University of Southern California Dissertations and Theses

From fuel cells to single cells: electrochemical measurements of direct electron transfer at microbial-electrode interfaces

(USC Thesis Other)

From fuel cells to single cells: electrochemical measurements of direct electron transfer at microbial-electrode interfaces

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content FROM FUEL CELLS TO SINGLE CELLS: ELECTROCHEMICAL
MEASUREMENTS OF DIRECT ELECTRON TRANSFER AT
MICROBIAL-ELECTRODE INTERFACES
by
Benjamin J. Gross
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulllment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHYSICS)
August 2015
Copyright 2015 Benjamin J. Gross
In memory of Lieutenant Joshua W. Gross
ii
Acknowledgments
I would like to rst and foremost thank my adviser and mentor Moh El-Naggar.
In my third year of this program I had suered a great personal tragedy and had
lost all direction. Were it not for Moh taking me in when he did I would likely
never have completed my Ph.D. Moh has shown me innite patience over the years
in the face of countless failures, oversights, frustrations and spelling mistakes. He
provided me a place to work, resources and direction when I had none. He kept
faith in my project even when I lost it, and pushed me to do better work than I
ever thought I was capable of. I am a better student and a better person because
of him.
I would like to thank the rest of my committee, Stephan Haas, Vitaly Kresin,
James Boedicker, and Steve Finkel for their time and energy in helping me complete
this work.
I would also like to thank all my lab mates who have shared this space and
helped to make it my home: Ian McFarlane, Sahand Pirbadian, Tom Yuzvinsky,
Edmond (Kar Man) Leung, Matt (Shuai) Xu, Yamini Jangir and Hyesuk Byun,
Jerey Huan, and James Lu, and Julia Lazzari-Dean thank you all for your support
and for making this a fun and productive work place.
I would like to thank all the wonderful friends who have been with me through
thick and thin: Ari Magdar, Christopher Kaddoura, Michael Jarret, Chanon
iii
Rosefelt-Finley, Michael Tetro, Joseph Perez-Marchese, Hanina Rosenstein and
the whole Stettin family, Kira Muratova, and Mark Phong. Without you guys my
life would be empty indeed.
I would like to thank my family. My parents, Walter Gross and Karen Gross,
whose love and support made me the person I am today. Special thanks to
Nathaniel Gross, Rebecca Gross and Elizabeth Gross, who are the best siblings
anyone could ever ask for. To Grandma Gretchen and Phillis whose love and
encouragement can be felt across a continent, to Grandpa Red and Robert, and
to Matthew Gross, Norman Gross, Sarah Freeman, Robert Freeman, Melinda
Habekost, Carl Habekost, Janine Freeman, Pamela Freeman, Garrett Barboza,
Cole Habekost, and Katie Habekost.
I would also like to thank our funding agencies for providing us with the
resources to complete this work: The Air Force, and The U.S. Department of
Energy. Special thanks to the Nanoelectronics Research Facility at the University
of California, Los Angeles. Finally, I would like to thank the University of South-
ern California Department of Physics and Astronomy for being my home for the
last nine years.
iv
Contents
Dedication ii
Acknowledgments iii
Abstract vii
1 Introduction 1
2 Background 11
2.1 Oxidative phosphorylation as an energy harvesting strategy for life . 11
2.2 Shewanella oneidensis MR-1 . . . . . . . . . . . . . . . . . . . . . . 13
2.3 Outer Membrane Cytochromes . . . . . . . . . . . . . . . . . . . . . 14
2.4 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.5 Microbial Fuel Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3 Electrochemical Analysis of the Eect of Calcium on Microbial
Fuel Cell Current Production 25
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1.1 Microbial Fuel Cell Optimization . . . . . . . . . . . . . . . 26
3.1.2 The Biological Role of Calcium . . . . . . . . . . . . . . . . 27
3.1.3 Electrochemical Impedance Spectroscopy . . . . . . . . . . . 28
3.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.1 Microbial Fuel Cell Operations . . . . . . . . . . . . . . . . 29
3.2.2 Electrochemical Impedance Spectroscopy Measurements . . 30
3.2.3 Equivalent Circuit Model . . . . . . . . . . . . . . . . . . . . 30
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3.1 Microbial Fuel Cell Data . . . . . . . . . . . . . . . . . . . . 31
3.3.2 Bode Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
v
4 Microbial Fuel Cell Studies of Shewanella oneidensis MR-1 and
Mutants Disrupted in Extracellular Appendage Production 36
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.1.1 Polarization Resistance Measurements . . . . . . . . . . . . 38
4.1.2 Cultivation of Bacteria for MFC Testing . . . . . . . . . . . 40
4.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.1 Microbial Fuel Cell Mutant Response . . . . . . . . . . . . . 43
4.2.2 Polarization Resistance . . . . . . . . . . . . . . . . . . . . . 44
4.2.3 Scanning Electron Microscopy . . . . . . . . . . . . . . . . . 46
4.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5 Development of a Single Cell Extracellular Electron Transfer Mea-
surement Technique: Design, Construction and Electrochemical
Validation. 49
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.2 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.2.1 Infrared Optical Trap . . . . . . . . . . . . . . . . . . . . . . 52
5.2.2 Electrochemical Chip: Concept and Fabrication . . . . . . . 55
5.2.3 Perfusion Chamber and Assembly . . . . . . . . . . . . . . . 59
5.3 Electrochemical Characterization of the Microelectrodes . . . . . . . 60
5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6 Single Cell Extracellular Electron Transfer Measurements of MR-
1, OMC, and bfe and the Importance of the Flavin Export
System on Current Production 66
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.2.1 Growth protocol . . . . . . . . . . . . . . . . . . . . . . . . 67
6.2.2 Trapping events . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
7 Conclusion 73
Bibliography 77
vi
Abstract
Metal-reducing bacteria gain energy by extracellular electron transfer to external
solids, such as naturally abundant minerals, which substitute for oxygen or the
other common soluble electron acceptors of respiration. By performing electron
transfer to synthetic electrodes instead of minerals, these microbes can be used
as biocatalysts for conversion of diverse chemical fuels to electricity. This ability
enables the emerging technology of microbial fuel cells (MFCs), where living cells
utilize complex or mixed biofuels to produce electricity. The exact mechanism of
this extracellular electron transport, however, is not yet fully understood.
In the rst part of this work I focus on the role of calcium in MFCs innocu-
lated with Shewanella oneidensis MR-1. Calcium is known to play important roles
in cellular metabolism, including aggregation of cells onto surfaces. It has been
shown that the addition of calcium to MFCs results in increased current. Here we
use electrochemical impedance spectroscopy (EIS) and equivalent circuit model-
ing to determine that calcium is not simply increasing the current by abiotically
reducing the solution resistance, and may prove useful as a biological optimization
parameter.
In the next section I focus on the role of pili in MFC current production. She-
wanella oneidensis MR-1 produce type IV pili which may play an important role
in biolm formation and current production in MFCs. MFCs have permitted the
vii
measurement of current output from mutants lacking the ability to produce certain
extracellular appendages thought to play a role in current production. Our results
prove ambiguous due to the bulk nature of MFC measurements which cannot
determine whether dierences in current production are due to factors uniformly
impacting the entire culture, or heterogeneities in the phenotypical behavior of
dierent cells.
In order to address these issues, we describe an experimental platform for single
cell respiration measurements. The design integrates an infrared optical trap, per-
fusion chamber, and lithographically fabricated electrochemical chips containing
potentiostatically controlled transparent indium tin oxide microelectrodes. Indi-
vidual bacteria are manipulated using the optical trap and placed on the micro-
electrodes, which are biased at a suitable oxidizing potential in the absence of any
chemical electron acceptor. The potentiostat is used to detect sub-pA currents
associated with single cell direct electron transfer (DET) events.
Finally, I demonstrate the system with single cell measurements of the dissim-
ilatory metal reducing bacterium Shewanella oneidenis MR-1, which resulted in
respiration currents ranging from 15 fA to 100 fA per cell under our measurement
conditions. Mutants lacking the outer membrane cytochromes necessary for extra-
cellular respiration did not result in any measurable current output upon contact.
Mutants lacking the
avin export system (bfe) also did not produce current at
the single-cell level. Only one out of 28 contact events showed a current increase
correlating to contact with the electrode of 10 fA, which is a the detection limit
of the instrument. This is consistent with the previously proposed role for
avins
in EET, although debate continues on whether the
avins act as secreted electron
shuttles or as bound co-factors to the outer membrane cytochromes.
viii
In addition to the application for extracellular electron transfer studies, the
ability to electronically measure cell-specic respiration rates may provide answers
for a variety of fundamental microbial physiology questions.
ix
Chapter 1
Introduction
Living things rely on a complex symphony of chemical reactions in order to stay
alive and ultimately reproduce. This century has seen a rapid increase in tech-
niques for studying these reactions that have allowed us to make breakthroughs
in understanding living systems. New theories, including quantum mechanics and
modern chemistry have made possible detailed quantitative measurements of bio-
logical systems that have put biology in a new framework that has allowed us to
interpret biological systems in terms of the underlying molecular dynamics.
Fundamental to all of lifes processes is the ability to manipulate the
ow of
electrons in metabolic pathways [5, 68]. Charge transfer is a ubiquitous process
throughout all lifeforms in which a series of proteins, with metallic cofactors, which
create a conductive pathway for electrons to
ow [34]. All living things, from
elephants to bacteria use the
ow of electric charges to harness energy from the
environment to do virtually all processes in the cell which require physical work.
Plants convert sunlight into sugars using chains of enzymes in which carbon dioxide
is transformed into long carbohydrates by charge transfer reactions. Those same
organic compounds can later be broken down by cells which use the
ow of high
energy electrons released from these molecules to generate adenosine tri-phosphate
(ATP), the energy carrying chemical currency for life [63]. Combined, these two
redox activities serve to convert sunlight into biological functions. Without charge
transfer, life could not exist. One of the most common strategies for releasing the
1
Figure 1.1: Redox tower shows the available free energy from dierent elecron
donor-acceptor pairs.
stored energy from organic molecules to generate ATP is the process of respiration
and I will look at it in great detail in this work.
When an organism respires, it is taking advantage of the dierence in potential
energy between an electron stored in an organic fuel and that of an electron bound
in an electron accepting molecule. Figure 1.1 is a redox tower, which shows the
potential dierence between products and reactions for some common chemical
reactions involved in respiration. The greater the vertical distance between the
fuel source and the electron acceptor, the more energy is available to the organism
by completing that reaction. Take for instance glucose and oxygen. The use of
oxygen as the electron acceptor is the most energetic use of an organic fuel molecule
for ATP synthesis, as opposed to sulfur reduction, where the potential energy
2
dierence between the fuel and the electron acceptor is much lower (1.1). The
higher the energy gradient of the electrons released from these reactions, the more
ATP can be generated by the organism per molecule of fuel. However, oxygen has
not always been available in the environment. This is because until approximately
2.45 billion years ago, there were no photosynthetic organisms releasing molecular
oxygen into the environment as a byproduct of photosynthesis [31]. Species that
evolved early on in our history required other methods for harvesting energy.
Respiration is often broadly categorized in two ways: aerobic and anaero-
bic [13]. While both use dierent chemical pathways, the basic process is the
same. Fuel is oxidized releasing high energy electrons that are used to power a
series of enzymes that generate ATP. When the energy has been used up, the elec-
trons reduce a terminal electron acceptor and leave the cell, making room for the
next high energy electrons to continue the process. While all aerobic respiration
involves the reduction of molecular oxygen to generate ATP, anaerobic respira-
tion is a term which covers a variety of energy generating strategies, commonly
found in single celled organisms including bacteria and archaea, including sulfur
reduction, nitrogen xation and metal reduction as alternative means of ATP pro-
duction [23, 64, 90, 92, 100]. In eukaryotes, mitochondria serves as the organelle
responsible for respiration, while in prokaryotic species respiration happens at the
cell membrane [13]. In both cases, a membrane is used to create a proton gradi-
ent, and the
ow of ions back across the membrane through specialized proteins
which act as a turbine powered by the
ow of charges recharge ADP into ATP.
Anaerobic respiration generally does not produce much energy relative to aerobic
respiration. For instance, sulfur reduction generates as few as 2 ATP molecules
per oxidized organic fuel molecule, depending on the specics of the metabolism
3
involved, compared to glucose and oxygen which generate between 36 and 38 ATP
molecules per reaction through glycolysis and the citric acid cycle [13].
However, what anaerobic respiration lacks in eciency, it makes up for with
diversity. Aerobes are conned to those areas of the biosphere where molecular
oxygen is abundant. Anaerobes, on the other hand, are under no such constraints.
They can be found in the roots of plants, xing nitrogen and creating vital nutrients
for the plant [13]. They can be found inside the intestinal tract of human beings,
to digest vitamines, or in termites helping them digest the wood frame of your
house. Virtually anywhere that fuel and electron acceptors can be found on Earth,
you will also nd a species of bacteria thriving.
One of the most unique types of anaerobic respiration from a physics stand-
point are what are colloquially referred to as rock breathers or metal reducers [27].
These are species that have evolved pathways to reduce solids, such as iron oxide,
outside of the cell membrane as the terminal electron acceptor in their respira-
tion pathway. In most cellular systems, the chemistry takes place inside the cell,
and chemicals are transported back and forth across the membrane by various
pumps and pores. Metal reduces, such as Shewanella oneidensis MR-1 (MR-1),
and Geobacter sulfurreducens DL-1 (DL-1), which are found in lake bed sediment
layers, have instead evolved a method of conducting electrons across the insulat-
ing lipid membranes directly to the surface of solid conductive materials. This is
particularly interesting because these bacteria oer us a window into the living
vital systems of cells through electrical measurements, as well as opening up new
exciting technologies that fuse biological and electrical systems.
Chemists have developed techniques to manipulate solid electrodes in chemi-
cal solutions to control the
ow of electrons in and out of the solution, and thus
manipulate the electrochemical conditions for various reactions. Electrochemistry
4
uses a system of electrodes to control the potential dierence between a bulk solu-
tion and a conductive electrode. The same technique can be used to mimic the
environmental conditions that are favored by metal reducing bacteria. In the same
way that microbes use the potential energy dierence between a fuel and an elec-
tron acceptor, so too can you bias an electrode at the equivalent potential, thus
replacing one of the molecules in the reaction. In so doing, we can provide the elec-
tron acceptor they need, and collect precise data about their respiration, including
which potentials are favored, and how much they respire under various chemical
changes in their environment, as well as investigate the impact of various genetic
mutations. By combining these techniques, we can begin to unravel the mystery of
direct electron transfer in bacteria, and open up a new frontier in bioengineering.
One of the rst technologies proposed which take advantage of this process are
Microbial Fuel Cells (MFCs). MFCs were rst introduced in 1911, before metal
reducers were discovered [82]. The early systems required the introduction of elec-
tron mediators, chemicals which diuse across the cell membrane and are reduced
intracellularly and then diuse back across the cell to the electrode. This process
is constrained by diusion rates of the mediators which constrains the eciency of
the fuel cell. It took until 1999 when the concept of metal reducers as the biocat-
alysts inside a fuel cell was introduced which eliminated the need to add electron
carrying molecules to generate current [47]. While inecient compared to other
abiotic designs, these systems oer several advantages that traditional fuel cells
do not. MFCs have the ability to operate under a wide variety of environmen-
tal conditions due to the robust nature of the metabolism of the microbe. This
could lead to applications where MFCs are deployed in areas such as under the
ocean, and harvest ocean sediment for organic fuels [54]. This metabolic path-
way can also be reversed, taking electrons from the electrode to synthesize organic
5
fuels [35, 36, 75, 83, 88, 94]. Furthermore, the same process by which MFCs cat-
alyze oxidation of fuel organic in natural environments may also serve to treat
waste products generated by human activity [43].
Waste water remediation is an energy intensive process. Both organic and
inorganic contaminants must be removed before waste water can be reintroduced
into the biosphere. Some estimates claim that there is over seven times as much
unclaimed potential energy in the organic waste as is spent cleaning the water [93].
Work has been done to use waste water as the organic fuel for a microbial fuel
cell [43]. By letting the microbes catalyze the reactions, expensive chemical treat-
ment techniques may one day be avoided entirely.
Because of these exciting possibilities, there has been a lot of research into the
exact mechanisms involved in extracellular electron transfer. This unique pathway,
in which electrons are transferred by the cell extracellularly to solid electron accep-
tors, has been shown to involve a network of proteins called cytochromes which
have iron heme groups that act as charge carriers [86]. Once the electron is outside
the cell, several methods have been proposed as to how the electrons move from
the terminal heme of the cytochrome to the solid electron acceptor.
One proposed mechanism is the secretion of chemical charge carrying shuttles.
Flavin molecules have been shown to be secreted by Shewanella species [62, 98]
and removing the
avins dramatically reduces the current produced in MFCs.
It has been suggested that these molecules diuse back and forth between the
electrode surface and the cell surface, shuttling electrons as they go [15]. Okamoto
et al. proposed an alternative role for
avins as bound co-factors to multiheme
cytochrhomes that alter the energetics for interactions between cytochromes and
external surfaces. Although
avins are known to be important in Shewanella,
Geobacter species do not use
avins, so it is not the whole story.
6
Figure 1.2: In vivo imaging of Shewanella oneidensis MR-1 with a nanowire
between two cells. The cells were stained with the non-specic protein dye NanoOr-
ange.
Figure 1.2 shows an image of Shewanella oneidensis MR-1 stained with the
non-specic protein dye NanoOrange. The long string in the center is a nanowire.
These were rst discovered in Shewanella in 2006 and in Geobacter in 2005 [33,
84]. Since then, it has been shown that these wires are conductive over several
microns [29, 81]. The exact purpose of nanowires is not yet known. They might
serves as a conductive pathway between multiple cells in a biolm. This might
serves to connect cells near rich sources of electron acceptors to those farther
7
way, thus increasing the eciency of use of such a resource. Along with cell-to-
cell interactions, nanowires might also serve to connect directly to solid electron
acceptors.
Lastly, there is direct electron transfer (DET) which is when the membrane
of the cell, and the terminal conductive ends of the cytochromes come into direct
contact with the solid electron acceptor and transfer electrons.
In chapter 2 we will introduce some background concepts. We will begin with a
detailed overview of oxidative phosphorylation. We will look at a general overview
of the pathways involved, and then examine in some detail the dierent mechanisms
that various organisms have employed to achieve ATP synthesis. Next we will
examine metal reducing bacteria, and specically the cytochrome pathways that
allow these microbes to reduce solid electron acceptors. This will be followed by
an overview of electrochemistry, and specically three electrode systems and basic
fuel cells, as this is the principle means by which data are collected and analyzed
in this work.
This thesis is focused on electron transfer reactions taking place between solid
electrode surfaces and dissimilatory metal reducing bacteria (DMRB). Electro-
chemical techniques, along with mutants provided by collaborators, provide a set
of tools with which the electrical currents between the electrode and the bacteria
can provide insight into several areas of interest. We describe both biotic and abi-
otic strategies for enhancing MFC performance. Chapter 3 describes our studies of
how CaCl
2
impacts MFC performance. This is followed by a study of the impact of
MFC performance of mutants lacking various extracellular appendages proposed
to play a role in EET. The results prove ambiguous which motivates chapters 5
and 6 wherein we describe the construction of a novel measurement device for
8
studying DET on a per-cell basis and then use the instrument to examine the role
of excreted
avins in DET.
MFC optimization has been a topic of great interest in recent years, and in
chapter 3 we will explore the question of whether current production increases
in MFCs, as a result of the addition of CaCl
2
, is a biological aect, or simply a
decrease in the solution resistance of the MFC anode chamber, resulting in higher
current for abiotic reasons. We will use measurements and modeling techniques
designed to interpret electrochemical behaviors as equivalent circuits and infer that
the solution resistance of an MFC is not impacted by the addition of CaCl
2
and
that the observed increase in current production must be due to biological eects.
Chapter 4 will reveal some of the limitations of MFCs and provide the motiva-
tion for the later work on single cells. One area of great interest has been nanowire
production. It has been proposed that nanowires are pili structure decorated with
cytochromes and this has been shown in DL-1, however in MR-1 this remained
an unanswered question until recently when it was shown that nanowires in She-
wanella are membrane extensions [80]. In order to test the role of pili in MFC
current production, MFCs were inoculated with wild type bacteria, as well as
mutants lacking specic genes for creating pili. The current output of the MFCs
was measured, as well as polarization resistance curves to determine which mutants
produced the most power. Scanning electron microscopy (SEM) shows that it is
impossible to determine whether cell attachment was impacted by the mutations.
The basic question that cannot be answered by MFCs is whether the individual
cells in the fuel cell are respiring less, or whether less cells are attached to the
anode. Data gathered in bulk cannot distinguish between the two eects. This
presents a real problem if we wish to move forward. A new technique is called for.
9
In chapter 5 we describe a novel technique for measuring direct electron transfer
of single cells. The system contains an optical trap along with a lithographically
fabricated transparent microelectrode array and a vacuum sealed perfusion cham-
ber. The perfusion chamber is vacuum sealed against the transparent electrode
array and a highly diluted concentration of cells is injected into the system. The
optical trap is used to select individual planktonic cells which are brought into
contact with micron scale electrodes. A potentiostat is used to control the electro-
chemical properties of the electrode and to measure small currents, as low as 10
fA, that correspond to direct contact with an individual bacterium. We perform
electrochemical tests on the electrode array , as well as atomic force microscopy
(AFM) imaging of microelectrodes, to verify that it is sensitive enough for DET
measurements on the order of 10 fA.
The system is then used in chapter 6 to test single cell DET of three strains
of Shewanella; the wild type, a mutant lacking surface proteins that have been
shown to be necessary for DET to solid electrodes as a biological control, and a
mutant lacking the ability to export
avins. Our ndings indicate that both outer
membrane cytochromes and extracellular
avins are required for DET. Wild type
showed a higher probability of generating reduction currents when in contact with
the electrode surface than did either mutant.
We have created a new tool which can be used on a variety of questions that
are more readily answered through single cell measurements. This can provide
additional insight that bulk techniques lack and help generate future models of
DET formation. While single cell measurements are still relatively new and subject
to a number of variables which can be dicult to control, we provide suggestions
to improve the system, as well as some thoughts on possible future measurements
that are appropriate for this type of system.
10
Chapter 2
Background
Biophysics is a highly interdisciplinary subject which relies on concepts and tech-
niques from a variety of subjects. This work in particular requires the reader
to have a basic understanding of cellular metabolism, electrochemistry, fuel cell
specications, optics, and photolithography for example. Here we take some time
to familiarize the reader with the basics from the most recurring subjects in this
work. We begin with cellular respiration, metal reducing bacteria and the specic
protein structures that make them unique, followed by an overview of basic elec-
trochemical concepts and techniques, and nally discuss fuel cells. While other
subjects will come up at later points, these three are required throughout most of
this work.
2.1 Oxidative phosphorylation as an energy har-
vesting strategy for life
All lifeforms reproduce. Reproduction is fundamentally the process of obtaining
raw materials from the environment, and ordering them into a new version of
the original system. In order to accomplish this task, living systems have devel-
oped strategies for harvesting stored energy from the environment and convert-
ing them into a universal energy storage mechanism known as adenosine triphos-
phate (ATP). ATP is a molecule with high energy phosphate bonds that releases
11
Figure 2.1: Electron transport pathway for ATP synthesis. Organic fuels are
oxidized, releasing protons and electrons which travel through the quinone to the
electron transport pathway. The electrons are used by the electron transport path-
way to produce a proton gradient which causes protons to
ow through the ATP
synthase complex to form ATP.
energy when a single phosphate group is hydrolyzed, converting ATP into adeno-
sine diphosphate (ADP). This releases energy which is then used to power the
energy demanding processes of life. Respiration uses the process of oxidative phos-
phorylation to generate ATP from ADP molecules.
Oxidative phosphorylation is the process by which organic fuels are oxidized,
releasing high energy electrons that travel through a series of proteins situated
between two intracellular spaces, separated by a lipid membrane, called the electron
transport chain and ultimately end up in an electron acceptor [79]. The electron
transport chain uses the energy from the electron to create a proton gradient across
a membrane. The hydrogen ions
ow from high concentration to low concentration
across the membrane through the ATP synthase complex which generates ATP
12
(Figure 2.1). The terminal electron acceptor for the electron transport chain varies
depending on the strategy employed by the organism. While aerobes use oxygen,
anaerobes have a variety of electron acceptors including some species that use solid
minerals such as iron oxide.
2.2 Shewanella oneidensis MR-1
Shewanella oneidensis MR-1, which was rst discovered in 1988, is a widely stud-
ied species of bacteria belonging to a class of bacteria known as dissimilatory metal
reducing bacteria, or DMRBs [71]. It was known that iron and manganese oxides
in Lake Oneida, were reduced at a very high rates compared to the known rates
of purely abiotic chemical processes that could account for such metal oxide redox
reactions, and it had been known for many years that bacterial metabolic activity
was involved in the manganese cycle [9, 14, 17, 38]. The chemical reaction involved
in the reduction of metal oxides coincides nicely with the thermodynamic require-
ments for an anaerobically respiring microorganism to maintain its life functions
and it was suspected that anaerobic respiration by a species of bacteria may be
the principle cause of the manganese reduction observed [26]. The discovery of
metal reducers, including MR-1, helped explain this process in aquatic systems,
and opened a new frontier of research that is thriving to this day.
Shewanella is a facultative anaerobe able to respire oxygen if it is available in
the environment, but will use any of a large number of possible electron acceptors
in the absence of oxygen [72]. These electron acceptors need not be soluble. In
fact, one of the most remarkable features of MR-1 is its ability to reduce solid phase
electron acceptors, such as carbon electrodes, or indium tin oxide lms, which will
be important to us later on.
13
Figure 2.2: a) Representation of the MtrDEF cytochrome pathway for extracellular
electron transport. b) MtrF crystal structure, PDB ID code 3PMQ. Clarke et al.
2011
MR-1 respires much like any other organism by oxidizing one of several organic
compounds that act as electron donors, and in the process releasing high energy
electrons. What separates MR-1 from most strains is that in some cases, the
electrons are transferred to the acceptor and away from the cell directly without
the presence of any soluble electron acceptor passing back and forth across the
membrane. This means that it is capable of directly transferring electrons onto
insoluble surfaces. This behavior has been called Extracellular Electron Transfer
(EET). It has been shown that EET is dependent on specialized proteins known
as cytochromes located on the outer membrane of the cell.
2.3 Outer Membrane Cytochromes
The ability of DMRBs to reduce solid phase electron acceptors has been tied to
the presence of membrane bound cytochromes, which are proteins containing iron
14
functional groups known as heme groups [86]. The structures is made up of Mtr-
CAB and MtrDEF protein complexes (gure 2.2) [20]. Electrons which have been
used for ATP synthesis through respiration are channeled from the inner mem-
brane through a series of cytochromes to the outer membrane beginning at MtrD,
which is a deceheme c-cytochrome situated inside the porin MtrE and nally to
MtrF which is a decaheme cytochrome bound to the outer membrane, or to the
MtrA, MtrB and MtrC protein complex (gure 2.2a). In this system, both the
MtrCAB and MtrDEF chains are homologous of one another and carry out similar
functions [91].
It is fortunate that we have a crystal structures for two outer membrane
cytochromes, MtrF and OmcA, due to the inherent diculty of crystalizing pro-
tein [19, 25]. The structure of MtrF is shown in gure 2.2b. It consists of ten heme
groups containing iron in a staggered cross conguration separated by distances
of no more than 1 nm. These cytochromes are proposed to allow electrons to hop
from one heme site to another and from one cytochrome to the next [81].
2.4 Electrochemistry
Electrochemistry is the study of chemical reactions which take place on or near
an electrode surface as a result of a transfer of charge between a chemical and
an electrode. In the majority of cases, the chemical is in solution, the electrodes
are made of a solid conductive material, and the chemical reactions are reduction-
oxidation (redox) reactions. Reduction happens when a species absorbs a net
negative charge, usually in the form of an electron (although in many biological
cases the addition of a hydrogen atom results in reduction due to the hydrogen
donating the electron to the rest of the molecule) and oxidation occurs when a
15
substance loses a positive charge (often due to interactions with oxygen, hence the
name). An electrolyte (e.g. salt) is normally present to reduce the solution resis-
tance to charge transfer. The electrodes can be made from a variety of materials.
Common examples include metallic substances such as platinum, iron and gold,
various carbon based substances, as well as semiconductors such as Indium Tin
Oxide (ITO) which we will examine in greater detail in chapter 5.
When a large metallic substance is placed in an aqueous solution and equilib-
rium is achieved, the Fermi levels between the solution next to the electrode, and
the electrode itself become equal. If their Fermi levels are dierent before contact
is made, then charges must
ow in order to accommodate this change in energy
states. This
ow of charges results in a voltage dierence between those molecules
in the solution that are at the surface of the electrode, as shown in the graph in
gure 2.3a. This is called the electrochemical potential.
All chemical activity in electrochemistry takes place at the interface between
the electrode and the solution. When a redox active chemical is subjected to
an electrode at a particular potential, the Butler-Volmer equation holds for the
current generated through the electrode:
i =FAj
0
[C
O
(0;t)e
f(EE
0
0
)
C
R
(0;t)e
(1)f(EE
0
0
)
]
(Bard equation 3.3.11) [4] where i is the current, F is Faraday's constant, j
0
is
the exchange current density, is the transfer coecient, A is the electrochemi-
cally active surface area,C
O
(0;t) is the initial concentration of the oxidized species,
C
R
(0;t) is the concentration of the reduced species, E is the electrochemical poten-
tial, andE
0
0
is the formal potential of the reaction. These currents are the so called
faradaic currents that occur as a result of electron transfer between the solution
16
and electrode. The transfer coecient represents the fraction of the electrode-
electrolyte interface that is involved with lowering the free energy barrier for the
reaction to take place and is at the heart of electrode kinetics. It should be noted
that the Butler-Volmer equation holds only for chemicals that are in constant close
contact with the electrode. When other considerations, such as
uid dynamics, are
taken into account, this equation helps provide a starting point for quantitative
analysis, but does not provide the whole picture. What the Butler-Volmer equa-
tion does provide is a direct relationship between the physics of various reactions
to changes in electrochemical potential as well as the current
ux through the
electrode.
Potential dierences between the electrolyte solution and the electrode are most
signicant very near (within angstrom length scales) of the electrode surface. Fig-
ure 2.3a shows a typical potential prole of a solid in an electrolyte solution. The
prole changes drastically as you move away from the electrode surface, so any
measurement that does not take place very near the electrode surface is insu-
cient. If you attempt to measure the voltage between the chemical solution at the
surface of the electrode, and the electrode itself, you must introduce some mea-
surement apparatus, such as a probe from a volt meter. However, the presence
of the metal tip will disturb the energy states of the solution and at the distance
of angstrom from the electrode, likely impact the electrode as well. However, it
is possible to measure and control this potential indirectly. This is done with the
help of a reference electrode.
A reference electrode is a system involving the interface between two chemical
species that are stable with fast kinetics whose electrochemical potential does not
shift over a large range of applied voltages and has a high impedence so virtually
no current
ows through it. This work uses the Ag/AgCl reference electrode in
17
Figure 2.3: Standard a) Top shows a model of a solid conductive electrode in
contact with a solution (blue) containing a redox active chemical (yellow). b)
Three electrode system diagram for standard electrochemical measurements. c)
Ag/AgCl reference electrode schematic.
which Ag
+
+ e

