The Pennsylvania State University

The Graduate School

Department of Civil and Environmental Engineering

NITROGEN REMOVAL IN A SINGLE-CHAMBER MICROBIAL WITH

NITRIFYING ENRICHED AT THE AIR CATHODE

A Thesis in

Environmental Engineering

by

Hengjing Yan

© 2010 Hengjing Yan

Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

August 2010 ii The thesis of Hengjing Yan was reviewed and approved* by the following:

John M. Regan Associate Professor of Environmental Engineering Thesis Advisor

Bruce E. Logan Kappe Professor of Environmental Engineering

Mary Ann Bruns Associate Professor of Soil Microbiology

Peggy Johnson Professor of Civil Engineering Head of the Department of Civil and Environmental Engineering

*Signatures are on file in the Graduate School

iii ABSTRACT

The implementation of removal in microbial fuel cells (MFCs) is of interest for the treatment of most real waste streams. Current designs are based on cathodic biological with side-stream or combined sustained by direct or upstream aeration, which negates the energy-saving benefits of MFC technology. The commonly used two-chamber

MFC design also suffers from leaking through the cation exchange membrane

(CEM), resulting in moderate ammonia removal. This research was aimed at achieving simultaneous nitrification and denitrification, without extra energy input for aeration, using a single-chamber air-cathode MFC with a nitrifying biofilm pre-enriched at the cathode and sustained by oxygen passively diffusing through the cathode.

MFCs were operated in batch-fed mode, with two different materials, either Nafion or

diethylamine-functionalized polymer (DEA), used as a Pt catalyst binder at the cathode. With

pre-enriched nitrifying biofilm, duplicate DEA and Nafion MFCs achieved significantly higher

ammonia removal efficiency (up to 96.8 ± 0.2% and 90.7 ± 0.3%, respectively) than a Nafion-

control MFC without nitrifier pre-enrichment (54.5% initially due to assimilation and

volatilization through the air cathode, and up to 75.1% as a nitrifying biofilm developed). The net

production of and were measured and the maximum total nitrogen (TN) removal

efficiencies were 75.1%, 79.1%, and 93.9% for the Nafion-Control, Nafion, and DEA MFCs,

respectively. The nitrifying biofilm MFCs also produced greater maximum power densities, with

945 ± 42 mW/m2 cathode area from Nafion MFCs and 900 ± 25 mW/m2 from DEA MFCs,

compared to 750 mW/m2 from the Nafion-control MFC. The polarization experiment hindered the nitrification process in subsequent batches, possibly due to elevated pH at the cathode with high current densities. Coulombic efficiencies (CEs) in nitrifying biofilm MFCs were lower than in the control MFC, probably due to greater oxygen flux at the nitrifying biofilm air cathode.

iv Overall, the use of single-chamber air-cathode MFCs to achieve simultaneous nitrification and denitrification without an investment of aeration energy was successful at the tested ammonia loadings.

v

TABLE OF CONTENTS

LIST OF FIGURES ...... vii

LIST OF TABLES...... viii

LIST OF ABBREVIATIONS...... ix

ACKNOWLEDGEMENTS...... x

CHAPTER 1 INTRODUCTION ...... 1

1.1 Microbial fuel cells for bioenergy production from wastewater...... 1 1.2 Nitrogen removal in environmental systems...... 2 1.3 Objectives...... 5

CHAPTER 2 LITERATURE REVIEW ...... 7

2.1 Nitrogen removal in microbial fuel cells ...... 7 2.2 The enhancement of AOB attachment on air cathodes...... 11

CHAPTER 3 MATERIAL AND METHODS...... 14

3.1 Experimental setup...... 14 3.1.1 Reactor setup...... 14 3.1.2 Air cathode and catalyst binder manufacture ...... 15 3.2 Inoculation ...... 17 3.2.1 Enrichment stage...... 17 3.2.2 MFC operation stage ...... 17 3.3 Analyses...... 18 3.3.1 Chemical analyses...... 18 3.3.2 Electrochemical analyses ...... 19 3.3.3 Calculations...... 20

CHAPTER 4 RESULTS...... 22

4.1 Nitrification...... 22 4.1.1 Enrichment stage...... 22 4.1.2 MFC operation stage ...... 24 4.3 Electrochemical performance...... 27 4.4 Coulombic efficiency and sCOD Removal...... 31

CHAPTER 5 DISCUSSION...... 32

5.1 Simultaneous nitrification and denitrification in single-chamber air-cathode MFCs...... 32 5.1.1 Reduction of ammonia loss through air cathodes ...... 32

vi 5.1.2 Nitrifier biofilm enrichment effects on nitrification and maximum power generation ...... 32 5.1.2 Comparing DEA and Nafion catalyst binders...... 33 5.1.3 Denitrification in MFC status...... 34 5.1.4 pH change due to nitrification and denitrification...... 35 5.2 The effect of the polarization experimenst on nitrification...... 36 5.2 The effect of N removal process on Coulombic efficiencies ...... 36

CHAPTER 6 CONCLUSION...... 39

CAPTER 7 FUTURE WORK ...... 40

REFERENCES ...... 42

vii

LIST OF FIGURES

Figure 1-1: Redox cycle for nitrogen...... 4

Figure 1-2: Plots of ammonia oxidation rates versus pH at 30 ºC...... 5

Figure 2-1: Schematic of the basic components of a ...... 8

Figure 2-2: Schematic of microbial fuel cells for nitrogen removal...... 10

Figure 2-3: Profile of an air cathode with nitrifying biofilm...... 12

Figure 3-1: Schematics of the enrichment reactors and the single-chamber air-cathode MFCs for simultaneous nitrification and denitrification...... 15

Figure 3-2: Diethylamine functionalization of PEMAGMA ...... 16

Figure 3-3: Reactors in MFC operation stage...... 18

Figure 4-1: Ammonia concentrations in enrichment reactors...... 22

Figure 4-2: Nitrite concentrations in enrichment reactors ...... 23

Figure 4-3: Nitrate concentrations in enrichment reactors...... 23

Figure 4-4: Ammonia removal efficiencies and ammonia concentrations in the influent and effluent in Nafion-Control, Nafion and DEA MFCs...... 25

Figure 4-5: Nitrate and nitrite concentrations in Nafion-Control, Nafion and DEA MFCs ....26

Figure 4-6: pH change in MFC status...... 27

Figure 4-7: LSV curve of new binder (DEA) cathodes and Nafion cathodes...... 28

Figure 4-8: EIS curve of new binder (DEA) cathodes and Nafion cathodes ...... 29

Figure 4-9: Cell voltage output of Nafion-Control, Nafion, and DEA MFCs ...... 29

Figure 4-10: Power densities of Nafion-Control, Nafion, and DEA MFCs in batch 12...... 30

Figure 4-11: Coulombic efficiencies (CE) of Nafion-Control, Nafion, and DEA MFCs...... 31

viii

LIST OF TABLES

Table 1-1: Chemical formula of nitrification reactions...... 4

Table 4-1: pH change in enrichment reactors ...... 24

Table 4-2: The maximum and mean total nitrogen (TN) removal in Nafion-Control, Nafion and DEA MFCs...... 26

Table 5-1: Comparing ammonia oxidation with nitrate and nitrite removal...... 35

ix LIST OF ABBREVIATIONS

Anammox Anaerobic Ammonia Oxidation

AOA Ammonia-Oxidizing Archaea

AOB Ammonia-Oxidizing Bacteria

CE Coulombic Efficiency

DGGE Denaturing Gradient Gel Electrophoresis

DO Dissolved Oxygen

EIS Electrochemical Impedance Spectroscopy

FISH Fluorescent in situ Hybridization

HRT Hydraulic Retention Time

LSV Linear Sweep Voltammetry

NCC Net Cathode Compartment

NOB Nitrite-Oxidizing Bacteria

MFC Microbial Fuel Cell

PEM Proton Exchange Membrane

PTFE Polytetrafluoroethylene

SRT Solid Retention Time

TEA Terminal Electron Acceptor

TN Total Nitrogen

WWTP Wastewater Treatment Plant

x

ACKNOWLEDGEMENTS

First of all, I would like to express my deepest sense of gratitude to my advisor, Dr. Jay

Regan, for his patient guidance, encouragement and continuous support throughout the course of

this study. I would also like to thank Dr. Bruce E. Logan and Dr. Mary Ann Bruns for their

suggestions and serving as my committee members.

