Luận án tiến sĩ Triết học: Simultaneous carbon and nitrogen removal accompanied by energy recovery from wastewater in a coupled microbial fuel cells system

ACCOMPANIED BY ENERGY RECOVERY FROM

WASTEWATER IN A COUPLED MICROBIAL FUEL CELLS SYSTEM

BY

NGUYEN HOANG DUNG

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY (ENGINEERING AND TECHNOLOGY)

SIRINDHORN INTERNATIONAL INSTITUTE OF TECHNOLOGY THAMMASAT UNIVERSITY

ACADEMIC YEAR 2022

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FUEL CELLS SYSTEM

was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy (Engineering and Technology)

(Professor Pruettha Nanakorn, D.Eng.)

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Dissertation Title SIMULTANEOUS CARBON AND NITROGEN REMOVAL ACCOMPANIED BY ENERGY RECOVERY FROM WASTEWATER IN A COUPLED MICROBIAL FUEL CELLS SYSTEM

Author Nguyen Hoang Dung

Degree Doctor of Philosophy (Engineering and Technology)

Faculty/University Sirindhorn International Institute of Technology/ Thammasat University

Dissertation Advisor Professor Sandhya Babel, D.Tech.Sc Academic Years 2022

ABSTRACT

For over a decade, microbial fuel cell (MFC) has received much attention as a pioneering technology for wastewater treatment, owing to its ability to generate electricity from organic matter However, most wastewater has the presence of nitrogen and the requirement to remove nitrogen as a contaminant has caused limitations in energy recovery from organic carbon Despite the advantages of integrating biological nitrogen removal (BNR) into MFC and the promising results in organic carbon removal, achieving high nitrogen removal efficiency while optimizing energy recovery from organic matter remains a challenge It is difficult to select an external resistance (ER) value that satisfies both goals The chambers of a stacked MFC system can be sequencing-batch operated to achieve specific goals System can configured accordingly with suitable conditions for each chamber to achieve the goal of energy recovery from organic carbon and nitrogen removal

In this study, BNR was integrated into a coupled MFC with four chambers sequencing-batch operated reactor With ER close to its internal resistance, the first MFC (N-MFC) was responsible for power generation from organic carbon and ammonium oxidation in the input wastewater The ER of the second MFC (D-MFC)

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was set at a small value (10 Ω) to have a high current density, facilitating nitrogen removal in presence of minimum carbon as required for the denitrification The study evaluated the removal efficiency of carbon and nitrogen accompanied by power generation by applying three different sequencing-batch operation modes to four chambers of a coupled MFC In the first mode, wastewater transferred from the N-MFC anode chamber to the cathode chamber, then to the D-MFC anode chamber, and lastly to the D-MFC cathode chamber In the second and the third modes, the wastewater was fed into anode chamber of the N-MFC The D-MFC received the N-MFC output (in order from anode chamber to anode chamber, from cathode chamber to cathode chamber) The output of the cathode chamber of the D-MFC was the effluent of the coupled MFC system, while the output of the anode chamber of the D-MFC came back to the cathode chamber of the N-MFC The distinction between the second and third modes is the different dissolved oxygen (DO) in the cathode chamber of N-MFC to control the nitrification process The study provided a solution for optimizing electrical energy recovered from organic matter in parallel with efficient nitrogen treatment and better understanding of integrating BNR process into MFC technology Depending on each operation mode, the following specific objectives are proposed: (i) to investigate the effect of DO, and initial ammonium concentration for ammonium oxidizing and power generation from N-MFC; (ii) to assess the ability of ammonium diffusion through cation exchange membrane (CEM) in N-MFC; (iii) to investigate the effect of the COD/N ratio for nitrogen removal in D-MFC; (iv) to evaluate the coulombic efficiency of the system; (v) to evaluate the ratio of autotrophic denitrification in cathodic chamber of D-MFC

The following is a summary of the results and findings of this study

(i) In the N-MFC, the second operational mode produced more power than the first operational mode by addressing the flaws that impeded electricity production in the first operational mode Overall, power generation decreases as DO at the cathode decreases, while variations in nitrogen input showed no great influence on power generation of the N-MFC Ammonium was completely oxidized to nitrate as the major product with very small amounts of nitrite detected under high DO at the cathode chamber In the third operational mode with low DO at the cathode chamber of the N-MFC, nitrite was the main product of the ammonium oxidation process

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(ii) The input COD of the anode chamber and the DO concentration in the cathode chamber affect the electricity generation of the N-MFC, directly influencing the diffusion of cations (such as ammonium) from the anode chamber to the cathode chamber in order to balance the charge In addition, the ER of N-MFC in the second operational mode (50 Ω) is lower than that in the third operational mode (100 Ω), resulted in more favorable current production and more ammonium diffusion for charge balance

(iii) The nitrogen removal efficiency at the D-MFC increased when the COD/N ratio of wastewater entering the D-MFC increased The first operational mode, which used both the anode and cathode chambers for denitrification, enhanced nitrogen removal efficiency In the second and third operational modes, the denitrification process only occurred in the cathode chamber of D-MFC The nitrogen removal efficiency at the cathode chamber of the D-MFC was higher in the third operational mode than in the second operational mode for the same COD/N ratio input to the D-MFC, which is the result of a higher reduction rate of nitrite than nitrate

(iv) In the first operational mode, an increase in the COD input of the anode chamber resulted in a decrease in the anodic coulombic efficiency The anodic coulombic efficiencies of N-MFC in the second operational mode was higher than that in the first operational mode The anodic coulombic efficiencies of N-MFC in the third operational mode were relatively low because of low DO in the cathode chamber

(v) In all operational mode, the high ratio of autotrophic denitrification in the cathode chamber of the D-MFC demonstrated that autotrophic denitrification was the primary process assisting in nitrogen removal

In conclusion, this study showed that a properly configured and operated coupled MFC can effectively remove carbon and nitrogen with energy recovery from wastewater By taking advantage of ammonium diffusion across the CEM to improve the operating method, the second operational mode outperformed the first operational mode with respect to power generation and coulombic efficiency The suitable setup of the system allowed the N-MFC to oxidize over 75% of organic matter input and isolate nitrogen input simultaneously, giving favorable environmental conditions for generating the energy from wastewater primarily in the N-MFC The nitrogen isolation efficiency of the N-MFC depends on input organic matter of the anode chamber, the

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DO concentration in the cathode chamber, and the ER In the second and the third operational mode, the main mechanism for nitrogen removal at the D-MFC was autotrophic denitrification When comparing the second and the third operational modes, the power generation was higher in the second mode, which followed a conventional nitrification/denitrification system However, the third mode with shortcut nitrification-denitrification was more energy-efficient and better in nitrogen removal

Keywords: Biodegradation, Anaerobic process, Nitrogen removal, Microbial fuel cell, Energy recovery

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Prof Sandhya Babel, for her patient guidance, continuous support, and valuable encouragement throughout my Ph.D journey Her expertise, kindness, and dedication have been uncountable to me

