APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2008, p.

73297337
0099-2240/08/$08.000 doi:10.1128/AEM.00177-08
Copyright 2008, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 23

Microbial Biofilm Voltammetry: Direct Electrochemical Characterization

of Catalytic Electrode-Attached Biofilms
Enrico Marsili,1 Janet B. Rollefson,2 Daniel B. Baron,1
Raymond M. Hozalski,3 and Daniel R. Bond1,4*
BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 551081; Department of Biochemistry, Molecular Biology and
Biophysics, University of Minnesota, Minneapolis, Minnesota 554552; Department of Civil Engineering, University of Minnesota,
Minneapolis, Minnesota 554553; and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 554554
Received 18 January 2008/Accepted 21 September 2008

Anaerobic metal-reducing Geobacteraceae catalyze the

transfer of electrons from reduced electron donors, such as
acetate, to oxidized electron acceptors such as Fe(III). While
electron transport is confined to the inner membrane of most
respiratory bacteria (66), Fe(III) and Mn(IV) exist primarily in
the environment as insoluble precipitates at circumneutral pH.
To reduce minerals of various crystallinity, redox potential,
and surface charge, metal-reducing bacteria must possess a
complete mechanism for transporting electrons across the inner membrane, periplasm, outer membrane, and outer surfaces (23, 38, 48, 54, 57, 58). Soluble redox-active shuttles or
chelators can facilitate respiration beyond the outer membrane
in some organisms (12, 34, 55, 56, 63), but no evidence for the
secretion of such a compound has been observed in Geobacteraceae.
This electron transfer capability has recently been utilized in
devices known as microbial fuel cells (MFCs), where electrodes serve as electron acceptors to support growth (11, 18,
35, 36, 62). However, variations in electrode surfaces, operating temperatures, electron donor concentrations, and culture
conditions limit comparison between electrode-reducing bacteria. In addition, MFCs aim to maximize electrical power
production per unit volume, using large anodes consisting of
carbon felt (19, 39, 50) or carbon paper (43) in small reactors

(with surface/volume ratios between 25 and 640) (19, 25, 29,

43). In such systems, the actual surfaces accessible to bacteria
are not known, multiple dead zones or diffusion-limited regions exist (19), high-surface-area electrodes increase capacitive current and reduce the sensitivity of analyses (53, 68), and
irregular surfaces prevent mathematical modeling or imaging
of attached biomass (22).
Electrochemical techniques typically used to study purified
redox proteins (3, 4, 7) and cell extracts (26, 45) could likely be
adapted for the study of metal-reducing bacteria. While protein voltammetry is limited by the success of purification and
immobilization of enzymes (17, 49), metal-reducing bacteria
represent a self-assembling multienzyme system that naturally
interfaces with electrodes. In this report, we describe procedures for analysis of G. sulfurreducens biofilms on small carbon
electrodes of defined roughness in potentiostat-controlled
three-electrode cells. Several direct current and alternating
current techniques could be applied to the characterization of
the biofilm-electrode interface and have produced insights into
electron transfer mechanisms between living cells and electrodes. Application of these techniques in routine characterization of other living bacteria and biofilm communities will
allow quantitative comparisons between electron transfer rates
and mechanisms used by these organisms.

* Corresponding author. Mailing address: BioTechnology Institute,

University of Minnesota, 140 Gortner Laboratory, 1479 Gortner Ave.,
St. Paul, MN 55108. Phone: (612) 624-8619. Fax: (612) 625-1700.
E-mail: dbond@umn.edu.
Supplemental material for this article may be found at http://aem
.asm.org/.

Published ahead of print on 10 October 2008.

