Luận văn - Kha Lil

CAN THO UNIVERSITY COLLEGE OF NATURAL SCIENCES DINH KHA LIL STUDY ON THE EFFECT OF SUBSTRATE ON POWER GENERATION OF DUAL CHAMBERED MICROBIAL FUEL CELLS USING WASTEWATER AT LUODONG WASTEWATER TREATMENT PLANT, TAIWAN MASTER OF SCIENCE THESIS PROGRAM: THEORETICAL CHEMISTRY AND PHYSICAL CHEMISTRY 2021





CAN THO UNIVERSITY COLLEGE OF NATURAL SCIENCES DINH KHA LIL STUDY ON THE EFFECT OF SUBSTRATE ON POWER GENERATION OF DUAL CHAMBERED MICROBIAL FUEL CELLS USING WASTEWATER AT LUODONG WASTEWATER TREATMENT PLANT, TAIWAN MASTER OF SCIENCE THESIS PROGRAM: THEORETICAL CHEMISTRY AND PHYSICAL CHEMISTRY Advisors: Assoc. Prof. TRAN VAN MAN, Ph.D. DAI HUE NGAN, Ph.D. August 2021

Master of Science Thesis course 25 – 2021 Can Tho University ACKNOWLEDGMENTS This thesis is a fruit of the collaboration of many people who shared their time, enthusiasm, knowledge, skills, and friendship. Everyone proved essential and unique, and everyone was so kind and helpful. For these reasons and more, I gratefully acknowledge you with the hope that this thesis will help develop environmentally friendly MFCs in the future. First of all, I would like to thank the lecturers in the College of Natural Sciences of Can Tho University for all the learnings they have imparted to me. I would like to express my utmost gratitude to my supervisor, Associate Professor Tran Van Man, for his endless guidance, support, motivation, encouragement, and patience during my M.Sc. studies. Thank you very much, Prof. Tran, for giving me this excellent opportunity. Under your supervision, I successfully surpassed many difficulties and learned a lot. You have given me enough freedom during my research to encourage me to become an independent thinker. You are a great supervisor. I would also like to express my sincere thanks to my co-supervisor, Dr. Dai Hue Ngan, for her valuable discussions, constructive suggestions, contributions, help, patience, and endless support. I express my deepest gratitude to all my co-authors who have shared the stressful times and supported me with their knowledge. A huge thanks to Professor Chin-Tsan Wang for the beautiful internship experience in the Thermofluid Bio-Energy Lab (TFBE Lab), National Ilan University (NIU), Taiwan. I would like to thank my labmate, Imee A. Saladaga, from the Eastern Visayas State University, Philippines. Her friendship and the time spent discussing and working together helped a lot in my research progress and gave me the confidence to trust my experiments. Finally, I would like to acknowledge the support from TFBE Lab, College of Engineering, NIU, and Applied Physical Chemistry Laboratory (APC Lab) VNUHCM-University of Science Vietnam. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences i

Master of Science Thesis course 25 – 2021 Can Tho University ABSTRACT For about two decades now, microbial fuel cells (MFCs) have been an emerging technology that has gained attention for its new wastewater treatment and energy generation, especially its ability to convert chemical energy from a broad range of substances into electricity. However, MFC has not been widely commercialized due to low efficiency. Studies have shown that substrate loading is an essential factor in scaling up. Therefore, this study investigates the effect of substrate type and concentration on honeycomb MFCs (HCMFCs). The effect of different concentrations ranging from 10 to 40 mM of lactate and acetate (1:1 ratio) substrates has been was investigated. Power efficiency was analyzed using polarization and power density curves. Results showed that the performance of MFCs and biofilm formation are affected by the substrates. Scanning electron microscopy showed some changes in biofilm formation. Mixing lactate and acetate at 30 mM gave the best performance with a power density of 956.75 mW/m2 and chemical oxygen demand removal of 87.8%. Furthermore, effective substrate degradation, having COD removal of 91.4%, was observed with acetate. Keywords: Acetate, biofilm, energy, lactate, microbial fuel cell, substrate, wastewater treatment Major in Theoretical chemistry and Physical chemistry College of Natural Sciences ii

Master of Science Thesis course 25 – 2021 Can Tho University DECLARATION I hereby declare that this thesis represents my own work which has been done and has not been previously included in a thesis or dissertation submitted to this or any other institution for a degree, diploma or other qualifications. Signature:____________ Date: August 2021 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences iii

Master of Science Thesis course 25 – 2021 Can Tho University CONTENTS ACKNOWLEDGMENTS ................................................................................i ABSTRACT......................................................................................................ii DECLARATION ............................................................................................iii LIST OF TABLE ............................................................................................vi LIST OF FIGURE .........................................................................................vii LIST OF ABBREVIATION ........................................................................viii Chapter 1: INTRODUCTION........................................................................1 1.1 REASONS FOR CHOOSING THE TOPIC .............................................1 1.2 RESEARCH SIGNIFICANCE ................................................................. 3 1.3 RESEARCH NOVELTY .......................................................................... 3 Chapter 2: LITERATURE REVIEW............................................................4 2.1 INTRODUCTION TO MICROBIAL FUEL CELL ................................... 4 2.2 MICROBIAL FUEL CELL DESIGNS ....................................................... 5 2.3 ANODE MATERIALS ............................................................................. 10 2.4 MEMBRANES EXCHANGE ................................................................... 14 2.5 CATHODE MATERIALS ........................................................................ 15 2.6 ELECTROACTIVE (EA) BIOFILMS: THE MICROBIAL ELECTROCATALYSTS OF BIOELECTROCHEMICAL SYSTEMS ........ 16 2.6.1 Direct electron transfer ........................................................................... 17 2.6.2 Mediated electron transfer ...................................................................... 17 2.6.3 Indirect electron transfer......................................................................... 17 Chapter 3: EXPERIMENTAL ..................................................................... 19 3.1. RESEARCH MATERIAL........................................................................ 19 3.1.1 Chemicals and equipment....................................................................... 19 3.1.1.1 Equipment ............................................................................................ 19 3.1.1.2 Chemicals and material ....................................................................... 19 3.1.2 Source of microorganisms and wastewater quality ................................ 19 3.1.3 Research time.......................................................................................... 19 3.1.4 Location of study .................................................................................... 20 3.2 EXPERIMENTAL DESIGN ..................................................................... 20 3.2.1 MFC system design ................................................................................ 20 3.2.2 Reynolds number (Re) ............................................................................ 21 3.2.3 Shear rate and flat plate boundary layer thickness (δ)............................ 21 3.2.4 MFCs inoculation and operation ............................................................ 23 3.3 RESEARCH METHODS .......................................................................... 23 3.3.1 Electrochemical analysis ........................................................................ 23 3.3.2 Chemical characterization ...................................................................... 24 3.3.3 Replica plating method ........................................................................... 25 3.3.4 Scanning electron microscopy analysis of electrode biofilm ................. 25 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences iv

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 4: RESULTS AND DISCUSSION ................................................ 27 4.1 EFFECTS OF SUBSTRATE PARAMETERS FOR PERFORMANCE ON MFC ............................................................................................................ 27 4.2 EFFECTS OF SUBSTRATE PARAMETERS FOR ANODIC BIOFILM GROWTH ........................................................................................................ 30 4.3 THE OVERSHOOT PHENOMENON ...................................................... 35 4.4 INTERNAL RESISTANCE OF MFC ....................................................... 36 4.5 SUBSTRATE DEGRADATION AND COULOMBIC RECOVERY ...... 38 Chapter 5: CONCLUSION AND SUGGESTIONS ................................... 42 5.1 CONCLUSION.......................................................................................... 42 5.2 SUGGESTIONS ........................................................................................ 42 REFERENCES............................................................................................... 43 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences v

Master of Science Thesis course 25 – 2021 Can Tho University LIST OF TABLE Table 4. 1 Internal resistance of MFC with respect to the substrate different ......38 Table 4. 2 The substrate consumption analysis by HPLC .....................................41 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences vi

