在進料各種不同廚餘來源的間歇式連續攪拌反應器中，發現到體積產氫速率（rH2）與氫氣產量（YH2）分別大約是2 L-H2 L-1 d-1 與2 mmol-H2 g-COD-1。經過廚餘成份分析與反應槽操作結果，乳酸很容易因廚餘不當貯放即形成，或是在反應槽中經由Lactobacillus sp. （由分子生物檢測得知）的作用而形成高濃度的乳酸（6~12 g L-1）。因此本研究的目的為建構一微生物燃料電池，以達成產氫出流中高濃度有機酸再利用，提昇總能源回收效率。本論文分別研究Shewanella decolorationis NTOU1於膽紅素氧化酶於陽／陰極上的反應機制，期能提供一些深入的資訊以利未來的發展。
第四章的研究在探討S. decolorationis NTOU 產電的最佳條件。在經過72小時，使用0.1 cm厚之碳布電極的電化學反應器中，發現初始pH = 7以及30 oC是產電的最佳條件，但根據電子平衡的計算，最佳的庫侖效率（R）則在37 oC之下（細胞生長較差）中發現。此外，與乳酸相比，丙酮酸更易於被分解產生電流，但醋酸則較不易。這些結果說明了為何在諸多反應條件下，不常觀測到丙酮酸的累積，反之醋酸為最主要殘留的副產物。在S. decolorationis NTOU1 與S. putrefaciens ATCC8071的比較實驗之中發現，使用1.4 cm厚的碳布電極時，S. decolorationis NTOU1擁有更優越的產電能力與適應力，可視為一有潛力的產電菌種。
在第五章中，為了釐清何者為氧氣強化S. decolorationis NTOU1 產電量的重要因素，在此設計了一組實驗設計以做驗證（即：添加／不添加氧氣與添加／不添加氮源）。在六天的實驗過程中，所有的實驗都以定電位儀設定在+0.4 V（vs. Ag/AgCl），且添加35 mM的乳酸做為基質。不論有無添加氮源與否，較大的產電量皆發現在有氧的條件下。同時，尼古丁醯胺腺嘌呤二核苷酸（NADH）產生量（Q）被發現與產電量有顯著的正相關關係，但最高的R（18%）則在無添加氮源的厭氧條件下被發現。這些結果指出NADH的生成量是主導產電量增加的主要因素，但氧氣還原與細胞生長則會消耗部份的NADH電子當量而使得電子回收率降低。其他因素如細胞生長量及乳酸消耗率等等，皆不如NADH產生量的影響來得顯著。使用高效能液相層析儀分析，可以在好氧反應72 小時後的醱酵液之中發現一些氧化還原物質（如：0.5 uM的核黃素），但在厭氧反應下則沒有測得。然而，使用循環伏安法檢測，在兩條件下測得的氧化還原訊號是相近的。由此可推論氧氣可使得核黃素更容易分泌至胞外，但高濃度的胞外核黃素，並非增加產電量的最主要因素。更進一步地，從循環伏安圖的S型曲線可得知，可得知核黃素即使是在厭氧條件，皆粘著在細胞表面的狀況下，仍然能擔任稱職的電子傳遞媒角色。
　　在第六章之中，固定膽紅素氧化酶的實驗選用了聚乙二胺（PEI）做為固定劑。在製備程序中先用過碘酸將膽紅素氧化酶的表面的醣基氧化產生醛基，再使醛基與聚乙二胺的一級胺以席夫鹼的型式鍵結，最後將修飾後的酵素（BOD×PEI）以物理吸附方式固定在電極表面。此修飾後的電極在飽和溶氧，且添加0.2 mM的2,2'-聯氮基-雙-(3-乙基苯并噻唑啉-6-磺酸）（ABTS）的條件下，可產生90 mA cm-2的氧氣還原電流。在無氧的情形下，由循環伏安法觀之，可發現ABTS會因電荷相吸作用被吸附在電極表面，且在0.4及0.6 V（vs. Ag/AgCl）的電位區間發現兩對氧化還原反應訊號。這結果顯示ABTS被吸附在電極表面時並不安定。與使用戊二醛進行交聯法相比較，發現BOD×PEI法可以在添加較低濃度的ABTS時，即達到最大電流值。此結果佐證了因陽離子吸附作用使然，ABTS被濃縮在電極表面的現象。BOD×PEI亦施用在由碳布電極與碳素粒子Vulcan VC-72R構成的多孔性電極上。在飽和溶氧且輔以1500 rpm攪拌之下，最大電流值可達到1 mA cm-2。

As the results of operating intermittent-continuous-stirring-tank reactors (I-CSTR) fed with various sources of kitchen waste, the volumetric hydrogen production rate (rH2) and hydrogen yields (YH2) were found to be ca. 2 L-H2 L-1 d-1 and 2 mmol-H2 g-COD-1, respectively. As the results of kitchen-waste components and reactor performance, lactate might be readily accumulated to a high level (6-12 g L-1) due to inadequate storage of kitchen waste, or produced via Lactobacillus sp. (evidenced by molecular methods) existing in the fermentors. Therefore, the objective of this study was to develop a microbial fuel cell to reutilize high-strength organic acids, especially lactate, in the effluent of hydrogen fermentation. The electron-transfer mechanisms of two biocatalyst, Shewanella decolorationis NTOU1 and bilirubin oxidase (BOD), will be studied in this thesis to provide insight information in favor of future development.
