博碩士論文 104326013 詳細資訊




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姓名 黃奕傑(Yi-Jie Huang)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 胞外溶解鎘的化學物種組成對於非抗性細菌生長之影響
(Effect of extracellular cadmium speciation on the growth of non-resistant bacteria)
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摘要(中) 摘要
台灣由於灌排不分的關係,常使得承受水體與其周遭環境容易因偷排而受到諸如銅、鋅、鎘、鉛等親硫金屬的污染。這些污染對環境所造成的衝擊雖然與所排出的重金屬的總濃度有關,但更關鍵的是這些重金屬在特定環境條件下所呈現出的生物有效性;而此生物有效性不僅可左右重金屬對於生態系統所帶來的毒性風險程度外,也已被懷疑可能與抗生素抗藥性在環境中的誘發與持續留佈有關。由此可知,明確掌握重金屬的生物有效性將能更準確的評估生態健康風險,並讓環境管理與整治更有效率。有鑑於此,本研究旨在探討親硫金屬鎘的生物有效性,希望在藉由調控培養液組成的手段下,利用不具鎘抗性且在親緣關係上相距甚遠的Escherichia coli K-12 (革蘭氏陰性菌)與Bacillus subtilis 168 (革蘭氏陽性菌)為模式細菌,以細胞的生理表徵為指標,釐清何種型態的鎘離子是最容易被這些非抗性細菌予以攝取,並進一步產生毒性。由於鎘對於一般淡水生態系統的單細胞生物體而言就如同汞一樣,可視為是單純的毒性物質,因此為了驗證「溶解鎘主要是以自由型態、非錯合的鎘離子(即Cd2+)被菌株以主動運輸的方式攝取」之假說,本研究改變培養液中的氯離子濃度使得自由鎘離子除了成為系統中的優勢鎘物種外也呈現出濃度梯度,進行「飢餓」與「非飢餓」細胞的鎘暴露試驗,並同時利用菌株的存活率(與生長抑制程度)以及螯合劑清洗細胞的方式,實際量化菌株對於鎘的攝取程度。研究的結果確實與假說相符:當實驗條件在Cd2+濃度越高情況下不管是陽性菌或是陰性菌都出現抑制與死亡增加的情況,並且藉由飢餓實驗的結果得知當細胞在缺乏能量的時候死亡率顯著下降,表明試驗菌株可能是藉由主動運輸的攝取方式讓鎘進入細胞,暗示著若環境中的鎘污染主要是以自由型態的鎘離子存在時,將可能對生態帶來較高的污染與風險,因此需提高處理層級。
關鍵字:生物有效性、胞外化學物種組成、非金屬抗性細菌、鎘攝取機制、主動運輸
摘要(英) Abstract
Due to the current water resource policy in Taiwan that allows irrigation canals to receive the effluent from wastewater treatment systems of nearby house-holds and/or industrial manufactures, it is reported that a considerable amount of sediment and farmland have been contaminated with chalcophile heavy metals (e.g., copper, zinc, cadmium and lead) resulting from frequent incidents of illegal discharge of untreated wastewater. While the impact of a pollutant on the environment is related to its total emitted concentration, it is the bioavailability of a heavy metal under specific environmental conditions that plays the key role in determining the potential risk exerted on the ecosystem health. Hence, a better understanding of the bioavailability of a heavy metal will lead to a more accurate assessment on the ecological health risk and more efficient environmental management and remediation. This knowledge may also provide insight into the contemporary mechanism (i.e., co-selection pathways) of antibiotic resistance development in the environment. In this study, laboratory experiments were conducted to investigate the bioavailability of Cd(II) to two phylogenetically distinct prokaryotes, namely Escherichia coli K-12 (a Gram-negative bacterium) and Bacillus subtilis 168 (a Gram-positive bacterium), using growth inhibition and cell death as an indicator of Cd(II) uptake. Specifically, chemistry of the assay medium was manipulated by varying chloride and organic carbon concentrations to test the hypothesis that the uncomplexed, free Cd(II) ion (i.e. Cd2+) is the primary permeant to the cell via active transport. In addition, cell-washing was performed to quantify the intracellular quantity of Cd(II). Results of the study were indeed consistent with the hypothesis: the increase in Cd2+ concentration resulted in increasing inhibition and death of both Gram-positive and negative bacteria. Moreover, the death rate decreased significantly in starving cells. It is thus suggested that the tested bacteria may take up Cd(II) into their cell by active transport. The obtained results also indicated that if Cd(II) mainly appears in the form of free Cd ions, it may cause higher pollution and toxicity risk to the ecosystem, and hence it is necessary to improve the quality of wastewater treatment process.
Keywords: Bioavailability, extracellular speciation, non-resistant bacteria, Cd uptake, active transport
關鍵字(中) ★ 生物有效性
★ 胞外化學物種組成
★ 非金屬抗性細菌
★ 鎘攝取機制
★ 主動運輸
關鍵字(英) ★ Bioavailability
★ extracellular speciation
★ non-resistant bacteria
★ Cd uptake
★ active transport
論文目次 目錄
摘要 I
Abstract II
致謝 IV
目錄 V
圖目錄 VIII
表目錄 X
第一章 前言 1
1.1 研究動機 1
第二章 文獻回顧 5
2.1 FIAM 自由活性離子模型 (Free-Ion Activity Model) 5
2.2鎘金屬生物有效性 7
2.3銅金屬生物有效性 8
2.4鋅金屬生物有效性 9
2.5鉛金屬生物有效性 11
2.6鎘細胞毒性 13
2.7鎘的循環與環境 14
第三章 材料與方法 17
3.1實驗藥品與儀器 17
3.1.1實驗用藥品 17
3.1.2實驗用儀器 18
3.2 化學物種組成模擬軟體 18
3.3實驗用模式生物 19
3.4試驗培養液的成分及製備 19
3.5 E. coli K-12 及 Bacillus subtilis 168 實驗菌液準備 27
3.6微生物毒性試驗方法 28
3.6.1 平板計數法 31
3.6.2 光學密度生長曲線法 31
3.6.3 Live/Dead 微生物存活率試驗 32
3.7測量胞內外鎘含量之細胞洗滌試驗 34
第四章 結果與討論 36
4.1鎘物種於試驗培養液之組成模擬 36
4.2 暴露實驗前期準備 39
4.2.1菌株於試驗培養液之生長曲線 39
4.2.2鎘於試驗培養液對菌株之最低生長抑制濃度 39
4.3 Live/Dead kit測試 42
4.4鎘物種組成對細菌的毒性影響探討 46
4.4.1不同氯離子濃度下 E.coli K12 毒性試驗結果探討 46
4.4.2 不同氯離子濃度下 Bacillus subtilis 毒性試驗結果探討 51
4.5 E. coli 對鎘的攝取方式 54
4.6 測量進入細胞中的鎘之探討 59
4.7 環境意義 60
第五章 結論與建議 62
5.1 結論 62
5.2 建議 63
參考文獻 64
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指導教授 林居慶(Chu-Ching Lin) 審核日期 2018-8-9
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