Article
pubs.acs.org/est
Temporal-Spatial Changes in Viabilities and Electrochemical
Properties of Anode Biofilms
Dan Sun,† Shaoan Cheng,*,† Aijie Wang,‡ Fujian Li,† Bruce E. Logan,§ and Kefa Cen†
†
State Key Laboratory of Clean Energy Utilization, Department of Energy Engineering, Zhejiang University, Hangzhou 310027, P.R.
China
‡
Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, China Academy of Sciences,
Beijing, China
§
Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802,
United States
S Supporting Information
*
ABSTRACT: Sustained current generation by anodic biofilms
is a key element for the longevity and success of
bioelectrochemical systems. Over time, however, inactive or
dead cells can accumulate within the anode biofilm, which can
be particularly detrimental to current generation. Mixed and
pure culture (Geobacter anodireducens) biofilms were examined
here relative to changes in electrochemical properties over
time. An analysis of the three-dimensional metabolic structure
of the biofilms over time showed that both types of biofilms
developed a live outer-layer that covered a dead inner-core.
This two-layer structure appeared to be mostly a result of
relatively low anodic current densities compared to other
studies. During biofilm development, the live layer reached a
constant thickness, whereas dead cells continued to accumulate near the electrode surface. This result indicated that only the live
outer-layer of biofilm was responsible for current generation and suggested that the dead inner-layer continued to function as an
electrically conductive matrix. Analysis of the electrochemical properties and biofilm thickness revealed that the diffusion
resistance measured using electrochemical impedance spectroscopy might not be due to acetate or proton diffusion limitations to
the live layer, but rather electron-mediator diffusion.
■
of ∼5 mS/cm measured for G. sulf urreducens biofilms.10,11,13
There is currently no consensus on which of these two models
is correct. Therefore, additional insights into the structure of
the biofilm relative to its electrochemical properties may help
further our understanding of the mechanism of electron
transfer in these biofilms, as well as lead to improvements in
BES performance.12
The growth of bacteria in thick electrogenic biofilms can be
affected by biofilm conductivity and rates of diffusion of
substrates and end products.6,9,14−18 Three different conditions
have been considered important relative to bacteria location
within the biofilm (i.e., nearer the anode interface or solution
interface) and metabolic activity. First, if the biofilm electrical
conductivity limits growth, then the metabolic activity of the
outer cells (at the solution interface) could become restricted
by long distance EET due to charge transfer resistances. In this
case, the metabolic activity would decrease with distance from
INTRODUCTION
Bioelectrochemical systems (BESs), such as microbial fuel cells
(MFCs) used for electricity generation and microbial
electrolysis cells (MECs) for hydrogen production, are
technologies in which exoelectrogens generate electrical current
by oxidizing organic matter and using the anode as their
terminal electron acceptor.1 Anodic microorganisms, such as
Geobacter sulfurreducens and Geobacter anodireducens,2 as well as
other bacteria in mixed microbial communities, attach to the
anode surface and form a thick biofilm.3 Long-distance
extracellular electron transfer (EET) occurs in these thick
biofilms at distances of tens or even hundreds of microns
distant from the electrode surface.4−6 There are two competing
models proposed for long-distance EET through the electrically
conductive biofilms: electron hopping; and metallic-like
conductivity.7−12 According to the electron hopping model,
electrons are relayed through the biofilm by electron hopping/
tunneling using mediators positioned in the exopolymer matrix
and on pili.8 In contrast, the metallic-like conduction model
describes electron transport as occurring by delocalized
electrons in the pili (nanowire) network within the biofilm.10
Both of these models support the high electronic conductivity
© 2015 American Chemical Society
Received:
Revised:
Accepted:
Published:
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March 15, 2015
March 26, 2015
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the anode.19−21 Second, if accumulation of metabolic end
products limited growth, for example proton accumulation
leading to low pH,14 then growth at the solution interface
would be favored. Third, if diffusion of substrate to the biofilm
was important, then bacterial growth at the solution interface
would again be favored for improved metabolic activity.6,17 So
far, most previous studies have generally shown that the entire
biofilm remained metabolically active.4,5,10,16,22−25 However,
metabolic activities relative to temporal changes and spatial
structure, and current densities and electrochemical properties
have not been previously investigated. The activity of the
biofilm can change over time, and thus it is possible that biofilm
viability might change as well. Changes in either EET pathways
or inherent biofilm conductivity could affect its electrochemical
activity, and thus current densities over time.
