AN ABSTRACT OF THE DISSERTATION OF
Shoutao Xu for the degree of Doctor of Philosophy in Biological and Ecological
Engineering presented on June 15, 2012.
Title: Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced
Performance of Microbial Electrochemical Systems Using Nano-Decorated Anodes
Abstract approved:
Hong Liu
Frank W.R. Chaplen
Microbial electrochemical systems (MESs) have attracted much research attention in
recent years due to their promising applications in renewable energy generation,
bioremediation, and wastewater treatment. In a MES, microorganisms interact with
electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the
cathode.
The bacterial community of a high power mixed consortium MESs (maximum power
density is 6.5W/m2) was analyzed by using denature gradient gel electrophoresis (DGGE)
and 16S DNA clone library methods. The bacterial DGGE profiles were relatively
complex (more than 10 bands) but only three brightly dominant bands in DGGE results.
These results indicated there are three dominant bacterial species in mixed consortium
MFCs. The 16S DNA clone library method results revealed that the predominant
bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter
sp. These three bacterial species reached to 88% of total bacterial species. This result is
consistent with the DGGE result which showed that three bright bands represented three
dominant bacterial species.
Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial
fuel cell by conventional plating techniques with ferric citrate as electron acceptor under
anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it
was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the
most closely related species. The bacterial strain SX-1 produced electricity from citrate,
acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density
of 205 mA/m2 generated from citrate. Cyclic voltammetry analysis indicated that
membrane associated proteins may play an important role in facilitating electron transfer
from the bacteria to
the electrode. This is the first study that demonstrates that
Citrobacter species can transfer electrons to extracellular electron acceptors. Citrobacter
strain SX-1 is capable of generating electricity from a wide range of substrates in MFCs.
This finding increases the known diversity of power generating exoelectrogens and
provids a new strain to explore the mechanisms of extracellular electron transfer from
bacteria to electrode. The wide range of substrate utilization by SX-1 increases the
application potential of MFCs in renewable energy generation and waste treatment.
Anode properties are critical for the performance of microbial electrolysis cells
(MECs). Inexpensive Fe nanoparticle modified graphite disks were used as anodes to
preliminarily investigate the effects of nanoparticles on the performance of Shewanella
oneidensis MR-1 in MECs. Results demonstrated that average current densities
produced with Fe nanoparticle decorated anodes were up to 5.9-fold higher than plain
graphite anodes. Whole genome microarray analysis of the gene expression showed that
genes encoding biofilm formation were significantly up-regulated as a response to
nanoparticle decorated anodes. Increased expression of genes related to nanowires,
flavins and c-type cytochromes indicate that enhanced mechanisms of electron transfer
to the anode may also have contributed to the observed increases in current density. The
majority of the remaining differentially expressed genes were associated with electron
transport and anaerobic metabolism demonstrating a systemic response to increased
power loads.
The carbon nanotube (CNT) is another form of nano materials. Carbon nanotube
(CNT) modified graphite disks were used as anodes to investigate the effects of
nanostructures on the performance S. oneidensis MR-1 in microbial electrolysis cells
(MECs). The current densities produced with CNT decorated anodes were up to 5.6-fold
higher than plain graphite anodes. Global transcriptome analysis showed that cytochrome
c genes associated with extracellular electron transfer are up-expressed by CNT
decorated anodes, which is the leading factor to contribute current increase in CNT
decorated anode MECs. The up regulated genes encoded to flavin also contribute to
current enhancement in CNT decorated anode MECs.
©Copyright by Shoutao Xu
June 15, 2012
All Rights Reserved
Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance
of Microbial Electrochemical Systems Using Nano-Decorated Anodes
by
Shoutao Xu
A DISSERTATION
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented June 15, 2012
Commencement June 2013
Doctor of Philosophy dissertation of Shoutao Xu presented on June 15, 2012.
APPROVED:
Co-Major Professor, representing Biological and Ecological Engineering
Co-Major Professor, representing Biological and Ecological Engineering
Head of the Department of Biological and Ecological Engineering
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Shoutao Xu, Author
ACKNOWLEDGEMENTS
I would like to thank all people who have helped and inspired me during my
doctoral study. Foremost, I would like to express my sincere gratitude to my advisors Dr.
Hong Liu and Dr. Frank Chaplen for their continuous support of my Ph.D. study and the
research of Microbial Fuel Cells at Oregon State University. Hong inspired me to devote
myself to the field of Bio-energy with her great patience and enthusiasm towards
scientific educations. Frank was always available and willing to help me with my study
especially during the period of Hong’s sabbatical leave. Thanks to his kindness and
assistance, my study at OSU became smooth and rewarding.
Besides my advisors, I would like to thank everyone in my dissertation committee: Dr.
Martin Schuster, Dr. Clare Reimers, and Dr. Mark Dolan. Due to their encouragement
and insightful comments on my research, I could always have the courage and knowledge
to overcome difficulties in my research. I benefited greatly from Martin’s valuable
suggestions on my writing skills and his generous help regarding microarray data analysis.
Clare’s advice helped me build a solid foundation of Electrochemistry. Also, it was a
great honor to have Mark as my committee member. His teaching gave me an insight into
the world of environmental engineering and enhanced the depth and width of my research.
I thanked Dr. Yanzhen Fan for always giving me guidance about the designs of the
reactors in my experiments.
It was also important for me to say thanks to my current and previous lab-mates:
Keaton Lesnik, Kuhuan Chien, Cheng Li, Corale Abourached, Anthony Janicek,
Hongqiang Hu, Jeremy Chignell, Yudith Nieto, and Wengguo Wu. I had a wonderful
time enjoying doing research with them. Their friendship and help made me confident of
my ability to do research as a scientist. In addition, I cherished and appreciated the
friendly environment in our BEE department. Faculty members, especially Dr. John Bolte,
Dr. John Selker, Dr. Roger Ely, Dr. Ganti Murthy, and Desiree Tullos made BEE an
excellent department for our students.
My deepest gratitude went to my father, Fuqin Wang, and my mother, Jinzhi Wang
for their endless care, love and support throughout my life. Also I would like to say
thanks to my best-loved wife, Songhua Zhu. She always supported me and never
complained that I could spend so little time accompanying with her. This dissertation was
also for my lovely son Gabriel Hong-Yi Xu.
TABLE OF CONTENTS
Page
1 General Introduction………………………………………………………..
1
2 Bacterial Community Analysis of Mixed Consortium in Microbial
Electrochemical Systems…………...............................................................
13
3 New Exoelectrogen Citrobacter sp. SX-1 Isolation and
Characterization…………………………………………………………….
27
4 Enhanced Performance and Mechanism Study of Microbial Electrolysis
Cells using Fe Nanoparticles Decorated Anodes…………………………... 45
5 Global Transcriptome Analysis of Response of Shewanella oneidensis
MR-1 to CNT Nanostructure Decorated Anodes in Microbial
Electrochemical System................................................................................
66
6 Summary……………………………………………………………………
80
7 Bibliography ……………………………………………………………….
82
8 Appendices ………………………………………………………………… 93
LIST OF FIGURES
Page
Figure
1-1
Generalized schematics: (a) Microbial fuel cell (MFCs)(b) Microbial
electrolysis cell (MECs) …………...……………… …………................
2
1-2
Mechanisms for extracellular electron transport in a MFCs anode..........
7
2-1
Polarization curves of high power mixed culture MFCs………………..
20
2-2
DGGE result of high power generation mixed culture MFCs....………...
21
2-3
Bacterial species and percentage of bacterial community of mixed
culture MFCs ………….…………………………………………...........
22
2-4
Phylogenetic trees of bacterial species from mixed culture MFCs...........
22
2-5
Dominant bacterial species identification in DGGE…………………….
23
2-6
Current density generated by different dominant isolates……………….
24
3-1
Phylogenetic tree of strain SX-1 and closely related species based on
16S rDNA sequences……………………………………………………
35
3-2
SEM image of planktonic cells of Citrobacter sp. SX-1..………………
35
3-3
Electricity generation by Citrobacter sp. SX-1 in a single chamber
MFCs with sodium citrate ……………………………….……………...
36
Electricity generation of Citrobacter sp. SX-1 using different
substrates in single chamber air-cathode MFCs at 1 KΩ………............
37
Power and voltage generation by Citrobacter sp. SX-1 as a function
of current density …………………………………………………..…...
38
3-6
Four current generating stages for CV analysis. …………………...…...
39
3-7
Cyclic Voltammetry analysis of of Citrobacter sp. SX-1 biofilms …….
41
4-1
SEM images of Fe NPs decorated anodes……………………………....
50
4-2
Effects of Fe-NP decorated anodes on the current density in MECs ……
51
4-3
Differentially expressed genes grouped by functional classification in
Fe NP decorated anodes………………... ………………………………
53
5-1
SEM images of CNT decorated anodes………………………………....
70
5-2
Effects of CNT decorated anodes on the current density in MECs……...
73
5-3
Differentially expressed genes grouped by functional classification in
CNT decorated anodes …………………………………………………..
74
3-4
3-5
LIST OF TABLES
Table
1-1
Taxa of bacteria, current density generated and reactor configuration
used in microbial electrochemical cell experiments..………….................
Page
4
4-1
Expression levels of genes related to biofilm formation……..………..
55
4-2
Genes related to anaerobic growth and electron transfer with
significantly change expression level………………………………….....
56
4-3
Expression level of flavin and cytochrome genes....……………………..
58
4-4
Other genes with significantly changed expression level...........................
59
5-1
Expression level of cytochrome c as response to CNT decorated anodes..
76
5-2.
Expression level of genes related to flavin synthesis as response to CNT
decorated anode in CNT decorated anodes…….........................................
78
1
1
Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance
of Microbial Electrochemical Systems Using Nano-Decorated Anodes
Chapter 1
General Introduction
1.1 Microbial Fuel Cells and Microbial Electrolysis Cells
Microbial electrochemical systems (MESs) have drawn the attention of researchers
in recent years due to their promising applications in a variety of scientific fields, such as
renewable energy generation, bioremediation, and wastewater treatment. In a MES,
microorganisms interact with electrodes, catalyzing oxidation and reduction reactions at
the anode and the cathode.
The most-described type of MESs is the microbial fuel cells (MFCs), in which
useful power is generated directly using the catalytic action of active microorganisms
(Kim, 2002; Cheng and Logan, 2007; Cheng et al., 2006). In a typical two-chamber
MFCs (Figure 1a), organic matter is oxidized by electrochemically active
microorganisms in the anode chamber to release protons and electrons. Protons diffuse
into the cathode chamber through a proton exchange membrane. Meanwhile, electrons
are transferred to the anode through various mechanisms (Logan et al., 2006; Lovley
2
2006; Rabaey et al., 2003) and then travel to the cathode, where normally they combine
with oxygen and protons to form water.
Another common used type of MES is the microbial electrolysis cell (MECs), in
which hydrogen is produced instead of electricity by applying a circuit voltage to the
MFCs system and maintaining anaerobic conditions in the cathode chamber (Liu et al.
2005; Logan et al. 2008; Rozendal et al. 2006; Schaller et al. 2009) (Figure 1b). A
cathode potential of at least - 410 mV (vs standard hydrogen electrode at pH 7.0) is
required to produce hydrogen at the cathode. Therefore, a theoretical potential of 110 mV
(i.e., 410-300 mV) is needed, but voltages of >0.2 V are normally applied due to various
overpotentials.
a
e-
e-
R
b
e-
e-
PS
H2O
Cathode
H+
PEM
Bacteria
Anode
Cathode
PEM
Bacteria
Anode
H+
H2
Fig 1-1. Generalized schematics: (a) Microbial fuel cells (MFCs), where electricity is
captured through the resistance R; (b) Microbial electrolysis cells (MECs), where
hydrogen gas is produced in the cathode chamber. R for resistor, PS for power supply and
PEM for proton exchange membrane
The fundamental feature shared by microbial electrochemical systems (MESs)
(MECs and MFCs) is the microbe-catalyzed electron transfer from organic matter to
electrodes (anodes) (Rebaey et al., 2004).
Therefore, different types of MESs can be
3
utilized to investigate the phenomena and mechanisms of interactions between microbes
and electrodes.
1.2 Bacteria and Electron Transfer Mechanisms in MESs
The electrochemically active bacteria in MFCs are thought to be iron-reducing
bacteria such as Shewanella and Geobacter species (Gorby et al., 2006). They have great
importance in the natural environment, principally in metal oxidation and reduction.
However, recent studies have shown that the diversity of bacterial communities is much
greater than these model iron reducers on MFCs anodes (Reimers et al. 2001; Liu et al.
2004b; Kim et al. 2005). Over 20 electrochemically active bacterial species, those can
transfer electrons exocellularly to electrodes, have been reported in the past 10 years
(Logan 2009; Liu et al. 2010). These bacteria have been categorized into diverse genetic
groups so far, including alphaproteobacteria (Rhodopseudomonas, Ochrobactrum and
Acidiphilium) (Malki et al. 2008; Xing et al. 2008; Zuo et al. 2008) betaproteobacteria
(Rhodoferax) (Chaudhuri et al. 2003), gammaproteobacteria (Shewanella, Pseudomonas,
Klebsiella, Enterobacter and Aeronomas) (Kim et al. 2002; Pham et al. 2003; Rabaey et
al. 2005; Bretschger et al. 2007; Zhang et al. 2008; Chung et al. 2009; Rezaei et al. 2009),
deltaproteobacteria (Geobacter, Geopsychrobacter, Desulfuromonas, and Desulfobulbus)
(Bond et al. 2002; Bond and Lovley 2003; Holmes et al. 2004a,b; 2006),
Epsilonproteobacteria (Arcobacter) (Fedorovich et al. 2009) Firmicutes (Clostridium and
Thermincola) (Park et al. 2001; Wrighton et al. 2008), Acidobacteria (Geothrix) (Bond
and Lovley 2005), and actinobacteria (Propionibacterium) (Wang et al., 2008). A wider
range of electrochemically active bacteria are expected to be discovered.
