為了在維流體系統中發展一個可以取代菌膜且有效固定微生物的方法，本論文提出了以微載體包覆微生物的方式，將微生物濃縮在一特定位置或區域內，一方面可促進微生物生長，另一方面結合連續式操作，補充具活性的微生物，維持系統運作的效能。因此本研究中設計並製備了一微流體式微生物燃料電池，結合了微載體填充槽，來研究微載體與微生物燃料電池效能之間的關係。
本論文分成三個部分。由於微液滴系統是一個具有潛力可發展成量產製備微載體的平台，因此了解如何在此系統下準確控制液滴和微載體的生成是很重要的。所以首先論文的第一部分以實驗法研究高分子電解質(褐藻酸鈉)其液滴在不同無因次群(毛細係數、相對流量、相對黏度、深寬比)操控下的生成情形，並利用偏最小二乘法(PLS)回歸求得一經驗方程，用以預測褐藻酸鈉微液滴的尺寸。
接著在第二部分中研究了如何將高分子電解質液滴生成並聚合成微載體，且探討在高分子電解質液滴中添加顆粒狀溶質(PS微粒子、小球藻與大腸桿菌)對於高分子電解質溶液性質以及液滴/微載體生成的影響，最後將其應用於微生物培養。實驗結果顯示，除了介面張力和剪切力之外，溶液膠體穩定性與顆粒溶質的形態對於液滴生成也是很重要的。溶液膠體穩定性越好，有助於液滴生成之預測。若添加了顆粒溶質後增加了分散相與微流道壁的親和力，容易使得液滴生成模式從Dripping區域變換到Jetting區域。添加尺寸與形態越均勻的顆粒溶質，液滴生成與液滴大小幾乎與不添加顆粒狀溶質時狀態一樣，不受其影響。最後以PDDA為材料合成微載體後進行微生物培養測試，發現大腸桿菌包覆在PDDA的生長活性與沒有包覆、在懸浮溶液系統的情形一樣。然而小球藻被包覆在PDDA之後，因為此材料中的四級銨結構阻礙了小球藻的養分，因此無法在此材料中順利增長。
微型微生物燃料電池被視為極具潛力的平台，可用來篩選適合的微生物、碳源及許多操作參數，用以調整大型微生物燃料電池操作的最佳化條件。然而微型燃料電池的操作仍有一些困難，像是低功率輸出及低庫倫效率。因此在第三部分中，PDDA微載體被應用於空氣陰極微型微生物燃料電池中，來研究微載體對於微型燃料電池的影響。首先研究不同種類的純菌樣品(Shewanella oneidensis MR-1, Aeromonas hydrophila NIU01 and Proteus hauseri ZMd44)在此系統的效應。由於此材料的導電度低，造成包覆在微載體的三種純菌系統中的電荷阻抗都增加了。另外A. hydrophila NIU01 and P. hauseri ZMd44此兩種純菌在含有醋酸鈉為碳源的培養液中，生長狀態不佳，造成在微載體系統的功率隨著培養時間，分別降低了71%及45%。另一方面，S. oneidensis MR-1則是呈現旺盛的生長情形，且包覆在微載體後，微型微生物燃料電池的效能呈現相對較穩定的功率，大約維持在9.59 μW/m2。
接著，本研究以微型微生物燃料電池培養一混菌樣品來深入研究其效能。其中我們利用了顯微鏡(共軛焦顯微鏡、電子顯微鏡)、循環伏安法、交流阻抗分析儀以及連續式操作來檢測微載體的生物相容性、陽極的氧化還原提升之能力以及減少內電阻的能力等性質。對於混菌樣品而言，在包覆於微載體培養168小時後，其功率輸出高於菌膜系統250%，內電阻相較於菌膜系統降低了56%。即使其生長狀態與A. hydrophila NIU01或P. hauseri ZMd44類似，它的效能依然在微載體的協助下改善且優於其他兩種純菌的表現，對於在微生物燃料電池領域中，具有更實際的應用優勢。
最後，本研究提出一簡易的方式製備以高分子電解質為材料的微載體，且具有量產之優勢。此外，微載體與空氣陰極微型微生物燃料電池結合後，有潛力可用於快速篩選具產電活性的微生物，以及其他重要的操作參數像是有機物負載量和營養成分等。另外，成熟的微生物培養技術亦可適用於微載體系統，期望能取代原位菌膜，減少系統啟動的時間，並協助更換補充具活性的微生物來源。

To develop effective methods for retaining microorganisms in microscale devices without developing in-situ biofilm, microcarrier is proposed as a promising method in this study for concentrating functional microorganisms at desired locations and facilitating the establishment and replenishment of microorganism cells in continuous microfluidic bioprocess. Hence, a microfluidic-based microbial fuel cell (μMFC) with packed polyelectrolyte microcarriers is designed, fabricated and investigated with regard to the performance of MFC after associated with microcarriers.