In this study, spatial metabolic structure, biological properties
(morphology, biomass, and microbial community), and
electrochemical properties (current generation, linear sweep
voltammetry, nonturnover cyclic voltammetry, electrochemical
impedance spectroscopy analyses for charge transfer and
diffusion resistance) were simultaneously investigated throughout the biofilm development process. Both mixed culture and
pure culture biofilms were examined. A pure culture of G.
anodireducens was studied because this strain was isolated from
a mature biofilm of an MFC anode, as opposed to other
Geobacter strains that have been isolated under iron reducing
conditions.26 G. anodireducens SD-1 forms thicker biofilms than
G. sulf urreducens PCA, a species which has previously been
studied and indicated to be predominant in several mixedculture biofilms.2,26−28 However, these two species share more
than a 98% similarity based on 16S rRNA sequences, so there is
no certainty about which strain was really predominant in
previous studies in anode biofilms. Mini-BESs were used here
to cultivate biofilms.27 These reactors have low internal
resistance, they have shown to be useful for studying both
pure and mixed cultures,27,29 and their simple construction and
operation enabled us to simultaneously operate dozens of
reactors in order to independently examine multiple biological
and electrochemical properties of the biofilms over real time. As
a large number of reactors can be started up at the same time,
individual reactors could be sacrificed and analyzed over time to
avoid changes in the biofilms that would occur when repeatedly
analyzing reactors using disruptive chemicals (such as
fluorescent dyes) or exposing the biofilms to different
potentials during various electrochemical analyses.5,6
were used to record electrode potentials in some tests. All
electrode potential values are reported in this study vs SHE.
Before being inserted into the BESs, the Ag/AgCl electrodes
were immersed in 70% ethanol for 6 h to sterilize them.
Two-chamber BESs32 and air-cathode BESs33 were constructed as previously described for additional tests of the
metabolic structure of anode biofilms in BESs with different
architectures. The electrodes were the same with those in miniBES except that an air cathode was used in the single-chamber,
air-cathode BES.34 A two-chamber BES was created by
separating the electrode using an anion exchange membrane
(AMI-7001S; Membrane International, NJ), and it was
operated at a slightly higher applied voltage (0.9 V) to increase
the rate of biofilm growth. The air-cathode BES produced
voltage, and was operated with a 1000 Ω external resistance for
half a month.
Mini-BES Operation and Biofilm Growth. The initial
inocula were the effluents from the two mini-BESs that were
inoculated with G. anodireducens SD-1 or domestic wastewater,
and operated for a 1 week period as previously described.27
These two inocula were diluted to the same cell density (based
on cell counts), and then used to inoculate BESs (10% v/v).
Except as noted (in electrochemical tests without an electron
donor), BESs were operated with a 50 mM phosphate buffer
solution (PBS) nutrient medium that contained (per liter): 1 g
acetate, 2.45 g NaH2PO4·H2O, 4.58 g Na2HPO4, 0.31 g NH4Cl,
0.13 g KCl, 12.5 mL metal salts and 5 mL vitamins (pH 7;
conductivity = 7.5 mS/cm).27 All mini-BESs were filled with
100% N2 in the headspace, and sterilized by autoclaving before
being inoculated. All reactors were operated in fed-batch mode
at 30 °C. The cycle time of the mini-BES was 1 day, except for
the first cycle (2−3 days). For each culture, three mini-BESs
were used at each time-point to examine the metabolic
structure, protein content of the anode, and electrochemical
properties. Based on the current generation and visual biofilm,
the BESs were examined at the end points of cycles 1, 2, 3, 5,
12, 30, 45, 60 for both types of inocula, and also at cycle 78 for
the mixed culture. In addition, other BESs used in the
experiments (mini-BES with 0.9 V applied voltage, mini-BES
with small anode, mini-BES inoculated with G. sulf urreducens
PCA, a two-chamber BES and an air-cathode BES) were also
examined once they had produced stable and reproducible
cycles of current generation. Standard anaerobic techniques
were used throughout all tests. Biofilm growth was monitored
based on the protein content extracted from the entire anode
using a Bicinchoninic acid protein assay kit.
