Analysis of Energy Losses of Microbial Fuel Cells (Mfcs) and Design of an Innovative Constructed Wetlands-MFC

Analysis of Energy losses of Microbial Fuel Cells (MFCs) and Design of an Innovative Constructed Wetlands-MFC

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of the Ohio State University

Ke Li

Graduate Program in Food, Agricultural and Biological Engineering

The Ohio State University

2017

Thesis Committee:

Ann D. Christy, PhD., P.E., Advisor

Lingying Zhao, PhD Co-Advisor

Olli H. Tuovinen, PhD

Copyrighted by

Ke Li

2017

Abstract

A Microbial Fuel Cell (MFC) is a device used to harvest electrons from living microorganisms to generate electrical power. After decades of development, new architectures have been developed and new materials have been applied to MFC systems.

Improvements to this promising technology have been extensively reported. However, scientists and engineers are still facing difficulties on enhancing energy output and increasing the MFC system efficiencies. In addition to physical resistances caused by

MFC materials, there are still many unknown factors affecting the electron transfer pathway used by microorganisms in MFC environments. To increase the performance, a series of technologies have been integrated into MFCs. For instance, MFC technology has been combined with other technologies such as algae pounds, anaerobic digesters and constructed wetlands, to increase substrate utilization efficiency.

As one example, CW-MFCs have already been studied for wastewater treatment and electricity generation. However, the system efficiencies presented by current models are low, and new designs need be explored to reduce the energy cost during installation and operation of CW-MFC systems. The first objective of this thesis research was to review the development of MFCs and relevant technologies, then to evaluate the energy loses and efficiencies in MFC systems, leading to a comprehensive understanding of MFC system from a thermodynamic viewpoint. The second objective was to design and ii

construct a down-flow Constructed Wetland-Microbial Fuel Cell combined system with a semi-air cathode and analyze its performance. This innovative design will be able to enhance the system efficiencies of CW-MFCs by reducing external energy requirements, and this research will provide foundational work for further CW-MFC explorations.

Keywords: energy losses, system efficiency, electron transfer, constructed wetland, microbial fuel cell, power generation

iii

Acknowledgments

First, I would like to thank my parents. Their support and encouragement were really important for me. Otherwise, I would not have been able to have such a good opportunity to go abroad and work with these excellent people.

I appreciate the effort of my graduation committee: Dr. Ann Christy, Dr. Lingying Zhao and Dr. Olli Tuovinen provided their constant support and insightful suggestions towards my work. Especially Dr. Christy, her patient mentorship helped me a lot during my thesis writing and study. Drs. Beenish Saba and Young Woon Kang also provided their expertise and professional opinions to help me.

I would like to thank Ms. Candy McBride, the graduate coordinator in the Department of

Food, Agricultural and Biological Engineering. She gave me a lot of help during these two years. And I really appreciate the Ohio State University for providing such a good opportunity to work with these wonderful people.

I would like to thank the staff at the Wilma H. Schiermeier Olentangy River Wetland

Research Park who allowed me to obtain wetland materials from there for my research

resources, especially Brent Macolly, who provided really useful background information about wetland plants.

Finally, I thank Christopher Gecik, for helping me with fabrication of the electrodes I used during my study.

Vita

March 1991 ...... Wuhan China

2014...... B.S. Jianghan University

2016 to present ...... Graduate Teaching Associate, Department

of Food, Agricultural and Biological

Engineering, The Ohio State University

Fields of Study

Major Field: Food, Agricultural and Biological Engineering

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita ...... vi

List of Tables ...... ix

List of figures ...... x

Chapter 1: Introduction ...... 1

1.1 Microbial fuel Cells ...... 2

1.2 Energy recovery technology: Constructed Wetland-Microbial Fuel Cell (CW-MFC) combined system ...... 7

1.3 Research objectives ...... 8

Chapter 2: Energy losses and system efficiencies of Microbial Fuel Cells: An Overview ...... 10

2.1 Introduction ...... 10

2.2 Energy losses during biotransformation ...... 15

2.2.1 Unused organic components ...... 16

2.2.2 Alternative metabolic pathway ...... 18

2.2.3 Excessive biomass accumulation ...... 19

2.3. Energy losses during electron transportation ...... 20

2.3.1 The effects of electrodes ...... 20

vii

2.3.2 The effects of membranes ...... 21

2.4 Other consideration ...... 22

2.5 Summary and Conclusions ...... 23

Chapter 3. Down-flow Constructed Wetland-Microbial Fuel Cell system with semi-air cathode ...... 24

3.1 Abstract ...... 24

3.2 Introduction ...... 25

3.2.1 Constructed wetlands-MFC ...... 25

3.2.2 Issues in CW-MFC systems ...... 26

3.2.3 Introduction to this research ...... 27

3.3 Material and methods ...... 29

3.3.1 Configuration and setup of CW-MFC ...... 29

3.3.2 Assembly of down-flow CW-MFC ...... 33

3.3.3 Step tests ...... 34

3.3.4 Organic Waste Treatment test ...... 37

3.3.5 Operation and analysis ...... 37

3.4 Results and Discussion ...... 37

Chapter 4. Conclusions ...... 43

References ...... 46

Appendix A: Experimental data ...... 51

viii

List of Tables

Table 3.1 Substrate components ...... 32

Table 3.2 Step test external resistance and switch combinations ...... 35

Table 3.3 Voltage and power output of four systems ...... 40

Table A.1 Resistance decade box data for the step test with variable time intervals ...... 51

List of Figures

Figure 1.1 Estimated U.S. Energy Consumption in 2016 (Lawrence Livermore National Laboratory, 2017) ...... 2

Figure 1.2 A Schematic indicating the operating principle of a Microbial Fuel Cell (Rabaey & Verstraete, 2005) ...... 3

Figure 1.3: Schematics of Direct Electron Transfer mechanism: (a) Membrane bound cytochromes, (b): Direct Electron Transfer mechanism via electronically conducting nanowires (pili) (from Rinaldi et al., 2008) ...... 4

Figure 1.4: Schematic of Mediated Electron Transfer mechanism via added (exogenous) or secreted (endogenous) mediators. (modified from Rinaldi et al., 2008) ...... 4

Figure 2.1. Six types of over-potential during electricity generation in a MFC. (Rabaey and Verstraete, 2005) ...... 12

Figure 2.2 Schematic of energy flux in microbial fuel cell (Schröder, 2007) ...... 13

Figure 2.3 Schematic potential losses for a cathodic reaction displaying activation, ohmic, mass transport and parasitic regions (Rismani-Yazdi, 2008) ...... 14

Figure 3.1- Schematic of constructed wetland (from Ebrahimi et al., 2015) ...... 25

Figure 3.2: Schematic of down-flow CW-MFC with semi air cathode ...... 28

Figure 3.3 Photograph of experimental tank...... 29

Figure 3.4 Schematic of Olentangy river wetland research park (Douglass, 2014) ...... 30

Figure 3.5: Photograph of example electrode used in this study ...... 32

Figure 3.6: Photograph of two experimental down-flow CW-MFCs with semi-air cathodes...... 33

Figure 3.7: Resistance decade box (model RS-500, Eleno Electronics, Inc) ...... 34

Figure 3.8: Voltage and power outputs of CW-MFC system 113 step test with variable time intervals (resistance changed only after voltages stabilized) ...... 38

Figure 3.9: Current density and power density versus external resistance during System 113 step test with variable time intervals (resistance changed only after voltages stabilized)...... 39

Figure 3.10: Stabilized average voltage of four CW-MFC systems ...... 41

Figure 3.11: Voltage performance of four individual systems after first feeding test ...... 42

Figure A 1: Averaged voltage and power outputs of CW-MFC systems’ step test ...... 52

Figure A 2: Voltage and power outputs of CW-MFC system 114 step test ...... 53

Figure A 3: Voltage and power outputs of CW-MFC system 115 step test ...... 54

Figure A 4: Voltage and power outputs of CW-MFC system 116 step test ...... 55

Figure A 5: Averaged current density and power density versus external resistance during step test ...... 56

Figure A 6: Current density and power density versus external resistance of system 114 during step test ...... 57

Figure A 7: Current density and power density versus external resistance of system 115 during step test ...... 58

Figure A 8: Current density and power density versus external resistance of system 116 during step test ...... 59

Chapter 1: Introduction

The challenging problem of growing worldwide demands for energy combined with concerns about pollution and climate change are creating opportunities for researchers who work at the intersection of energy and the environment. The widespread use of fossil fuel resources has contributed to many of these issues. Although clean energy technologies, such as wind energy, solar energy, and geothermal (earth heat) energy have been increasingly utilized in recent years, the overall consumption of energy resources is still heavily weighted toward fossil fuels with only a small amount of renewable energy usage. In addition, by relying on current power plant technologies, a large portion of energy is rejected as thermodynamic losses (Figure 1.1). Thus, it is important to find a better way to produce and utilize clean and sustainable energy.

