eScholarship provides open access, scholarly publishing
services to the University of California and delivers a dynamic
research platform to scholars worldwide.
Lawrence Berkeley National Laboratory
An Electrode-based approach for monitoring in situ microbial activity during subsurface
Lawrence Berkeley National Laboratory
LBNL Paper LBNL-2961E
Environmental Science and Technology, 44, 47-54, 2010
An electrode-based approach for monitoring in situ
microbial activity during subsurface bioremediation
Kenneth H. Williams1*, Kelly P. Nevin2, Ashley Franks2, Andreas Englert3, Philip E.
Long4, and Derek R. Lovley2
1Lawrence Berkeley National Laboratory, Berkeley, CA 94720
2Department of Microbiology, University of Massachusetts, Amherst, MA 01003
3Hydrogeology Department, Ruhr University, Bochum, Germany
4Pacific Northwest National Laboratory, Richland, WA 99352
*Corresponding author e-mail: email@example.com; phone: 510.701.1089
Current production by microorganisms colonizing subsurface electrodes and its
relationship to substrate availability and microbial activity was evaluated in an aquifer
undergoing bioremediation. Borehole graphite anodes were installed downgradient from
a region of acetate injection designed to stimulate bioreduction of U(VI); cathodes
consisted of graphite electrodes embedded at the ground surface. Significant increases in
current density (≤50 mA/m2) tracked delivery of acetate to the electrodes, dropping
rapidly when acetate inputs were discontinued. An upgradient control electrode not
exposed to acetate produced low, steady currents (≤0.2 mA/m2). Elevated current was
strongly correlated with uranium removal but minimal correlation existed with elevated
Fe(II). Confocal laser scanning microscopy of electrodes revealed firmly attached
biofilms, and analysis of 16S rRNA gene sequences indicated the electrode surfaces were
dominated (67-80%) by Geobacter species. This is the first demonstration that electrodes
can produce readily detectable currents despite long-range (6 m) separation of anode and
cathode, and these results suggest that oxidation of acetate coupled to electron transfer to
electrodes by Geobacter species was the primary source of current. Thus it is expected
that current production may serve as an effective proxy for monitoring in situ microbial
activity in a variety of subsurface anoxic environments.
There is a pressing need to monitor microbial activity in subsurface environments
because of its influence on groundwater chemistry and its role in controlling contaminant
fate and transport. Many of the approaches for estimating rates of microbial processes in
near-surface environments are not applicable to the subsurface. For example, it is
common to measure rates of microbial processes in short-term incubations of cores of
aquatic sediments injected with radiolabeled tracers of organic substrates or electron
acceptors . However, such an approach is not readily feasible for subsurface
environments, due to drilling costs and difficulty in obtaining undisturbed cores, and
because radiotracer measurements can significantly overestimate rates of metabolism in
subsurface environments .
This has led to the search for in situ measurements that can serve as a proxy for
microbial activity. One of the most successful has been measuring concentrations of
dissolved hydrogen to determine which anaerobic terminal electron-accepting processes
predominate in subsurface zones of interest . With this method, it is possible to
establish the terminal electron-accepting process, which often cannot be determined with
standard geochemical measurements. Unfortunately, hydrogen measurements provide no
indication of the rates of the microbial processes.
In some instances, it is possible to estimate rates of microbial processes in the
subsurface by monitoring changes in relevant groundwater constituents at sampling
points along groundwater flow paths  or in push-pull studies . However, these
approaches require extensive sampling and analytical expertise. Furthermore, these
approaches do not work when trying to estimate rates of multiple anaerobic respiratory
processes. For example, monitoring the accumulation of Fe(II) in groundwater greatly
underestimates Fe(III) reduction rates because most of the Fe(II) produced during Fe(III)
oxide reduction remains in solid phases or sorbed to mineral surfaces [6, 7]. Such
considerations indicate that novel approaches to the in situ measurement of anaerobic
processes in subsurface environments are required.
Acetate is expected to be an important intermediate in the metabolism of complex
organic matter in anoxic subsurface environments  and thus rates of acetate
metabolism are likely to approximate rates of anaerobic respiration. Furthermore, both
acetate and simple organic compounds, such as lactate and ethanol that are fermented to
acetate, are frequently added to groundwater to stimulate anaerobic respiration during
bioremediation of metals and chlorinated solvents [9-12]. Some microorganisms, most
notably members of the family Geobacteraceae, can oxidize acetate with electron
transfer to electrodes, producing current [13-15]. In natural systems, Geobacteraceae
colonize graphite electrodes buried in anoxic sediments, producing current from the
oxidation of acetate and products released from the fermentation of organic matter [13,
16]. Current levels are expected to be proportional to the rate of acetate production up to
the point where additional increases in acetate concentration do not yield additional
increases in current production due to limitations in the rate of electron transfer at both
anode and cathode [17, 18].
