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Article
Self-Sustained Phototrophic Microbial Fuel Cells
Based on the Synergistic Cooperation between
Photosynthetic Microorganisms and Heterotrophic Bacteria
Zhen He, Jinjun Kan, Florian Mansfeld, Largus T. Angenent, and Kenneth H. Nealson
Environ. Sci. Technol., 2009, 43 (5), 1648-1654• DOI: 10.1021/es803084a • Publication Date (Web): 02 February 2009
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Environ. Sci. Technol. 2009, 43, 1648–1654
Self-Sustained Phototrophic
Microbial Fuel Cells Based on the
Synergistic Cooperation between
Photosynthetic Microorganisms and
Heterotrophic Bacteria
Z H E N H E , †,‡ J I N J U N K A N , †
FLORIAN MANSFELD,‡
LARGUS T. ANGENENT,§ AND
K E N N E T H H . N E A L S O N * ,†
Department of Earth Sciences, University of Southern
California, Los Angeles, California 90089, Mork Family
Department of Chemical Engineering and Materials Science,
University of Southern California, Los Angeles, California
90089, and Department of Biological and Environmental
Engineering, Cornell University, Ithaca, New York 14853
Received October 31, 2008. Revised manuscript received
January 12, 2009. Accepted January 13, 2009.
A sediment-type self-sustained phototrophic microbial fuel
cell (MFC) was developed to generate electricity through the
synergistic interaction between photosynthetic microorganisms
and heterotrophic bacteria. Under illumination, the MFC
continuously produced electricity without the external input of
exogenous organics or nutrients. The current increased in
the dark and decreased with the light on, possibly because of
the negative effect of the oxygen produced via photosynthesis.
Continuous illumination inhibited the current production while the
continuous dark period stimulated the current production.
Extended darkness resulted in a decrease of current, probably
because of the consumption of the organics accumulated
during the light phase. Using color filters or increasing the
thickness of the sediment resulted in a reduction of the oxygeninduced inhibition. Molecular taxonomic analysis revealed
that photosynthetic microorganisms including cyanobacteria
and microalgae predominated in the water phase, adjacent to
the cathode and on the surface of the sediment. In contrast,
the sediments were dominated by heterotrophic bacteria,
becoming less diverse with increasing depth. In addition, results
from the air-cathode phototrophic MFC confirmed the lightinduced current production while the test with the two-chamber
MFC (in the dark) indicated the presence of electricigenic
bacteria in the sediment.
Introduction
In our electricity-based society, generating electricity directly
from sunlight should be one of our major goals, given that
sunlight is both abundant and virtually free. To this end,
phototrophic microbial fuel cells (MFCs), although usually
at low conversion efficiency, represent an approach for the
* Corresponding author phone: (213) 821-2271; fax: (213) 7408801; e-mail: [email protected].
†
Department of Earth Sciences, USC.
‡
Mork Family Department of Chemical Engineering and Materials
Science, USC.
§
Cornell University.
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conversion of solar into electric energy, and have recently
drawn increased attention because of their potential application in a certain areas, such as providing power for
remote sensors (1-4). Unlike traditional MFCs whose
operation is independent of light, phototrophic MFCs require
sunlight to drive the production of energy rich chemicals
(reducing equivalents) that are used for electricity production;
for example, photosynthetic microorganisms convert solar
energy into chemical energy, which is then converted into
electric energy, by either microorganisms or metal catalysts.
The study of phototrophic MFCs is in its nascent stages.
With the external addition of organic compounds, Rosenbaum et al. (2) successfully operated an electrochemical cell
to generate electricity from hydrogen that was produced by
a two-step process in which Escherichia coli K 12 fermented
glucose into organic acids and alcohols and Rhodobacter
sphaeroides photo-oxidized these products to produce
hydrogen. A similar process was investigated in a singlechamber MFC in which only R. sphaeroides (fed on succinate)
was used as the anodic bacterium (3). In the latter study, the
power output was dependent on both light and the nature
of the nitrogen source. When a mixed phototrophic consortium was tested in a two-chamber MFC fed with acetate
(4), the authors proposed that electron mediators were
excreted by certain microorganisms to promote electron
transfer to the anode.