I am thankful to our lab members, Sokhee Jung, Heather Hunt, and Nick Rose for their generous assistance during this time. I would also like to thank Dr. Tomonori Saito, for his help in making the new cathode binder material and valuable suggestions for this research. Also, I would give many thanks to Drs. Bob Parette and Judodine Patterson for training me on Ion

Chromatography and helping me in solving problems when doing this. I am thankful to Dr.

Defeng Xing for helping me with the DGGE analysis of Dr. Zhiyong Ren’s samples.

It is an honor for me to be companied by colleagues here. Especially thanks to Fang

Zhang, Yimin Zhang, and Roland Cusick for their encouragement on both my study and experiment. I would also like to acknowledge the KAUST for financially supporting this research and my graduate study.

Finally, I want to express my deep appreciation to my parents, for their consistent love, understanding and encouragement. Without their support, I could not have finished my study and research here. Also, I would like to give my special thanks to all my friends in Penn State whose company makes my life here more colorful.

Hengjing Yan

July, 2010

1

CHAPTER 1

INTRODUCTION

1.1 Microbial fuel cells for bioenergy production from wastewater

In the past few centuries, the fast global industrialization and population and economic growth succeeded at the expense of rapid energy consumption of available sources on the planet.

In 2001, worldwide primary energy consumption reached 425 × 1018 J, of which 86% was

obtained from fossil fuels, with oil, coal and natural gas contributing roughly equal parts [1].

However, as a non-renewable resource, fossil fuels are being depleted much faster than new ones

are being formed, and the release of stored carbon in fossil fuels is increasing the concentration of

carbon dioxide in the atmosphere, which was reported to accelerate global climate change. Hence,

alternative renewable carbon-neutral energy sources need to be investigated to meet humans’

increasing energy needs. Nuclear fission and solar energy are two of the alternative approaches

that have received a lot of attention, yet their applications are still limited due to 1) the safety

concerns of uranium mining and long-term nuclear waste storage, and 2) lack of efficient

methods of solar energy storage.

Microbial fuel cell (MFC) technologies emerged as a new promising approach for generating electricity from biomass using bacteria [2]. Although the observation of electricity generation by bacteria was first reported a century ago [3], scarce research advances in MFCs were achieved until the study of fuel cells became increasingly popular in 1990s [4]. At the end of last century, MFCs without exogenous mediator addition succeeded [5, 6] and promoted the

2 development of research in this field. MFC technologies applied in wastewater treatment can help eliminate the large energy demands of this industry and enable energy capture in the form of electricity, hydrogen gas, or some other energy carrier or valuable reduced product. It was calculated that an MFC design instead of the aeration basin in the treatment process could save up to 22% of the energy demand for the North Toronto Wastewater Treatment plant [2]. In addition,

MFCs can also be used for removing chemicals in wastewater [7-9], bioremediation [10-12] in other water systems, or as biosensors [13-17].

1.2 Nitrogen removal in environmental systems

Due to eutrophication in freshwater systems induced by anthropogenic discharge of nutrient elements such as N and P, nitrogen removal has become one of the key goals of wastewater treatment plants (WWTPs). Nitrogen can be removed from wastewater by microbial assimilation for synthesis of proteins and nucleic acids followed by biomass wasting, and by coupling nitrification and denitrification (Figure 1-1). Presently, nitrification is the primary way to remove ammonia from wastewater, involving the sequential oxidation of ammonia (herein

+ - referring to NH3 and NH4 ) to nitrite (NO2 ) through a hydroxylamine intermediate and then

- nitrite to nitrate (NO3 ) by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria

(NOB), respectively. Recently the existence of ammonia-oxidizing archaea (AOA) was

discovered, and they have been detected in WWTPs [18], but their relative contribution to

ammonia removal is presently unknown. The resultant nitrate can then be reduced sequentially to

nitrite, (NO), nitrous oxide (N2O), and nitrogen gas (N2) by denitrification, which is catalyzed by facultative bacteria under anoxic conditions as they use nitrate as their electron acceptor. An emerging approach to nitrogen removal in wastewater involves the coupling of partial ammonia oxidation to nitrite and anaerobic ammonia oxidation (anammox), which is

3 catalyzed by the recently discovered anammox bacteria that use ammonia as an electron donor, nitrite as an electron acceptor, and inorganic carbon as a carbon source [19-21]. While this approach has several potential benefits, such as reducing oxygen requirements associated with only oxidizing half of the ammonia by AOB and not oxidizing the nitrite by NOB, the implementation of this process is at its infancy and has the concern of process stability due to the extremely slow growth of anammox bacteria.

Both groups of nitrifiers, AOB and NOB, are aerobic chemolithoautotrophs, using

oxygen as their electron acceptor, ammonia or nitrite as their electron donor, and carbon dioxide

as their sole carbon source. Some strains are reportedly capable of mixotrophic growth, using

soluble organics as their carbon source. The two-step nitrification reactions of ammonia and

nitrite oxidation are shown in (Table 1-1), with an overall reaction consisting of ammonia

oxidation to nitrate. The oxidization of ammonia to nitrite by AOB is often the rate-limiting step

of nitrification because the growth of AOB is slower and affected by many factors such as pH,

temperature, oxygen, and ammonia concentration. AOB are easily affected by pH (Figure 1-2) in

insufficiently buffered systems because their reaction is an acidification process (Table 1-1). The

low growth rate of AOB make it very difficult to get a thick biofilm produced within a short

period in ammonia-rich wastewater containing no organic compounds [22]. One possible way to

enhance this is to modify the biofilm attachment surface, such as the grafting method to improve

the adhesivity of bacteria to a polyethylene membrane [23].

4

Table 1-1: Chemical formula of nitrification reactions

Figure 1-1: Redox cycle for nitrogen [24]

5

Figure 1-2: Plots of ammonia oxidation rates versus pH at 30 ºC. Open squares and circles: data from two different batches of Nitrosomonas biomass incubated with 0.37 -1 -1 mg NH3-N L . Solid symbol: data from incubations with 5 mg NH3-N L [25].

1.3 Objectives

In this study, a single-chamber MFC with a nitrifying biofilm enriched at the air cathode

was investigated for nitrogen removal coupled with electricity generation. Nitrifying bacteria

were pre-enriched on the air cathode before exoelectrogenic bacteria were inoculated into the

MFCs. To enhance the enrichment of a nitrifying biofilm, a new catalyst binder, diethylamine-

functionalized polymer (DEA), was used and compared to regular Nafion-binder systems.