Metabolic Structure of the Anode Biofilm. Anodes were
stained using a LIVE/DEAD BacLight Bacterial Viability Kit
(Invitrogen, CA)10,35 and examined with a confocal laser
scanning microscope (CLSM) (LSM710 NLO, ZEISS) with a
water objective (LD LCI Plan-Apochromat 25 × /0.8 Imm
Korr DIC). The three-dimensional biofilm structure (z-stack)
was reconstructed and analyzed using the software ZEN 2009
Light Edition. Anode samples were rinsed in sterile 50 mM PBS
to eliminate the original medium, stained for 20 min, and then
rinsed in sterile 50 mM PBS twice to eliminate excess dye. The
anode was then placed in a dish, immersed in sterile 50 mM
PBS, and observed using the CLSM. For each sample, at least
two CLSM images were taken.
Electrochemical Analysis. Linear sweep voltammetry
(LSV), electrochemical impedance spectroscopy (EIS) and
nonturnover cyclic voltammetry (CV) were used to examine
the biofilm electrochemical characteristics with a potentiostat
■
MATERIALS AND METHODS
Mini-BES Construction. Seventy mini-BESs were constructed as previously described.29 Anodes were 1.5 × 1 × 0.3
cm graphite plates (4.5 cm2 surface area, except as noted). All
anodes were successively polished using grit type P400 and
P1500 sandpaper and 0.05 μm Al particles. Anodes with a
smaller available surface area of 0.75 cm2 were produced by
covering part of the anode surface using an insulating layer of
epoxy. The cathodes were 1.5 × 1 cm stainless steel mesh
(Type 304, mesh size 90 × 90). An added voltage of 0.7 V was
applied here to each BES using a power supply (except as
noted), which is typical of other BES studies.30,31 All MECs
were connected in parallel to the power supply, with each
circuit containing a 10 Ω resistor to monitor voltage. Current
was calculated using Ohm’s law (I = U/R), and current density
was normalized by the anode area. Ag/AgCl reference
electrodes (+200 mV vs a standard hydrogen electrode, SHE)
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(CHI660D; Chenhua, China; EC-Lab V10.02 software). The
anode was the working electrode, the cathode was the counter
electrode, and an Ag/AgCl electrode was used as the reference
electrode. Before tests, the reactors were emptied, sparged with
N2, and then refilled with fresh medium for EIS and LSV tests,
or 50 mM PBS for nonturnover CV tests. EIS was conducted 2
h after connecting the circuit at a set potential equal to the
potential of −0.15 V, over a frequency range of 200 kHz to 10
mHz, with a sinusoidal perturbation of 5 mV amplitude. The
EIS spectra (Supporting Information Figure S1) with two
circles were fitted to an equivalent circuit containing a solution
resistance (Rs), two charge transfer resistances (Rct1 and Rct2), a
diffusion resistance (Rd), and double layer capacitance (Q)
(Supporting Information Figure S2).27 For LSV analyses, the
reactors were set at open circuit conditions for 1 h, and then
scanned from −0.50 to +0.30 V at a rate of 1 mV/s. For CV
tests, the reactors were rinsed with 50 mM PBS for 10 min
(twice), refilled with 50 mM PBS, set at open circuit conditions
for 1 h, and then scanned from −0.50 to +0.30 V at a rate of 1
mV/s. After all electrochemical tests were finished, electrode
potentials were adjusted for the accuracy of the Ag/AgCl
electrode, and the corrected potentials adjusted (vs Ag/AgCl)
and presented in all results.
Community Analysis of the Mixed Culture Biofilm.
