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ARTICLE
Simultaneous Analysis of Physiological and
Electrical Output Changes in an Operating
Microbial Fuel Cell With Shewanella oneidensis
Justin C. Biffinger,
1
Ricky Ray,
2
Brenda J. Little,
2
Lisa A. Fitzgerald,
1
Meghann Ribbens,
3
Steven E. Finkel,
3
Bradley R. Ringeisen
1
1
Chemistry Division, Naval Research Laboratory, 4555 Overlook Avenue, SW, Code 6113,
Washington 20375, District of Columbia; telephone: 202-767-2398;
fax: 202-404-8119; e-mail: justin.biffinger@nrl.navy.mil
2
Oceanography Division, Naval Research Laboratory, Building 1009,
John C. Stennis Space Center, Slidell, Mississippi
3
Molecular and Computational Biology Section, Department of Biological Sciences,
University of Southern California, Los Angeles, California
Received 26 November 2008; revision received 7 January 2009; accepted 8 January 2009
Published online 15 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22266
ABSTRACT: Changes in metabolism and cellular physiology
of facultative anaerobes during oxygen exposure can be
substantial, but little is known about how these changes
connect with electrical current output from an operating
microbial fuel cell (MFC). A high-throughput voltage based
screening assay (VBSA) was used to correlate current output
from a MFC containing Shewanella oneidensis MR-1 to
carbon source (glucose or lactate) utilization, culture con-
ditions, and biofilm coverage over 250 h. Lactate induced an
immediate current response from S. oneidensis MR-1, with
both air-exposed and anaerobic anodes throughout the
duration of the experiments. Glucose was initially utilized
for current output by MR-1 when cultured and maintained
in the presence of air. However, after repeated additions of
glucose, the current output from the MFC decreased sub-
stantially while viable planktonic cell counts and biofilm
coverage remained constant suggesting that extracellular
electron transfer pathways were being inhibited. Shewanella
maintained under an anaerobic atmosphere did not utilize
glucose consistent with literature precedents. Operation of
the VBSA permitted data collection from nine simultaneous
S. oneidensis MR-1 MFC experiments in which each experi-
ment was able to demonstrate organic carbon source
utilization and oxygen dependent biofilm formation on a
carbon electrode. These data provide the first direct evidence
of complex cellular responses to electron donor and oxygen
tension by Shewanella in an operating MFC at select time
points.
Biotechnol. Bioeng. 2009;103: 524–531.
Published 2009 Wiley Periodicals, Inc.
y
KEYWORDS: microbial fuel cell; Shewanella; biofilm; cellular
physiology; glucose; lactate
Introduction
Microbial fuel cells (MFCs) are electrochemical devices
capable of generating an electrical current directly from the
oxidation of carbon electron sources using bacterial
metabolic pathways. These devices are currently being
developed for a variety of applications ranging from the
generation of electricity using wastewater (Aelterman et al.,
2006; Angenent et al., 2004) to autonomous power sources
for sensors (Shantaram et al., 2005) and beacons (Tender
et al., 2008). Different bacterial strains have evolved a variety
of strategies for delivering electrons to solid electron
accepting materials (metal oxides, carbon electrodes). For
example, direct contact with the electrode surface is required
for Geobacter sulfurreducens to generate current from a MFC
(Reguera et al., 2006) while Shewanella oneidensis MR-1 can
deliver reducing equivalents to electron accepting surfaces
without direct contact (Lies et al., 2005) using redox
mediators (Marsili et al., 2008; von Canstein et al., 2008).
Several reviews have been published recently that summarize
the significant progress in understanding electron transport
pathways within electrochemically active bacteria (EAB)
(Chang et al., 2006; Fredrickson et al., 2008; Hernandez and
Newman, 2001; Lovley, 2008; Schro
¨
der, 2007). However,
few studies have been published that address the complex
Correspondence to: J.C. Biffinger
Contract grant sponsor: Office of Naval Research
Contract grant numbers: 62123N; N0001409AF00002
Contract grant sponsor: Air Force Office of Scientific Research (MURI Program)
FA9550-06-1-0292
524 Biotechnology and Bioengineering, Vol. 103, No. 3, June 15, 2009 Published 2009 Wiley Periodicals, Inc.
y
This article is a US Government
work and, as such, is in the public domain in the United States of America.
real-time cellular physiological changes that determine how
EAB interact with anodes during MFC operation (Lanthier
et al., 2008).