Bacterial strain and culture media. G. sulfurreducens strain PCA (ATCC

51573) was subcultured in our laboratory at 30C using a vitamin-free anaerobic
medium containing the following per liter: 0.38 g KCl, 0.2 g NH4Cl, 0.069 g
NaH2PO4 H2O, 0.04 g CaCl2 2H2O, 0.2 g MgSO4 7H2O, and 10 ml of a
mineral mix [containing 0.1 g MnCl2 4H2O, 0.3 g FeSO4 7H2O, 0.17 g
CoCl2 6H2O, 0.1 g ZnCl2, 0.04 g CuSO4 5H2O, 0.005 g AlK(SO4)2 12H2O,

MATERIALS AND METHODS

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While electrochemical characterization of enzymes immobilized on electrodes has become common, there is
still a need for reliable quantitative methods for study of electron transfer between living cells and conductive
surfaces. This work describes growth of thin (<20 m) Geobacter sulfurreducens biofilms on polished glassy
carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron
transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active
species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic
electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached
to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an
adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of
this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer
phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a
framework for comparisons with other species or communities.

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APPL. ENVIRON. MICROBIOL.

0.005 g H3BO3, 0.09 g Na2MoO4, 0.12 g NiCl2, 0.02 g NaWO4 2H2O, and 0.10 g
Na2SeO4 per 1 liter]. Acetate was provided as an electron donor at 20 mM. All
media were adjusted to pH 6.8 prior to the addition of 2 g/liter NaHCO3 and
were flushed with oxygen-free N2-CO2 (80:20 [vol/vol]) prior to sealing with butyl
rubber stoppers and autoclaving. All experiments were initiated by inoculating
with 40% (vol/vol) of cells within 3 h of reaching maximum optical density (0.6)
in the medium described.
Electrode preparation. Glassy carbon (Toko America, New York, NY) was
machine cut into 2- by 0.5- by 0.1-cm electrodes. Freshly cut glassy carbon
electrodes were polished using aluminum oxide-silicon carbide sandpaper with a
grit designation of between P400 and P4000 (3M, Minneapolis, MN), as indicated in the ISO/FEPA standard (http://www.fepa-abrasives.com/). Mirror-polished electrode surfaces were obtained with 0.05-m alumina powder (CH Instruments, Austin, TX). Polished electrodes were sonicated to remove debris,
soaked overnight in 1 N HCl to remove metals and other contaminants, washed
twice with acetone and deionized water to remove organic substances, and stored
in deionized water. After each experiment, electrode surfaces were cleaned with
an additional 1 N NaOH treatment (to remove biomass), and the entire surface
was refreshed through sandpaper polishing and cleaning as described above to
remove immobilized electron transfer agents. These working electrodes were
attached to 0.1-mm Pt wires via miniature nylon screws that ensured electrical
contact throughout the experiment.
Electrode cell assembly. Platinum wires from the working electrode were
inserted into heat-pulled 3-mm glass capillary tubes (Kimble, Vineland, NJ) and
soldered inside the capillary to copper wires. Counter electrodes consisted of a
0.1-mm-diameter Pt wire (Sigma-Aldrich, St. Louis, MO) that was also inserted
into a 3-mm glass capillary and soldered to a copper wire. The resistance of each
electrode assembly was measured, and electrodes with a total resistance of higher
than 0.5 were discarded. Reference electrodes were connected to bioreactors
via a salt bridge assembled from a 3-mm glass capillary and a 3-mm Vycor frit
(BioAnalytical Systems, West Lafayette, IN). Electrode capillaries were inserted