One of the more promising renewable energy technologies is the Microbial Fuel Cell

(MFC), a biotechnology developed to generate electricity from organic components

(Logan, 2008; Rabaey and Verstraete, 2005). This technology would come under the

"Biomass" category in Figure 1.1's energy flow diagram, along with other biomass consuming technologies such as anaerobic digestion, fermentation, cellulosic ethanol production, and wood burning. The characteristics of MFCs enable them to treat biomass and various organic wastewaters while directly generating electrical energy.

Figure 1.1 Estimated U.S. Energy Consumption in 2016 (Lawrence Livermore National Laboratory, 2017)

1.1 Microbial Fuel Cell

The Microbial Fuel Cell (Figure 1.2) is a device which is able to generate electricity by harvesting electrons metabolized by selected microbes (Logan, 2008; Rabaey and

Verstraete, 2005).

Figure 1.2 A Schematic indicating the operating principle of a Microbial Fuel Cell (Rabaey & Verstraete, 2005)

Electrons are generated via anaerobic respiration by microorganisms in the anodic compartment of the MFC, are transferred from electron donors (oxidizable substrates) to the anodic electrode via either direct electron transfer (Figure 1.3) or mediated electron transfer via added (exogenous) or secreted (endogenous) mediator compounds (Figure

1.4) (Schröder, 2007). Electrons then flow to the cathodic electrode through an external circuit, combining with electron accepters, such as oxygen and ferricyanide (Logan,

2012). During this process, the electricity can be either harvested or utilized via the external circuit. Typical energy harvesting systems include capacitor-based systems, charge pump-based systems, and boost converter-based systems (Wang et al., 2015).

Alternatively, the energy can be directly used to power small devices such as LED lights or remote sensors (Donovan et al., 2011).

Figure 1.4: Schematic of Mediated Electron Transfer mechanism via added (exogenous) or secreted (endogenous) mediators. (modified from Rinaldi et al., 2008)

Substrate can be fed either continuously or intermittently during the operation of MFC systems (Logan et al., 2006). Wastewater treatment is an expensive and energy consuming process, typically including primary physical separation, secondary activated sludge systems, and sometimes tertiary polishing systems (Tchobanoglous et al., 2003).

The organic components in some wastewaters plus microbial biomass generated in the activated sludge process can be used as substrate or feedstock to an MFC, acting as a resource and not simply a waste. This approach can be considered a partial solution for wastewater treatment, or an integrated part of a larger waste treatment system. The benefit of the MFC is that the electricity it produces can offset some of the energy required to run the more conventional wastewater treatment operations. For example, fermented primary sludge from domestic wastewater treatment has been utilized for electricity generation using single-chamber air-cathode MFCs (Yang et al., 2013). Swine wastewater has been used for electricity generation with MFCs as well.

Due to the characteristics of the MFC, it is considered to be a potential solution to the current global energy and environment crises, but its power output is still relatively low.

Research on finding ways to improve performance is needed to elevate MFC technologies from the laboratory to commercialization (Logan, 2012). Different types of applications have already been reported during the last decades. Conversion of wastewater into bioenergy is one type of application of MFC that has been widely documented (Logan & Rabaey, 2012).

Researchers studying in different disciplines are contributing to the development of the

MFC. Material scientists and engineers study electrodes and membranes such as the effects of carbon electrode morphology on MFCs (Sanchez et al., 2015). Microbiologists study microbial metabolism and community interactions (Jung & Regan, 2007). Chemists study the MFC's electrochemical redox reactions (Dušek et al., 2008). Environmental engineers (Logan, 2008) study the treatment effectiveness of MFCs, and electrical engineers study the energy harvesting processes (Alaraj et al., 2014; Corbella et al., 2015).

Most MFC investigations described in the literature are based on five main types of studies:

1. The enhancement of physical components used in the MFC such as electrodes,

membranes, and external resistances

2. Architecture of the MFC device

3. Combination of the MFC with other technologies

4. Microorganisms and microbiomes within the MFC system (Inglesby & Fisher,

2012)

5. Different substrates, anolytes, and catholytes

During MFC operation, all these five aspects will significantly affect the overall performance of the system. Many literature reviews have already provided information and discussions about those topics. However, to increase the performance of the system, it is necessary to understand the pathways of energy conversion and transportation of electrons. Energy losses along these pathways reduce the system efficiencies of the MFC.

More details about these topics are presented in the next chapter which provides a discussion about energy losses and system efficiency during the operation of MFC systems.

1.2 Energy recovery technology: Constructed Wetland-Microbial Fuel Cell (CW- MFC) combined system

Microbial Fuel Cells have been studied for many years, and various types of applications have been documented, while energy loss is still one of the limitations of MFC systems.

For example, substrate in the anodic compartment is difficult to be utilized sufficiently, and operation of substrate feeding delivery systems consume extra energy. The cost of the proton exchange membrane in MFC construction is another limitation of this system.

Integrating MFC systems with other similar engineered systems is a widely used practice to recover the energy losses and reduce the economic limitations. The combined

Constructed Wetland-Microbial Fuel Cell (CW-MFC) is one integrated technology that can be used for electricity generation and simultaneous wastewater treatment.

Full-scale Constructed Wetlands (CWs) can be applied to wastewater treatment, flood control, and ecology remediation. Compared to other engineered systems with similar functions, CW's advantages include low-cost plus ease of operation and maintenance.

They have been employed to treat domestic sewage, agricultural wastewaters, and other applications for decades (Liu et al., 2015). However, the CW system also has some limitations, for example, when applying high organic loading rates to a CW, substrate clogging can be a problem leading to system maintenance issues (Ruiz et al., 2010).

Therefore, CWs have been combined with other systems such as membrane bio-reactors,

anaerobic digesters, and Microbial Fuel Cells to overcome these drawbacks (Liu et al.,

2015).

Constructed Wetlands and MFCs have many structural and functional similarities. For example, both CW systems and MFC systems consist of an anaerobic zone and an aerobic zone, which means the required redox gradient for MFC operation can be also be found in a CW. In addition, these two systems have been used successfully for organic waste treatment. The same organics applied into the CW can be used as electron donors for the MFC. The combination of those two promising technologies can potentially overcome the limitations of both the MFC and CW. Different architectures of CW-MFC systems have been developed in recent years. The CW-MFC systems have been applied to treat synthetic wastewater under different organic loading rates (Villasenor et al.,

2013). In one study, a maximum of 93.15% dye removal rate was achieved after 96 hours of operation (Yadav et al., 2012). These results from related research studies indicate the feasibility of simultaneous bioelectricity generation and wastewater treatment with CW-

MFC systems.

1.3 Research objectives

The first objective of this project was to develop a comprehensive analysis of energy losses and system efficiency of MFCs from a thermodynamics viewpoint. A review of refereed journal articles discussing energy loss pathways can provide ideas for other

scholars when studying MFCs from a novel aspect.