Both environmental bacterial consortia and single strain
MFCs are reported in the literature (Logan et al., 2006).
Working with single strains allows mechanistic and
physiological details to be observed directly. Two bacterial
families, Geobacteracea and Shewanellacea, are commonly
used in pure culture MFC research. S. oneidensis MR-1 is
a facultative, anaerobic g-proteobacterium capable of
dissimilatory metal reduction (Myers and Nealson, 1988)
as well as generating current within MFCs (Bretschger et al.,
2008; Kim et al., 2002; Ringeisen et al., 2006). Shewanella
was chosen for this work because of its adaptability to
aerobic and anaerobic environments.
Glycolytic aerobic metabolic pathways in Shewanella have
been identified through genomic and proteomic studies
(Beliaev et al., 2005; Driscoll et al., 2007; Fang et al., 2006;
Leaphart et al., 2006; Serres and Riley, 2006; Wan et al.,
2004) and experimental evidence linking current output
with glucose metabolism was recently reported using aerobic
cultures of S. oneidensis DSP10 in a miniature MFC
(Biffinger et al., 2008). Prior to the aforementioned work,
S. oneidensis was considered limited in the range of organic
electron sources (e.g., formate, lactate, pyruvate, amino
acids) that could be used for anaerobic metal reduction or
current output from MFCs (Nealson et al., 2002). Bacterial
metabolic pathways dictate how different types of organic
electron sources (carbohydrates, linear carboxylic acids,
polysaccharides) are utilized for current output from MFCs.
Therefore, in situ monitoring of both cellular and culture
environment conditions is important for improving the
long-term survivability of MFC devices.
Since removing electrode samples from a continuously
operating MFC is not practical and running multiple
laboratory scale MFCs under identical conditions for
sampling electrodes is unfeasible, no research has been
reported on the study of microbial cellular changes within
an operating MFC. Direct real time measurements of biofilm
formation and coverage have been analyzed by nuclear
magnetic resonance (NMR) (McLean et al., 2008) and
confocal microscopy (Teal et al., 2006) on transparent
supports. However experiments performed on non-con-
ductive surfaces may not be germane to the conditions in an
operating MFC (Lanthier et al., 2008). Indirect real time
analysis of biofilm formation by electrochemical impedance
spectroscopy (EIS) (Manohar et al., 2008; Ramasamy
Ramaraja et al., 2008) or utilizing pre-formed biofilms on
electrodes placed in MFCs (Venkata Mohan et al., 2008)
have also been used to monitor biofilm dynamics but are
difficult to relate to actual biofilm coverage in an operating
MFC.
In this study, a voltage based screening assay (VBSA) was
used to monitor voltage output from EAB under both
closed and open circuit conditions (Biffinger et al., 2009).
Additionally, the use of a high-throughput assay for
monitoring current output from bacteria provided a
pathway to correlate electrical current output with cellular
and metabolic changes; factors that have not been studied
within an operating MFC to date. The VBSA was used to
monitor real-time current output as it relates to anaerobic
and air-exposed cultures, planktonic cell concentration, and
extent of biofilm formation on a carbon electrode at defined
time points during the experiment. The combination of
these data resulted in a physiological description of how
Shewanella respond to glucose and lactate in the presence of
oxygen in an operating MFC.