The mixed culture biofilm grew in the 30th cycle was analyzed
the bacterial community by the MiSeq Illumina sequencing
technology. DNA was extracted, amplified and purified as
previously described,26 except that paired primers in the
variable regions V4 (F: 5′-AYTGGGYDTAAAGNG-3′ and R:
5′-TACNVGGGTATCTAATCC-3) were used for the PCR
amplification. The MiSeq Illumina sequencing was conducted
and analyzed as described previously.36
Figure 1. Current densities of G. anodireducens SD-1 and mixed
cultures (MC). (a) Maximum current density recorded for each fed
batch cycle with an applied voltage of 0.7 V. The data indicate standard
deviations (error bars) based on the mean of triplicate reactors. (b)
Peak current densities recorded for different cycles in LSVs (1 mV/s;
Supporting Information Figure S3 for details).
cells. The biofilm structure showed a consistent evolution of a
live outer-layer on top of a dead inner-core layer for both types
of biofilms (Figure 2 and Supporting Information Figure S4).
This structure indicated that live cells were only present on the
top of the biofilm, rather than throughout the entire biofilm,
showing that the entire biofilm was not metabolically active.
The lack of a viable biofilm near the electrode cannot be due to
incomplete penetration of the dye as the thickness of the live
biofilm varied over time, and a dead layer was observed even in
very thin biofilms. This finding of the lower part of the biofilm
being metabolically inactive was unexpected given published
biofilm models and previous observations using live/dead
staining of electroactive biofilms.4,5,10,16,19−25
The two-layer structure of the biofilm with dead cells at the
electrode-biofilm interface demonstrated that continued
metabolic activity of the outer layer cells, and therefore that
respiration even by cells on the outer layer of thick biofilms was
not limited by electrical conductivity.6,37 The presence of this
dead inner core layer also showed that long-distance EET
within biofilms did not rely on metabolic activity of all cells in
the biofilm since electrons had to have been transported
through the dead layer of biofilm to the electrode. This inactive
base layer did not appear to result from any diffusion limitation
of chemicals into or out of the biofilm6 since this same deadlive two layer structure was observed in the thin and newly
developing biofilms (cycle 1, Figure 2a and b) as well as thicker
and more mature biofilms. The thickness of the live outer-layer
reached an apparent limit of ∼10−15 μm based on comparison
of the maximum current densities in fed-batch tests as well as
peak electrochemical activities in LSVs. As biofilm thicknesses
increased, the size of the live layer remained constant, while the
thickness of the dead inner layer continued to increase from 15
to 30 μm for G. anodireducens biofilms (Figure 2e and g), and
■
RESULTS AND DISCUSSION
Operation and Maximum Current Densities over
Time. Current generation of G. anodireducens SD-1 biofilms
changed over time, showing four different stages relative to
current generation: a rapid increase in current over the first few
cycles; a peak current after around 12 cycles; a decline in
current over the next 10−15 cycles; and a final stable current
over the last 30 cycles (Figure 1). Current generation by the
mixed culture increased more slowly, and reached a stable
current after ∼15 cycles. In the last 30 cycles, the average peak
current densities measured for the mixed culture averaged 2.58
± 0.09 A/m2 over cycles 30−60, which was significantly larger
than that obtained for the pure culture of G. anodireducens of
2.07 ± 0.08 A/m2 (t test, p < 0.001) (Figure 1a).
The highest current densities that these biofilms could
produce were evaluated over time based on peak currents
measured in LSV tests (Figure 1b and Supporting Information
Figure S3). The same general trends in peak current densities
relative to maximum current densities were observed over time
between the two different cultures, with the pure culture
producing the highest peak current density of 5.29 A/m2 in the
earlier fed batch cycles, and then decreasing to lower peak
current densities in the later cycles. The G. anodireducens
biofilms produced an average peak current density 4.25 ± 0.33
A/m2 in cycles 30−60 (3 LSVs), that was similar to 4.47 ± 0.14
A/m2 for the mixed cultures (2 LSVs for cycles 60 and 78) (t
test, p = 0.46).
Spatial and Real-Time Metabolic Structure of Anode
Biofilm. The changes in biofilm viability were examined over
time using fluorescent staining to distinguish live versus dead
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Figure 2. 3D metabolic-structure images of anode biofilms operated with anodic current density of <2.6 A/m2 (0.7 V applied voltage). Biofilms were
LIVE/DEAD viability staining, and observed by CLSM. Live cells were imaged as green, whereas dead cells were imaged here as red. The images in
the left panel were the biofilms of G. anodireducens SD-1 operated in the cycle 1(a), 5(c), 12(e), and 30(g), whereas images in the right panel were
the biofilms of the mixed culture operated in the cycle 1(b), 5(d), 30(f) and 60(h). The ortho images were showed in Supporting Information Figure
S4.
from 35 to 80 μm for the mixed culture biofilms (Figures 2f and
h). This increase in the dead biofilm thickness over time can
help to explain why the maximum and peak current densities
peaked and then decreased to a lower stable current over time.