$ Ag and AgCl$ Ag
+
+ Cl

which are very reversible and
result in a stable potential of 0.23 V vs. the dened Standard Hydrogen Electrode
(SHE), which is taken as 0 V by convention. This electrode is very stable under
a wide range of pH values, from 1 to over 13. Figure 2.3 C shows a Ag/AgCl
reference electrode which consists of a Ag wire coated with AgCl salt. The wire
and salt are placed inside a glass tube containing a KCl solution (1M) which
balances the concentration of Cl

ions and maintains the salt concentration on
the wire. The bottom of the tube contains a porous glass frit which provides a
conductive pathway between the bulk solution and the reference electrode.
The working electrode is the electrode where the measurements take place.
It is in the same electrolyte solution as the reference electrode and the two are
connected by an external volt meter, the voltage dierence between the two can
be measured. Thus all voltages in electrochemistry are reported as the voltage
dierences between the voltage of the working/solution and that of the reference
18
electrode. Any change in the voltage is due to changes at the working electrode
due to the stable nature of the reference.
Varying the potential at the electrode surface will provide us with a means
of manipulating redox chemistry in the solution. This is commonly done with a
three electrode system which involves a working, reference and counter electrode
(gure 2.3b). The three electrodes are connected to a potentiostat, which is an
operational amplier with precise feedback and measurement capabilities, that
can simultaneously measure the potential dierence between the reference and
the working electrode, while sourcing current through the counter and working
electrodes. An amp meter measures the current
owing between the counter and
working electrodes, and in conjunction with the potential reading, the potentiostat
can adjust the
ow of current in such a way that the voltage changes to a desired
value, or in some cases, vice versa. It should be noted that the potential dierence
between the counter electrode and the solution is unknow, however, we are not
generally interested in this value, as our experiments take place at the working
electrode surface.
The ability to control the voltage and current
owing across the working elec-
trode interface provides electrochemists with a wide variety of tools for studying
electrochemical reactions. In general, all of these techniques involve dening a
potential wave function and measuring the current. In one of the simplest exam-
ple, chronoamperometry, a potential is set at a xed value, and the current is
monitored over some period of time as a reaction takes place. Cyclic Voltam-
metry (CV) involves sweeping a potential back and forth between a negative, or
reducing potential, and a positive or oxidizing potential and studying the behav-
ior of the current. Electrochemical Impedance Spectroscopy (EIS) is a technique
that involves inducing sinusoidal potential waveforms and monitoring the current
19
as the frequency of the waveform is changed over a wide range of frequencies.
This technique probes the resonance behavior of the chemical species, and when
combined with modeling techniques, can infer which aspects of the experiment
contribute currents at which frequencies. There are a host of other techniques that
have been developed, including Dierential Pulse Voltammetry (DPV) , Staircase
Voltammetry, Polarization Resistance measurements and many others.
Many factors come into play when attempting to understand an electrochemical
interaction. Various forces control and limit the availability of redox active chemi-
cal species at the surface of the electrode. These factors are generally governed by
the Nernst-Planck equation given by
J
j
=D
j
rC
j

z
j
F
RT
D
j
C
j
r +C
j
v
(Bard equation 4.1.9) [4] J
j
is the
ux of species j, D
j
is the diusion constant of
species j,C
j
is the concentration of species j,z
j
is the charge on species j. The rst
accounts for diusion, the second term controls for the electrostatic forces acting
on species j, and the nal term involves convection currents moving at velocity
v. This equation is generally dicult to solve analytically, however special cases
which correspond to specic experimental conditions where one or more terms are
set to zero, can be solved.
There is also the matter of how a specic species interacts with the surface of
the electrode directly. Depending on their anity to stay attached to the electrode
or not, the kinetics of a reaction can be greatly aected. The choice of electrode
can play a signicant role in this due to the various species anity for various
materials. Furthermore the geometry of the electrode is important as well. While
large surface electrodes are the most common, for studying electrochemistry of
20
micron scale systems, micron scale electrodes are needed. When the size of the
electrode is reduced suciently, the geometry of the electrode changes the behavior
of the diusion of redox species. Microelectrodes oer many advantages which will
be used in chapter 5.
2.5 Microbial Fuel Cells
Fuel cells are electrochemical devices which can be used to generate electrical power
from a redox reaction. They have received a great deal of attention in the past few
decades due to growing interest in green energy technologies [37, 50, 103]. Fuel
cells consist of an anode chamber, in which an electrode and electrolyte solution
containing an electron donating fuel source, a cathode chamber with an electron
acceptor (often oxygen), a proton exchange membrane separating the anode from
the cathode. The anode and cathode are connected to an electrical load (gure
2.5). A catalyst is needed in both the anode and cathode chamber to facilitate
the reduction and oxidation half reactions which drives the
ow of charges. While
fuel cells have the potential to provide inexpensive and clean energy, the catalyst
is often the least cost ecient step. In many cases, the catalyst is platinum which
means that the downside for fuel cells is the cost of materials.
Fuel cells function by separating a chemical reaction into two half reactions. A
fuel is oxidized at the anode, releasing protons and electrons that transfer to the
anode. The protons diuse across a cation exchange membrane which separates
the anode and cathode electrolytes and permits the hydrogen cations to diuse
into the cathode chamber. Charges that
ow into the anode
ow through a lead
connected to a load and nally to the cathode, where they reduce an electron
21
acceptor, generally oxygen, and form H
2
O. The electromotive force of any fuel cell
is given by the Nernst equation which states
E
cell
=E
0
cell

RT
nF
ln
a
O
a
R
(Bard equation 2.1.40) [4] whereE
cell
is the total electromotive force generated by
the full cell reaction,E
0
cell
is the standard potential of the cell, R is the gas constant,
T is the temperature, n is the number of electrons per mol of reactants and F is
the charge on a mole of electrons (94000 C). The logarithmic part of the equation
is governed by the relative activities of the fuel cell, and those are greatly aected
by the catalysts involved in both the anode and cathode half reactions. Therefore
one strategy for increasing the power production of a fuel cell is to increase the
eciency of the catalysts, and biological systems oer a wide variety of enzymes
which have evolved to perform electron transfer reactions.
Biocatalysts have been looked at as a means of providing a cheap and robust
alternative to abiotic catalysts. Life forms have evolved a wide variety of enzymes
which facilitate redox reactions. One approach to harnessing their power is through
enzymatic fuel cells. Enzymatic fuel cells use enzymes harvested from organisms
and injected into the anode chamber, or xed to the surface of the anode material.
However, enzymes are not self-sustaining. Like all proteins, they denature and
break down. Furthermore, it can be dicult to isolate the correct enzyme cocktail
and maintain their catabolic activity.
Microbial fuel cells (MFCs) are fuel cells that use DMRBs, such as MR-1, DL1,
and related strains, as a biocatalyst which couples these organisms DET path-
ways with the oxidation of fuel and the reduction of the anode. Unlike enzymatic
fuel cells, the microbes are alive and capable of replenishing denatured proteins.
22
Figure 2.4: Example of MFC current vs. time data in normal opperation. Three
feeding cycles are shown which correspond to spikes in current.
Microbes oer many benets as catalysts. They can function at room temperature,
as opposed to most abiotic fuel cells which operate in higher temperature regimes
(>60

C), and can function in normal pH ranges. MFCs also take advantage of
DMRBs robust metabolism and can use a large variety of organic materials as
fuels.
MFCs oer another electrochemical technique to study DMRBs. Fuel cells can
be constructed using various materials, bacterial growth media, dierent resistive
loads, and inoculated with a variety of strains of DMRBs. The primary data taken
23
Figure 2.5: MFC schematic showing the anode chamber on the left with the anode
(grey) and metal reducing bacteria (red). The cathode is bubbled with oxygen
(white). Electrons
ow through the load, while protons cross the proton exchange
membrane (yellow) to complete the circuit
from MFCs are current vs. time plots which indicate the eciency with which the
system turns over fuel. Figure 2.4 shows the current vs. time point for an MFC
inoculated with MR-1. The current spikes happen directly after the anode chamber
is injected with lactate. The current rises after the feeding and then falls as the
lactate is consumed. Further feedings result in renewed current production in the
MFC. Many variables play a role in MFC current production. Gene knockouts,
in which the genes for proteins involved in EET are removed from the genome,
provide a comparison technique to determine which proteins are responsible for
increased current production, which inhibit DET, and which have no eect. From
these measurements we can infer the relationship between physiological parameters
and eciency in MFC turnover.
24
Chapter 3
Electrochemical Analysis of the
Eect of Calcium on Microbial
Fuel Cell Current Production
3.1 Introduction
One of the major drawbacks of MFCs is that they suer from low power den-
sity [54]. Because of this, much of the current research into MFCs involves dierent
strategies for performance optimization [6, 7, 44, 52, 52, 62, 69, 97, 98]. Several
strategies have been investigated which broadly aect MFC performance in two
ways: abiotically and biologically. While abiotic parameters are important to
understand, the biological factors are more interesting for several reasons. Biologi-
cal factors are the most likely bottleneck for current production, given that abiotic
fuel cells are already more ecient. One reason for this is that the organism must
use some of the free energy available in the fuel cell to support its metabolism.
Second, the abiotic parameters are limited in how they can be adjusted due to
the adverse aect they can have on cellular metabolism. For instance, signi-
cantly altering the pH or salinity of the anode compartment can kill the bacteria.
Finally, MFCs provide quantitative in-vivo measurements of the metabolic activity
of DMRBs, which make them interesting as subjects of pure research. Therefore
25
biological factors are of particular interest in optimizing current production in
MFCs.
3.1.1 Microbial Fuel Cell Optimization
Several parameters have been investigated for their role in current production in
MFCs. The relationship between pH and current production has been investigated
in MFCs for two reasons: rst acidity will increase the driving force for oxygen
reduction by 59 mV per pH unit based on the Nernst Equation, and secondly
because of the eects pH will have on microbial physiology [7]. It was shown that
increased acidity triggers the production of
avins in MR-1 around pH 5 which in
turn increases current output in MFCs, although it was unclear why the
avins
were released, either as a stress response or due to cells lysing. Flavins are a
popular additive due to MR-1s known relationship between
avins and insoluble
electron acceptor reduction [97].
Surfactants have been investigated as additives to MFCs and shown to increase
power production as well. However, it is suggested that they may simply reduce the
conductivity of the MFC anode chamberl [101]. Other strategies have included the
addition of NaCl to MFCs to increase the ionic conduction of the anode medium
and it was shown that high concentrations of NaCl (1500 M) do have an impact
on the current production due to the increase in conductivity of the anode media
salinities [44, 52, 69]. However, very few strains of bacteria are capable of direct
electron transfer at high salinities. Other factors shown to have an impact on
current production include decreasing the distance between the anode and cathode,
thus reducing the diusion distance for cations between the two [52]. These
strategies, unlike
avins, involve abiotic factors, mostly due to solution resistance
and diusion limitations which increase the current production.
26
3.1.2 The Biological Role of Calcium
Calcium has long been understood to play a critical role in biological functions
across a wide range of species. Eukaryotes use calcium as a signal to send mes-
sages from the cell exterior to the interior [24]. Prokaryotes have been shown to
have several uses for calcium ions including signaling and regulation of various
transcription factors and other enzymes [77]. However, calcium has been dicult
to study in prokaryotes due to the small cell size, and the toxicology of the reagents
involved, but it is clear that calcium does act as a regulator in many species of
prokaryotes [24].
Calcium is also known to enhance cell aggregation in biolms in several species
including Halobaterium salinarum, Vibrio cholera, Pseudomanas aeruginosa, and
Shewanella oneidensis MR-1 [8, 46, 95]. In MR-1 Ca
2+
has been shown to cause
aggregation of cells and the expression of outer membrane proteins including MtrF
under aerobic conditions, but disaggregation upon the removal of dissolved oxy-
gen from the environment [30]. Ca
2+
is also shown to reduce the rate of U(VI)
microbial reduction [51]. Pellicle formation is similarly aected by the presence
of Ca
2+
which resulted in increased expression of surface proteins necessary for
cell aggregation [49]. Ca
2+
has also been shown to be closely associated with the
heme groups in MtrF [19]. Ca
2+
thus have the potential to be a critical factor in
determining current output from MFCs. However, simply measuring the current
output from MFCs with and without Ca
2+
ions is insucient to determine if this is
the case. Any current increase associated with the addition Ca
2+
of could simply
be the result of decreased solution resistance in the anode chamber of the MFC, as
in the case of surfactants and NaCl [44, 52, 69, 101]. This possibility needs to be
ruled out before any conclusions can be formed about the biological signicance of
Ca
2+
.
27
3.1.3 Electrochemical Impedance Spectroscopy
Electrochemical Impedance Spectroscopy (EIS) is one of many electrochemical
measurement techniques used to understand electron transfer mechanisms between
solutions and electrodes. Unlike voltammetry techniques, which sweep the elec-
trochemical potential over a large range of values, EIS involves a small sinusoidal
potential perturbation around a xed potential, E
0
, and generally less than 5 mV.
The function E
t
= E
0
sin(!t) is applied to the system and the frequency of the
waveform is the variable that is swept across a large range, often between 10
8
Hz
to 10
2
Hz. The current prole generated by the EIS measurement directly mea-
sures the frequency response of the electrochemical cell. Time dependent current
and voltages are measured and used to calculate the impedance of the cell for a
range of frequencies.
Bode plots are a popular technique for plotting EIS data. Bode plots are gen-
erated by plotting the log of the frequency on the x-axis, and the amplitude of
the impedance on the y-axis. This way the relationship between frequency and
impedance response can be visualized. However, this plot represents the sum of
all factors contributing to the impedance. For this reason EIS data are generally
interpreted as a linear superposition of several factors inside the cell. Ignoring the
eects of mass transport for the time being, EIS depends on the solution resis-
tance, capacitive charge buildup between the working electrode and the solution,
the electrochemical properties of the chemicals in the solution, especially those
specically absorbed onto the electrode surface, and any faradaic currents gener-
ated by redox reactions at the electrode. Each of these parameters contributes to
the overall impedance of the system. The faradaic currents in an MFC are the
result of DET, as well as some of the absorbed species on the electrode due to
biolm formation.
28
In order to separate which eect each of the physical parameters has on the
overall impedance, a simplifying assumption is made that the total behavior of
the system can be deconstructed into the separate contributions of each factor.
This allows for the data to be separated and treated as modular components and
modeled using an equivalent circuit. This equivalent circuit is designed to mimic
the behaviors of the electrochemical cell as they are best understood by the exper-
imenter. Toward that end, the experimenter postulates an equivalent circuit and
theoretically applies the same waveform to the circuit. The impedance behavior
is adjusted by altering the values of the individual circuit elements in order to
increase the accuracy of the t. Assuming the equivalent circuit is accurate; this
allows the experimenter to infer the values of each of the physical factors taken
into account by the equivalent circuit components, such as the capacitive nature
of the electrode/solution interface, or the solution resistance of the cell.
3.2 Experimental
3.2.1 Microbial Fuel Cell Operations
A Dual-compartment MFC system, previously described [12], were used to evaluate
the eects of Ca
2+
on MFC performance. The two compartments were separated
by a proton exchange membrane (Naon 424, DuPont). The anode (graphite felt
GF-S6-06, Electrolytica) compartment was kept anaerobic by passing N
2
gas at a
rate of 20 mL/min. Aerobic conditions were maintained in the cathode (graphite
felt electroplated with Pt, 0.15 mg/cm
2
) compartment by passing puried air at
a rate of 40 mL/min. After growth in the minimal media [12], the cells were
harvested and injected into the MFC anode compartment for a nal optical density
at 600 nm (OD
600
) of 0.4 over a 1 cm pathlength. Each experimental condition was
29
evaluated in triplicate using three MFCs simultaneously, and each MFC voltage
was recorded every 2 min across a 10 ohm resistor by a high-impedance digital
multimeter (Keithley Instruments, model 2700). The MFCs were operated under
batch conditions. After addition of bacteria and the establishment of a baseline
voltage, lactate (2 mM) was added as the electron donor in the anode compartment,
and additional lactate feedings were performed when the cell voltage dropped to
baseline values (Figure 3.2). Three conditions were tested: a control with 0 M
CaCl
2
, cells grown in 1400 M CaCl
2
, and a delayed addition of M CaCl
2
to the
control after the third feeding cycle.
3.2.2 Electrochemical Impedance Spectroscopy Measure-
ments
EIS measurements were performed for the whole MFC in two electrode mode at the
open circuit potential (OCP), as previously described [42, 60], using a Gamry Ref-
erence 600 potentiostat (Gamry Instruments) equipped with the EIS300 software
package. For each experimental condition, the EIS measurements were performed
in the frequency range 100 kHz - 100 MHz with an ac amplitude of 1 mV.
3.2.3 Equivalent Circuit Model
The equivalent circuit model used in this work was proposed by He et al. and is
comprised of three distinct components: the anode, cathode and electrolyte equiv-
alent circuit elements, each in series with one another (Figure 3.1) [42]. The anode
and cathode elements both consist of a capacitor and resistor in parallel. The resis-
tors for both represent the polarization resistance of each electrode which results
due to chemical reactions taking place at the electrode surface. The capacitors
30
Figure 3.1: .Equivalent circuit model which is made of three components. The
anode component shows a capacitor (CPE) and resistor, R
anode
, in parallel. The
anode components are in series with the solution resistance, R
series
which is in turn
in series with the cathode components, CPE and R
cathode
.
represent the inhomogeneous nature of the electrode-solution interface that results
due to variability in surface roughness, absorption and formation of a thin biolm
which does not uniformly cover the anode surface. Both the anode and cathode
components are in series with the solution resistance of the electrochemical cell.
This is the sum of all resistances that exist due to charge transfer away from the
electrode surface. This is the resistance factor that this work is concerned with,
because a drastic change in this value as a result of the addition of Ca
2+
would be
indicative of a decrease in solution resistance causing an increase in MFC currents.
3.3 Results
3.3.1 Microbial Fuel Cell Data
Three experimental conditions (0M CaC
2
, 1400M CaCl
2
, and delayed addition
of 1400 M CaCl
2
) were tested in an MFC platform with 25 mL working volume
31
compartments and an aqueous oxygen reduction cathode [12]. The current vs. time
results from this platform were in agreement with the results obtained from mini-
MFC apparatus done by collaborators [32]. MFCs inoculated with 1400M CaCl
2
produced as much as 50% as much current as the control. Furthermore,the delayed
innoculated control, which was inoculated with 1400M CaCl
2
after three feeding
cycles, also showed an increase in current above the rst three feedings shown in
gure 3.2.
3.3.2 Bode Plots
The EIS measurements for all three experimental conditions are shown in gure
3.3. The spectra were t to a two time constant equivalent circuit [42] with a
solution resistance R
s
, anodic polarization resistance R
a
, cathodic polarization
resistance R
c
, and two constant phase elements anodic and cathodic CPE (gure
3.1). To address the eect of Ca
2+
addition on the solution resistance, R
s
was
used since it contains contributions from the resistances of catholyte and anolyte,
where the Ca
2+
addition took place, in addition to the resistance of the membrane.
The R
s
values resulting from the t are included in gure 3.3, and are of the same
order as the values previously obtained using this same system (Manohar et al.,
2008). However, the ts do not reveal a correlation between Ca
2+
addition and the
solution resistance. The experiment performed using cells grown in1400 MCaCl
2
resulted in R
s
= 27.5 , which is even higher than the control without Ca
2+
(R
s
= 20.6 ). At the same time, delayed addition of 1400 M CaCl
2
to this control
decreased. (R
s
= 18 ).
32
Figure 3.2: Current vs. time data for MFCs grown with 1400 M CaCl2 (Red)
and with no CaCl2. Blue arrows indicate feedings of 2 mM lactate and the orange
arrow shows where1400M CaCl2 was added to the anode chamber of the control
MFC. Grey data is from disconnecting the MFC to take an EIS measurment.
33
Figure 3.3: Electrochemical impedance spectroscopy (EIS) measurements for whole
MFCs under three experimental conditions. (A) S. oneidensis MR-1 grown in 0
M CaCl
2
. (B) Culture grown in 1400 M CaCl
2
. (C) After delayed addition of
1400 M CaCl
2
. The solution resistance (R
s
) obtained by tting the EIS spectra
is indicated for each experimental condition.
3.4 Discussion
We have shown that the addition of CaCl
2
to MFCs causes an increase in power
generation. This is true whether the cultures were grown in CaCl
2
or whether
the MFC was innoculated with CaC
2
after several feedings. Cultures grown in
CaCl
2
produced the highest current, yet EIS showed that the solution resistance
was higher than that of the control. This could be due to several factors, including
thicker biolms due to the role of CaCl
2
in cell aggregation. MFCs innoculated
34
with CaCl
2
after three feedings showed an increase in current production, but a
decrease in solution resistance. If current increases due to CaCl
2
were the result
of reduced solution resistance, we would expect both the culture grown in CaC
2
as well as the delayed innoculated culture to show decreases in solution resistance.
However, the solution resistance does not show this trend, implying that CaCl
2
does not impact the solution resistance. We hypothesize that the increased current
is therefore due to the increased cell aggregation previously observed in bacterial
culture [46] which may have lead to higher biomass density on electrodes.
3.5 Conclusion
CaCl
2
has been shown to play a signicant role in cellular metabolism. It is known
to be important in both eukaroytes as well as bacteria. It plays a role in signalling
and cell aggregation, such as biolms. MFCs innoculated with CaC
2
show an
increase in current production, and this work showed that the impact of CaCl
2
on current Shewanella oneidensis MR-1 was determined to be primarily due to
biological factors. EIS data show that solution resistance is not impacted, and
MFCs with the highest current production in the presence of CaCl
2
showed the
highest solution resistance. This implies that cell aggregation may be the key factor
contributing to increased current production, which might simultaneously increase
the resistivity of the MFC, while also increasing the number of cells participating
in DET.
35
Chapter 4
Microbial Fuel Cell Studies of
Shewanella oneidensis MR-1 and
Mutants Disrupted in
Extracellular Appendage
Production
4.1 Introduction
In the previous chapter, we saw an example of MFCs being used to make biological
inferences about microbe-electrode interfaces. In this chapter we will attempt
to extend this technique to determine what, if any, conclusions can be formed
about the relationship between extracellular appendeges and current production
in fuel cells. This work uses mutants lacking the genes that encode certain protein
structures found on Shewanella oneidensis. However, understanding the impact of
genetic alterations on MFC current production is a more dicult problem because
of possible eects of pleiotropy when a gene in
uences multiple phenotypical traits,
and so we will see that while current production and power generation can be
quantied, extending these observations to form hypotheses about the biological
signicance of the results has limitations.
36
Bacterial type IV pili are polymeric protein laments found on all Gram-
negative bacterial surfaces, and are known to play a crucial role in diverse cellular
functions, including motility, surface attachment, biolm formation, adhesion to
host cells, signaling, DNA uptake, and pathogenicity [21]. DMRBs are known to
produce nanowires that are involved in extracellular electron transfer [33]. The
composition of bacterial nanowires was found in Geobacter sulfurreducense to be
type IV pili and so it was suggested the same must be true for MR-1. It was
not until recently that the composition of nanowires in Shewanella oneidensis was
found to be membraneous [80]. However, the role of pili in Shewanella oneidensis
has not been determined.
Previous studies targeting the biomolecular composition and physical mecha-
nism of nanowires focused on the DMRBs Geobacter and Shewanella. Conductive
laments produced by Geobacter consist of PilA, the structural subunit of the
type IV pilus [59, 84]. In addition to direct measurements of conductance in
Geobacter pili, the presence of conductive pili has been correlated with improved
electricity generation in MFCs, and enhanced reduction of solid-phase iron
oxides [84]. Furthermore, these pili may serve a nonconductive role in biolm
development, which also aects overall EET to solid electron acceptors [85].
Direct measurements of conductance in individual Shewanella nanowires, using
scanning probe techniques and nanofabricated devices, suggest that electron
transport proteins (c-type cytochromes) are necessary for electron transport along
Shewanella nanowires [28, 29]. A recent study [11] also examined the eect
of Shewanella oneidensis MR-1 pili on MFC current generation by screening
mutants disrupted in
agella, and mutants disrupted in one or both of the two
type IV pilin systems in Shewanella (msh and pi), using a high-throughput
37
system known as a voltage-based screening assay (VBSA) [6]. Using this tech-
nique, it appeared that Shewanella type IV pili were not required for current
generation in MFCs, which contradicts the nanowire model. Even more surprising
was the nding that
agella and type IV pili mutants (
g andpilM-Q)
produced even more current than the wild-type, at least under the xed and
relatively high MFC external resistance (R
ext
= 100 k
) used in the previous study.
To help shed further light on the role of type IV pili in Shewanella MFCs,
we present a more detailed electrochemical analysis of MFC performance using
wild-type Shewanella oneidensis MR-1, mutants lacking
agella or the type IV
pili biogenesis systems, in addition to new mutants lacking the specic structural
subunits of type IV pili. However, ambiguities arose when analysing the data
which cast doubt on what role the deletion of the pili played. The dierence in
current production, as well as the cell polarization behavior spanning a range of
MFC external resistances could be due to either poor biolm formation, or low
cellular respiration rates. This work highlights the need for a more direct method
of examining the behavior of individual cells.
4.1.1 Polarization Resistance Measurements
Polarization resistance measurements are a common electrochemical technique
used for analyzing the behavior of fuel cells. These measurements involve dynam-
ically controlling the resistance between the anode and cathode of the cell while
measuring the current. The resistance begins at open circuit and is lowered to
short circuit shown in blue in 4.1. The current vs. potential prole reveals the
behavior of the cell at various resistances. The power can be calculated by multi-
plying the voltage times the current, shown in red in 4.1. The peak value represents
38
Figure 4.1: Example of a polariation resistance measurement. Black are the raw
data, taken from open circuit potential (Left y intercept) to short circuit (right
x intercept). Blue curve is the power calculated from the polarization curve. i)
shows the open circuit voltage, ii shows the low current, high resistance regime,
iii shows the peak power, iv shows the Ohmic regime, v shows the short circuit
regime
the resistance at which the power output of the fuel cell is optimal. The weaker
the fuel cell performance, the faster the drop o to short circuit current values and
the smaller peak value of the power curve.
39
4.1.2 Cultivation of Bacteria for MFC Testing
Strain Description Source
MR-1 Shewanella oneidensis wild type Myers et al. 1988