through ports in a custom-made Teflon lid that was sealed with an O ring gasket.
This lid fit onto a 20-ml conical electrochemical cell (BioAnalytical Systems,
West Lafayette, IN), which had been previously washed in 3 N HNO3 (Fig. 1A).
After the addition of a small magnetic stir bar, the cell was autoclaved for 15
min. Following autoclaving, the salt bridge was filled with 0.1 M Na2SO4 in 1%
agar. A saturated calomel reference electrode (Fisher Scientific, Pittsburg, PA)
was placed at the top of this agar layer and covered in additional Na2SO4 to
ensure electrical contact (60). The reactors were placed in an in-house-built
water bath to maintain cells at 30C (which introduced less electrical noise than
large circulators, incubators, or temperature-controlled rooms) (Fig. 1B). To
maintain the strict anaerobic conditions required by bacteria, all reactors were
operated under a constant flow of sterile humidified N2-CO2 (80:20 [vol/vol]),
which had been passed over a heated copper column to remove trace oxygen.
Each reactor was located above an independent magnetic stirring unit.
Autoclaved bioreactors flushed free of oxygen, filled with sterile growth medium, and incubated at 30C were analyzed before each experiment to verify
anaerobicity and the absence of redox-active species. Electrochemical cells showing residual peaks in differential pulse voltammetry (DPV), anodic current in
cyclic voltammetry (CV), or baseline noise were discarded as having possible
electrode cleanliness or connection noise issues. These autoclaved, verified bioreactors were then used for growth of G. sulfurreducens cultures.
A typical bioreactor was inoculated with 40% (vol/vol) of stationary phase G.
sulfurreducens cells. After inoculation, a potential step of 0.24 V versus the
standard hydrogen electrode (SHE) was applied and the reactors were incubated
until the current stabilized (72 h). The potential step was 200 mV higher than
the midpoint potential, sustaining a half-maximal anodic current (as determined
by preliminary CV; see below) (9).
Electrochemical instrumentation. A 16-channel potentiostat (VMP, BioLogic, Knoxville, TN) was connected to the three-electrode cells described above
(Fig. 1B). Software from the same producer (EC-Lab v9.41) was used to run
simultaneous multitechnique electrochemistry routines, which include CV, DPV,

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FIG. 1. Cartoon of three-electrode bioelectroreactor (A) and connection scheme to multichannel potentiostat (B).

VOL. 74, 2008

MICROBIAL BIOFILM VOLTAMMETRY

RESULTS AND DISCUSSION

Colonization of electrodes. In MFCs, the half-cell potential
at the anode varies with the activity of the bacterial biofilm and
the chosen external resistance (37, 43). Potentiostat-controlled
electrodes at a sufficiently positive potential are equivalent to
anodes in which the electron acceptor is nonlimiting, reducing
the technical complexity and simplifying the conceptual model
of electron transfer. However, at high oxidative potentials,
conformational change, unfolding, and irreversible process
could alter the catalytic abilities of enzymes, decreasing the
anodic (oxidation) current (27, 52). In all experiments described, the potential step used was 0.24 V versus SHE. This
potential was chosen based on preliminary CV experiments,
which showed that the oxidation current from cells was
independent of applied potential in the range of 0.1 to 0.3 V
versus SHE.
After inoculation, the potential step was applied, and a rapidly increasing anodic (oxidation) current was detected (Fig.
2). As this rate of increase was many times higher than what
could be explained by known growth rates of G. sulfurreducens,
it was likely due to cell attachment to the electrode. The
attachment phase was most rapid when fumarate-limited cells
(compared to mid-log-phase cells) were used as the inoculum,

FIG. 2. Characteristic growth curve for G. sulfurreducens inoculated into a bioreactor containing a 2.5-cm2 glassy carbon electrode
(P400 grit polishing treatment). Analysis breaks indicate when CV
and DPV were performed. Medium change breaks indicate where
planktonic cells and culture medium were removed and replaced with
fresh medium. Raw data are shown with no smoothing or noise-reduction transformations.

consistent with reports linking acceptor limitation to the expression of enzymes important to metal reduction (21, 23).
This was followed by a growth phase, characterized by an
exponential increase in current, which doubled at a rate typically observed for G. sulfurreducens reducing Fe(III)-citrate or
fumarate (doubling time, 7 h).
Within 72 h, electrodes reached a saturation phase characterized by a stable maximum electron transfer rate with a
standard deviation between biological replicates of less than
3% (n 6). Confocal imaging of electrodes always revealed
uniform thin films of 15 m with few structural features such
as pillars (see Fig. S1 in the supplemental material). During the
attachment and growth phases, electrodes were tested for the
effect of voltammetry analyses on the rate of growth or current
plateau. Repeated CV and DPV (see Materials and Methods)
did not alter key midpoint potentials, colonization rates, or
final current densities (Fig. 2).
In order to minimize contribution of other electron transfer
agents, we used a medium lacking redox-active species, such as
vitamins (i.e., riboflavin), redox indicators (resazurin), and
chemical reductants (cysteine and dithiothreitol) (47, 64). In
our recent study (47), we showed that flavin concentrations in
the nM range can be detected under the experimental conditions applied in the present work. When the medium in reactors was replaced, removing planktonic cells and any potential
microbially produced soluble electron transfer agent, the electron transfer rate to electrodes remained unchanged. In addition, removal of nitrilotriacetic acid-chelated minerals from
the medium (once electrode colonization had occurred) did
not affect current production. These findings created stringent
conditions for collection of baseline data in the absence of
most common soluble redox-active compounds. A systematic
examination of the effects of commonly present redox species