An increase in the thickness of the inactive biofilm layer could
have reduced peak or maximum current densities as a result of a
less electrical conductivity of the dead biofilm compared to the
viable biofilm, as well as greater distances needed for electron
transport to live cells.
Effects on Metabolic Structure of Anode Biofilm. The
different two-layer structure of the biofilm observed here,
compared to previous studies,4,5,10,16,22−25 could have been due
to three different factors: differences in current densities among
the studies; the use of hydrogen gas by the anode biofilms in a
single-chamber MEC compared to its absence in two-chamber
tests; and the use of G. anodireducens rather than G.
sulf urreducens. Our results suggest that of these three
possibilities, a difference in anodic current densities was most
likely reason for development of a biofilm with two layers. In
several studies with G. sulfurreducens with a relatively high
anodic current density of 3.5 A/m2, the entire biofilm on the
graphite rod was observed to be viable (using the same staining
approach as used here).5,16,22 However, in some other studies
with relatively lower anodic current densities of 0.85 to 1.85 A/
m2, the biofilm appeared to have a dead inner-core layer,
although the development or implications of this layer were not
further discussed in these studies.4,20,38 Here, current densities
averaged 2.1 A/m2 for the G. sulf urreducens biofilms and 2.6 A/
m2 for the mixed culture biofilms at an applied voltage of 0.7 V.
In order to test whether a higher current density would affect
the metabolic structure of the biofilm, we increased the current
density using two different approaches: using a higher applied
voltage of 0.9 V; or by using smaller anodes (projected surface
area of 0.75 cm2 compared to 4.5 cm2). Although reducing the
anode surface area decreases the total current, it increases the
current that must be produced by using a smaller anode relative
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Figure 3. 3D metabolic-structure images of anode biofilms operated with anodic current density of >3.8 A/m2. (a) G. anodireducens SD-1 (∼3.8 A/
m2, 0.9 V applied voltage), (b) the mixed culture (∼4.1 A/m2, 0.9 V applied voltage), (c) G. anodireducens SD-1 (∼5.2 A/m2, smaller anodic area),
and (d) the mixed culture (∼5.6 A/m2, smaller anodic area). Live cells were imaged as green, whereas dead cells were imaged here as red.
that the differences in metabolic structure were due to changes
in current density and did not result from differences in biofilm
growth or the rate of change in the current.
Hydrogen gas could have been a factor in the development of
a two-layer biofilm as hydrogen gas is produced on the cathode
and released into solution. Hydrogen gas is sparingly soluble,
but it can be used by the anodic biofilm for current generation.
Thus, hydrogen gas utilization might benefit microbes on the
water-side of the biofilm compared to those near the electrode.
To test whether hydrogen gas evolution was responsible for the
development of a dead inner layer on the anode, biofilms were
examined in two other reactors: a two-chamber BES, which is
the most common BES construction in anode biofilm studies,
with the two chambers separated by an anion exchange
membrane that minimizes hydrogen gas crossover from the
cathode to the anode; and a single-chamber, air-cathode MFC.
Even with these two different architectures, the same two-layer
metabolic structure was observed for the biofilms (Supporting
Information Figure S5a, b and c). In the two-chamber BES
studies, biofilms of G. anodireducens were thicker than those of
the mixed culture, and the current densities reached a
maximum of <2.7 A/m2 compared to that of <1.8 A/m2 for
the mixed culture. However, current densities decreased over
time, likely due to use of two chambers which can result in pH
gradients between the chambers. At the end of the batch cycles,
the pHs for G. anodireducens tests were ∼6.5 in the anode and
∼8.3 in the cathode, and they were ∼6.6 for the anode and
∼8.1 for the cathode in mixed culture tests. These results
showed that hydrogen evolution in the single-chamber BES
design was not the reason for the development of the dead
inner layer biofilm on the anode.