g MR-1 mutant lacking the
agellin
structural genes
Bouhenni et al. 2010
mshA-D MR-1 mutant that lacks the
structural mshDCAB pilin genes
Fitzgerald et al.2012
mshH-Q MR-1 mutant lacking the Msh
biogenesis genes
Bouhenni et al. 2010
pilA MR-1 mutant lacking structural
component of type IV pili
Pirbadian et al. 2014
pilM-Q MR-1 mutant lacking type IV
pilin biogenesis genes
Bouhenni et al. 2010
pilM-Q/
mshH-Q
MR-1 mutant that lacks both
Msh and type IV pilin biogenesis
genes
Bouhenni et al. 2010
40
A list of the strains used in this study is provided in table 1. For each strain, a
starter culture was grown in LB broth from a glycerol stock frozen at -80

C. The
starter culture was used to inoculate a dened PIPES-buered minimal medium
as detailed previously (Bretschger et al. 2007), using 18 mM lactate as the sole
carbon source. All cultures were grown aerobically at 30

C and agitated at a rate
of 150 rpm. Cells were harvested and injected into the MFC anode compartment
for a nal OD600 of 0.4, using the buer as the diluting medium when needed.
Electrochemical tests were performed in a dual-compartment MFC system,
previously described in detail [12]. The two compartments were separated by a
proton exchange membrane (Naon 424, DuPont). The anode (graphite felt GF-
S6-06, Electrolytica) compartment was kept anaerobic by passing N
2
gas at a
rate of 20 ml/min. Aerobic conditions were maintained in the cathode (graphite
felt electroplated with Pt, 0.15 mg/cm2) compartment by passing puried air at
a rate of 40 ml/min. The PIPES buer solution was used in both the anode
compartment (inoculated to an OD600 of 0.4), and the sterile aqueous oxygen-
reduction cathode compartments. Each strain was evaluated using a minimum of
three MFCs simultaneously, and current-time (I-t) data were collected by recording
each MFC voltage (V) every 2 minutes across a 10
resistor by a high-impedance
digital multimeter (Keithley Instruments, model 2700), and calculating the current
from Ohm
s law, I = V/R. The MFCs were operated under batch conditions.
After addition of bacteria and the establishment of a baseline voltage, lactate (2
mM) was added as the electron donor in the anode compartment, and additional
lactate feedings were performed when the cell voltage dropped to baseline values.
A minimum of 3 feedings were performed for each MFC test. In addition to the
I-t data at a xed external resistance, the MFC polarization was examined by
performing MFC potential sweeps (V-I) using a Gamry Reference 600 potentiostat
41
(Gamry Instruments) as described previously [60]. Polarizations began at the
open-circuit cell voltage (V
oc
), where I = 0, and continued at a scan rate of 0.2
mV/s until the short-circuit cell voltage V
sc
= 0, where I = I
max
. In addition, the
polarization measurements were used to calculate the power-current (P-I) curves.
To detect the levels of the extracellular ribo
avin in the MFC anode compart-
ments, supernatants were collected from a number of strains (WT MR-1,
g,
pilA, mshA-D) and cells were removed by centrifugation and ltration using
a 0.2 m lter. The ribo
avin concentrations were tested via High Performance
Liquid Chromatography (HPLC), as described in previous work [11].
Cell attachment was examined by scanning electron microscopy (SEM), anodes
were removed from the MFCs after tests were completed. The anodes and asso-
ciated cells were subjected to a serial dehydration protocol using increasing con-
centrations of ethanol (10, 25, 50, 75, and nally 100% vol/vol ethanol). The
dehydrated samples were then critical-point dried and examined using a JEOL
JSM-7001F-LV eld emission SEM.
42
Figure 4.2: A) Current vs. time data for MFCs innoculated with mutants. Arrows
indicate injection of 2 mM lactate. B) Average peak current for triplicate MFCs
for each mutant.
4.2 Results and Discussion
4.2.1 Microbial Fuel Cell Mutant Response
The MFCs responded quickly at the beginning of each batch cycle, by achieving
peak current typically within 10 minutes of adding lactate as the electron donor
(4.2A). However, signicant dierences in the peak current were observed between
the strains tested in this study. Figure 4.2A compares the current output (I-t
during three feeding cycles) for MFC experiments operated using wild-type MR-1
and the two mutants lacking the Pil and Msh structural proteins, with a 10

external resistor. In this experiment, the pilA and mshA-D strains produced
less than 10% of the peak current, compared to wild-type MR-1. Of all the strains
tested, the mutant lacking
agella (
g) as well as the mutant lacking the mshH-Q
proteins achieved a peak current statistically comparable to MR-1. The remaining
strains, disrupted in the biogenesis and/or structural subunits of type IV pili, all
43
Figure 4.3: A) Polarization resistance measurements for MR-1 and mutants. B)
Power density curves calculated from data in A.
produced signicantly less current than the MR-1 and
g strains (4.2A), with
the pilA strain producing less than 2% of the wild-type peak current.
4.2.2 Polarization Resistance
While the observation of comparable peak current output from the wild-type and