These considerations suggest that the rate of current production from graphite
electrodes in the subsurface might serve as a proxy for monitoring rates of microbial
activity. This possibility was suggested on the basis of the linear dependence between
current and acetate concentration (when ≤2.3 mM) observed during column experiments
using Geobacter sulfurreducens . These results corroborated those obtained using
more conventional microbial fuel cells in pure culture laboratory studies , which
show a similar dependence between acetate availability and current production.
There are many unknowns to the functioning of an electrode deployed in the
subsurface that are dependent upon the activity of natural microbial consortia. For
example, studies with laboratory and benthic microbial fuel cells have emphasized the
importance of close proximity between anode and cathode to promote optimal proton
transfer and maximum current output , suggesting that substantial distances between
subsurface anodes and air-coupled cathodes may not allow for significant current
production. Furthermore, the ability of aquifer microorganisms to colonize electrodes
and produce current has not previously been evaluated. Finally, the current production
capacity of subsurface microorganisms varies significantly, even among Geobacter
species , and potentially confounding current production from reduced metabolic end
products, such as Fe(II) and sulfide, interacting with electrodes must also be assessed.
This last concern is largely alleviated during remediation scenarios where organic
carbon amendments are designed to stimulate subsurface microbial activity. Such is the
case at Department of Energy study sites near Rifle, Colorado and Oak Ridge, Tennessee
where organic carbon additions to groundwater have repeatedly demonstrated the ability
to remove uranium from groundwater by stimulating the activity of metal reducing
microorganisms, such as Geobacter [9, 11, 12, 20]. Here we report on current production
using subsurface electrodes during in situ bioremediation at the Rifle site and
demonstrate a clear correspondence between current levels and both the availability of
acetate and the removal of uranium from groundwater. These results serve as the first
validation that such an approach may be employed as part of field bioremediation
activities capable of providing real time, semi-quantitative information relevant to
optimized uranium bioremediation.
Materials and Methods
A comprehensive description of the Rifle site has been presented elsewhere [12,
21]. Briefly, the site is located on a flood plain in Northwestern Colorado (USA)
consisting of an aquifer comprised of approximately 6.5 m of unconsolidated sands, silts,
clays and gravels deposited by the adjacent Colorado River. Elevated concentrations of
uranium (1-1.5 µM) in groundwater result from residual contamination by mill tailings.
Groundwater amendment, sampling, and transport properties
Acetate amendment experiments were conducted over consecutive summers
within the same experimental gallery (Fig. S1). In both cases, upgradient groundwater
was pumped into a storage tank and amended with sodium acetate and bromide to
achieve aquifer concentrations of 5 mM and 1.5 mM, respectively. Injection occurred
within ten boreholes and lasted 31 and 110 days during 2007 and 2008, respectively.
Amended groundwater was continuously injected at a rate of 16 L per injection well per
day. Both experiments involved short-duration intervals (6-8 days) bracketing the first
and second amendment tanks where neither acetate nor bromide was injected referred to
as a groundwater flush.
Groundwater samples were obtained from monitoring wells up and downgradient
from the injection galleries (Fig. S1). Acetate, bromide, and sulfate were measured using
an ion chromatograph (ICS-1000, Dionex) equipped with an AS-22 column. Fe(II) and
dissolved sulfide were filtered (0.2 µm) and measured immediately using the 1,10
Phenanthroline and Methylene Blue colorimetric methods, respectively (Hach Company,
Loveland, CO). Dissolved uranium was quantified using kinetic phosphorescence
analysis (Chemchek Instruments, Richland, WA).
An analytical solution of the convection dispersion equation (CDE) was fit to the
bromide breakthrough curves observed at D03 and D09 during the 2007 experiment using
established approaches ; the two injection peaks resulting from the groundwater flush
were combined in a single fitting procedure using superposition. Injection tank bromide
concentrations, injection volumetric flux, groundwater levels, and estimates of hydraulic
conductivity were used to estimate bromide concentrations in the vicinity of the injection
gallery during the first and second peak, after which the final CDE fit was obtained by
including the exact timing and concentration ratio between first and second peak.
Sensor design, deployment, and current monitoring
Anodic electrodes were installed within boreholes (5 cm; inner diameter), while
cathodes consisted of air-coupled electrodes embedded at the ground surface (Fig. 1). All
electrodes were composed of graphite cylinders (3 cm diameter), with baffles machined
into the base of each to increase surface area (220 cm2). All connections were made with
watertight connectors using marine-grade wire screwed into holes drilled into the
graphite and sealed with silver and marine epoxy. The borehole and surface electrodes
were connected using 560 Ω resistors, and charge balance and completion of the circuit
occurred through the sediments and pore fluid. Current was calculated at 30-minute
intervals using the voltage drop across the resistor and measured with a high impedance
Electrodes were deployed within the same upgradient (U03) and downgradient
monitoring boreholes (D03 and D09) during the consecutive field experiments (Fig. S1).
The 2007 experiment utilized electrodes deployed at a single depth located 5 m below
ground surface (bgs). The 2008 experiment utilized electrodes deployed at depths of 4, 5,
and 6 m bgs in D03 and D09; the control electrode in U03 was located at 5 m bgs. The
two monitoring wells were located 2.5 and 8.5 m, respectively, downgradient from the
region of acetate injection.