I am also deeply grateful to my thesis committee members, Prof Nipon Pisutpaisal, Assoc Prof Paiboon Sreearunothai, Assoc Prof Rachnarin Nitisoravut, Dr Warunsak Liamlaem and Assoc Prof Jenyuk Lohwacharin, for their insightful feedback and constructive criticism Their comments have been instrumental in shaping my research

I thank my colleagues and friends, Dr Tuan Anh Ta, Dr Chamath D.Y Yahampath Arachchige Don, and Mr Tan Thong Nguyen, for contributing to the experimental model construction, proofreading, and helpful brainstorming

I am indebted to the staff and faculty of Sirindhorn International Institute of Technology (SIIT), Thammasat University, for providing a stimulating research environment and many personal growth opportunities This work would not have been possible without the full financial support of SIIT through an EFS scholarship in the School of Bio-Chemical Engineering and Technology

Finally, I would like to give my heartfelt appreciation to my family, especially to my father Van Nghia Nguyen, my mother Hong Phuong Ha, my brother Trung Hieu Nguyen, my sister Ngoc Lan Nguyen, my brother Hoang Long Nguyen, for their unwavering love throughout my abroad study Their sacrifices and understanding have been my constant source of strength

Thank you all for your inspiration

Nguyen Hoang Dung

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2.5 Influencing factors for carbon and nitrogen removal in an MFC 27

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2.6 Challenges in using MFC for carbon and nitrogen removal 30

CHAPTER 3 RESEARCH METHODOLOGY 32

CHAPTER 4 RESULTS AND DISCUSSION 43

4.4.3 Shortcut nitrification 754.4.4 Denitrification at the D-MFC 764.5 The advantages and disadvantages of three different operational modes:

4.6 Characterization of electrode surfaces 81

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 84

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LIST OF TABLES

2.1 Redox reactions with theoretical potentials standard conditions in MFC 82.2 The advantages and disadvantages of some electrode materials 132.3 The performance with pros and cons of various MFC configurations for

simultaneous removal of carbon and nitrogen 192.4 Some microorganisms and their roles in MFC system 274.1 COD output of each chamber of MFC 494.2 Nitrogen parameter outputs of each chamber of MFC 514.3 A comparion of three different operational modes with same influent

4.4 EDS analysis before and after operation 83

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LIST OF FIGURES

2.1 Diagram of MFCs technology principles in wastewater treatment 9

2.3 Shortcut nitrification-denitrification process in MFCs 222.4 Mechanism of heterotrophic anodic denitrification process in MFCs 243.1 The main components of the coupled MFCs 323.2 The procedure for pretreatment of CEM 333.3 The procedure for pretreatment of electrode 333.4 Schematic diagram of start-up stage 353.5 Summary of operational stage 363.6 Schematic diagram of the first operational mode 373.7 The second mode operating schematic of the coupled MFC system 383.8 The third mode operating schematic of the coupled MFC system for shortcut nitrification-denitrification 404.1 Cell votage progression (Rext = 500 Ω) during 30 days start-up in both MFC

4.8 Cell voltage of N-MFC and D-MFC during the second operational mode

(A, B, C – nitrogen input of 50, 75, 100 mg L-1, respectively) 584.9 Chemical analysis of the effluent from each chamber of the coupled MFC

system in the second operational mode (n.d: not detectable) 614.10 Output pH of chambers in the coupled MFC with different nitrogen influents 644.11 Cell voltage progression of the coupled MFC system during operation

(1, 2, 3, 4 correspond to the first to fourth operating conditions) 664.12 Cell voltage vs current of N-MFC when conducting polarization test

(1, 2, 3, 4 correspond to the first to fourth operating conditions) 714.13 Chemical analysis of the effluent from each chamber of the coupled MFC system in four operating conditions (n.d: not detectable) 734.14 The pH of each chamber’s output under various operating conditions 784.15 SEM images of electrodes (O – before operation; A, B, C, D – anode of

N-MFC, cathode of N-MFC, anode of D-MFC, cathode of D-MFC after

operation, respectively) 82

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MET Mediated Electron Transfer MFC Microbial Fuel Cell

NOB Nitrite-Oxidizing Bacteria

SND Simultaneous Nitrification-Denitrification TOC Total Organic Carbon

UASB Upflow Anaerobic Sludge Blanket

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CHAPTER 1 INTRODUCTION

1.1 Problem statement

Organic matter contains carbon, hydrogen, oxygen, and other trace elements and is the primary pollutant in wastewater The release of wastewater with a significant quantity of organic matter endanger the water ecosystem because the degradation of these organic compounds utilizes dissolved oxygen (DO) in water, causing a lack of DO for aquatic life (Nora’aini et al., 2005) Besides carbon, nitrogen is also a notable parameter in wastewater High nitrogen concentrations cause eutrophication in ponds, lakes, canals, rivers, and coasts As a result, the planktonic algae overgrowth depletes the DO in water, clogs water intakes, blocks light to deeper waters, and destroys the ecosystem (Howarth and Marino, 2006) An excessive amount of nitrate in groundwater can cause methemoglobinemia, which interferes with the circulation of oxygen in the blood (Feleke and Sakakibara, 2002) Besides, the water bodies where wastewater is received may be the influent of the water treatment plant, supplying water for humans As a result, the cost of water treatment to meet standards for drinking or production Therefore, wastewater treatment is essential before discharging it into the environment Carbon and nitrogen criteria always received special attention from the designers of wastewater treatment plants when choosing technology

Most organic compounds in wastewater are easily degraded by microorganisms In most wastewaters, nitrogen is encountered mainly as ammonium and organic nitrogen (Rittmann and McCarty, 2001) Organic nitrogen is converted to ammonium through the ammonification process made by heterotrophic bacteria Part of the ammonium in wastewater is used as the N source to synthesize new biomass Nitrification and denitrification processes can eliminate the rest The nitrification process consists of successive nitrogen oxidation steps, which are nitritation and nitratation Nitritation is the process in which ammonia-oxidizing bacteria (AOB) oxidize ammonium to nitrite, while nitratation involves nitrite-oxidizing bacteria (NOB) converting nitrite to nitrate The denitrification process includes consecutive nitrogen oxides reduction steps, the desired final product of which is nitrogen gas, removed from

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the wastewater Denitrification bacteria use the biodegradable organic carbon as the source of electrons in these processes Many technologies can currently be selected for wastewater treatment, most of which use biotechnology because of its sustainability and low cost Conventional technologies, like the anoxic-oxic (AO) system based on the activated sludge processes are favored for its ability to remove organic substance and nitrogen simultaneously However, these treatment methods consume a significant quantity of energy for aeration, which accounts for nearly half of the total operational energy (Drewnowski et al., 2019) This energy is required to provide oxygen for the oxidation of organic matter and results in the generation of substantial sludge that needs to be properly managed Anaerobic biological technology, such as upflow anaerobic sludge blanket (UASB) reactors, offers advantages including not requiring air supply, low solid yield, methane production as biofuel However, anaerobic treatment has low organic removal efficiency and is very limited in nitrogen removal, resulting in low-quality effluent and needs additional treatment for the remaining pollutant Slow startup and high-temperature requirements are also disadvantages of anaerobic treatment (Chong et al., 2012) In addition, produced biogas needs to be pretreated (separation and purification) before being used to generate electricity In terms of cost-benefit analysis, this is not feasible for small wastewater treatment plants (Gude, 2016) The anammox microorganisms derive their energy from the reaction of ammonium and nitrite to generate nitrogen gas Therefore, anammox processes with the participation of AOB and anammox bacteria have some outstanding benefits such as oxygen-saving for nitrification, eliminating external carbon source requirement for denitrification, and low sludge production (Cho et al., 2010) Nevertheless, its drawback is the complicated operational control The anammox biomass not only grows extremely slowly but is also easily washed out during operation (Huynh et al., 2019)