The development of the dead inner layer also cannot be
attributed to the use of G. anodireducens rather than G.
sulf urreducens. When mini-BESs were inoculated with a pure
culture of G. sulfurreducens, the same metabolic structure of a
live outer-layer and a dead inner-core was observed, under
conditions which resulted in a maximum anodic current density
of ∼1.7 A/m2 in the mini-BES (Supporting Information Figure
S 5d).
to its area, and therefore it substantially increases the anode
current density (current per anode area).39 The current
densities of G. anodireducens increased to ∼3.8 A/m2 with an
applied voltage of 0.9 V, and to ∼5.2 A/m2 with the smaller
anodic area. With either of these changes, we observed that the
entire biofilm (∼10 μm thick) of G. anodireducens was alive,
with no dead cell layer present, and very few dead cells
observed (Figures 3a and c). Using the same two operating
conditions for the mixed cultures, the current densities were
∼4.1 A/m2 with an applied voltage of 0.9 V, and ∼5.6 A/m2
with a smaller anodic area. Although dead cells were not
completely absent, the ratio of live cells appeared to
significantly increase with current density, and the dead inner
layer disappeared (Figures 3b and d). Thus, we speculate that a
high anodic current density was important for achieving a
higher ratio of live cells and more metabolically homogeneous
biofilm. The observation that the biofilm viability was improved
with higher current densities also supports a lack of diffusional
limitations producing a dead biofilm layer, as higher current
densities should have favored a thinner active biofilm layer and
a thicker dead biofilm. While there were changes in the anode
potential as a result of increasing the applied potential, it is
more likely that the higher current was the main reason for the
different metabolic structure. A higher current means that more
electrons per unit of time were generated by the biofilm, which
further supported the observation of more viable cells in the
biofilm, and higher electrochemical activity from the change in
applied potential. Anode potentials and current became more
positive at the higher applied potential, with ca. −0.20 at 0.7 V
compared to ca. −0.15 at 0.9 V, and it increased to ca. 0 at 0.7
V with the smaller anodic area. These changes in potential
could also have affected the biofilm growth and current
generation, but higher anode potentials have not always been
shown to produce more active biofilms.26,40 Additional studies
may be needed to further clarify the separate issues of the
impacts of current and potential on these biofilms. Differences
in time for biofilm development were not a factor. In these
three kinds of BESs, the startup time (3−5 days) and the time
to produce a stable current were similar (∼7 days), suggesting
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Anode Biofilm Growth. The amount of biofilm biomass
was evaluated based on the total protein content of the anode.
The results showed that biofilms of G. anodireducens grew faster
than that of the mixed culture, and that they reached a constant
density based on protein per anode area, with 8.17 ± 0.26 g/m2
for G. anodireducens (cycles 30−60), and 7.10 ± 0.04 g/m2 for
the mixed cultures (cycles 60−78) (Figure 4a). Constant
Figure 5. Anode resistances (solution resistance, Rs; charge transfer
resistance, Rct; and diffusion resistance, Rd) of (a) G. anodireducens SD1 and (b) mixed cultures.
(cycle 12 for G. anodireducens and cycle 30 for the mixed
culture) (Figure 2 and Figure 5).
The EIS results above, combined with biofilm metabolic
structure results, do not support a conclusion that diffusion
resistances measured using EIS arise from mass transfer
resistances related to diffusion of electron donors to the
biofilm or proton diffusion away from the biofilm, as suggested
in previous studies.6,14,16,17 Instead, these data support a view
that the diffusion resistance arose from long-range EET limited
by electron diffusion within the biofilm, for several reasons.
First, the EIS results show very little diffusion resistance for
young biofilms <15 μm in total thickness, suggesting that the
diffusion resistance in the live layer (10−15 μm saturation
thickness, Figure 2) in the mature phase might be negligible.
Second, as indicated above, diffusion resistance increased with
the development of a thick layer of dead biofilm rather than the
live one, but the importance of diffusion of chemical species
(electron donor or protons) only were important relative to the
live layer. Thus, we speculated that electron diffusion was
responsible for the diffusion behavior observed using EIS and
not mass transfer of protons or substrate. Third, a loose biofilm
structure should reduce diffusion resistances associated with
mass transfer of the electron donor and protons, but a less
dense biofilm would be expected to have higher electrical
resistance for long-range EET. Thus, our observation that the
diffusion resistances of the loose biofilms of the mixed culture
were higher than those of the more dense biofilms of G.
anodireducens favor a conclusion that diffusion resistance was
due to EET rather than chemical diffusion.