Master of Science Thesis course 25 – 2021 Can Tho University LIST OF FIGURE Figure 2. 1 Diagram of the basic components of MFC [19]....................................4 Figure 2. 2 MFC types in studies: (A) device with a salt bridge (pointed by arrow) which is easily assembled [36]; (B) single-chamber, simple “tube” arrangement of air-cathode [37]; (C) stacked MFC, with one out of two ceramic supports removed [38] ...............................................................................................................7 Figure 2. 3 MFC operated continuously: (A) upward flowing, tubular type MFC with inner graphite bed anode and outer cathode [43]; (B) flat plate design where a serpentine pattern for fluid flow [45]......................................................................9 Figure 2. 4 Extracellular electron transfer (EET) mechanisms [96]......................16 Figure 3. 1 Perspective view of recirculation dual chambers HCMFC ................21 Figure 3. 2 The schematic diagram of hydrodynamic boundary layer in the MFC [16].............................................................................................................................22 Figure 4. 1 Polarization curves (A) different substrate types at 10 mM (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations...................27 Figure 4. 2 Power density curves (A) different substrate types at 10mM, (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations...................28 Figure 4. 3 Voltage output of the MFCs during bacteria acclimation using a 1000- Ω external resistance.................................................................................................31 Figure 4. 4 SEM images of anodes with electroactive bacteria biofilm grown with different substrates at 10 mM concentrations: (A) acetate, (B) lactate, and (C) acetate and lactate .....................................................................................................33 Figure 4. 5 SEM images of anodes with electroactive bacteria biofilm grown with different substrate concentrations: (A) acetate and lactate at 20 mM, (B) acetate and lactate at 30 mM, and (C) acetate and lactate at 40 mM concentration .........34 Figure 4. 6 The equivalent circuit of MFC..............................................................36 Figure 4. 7 Nyquist plots of MFCs with with EC-Lab software ...........................37 Figure 4. 8 COD removal efficiency for MFCs using (A) different substrate types at 10mM and (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations ...........................................................................................................39 Major in Theoretical chemistry and Physical chemistry College of Natural Sciences vii

Master of Science Thesis course 25 – 2021 Can Tho University LIST OF ABBREVIATION Abbreviations Full terms AEM Anion Exchange Membrane CEM Cation Exchange Membrane COD Chemical Oxigen Demand DET Direct electron transfer EA Electrochemically active EAB Electrochemically active bacteria EIS Electrochemical impedance spectroscopy EET Extracellular electron transfer GAC Granular activated carbon HPLC High-performance liquid chromatography HCMFC Honeycomb Microbial Fuel Cells MEC Microbial Electrolysis Cell MFC Microbial Fuel Cells PEM Proton exchange membrane SEM Scanning Electron Microscope Major in Theoretical chemistry and Physical chemistry College of Natural Sciences viii

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 1: INTRODUCTION 1.1 REASONS FOR CHOOSING THE TOPIC Fossil fuels are non-renewable energy sources – their use is at a much higher rate than their creation. The consumption of fossil fuels worsens environmental concerns, which urges the world to use renewable energy sources. MFC technology has shown promising results in wastewater treatment and energy recovery applications [1]. Studies have shown that in MFCs, the extracellular microbes have an essential role in the electron transfer outside the cell. Exoelectrogenic bacteria in the anode compartment oxidize the substrate, converting the chemical energy in the organic compounds to electrical power [2]. In contrast to traditional fuel cells, MFCs can function using a wide range of operating conditions and many types of substrates, making the technology versatile [3]. The microorganisms in electrically active biofilms are the main actors in any biochemical reactor. They act as mediators in the essential processes, which are complex electrochemical and catalytic reactions. The electrochemically active bacteria (EAB) grow and develop on an anode electrode, oxidize organic matter, and transfer electrons to the anode by several mechanisms [4]. The microbial community that develops in an MFC anode chamber is naturally diverse. They are composed of the common electrochemical active bacteria such as Geobacter or Shewanella and other species [5]. However, microorganisms are susceptible to changes in their environment. Small fluctuations in the environmental factors cause biomes to change structure and diversity, which affects the MFC performance [6]. A new approach is to use a community of indigenous microorganisms that grows and thrives in the wastewater to be treated. Many studies have analyzed the diversity of microbial communities in wastewater. Lei Zhang et al., 2019, examined the microbiological community structure of activated sludge in the urban wastewater treatment system (Zhuzhou City, China). Results showed that Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, Actinobacteria, and Firmicutes were the most dominant phyla of the five activated sludge samples [7]. Yang et al., 2011, reported Proteobacteria, Bacteroidetes, and Firmicutes as the most significant phylum clusters in wastewater treatment sludge [8]. Many bacterial species have been detected in wastewater. These are Salmonella, Shigella, Escherichia coli, Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 1

Master of Science Thesis course 25 – 2021 Can Tho University Aeromonas, Legionella, pneumonia, Leptospira, Achromobacter, Bacillus, Enterococcus, Erwinia, Pseudomonas, Shigella, Staphylococcus, Streptococcus, Burkholderia, Shewanella, Enterobacter cloacae, Enterococcus, Klebsiella Proteus vulgarrison, and Shigellagenera [9, 10]. Most of them are found to be EAB and were applied in MFC studies. The use of diverse existing indigenous microorganism cultures is a practical approach. Furthermore, the substrate is one of the most critical factors affecting electricity generation in MFCs because it serves as the nutrient and energy source essential for the microorganisms’ growth. The substrate affects the bacterial diversity in the anode biofilm of MFC [11]. Recent studies have mainly focused on available carbon sources, such as bamboo fermentation effluent [12], pretreated latex wastewater [13], and seafood industrial wastewater [14]. A fermentation step is required to break these complex molecular substrates into simpler molecules before the anode bacteria can consume them to generate electricity. Lactate and acetate are well-known fermentation products that are simple forms of carbon easily used by anodic bacteria. Therefore, in this study, acetates and lactates were applied to examine the effect of substrate type and concentration on the biofilm formation on anode electrode in honeycomb MFC (HCMFC) by culturing a mixture of bacteria available in domestic wastewater treatment plants. The combination of native bacteria available in real wastewater with the application of continuous flow brings MFC technology closer to practical application by operating it using existing conditions in wastewater treatment plants. The HCMFC design is very beneficial in maintaining a uniform, symmetrical, and homogeneous flow of fluids inside the reactor. This design affects the mass transfer of the substrate and ions and ultimately improves the efficiency of MFCs. Until now, the use of honeycomb MFCs as a flow rectifier is relatively new. The studies that have been published include the analysis of MFC performance and its relationship with flow properties and biofilm formation. The reactors achieved their maximum voltage and current density with reduced internal resistance when the optimal flow rate was applied to the reactors at 40 mL/min [15]. The hydrodynamic boundary layer's influence on the biofilm formation and the mass transfer of the substrate in MFCs were observed. The results show that a thin hydrodynamic boundary with a boundary layer thickness of 1.6 cm creates a high voltage output [16]. Furthermore, the effect of the distance of electrodes on the performance of HCMFCs has also been studied [17]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 2

Master of Science Thesis course 25 – 2021 Can Tho University 1.2 RESEARCH SIGNIFICANCE (1) It presented an innovative attempt to investigate various substrate types and concentrations in microbial fuel cell technology, identify suitable substrate type and concentration, and determine its efficiency in generating power. (2) It has shown the feasibility of using domestic wastewater with mixed microbiological culture in MFCs with recirculation to generate electricity and treat wastewater simultaneously. (3) Electrochemical analysis, quantitative analysis and scanning electron microscopy techniques were performed to present and prove the research results. 1.3 RESEARCH NOVELTY (1) This study presented the effect of substrates on the formation of biofilm and the performance of MFC. The following results were found out: a substrate concentration that is too high decreases the MFC performance significantly; the substrate greatly influences the formation of biofilms; and, a mixed microbial community is favorable for the robustness of the MFC. (2) This study evaluated electrochemical factors, substrate consumption, and thickness of cultivated biofilms to emphasize the effects of different substrate types and concentrations on the MFC performance and efficiency. (3) The results of this study can be applied to continuous MFC systems using similar wastewater sources. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 3

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 2: LITERATURE REVIEW 2.1 INTRODUCTION TO MICROBIAL FUEL CELL Microbial fuel cells use bacteria to catalyze inorganic and organic matter oxidation and generate current [18]. The actions of bacteria result in electron production from these substrates transferred from the negative anode to the positive cathode through a conductive material and a load or resistor (Figure 2. 1). Figure 2. 1 Diagram of the basic components of MFC [19] The substrate of the system must be replenished in a continuous or intermittent manner; otherwise, it is categorized as a bio-battery. Electron mediators or shuttles transfer electrons to the anode [20] through electron transfer directly associated with the membrane, or through nanowires formed by the bacteria [21], or possibly through some other unexplored ways. Chemical mediators, such as neutral red or anthraquinone-2,6-disulfonate can be used in the MFC to facilitate electricity generation by bacteria that cannot use the electrode without aid [22]. Suppose the system has no added exogenous mediators. In that case, the MFC is considered as “mediator-less” even if the electron transfer mechanism may be unknown [23]. Oxygen from the air combines protons from the anode that diffused across a separator with most electrodes that reach the cathode and produce water in the process [22]. Ferricyanide or Mn (IV) are among the chemicals used as oxidizers. Still, it has the disadvantage of needing replenishment or regeneration [20]. Bacteria can help catalyze metal ions’ reoxidation using Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 4

