臺灣博碩士論文加值系統

微生物燃料電池為一種再生能源，電池中具有電化學活性之微生物，能氧化廢水中的營養物質，達到處理污染物同時又產生電能之效果，因而此技術逐漸受到關注。除了作為再生能源之角色，縮小其規格製成微流道微生物燃料電池具有應用於生物傳感器的潛力，能透過微生物快速反應及產電之優勢，進行重金屬污染物之檢測。本研究以碳紙作為電活性微生物附著之載體（即本系統之電極），於陽極電極上培養指標性產電菌Shewanella oneidensis，Pseudomonas delhiensis及Pseudomonas citronellolis三株菌株，並以醋酸鈉合成廢水作為陽極液，於其中添加相異濃度之六價鉻及二價銅，注入微流道中觀測三株產電菌產生的電壓所受到的抑制程度，進而建立重金屬濃度與減少電壓之關聯，成功於不同濃度區間建構出能夠快篩重金屬污染物存在之感測系統。透過與其他常見之微生物毒性檢測方法，包括吸光值OD600、結晶紫染色、Microtox○R等三種方法詳細比較後，確立本研究方法之可行性。
結果顯示，六價鉻於0 mM－0.125 mM區間、二價銅於0.125 mM－1 mM區間，菌株能夠有效的反應其毒性，建立良好的重金屬濃度－抑制程度關係，R2可達0.99，三株菌株於六價鉻都能作為生物傳感器之反應媒介，而二價銅部分又以Pseudomoans delhiensis 有較佳之反應且有較廣之濃度區間。本研究所建立之微流道方法與吸光值OD600之重金屬毒性反應趨勢相似，皆能以類似微生物生長公式之自然對數型式表示，確立微流道作為微生物傳感器快篩水體重金屬污染物之潛力。

The microbial fuel cell (MFC) as one of green energy harvesting techniques can reach the goal of simultaneous pollutants removal and power generation through electroactive bacteria (EAB). In addition, with the advantages of shorter reaction time, microfluidic microbial fuel cell (mMFC) has been developed by combining small-scale MFC and laminar flow, and it has potential to be applied in biosensors to detect pollutants.
In this study, laminar-flow based mMFC is constructed to monitor environmental toxicity of heavy metal by electroactive microorganisms. Carbon paper is used as the anode electrode, with EAB such as Shewanella oneidensis, Pseudomonas delhiensis and Pseudomonas citronellolis cultivated on it. Synthetic wastewater with sodium acetate was the anolyte and different concentrations of Chromium and Copper were added for observing the relationship between extent of inhibition and heavy metal concentrations by measuring open circuit and closed circuit voltages of mMFC. Consequently, we successfully constructed a fast-screening system for heavy metals in different concentration ranges. Common heavy-metal toxicity testing methods, including measurement of bacterial growth using absorbance at 600 nm wave length, crystal violet staining, and Microtox, were measured in this work for comparison and verifying the feasibility of our newly constructed system.
Results showed that around 0 mM－0.125 mM of Chromium and 0.125 mM－1.0 mM of Copper, accurate relationship was observed between the concentrations and the extent of inhibition of EAB, achieving R2 = 0.99. For Copper, Pseudomonas delhiensis had better performance and wider concentration ranges. In addition, after compared with other three methods, laminar-flow based mMFC constructed in this study was observed to perform similar trend with bacterial growth measurement at OD600. Both of the methods can be formulated as natural logarithm similar to bacterial growth curves. In conclusion, we confirmed the potential of laminar-flow based mMFC as biosensor for fast-screening of toxicity substance in wastewater.

目錄

論文口試委員審定書 i
致謝 ii
中文摘要 iv
Abstract v
第 一 章 緒論 1
1.1 研究背景 1
1.2 研究動機與目的 2
1.3 研究架構圖 4
第 二 章 文獻回顧 5
2.1 生物感測 5
2.1.1 生物傳感器及其演進 5
2.1.2 生物傳感器之應用 6
2.2 生物電化學之理論與發展 7
2.2.1 生物電化學系統之沿革 9
2.2.2 微生物燃料電池之產電原理 9
2.2.2 電子傳遞機制 13
2.3微型微生物燃料電池 16
2.3.1微型微生物燃料電池之發展及應用 16
2.3.2微型微生物燃料電池之影響因子 17
2.4 重金屬對微生物之影響 20
2.4.1 鉻的來源及對微生物之影響 20
2.4.2 銅的來源及對微生物之影響 22
第 三 章 材料與方法 25
3.1 實驗藥品與設備 25
3.1.1 實驗藥品 25
3.1.2 實驗設備與儀器 26
3.2 微流道微生物燃料電池系統 27
3.2.1 菌種來源與馴養 28
3.2.1.1菌液活化 29
3.2.1.2 菌種介紹 30
3.2.2 微流道微生物燃料電池組態 33
3.2.3 微流道微生物燃料電池運行 33
3.2.4 溶液層流條件與擴散層 35
3.3 微流道微生物燃料電池之重金屬毒性試驗 38
3.3.1 陽極菌微流道對重金屬毒性測試之運行 38
3.3.2 陰極菌微流道重金屬毒性測試 41
3.4 電極特性分析 44
3.4.1 循環伏安法 44
3.4.2 螢光顯微鏡 47
3.5吸光值重金屬毒性試驗 48
3.5.1吸光值測量細胞濃度 48
3.5.2 懸浮微生物之重金屬毒性測試 49
3.5.3 微生物膜重金屬毒性測試 49
3.6細菌冷光法毒性試驗 51
第 四 章 結果與討論 53
4.1重金屬毒性試驗之表現 53
4.1.1微流道之重金屬毒性試驗 53
4.1.2 吸光值重金屬毒性試驗 60
4.1.2.1 懸浮微生物之毒性試驗 60
4.1.2.2 結晶紫染色之微生物膜重金屬毒性試驗 63
4.1.3 細菌冷光法之重金屬毒性試驗 64
4.2 重金屬毒性試驗方法之比較 66
4.3 電極特性分析 74
4.3.1 循環伏安法 74
4.3.2 螢光顯微鏡 75
第 五 章 結論與建議 77
5.1 結論 77
5.2 建議 78
第 六 章 參考文獻 81

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