Materials and Methods
Solutions and Media
A stock solution of 1.95 M sodium lactate was adjusted to
pH 7.0 and sterilized by autoclaving for 15 min at 1218C. A
D-glucose (1 M) stock solution was sterilized with a 0.2 mm
cellulose nitrate filter. Luria-Bertani (LB) Broth (Miller) and
LB/agar (Difco LB-Agar, Miller) was used for liquid cultures
and plates, respectively (Fisher Scientific, Inc, Pittsburgh,
PA). The solvent for each solution was Millipore 18 MV
water. Serial dilutions for viable planktonic cell concentra-
tion measurements were performed in phosphate buffered
saline (pH 7.0) with 0.03% Triton-X100 (Sigma–Aldrich,
Milwaukee, WI).
Cell Culture Conditions
S. oneidensis MR-1 (obtained from Dr. Kenneth Nealson
(University of Southern California, Los Angeles, CA)) was
grown from a single colony isolated from a LB/agar
plate inoculated from a 808C glycerol stock culture. A
single colony was transferred to 50 mL of LB broth and
incubated aerobically at 258C with gentle shaking (100 rpm).
Experimental cultures were sub-cultured after 20 h of
growth three times before being used in VBSA experiments.
Anaerobic S. oneidensis MR-1 cultures were created from a
MR-1 culture, which was incubated aerobically for 48 h.
These aerobic stationary phase MR-1 cultures were then
degassed with a nitrogen purge and shaken (100 rpm) in an
anaerobic chamber for 24 h at 258C prior to MFC
experimentation.
VBSA Construction and Data Acquisition
Dimensions and fabrication of the VBSA were published
previously (Biffinger et al., 2009). The anodes were single-
sided carbon-coated titanium flags and the cathode system
was graphite paper in a 50 mM potassium ferricyanide
(dissolved in 100 mM phosphate buffer at pH 7.0). Each
experiment was completed in a nine-well VBSA apparatus
depicted in Figure 1. Experiments with no addition of
glucose or lactate were labeled as blank. Planktonic cell
concentrations of each well were determined from serial
dilution of aliquots in phosphate buffered saline with 0.03%
Biffinger et al.: Real-Time Analysis of a Microbial Fuel Cell 525
Biotechnology and Bioengineering
Trition-100 and plated onto LB/agar with average cell
counts reported for glucose, lactate, and blank during the
experiment. Once the electrode was removed for environ-
mental scanning electron microscopy (ESEM) fixation, the
well was no longer used for planktonic cell concentration
determination. The voltages across a 100 kV resistor bank
(in a custom nine-resistor bank made for simultaneous
measurements) were recorded with a personal data acqui-
sition device (I/O tech, personal daq/54) every 4 min. Ohm’s
law was used to convert voltage to current. Anaerobic
(performed in a Coy instruments anaerobic chamber) and
aerobic (or air-exposed) experiments were performed at
238C.
Imaging S. oneidensis Biofilms
Environmental scanning electron microscopy (ESEM) of
carbon surfaces on the titanium anodes was performed at
the Naval Research Laboratory, Stennis Space Center,
(NRLSSC). Unattached biomass was removed by washing
each anode with three separate 1 mL aliquots of phosphate
buffered saline solution at the Naval Research Laboratory,
Washington, DC (NRLDC). Each anode was placed in 2 mL
of 4% cacodylate buffered glutaraldehyde fixative (Ray et al.,
1997) in water at NRLDC and fixed for at least 24 h at 48C
prior to shipment to NRLSSC without further manipula-
tions. Anaerobic samples were fixed in the anaerobic hood
using degassed 4% cacodylate buffered glutaraldehyde
fixative. For collecting ESEM images, each anode was
removed from the fixative and washed with 50 mL of
distilled water. After 2 min of gentle rinsing, each anode was
placed on a mounting stub on the Peltier cooling device
inside the ESEM chamber. The anodes were kept wet/moist
by using the Peltier cooling device maintained at 48C and a
chamber water vapor pressure between 4.5 and 5.5 torr.
Water vapor was allowed to condense on the cooled anodes
to keep it moist while performing ESEM imaging. Liquid
water was removed from the top layer, several microns thick,
to view the biofilm on the carbon surface of each titanium
anode. A gaseous secondary electron detector (GSED) was
used to collect the ESEM images of the wet/moist sample
surface.