The hypotheses of this study were:

1. The partially positive-charged DEA binder will improve the nitrogen removal

efficiency/stability of this system relative to Nafion binder; and

6 2. The pre-enrichment of nitrifier biofilm on the cathode will improve the

nitrogen removal efficiency/stability of this system.

This thesis is organized with a brief literature review in Chapter 2, materials and methods in Chapter3, experiment results in Chapter 4, discussion in Chapter 5, conclusions in Chapter 6, and a summary of future work in Chapter 7. Additional research to which I contributed during my study for the Master’s degree, which is not included in this thesis, was microbial community analysis of MFC anode biofilm communities using 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) technology for community screening and sequencing of prominent

DGGE bands. Part of this work is in print [26], and a second manuscript is in preparation.

7

CHAPTER 2

LITERATURE REVIEW

2.1 Nitrogen removal in microbial fuel cells

In an MFC system, organic substrates are oxidized by bacteria growing on the anode electrode with electrons delivered to the anode and protons released into the solution (Figure 2-1).

Electrons transfer through the outer circuit and then are consumed at the cathode electrode by reduction of a terminal electron acceptor (TEA) such as oxygen (Figure 2-1) [27, 28], ferricyanide [29, 30], perchlorate [12], chlorinated organic compounds [31], and nitrate [32]. The reduction can involve a chemical catalyst such as Pt or Co, or a microbial catalyst in what is termed a biocathode. There have been numerous reviews of MFC design, monitoring, and performance [33-37]. The reader is directed to those articles, as this literature review will focus on issues pertinent to nitrogen transformations and removal in MFCs.

Nitrate removal in an MFC can be achieved by simple nitrate reduction in conjunction with soluble electron donor oxidation or with actual biocathodes. In 2004, Gregory et al first reported a Geobacter species capable of using a graphite electrode as direct electron donor during nitrate reduction to nitrite [10]. Later, Clauwaert et al [32] and Virdis et al [9] found that cathodic denitrification without intermediate H2 production was coupled with anodic oxidation of organic carbon using MFCs with biocathodes. In a single-chamber air-cathode MFC system for nitrate removal [38], however, bacteria on the cathode reportedly were not involved in accepting electrons from the circuit to reduce the nitrate, since no electricity was generated when the MFC fed with nitrate was moved to a glove box (no oxygen). Appropriate stable cathode pH (at 7.2)

- -3 was demonstrated to have increased nitrate removal from 0.22 to 0.50 kg NO3 -Nm net cathode

8 compartment (NCC) d-1 compared to a non-pH control condition in Clauwaert et al’s reactor [39].

In an MFC removing both acetate and nitrate, methane (CH4) production and biomass formation

at the anode and nitrous oxide accumulation at the cathode were reported to be responsible for the

reduction of Coulombic efficiency (CE) during continuous feeding conditions [40]. A lower

cathodic potential significantly reduced the fraction of electron transferred and released as N2O for a given current [40].

Figure 2-1: Schematic of the basic components of a microbial fuel cell. (1: anode electrode; 2: cathode electrode; 3: proton exchange membrane (PEM); 4: air sparger) (Modified from Logan’s book [2])

However, in order to achieve nitrification in MFCs, oxygen is required for the system.

Two-chamber MFCs with aerated cathode chambers (Figure 2-2A) were tested for both ammonia removal and electricity generation [41-43]. It was reported that there was ammonium leakage from anode to cathode chamber across the proton exchange membrane (PEM), which resulted in only moderate ammonia removal for the whole system [42]. Another shortcoming of this system is that the air sparging imposes an extra energy cost that MFC operation aims to eliminate. Other

9 than directly aerating the cathode chamber, Virdis et al [9] achieved both nitrification and denitrification by combining a sidestream of an aerated nitrification tank with a two-chamber

MFC (Figure 2-2B) to reduce nitrate on the anoxic biocathode. This system still had the same problems of ammonium leakage and the extra energy cost for aeration. Their recent design [44] is the first to incorporate both nitrification and denitrification in the cathode chamber. Instead of aerating the sidestream tank, they sparged the cathode influent to provide substantial dissolved oxygen (DO) for nitrifiers (Figure 2-2C). The ammonia leakage problem was solved since the nitrification occurred in the cathode chamber, yet their method of oxygen supply still consumes

energy and needs to be strictly controlled to provide permissive conditions for both nitrification

and denitrification.

At the same time, single-chamber air-cathode MFCs (Figure 2-2D), which allow oxygen in the atmosphere to passively diffuse into the solution, were tested for ammonia removal in

MFCs [41, 42]. In tests using swine wastewater [41], 83 ± 4% ammonia removal with 86 ± 6% soluble COD removal was found over a 100-h batch cycle. The increase of nitrate concentrations from 3.8 ± 1.2 to 7.5 ± 0.1 mg NO3-N/L indicated that nitrification occurred sustained by oxygen diffusion into the single-chamber MFC through the air cathode. Later, Kim et al [42] found 60% ammonia removal for a single-chamber air-cathode MFC fed acetate in five days. However, it was also found that the main mechanism for removal was not due to biological processes, but rather to volatilization of ammonia at the cathode. During the operation of the MFC, protons were consumed at the cathode and the solution pH near the cathode was raised, resulting in conversion of ammonium ion to ammonia. Since this system contained no buffer solution and only one diffusion layer on the cathode (the diffusion layer will help prevent water loss and ammonia volatilization through the cathode), ammonia easily volatilized and was lost through the cathode to the air.

10

Figure 2-2: Schematic of microbial fuel cells for nitrogen removal. (A: two-chamber MFCs with aerated cathode; B: the continuous system of a two-chamber MFC with an aerated nitrification sidestream [9]; C: the continuous system of a two-chamber MFC with aerated influent for cathode chamber [44]); D: single-chamber air-cathode MFCs (only one PTFE diffusion layer on the cathode).)

Electricity production from ammonium with an ammonium removal efficiency up to 69.7

± 3.6% in a rotating-cathode microbial fuel cell was reported by He et al [45]. However, the CE

obtained in this system was very low and could be explained with primary production of the

autotrophic nitrifiers sustaining heterotrophic exoelectrogens. Also, ammonia oxidation at an

anaerobic anode has the fundamental problem of the only know mechanism of the ammonia

monooxygenase requiring molecular oxygen.

Hence, the critical step in the research of nitrogen removal in MFCs is to design a concise system that can achieve simultaneous nitrification, denitrification, and net energy generation in a

11 system with the lowest energy cost for both construction and operation. Two-chamber MFCs encounter ammonia leakage from the anode to the cathode chamber, which will lower the ammonia removal efficiency of the whole system. Aerating the cathode or influent requires extra energy cost for MFC operation. The single-chamber air-cathode MFC in Kim et al’ s research [42]

eliminates the active aeration requirement, but has the potential of ammonia volatilization

through the air cathode with one polytetrafluoroethylene (PTFE) diffusion layer when there is no

buffer strength in the solution. If the air cathode is coated with more PTFE diffusion layers (up to

four layers are commonly used) and the solution contains adequate buffer strength (50 mM PBS

or more), the ammonia volatilization might be reduced.