To maintain integrity and expedite colonization, electrodes were placed within
permeable housings (Fig. 1) and surrounded by site sediments; nylon mesh was used to
retain sediments within the housing. Sediment porosity (30%) yielded a graphite surface
area exposed to the pore fluids of 66 cm2. Prior to acetate amendment, the contact
resistance between anode and cathode ranged from 1-2 kΩ. Electrodes were removed
from D09 for confocal laser scanning microscopy and microbial community analysis
during both experiments. In 2007, the single electrode was removed after 29 days; in
2008, two electrodes were removed 261 days after beginning acetate injection.
Confocal laser scanning microscopy and 16S rRNA microbial community analysis
The spatial extent and thickness of biofilms associated with the borehole
electrodes was examined with confocal laser scanning microscopy. After recovery from
the boreholes, electrodes were placed at 4°C and shipped by overnight courier for
analysis. Prior to analysis, loosely bound material was removed by gentle washing with
isotonic buffer . Duplicate biofilm samples were fluorescently stained with either the
LIVE/DEAD BacLight bacterial viability kit (L7012, Molecular Probes, Inc., Eugene,
OR) or a generic nucleic acid binding stain (Syto9; Molecular Probes) and examined as
described . The image stacks were representative of the entire electrode as each
image was obtained from twenty random locations across the surface.
Biofilm samples for microbial community analysis were frozen on dry ice and
shipped by overnight courier for analysis. They were rinsed with sterile isotonic buffer
prior to analysis to remove sediments. A sterile razor blade was used to remove a
mixture of biomass plus graphite before extraction of DNA from the sample, PCR
amplification, and cloning of the 16S rRNA gene as described . Assembly of the
clone library was derived from a total of 48 clones, and at least half the electrode surface
was used for the analysis.
Results and Discussion
Current Production Associated with Acetate Amendment
An electrode deployed in well upgradient from the region of acetate injection
(U03) produced continuous, low levels of current (Fig. 2). In contrast, electrodes
deployed in downgradient wells D03 and D09 produced significant amounts of current in
the presence of elevated acetate concentrations. The maximum current densities of ca. 40
mA/m2 produced by the D03 and D09 electrodes were higher than the ca. 10 mA/m2 and
25 mA/m2 produced in freshwater  and marine  sediment microbial fuel cells
(MFC’s), respectively, and somewhat lower than laboratory-scale Geobacter
sulfurreducens MFC’s fed equivalent concentrations of acetate .
The initial increase in current from the D03 and D09 electrodes corresponded
with the initial appearance of acetate (Fig. 2). This was particularly evident for D03,
which had higher acetate concentrations. Bromide breakthrough data indicated that
upgradient consumption was the primary cause of diminished acetate delivery to D09
(Fig. 3). Maximum current densities in D03 and D09 were comparable even though
acetate concentrations were significantly higher in D03 at peak current. This suggests
that at high acetate concentrations factors other than acetate availability may limit current
production, as previously observed in laboratory incubations [15, 17, 25, 26]; the
inefficient reduction of molecular oxygen at the cathode was another likely contributing
Although the D09 electrode was removed for microbial analysis, the D03
electrode was retained for longer-term analysis of current production. During the
groundwater flush, acetate concentrations rapidly fell with a corresponding drop in
current (Fig. 2). Current rebounded when acetate was reintroduced, reaching a maximum
current density comparable to that observed prior to the flush. This was despite the fact
that acetate did not rebound to pre-flush levels, further emphasizing the observation that
factors other than acetate availability likely limit maximum current output. When
amendment ceased and acetate dropped below detectable limits, current densities
declined to levels slightly higher than those observed at the upgradient control.
Additional insight into the temporal current patterns was gained from analysis of
the CDE fits, which exhibited good (D03) to fair (D09) correspondence with
breakthrough of bromide (Fig. 3). The fits suggest that conservative transport (as
bromide) and current production are not related in a simple linear fashion. Such a
relationship would not be expected, however, as current production may not be treated in
an identical fashion to bromide breakthrough, given that it results from microbial activity
stimulated by transport of a non-conservative species (acetate) to the electrode. In
comparing the D03 electrode response to the CDE fit (Fig. 3C), a clear lag (4-5 days)
existed between delivery of amendments during the first peak and onset of current flow.
This lag – also observed at D09 – likely corresponded to the time required for
colonization of the electrode and onset of electron transfer tied to acetate oxidation. In
contrast, current production associated with the second injection peak began with no
discernible lag and began before arrival of maximum amendment concentrations (Fig.
3C). Such an effect would be expected given the existence of an affixed and pre-
conditioned microbial community capable of immediate current production following
resumption of acetate delivery.
The temporal change in current density in response to changes in acetate
concentration was remarkably similar to previously described changes in the metabolic
activity of Geobacter species during a previous field experiment at the Rifle site . In
that study, monitoring transcript levels of the Geobacter-specific citrate synthase gene
(gltA) indicated rates of metabolism that (a) increased sharply when acetate was
introduced into the system, (b) decreased when acetate additions were temporarily
disrupted, (c) increased again when acetate additions were resumed, and (d) declined
when acetate inputs were stopped.