Master of Science Thesis course 25 – 2021 Can Tho University dissolved oxygen, such as the reduction of Mn (IV) to Mn (II) [24]. MFC characteristics are defined by the sustainable catalysis of microbes on the anodic electron production and subsequent using up of electrons at the cathode. Hence, an Mg alloy slab used as a sacrificial anode makes the system unqualified to be categorized as an MFC since bacteria are inessential in the fuel’s catalyzed oxidation. Furthermore, systems that use enzymes or other catalysts that are not directly produced by bacteria sustainably are not considered MFC. Instead, these are categorized as enzymatic biofuel cells, and several papers review these extensively [25]. Operating MFCs employing a mixed culture of bacteria has substantially produced power densities than those operating with pure cultures [20]. In one study, though, an MFC generated high power employing pure bacterial culture. However, a test on the same MFC, which uses acclimated mix cultures, was not performed. Furthermore, the cells were grown outside of the MFC [26]. Examinations conducted on existing MFC microorganism communities have shown that these are incredibly diverse in nature [27]. Research is increasing on MFCs constructed with various materials and configurations. The operating conditions also differ in temperature, pH, electron acceptor, operative time, surface areas of electrodes, and reactor size. Studies report potentials using different reference states and occasionally only using a single resistor (load). These and sometimes the lack of essential data such as resistance that is internal to the system, or power densities obtained from polarization curves using differing methods, has created a challenge in interpreting and comparing results among studies [28]. 2.2 MICROBIAL FUEL CELL DESIGNS Currently, many types of MFC designs exist that are researched and developed. Each design has its own advantages and disadvantages and is suitable for specific uses. Various factors are considered in designing MFCs. The size, shape, and configuration of reactors widely differ and are wholly decided upon by the designer. There is no existing recommended standard design yet. MFC overall performance is significantly affected by reactor configurations, including the volume, oxygen supply, area of the membrane, and spacing between electrodes. Among the studied structures, double chamber H-type MFC is typically used because of its ion exchange membrane, facilitating proton diffusion and limiting the crossover of substrate and oxygen [29]. It is up to the designer to decide the aims of the project and plan the design accordingly. Presently, the available reactor designs are horseshoe- Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 5

Master of Science Thesis course 25 – 2021 Can Tho University shaped, cylindrical, cubed, dual- and single-chamber, and H-type. Some are made of glass, while others are made up of a variety of plastic. Also, sizes range widely, having reactor volumes of a few square millimeters and others of up to a square meter with volumes ranging from microliters to thousands of liters. The fuel cell design is an essential element in the MFC/microbial electrolysis cell (MEC). From a two-chamber design, single-chamber cells have been created to eliminate the membrane [30]. Furthermore, single-chamber MFCs have shown promising results; however, dual-chamber is still widely studied. Dual-chamber cells are easier to construct than single-chamber reactors. A simple MFC device can either be dual- or single-chambered, based on the anode and cathode chambers assembly. Several adaptations of MFC design and structure have been made from these two common designs [31]. H-shaped fuel cells, which usually consist of two bottles connected by a tube containing a separator, usually a cation exchange membrane (CEM) such as Nafion [22] or Ultrex [32], or a plain salt bridge [33] (Figure 2. 2A). The vital consideration to this design is choosing a membrane that allows proton transfer between the chambers but hinders the substrate or electron acceptor (usually oxygen) in the cathode chamber from crossing. Using a glass tube heated and bent to a U-shape is a cost-effective method to connect the bottles. Agar and salt are used as a CEM in the U-shaped glass tube and are inserted through the bottles’ lids (Figure 2. 2A). However, it was observed that MFCs using salt bridge generates low power because of high internal resistance. H-shaped MFC devices are generally accepted for basic parameter research. An example is testing the power produced using new materials or new microbes developing from some compound decomposition. However, this MFC type typically generates low power densities. The relative surface area of the cathode to that of the anode [34] and the membrane surface [35] affect the power generation. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 6

Master of Science Thesis course 25 – 2021 Can Tho University A BC Figure 2. 2 MFC types in studies: (A) device with a salt bridge (pointed by the arrow) which is easily assembled [36]; (B) single-chamber, simple “tube” arrangement of air-cathode [37]; (C) stacked MFC, with one out of two ceramic supports removed [38] Limitations on the power densities generated in these systems are typically due to the electrodes' considerable internal resistance and losses. Therefore, upon comparing the systems’ power production, it is logical to compare using anodes, cathodes, and membranes having the exact sizes [35]. Ferricyanide, the cathodic electron acceptor, improves power generation because of the high concentrations of electron acceptors. In an H-shaped reactor using Nafion as CEM, compared to a Pt-catalyst and dissolved oxygen in the cathode, ferricyanide increased the power produced by 1.5 to 1.8 times [35]. MFCs with the highest power densities and low internal resistances that have been published so far reported the use of ferricyanide at the cathode chamber [20]. Even though this chemical is excellent as a catholyte for system Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 7

Master of Science Thesis course 25 – 2021 Can Tho University performance, it is not a sustainable practice since it is not chemically regenerated. Therefore, there exist restrictions on the use of ferricyanide in basic laboratory research only. Several studies have also explored using cathode directly in contact with air (Figure 2. 2B, C), either with or without a membrane [39]. In one study, a separator based on kaolin clay and cathode made of graphite was connected to combine the separator and cathode structure [40]. MFCs using air-cathodes improved power densities significantly compared to MFCs with aqueous-cathodes. The most straightforward configuration involves placing the anode and cathode on either side of a tube, sealing the anode against a flat plate, and exposing the cathode to air on one side and water on the other (Figure 2. 2B). A membrane’s purpose in an air-cathode device is to prevent water leakage through the cathode. However, it also decreases oxygen diffusion into the anode chamber. The bacterial use of oxygen in the anode chamber can lower Coulombic efficiency, which is the fraction of electrons recovered over the maximum number to possibly be recovered [39]. Although hydrostatic pressure on the cathode will cause water leaks, this can be minimized using coatings, such as polytetrafluoroethylene (PTFE), on the outside of the cathode. These coatings allow the diffusion of oxygen but limit the bulk loss of water [41]. The systems mentioned so far are batch-operated devices. Several other basic designs also exist that provide flow continuously through the anode chamber. Some designs include an outer cylindrical reactor and a concentric inner cathode tube [42]. Some are the other way around having an internal cylindrical anode filled with granular media and outer cathode [43] (Figure 2. 3A). Another design variation is an upward flowing fixed-bed biofilm reactor, having flowing fluid continuously through permeable anodes on the way to a membrane that separates the anode from the cathode chamber [44]. System designs resembling hydrogen fuel cells have been employed, where a CEM is placed between the cathode and anode (Figure 2. 3B). Stacking systems as a series of flat plates or linking together in series can increase overall system voltage (Figure 2. 2C) [38]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 8

Master of Science Thesis course 25 – 2021 Can Tho University A B Figure 2. 3 MFC operated continuously: (A) upward flowing, tubular type MFC with inner graphite bed anode and outer cathode [43]; (B) flat plate design where a serpentine pattern for fluid flow [45] Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 9

Master of Science Thesis course 25 – 2021 Can Tho University Sediment MFCs have been developed. These are created by putting an electrode inside marine sediment abundant in sulfides and organic substances and the other electrode placed in the overlying oxic water. With these, electricity is produced sufficiently to provide power to some marine devices [46]. Graphite disks [22] and platinum mesh electrodes [46] have been used as electrodes. “Bottlebrush” cathodes have a high surface area and are corrosive-resistant. Therefore, these find applications for seawater batteries and are promising for long-term operation [47]. H-tube dual-chamber systems have also been applied to sediment MFCs to study bacterial communities [22]. Modifications have also been done to produce hydrogen. Using a slight external potential in the MFC, the potential produced at the anode by the bacteria was assisted, making cathodic hydrogen generation possible [48]. These are called bioelectrochemical assisted microbial reactors (BEAMRs) or bio-catalyzed electrolysis systems and are not considered as real fuel cells since they are operated to generate not electricity, but hydrogen. Having a second chamber for hydrogen gas capture would make it possible to develop various designs for hydrogen generation. 2.3 ANODE MATERIALS There are several specific characteristics that anode electrodes must possess for improved biofilm and material surface interaction. According to reviews, the most important are the surface area developed, electrical conductivity, biocompatibility, high mechanical strength, environmental friendliness, corrosion resistance, and low cost [49]. The electrode materials have a great influence on the performance of the MFC [50]. Carbon materials are used as electrodes in MFC because they are non-corrosive, highly biocompatible, and exhibit some distinctive surface characteristics of electrode materials. Modification of the electrode material has been shown to be an effective way to improve the performance of the MFC [51]. Examples of materials adopted as electrodes that fit all of the characteristics mentioned are carbonaceous and materials based on metals [52]. Carbonaceous materials, for example, carbon brush, carbon rod, carbon mesh, carbon veil, carbon cloth, carbon paper, carbon felt, granular activated carbon, granular graphite, carbonized cardboard, graphite plate, reticulated vitreous carbon, stainless steel scrubber, stainless steel plate, silver sheet, copper sheet, nickel sheet, titanium plate, and gold sheet, stainless steel mesh. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 10