2.2 The enhancement of AOB attachment on air cathodes

In a previous study of a single-chamber MFC treating swine wastewater, the AOB

Nitrosomonas europaea was found on the air cathode at a density of 2040 ± 720 amoA

copies/cm2 and in suspension at 220 ± 70 amoA copies/mL but not on the anode [42], suggesting

that nitrification could be occurring by AOB on the cathode supported by oxygen diffusion

through the cathode electrode. However, in a single-chamber MFC in which the hydraulic

retention time (HRT) is the same for the anode and cathode, it is necessary to achieve an

ammonia removal rate at the cathode that matches the COD removal rate at the anode. Since

nitrifying bacteria have considerably lower growth rates than heterotrophs, it is very difficult for

them to successfully survive in a mixed-culture MFC system provided with substantial organic

substrates, as aerobic heterotrophs would be expected to outcompete them at the cathode.

Therefore, the enrichment of a thick AOB biofilm before the inoculation of heterotrophs is

preferred.

12 A typical air cathode is made of a piece of carbon cloth with one carbon base layer [28] and four PTFE diffusion layers coated on the air-facing side and one Pt catalyst layer on the solution-facing side (Figure 2-3). When oxygen passively diffuses into the MFC through the air cathode, AOB are likely to be enriched on the catalyst layer. Therefore, the molecular structure of the catalyst binder, which binds catalyst Pt to the carbon cloth, will affect the attachment of AOB.

The typical catalyst binder used for an air cathode is the commercially available Nafion (Aldrich).

In a previous study, air cathodes with Nafion catalyst binder could get a visible biofilm of heterotrophs [46], but its compatibility specifically for AOB adherence was not tested to our best acknowledge, apart from the previously mentioned detection of AOB in a mixed-culture air- cathode biofilm.

Figure 2-3: Profile of an air cathode with nitrifying biofilm.

Tsuneda et al [47] reported a novel porous hollow-fiber membrane, with polymer chains containing a diethylamino group immobilized on the pore surface, that can collect proteins at a high rate and high capacity because of convective transport and multi-layering of proteins. Then

13 Hibiya et al [22, 48, 49] demonstrated that diethylamine functionalization onto hollow-fiber

membranes enhanced the adhesivity of nitrifying bacteria in a continuously fed fluidized bed

bioreactor and the biofilm exhibited a much higher nitrification rate per unit area than other

, which might have been due to particular properties of the grafted membrane surface,

such as positive charge and soft polymer chains. Hence, a suitable modification of the catalyst

binder, such as grafting diethylamine groups, might be an effective way to improve nitrifying

biofilm formation on the cathode surface.

14

CHAPTER 3

MATERIAL AND METHODS

3.1 Experimental setup

To help AOB survive in a mixed-culture single-chamber MFC system, a nitrifier pre- enrichment stage was carried out in which reactors were fed with inorganic medium only and set in open-circuit status. Two different cathode binders were tested with duplicates for each as well as abiotic controls. The abiotic controls were used to quantify nitrogen removal due to non- biological processes. After the development of nitrifying biofilms on cathode electrodes, organic substances were added to the feed medium, reactors were reinoculated, and MFCs were connected to an external resistance.

3.1.1 Reactor setup

Single-chamber air-cathode MFC reactors consisting of an anode and cathode placed on the opposite ends of a plastic (Plexiglas) cylindrical chamber 4 cm long by 3 cm in diameter

(empty bed volume of 28 mL) [27] were used in the nitrifying biofilm enrichment stage

(Figure 3-1A). Anodes were made of non-wet proof carbon cloth (type A, E-TEK), while cathodes were wet-proofed (30%) carbon cloth (type B, E-TEK) coated with a carbon base layer and four PTFE layers on the air-facing side and a 0.5 mg-Pt/cm2 catalyst layer on the solution- facing side [28]. Two different materials were used as binders for the Pt catalyst layer, either

Nafion (Aldrich, Batch #: MKAA1175) (reactors Nafion-1 and Nafion-2) or a diethylamine- functionalized polymer (DEA, reactors DEA-1 and DEA-2). The DEA binder provided partial

15 positive charges and soft polymer chains to the cathode surface, which were hypothesized to enhance the attachment of ammonia-oxidizing bacteria (AOB).

Two abiotic controls (Nafion-Control and DEA-Control) were used to monitor ammonium loss through the air cathode without biological processes involved. During the enrichment stage, all the reactors were operated in open-circuit mode. When enrichment reactors were changed into batch MFCs, the reactor chambers and air cathodes enriched with nitrifying biofilm were preserved, while the carbon-cloth anodes were replaced by graphite fiber brush anodes [50] with a 2.5 cm OD that were made of carbon fibers (PANEX33 160k, ZOLTEK)

(Figure 3-1B). Then MFCs were operated with an external resistor of 1000 Ω.

Figure 3-1: Schematics of the enrichment reactors (A) and the single-chamber air-cathode MFCs for simultaneous nitrification and denitrification (B)

3.1.2 Air cathode and catalyst binder manufacture

To prepare DEA binder, (Figure 3-2), poly(ethylene-co-methyl acrylate-co-glycidyl

methacrylate) (PEMAGMA, Aldrich; 67 wt% poly(ethylene), 25 wt% poly(methyl acrylate), 8

wt% poly(glycidyl methacrylate)) was functionalized with diethylamine (Aldrich, >99.5%).

16 PEMAGMA (3.5 g, 2.0× 10-3 mol of epoxy groups) was dissolved in tetrahydrofuran (75 mL, 5 wt% solution) in a round-bottomed flask equipped with a magnetic stirrer. Diethylamine (4.14 mL, 0.04 mol) was injected to the polymer solution via a syringe. The reaction mixture was stirred at 50 ºC for 12h. Then, the reaction mixture was precipitated into methanol and filtered.

The isolated polymer was dried in a vacuum oven at 40 ºC for 24 h. 1H NMR analysis confirmed quantitative conversion of epoxy groups on poly(glycidyl methacrylate) to diethylamine groups, thus poly(ethylene-co-methyl acrylate-co-3-(diethylamino)-2-hydroxypropyl methacrylate) (DEA polymer) was successfully synthesized.

Figure 3-2: Diethylamine functionalization of PEMAGMA

The cathodes were fabricated via brush application of the binder solution containing platinum catalyst (0.5 mg/cm2 Pt) with carbon black on wet-proofed carbon cloth (type B-1B, E-

TEK) with four PTFE diffusion layers as previously described [28]. The catalyst (10% Pt on

Vulcan XC-72, BASF Fuel Cell Inc.) with 400 μL of 5 wt% polymer solution per cathode was

applied to each cathode with 400 μL of 5 wt% polymer solution per cathode. The Nafion solution

(Aldrich) was composed of a methanol and water solvent mixture, while DEA polymer solution was composed of a tetrahydrofuran solvent.

17 3.2 Inoculation

3.2.1 Enrichment stage

At the beginning of nitrifier enrichment, each reactor except the abiotic controls was inoculated with 2 mL of mixed liquor taken from the aerated nitrification tank of the

Pennsylvania State University Wastewater Treatment Plant and 3 ml cultured suspension of

Nitrosomonas europaea (ATCC19718). The mixed culture in the inoculum was expected to help the formation of biofilm on the cathode, since previous attempts to grow pure culture N.s

europaea biofilm on the air cathode surface failed. N. europaea were cultured in a flask incubated

in a 28 °C shaker. Sterile 0.3 M K2CO3 solution was periodically added to the culture to control pH around 7.5. All reactors were operated in a 30 °C thermostatic chamber in fed-batch mode,

-1 -1 -1 with autoclaved medium containing 0.764 g L NH4Cl, 1.78 mg L Na2CO3, 3.12 mg L MgCl2,

-1 -2 -1 -3 -1 0.79 mg L CaCl2, 4.26×10 mg L FeSO4, and 3.76×10 mg L CuSO4 in phosphate buffer (50

mM PBS, pH 7.0) (a modification to the ATCC 19718 recipe). For abiotic controls, reactors were

also autoclaved at the beginning. Reactors were covered to prevent the growth of photosynthetic

organisms and UV inhibition of AOB. When there was above 90% ammonia removal in any

reactor, all of the reactors were refilled with fresh sterile medium only. The enrichment lasted 60

days (four batches).