One concern in using current as a proxy for microbial activity is the potential for
abiotic current production from reduced species, such as Fe(II) and dissolved sulfide.
Although sulfide was not detected during the 2007 field experiment, aqueous Fe(II) did
accumulate during acetate amendment (Fig. 2). In contrast to the clear relationship
between acetate availability and current, there was no clear relationship between Fe(II)
and current. For example, the dip in current from day 19-22 was associated with the
greatest increase in Fe(II), and high concentrations of Fe(II) from day 45 onward
corresponded to periods of low current density. In contrast to Fe(II), there was a strong
relationship between current levels and the removal of U(VI) from the groundwater (Fig.
2). As current densities reached high levels, U(VI) declined substantially. When current
declined, U(VI) concentrations rebounded to influent levels.
The field-derived relationships between Fe(II) and current production were
corroborated by laboratory experiments in which inoculated (Geobacter sulfurreducens)
and uninoculated fuel cells were poised at +300 mV (vs. Ag/AgCl) and supplemented
with 5 mM FeCl2. Whereas acetate addition to the inoculated fuel cell generated a
steady-state current of 12 mA, no additional increase in current was detected following
Fe(II) addition (data not shown). Similarly, no increase in current was observed after
Fe(II) addition to the uninoculated control. These results suggest that elevated
concentrations of Fe(II) are insufficient to enable significant current production above
background levels. Rather, current production during microbial Fe(III) reduction appears
to result primarily from electron transfer to electrodes by electrode-respiring
microorganisms suggesting sensitivity of the method to microbial activity rather than
accumulation of a reduced by-product.
Several possibilities for the apparent lack of Fe(II)-mediated current production
exist. Graphite fuel cells optimized for the abiotic oxidation of Fe(II) have demonstrated
that large current densities are achievable at high ionic strength and low pH (2-4 M
H2SO4) . In contrast, the circumneutral pH and low ionic strength conditions of the
laboratory and field experiments may have minimized rates of Fe(II) oxidation. Indeed,
rates of electron transfer for Fe(II)/Fe(III) couples on glassy carbon surfaces decrease by
10-100 fold with decreases in ionic strength from 1 to 0.01 M , with the latter value
characteristic of our studies. Just as an enzymatic catalyst enables charge transfer during
acetate amendment, the presence of a similar catalyst – or catalytic effect, such as
electrode pretreatment – may be required to enable high rates of Fe(II)-mediated electron
transfer. Even under ideal conditions using polished and/or hydrogenated glassy carbon
electrodes, measured electron transfer rate constants for different iron compounds vary
dramatically, with rate constants for compounds relevant to our field and laboratory
experiments (e.g. Fe2+/3+) three orders of magnitude lower than those of more reactive
iron couples (e.g. Fe(CN)63-/4-) . The unpolished and untreated graphite electrodes
used here would thus be expected to only marginally enable the Fe(II) oxidation process.
Lastly, adsorption of non-catalytic, passivating organics to the electrodes could have
further degraded their capacity for mediating significant rates of Fe(II) oxidation.
Geobacter species were the most abundant subsurface microorganisms during the
active phase of U(VI) removal with Geobacter 16S rRNA gene sequences accounting for
76-98% of the sequences recovered in groundwater samples . This finding is
consistent with the concept that Geobacter species are responsible for acetate-stimulated
U(VI) reduction at the Rifle site and the finding that Geobacter species are consistently
the most abundant microorganisms in groundwater during such field studies [9, 12, 27,
30, 31]. Analysis of the 16S rRNA gene sequences extracted from the D09 electrode -
harvested as current density peaked and U(VI) removal commenced - revealed a
predominance of Geobacter species (Fig. 4), which accounted for 67% of the sequences
recovered. The more minor sequences were distributed in the gamma (16%), beta (12%),
and alpha (2%) Proteobacteria and Firmicutes (2%). The Geobacter sequences on the
electrode were most similar (96% similarity) to those of Geobacter strain M18, which
was recovered in culture from the Rifle site and has a 16S rRNA gene sequence matching
one that predominates in the groundwater during uranium bioremediation. These results
suggest that the Geobacter species colonizing the electrode were similar to those
involved in U(VI) reduction. If so, the physiological responses of Geobacter species
predominating on the electrode may be similar to those of Geobacter species primarily
responsible for U(VI) reduction.