Results and Discussion
The combination of time course results from ESEM images
of electrode surfaces, viable planktonic cell densities, and
electrical current output generates a broader understanding
of how S. oneidensis interact with carbon electrode surfaces
in an operating batch MFC. Correlating the three
parameters mentioned previously was made possible by
using a small modular array of identical MFCs operated in
parallel. These data demonstrate distinct cellular differences
Figure 1. General diagram of the nine-well VBSA experimental setup for both
aerobic and anaerobic studies. Electrodes were removed and chemically fixed at
time 1 (t
1
), time 2 (t
2
), and time 3 (t
3
) with the carbon electron source indicated in each
well.
Figure 2. Average current output from S. oneidensis MR-1 containing VBSA
exposed to (a) aerobic or (b) anaerobic atmospheres with 10 mM lactate as the sole
electron carbon source with baseline correction. Secondary axis reports planktonic
cell count with time in colony forming units (CFU)/mL. Solid vertical lines indicate when
lactate was added and block arrows designate when anode was removed and
chemically fixed for ESEM at t
1
, t
2
, and t
3
.
526 Biotechnology and Bioengineering, Vol. 103, No. 3, June 15, 2009
with carbon source utilization and oxygen tension as well as
providing insight into the role cellular physiology plays on
current output from an operating S. oneidensis MFC.
Lactate Metabolism by S. oneidensis
Current output (Fig. 2) from S. oneidensis MR-1 was
correlated to both planktonic cell density (Fig. 2) and
biofilm formation (Fig. 3) with lactate as the sole electron
source. Subsequent additions of lactate over the first 170 h
for air exposed anodes resulted in a four-fold current
increase (Fig. 2a). The remaining 100 h of the experiment
resulted in a doubling of the current output. In general, the
current output doubled from successive additions of lactate
to air-exposed MR-1. The maximum current generated by
anaerobic MR-1 with lactate (Fig. 2b) was eight-fold less
than air-exposed MR-1 (Fig. 2a). However, there were rapid
current responses (< 4 min) from lactate additions for
anaerobic cells.
The planktonic cell density remained constant for lactate
(8 10
8
CFU/mL) in both the presence and absence of air
(Fig. 2a and b, respectively). Planktonic cell density in the
blank anode decreased exponentially after 70 h, correlating
with LB nutrient depletion. Biofilm formation was weak for
all blank electrodes under aerobic (Fig. 3a–c) and anaerobic
(Fig. 5a–c) atmospheres. Electrodes from air-exposed
anode chambers (Fig. 3g–i) showed significant biofilm
coverage with a complete lawn of MR-1 formed over the
entire anode surface after 220 h of operation (Fig. 3h).
Anaerobic MR-1 did not form a substantial biofilm in the
presence of lactate (Fig. 3d–f).
The gradual increase in current with time (Fig. 2a) corre-
lated with the formation of biofilm for MR-1 (Fig. 3g–i).
This gradual current increase is typically described as a
conditioning period where the bacteria modify the electrode
surface for either bacterial attachment or mediator release.
However, when using air-exposed anodes this gradual
increase in current should also be attributed to a decrease in
oxygen concentration at the electrode surface, which would
Figure 3. ESEM images of the chemically fixed carbon anode surfaces (designated by block arrows in Fig. 2) from acellular (a–c) and S. oneidensis MR-1 anaerobic (d–f)or
air exposed (g–i) anode chambers with lactate as the sole carbon electron source. Scale bar is 10 mm.
Biffinger et al.: Real-Time Analysis of a Microbial Fuel Cell
527
Biotechnology and Bioengineering
eliminate the competitive oxygen reduction reaction and
increase the Coulombic efficiency of the MFC. The
Coulombic efficiency doubled as substantial biofilm was
formed on the electrode surface (Fig. 2a). This concept of
oxygen gradients in biofilms was first demonstrated using
direct microelectrode measurements showing a decrease
in oxygen concentration with increasing biofilm thickness
(Rasmussen and Lewandowski, 1998) and is consistent with
these results.