Master of Science Thesis course 25 – 2021 Can Tho University Carbon brush Carbon fibers are twisted on a titanium core in carbon brush materials [53]. This makes the surface area reasonably significant and the ratio of area to volume optimal. Electrical conductivity can be high due to the titanium metal at the center, but this also increases the cost of the material. Carbon brushes are frequently applied as anode electrodes, and efforts in research aim to lower the overall costs of these [54]. Carbon rods Carbon rods are typically utilized to collect current, not as electrodes in the anode, because of their low surface area [55]. These are reasonably priced for MFC use. Carbon mesh Carbon mesh is among the commercially available carbonaceous materials, with relatively low cost and low electrical conductivity [56]. Its disadvantage is mainly its low mechanical strength, which causes low durability in conditions under high flow. Although its porosity is low, carbon mesh can be created into a 3-D electrode by folding it. Carbon veil It is a carbonaceous material that is highly inexpensive and has comparatively high electrical conductivity and porosity [57]. It is imperative since it allows bacteria to access and colonize all of the sites on the material available. Although a single layer of this material breaks easily, however, it is versatile. Therefore, it can produce robust and porous 3-D electrodes by folding [58]. Carbon cloth It is a type of carbonaceous material frequently utilized as an MFC anode material [59]. This provides high surface area, relative porosity, electrical conductivity, flexibility, and mechanical strength, forming complex 3D structures. However, this material is generally expensive [60]. Carbon paper It is a planar type of carbonaceous material that has relatively high porosity. However, it is fragile and expensive. It is mainly applied in lab-scale batch conditions [61]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 11

Master of Science Thesis course 25 – 2021 Can Tho University Carbon felt It is a type of carbonaceous material typically employed as an MFC anode. It is high in porosity and electrical conductivity. It allows bacteria to penetrate through its structure and form biofilms internally through its large pores. It is relatively cheap with high mechanical strength that depends on the material’s thickness [62]. Granular activated carbon (GAC) GAC is biocompatible and low cost, which makes it applicable to MFC anode applications [55]. It possesses high porosity and consequently low electrical conductivity. Because of this, it is typically used packing instead of as an anode that is stand-alone. To intensify conductivity, this material has to be packed. This leads to a possible problem of clogging for MFCs with continuous flow. The overall surface area is relatively high, but its available surface area for bacteria is low since most of the surface area is nanometric in size. Typically, to serve as a current collector, carbon rods are joined with GAC [55, 63]. Its high surface area can be used to adsorb organic pollutants and heavy metals, which can further purify wastewater. Granular graphite The granular graphite and GAC properties are similar except for the much smaller surface area of granular graphite because of the lack of activation. Consequently, granular graphite possesses electrical conductivity that is much higher [63]. It is also used in packing instead of stand-alone, just like GAC. Carbonized cardboard This material is high in electrical conductivity, high porosity, and very inexpensive. It is a 3-D material that consists of a single wall corrugated cardboard made from recycled paper from a flute layer inserted between two liner layers and underwent thermal treatment at 1000 ºC for one hour under an inert atmosphere. A rigid wall to which it is attached serves as its support [64]. Graphite plate (or sheet) This very simple electrode provides high electrical conductivity at relatively inexpensive. It has a small surface area and a surface-to-volume ratio. As a result, lower output levels are generated compared to structured or porous materials [65]. It has high mechanical strength, which explains its frequent use as support for modified structures. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 12

Master of Science Thesis course 25 – 2021 Can Tho University Reticulated vitreous carbon It has unique characteristics due to its high conductivity and high porosity, allowing the bacteria to develop biofilm penetrating through the entire electrode structure. However, it is not durable and very high in cost for MFC applications [44]. Other carbonaceous materials, which include electrospun carbon fibers [66], activated carbon nanofibers [67], and carbonized plant stems [68] have also been employed as an electrode in the anode. As Figure 1.4 suggests, several metallic materials have been applied as MFC anodic electrodes. These include stainless steel (in the forms of plate, mesh, foam, or scrubber) used due to its conductivity, robustness, and low-cost price [69]. In recent years, other metals such as copper, nickel, silver, gold, and titanium were also successfully applied as electrode materials in the anode [65, 70]. It has been found that copper and nickel ions, produced by the electrodes, have adverse effects on microbes, which harm biofilm formation. Yet, high and stable performance has been published [70]. Modifying the chemistry at the surface can produce better bacteria attachment, such as (a) positive surface charge typically favored [71]; (b) hydrophilicity/hydrophobicity, the former typically favored in the attachment of bacteria [61]; (c) functional groups containing O and N that enable bacteria- surface interaction [72] and (d) mediators that are immobilized [73]. Surface morphology and roughness controllable at the nano and microscopic levels can affect bacterial attachment [72]. Chemical and/or morphological surface areas may be affected by surface coatings [74], various chemical [56], thermal [75], and electrochemical treatments [76] have been studied as well. Mainly, alterations on the electrodes have increased power outputs. It has been observed that surface morphology is crucial for biofilm development and the MFC current generation. Studies have evolved from flat surfaces to three- dimensional electrode materials to increase the surface area that is available for bacteria to colonize and grow, as well as enhance the bacteria-electrode interface [77]. In theory, increasing surface area is expected to result in an increase in current generated. A study reported that although a carbon felt anode generated more significant current compared to a flat carbon graphite anode, the study did not correlate between the current and the actual surface area. One possible reason could be that the bacteria did not achieve complete colonization in all available regions. A more extended period may be needed to allow for further colonization [78]. Another study showed successful Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 13

Master of Science Thesis course 25 – 2021 Can Tho University biofilm formation on both 2-D carbon cloth and 3-D carbon felt and current generation. The study found that similar performance levels were seen even though the 3-D felt has a greater surface area. It was also noticed that the biofilm did not penetrate the whole electrode but remained only on the external surfaces [79]. Another challenge in complex 3-D surfaces is the limitations of the reactants’ and products’ diffusion transport phenomena and the pH gradients [80]. In the long-term operation, these factors have been shown to have a tremendous effect. Finally, clogging in the anode electrodes is an issue in designing the bioreactor and the overall system [77]. 2.4 MEMBRANES EXCHANGE In a dual-chamber design, the anode and the cathode compartments are separated by an ion-selective membrane, allowing proton transfer from the anode to the cathode and preventing oxygen diffusion in the anode chamber from the cathode compartment. The membrane in the MFCs plays an important role in MFC performance. There are several significant types of ion exchange membranes used in MFC systems: cation exchange membranes (CEM), anion exchange membranes (AEM), and polarized membranes (PBM) [81]. The most commonly used material for PEM affects the internal resistance and concentration of the polarisation loss of the MFCs system and influences the power output of the MFCs. The bioreactor architecture, material type, and reactor geometry determine the device's performance and cost. Studies aim to find the optimal combination of materials and design that will result in a high performance, low cost, and multiple functions to establish a standard for convenient and economically feasible scaling up. One such effort is the use of CEM such as Nafion that was derived from the existing technology on hydrogen PEMFC [82] or treatment systems on water using membranes [44]. Since Nafion is expensive, most of the research is concentrated on possible alternatives, including investigations on materials such as nylon and glass fibers, j-cloth, biodegradable plastic bags, and ceramics [82]. Several waste materials have also been unusually tested, such as laboratory gloves and natural rubber [83]. Results show that these materials offer benefits in terms of membrane fouling. The principles of MFC operation are on the use of similar metals in different solutions or different metals in similar solutions since liquid electrolytes are employed in the anode and cathode, as long as the cathode is Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 14