3.2.2 MFC operation stage

To move into MFC status, the nitrifier-enriched reactors Nafion-1&2 and DEA-1&2 as

well as Nafion-Control (Figure 3-3) were inoculated with 14 mL of a suspended exoelectrogenic

microbial consortium taken from a bottle air-cathode MFC fed with sodium acetate that had been

18 running for more than 1 year. MFCs were operated in batch mode with medium containing 1 g L-1

-1 -3 -1 -3 -1 -4 -1 CH3COONa, 0.382 g L NH4Cl, 1.78×10 g L Na2CO3, 3.12×10 g L MgCl2, 7.87×10 g L

-5 -1 -6 -1 CaCl2, 4.26×10 g L FeSO4, 3.76×10 g L CuSO4, trace minerals and vitamins [51] in phosphate buffer (50 mM PBS, pH 7.0). Therefore, the COD:N ratio of the medium was about

7.8:1, which was close to real wastewater condition. Reactors were also covered to prevent light effects except during medium changes. In the first batch, since the inoculum was half of the reactor volume, double-strength growth medium was used. Reactors were re-fed with complete replacement of the medium when the voltage decreased below 10 mV, giving batch durations of two to four days.

Figure 3-3: Reactors in MFC operation stage (Nafion-Control, Nafion-1&2 and DEA-1&2 )

3.3 Analyses

3.3.1 Chemical analyses

During the enrichment stage, 0.4 ml samples were taken from each reactor every two or

three days (no solution refills to make up for the sample volume) using 3 mL syringes (VWR

International, LLC., Cat. No. BD309586) and filtered immediately through a 0.2 μm pore diameter PTFE syringe filter (VWR International, LLC., Cat. No. 28145-491). The concentration

+ of ammonia (referring to NH4 -N and NH3-N in this paper) was measured using Standard

19 Methods 10031 [52] (HACH Company, Loveland. CO) by diluting 10 μL original sample in 200

- μL deionized water (HACH Company, Loveland. CO, Cat. No. 272-42). Nitrite (NO2 -N) and

- nitrate (NO3 -N) concentrations were quantified by ion chromatography (Dionex DX-120, Dionex

Co., Sunnyvale, CA) using an AS16 4-mm column with 1.0 mM NaOH eluent by diluting 200 μL original sample in 6 mL deionized water. Bulk solution pH in each MFC was tested before and after each batch using a calomel pH electrode (VWR International, LLC., Cat. No.14002-774) and pH meter (Fisher Scientific, AB15). In MFC status, 1.0 ml samples were taken from reactors

+ before and after each batch, and pH was tested at the same time. In addition to NH4 -N/NH3-N,

- - NO2 -N, and NO3 -N, soluble COD was also measured using Standard Methods 5220 [52]

(HACH COD system).

3.3.2 Electrochemical analyses

The voltage drop (V) across the external resistor in the circuit was measured at 10-min intervals using a multimeter (Keithley Instruments, Cleveland, OH), and current (I) was calculated using Ohm’s Law. Power density (P) was obtained using P = IV / A, where A is the

projected surface area of the cathode (7 cm2). To obtain the maximum power densities of MFCs,

polarization experiments were carried out in batch 11 and 12. During these tests, the external

resistance was changed from 1000 Ω to 500 Ω, 200 Ω, 150 Ω, 100 Ω, 80 Ω, 60 Ω, and 40 Ω

gradually with each resistance for 20 minutes and the voltage was measured by multimeter

(RadioShack, Cat. No. 22-813).

Electrochemical Impedance Spectroscopy (EIS) tests were carried out for both Nafion and DEA cathodes before they were inoculated with bacteria. The frequency range was set between 100 kHz to 5 mHz with an ac signal of 10 mV amplitude. Impedance spectra were recorded using the cathode as the working electrode and the Pt plate anode as the counter

20 electrode, with a Ag/AgCl reference electrode positioned closer to the cathode. Before the EIS test, one Linear Sweep Voltammetry (LSV) test at a scan rate of 3 mV/s and two at 1 mV/s were carried out to help the system get steady. In the LSV tests, the working electrode was the cathode and the range of voltage scanned was between 0.7 V to -0.3 V versus the Ag/AgCl reference electrode.

3.3.3 Calculations

Coulombic efficiency (CE) was calculated based on COD removal, current generation and reactor volume (Vr) as

t 8∫ Idt CE = 0 (3.1) FV ΔCOD r

where t = batch time, F = Faraday’s constant, ΔCOD = the change of COD concentration from

the beginning to the end of each batch, and Vr = 26 mL (the graphite fiber brush took approximate

2 mL water volume space).

The ammonia removal efficiency (η + ) was calculated every batch as 4 −NNH

+ + 4 Start 4 −−− ]NNH[]NNH[ End η + = × %100 (3.2) 4 −NNH + 4 − ]NNH[ Start

+ + + where 4 − N]NH[ Start and 4 − N]NH[ End are the NH4 -N concentrations in the bulk solution at the beginning and end of a batch, respectively.

The total nitrogen (TN) was defined as

− − 2 3 −+−+= ]NNO[]NNO[TKNTN (3.3)

21 − where TKN = Total Kjeldahl Nitrogen (organic and reduced nitrogen), and 2 − ]NNO[ and

− 3 − ]NNO[ are nitrite and nitrate concentrations, respectively. Since samples were all filtered

+ before tests, insoluble organic N can be ignored. We took 4 − N]NH[ instead of TKN here.

Therefore the total nitrogen (TN) removal efficiency (η −NTN ) was calculated as

start − ]TN[]TN[ end η −NTN = × %100 (3.4) ]TN[ start

22

CHAPTER 4

RESULTS

4.1 Nitrification

4.1.1 Enrichment stage

+ In the 60-day enrichment stage, concentrations of NH3-N/NH4 -N were stable in the uninoculated Nafion-Control and DEA-Control reactors, but decreased in Nafion 1&2 and DEA

1&2 reactors at an increasing rate from batch 1 to batch 3 (Figure 4-1). In the beginning of batch

4, the ammonia removal in DEA 1&2 was slow but increased greatly later. The ammonia removal

was consistently more rapid in the DEA reactors than in the Nafion reactors. Nitrite (Figure 4-2)

and nitrate (Figure 4-3) production and pH decrease (Table 4-1) indicative of ammonia oxidation

were observed in the biotic reactors, but not in the abiotic controls.

Figure 4-1: Ammonia concentrations in enrichment reactors

23

Figure 4-2: Nitrite concentrations in enrichment reactors

Figure 4-3: Nitrate concentrations in enrichment reactors

24

Table 4-1: pH change in enrichment reactors*

* Effluent pH in DEA and Nafion reactors are the average pH of their duplicates.

It was also notable that, during the enrichment, the duplicated Nafion 1 and Nafion 2 reactors had increasing difference in ammonia removal. In the Nafion 1 reactor, the ammonia concentration decreased faster and the removal rate nearly caught up with the DEA reactors.

