臺灣博碩士論文加值系統

　　土壤中存在有各式各樣的微生物，其在土壤化學、土壤肥力和作物生長上扮演非常重要的角色。傳統上常見之鑑別微生物的方法是透過選擇性培養基去篩選，較少見透過電化學快速、簡便的偵測方法來分析各菌種特性。因此，本論文研究目的要以較具有電化學活性的 Fe(CN)63- 及 Ru(NH3)63+ 當作探針化合物，利用六種代表性黏土礦物修飾三極式網版印刷電極 (TESPE)，來探討地衣芽孢桿菌 (革蘭氏陽性菌，G+)、釀酒酵母菌 (真菌) 及大腸桿菌 (革蘭氏陰性菌，G–) 菌體表面的電化學反應，並進一步搭配超音波震盪以瞭解不同細菌種類特性。試驗結果指出，PFl-1 與地衣芽孢桿菌混合液修飾電極對 Fe(CN)63-偵測到的氧化還原波峰電流量隨第一圈到第五圈逐漸下降，且氧化波峰形狀較為尖銳，由電極表面的掃描式電子顯微鏡圖可明顯看到地衣芽孢桿菌的形狀。SWy-1 與菌混合修飾電極對 Fe(CN)63- 偵測到的還原波峰形狀較為駝形，其還原波峰電流量隨掃描第一圈到第五圈呈現有規則性逐漸減少的現象。SWy-1 與地衣芽孢桿菌混合修飾電極對 Ru(NH3)63+ 測到的氧化還原波峰電流量比 SWy-1 修飾電極所測到的小很多。由黏粒修飾電極的電化學分析結果顯示以 Fe(CN)63- 作為探針分子較 Ru(NH3)63+ 能顯現地衣芽孢桿菌在電化學反應上的特色。
　　由酵母菌搭配六種黏土礦物對 Fe(CN)63- 偵測的五圈循環伏安圖與大腸桿菌的情形有所不同，以第五圈伏安圖之還原波峰電流量來看，酵母菌均略大於大腸桿菌，且由第一圈還原波峰電位與第五圈還原波峰電位相比較，酵母菌加六種黏土礦物對 Fe(CN)63- 測到的五圈還原波峰之電位大部分都往更負電位偏移，但大腸桿菌就未有此現象。六種黏土礦物加大腸桿菌修飾電極對 Fe(CN)63- 測到第五圈的還原波峰電位都接近0 V。由上結果顯示，以 Fe(CN)63- 作為探針對兩種菌電化學特性上的探討得知，兩種菌外層的電化學反應確實有所不同，且隨著不同黏土礦物的交互作用會呈現不同的電化學反應。
　　將以上三種菌進行超音波震盪處理，以期望獲得更好的訊號來分辨三種菌的特性。由掃描式電子顯微鏡圖發現黏粒與菌經超音波處理後分布得更為均勻，導致修飾膜層的孔隙度減少，因此降低了以 Ru(NH3)63+ 作為探針的波峰電流量，但以 Fe(CN)63- 作為探針所偵測到的還原波峰電流量之變化趨勢在不同菌種間有所不同。釀酒酵母菌對膨脹性黏土礦物 (SHCa-1、SWy-1和SWa-1) 有略為增加的情形，對非膨脹性黏土礦物 (KGa-1、PFl-1和VTx-1) 則無；大腸桿菌 (G–) 則因細胞壁構造較易遭超音波破壞，而使還原波峰電流量皆略為增加，其中以KGa-1增加的幅度最大，其次為VTx-1；地衣芽孢桿菌 (G+) 則無明顯的規律。因此，超音波處理搭配六種黏土礦物加菌修飾電極的電化學分析讓這三種菌膜層特性上的差異更能夠顯現出來，其中以PFl-1表現的差異性最大，證明此電化學分析方法在菌種鑑定上的可行性。