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and chronoamperometry (CA). Scan rates higher than 100 mV/s and all electrochemical impedance spectroscopy (EIS) experiments were performed using a
Gamry PCI4 Femtostat (Gamry Instruments, Warminster, PA), and EIS data
fitting was performed with E-Chem Analyst v5.1. Postacquisition analysis of CV
data was performed with the software Utilities for Data Analysis (UTILS), kindly
provided by D. Heering (v1.0; University of Delft, The Netherlands [http://www
.tudelft.nl/]). All measurements, with the exception of CA, were performed in
succession without stirring enabled. The parameters for DPV were as follows: Einitial
(Ei) 0.558 V versus SHE and Efinal (Ef) 0.242 V versus SHE; pulse height,
50 mV; pulse width, 300 ms; step height, 2 mV; step time, 500 ms; scan rate, 4
mV/s; current averaged over the last 80% of the step (1 s, 12 points); accumulation time, 5 s. The parameters for CV were as follows: equilibrium time, 5 s;
scan rate, 1 mV/s; Ei 0.558 V versus SHE; Ef 0.242 V versus SHE;
current averaged over the whole step (1 s, 10 points). For CA, the parameters
were Eapplied (E) 0.242 V versus SHE.
EIS was conducted while maintaining a direct current voltage between the
working electrode and the reference electrode (potentiostatic EIS) as electron
transfer processes by Geobacter isolates were observed to be voltage dependent
(62). EIS was performed at four potentials, 0.04, 0.06, 0.16, and 0.26 V
versus SHE, with a perturbation amplitude of 10 mV. The frequency was varied
from 105 to 0.01 Hz, as most biological charge transfer phenomena are observed
in this range (10). Data were then fitted with a simple empirical model (62), using
a constant phase element to account for irregularities in the morphology and
chemistry of the glassy carbon surface. EIS was typically performed only at the
end of incubations as extensive characterization required electrodes to be held at
multiple potentials for long periods of time.
Confocal and SEM analyses. A S3500N scanning electron microscope (SEM)
(Hitachi, Japan) was used to image freshly polished (SEM) electrodes. After
being thoroughly cleaned with deionized water, 2- by 0.5- by 0.1-cm graphite
electrodes freshly polished with P400 and P4000 sandpapers were air dried and
fixed through graphite tape to a sample stub. The sample stubs were placed
directly into the SEM vacuum compartment and examined at an accelerating
voltage of 5 kV.
A Nikon C1 spectral imaging confocal microscope (Nikon, Japan) with a 60
lens was used to image biofilm-covered electrodes. Immediately following harvesting, the biofilm-covered electrodes were gently washed in growth medium to
remove planktonic cells and then stained with the LIVE/DEAD Baclight bacterial viability kit (Invitrogen Corp., Carlsbad, CA), incubated for 15 min in the
dark, rinsed with growth medium, and placed on a microscope slide. The coverslip was supported by glass spacers thicker than the electrode to prevent
damage to biofilm structure. Two laser wavelengths, 488 and 561 nm, were used
to excite the two stains.