4
Table 1. Taxa of bacteria, current density generated and reactor configuration used in
microbial electrochemical cell experiments. (Liu et al. 2010)
Taxon
Microorganisms
α-proteobacteria
β-proteobacteria
γ-proteobacteria
δ-proteobacteria
Firmicutes
Acidobacteria
Actinobacteria
2
Current Density (A/m )
Rhodopseudomonas palustris DX-1
0.03
Ochrobactrum anthropi YZ-1
0.71
Acidiphilium sp. 3.2sup5
3.00
Rhodoferax ferrireducens
0.031
Shewanella putrefaciens IR-1
0.016
Shewanella oneidensis DSP10
0.013
Shewanella oneidensis MR-1
0.018
Pseudomonas aeruginosa KRA3
0.017
Escherichia coli K12 HB101
1.00
Klebsiella pneumoniae L17
1.20
Enterobacter cloacae
0.13
Aeromonas hydrophila PA3
0.30
Geobacter metallireducens
0.65
Geobacter sulfurreducens
8.00
Desulfuromonas acetoxidans
Geopsychrobacter
Electrodiphilus strain A2
0.005
0.066
Desulfobulbus propionicus
0.03
Lactococcus lactis
0.03
Thermincola sp. strain Jr
0.20
Clostridium butyricum EG3
0.22
Thermincola ferriacetica Z-0001
0.40
Brevibacillus spp. PTH1
0.009
Desulfitobacterium hafniense DCB2
1.10
Geothrix fermentans
Propionibacterium freudenreichiiET-3
0.097
1.20
The electrochemically active bacterial species that possess the ability to transfer
electrons outside of the cell are called exoelectrogens in the MESs research field. The
different exoelectrogens have demonstrated a wide-ranging power generation ability in
MFCs. For example, Shewanella oneidensis MR-1 has demonstrated the ability to
2
generate 0.018 A/m current density in single chamber MFCs; while the Geobacter
5
2
sulfurreducens species has generated current densities as high as 8.0 A/m , which is 400
times higher than the one generated by Shewanella oneidensis MR-1. However, mixedculture communities have generated much higher power densities than their pure-culture
counterparts in MFCs, perhaps due to synergistic interactions within the anode bacterial
communities and the participation of currently unknown bacteria species and mechanisms
(Jung and Regan 2007). The dominant bacteria species vary in mixed cultures bacteria
communities in MFCs due to the enrichment of different substrates.
Traditional methods of extroelectrogen study depend on cultivation hampered novel
exoelectrogens discovery because the inadequacy of defined media underestimates the
actual microbial diversity in MFCs; Slow growth rate and unknown growth requirements
of anaerobic microorganisms make some anaerobic extroelectrogens cultivation difficult.
However, these limitations have been overcome by using molecular biological methods
based on DNA/RNA analysis. Molecular biological techniques are now widely applied to
assess the diversity of microbial communities by analyzing the 16S rDNA sequence. The
most commonly used molecular biological techniques for bacterial community analysis
include denaturing gradient gel electrophoresis (DGGE), restriction fragment length
polymorphism (RFLP) and 16S rDNA sequencing. These methods are much less time
consuming than traditional isolation and cultivation methods.
Isolated exoelectrogens were utilized to explore the mechanism of electron transfer
to the anode. However, the mechanisms of electron transfer to extracellular electron
acceptors are not well understood. Three mechanisms have been proposed for exocellular
transport of electrons by exoelectrogens to anodes in MFCs so far. Some exoelectrogenic
bacteria can transfer electrons to electrodes through soluble redox compounds (Bond and
6
Lovley 2005). These compounds include artificial mediators and mediators secreted by
exoelectrogenic bacteria. A variety of chemicals have been used to facilitate the shuttling
of electrons from inside of cell to electrodes outside the cell. These exogenous mediators
include, neutral red (Park et al. 1999), anthraquinone-2-6, disulfonate (AQDS), thionin,
potassium ferricyanide (Bond et al. 2002), methyl viologen, and others (Logan 2004;
Rabaey and Verstraete 2005). Some bacteria can secrete their own chemical mediator,
for example, Pseudomonas aeruginosa secretes pyocyanin and phenazine-1-carboxamide
to transfer electrons toward the MFCs anode (Rabaey et al. 2005). Another evidence for
mediator production by bacteria is Geothrix fermentans. When the medium was replaced
in a MFCs that had stable power generation with this bacteria, power dropped by 50%
and required 10 days to resume the original level.
Some bacteria can directly transfer electrons to anodes via outer cell membrane
proteins (Myers and Myers 1992). The outer cell membrane protein cytochrome c is
thought to play a critical role in to transferring electrons to anodes. Ly et al (2011)
isolated the haem protein cytochrome c, and demonstrated that electric field effects may
be functional for the natural redox processes of cytochrome c in the respiratory chain.
Shewanella oneidensis is the example of a bacterium that is capable of transfer electron to
anode via outer cell membrane proteins. When Shewanella oneidensis adhered to an iron
surface, the greater force has showed benefits to grow cells because closer contact
required for electron transfer from cell bound cytochromes (Lower et al. 2001).
7
Fig.1-2. Mechanisms for extracellular electron transport in an MFCs anode (1) direct
contact (top in green); (2) by nanowires (middle in purple); and (3) self-produced
mediators (bottom in blue). (Logan, 2009)
More and more evidence supports the involvement of bacterial nanowires in
extracellular electron transport (Reguera et al. 2005; 2006; Gorby et al. 2006). Nanowires
are conductive appendages produced by both Geobacter and Shewanella species (Gorby
and Beveridge 2005). The conductivity of the appendages was examined and confirmed
by using conductive scanning tunneling microscopy (STM) (Gorby et al. 2006).
Nanowires can carry electrons from the cell to the anode surface of MFCs.
The solid component of the extracellular biofilm matrix has high efficiency on
extracellular electron transfer compared with other extracellular electron transfer
8
mechanisms and recently, Torres et al. (2010) hypothesized that the solid component of
the extracellular biofilm matrix formed by exoelectogens is another way bacteria transfer
electrons to electrodes. This hypothesis was based on kinetic analysis of each EET
mechanism reported in available literature (Torres et al. 2010).
1.3 Anode electrodes
In MFCs/MECs anode electrodes are a critical component because exoelectrogens
adhere to the surface of anodes to transfer electrons to the electrode. The characteristics
of anodes have significant effects on electron transfer rate from bacteria to anode
electrodes in MFCs. The requirements of an anode material are: it should be highly
conductive, non-corrosive, have a high specific surface area (area per volume), high
porosity, be non-fouling, inexpensive, and easily scaled to larger sizes. Of these
properties, the most important one that is different from other biofilm reactors is that the
material must be electrically conductive. Normally they are made of various carbon
materials, including carbon fiber, carbon clothe, and carbon paper due to their stability,
high conductivity and high specific surface-area. Nevertheless, they have little
electrocatalytic activity for the anode microbial reactions and thus a modification of the
carbon materials is the main approach for improving their performance. Consequently,
there is a great need to develop a new type of anode material for MFCs/MECs.
It is a great challenge to develop a new anode material to further increase the power
density of a MFCs. The nature of the catalytic mechanism of a MFC’s anode involves not
only a biological but also an electrocatalytic process. An optimal nanostructure with a
high specific surface area favorable for both catalytic processes could play a critical role
in improving the power density of the MFCs, such a structure needs to host the bacteria
9
with high bioactivity while enhancing the electron-transfer rate. Schröder et al (2008).
employed PANI to modify a platinum anode for MFCs and achieved a current density 1
order of magnitude higher than the previously reported value. PANI/inorganic
composites are also reported to have better conductivity. Qiao, et al also applied a new
mesoporous TiO2 electrode material with uniform nanopore distribution and a high
specific surface area to anode, in comparison to previously reported work with E. coli
MFCs, the composite anode delivers 2-fold higher power density (Qiao et al., 2008).
Thus, it has great potential for use as the anode in a high-power MFCs and may be a new
approach for improving performance of MFCs.
1.4 Other parts of MESs
1.4.1 Membranes and ion transport
The ion exchange membrane is another one of the critical components in twochamber MESs systems. It separates anode and cathode chambers and at the same time
maintains the electron neutrality of the system, i.e., transport of electrons to the cathode
needs to be compensated by transport of an equal amount of positive charge to the
cathode chamber. A proton exchange membrane (PEM), such as Nafion, a per-fluorinated
sulphonic acid membrane (PFSAs) consisting of a hydrophobic fluorocarbon backbone to
which hydrophilic sulfonate groups (-SO3-) are attached, is commonly used in chemical
fuel cell systems. For MFCs systems, however, mainly cation species like Na+ and K+
other than proton are often responsible for the dominant transport of positive charge
through the cation exchange membrane (CEM) to maintain electroneutrality due to the
low proton concentration in any aqueous medium with near neutral pH (Rozendal et al.,
2006). Consequently, the pH increases in the cathode chamber due to the consumption of
10
protons and decreases in the anode chamber because of the accumulation of protons
(Rozendal et al. 2007). This pH change in two-chamber systems results in a decrease of
the cathode potential and performance. The application of anion exchange membrane
(AEM) has also been explored in MFCs systems (Cheng and Logan, 2007, Kim and
Logan, 2007), where it has been proposed that protons are transferred via pH buffers, like
phosphate anions.
1.4.2 Cathodes and Catalysts
The cathode is another challenge for making MFCs commercially available
technology because the chemical reaction that occurs at the cathode is difficult to
engineer as the electrons, protons and oxygen must all meet at a catalyst in a tri-phase
reaction (solid catalyst, air, and water). The catalyst must be on a conductive surface and
must be exposed to both water and air so that protons and electrons in these different
phases can reach the same point. The most commonly used material for a cathode is
commercially available carbon paper pre-loaded with a Pt catalyst on one side. When it is
used in a MFC, the side that contains the catalyst faces the water and the uncoated side
faces air. To reduce the high cathode cost associated with platinum catalyst, other preciousmetal-free cathodes such as (NiMo and NiW) were developed by electrodepositing on a
carbon fiber. They have achieved comparable performance with Pt catalyst with same
loading at a much lower cathode fabrication cost (Hu, 2010).
The requirements of cathode for MECs are quite similar to the requirements of
cathode for MFCs but easier than cathode for MFCs for the manufacturing process,
because the cathode in MECs is not necessarily to exposed to air. Therefore, it can be
made of the exact same materials of cathode in MFCs except a waterproof layer.
11
Recently, some researchers have attempted to apply microorganisms as a biocatalyst to
precede the combination of electron with oxygen in the cathodes.
1.5 Dissertation overview
Low power densities in MESs limit practical applications. The improvement of
MESs performance requires a detailed understanding of the physiology and ecology of
microorganisms in MESs, including the mechanism of electron transfer to the anode from
the microorganism.
This dissertation focuses on the problem of the low power density of MESs. The
bacterial community structure of a high power generated mixed culture communities in
MFCs will be identified firstly, and then one of major exoelectrogens will be isolated and
characterized. This information will be helpful to understand the physiology and ecology
of exoelectrogens in MESs. Consequently, they will be beneficial to improve power
density of MFCs. Lastly, the nanostructure decorated anodes in MECs will be utilized to
improve the power density. The power enhancement mechanism will be explored by
using a whole genome microarray. They are presented here as four papers.
In the first paper, the cultivation independent molecular biological techniques,
denaturing gradient gel electrophoresis (DGGE) and 16S rDNA clone library, are utilized
to analyze the bacterial community structure of a higher power mixed culture MFCs. The
analyzed results provide fundamental information for isolating the dominant bacteria in
mixed culture MFCs. Two of dominant bacterial species has been isolated and used aone
to test power generation in MFCs. The possible interaction among different bacterial
species in mixed culture is discussed.
12
In the second paper, one
isolated exoelectrogenic bacterial strain SX-1 is
characterized. It is identified as a member of the Citrobacter genus and power generation
is tested ultilizing a wide range of different substrates. The electron transfer mechanism is
explored using Cyclic Voltammetry (CV). This study increases the known diversity of
power generating exoelectrogens and provides a new strain to explore the mechanisms of
extracellular electron transfer from bacteria to electrodes.
The third paper shows effects on MESs performance by Fe nanoparticle decorated
anodes in the MESs. The average current density produced with Fe nanoparticle
decorated anodes increased up to 5.9-fold higher than plain graphite anodes. A whole
genome microarray is utilized to analyze the possible mechanism of enhanced current
density as responded to nanoparticle decorated anodes.
The fourth paper describes the effects of carbon nanotube (CNT) modified anode on
the performance S. oneidensis MR-1 in MESs. Results demonstrate that current densities
produced with CNT decorated anodes are up to 5.6-fold higher than plain graphite anodes.
The possible mechanisms of enhanced current density by CNT decorated are explored.
13
Chapter 2
Bacterial community analysis of mixed consortium in higher
power density MESs
Shoutao Xu and Hong Liu
1 Introduction:
The improvement in the performance of mixed culture MFCs requires an
understanding of the ecology in microbial communities of MFCs. Many researchers have
attempted to characterize microbial populations and activities to elucidate the behaviors
and ecology of microorganisms in MFCs (Wrighton et al. 2008; Fedorovich et al. 2009).
In order to study the microbial ecology of the mixed culture in a MFC and select the
appropriate isolation medium for dominant bacterial species in the mixed culture, the
fundamental steps are to analyze the bacterial diversity in the mixed culture of MFCs and
identity the dominant bacterial species in bacteria communities in MFCs.
14
As for the identification of bacterial communities, typically, there are two general
methods. The first method for identification of bacterial community is the traditional
cultivation processes using selective nutrients to promote the growth of different types of
bacteria within the samples (Amman et al. 2000). The community structure can then be
assessed by identifying the isolates from the dominant colonies that were cultured. This
can often be costly and laborious as each isolate has to be further studied by examining
its physiology, taxonomy and reactivity to stains (Adwards et al. 1989).
The second method relies on utilizing molecular techniques to analyze bacterial
community DNA. Several molecular methods involving the extraction and analysis of
DNA from entire bacterial communities are used to identify genetic fingerprints of
bacteria. These methods, including the cloning and sequencing of 16S rDNA, automated
ribosomal intergenic spacer analysis (ARISA), terminal restriction fragment length
polymorphism (T-RFLP), and denaturing gradient gel electrophoresis (DGGE), generate
profiles of bacterial community structures. They can rapidly assess complex communities
from various environments (Amman et al. 2000).
Due to the conservative characteristic of 16S rDNA in bacteria during the process
of evolution, 16S rDNA sequencing can be used to identify different species of bacteria.
Therefore, the molecular techniques of denaturing gradient gel electrophoresis (DGGE)
with PCR and 16S rDNA clone library are used for analysis of the microbial diversity.
These methods are more convenient and save time compared to traditional
isolation/cultivation methods for microorganism analysis.
In this work, a biofilm bacterial community from an anode of a MFCs wase studied
by using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S
15
rRNA genes followed by cloning and sequencing of 16S rDNA. The results provided
essential information for dominant bacterial isolation in mixed culture MFCs.
2 Materials and methods
2.1 High power generation of mixed culture MFCs
Mixed cultures were originally inoculated from domestic wastewater (Corvallis
Wastewater Treatment Plant, Corvallis, OR) to MFCs after operating for 2 years using a
defined medium solution (Lovely, 2002) with sodium acetate as the carbon source. A
new MFCs was inoculated from the operating MFCs. The polarization curves were
performed to measure the power generation when maximal stable power were established
(normally 3-5 batches post-inoculation); and the genomic DNA from the microbial
biofilm on the anode was extracted under the sterile conditions for further bacterial
community analysis.