This dissertation contains three parts. Since the droplet microfluidic system is the technique for mass production of microcarriers, it is critical to understand how to control the droplet/particle formation precisely in the droplet microfluidic system. Therefore, in the first part of this study, the generation of polyelectrolyte (alginate) micro-droplets is experimentally investigated by varying dimensionless groups (capillary number, relative flow rate, relative viscosity and aspect ratio). Then a reliable empirical model for predicting the size of alginate droplets is built by partial least square (PLS).
In the second part, this study experimentally investigates the generation of polyelectrolyte droplets, and subsequently, cured microcarriers for application in microorganism cultivation. This study examines the effects of particulate solutes (polystyrene microparticles, Chlorella vulgaris, and Escherichia coli) on polyelectrolyte solution properties (zeta potential, contact angle, and interfacial tension) and the droplet/microcarrier formation. The results indicated that, except for interfacial tension and shear stress, the colloidal stability and morphology of particulate solutes should also be considered when generating droplets. A particulate solute resulting in a dispersed phase with colloidal stability was beneficial for the predictable droplet generation. A particulate solute increasing the affinity between the disperse phase and the channel wall hastened the droplet generation to shift from the dripping region to the jetting region. Adding particulate solutes with consistent size and morphology into the dispersed phase was less likely to affect the droplet formation and the droplet size. The droplets containing microorganisms were cured to generate poly-DDA (PDDA) microcarriers, and E. coli cultivated in PDDA microcarriers had the same viability as those cultivated in suspension. However, C. vulgaris cultivated in PDDA microcarriers failed to proliferate possibly due to the blockage of the nutrient intake by the quaternary ammonium cation of DDA.
Micro-scale MFCs are considered promising platforms for screening suitable microorganisms, carbon sources, and many operational parameters for aiding the optimization of large-scale MFCs. However, micro-scale MFCs still have challenges such as low power output and Coulombic efficiency. In the third part, the application of PDDA microcarrier in an air-cathode μ-Liter Microbial Fuel Cell (μMFC) is demonstrated. The performance of the μMFC is investigated by using polyelectrolyte microcarriers encapsulating various pure culture microorganisms (Shewanella oneidensis MR-1, Aeromonas hydrophila NIU01 and Proteus hauseri ZMd44). Due to the low electric conductivity of this polyelectrolyte polymer, the resistance of charge transfer is increased after encapsulating three strains of pure culture exoelectrogens in microcarriers. For A. hydrophila NIU01 and P. hauseri ZMd44, since they were retarded in the medium containing sodium acetate as a carbon source, the growth state is relative weak and the power performance decreases to 71% and 45%, respectively, in the microcarrier system along the cultivation period. By contrast, S. oneidensis MR-1 shows a vigorous growth state in μMFCs and performs a relative stable power generation at around 9.59 μW/m2 after encapsulating in microcarriers.
The performance of mixed culture microorganisms in μMFCs is also further investigated. The microcarrier’s biocompatibility, ability in enhancing the redox activity of anode, and the capabilities in reducing internal resistance and sustained electricity generation were examined with a series of assays including microscopy (both confocal fluorescence and electronic), cyclic voltammetry, AC impedance analyzer and continuous operation in the air-cathode μMFC for 168 hours. For mixed culture microorganism, the polyelectrolyte microcarrier has proven to increase its power output to 250% and to reduce internal resistance by about 56% compared with biofilm during the 168-hour operation. Even though the growth state of mixed culture microorganisms is as similar as that of A. hydrophila NIU01 or P. hauseri ZMd44, its performance is improved and better than these two pure strains after encapsulating in microcarriers. Eventually, mixed communities show its predominance for more practical applications in the field of microbial fuel cell.
In conclusion, the fabrication process of the polyelectrolyte microcarrier is simple and has the potential to scale up into mass production. The proposed polyelectrolyte microcarrier and air-cathode μMFC have great potential not only in rapid screening of electrochemically active microorganisms but also other critical parameters such as organic loading rate and nutrient components. Additionally, microcarriers containing a mature microorganism culture can replace in situ biofilms in microfluidic bioprocesses to reduce the startup duration and facilitate the replenishment of functional microorganisms.

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