agella mutant strains was reported previously, the diminished current from all
type IV pili mutants stands in contrast to the previous study [11], which indicated
that pili are not essential for EET to minerals or MFC anode surfaces. However,
the overall current output of an MFC depends strongly on the external resistance
imposed on the device. The previously used high-throughput voltage-based screen-
ing assay (VBSA) monitored the cell voltage across a 100 k
external resistance.
This value is signicantly higher than the condition chosen to examine the high-
current regime in gure 4.2: R
ext
= 10
which is much less than the internal
resistance, R
int
, of this MFC system [61]. Therefore, to examine whether the dis-
crepancy is due to dierences in the polarization behavior caused by the dierent
44
external resistances, we measured the MFC polarization curves for all the strains
using a potentiostat. Polarization curves are recognized as powerful tool for analy-
sis of MFCs [55], and provide a more complete picture by characterizing the MFC
behavior from open-circuit to short-circuit conditions, eectively spanning a range
of external loads.
The cell polarizations, for all the strains examined in this study, are presented in
gure 4.3. The wild-type and mutant strains had comparable open-circuit voltages
(V
oc
) as shown in gure 4.3A. However, in the higher electron transfer regime,
dierences could be observed between the pili mutants and the MR-1/
g strains
which matched the current output data from the MFCs in 4.2. Mutants lacking
pili performed worse under all conditions. This nding further reinforces the need
to examine the MFC polarization behavior, spanning dierent electron transfer
regimes, when making comparative evaluations between dierent microbial strains.
The characteristic power curves for each strain are shown in gure 4.3B, and are
consistent with the nding that the disruption of type IV pili diminished power
output in Shewanella oneidensis MR-1 MFCs.
The eect of type IV pili on current generation in Shewanella MFCs may be
attributed to a number of causes. Previous studies [62, 98] found a role for soluble
redox shuttles, specically
avins, in mediating EET between Shewanella and
anodes. A subset of the evaluated strains were analyzed for extracellular
avin
concentration at the end of MFC testing. The concentration of ribo
avin from the
MR-1 anode compartment was 40 nM; less than the measured concentrations in
the lower current-producing mshA-D and pilA strains, which were found to be
70 nM and 220 nM, respectively. These data suggest that this mediator does not
account for the observed EET dierences under the MFC conditions investigated
here.
45
4.2.3 Scanning Electron Microscopy
The presence of type IV pili, Msh pili, and
agella is known to in
uence cell attach-
ment and subsequent biolm formation [58], which in turn can aect the overall
MFC current production by involving more cells in direct EET to anodes [65].
SEM imaging, however, did not reveal a clear correlation between the density of
attached cells and the observed current generation. While the non-motile strain
(
g) has been identied as decient in biolm formation [18, 58] relative to MR-1,
the current densities of these two strains were comparable. In addition, none of the
anodes examined revealed a high degree of biolm development making it ambigu-
ous whether biolm attachment had anything to do with the dierence in current
production. On the other hand, the mshA-D anode showed no attached cells
at all, yet a similar current production to the pilA strain. It should be noted,
however, that SEM imaging may underestimate overall biolm formation, since
well-adhered cells are most likely to survive the sample preparation procedure.
Recent reports utilized single-cell [53] and microscopic methods [65] to dis-
tinguish between single-cell and multi-cellular EET. The diminished current-
generation by mutants lacking pili, reported here, along with recent measurements
of conductance in individual MR-1 laments, motivate further studies to determine
whether these pili in
uence MFC performance by only aecting cell attachment
and subsequent biolm formation, or also as direct EET conduits from individual
cells.
46
Figure 4.4: Scanning electron microscopy images of A) MR-1, B)
g, C) pilA,
D) pilM-Q/mshH-Q
4.3 Conclusion
In conclusion, peak current measurements and polarization curves revealed that
the disruption of type IV pili decreased current generation in Shewanella oneiden-
sis MR-1 microbial fuel cells. The diminished overall extracellular electron transfer
rate was not caused by decreased levels of extracellular ribo
avin, a known solu-
ble redox shuttle. Scanning electron microscopy did not reveal a clear correlation
47
between the extent of cell attachment, which can be in
uenced by pili and
agella,
and the overall MFC current. The results suggest a possible contribution to extra-
cellular electron transfer from type IV pili, however it remains unclear whether this
is due to poor biolm formation, i.e. fewer cells contributing to current production,
or whether the individual cells were respiring less. This motivates further studies to
quantify this contribution on a per cell basis. Towards this goal, we developed new
measurement platforms combining optical trapping and in situ electrochemistry
approaches to gain a mechanistic understanding of single-cell direct extracellular
electron transfer which we detail in chapter 5.
48
Chapter 5
Development of a Single Cell
Extracellular Electron Transfer
Measurement Technique: Design,
Construction and Electrochemical
Validation.
5.1 Introduction
In the previous chapter we saw that genetic studies using MFCs can lead to ambi-
guities resulting from the bulk nature of the measurements. Given the wealth of
knowledge that can be gained from genetic studies, single cell techniques are crit-
ical for studies that compare wild-type to mutant strains lacking putative EET
proteins. Gene products hypothesized to perform EET (e.g. surface cytochromes
or type IV pili) may also impact the cellular surface charge, attachment ability, and
biolm forming properties. Bulk techniques therefore cannot distinguish whether
a decrease in anodic current is due to diminished EET directly through a spe-
cic protein, or indirectly by disrupting cellular attachment to electrodes and the
subsequent biolm development.
49
The eect of heterogeneity is particularly important in MFCs that contain a
mixture of attached and planktonic cells, keeping in mind that a wide statistical
distribution of cellular respiration current is possible even within each of these two
sub-groups. The causes of microbial heterogeneity include basic genotypic vari-
ability resulting from mutations, as well as phenotypic variability resulting from
progression through the cell cycle or as a physiological response modulated by the
local environment and its history [22]. As we seek a more complete understand-
ing of the maximum power densities possible from microbial technologies such as
microbial fuel and electrosynthesis cells, there is a clear need for techniques that
quantify both the expected per-cell and statistical distribution of EET in microbial
cultures.
Only a handful of recent studies address these issues surrounding bulk electro-
chemical techniques. McLean et al. quantied the average per-cell EET to graphite
electrodes by combining live noninvasive imaging with an optically accessible MFC,
allowing an accurate cellular count as the anode's Shewanella oneidensis MR-1
population developed from separate cells to mature biolms [65]. This approach
revealed a current per cell range from 50-200 fA, depending on the MFC resis-
tance and growth phase. Another study obtained single cell measurements of EET
from a dierent DMRB, Geobacter sulfurreducens DL-1, using microscale Ti/Au
electrode arrays [45]. When active cells transiently came into contact with these
electrodes, a short circuit current of 92( 33)fA per cell was observed in about 30%
of the contact events. Finally, Liu et al. reported an innovative approach taking
advantage of optical tweezers to attach single Shewanella oneidensis MR-1 cells
to electrodes, revealing a current output of 200 fA per cell at a working electrode
potential of +200 mV vs. Ag/AgCl [53]. To date, however, there is no technical
50
description of a complete instrument or standardized measurement procedure for
detecting single cell extracellular respiration.
Here we present the rst complete description of a system inspired by, and
combining elements from, the above-mentioned studies of specic respiration rates.
The integrated system combines live imaging, an infrared optical trap, and a new
electrochemical chip concept containing an array of indium tin oxide (ITO) micro-
electrodes that serve as `landing pads' for individually trapped bacteria. The
microelectrode potentials are controlled by a potentiostat, allowing the detection
of the respiration current concomitant with landing a cell on a specic microelec-
trode. The entire system can be built from standard commercially available optical
and electrochemical components, in combination with standard lithographic micro-
fabrication techniques. The microfabrication procedure was optimized to address
several challenges stemming from the biocompatibility requirement and high cur-
rent sensitivity needed to detect single cells. Furthermore, the biological growth
conditions and measurement protocol are described in detail. In addition to allow-
ing single cell measurements of EET from a variety of organisms and mutants,
the reported system may nd wider applicability for general microbiology studies,
including the analysis of how respiration rates in heterogeneous microbial cultures
are impacted by specic environmental factors.
5.2 Experimental Setup
The single cell extracellular respiration platform integrates three main components:
(1) An infrared optical tweezers system for positioning individual cells, (2) a cus-
tom transparent electrochemical chip containing an array of indium tin oxide (ITO)
51
microelectrodes on standard coverslips, and (3) A vacuum-sealed perfusion cham-
ber for loading and handling of bacterial cultures in physiological media. In what
follows, we detail the design criteria and construction of each of these components.
5.2.1 Infrared Optical Trap
Optical trapping stemmed from a series of pioneering experiments by Ashkin, [2]
demonstrating the eect of laser-induced optical forces on controlling micron-sized
particles in both air and liquid. The technique has been extensively used to charac-
terize biological samples, ranging from DNA and single protein molecules to whole
cells [73].
Our optical tweezers system is constructed from commercial components (com-
bined as #OTKB, ThorLabs), and is based on previously published designs [1].
The system (Figure 5.1) is assembled in an inverted conguration, ideal for in vivo
biological work, and equipped with beam directing/steering components, a manual
translation stage in combination with three piezo-actuators (20 m of travel at 20
nm resolution driven by three #TPZ001 piezo drivers, ThorLabs), a 100X Nikon oil
immersion objective (1.25 numerical aperture, 0.23 mm working distance), and a
12801024 pixel CCD camera interfaced via USB to a computer for video imaging.
The sample is illuminated for imaging by a single emitter white light LED through
a 10X air condenser lens (0.25 numerical aperture, 7 mm working distance). The
trapping laser source is a 980 nm single mode ber-pigtailed laser diode (1.1 m
spot size, 330 mW maximum power, #PL980P330J, ThorLabs) mounted in a laser
diode mount (#LM14S2, ThorLabs) and controlled using a laser diode current
driver and temperature controller (#LDC210C and #TED200C, ThorLabs). The
laser output is collimated using a FiberPort (#PAF-X-7-B, ThorLabs). Dichroic
52
Figure 5.1: Optical layout of the trapping setup for single cell extracellular respi-
ration measurements. The infrared laser (980 nm) traps individual bacteria within
a perfusion chamber sealed against an electrochemical chip containing indium tin
oxide (ITO) microscale working electrodes. The microelectrodes are biased at a
suitable electron-accepting potential, relative to an integrated Ag/AgCl reference
electrode, using a potentiostat. The chip also contains an ITO counter electrode
to complete the standard 3-electrode electrochemical cell. The trap is used to land
individual bacteria on the microelectrodes, and the single cell respiration current
is measured using the potentiostat.
mirrors are used to transmit visible light (e.g. to the camera for imaging), while
re
ecting the infrared laser towards the sample for trapping.
The laser wavelength (980 nm) was chosen to minimize damage to the cells
during manipulation before placement on the microelectrodes. Previous studies
have characterized possible photodamage to the trapped cells [67, 74]. The damage
can be largely mitigated by using the near infrared region (790-1064 nm), with
a photodamage minimum at 970 nm. In addition, since oxygen is implicated
in the photodamage pathway, anaerobic conditions reduce cell damage down to
53
background levels [74]. This is ideal for our purposes, since our goal is to perform
EET measurements under anaerobic conditions to exclude oxygen as an alternate
electron acceptor. By taking appropriate precautions including a near-IR laser,
anaerobic conditions, and < 100 mW of trapping power at the sample plane,
previous studies have manipulated individual E. coli cells for durations up to an
hour [67] (in excess of the time needed to perform EET measurements) and even
conrmed the long-term viability by observing the division of trapped cells [3].
These conditions allowed us to trap individual Shewanella oneidensis MR-1 cells,
which remained viable judging by their continued motility after turning o the
trap.
54
5.2.2 Electrochemical Chip: Concept and Fabrication
Our chip concept facilitates respiration measurements from individual bacteria in
a standard three-electrode electrochemical conguration. An array of nine ITO
working electrodes (WEs) are lithographically patterned on standard glass cover-
slips, in addition to a central counter electrode (CE) (Figure 5.2). An Ag/AgCl
reference electrode with porus te
on tip (#CHI111P, CH Instruments) is connected
just upstream of the chamber.
Since our goal is to position individual bacteria on dened electrodes, the major-
ity of the chip's surface is passivated with a layer of SU-8 epoxy based photoresist
(#2000.5, MicroChem), except for exposed ITO regions that allow contact to the
biological growth medium by the central 0.5 mm radius CE and dened 15
15 m windows at the WE tips. In addition to discouraging accidental contact
with more than one (or few) individual bacteria, the small exposed WE window
translates to a small background electrochemical current before cellular contact.
The small baseline current is critical, allowing us to select a small total current
measurement range (e.g. < 60 pA) by the potentiostat (Reference 600, Gamry),
which in turn facilitates detection of the sub-pA currents expected from individual
bacteria (e.g. 10 fA steps can be detected in the 60 pA total range using the
potentiostat's 16-bit analog/digital converter). Figure 5.2a is a picture of the fab-
ricated chip, with an inset image (atomic force microscopy, see below) showing the
ITO window at a WE's tip, which serves as the cellular landing pad. Figure 5.2b is
a schematic of the same chip. Note the centralized design, allowing minimal trans-
lational motion while switching between WEs on the optical stage, while keeping
all the WEs equidistant from the central CE. Next we detail the microfabrication
work
ow leading to the nished chip.
55
Figure 5.2: (a) The microfabricated electrochemical chip containing an array of
ITO electrodes. Inset shows a tapping mode atomic force microscopy image of one
of the windows fabricated at the tips of the nine working electrodes (WEs). These
windows provide microscale openings to the ITO underlying an SU-8 passivation
layer. (b) Schematic of the device, showing the nine WEs as well as the central
counter electrode (CE) in yellow, as well as the overlying passivation layer in purple.
Inset shows a larger view of one of the windows, which allows the attachment of
optically trapped bacteria on ITO.
56
The microfabrication process is divided into three stages, each requiring a
lithography mask, as shown in Figure 5.3). The rst stage (steps 1-5 in Figure 5.3)
results in the desired ITO pattern on the coverslip. The second stage (steps 6 and
7) produces a preliminary SU-8 passivation layer, which keeps only desired ITO
features exposed. Finally, the third stage (steps 8-9) produces the second SU-8
passivation layer, which dene the small 15 15 m WE windows. The chip is
then sealed against the perfusion chamber (step 10), ahead of optical trapping and
electrochemical measurements (step 11).
Before microfabrication, the glass coverslips are cleaned with a series of son-
ication steps in acetone, isopropyl alcohol, and distilled de-ionized (DDI) water,
for ve minutes each, and then nally rinsed in DDI water and dried using N
2
gas. Any residual water is removed by heating the chips at 150

C for ten minutes
on a hot plate. To remove all organic matter, the coverslips are then exposed to
an O
2
plasma (Tegal Plasma Asher 421) for two minutes at 200 W with a re
ec-
tive power of 5 W. The coverslips are then treated with hexamethyldisilazane
(HMDS) vapor in an HMDS vapor deposition tank to promote the adhesion of
photoresist in the subsequent steps.
To begin the rst stage (steps 1-5 in Figure 5.3), positive photoresist (AZ 5214
IR, Clariant) is spin coated on the coverslips for 5 s at 500 rpm, using a ramp
of 100 rpm/s, to remove the bulk of the photoresist, and then at 3,000 rpm for
30 s. The resulting photoresist layer is 1.6 m thick. The chips are then baked
for 2 min at 100

C in order to remove the solvent and set the resist. The rst
photomask, which denes the ITO WEs and CE, is aligned with each chip using
a Karl Suss Aligner MA6 set for 15 s exposure time at 8 W lamp power, with
an alignment gap of 30 m. The exposed chips are then submerged for 1 min
in a solution of 5 parts DDI water to 1 part positive photoresist developer (AZ
57
400K, Clariant). The chips are rinsed in DDI water and plasma cleaned again
as described above. The result is a coverslip with photoresist present everywhere
except where conductive electrode contacts are desired. A 300 nm thick ITO lm is
then deposited (2,400 s at 180 W in Denton Discovery Sputterer 550) on the entire
surface. The thickness was chosen based on prior work showing favorable electrical
properties at this thickness while still maintaining high optical transmittance for
both visible and infrared light [39]. After ITO deposition, the chip is submerged in
acetone and sonicated for 5 min to lift o the resist. It is then cleaned again using
isopropyl alcohol and DDI water. At this point, the chip consists of the desired
ITO array pattern on the coverslip. The chips are then baked at 400

C in N
2
.
Annealing helps create oxygen vacancies, which donate two free electrons, as well
as increases the density of the crystal structure.
In the second stage of microfabrication (steps 6 and 7 in Figure 5.3), SU-8
2000.5 negative photoresist (MicroChem) is spin coated on the chips for 5 s at 500
rpm, using a 100 rpm/s ramp, and then for 30 s at 1,800 rpm with 1,000 rpm/s
ramp. The chips are then baked at 90

C for 1 min in order to remove the solvent.
The second photomask, which denes the openings for ITO features, is aligned
with each chip using the Karl Suss Aligner set for 23 s exposure with a 23 m
gap on soft contact. The chips are post baked for 10 min at 90

C in order to
cross link the SU-8 polymer and harden the exposed resist. After post baking, the
chips are submerged in SU-8 developer for 1 minute, rinsed with isopropyl alcohol
for 10 s, rinsed with DDI water, and developed again for 10 s in fresh developer.
The second development step ensures the removal of any traces which might have
reattached from the solution during the rst development step.
Large area development of SU-8 can often introduce microscale
aws (e.g.
cracks or holes) that may leave ITO exposed in undesirable areas. The third stage
58
of microfabrication (steps 6 and 7 in Figure 5.3) addresses this issue by applying
a second SU-8 passivation layer on top of the rst. Using a dierent photomask,
the second layer features smaller 15 15 m windows designed to t within the
larger windows of the rst layer. The coating and exposure details are similar to
the second stage, described above, except that the exposure time is reduced to 21
s from 23 s. The reduced exposure time ensures the unexposed SU-8 (now rela-
tively small 15 15 m) features are developed away more easily, avoiding any
surface residue that may passivate the desired window. Furthermore, the chip is
now sonicated during development for 10 s, unlike the rst layer of SU-8. After
development, the chips are treated with O
2
plasma in the Tegal Plasma Asher 421
once more, but at a lower power (100 W forward power with re
ective power 5W
for 1 min) so as not to damage the thin SU-8 layer. The result is a ready-to-use
mostly passivated surface with small exposed windows at the tips of the ITO WEs,
and a large exposed central ITO CE.
5.2.3 Perfusion Chamber and Assembly
A perfusion chamber, for loading and handling of growth medium and bacterial cul-
tures, was designed and constructed to interface to the electrochemical chip. The
key design criterion is a good seal, in order to minimize oxygen permeability into
the system. While Shewanella oneidensis MR-1 is a facultative anaerobe, capable
of growing with both oxygen and other soluble or insoluble electron acceptors, the
availability of any alternate oxidant in the system will minimize the EET current to
the microelectrodes. The round polycarbonate chamber body (Figure 5.4a) seals
against the bottom chip and a top coverslip by pulling a vacuum between two con-
centric o-rings at the bottom and top interfaces. Figure 5.4b shows the chip (with
attached 32 gauge stranded wires that connect to the potentiostat) and chamber
59
(with inlet, outlet, reference electrode and vacuum line) assembly mounted on top
of the optical trap's microscope stage. Finally, Figure 5.4c shows the whole inte-
grated system, which is enclosed within a home-made metallic enclosure serving
as a Faraday cage to minimize any external interference detrimental for sub-pA
measurements.
5.3 Electrochemical Characterization of the
Microelectrodes
The microelectrodes' fabrication and electrochemical activity were characterized in
advance of the biological measurements. Tapping mode atomic force microscopy
(TM-AFM) was used to conrm that the lithographically fabricated WE win-
dows developed properly, using a Bruker Innova AFM and Olympus silicon probes
(AC240TS) with 2 N/m spring constant and 92nm tip radius of curvature. Fig-
ure 5.3a contains the AFM topography prole of a typical window, showing a
central smooth (1 nm RMS roughness) ITO bottom surface with a sharp drop
o at the edge of the window, and the relatively rough (5 nm RMS roughness)
surface of the SU-8 passivation layer. The AFM images also revealed that the
square design developed with rounded edges, rather than sharp corners, making
the window geometry closer to a circle with 8.4 m diameter.
After ensuring the successful microfabrication of the ITO WEs, we turned our
attention to conrming the electrochemical activity of these microelectrodes using
the standard ferri/ferro cyanide redox couple (Fe
3
CN
6
3
+e