Master of Science Thesis course 25 – 2021 Can Tho University not exposed to open air. Ions are contained in the liquid solutions, which creates the need for an ion-exchange membrane. Therefore, in theory, a membrane is not a necessity for MFCs [39], given that the anode and cathode are either dissimilar (electrochemically separated) or identical but placed at a distance apart to avoid short-circuit. Membrane-less MFC was therefore born [84]. While this eliminates the need for high-cost membranes and fouling problems, the downside involves oxygen diffusion, which creates adverse competition with the anode on the available electrons. Most MFCs have been designed to have rigid, inert structural materials for housing anode and cathode half-cells, regardless of the presence of membranes. Currently, studies have emerged with the application of 3-D printing in the fabrication of MFCs [85]. 3-D printing of MFC bioreactors also has the benefit of creating complete reactors. As a result, these products can be applied to various applications and environments [86]. The challenge of electrode spacing can be addressed with material type. Porosity, strength, chemical inertness, and longevity are the factors that may address this challenge while hindering oxygen penetration. The materials that have been studied so far are the canvas [84], photocopy paper [87], microporous filtration membranes [88], and nylon-infused membrane [89]. The anolyte diffusion through the membrane to the cathode causes membrane fouling and inhibition of the proton's passage to the cathode. This decreases the power output of the MFCs. In addition, the passage of catholyte to the anode chamber affects the performance of the bacteria for electricity generation in MFC. Furthermore, the membrane increases the MFC’s internal resistance and the diffusion of ions or electrolytes, which affects the current generation of the MFCs. 2.5 CATHODE MATERIALS Ferricyanide (K3[Fe(CN)6]) has achieved popularity as an electron acceptor in lab-scale MFCs due to its good performance [40]. This is because the substance possesses a low overpotential when used with a plain carbon cathode, which results in a cathode working potential similar to its open circuit potential. The most significant setback is the inadequate reoxidation by oxygen because this creates the need for regular replacement of the catholyte [43]. Furthermore, adverse long-term effects may be observed when ferricyanide diffuses across the CEM to the anode chamber. The most well-suited electron acceptor for MFC applications is oxygen because of its availability, free oxidation potential, sustainability, and clean Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 15

Master of Science Thesis course 25 – 2021 Can Tho University waste product (water). The material used as cathode impacts performance tremendously and is chosen based on application. For instance, plain graphite disk electrodes have been applied in sediment fuel cells [43]. Because of the meager reduction rates of oxygen in plain carbon, overpotential is large, limiting its use to devices that can accept low performance. Studies show that the reduction of oxygen in carbon cathodes under seawater is supported by microbes [24]. Such processes have been seen in stainless steel cathodes in which the biofilm of bacteria quickly reduces oxygen [90]. To improve oxygen reduction kinetics, catalysts such as platinum are usually applied for dissolved oxygen [91] or gas diffusion (open-air) cathodes [92]. To lower MFC costs, the expensive Pt can be loaded as little as 0.1 mg cm–2 [93]. However, Pt stability should be studied in long-term applications. Alternative catalysts which are low-cost need to be explored. At present, studies on MFC cathode applications on catalysts without noble metals and are employed in pyrolyzing iron (II) phthalocyanine or CoTMPP [93] were suggested. 2.6 ELECTROACTIVE (EA) BIOFILMS: THE MICROBIAL ELECTROCATALYSTS OF BIOELECTROCHEMICAL SYSTEMS EAB has been identified in many natural ecosystems such as soils, sediments, seawater or freshwater and in samples collected from a wide range of different microbially-rich environments (sewage sludge, activated sludge, or industrial and domestic effluents). The microbes transfer the electrons to the electrode through various electron transfer mechanisms. However, the electron transfer mechanism plays a vital role in maximizing the microbe to electrode interaction and helps provide an understanding of how such systems operate in the MFC [94]. Researchers have proposed three kinds of extracellular electron transfer (EET) mechanisms [95] (Figure 2.4). Depending on the mechanisms involved, the distances of EET may vary greatly, from the nanometer-scale in the case of ET across the cell envelope to lengths exceeding one centimeter for cable bacteria [96]. Figure 2. 4 Extracellular electron transfer (EET) mechanisms [96] Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 16

Master of Science Thesis course 25 – 2021 Can Tho University 2.6.1 Direct electron transfer The first transfer mechanism uses direct electron transfer (DET) between electrons carriers in the bacteria and the solid electron acceptors [97]. The mechanism is carried out by the presence of outer membrane cytochromes that can interact directly with the solid surface to carry out respiration [98]. DET can occur through direct physical contact between the cell and an electrode without the involvement of any diffusable redox compounds. This is achieved if the microbe contains redox-active proteins on the outer surface of the cell membrane or cell envelope, e.g., cytochromes [99], flavoproteins [100], or multi-copper proteins [101], which allow transport of electrons between the inside of the cell and an external environment. According to studies reported so far, three different mechanisms accomplishing this type of electrical connection have been proposed [102]: (i) ET through electrically conductive pili [103], (ii) ET between redox proteins bound to the outer cell surfaces [104], or (iii) ET through abiotic conductive materials [105]. 2.6.2 Mediated electron transfer The second transfer mechanism employs an electron shuttle between bacteria and electrodes. The mediated electron transfer (MET) has redox mediators involved in the shuttling of electrons between bacteria and electrodes [98]. MET takes place by the presence of redox-active mediating compounds, which shuttle electrons between an external donor/acceptor and a microorganism. Some compounds shown to be effective electron shuttles including both inorganic and organic compounds have been identified potassium ferricyanide [106], flavin mononucleotide [107], neutral red [108], phenazines, phenoxazines, phenothiazines, and quinones [109], 9,10-anthraquinone-2,6-disulfonic acid disodium salt, safranine O, resazurin, methylene blue, and humic acids [110]. As multiple prior studies have proven that bacteria excrete various primary and secondary metabolites, which may be involved in EET as diffusible mediators [111]. Thus, these disadvantages lead to the general relinquishment of this approach. Because MET uses natural electron shuttles and DET mechanisms, it is generally established that artificial mediators are no longer significantly needed [25]. 2.6.3 Indirect electron transfer The third type of mechanism is indirect electron transfer (IET), which is based on the electrochemical synthesis of a wide range of microbial electron donors and acceptors. Here, the compounds used as Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 17

Master of Science Thesis course 25 – 2021 Can Tho University electron donors or acceptors undergo irreversible redox processes, creating new compounds such as hydrogen or formic acid. Additionally, electroactive (metabolic) substances can be secreted by microorganisms and transfer electrons between the microbes and electrodes [96]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 18

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 3: EXPERIMENTAL 3.1. RESEARCH MATERIAL 3.1.1 Chemicals and equipment 3.1.1.1 Equipment - Data acquisition system, JIEHAN 5000, JIEHAN – Taiwan. - COD Thermoreactor, Suntex – Taiwan - Handheld colorimeter, pHotoFlex STD – Germany. - Field Emission SEM- JEOL JSM-6500F – USA. - Multifunctional electrochemical analyzer, JIEHAN 5900, JIEHAN – Taiwan. - HIOKY 3522-50 LCR Hi-TESTER – Japan - Agilent 1200 HPLC system, Agilent Technologies – USA. - pH meter SP-701, Suntex – Taiwan. - Pump Head for High-Performance Precision Tubing, Masterflex L/S – USA. 3.1.1.2 Chemicals and material - LB broth, Miller (Luria-Bertani), Fisher – USA - Acetic acid analytical standard, Sigma Aldrich – Germany - Lactic acid analytical standard, Sigma Aldrich – Germany - Sodium phosphate monobasic (NaH2PO4), Nihon shiyaku reagent – Taiwan - Sodium acetate (CH3COONa), Nihon shiyaku reagent – Taiwan - Sodium lactate solution 50% (C3H5NaO3), Nihon shiyaku reagent – Taiwan - Gutaraldehyde, Nihon shiyaku reagent – Taiwan - COD Vials Kit, K-7360S, CHEMetrics – USA - Graphite felt – CeTech Co., Ltd., Taiwan - Nafion 117 – DuPont Co., USA 3.1.2 Source of microorganisms and wastewater quality Research was conducted with microorganisms in the treated wastewater sample at LouDong Wastewater Treatment Plant, Yilan, Taiwan. The wastewater had the following properties: COD: 273 mg/L, bacterial count in LB agar: 1.86 108 CFU/mL, pH: 7.42. 3.1.3 Research time This study was conducted from July 27th, 2019 to December 27th, 2020. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 19