The second objective was to design, construct, and test an innovative structure: a down-

flow constructed Wetland-Microbial Fuel Cell (CW-MFC) system with a semi-air

cathode. The hypothesis of this study was that a down-flow CW-MFC system with a semi-air cathode would perform better than traditional CW-MFC designs documented in the literature. The advantages of the proposed innovative CW-MFC were hypothesized to be the following:

1. Down-flow design will allow this system to operate without extra energy input

into the system, thus saving energy and increasing system efficiencies.

2. Semi-air cathode will provide more accessible surface area for the cathodic

electrode to interact with oxygen which serves as a terminal electron acceptor.

In chapter 2, a more comprehensive understanding of energy losses during the operation of MFC systems is provided. Energy losses in MFC systems are classified into two categories: energy losses during biotransformation and energy losses during electron transportation. Chapter 3 describes the design, construction, and testing of four replicate down-flow CW-MFC systems with semi-air cathodes. Chapter 4 summarizes the conclusions from this research and proposes future studies that could build upon the work presented in this thesis.

Chapter 2: Energy losses and system efficiencies of Microbial Fuel Cells: An overview

2.1 Introduction

Microbial Fuel Cells (MFCs) can directly generate electricity by converting biochemical energy from organic compounds via microbial anaerobic respiration. This ability to degrade organic substrates gives the MFC excellent potential to deal with various environmental contaminant issues. Substrates like glucose, cellulose, acetate and other more complex organics including domestic wastewater are reported to have been used for

MFC studies (Ahn & Logan, 2010) . Different architectures range from the most common two-chambered MFCs (Logan et al., 2006) to single chamber MFCs (Abourached et al.,

2014) to air cathode MFCs (Fan et al., 2007).

The weak overall performance of MFCs is limiting the development and commercialization of this technology. Due to the complexity of the combined microbial and electrochemical processes in a MFC, changing any single factor can cause unpredictable interactions with other correlated factors. In an ideal situation, available voltage can be predicted by the Nernst equation:

= ln ( ) (1) 𝑜𝑜 𝑅𝑅𝑅𝑅 𝐸𝐸𝑡𝑡ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑜𝑜 𝐸𝐸 − 𝑛𝑛𝑛𝑛 Π 10

In the Nernst equation, is the ideal theoretical ideal voltage(V), is the 𝑜𝑜 𝑡𝑡ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 standard cell voltage (V),𝐸𝐸 R is the ideal gas constant (R=8.314 j 𝐸𝐸), T is the −1 −1 temperature (K), n is the number of electrons transferred in the during𝑚𝑚𝑚𝑚𝑚𝑚 𝐾𝐾the electricity generation process (dimensionless), F is the Faraday’s constant (F=96485 C ), and −1 ∏ is the chemical activity of the reaction product divided by those of the reactants𝑚𝑚𝑚𝑚𝑚𝑚

(dimensionless).

In practice, the measured voltage across an MFC will always be lower than the

𝑡𝑡ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 value due to potential losses. This loss in voltage is called over-potential. The potential𝐸𝐸

losses during electron transfer in a MFC can be categorized as the following six types

(Rabaey & Verstraete, 2005) (Figure 2.1):

(1) Loss due to bacterial electron transfer.

(2) Losses due to electrolyte resistance.

(3) Losses at the anode.

(4) Losses at the MFC's external resistance (useful potential difference) and

membrane resistance losses.

(5) Losses at the cathode.

(6) Losses due to electron acceptor reduction.

Figure 2.1. Six types of over-potential during electricity generation in a MFC. (Rabaey and Verstreate, 2005)

Energy losses in the anodic and cathodic compartments can be explained by different mechanisms. Possible energy losses in the anodic compartment have been illustrated based on the analysis of energy flux in anodic compartment (Figure 2.2).

Figure 2.2 Schematic of energy flux in microbial fuel cell (Schröder, 2007)

In this model, the microorganisms facilitate the conversion of energy contained in the substrate into electricity, but a distinct portion of the Gibbs free energy, is used for

𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 microbial survival and reproduction (Schröder, 2007). ∆𝐺𝐺

= (2)

𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 where indicates the∆ 𝐺𝐺electric ∆energy𝐺𝐺 that− ∆ can𝐺𝐺 be harvested, denotes the total

𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 available∆𝐺𝐺 energy can be catalyzed by the microbes, and is ∆the𝐺𝐺 energy utilized by

𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 microorganisms in the anodic compartment. ∆𝐺𝐺

Due to this explanation about energy flux. Dr. Schröder suggested that the terminology

“biotransformation” instead of “biocatalysis” should be used to describe the function of

microorganisms in the anodic compartment of MFCs.

The main cathodic limitations include activation losses, ohmic losses, and mass transport

losses which have been documented (Rismani-Yazdi et al., 2008). These mechanisms also contribute to anodic energy losses (Figure 2.3). The energy losses in the anode and cathode collectively limit the performance of MFCs (Rismani-Yazdi et al., 2008) as

shown in equation (3):

= [( + + ) + ( + + ) ] (3)

𝑜𝑜𝑜𝑜 𝑡𝑡ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜ℎ𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜ℎ𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 where𝑉𝑉 𝐸𝐸 − is the𝜂𝜂 thermodynamically𝜂𝜂 𝜂𝜂 𝑐𝑐𝑐𝑐𝑐𝑐 predictedℎ𝑜𝑜𝑜𝑜𝑜𝑜 voltage,𝜂𝜂 𝜂𝜂 is the𝜂𝜂 activation𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 loss due

𝑡𝑡ℎ𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑎𝑎𝑎𝑎𝑎𝑎 to reaction𝐸𝐸 kinetics, is the ohmic loss from ionic and 𝜂𝜂electronic resistances, and

𝑜𝑜ℎ𝑚𝑚𝑚𝑚𝑚𝑚 is the concentration𝜂𝜂 loss due to mass transport limitation.

𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝜂𝜂

Figure 2.3 Schematic potential losses for a cathodic reaction displaying activation, ohmic, mass transport and parasitic regions (Rismani-Yazdi et al., 2008).

The energy losses in other pathways and their solutions have been rarely reported. The goal of this chapter was to comprehensively discuss energy losses during the operation of

MFCs to stimulate ideas for improving MFC designs.

2.2 Energy losses during biotransformation

The first step of electricity generation is the microbial metabolism of organic substrates in the anodic compartment of the MFC. Energy losses in this process include:

1. Unused organic components: The efficiency of substrate utilization is an

important factor which can directly affect the performance of the MFC systems.

Unused organic components contain a large amount of energy.

2. Alternative metabolic pathways producing unwanted byproducts: Other metabolic

pathways such as fermentation will generate products such as methane gas instead

of free electrons. Methanogenesis in MFCs will not reduce the efficiency of

substrate utilization but will sharply decrease electron generation, and also affect

the microbiome or other biochemical reactions in the anodic compartment

(Rismani-Yazdi et al., 2013).

3. Excessive biomass accumulation in the anodic compartment: Complex

communities of microorganisms inoculated into MFC systems give better

performance than single species cultures (Rabaey et al., 2003). Some microbial

species within the community play important roles other than electron generation

such as delivering electrons to the anodic electrode. The presence of other

microorganisms in the anodic chamber may not have a positive impact on

electricity generation, and thus energy consumed by these microorganisms

contribute to energy losses during the electricity generation process.

2.2.1 Unused organic components

The characteristics of the MFC enable it to utilize various types of substrates, including carbohydrates, fatty acids, amino acids, and a wide range of organic-containing wastewaters such as domestic wastewater (Logan, 2008), swine wastewater (Min et al.,

2005), beer brewery wastewaters (Wang et al., 2008), and food processing wastewaters

(Mansoorian et al., 2013). Among all these available sources, the most common types of substrates reported in the literature are monosaccharides, polysaccharides, and short chain fatty acids. However, it is difficult to compare research results for all of these simple organic substrates due to the various operation conditions of different studies. This study attempts to perform a quantitative comparison among factors of single substrates, specifically carbohydrates, organic fatty acids, and organic-containing wastewaters by discussing the factors that may affect energy losses of substrates in MFCs based on prior research studies.