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on electron transfer by Geobacter spp. will be the focus of a

future report.
The effect of microscale surface roughness on bacterial attachment and direct electron transfer has not been reported.
Figure 3 shows the surface features of carbon electrodes polished with different sandpapers. When G. sulfurreducens biofilms were grown on electrodes polished with increasing grit
size, the maximum current density increased over 100% for the
same geometric area (Fig. 3). Polishing with particles smaller
than 5 m did not significantly reduce the maximum current.
Therefore, even micrometer scale features have to be controlled to allow reproducibility and comparisons between cultures and electrodes in this and future studies.
CV. CV is the principal diagnostic method in protein film
voltammetry, which studies immobilized enzymes on rotating
and stationary carbon electrodes. When in the presence of a
suitable substrate and slowly cycled through a range of applied
potentials at low scan rates, enzymes have time to undergo
multiple turnovers at each sampled potential (57, 33). As scan
rates increase, slow reactions may not have time to occur
before the potential changes to the next step, and concentration gradients representative of steady-state conditions may
not have time to establish. Thus, as the scan rate increases, the
kinetics of interfacial electron transfer between redox protein(s) and electrodes affects the voltammetric response more

strongly and can hide the kinetics of continuous enzymatic

turnover. As the purpose of this study was to establish methods
for measuring catalytic (turnover) kinetics in viable bacterial
biofilms, in the absence of prior knowledge regarding interfacial electron transfer rates, low scan rates represented the most
conservative experimental conditions. Parameters for the study
of nonturnover voltammetry at high scan rates will be discussed in subsequent reports.
Application of CV to living bacteria required limiting the
potential range to prevent harmful oxidizing or reducing conditions, as well as selection of informative scan rates. While it
is known that application of potentials can disrupt biofilms
either by producing hydrogen (28) or by inducing unfolding/
oxidation of adsorbed proteins at oxidizing potentials (52), G.
sulfurreducens biofilms were not harmed by exposure to potentials between 0.55 and 0.24 V versus SHE. For example,
routine CV did not slow or impede biofilm development, as the
anodic current after such analyses was 100.1% 3.4% (n 6)
of the current recorded before analyses. This was not unexpected, as Geobacter spp. are commonly exposed to metals in
this potential range. Future work with stricter anaerobes, or
organisms adapted to higher-potential environments, should
independently verify the potential range which is not harmful
to electron transfer.
Stable catalytic features with a high signal-to-noise ratio
were observed when CV was performed at low scan rates (1
mV/s). Under these conditions, the rate of electron transfer
rose rapidly at a potential higher than 0.3 V, reaching a
limiting current above a potential of 0.1 V (Fig. 4). This
response, commonly called a reversible catalytic wave (7),
indicated continuous regeneration of the entire series of proteins catalyzing electron transfer from the substrate to the
electrode. In preliminary investigations, each increase in the
scan rate significantly increased the ohmic current and shifted
the location of peaks in voltammetric waves, as expected from
kinetic effects (see Fig. S2 in the supplemental material). However, increasing the scan rate by fivefold did not significantly
alter the limiting current, demonstrating that at low scan rates,
conditions were within a timescale sufficient to establish sustained catalysis by cells at each applied potential. Since the
most informative scan rate could vary for each experimental
setup, scan rates used in this report should be taken only as an
aid in choosing conditions for new organism or electrode surface studies.
The inflection point(s) in such catalytic waves was visualized
as a peak using first-derivative analysis of voltammetry data
(using software described in Materials and Methods) (2, 16).
Such analysis of four independent biofilms is shown in Fig. 4.
One dominant inflection point could be detected by this analysis, centered at about 0.15 V, which increased in height with
the age of electrode-attached culture, while smaller intensity
peaks, centered at 0.220 V and 0.020 V versus SHE, could
also be detected. The dominant peak revealed in derivative
analyses was consistent with the presence of a single oxidation/
reduction event in electron transfer that was rate limiting (1, 9,
15). As shown in Fig. 4, these values were highly repeatable
between individual biological replicates.
Once a biocatalyst is adsorbed, it is also possible to determine its response to increasing concentrations of substrate
(24). To test this approach, the electron donor was first re-

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FIG. 3. SEMs of glassy carbon polished with P400 versus P4000 grit
treatment (top). Bar graph (below) shows maximum current measured
(n 2 for each roughness) during CA after 72 h of growth. Also
indicated (f, right y axis) is the average grit size of each polishing
treatment.