$Fe
3
CN
6
4
) in our
system, rather than the bacteria (which are characterized in the next chapter).
Figure 5.5 shows the cylic voltammetry (CV) from two separate WE windows
exposed to a solution of 10 mM K
3
Fe(CN)
6
in 1 M KNO
3
. The CVs exhibited
60
the classic expected ultramicroelectrode[4] (UME i.e. with a critical dimension
smaller than the length scale of the surrounding depletion later) behavior with no
peaks but a sigmoidal quasi-reversible transition between the oxidized and reduced
states. Under these conditions, starting in the fully oxidized state and applying
a reductive potential results in a maximum current described by i =nAFm
O
C

O
,
wheren=1 for this one-electron process,F is Faraday's constant,A is our nominal
electrode area, and C

O
is the bulk concentration of ferricyanide in our CV mea-
surement. m
O
is a geometric mass-transfer coecient given by
4D
O
r
O
for a UME
disk shape with radiusr
O
andD
O
is the ferricyanide diusion coecient, which is
on the order of 10
9
m
2
/s from prior studies [4, 40, 96]. This calculation, which
is intended for heuristic comparison since the disk shape only approximates our
rounded-corner WEs and because of the inherent window-to-window size variabil-
ity (up to a few microns) in our SU-8 photolithography process, reveals an expected
current on the order of tens of nA. This expected range is in good agreement with
the results in Figure 5.5 for two separate WEs, conrming that most, if not all,
the surface area of the ITO windows is electrochemically active. Shorting the two
WEs gave the expected additive current result, with no extraneous contributions.
With the electrochemical validation in hand, we now describe the cellular EET
measurements.
5.4 Conclusion
We described an integrated experimental system that combines optical trapping,
transparent electrochemical chips displaying potentiostatically controlled indium
tin oxide microelectrodes, and a biological perfusion chamber for single cell mea-
surements of extracellular electron transfer in bacteria. The system is capable
61
of manipulating individual bacteria and detecting their specic extracellular res-
piration rates down to currents < 100 fA. The trapping, microfabrication, and
measurement protocols were optimized to address several challenges surrounding
the biocompatibility, feature resolution, and electrochemical sensitivity needed to
detect single cell response. The system helps dene the fundamental limit of res-
piration in microbial cultures and biolms, down to the single cell level, which
is critical for predicting the maximum power densities of technologies that rely
on electron exchange between bacteria and electrode surfaces, including microbial
fuel and electrosynthesis cells. Applications of this instrument also include dis-
covering the extracellular electron transfer pathways in living cells, by studying
specic mutations, and quantifying the impact of environmental factors on the
heterogeneity inherent in microbial populations.
62
Figure 5.3: Microfabrication work
ow for the electrochemical chip. Steps 1-5 show
ITO deposition and patterning. Steps 6-9 detail the passivation of the surface
with two SU-8 layers, while leaving microscale windows to the ITO for cellular
attachment. Steps 10 and 11 show perfusion chamber assembly and trapping.
63
Figure 5.4: (a) Schematic of the perfusion chamber sealed against the bottom
electrochemical chip and a coverslip allowing illumination from the top. A seal
is achieved against both surfaces by pulling a vacuum between dual o-rings. (b)
The entire chamber-chip assembly mounted on a microscope stage. The reference
electrode/inlet, vacuum line, and stage are labeled with i, ii, and iii, respectively
(c) The integrated platform combining the perfusion chamber and electrochemical
chip, labeledi, with the optical trapping system. The translation stage and infrared
laser ber are indicated by ii and iii.
64
Figure 5.5: Cyclic voltammetry demonstrating the electrochemical activity of the
lithographically patterned ITO working electrodes exposed to 10 mM K
3
Fe(CN)
6
in 1 M KNO
3
, and using the chip's central ITO counter electrode. Potential is
shown against Ag/AgCl (1M KCl). Scan rate 10 mV/s.
65
Chapter 6
Single Cell Extracellular Electron
Transfer Measurements of MR-1,
OMC, andbfe and the
Importance of the Flavin Export
System on Current Production
6.1 Introduction
In the previous chapter we described the development of a single cell DET measure-
ment system involving optical trapping and custom microelectrodes and validated
the electrochemical properties of the electrode for sub-pA electrochemical measure-
ments. In this chapter we perform single cell trapping and highlight the importance
of specic genes in DET through the use of mutants. The ability to produce outer
membrane cytochromes, as well as the ability to extracellularly export
avins, is
investigated for their importance in DET in single cell contact events with the
electrode surface.
Wild-type Shewanella oneidensis MR-1, a markerless multi-deletion mutant
OMC, which lacks all ve of the outer membrane cytochromes encoded in the
genome, some of which are known to play a role in EET, [16], as well as bfe
66
strain which lacks the bacterial
avin adenine dinucleotide [FAD] exporter [48],
were tested using single cell trapping. This work shows the importance of both

avins and extracellular cytochromes in DET. Single cell measurements highlight
how the heterogeneity of individual cell behavior varies between the strains, and
allows us to infer a role for
avins in DET.
6.2 Materials and Methods
6.2.1 Growth protocol
All three strains tested in this work were rst grown aerobically in 50 mL LB
medium from a frozen (80

C) stock up to an optical density at 600 nm (OD
600
)
of 1.5. These aerobic pre-cultures were inoculated at 1% (v/v) into anaerobic serum
bottles containing 100 mL PIPES-buered (pH 7.0) M1 medium with 18 mM lac-
tate and 30 mM fumarate as the electron donor and acceptor, respectively. [12]
Prior to inoculation, anaerobic conditions were reached by purging with N
2
for
one hour and the serum bottles, sealed with butyl stoppers and aluminum seals,
were sterilized by autoclaving at 120

C for 15 min. Both MR-1 and OMC anaer-
obic cultures were grown to mid-exponential phase (OD
600
0.07), while bfe was
grown to late exponential phase(OD
600
0.09) due to delayed CFU production,
after which cells were injected into a dened non-growth trapping medium (at 10
6
fold dilution for low cell density) that lacks fumarate as well as the vitamins, min-
erals, and amino acid trace solutions of M1 medium. Fumarate was removed to
ensure that no soluble electron acceptor diminishes EET, while the trace solutions
were removed to avoid their possible redox activity at the microelectrode surfaces.
The low cell density in the trapping medium reduces the likelihood of cells con-
tacting or accidentally landing on the microelectrodes before and during single cell
67
Figure 6.1: a) Current vs. time raw data (black) with the background (red) shown
for 10 min prior to contact and extended for 10 min post contact. b) Baseline
subtracted data.
measurements. Despite the low cell density, it should be noted that we did not
encounter diculty nding cells in the vicinity of WEs, likely due to a previously
reported phenomenon where cells congregate near insoluble electron acceptors [41].
Finally, this trapping culture is introduced into the chamber-chip assembly that is
integrated into the optical trap.
6.2.2 Trapping events
A potential of +400 mV vs. Ag/AgCl (1M KCl) was applied to the chips' WEs,
and the system was left to stabilize for up to three hours before trapping, in
order to achieve the low electrochemical background (typically < 60 pA and a
current change rate < 10 fA s
1
) necessary for detecting the expected respiration
currents, as discussed above. Such a background allowed a simple detrending pro-
cedure to quantify the eect of single cell respiration, where the current before
68
Figure 6.2: Current measurements for four distinct a) Shewanella oneidensis MR-1
cell contacts, and b) OMC cell contacts with microelectrodes biased at +400 mV
vs. Ag/AgCl (1M KCl). Contact happens at t=0 in all cases. c) shows an image
of a single Shewanella oneidensis MR-1 cell (indicated with white circle) trapped
on the microelectrode window. 5 m scale bar.
cell attachment is t with a linear or low-order polynomial and this trend is sub-
tracted from the time series (6.1). Prior to the trapping experiments we conrmed,
using standard plating and colony counting methods, that this stabilization time
in the acceptor-free medium did not result in any loss of viability. The WE poten-
tial was chosen to be compatible with Shewanella's direct EET pathways, being
more positive than the redox potential window of the outer membrane multiheme
cytochromes [89, 99].
6.3 Results and Discussion
In a typical scenario, a single cell is trapped in the vicinity of a WE after the
stabilization time and the stage's piezo-actuators are used to land the cell on the
ITO window. Out of 27 wild-type Shewanella oneidensis MR-1 cells landed in this
69
manner, 4 cells resulted in a clear current increase above background, all in the
range of 15 fA to 100 fA and sustained over> 10 min (Figure 6.2). Electrochemical
data were recorded for up to 15 min or until cells appeared to detach from the
surface. An increase in current after attachment was observed in a few additional
cases, but with a much slower rise time (> 15 min post attachment) than Figure 6.2,
making it dicult to denitively assign these responses to respiration rather than
instrument drift, using our simple detrending procedure. Our observation that
only a fraction of the cells is actively respiring at electrodes is consistent with a
previous study not based on optical trapping [45]. It remains unclear whether such
a percentage of actively electrode-respiring cells re
ects the typical heterogeneity
in these cultures, or whether it stems from the particular growth procedure used in
this and previous studies; our nding clearly motivates additional single cell studies
of the statistical distribution in EET as a function of various growth conditions.
In comparison, no EET current was detected from any of 20 OMC cells tested
under identical conditions( 6.2 b), further validating our measurement system, as
the outer membrane multiheme cytochromes are known to be necessary for medi-
ating EET [66, 70]. Similarly, 27 out of the 28 bfe cells tested showed no current
response either. Only one contact showed a slight increase of 10 fA after contact,
which is the detection limit of the instrument. This is consistent with previous
work done with bfe which showed that the strain produced approximately 25%
of the current of MR-1 in batch MFCs [48].
The suggested strategies that DMRB are reported to employ for EET fall
into two broad categories: indirect, and direct mechanisms. Indirect mechanisms
include naturally-occurring [56] or biogenic soluble mediators [62, 76, 98], such as
ribo
avin in Shewanella. Direct mechanisms take advantage of cell-surface contact,
using using multiheme c cytochromes located on the cell exterior [66, 70]. While it
70
has been shown that
avins are critical for EET in Shewanella, the exact mecha-
nisms involved are not yet fully understood [62, 76, 98]. It has been suggested that

avins are reduced by cytochromes on the cell surface, diuse away from the cell,
and are oxidized at the surface of the electrode, thus acting as shuttles [15]. How-
ever, others have suggested that
avins bound to outer membrane cytochromes
can account for their importance in DET [78]. Our experimental system probes
EET under direct contact conditions, and our observations that mutants disrupted
in
avin export are also disrupted in EET suggests that
avins play a role even
under direct contact conditions, consistent with the notion of cytochrome bound