However, in Nafion 2, ammonia removal was still slow in the last batch of enrichment, indicating incomplete formation of a nitrifying biofilm. The initial concentrations of nitrite in all reactors during batch 1 were around 50 mg N/L, which did not come from the feeding medium but rather the N. europaea culture (indicating AOB activity during the pure culture stage).

4.1.2 MFC operation stage

During the first 10 cycles of fed-batch MFC operation, ammonium removal efficiencies in DEA and Nafion MFCs were 88.8±2.6% to 96.8±0.2% and 75.3±11.1% to 90.7±0.3%, respectively, compared to 49.0% to 75.1% in the Nafion-Control MFC (Figure 4-4). With initial

+ + NH4 -N concentration between 89.6 and 123.9 mg N/L in the influent medium, the NH4 -N

25 concentration in the effluent of DEA MFCs after each cycle was below 11.2±2.5 mg N/L, while

+ Nafion-Control and Nafion MFCs had more unstable effluent NH4 -N concentrations, ranging

from 7.3 to 30.6 mg N/L and 18.2 to 63.2 mg N/L, respectively. The difference between Nafion 1

and Nafion 2 in ammonia removal also occurred in MFC status, with Nafion 1 having greater

ammonia removal efficiency than Nafion 2.

- The NO2 -N concentration in the Nafion-Control MFC was between 5.5 and 11.2 mg N/L

during cycles 3 to 6 but below detection (<1.2 mg N/L) in DEA and Nafion MFCs (Figure 4-5).

Nitrate concentrations of 7.9 to 29.9 mg N /L and 13.7 to 27.9 mg N /L were found in the effluent

- of DEA and Nafion MFCs, while in the Nafion-Control MFC, NO3 -N production was only

- detected in cycle 7 (5.2 mg N/L). Consistent with ammonia removal, the net production of NO3 -

N in the two duplicated Nafion MFCs also showed differences, with Nafion 1 higher than Nafion

2. The maximum total nitrogen (TN) removal efficiencies were 75.1%, 79.1%, and 93.9% for

Nafion-Control, Nafion, and DEA MFCs, respectively (Table 4-2).

Figure 4-4: Ammonia removal efficiencies (hollow symbol with solid line) and ammonia concentrations in the influent (solid symbol only) and effluent (hollow symbol only) in Nafion-Control, Nafion and DEA MFCs

26

Figure 4-5: Nitrate (solid symbol) and nitrite (hollow symbol) concentrations in Nafion-Control, Nafion and DEA MFCs

Table 4-2: The maximum and mean total nitrogen (TN) removal in Nafion-Control, Nafion and DEA MFCs

In batches 11 and 12, a polarization experiment was carried out to test the maximum

power densities of the MFCs. However, when the external resistance was incrementally changed

from 1000 Ω down to 40 Ω, the ammonia removal efficiency in each MFC continuously dropped,

and until batch 13 it had decreased by up to 21 to 46% of the efficiency in batch 10 just before the

polarization test. After batch 13, with no additional polarization experiments, the ammonia

removal abilities of both DEA and Nafion 1 MFCs started to recover and finally reached their

previous level. The ammonia removal efficiency in Nafion 2 MFC also recovered partially from

27 batch 13; however its difference between Nafion 1 increased. In the Nafion-Control MFC,

ammonia removal efficiency dropped to and then stayed around 40-50% after batch 11.

There was a pH increase in all the reactors during the first three batches of MFC status,

with the change in Nafion-Control the most obvious. Then the pH change in Nafion and DEA

MFCs stayed relatively small. The Nafion-Control started to have pH decrease from batch 4

(Figure 4-6).

10 DEA Nafion Nafion-Control Initial 9

8

pH 7

6

5

0123456789101112 Batch

Figure 4-6: pH change in MFC status

4.3 Electrochemical performance

LSV and EIS curves of DEA and Nafion cathodes before the inoculation of any bacteria

showed that under abiotic conditions, DEA cathodes showed less activity near the oxygen

reduction potential (Figure 4-7) and larger charge transfer resistance than Nafion cathodes

(Figure 4-8). However, after the inoculation of exoelectrogenic bacteria and the addition of

28 sodium acetate to the feed medium, the big difference in charge transfer resistance between DEA

and Nafion cathodes did not affect observable differences in voltage output or power generation.

All MFCs started to produce relatively similar maximum cell voltages from the third batch

(Figure 4-9), which were 528, 561, and 548 mV for DEA, Nafion, and Nafion-Control MFCs,

respectively. In successive batches, it was found that in the Nafion-Control MFC the duration of

voltage output above 50 mV in each batch had increased from 34 to 62 hours by batch 11, while

in DEA MFCs this time period had just slightly increased from 27 to 35 hours. The duplicated

Nafion MFCs did not show obvious differences in duration above 50 mV voltage output in the

first 11 batches. However, longer operation showed that this duration in Nafion 2 increased

greatly and nearly caught up with the Nafion-Control after more than 20 batches, while in Nafion

1 it stayed similarly with the DEA MFCs. Also, the maximum voltage output of Nafion 2 and

Nafion-Control decreased with successive batches, although not significantly.

0.05 Nafion 0.03 New Binder

0.00

-0.03 Im (A)

-0.05

-0.08

-0.10 -0.40 -0.18 0.04 0.26 0.48 0.70 Vf (V vs. Ref)

Figure 4-7: LSV curve of new binder (DEA) cathodes and Nafion cathodes (Reference electrode : Ag/AgCl)

29

100.00

Nafion

80.00 New Binder

60.00

40.00 -Zigmag (ohm)

20.00

0.00 0.00 50.00 100.00 150.00 200.00 250.00 Zreal (ohm)

Figure 4-8: EIS curve of new binder (DEA) cathodes and Nafion cathodes

Nafion Control Nafion DEA 0.6

0.5

0.4

0.3

0.2 Cell votalge (V). votalge Cell 0.1

0.0 0 2 4 6 8 10121416182022 Time (d)

Figure 4-9: Cell voltage output of Nafion-Control, Nafion, and DEA MFCs

30

1.2

1.0 ) 2

0.8

0.6 Nafion Control 0.4 Nafion Power density (W/m 0.2 DEA 0.0 0 0.1 0.2 0.3 0.4 0.5 0.6 Current density (A/m2)

Figure 4-10: Power densities of Nafion-Control, Nafion, and DEA MFCs in batch 12

During normal MFC operation (Rexternal=1000 Ω), the MFCs had slightly different power

densities, with Nafion MFCs the highest (399 ± 18 mW/m2) and DEA MFCs slightly lower (363

2 2 ± 4 mW/m ). The power density of the Nafion-Control at Rexternal=1000 Ω was 313 mW/m . The

Nafion MFCs also produced the highest maximum power density at Rexternal=100 Ω, which was

averaged 945 ± 42 mW/m2, with Nafion 1 higher than Nafion 2. The duplicate DEA MFCs obtained more consistent maximum power densities with each other, which was 900 ± 25 mW/m2

at Rexternal=100 Ω. The maximum power density achieved in the Nafion-control was the lowest

2 (750.4 mW/m at Rexternal = 80 Ω) (Figure 4-10).