2.2 Denaturing gradient gel electrophoresis (DGGE).
DGGE with PCR is a method of analysis of bacterial community composition
based on different GC percentage of 16s rDNA in different bacterial species. PCR-DGGE
comprises three steps: (i) extraction of total community DNA from the sample; (ii) PCRcontrolled amplification using specific oligonucleotide primers; and (iii) separation of the
amplicons using DGGE. For this purpose, a reproducible and efficient method for total
DNA extraction is indispensable and needs to be evaluated and optimized depending on
the nature of the sample. In the subsequent PCR step, multiple PCR primer sets with
different resolution can be used. In most PCR-DGGE applications on bacteria, universal
or specific primers are targeting the 16S rRNA gene. Upon electrophoresis of the PCR
16
amplicons and gel staining (using ethidium bromide, silver staining or SYBR green),
DGGE gels are digitally captured and further analyzed using computer software packages.
The richness of DNA fragment bands in DGGE indicates the bacterial diversity of the
sample.
The detailed steps are as followed. Biofilms were scratched from the anodes of high
power generated MFCs fed with sodium acetate. Bacterial genomic DNA was extracted
from the biofilm samples using the DNeasy blood and tissue kit (Qiagen) according to the
manufacturer’s instructions. The universal primer set was used to amplify the 16S rDNA
from the extracted genomic DNA, then the V3 region of bacterial 16S ribosomal DNA
(rDNA) was amplified with GC-clamp primer (Muyzer et al. 1993) for DGGE. PCR
amplification was performed in a thermocycler. DGGE of the PCR products was carried
out in a DcodeTM Universal Mutation Detection System). The 8% (w/v) polyacrylamide
gels contains from 30% to 55% denaturing gradients. Electrophoresis was conducted
using a 1×TAE (Tris-Acetate-EDTA) buffer at 130V and 60°C for 5 hours. After
electrophoresis, the gel was stained with ethidium bromide in 1×TAE buffer for 15
minutes and washed in 1×TAE buffer for 10 minutes. The fragments were visualized
under a UV transilluminator. The richness of single band in DGGE gel picture
preliminarily showed that the bacterial diversity in mixed culture since the single band in
gel represents one bacterial species.
2.3 16S rDNA clone library construction method
16S rDNA-DGGE fingerprinting has proven to be particularly useful as an initial
investigation into bacterial communities and is suitable for identifying the predominant
bacteria that comprise 1% or more of cells within a given sample. In the 16s rDNA clone
17
library construction process, the first step is the extratction of the total genomic DNA,
then the genomic DNA of the mixed culture biofilm was used as template for PCR
amplification of approximately 1500 bp of 16S rDNA with universal primers. The PCR
products of 16S rDNA were purified and inverted into pGEM-T Easy vector system
before they were transformed into competent E. coli. The transformed cells were spread
on Luria-Bertani (LB) plates containing the selective antibiotic ampicillin and X-Gal and
incubated overnight at 37 OC. after the more than 80 inverted 16s DNA fragment E. coli
colonies were picked up and inoculated in the 1-3 ml liquid LB medium and left to grow
for 16 hours, The plasmid DNA were extracted and sequenced. The sequences were
compared directly to all known sequences deposited in GenBank databases using the
basic local alignment search tool (BLAST).
2.4 Bioinformatics Analysis
16S rDNA sequencing results of more than 80 colones were queried against the
GenBank and Ribosomal Database Project (RDP) databases using BLAST and
SEQUATCH algorithms (Cole, et al. 2007; Wang et al. 2007; Cole, et al. 2009). The
neighbor-joining trees were constructed with the Molecular Evolutionary Genetics
Analysis package (MEGA, Version 4) based on 1000 bootstrap analysis (Tamura et al.
2007).
2.5 Dominant bacterial species band in DGGE identification
The DGGE results gave the information of dominant bacterial richness of bacterial
species in mixed culture based on the theory that single band represents one bacterial
species, which showed the diversity of bacterial community in the mixed culture of a
MFC. 16S rDNA clone library results provided the whole picture of bacterial community,
18
including the bacterial species name and bacterial species percentage in the mixed culture.
The known pure bacterial species which have been sequenced can be used as markers to
identify dominant bacterial species in DGGE. The whole experimental procedure is
similar to the procedure of the mixed culture DGGE steps. The first step is to extract the
collect the mixed culture biofilm genomic DNA and pure bacterial species marker
genomic DNA. Then, the universal primer set was used to amplify the 16S rDNA from
the extracted genomic DNA. After that, the V3 region of bacterial 16S ribosomal DNA
(rDNA) was amplified with GC-clamp primer (Muyzer et al. 1993) for DGGE. The
subsequent steps were similar to the mixed culture DGGE experimental procedure. The
dominant bacterial bands in DGGE were determined by comparisons of the single band
position in the mixed culture DGGE with single pure bacterial species location in DGGE.
2.6 Dominant bacterial species isolation and power generation
The bacterial community of mixed culture in MFCs were predominantly composed
of Geobacter sp, Arcobacter sp , and Citrobacter sp. Two dominant bacterial species
have been isolated.
Citrobacter sp SX-1 was isolated from the anode biofilm by using a solid agar
medium containing NH4Cl, 6mM; KCl, 2mM; CH3COONa (electron donor), 30 mM;
FeC6H5O7 (electron acceptor), 20mM; minerals and vitamins (Lovley and Phillips 1988).
Single colonies were picked up after culturing on the plate for 36 hours at 30 °C, and
transferred two times on the agar plate for purification. Then the isolates grew in a liquid
medium solution in anaerobic tubes, containing the same constituents as the solid
medium. All isolation process was operated in a glove box anaerobic chamber (Coy
Laboratory Products, Grass Lake, MI).
19
Arcobacter sp SX-2 was isolated from the anode biofilm of a MFC fed with sodium
acetate by using the same method of isolation of pure bacterial species Citrobacter XS-1
except using Trypticase Soy Agar with 5% Sheep Blood solid (from American type
culture collection (ATCC)) agar medium instead of Citrobacter sp medium. Single
colonies were picked up after culturing on the plate for 36 hours at 30 °C, and transferred
twice on the agar plate for purification. Then the isolates grew in a liquid medium
solution microaerobically.
Geobacter sp have been trying to isolate by using three different methods. They are
the colony pickup after different condition enrichment; Goebacter medium isolation and
dilution to extinction by multiple channel mini MFCs isolation, however, the Geobacter
sp. has not been isolated yet due to some limits so far. But they provide valuable
information for further isolation of Geobacter sp.
Single chamber MFCs were used to evaluate power generation by different isolates.
The MFCs were constructed as described previously (Liu and Logan, 2004) and modified
with 3 cm2 carbon cloth anodes and 7 cm2 carbon cloth/Pt cathodes. The total liquid
volume of each MFCs was about 15 ml with electrode spacing about 1.7 cm. All MFCs
were operated in an autoclaved closed plastic box and sterile cotton was attached to the
outer surface of the air cathodes to prevent contamination. A MFCs without bacterial
culture was used as control. MFCs were inoculated with 3 ml late exponential phase
cultures of SX-1 in the medium solution reported previously (Liu and Logan 2004).
3 Results and discussion
3.1 Power production by mixed culture MFCs.
20
A polarization curve was used to characterize current as a function of voltage in the
MFCs. The polarization curves are performed by varying a series of external resistances.
The results are shown in Figure 2.1. With MFCs acclimated to 25 Ω external resistance,
the maximum power density was achieved at 6.5W/m2 based on the polarization data. At
this point, the current density is 2.1mA/cm2. The power density was three times higher
than previous report (2.0 W/m2) using phosphate buffer (Fan et al. 2007). Power density
was also 2 times higher than (2.8 m W/m2) using a bicarbonate buffer ( Fan et al. 2007).
7
power density
6.5w/m2 at current
density2.08 mA/cm 2
Power
Voltage
0.60
0.50
5
0.40
4
0.30
3
Voltage (V)
Power density (W/m2)
6
0.70
0.20
2
0.10
0.4 0.5 0.5 0.6 0.7 0.8 1.0 1.1 1.3 1.5 2.1 2.2 2.3 2.4
Current density (mA/cm2)
Figure 2-1: Polarization curves of high power mixed culture MFCs.
3.2 Bacterial community structure assessed by PCR-DGGE
Bacterial DGGE profiles for higher power generating MFCs mixed culture setups
were relatively complex (more than 10 bands). However, there are three brightly stained
bands and other less intense bands (Fig. 2-2). These results indicated there were probably
three dominant bacterial species in mixed culture MFCs.
21
1
2
Figure 2.2 DGGE result of high power generation mixed culture MFC (1 mixed
culture; 2. pure culture control).
3.3 16S rDNA clone library results
A total of 83 colonies were sequenced for 16S rDNA clone library construction. They
represented the bacterial community structure in mixed culture in MFCs. The results
revealed that the predominant bacterial species in mixed culture are Geobacter sp (66%
of the mixed culture) Arcobacter sp (12% of the mixed culture) and Citrobacter sp (11%
of the mixed culture). Those three genuses comprised 88% of the total bacterial species
(Fig. 2.3). This result is consistent with the DGGE result which showed that three bright
bands in the DGGE gel represented three dominant bacterial species in the mixed culture.
The phylogenetic analysis of the colonies (83 colony sequences) is shown in Figure 2.4.
22
Anaerovorax
2%
Pseudomonas
1%
others
6%
Geobacter
Arcobacter
Clostridium
2%
Citrobacter
Clostridium
Citrobacter
11%
Anaerovorax
Pseudomonas
others
Arcobacter
12%
Geobacter
66%
Figure 2.3 Bacterial species and percentage of bacterial community of mixed
culture MFC
'< 1% Alcaligens>'
100
'<1% Commamonas>'
100
'<1% Pseudomonas aeruginosa> '
'<1 % Klebsiella>'
85
82
100
'<11% Citrobacter>'
'<1% Wolinella>'
100
100
99
'<12% Arobacter>'
'<2% Anaeroborax>'
'<2 % Clostridium >'
87
'<1% unclear bacteria>'
'<66%Geobacter>'
0.02
Figure 2.4 Phylogenetic trees of bacterial species from mixed culture MFC
3.4 Dominant bacterial band in DGGE identification
23
The dominant bacterial species, including Goebacter sp, Citrobacter sp and Arcobacter
sp in the mixed culture, are shown in Figure 2.5. The upper band is Arcobacter sp; and
Citrobacter sp is located in the bottom band of DGGE. Geobacter sp is the middle one.
The analysis of the bacterial community using denaturing gradient gel electrophoresis
(DGGE) of PCR-amplified 16S rRNA gene fragments and 16S rDNA clone library
construction method showed great phylogenetic diversity of mixed culture in MFCs, with
the identification of sequences derived from bacteria of the taxa deltaproteobacteria
(Geobacter sp), gammaproteobacteria (Citrobacter sp) , and
epsilonproteobacteria
(Arcobacter sp). The result is consistent with the conclusion that among the isolated
exoelectrogens, gammaproteobacteria and deltaprotebactia appear to be dominant among
others (Parot et al. 2009; Liu et al. 2010).
A
B
C
D
A: Citrobacter sp.
B: Geobacter sp.
C: Arobacter butzmeri
D: mixed culture control
Figure 2.5 Dominant bacterial species identification in DGGE
3.5 The dominant bacterial species isolation and power generation
24
Two dominant bacterial species Citrobacter sp XS-1 and Arcobacter sp XS-2 have been
isolated and tested for power generation. The power densities generated by the two
isolates are shown in the Fig 2.6. Current densities generated by isolate Citrobacter sp
and Arcobacter sp were 98 mA/m2 and 20 mA/m2, respectively, which were much less
than that (2.1A/m2) generated by the mixed culture from which they were isolated.
2500
Current density (mA/m2)
2000
1500
1000
500
0
Mixed culture
Citrobacter sp
Arcobacter Sp
Figure 2.6 Current density generated by different dominant isolates
The maximum current density generated by mixed culture is much higher than the
one produced by pure bacterial species Citrobacter sp XS-1 and Arcobacter sp XS-2 in
the same structure MFCs. Although the Geobacter sp in our mixed culture has not been
isolated, a previous study showed that maximum current density generated by Geobacter
sp was 0.80 A/m2 (Bond and Lovley 2003). The current density results seem partially
supporting the conclusion that mixed-culture generated higher power densities than their
pure-culture counterparts in MFCs (Jung and Regan 2007). This result suggests there
may be synergistic interactions within the anode bacterial communities.
25
Geobacter sp are strictly anaerobic bacteria which were enriched in most of the anode
biofilms (George 2005); Citrobacter species are facultative anaerobic bacteria (George
2005) which can be found in a wide variety of habitats, including in soil, water and
wastewater; Arcobacter sp are micro-aerobic bacteria, including both environmental
nonpathogens and opportunistic human pathogens. They are able to grow in aerobic
conditions but in the optimal growth under micro-aerobic conditions (George 2005).
Arcobacter sp has been discovered in the MFCs by Fedorovich (Fedorovich et al. 2009).
It is interesting that the mixed culture bacterial community composed of primarily these
three bacterial species has demonstrated to generate high power at non-strictly anaerobic
condition, while leading dominant bacterial species Geobacter sp (66%) is strict
anaerobic bacteria and generated powder in the MFCs only under strict anaerobic
condition (George 2005). Therefore, it is possible that microaerobic bacteria such as
Arcobacter sp and facultative anaerobic bacteria Citrobacter sp can facilitate to create
anaerobic condition for Geobacter sp when they utilized oxygen for growing in the
mixed culture. Syntrophic communities study have showed that anaerobic bacteria and
methanogenic archaea form compact microbial structures that operate like an organ rather
than a set of microorganisms functioning independently (Stams and Plugge, 2009). Some
substrates have been degradeted within these communities while they are not able to be
fermented by individual species alone. interspecies electron transfer also have been
presented in these communities (Stams and Plugge, 2009). Summers et al (2010) also
discovered that direct exchange of electrons happened within coculture of Geobacter
metallireducens and Geobacter sulfurreducens. Therefore, we believe that there are
26
more complex synergistic interactions between different bacteria species in mixed culture
in the MFCs.
Acknowledgements
The authors acknowledge support from the U.S. National Science Foundation
(CBET 0955124). We also thank Keaton Lesnik for the review of this manuscript.
