil by M300 biochar, transforming the bioavailable Cu(II) in the soil system into a less accessible form for microorganisms. The correlation analysis and redundancy analysis confirm a strong positive correlation (p < .05) between bioavailable copper content and ARGs/MGEs, further suggesting that the heavy metal Cu(II) in biogas slurries and residues contributes to the abundance of ARGs/MGEs in the soil to some extent. Unfortunately, significant changes in bacterial community composition in the simulated soil due to the application of biochar were not observed in the next-generation sequencing analysis. However, based on the above results, it can be concluded that biochar, specially modified biochar, may reduce antibiotic resistance in the soil after the application of biogas slurries and residues as fertilizer through different mechanisms.

關鍵字(中)

★ 生物炭
★ 沼液沼渣
★ 抗生素抗性基因
★ 土壤縮模試驗

關鍵字(英)

★ biochar
★ biogas slurries and residues
★ antibiotic resistance gene
★ soil microcosm

論文目次

摘要 i
Abstract iii
致謝 v
目錄 vi
圖目錄 ix
表目錄 xi
第一章研究緣起與目的 1
1.1 研究緣起 1
1.1.1 抗生素與抗生素抗藥性 1
1.1.2 畜牧業與抗生素抗藥性之關聯 1
1.1.3 禽畜糞再製成肥料對土壤中ARGs豐度影響 3
1.1.4 生物炭 4
1.1.5 生物炭減輕環境中抗生素抗藥性 4
1.1.6 改質生物炭對重金屬生物有效性及抗生素抗藥性之影響 5
1.2 研究目的 6
第二章材料與方法 7
2.1 研究流程與步驟 7
2.2 常規與錳改質生物炭合成方法 8
2.3 生物炭特性分析方法 8
2.3.1 pH 8
2.3.2 元素分析 9
2.3.3 比表面積與孔徑分布分析 9
2.3.4 掃描式電子顯微鏡-X射線能譜儀分析 9
2.3.5 傅立葉轉換紅外線光譜儀分析 9
2.3.6 X 射線粉末繞射儀分析 10
2.4 水相吸附實驗 10
2.4.1 動力學吸附實驗及分析 10
2.4.2 等溫吸附實驗 11
2.5 土壤縮模試驗架設 12
2.5.1 沼液沼渣樣品採集與保存 12
2.5.2 試驗土壤樣品採集與保存 12
2.5.4 縮模試驗架設流程 12
2.6 沼液沼渣及土壤之物化分析 13
2.6.1 沼液沼渣重金屬分析 13
2.6.2 土壤pH 13
2.6.3 土壤含水量 14
2.6.4 土壤重金屬總量分析─微波消化法 14
2.6.5 土壤重金屬分析─序列萃取法 14
2.6.6 土壤總有機碳、總有機氮分析─燃燒法 16
2.7 分子生物實驗 16
2.7.1 DNA提取 16
2.7.2 目標基因定量─qPCR分析 16
2.8 分子生物研究數據與統計分析 21
2.8.1 qPCR數據處理 21
2.8.2 縮模試驗ARGs顯著性分析 21
2.8.3 熱點圖分析(heatmap) 22
2.8.4 Spearman等級相關係數分析 22
2.8.5 目標基因衰減係數 22
2.8.6 冗餘分析(Redundancy analysis, RDA) 23
2.8.7 細菌群落分析 23
2.8.8 網絡分析 (Network analysis) 23
2.9 研究儀器設備及試劑 24
第三章結果與討論 26
3.1 生物炭基本特性分析 26
3.2 生物炭吸附重金屬鋅之吸附機制探討 34
3.2.1 動力學吸附 34
3.2.2 等溫吸附研究 36
3.3 沼液沼渣土壤縮模試驗 38
3.3.1 試驗土壤基本特性 38
3.3.2 縮模試驗土壤樣品之環境參數分析 38
3.3.3 縮模試驗土壤樣品ARGs/MGE豐度概況 43
3.3.4 縮模試驗各實驗組於不同時間之目標基因豐度 44
3.3.5 目標基因豐度削減程度 53
3.3.6 環境參數與目標基因spearman相關及冗餘分析 55
3.3.7 菌種分析結果 66
3.3.8 網絡分析 71
3.4 環境意義 72
第四章結論與建議 74
4.1 結論 74
4.2 建議 75
參考文獻 76
附錄 88

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指導教授

林居慶(Chu-Ching Lin)

審核日期

2023-7-31

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