Self-sustained phototrophic MFCs obviate the need for
external input of exogenous energy sources and are complete
solar-to-electric energy systems. In such systems, organic
compounds or other reductants (e.g., hydrogen) are supplied
via photosynthesis. Early efforts to develop the self-sustained
phototrophic MFCs began in the 1980s with a “living
electrode” that utilized cyanobacteria to produce the electron
donor (hydrogen). This system produced only ∼1 µA under
constant illumination (5). In another study, cyanobacteria
were used to generate current under light through the
oxidation of endogenous glycogen produced in the dark (6).
Current production during illumination was significantly
enhanced with nitrogen gas bubbling, suggesting that oxygen
accumulation during photosynthesis was inhibitory. When
carbonyl cyanide m-chlorophenylhydrazone (CCCP), an
inhibitor of photosynthesis, was added, electricity generation
was inhibited (7).
In nature, photosynthetic organisms are usually found
living together with other microbes, often in communities
containing heterotrophs that utilize products produced by
the photosynthetic partners (8). Self-sustained phototrophic
MFCs based on such syntrophic communities have been
reported in which MFC systems in rice paddy fields were
found to produce electricity by rhizosphere populations
oxidizing organic carbon delivered to the rhizosphere (9, 10).
This process has also been applied to other aquatic plants
(11). Similar synergistic interactions are thought to exist
between photosynthetic microorganisms (e.g., cyanobacteria
or microalgae) and heterotrophic bacteria, for instance in a
microbial mat (12). In a recent study, algal photobioreactors
were used to supply organic matter produced via photosynthesis to a MFC for electricity generation, which is an
example of “an indirect synergistic relationship” between
photosynthetic organisms and electricigens (13).
In this paper, we report the production of electricity from
a self-sustained sediment phototrophic MFC that was
operated with a mixed microbial community consisting of
photosynthetic microorganisms and heterotrophic bacteria.
Electricity was constantly generated with no input of organic
compounds or nutrients. The effects of light or dark duration
10.1021/es803084a CCC: $40.75
 2009 American Chemical Society
Published on Web 02/02/2009
FIGURE 1. Schematics of the MFCs used in this study: (A)
sediment phototrophic MFC; (B) air-cathode phototrophic MFC;
(C) two-chamber MFC. CEM: cation exchange membrane.
and light wavelength on current generation were investigated.
The microbial communities on the anode and cathodes were
analyzed using molecular techniques. In addition, we
examined the electricity production from an air-cathode
phototrophic MFC and a traditional two-chamber MFC fed
with glucose (in the dark), both of which were inoculated
with the anodic microbes of the sediment phototrophic MFC.
Materials and Methods
Phototrophic MFC Setup and Operation. A sediment MFC
was built in a 1-L glass beaker that was open to the
atmosphere (Figure 1A) and has been operated for more
than 7 months for data collection. The anode, made of round
graphite felt (project surface area of ∼78 cm2, Electrolytica
Inc., Amherst, NY), was placed on the bottom. The cathode,
a piece of graphite plate (surface area of ∼84 cm2, POCO
Graphite Inc., Decatur, TX), was hung about 12 cm above the
anode and connected to the anode using insulated copper
wire. Sediment and lake water from Mono Lake, CA (that has
been stored in the laboratory at room temperature for more
than one year), were mixed with tap water (20% of total
volume), and this mixture was used to fill the glass beaker,
producing a sediment layer of ∼0.5 cm (4% v/v) above the
anode and a total water volume of ∼950 mL in the beaker.
The composition of Mono Lake water can be obtained in a
previous report (14). To compensate for the water loss due
to evaporation and photoelectrolysis, 10 mL of tap water
was added into the MFC once a day. Illumination was
achieved via a full spectrum light bulb (24 W, BlueMax
Lighting, Jackson, MI) that was installed 20 cm away from
the MFC and controlled by a timer with an on/off period of
8/16 h. The dark condition was created by placing MFCs in
the dark room and using aluminum foil to surround them.
Blue and red filters were purchased from Anchor Optics
(Barrington, NJ). The cell voltage across a 1000 ohm resistor
was recorded every 30 s by a digital multimeter (2700, Keithley
Instruments, Inc., Cleveland, OH). The concentration of
dissolved oxygen was measured using a DO meter (Control
Company, Friendswood, TX). The pH was measured by a
benchtop pH meter (UB 10, Denver Instrument, Denver, CO).