Carbohydrates

Carbohydrates, including monosaccharides and polysaccharides, are common electron donors used as substrates in MFC systems. Monosaccharides such as glucose, xylose, and galactose (especially glucose) are widely used in MFC studies. Theoretically, there are 24 electrons that can be released from one glucose molecule, however, due to incomplete

oxidation of glucose substrate, those theoretical values can rarely be achieved during the

electricity generation process in MFCs (Schröder, 2007).

Polysaccharides including starch and cellulose are some of the most commonly used

substrates in MFC studies. The structure and other characteristics are very different

between starch and cellulose. In a comparison of different polysaccharide-fed MFCs,

Ahmad et al. (2013) found that the hydrolysis rate of these polysaccharides was one of the major factors affecting MFC performance. MFCs using starch and soluble forms of cellulose generate higher power densities than those systems using other forms of polysaccharide substrates. This may be attributed to the faster hydrolysis rates of α-1-4- glycosidic bonds in starch as compared with the slower hydrolysis rates of the β-1-4 glycosidic bonds found in cellulose and chitin (Ahmad et al., 2013). Some microorganisms, such as those found in the digestive systems of ruminant animals, are selectively better at metabolizing cellulose than most microbes. Cellulose and bovine rumen fluid were successfully used in MFCs (Rismani-Yazdi et al., 2007).

Short chain organic fatty acids

Organic fatty acids, especially short chain fatty acids such as acetates (Lee et al., 2008), butyrates, and propionates have been used successfully as substrates in MFC studies

(Chae et al., 2009). Acetate-fed systems presented higher energy-conversion efficiency

(ECE) and potential efficiency (PE) then glucose-fed systems (Lee et al., 2008) Unlike glucose, short fat acids are non-fermentable resources can be used as substrate more

17 efficiently during the operation of MFC systems. Acetate-fed-MFC showed the highest

CE (72.3%), followed by butyrate (43.0%), propionate (36.0%) and glucose (15.0%)

(Chae et al., 2009).

Organic Wastewaters

Many different types of wastewater have been applied to MFC studies, including domestic wastewater (Logan, 2008), brewery industry wastewater ( Wang et al., 2008), dye wastewater (Fang et al., 2013), food processing wastewater (Mansoorian et al., 2013) and other organic-containing wastewaters. The microorganisms in the anodic component are able to utilize organic components like carbohydrates and short fat acids as substrates to generate electrons, and the electrons released by the microorganisms can be either delivered to cathodic compartment via external circuit or interact with pollutants in anodic compartment. Dye wastewater decolorization by MFC is a good example (Sun et al., 2009).

2.2.2 Alternative metabolic pathways

Changes in redox potential can trigger use of alternative metabolic pathways by affected microorganisms. The first step of electricity generation in MFCs is the utilization of substrates. Theoretical energy from reactions involving organic compounds can be calculated assuming full reduction in the present of oxygen. For example, in the breakdown of glucose, 2895 Joules of energy and 24 electrons can be released in this process:

+ 6 6 + 6 + 6 = 2895 −1 𝐶𝐶6𝐻𝐻12 𝑂𝑂2 → 𝐻𝐻2𝑂𝑂 𝑂𝑂2 𝐶𝐶𝑂𝑂2 ∆𝐺𝐺 − 𝑘𝑘𝑘𝑘 ∙ 𝑚𝑚𝑚𝑚𝑚𝑚

Lower redox potentials will trigger alternative pathways of electron release. Furthermore,

under the absence of electron acceptors, methanogenic fermentation will be the dominant

metabolic pathway. The performance of cellulose-fed MFCs were studied with and without methanogenesis suppression (Rismani-Yazdi et al., 2013).

2.2.3 Excessive biomass accumulation.

Microbial communities in the anodic compartment play an essential role in MFC

systems. Researchers have studied electrochemical and microbial community dynamics

in MFCs using various substrates and inoculants. MFC electrochemical performance was

observed to have stabilized and microbial communities to have converged after two

months of operation (Yates et al., 2012). Species in such complex microbial communities may have different roles and responsibilities. Before the community reached stabilization, energy was consumed to support the increases in biomass. The accumulation of biomass and unwanted biofilms can affect the overall performance of MFC systems. Dead cells were observed to form an inner core attached on the surface on the electrode, serving to deliver electrons to the electrode while also causing increased ohmic losses. Only the live outer-layer contributed to electron generation (Sun et al., 2015).

2.3 Energy losses during electron transportation

The physical components of an MFC play an important role in energy harvesting and losses. For example, anode electrodes serve as electron accepters in the anodic compartment. Electrons are delivered via DET (direct electron transportation) or IDT

(indirect electron transportation) to anodic electrode, then can be delivered through external circuits, eventually reaching the cathodic electrode and combing with terminal electron accepters. Anodic losses can occur during mass transport of substrates within bulk liquid anolyte, diffusion of substrate across accumulated biomass, selective transport of both substrate and electrons across microbial membranes, and electron delivery to the electrode. Analagous mechanisms exist within the cathodic compartment, even when using inorganic catholytes. Cathodic losses can occur during mass transport of electron acceptor reactants within bulk liquid catholyte and delivery of reactants to the electrode.

During this process, resistance of physical materials such as electrodes in anodic compartment and cathodic compartment and external resistances (Rismani-Yazdi et al.,

2011) will also affect the performance of MFCs.

2.3.1 The effects of electrodes

Both anodic and cathodic electrodes play important roles in electron transportation, and the properties of these electrodes will affect the energy transportation efficiency.

Accessible surface area of the anodic electrode for microorganism attachment also is well documented to be directly proportional to power output (Alatrakchi et al., 2014). The two types of electrodes in MFCs, anodes and cathodes, are usually studied separately because

of their different roles in the redox reaction. Different types of anode designs have been documented in recent years. For example, carbon nanotubes (Qiao et al., 2007) have been studied in MFCs, and different morphology of carbon has been discussed (Sanchez et al.,

2015). Zhou et al. (2011) stated that there are five important properties for good anode

materials. They are: (a) good electrical conductivity and low resistance, (b) bio

compatibility, (c) chemical stability, (d) large surface area, and (e) good mechanical

strength and toughness.

Factors such as electrode spacing, surface area ratio (ratio of surface area to compartment

volume might cause decreasing of efficiency), manufacturing method (Logan, 2012), the

use of air cathodes, and MFC configuration will also affect performance (Fan et al.,

2007).

2.3.2 The effects of membrane

Membrane, as another important physical component of the MFC, is widely studied

( Leong, 2013). Important factors associated with membranes in MFCs include membrane resistance, oxygen diffusion rate, and substrate crossover resistance. Because

the membrane is often the most expensive component within an MFC, membrane-less

MFCs are also being developed aiming to reduce the cost of MFCs (Ghangrekar &

Shinde, 2007).

2.4 Other considerations

Additional factors such as pH, temperature, and loading rate which can affect overall

performance have also been documented in the literature (Jadhav & Ghangrekar, 2009).

For instance, concentration of substrate or salt levels within a given substrate are also

important to the performance of MFC (Zhu et al., 2015). In addition, the architecture of

the MFC is another important factor affecting the overall performance and system

efficiency. There are many new types MFC which have been reported other than

conventional two chamber MFCs or single chamber MFCs, like Constructed Wetlands-

MFCs (Srivastava et al., 2015) and Anaerobic Digester-MFCs (Inglesby & Fisher, 2012).