31 4.4 Coulombic efficiency and sCOD Removal

The Coulombic efficiencies (CE) in all the reactors started from approximately 10% in the first batch, and then increased to 45%, 26%, and 25% for Nafion-Control, Nafion, and DEA

MFCs after 10 batches (Figure 4-11). The Nafion-Control MFC had a higher CE than the nitrifier-enriched MFCs, and among the nitrifier-enriched MFCs the Nafion MFCs had slightly higher values than DEA MFCs.

The soluble COD (sCOD) in the effluent was always below 72 mg COD/L in all MFCs, with an initial concentration of 770 mg COD/L in each batch. The average sCOD removal efficiencies for Nafion-Control, Nafion, and DEA reactors were 94.6%, 95.3%, and 96.2%, respectively. The duplicated Nafion MFCs showed relative consistent performance in sCOD removal and CEs.

100 Nafion Control 80 Nafion DEA 60

40

20 Coulombic efficiency (%) efficiency Coulombic

0 01234567891011 Batch

Figure 4-11: Coulombic efficiencies (CE) of Nafion-Control, Nafion, and DEA MFCs

32

CHAPTER 5

DISCUSSION

5.1 Simultaneous nitrification and denitrification in single-chamber air-cathode MFCs

5.1.1 Reduction of ammonia loss through air cathodes

Both of the duplicate DEA reactors and one of the Nafion single-chamber air-cathode

MFCs in this research reached higher ammonia removal efficiencies (96.8 ± 0.2% and 95.2%) within shorter batch times than Min et al’s (ammonia removal efficiency was 83 ± 4%) [41] and

Kim et al’s (60%) [42] single-chamber air-cathode MFCs. Also, with three more PTFE layers on the air-facing side of the Nafion air cathode and well-buffered systems, ammonia loss due to its volatilization at the cathode was greatly reduced, by comparing the performance of the Nafion-

Control system with the nitrifier MFCs. Passively diffused oxygen was adequate for cathode oxidation at a current density of 76 mA/m2 and an ammonia oxidation rate of 53.8 mg N/L in 72 hours. The ammonia oxidation efficiency was obtained by subtracting the ammonia removal efficiency of Nafion-Control from that of the nitrifier MFCs, assuming that the ammonia removal in Nafion-Control was not related with ammonia oxidation process, but only ammonia assimilation by microbes and ammonia loss through the air cathodes.

5.1.2 Nitrifier biofilm enrichment effects on nitrification and maximum power generation

In single-chamber air-cathode MFCs, facultative heterotrophic bacteria can easily form biofilm on air cathodes, where in a nitrogen-removal MFC AOB and NOB are also expected to locate. The ammonia removal efficiencies in both DEA and one of the Nafion MFCs were up to

33 134% higher than the Nafion-Control, demonstrating that an enrichment of nitrifying bacteria

with inorganic medium only before MFC status greatly helped AOB and NOB in the competition

with heterotrophs on the cathodes. Their performance in ammonia removal in fed-batch MFC

status were relative stable over more than two months’ operation except following the

polarization experiment (discussed later), suggesting that once the nitrifier biofilm is well developed on the cathode surface, it will not be easily washed out or outcompeted by facultative heterotrophs. However, if the enrichment stage is not successful, such as in the Nafion 2 reactor, it is difficult for a nitrifier biofilm to mature in later MFC status by itself and its ability to recover from unexpected disturbances, like the effect of the polarization experiment on ammonia removal during batch 11 and 13, is low. Even the Nafion-Control system, which had no pre-enrichment of nitrifiers on the cathode, showed some evidence of nitrification during MFC operation through the production of nitrite and nitrate. However, this system never established the extent of ammonia removal observed in the nitrifier-enriched systems.

The enrichment process also seemed to improve the maximum power densities of MFCs,

regardless of the catalyst binder. This is despite the fact that the previous LSV and EIS tests

indicated a significant difference in electrochemical performance between DEA and Nafion

cathodes under abiotic conditions. Protons were continuously being produced along with

ammonia oxidation within the nitrifier biofilm, which serves to relieve the proton consumption

due to the oxygen reduction at the cathode, thus lowering the charge transfer resistance of the

system.

5.1.2 Comparing DEA and Nafion catalyst binders

The DEA binder accelerated nitrifier biofilm enrichment and enhanced the stability

compared to the Nafion binder, though this is only based on the performance of duplicate reactors.

34 The duplicated Nafion MFCs showed obvious differences in ammonia removal but relatively

consistent electrochemical performance (such as voltage output and power generation) and

Coulombic efficiency, suggesting that a marginally successful enrichment of nitrifier biofilm

before the MFC status in the Nafion 2 MFC was the most likely reason. Also, although the

Nafion 1 reactor eventually caught up with DEA MFCs in ammonia removal at the end of the enrichment stage, the DEA binder system showed faster start up of AOB and NOB attachment on the cathode.

The portion of the positive charge that DEA binder provided can actually be adjusted by

tuning the percentage of diethylamine groups in this molecule during its preparation. More partial

positive charge may attract more AOB to the surface, but it is unknown whether there could be a

deleterious effect that could lower the activity of attached cells. The percentage of diethylamine

groups in this research (8 wt %) was demonstrated appropriate for enhancing AOB attachment.

During the first few batches of MFC status, DEA MFCs produced approximate 15 mV

lower maximum cell voltage than Nafion MFCs; however, with batch succession, this difference

decreased due to the maximum voltage drop in Nafion MFCs. This was probably because the

biofilm on the Nafion cathode developed thicker than on DEA cathodes, and thicker biofilm

increased the charge transfer resistance near the cathode and thus lowered the voltage output after

long-term operation.

5.1.3 Denitrification in MFC status

The end-products of the denitrification process were not tested in this research, and in

MFC status with soluble organic electron donor, both nitrification and denitrification likely

occurred, making it impossible to calculate nitrate or nitrite reduction efficiencies by nitrogen

balance. However, since the enrichment of nitrifying bacteria was slow and no particular AOB or

35 NOB source was inoculated in the Nafion-Control MFC, we can probably assume that the

ammonia removal efficiency in the Nafion-Control during the first four batches (average 52.3%)

was not related with the ammonia oxidation process but rather to assimilation and volatilization

losses. Therefore, by subtracting 52.3% from the ammonia removal efficiencies in the DEA and

Nafion MFCs, their ammonia oxidation efficiencies can be obtained (Table 5-1). By comparing

- - this estimated ammonia oxidation with the total concentration of NO3 / NO2 , we can see roughly

- - 67% and 50% NO3 / NO2 removal in the DEA and Nafion MFCs, respectively. These might include denitrification, nitrate and nitrite assimilation, or other processes.

Table 5-1: Comparing ammonia oxidation with nitrate and nitrite removal*

* All data are mean values for the first 11 batches.

5.1.4 pH change due to nitrification and denitrification

The bulk solution pH obviously dropped in the enrichment stage in conjunction with

ammonia oxidation but stayed relatively constant in MFC status after 3 batches, which was

probably affected by nitrification and denitrification processes in these systems. During the

enrichment stage, nitrification was the main process occurring in the reactors since no organic

substrate was provided in the autoclaved feed medium, though reactors were initially inoculated

with mixed culture. Therefore, along with ammonia and nitrite oxidation, protons were produced

within the nitrifier biofilm and diffused into the bulk solution, causing the decrease of pH.

36 However, in MFC status, denitrification was expected to happen in these systems. Hence, protons produced from nitrification might be totally or partially consumed by nitrate and nitrite reduction, making the bulk solution pH not change as much as in the enrichment stage. The oxygen reduction at the cathode should also have consumed protons in MFC status; however, this was accompanied by an equivalent amount of protons produced from sodium acetate oxidation at the anode, which would made the final pH in bulk solution unchanged if the transport of protons was not obstructed from anode or cathode biofilms.