Air-Cathode MFC and Two-Chamber MFC Setup. An
air-cathode MFC was used to examine photoelectricity
production in the absence of Biofilm on the cathode (Figure
1B). A two-chamber MFC was built to detect the presence
of electricigenic bacteria in sediments (Figure 1C). The anode
inocula (4% v/v) of both MFCs were taken from the anode
biomass of the sediment phototrophic MFC (after six-month
operation). The detailed setup is described in the Supporting
Information.
FIGURE 2. Electric current productions from the sediment
phototrophic MFC under the full-spectrum light after one month
(A) and five months (B). The symbols of moon and sun represent
dark and light conditions, respectively.
DNA Extraction and Polymerase Chain Reaction (PCR).
Biomass and water samples were collected from the different
sites in the sediment MFC (Figure 5). Genomic DNA was
extracted by UltraClean Soil DNA kit (MO BIO Laboratories,
Carlsbad, CA) following the manufacturer’s instructions. DNA
concentration was estimated based on 260 nm absorbance
using a Spectrophotometer ND-1000 (NanoDrop Products,
Wilmington, DE). PCR amplification was performed in a 50
µL reaction containing approximately 25 ng of template DNA,
25 µL of PCR Mastermix (Qiagen), 0.5 mM (each) primer,
and distilled water. PCR program was performed with a
Mastercycler gradient (Eppendorf, Hamburg, Germany). PCR
primers used were 341f (GC) and 907r and the PCR program
followed the protocol described by Scäfer and Muyzer (15).
Agarose gel electrophoresis was used to detect and estimate
the concentrations of PCR amplicons.
Denaturing Gradient Gel Electrophoresis (DGGE) Sequencing and Phylogenetic Analysis. DGGE was performed
as previously described (16) except the linear gradient of the
denaturants was from 40 to 70% instead of 40 to 65%.
Representative bands were excised from DGGE gels and
incubated in diffusion buffer (0.25 M ammonium acetate, 10
mM magnesium chloride, and 0.1% SDS) at 50 °C for 30 min.
One microliter of supernatant was used to reamplify the band.
PCR products were purified by ExoSAP-IT (USB, Cleveland,
OH) and sequenced with primer 341f (no GC) by using Bigdyeterminator chemistry with a ABI PRISM3100 Genetic Analyzer
(Applied Biosystems, Foster City, CA). All sequences were
compared with GenBank database using BLAST, and the
closest matched sequences were obtained and included in
the downstream analysis. Phylogenetic trees were constructed
using MacVector 10.0 software package (MacVector Inc., Cary,
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FIGURE 3. Electric current productions the sediment
phototrophic MFC under the extended light (A) and in the
extended dark (B). The symbols of moon and sun represent
dark and light conditions, respectively.
NC). Briefly, sequence alignment was performed with the
program CLUSTAL W. Evolutionary distances were calculated
using the Jukes-Cantor method (17), and distance trees were
constructed using the neighbor-joining algorithm (18).
Bootstrap values were obtained based on the analysis of 1000
resampling data sets. Sequences of the partial 16S rRNA genes
of representative DGGE bands have been deposited in the
GenBankdatabaseunderaccessionnumbersFJ418944-FJ418972.
Epifluorescence Microscopy. Bacteria were collected,
stained by SYBR Gold (Invitrogen, Carlsbad, CA), and
observed following the protocol previously described (19).
Briefly, 0.5 mL of the MFC water was fixed by 0.5 mL of 4%
paraformaldehyde for 24 h and filtered onto a 0.02 µm poresize Al2O3 Anodisc 25 mm membrane filter (Whatman) with
approximately 10 kPa vacuum. The membranes were stained
with 2.5 × SYBR Gold solution (final concentration) in the
dark. The stained membrane filters were mounted on glass
slides and covered with coverslips. The total bacteria were
observed under blue excitation (485 nm), and autofluorescence cells (eukaryotic phytoplankton and cyanobacteria,
etc.) were imaged under green excitation (528-533 nm) on
a Zeiss Axioplan epifluorescence microscope (Zeiss, Germany) using a 100× Antiflex Neoflua oil object-lens.