avins playing a role in EET.
Finally, we discuss the magnitude of the observed respiration rates (15-100 fA
per cell), in light of previous EET studies and more recent measurements of the
Shewanella cytochrome complexes. The single cell currents detected here are con-
sistent with previous respiration measurements in whole planktonic and biolm
cultures (normalized by cell number), using O
2
and graphite electrodes as electron
acceptors, respectively [29, 65]. Furthermore, the respiration currents detected
here translate to an electron transfer rate up to 6:210
5
s
1
per cell. Recent mea-
surements of Shewanella's cytochrome complexes assembled into proteoliposomes
indicated average electron transfer rates up to 10
4
s
1
per complex to external min-
erals. [102] At this rate, the experimentally estimated average density of 10
3
-10
4
outer membrane cytochromes per cell [10, 57, 87] can easily satisfy our measured
extracellular respiration rates in whole cells. The type of measurement demon-
strated here therefore provides a gure of merit that can be used to quantitatively
assess the validity and impact of specic EET pathways.
71
6.4 Conclusion
We demonstrated the system with single cell respiration measurements of the
dissimilatory-metal reducing bacterium Shewanella oneidenis MR-1, which ranged
from 15 fA to 100 fA per cell, compared to no current output from mutants lack-
ing the outer-membrane cytochromes necessary for extracellular electron trans-
fer. The mutant OMC acted as a biological control and produced no current,
as is expected for this strain. Furthermore, the removal of excreted
avins also
showed reduced current compared to the wild type, indicating a strong relation-
ship between
avins and DET. Finally, this preliminary work shows the optical
trapping system described in chapter 5 to be a capable platform for measuring
single cell DET events, and calls for future single cell measurements.
72
Chapter 7
Conclusion
Dissimilatory metal reducing bacteria (DMRBs) unique ability to reduce solid
phase electrodes has been a topic of intense study for many reasons, ranging from
applications in electric power generation and wastewater remediation, to funda-
mental questions about biology and the origins of life. Fuel cells are a relatively
simple platform which takes advantage of DMRBs ability to interact with an elec-
trode surface to generate power. DMRBs interact with the surface of solids through
a series of c-cytochromes containing heme groups which act as charge carriers that
create a conductive bridge between the periplasm and the outer membrane. This
unique adaptation allows for electron transfer reactions involved in respiration to
take place over long distances. There are three proposed mechanisms involved
in DET; direct contact, the growth of conductive nanowire appendages, and the
excretion of redox active molecules that act as shuttles. However, the exact inter-
play between these mechanism is not yet understood.
This work examined fuel cells as instruments for understanding the interactions
between electrodes and bacteria. First this work looked at how Ca
+
ions aect
current production and examined whether or not current increases correspond-
ing to the addition of CaCl
2
into the anode of an MFC was the result of simple
abiotic reduction of the solution resistance in the fuel cell, or rather a biological
response increasing the rate of electron transfer to the anode. EIS measurements,
in conjunction with equivalent circuit modeling, revealed that the CaCl
2
did not
correspond to a signicant change in solution resistance, and thus CaCl
2
is likely
73
associated with biological factors, including biolm formation which resulted in
increased current production.
Next this work attempted to extend MFC operations to examine the role of type
IV pili on current production. At the time, it was known that nanowires found in
Geobacter were type IV pili and the same was assumed about Shewanella. In order
to examine the importance of these pili, mutants were generated which lacked either
the substructural proteins that serve as the building blocks for pili, or the biogenesis
systems that form the pili on the outer membrane. Both types of mutants produced
less current in MFCs under identical conditions. However, polarization resistance
measurements were unable to determine any signicance in power production in
MFCs as a result. Furthermore, SEM images of the anode bers revealed sparse
biolm formations and reduced cell size in some of the mutants. This raised a
fundamental question about the MFC results. Is the decrease in current output
from one mutant vs. another the result of a thinner biolm, in other words fewer
cells interacting with the anode of the MFC, or was each individual cells ability to
reduce the anode diminished?
Bulk measurements are fundamentally limited in their ability to answer ques-
tions of this nature. While the obvious thing to do is to normalize the total current
production by the total number of cells attached to the electrode surface, there is
no way to tell if each cell is contributing in a homogeneous fashion, or whether
some cells are producing more current. If one in ten cells produces ten times the
current of the others, an average will fail to make this distinction.
In chapter 5 we detail a method for a examining single cell electron transfer to
electrodes. The system involves an optical trap, created by focusing an infrared
laser through a 100x microscope objective which creates a parabolic potential well
capable of trapping and manipulating micron scale objects. Such traps have been
74
used for many years and their ecacy in biological systems has been examined and
shown to have a negligible impact on biological functions. In order to measure the
current of a trapped cell, a microelectrode array was lithographically patterned
onto microscope coverglass. The coverglass was sputtered with ITO, a transparent
heavily doped semiconducive material which has high transparency for both visible
light, as well as infrared light used by the trap. The ITO electrode array contained
nine working electrodes centered around a central large counter electrode. The
working electrodes were pacied using SU-8, a polymer based negative photoresist
that is transparent as well. The SU-8 pattern allowed for 15m x 15m windows.
These windows served as landing pads for the trapped bacteria. The electrodes
were connected to a potentiostat set at a constant biasing potential of +400 mV
vs. Ag/AgCl. A vacuum sealed perfusion chamber was attached to the top of the
electrode array which held the bulk solution with DMRB cells suspended. The
entire system was protected by a faraday cage. This system allowed for trapped
cells to be landed on electrodes and for currents as low as 10 fA to be detected.
In chapter 6 we provided the rst experimental system to achieve this level
of sensitivity for single cell DET measurements. MR-1 produced heterogeneous
currents in 4 out of 27 contact events, ranging from 15 to 100 fA while OMC
showed no current in 20 contact events. bfe mutants showed only one current
increase of 10 fA out of 28 contact events. Previous work with MR-1 only gave
higher end currents of 50 - 200 fA [53]. This work futher examined the importance
of outer membrane cytochromes and
avins in a single cell system for the rst
time, validating previous results that showed them to be critical for DET.
Single cell electrochemical trapping techniques are still in their infancy. While
these results are promising, there are variables that remain unaccounted for. For
the low volumes involves, on the order of 1 mL, there is no reliable way to test for
75
oxygen contamination of the chamber during experiments. Shewanella is known
to use oxygen as an alternative acceptor, however the impact of oxygen on DET
is not well understood. One proposed solution would be to construct an anaerobic
chamber around the entire trapping system. Other techniques could involve reduc-
tants, such as cysteine, to reduce oxygen from the system. There are concerns,
however, that the addition of any redox active chemical species into the chamber
could impact the sensitivity of the instrument, as well as provide alternative elec-
tron acceptors for Shewanella oneidensis. These improvements would allow for a
more systemic study of the impact of alternative electron acceptors on DET.
Despite these limitations, single cell trapping oers many exciting possibilities
for future measurements. Any new mutant can be screened for their ability to
reduce solid electrodes. Furthermore, nanowires have still not been denitively
shown to act as long distance charge carriers, although their composition and con-
ductivity strongly suggest this as a role they would play. Cells could be trapped
and held near electrodes, and nanowire growth could be induced as shown previ-
ously [80] to make contact with the electrode. This would involve including
u-
orescence measurements as nanowires cannot be resolved with the current optics
in the system. Current increases as a result of contact with the nanowire would
demonstrate that nanowires do in fact act as charge transfer mechanisms for She-
wanella oneidensis. This work hopes to provide a blueprint for studying DET
systems from a bottom up perspective, providing insights into the building blocks
of larger batch systems already in use.
76
Bibliography
[1] Appleyard, D. C., Vandermeulen, K. Y., Lee, H., and Lang, M. J. (2007).
Optical trapping for undergraduates. American Journal of Physics, 75(1):5{14.
[2] Ashkin, A. (2000). History of optical trapping and manipulation of small-
neutral particle, atoms, and molecules. IEEE Journal of Selected Topics in
Quantum Electronics, 6(6):841{856.
[3] Ashkin, A., Dziedzic, J. M., and Yamane, T. (1987). Optical trapping and
manipulation of single cells using infrared-laser beams. Nature, 330(6150):769{
771.
[4] Bard, A. J. and Faulkner, L. R. (2001). Electrochemical methods : fundamentals
and applications. Wiley, New York, 2nd edition.
[5] Beratan, D. N., Onuchic, J. N., Winkler, J. R., and Gray, H. B. (1992).
Electron-tunneling pathways in proteins. Science, 258(5089):1740{1741.
[6] Binger, J., Ribbens, M., Ringeisen, B., Pietron, J., Finkel, S., and Neal-
son, K. (2009). Characterization of electrochemically active bacteria utilizing a
high-througput voltage-based screening assay. Biotechnology and Bioengineer-
ing, 102(2):436 { 444.
[7] Binger, J. C., Pietron, J., Bretschger, O., Nadeau, L. J., Johnson, G. R.,
Williams, C. C., Nealson, K. H., and Bradley, R. R. (2008). The in
uence of
acidity on micrrobial fuel cells containing Shewanella oneidensis. Biosensors
and Bioelectrnics, 24:900 { 905.
[8] Bilecen, K. and Yildiz, F. (2009). Identication of a calcium-controlled negative
regulatory system aecting Vibrio cholerae biolm formation. Environmental
Microbiology, 11:2015 { 2029.
[9] Boogerd, F. C. and Vrind, J. P. M. (1987). Manganese oxidation by Leptothrix
discophora. Jouranl of Bacteriology, 169:489{494.
77
[10] Borloo, J., Vergauwen, B., De Smet, L., Brige, A., Motte, B., Devreese, B.,
and Van Beeumen, J. (2007). A kinetic approach to the dependence of dissimi-
latory metal reduction by Shewanella oneidensis MR-1 on the outer membrane
cytochromes c Omca and Omcb. Febs Journal, 274(14):3728{3738.
[11] Bouhennni, R. A., Vora, G. J., Binger, J. C., Shirodkar, S., Brockman, K.,
Ray, R., Wu, P., Johnson, B. J., M., B. E., Marshall, M. J., Fitzgerald, L. A.,
Little, B. J., Fredrickson, J. K., Beliaev, A. S., Ringeisen, B. R., and Saarini,
D. A. (2010). The roll of Shewanella oneidensis MR-1 outer surface structures
in extracellular electron transfer. Electroanalysis, 22(7 - 8):856 { 864.
[12] Bretschger, O., Obraztsova, A., Sturm, C. A., Chang, I. S., Gorby, Y. A.,
Reed, S. B., Culley, D. E., Reardon, C. L., Barua, S., Romine, M. F., Zhou, J.,
Beliaev, A. S., Bouhenni, R., Saarini, D., Mansfeld, F., Kim, B.-H., Fredrick-
son, J. K., and Nealson, K. H. (2007). Current production and metal oxide
reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl. Envi-
ron. Microbiol., 73(21):7003{7012.
[13] Brock, T. D., Madigan, M. T., and Martinko, J. M. (2006). Brock Biology of
Microorganisms. Pearson Education, Inc., New Jersey.
[14] Bromeld, S. M. and David, D. J. (1976). Sorption and oxidation of manga-
neous ions and reduction of manganese oxide by cell suspensions of a manganese
oxidizing bacterium. Soil Biology and Bichemistry, 8:37{43.
[15] Brutinel, E. D. and Gralnick, J. A. (2012). Shuttling happens: soluble
avin
mediators of extracellular electron transfer in Shewanella. Applied Microbiology
and Biotechnology, 93:41 { 48.
[16] Bucking, C., Popp, F., Kerzenmacher, S., and Gescher, J. (2010). Involvement
and specicity of Shewanella oneidensis outer membrane cytochromes in the
reduction of soluble and solid-phase terminal electron acceptors. Fems Microbi-
ology Letters, 306(2):144{151.
[17] Burdige, D. J. and Nealson, K. H. (1985). Microbial manganese reduction by
enrishment cultures from coastal marine sediments. Applied and Environmental
Microbiology, 50:491{497.
[18] Carmona-Martines, A. A., Harnisch, F., Fitzgerald, L. A., Binger, J. C.,
Ringeisen, B. R., and Schroder, U. (2011). Cyclic voltammetric analysis of
the electron transfer of Shewanella oneidensis MR-1 and nanolament and
cytochrome knock-out mutants. Bioelectrochemistry, 81(2):74 { 80.
[19] Clarke, T. A., Edwards, M. J., Gates, A. J., Hall, A., White, G. F., Bradley, J.,
Reardon, C. L., Shi, L., Beliaev, A. S., Marshall, M. J., Wang, Z., Watmough,
78
N. J., Fredrickson, J. K., Zachara, J. M., Butt, J. N., and Richardson, D. J.
(2011). Structure of a bacterial cell surface decaheme electron conduit. PNAS,
108(23):9384 { 9389.
[20] Coursolle, D. and Gralnick, J. A. (2010). Modularity of the mtr respiratory
pathway of Shewanella oneidensis strain MR-1. Molecular Microbiology, 77:995
{ 1008.
[21] Craig, L., Pique, M. E., and Tainer, J. A. (2004). Type IV pilus structure
and bacterial pathogenicity. Nature Reviews Microbiology, 2(5):326 { 328.
[22] Davey, H. M. and Kell, D. B. (1996). Flow cytometry and cell sorting of
heterogeneous microbial populations: The importance of single-cell analyses.
Microbiological Reviews, 60(4):641{696.
[23] Deppenmeier, U. (2002). Redox-driven proton translocation in methanogenic
Archaea. Cell Molecular Life Sciences, 59:1513{1533.
[24] Domingues, D. C. (2004). Calcium signalling in bacteria. Molecular Microbi-
ology, 54(2):291 { 297.
[25] Edwards, M. J., Baiden, N. A., Johs, A., Tomanicek, S. J., Liang, L., Shi, L.,
Fredrickson, J. K., Zachara, J. M., Gates, A. J., Butt, J. N., Richardson, D. J.,
and Clarke, T. A. The x-ray crystal structure of Shewanella oneidensis OmcA
reveals new insight at the microbe-mineral interface.
[26] Ehrlish, H. L. (1987). Manganese oxide reduction as a form of anaerobic
respiration. Geomicrobiology journal, 5:423.
[27] El-Naggar, M. Y. and Finkel, S. E. (2013). Live wires. Scientist, 27(5):38{43.
[28] El-Naggar, M. Y., Gorby, Y. A., Xia, W., and Nealson, K. H. (2008).
The molecular density of states in bacterial nanowires. Biophysical Journal,
95(1):L10 { L12.
[29] El-Naggar, M. Y., Wanger, G., Leung, K. M., Yuzvinsky, T. D., Southam,
G., Yang, J., Lau, W. M., Nealson, K. H., and Gorby, Y. A. (2010). Electrical
transport along bacterial nanowires from Shewanella oneidensis MR-1. Proc.
Natl. Acad. Sci. USA, 107(42):18127{18131.
[30] et al., M. J. S. (2008). Oxygen-dependent autoaggregation in Shewanella