The peak current densities obtained in LSVs showed good
agreement with changes in biomass (based on protein content),
which showed an increase to ∼3.5 g/m2 where the live layer
reached a constant thickness (cycle 12 for G. anodireducens;
cycle 30 for the mixed culture) (Figure 2 and Figure 4a) and
then a slight decrease with further biomass (Figure 4b). LSV
Figure 4. (a) Biofilm growth of G. anodireducens SD-1 and the mixed
culture (MC) based on protein density over time. (b) Linear behavior
between the biofilm biomass and peak current density in LSVs
(Supporting Information Figure S3 for details).
biofilm densities were therefore reached in both cases,
consistent with the results showing the development of
relatively stable maximum current and peak current densities
in the later cycles. Although the biomass concentration of G.
anodireducens was higher than that of the mixed culture on the
basis of protein content (Figure 4a), the CLSM images showed
that the biofilms of the mixed culture biofilms were thicker than
G. anodireducens biofilms (Figure 2). Taken together, these data
suggest that the biofilms of G. anodireducens were more dense
than mixed culture biofilms. At the end of the study, the protein
volume-density of mixed cultures (ca. 110 mg/cm3) was
calculated to be about half of that of G. anodireducens biofilms
(ca. 240 mg/cm3) (Figures 2g and h). As a result, we observed
that small fragments sloughed off in the aged biofilm of the
mixed culture, consistent with the CLSM image (Figure 2h).
Electrochemical Properties of Anode Biofilms. The
components of the internal resistance of the BESs were
examined using EIS (Figure 5). The total internal resistance
decreased in the first few cycles, and then reached a stable value
of 119 ± 7 Ω for G. anodireducens (cycles 30, 45 and 60),
compared to 169 ± 1 Ω for the mixed culture (cycles 60 and
78). In the first cycle, there was minimal diffusion resistance for
both types of biofilms, with predominantly charge transfer
resistance. Over time, there was an increase in the diffusion
resistance, which occurred in concert with an increase in the
dead biofilm thicknesses. The anode resistance reached a
minimum when the live layer remained constant in thickness
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predominant genus on the anode biofilm in the mixed culture
BES was Geobacter (96% of sequences, based on a >97%
similarity) (Supporting Information Figure S6). While the
specific Geobacter species cannot be positively identified in the
mixed culture species BESs, it is clear that the low numbers of
non-Geobacter species (4%) in the mixed culture biofilm had no
dramatic effect on the performance relative to maximum
current densities (Figure 1), although differences in the biofilm
communities did impact biofilm densities and current densities
measured over time.
Implications. The same metabolic structure, consisting of a
live outer-layer and dead inner-core, was observed for both pure
and mixed culture anodic biofilms, at all stages of biofilm
development over time, in single chamber BESs as well as in
two-chamber BESs. This live/dead stratification is different
from the assumed or observed structure of the entire biofilm
proposed in the literature.4,5,10,16,22−25 This two-layer structure
indicated that (1) only the live cells located at the top layers
contribute to substrate oxidization and electron generation; (2)
the electrochemical activity of the outer layer cells is impaired,
but not prevented, by the dead cell layer; and (3) that longdistance EET within the biofilms does not require metabolic
activity of the whole biofilm since electrons can be transported
through dead-cell region to the electrode. In previous studies
when dead cells were observed near the electrode surface it was
suggested that development of this dead biofilm layer was a
result of diffusion limitations of either the electron donor to the
cells, or the accumulation of protons which lowered the pH.6,14
However, even in relatively thin biofilms, which were shown to
have minimal diffusion resistance using EIS, this same two-layer
structure also developed. The consistent presence of the dead
layer even in thin biofilms suggests that the cell’s metabolic
activity is not restricted by the diffusion processes for either
thin or thick biofilms.