Results
Effects of Light/Dark Cycles on Current Generation. Current
production by the sediment phototrophic MFC varied during
the operating period. In the first month, current generation
increased when the light was on and decreased in the dark
(Figure 2A). However, the peak current of 0.041 ( 0.002 mA
always appeared several hours after the light was switched off.
This trend changed slowly over the operating period. The
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FIGURE 4. Electric current productions of the sediment
phototrophic MFC under red light (A) and blue light (B). The
symbols of moon and sun represent dark and light conditions,
respectively.
occurrence of the peak current was delayed until the end of the
dark period while the lowest point current decreased eventually
to a negative value with the light on (data not shown). After five
months of operation, the pattern of current production was
nearly reversed from that was seen initially, with the increase
or decrease of the current immediately occurring when the
light was switched off or on (Figure 2B). Upon illumination, the
current decreased rapidly to -0.045 ( 0.003 mA while in the
dark the current started to increase and reached the highest
value of 0.054 ( 0.002 mA. When the illumination period was
extended from 8 to 80 h, the current decreased first as usual
and then rose to levels close to zero (Figure 3A). When the light
was reset to the on/off mode, the current-generating profile
was restored in 10 days (Figure 3A). In contrast, a longer dark
period improved the peak current to 0.104 mA after 50 h,
followed by a decrease to 0.038 mA in 36 h (Figure 3B). Switching
on the light resulted in an increase in current to 0.078 mA,
followed by a decrease. In the following 15 days, the currentgenerating profile returned to its normal mode (Figure 3B).
Current Production with Red or Blue Light. Photosynthetic microorganisms contain pigments that conduct light
absorption at different wavelengths, which will subsequently
affect their metabolic activities. To examine the effect of light
wavelength, light filters were used to create red (620-750
nm) or blue (450-495 nm) light. The application of light
filters affected the sediment phototrophic MFC considerably.
When the red light was off, the current increased quickly to
0.060 ( 0.002 mA in about 4 h and then started to slowly
decrease (Figure 4A). The decrease was accelerated when
the red light was on and slowed down at the end of the
illumination period. With blue light the shape of currentgenerating curves became irregular (Figure 4B): the current
FIGURE 5. DGGE fingerprints of bacterial communities (left) and the schematic of the sampling sites in the sediment phototrophic
MFC (right): (A) biomass on the cathode surface; (B) upper-level water; (C) middle-level water; (D) bottom-level water; (E) sediment
surface; (F) sediment; (G) biomass inside the anode; (H) original inoculum (not shown). Numbers represented bands that were
excised and sequenced for further analysis.
still increased and reached a maximum in the dark; however,
with blue light the current fluctuated with a decrease first
and then an increase. The current did not drop below zero
when either red or blue light was provided (Figure 4).
Bacterial Communities on the Electrodes. Bacterial
community profiles of the sediment phototrophic MFC were
fingerprinted by DGGE (Figure 5), and phylogenetic affiliations of the representative DGGE band sequences were
shown in Figure 6. The bacterial communities in the sediment
phototrophic MFC have become notably different from its
original inoculum that mainly contained low GC Gram
positive bacteria (bands 25-28) and uncultured Alphaproteobacterium (band 29). The bacterial communities from
the cathode and the sediments appeared to have the highest
complexity, sharing similar cyanobacterial groups while more
homogeneous photosynthetic groups of organisms (cyanobacteria/plastids) existed in water columns as shown by
similar DGGE band patterns among upper, middle, and
bottom layers of water (bands 1, 2, 10-13). However,
cyanobacterial groups could not be differentiated by the
sequences amplified with the DGGE primers applied in this
study. On the cathode surface, bacteria that belong to
uncultured Bacteroidetes were also highly representative on
the gels (bands 3-6, 8). It seemed that community diversity
became less with increasing depth of the sediment (Figure
5E-G), with the top (surface) layer showing abundant
cyanobacteria and Bacteroidetes (bands 15, 16), as well as
a few others. In contrast, the middle layer of the sediment
was dominated by low GC Gram positive bacteria (bands 20,
21) and uncultured Alphaproteobacterium (band 9), and the
bottom of the sediment (or the anode) was occupied by
Firmicutes and Gammaproteobacterium (closely related to
Alkalilimnicola ehrlichei).