Master of Science Thesis course 25 – 2021 Can Tho University 3.1.4 Location of study Experimental setup and monitoring of the development of biofilms were done at the Thermofluid Bio-Energy Lab (TFBE Lab), College of Engineering, National Ilan University (NIU), Taiwan. Biological morphological analysis of biofilms were done in the SEM Lab, College of Engineering, National Taiwan University Science Technology (NTUST), Taiwan. 3.2 EXPERIMENTAL DESIGN 3.2.1 MFC system design Dual-chambered reactors were assembled with polymethyl methacrylate (PMMA) sheets. The anode compartment with a total working volume of 1 L consisted of several chambers: A flow chamber (10 cm  10 cm  12 cm) in the middle of the reactor, one reservoir with a honeycomb structure (5 cm  5 cm  8 cm) on each side of the flow chamber and one surge tank (7 cm  5 cm  8 cm) next to each honeycomb reservoir (Figure 3.1). The flow chamber contains the anode electrode. The honeycomb structure was made up of equal-length plastic cylindrical tubes (5 cm) and radius (0.025 cm) arranged to create a uniform and straightened substrate flow. Another chamber with a working volume of 0.125 L (5 cm  5 cm  6 cm) attached to the anode flow chamber served as the cathode compartment. It is separated from the anode by a Nafion-117 PEM film (projected area of 5 cm  5 cm; DuPont Co., USA). To remove impurities, the PEM was soaked in a 3% H2O2 solution at 75 °C for 1 h, washed with deionized water, immersed in 0.5 M H2SO4 at 75 °C for 1 h, and rinsed with deionized water. The membranes were submerged in deionized water to preserve membrane conductivity by filling the anode and cathode compartments [16]. Graphite felts with titanium wire head (projected area of 5 cm  5 cm, CeTech Co., Ltd., Taiwan) were used as anode and cathode electrodes, with electrode spacing at 0.0 cm [17]. To enhance bacterial attachment, these were treated in 10% H2O2 solution at 90–100 °C for 3 h and air-dried for two days. A titanium wire was inserted into the electrode and connected to a 1000-Ω external resistance during cell operation [112]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 20

Master of Science Thesis course 25 – 2021 Can Tho University Figure 3. 1 Perspective view of recirculation dual chambers HCMFC 3.2.2 Reynolds number (Re) Reynolds number (Re) is the ratio of the inertial force to the viscous force. It is a dimensionless quantity used to probe different fluid flow situations in fluid mechanics. It is used to classify fluid systems in which viscosity is important in controlling a liquid's velocity or flow pattern. It is defined in Equation 3. 1 [113]: Re  VDT Equation 3. 1  where  represents the density of water (997 kg/m3); V indicates the inlet flow velocity (6.67×10–4 m/s, corresponding to the flow rate of 40 ml/min); DT represents the hydraulic diameter (10 cm) and  is the viscosity of water (8.9×10–4 kg/ms). 3.2.3 Shear rate and flat plate boundary layer thickness (δ) A boundary layer is a thin layer of viscous fluid close to the solid surface of a wall in contact with a moving stream in which (within its thickness δ) the flow velocity varies from zero at the wall (where the flow “sticks” to the wall because of its viscosity) up to Ue at the boundary, which approximately (within 1% error) corresponds to the free stream velocity (see Figure 3.2). Strictly speaking, the value of δ is arbitrary because the friction Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 21

Master of Science Thesis course 25 – 2021 Can Tho University force, depending on the molecular interaction between fluid and the solid body, decreases with the distance from the wall and becomes equal to zero at infinity. The thickness of flat plate boundary layer thickness defined by Equation 3. 2 [114]:  5 x Equation 3. 2 V where x is the electrodeposition of 9 cm. (Corresponding to the hydrodynamic boundary layer thickness of 5 cm), and the schematic diagram of the hydrodynamic boundary layer applied to MFC is shown in Figure 3. 2. The hydrodynamic boundary layer thicknesses were chosen based on the different flow fields. The hydrodynamic boundary of thickness 5 cm occurred at the hydrodynamically fully developed region. In this region, the velocity profile was fully developed and remained unchanged. Figure 3. 2 The schematic diagram of the hydrodynamic boundary layer in the MFC [16] The shear rate can influence the mass transfer and biofilm formation and is determined as shown in Equation 3. 3 [115]: G  V Equation 3. 3 d where V is the flow velocity at the hydrodynamic boundary layer (0.99 times less than inlet flow velocity) (m/s), and d represents the thickness of the hydrodynamic boundary layer (m). In this study, the applied flow was 40 mL/min, and the hydrodynamic boundary layer will be valid 5 cm. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 22

Master of Science Thesis course 25 – 2021 Can Tho University 3.2.4 MFCs inoculation and operation The anolyte was composed of a mixture of domestic wastewater taken from Lou Dong wastewater treatment plant, Yilan City, Taiwan, and 100 mM phosphate-buffered saline (PBS) solution in a 1:1 ratio. The wastewater had the following properties: COD: 273 mg/L, bacterial count in LB agar: 1.86 108 CFU/mL, pH: 7.42. PBS was composed of the following: Na2HPO4, 9.16 g/L; NaH2PO4·2H2O, 5.56 g/L; NH4Cl, 0.638 g/L; and KCl, 0.278 g/L with the pH value of 7.0 [112]. All MFCs were operated and tested at 25 °C with the anolyte continuously recirculated at 40 mL/min using a peristaltic pump (Masterflex, model no. 7524-50, Cole-Parmer Instrument Company, USA) [16]. The catholyte was a 1:1 mixture of 100 mM K3[Fe(CN)6] (32.924 g/L) and 100 mM PBS solution. Six MFCs having different substrates and concentrations were employed and labeled as follows: MFC1 – acetate, 10 mM; MFC2 – lactate, 10 mM; MFC3 – acetate and lactate, 10 mM; MFC4 – acetate and lactate, 20 mM; MFC5 – acetate and lactate, 30 mM; and, MFC6 – acetate and lactate, 40 mM. The anolyte and catholyte were replaced with new solutions at the end of each fed-batch cycle, indicated by a decline of production voltage to 10% of the maximum generated voltage. MFC operation was conducted for 30 days to ensure that steady voltage is achieved for at least three consecutive cycles before output measurements. 3.3 RESEARCH METHODS 3.3.1 Electrochemical analysis The current (I), unit: ampere (A) was calculated using Ohm’s law, I = U/R, where U represents the voltage output, unit: volts (V) and R is the external load resistance, unit: Ohm (Ω). Power (P), unit: watts (W) was calculated by charging the voltage and amperage: ������ = U × I. The power density was calculated as: ������ = ������ / Aanode, where Aanode is the total surface area of the anode electrode in square meters (m2). Electrical impedance spectroscopy (EIS) is a technique used in many applications, such as food product screening, corrosion monitoring, coating quality inspection, and cement sizing. It is also used in the characterization of solid electrolytes and human body analysis. It is also used to monitor battery and fuel cell performance, among many other applications [116, 117]. Electrochemical Impedance Spectroscopy (EIS) was done by applying a 10- mV AC signal using HIOKY 3522-50 LCR Hi-TESTER (Japan) within a frequency range of 100 kHz to 0.1 Hz. The signal was chosen to be small enough to prevent the biofilm from detaching from the electrode and minimize Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 23

Master of Science Thesis course 25 – 2021 Can Tho University the disturbance on the system’s stability [48]. An equivalent circuit was used to simulate EIS spectra and calculate internal resistance values in EC-Lab software. 3.3.2 Chemical characterization Chemical oxygen demand (COD) is defined as the mass of oxygen equivalent to the amount of dichromate consumed by suspended substances and dissolved in a water sample when the water sample is treated with that oxidant under specified conditions [118]. All the inorganic and organic components of the sample are oxidized. Still, in most cases, the organic composition dominates and is given more attention. If the individual or inorganic COD is to be measured, additional steps need to be taken to distinguish one from the other. COD test can determine the amount of organics contained in water. The decomposition time is determined, affecting the sample oxidation level, reagent strength, and sample COD concentration. COD is often used as a measurement of pollutants in wastewater. In this study, the COD removal capability analysis was performed using the TR-1100 COD thermal reactor (Suntex Instruments Co., Ltd., Taiwan). Anolyte liquid samples were taken at the beginning and end of each feeding cycle, usually taking 24 hours per cycle. The filtered 2-mL samples were taken and then added to the COD vial. These were heated to 150°C for 2 hours and cooled down for another 2 hours. Finally, the sample concentrations were measured using a spectrophotometer (pHotoFlex, WTW) [119]. The concentrations of the substrate were measured by Agilent 1200 HPLC system with a RI detector. The column flow rate is increased after the column has reached the operating temperature. First, the column was allowed to heat up while maintaining a slow flow rate (0.2 mL/min). Then the operating flow rate was increased once the column had reached the recommended operating temperature. The row was not allowed to speed up beyond the maximum specified speed for the column. The column was not allowed to work at the maximum row rate for extended periods. The standard flow rate for the Bio-Rad HPX-87 H column was used with sulfuric acid as the mobile phase at a flow rate of 0.6 mL/min [120]. The retention time of lactic and acetic acids is 12.1 min and 14.7 min, respectively. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 24

