應用電活性微生物於微流道微生物燃料電池監測水中重金屬毒性之探討

Abstract

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.