Jonathan et al. (2017) introduced integrating microbial electrochemical technologies with

anaerobic digestion from different perspectives. They proposed six types of operations

integrating of those two technologies, namely (1) Using Bioelectrochemical systems as a

downstream unit, (2) Monitoring anaerobic digestion process stability with

bioelectrochemical systems, (3) Feedstock pretreatment of anaerobic digestion by using

bioelectrochemical systems, (4) Direct integration of bioelectrochemical systems in

anaerobic digestion to facilitate in situ electromethanogenesis for augmenting biogas

yield, (5) Upgrading anaerobic digestion biogas quality by using bioelectrochemical

systems, and (6) Using bioelectrochemical systems as an add-on unit in the anaerobic digestion recycle stream for toxicity removal and nutrient recovery.

2.5 Summary and Conclusions

In this chapter, different types of energy losses in both the anodic and cathodic compartments have been discussed. As materials with better qualities were applied to

MFCs, energy losses during biotransformation were revealed to cause other issues in

MFC studies. Unused organic components, alternative metabolic pathways, and excessive biomass accumulation contribute to much of the energy losses observed during MFC operation.

Thus, understanding the functions and phylogenetics of microorganisms in MFCs will be one of the keys to future MFC studies. Based on advances in genetic and molecular techniques, more biological information can be explored leading to better MFC designs.

CHAPTER 3. Down-flow Constructed Wetland-Microbial Fuel Cell system with semi-air cathode

3.1 Abstract

Constructed Wetlands (CW) and Microbial Fuel cells (MFC) both are promising technologies for solving environmental problems. Due to the structural and functional similarities of these two systems, MFCs have been integrated into CWs thereby improving their wastewater treatment capacity while generating electrical power at same time. However, there are still limitations of this combined Constructed Wetland-

Microbial Fuel Cell(CW-MFC) system, such as low energy efficiency and complicated installation issues. The aim of this study was to design, construct, and assess the performance of a down-flow CW-MFC system with semi-air cathode. Four batch scale

CW-MFC systems were built for this study. Then, substrates were fed to test the system's potential for wastewater treatment. During step tests, these down-flow CW-MFC systems performed well, and the output voltages indicated the future potential of this new modification of an CW-MFC. However, after two weeks of operation, output voltages of these systems became unstable, and gradually decreased. After summarizing the results of this study, several suggestions were made for future studies of CW-MFC systems. The

CW-MFC technology is still full of challenges but has a promising future for sustainable simultaneous wastewater treatment and electricity generation.

3.2 Introduction

3.2.1 Constructed wetlands-MFC

A Constructed Wetland (CW) is an engineered system which has been studied for wastewater treatment, phytoremediation, and water quality improvement. This green technology has demonstrated outstanding performance as a sustainable solution for various environmental problems (Wu et al., 2015), however it is difficult to achieve current pollutant discharge requirements while operating a CW alone (Álvarez et la.,

2008). Therefore, other relevant technologies, such as anaerobic digestion, have been combined with CWs to enhance the overall system performance (Liu et al., 2015).

Figure 3.1- Schematic of constructed wetland (from Ebrahimi et al., 2015)

Due to some similarities between CWs and MFCs, researchers have recently proposed merging these two systems together. The combined CW-MFC system potentially can

increase the efficiency of wastewater treatment of the CW alone, plus generate electrical

power at same time (Doherty et al., 2015). Structurally, both CWs and MFCs are characterized by having an anaerobic zone and an aerobic zone. Inclusion of CW plants has been reported to significantly increase the abundance and diversity of the microbial community and power generation performance of the associated MFCs (Lu et al., 2015).

CW-MFC systems have been studied for various applications including treatment of

industrial wastewaters from textile manufacturing. For instance, 93.15% dye removal has

been reported (Yadav et al,, 2012). A decolorization efficiency of 91.24% has also been observed in other CW-MFC dye treatment systems (Fang et al., 2013).

3.2.2 Issues in CW-MFC systems

Integrating CWs with MFCs has some advantages over a traditional CW system, for

example, a CW-MFC performed better on chemical oxygen demand (COD) removal than

a traditional open CW system (Srivastava et al., 2015). However, research and

development of the CW-MFC technology is still facing challenges. Although many

newly developed molecular biotechnologies have been applied to the study of microbial

communities in CW-MFCs, the functional microbial community in this complex eco-

system has not been fully understood. As one of the main factors affecting performance

of the combined system, the anaerobic zone's microbiome will determine the efficacy and

even the feasibility of the system. More genetic and molecular information about those

microbiomes is needed. (Lu et al. 2015) studied the syntrophy, or nutritional

interdependence, between wetland plants and the microorganisms in combined CW-MFC

systems by using high throughput sequencing analysis.

The architecture of the combined CW-MFC and other operating conditions should also be considered when designing a real world or large scale application of the technology. Up-

flow (Liu et al., 2013) and horizontal flow (Mantovi et al., 2003) are the two most commonly used feeding systems documented in CW-MFC studies due to the requirement of maintaining an anaerobic zone. The distance between electrodes affects MFC electricity generation, with greater ohmic losses being associated with longer distances

(Ghangrekar & Shinde, 2007). However, it is common for electrode spacing in up-flow and horizontal CW-MFC systems to be relatively large in order for the anode electrode to be placed within the anaerobic zone and the cathode in the aerobic zone. Continuously pumping substrate into up-flow or horizontal flow systems requires more energy input than will be harvested by the systems' MFC circuits. As a consequence, the net energy outputs of these combined CW-MFC systems will be negative. In addition, controlling the substrate loading rate requires extra management components which makes installation even more challenging.

3.2.3 Introduction to this research

To explore possible improvements to the CW-MFC technology, a down-flow CW-MFC system was designed and tested. The down- flow design was proposed to allow the CW-

MFC system to use gravity to induce flow, thereby avoiding the need for extra energy inputs. Unlike the up-flow and horizontal flow designs which intrinsically maintain a

separate anaerobic zone, the down-flow system produces a smaller oxygen gradient

between the top and bottom of the CW-MFC system. To maximize the oxygen gradient

available between the two MFC electrodes, a perpendicular semi-air cathode was installed into each system. Oxygen, which plays the role of terminal electron acceptor, can easily access the portion of the cathodic electrode exposed to air. Therefore, the long distance between anodic and cathodic electrodes often observed in more conventional

MFC design is not necessary in this case.

In this study, four down-flow CW-MFC systems with semi-air cathodes were developed.

After step tests, the stabilized systems were fed with organic substrate to examine the waste water treatment performance in these systems. Due to the innovative down-flow design, it was hypothesized that down-flow CW-MFC systems could potentially be more efficient than traditional CW-MFC designs.

Figure 3.2: Schematic of down-flow CW-MFC with semi air cathode 28

3.3 Material and Methods

3.3.1 Configuration and setup of CW-MFC

Each of the four systems were built in a 5-L clear acrylic open tank (Figure 3.3). The bottom dimensions were 5.3 inches (13.46 cm) by 5.7 inches (14.48 cm). The top dimensions were 6.3 inches (16.00cm) by 6.7 (17.02 cm)inches, and the height was 6.5 inches (16.51 cm).

Figure 3.3 Photograph of experimental tank

The gravel selected for use in the systems was a decay resistant product (0.5 cubic feet

(14.16 L), Earth Essentials by Quikrete, No.1275-19) with homogeneous particle sizing.

The gravel samples were washed using tap water, saturated in 75% ethanol for 24 hours to remove any biological contamination, then carefully rinsed with DI water. After cleaning, all gravel samples were placed on the bottom of the acrylic tanks.

Figure 3.4 Schematic of Olentangy river wetland research park (Douglass, 2014)

Wetland plants were collected from the constructed mesocosm wetland systems at the

Olentangy River Wetland Research Park (The Ohio State University, Columbus, Ohio).