27
Chapter 3
New Exoelectrogen Citrobacter sp. SX-1 Isolated from a Microbial Fuel Cell
Shoutao Xu and Hong Liu
Published in
Journal of Applied Microbiology, 111(5):1108-1115 (2011)
ABSTRACT
Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel
cell by conventional plating techniques with ferric citrate as electron acceptor under
anaerobic condition. Phylogenetic analysis of the 16S rDNA sequence revealed that it
28
was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the
most closely related species. The bacterial strain SX-1 produced electricity from citrate,
acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density
of 205 mA m-2 generated from citrate. Cyclic voltammetry analysis indicated that
membrane associated proteins may play an important role in facilitating the electrons
transferring from bacteria to electrode. This is the first study that demonstrates that
Citrobacter species can transfer electrons to extracellular electron acceptors. Citrobacter
strain SX-1 is capable of generating electricity from a wide range of substrates in MFCs.
This finding increases the known diversity of power generating exoelectrogens and
provided a new strain to explore the mechanisms of extracellular electron transfer from
bacteria to electrode. The wide range of substrate utilization by SX-1 increases the
application potential of MFCs in renewable energy generation and waste treatment.
Keywords: Microbial fuel cell; Exoelectrogen; Citrobacter sp. SX-1; Extracellular
electron transfer
29
Introduction
Microbial Fuel Cells (MFCs) technology has become an active research area
recently as a promising approach for renewable energy generation, wastewater treatment,
and bioremediation (Rabaey and Verstraete 2005; Lovley 2006; Fan et al. 2008; Logan
2009; Liu et al. 2010). The key feature of MFCs system is the microbe-catalyzed electron
transfer from organic matter to anodes. Many studies have shown the presence of diverse
bacterial communities on MFCs anodes (Reimers et al. 2001; Liu et al. 2004b; Kim et al.
2005). Over 20 exoelectrogens, i.e. microbes that can transfer electrons exocellularly to
electrodes have been reported in the past 10 years (Logan 2009; Liu et al. 2010). The
exoelectrogens so far belong to diverse genetic groups, including alphaproteobacteria
(Rhodopseudomonas, Ochrobactrum and Acidiphilium) (Malki et al. 2008; Xing et al.
2008; Zuo et al. 2008) betaproteobacteria (Rhodoferax) (Chaudhuri et al. 2003),
gammaproteobacteria
(Shewanella,
Pseudomonas,
Klebsiella,
Enterobacter
and
Aeronomas) (Kim et al. 2002; Pham et al. 2003; Rabaey et al. 2005; Bretschger et al.
2007; Zhang et al. 2008; Chung et al. 2009; Rezaei et al. 2009), deltaproteobacteria
(Geobacter, Geopsychrobacter, Desulfuromonas, and Desulfobulbus) (Bond et al. 2002;
Bond and Lovley 2003; Holmes et al. 2004a,b; 2006), Epsilonproteobacteria (Arcobacter)
(Fedorovich et al. 2009) Firmicutes (Clostridium and Thermincola) (Park et al. 2001;
Wrighton et al. 2008), Acidobacteria (Geothrix) (Bond and Lovley 2005), and
actinobacteria (Propionibacterium) (Wang et al., 2008). A wider range of
exoelectrogenic species are expected to be discovered.
Three mechanisms have been proposed for exocellular transport of electrons by
exoelectrogens without artificial mediators. Some exoelectrogenic bacteria can transfer
30
electrons to electrodes through soluble redox compounds excreted by microorganisms
(Bond and Lovley 2005; Rabaey et al. 2005) and others can directly transfer electrons to
anodes via outer cell membrane proteins (Myers and Myers 1992). Recently, more and
more evidence supports the involvement of bacterial nanowires in extracellular electron
transport (Reguera et al. 2005; 2006; Gorby et al. 2006). In spite of the discovery of
many bacterial species that can transfer the electrons to electrode without the need of
artificial mediators, the investigation of extracellular electron transfer mechanisms was
mainly focused on a few species, such as those from Geobacter and Shewanella genera
(Kim et al. 1999; 2002; Reguera et al. 2005; 2006; Gorby et al. 2006). The electron
transfer mechanisms for many of the isolated exoelectrogens species are still not well
studied.
In the present study, we isolated a new exoelectrogen strain SX-1 from a MFC, a
strain phylogenetically related to Citrobacter sp. Power generation from various carbon
sources by this strain was evaluated using single chamber MFCs. Plausible extracellular
electron transfer mechanisms were also discussed based on the characterization of anodic
biofilms by cyclic voltammetry (CV).
Materials and methods
Bacterial strain SX-1 isolation
Bacterial strain SX-1 was isolated from the anode biofilm of a MFC fed with sodium
acetate operated in fed-batch mode over a period of six months. The original source of
the inoculum is wastewater from a local waste water treatment plant. Bacterial cells were
released from the carbon cloth anode by shaking the cloth in a sterile flask with 15 mL
sterile 1% NaCl solution and glass beads (2 mm diameter). The suspension was then
31
serially diluted from 10 times to 105 times and plated on a petri dish with a solid agar
medium containing NH4Cl, 6mM; KCl, 2mM; CH3COONa (electron donor), 30 mM;
FeC6H5O7 (electron acceptor), 20mM; minerals and vitamins (Lovley and Phillips 1988).
Single colony was randomly selected after culturing on the plate for 36 hours at 30 °C,
and purified on a new agar plate following a procedure reported previously (Chung and
Okabe, 2009). Then the isolate was picked up and grew in a liquid medium solution in
anaerobic tubes containing the same constituents as the solid medium for further analysis.
All isolation process was operated in a glove box anaerobic chamber (Coy Laboratory
Products, Grass Lake, MI).
16S rDNA sequencing and phylogenetic analysis
Bacterial genomic DNA was extracted from the isolated strain SX-1 using the DNeasy
tissue Kits (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The 16S
rDNA was amplified by PCR using a pair of universal primers: 27F (5’AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTTGTTACGACTT3’) (Park et al. 2001). The conditions used for PCR were described previously
(Ferna´ndez et al. 1999). PCR products were purified, ligated and cloned according to a
procedure described in a previous report (Xing et al. 2008). Plasmids were isolated from
randomly selected clone colonies with a QIAprep Spin Miniprep kit (Qiagen, Valencia,
CA), and three plasmid inserts were then sequenced in both directions using an ABI 3730
DNA sequencer (Applied Biosystems, Carlsbad, CA)United States). Due to the identical
16S rDNA sequence of the three colones, only one was queried against the GenBank and
Ribosomal Database Project (RDP) databases using BLAST and SEQUATCH algorithms
(Cole, et al. 2007; Wang et al. 2007; Cole, et al. 2009). The 16S rDNA sequence of
32
strain SX-1 and closely related type strains were aligned using CLUSTALX software
(Thompson et al. 1997). The neighbor-joining trees were constructed with the Molecular
Evolutionary Genetics Analysis package (MEGA, Version 4) based on 1000 bootstrap
analysis (Tamura et al. 2007).
SEM
The morphologies of SX-1 cells growing overnight in anaerobic tubes were examined
with a scanning electron microscope (FEI Quanta 600 FEG, FEI Company, Hillsboro,
OR). The suspended cells were fixed in a 2.5% glutaraldehyde and 0.1M phosphate
buffer solution and dehydrated with a graded ethanol series from 30% to 100%. After
dehydration, the samples were dried in a critical point dryer and then sputter-coated with
Au/Pd for SEM examination (Liu and Logan. 2004).
Evaluation of power generation by SX-1 in MFCs
Single chamber MFCs were used to evaluate power generation by SX-1 using various
substrates. The MFCs were constructed as described previously (Liu and Logan 2004)
and modified with 3 cm2 carbon cloth anodes and 7cm2 carbon cloth/Pt cathodes. The
total liquid volume of each MFCs was about 15 ml with electrode spacing about 1.7 cm.
All MFCs were operated in an autoclaved closed plastic box and sterile cotton was
attached to the outer surface of the air cathodes to prevent contamination. A MFC
without bacterial culture was used as control. MFCs were inoculated with 3 ml late
exponential phase cultures of SX-1 in the medium solution reported previously (Liu and
Logan 2004). Seven substrates, including citrate, acetate, glucose, sucrose, glycerol, and
lactose were evaluated individually for power generation in a fed-batch mode in a
temperature control chamber (30 ±2°C). For each substrate, at least 8 batches were run to
33
investigate the effect of biofilm formation on current output at fixed external resistance of
1kΩ and the same initial substrate concentration of 30 mM. Phosphate buffer (100mM)
was used to maintain the solution pH and conductivity. The MFCs with sodium citrate as
electron donors were also examined for maximum power output by varying the external
resistance from 20 kΩ to 4 kΩ. A data acquisition system was used to record voltage data
during the MFCs operation (Keithley 2700, Keithley Instruments Inc., Cleveland, OH). It
took about 15 to 30 minutes for the MFCs to stabilize depending on the external
resistance. At each resistance, we collected at least five data at the steady condition to
make the polarization curves. Averaged voltages were used to calculate the power density
(mW m-2) according to P=IV/A, where I was the current, V was voltage, and A was crosssectional area of the anode.
CV analysis
CV was used to characterize the oxidation and reduction reactions on the anodic surface
of the MFCs fed with citrate and operated at 7 KΩ. This external resistance was selected
because the maximum power density was obtained at this resistance based on the
polarization experiment. We assumed the biofilm on the anode of MFCs were welldeveloped when stable power output was obtained after 3 batches of operation. The
MFCs were then used directly for CV analysis at four current generating stages of the
fourth batch: (1) initial exponential current increasing stage (middle point of stage about
4 h after media change); (2) current plateau stage (middle point of stage about 8 h after
media change); and (3) current decreasing stage (middle point of stage about 16h after
media change); (4) right after the complete replacement of medium solution (Figure 6).
The anode was used as working electrode, the cathode as counter electrode, and an
34
Ag/AgCl electrode was selected as the reference. The CV curves were scanned from 200
to -600 mV at a rate of 5 mV s-1 using a potentiostat (G300, Gamry Instrument
Inc.,Warminster, PA). Control experiment was also conducted using new anode (without
biofilm) and new medium solution.
Nucleotide sequence accession number
The 16S r DNA sequence determined in this study has been deposited in the GenBank
database under accession number HQ845373.
Results
Identification of the strain SX-1
An almost-complete 16S rDNA sequence (1,475 bp) of strain SX-1 was obtained and
subjected to comparative analysis with the 16S rDNA of closely related reference strains.
A phylogenetic analysis revealed that strain SX-1 should be assigned to the genus
Citrobacter and was most closely related to Citrobacter sp. sdy-48 EJ463782 (99.0%
sequence similarity) and Citrobacter sp. yy-21 FJ463779 (97.5% sequence similarity).
These three strains formed a distinct sub cluster in the neighbor-joining, in which the new
isolate and Citrobacter sp. sdy-48 EJ463782 formed a distinct subline (Figure 3.1).
The SEM image illustrated that the strain SX-1 is a rod-shaped bacterium 0.3-0.6 µm
wide and 1.2-1.7 µm long (Figure 3.2). Strain SX-1 colonies on nutrient agar appeared 23 mm in diameter, smooth moist opaque with a shiny surface after 5 days incubation
under anaerobic condition.
The fact that strain SX-1 grew both aerobically and
anaerobically using FeC6H5O7 as electron acceptor suggests that the strain is facultatively
anaerobic. Strain SX-1 showed robust growthD on citrate as a carbon source. These
35
properties were similar to those of genus Citrobacter described in Bergey’s Manual of
Systematic Bacteriology (George 2005).
53
Strain SX-1
36
Citrobacter sp. sdy-48 (FJ463782)
Citrobacter sp. yy-21 (FJ463779)
92
Citrobacter koseri E639 (ATCC 25408)
Citrobater koseri CDC 3613-63(AF025372)
44
76
Citrobacter koseri CDC 8132-86(AF025366)
100
Salmonella enterica SL483 (CP001138)
Salmonella enterica AKU12601(AY696668)
Shigella sonnei Ss046(CP000038)
100
88
E. coli C2 (AF403733)
E. coli E24377A(CP000800)
0.001
Figure 3.1: phylogenetic tree of strain SX-1 and closely related species based on 16S
rDNA sequences. The tree was constructed using the neighbor-joining method.
1 µm
Figure 3.2: SEM image of planktonic cells of Citrobacter sp. SX-1
36
Electricity production by strain SX-1 in MFCs
The isolated strain SX-1 was first tested for its ability to generate current from sodium
citrate at 1kΩ. Current gradually increased to a maximum 50 mA m-2 after cell
inoculation and then decreased (Figure 3.3). After the MFCs was refilled with new
substrate solution, the current recovered rapidly and reached a higher level than the first
batch. After 4 batches operation, the maximum current output of each batch became
stable. The highest current density achieved in the MFCs with Citrobacter sp. SX-1 at 1
KΩ was 98 mA m-2 (Figure 3.3).
Current density (mA m-2)
120
100
80
60
40
20
0
0
50
100
150
200
Time (h)
Figure 3.3: Electricity generation by Citrobacter sp. SX-1 in a single chamber MFCs
with sodium citrate (30 mM) as substrate at 1 KΩ.
37
Current generation by SX-1 from other substrates, including glucose, lactose, sodium
acetate, glycerol, and sucrose, were also assessed at an external resistance of 1KΩ
(Figure 3.4). When repeatable cycles of current output were obtained for these substrates,
glycerol generated the highest maximum current density of 58 mA m-2, followed by
lactose and sucrose with 29 mA m-2 and 27 mA m-2, respectively. Glucose and acetate
produced the lowest maximum current density of 9.6 and 4.3 mA m-2, respectively. These
results indicated that strain SX-1 can utilize a wide range of substrates for electricity
generation in MFCs, but with different power generation potentials.
70
Current density (mA m-2)
60
50
40
30
20
10
0
Glucose
Lactose
Glycerol
Acetate
Sucrose
Figure 3.4: Electricity generation of Citrobacter sp. SX-1 using different substrates in
single chamber air-cathode MFCs at 1 KΩ. (The error bars stand for the standard
deviation of 3 replicates)
38
Polarization experiment was further conducted to determine the maximum power
density generated by strain SX-1 in the single chamber MFCs with citrate as substrate. A
maximum power density of 88.1 mW m-2 was obtained at current density of 205 mA m-2
at an external resistance of 7 KΩ (Figure 3.5).
0.7
100
Voltage
80
Voltage (V)
0.5
70
0.4
60
50
0.3
40
0.2
Power density (mW m-2)
90
Power
0.6
30
0.1
20
80
110
140
170
200
230
Current density (mA m-2)
Figure 3.5: Power and voltage generation by Citrobacter sp. SX-1 as a function of
current density using sodium citrate (30 mM) as substrate. The error bars stand for the
standard deviation of the 3 voltage/power outputs obtained in three MFCs
39
Current density (mA m-2)
250
Stage 2
200
Stage 3
A
150
Stage 4
Stage 1
100
A
50
0
50
55
60
65
70
75
80
85
Time (h)
Figure 3.6: Four current generating stages for CV analysis: current increasing stage (stage
1); current plateau stage (stage 2); current decreasing stage (stage 3); and: right after the
replacement of medium solution (stage 4). The MFCs was operated at 7 KΩ with sodium
citrate (30 mM) as substrate.
Cyclic Voltammetry
To determine the presence of redox active compounds produced by SX-1 and the
location of these compounds, CV scan of the anodic biofilms at four current output stages
and the supernatant of MFCs medium solution at the end of the batch experiment were
performed. Reduction peaks at range of -310 – -350 mV and oxidation peaks at range 100 – -150 mV were observed at all current generating stages (Figure 3.6, Figure 3.7A),
suggesting the presence of redox active compounds may involve in extracellular electron
transfer by Citrobacter sp. SX-1. The amplitude of the peaks increased according to the
growth stage of the batch and the highest peaks were present after the current plateau
stage, which indicated the redox active compounds mainly were secreted in the current
plateau stage. While the current density at stage 3 (deceasing stage) was lower than that
at stage 2 (current plateau stage), the redox peak at stage 3 was higher than that in stage 2.
40
A plausible explanation for this pattern was that redox active compounds were
continuously being secreted and accumulated after the current peak was reached,
resulting in more redox compounds present after the plateau stage. But, since most of the
carbon source had already been being used up, many of the redox compounds at this
stage had not been as active as those at current plateau stage due to much less electrons
were available to be transferred. The bigger peak amplitude in stage 4 than in stage 1
indicates that the redox active compounds were continuously being secreted and
accumulated in the biofilm even after stable power was generated at 7 kΩ. The slightly
change of the peak locations at different stages was possible due to the solution chemistry
change during the current generation process or the slightly location change of reference
electrode during the measurement (Figure 3.7B). When the medium solution in the MFCs
was replaced by fresh medium at the end of the batch, the oxidization and reduction
peaks were still presented in the CV (Figure 3.7B), but no peaks were observed when the
supernatant and fresh mediums were tested in a MFCs with no biofilm (Figure 3.7C).
These results suggest the compounds involved in the electron transfer were located not in
the supernatant (solution) but in the biofilm.
41
3.0E-04
A
2.0E-04
Current (A)
1.0E-04
0.0E+00
-1.0E-04
Current plateau stage
-2.0E-04
Current increasing stage
-3.0E-04
Current decreasing stage
-4.0E-04
-0.60
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
0.10
0.20
Potential (V) vs Ag/AgCl
3.0E-04
2.0E-04
B
Current (A)
1.0E-04
0.0E+00
-1.0E-04
-2.0E-04
Current decreasing stage
-3.0E-04
Biofilm with fresh medium
-4.0E-04
-0.60 -0.50 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20
Potential (V) vs Ag/AgCl
0.0E+00
-5.0E-05
C
Current(A)
-1.0E-04
-1.5E-04
-2.0E-04
-2.5E-04
-3.0E-04
Supernatant
-3.5E-04
Fresh medium
-4.0E-04
-0.60
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
0.10
0.20
Potential (V) vs Ag/AgCl
Figure 3.7: Cyclic Voltammetry analysis of Citrobacter sp. SX-1 biofilms at: (A) current
increasing stage, current plateau stage and decreasing stage (end of the batch); (B) end of
the batch and replaced with fresh medium and (C) controls.
42
Discussion
Citrobacter
species,
belonging
to
Gammaproteobacteria,
Enterobacteriales,
Enterobacteriaceae are facultative anaerobic bacteria (George 2005). They can grow with
oxygen as an electron acceptor but also with a variety of electron accepters such as Fe (III)
in the absence of oxygen (George 2005). Citrobacter species can be found in a wide
variety of habitats, including in soil, water and wastewater. Several Citrobacter species
have been studied as important bioremediation bacterium for heavy metal removals,
sulfate reduction, phenol degradation and chlorophenol degradation (Macaskie et al. 1995;
Narde et al. 2004; Qiu et al. 2009). Citrobacter species have also been found on the
anodic biofilm of a diesel-degrading MFCs (Morris et al. 2009). However, there was no
study reported that Citrobacter species can transfer electrons to extracellular electron
acceptors. This finding that Citrobacter strain SX-1 can generate electricity in MFCs
increased the diversity of power generating exoelectrogens and provided a new strain to
explore the mechanisms of extracellular electron transfer from bacteria to electrode.
Among the isolated exoelectrogens, gammaproteobacteria and deltaprotebactia appear to
be dominant among others (Parot et al. 2009; Liu et al. 2010). The isolated strain
Citrobacter sp SX-1 phylogenetically belongs to gammaproteobacteria, which provides
another evidence to support this observation.
Most exoelectrogens utilize a limited range of substrates for power generation. For
example, Shewanella oniedensis MR-1 can oxidize lactate under anaerobic condition but
cannot utilize acetate for electricity generation (Kim et al. 2002); Geobacter
sulfurreducens can completely oxidize acetate for power generation, but it cannot utilize
simple sugars (Bond and Lovley 2003; Caccavo et al. 1994)). In comparison, strain SX-1
43
not only can oxidize citrate for power generation, but also utilize a wide range of
substrates for power generation, including glycerol, glucose, lactose, sucrose and acetate.
Interestingly, the higher current density generated by strain SX-1 from glycerol, a main
by-product of biodiesel production process, suggests that strain SX-1 may be potentially
used for harvesting energy from biodiesel wastes using MFCs. However, the current
density generated by strain SX-1 (205 mA m-2) is lower than that (805 mA m-2 at 1 kΩ)
generated by the mixed culture from which the SX-1 was isolated, indicating the
existence of other higher power generating bacteria and/or complex ecology in the mixed
culture community.
Understanding mechanisms of microbial extracellular electron transfer is critical for
enhancing the electron transfer rate from bacteria to electrode through metabolic or
genetic engineering of exoelectrogens. Since the spent supernatant solution of MFCs run
with SX-1 showed no redox properties, the redox compounds produced by SX-1 may
have been retained in the biofilm. Alternatively, SX-1 may utilize one or both of the
other two known extracellular electron transfer mechanisms. For example, it is possible
that strain SX-1 transfers electrons to the anode via cell-bound outer membrane proteins.
The two distinct oxidation and reduction peaks observed at -100mv- -150 mV and -310-350 mV of the SX-1 biofilm have a midpoint potential range from -205mV to -250mV,
which is close to the redox potential of some c-type cytochromes (Field et al. 2000; Meitl
et al. 2009), which are well-known to play an important role in extracellular electron
trasnfer (Eggleston et al. 2008; Field et al. 2000; Meitl et al. 2009). Field et al. (2000)
reported that the c-type cytochromes CymA had the midpoint potentials of -229 mV.
Meitl et al. (2009) and Eggleston et al. (2008) reported that the c- type cytochromes
44
OmcA in Shewanella oneidensis MR-1 had the midpoint potentials of -201 mV and -208
mV.
While it’s possible that SX-1 may also have conductive appendages to enhance the
extracellular transfer electron, similar to the nanowires discovered in some Geobacter
and Shewanella species (Reguera et al. 2005; 2006; Gorby et al. 2006), further
investigation on the presence of these appendages on the cell surface of SX-1 is needed.
Acknowledgements
The authors acknowledge support from the U.S. National Science Foundation
(CBET 0955124). We also thank Teresa Sawyer for her helps with the SEM analysis and
Jeremy Chignell and Yanzhen Fan for their review of this manuscript.
45
Chapter 4
Enhanced Performance and Mechanism Study of Microbial Electrolysis Cells
Using Fe Nanoparticles Decorated Anodes
Shoutao Xu, Hong Liu,Yanzhen Fan, Rebecca Schaller, Jun Jiao, Frank Chaplen
Published in
Applied Microbiology and biotechnology 93(2):871-880 (2012)
46
ABSTRACT
Anode properties are critical for performance of microbial electrolysis cells (MECs). In
the present study, Fe nanoparticle modified graphite disks were used as anodes to
investigate the effects of nanoparticles on the performance of Shewanella oneidensis MR1 in MECs. Results demonstrated that average current densities produced with Fe
nanoparticle decorated anodes were up to 5.6-fold higher than plain graphite anodes.
Whole genome microarray analysis of the gene expression showed that genes encoding
biofilm formation were significantly up-regulated as response to nanoparticle decorated
anodes.
Increased expression of genes related to nanowires, flavins and c-type
cytochromes indicate that enhanced mechanisms of electron transfer to the anode may
also have contributed to the observed increases in current density. The majority of the
remaining differentially expressed genes were associated with electron transport and
anaerobic metabolism demonstrating a systemic response to increased power loads.
Keywords: Microbial electrochemical system, microbial fuel cell, microbial electrolysis
cell, nanotechnology, differential gene expression, DNA microarray
47
Introduction
Microbial electrochemical systems (MESs) have been intensively studied since Lewis
achieved practical advances in this field (Logan 2007), however they attracted much
research attention in recent years due to their promising applications in renewable energy
generation, bioremediation, and wastewater treatment. In a MES, microorganisms
interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the
anode and the cathode. The most-described type of MESs is microbial fuel cells (MFCs),
in which useful power is generated from electron donors, typically biodegradable organic
materials (Logan et al. 2006). Various novel MESs have recently been developed to
produce hydrogen (microbial electrolysis cells (MECs)) (Liu et al. 2005; Logan et al.
2008; Rozendal et al. 2006; Schaller et al. 2009), to power an autonomous sensor or
sensor network (Ringeisen et al., 2006), to reduce bioremediation targets (Lovley 2006;
Shea et al. 2008); Sukkasem et al. 2008), and to desalinate water (Cao et al. 2009). The
key feature shared by these systems is the microbe-catalyzed electron transfer from
organic matter to electrodes (anodes) (Rabaey et al 2004). Enhancing the anodic current
output, which highly depends on the performance of the electrodes, is critical for the
successful application of all these processes (Logan et al. 2007; Park and Zeilus 2002;
2003).
Nanomaterials have received much attention from researchers in the context of
microbiology due to their unique physical, electrical, and chemical properties, which
facilitate the study of interactions between bacteria and surfaces (Liu 2006). Previous
studies have demonstrated that electrodes decorated with different nanostructures such
as carbon nanotubes , nanostructured polyaniline/titanium dioxide composite and titania
48
nanotubes have the potential to enhance power generation in MFCs (Morozan et al. 2007;
Qiao et al. 2008; Quan et al. 2005). Furthermore, our recent study shows nanoparticle
(NP) decorated anodes greatly increased the electrochemical electron transfer rate in
MFCs (Fan et al. 2011). However, Fe NPs, as an ideal candidate for decorating electrodes
because of respectively low price and high conductivity compared to other materials has
not been focused to study in MECs.
S. oneidensis MR-1, an important electrochemically active bacterial strain, has been
exploited for the electricity generation in MFCs (Logan et al. 2007; Logan 2009; Park
and Zeikus 2002). The availability of genome sequence for this strain makes it possible to
use transcriptome assays to globally measure the responses to different growth conditions
and environmental stresses (Murray et al. 2001). S. oneidensis MR-1 gene expression
response to heat shock, cold shock, Cr (VI) and U (VI) reduction, chromate stress, and
iron and acid tolerance have been studied in the past several years (Bencheikh-Latmani et
al. 2005; Brown et al. 2006; Gao et al.2006; Gao et al. 2004; Yang et al. 2008). However,
no studies have been focused on the Shewanella gene expression response to NPs in
MESs.
The mechanism of increased electron transfer rate exhibited by nanoparticle decorated
electrodes is not well understood yet. In the present study, graphite disks decorated with
Fe NPs were used as anodes to explore the effects of nanostructures on current generation
in a multi-anode MECs. DNA microarrays were utilized to investigate differences in the
global gene expression profile of S. oneidensis MR-1 grown on plain versus Fe NPs
decorated anodes.
Materials and methods
49
Bacterial cultures
S. oneidensis MR-1 was purchased from American Type Culture collection (ATCC
700550) and stored as freezer stocks at -80°C. Stock cells of S. oneidensis MR-1 was
grown aerobically using a medium containing 30 g/L Trypticase Soy Broth (BD 211825,
Becton, Dickinson Company Franklin lakes, NJ.) at 30°C. Cells in early stationary
growth phase (optical density of 1.5-1.6 at 600nm; (Spectrophotometer UV-1700,
Shimadzu, Columbia, MD) were washed two times then injected into the chamber of the
MECs for current production. Trypticase Soy Broth medium with 30 mM sodium lactate
as electron donor was used in MECs. Phosphate buffer (100mM) was used to maintain
the solution pH 7 and solution conductivity at 15 mS/cm.
Characterization of nanostructured anodes
Superfine isomolded graphite disks (1.6 cm x 0.5 cm, GraphiteStore.com, Inc.) were
polished with ultrafine sand paper (2000 grit, 3M Company) as the base for the NP
decorated and control anodes. Fe NPs decorated anodes were fabricated by thermal
annealing of metal thin film coated graphite disks. Thin films of Fe were firstly deposited
on the polished graphite disk by using sputter coating for 9.5 min, and the samples then
were annealed in the Chemical vapor deposition (CVD) at 750°C for 20 min to form Fe
NPs.
Fe-NPs were produced with a diameter of 200±100 nm and a density range
(distance between NPs) of 200±100 nm with a coating time of 9.5 min. SEM images of
Fe NPs decorated anode and control surfaces were shown in the Figure 4.1.
50
B
A
500 nm
500 nm
Figure 4.1, SEM images of Fe NPs decorated anodes. (A) Graphite disk control; (B) Fe
Nanoparticle decorated.
Multiple channel MECs construction and operation
A MECs with removable multiple anodes with each effective anode area 0.7 cm2 was
constructed and used to evaluate the effects of nanostructure on current density of
according to a previous report (Fan et al. 2011). The cathode was made of wet-proof
(30%) carbon cloth (type B, E-TEK Division, Inc., USA) coated with Pt/C (20%, E-TEK
Division, Inc., USA) following a previously reported procedure (Liu et al. 2005). The
final platinum loading was 0.5 mg/cm2 per projected cathode area. The size of cathode
(150 cm2) was 25 times larger than the total effective area of the 8 graphite disk anodes
(5.6 cm2) to prevent cathode limitations on the performance of the MECs system. All
MECs with Fe-NP decorated anodes and control anodes were tested for current
generation for 24 h with medium (without bacteria) before injecting the bacterial cells in
order to determine whether the Fe-NP decorated anodes demonstrate chemical current
generation compared to control anodes. All the testing anodes were set up in the MECs
51
at once so that the S. oneidensis MR-1 culture was applied to all of them simultaneously.
Short electrode spacing (1.7 cm) was used in the MECs design to reduce the internal
resistance. A voltage of 0.6 V was applied the MECs for current generation after the cells
of S. oneidensis MR-1 were inoculated into MECs at early stationary phase. A multimeter
with a data acquisition system (2700, Keithly, USA) was used to monitor the current
change by measuring the voltage (every 300 seconds) drop through a resistor at 300 ohm.
Fig 4. 2 Effects of Fe-NP decorated anodes on the current density in MECs. Control is
plain graphite disk anode; Fe-NP is the anode with Fe-NP decoration.
Microarray analysis
Biofilms for whole gene microarray analysis were aseptically removed from the plain
and Fe-NP decorated anodes of MECs after 110 hours further incubation at 30oC when
current density obviously started to decease. Total RNA was extracted using Trizol
(Ambion inc., Austin,TX) following the manufacturer’s instructions. Integrity of the
52
RNA samples was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technology
Inc., Palo Alto, CA). The same amount of RNA (200 ng) from each sample was used for
further analysis after amplification by using the MessageAmpTM II-Bacteria Prokaryotic
RNA Amplification kit (Ambion, Austin,TX) according to the manufacturer’s
instructions. All of the 4295 genes (resource from NCBI) represented on the S. oneidensis
MR-1 whole-gene microarrays were ordered from Roche NimbleGen Inc. (Madison, WI).
Biological triplicates of biofilms on the Fe-NP decorated anodes and control were
analyzed, respectively. cDNA synthesis, labeling and hybridization were carried out by
the CGRB Core Laboratories at Oregon State University. DNASTAR ArrayStar TM 3
software was used to identify genes that were up- or down-regulated more than 2-fold
when grown on the nanoparticle modified anodes using the unpaired two sample t-test
with a cutoff p-value of 0.05. The complete microarray data set generated in this study is
deposited
for
public
access
in
the
Gene
Expression
Omnibus
(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE31535.
Results
Enhancement of current generation by using NP decorated anodes in MECs
The current density generated by Fe-NP decorated anode MECs with S. onidensis MR-1
increased to approximately 43 µA/cm2 20 h following inoculation and then slowly
decreased.
Figure 4.2 shows that the maximum current density achieved by NP
decorated anodes was 8.3 times higher than that (5.1 µA/cm2) generated by the control
(plain graphite disk). The average current density improvement of 110 hours was 5.9
times of that generated by the control. The current densities generated in MECs in the
53
absence of bacteria were negligible (0.3 µA/cm2), which demonstrated that the current
enhancement observed with the Fe-NP decorated anodes was biologically-derived.
Global transcriptome analysis
Whole-genome DNA microarrays were used to attain a comprehensive, general overview
of the transcriptional response to Fe-NP decorated anodes by S. oneidensis MR-1 in MEC.
Of a total 4,295 genes assayed, 392 (188 induced and 204 repressed) exhibited significant
(P < 0.05) differential expression at a 2-fold (log (expression ratio 1) level in 3
replicates in response to Fe-NPs. These total gene numbers present 9% of the 4,295 open
reading frames (ORFs) presented on the array.
125
Up-Regulated
100
Number of Genes
75
50
25
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
-25
-50
-75
Down-Regulated
Fig 4.3 Differentially expressed genes grouped by functional classification according to
the TIGR S. oneidensis genome database (www.tigr.org). Columns: 1, amino acid
biosynthesis; 2, biosynthesis of cofactors, prosthetic groups, and carriers; 3, cell
envelope; 4, cellular processes; 5, central intermediary metabolism; 6, DNA metabolism;
7, energy metabolism; 8, fatty acid and phospholipid metabolism; 9, other categories; 10,
protein fate; 11, protein synthesis; 12, purines, pyrimidines, nucleosides, and nucleotides;
13, regulatory functions; 14, signal transduction; 15, transcription; 16, transport and
binding proteins; 17, unknown function; 18, hypothetical proteins.
54
Figure 4.3 summarizes the overall genomic response of S. oneidensis MR-1 to Fe-NP
decorated anodes by grouping the differentially expressed genes into their functional role
categories, based on The Institute for Genomic Research’s annotation (Version 8) of the
MR-1 genome sequence. The wide distribution of putative functional roles attributed to
the differentially expressed genes indicated the extent of the molecular response of
S.oniedenis MR-1 to the NP decorated anodes. Among genes with defined function, a
large number of up-regulated genes were associated with cellular processes (group 4),
energy metabolism (group 7), other categories (group 9) and hypothetical proteins (group
18). While those genes encoding proteins involved in amino acid biosynthesis (group 1),
cellular processes (group 4), substrate transport (group 16), and hypothetical proteins
(group 18) were among the most down-regulated genes. Two groups of genes that were
the most noticeable among the functional gene groups: genes related to energy
metabolism (group 7) and genes related to amino acid biosynthesis (group 1). The ratios
of up-regulated genes to down-regulated genes in these functional groupings were much
higher than other functional gene groupings: there were 21 up-regulated genes, and 7
down-regulated genes in energy metabolism gene group, and 2 up-regulated and 22 down
regulated genes in the amino acid biosynthesis group.
Genes related to biofilm formation
In order to determine whether there was a correlation between the observed biofilm
enhancements by NP decorated anodes and the genes related to biofilm formation, the
significant modified genes related to biofilm formation were examined and summarized
in the Table 4.1. These included SO_3223 and SO_3228 genes encoding the flagellum
proteins (Thormann et al. 2004), which has critical impacts on initial attachment to the
55
surface as swimming motility functions, were up-regulated over 1.0 and 0.9 fold,
respectively. The gene clusters SO_4178-SO_4180, which are thought to be essential for
formation of a three-dimensional biofilm, were up-regulated from 1.2 to 1.3 fold
(Thormann et al. 2004). In addition, the genes SO_4105 encoding IV pilus, which is
known to represent a critical factor for biofilm formation on a biotic surfaces (Thormann
et al. 2004), were also induced 0.8 fold (Table 4.1). The genes related to flagellum
motility (SO_4286, SO_1529, and SO_1530) were also induced slightly (Thormann et al.
2004).
Table 4.1
Locus Tag
Expression levels of genes related to biofilm formation
Gene product descriptions
Fold change
SO_3228
flagellar basal-body MS-ring and collar protein, FliF
1.0
SO_3223
flagellar hook-length control protein FliK
0.9
SO_4103
MshA minor pilin protein, MshD
0.5
SO_4105
MSHA major pilin protein MshA
0.8
SO_4178
expressed protein of unknown function, MxdC
1.2
SO_4179
inner membrane family 2 glycosyltransferase, MxdB
1.3
SO_4180
diguanylate cyclase-like protein, MxdA
1.0
a: fold changes were presented after log2
b: positive number represented up-regulated and negative represented down-regulated
Genes related to energy metabolism
A closer consideration of energy metabolism genes was undertaken as anaerobic
metabolism for electron generation and electron transport functions may play critical
roles in enhanced current density generation in MECs. Genes associated with energy
56
metabolism and with significantly modified gene expression levels are summarized in the
Table 4.2. Several of the up-regulated genes were related to formate dehydrogenase
proteins. Most notably, four genes related to formate dehydrogenase were up-regulated
more than 2-fold: SO_0102, SO_0103, SO_0104 and SO_0107. The other genes
expression related to formate dehydrogenase proteins SO_4513, SO_4514, SO_4515 also
significantly increased. Another interesting gene is that encoding the cytochrome c
oxidase protein, gene SO_4609, which was up-regulated 1.1 fold, while the gene
SO_0479 related periplasmic octaheme cytochrome c, MccA was down-regulated.
However, SO_1519, the gene encoding lactate dehydrogenase was down-regulated 1.2
fold, despite lactate being the primary carbon source in the media.
Table 4. 2 Genes related to anaerobic growth and electron transfer with significantly change expression level
Locus Tag
Gene product descriptions
Fold
change
SO_0101
nitrate-inducible formate dehydrogenase, molybdopterin-binding
1.8
subunit, FdnG
SO_0102
formate dehydrogenase, nitrate-inducible, iron-sulfur subunit
2.1
SO_0103
formate dehydrogenase, nitrate-inducible, cytochrome b556 subunit
2.4
SO_0104
formate dehydrogenase accessory protein, FdhE
2.2
SO_0107
formate dehydrogenase accessory protein fdhD
2.4
SO_0397
quinol:fumarate reductase, menaquinol-oxidizing subunit, FrdC_2
1.2
SO_0452
thioredoxin 2
1.5
SO_0903
Na-translocating NADH-quinone reductase subunit B, NqrB_1
1.2
SO_1012
NADH-ubiquinone oxidoreductase subunit K, NuoK
1.2
SO_1013
NADH-ubiquinone oxidoreductase subunit J, NuoJ
1.2
SO_1363
hydroxylamine reductase
1.3
57
SO_2417
ferredoxin, cofactor maintenance protein, YfaE
1.0
SO_3922
formate dehydrogenase, cytochrome b, Fdh
-1.2
SO_4513
formate dehydrogenase, molybdopterin-binding subunit, _2
1.5
SO_4514
formate dehydrogenase, FeS subunit, FdhB_2
1.2
SO_4515
formate dehydrogenase, cytochrome b subunit, FdhC_2
1.4
SO_4609
aa3 type cytochrome c oxidase, subunit III, CoxC
1.1
SO_0479
periplasmic octaheme cytochrome c, MccA
-1.2
SO_0847
periplasmic nitrate reductase, ferredoxin component, NapG
-1.5
SO_1519
L-lactate dehydrogenase, iron-sulfur cluster-binding protein, LldF
-1.0
SO_1251
ferredoxin, 4Fe-4S
-1.1
SO_3741.1
hypothetical inner membrane protein
-1.1
a: fold changes were presented after log2
b: positive number represented up-regulated and negative represented down-regulated
Flavin and cytochrome related genes
Flavins can be secreted by Shewanella species as electron shuttle to facilitate
extracellular electron transfer (Cansterin et al. 2008). C-type cytochromes play the
important roles on the process of extracellular electron transfer (Shi et al. 2007). The
genes related to flavins synthesis and the genes encoding the cytochromes electron
transport proteins were shown in the table 4.3, the genes SO_1414, and SO_3058 related
to flavins synthesis was up-regulated to 0.3 and 0.1 folds respectively. The genes related
to s C-type cytochromes SO_0135, SO_4105, SO_0169, SO_1778, are increased to 1.0,
0.8, 0.5, 0.4, 0.1 folds respectively. However, most of genes have shown no significant
changes (less one-fold change) as response to nano particle decorated anode in MECs.
58
Table 4.3 Expression level of flavin and cytochrome genes
Locus Tag
Gene product descriptions
Fold
change
SO_1414
flavocytochrome c flavin subunit, putative
0.3
SO_3468
riboflavin synthase subunit alpha
-0.2
SO_3058
flavocytochrome c, flavin subunit
0.1
SO_4105
MSHA pilin protein MshA
0.8
SO_0169
general secretion pathway protein, GspG
0.5
SO_1778
outer membrane decaheme cytochrome c lipoprotein, MtrC
0.4
SO_1779
outer membrane decaheme cytochrome c, OmcA
0.1
SO_0135
lipoprotein of unknown function DUF333
1.0
SO_0136
conserved hypothetical inner membrane protein
0.1
a: fold changes were presented after log2
b: positive number represented up-regulated and negative represented down-regulated
Other genes with significantly modified expression levels
These genes over 1.5 fold change and possibly related to current enhancements were
shown in Table 4.4. SO_3104 (expressed inner membrane protein) was over expressed
and the gene SO_2194, which encodes outer membrane protein (OmpA family protein)
was repressed as response to nanoparticle decorated anode, however it is unclear the
relationships of this modified membrane protein genes with the current enhancement.
Another interesting phenomenon was possible co-regulation of several gene clusters
indicating possible operon associations. This includes three sets of genes that were
induced more than 1.5 fold: SO_0108/SO_0109 and SO_1042/SO_1043/SO_1044. The
59
consistency of expression of these genes under the NP conditions provides basic evidence
to support operon structure. However, the correlation of these significant changed gene
with current density enhance are unclear, which showed there are unknown multiple and
complex responses of S. oneidensis MR-1 to nanoparticle anode of MECs.
Table 4.4 Other genes with significantly changed expression levels
Locus Tag
SO_0101
Gene product descriptions
nitrate-inducible formate dehydrogenase, molybdopterin-binding
Fold change
1.8
subunit, FdnG
SO_0108
integral membrane protein of unknown function DUF39, YedE
1.7
SO_0109
SirA family protein, YedF
1.7
SO_0275
N-acetyl-gamma-glutamyl-phosphate reductase
-3.1
SO_0277
ornithine carbamoyltransferase
-2.1
SO_0279
argininosuccinate lyase
-2.4
SO_0404
zinc dependent metalloprotease domain lipoprotein
2.1
SO_0956
alkyl hydroperoxide reductase, F subunit
-2.0
SO_1042
amino acid ABC transporter, ATP-binding protein
-1.8
SO_1043
amino acid ABC transporter, permease protein
-2.7
SO_1044
amino acid ABC transporter, periplasmic amino acid-binding protein
-2.7
SO_1072
chitin-binding protein, putative
2.1
SO_1405
transglutaminase family protein
2.0
SO_1822
TonB-dependent receptor, putative
-2.2
1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)
SO_2069
-1.9
methylideneamino] imidazole-4-carboxamide isomerase
SO_2070
amidotransferase HisH
-2.6
SO_2071
imidazole glycerol-phosphate dehydratase/histidinol phosphatase
-2.7
60
SO_2072
histidinol-phosphate aminotransferase
-2.8
SO_2073
histidinol dehydrogenase
-3.0
SO_2194
OmpA family protein
-3.6
SO_2195
inter-alpha-trypsin inhibitor domain protein
-2.7
SO_2767
asparagine synthetase B
-2.6
SO_2945
prophage LambdaSo tail fiber protein
2.0
SO_2963
prophage LambdaSo, major capsid protein, HK97 family
2.0
SO_2987
prophage LambdaSo hypothetical protein
1.9
SO_2988
prophage LambdaSo expressed protein of unknown function
1.8
SO_3104
expressed inner membrane protein
1.31
SO_3408
conserved hypothetical inner membrane protein
-1.8
SO_3585
azoreductase, putative
-1.8
SO_3586
glyoxalase family protein
-1.7
SO_3687
curli production assembly/transport component CsgE, putative
-1.8
SO_3819.1
hypothetical ammonia permease
-1.9
SO_4014
AcrB/AcrD/AcrF family protein
1.8
SO_4015.1
type I secretion system, membrane fusion protein, RND family
1.8
SO_4054
5,10-methylenetetrahydrofolate reductase
1.8
SO_4245
N-acetylglutamate synthase
-2.2
SO_4525.1
hypothetical transcriptional regulator, LysR family
-1.7
SO_4527
integral membrane domain protein
-1.9
SO_4705
transcriptional regulator, putative
-1.9
a: fold changes were presented after log2
b: positive number represented up-regulated and negative represented down-regulated
Discussion
61
The addition of NP to anodes significantly impacts current densities with the elemental
composition being a critical factor. The chemical composition of NPs significantly affects
the current enhancement in MECs (Fan et al. 2011), because the chemical composition of
NP properties is a critical factor in determining the conductivity, which significantly
affects the efficiency of electron transfer from bacteria to electrode. Fe is an ideal
candidate for NP formation for current enhancement in MESs because of high
conductivity and low price compared to gold and other conductive materials. Gold NPs
has also been studies for enhancement of electricity conductivity (Bao et al. 2008).
However, the disadvantages of the high price of gold limit their practical application in
MECs (Fan et al. 2011). The different size and density of the same composition of NP
also has essential effects on the current density enhancement (Fan et al. 2011). In this
study, the results showed the current density enhancement with Fe-NP size range of
200±100 nm and density range of 200±100 nm is considerable enhancement consequence.
There are a number of reports of studies of current density enhancement in MFCs
by nanostructured anodes (Fan et al. 2011; Qiao et al. 2008; Sharma et al. 2008; Tsai et al.
2009). Based on bacterial behavior on nanostructured anode surface, Qiao et al. (2008)
believes that Escherichia coli cells on the nanostructured electrode surface produce hairlike structures, similar to pili that could facilitate the electron transfer between the cells
and electrode. The production of hair-like structures is believed to be stimulated by
nanostructures and could play the key role on current density enhancement in piliproducing bacteria in MFCs (Qiao et al. 2008). S. oneidensis can produce electrically
conductive bacterial nanowires, which have similar structure to the hair-like structures in
E.coli (Gorby et al. 2006; Reguera et al. 2005). A number of possible genes related
62
nanowires of S. oneidensis have been identified, including mshA (SO_4105), and gspG
(SO_0169) (Konstantinidis et al. 2009), They are up-regulated 0.8 and 0.5 fold in the
current study respectively, even though they are not significant induced. This result
supported the increased nanowire genes had contributions to enhance current density in
MFCs by NPs decorated anodes.
Based on the previous research on electron transfer mechanisms for S. oneidensis, it is
known that self-produced mediators (flavins) and multihaem c-type cytochromes (c-Cyts)
may play critical roles in the electron transfer of S. oneidensis to extracellular insoluble
electrode (Canstein et al. 2008; Myers and Myers 1992; Scott and Nealson 1994; Shi et al.
2007). The flavins secreted by S. oneidensis can facilitate to transfer electrons from
bacteria to solid anode electrodes by acting as extracellular electron shuttles (Canstein et
al. 2008). In this study, the genes encoding flavins (SO_1414, SO_3468, and SO_3058)
had no significant increase (less than 1 fold change) as a response to nanoparticle
decorated anode in MECs, which suggests that increased flavin gene expression does not
significantly contribute to the current density enhancements in nano-particle decorated
anode. The genes encoding cytochromes electron transport proteins (SO_4104, SO_0417,
SO_1778/79, SO_0135/36) had no significant increase (less than 1-fold change).
However, those genes were up-regulated slightly. These results suggest the increased
expression flavin and c type cytochromes genes had partial contributions even not
significantly to enhance current density in MFCs by NPs decorated anodes.
The thickness of bacterial biofilm on the anode can affect the power generation in
MFCs (Biffinger et al. 2007; Heydorn et al. 2000), since increased biofilm thickness on
the anode represents more active bacteria involving in the process of electron generation
63
and transfer in MFCs. Biofilm formation related genes included flagellar genes IV, pilus
genes and other genes. The flagellar related genes SO_3228, SO_3229 (fliB and fliK) and
IV pilus genes SO_4105 (mshA) were induced from 1.9 to 0.9 fold; the genes (SO_4178
-4180) related to formation of a three-dimensional biofilm were up-regulated from 1.0 to
1.3 folds, even though the genes related bacterial motility protein (SO_ 4286, SO_4287,
SO_1529 and SO_1530) are induced slightly (less than 0. 5 fold). However, the motility
genes most probably play a role for initial biofilm attachment on the anode surfaces
(Thormann et al. 2004; Thormann et al. 2006), with the rest of biofilm genes being more
essential for biofilm maintenance in mature cultures as were presented at harvest in this
study. This enhancement of biofilm density under conditions of increase current density
has been observed for gold NP decorated anodes using confocal light microscopy (data
not shown) but has not yet been confirmed for Fe NP decorated anodes. In any event, an
increase in the expression level of genes related biofilm formation therefore also
supported the possibility of enhanced biofilm formation on NPs decorated anodes, thus
facilitating electron transfer from bacteria to anodes.
Beliaev et al. described a model of the Fe reduction pathway in S oneidensis MR-1 using
solid substrates as the electron acceptor (Beliaev et al. 2001). This model proposes that
electrons are generated and released in cytoplasm then transferred to the quinone pool,
e.g., menaquinone in the cytoplasmic membrane. The reduced menaquinone in turn
reduces CymA, and from there electrons are then transferred to a periplasmic shuttle (e.g.
CctA) and then to MtrA, a periplasmic soluble decaheme cytochrome c located in
associated with the outer membrane via interaction with MtrB. Finally, surface displayed
outer membrane proteins decaheme cytochromes (OmcA, MtrC, or MtrF) can transfer
64
electron to Fe or other solid substrates (Beliaev et al. 2001)). Bencheikh-Latmani et al.,
demonstrated that the same electron transport pathway may be used for more than one
electron acceptor through studies of the response of S. oneidensis MR-1 to U(VI) and
Cr(VI) under anaerobic conditions, particularly focused on the critical genes
(SO_1777:mtrA, SO_1776:mtrB, SO_1778:mtrC) (Bencheikh-Latmani et al. 2005).
However, our studies showed that these genes had no significant expression changes in
response to nanostructured anodes. Contrastingly, one of expressed inner membrane
protein genes SO_3104 and one of cytochromes genes SO_4609 encoding cytochrome c
oxidase subunit III demonstrated a 1.3-fold and 1.1-fold up-regulation respectively in this
study, while the gene SO_2194 which encodes outer membrane protein (OmpA family
protein) was down regulated 3.6 fold. Our results therefore suggest that S. oneidensis
MR-1 does not utilize the same set of core critical genes (SO_1777: mtrA, SO_1776:
mtrB, SO_1778: mtrC) products to transfer electrons in all instances. This is the similar
result with the study of Bretschger et al. (2007), which indicated that the electron
transport system in S. oneidensis MR-1 is complex, with several different proteins able to
participate in electron transfer to the anode of MECs.
It should also be mentioned that the gene (SO_1519) encoding lactate
dehydrogenase repressed (-1.2 fold) here suggesting that lactate was depleted at the time
of cell harvest from the MECs and that other pathways of anaerobiosis had been activated
including those associated with amino acid uptake and consumption; the growth media
was a complex mixture including yeast extract. In particular, the catabolic pathway for
histidine of S. oneidensis MR-1, a pathway that may formate as an intermediate, is upregulated, with the genes for histidine ammonia lyase, SO_0098, SO_3057 and SO_4374
65
up-regulated 1.3, 1.1, and 1.1-fold, respectively. The possible production of formate
during histidine degradation may in turn explain the high levels of formate
dehydrogenates as evidenced by upregulation of SO_0102, SO_0103, SO_0104 and
SO_0107, annotated as formate dehydrogenase proteins. These genes had the highest
levels of up-regulated expression as response to Fe NP decorated anodes (greater than 2fold).
Acknowledgements
This research was partially supported by the U.S. National Science Foundation CBET
0828544 and the funds from ONAMI/DOD (ARL-DOD Cooperative Agreement#
W911NF-07-2-0083). We thank Barbara Gvakharia and Caprice Rosato for helpful
suggestions. We also thank Margaret Romine of Pacific Northwest Laboratories for
valuable comments on manuscripts. We also thank anonymous reviewers for significant
suggested improvements imparted as part of prior review of this manuscript.
66
Chapter 5
Global Transcriptome Analysis of the Response of Shewanella oneidensis MR-1 to
Carbon Nanotube Decorated Anodes in Microbial Electrochemical Systems
Shoutao Xu, Yanzhen Fan, Rebecca Schaller, Frank Chaplen, Jun Jiao, Hong Liu*
67
Abstract:
Shewanella oneidensis MR-1 is an important model microorganism for metabolic studies
on the effects of different environmental factors because of its diverse respiratory
capabilities. Carbon nanotube (CNT) modified graphite disks were used as anodes to
investigate the effects of nanomatrials on the performance of S. oneidensis MR-1 in
microbial electrolysis cells (MECs). The current densities produced with CNT decorated
anodes were on average 5.6-fold higher than plain graphite anodes. Whole genome
microarray analysis of gene expression showed that up-regulation of cytochromes c genes
associated with extracellular electron transfer are strongly correlated to current increases
in CNT decorated anodes. Genes involved in flavin biosynthesis also contribute to
current increase in CNT decorated anode MECs.
Keywords: Microbial electrochemical system; microbial fuel cell; microbial
electrolysis cell; carbon nanotube, gene expression; DNA microarray.
68
Introduction:
Shewanella oneidensis MR-1 is an important model microorganism for metabolic
studies of the effects of different environmental factors because of its diverse respiratory
capabilities. It has been used for transcriptome analysis to investigate the responses to
different growth conditions and environmental stresses (Murray et al., 2001). Gene
expression patterns under different conditions such as heat shock, cold shock, Cr (VI), U
(VI) reduction, chromate stress, iron and acid tolerance have been studied previously
(Bencheikh-Latmani et al. 2005; Brown et al. 2006; Gao et al.2006; Gao et al. 2004;
Yang et al. 2008). More recently, it has been exploited as a model species for power
generation in microbial electrochemical systems (MESs),which have potential
applications in renewable energy generation, bioremediation, and wastewater treatment
(Logan et al. 2007; Logan 2009; Park and Zeikus 2002; Fan et al 2011; Xu et al 2012).
In a MES, electrochemically active microorganisms oxidize organic matter in the an
ode chamber to release electrons. Electrons are then transferred to the anode electrode thr
ough various mechanisms (Logan et al., 2006; Lovley 2006; Rabaey et al., 2003) and fina
lly travel to the cathode electrode and combine with the terminal electron acceptor. The
key feature of MESs is the microbe-catalyzed electron transfer from the organic matter to
the anode (Rabaey et al 2004). Enhancing the current output, which highly depends on
the performance of the anode electrode, is critical for the successful application of MESs
(Logan et al. 2007; Park and Zeilus 2002; 2003). Several nanomaterials including TiO2,
gold (Au) and palladium (Pd) nanoparticles have been utilized to decorate electrodes to
enhance power density in MESs (Qiao et al. 2008; Quan et al. 2005; Fan et al. 2011). The
possible mechanisms for increased current densities have been studied by using Fe
69
nanoparticle-decorated anodes (Xu et al 2012).
Carbon nanotubes (CNT) are cylindrically shaped nanomaterials with extremely
high surface area, excellent electrical conductivity and chemical inertness (He et al.
2005a; Serp et al. 2003). These unique properties make CNT a promising electrode
material (Liang et al. 2008). The biocompatibility of microorganisms and carbon
nanostructures has been evaluated (Morozan et al. 2007). The effects of CNT on anodic
biofilms have been tested in MFCs (Liang et al. 2011). However, no studies have been re
ported on global transcriptome analysis for the response of S. oneidensis MR-1 to CNT
decorated anode in MECs.
In the present study, the graphite disks decorated with CNT were used as anodes to
investigate the effects of nanomaterials on current generation in multi-anode MECs.
DNA microarrays were used to analyze differences in the global gene expression profile
of S. oneidensis MR-1 grown on plain versus CNT decorated anodes.
Materials and methods
Bacterial cultures: S. oneidensis MR-1 was purchased from American Type Culture
collection (ATCC 700550) and stored as freezer stocks at -80°C. Stock S. oneidensis
MR-1 cells were grown aerobically using M1 medium at 30°C. Cells in early stationary
growth phase (optical density of 1.5-1.6 at 600nm; (Spectrophotometer UV-1700,
Shimadzu, Columbia, MD) were injected into the chamber of the MECs for current
production. Sodium lactate (final concentration 30mM) was added as the additional
electron donor.
Fabrication and characterization of nanostructured anodes: Superfine isomolded
graphite disks (1.6 cm x 0.5 cm, GraphiteStore.com, Inc.) were polished with ultrafine
70
sand paper (2000 grit, 3M Company) as the base for the CNT decorated and control
anodes. Aligned multi-walled carbon nanotube samples were synthesized via plasma
enhanced chemical vapor deposition (PECVD). Samples, superfine isomolded graphite
pillars with a 1.6 cm diameter and 0.5 cm thickness, were sputtered with a thin film of Ni
in a CRC 300 magnetron sputter coater for 1 to 2 min. They were then placed in the
PECVD reactor where the Ni was annealed into small catalyst islands, C2H2 was bled
into the chamber for growth, and an NH3 plasma was used to vertically align the growth
of the multi-walled carbon nanotubes (MWCNTs). The temperature and time were varied
between 725°C and 775oC and 15 to 60 min to control the length of the tubes and density
of samples. Samples were then analyzed in a FEI Sirion field emission scanning electron
microscope (FESEM). SEM images of CNT decorated anode and control surfaces were
shown in the Figure 5.1.
A
B
Figure 5.1, SEM images of CNT decorated anodes. (A) Graphite disk control; (B) CNT
decorated anode.
Multiple channel MECs construction and operation: A MECs with multiple
removable anodes each with an effective anode area of 0.7 cm2 was constructed and used
to evaluate the effects of nanostructures on the current density of according to a previous
71
study (Fan et al, 2011). The cathode was made of wet-proof (30%) carbon cloth (type B,
E-TEK Division, Inc., USA) coated with Pt/C (20%, E-TEK Division, Inc., USA)
following a previously reported procedure (Liu et al. 2005). The final platinum loading
was 0.5 mg/cm2 per projected cathode area. The size of the cathode (150 cm2) was 25
times larger than the total effective area of the 8 graphite disk anodes (5.6 cm2) to prevent
cathode limitations on the performance of the MECs system. All MECs with CNT
decorated anodes and control anodes were tested for current generation for 24 h with
sterile a medium (without bacteria) before injecting the bacterial cells in order to
determine whether the CNT decorated anodes demonstrate chemical current generation
compared to control anodes.