5.2 The effect of the polarization experiments on nitrification

Polarization experiments carried out in batches 11 and 12 showed great impact on ammonia removal. In the tests, when small external resistances were connected to each MFC, larger current was generated. This would promote faster oxygen consumption at the cathode and a larger pH gradient within the cathode biofilm. In this case, an oxygen deficiency to AOB and

NOB near the cathode might occur and inactivate nitrifiers. At the same time, the rapid elevation of pH within the biofilm would hinder nitrification. After the polarization experiment, the ammonia removal in Nafion-Control and Nafion 2 MFCs could never recovered to their previous levels, which suggests that some nitrifiers on the cathode might have already been killed during the polarization experiment rather than temporarily inactivated or experiencing oxygen limitation.

5.2 The effect of N removal processes on Coulombic efficiencies

With sodium acetate (CH3COONa) as the electron donor, the total redox reaction with cell synthesis for aerobic heterotrophs is

- + - 3 COOCH25.10 + 0.088O 2 + 0.033NH 4 + 0.005CO 2 = 275 NOH0.033C + 0.096HCO 3 + 2 O0.093H

37 (5.1)

assuming the portion of the total acetate-derived electrons used for synthesis, fs,heterotrophs, is 0.65

(the regular range of fs for aerobic heterotrophs is from 0.6 to 0.7 [53]) and ammonium is their N source. Equation 5.1 showed that direct oxygen reduction by aerobic heterotrophic bacteria will

- consume 1.43 mol CH3COO /mol O2.

When there is full nitrification from ammonium to nitrate and complete denitrification

from nitrate to dinitrogen in the system, the total redox reactions with cell synthesis are

+ + − 0NH3.10 4 + 2 + 2 = + NO125.0H250.0CO20.000.225O 3 + 275 NOH0.005C + 0.005HCO 3 + 2 O0.12H

(5.2) and

- − + - 3 + NO128.0COOCH25.10 3 + 2 + 0.127HO0.034H = 2 + 275 + 2 + HCO128.0CO046.0NOHC016.0N056.0 3 (5.3)

respectively, by assuming fs,nitrifier= 0.10, fs,denitrifier = 0.44 [53], ammonium as the N source for nitrifiers, and nitrate as the N resource for denitrifiers. Equation 5.2 and 5.3 showed that the full

- nitrification and denitrification will consume 0.54 mol CH3COO /mol O2.

Similarly, for ammonium oxidation and nitrite reduction (i.e., without NOB activity), the total reactions are

+ - + − 0.167NH 4 0.225O 2 ++ 0.02CO 2 + 0.005HCO 3 = 0.333H + 0.167NO 2 + 275 NOH0.005C ++ 2O0.162H

(5.4) and

- − + - 0.125CH3COO + 0.204NO2 0.204H =+ 0.040CO2 + 275 0.093NNOH0.017C 2 ++ 0.128HCO3 + 2O0.167H (5.5)

respectively, by assuming fs,nitrifier= 0.10, fs,denitrifier = 0.44 [53], ammonium as the AOB N

source, and nitrite as the denitrifier N resource. Equation 5.4 and 5.5 showed that the

- ammonium oxidation and nitrite reduction will consume 0.45 mol CH3COO /mol O2.

38 The calculations above indicated that CEs in the system should be benefited by nitrification and denitrification processes theoretically, compared to direct oxygen reduction by aerobic heterotrophic bacteria.

However, in the test, the CEs of DEA and Nafion MFCs were slightly lower than that of

the Nafion-Control. The possible reason for this might be a larger oxygen flux through the air

cathode in the nitrifying systems. There are two possible contributors for a larger oxygen flux in

these systems: 1) Smaller biofilm thickness. By naked eye detection, the nitrifier MFCs had

thinner cathode biofilms than the control by batch 10. Since it was reported that CE could

decrease immediately by removing the cathode biofilm [46], thinner biofilm at air cathodes might

result in smaller barrier of oxygen diffusion into the solution. 2) Low current densities. The

difference in the time period above 50 mV voltage output made the DEA and Nafion MFCs have

longer time in low current density status than the Nafion-Control. In that case, oxygen

consumption by cathode oxidation was lower and more oxygen was diffusing into the solution.

Based on this problem, a continuous-flow system might be a good improvement to the batch

mode.

39

CHAPTER 6

CONCLUSION

Single-chamber air-cathode MFCs were demonstrated to have achieved simultaneous nitrification and partial denitrification without aeration, with a mixed culture at the anode and a nitrifier biofilm enriched at the cathode. The highest ammonia removal efficiency of 96.8 ± 0.2% was achieved in DEA-binder MFCs within 72 hours batch time, of which 52% was assumed not to be related with ammonia oxidation based on the control system. A maximum power density of

945 ± 42 mW/m2 was obtained in the Nafion MFCs, compared to 750 mW/m2 in the control

MFC. The maximum total nitrogen (TN) removal efficiencies were 75.1%, 79.1% and 93.9% for

Nafion-Control, Nafion and DEA MFCs, respectively. An estimated average of 67% and 50% nitrate/nitrite was removed in DEA and Nafion MFCs, respectively. For catalyst binders, DEA enhanced the enrichment of AOB on the air cathode, while Nafion reactors showed unstable performance in N removal in duplicate systems. Polarization experiments were found to have side effects on ammonia removal, probably due to instantaneous oxygen deficiencies and a large pH elevation within the nitrifier biofilm. Coulombic efficiencies in nitrifier biofilm MFCs were lower than the control probably due to greater oxygen flux at the air cathode. Bulk solution pH showed an obvious decrease in enrichment stage due to nitrification, but was relieved in MFC status due to the simultaneous nitrification and denitrification in the system.

40

CAPTER 7

FUTURE WORK

This work has successfully demonstrated nitrogen removal in single-chamber air-cathode

MFCs with enrichment of nitrifier biofilm. The problem of ammonia loss through the air cathode reported in previous research has been reduced here and possible ways of enhancing nitrifier enrichment were attempted. However, there are still challenges to be addressed for improving the performance of the system:

1. It would be interesting to study the distribution of AOB, NOB and

heterotrophic bacteria on the air cathode of DEA, Nafion and Nafion-Control

MFCs. We showed here that nitrifier biofilm enrichment improved ammonia

removal and the DEA binder enhanced consistent performance of MFCs

relative to Nafion, so it would be important to investigate the difference of

microbial distribution in the cathode biofilm between different catalyst

binders and reactors using fluorescent in situ hybridization (FISH).

2. Continuous flow experiments might be able to solve the side effect of

asynchronous electrochemical performances of different MFCs on Coulombic

efficiencies. Also ammonia removal rate and denitrification rate could be

tested in this system.

3. The cathode surface area could be enlarged to further enhance the ammonia

removal efficiency of the system. This might require a new construction

design of the single-chamber air-cathode MFC.

41 4. The ratio of diethylamine groups in the DEA binder needs to be optimized to

enhance the attachment of AOB on the air cathode. It is expected that with a

greater ratio of diethylamine groups, the attachment of AOB will be enhanced,

but too much diethylamine will provide too strong partial positive charge that

might inactive microbes.

5. The direct end-products of denitrification, such as N2O and N2, need to be

measured to investigate the denitrification efficiencies.

42

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