Sediments were collected together with the wetland plants from their original environments. Species of the sampled wetland plants were identified as follows:

1. Common spikerush (Eleocharis palustris), which was the major species

colonized in the systems

2. Arrow Arum (Peltandra virginica) 30

3. Bottlebrush Sedge (Carex comosa)

Each electrode was a single graphite sheet (McMaster Carr), which was1/4 inches (0.63 cm) in thickness, 4 inches (10.16 cm) in width and 6 inches (15.24 cm) in length. A hole, which was 1/6 inches (0.42 cm) in diameter and1/4 inches (0.63 cm) in depth, was drilled on the top of each graphite electrode. The total surface area of each electrode is 53 square inches (0.034 square meters) After drilling, all of the electrodes were washed under flowing tap water to remove all the debris from drilling process and soaked in 1 M HCl for 24 hours. Then they were rinsed by DI water and soaked in 1M NaOH for 24 hours.

After being rinsed again, they were dried in an oven at 60。C for 60 minutes, and finally

cooled to room temperature.

Plastic-sheathed cooper wire was inserted into the hole drilled in each electrode, attached

with silver epoxy adhesive (AA-DUCT 902, Atom Adhesives), then allowed to set over

48 hours. Finally, a chemical resistant epoxy layer (AA-BOND 28, Atom Adhesives) was applied at the connection site between wire and electrode to protect the silver epoxy adhesive from being degraded in the CW-MFC environment.

Figure 3.5: Photograph of example electrode used in this study

To test the wastewater treatment performance of these systems, a synthetic substrate with a known formula was applied in this study. The substrate ingredients are listed in Table

3.1.

Table 3.1: Substrate components

Chemical compound Concentration

Glucose 5g/L

Tryptone 15g/L

Yeast extract 5g/L

K2HPO4 0.45g/L

KH2PO4 5g/L

NH4Cl 0.729g/L

NaCl 0.9g/L

MgSO4 0.09g/L

3.3.2 Assembly of down-flow CW-MFC

The gravel was washed by running tap water through the material until the effluent ran clear, sterilized at 121 °C for 30 minutes, then dried in an oven at 60 °C for 120 minutes, and cooled to room temperature. Sterilized gravel was placed on the bottom of the containers. Then wetland sediments were placed on the top of the gravel, installing the

anodic electrode 1.5inches (3.81cm) above gravel level. Approximately 2L of sediment

was added until the anodic electrode was covered by 2 inches (5.08 cm) of the sediment.

Distilled water was then added to fully saturate the sediment. Finally, the cathodic

electrode was installed perpendicular to the horizon and semi submerged in the sediment

(Figures 3.2 and 3.6).

Figure 3.6: Photograph of two experimental down-flow CW-MFCs with semi-air

cathodes.

3.3.3 Step tests

Four sets of step tests were conducted prior to the experiment to determine the optimal

external resistance for the CW-MFC systems. The resulting polarization curves which

present voltage-current and power-current characteristics were to be compared with

expected properties of a typical fuel cell (Sirinivasan, 2006). The step tests were

performed using resistance decade boxes (Model RS-500, Eleno Electronics, Inc.) shown in Figure 3.7, comprised of resistance switches ranging from 1 Mega-Ohm to 5 Ohm.

Figure 3.7: Resistance decade box (model RS-500, Eleno Electronics, Inc)

The switch combinations are shown in Table 3.2. The step test started from 1M. One of the tests waited between changing resistances, maintaining each resistance step until readings became stable. The times intervals ranged from 20 minutes to 257 minutes. The other three step tests were conducted under set interval times of 30 minutes, 60 minutes, and 120 minutes.

Table 3.2: Step test external resistance and switch combinations

Step number Resistance (Ohm) Switch combination

1 1M 1M

2 0.5M 200k+300k

3 250k 200k+40k+10k

4 100k 100k

5 50k 40k+10k

6 25k 20k+3k+2k

7 10k 10k 8 7.5k 4k+3k+400+100

9 5k 3k+2k

10 2.5k 2k+400+100

11 1k 1k

12 900 400+300+200

13 800 400+300+100

14 700 400+300

15 600 400+200

16 500 300+200

17 400 300+100

18 300 300

19 200 200

20 100 100

21 50 30+20

22 25 20+4+1

23 10 10

24 5 4+1

3.3.4 Organic Waste Treatment test

The feeding operation was conducted after the stabilization of all four down-flow CW-

MFC systems. Initially, 50 ml of substrate (Glucose 5g/L, Tryptone 15g/L, Yeast extract

5g/L, K2HPO4 0.45g/L, KH2PO4 5g/L, NH4Cl 0.729g/L, NaCl 0.9g/L, MgSO4 0.09g/L)

were fed to the four systems separately. No significant change was observed. CW-MFC

is a natural system which needs more reaction time than traditional MFC, so the first

feeding volume of substrate was only 50 ml (1% to the total system volume) to give the

system sufficient time to consume the substrate. The next feeding operation was

conducted after 48 hours. No significant change was observed during the 48 hours. After

three rounds of feeding operation, 100 ml substrate was fed to the four systems

separately.

3.3.5 Operation and analysis

Each CW-MFC system was connected to a data acquisition unit (DATAQ Instruments

Inc., Akron, OH), and the data were recorded on a computer on a 10 second interval. The four replicate systems were connected to this data acquisition unit on circuits labelled

113, 114,115, and 116. Power was calculated according to Ohm’s law, and power density was normalized on a per anode area basis (Logan, 2012):

Power, P=I X V (1)

Power Density = P/A or I X V/ A (2)

Where V is voltage(V); I (I =V/R) is the current (Amps), and A indicates the surface area of the anode electrode.

3.4 Results and Discussion

After four rounds of step tests, the results indicated that 1000 ohm was the optimal value for external resistance of the down-flow CW-MFC system; this was the resistance that produced the highest power density. Step tests of four systems yielded similar current density and power density curves regardless of interval time between changes in resistance. The Appendix presents graphs of average results as well as individual test results for systems 114, 115, and 116. The results for system 113 were chosen to highlight here because those data were most similar to the expected fuel cell polarization curve.

Figure 3.8 presents the relationships among voltage, current, and power of system 113 during the operation of the step test with variable interval times. Data were recorded when the values of voltage for all four CW-MFC experimental units did not significantly change. The orange curve with triangular data points describes the relationship between power output and current of the systems. The blue curve with circular data points shows the relationship between voltage output and current of the system. The shapes of these two curves indicate that there are reactions or responses other than linear current / voltage relationships.

Figure 3.8: Voltage and power outputs of CW-MFC system 113 step test with variable time intervals (resistance changed only after voltages stabilized)

Figure 3.9 documents the result of System 113 from the step test with variable time

intervals (resistance changed only after voltages stabilized), and indicates that the system

produced the highest current density of 7.04 mA/m2 at an external resistance of 500 ohm,

and the highest power density of 1.09 mW/m2 at an external resistance of 1000 ohm. The polarization curves of systems 114 and 115 presented patterns similar to system 113, however system 116 was dissimilar to the other three. To maximize power output, 1000 ohm was chosen as the proper value for external resistance in this research study.

Figure 3.9: Current density and power density versus external resistance during System 113 step test with variable time intervals (resistance changed only after voltages stabilized).

After applying a constant external resistance of 1000 ohm for 48 hours, the outputs of all systems stabilized. Seven groups of readings were recorded for voltage output calculations. Figure 3.10 indicates the average stabilized voltage values of the four CW-

MFC systems individually with system 113, 114, 115, and 116 labeled as 1, 2, 3, and 4 respectively. System 116 provided the best performance among the four systems with an average voltage output of 318.9 mV, maximum voltage output of 324.70 mV, and minimum voltage output of 314.10 mV (Table 3.3).

Table 3.3 Voltage and power output of four systems

System Voltage(mV) Average Power Average power

output (uW) density(mW/m2)

Maximum Average Minimum

113 222.70 208.33 200.90 41.85 1.23

114 265.30 254.49 248.10 63.14 1.86

115 234.30 199.23 184.00 33.66 0.99

116 324.70 318.90 314.10 103.55 3.05

All four systems presented power density values similar to the ranges reported in other

researchers' work including a CW-MFC system that was reported with maximum power

density of 0.852mW/m-2 (Fang et al., 2013) and a vertical-flow CW-MFC system with

12.4 L liquid volume producing a maximum power density of 12.42mW/m-2 (Liu et al.,

2013).