We obtained additional results which suggested that the
anodic current density contributed to the development of a
two-layer structure compared to biofilms consisting primarily of
viable cells. By increasing the anodic current densities from
<2.6 A/m2 to >3.8 A/m2, a biofilm structure developed that
contained a much higher percentage of metabolically active
cells. Previous studies focused on studying the anode biofilms
typically have used conditions where current generation is
limited by the anode, for example by using relatively larger
cathodes than anodes or setting an anode potential. When the
current densities were higher in these studies compared to
those measured here, most of the biofilm remained viable.5,16,22
This impact of current density suggests that maintaining a high
anodic current density, which will create a high electrochemical
pressure condition for biofilm growth, may be necessary to
inhibit accumulation of dead cells. Different approaches could
be used to avoid a dead core biofilm, such as setting a high
anodic current density to raise the live/dead ratio, or biofilm
cleaning to keep the biofilm thin, therefore avoiding a large
accumulation of dead cells.
There was no support for the view that the diffusion
resistance measured in EIS tests was due to electron donor or
proton diffusion limitations,6,14,16,17 as the diffusion resistance
was absent in the EIS until the whole biofilm was thicker than
∼15 μm. In thicker biofilms, the live layer was always about this
thickness (10−15 μm) but diffusion resistance only occurred as
the dead biofilm layer thickness increased. Moreover, the
diffusion resistances of the loose biofilms were higher than
those of the more dense biofilms. Thus, our results supported a
and protein content results indicated current generation in
proportion to live biomass rather than total biomass. The
saturation in the maximum current densities with higher total
protein content, along with the increased diffusion resistances
observed in EIS results for the latter cell cycles, suggested that
accumulation of the dead cells impeded, but did not prevent,
electron transfer through biofilms. The midpoint potentials
were observed at −0.18 to −0.25 V for G. anodireducens and
−0.23 to −0.25 V for the mixed culture (Supporting
Information Figure S3).
CVs of G. anodireducens and the mixed culture biofilms were
examined over time under nonturnover conditions (no electron
donor, no acetate) to investigate the redox potentials and
kinetics of reversible redox-active proteins in biofilms (Figure
6). The G. anodireducens showed a typical double peak
Figure 6. Nonturnover CV (1 mV/s) of G. anodireducens SD-1 (a) and
the mixed culture (b) over time (CVs were conducted in all testing
time-points, but, in order to simplify the figure, only five representative
growth cycles for each culture were selected to provide in this figure).
voltammetric signature.19,24,41 The position of the main
oxidization peak in CVs converged to −0.15 to −0.12 V,
whereas the position of the small peak was showed at a more
positive anode potential range of −0.08 to −0.06 V. The mixed
culture showed only one oxidization peak at a similar position
of the main peak of G. anodireducens (−0.13 to −0.16 V) except
that in the cycle 78 (Figure 6). The redox peak height in
nonturnover CVs is associated with the amount of redox active
components (mediators) present within the biofilms. Thus, the
peak current densities measured in nonturnover CVs increased
with the total biomass rather than the live biomass, suggesting
that the redox active components are involved in EET through
the diffusive behavior.
Comparison of G. anodireducens and Mixed Culture
Biofilms. The composition of the mixed culture biofilm was
examined in cycle 30. Anodic bacterial communities in BESs
inoculated with a wide variety of environmental and engineered
reactor samples, and fed acetate, have typically been shown to
converge to the genus Geobacter.26,40,42 As expected, the
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■
ASSOCIATED CONTENT
S Supporting Information
*
Figures provided include Nyquist plots of EIS spectra, the
equivalent circuit used for EIS, LSVs over time, the ortho
CLSM images of anode biofilms, 3D CLSM images of the
biofilms grown in the absence of hydrogen gas and biofilms of
G. sulf urreducens, and bacterial communities of the mixed
culture anode biofilms. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-571-87952038; fax: +86-571-87952038; e-mail:
[email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Shelong Zhang for assistance with the CLSM
imaging. This research was supported by the National Natural
Science Foundation of China (grant nos. Nos. 51278448,
51478414 and 51408541), China Postdoctoral Science
Foundation (grant nos. 2013M541773 and 2014T70573),
and the National High Technology Research and Development
Program of China (863 Program) (grant nos. 2011AA060907
and 2012AA051502).
■
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