Confocal laser scanning microscopy revealed that the biofilms were 25-50 µm
thick (Fig. 5). Based on background fluorescence, the stained images were not
significantly influenced by the presence of fluorescent minerals (Fig. S2). The biofilms
were morphologically similar to those formed by G. sulfurreducens on graphite surfaces
in laboratory studies [15, 32]. When treated with the LIVE/DEAD BacLight viability kit,
30-40% of the cells in the biofilm stained green, suggesting viability (Fig. 5A). It has
been proposed, however, that some cells staining red with this kit may also be viable
, suggesting that a higher proportion of cells were capable of contributing to current
production. The dense accumulation of cells observed using the general nucleic acid
binding stain (Fig. 5B) suggests this is a possibility. Previous studies with G.
sulfurreducens have suggested that cells at distances up to 75 µm from the electrode
surface may contribute to current production, possibly via electron transfer through
conductive filaments [32, 34], outer-surface c-type cytochromes and/or multicopper
proteins [24, 31, 35], or a combination of mechanisms .
Current Production Following Long-Term Acetate Amendment
Electrode-based monitoring during acetate amendment was repeated in 2008 and
a similar correspondence between groundwater acetate concentrations and current was
observed (data not shown). Unfortunately, changes in injection approach and decreases
in aquifer permeability made continuous and uniform delivery of acetate problematic,
thus confounding reliable interpretation of current patterns during acetate amendment.
The 2008 experiment did provide the opportunity to evaluate long-term current
production following prolonged organic input. After acetate concentrations fell to levels
below detection, the D03 and D09 electrodes yielded low but steady current densities of
3-6 mA/m2 for 100+ days (data not shown). These lower values suggest a slower rate of
microbial metabolism during this period than was observed during periods of high acetate
concentration, consistent with the lower availability of acetate. However, current
densities were significantly higher than the 0.05-0.2 mA/m2 values observed in the
upgradient control well not exposed to acetate. This is consistent with the concept that
following prolonged periods of acetate addition there are substantial quantities of
moribund biomass that can promote prolonged microbial metabolism .
Two electrodes deployed in D09 were removed after 261 days of current
production. The maximum biofilm thickness was 5-10 µm and ca. 10% of the cells in the
biofilm stained green with the viability stain (Fig. S3). This thinner biofilm was
consistent with the lower current output and the general correspondence expected
between biofilm thickness and current production . As with the 2007 electrode,
Geobacter species predominated, accounting for more than 80% of the sequences (Fig.
S4). The predominant Geobacter species were most closely related to the subsurface
isolates G. psychrophilus and G. chapellei, which were isolated from an acetate-impacted
aquifer  and a deep subsurface site , respectively. This contrasts with the
predominance of Geobacter species most closely related to strain M18 on the 2007
electrode suggesting growth of different Geobacter species in the subsurface may be
favored under different environmental conditions.
Implications for future monitoring studies.
These results demonstrate that electrodes deployed in anoxic subsurface
environments can serve as electron acceptors for microbial metabolism even when anode-
cathode separations are large. This is the requisite first step for ultimately using
electrode-based approaches for quantifying rates of microbial metabolism in the
subsurface. In the interim, monitoring temporal changes in current appears to be a useful
tool for ensuring appropriate concentrations of electron donor are being supplied to a
desired location, such as occurs during the remediation of chlorinated solvents,
perchlorate, and other metal and radionuclide contaminants that rely on electron donor
amendments to stimulate desired metabolic processes.
Now that is it is known that sufficient current can be produced from subsurface
electrodes to readily detect microbial metabolism in situ - at least at the elevated levels
associated with the introduction of allochthonous organic compounds - this raises the
possibility of monitoring rates of microbial metabolism in aquifers contaminated with
organics such as petroleum hydrocarbons or landfill leachate. These are contaminants
that Geobacter species and other organisms can oxidize with the reduction of Fe(III), and
hence their oxidation coupled to electrodes might also be expected . To further
expand the applicability of the method, studies are warranted at sites having different
lithological properties (e.g. low permeability silts and fractured rock) and where
bioremediation activities targeting other metals and radionuclides (e.g. chromium and
technetium) via stimulation of metal-reducing microorganisms are underway (e.g.
Hanford, Washington and Oak Ridge, Tennessee).
The predominance of Geobacter species on the electrodes, coupled with the
known ability of Geobacter species to effectively couple the oxidation of organic
compounds to electron transfer to electrodes, suggests that Geobacter species played an
important role in the current production at the Rifle site. The current production patterns
in response to changes in acetate availability were similar to previously documented
responses in metabolic rates of Geobacter species at the Rifle site during similar
fluctuations in acetate concentration [27, 31]. Furthermore, high currents were associated
with the rapid removal of U(VI) from groundwater, an activity previously attributed to
Geobacter species reducing soluble U(VI) to insoluble U(IV) .
Additionally, colonization of subsurface electrodes at the Rifle site by Geobacter
species was similar to the abundance of Geobacter species found on the anodes of
sediment microbial fuel cells [13, 16, 24], as well as the colonization of anodes by
Geobacter species in laboratory systems capable of producing high current densities
when inoculated with sewage and amended with acetate [39-41]. However, a wide
diversity of microorganisms are capable of electron transfer to electrodes via a variety of
mechanisms , and it remains quite possible that in other subsurface environments
microorganisms other than Geobacter species could be the primary current producers.