Figure 3.10: Stabilized average voltage of four CW-MFC systems

It was observed that the electricity generation performance often decreased after each feeding operation (Figure 3.11). This was most marked for systems 113 and 116. The other two systems experienced an initial rise in voltage that then decreased over time.

System 115 was the most stable of the four systems studied, maintaining its initial voltage within plus or minus 10% over the entire experimental run.

Figure 3.11: Voltage performance of four individual systems after first feeding test.

CHAPTER 4. Conclusions

Combining two engineered systems, CW and MFC, with a gravity driven down-flow

feeding system shows promise. No extra pump or other unit is needed, thus saving the

energy for the feeding operation. Conventional CW-MFCs, generally with up-flow or horizontal-flow feeding systems, can be improved by using this down-flow design.

Another improvement made during this study was the reduction in distance between the

anodic electrode and cathodic electrode by installing a perpendicular semi air-cathode.

Comparing this new design to conventional CW-MFCs with fully submerged cathode

electrodes, the semi air cathodes can more easily access oxygen in the air (electron

accepter). Because of this design feature, the installation of electrodes during CW-MFC construction will cost less money and be less energy intensive, and maintenance costs of the system are predicted to be less during long term operation.

Electrical power generation deceased after four rounds of substrate feeding. There are two possible reasons for this phenomenon: the complex biological and ecological environment within the CW-MFC system and substrate residues in the aerobic zone.

During the construction of these down-flow CW-MFC systems, many native wetland faunae were observed to have been accidentally collected when the wetland sediments

were sampled from the Olentangy River Wetland Research Park. These included several

species of worms and insects. Their presence might have affected the performance of the

system, especially after feeding substrate, and their death and subsequent decay might

have caused shortages of oxygen in the aerobic zone. In this case, the redox gradient

would have been reduced causing the electricity generation to become less efficient or

even to stop entirely. Identification of those species will be necessary in further studies.

To illustrate the function and biological niche of those species, both morphological

identification and biomolecular identification should be executed.

The feeding operation was conducted by directly pouring substrate onto the top of the

system, and the organic containing substrate residue may have remained in the aerobic

zone, reducing the redox gradient, and thus affecting the overall performance of the

system. In future studies, it is recommended that a feeding pipe be installed in CW-MFCs with down flow design. Then the substrate will be able to reach the anodic electrodes but will not affect the aerobic zone which functions as the cathodic compartment in a two- chamber (non-CW) MFC. With this improvement, the short distance between the installed electrodes can be maintained while feeding substrate without any extra pumping energy required.

Given the results of this research, the development of down-flow CW-MFC systems with semi air-cathodes can be improved. Suggestions for other researchers include installing the cathodic electrode on the top of the CW-MFC and more closely evaluating the native

flora and fauna that co-exist in wetlands. Studies are needed to determine the optimum

distance between electrodes; larger distances between electrodes will provide higher redox potential therefore enhancing the performance of CW-MFCs, while shorter distances between electrodes will reduce mass transport limitations.

Further exploration of down-flow substrate feeding systems is needed as long as electron

acceptors on the cathode side will not be disturbed. Design improvements in material

selection and proper sizing of electrodes will increase efficiency and performance. This

newly developed CW-MFC system illustrates one practice of integrating MFCs into other

systems or combining MFCs with other related devices, making both CW and MFC

technologies more sustainable.

References

Abourached, C., Catal, T., & Liu, H. (2014). Efficacy of single-chamber microbial fuel cells for removal of cadmium and zinc with simultaneous electricity production. Water Research, 51, 228-233.

Ahmad, F., Atiyeh, M. N., Pereira, B., & Stephanopoulos, G. N. (2013). A review of cellulosic microbial fuel cells: Performance and challenges. Biomass and Bioenergy, 56, 179-188.

Ahn, Y., & Logan, B. E. (2010). Effectiveness of domestic wastewater treatment using microbial fuel cells at ambient and mesophilic temperatures. Bioresource Technology, 101, 469-475.

Alaraj, M., Ren, Z. J., & Park, J.D. (2014). Microbial fuel cell energy harvesting using synchronous flyback converter. Journal of Power Sources, 247, 636-642.

Alatrakchi, F. A., Zhang, Y., & Angelidaki, I. (2014). Nanomodification of the electrodes in microbial fuel cell: Impact of nanoparticle density on electricity production and microbial community. Applied Energy, 116, 216-222.

Álvarez, J. A., Ruíz, I., & Soto, M. (2008). Anaerobic digesters as a pretreatment for constructed wetlands. Ecological Engineering, 33, 54-67.

Chae, K. J., Choi, M. J., Lee, J. W., Kim, K. Y., & Kim, I. S. (2009). Effect of different substrates on the performance, bacterial diversity, and bacterial viability in microbial fuel cells. Bioresource Technology, 100(14), 3518-3525.

Corbella, C., Guivernau, M., Vinas, M., & Puigagut, J. (2015). Operational, design and microbial aspects related to power production with microbial fuel cells implemented in constructed wetlands. Water Research, 84, 232-242.

Doherty, L., Zhao, Y., Zhao, X., Hu, Y., Hao, X., Xu, L., & Liu, R. (2015). A review of a recently emerged technology: Constructed wetland--Microbial fuel cells. Water Research, 85, 38-45.

Donovan, C., Dewan, A., Peng, H., Heo, D., & Beyenal, H. (2011). Power management system for a 2.5W remote sensor powered by a sediment microbial fuel cell. Journal of Power Sources, 196, 1171-1177.

Douglass, J.F., Radosevich, M., Tuovinen, O.H. (2014). Mineralization of atrazine in the river water intake and sediments of a constructed flow-throughwetland. Ecological Engineering, 72, 35-30.

Dušek, J., Picek, T., & Čížková, H. (2008). Redox potential dynamics in a horizontal subsurface flow constructed wetland for wastewater treatment: Diel, seasonal and spatial fluctuations. Ecological Engineering, 34, 223-232.

Fan, Y., Hu, H., & Liu, H. (2007). Enhanced Coulombic efficiency and power density of air-cathode microbial fuel cells with an improved cell configuration. Journal of Power Sources, 171, 348-354.

Fang, Z., Song, H. L., Cang, N., & Li, X. N. (2013). Performance of microbial fuel cell coupled constructed wetland system for decolorization of azo dye and bioelectricity generation. Bioresource Technology, 144, 165-171.

Ghangrekar, M. M., & Shinde, V. B. (2007). Performance of membrane-less microbial fuel cell treating wastewater and effect of electrode distance and area on electricity production. Bioresource Technology, 98, 2879-2885.

Inglesby, A. E., & Fisher, A. C. (2012). Enhanced methane yields from anaerobic digestion of Arthrospira maxima biomass in an advanced flow-through reactor with an integrated recirculation loop microbial fuel cell. Energy & Environmental science, 5, 7996-8006.

Jadhav, G. S., & Ghangrekar, M. M. (2009). Performance of microbial fuel cell subjected to variation in pH, temperature, external load and substrate concentration. Bioresource Technology, 100, 717-723.

Jonathan W.C., Wong, R.D. Tyagi and Ashok Pandey. (2017). Current development in biotechnology and bioengineering: solid waste management. Elsevier B.V.

Jun X., Wan. R. W. D., Mostafa G., Kien B. L., Manal I. (2013). Ion exchange membranes as separators in microbial fuel cells for bioenergy conversion: A comprehensive review. Renewable and Sustainable Energy Reviews, 28, 575-587.

Jung, S., & Regan, J. M. (2007). Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors. Appllied Microbiol Biotechnology, 77, 393-402.

Lee, H. S., Parameswaran, P., Kato-Marcus, A., Torres, C. I., & Rittmann, B. E. (2008). Evaluation of energy-conversion efficiencies in microbial fuel cells (MFCs) utilizing fermentable and non-fermentable substrates. Water Research, 42, 1501- 1510.