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FIG. 4. (A) Low scan rate CV of G. sulfurreducens biofilms after

inoculation (0 h) and at maximum current production (72 h). (B) First
derivatives of cyclic voltammograms of biofilms (P400 grit polishing
treatment) (n 4) showing the midpoint potential detectable in catalytic waves of mature biofilms.

moved from the medium, and electrodes were incubated at

0.24 V for 36 h until no residual anodic (oxidation) current was
observed. At this point, the effect of lowering substrate concentrations could be determined. The anodic-limiting current
at 0.1 V was determined from CV and was found to increase
linearly with the acetate concentration when acetate was 200
M. At acetate concentrations higher than 3 mM, the anodiclimiting current did not change (Fig. 5). The midpoint potential of catalytic waves remained centered at the same position
for all concentrations, suggesting a similar electron transfer
mechanism and rate-controlling reaction, even at low current
densities. This dose-response test detected acetate at concentrations as low as 3.5 M (signal-to-noise ratio, 3).
DPV. Although CV is the most informative method used to
investigate catalytic substrate oxidation by adsorbed enzymes,
it has a low detection limit, and subtraction of the ohmic
(capacitive) current is necessary to reveal small features (9).
Furthermore, when electron transfer between adsorbed enzymes and electrodes is slow, as is expected for complex electron transfer chains studied in stationary electrodes, CV re-

quires substantial time, and proper derivative analysis requires

postprocessing of data (40). In comparison, pulse methods
have the potential to reveal characteristic peaks while cancelling out capacitive current, even at higher scan rates, and are
often used as complementary techniques to CV (20, 44, 65).
Preliminary experiments with G. sulfurreducens biofilms indicated that DPV could also be used to monitor biofilms nondestructively, across a range of scan rates (up to 50 mV/s) and
pulse heights (up to 100 mV). The parameters chosen (see
Materials and Methods) represent a compromise between the
time of analysis and sensitivity. When performed on mature
biofilms, DPV always revealed a broad peak, which increased
in height with the age of the biofilm. These voltammograms
were highly reproducible, with the peak centered at 0.105
0.005 V versus SHE (Fig. 6).
Additional peaks at lower and higher potential with respect
to the main peak were also detected. The results were consistent with multiple redox centers exposed on the surfaces of G.
sulfurreducens cells that spanned a range of potentials (from
approximately 0.2 to 0.1 V). In recent studies with
Shewanella biofilms grown on similar electrodes, we observed
strikingly different behavior, as DPV revealed a large, less
convoluted peak at 0.21 V. These Shewanella cells were
found to secrete redox-active flavins that mediated electron
transfer (47), and changes in the DPV peak height could be
used to monitor both the accumulation of this redox-active
mediator at the electrode and its loss when the medium was
changed to remove soluble compounds. As the DPV peak
height was unaffected by medium changes in experiments with
Geobacter biofilms, this represented additional support for accumulation of an attached electron transfer agent and lack of
soluble mediators within G. sulfurreducens biofilms. These results demonstrate the utility of combining sweep and pulse
methods in the study of living systems.
EIS. While originally applied to investigation of solid-solid
interfaces, EIS is today the most common alternating current
method applied to the characterization of aqueous biointer-

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FIG. 5. Low scan rate CV (1 mV/s) of G. sulfurreducens at different

concentrations of electron donor. The biofilm was poised at oxidizing
potential without an electron donor until no anodic current was observed. Then electron donor was added, and the limiting current was
recorded (inset). The midpoint potential of catalytic curves was constant with increasing concentration of the electron donor.