Although current production from subsurface electrodes may eventually be used
to quantify rates of subsurface microbial metabolism, it cannot unambiguously discern
the terminal electron accepting process associated with those rates. By coupling
hydrogen measurements - which can identify the terminal electron accepting process
associated with current measurements - the possibility exists of gaining a more complete
understanding of subsurface anaerobic microbial processes than is presently available
with other approaches.
Acknowledgments. Funding was provided by the Environmental Remediation Science
Program, Office of Biological and Environmental Research, U.S. Department of Energy
(DE-AC02-05CH11231), Cooperative Agreement DE-FC02ER63446, and the Office of
Naval Research N00014-07-1-0966. We thank Mike Wilkins, Hila Elifantz, and Lucie
N’Guessan for their assistance with field experiments and Sarah Morris for quantifying
groundwater uranium concentrations.
Supporting Information. This document contains supporting information consisting of
four figures intended to supplement the material presented in the manuscript. These
include the electrode layout, as well as additional confocal laser scanning microscopy and
microbial community analysis results.
We describe a novel electrode-based technique for monitoring the in situ activity of
microorganisms in anoxic subsurface environments.
(1) Fossing, H.; Jorgensen, B. B., Oxidation and Reduction of Radiolabeled Inorganic
Sulfur-Compounds in an Estuarine Sediment, Kysing Fjord, Denmark. Geochim.
Cosmochim. Acta 1990, 54, (10), 2731-2742.
Chapelle, F. H.; Lovley, D. R., Rates of Microbial-Metabolism in Deep Coastal-Plain
Aquifers. Appl. Environ. Microb. 1990, 56, (6), 1865-1874.
Lovley, D. R.; Chapelle, F. H.; Woodward, J. C., Use of Dissolved H2 Concentrations to
Determine the Distribution of Microbially Catalyzed Redox Reactions in Anoxic
Groundwater. Environ. Sci. Technol. 1994, 28, (7), 1205-1210.
Chapelle, F. H., Ground-water microbiology and geochemistry. 2 ed.; John Wiley &
Sons: New York, 2001; p 424.
Istok, J. D.; Humphrey, M. D.; Schroth, M. H.; Hyman, M. R.; OReilly, K. T., Single-
well, ''push-pull'' test for in situ determination of microbial activities. Ground Water
1997, 35, (4), 619-631.
Hansel, C. M.; Benner, S. G.; Fendorf, S., Competing Fe(II)-induced mineralization
pathways of ferrihydrite. Environ. Sci. Technol. 2005, 39, (18), 7147-7153.
Roden, E. E.; Urrutia, M. M., Influence of biogenic Fe(II) on bacterial crystalline Fe(III)
oxide reduction. Geomicrobiol. J. 2002, 19, (2), 209-251.
Lovley, D. R.; Chapelle, F. H., Deep Subsurface Microbial Processes. Rev. Geophys.
1995, 33, (3), 365-381.
Cardenas, E.; Wu, W. M.; Leigh, M. B.; Carley, J.; Carroll, S.; Gentry, T.; Luo, J.;
Watson, D.; Gu, B.; Ginder-Vogel, M.; Kitanidis, P. K.; Jardine, P. M.; Zhou, J.; Criddle,
C. S.; Marsh, T. L.; Tiedje, J. A., Microbial communities in contaminated sediments,
associated with bioremediation of uranium to submicromolar levels. Appl. Environ.
Microb. 2008, 74, (12), 3718-3729.
He, J. Z.; Sung, Y.; Dollhopf, M. E.; Fathepure, B. Z.; Tiedje, J. M.; Loffler, F. E.,
Acetate versus hydrogen as direct electron donors to stimulate the microbial reductive
dechlorination process at chloroethene-contaminated sites. Environ. Sci. Technol. 2002,
36, (18), 3945-3952.
Wu, W. M.; Carley, J.; Luo, J.; Ginder-Vogel, M. A.; Cardenas, E.; Leigh, M. B.;
Hwang, C. C.; Kelly, S. D.; Ruan, C. M.; Wu, L. Y.; Van Nostrand, J.; Gentry, T.; Lowe,
K.; Mehlhorn, T.; Carroll, S.; Luo, W. S.; Fields, M. W.; Gu, B. H.; Watson, D.; Kemner,
K. M.; Marsh, T.; Tiedje, J.; Zhou, J. Z.; Fendorf, S.; Kitanidis, P. K.; Jardine, P. M.;
Criddle, C. S., In situ bioreduction of uranium (VI) to submicromolar levels and
reoxidation by dissolved oxygen. Environ. Sci. Technol. 2007, 41, (16), 5716-5723.
Anderson, R. T.; Vrionis, H. A.; Ortiz-Bernad, I.; Resch, C. T.; Long, P. E.; Dayvault, R.;
Karp, K.; Marutzky, S.; Metzler, D. R.; Peacock, A.; White, D. C.; Lowe, M.; Lovley, D.