oneidensis MR-1. Environmental Microbiology, 10:1861 { 1876.
[31] Farquhar, J., Bao, H., and Thiemens, M. (2000). Atmospheric in
uence of
earth's earliest sulfur cycle. Science, 289(5480):756 { 758.
79
[32] Fitzgerald, L. A., Petersen, E. R., Gross, B. J., Soto, C. M., Ringeisen, B. R.,
El-Naggar, M. Y., and Binger, J. C. (2012). Aggrandizing power output from
Shewanella oneidensis MR-1 microbial fuel cels using calsium chloride. Biosen-
sors and Bioelectronics, 31:492 { 498.
[33] Gorby, Y. A., Yanina, S., McLean, J. S., Rosso, K. M., Moyles, D.,
Dohnalkova, A., Beveridge, T. J., Chang, I. S., Kim, B. H., Kim, K. S., Cul-
ley, D. E., Reed, S. B., Romine, M. F., Saarini, D. A., Hill, E. A., Shi, L.,
Elias, D. A., Kennedy, D. W., Pinchuk, G., Watanabe, K., Ishii, S., Logan, B.,
Nealson, K. H., and Fredrickson, J. K. (2006). Electrically conductive bacterial
nanowires produced by Shewanella oneidensis strain MR-1 and other microor-
ganisms. Proc. Natl. Acad. Sci. USA, 103(30):11358{11363.
[34] Gray, H. B. and Winkler, J. R. (2010). Electron
ow through metalloproteins.
Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1797(9):1563 { 1572.
[35] Gregory, K. B., Bond, D. R., and Lovely, D. R. (2004). Graphite electrodes as
electron donors for anaerobic resipration. Environmental Microbiology, 6:596{
604.
[36] Gregory, K. B. and Lovely, D. R. (2005). Remediation and recovery of uranium
from contaminated subsurface environments with electrodes. Environmental
Science and Technology, 39:8943 { 8947.
[37] Grubb, W. T. and Niedrack, L, W. (1960). Batteries with solid ion-exchange
membrane electrolytes'. Journal of Electrochemical Society, 107:131{135.
[38] H. L. Ehrlich, S. H. Y. and Jr., J. D. M. (1973). Bacteriology of manganese
nodules. Zeitschrift fur Allgemeine Mikrobiologie, 13:39{48.
[39] Hao, L., Diao, X. G., Xu, H. Z., Gu, B. X., and Wang, T. M. (2008).
Thickness dependence of structural, electrical and optical properties of indium
tin oxide (ITO) lms deposited on PET substrates. Applied Surface Science,
254(11):3504{3508.
[40] Harned, H. S. and Hudson, R. M. (1951). The dierential diusion coecient
of potassium ferrocyanide in dilute aqueous solution at 25-degrees. Journal of
the American Chemical Society, 73(11):5083{5084.
[41] Harris, H. W., El-Naggar, M. Y., and Nealson, K. H. (2012). Shewanella
oneidensis MR-1 chemotaxis proteins and electron-transport chain components
essential for congregation near insoluble electron acceptors. Biochemical Society
Transactions, 40(6):1167{1177.
80
[42] He, Z. and Mansfeld, F. (2009). Exploring the use of electrochemical
impedance spectroscopy (EIS) in microbial fuel cell studies. Energy & Envi-
ronmental Science, 2:215 { 219.
[43] Hong, L., Ramnarayanan, R., and Logan, B. E. (2004). Production of elec-
tricity during wastewater treatment using a single chamber microbial fuel cell.
Environmental science and technology, 38(7):2281 { 2285.
[44] Huang, J., Sun, B., and Zhang, X. (2010). Electricity generation at high ionic
strength in microbial fuel cell by a newly isolated Shewanella maris
avi EP1.
Applied Microbiology and Biotechnology, 85:1141 { 1149.
[45] Jiang, X. C., Hu, J. S., Petersen, E. R., Fitzgerald, L. A., Jackan, C. S.,
Lieber, A. M., Ringeisen, B. R., Lieber, C. M., and Binger, J. C. (2013).
Probing single- to multi-cell level charge transport in Geobacter sulfurreducens
DL-1. Nature Communications, 4.
[46] Kawakami, Y., Ito, T., Kamekura, M., and Nakayama, M. (2005). Ca
2+
- dependent cell aggregation of halophilic archaeon, Halobacterium salinarum.
Jouranl of Bioscience and Bioengineering, 100(6):681 { 684.
[47] Kim, B. H., Kim, H, J., Hyun, M. S., and Park, D, H. (1999). Direct elec-
trode reaction of Fe(III)-reducing bacterium, Shewanella putrefaciens. Journal
of Microbiology and Biotechnology, 9(2):127 { 131.
[48] Kotloski, N. J. and Gralnick, J. A. (2013). Flavin electron shuttles dominate
extracellular electron transfer by Shewanella oneidensis. mBio, 4(1):e00553{12.
[49] Liang, Y., Gao, H., Chen, J., Dong, Y., Wu, L., He, Z., Liu, Z., Qui, G., and
Zhou, J. (2010). Pellicle formation in Shewanella oneidensis. BMC Microbiology,
10:291 { 302.
[50] Linden, D. (1984). Handbook of Batteries and Fuel Cells. McGraw-Hill Inc,
NewYork.
[51] Liu, C., Jeon, B.-H., Zachara, J. M., and Wang, Z. (2007). In
uence of
calcium on microbial reduction of solid phase uranium(VI). Biotechnology and
Bioengineering, 97(6):1415 { 1422.
[52] Liu, H., Cheng, S., and Logan, B. (2005). Power generation of fed-batch
microbial fuel cells as a function of ionic strength, temperature and reactor
conguration. Environmental Science and Technology, 39:5488 { 5493.
[53] Liu, H. A., Newton, G. J., Nakamura, R., Hashimoto, K., and Nakanishi,
S. (2010). Electrochemical characterization of a single electricity-producing
81
bacterial cell of Shewanella by using optical tweezers. Angewandte Chemie-
International Edition, 49(37):6596{6599.
[54] Logan, B. E. (2008). Microbial Fuel Cells, volume 1. Wiley, John and Sons,
Incorperated, Hoboken, New Jersey.
[55] Logan Bruce E, Bert Hamelers, R. R. e. a. (2006). Microbial fuel cells:
Methodology and technology. Environmental Science and Technology, 40:5181{
5192.
[56] Lovely, D. R., Coates, J. D., Blunt-Harris, E. L., Phillips, E. J. P., and Wood-
ward, J. C. (1996). Humic substances as electron acceptors for microbial respi-
ration. Nature, 382(6590):445 { 448.
[57] Lower, B. H., Shi, L., Yongsunthon, R., Droubay, T. C., McCready, D. E.,
and Lower, S. K. (2007). Specic bonds between an iron oxide surface and outer
membrane cytochromes MtrC and OmcA from Shewanella oneidensis MR-1.
Journal of Bacteriology, 189(13):4944{4952.
[58] M., T. K., Saville, R. M., Shukla, S., Pelletier, D. A., and Spormann, A. M.
(2004). Initial phases of biolm formation in Shewanella oneidensis MR-1. Jour-
nal of Bacteriology, 186(23):8096 { 8104.
[59] Malvankar, N. S., Vargas, M., Nevin, K. P., Franks, A. E., Leang, C., Kim,
B. C., Inoue, K., Mester, T., Covalla, S. F., P., J. J., Rotello, V. M., Tuomi-
nen, M. T., and Lovely, D. R. Tunable metallic-like conductivity in microbial
nanowire networks. Nature Nanotechnology, 6(9):573 { 579.
[60] Manohar, A. K., Bretschger, O., Nealson, K. H., and Mansfeld, F. (2008).
The polarization behavior of the anode in a microbial fuel cell. Electrochimica
Acta, 53(9):3508 { 3513.
[61] Manohar, A. K. and Mansfeld, F. (2009). The internal resistance of a microbial
fuel cell and its dependence on cell design and operating conditions. Electrochim-
ica Acta, 54(6):1664 { 1670.
[62] Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A.,
and Bond, D. R. (2008). Shewanella secretes
avins that mediate extracellular
electron transfer. Proc. Natl. Acad. Sci. USA, 105(10):3968{3973.
[63] Maruyama, K. (1991). The discovery of adenosine triphosphate and the estab-
lisment of its structure. Journal of the History of Biology, 24(1):145 { 154.
[64] Matias, P. M., Pereira, I. A., Soares, C., and Carrondo, M. A. (2005). Sul-
phate respiration from hydrogen in desulfovibrio bacteria: a structural biology
overview. Progress in Biophysics and Molecular Biology, 89:292{329.
82
[65] McLean, J. S., Wanger, G., Gorby, Y. A., Wainstein, M., McQuaid, J., Ishii,
S. I., Bretschger, O., Beyenal, H., and Nealson, K. H. (2010). Quantication of
electron transfer rates to a solid phase electron acceptor through the stages of
biolm formation from single cells to multicellular communities. Environmental
Science and Technology, 44(7):2721{2727.
[66] Mehta, J., Coppi, M. V., Childers, S. E., and Lovley, D. R. (2005). Outer mem-
brane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in
Geobacter sulfurreducens. Applied and Environmental Microbiology, 71(12):8634
{ 8641.
[67] Min, T. L., Mears, P. J., Chubiz, L. M., Golding, I., Chemla, Y. R., and Rao,
C. V. (2009). High-resolution, long-term characterization of bacterial motility
using optical tweezers. Nature Methods, 6(11):831{871.
[68] Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen
transfer by a chemi-osmotic type of mechanism. Nature, 191(478):144{148.
[69] Mohan, Y. and Das, D. (2009). Eect of ionic strength, cation exchanger and
inoculum age on performance of microbial fuel cells. International Journal of
Hydrogen Energy, 34:7542 { 7546.
[70] Myers, C. R. and Myers, J. M. (1992). Localization of cytochromes to the
outer-membrane of anaerobically grown Shewanella-putrefaciens MR-1. Journal
of Bacteriology, 174(11):3429{3438.
[71] Myers, C. R. and Nealson, K. H. (1988). Bacterial manganese reduction
and growth with manganese oxide as the sole electron-acceptor. Science,
240(4857):1319{1321.
[72] Nealson, K. H., Belz, A., and McKee, B. (2002). Breathing metals as a way
of life: geobiology in action. Antonie Van Leeuwenhoek International Journal of
General and Molecular Microbiology, 81(1-4):215{222.
[73] Neuman, K. C. and Block, S. M. (2004). Optical trapping. Review of Scientic
Instruments, 75(9):2787{2809.
[74] Neuman, K. C., Chadd, E. H., Liou, G. F., Bergman, K., and Block, S. M.
(1999). Characterization of photodamage to Escherichia coli in optical traps.
Biophysical Journal, 77(5):2856{2863.
[75] Nevin, K. P., Woodard, T. L., Franks, A. E., Summers, Z. M., and Lovely,
D. R. (2010). Microbial electrosynthesis: Feeding microbes electricity to convert
carbon diozide and water to muticarbon extracellular organic compounds. mBio,
1(2):e00103{10.
83
[76] Newman, D. K. and Kolter, R. (2000). A role for excreted quinones in extra-
cellular electron transfer. Nature, 405(6782):94 { 97.
[77] Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F. N., Toth, L., Trinei,
M., Modha, K., and Norman, R. I. (1996). Calcium signalling in bacteria.
Journal of Bacteriology, 178(13):3677 { 3682.
[78] Okamoto, A., Hashimoto, K., Nealson, K., and Nakamura, R. (2012). Rate
enhancement of bacterial extracellular electron transport involves bound
avin
semiquinones. PNAS, 110(19):7856 { 7851.
[79] Oster, G. and Wang, H. (2000). Reverse engineering a protein: The
mechanochemistry of ATP synthase. Biochemica et Biophysica Acta, 1458:482{
510.
[80] Pirbadian, S., Barchinger, S. E., Leung, K. M., Byun, H. S., Jangir, Y.,
Bouhenni, R. A., Reed, S. B., Romine, M. F., Saarini, D. A., Shi, L., Gorby,
Y. A., Golbeck, J. H., and El-Naggar, M. Y. (2014). Shewanella oneidensis MR-
1 nanowires are outer membrane and periplasmic extensions of the extracellular
electron transport components. Proc. Natl. Acad. Sci. USA, 111(35):12883{
12888.
[81] Pirbadian, S. and El-Naggar, M. Y. (2012). Multistep hopping and extracel-
lular charge transfer in microbial redox chains. Physical Chemistry Chemical
Physics, 14:13802{13808.
[82] Potter, M. C. (1911). Electricla eects accompanying the decomposition of
organic compounds. Proceedings of the Royal Society of London, Series B, 84:260
{ 276.
[83] Rabaey, K. and Rozendal, R. A. (2010). Microbial electrosynthesis - revisit-
ing the electrical route for microbial production. Nature Reviews Microbiology,
8(10):706{716.
[84] Reguera, G., McCarthy, K. D., Mehta, T., Nicoll, J. S., Tuominen, M. T., and
Lovley, D. R. (2005). Extracellular electron transfer via microbial nanowires.
Nature, 435:1098 { 1101.
[85] Reguera, G., Pollina, R. B., Nicoll, J. S., and Lovely, D. R. (2007). Possi-
ble nonconductive role of Geobacter sulfurreducens pilus nanowires in biolm
formation. Journal of Bacteriology, 189(5):2125 { 2127.
[86] Richardson, D. J., Butt, J. N., Fredrickson, J. K., Zachara, J. M., Shi, L.,
Edwards, M. J., White, G., Baiden, N., Gates, A. J., Marritt, S. J., and Clarke,
T. A. (2012). The porin-cytochrome' model for microbe-to-mineral electron
transfer. Molecular Microbiology, 85(2):201{212.
84
[87] Ross, D. E., Brantley, S. L., and Tien, M. (2009). Kinetic characterization of
OmcA and MtrC, terminal reductases involved in respiratory electron transfer
for dissimilatory iron reduction in Shewanella oneidensis MR-1. Applied and
Environmental Microbiology, 75(16):5218{5226.
[88] Ross, D. E., Flynn, J. M., Baron, D. B., Gralnick, J. A., and Bond, D. R.
(2011). Towards electrosynthesis in Shewanella: Energetics of reversing the Mtr
pathway for reductive metabolism. PLoS ONE, 6(2):e16649.
[89] Roy, J. N., Babanova, S., Garcia, K. E., Cornejo, J., Ista, L. K., and
Atanassov, P. (2014). Catalytic biolm formation by Shewanella oneidensis
MR-1 and anode characterization by expanded uncertainty. Electrochimica Acta,
126:3{10.
[90] Schafer, G., Engelhard, M., and Muller, V. (1999). Bioenergetics of archada.
Microbiological Molecular biology Reveiw, 63:570{620.
[91] Shi, L. e. a. Isolation of a high-anity functional protein complex between
omca and mtrc: Two outer membrane decaheme c-type cytochromes of She-
wanella oneidensis MR-1. Applied and Environmental Microbiology, 188:4705 {
4714.
[92] Shima, S., Warkentin, E., Thauer, R. K., and Ermler, U. (2002). Structure and
function of enzymes involved in methanogenic pathway utilizing carbon dioxide
and molecular hydrogen. Journal of bioscience and Bioengineering, 93:519{530.
[93] Shizas, I. and Bagley, D. M. (2004). Experimental determination of energy
content of unkown organics in municipal wastewater streams. Journal of Energy
Engineering, 130(2):45{53.
[94] Strycharz, S. M., Woodard, T. L., Johnson, J. P., Nevin, K. P., and Standord,
R. A. e. a. (2008). Graphite electrode as a sole electron donor for reductive
dechlorination of tetrachlorethene by Geobacter lovleyi. Applied Environmental
Microbiology, 74:5943{5947.
[95] Turakhia, M. H. and Characklis, W. G. (1989). Activity of Pseudomonas
aeruginosa in biolms: eect of calcium. Biotechnology and Bioengineering,
33(4):406 { 414.
[96] Unocic, R. R., Sacci, R. L., Brown, G. M., Veith, G. M., Dudney, N. J.,
More, K. L., Walden, F. S., Gardiner, D. S., Damiano, J., and Nackashi, D. P.
(2014). Quantitative electrochemical measurements using in situ ec-S/TEM
devices. Microscopy and Microanalysis, 20(2):452{461.
85
[97] Velasquez-Orta, S., Head, I. M., Curtis, T. P., Scott, K., Lloyd, J. R., and von
Canstein, H. (2010). The eect of
avin electron shuttles in microbial fuel cells
current production. Applied Micribiology and Biotechnology, 85:1373 { 1381.
[98] von Canstein, H., Ogawa, J., Shimizu, S., and Lloyd, J. R. (2008). Secretion
of
avins by Shewanella species and their role in extracellular electron transfer.
Applied and Environmental Microbiology, 74(3):615{623.
[99] Wang, V. B., Kirchhofer, N. D., Chen, X. F., Tan, M. Y. L., Sivakumar,
K., Cao, B., Zhang, Q. C., Kjelleberg, S., Bazan, G. C., Loo, S. C. J., and
Marsili, E. (2014). Comparison of
avins and a conjugated oligoelectrolyte in
stimulating extracellular electron transport from Shewanella oneidensis MR-1.
Electrochemistry Communications, 41:55{58.
[100] Weber, K. A., Achenbach, L. A., , and Coates, J. D. (2006). Microorganisms
pumping iron: anaerobic microbial iron oxidation and reduction. Nature Reviews
Microbiology, 4:752{764.
[101] Wen, Q., Kong, F., Ma, F., Ren, Y., and Pan, Z. (2011). Improved perfor-
mance of air-chathode microbial fuel cell through addition of tween 80. Journal
of Power Sources, 196:899 { 904.
[102] White, G. F., Shi, Z., Shi, L., Wang, Z. M., Dohnalkova, A. C., Marshall,
M. J., Fredrickson, J. K., Zachara, J. M., Butt, J. N., Richardson, D. J., and
Clarke, T. A. (2013). Rapid electron exchange between surface-exposed bacterial
cytochromes and Fe(III) minerals. Proc. Natl. Acad. Sci. USA, 110(16):6346{
6351.
[103] Williams, K. R. (1966). An Introduction to Fuel Cells, volume 113. Elsevier
Publishing Company, New York.
86