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FIG. 6. (A) DPV (scan rate, 4 mV/s; pulse height, 50 mV) under
catalytic (presence of acetate) conditions at inoculation and after 72 h
of growth. (B) The shape of peaks and average potential for DPV of
mature biofilms (72 h) between four independent biological replicates.

faces (10, 13). In most EIS methods, a small sinusoidal potential perturbation is applied to the sample. The frequency of this
perturbation is changed in the range between few mHz and 105
Hz. The resulting sinusoidal current is analyzed via fast Fourier
transform techniques to calculate the impedance (Z) of the
interface in the frequency domain to estimate charge transfer
resistance (32), diffusion at surfaces covered by protein monolayers (20), charge transfer time constants (59), and mechanisms of electron transfer (8).
Applications of EIS are numerous in microbially influenced
corrosion, in which the sample is studied at the open circuit
potential (42, 51). However, we have shown that electron
transfer processes by G. sulfurreducens are voltage dependent
(example shown in Fig. 3). Thus, while EIS is typically performed at open-circuit potentials, potentiostatic EIS (where
the sinusoidal potential is applied while maintaining a given
potential at the working electrode) was more likely to reveal
electron transfer characteristics of active cells attached to electrodes.
Figure 7 shows a comparison of a glassy carbon electrode

before and after colonization by G. sulfurreducens. When EIS

was performed at 0.16 V versus SHE, the presence of G.
sulfurreducens reduced the charge transfer resistance (inferred
from the Z value at low frequencies), with a main time constant in the 1 to 10 Hz frequency range. At high rates of
electron transfer, a second, slower time constant was observed
in the 0.01 to 0.1 Hz range. The addition of a second time
constant to the model did not significantly improve the dispersion of residuals, likely due to the low frequency of the second
time constant.
The importance of potentiostatic EIS was illustrated by the
significant influence that the imposed potential had on the
charge transfer resistance and time constants for G. sulfurreducens biofilms (Fig. 8; Table 1). When the imposed potential
was similar to the midpoint potential of the catalytic wave
observed in CV, the lowest resistances and time constants were
measured. At potentials outside of this range, differences in
charge transfer were much smaller (in agreement with CV
results), resulting in higher apparent values for the charge
transfer resistance and time constant. Across the potential
range explored, the solution resistance (R 17.2 0.4 ),
double-layer capacitance (Cp 3.0 0.9 mF), and nonideality
coefficient (a 0.72 0.06) did not change appreciably. These
results again highlighted how data from one method (CV)
could be used to select parameters in a complementary method
(EIS). They also demonstrated the importance of investigating
the proper analysis parameters for each bacterial system, as
another strain with a different midpoint potential for its catalytic process would need to be studied at a different imposed
voltage to obtain the fastest time constant.
Implications. Many studies providing voltammetry data
from bacterium-electrode interactions have now been published. While reports differ in their pretreatment of bacteria,
biofilm age, growth medium, environmental conditions, electrode materials, and electrochemical parameters, a general
trend of catalytic wave-like behavior is clear (14, 31, 53, 68),
showing that many bacteria and communities behave in a man-

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FIG. 7. EIS of G. sulfurreducens biofilms at V 0.16 V versus

SHE; modulus of impedance (Z) after inoculation (F) and at 72 h (E);
phase angle V-I after inoculation (f) and at 72 h (). Biofilm development results in lower charge transfer resistance at the electrodebiofilm interface, with two distinct charge transfer processes around 1
and 0.03 Hz.

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TABLE 1. EIS parameters for biofilms at 72 ha

Potential vs
SHE (V)

Charge transfer
resistance ()

Characteristic
frequency (Hz)

Time
constant (s)

0.042
0.058
0.158
0.258

23,500
1,050
204
2,440

0.01
0.032
0.5
0.04

100
32
2
25

a
Data were fitted with the single time constant model described in Materials
and Methods. Values are averages of data from two separate experiments.