R., Stimulating the in situ activity of Geobacter species to remove uranium from the
groundwater of a uranium-contaminated aquifer. Appl. Environ. Microb. 2003, 69, (10),
Bond, D. R.; Holmes, D. E.; Tender, L. M.; Lovley, D. R., Electrode-reducing
microorganisms that harvest energy from marine sediments. Science 2002, 295, (5554),
Bond, D. R.; Lovley, D. R., Electricity production by Geobacter sulfurreducens attached
to electrodes. Appl. Environ. Microb. 2003, 69, (3), 1548-1555.
Nevin, K. P.; Richter, H.; Covalla, S. F.; Johnson, J. P.; Woodard, T. L.; Orloff, A. L.;
Jia, H.; Zhang, M.; Lovley, D. R., Power output and columbic efficiencies from biofilms
of Geobacter sulfurreducens comparable to mixed community microbial fuel cells.
Environ. Microbiol. 2008, 10, (10), 2505-2514.
(16) Tender, L. M.; Reimers, C. E.; Stecher, H. A.; Holmes, D. E.; Bond, D. R.; Lowy, D. A.;
Pilobello, K.; Fertig, S. J.; Lovley, D. R., Harnessing microbially generated power on the
seafloor. Nat. Biotechnol. 2002, 20, (8), 821-825.
Tront, J. M.; Fortner, J. D.; Plotze, M.; Hughes, J. B.; Puzrin, A. M., Microbial fuel cell
biosensor for in situ assessment of microbial activity. Biosens. Bioelectron. 2008, 24, (4),
Zhao, F.; Harnisch, F.; Schrorder, U.; Scholz, F.; Bogdanoff, P.; Herrmann, I.,
Challenges and constraints of using oxygen cathodes in microbial fuel cells. Environ. Sci.
Technol. 2006, 40, (17), 5193-5199.
Logan, B. E.; Hamelers, B.; Rozendal, R. A.; Schrorder, U.; Keller, J.; Freguia, S.;
Aelterman, P.; Verstraete, W.; Rabaey, K., Microbial fuel cells: Methodology and
technology. Environ. Sci. Technol. 2006, 40, (17), 5181-5192.
N'Guessan, A. L.; Vrionis, H. A.; Resch, C. T.; Long, P. E.; Lovley, D. R., Sustained
removal of uranium from contaminated groundwater following stimulation of
dissimilatory metal reduction. Environ. Sci. Technol. 2008, 42, (8), 2999-3004.
Mouser, P. J.; N'Guessan, A. L.; Elifantz, H.; Holmes, D. E.; Williams, K. H.; Wilkins,
M. J.; Long, P. E.; Lovley, D. R., Influence of Heterogeneous Ammonium Availability
on Bacterial Community Structure and the Expression of Nitrogen Fixation and
Ammonium Transporter Genes during in Situ Bioremediation of Uranium-Contaminated
Groundwater. Environ. Sci. Technol. 2009, 43, (12), 4386-4392.
Lapidus, L.; Amundson, N. R., Mathematics of Adsorption in Beds .6. The Effect of
Longitudinal Diffusion in Ion Exchange and Chromatographic Columns. J. Phys. Chem.
1952, 56, (8), 984-988.
Butler, J. E.; Kaufmann, F.; Coppi, M. V.; Nunez, C.; Lovley, D. R., MacA a diheme c-
type cytochrorne involved in Fe(III) reduction by Geobacter sulfurreducens. J. Bacteriol.
2004, 186, (12), 4042-4045.
Holmes, D. E.; Bond, D. R.; O'Neill, R. A.; Reimers, C. E.; Tender, L. R.; Lovley, D. R.,
Microbial communities associated with electrodes harvesting electricity from a variety of
aquatic sediments. Microbial Ecol. 2004, 48, (2), 178-190.
Franks, A. E.; Nevin, K. P.; Jia, H. F.; Izallalen, M.; Woodard, T. L.; Lovley, D. R.,
Novel strategy for three-dimensional real-time imaging of microbial fuel cell
communities: monitoring the inhibitory effects of proton accumulation within the anode
biofilm. Energ. Environ. Sci. 2009, 2, (1), 113-119.
Torres, C. I.; Marcus, A. K.; Rittmann, B. E., Proton transport inside the biofilm limits
electrical current generation by anode-respiring bacteria. Biotechnol. Bioeng. 2008, 100,
Holmes, D. E.; Nevin, K. P.; O'Neil, R. A.; Ward, J. E.; Adams, L. A.; Woodard, T. L.;
Vrionis, H. A.; Lovley, D. R., Potential for quantifying expression of the Geobacteraceae
citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on
current-harvesting electrodes. Appl. Environ. Microb. 2005, 71, (11), 6870-6877.
Lee, J.; Darus, H. B.; Langer, S. H., Electrogenerative oxidation of ferrous ions with
graphite electrodes. J. Appl. Electrochem. 1993, 23, (7), 745-752.
