不同料源製成之市售堆肥其抗生素抗性基因含量調查

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鄭念媛(Nien-Yuan Cheng) 查詢紙本館藏

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論文名稱

不同料源製成之市售堆肥其抗生素抗性基因含量調查
(Survey of antibiotic resistance gene levels harbored in commercially available composts derived from different feedstock)

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摘要(中)

環保署與農委會近年來大力推動畜牧糞尿資源化政策，建議畜牧業者可不再透過之前的三段式處理，只需經過一般的厭氧消化程序，即可將牧場所產生的畜牧排泄物轉化為沼渣沼液，並當作肥料施用於農地，讓原本該被排放到承受水體而可能造成污染擴散的畜牧糞尿能被「農地農用」，呼應當前的「循環經濟」政策。不過，既有的文獻已指出，傳統的中、常溫厭氧消化處理對於禽畜糞尿所含的抗生素抗性基因(ARG)濃度大多無法有效削減，而ARG濃度在環境持續升高的後果，已被國際權威組織預測將嚴重威脅現代醫學的成就，某種程度暗示著如要推動沼液沼渣作為農地肥分使用的政策時，應先審慎調查確認是否將因此而衍生出土壤環境的抗藥性增長問題。有鑒於ARG的濃度尚未有法規規範，本研究針對行之有年的「堆肥」進行ARG的含量調查，藉此讓沼液沼渣所造成的環境抗生素抗藥性的風險評估有參考對比值。
本研究所選的28件堆肥樣品涵蓋非禽畜糞、牛糞、豬糞、雞糞等不同原料，以基因即時定量技術，定量樣品中的目標基因(包括各個ARG、移動基因元件以及16S rRNA基因)，並以統計分析各類樣品彼此之間的相關性與差異性。調查結果顯示: (1)含禽畜糞便的堆肥中抗生素抗藥性基因含量比非禽畜糞的堆肥來的高；(2)不論是非禽畜糞便還是含禽畜糞之堆肥其主要以磺胺類抗生素抗藥性基因以及第一類整合子基因為主；(3)堆肥中的重金屬鋅、銅與抗生素抗藥性基因呈現負相關；(4)堆肥中殘留的抗生素與抗生素抗藥性基因之間是具有相關性；(5)當堆肥進入土壤環境後主要是以四環素類、磺胺類抗生素抗藥性基因以及第一類整合子基因為主；(6)堆肥中的抗生素抗藥性基因及第一類整合子進入土壤環境後，其在環境中的宿命不會因其料源不同而有所差異。這些結果說明不同料源的堆肥中的抗生素抗藥性基因及第一類整合子的豐度確實有所不同，但其對於抗生素耐藥性基因進入環境之影響並不會因為其原料不同而有所差異。此外，對於堆肥中抗生素抗藥性基因及第一類整合子在環境中降解速率值得加以追蹤及確認。

摘要(英)

In recent years, the Environmental Protection Agency and the Committee of Agriculture have vigorously promoted the policy of resource utilization of livestock manure and urine, suggesting that livestock farmers can no longer go through the previous three-stage treatment, but only need to go through the general anaerobic digestion process to convert the livestock excrement produced by the pasture. It is used as biogas residue and applied to agricultural land as fertilizer, so that livestock manure that should have been discharged into receiving water bodies and may cause pollution to spread can be used for "farmland agriculture", echoing the current "circular economy" policy. However, the existing literature has pointed out that the traditional anaerobic digestion treatment at room temperature cannot effectively reduce the concentration of antibiotic resistance genes (ARG) contained in livestock manure and urine, and the consequence of the continuous increase of ARG concentration in the environment has been It is predicted by international authoritative organizations that it will seriously threaten the achievements of modern medicine. To some extent, it implies that if we want to promote the policy of using biogas residue as agricultural land fertilizer, we should conduct a careful investigation to confirm whether the drug resistance of the soil environment will be derived from its growth issues. Because the concentration of ARG has not yet been regulated by regulations, this study conducted a survey on the content of ARG in the long-established "compost", to provide a reference and comparative value for the risk assessment of environmental antibiotic resistance caused by biogas slurry and residue.
The 28 compost samples selected in this study covered different raw materials such as non-poultry animal manure, cow manure, pig manure, chicken manure, etc. The target genes (including various ARGs, mobile genetic elements, and 16S rRNA genes) in the samples were quantified by real-time gene quantitative technology, and statistically analyzed the correlation and difference between various samples. The survey results showed that: (1) the content of antibiotic resistance genes in the manure compost was higher than that of the non-manure compost; (2) both non-manure and manure compost mainly contained sulfonamides antibiotic resistance genes and intI1; (3) heavy metals zinc and copper in compost are negatively correlated with antibiotic resistance genes; (4) the residual antibiotics in compost and antibiotic resistance genes are closely related. (5) When the compost enters the soil environment, it is mainly composed of tetracycline and sulfonamide antibiotic resistance genes and intI1; (6) The antibiotic resistance genes and intI1 in compost After entering the soil environment, its fate in the environment will not vary due to different sources. These results indicate that the abundance of antibiotic resistance genes and intI1 in composts from different feedstock are indeed different, but their impact on the entry of antibiotic resistance genes into the environment will not be affected by different raw materials. difference. In addition, the degradation rates of antibiotic resistance genes and intI1 in compost in the environment deserve to be tracked and confirmed.