In certain wastewater types where the C/N ratio is imbalanced due to carbon source deficiency, achieving complete nitrogen removal is not possible (Smith and Solley, 2002) Therefore, the addition of external carbon sources such as methanol, ethanol, molasses becomes necessary to achieve the desired C/N ratio for heterotrophic denitrification This has resulted in concerns about increased treatment costs as well as safety issues in chemical transport and storage Even if the C/N ratio is theoretically sufficient, conventional technology still needs very high internal recirculation from

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aerobic to anoxic for high nitrogen removal efficiencies, resulting in increased pumping cost Also, the aerobic tank's circulating flow with a high DO concentration causes an adverse environment for denitrification at the anoxic tank Another limitation of the DO presence in the anoxic tank is that aerobic digestion of readily biodegradable organic carbon wastes the electron supplier for denitrification (Sander et al., 2017)

Lately, microbial fuel cells (MFCs) technology provides an encouraging solution to treat wastewater sustainably, as it can directly generate electricity using wastewater (Priya et al., 2022; Sun et al., 2016) Most MFC reactors consist of anode and cathode electrodes placed separately in two chambers An ion-exchange membrane is employed to split up electrolyte solutions in two chambers Microorganisms can be used in MFC as biological catalysts, oxidizing organic substances in the electrolyte solution and producing electrons flow from anode to cathode, while ions diffuse across the membrane in the opposite direction to maintain a neutral charge The ability of MFCs to harness the energy stored in the chemical bonds of organic matter present in wastewater and convert it into electrical power aligns with the new perspective of considering wastewater as a valuable resource In terms of wastewater treatment, MFCs can remove carbon and nitrogen simultaneously through a series of redox reactions at the two electrodes Electrons produced at the anode through carbon oxidation reactions flow via an electrical circuit to the cathode, where they are utilized in the reduction reaction, combining with nitrate to form nitrogen gas Thanks to the bacteria and electrolytes separation in two chambers, the amount of organic carbon oxidized by aerobic microorganisms is minimized, thus reducing the C/N ratio requirement compared to conventional technologies (Virdis et al., 2011) The denitrification process that occurs at the cathode of MFCs includes both bioelectrochemical denitrification (autotrophic denitrification) and conventional denitrification (heterotrophic denitrification) (Zhang and He, 2012) In particular, autotrophic denitrification has some benefits compared with the heterotrophic system such as lower biomass formation, less organic matter required, no need further steps for removing the excess substrate (Van Rijn et al., 2006) Besides, the employment of anaerobic microorganisms to oxidize the carbon at the anode reduces sludge generation as well as aeration energy of the whole MFCs system (since oxygen is only required for the nitrification process), while electrical power generated has been shown to partially support the pumping

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system (Zhang and He, 2012) Wang et al (2013) reported that recovered energy from MFCs helped to save 30–50% in operational costs

Because of these apparent advantages, MFCs have attracted many researchers with a significant quantity of publications in the field of treating wastewater (Santoro et al., 2017) Most of the research aims to improve the power recovered from wastewater and the efficiency of removing carbon and nitrogen Some modified configurations of MFCs have been applied, such as two-chambers MFC (Virdis et al., 2008), single-chamber MFC with the exposed-air cathode (Hussain et al., 2016), single-chamber MFC with the rotating cathode (Zhang et al., 2013), membrane-less MFC (Zhu et al., 2013), tubular dual-cathode MFC (Zhang and He, 2012), oxic/anoxic-cathode MFC (Xie et al., 2011), stacked five-units MFC (Park et al., 2017) In parallel, many types of ion-exchange membranes and electrode materials are also tested, and certain performance improvements are achieved Although the results showed a good carbon removal efficiency above 80%, the nitrogen removal efficiency fluctuated significantly and ranged from 36% to 97%, depending on experimental conditions The parameters affecting the nitrogen removal in MFC were investigated and evaluated such as carbon sources (Feng et al., 2013), C/N ratio (Huang et al., 2013a), pH (Clauwaert et al., 2009), DO (Virdis et al., 2010), and electrolyte conductivity (Puig et al., 2012) Because successful nitrogen removal in wastewater involves nitrification and denitrification processes, researchers found some methods to integrate these two processes into MFC technology Some studies conducted nitrification and denitrification separately by using an independent nitrification reactor (Virdis et al., 2008), using a rotating cathode or dual cathode (Xie et al., 2011; Zhang and He, 2012; Zhang et al., 2013) Some other research performed simultaneously these two processes by controlling aeration in the cathodic chamber (Virdis et al., 2010; Wu et al., 2017) or using the air cathode (Hussain et al., 2016)

Although nitrite has been proven capable of replacing nitrate to become the primary electron acceptor from the cathode (Puig et al., 2011; Virdis et al., 2008), the shortcut nitrification/denitrification process in MFC has not been fully understood Theoretically, this process could decrease oxygen required for nitrification by 25% and carbon needed for denitrification by 40% compared with nitrogen removal via nitrate In addition, the nitrite reduction rate can be up to 2 times faster than nitrate, while sludge

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generation is also decreased by 40% in the nitritation/denitritation process (Beylier et al., 2011) In MFCs, nitrite's electron utilization is better than that of nitrate, resulting in higher coulombic efficiency on cathodic reaction (Virdis et al., 2008) At high temperature (> 30oC), AOB biomass may dominate over NOB biomass, favoring nitrite accumulation for denitritation process (Hellinga et al., 1998) Therefore, industrial wastewater released with high temperature as dyeing wastewater may be suitable for the shortcut nitrification/denitrification process without wastewater heating

Most MFC reactors were designed with a separator between the two chambers to prevent crossover processes that could have adverse effects, drastically reducing MFC performance (Santoro et al., 2017) The fundamental requirement of the separator is to be conductive for ions to balance the overall charge transfer during the operation of the MFC Therefore, ion exchange membranes are a good choice for separators, such as cation exchange membranes (CEMs) However, they are pricey, accounting for the significant investment costs of MFC systems In addition, the poor selectivity of CEMs and the overwhelming concentrations of other cations such as Na+, K+, and NH4+

compared to protons in wastewater resulted in low protons transfer efficiency (Rozendal et al., 2006) This creates a pH imbalance between the two chambers, which leads to a decrease in MFC performance (Nguyen and Babel, 2022) However, from another perspective, this weakness can be exploited to an advantage by using ion exchange membranes to isolate nitrogen from influent wastewater This allows an MFC reactor to be fully configurable to optimize energy recovery from organics while the isolated ammonium oxidation contributes to pH balance Recent research also explored membrane penetration of nitrogen and took advantage of them to aid in nitrogen removal in a dual-chamber MFC (Jin et al., 2022)