Abstract (if available)

Abstract Metal‐reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. By performing electron transfer to synthetic electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. This ability enables the emerging technology of microbial fuel cells (MFCs), where living cells utilize complex or mixed biofuels to produce electricity. The exact mechanism of this extracellular electron transport, however, is not yet fully understood. ❧ In the first part of this work I focus on the role of calcium in MFCs innoculated with Shewanella oneidensis MR-1. Calcium is known to play important roles in cellular metabolism, including aggregation of cells onto surfaces. It has been shown that the addition of calcium to MFCs results in increased current. Here we use electrochemical impedance spectroscopy (EIS) and equivalent circuit modeling to determine that calcium is not simply increasing the current by abiotically reducing the solution resistance, and may prove useful as a biological optimization parameter. ❧ In the next section I focus on the role of pili in MFC current production. Shewanella oneidensis MR-1 produce type IV pili which may play an important role in biofilm formation and current production in MFCs. MFCs have permitted the measurement of current output from mutants lacking the ability to produce certain extracellular appendages thought to play a role in current production. Our results prove ambiguous due to the bulk nature of MFC measurements which cannot determine whether differences in current production are due to factors uniformly impacting the entire culture, or heterogeneities in the phenotypical behavior of different cells. ❧ In order to address these issues, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect sub-pA currents associated with single cell direct electron transfer (DET) events. ❧ Finally, I demonstrate the system with single cell measurements of the dissimilatory metal reducing bacterium Shewanella oneidenis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. Mutants lacking the flavin export system (Δbfe) also did not produce current at the single‐cell level. Only one out of 28 contact events showed a current increase correlating to contact with the electrode of 10 fA, which is a the detection limit of the instrument. This is consistent with the previously proposed role for flavins in EET, although debate continues on whether the flavins act as secreted electron shuttles or as bound co‐factors to the outer membrane cytochromes.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

From cables to biofilms: electronic and electrochemical characterization of electroactive microbial systems

PDF

Bacterial nanowires of Shewanella oneidensis MR-1: electron transport mechanism, composition, and role of multiheme cytochromes

PDF

From single molecules to bacterial nanowires: functional and dynamic imaging of the extracellular electron transfer network in Shewanella oneidensis MR-1

PDF

Electronic, electrochemical, and spintronic characterization of bacterial electron transport

PDF

Microbially synthesized chalcogenide nanostructures: characterization and potential applications

PDF

Electron transfer capability and metabolic processes of the genus Shewanella with applications to the optimization of microbial fuel cells

PDF

Electrochemical studies of outward and inward extracellular electron transfer by microorganisms from diverse environments

PDF

Electrochemical studies of subsurface microorganisms

PDF

Identification of a new bacterial sensing mechanism: characterization of bacterial insoluble electron acceptor sensing

PDF

Survival and evolution of Shewanella oneidensis MR-1: applications for microbial fuel cells

PDF

Microbial interaction networks: from single cells to collective behavior

PDF

Kinetic Monte Carlo simulations for electron transport in redox proteins: from single cytochromes to redox networks

PDF

Applications of advanced electrochemical techniques in the study of microbial fuel cells and corrosion protection by polymer coatings

PDF

Electrochemical investigations and imaging tools for understanding extracellular electron transfer in phylogenetically diverse bacteria

PDF

Electrokinetic transport of Cr(VI) and integration with zero-valent iron nanoparticle and microbial fuel cell technologies for aquifer remediation

Asset Metadata

Creator Gross, Benjamin J. (author)

Core Title From fuel cells to single cells: electrochemical measurements of direct electron transfer at microbial-electrode interfaces

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Physics

Publication Date 06/19/2015

Defense Date 04/15/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag direct electron transfer,microbial fuel cells,OAI-PMH Harvest,optical trapping,Shewanella oneidensis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor El-Naggar, Mohamed Y. (committee chair), Boedicker, James Q. (committee member), Finkel, Steven E. (committee member), Haas, Stephan W. (committee member), Kresin, Vitaly V. (committee member)

Creator Email benjamin.gross.phd@gmail.com,benjamjg@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-573743

Unique identifier UC11299118

Identifier etd-GrossBenja-3486.pdf (filename),usctheses-c3-573743 (legacy record id)

Legacy Identifier etd-GrossBenja-3486.pdf

Dmrecord 573743

Document Type Dissertation

Format application/pdf (imt)

Rights Gross, Benjamin J.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA