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Enhancing Factors of Electricity Generation in a Microbial Fuel Cell Using

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Article  in  Journal of Microbiology and Biotechnology · October 2012

DOI: 10.4014/jmb.1204.04010 · Source: PubMed

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J. Microbiol. Biotechnol. (2012), 22(10), 1395–1400
http://dx.doi.org/10.4014/jmb.1204.04010
First published online July 12, 2012
pISSN 1017-7825 eISSN 1738-8872
Enhancing Factors of Electricity Generation in a Microbial Fuel Cell Using
Geobacter sulfurreducens
Kim, Mi-Sun1,2*, Jaehwan Cha1, and Dong-Hoon Kim1
1
Clean Fuel Research Center, Korea Institute of Energy Research, Daejeon 305-343, Korea
2
Division of Renewable Energy Engineering, University of Science and Technology, Daejeon 305-350, Korea
Received: April 5, 2012 / Revised: May 29, 2012 / Accepted: June 1, 2012
In this study, we investigated various cultural and
operational factors to enhance electricity generation in a
microbial fuel cell (MFC) using Geobacter sulfurreducens.
The pure culture of G. sulfurreducens was cultivated using
various substrates including acetate, malate, succinate,
and butyrate, with fumarate as an electron acceptor. Cell
growth was observed only in acetate-fed medium, when
the cell concentrations increased 4-fold for 3 days. A high
acetate concentration suppressed electricity generation. As
the acetate concentration was increased from 5 to 20 mM,
the power density dropped from 16 to 13 mW/m2, whereas
the coulombic efficiency (CE) declined by about half.
The immobilization of G. sulfurreducens on the anode
considerably reduced the enrichment period from 15 to
7 days. Using argon gas to create an anaerobic condition
in the anode chamber led to increased pH, and electricity
generation subsequently dropped. When the plain carbon
paper cathode was replaced by Pt-coated carbon paper
(0.5 mg Pt/cm2), the CE increased greatly from 39% to
83%.
Keywords: Microbial fuel cell, Geobacter sulfurreducens,
electricity generation, coulombic efficiency, substrate specificity,
immobilization
In a natural environment, dissimilatory metal-reducing
bacteria use ferric oxide as a terminal electron acceptor
which cannot diffuse across the cell membrane and into the
cell. It is known that they transfer electrons to iron oxide
outside the cell via direct contact by outer membrane
cytochromes or use of excreted mediators [19, 21]. On the
same principle, they transfer electrons from the intracellular
cell to the extracellular anode instead of iron oxide as their
electron acceptor in microbial fuel cells (MFCs). These
electrons flow through a circuit to the cathode, where they
*Corresponding author
Phone: +82 42 860 3554; Fax: +82 42 860 3739;
E-mail: bmmskim@kier.re.kr
combine with protons and oxygen. Consequently, the
extracellular electron transfer of microorganisms makes
MFCs produce electricity from organic matter. This is the
reason why we call metal-reducing bacteria in MFCs
electrochemically active bacteria or exoelectrogens [8, 16].
In the beginning of MFC studies, exogenous electron
mediators were considered essential for efficient electricity
production [2]. These electron-shuttling mediators accept
electrons within a cell or on the cell surface and release
them at the anode. Unfortunately, the toxicity and instability
of synthetic mediators limit their applications in MFCs.
However, Kim et al. [13] first demonstrated electricity
production by a bacterium in the absence of an exogenous
mediator. The mediatorless MFC is very attractive because
it is operationally stable and yields high columbic efficiency.
Metal-reducing bacteria of Shewanella, Geobacter, and
Rhodoferax species have been reported to directly transfer
electrons to the electrode in mediatorless MFCs [5, 7, 15, 18].
Among many organisms discovered in MFCs, Geobacter
species are well-known representatives of exoelectrogens
that can generate electricity from organic matter [9, 14].
The complete genome of G. sulfurreducens was recently
sequenced to provide the physiological properties of
Geobacter species [17]. Bond and Lovley [5] demonstrated
that G. sulfurreducens can completely oxidize acetate by
using an electrode as the sole electron acceptor. They
achieved the high electron recovery of 95% with power
density of 49 mW/m2 using a two-chamber MFC. This result
suggests that G. sulfurrenducens can directly attach to
electrodes and transfer electrons via c-type cytochromes at
the outer membrane. Furthermore, a conductive pili termed
a nanowire was discovered in G. sulfurreducens [22]. It
provides a new insight into extracellular electron transfer
via microbial nanowires.
Since there is no need to add an artificial mediator,
mediatorless MFCs are advantageous in various applications
such as biosensors, wastewater treatment, remote power
sources, and bioremediation [10]. To further develop MFC
technologies, it is necessary to better understand the
 ENHANCING FACTORS OF ELECTRICITY GENERATION IN MFC 1396

physiological characteristics of microbial communities in

MFCs. Although microbial diversity and its electron
transfer mechanisms in MFCs have been broadly studied,
there is still a lack of information about the optimal growth
conditions of pure exoelectrogen cultures. In addition, the
mixed cultures are generally complex and make it difficult
to elucidate the response of an MFC. This study, therefore,
focused on the physiological characteristics of the
representative exoelectrogen G. sulfurreducens under
various growth and operational conditions. The object of
this study was to investigate the effect of substrate
specificity, substrate concentration, cell immobilization,
anodic pH, and electrode modification with Pt catalyst on
the electrochemical activity of G. sulfurreducens.
Fig. 1. Photograph of a two-chambered MFC.
MATERIALS AND METHODS each; working volume: 20 ml each) served as anode and cathode
chambers and were separated by a proton exchange membrane
Microorganisms and Culture Conditions
(12 cm2, Nafion 117; DuPont Co., USA) held in place by rubber
G. sulfurreducens (ATCC 51573) was obtained from the American
gaskets (Fig. 1). The chambers were fitted with ports for fixing the
Type Culture Collection (ATCC). The growth medium contained the
electrodes, adding solution components, passing gases, and sampling.
following (per liter of deionized water): 0.1 g KCl, 1.5 g NH4Cl,
Prior to use, all components of the MFCs were pretreated to
0.6 g NaH2PO4, 2.5 g NaHCO3, 10 ml of Wolfe’s mineral solution
prevent possible metal and organic contamination. The acrylic plates
[12], and 10 ml of Wolfe’s vitamin solution [12]. The sterilized
were immersed in 0.4% (w/v) NaClO and washed carefully in Milli-
medium was controlled to adjust the final pH to 6.8 for the optimal
Q water. The rubber gaskets and Teflon tubes were sterilized using
condition for microbial growth [14]. Batch cultivation was performed
70% ethanol. The Nafion membrane was pretreated, rendered in the
using inoculation of 10% in a 150 ml serum bottle, into which
H+ form, by soaking it in 0.5 M H2SO4 for 30 min, after which it
20 mM acetate and 50 mM fumarate were added as an electron
was stored in Milli-Q water. The electrodes were soaked in 1 N HCl
donor and acceptor, respectively. After inoculation, the bottle was
for 30 min and in 1 N NaOH for 30 min and stored in Milli-Q water
flushed with a N2-CO2 gas (80:20) to remove oxygen, and it was
before use.
kept at 30oC. The color of the culture medium changed from yellow
Each chamber contained a plain carbon paper electrode (11.5 cm2,
to orange when acetate was oxidized by G. sulfurreducens. The
without wet proofing, TGPH-090; E-Tek, USA) as the anode and
inoculums were transferred two times for each 72 h anaerobically.
cathode, if not otherwise specified. The anode and cathode were
Before adding it to the anode chamber of MFC, the culture of
spaced 1.5 cm apart. Both electrodes were attached to platinum
G. sulfurreducens, which was harvested during the late-exponential
wires with silver paste (Dotite, Fujikura Kansei Co., Japan) and
phase, was washed twice with the growth medium lacking acetate
epoxy (Devcon, ITW Performance Polymers Co., USA) to connect
and fumarate. The optical density of inoculums was 0.1 at 660 nm.
them through the external wire. The resistance of the carbon paper
To investigate the substrate specificity of G. sulfurreducens, the
connecting the platinum wire was between 0.4 and 0.7.
culture was centrifuged twice with the growth medium lacking
acetate and was then inoculated into a 50 ml serum bottle (10%
Operation of MFC
inoculation). The optical density at 660 nm of the initial inoculums
The anode chamber of each MFC was filled with the G. sulfurreducens
was adjusted to 0.07, while the initial pH was 7. Four different
culture and the same medium that was used for cell growth, except for
organic acids of succinate, butyrate, malate, and acetate of 20 mM
fumarate. The G. sulfurreducens culture, which was harvested during
were used as electron donors in the serum bottles. The experiments
the late-exponential phase, was washed twice with the growth
were carried out in duplicate, and the values were averaged.
medium lacking acetate and fumarate, before use. If not otherwise
To evaluate the effect of bacterial immobilization on electricity
specified, 5 mM acetate was used as the electron donor and carbon
production, G. sulfurreducens was inoculated into the MFCs using
source. The cathode chamber of each MFC was filled with the
carbon paper and carbon felt as the anodes. The mixture of
solutions containing (per liter of deionized water) 0.1 g KCl, 0.6 g
inoculums and growth medium was continuously stirred using a
NaH2PO4, 2.9 g NaCl, and 4.8 g Tris-HCl. The cathode chamber
magnetic bar, and was then replaced by fresh medium every 72 h
was continuously aerated to supply oxygen as an electron acceptor,
for 10 days. After immobilization of G. sulfurreducens, the carbon
while the anode chamber was continuously aerated with argon (Ar)
paper and carbon felt were directly used as an anode in the MFC
gas to prevent oxygen diffusing from the cathode to the anode and
experiments.
to mix the anolyte. The gases were filtered through 0.2 µm-pore-size
membranes positioned in front of the MFC, and their flow rates
Preparation and Construction of MFC
were maintained at 7 ml/min. All of the stock solutions were flushed
Two-chambered MFCs were constructed as reported in a previous
carefully with a N2-CO2 gas (80:20) before use. MFC experiments
study [2]. Two acrylic rectangular chambers (total volume: 25 ml
were carried out at 30oC in a temperature-controlled room.
1397 Kim et al.
Analysis and Calculation
The voltage over the external resistance was recorded by a digital
multimeter (PC20; Sanwa Co., Japan) interfaced to a personal
computer. The current I in amperes (A) was calculated using Ohm’s
law, I = V/R, where V is the measured voltage in volts (V), and R
is the known value of the external load in ohms (Ω). The external
resistance used in the experiment was 500 Ω, if not otherwise
specified. The recovery of electrons, that is, the coulombic efficiency
(CE), is calculated as
t
Ms ∫0b I dt
Coulombic efficiency ( CE) = -----------------------
-
F b vAn ∆c
where ∆c is the substrate concentration change over the batch cycle
over a time (tb), Ms is the molecular weight of the substrate, F is
Faraday’s constant, b is the moles of electrons defined for the
substrate based on a half reaction, and vAn is the volume of liquid in
the anode chamber [16].
Organic acids such as acetate, malate, succinate, and butyrate
were analyzed using a high-performance liquid chromatograph (Model
VP; Shimadzu Co., Japan) equipped with a sulfonated divinyl benzene-
styrene copolymer column (300 mm × 7.8 mm, Aminex HPX-87H;
BioRad, USA) and a photometric detector (216 nm). An aqueous
solution of 10 mM H2SO4 was used as an eluting solution at
0.6 ml/min, and the column temperature was maintained at 30oC.
The cell growth was determined by measuring the OD at 660 nm in
a 3 ml cuvette with a 1 cm light path.
RESULTS AND DISCUSSION
Substrate Specificity of G. sulfurreducens
Pure cultures of G. sulfurreducens were cultivated using
various substrates including acetate, malate, succinate, and
butyrate to investigate their substrate specificity. Before
use, the cultures were centrifuged twice with a growth
medium lacking organic acid and were then inoculated into
Fig. 2. pH (open symbols) and optical density at 660 nm (closed
symbols) in serum bottles cultivating G. sulfurreducens using
various substrates, including acetate (circles), malate (squares),
succinate (diamonds), and butyrate (triangles), with fumarate as
an electron acceptor.
a serum bottle using fumarate as an electron acceptor. Fig. 2
depicts the pH variation and the cell concentrations, which
were measured as the optical density at 660 nm (OD660).
During 7 days, the initial pH decreased slightly from 7 to
6.6, regardless of the substrate type. However, the growth
of G. sulfurreducens was observed only when acetate was
added. The cell concentration (OD660) in the bottle into
which acetate was added increased significantly from 0.1
to 0.4 for 3 days and became stable by 7 days. In contrast,
the cell concentrations decreased from 0.1 to 0.05, 0.07,
and 0.06 for 7 days when malate, succinate, and butyrate
were used, respectively. These results are direct evidence
that the culture of G. sulfurreducens prefers acetate as the
carbon and energy source for growth. This is consistent
with the results of a previous study that showed the
Geobacteraceae family was the predominant bacteria when
acetate was used as the fuel in an MFC using marine
sediment as the inoculum [4].
Effect of Acetate Concentration on Electricity Generation
The MFCs inoculated with G. sulfurreducens cultures were
operated using different acetate concentrations. As shown
in Fig. 3, the 5 mM acetate-fed MFC went through 8 batch
cycles for 20 days, whereas the 20 mM acetate-fed MFC
passed through only 3 batch cycles during the same period.
When 5 mM acetate was added to an MFC, the average
power density of 1.2 mW/m2 at the first batch cycle was
greatly increased to 15.2 mW/m2 at the sixth cycle. After
15 days of operation, the power density of the 5 mM
acetate-fed MFC became stable at 16.2 mW/m2. In
contrast, the power density of the 20 mM acetate-fed MFC
increased from 1.5 to 13.4 mW/m2 during only 3 batch
cycles, because it took a longer time for substrate of high
concentration to be degraded per batch cycle. Regardless
of acetate concentration, it took about 15 days for MFCs to
reach a steady state in current generation.
At the steady state, the 5 mM acetate-fed MFC achieved
the CE of 46.2%, which was two times higher than that in
the 20 mM acetate-fed MFC. The power generation of
MFCs generally increases with substrate concentrations,
but this result suggests that high substrate concentrations
can inhibit power production. Sharma and Li [24] reported
that high substrate concentrations limited the power
generation in MFCs inoculated by mixed cultures. They
observed that the cell voltage had a reverse correlation
with substrate concentration when the acetate concentration
was higher than 8 mM. There are two possible reasons for
the lower CE at high acetate concentrations. First, the
electron sink for bacterial growth might reduce the CE of
MFCs [1]. A high substrate concentration facilitates the
cell growth of G. sulfurreducens, and it causes much of the
electrons to be consumed for biomass production instead
of electricity generation. Second, an excessive amount of
substrate might inhibit the electron transfer activity via

Fig. 3. Cell growth of G. sulfurreducens and power density in
(A) the 5 mM acetate-fed MFC and (B) 20 mM acetate-fed
MFC: circles, power density; triangles, optical density; and
inverted triangles, pH.
direct contact between the enzyme in the outer membrane
and the anode [24]. At high substrate concentrations, more
than one substrate could bind to the active site of an
enzyme and consequently block the enzyme activity for
electron transfer to the anode.
Immobilization of G. sulfurreducens for Effective MFC
Operation
It is known that G. sulfurreducens can directly transfer
electrons to the anode via bacterial surface redox active
proteins or conductive pili. Accordingly, biofilm formation
on the anode is important to allow direct electron transfer
to be successfully conducted. However, it usually takes
several weeks or even several months to establish a stable
biofilm from a pure or mixed culture [3]. In this work,
therefore, the cultures of G. sulfurreducens were immobilized
onto the anode to reduce the enrichment period and
enhance electricity generation. Fig. 4 shows the effect of
immobilization of G. sulfurreducens on carbon paper and
carbon felt on electricity generation. When the suspended
culture of G. sulfurreducens and 20 mM acetate were
injected into an MFC, it took more than 15 days to reach a
steady condition. However, the MFC using a G. sulfurreducens-
immobilized anode became stable in 6-8 days. Compared
with the use of carbon paper, the immobilization of G.
ENHANCING FACTORS OF ELECTRICITY GENERATION IN MFC 1398
Fig. 4. Effect of immobilization of G. sulfurreducens onto carbon
paper (squares) and carbon felt (diamonds) on power density.
Free cells were inoculated into the anode chamber of the MFC as a control
(circles).
sulfurreducens on the carbon felt was favorable to high
power production at the beginning of MFC operation. In a
report by Cha et al. [6], it was shown that the MFCs using
carbon felt anode produced higher power density than
those using carbon cloth. They suggested that the high
porosity of carbon felt can make bacteria be easily attached
to the electrode and facilitate more rapid substrate diffusion
to the biofilm.
Effect of Argon Gas Purging on Anolyte pH and
Electricity Generation
In this study, the anode chamber was continuously aerated
with argon gas to prevent oxygen diffusing from the
cathode to the anode and to mix the anolyte. The flow of
argon gas in the anode chamber resulted in the liberation of
carbon dioxide into the atmosphere. It made a change in
the equilibrium condition for carbon dioxide between liquid
and gas phases. Consequently, the anodic pH increased to
9.5 after 27 days of operation, and the electricity generation
stopped, as shown in Fig. 5. This result shows that current
generation by G. sulfurreducens is severely inhibited at pH
levels over 9. When 1 M HCl was added to the anode
chamber to adjust the pH level, electricity was stably
produced over 35 days.
The effect of anodic pH on current generation has been
investigated in several studies. However, the optimal pH
for maximizing the power output varied according to the
type of microorganisms and reactor configuration. Gil et al.
[11] determined the best performance at pH 7 using a two-
chambered MFC, which used wastewater as the electron
donor and sludge as the bacterial source. In contrast, the
acidophilic condition of pH 6 in the anodic chamber showed
the highest power density, where the anaerobic mixed
consortia from the biofilm reactor producing fermentative
1399 Kim et al.
Fig. 5. Effect of argon gas purging on the anolyte pH and
electricity generation.
Compared with the control condition, 1 M HCl was added to the anode
chamber to adjust the pH level: circles, cell voltages with pH control;
squares, cell voltages without pH control; triangles, pH variation with pH
control; and inverted triangles, pH variation without pH control.
H2 was used as the inoculum [25]. Zhuang et al. [26]
reported that a tubular MFC produced the maximum power
density with an anodic pH of 10 and a cathodic pH of 2.
They suggested that the suppression of methanogenesis
with highly alkaline anolyte (pH 10) contributed to better
coulombic efficiency. Similar to our study, Nimje et al.
[20] evaluated the effect of anodic pH on current
generation using a pure culture of Enterobacter cloacae.
When the anodic pH was increased from 6.5 to 9.5, the
maximum power density decreased from 36 to 6.5 mW/m2
with an increase of internal resistance. These results,
therefore, indicate that appropriate pH control, considering
the microorganism types and reactor configurations, is a
critical factor to improve the performance of MFCs.
Enhancing Electricity Production Using a Pt-Containing
Carbon Cathode
The slow rate of oxygen reduction on the surface of the
carbon electrode leads to a high cathodic overpotential,
which limits the performance of MFCs. Several studies
have suggested strategies to reduce cathodic overpotentials
by using a mediator, modifying the electrode with a
catalyst and optimizing the operational conditions in the
cathode chamber [23]. This study investigated the effect of
a Pt-coated cathode on the electricity production of MFCs
using G. sulfurreducens. As shown in Fig. 6, an MFC was
continuously run in acetate-fed batch mode, where the
plain carbon paper cathode was replaced by Pt-coated
carbon paper (0.5 mg Pt/cm2) after 38 days of operation.
Using the Pt-coated cathode, the power density was greatly
increased from 3.5 to 75 mW/m2, and the CE also
increased from 39% to 83%. This indicates that electricity
Fig. 6. Effect of a Pt-coated cathode (0.5 mg Pt/cm2) on electricity
generation.
The arrow indicates replacement of a plain carbon paper cathode by a Pt-
coated carbon paper: circles, cell voltages; and triangles, optical density.
production of MFCs using G. sulfurreducens is highly
associated with cathodic overpotential for oxygen reduction.
Acknowledgment
This Research (Paper) was performed for the Hydrogen
Energy R&D Center, one of the 21st Century Frontier
R&D programs funded by the Ministry of Education
Science Technology of Korea.
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