Master of Science Thesis course 25 – 2021 Can Tho University 3.3.3 Replica plating method Replica plating is the technique in which each colony/replica is inoculated into another plate according to a numbering scheme. This method allows each copy to be examined using a variety of methods while at the same time keeping a master plate from which the copies can be selected. It is a technique that enriches culture, clones isolates and colony-forming (viability) assays, start cultures for mutation isolation, and determines growth rate in liquid culture. In this study, the bacterial density in the wastewater was analyzed using 100 μ L samples on LB agar, and the plate was incubated at 37oC for 24 hours. Then proceed to hook and conclude the density of bacteria in the sample analyzed. 3.3.4 Scanning electron microscopy analysis of electrode biofilm Scanning electron microscopy (SEM) is widely used to inspect the surface texture quality of trace specimens using either the secondary electron or terrain mode. SEM images are characterized by high resolution (up to 10 nm) and considerable depth of field. SEM is widely used in materials science for research, quality control, and fault analysis of materials. In modern materials science, investigations of nanotubes and nanofibers, high- temperature superconductors, neutral architecture, and alloying strength are mainly based on the use of SEM for research and investigation. In this study, SEM is used for the morphology analysis of biofilm attached to the anode electrode. Treatment of the surface of a solid (dried) biological sample with minimal changes is essential. The preparation needs to be gentle enough so that “significant” structural detail, in the nanometer range, of the solid components can be studied by SEM. In the process of solvent evaporation from a liquid sample to a gaseous atmosphere, such as drying with air, the surface tension on the sample surface is enormous. It leads to shrinkage and collapse of structures cells so that no visible surface features are present. Interestingly, red blood cells have an extremely smooth surface and do not have a significant structure in the 10−3 – 10−6 m size range, making any SEM assessment over the 1–10 nm range impossible. During normal air drying of blood cell suspensions from water, the surface tension is large enough to flatten the white blood cural changes in the micrometer range after Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 25

Master of Science Thesis course 25 – 2021 Can Tho University drying with air. However, molecular characteristics in the 1-10 nm range are obscured by surface tension. To evaluate the examine biofilms on the anode electrode surface, the electrode (area ~ 0.5 cm2) was taken without touching its surface. Samples were fixed in 3% glutaraldehyde in either 0.1 M phosphate buffer (usually at a pH of around 7.0) 3% at 4oC for 2 hours. After fixation, the samples were dehydrated in aqueous ethanol using: 30%, 50%, 70%, 80%, 90% and 100% for for 3 x 20 minutes in each solution. Subsequent dehydration was naturally in biosafety cabinets before coating with platinum using a JEC-3000FC sputter coater [121]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 26

Master of Science Thesis course 25 – 2021 Can Tho University Chapter 4: RESULTS AND DISCUSSION 4.1 EFFECTS OF SUBSTRATE PARAMETERS FOR PERFORMANCE ON MFC The voltage from the MFCs was allowed to stabilize for three consecutive cycles, known as the acclimation phase, in which the biofilm in the anode matures. The polarization curves showing current density as a function of voltage are presented in Figure 4.1. The graph shows that the MFCs do not significantly differ in the maximum voltage generated, which is about 720 ± 20 mV. Figure 4. 1 Polarization curves (A) different substrate types at 10 mM (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 27

Master of Science Thesis course 25 – 2021 Can Tho University An in-depth analysis of reactor performance was done using the power density curves in Figure 4.2. The polarization and power curves determine the reactor’s performance under external load and show losses in the reactor (activation, ohmic, and concentration) [122]. Figure 4. 2 Power density curves (A) different substrate types at 10mM, (B) mixtures of acetate and lactate (1:1 ratio) at different concentrations Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 28

Master of Science Thesis course 25 – 2021 Can Tho University The power density was calculated from the current density data. The influence of substrate type can be seen from the results in Figure 4.2A. The graph shows a maximum power density of 554.9 mW/m2 and 593.4 mW/m2 produced from lactate and acetate, respectively. The maximum power density of 855.8 mW/m2 was achieved using the MFCs with a mixture of acetate and lactate at the same concentration (10 mM). These results show that using a mixture of the two has superior performance than using each substrate individually. This difference in performance is due to the microbial community’s structure and composition developed according to the substrate, as suggested by a previous study [123]. To investigate the effects of substrate concentration on power generation performance, four MFCs containing both acetate and lactate at a 1:1 ratio were operated batch-wise with recirculation and fed with substrates at concentrations of 10 mM, 20 mM, 30 mM, and 40 mM, respectively. It can be seen from Figure 4.2B that as substrate concentration was increased from 10 to 30 mM, power density increased because of the increased availability of organic supply. Power density increased and peaked at around 30 mM of 956.75 mW/m2. The decrease in power density at 40 mM suggests that a high substrate concentration harms microorganisms’ growth, which leads to a reduction in MFC performance. At high concentrations of substrate, the substrate inhibits microbial growth, which decreases its rate. This substrate inhibition may be competitive or non-competitive similar to enzyme kinetics. If the rate-limiting step during microbial growth is a single-substrate enzyme- catalyzed reaction, then the inhibition of the enzyme activity also prevents microbial growth in a similar pattern [124]. High power output observed at laminar velocities was due to the sufficient distribution of substrate in the anode leading to an effective transfer of organic supply to the bacteria. This leads to more electrochemical activity and, consequently, power production improvement. A laminar flow could eventually lead to mature and robust biology with maximum performance [125]. In Figure 4.2, the MFCs with mixed substrate have the least power loss and recovers more quickly in the power density curve. It means that the bacterial culture grown in both acetate and lactate is more resilient in load condition changes than those grown in a single substrate. Studies have shown that a mix of cultures is better than a single culture [126]. The result may indicate that the bacterial culture grown with the mixed substrate is not dominated by a single bacteria type but a rich culture of different kinds of bacteria. Mixed substrate leads to the growth of a mixed bacterial culture seen Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 29

Master of Science Thesis course 25 – 2021 Can Tho University in previous studies as more robust than pure culture [127]. It was observed that the higher the substrate concentration used was, the higher the power losses and the slower the MFC power output recovery, and the less robust the MFC was. 4.2 EFFECTS OF SUBSTRATE PARAMETERS FOR ANODIC BIOFILM GROWTH Voltage output was continuously monitored to investigate the growth of microorganisms and biofilm formation at the anode with Jiehan 5020 (Jiehan Technology Corporation, Taiwan), an automatic data logging system connected with MFCs and 1000-Ω external resistors at a sampling rate of 5 minutes per point. Figure 4.3 presents the voltage output over time for MFCs at each substrate type with a 1000-Ω external resistance. The results showed that at a substrate concentration of 10 mM (MFC1, MFC2, and MFC3), the mixture of acetate and lactate (MFC3) caused the best microorganism growth, as evident by higher output voltage values. Output voltage started to become stable after 7 days for 30 mM (MFC5) and 40 mM (MFC6) substrate concentrations. The other biofilms grown in lower substrate concentrations took more time to achieve stability around 10 days into the acclimation process. The current output depends only on the microbial community profile, the electrode materials, and the substrate type. The microbial communities identified in the various studies were very diverse [128]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 30

Master of Science Thesis course 25 – 2021 Can Tho University Figure 4. 3 Voltage output of the MFCs during bacteria acclimation using a 1000-Ω external resistance Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 31

Master of Science Thesis course 25 – 2021 Can Tho University Biofilm develops from the aggregation of a complex mass of microbial communities. The biofilm is formed in three phases: attachment on the electrode surface, maturation, and dispersion [129]. The microbe’s adhesive and protective matrix excretions attach to a solid substrate via self- immobilization growth. The biofilm is vital in the electrochemical process through its anodic reaction (oxidation). Furthermore, the electron transfer mechanism is favored in cell-to-cell contact in high-density biofilms [130]. The anode electrodes of the MFC must contain stable and consistent biology to generate boosted energy [131]. The factors affecting bacterial biofilms’ performance and stability are biofilm volume, roughness, cluster size, diffusion distance, and fractal dimension [132]. A scanning electron microscope (SEM) was used to analyze the anode electrode’s biological morphology in this study. After one month of stable MFC operations, the anode electrodes were removed from all six MFCs and washed using distilled water. The electrodes containing biofilm were cut using sterilized scissors and tweezers to the 0.5 cm2 sample pieces. Colonies were seen in patches among the graphite felt fibers. ‘Clumpy’ growth with rod-like bacteria was observed for biofilm using acetate substrate (Figure 4.4A). In contrast, a smooth biofilm was observed on the anode’s surface using a lactate substrate (Figure 4.4B). These only show that the type of substrate determines the dominant bacteria types in the mixed culture. A combination of smooth and clumpy growth was seen on the anode using a mixture of acetate and lactate substrates (Figure 4.4C), indicating that both acetate- and lactate-loving bacteria are present. The dominant group of microbial communities was dependent on substrate type [133]. The same mixed morphology was seen in MFCs with acetate and lactate at various substrate concentrations (Figure 4.5). A mixed morphology is expected to contribute to better MFC performance due to the presence of mixed bacterial culture, which has been observed from a previous study to enhance current by as much as six times compared to pure culture [134]. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 32