In Chapter 4, the experiments were to evaluate the optimal conditions for S. decolorationis NTOU1 for electricity production using lactate as a substrate. In experiments using 0.1-cm-thick carbon felt, an initial pH of 7 and 30 oC yielded the greatest charge production (486 C) within 72 h, but the highest Coulombic efficiency (19%) was obtained at 37 oC, due to lower biomass growth, as determined from electron balance calculations. In addition, pyruvate was more readily degraded by S. decolorationis NTOU1 than was lactate; acetate was the most difficult to degrade. This result illustrated the acetate will be accumulated during the bio-anode process inoculated with Shewanella sp. In experiments using 1.4-cm-think carbon felt, S. decolorationis NTOU1, which is a potential strain for further applications, was found to have better adaptability and stronger charge production than S. putrefaciens ATCC8071.
In Chapter 5, to clarify the major factor which caused oxygen-enhancing charge production of Shewanella decolorationis NTOU1 towards a polarized anode, a series of experimental runs (i.e., with/without ambient air flushing and ammonia addition as nitrogen source) was conducted in this study. Within six-day operation at +0.4 V vs. Ag/AgCl and starting with 35 mM of lactate, consistently the electrical charge production under the aerobic condition was higher than that under the anaerobic condition. In all the experimental runs, the values of nicotinamide adenine dinucleotide (NADH) production were found to be correlated positively and significantly with the charge production, but the highest Coulombic efficiency of 18% was observed under the anaerobic conditions without ammonia addition while the lowest charge production occurred. Those results indicate that NADH production enhanced by oxygen is the leading cause of the increase of the charge production, but the biomass production and the oxygen reduction would both consume NADH electrons and lead to lower electron recoveries. In addition, whether under constant aerobic or anaerobic, or alternating aerobic/anaerobic conditions, chronoamperometric results made it possible to rule out other factors, like lactate uptake rate or cell growth, which might increase the charge production under aerobic conditions. By using high performance liquid chromatography, some diffusive flavins (e.g., 0.5 uM of riboflavin) were found under the aerobic condition, but were not found under the anaerobic one. However, from results of cyclic voltammetry (CV), the signals of flavins were found to be approximately the same under both conditions. Although it is inferred that oxygen renders the flavins secreted extracellularly, that is not the major effect of oxygen for boosting the charge production. Furthermore, bound flavins under anaerobic condition were found to be effectively electrocatalytic according to sigmoidal CV result.
In Chapter 6, BOD was bonded with polyethyleneimine (PEI) by oxidizing saccharide shells of BOD with periodate and forming a Schiff base with PEI (BOD×PEI), which physically adsorbed onto a glassy carbon electrode (GCE) surface. With 0.2 mM of 2,2’-azinobis (3-ethylbenzothiazolin-6-sulfonate) (ABTS) and saturated oxygen addition, the GCE/BOD×PEI exhibited a catalytic current of 90 mA cm-2. Under an oxygen-free condition, the ABTS was found to be absorbed in the BOD×PEI film, and two couples of redox signals were observed between 0.4 and 0.6 V (vs. Ag/AgCl), indicating that the ABTS2-/ABTS.- pair was unstable due to the anionic-charge difference. The concentration of ABTS at which the maximum current was obtained for the BOD×PEI method was lower than that for the glutaraldehyde-cross-linking method, indicating that the anion-absorption effect makes ABTS condense closed to electrode surface, which boosts the current density. The BOD×PEI method was applied to a porous electrode comprising carbon felt and attached Vulcan XC-72R, and a current density of ca. 1 mA cm-2 was obtained under a condition of saturated oxygen, 0.2 mM of ABTS, and a stirring rate of 1500 rpm.

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