Despite the advantages of integrating biological nitrogen removal (BNR) into MFC and the promising results in organic carbon removal, achieving high nitrogen removal efficiency while optimizing energy recovery from organic matter remains a challenge A dual-cathode chamber MFC attained a total nitrogen removal of 99.9%, but its power generation was low at 294.9 mW m-2 despite DO conditions of 3.5 (Li et al., 2016) A single MFC reactor, operated with an external resistance (ER) of 10 Ω, can attain a peak power density of 4.2 W m-3 and an overall nitrogen removal of less than 80% (Wu et al., 2017) Theoretically, these two objectives are difficult to

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accomplish concurrently in an MFC reactor Firstly, high nitrogen removal occurs at the lowest possible ER of an MFC (Li et al, 2014) However, to obtain maximum power density, ER needs to be set close to internal resistance (IR) of the MFC system (Chen et al., 2019; Li et al., 2016; Park et al., 2017) Basically, utilizing nitrate or nitrite as electron acceptors at the cathode causes the IR to be high compared to utilizing oxygen for the same purpose (Guo et al., 2020; Li et al., 2016; Zhang et al., 2020) As a result, it is difficult to select an ER value that satisfies both goals Secondly, O2/H2O has a higher redox potential (+0.82 V) than that of NO3-/N2 (+0.75 V) and NO2-/N2 (+0.35 V) (Nguyen and Babel, 2022) Therefore, oxygen reduction can produce a higher cell voltage than denitrification at the cathode However, the presence of oxygen at the cathode has a negative impact on the denitrification process, decreasing the nitrogen removal efficiency Finally, conducting simultaneous nitrification-denitrification (SND) in an MFC reactor requires complex control of operating parameters and does not benefit in the power generation from organic material biodegradation (Virdis et al, 2010) A dual-cathode MFC (Li et al, 2016; Zou et al, 2018) or a coupled MFC system (Chen et al, 2019; Xie et al, 2011) has been used to separate the nitrification and denitrification processes It has been reported that a stacked MFC with the effluent of one unit being the influent of the next unit can help improve electricity generation by decreasing IR within the system (Jadhav et al., 2021; Walter et al., 2016) Five stacked MFC units successfully treated low-strength wastewater and reduced the hydraulic retention time (HRT) to 2.5 h (Park et al, 2017)

To address these limitations, this study aimed to operate a coupled MFC to treat synthesis wastewater The chambers of a coupled MFC system can be sequencing-batch operated with some specific goals established to design a proper configuration and control the favorable conditions for each chamber (Nguyen and Babel, 2022) Setting suitable levels of different parameters in each chamber is required to achieve the goal of energy recovery from organic carbon and nitrogen removal With this approach, in this study, BNR was integrated into a coupled MFC with four chambers sequencing-batch operated to enhance nitrogen removal and energy recovery To overcome the current gap in achieving this dual objective, distinct ERs were built for the two MFC reactors of a coupled MFC system The first MFC (N-MFC) was responsible for power generation from organic carbon and ammonium oxidation in the input wastewater The

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second MFC (D-MFC) facilitated nitrogen removal in presence of minimum carbon as required for the denitrification process

1.2 Research objectives

This study evaluates the removal efficiency of carbon and nitrogen accompanied by power generation from synthetic wastewater Different sequencing-batch operation modes are applied to four chambers of a coupled MFC for optimizing recovery of electrical energy from organic matter in parallel with efficient nitrogen treatment Results of study will offer a deeper understanding of integrating the BNR process into MFC technology Batch-sequencing operation is expected to bring a potential technical solution Depending on operation mode, the following specific objectives are proposed

(1) To investigate the effect of DO, and initial ammonium concentration for ammonium oxidation and power generation from N-MFC

(2) To assess the ability of ammonium diffusion through CEM in N-MFC (3) To investigate the effect of the COD/N ratio on nitrogen removal in D-MFC (4) To evaluate the coulombic efficiency of the system

(5) To evaluate the ratio of autotrophic denitrification in cathodic chamber of MFC

D-1.3 Scope of research

To meet the aforementioned objectives, two dual-chamber MFC reactors were constructed at a laboratory scale All experiments were conducted in sequencing-batch mode with synthesis wastewater Influent synthesis wastewater COD concentration was varied within 1000-2000 mg L-1, while ammonium nitrogen was between 50-100 mg L-1 Bio-catalysts in the anode and the cathode were inoculated by anaerobic and facultative microorganisms, respectively DO in the cathode chamber of N-MFC was controlled at three different values of 0.6 mg L-1, 1.2-1.24 mg L-1 and 4 mg L-1 The temperature of the cathode chamber of N-MFC was maintained at room temperature and 37oC Observed parameters are pH, COD, NH4+-N, NO2--N, and NO3--N from the output of each chamber and cell voltage generated from each MFC reactor.

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Reductant Anodic reaction Ean (V vs SHE) Acetate CH3COO- + 4H2O  2HCO3- + 9H+ + 8e- -0.279 Glucose C6H12O6 + 12H2O  6HCO3- + 30H+ + 24e- -0.414 Metanol CH3OH + 2H2O  HCO3- + 7H+ + 6e- -0.453 Glycerol C3H5(OH)3 + 6H2O  3HCO3- + 17H+ + 14e- -0.385 Carbon C + 2H2O  CO2 + 4H+ + 4e- -0.207 Oxidant Cathodic reaction Ecat (V vs

SHE) Nitrate NO3- + 2H+ + 2e-  NO2- + H2O +0.433

2NO3- + 12H+ + 10e-  N2 + 6H2O +0.749 Nitrite NO2- + 2H+ + e-  NO- + H2O +0.350 Nitric oxide NO- + H+ + e-  0.5N2O + 0.5H2O +1.175 Nitrous oxide 0.5N2O + H+ + e-  0.5N2 + 0.5H2O +1.355 Proton 2H+ + 2e-  H2 0 Oxygen O2 + 4H+ + 4e-  2H2O +0.815

O2 + 2H+ + 2e-  H2O2 +0.280

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Figure 2.1 Diagram of MFCs technology principles in wastewater treatment

The anodic oxidation reactions and the cathodic reduction reactions are referred to as half-cell reactions Its potentials can be calculated following Logan et al (2006)

ln redox

MG G RT

(mol/L) is the molar concentration of oxidants For example, if the MFCs reactor is setup following Figure 2.1, we have

CH COOE E

P H OE E

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assume that the ideal condition are [NO3-] = [H2O] = [CH3COO-] = [HCO3-] = 1 M, PN2

= 1 atm, pH = 7 ([H+] = 10-7 M) and do not change with time E0 can be calculated from given G0 (Alberty, 2006), we have the same results as shown in table 2.1

OvervoltageE E     IR

where a, |c| (V) are the overpotentials of the anode and cathode respectively, I (A) is the produced current, and RΩ is the ohmic resistance of the MFC system Besides, the actual cell voltage also can be calculated as

where OCV (V) is the open-circuit voltage measured immediately after the circuit removes, I (A) is the produced current, Rint is the IR of the MFC system