Master of Science Thesis course 25 – 2021 Can Tho University Figure 4. 4 SEM images of anodes with electroactive bacteria biofilm grown with different substrates at 10 mM concentrations: (A) acetate, (B) lactate, and (C) acetate and lactate Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 33

Master of Science Thesis course 25 – 2021 Can Tho University Figure 4. 5 SEM images of anodes with electroactive bacteria biofilm grown with different substrate concentrations: (A) acetate and lactate at 20 mM, (B) acetate and lactate at 30 mM, and (C) acetate and lactate at 40 mM concentration The electrical conductivity of biofilms plays an essential role in the electricity generation in MFC. These charge transfer losses in the anode indicate biofilm activity, which may be caused by the difference in EAB present and the anodophilic properties of the biofilm due to substrate fed, demonstrated by EIS analysis. The previous studies reported that the generated electrons move from bacteria/biofilm to an anode surface through the Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 34

Master of Science Thesis course 25 – 2021 Can Tho University following mechanisms: indirect/mediated electron transfer (MET), direct electron transfer (DET), and inter-spacial electron transfer [135]. During a direct electron transfer, the electrons must reach the cell's outer membrane, which should be physically in contact with the anode. Biofilms from electrical substances or conductive nanowires (pili/flagella) are formed on the anode's surface [136]. Das et al. reported bacterial pili on the anode electrode surface using the same wastewater as the bacteria source [129]. Microbial nanowires provide the ability to enhance the interaction between bacteria and electrodes significantly and play an essential role in the microorganisms' electronic interactions with inorganic electron acceptors in their environment and the electron exchange between species [137]. Electronic mediators penetrate bacterial cells during an indirect electron transfer, separate electrons from the electrolytes' metabolic reactions, and supply them to the MFC's anode [138]. The biodegradable substrate serves as the electron source in the MFC [139]. Many bacteria species in the domestic wastewater have been identified as capable of self-synthesizing intermediates such as E. coli [140] and Shewanella [141]. The mixed microorganism communities in wastewater are so complex that it is difficult to unravel how electrons transfer. However, it could be seen that both MET and DET mechanisms were present in the bacteria culture used in this research, and they have enhanced the performance of the MFC vs. using a single bacteria culture. 4.3 THE OVERSHOOT PHENOMENON Through specific experimental design and data analysis, it was found that extracellular electron transfer mainly caused resistance in the anode, which reflects a power overshoot in the polarization curve. Figure 4.2 observed that voltage and current drop drastically at high current densities on the polarization curves after the maximum power point (the curve retracts or ‘bends inwards’), resulting in lower power produced than previously measured at lower current densities. It leads to underestimating the best possible performance of reactors at higher current densities, and the reason for this spike remains a concern in many MFC studies. This phenomenon, although not ideal, is widely seen in literature and is termed “power overshoot” or “Type D overshoot” [142]. This power overshoot is seen in a study using a mixed bacterial culture [143]. Type D overshoots could be avoided with the long-term use of external resistances. This suggests the crucial changes that occur during acclimation that affect the biofilm’s ability to generate current. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 35

Master of Science Thesis course 25 – 2021 Can Tho University In Figure 4.2, the MFCs seem to recover their power generation after the power dips, even during the ongoing polarization sweep. The supply and demand of electrons and ions are balanced at this period, and the current rises again. This recovery illustrates the bacterial community’s robustness and ability to adapt to changes in conditions, even in hostile environments [142]. The internal resistance influenced the overshoot phenomenon in MFCs. A healthy biofilm can reduce internal resistance and eliminate any power overshoot. In contrast, the destruction or inhibition of the microbial community on the anode can cause increased internal resistance, which decreases the performance of the MFCs and causes an overshoot. Furthermore, the presence of this in the polarization curve may indicate possible pollutants or toxic elements [144]. 4.4 INTERNAL RESISTANCE OF MFC Biofilm morphology is affected by external resistance (Rext) and has an integral part in MFC efficiency. Developing a biofilm below the higher-than- optimal Rext has a detrimental effect on its properties and performance. The highest energy efficiency was recorded from MFCs that matured using 1-kΩ Rext and produced low internal resistance (Rint) [145]. Hence in this study, a 1- kΩ Rext was used in the acclimation. The external resistance value may also be related to the overshoot in concordance with the study of Hong et al. (2011) that shows MFCs acclimated to resistances from 500 to 5000 Ω produced polarization curves with power overshoot [143]. High internal resistance is the main problem that limits the MFC's power output. Therefore, efforts to understand should be made to improve MFC performance [146]. An equivalent circuit shown in Fig. 4.6 was used to analyze the MFCs and calculate their corresponding internal resistance values [16], Rf (Ω) and its circuit components (Table 3.1). Nyquist plots (Figure 4.7) were generated, and the circuit components were calculated based on these. Figure 4. 6 The equivalent circuit of MFC According to Figure 4.6, the ohmic loss in MFCs is represented by R1. Its value is the linear resistance to the flow of electrons through different electrode materials and bonds and the resistance to the flow of ions through Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 36

Master of Science Thesis course 25 – 2021 Can Tho University the proton exchange membrane and concentration [15]. The charge transfer losses of the anode are represented by Ra, which reflects biofilms' function [147]. Two kinds of charge transfer resistances were observed in the anode: from the graphite felt sheet (Ra,g) and from the titanium conductor (Ra,t). The electrical double layer phenomenon of electrode materials and titanium conductor represented by capacitance Ca,g [148], and Ca,t indicated that the electrode is exposed to the anode's fluid. The charge transfer losses of the anode are represented by Rc and Cc, the electrical double layer [149]. Wc indicated concentration losses of the MFCs, which represents the diffusion of the substrate, oxygen, or other reactants in the MFCs [149]. Figure 4. 7 Nyquist plots of MFCs with EC-Lab software As shown in Table 4. 1, the ohmic losses R1 are similar. This is because the anolyte made of phosphate buffer solution (PBS) and the electrode material (graphite felt) were the same in all MFC. In general, reducing the distance between the anode and the cathode will reduce the distance traveled by the protons from the anode to the cathode. Therefore, the ohmic resistance of the MFC in the study was very small [150]. Similar values in Ca,g, Ca,t, and Cc,c indicate the insignificant difference in the electrical double layers at the interface with the anolyte/catholyte. The difference in internal resistance among the MFC, therefore, could be explained by the difference in charge transfer losses in the anode (Ra,t and Ra,g) and in the cathode (Rc), and by the concentration losses in the MFC (Wc). These charge transfer losses in the Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 37

Master of Science Thesis course 25 – 2021 Can Tho University anode (Ra,t and Ra,g) indicate biofilm activity which may be caused by the difference in EA biofilm present and the anodophilic properties of the biofilm due to substrate fed. The EA biofilm acclimates and colonizes onto the anode electrode and secrete matrix materials according to the type of EA biofilm [151]. This causes a difference in the anolyte and catholyte content, which causes a difference in the Wc and Rc values. Table 4. 1 Internal resistance of MFC with respect to the substrate different R1 (Ω) Ra,g (Ω) Ca,g, (F) Ra,t (Ω) Ca,t (F) Rc,g (Ω) Cc,c (F) Wc  Ohm s  MFC-1 2.41 2.34 0.05107 76.34 0.03343 19.15 3.74  10–5 385.30 MFC-2 2.79 9.59 0.03712 74.39 0.02265 20.43 9.73  10–5 532.90 MFC-3 1.79 3.63 8.02  10–4 75.84 0.02552 13.79 1.25  10–5 135.70 MFC-4 2.51 14.38 1.82  10–4 262.50 0.01619 16.92 3.23  10–5 247.93 MFC-5 2.53 3.51 5.66  10–5 265.80 0.01623 27.95 7.32  10–5 243.20 MFC-6 2.31 16.03 0.04376 661.10 0.02046 30.97 5.04  10–5 325.30 4.5 SUBSTRATE DEGRADATION AND COULOMBIC RECOVERY Microorganisms, such as bacteria in MFCs, decompose organic substances for cell maintenance and growth, leading to the production of electrons and protons, which causes bioelectricity [152]. MFC’s wastewater treatment efficiency was assessed by estimating the substrate’s chemical oxygen demand (COD) removal efficiency during operation. The COD removal efficiency was assessed after each MFC operation cycle. The results of Oh and Logan’s research (2005) have shown that the concentration of organic pollutants available in the sample used is proportional to the current generated in the MFCs. They also suggested that the power produced is higher when the sample’s COD value is also higher [153]. The initial COD readings varied because of the presence of different substrate types (Figure 4.8A). The MFC fed with acetate was found to have more than 90% COD removal efficiency, which is the highest obtained among the MFCs in this study. Major in Theoretical chemistry and Physical chemistry College of Natural Sciences 38