Since S. oneidensis does not need to be in contact
with electrode surfaces to deliver electrons at a distance,
planktonic cell density would impact current output
significantly for a Shewanella containing MFC. The
viable planktonic cell count remained essentially constant
throughout the air-exposed experiment, and significant
current was generated immediately, even with sparse biofilm
formation over the first 100 h of operation. We observed
little change in planktonic cell concentration with time
(Fig. 2a), but found a significant increase in biofilm coverage
on the anode (Fig. 3). This experiment demonstrated that
the decrease in oxygen concentration at the anode and
increased number of bacteria near the electrode surface is
primarily responsible for the gradual increase in current
from lactate. This colonization of the electrode is certainly
enhanced in the presence of oxygen when comparing ESEM
images from MR-1 exposed to air (Fig. 3g–i) and anaerobic
experiments (Fig. 3d–f).
There has been only one other study that has monitored
Shewanella growth and biofilm formation with an
active MFC carbon electrode as the sole electron acceptor
(Lanthier et al., 2008). Our results are consistent with their
observation that planktonic biomass is primarily responsible
for current output from anaerobic S. oneidensis containing
MFCs but is not consistent for air-exposed cultures.
It is clear from our results that substantial biofilms of
S. oneidensis MR-1 are formed with air-exposed anodes and
lactate (Fig. 3 g–i) in an operating batch MFC, while a
significant biofilm is not formed under anaerobic conditions
(Fig. 3 d–f). Therefore, a lack of biofilm formation yet
a sustained planktonic cell concentration over time
indicates Shewanella utilizes lactate as a food source and
our observations under anaerobic conditions indicate that
planktonic cells rather than direct cell-anode contact are
primarily responsible for current output.
Glucose Metabolism by S. oneidensis
Until recently, only a limited range of organic electron
sources were known which S. oneidensis could use for
anaerobic metal reduction or current output in a MFC
(Fredrickson et al., 2008). Lactate is one such electron source
that has been utilized for studies of current production from
a Shewanella MFC (Kim et al., 1999, 2002; Ringeisen et al.,
2006) and also was shown in the previous section. However,
the natural abundance of lactate is limited. Therefore, in
order to use a Shewanella-containing MFC in a variety of
applications, such as an autonomous power source for
sensors, we must understand the physiological role naturally
occurring electron sources might play on bacteria. The
results presented here demonstrate a simultaneous time
course analysis of the physiological and electrical output
changes S. oneidensis undergoes in an operational MFC with
glucose as the sole electron source.
S. oneidensis MR-1 cultured and exposed to air within a
MFC (Fig. 4a) can utilize glucose as an electron source for
current production. However, repeated additions of glucose
resulted in a gradual increase in current over the first 150 h
with a subsequent decrease in current after this time period
(Fig. 4a). The addition of glucose did result in smaller
current increases after 150 h for MR-1 but significantly less
than the maximum current of 17 mA recorded in the first
150 h. This result is consistent with similar experiments
using air-exposed S. oneidensis DSP10 cultures in a flowing
miniature MFC (Biffinger et al., 2008) with glucose as the
sole electron source. Significantly more of the electrode
surface was covered by MR-1 using glucose with oxygen-
exposure (Fig. 5g–i) than without (Fig. 5d–f). Planktonic cell
density remained high for air-exposed cells indicating that
glucose was being utilized by Shewanella during this
experiment.
Figure 4. Average current output from S. oneidensis MR-1 containing VBSA
exposed to (a) aerobic or (b) anaerobic atmospheres with 10 mM glucose as the sole
electron carbon source with baseline correction. Secondary axis reports planktonic
cell count with time in colony forming units (CFU)/mL. Solid vertical lines indicate when
glucose was added and block arrows designate when anode was removed and
chemically fixed for ESEM at t
1
, t
2
, and t
3
.