ner similar to the early stage Geobacter biofilms grown on

geometrically simple electrodes shown in this work. As a result,
we can confirm what early observations suggested, that
Geobacter bacteria behave as attached biocatalysts, stimulated
by changes across a narrow potential range. For example, in a
prior study, where we adsorbed washed Geobacter cells on
carbon paper electrodes (using pectin to retain free cells) (62),
similar features were reported even after a few hours of cells
being exposed to electrodes. These consistencies further suggest that these responses and potentials are not dramatically
influenced by such factors as stage of growth or electrode
material.
In particular, this study shows how the response of an intact
film to a range of applied potentials can be measured systematically and analyzed to produce data that are easily compared
between species, conditions, or electrode surfaces. As scan
rates are lowered, electron transfer kinetic effects (rates of
electron transfer between cell surfaces and electrodes) are
minimized, and enzymatic effects (related to microorganisms

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FIG. 8. EIS of mature G. sulfurreducens biofilms (72 h) performed

at different potentials. (A) Bode plot. Modulus of impedance (Z) at
0.04 (filled black circles), 0.06 (filled red circles), 0.16 (filled blue
circles), and 0.26 V (filled green circles) versus SHE; phase angle V-I
at 0.04 (open black circles), 0.06 (open red circles), 0.16 (open blue
circles), and 0.26 V (open green circles) versus SHE. Note that
frequency of the major charge transfer process changes with applied
potential. The second charge transfer process can be observed only at
0.16 V. (B) Nyquist plot. The relaxation times for the electron transfer process at the interface are summarized in Table 1.

activity) become primary factors. As the goal was to initially

characterize the catalytic behavior of the system, low scan rates
(1 mV/s) were chosen, since they permitted reactions with a
time constant on the order of 1 s to be active as turnover
processes at each imposed potential step. At high levels of
electron donor and low scan rates, catalytic voltammograms
should therefore be representative of steady-state conditions.
In addition, minimization of ohmic current aided in identification of inflection points in derivative analysis. The choice of
a relatively low scan rate was also supported by data from EIS
measurements, in which we found time constants on the order
of 0.1 and 1 to 10 s. For a system such as this, with what
appears to be sluggish interfacial electron transfer kinetics, the
potential difference between anodic and cathodic peaks for a
given redox couple could change significantly with even modest
changes in scan rate. Future work will focus on the use of
electrochemical techniques under nonturnover conditions to
better elucidate these electron transfer kinetics for a more
complete understanding of the interplay between microbial
catalytic abilities and interfacial electron transfer.
The high reproducibility between biological replicates and
the ability to perform experiments with a range of electrodes
provided a robust measurement of G. sulfurreducens current
densities, which approached 2 A/m2 for electrodes polished
with 35-m-grit-size particles. The catalytic wave consistently
observed for G. sulfurreducens is an independent demonstration that interior oxidative processes of this organism are
linked via a continuous pathway to surfaces and that the entire
collection of attached organisms (i.e., the biofilm) behaves as
an adsorbed catalyst. The midpoint potential of the catalytic
wave at 0.15 V supports a model with a dominant ratelimiting electron transfer reaction and shows that G. sulfurreducens respiration rate does not increase when cells are provided with an electron acceptor with a potential greater than 0
V. The latter result implies that the final step of electron
transfer (e.g., between a terminal external protein and the
electrode) is not rate limiting, as this process can always be
accelerated by additional applied potential (as described by the
standard rate law kf k0 eE) (9). The midpoint and the
limiting current potential found in this study are consistent
with G. sulfurreducens being adapted for the reduction of iron
oxides with a potential between 0.2 and 0 V versus SHE in
the environment and suggest that cells do not derive any additional energetic benefit from higher-potential electron acceptors.
While the study of an intact electron transport chain in G.
sulfurreducens represents perhaps a complex example of this
electrochemical approach, similar methods have been used to
characterize extracts from bacterial cells adsorbed onto, or

7336

MARSILI ET AL.

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21.

22.
23.

ACKNOWLEDGMENTS
We thank IREE and NSF (grant DBI-0454861) for their support, as
well as the BioTechnology Institute at the University of Minnesota.
We also thank Jeremy Alley for technical help with the experimental
setup, Dirk Heering for suggestions in the interpretation of data, and
Lewis Werner for the SEM images of bare electrodes.

24.

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