An indispensable factor in the operation of MFCs is the microbial capacity to transfer electrons to the surface of the electrode This type of microorganism is commonly referred to as electrochemically active bacteria or exoelectrogen Two primary electron transfer mechanisms are direct electron transfer (DET) (Fig 2.2) and mediated electron transfer (MET) described by Sun et al (2016) Besides transferring electrons directly to the electrode via outer membrane c-type cytochrome, the flavin bound to c-type cytochrome, Geobacter and Shewanella species can also form its biological nanowire, allowing them to transport electrons to the anode surface in further distances (Mook et al., 2013) These two species (e.g Geobacter metallireducens,

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Geobacter sulfurreducens, Shewanella oneidensis, Shewanella putrefaciens) were frequently detected in many MFCs studies MET is an indirect electron transfer performed by mediators, additionally provided externally or generated by certain microbes The mediators act as shuttles; they receive electrons from the microbe and transport them to the electrode's surface

Figure 2.2 DET mechanisms to anode

2.2 Design of MFCs

To date, the design of MFCs has reached many advances in the wastewater treatment study Researchers focused on MFCs configuration, electrode, separator, inoculum, and electrolyte solution to improve carbon and nitrogen removal efficiency, increase power productivity, minimize costs, and enhance the MFCs system's stability

2.2.1 Casing

The general feature of the materials used as the casing of the MFCs reactor in lab-scale is non-conductive, such as glass, plastic, polycarbonate, plexiglass The shapes of chambers are also varied The most common shapes used are cylindrical and

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rectangular Evaluating the impact of various geometries and dimensions on the performance of MFC reactors can be challenging (Krieg et al., 2014)

2.2.2 Electrode

Two electrodes play a fundamental role in electrochemical reactions, electron transfer, and EA biofilm formation Because MFCs reactor requires biocathode for nitrogen removal, to simplify model preparation, studies of MFCs for wastewater treatment often use the same material for both anode and cathode The electrode material is necessarily of good conduction to have the ability to transmit electrons, be stability to chemicals in wastewater, good biocompatibility to not inhibit the growth of bacteria, has a high specific surface area to create microbial attachment conditions (Santoro et al., 2017) Besides, the mechanical strength, price, and environmental impact should also be considered when choosing materials for electrodes The common materials used to fabricate electrode are carbonaceous-based material and non-corrosive metallic-based materials (Santoro et al., 2017) The advantages and disadvantages of these materials are listed in Table 2.2 To increase the microbial attachment to the electrode surface, increase EA biofilms formation and improve the MFCs performance, surface chemistry and surface morphology can be conducted on the electrode before installing Several methods for surface treatment of electrode have been reported, such as chemical treatments (methanol soaking, acid soaking) (Feng et al., 2010; Park and Zeikus, 2002), surface coatings with catalysts (Zhang et al., 2015), electrochemical treatments with strong oxidizing electrolytes (Zhou et al., 2012), thermal treatments (Cheng and Logan, 2007; Wang et al., 2009)

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Table 2.2 The advantages and disadvantages of some electrode materials Material Advantages Disadvantages Carbon cloth High surface area

Relatively high porosity High conductivity Flexibility

Mechanical strength

Relatively high cost

Carbon brush Relatively high surface area High conductivity

High cost due to its titanium core

Carbon rods Affordable cost Low surface area, so primarily utilized as current collectors rather than electrodes

Carbon mesh Relatively low-cost Low conductivity

Low mechanical strength Low porosity

Carbon veil Very cheap

Relatively high conductivity High porosity

Quite fragile

Carbon paper Relatively porous Expensive Fragile Granular

activated carbon (GAC)

Biocompatibility Low cost

Very porous High surface area

Relatively low conductivity

High mechanical strength

Low surface area and surface/volume ratio

Reticulated vitreous carbon (RVC)

Very conductive Great porosity

Quite fragile Very expensive Metallic-based

materials

(stainless steel, silver, nickel, copper, gold, titanium)

Very conductive Robust

Cheap

The release of copper and nickel ions from electrodes can be toxic to microbes

2.2.3 Separator

In principle, the separator is not an essential requirement Removing it will eliminate its high cost and membrane fouling, minimize IR and concentration

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polarization However, to prevent crossover processes that could have adverse effects, drastically reducing MFC's performance, most MFCs reactors were designed with a separator between the two chambers The separator must be conductive for ions to balance the overall charge transfer Therefore, the ion exchange membranes made from polymer were the good choice for separator Among them, the CEMs were used in most studies about MFCs Two common CEMs are Nafion (DuPont Inc., USA) and Ultrex CMI 7000 (Membranes Inc., USA) because they respectively contain hydrophilic sulfonate groups and sulphonic acid groups, which facilitates to transport of cations and protons (Li et al., 2011) However, the poor selectivity of CEMs and the overwhelming concentrations of other cations such as Na+, NH4+ compared to protons in wastewater resulted in the low protons transfer efficiency (Rozendal et al., 2006) This creates a pH imbalance between the two chambers, which leads to a decrease in MFC performance With the same volume of MFCs reactor, the large designed membrane area minimizes IR, resulting in increased power density (Logan et al., 2006) Anion exchange membranes (AEMs) with ammonium groups in polymer structure help anions travel between two chambers Surprisingly, AEMs have a better proton transfer ability than CEMs if phosphate is added to take the role of proton carrier and pH buffer (Li et al., 2011) However, the practice of AEMs results in the loss of substrates in the anodic chamber by diffusion of anions such as acetate across the membrane to arrive in the cathodic chamber, affecting electron production A bipolar membrane combined from CEM and AEM is suitable for desalination by favoring water splitting, but not proper for MFCs due to the use of two membranes at the same time dramatically increases IR (Li et al., 2011)

2.2.4 Inoculum and substrate

The formation of electroactive biofilms in the anode is particularly crucial in the MFCs reactor's operation, affecting bioelectrochemical performance It depends on the inoculum culture and the substrate used in the adaptation process (Santoro et al., 2017) A variety of inoculums can be used, from monocultures of bacteria to mixed culture, but their shared trait is that they require anaerobic conditions to grow Inoculation with complex mixed cultures helps MFCs better performance than using pure cultures because bacteria can use each other's mediators to accelerate the electron

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transfer rate Besides, mixed culture MFCs allows much broader substrate utilization because each microorganism is suitable with different substrates Du et al (2007) compiled a list of microbes that can transfer electrons and their substrate A wide range of biodegradable organic compounds can serve as substrates in the anodic chamber Its composition, characteristics, and concentration in electrolyte affect the efficiency of converting chemical energy into electricity Acetate owned simple structure is the common choice in most design of MFCs because it is less likely to participate in fermentation, methanogenesis, which does not contribute to electricity production and decrease coulombic efficiency Chae et al (2009) reported that MFCs used acetate as a substrate showed the highest power density and coulombic efficiency compared with butyrate, propionate, and glucose In addition, many different substrates in MFCs for electricity production were reviewed carefully (Pandey et al., 2016; Pant et al., 2010) With the goal of nitrogen removal, nitrifying and denitrifying bacteria were often cultured at the cathode Substrates such as ammonium, nitrite, and nitrate are often used for their growth and metabolism In practice, isolating to cultivate a single microbe requires many complicated techniques such as ultrasound, temperature, antibiotics, and fungicides (Santoro et al., 2017) It has also not proven effective in MFCs when compared with using mixed cultures Therefore, most MFCs studies used anaerobic sludge or activated sludge from the wastewater treatment plants as inoculum Another simple way to shorten acclimation time is ultilizing successfully cultured inoculum from a parent MFC Synthetic wastewater consists of substrates, nutrients, and some trace elements needed for microbial growth commonly used as electrolyte solutions in MFCs reactors Some recent studies also applied MFCs to treat actual wastewater treatment (Kim et al., 2008; Park et al., 2017; Ryu et al., 2013; Wen et al., 2010)