Liu, R., Zhao, Y., Doherty, L., Hu, Y., & Hao, X. (2015). A review of incorporation of constructed wetland with other treatment processes. Chemical Engineering Journal, 279, 220-230.

Liu, S., Song, H., Li, X., & Yang, F. (2013). Power Generation Enhancement by Utilizing Plant Photosynthate in Microbial Fuel Cell Coupled Constructed Wetland System. International Journal of Photoenergy, 2013, 1-10.

Logan, B. E. (2008). Microbial Fuel Cells, Wiley-Interscience. 216p .

Logan, B. E. (2012). Essential data and techniques for conducting microbial fuel cell and other types of bioelectrochemical system experiments. ChemSusChem, 5, 988- 994.

Logan, B. E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., . . . Rabaey, K. (2006). Microbial Fuel Cells: Methodology and Technology. Environmental Science & Technology, 40, 5181-5192.

Logan, B. E., & Rabaey, K. (2012). Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies. Science, 337, 686- 690.

Lu, L., Xing, D., & Ren, Z. J. (2015). Microbial community structure accompanied with electricity production in a constructed wetland plant microbial fuel cell. Bioresource Technology, 195, 115-121.

Mansoorian, H. J., Mahvi, A. H., Jafari, A. J., Amin, M. M., Rajabizadeh, A., & Khanjani, N. (2013). Bioelectricity generation using two chamber microbial fuel cell treating wastewater from food processing. Enzyme and Microbial Technology, 52, 352-357.

Mantovi, P., Marmiroli, M., Maestri, E., Tagliavini, S., Piccinini, S., & Marmiroli, N. (2003). Application of a horizontal subsurface flow constructed wetland on treatment of dairy parlor wastewater. Bioresource Technology, 88, 85-94.

Min, B., Kim, J., Oh, S., Regan, J. M., & Logan, B. E. (2005). Electricity generation from swine wastewater using microbial fuel cells. Water Research, 39, 4961-4968.

Rabaey, K., & Verstraete, W. (2005). Microbial fuel cells: novel biotechnology for energy generation. Trends in Biotechnology, 23, 291-298.

Rabaey, K., Lissens,G., Siciliano, S.D., Verstraete, W. (2003). A microbial fuel cell capable of converting glucose to electricity at high rate and efficiency. Biotechnology Letters, 25, 1531-1535.

Rinaldi, A., Mecheri, B., Garavaglia, V., Licoccia, S., Nardo, P. D., & Traversa, E. (2008). Engineering materials and biology to boost performance of microbial fuel cells: a critical review. Energy & Environmental science ,1, 417-429.

Rismani-Yazdi, H., Carver, S. M., Christy, A. D., Yu, Z., Bibby, K., Peccia, J., & Tuovinen, O. H. (2013). Suppression of methanogenesis in cellulose-fed microbial fuel cells in relation to performance, metabolite formation, and microbial population. Bioresource Technology, 129, 281-288.

Rismani-Yazdi, H., Christy, A. D., Carver, S. M., Yu, Z., Dehority, B. A., & Tuovinen, O. H. (2011). Effect of external resistance on bacterial diversity and metabolism in cellulose-fed microbial fuel cells. Bioresource Technology, 102, 278-283.

Rismani-Yazdi, H., Carver, S.M., Christy, A.D., Tuovinen, O.H 2008. Cathodic Limitations in Microbial Fuel Cells: An Overview. Journal of Power Sources 180(2): 683-694.

Rismani-Yazdi, H., Christy, A. D., Dehority, B. A., Morrison, M., Yu, Z., & Tuovinen, O. H. (2007). Electricity generation from cellulose by rumen microorganisms in microbial fuel cells. Biotechnology and Bioengineering, 97, 1398-1407.

Sanchez, D., Jacobs, D., Gregory, K., Huang, J., Hu, Y., Vidic, R., & Yun, M. (2015). Changes in Carbon Electrode Morphology Affect Microbial Fuel Cell Performance with Shewanella oneidensis MR-1. Energies, 8, 1817-1829.

Schröder, U. (2007). Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Physical Chemistry Chemical Physics, 9, 2619-2629.

Srinivasan S. 2006. Fuel cells: From fundamentals to applications. New York: Springer. 691p

Srivastava, P., Yadav, A. K., & Mishra, B. K. (2015). The effects of microbial fuel cell integration into constructed wetland on the performance of constructed wetland. Bioresource Technology, 195, 223-230.

Sun, D., Cheng, S., Wang, A., Li, F., Logan, B. E., & Cen, K. (2015). Temporal-spatial changes in viabilities and electrochemical properties of anode biofilms. Environmental Science & Technology, 49, 5227-5235.

Sun, J., Hu, Y., Bi, Z., & Cao, Y. Q. (2009). Simultaneous decolorization of azo dye and bioelectricity generation using a microfiltration membrane air-cathode single- chamber microbial fuel cell. Bioresource Technology, 100, 3185-3192.

Tchobanoglous, G., Burton, F.L., Stensel, H.D., Metcalf & Eddy. (2003) Wastewater engineering: treatment and reuse.

Villasenor, J., Capilla, P., Rodrigo, M. A., Canizares, P., & Fernandez, F. J. (2013). Operation of a horizontal subsurface flow constructed wetland--microbial fuel cell treating wastewater under different organic loading rates. Water Research, 47, 6731-6738.

Wang, H., Park, J. D., & Ren, Z. J. (2015). Practical energy harvesting for microbial fuel cells: a review. Environmental Science & Technology, 49, 3267-3277.

Wang, X., Feng, Y. J., & Lee, H. (2008). Electricity production from beer brewery wastewater using single chamber microbial fuel cell. Water Science and Technology, 57, 1117-1121.

Wu, H., Zhang, J., Ngo, H. H., Guo, W., Hu, Z., Liang, S., . . . Liu, H. (2015). A review on the sustainability of constructed wetlands for wastewater treatment: Design and operation. Bioresource Technology, 175, 594-601.

Yadav, A. K., Dash, P., Mohanty, A., Abbassi, R., & Mishra, B. K. (2012). Performance assessment of innovative constructed wetland-microbial fuel cell for electricity production and dye removal. Ecological Engineering, 47, 126-131.

Yang, F., Ren, L., Pu, Y., & Logan, B. E. (2013). Electricity generation from fermented primary sludge using single-chamber air-cathode microbial fuel cells. Bioresource Technology, 128, 784-787.

Yates, M. D., Kiely, P. D., Call, D. F., Rismani-Yazdi, H., Bibby, K., Peccia, J., . . . Logan, B. E. (2012). Convergent development of anodic bacterial communities in microbial fuel cells. ISME Journal, 6, 2002-2013.

Zhu, X., He, W., & Logan, B. E. (2015). Influence of solution concentration and salt types on the performance of reverse electrodialysis cells. Journal of Membrane Science, 494, 154-160.

Appendix A: Experimental data

Table A 1: Resistance decade box data for a step test with variable time intervals

Resistance Interval time (min) 1 M 40 0.5 M 30 250 K 124 100 K 63 50 K 68 25 K 45 10 K 133 7.5 K 42 5 K 133 2.5 K 42 1 K 49 900 93 800 148 700 74 600 69 500 100 400 77 300 76 200 257 100 20 50 215 25 168 10 195

Figure A1: Averaged voltage and power outputs of CW-MFC systems’ step test

Figure A2: Voltage and power outputs of CW-MFC system 114 step test

Figure A3: Voltage and power outputs of CW-MFC system 115 step test

Figure A4: Voltage and power outputs of CW-MFC system 116 step test

Figure A5: Averaged current density and power density versus external resistance during step test

Figure A6: Current density and power density versus external resistance of system 114 during step test

Figure A7: Current density and power density versus external resistance of system 115 during step test

Figure A8: Current density and power density versus external resistance of system 116 during step test with