528 Biotechnology and Bioengineering, Vol. 103, No. 3, June 15, 2009
Current output after addition of glucose for air-exposed
MR-1 was initially weak (<5 mA) but over the next 150 h
generated approximately 17 mA (Fig. 4a). Since improve-
ments in current output correlate with biofilm formation
with air exposed cultures, then maximizing biofilm
formation is a major factor in optimizing Shewanella
containing MFCs. In general, there were only sparse biofilms
formed when glucose was the sole electron source under all
conditions. Air-exposed MR-1 current vs. time data (Fig. 4a)
is consistent with Shewanella utilizing glucose upon
continued exposure to oxygen. The present experiments
show distinct cellular physiological responses from repeated
exposure to glucose as well as a gradual decrease in current
after 170 h consistent with previous results (Biffinger et al.,
2008). The viable planktonic cell concentration and biofilm
coverage remained constant throughout the air-exposure
experiments, suggesting that repeated additions of glucose
eventually down-regulates extracellular electron transport
pathways in favor of sustaining growth. The conservation of
energy for growth rather than biofilm formation on an
electrode by anaerobic S. oneidensis was reported recently,
although a complete picture of bacterial growth changes on
the electrode was not provided (Lanthier et al., 2008).
S. oneidensis is capable of reducing a wide range of
electron acceptors, but only a small number of electron
donors have been utilized effectively in anaerobic environ-
ments (Fredrickson et al., 2008). The initial current response
of S. oneidensis under anaerobic conditions to glucose is
attributed to the transition of aerobically cultured cells to an
anaerobic MFC environment. Since air exposed stationary
phase MR-1 cultures were degassed for anaerobic experi-
ments, the decrease in current with time after 170 h is
consistent with the decreased expression of proteins in
glycolytic pathways under low oxygen levels (Scott and
Nealson, 1994). This conclusion is also supported by the
repeated positive current responses to additions of glucose
in the presence of oxygen (Fig. 4a), while anaerobic MR-1
cells did not generate any current response with repeated
glucose additions (Fig. 4b). Planktonic cell densities also
decreased with time under anaerobic conditions and glucose
exposure, while air-exposed cells were able to maintain
their cell density (Fig. 4). All of these data indicate that
Figure 5. ESEM images of the chemically fixed carbon anode surfaces (designated by block arrows in Fig. 4) from acellular (a–c) and S. oneidensis MR-1 anaerobic (d–f)or
air exposed (g–i) anode chambers with glucose as the sole carbon electron source. Scale bar is 10 mm.
Biffinger et al.: Real-Time Analysis of a Microbial Fuel Cell 529
Biotechnology and Bioengineering
glucose was metabolized when exposed to air and not
utilized efficiently under anaerobic conditions in a single
experiment.
Conclusions
The miniature modular design of the VBSA resulted in the
first time-lapse analysis correlating cellular physiological
responses to current output from an operating MFC. Large
differences in current output and physiology were observed
between MFCs utilizing air-exposed and anaerobic MR-1
cultures exposed to glucose and lactate. The reduced
response in current generation from lactate-exposed
anaerobic S. oneidensis MR-1 was fivefold greater than the
current response from glucose-exposed anaerobic MR-1.
However, the sustainability of aerobic Shewanella cultures
in the presence of glucose, a naturally occurring electron
source, is a promising result for developing long-term
autonomous sensors. Nonetheless, the fact that sustained
current production has not been demonstrated when
glucose is the sole electron donor means that consortia
will still be necessary to achieve efficient energy harvesting
by MFCs. These results demonstrate, for the first time, the
ability to correlate current output in relation to carbon
source utilization, culture conditions, and biofilm coverage
in an operational MFC.
This work was funded by the Office of Naval Research (NRL 6.2
Program Element Number 62123N) and ONR program
N0001409AF00002 and the Air Force Office of Scientific Research
(MURI Program, #FA9550-06-1) to S.E.F. We thank the National
Research Council for L.A.F. research associateship.
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