All the testing anodes were set up in the MECs
simultaneously so that the S. oneidensis MR-1 culture was applied to all of them
simultaneously. Short electrode spacing (1.7 cm) was used in the MECs design to reduce
the internal resistance. A voltage of 0.6 V was applied the MECs for current generation
after the cells of S. oneidensis MR-1 were inoculated into the MECs in the early
stationary phase. A multimeter with a data acquisition system (2700, Keithly, USA) was
used to monitor the current change by measuring the voltage drop through a resistor.
Microarray analysis: Biofilms for whole gene microarray analysis were aseptically
removed from the plain and CNT decorated anodes of MECs after 80 hours further
incubation at 30oC when current density obviously reached to a stable phase. Total RNA
was extracted using Trizol (Ambion inc., Austin,TX) following the manufacturer’s
instructions. Integrity of the RNA samples was confirmed using the Agilent 2100
Bioanalyzer (Agilent Technology Inc., Palo Alto, CA). Total RNA (200 ng) from each
sample was amplified using the MessageAmpTM II-Bacteria Prokaryotic RNA
A
72
Amplification kit (Ambion, Austin,TX) according to the manufacturer’s instructions. The
S. oneidensis MR-1 whole-gene microarrays representing all of the 4295 genes (resource
from NCBI) were ordered from Roche NimbleGen Inc. (Madison, WI).
Biological
triplicates of biofilms on the CNT decorated anodes and control were analyzed. cDNA
synthesis, labeling and hybridization were carried out by the CGRB Core Laboratories at
Oregon State University. DNASTAR ArrayStarTM 3 software was used to identify genes
that were up- or down-regulated more than 2-fold when grown on the nanoparticle
modified anodes using the unpaired two sample t-test with a cutoff p-value of 0.05 with
bonferroni correction .
Results and discussion:
Enhancement of current generation using CNT decorated anodes in MECs: The
current density generated by CNT decorated anode MECs with S. onidensis MR-1
increased to 36 µA/cm2 10 hours post-inoculation and reached a maximum of 40 µA/cm2
at 29 hours inoculation, then gradually decreased. The current density stabilized at
30µA/cm2 after 78 h operation. Figure 5.2 shows that the current density curve generated
by CNT decorated anode in MECs. The average current density generated by CNT
decorated anodes was 5.6 times that of the control. The current density generated in the
MECs without bacteria was negligible (data not shown). This result demonstrates that the
current enhancement observed with the CNT decorated anodes was biologically-derived.
73
48
Control
Current density (µA/cm2)
41
CNT
34
27
20
13
6
-1
0
10
20
30
40
50
Time (Hours)
60
70
80
Figure 5.2 Effects of CNT decorated anodes on the current density in MECs (3 replicates)
. Control is plain graphite disk anode; CNT is the anode with CNT decoration. (Error bars
represent standard deviation)
It has been reported that nanostructured decorated anodes have significant impacts on
current densities (Fan et al. 2011; Qiao et al. 2006; Sharma 2008; Tsai et al. 2009). The
CNT decorated anodes had significantly enhanced current densities in MECs in this
study. This result is consistent with previous studies. Interestingly, the current density
curve generated by CNT decorated anode MECs is quite different from the current
density curve generated by Au NP and Pd NP decorated anodes using same bacterial
species (Fan et al., 2011). In the Au and Pd NP decorated anode MECs, the current
density curve was not significantly increased in the beginning compared with the control
but gradually increased after the inoculation; the current density reached the maximum
current density more than 50 hours after the inoculation. However, the current density
generated by CNT decorated anode MECs started to increase significantly immediately
after the inoculation. It reached a maximum current density 29 hours post-inoculation.
74
These results indicate that there might be different current density enhancement
mechanisms between CNT with other metal NP decorated anodes in MECs.
Global transcriptome analysis: Whole-genome DNA microarrays were used to attain a
comprehensive, general overview of the transcriptional response of S. oneidensis MR-1 of
S. oneidensis MR-1 to CNT decorated anodes in MECs. In a total of 4,295 genes analyzed,
457 genes (250 genes up-regulated and 207 genes down-regulated) exhibited significantly
(P < 0.05) changed expression at a 2-fold level in at least 3 replicates in the response to
CNT. The total number of the regulated genes present 11% of the 4,295 open reading
frames (ORFs) presented on the microarray. The total number of genes at significant
expression levels in response to CNT decorated anodes is close to that to Fe NP decorated
anodes in MECs (392, 9%) ( Xu et al. 2012).
100
80
Up-regulated
Number of Genes
60
40
20
0
-20
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
-40
-60
Down-regulated
-80
Fig 5.3 Differentially expressed genes grouped by functional classification according to
the TIGR S. oneidensis genome database (www.tigr.org). Columns: 1, amino acid
biosynthesis; 2, biosynthesis of cofactors, prosthetic groups, and carriers; 3, cell
envelope; 4, cellular processes; 5, central intermediary metabolism; 6, DNA metabolism;
7, energy metabolism; 8, fatty acid and phospholipid metabolism; 9, other categories; 10,
protein fate; 11, protein synthesis; 12, purines, pyrimidines, nucleosides, and nucleotides;
75
13, regulatory functions; 14, signal transduction; 15, transcription; 16, transport and
binding proteins; 17, unknown function; 18, hypothetical proteins.
The overall transcript genomic response of S. oneidensis MR-1 to CNT decorated
anodes was summarized in Figure 5.3 by grouping the differentially expressed genes into
their functional role categories, based on The Institute for Genomic Research’s annotation
(Version 8) of the MR-1 genome sequence. The wide distribution of putative functional
roles attributed to the differentially expressed genes indicated the extent of the molecular
response of S.oniedenis MR-1 to the CNT decorated anodes. A large number of upregulated genes were presented in genes associated with biosynthesis of cofactors,
prosthetic groups, and carriers (group 2), cell envelope (group 3), protein fate (group 10),
protein synthesis (group 11), and purines, pyrimidines, nucleosides, and nucleotides
(group 12) in this study. The number of up-regulated genes was two times more than that
of down-regulated genes in each functional group. Contrastingly, in the genomic response
S. oneidensis MR-1 to Fe NP decorated anodes (chapter 4), most of up-regulated genes
were associated with cellular processes (group 4), energy metabolism (group 7), other
categories (group 9) and hypothetical proteins (group 18). The down-regulated genes
presented in the amino acid biosynthesis (group 1), cellular processes (group 4), substrate
transport (group 16), and hypothetical proteins (group 18). While those genes encoding
proteins involved in amino acid biosynthesis (group 1), central intermediary metabolism
( group 5), DNA metabolism( group 6), fatty acid and phospholipid metabolism (group
8), and other categories (group 9) were among the most down-regulated genes in this
study. However, the two groups of genes that were the most noticeable between the
genomic response S. oneidensis MR-1 to CNT and Fe NP decorated anodes, which they
are the genes related to energy metabolism (group 7), and hypothetical proteins (group
76
18). These results indicated there are different and common genomic responses to S.
oneidensis MR-1 on CNT and Fe NP decorated anodes in MECs.
Electron transfer related genes: Based on previous electron transfer mechanism
studies on Shewanella, a serial of group proteins collectively described as the Mtr
pathway play the role to transfer electron rom the inner bacterial body to the outer
membrane (Beliaev et al. 2001, Shi et al. 2007, Brutinel and Gralnick, 2012). In the Mtr
pathway, firstly electrons generated by bacteria received by CymA, a tetraheme c-type
cytochrome anchored in the inner membrane, then electrons were transferred to MtrA, a
periplasmic decaheme c-type cytochrome. After that, electrons were transferred from
MtrA to MtrC, an outer-membrane decaheme c-type cytochrome is facilitated by MtrB, a
non-heme containing integral outer-membrane protein.Outer-membrane decaheme c-type
cytochrome MtrC and OmcA are in change of electron transfer to exreacellular electron
acceptor (Shi et al. 2007, Brutinel and Gralnick, 2012 ). These genes encoding electron
transport proteins are shown in the table 5.1. The critical genes SO_1777: mtrA, SO_1776:
mtrB, SO_1778: mtrC are unregulated 4.1 to 8.0 fold in response to CNT decorated
anodes, other cytochrome c genes related to electron transfer in MtrA pathway also are
significantly increased in response to CNT decorated anodes. These genes encoding outer
membrane proteins have different level increases, supporting that the increase of
cytochrome c gene expression contributes to the current enhancements of MECs as
response to CNT decorated anodes.
77
Table 5.1 Expression level of cytochrome c as response to CNT decorated anodes
Locus Tag
Gene product descriptions
Fold change CNT
SO_0165
general secretion pathway protein, GspC
3.1
SO_0167
general secretion pathway protein, GspE
4.5
SO_1776
outer membrane protein precursor MtrB
4.1
SO_1777
outer membrane decaheme cytochrome c
8.0
lipoprotein, MtrA
SO_1778
outer membrane decaheme cytochrome c
4.3
lipoprotein, MtrC
SO_1779
outer membrane decaheme cytochrome c,
6.3
OmcA
SO_0135
lipoprotein of unknown function DUF333
2.2
SO_0136
conserved hypothetical inner membrane protein
2.4
Flavin related genes: It is known that self-produced mediators play critical roles in the
electron transfer of S. oneidensis to extracellular insoluble electrode (Canstein von et al. 2
008; Myers et al. 1992; Scott et al. 1994; Shi et al. 2007). It has been proved that the
flavins secreted by S. oneidensis can facilitate electron transfer from bacteria to solid
anode electrodes by acting as extracellular electron shuttles (Cansterin von et al. 2008). T
wo critical genes (SO_1414, and SO_3468) related to flavin synthesis were shown increa
sed significantly (more than 2 folds) increase in this study (Table 5.2). This result support
s the hypothesis that flavin production increases as the response to CNT decorated anodes
in MECs, have significant contribution to the current density enhancements.
78
Table 5.2 Expression levels of genes related to flavin synthesis as response to CNT
decorated anode
Locus Tag
Gene product descriptions
Fold change CNT
SO_1414
flavocytochrome c flavin subunit, putative
2.2
SO_3468
riboflavin synthase subunit alpha
2.0
The amount of bacterial biofilm biomass on the anode can affect the power
generation in MFCs (Biffinger et al. 2007; Heydorn et al. 2000). Biofilm formation
related genes include flagellar gene IV, pilus genes and other genes; the flagellar related
genes (fliB and fliK) and IV pilus genes (mshA) were induced only from 1.05 to 1.34
fold as response of S. oneidensis MR-1 to CNT decorated anodes in this study. No
biofilm-associated genes presented significant changes in expression levels in this study,
indicating there is no direct connections between biofilm enhancements to CNT
decorated anodes in this study. This result is consistent with the Liang et al (2011)
conclusion that using CNT enhanced the electrochemical activity of anodic biofilms but
did not result in a significant increase of biomass in the anodic biofilms.
Our microarray results showed two significantly up-regulated gene groups: the
genes encoding proteins localized on the outer membrane and the genes involved in
flavin biosynthesis contributed to current density enhancement by CNT decorated anodes.
Among 457 significantly changed genes to CNT in this study, there are also a relatively
large number of genes encoding proteins with unknown functions, which are either up- or
down-regulated in the response to CNT decorated anodes, which indicated more
79
complicated responses of S. oneidensis MR-1 to CNT decorated anodes in MECs and
further study is needed .
80
Chapter 6
Summary
Microbial electrochemical systems (MESs) with a mixed culture initially inoculated
from Corvallis wastewater treatment plant have been studied for more than 6 years for
varying purposes, including power generation, hydrogen production, heavy metal
removal and wastewater treatment. Experiment results have shown that the mixed culture
is quite stable with excellent performance in MESs. Our community analysis using
denaturing gradient gel electrophoresis (DGGE) and 16S rDNA clone library construction
suggests that the mixed culture is composed predominantly of Geobacter sp (66%),
Arcobacter sp (12%) and Citrobacter sp (11%). These results not only improved our
understanding of the mixed culture community, but also guided our studies on the
cultivation and isolation of the different bacterial species.
Exoelectrogenic Citrobacter sp SX-1 was first isolated and characterized from a
MFC operated with the mixed culture. Citrobacter sp SX-1 has been demonstrated to
produce electricity from wide range of different substrates, including citrate, acetate,
glucose, sucrose, glycerol, and lactosein MFCs. Cyclic voltammetry analysis indicated
that membrane associated proteins may play an important role in facilitating the electrons
transferring from bacteria to electrode. The strain SX-1 increased the known diversity of
power generating exoelectrogens and provided a unique bacterial species for study in
renewable energy generation and waste treatment
81
To enhance the electron transfer from bacteria to anode, nano decorated anodes
including Fe NP and CNT were developed and characterized and evaluated in MECs
using Shewanella oneidensis MR-1 as a model species. Both nanostructures have
significantly increased current density compared with the control. Whole genome
microarray analysis elucidated the possible mechanisms of power enhancement in
response to these nano-decorated anodes.
These results benefit to understanding of physiology and ecology of mixed
cultures in MFCs and improve the efficiency of current generation in MESs, which will
facilitate the viability of niche applications for MESs in near future.
82
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APPENDICES
List of published papers during PhD study
1.
Shoutao Xu, Hong Liu, Yanzhen Fan, Rebecca Schaller, Jun Jiao and Frank
Chaplen. (2012) Enhanced performance and mechanism study
of microbial electrolysis cells using Fe nanoparticle-decorated anodes. Applied
Microbiology and Biotechnology, 93(2):871-880.
2.
Shoutao Xu, Hong Liu. (2011) New exoelectrogen Citrobacter sp. SX-1 isolated
from a microbial fuel cell. Journal of Applied Microbiology,111(5):1108-1115.
3.
Yanzhen Fan, Shoutao Xu, Rebecca Schaller, Jun Jiao, Frank Chaplen, Hong Liu
(2011) Nanoparticle decorated anodes for enhanced current generation in microbial
electrochemical cells. Biosensors and Bioelectronics, 26(5): 1908-1912.
4.
Tunc Catal, Shoutao Xu, Kaichang Li, Hakan Bermek, Hong Liu.
(2008) Electricity generation from polyalcohols in single-chamber microbial fuel
cells. Biosensors and Bioelectronics, 24(4):849-854.
5.
Chontisa Sukkasema, Shoutao Xu, Sunhwa Park, Piyarat Boonsawang, Hong Liu
(2008). Effect of nitrate on the performance of single chamber air cathode
microbial fuel cells. Water research, 42:4743-4750.
6.
Rebecca Schaller, Yanzhen. Fan, Shoutao Xu, Alan Fern, Frank. Chaplen, Hong
Liu, and Jun Jiao. (2009) Vertically Aligned Multi-walled Carbon Nanotube
Decorated Anodes for Microbial Fuel Cells. Proceedings of Materials Research
Society 2009, 1170: R05-13.