關鍵字(中)

★ 禽畜糞
★ 堆肥
★ 抗生素抗藥性基因
★ 農地土壤

關鍵字(英)

★ manure
★ compost
★ antibiotic resistance gene
★ agriculture soil

論文目次

致謝................................ I
摘要................................ II
ABSTRACT............................ III
目錄................................. V
表目錄............................... VII
圖目錄............................... VIII
第一章前言............................... 1
1.1 研究緣起及背景...................... 1
1.1.1 抗生素的作用........................ 1
1.1.2 抗生素抗藥性........................ 2
1.1.3 抗生素抗藥性對於畜牧業的影響.............. 3
1.1.4 抗生素抗藥性與畜牧廢棄物產品之關係... 5
1.1.5 抗生素抗藥性之於畜牧廢棄物之處理方式.... 6
1.1.6 抗生素抗藥性與土壤之關係................ 7
1.1.7 整合子(integron)與環境之關係........... 7
1.2 研究目的............................... 8
第二章研究方法.................................9
2.1 實驗流程................................ 9
2.2 堆肥之選取及收集.................. 10
2.3 土壤縮模試驗之架設................ 15
2.4 堆肥及土壤之物化分析.............. 15
2.4.1 含水量(water content) (AFS2901-1)...... 15
2.4.2 pH (NIEA S410.62C).............. 16
2.4.3 電導度(EC) (AFS2905-1)........... 16
2.4.4 土壤有機質測定(organic matter, OM)...... 16
2.4.5 有機肥料銅、鋅濃度分析─王水消化法(NIEA S321.65B).. 17
2.4.6 有機肥料中砷濃度分析─微波消化法......... 17
2.4.7 土壤中重金屬分析─微波消化法............ 17
2.4.8 土壤顆粒比重分析.................. 17
2.4.9 土壤最大保水力(maximum water-holding capacity).. 18
2.4.10 土壤粒徑分析─比重計法.............. 18
2.4.11 堆肥中抗生素殘留量分析............. 19
2.5 樣品中基因的提取及目標基因的定量........ 20
2.5.1 DNA提取.......................... 20
2.5.2 樣品DNA純化....................... 20
2.5.3 聚合酶連鎖反應(polymerase chain reaction, PCR)分析 20
2.5.4 目標基因之標準品製備................ 24
2.5.5 目標基因定量─qPCR分析................ 24
2.6 數據分析與統計分析................ 27
2.6.1 堆肥ARGs之顯著性分析................ 27
2.6.2 縮模試驗ARGs之顯著性分析................ 27
2.6.4 熱點分析(heatmap)................ 28
2.6.5 Spearman等級相關係數分析................ 28
2.6.6 冗餘分析(redundancy analysis, RDA)................ 28
2.7 試劑與儀器................ 28
第三章結果與討論................ 30
3.1 堆肥樣品之檢測................ 30
3.1.1 堆肥之物化特性................ 30
3.1.2 堆肥中的ARGs絕對豐度之調查................ 38
3.1.3 堆肥中ARGs及intI1相對豐度之調查................ 44
3.1.4 堆肥中抗生素殘留與ARGs之關係................ 49
3.1.5 有機肥料中ARGs與intI1之關係................ 52
3.2 堆肥土壤縮模試驗................ 57
3.2.1 堆肥土壤縮模試驗中抗生素抗藥性基因之絕對豐度................ 57
3.2.2 堆肥土壤縮模試驗中ARGs、intI1之相對豐度................ 63
3.2.3 縮模試驗中抗生素抗藥性基因變化率................ 68
3.2.4 堆肥土壤縮模試驗中ARGs與時間之關係................ 76
3.3 抗生素抗藥性在環境之意義及風險................ 79
第四章結論與建議................ 81
4.1 結論................ 81
4.2 建議................ 82
參考文獻 ................83
附錄................ 89

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指導教授

林居慶(Chu-Ching Lin)

審核日期

2022-9-22

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