2.3 Recent MFCs studies for nitrogen and carbon removal, power generation Clauwaert et al (2007) pioneered the utilization of nitrate as an electron acceptor at the cathode Denitrification was shown to be achievable without H2

formation The discovery has garnered significant interest among researchers regarding the removal of nitrogen using MFC biocathodes

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2.3.1 Nitrification and cathodic denitrification in MFCs

Virdis et al (2008) conducted nitrification and denitrification separately, using an independent nitrification reactor combined with a dual-chamber MFC Their study was the first MFC application with complete nitrogen removal in wastewater A synthetic wastewater consisting of acetate and ammonium was continuously supplied to the anode chamber of an MFC It then flowed to an external aerobic reactor where ammonium in the influent underwent oxidation to form nitrate, and finally entered the cathode chamber of the MFC Electrons produced at the anode from acetate oxidation reactions are transferred via an electrical circuit to the cathode where nitrate captures electrons and undergoes reduction to form nitrogen gas The system had positive results with a nitrogen removal rate of 0.41 kg m-3 d-1 (net cathode volume) and a maximum power output of 34.6 W m-3 The COD/N removal ratio of 4.48 was lower than the typical values for the conventional technology and ranged between 10.5 and 12.5 (Beylier et al., 2011) Although a nitrification reactor has high ammonium oxidation efficiency, the cathode chamber effluent still had 26.9 mg L-1 ammonium at an ER of 5 Ohm This is because some of the ammonium ions diffuse from the anode chamber via the CEM

Virdis et al (2010) overcame this weakness by controlling the aeration in the cathodic chamber,enabling SND processes The achieved total nitrogen (TN) removal efficiency of the system was 94.1%, leaving only a small amount of ammonium (1 mg L-1) remaining in the effluent

With varied MFC configurations, subsequent studies performed nitrogen removal using separate or SND methods Xie et al (2011) operated a coupled MFC One MFC had an oxic cathode and the other had an anoxic cathode The synthesis wastewater is (separately) continuously fed to the two anode chambers of the coupled MFC The anodic effluents were transported to the oxic cathode for nitrification before being moved to the anoxic cathode for denitrification Ammonium concentration in the effluent was high due to ammonium losses caused by diffusion to the anoxic cathode through the CEM To alleviate this, a portion of the effluent was recirculated to the oxic cathode for additional nitrification In this system, the TN removal efficiency was up to 97.3%, and the maximum power density was 14 W m-3

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To save aeration energy, some MFC configurations with passive nitrification were investigated Zhang and He (2012) constructed a tubular dual-cathode MFC with an outer cathode for nitrification and an inner cathode for denitrification The system used an AEM instead of a CEM to separate the anode and the inner cathode, avoiding ammonium dispersion The wastewater enters the anode and moves to the outer cathode and finally to the inner cathode This system achieved a maximum power density of 6.8 W m-3 The ammonium removal efficiency was 96%; however, the TN removal was only between 66.7 and 89.6%, depending on different nitrogen loading rates corresponding COD/N ratios of 2.81, 4.21, and 8.43 At a high nitrogen loading rate, the effluent contained significant nitrate levels, demonstrating a limitation in the bioelectrochemical denitrification Some of the nitrate ions may diffuse through the AEM from the inner cathode and back to the anode, conducting heterotrophic denitrification at the anode, whereas acetate can diffuse in the opposite direction This resulted in a low Coulombic efficiency and decreased the MFC's performance at a high nitrogen loading rate

A single-chamber MFC with a rotating cathode was another system developed to passively supply oxygen and minimize the pump energy (Zhang et al., 2013) At a COD/N ratio of 5:1, 91.5% and 82.1% of TN were removed under the continuous feeding mode and batch mode, respectively The system achieved a maximum power density of 585 mW m-3 Hussain et al (2016) experimented with a single-chamber MFC with an exposed-air cathode Non-conductive nylon cloth was employed as a separator between the anode and cathode At an influent COD of 2067 mg L-1 and ammonium of 200 mg L-1, their removal efficiency achieved were 84% and 55%, respectively, without output nitrate and nitrite The Coulombic efficiency of the system was about 65%, while the achieved power output was 75 mW L-1

Nitrogen removal in an MFC was also studied on a larger scale with a reactor volume of 7.2 L (Yu et al., 2011) Their research implements two different air supply methods on the MFC via an air diffuser and through a gas-permeable membrane The membrane aerated MFC system showed a better nitrogen removal at a low DO of 0.5 mg L-1 Ammonium can diffuse into the membrane biofilm and achieve SND based on the oxygen-permeability of the membrane Wu et al (2017) performed a similar experiment with a conductive, aerated membrane as the biocathode Their study

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reported a maximal TN removal efficiency of 80.82% at a low COD/N ratio of 2.8 The Coulombic efficiency also improved, leading to a maximum power density of 4.2 W m-3 Besides single MFC systems, a stacked MFC system, including five air-cathode MFC units connected in series, successfully removed 85% of COD and 94% of TN under a short hydraulic retention time (HRT) of 2.5 h (Park et al., 2017)

Apart from experiments with synthetic wastewater, some studies also applied MFCs to treat actual wastewater For instance, four MFC reactors using different electrode materials and separators were tested to treat piggery wastewater (Ryu et al., 2013) The COD removal rate was 0.523 kg m-3 d-1 (anode compartment volume) and the TN removal rate was 0.194 kg m-3 d-1 (cathode compartment volume) The maximum power density was relatively low at 1415.6 mW m-3 (with a CEM as the separator) because of the poor buffer capacity of real wastewater

The performance with pros and cons of various MFC configurations for nitrogen removal are listed in Table 2.3 When comparing the different configurations so far investigated and discussed above, the stacked air-cathode MFC system of Park et al (2017) excelled at removing COD and TN from actual domestic wastewater below the discharge limit This model has a short HRT and eliminates the requirement for aeration and external carbon supply Basically, a stacked MFC system is created by dividing a large MFC into many smaller modules, causing the water flow to be better distributed It also increasing the ratio of electrode surface area to system volume, and thus improving contaminant removal performance However, the cost to build a stacked MFC is higher than usual The recovery current from wastewater treatment by MFCs is relatively small, and a stacked MFC further divides that current, reducing its practicality Based on the benefits of MFCs technology in nitrogen removal, such as the energy efficiency and the capacity to treat wastewater with low C/N ratios, the shortcut nitrification/denitrification process has also attracted many researchers