Current Production by the Air-Cathode and Twochamber MFCs. The air-cathode phototrophic MFC generated a similar current profile to that of the sediment
phototrophic MFC, though at a lower level (Supporting
Information Figure S1). Two current peaks were observed
during a single light-dark cycle: the first current peak occurred
before the light was switched on, and then the current slightly
decreased and reached the second peak shortly after the light
was turned on. The reason for this phenomenon remains
unclear. The two-chamber MFC that was operated in the dark
produced current when glucose was added, indicating the
presence of electricigenic bacteria in the anode biomass of the
sediment phototrophic MFC (Supporting Information Figure
S2).
Discussion
Electricity was produced from a self-sustained sediment phototrophic MFC without the input of external carbon source.
The possibility that the voltage resulted from pH variation could
be excluded because of insignificant changes in pH between
the anode and the cathode during the illumination/dark process
(Supporting Information Table S1). A synergistic relationship
between photosynthetic microorganisms and heterotrophic
electricigenic bacteria may exist during the electricity-generating
process. DGGE sequences have revealed the dominant presence
of photosynthetic microorganisms (e.g., cyanobacteria and
microalgae) in the water column of the sediment MFC. The
microbial community in the sediment or the anode, however,
did not contain any detectable photosynthetic microorganisms
(sampling sites F and G in Figure 5). Although some photosynthetic bacteria are able to produce electricity from organic
compounds (20), it is unlikely that they directly contributed to
electricity production in this study considering (1) the great
distance between photosynthetic microorganisms and the
anode, and (2) a complex microbial community on the anode
that might obstruct the ion and electron transport that are
required for generating electricity. The major sequence (band
24) found on the anode was close to Alkalilimnicola ehrlichei,
which is a facultative bacterium and can grow as either
heterotroph or autotroph with nitrate or oxygen as the electron
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FIGURE 6. Phylogenetic analysis of DGGE band sequences obtained in the current study and most closely related representatives
from GenBank: (A) Proteobacteria; (B) Spirochates and Bacteroidetes; (C) other bacterial groups including Cyanobacteria/plastids,
low GC Gram positive, and Firmicutes. Scale bars indicated the number of substitutions per site. Bootstrap values were based on
1000 resampling data sets. Only bootstrap values relevant to the interpretation of groupings were shown.
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acceptor (21). Whether A. ehrlichei is capable of electricity
generation remains unknown, although other nitrate-reducing
bacteria, such as Shewanella spp. and Geobacter spp., have
been shown to be electrochemically active in MFCs (22, 23).
Another major band belongs to Firmicutes (band 23), several
of which have been shown to be positive for electricity
production in MFCs (24, 25). In addition, the electricity
production from the two-chamber MFC operated in the dark
suggested the presence of electricigenic bacteria in the sediment
(Figure S2). Thus, it is reasonable to hypothesize a synergistic
interaction in which these bacteria and perhaps others oxidized
organic matter or hydrogen produced by photosynthetic
microorganisms via photosynthesis, generating electricity from
this reaction.
Microscopic examination revealed cells of both photosynthetic eukaryotes and cyanobacteria (Figure S3), consistent with the identification of plastid sequences related to
eukaryotic algae. All these suggested that the phototrophic
MFC ecosystem contained eukaryotic microalgae, cyanobacteria, and heterotrophic bacteria. It is, however, unclear
whether cyanobacteria or algae have played a more important
role in electricity production. The research with pure cultures
of both cyanobacteria and algae is underway to clarify their
detailed functions in the synergistic relationship in phototrophic MFCs.
Unlike the phototrophic MFCs previously reported in
which electricity production was stimulated by sunlight (2-4),
the current generation in this study was indirectly dependent
on light, i.e., while sunlight was indispensable to current
generation, no increase in current generation was seen upon
illumination. Both the sediment and the air-cathode phototrophic MFCs of this study showed an increased electric
current in the dark and a decreased current with the light.