Chen, Q. Y.; Swain, G. M., Structural characterization, electrochemical reactivity, and
response stability of hydrogenated glassy carbon electrodes. Langmuir 1998, 14, (24),
Akob, D. M.; Mills, H. J.; Gihring, T. M.; Kerkhof, L.; Stucki, J. W.; Anastacio, A. S.;
Chin, K. J.; Kusel, K.; Palumbo, A. V.; Watson, D. B.; Kostka, J. E., Functional diversity
and electron donor dependence of microbial populations capable of U(VI) reduction in
radionuclide-contaminated subsurface sediments. Appl. Environ. Microb. 2008, 74, (10),
(31) Holmes, D. E.; Mester, T.; O'Neil, R. A.; Perpetua, L. A.; Larrahondo, M. J.; Glaven, R.;
Sharma, M. L.; Ward, J. E.; Nevin, K. P.; Lovley, D. R., Genes for two multicopper
proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different
expression patterns both in the subsurface and on energy-harvesting electrodes.
Microbiology+ 2008, 154, 1422-1435.
Reguera, G.; Nevin, K. P.; Nicoll, J. S.; Covalla, S. F.; Woodard, T. L.; Lovley, D. R.,
Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens
fuel cells. Appl. Environ. Microb. 2006, 72, (11), 7345-7348.
Shi, L.; Gunther, S.; Hubschmann, T.; Wick, L. Y.; Harms, H.; Muller, S., Limits of
propidium iodide as a cell viability indicator for environmental bacteria. Cytom. Part A
2007, 71A, (8), 592-598.
Reguera, G.; McCarthy, K. D.; Mehta, T.; Nicoll, J. S.; Tuominen, M. T.; Lovley, D. R.,
Extracellular electron transfer via microbial nanowires. Nature 2005, 435, (7045), 1098-
Nevin, K. P.; Kim, B. C.; Glaven, R. H.; Johnson, J. P.; Woodard, T. L.; Methe, B. A.;
Didonato, R. J.; Covalla, S. F.; Franks, A. E.; Liu, A.; Lovley, D. R., Anode biofilm
transcriptomics reveals outer surface components essential for high density current
production in Geobacter sulfurreducens fuel cells. PLoS One 2009, 4, (5), e5628.
Lovley, D. R., Microbial fuel cells: novel microbial physiologies and engineering
approaches. Curr. Opin. Biotechnol. 2006, 17, (3), 327-332.
Nevin, K. P.; Holmes, D. E.; Woodard, T. L.; Covalla, S. F.; Lovley, D. R.,
Reclassification of Trichlorobacter thiogenes as Geobacter thiogenes comb. nov. Int. J.
Syst. Evol. Microbiol. 2007, 57, 463-466.
Coates, J. D.; Bhupathiraju, V. K.; Achenbach, L. A.; McInerney, M. J.; Lovley, D. R.,
Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new,
strictly anaerobic, dissimilatory Fe(III)-reducers. Int. J. Syst. Evol. Microbiol. 2001, 51,
Ishii, S.; Watanabe, K.; Yabuki, S.; Logan, B. E.; Sekiguchi, Y., Comparison of
Electrode Reduction Activities of Geobacter sulfurreducens and an Enriched Consortium
in an Air-Cathode Microbial Fuel Cell. Appl. Environ. Microb. 2008, 74, (23), 7348-
Jung, S.; Regan, J. M., Comparison of anode bacterial communities and performance in
microbial fuel cells with different electron donors. Appl. Microbiol. Biotechnol. 2007, 77,
Liu, Y.; Harnisch, F.; Fricke, K.; Sietmann, R.; Schroder, U., Improvement of the anodic
bioelectrocatalytic activity of mixed culture biofilms by a simple consecutive
electrochemical selection procedure. Biosens. Bioelectron. 2008, 24, (4), 1006-1011.
Figure 1. Illustration of the sensor design for evaluating subsurface microbial activity via
current production from subsurface electrodes. Anodes consist of graphite electrodes
embedded within a sand pack (encapsulating mesh not shown) located within the aquifer,
while air-coupled cathodes are embedded in the soil.
Figure 2. Temporal changes in acetate, uranium, Fe(II), and current density at wells U03,
D03, and D09 during the 2007 experiment. The D09 electrode was removed for
microbial analysis on day 29; the 6-day acetate-free groundwater flush started on day 12.
Figure 3.: Comparison of conservative transport behavior and current production for D03
and D09: (A) and (B) measured bromide concentrations and fitted convection dispersion
equation (CDE) including estimates from the fitting procedure (v=velocity in m/d,
d=dispersivity in m and cn=bromide concentration at the injection in mM). (C) and (D)
current production versus the CDE fits.
Figure 4. Microbial community composition based on 16S rRNA gene sequences
extracted from the D09 electrode on day 29 during the 2007 experiment; Geobacter
clones were 96% similar to clade M18.
Figure 5. Confocal laser scanning microscopy (3-D projections and cross sections) of the Download full-text
D09 (5 m bgs) electrode recovered during peak current flow (day 29) in 2007. (A)
Results using the LIVE/DEAD BacLight viability kit, with putative viable cells
exhibiting green fluorescence and putative moribund cells staining red. (B) Results using
the Syto9 general nucleic acid binding stain.