　　In soils, various microorganisms have an important role in soil chemistry, soil fertility and crop growth. The traditional identification of soil microbes use the selective culturing methods, while they are not rapid or convenient method for analyzing bacterial characteristics comparing with the electrochemical analysis. In this research, Fe(CN)63- and Ru(NH3)63+ were used as chemical probes to study the electrochemical reaction of surface membrane in Bacillus licheniformis (Gram-positive bacteria, G+), Saccharomyces cerevisiae (fungus) and Escherichia coli (Gram-negative bacteria, G–) with various clay-modified three-electrode screen-printed electrode (TESPE). Furthermore, the ultrasonication was used to understand various bacterial characteristics. The results indicated that the redox peak currents of Fe(CN)63- at PFl-1 added bacterium modified electrode were decreased from first to fifth cycle and the shape of oxidative peak were sharper than other modified electrodes. Field emission SEM clearly showed the rob form of B. licheniformis on the electrode modified by PFl-1 added bacterium. At the SWy-1 added bacterium modified electrode, the reductive peak currents of Fe(CN)63- were found to regularly decrease from first to fifth cycle. Whereas, the decrease was not apperent for Ru(NH3)63+ at the electrode modified by SWy-1 added bacterium. Overall, Fe(CN)63- appeared a favorable probe for the electrochemical reaction of B. licheniformis on clay-modified electrodes.
　　Five cyclic voltammograms of Fe(CN)63- detected by six clay minerals added S. cerevisiae were different from that of E. coli modified electrodes. The reductive peak currents of Fe(CN)63- at six clay added S. cerevisiae modified electrodes slightly larger than that of E. coli. To compare five reductive peak potential of S. cerevisiae might shift more negative potential, but E. coli were not. Fifth reductive peak potential of Fe(CN)63- tended to 0 V at six clay added E. coli modified electrodes. Fe(CN)63- is a good probe to investigate the different electrochemical properties of microbes due to the different electroanalysis of membrane character affected by the interaction of different clay minerals.
　　Ultrasonication was used and expected to detect a better signal and distinguish three kinds of microbes. SEM profiles indicated the distribution of mrcrobes and clay minerals thought the ultrasonic treatment. The reducing peak current of Ru(NH3)63+ was affected by reducing the porosity of the modified film. Fe(CN)63- probe could indicated the changeable trend of reductive peak current of different microbes. For S. cerevisiae, the trend of reductive peaks increased slightly in expanding clay minerals (SHCa-1, SWy-1 and SWa-1), but non-expanding clay minerals (KGa-1, PFl-1 and VTx-1) were not. For E. coli (G–), the reductive peaks current are apparently increased at all kinds of clay modified electrode, especially KGa-1 increased largest due to the change of membrane character treated by ultrasonic wave. However, that are no obvious rule observed in B. licheniformis (G+). Thus, the electrochemical analysis of ultrasonic wave treatment coupling with different clay minerals added microbes modified electrode that better emerging the characteristics of experimental microorganisms. The probe of Fe(CN)63- determined by PFl-1 added microbes modified electrodes could prove feasible method of electreanalysis to investigate experimented microbes.

致 謝 i
摘 要 ii
Abstract iv
目 錄 vii
表 次 ix
圖 次 x
壹、前 言 1
貳、前人研究 4
一、 黏土礦物的結構與特性 4
二、 黏粒修飾電極與電化學分析 9
(一) 電化學分析方法介紹 9
(二) 黏粒修飾電極之應用 15
三、 土壤微生物與黏土礦物的交互作用 18
四、 超音波化學在黏土礦物上的應用 22
參、材料與方法 25
一、藥品 25
二、儀器 29
三、藥品配製 30
四、黏粒與B. licheniformis修飾電極的電化學分析研究 31
(一) 修飾電極的製備 31
(二) 黏粒與B. licheniformis修飾電極的電化學分析 32
(三) 掃描式電子顯微鏡觀測不同修飾電極表面 32
五、黏粒與S. cerevisiae及E. coli修飾電極的電化學分析研究 33
(一) 修飾電極的製備 33
(二) 黏粒與S. cerevisiae及E. coli修飾電極的電化學分析 34
(三) 掃描式電子顯微鏡觀測不同修飾電極表面 34
六、超音波處理之菌液與黏粒修飾電極的電化學分析研究 35
(一) 修飾電極的製備 35
(二) 超音波處理之菌液與黏粒修飾電極的電化學分析 36
(三) 掃描式電子顯微鏡觀測不同修飾電極表面 36
肆、結果與討論 37
一、以黏粒修飾電極探討地衣芽孢桿菌的電化學反應 37
(一) 修飾電極對鐵氰化鉀的電化學分析 40
(二) 修飾電極對氯化釕六氨錯合物的電化學分析 52
二、以黏粒修飾電極探討釀酒酵母菌及大腸桿菌的電化學反應 55
(一) 黏粒加菌修飾電極對鐵氰化鉀的電化學分析 60
(二) 黏粒加菌修飾電極對氯化釕六氨錯合物的電化學分析 69
三、以超音波處理之菌液與黏粒修飾電極的電化學反應 74
(一) 超音波黏粒加菌修飾電極對鐵氰化鉀的電化學分析 82
(二) 超音波黏粒加菌修飾電極對氯化釕六氨錯合物的電化學分析 98
伍、結 論 110
陸、參考文獻 112
附錄、電極代號說明 126

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