We hypothesize that during the extended dark period, current
first increased because of the oxidation of organic compounds
that accumulated during the light reactions, and then
decreased because of the depletion of easily metabolized
organics. We also hypothesize that the inhibition of current
production during long-time illumination was due to the
presence of dissolved oxygen produced by photosynthetic
microorganisms. In this scenario, photosynthesis supplied
both “food” (organic compounds) to heterotrophic bacteria
inhabiting the sediment and the anode, and “inhibitors” of
the anode reaction, allowing the anode bacteria to utilize
oxygen as an electron acceptor, rather than the anode of the
MFC. That is, with long-term illumination, the oxic zone
should expand downward in the sediment (26) and the
dissolved oxygen should inhibit the anodic reaction via
inhibition of obligate anaerobes, and/or allowing facultative
bacteria to switch from anode-respiration to oxygen-respiration. Alternatively, phototrophs may compete for nutrients
and/or even carbon source, and the presence of oxygen could
lead to a more rapid decrease of organics by bacteria not
involved with current production. None of these inhibitory
effects should be seen while in the dark.
It should be noted that the current of the sediment
phototrophic MFC decreased to negative values under
illumination (Figure 2B), indicating that the electrodes of
the MFC might be reversed to a certain degree, possibly
because the photosynthetic microorganisms on the bottom
of the MFC (anode) produce more oxygen than the organisms
on/around the cathode, and thus the anode functioned as
“a cathode” and the cathode was opposite. The measurement
of the dissolved oxygen (DO) in the upper- and bottom-level
water phase has confirmed that the DO concentration
adjacent to the bottom of the MFC was higher than that in
the upper-level water under the illumination (Supporting
Information Table S2). Microbial analysis showed that the
cathodic microbial community contained Proteobacteria and
Bacteriodetes (bands 3-9), some of which have also been
found in the anode of MFCs (27-29), indicating that these
microorganisms might perform the anodic reactions on the
cathode under a suitable condition. The air-cathode phototrophic MFC, however, had no Biofilm formed on the
cathode and did not show a negative current under the light
(Figure S1), suggesting that the cathodic Biofilm of the
sediment phototrophic MFC played a role in the reversed
current generation. In addition to oxygen effect, substrate
changes may also contribute to current reversal (30), though
the exact reason remains unclear.
The negative effect of oxygen can be alleviated by using
color filters to filtrate the full-spectrum light or increasing
the thickness of the sediment layer on the anode. Color filters
would not completely inhibit the activities of photosynthetic
microorganisms because most of them have a wide adsorption spectrum (31). Instead, color filters may lessen oxygen
evolvement by reducing light strength. However, it requires
further investigation on how the photosynthetic microorganisms in this study were affected by red or blue light. The
sediment layer in the MFC was about 0.5 cm above the anode.
When more sediment was added to increase the thickness
to 2 cm, the current did not decrease below the zero under
illumination (data not shown), suggesting that a better anoxic
condition was created adjacent to the anode with more
sediment. But a thicker sediment layer will cause problems
in transporting ions and electrons, as well as substrates to
the microbes in the deeper level. Hence, a balance between
inhibiting oxygen effect and improving ion/electron and
substrate transport should be established in determining the
thickness of the sediment layer applied to the anode.
The synergistic interaction between photosynthetic organisms and heterotrophic bacteria exists in many places,
such as coastal area, lagoon, and microbial mat. A proper
design of the phototrophic MFC systems will make it possible
to convert solar energy into electricity and power remote
sensors for monitoring environmental conditions. In addition, photosynthetic microorganisms can provide oxygen for
the cathode reaction (32). Future research will focus on
improving power output and optimizing the anode/sediment
layer to prevent oxygen inhibition.
Acknowledgments
We thank Shana Rapoport (University of Southern California)
for providing lake sediments as inocula and for her help with
the purchase of experimental materials. We also thank
anonymous reviewers for helpful comments.
Supporting Information Available
The setup of air-cathode MFC and two-chamber MFC is
described. The current production by the air-cathode phototrophic MFC is presented in Figure S1. The current
production with the addition of glucose by the two-chamber
MFC (in the dark) is presented in Figure S2. The epifluorescence images of the microorganisms in the water phase
of the sediment phototrophic MFC are shown in Figure S3.
The values of pH and the concentrations of the dissolved
oxygen are presented in Tables S1 and S2. These materials
are available free of charge via the Internet at http://
pubs.acs.org.
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