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Table 2.3 The performance with pros and cons of various MFC configurations for simultaneous removal of carbon and nitrogen

Two-chamber MFC and nitrification reactor

Electrode: Granular graphite Separator: CEM

Virdis et al (2008) Two-chamber MFC

Electrode: Granular graphite Separator: CEM

COD removal: 100% TN removal: 94.1%

Power density: 0.44-1.58 W m-3

Successfully perform SND in the cathode chamber

(2010) Coupled MFC

Electrode: Graphite felt Separator: CEM

COD removal: 98.8% TN removal: 97.3% NH4+ removal: 97.4% Power density: 14 W m-3

High nitrogen removal efficiency

Low Coulombic efficiency for glucose oxidation

High COD/N for denitrification of 15-16

Xie et al (2011)

Tubular dual-cathode MFC Electrode: Carbon cloth (anode, inner cathode), platinum coated carbon cloth (outer cathode)

Separator: CEM, AEM

COD removal: 85-99% TN removal: 66.7-89.6% Power density: 6.8 W m-3

Passive nitrification with no aeration

The energy production higher than energy consumption

Low performance at high nitrogen loading rate

Nitrate diffusion through AEM

Zhang and He (2012)

Single-chamber MFC with a rotating biocathode

Electrode: Carbon felt Separator: None

Total organic carbon (TOC) removal: 85.7%

TN removal: 91.5%

Power density: 585 mW m-3

Passive nitrification High nitrogen removal at COD/N of 5:1

Low Coulombic efficiency because of the open system

Zhang et al (2013)

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Table 2.3 The performance with pros and cons of various MFC configurations for simultaneous removal of carbon and nitrogen (Cont.)

Single-chamber air cathode MFC

Electrode: Carbon felt anode and gas diffusion cathode Separator: Nylon cloth

Low power production and nitrogen removal

Hussain et al (2016)

Two-chamber MFC with a clarifier

Electrode: Carbon paper Air supply through gas-permeable membrane Separator: None

COD removal: 97% TN removal: 52%

Large-volume reactor of 5 L

Inexpensive electrode No ion exchange membrane Nitrogen removal capability at low DO of 0.5 mg/L

Low nitrate reduction efficiency

Yu et al (2011)

Two-chamber MFC Electrode: Carbon felt

(anode), conductivity aerated membrane (cathode)

High nitrogen removal at

low COD/N ratio of 2.8 Low Coulombic efficiency 40.67%)

(9.27%-Wu et al (2017)

Five stacked MFCs with cathodes

air-Electrode: Graphite felt (anode), Pt coated carbon cloth (cathode)

Separator: Non-woven fabric

COD removal: 85%

TN removal: 94% Experiment with actual domestic wastewater The effluent met Korean discharge standard with short HRT of 2.5 h

(2017)

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Table 2.3 The performance with pros and cons of various MFC configurations for simultaneous removal of carbon and nitrogen (Cont.)

Loop configuration MFC Electrode: Granular graphite (anode), Pt coated graphite felt (cathode)

Separator: Plexiglas-wall, CEM

COD removal: 0.523 kg m-3 d-1

TN removal: 0.194 kg m-3 d-1 Experiment with actual piggery wastewater and large-volume reactor of 5 L

Need external buffer Ryu et al (2013)

MFC system with one anode chamber and two cathode chambers

Electrode: Carbon brushes (anode), carbon cloth (cathode)

Separator: CEM

TN removal: 99%

Power generation: 294.9 mW m2

-Successful shortcut nitrification with a nitrite/nitrate ratio of 3:1 High nitrogen removal Produce the net electric power of 0.007 kWh m-3

Li et al (2016)

Two-chamber MFC Electrode: Carbon felt Separator: AEM

TN removal: 91.71%

Power density: 149.76 mW m-2 No need for pH control

Microscale air-cathode MFC reactor

Electrode: Graphite felt (anode), activated carbon (cathode)

Higher power generation with anodic

denitrification

Substrate consumption due to denitrification Power generation significantly dropped with nitrate influent of 120 mg L-1

Ren et al (2020)

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2.3.2 Shortcut nitrification/denitrification process in MFCs

Since nitrite can be used to replace nitrate and become the primary electron acceptor of the cathode (Virdis et al., 2008), the shortcut nitrification/denitrification process in an MFC (Fig 2.3) is also of interest

Figure 2.3 Shortcut nitrification-denitrification process in MFCs

In MFC, electron utilization by nitrite is better than that of nitrate, resulting in a higher Coulombic efficiency for the cathodic reaction of up to 97.8% (Virdis et al., 2008) Puig et al (2011) conducted an experiment with nitrite or nitrate medium in the cathode chamber of an MFC reactor Their study indicated that nitrate and nitrite can be interchangeable in the autotrophic denitrification process at the cathode At the same generated current, the nitrogen removal rate with nitrite feeding was higher than with nitrate feeding (257 g m-3 d-1 compared to 203 g m-3 d-1) Similarly, the HRT at the cathode for nitrite feeding was lower than for nitrate feeding (2.76 h compared to 3.10 h), and the nitrogen removal efficiency achieved with nitrite feeding was 37% which was higher than with nitrate feeding (30%) Li et al (2014) employed an MFC reactor with the cathode chamber inoculated with anoxic sludge to perform the shortcut denitrification process However, most of the nitrite removed (over 80%) was converted into nitrate, resulting in a low TN removal rate of only 0.047 kg m-3 d-1 With sodium azide added to inhibit nitrite oxidation, the TN removal rate increased to 0.075 kg m-3

d-1 However, some process controls (DO, pH, temperature, organic matter) should be

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applied to accumulate nitrite or effectively curb the nitratation step rather than using the sodium azide inhibition Adding phosphate buffer also helped to improve the TN removal rate but did not have much impact on the COD removal A maximum power density of 8.3 W m-3 was attained The power generation was almost the same with and without sodium azide, but it decreased by 50% without the phosphate buffer

Li et al (2016) integrated shortcut nitrification and denitrification into a cathode MFC system The study included an oxygenated cathode for partial nitrification and an oxygen-depleted cathode for shortcut denitrification In the oxygenated cathode chamber, 98% of the ammonium was oxidized to nitrite and nitrate at a DO of 3.5 mg L-1 with a nitrite/nitrate ratio of 3:1 The aerobic cathode chamber's effluent was fed to the anoxic cathode chamber, to perform the autotrophic denitrification process with a COD/N ratio of 9.6:1 (calculated) The TN removal efficiency of their system was 99% Their study also found that the reduction rates of nitrite and nitrate with Pt-coated cathode were higher than those without Pt With partial nitritation, the anammox process can also serve as a shortcut to remove nitrogen in an MFC system It has the potential to be the primary nitrogen removal pathway in MFC (Kim et al., 2016) However, anammox bacteria grow even slower than autotrophic nitrifiers/denitrifiers Thus, a set of environmental conditions to exert some selection pressures for the growth of anammox bacteria in MFC reactor should be investigated further The shortcut nitrification/denitrification process significantly saves the organic carbon used as an electron donor source Consequently, the MFC system can utilize a larger amount of organic matter to produce electricity Further studies should focus on taking advantage of the shortcut nitrification/denitrification process to increase power generation in the MFC system Denitrification at the cathode has achieved remarkable progress in decreasing carbon requirement Theoretically, the anaerobic environment at the anode also facilitates the reduction of nitrate, thus the studies on anodic denitrification process are discussed in next section

dual-2.3.3 Heterotrophic anodic denitrification in MFCs

Heterotrophic anodic denitrification (HAD) in MFCs is a new approach to nitrogen removal (Fig 2.4) Drewnowski and Fernandez-Morales (2016) found that nitrate reduction at the anode did not negatively affect electricity production with a low-

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nitrate influent concentration A dual-chamber MFC was operated with oxygen reduction at the cathode and acetate oxidation combined with heterotrophic denitrification at the anode (Jin et al., 2019) The highest nitrate removal efficiency was 98.9% Results demonstrated that MFC operated based on HAD achieved a higher power density than MFC without HAD (11.9 W m-3 compared to 10.4 W m-3) With nitrate in the medium, the MFC had an IR of 100 Ohm, compared to 150 Ohm in the absence of nitrate A COD/N ratio of 5:1 obtained the optimal efficiency for both HAD and power generation Zhang et al (2020) employed a dual-chamber MFC for nitrogen removal, where the cathode facilitated the nitrification process and the HAD process was implemented Nitrate produced from ammonium oxidation at the cathode passed through the AEM to reach the anode for heterotrophic denitrification The highest nitrogen removal efficiency was 91.71% at a DO of 6.8 mg L-1 without pH control at both the anode and the cathode

Figure 2.4 Mechanism of heterotrophic anodic denitrification process in MFCs

Ren et al (2020) investigated the impact of different nitrate concentrations on HAD and power generation in a single-chamber MFC without a pH buffer The organic carbon source used in the study was glucose The nitrate reduction efficiency of 96% was achieved within 10 h Not adding nitrate caused a significant decrease in pH (to 5.1) at the anode, thus inhibiting power generation MFCs with nitrate in the medium (60 or 90 mg L-1) helped to control the pH in the neutral range through the HAD process,

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resulting in better MFC performance Although some of the organic carbon was utilized by denitrification process, the proportion of TOC consumption by electrogenesis was still higher than a nitrate-free MFC (9.6% and 12.9% for 60 or 90 mg L-1 influent nitrate concentration, respectively, compared to 6.3% for without influent nitrate) However, when the nitrate concentration increased to 120 mg L-1, insufficient substrate was observed Denitrifying bacteria strongly competed with exoelectrogens for the substrate, causing only 1.6% of TOC consumption to generate electricity

The HAD process in MFCs offers some benefits in pH balance and decreasing IR With an appropriate nitrate influent concentration and a proper C/N ratio, it can significantly improve the performance of MFCs without buffer In addition to pH balance, other mechanisms that promote the exoelectrogenic bacteria's activity in the HAD-MFC, need to be further investigated The nitrate concentration limit at anode also needs to be overcome for wastewater with high nitrogen influent

2.4 Microbial communities

The bacteria population at both anode and cathode are dominated by Proteobacteria, which accounted for a major proportion (Chae et al., 2009; Jin et al., 2019; Ren et al., 2020; Wang et al., 2018) The β-Proteobacteria were frequently detected in biofilm anode This subclass includes many bacteria capable of biodegrading organic compounds Chae et al (2009) reported Thauera genera accounting for 70.1% of total β-Proteobacteria in the acetate-enriched MFC Geobacter, Desulfovibrio, Pseudomonas, and Shewanella species are well-known anodophilic bacteria with electron transferability to the anode, contributing to electricity generation (Chae et al., 2009; Jin et al., 2019; Ren et al., 2020; Virdis et al., 2011) Geobacter and Desulfovibrio belong to subclass δ-Proteobacteria, while Pseudomonas and Shewanella belong to subclass γ-Proteobacteria Thauera and Pseudomonas genera are common denitrifiers, particularly capable of reducing nitrate under aerobic conditions (Jin et al., 2019; Virdis et al., 2011; Wang et al., 2018)

Although most aerobic denitrifiers are heterotrophic microorganisms, Pseudomonas can perform autotrophic denitrification using hydrogen as electron donor and CO2 as the carbon source (Ahn, 2006) Geobacter is not capable of denitrifying in the MFCs system When nitrate was present in the anode chamber, the proportion of

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Geobacter decreased markedly, while that of Thauera and Pseudomonas increased significantly (Jin et al., 2019) Other genera such as Dysgonomonas, Enterobacter, and Klebsiella were reported as the exoelectrogenic bacteria (Ren et al., 2020) The methanogens were reported to grow in anaerobic conditions at the anode, accounting for 1-6% of the total bacteria (Yu et al., 2011) The methanogenesis does not contribute to electricity production and decrease coulombic efficiency

At the cathode, AOB and NOB are genetically related in the subdivision Proteobacteria and α-Proteobacteria, respectively Among these AOB, Nitrosomonas genera was frequently detected in the MFC as a main nitrifying bacterium (Park et al., 2017; Wang et al., 2018) Nitrosomonas is also an autotrophic denitrifier that can perform nitrite reduction to nitrogen gas (Ahn, 2006) Besides Nitrosomonas, other genera such as Nitrosococcus, Nitrosopira, Nitrosovibrio, Nitrosolobus, Aquamicrobium, Phycisphaera, and Truepera were also found to be AOB (Ahn, 2006; Wang et al., 2018) Several well-known NOB are Nitrobacter, Nitrospira, Nitrospina, Nitrococcus, and Nitrocystis (Ahn, 2006) The ratio AOB to total bacteria increased with increasing DO concentration maintained in the cathode chamber (Yu et al., 2011)

Denitrifying microorganisms usually belong to the α-Proteobacteria and Proteobacteria (Ahn, 2006; Puig et al., 2011) Many denitrifiers are electro-active bacteria, such as Pseudomonas, Comamonas, Rhodopseudomonas, which can produce electricity in MFCs system (Virdis et al., 2011; Wang et al., 2018; Zhang et al., 2013) Most denitrifying bacteria prefer anoxic conditions, however, some can complete denitrification at high DO concentration, such as Pseudomonas, Paracoccus, Diaphorobacter, Limnobacter, Comamonas, Thauera (Virdis et al., 2011; Wang et al., 2018) Most aerobic denitrifying bacteria grow heterotrophically (Ahn, 2006) Some autotrophic denitrifiers were observed in the MFCs system, such as Oligotropha carboxidovorans, Rhodopseudomonas, Thermomonas (Puig et al., 2011; Wang et al., 2018) Ignavibacterium and Bellilinea can perform shortcut denitrification process with nitrite as electron acceptor (Puig et al., 2011; Wang et al., 2018) Park et al (2017) detected Nitratireductor and Acidovorax on the biocathode as denitrifying bacteria In the MFC reactor of Jin et al (2019), Azoarcus accounted for the largest proportion of the denitrifying genera Ren et al (2020) found that Petrimonas and Devosia genera

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