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Biotechnol. Prog.

2006, 22, 241246

241

A Comparison of Air and Hydrogen Peroxide Oxygenated Microbial Fuel Cell


Reactors
B. Tartakovsky* and S. R. Guiot
Biotechnology Research Institute, NRC, 6100 Royalmount Ave, Montreal, Quebec, Canada H4P 2R2

In this study, a two-compartment continuous flow microbial fuel cell (MFC) reactor was used
to compare the efficiencies of cathode oxygenation by air and by hydrogen peroxide. The MFC
reactor had neither a proton-selective membrane nor an electron transfer mediator. At startup,
the cathodic compartment was continuously aerated and the anodic compartment was fed with
a glucose solution. An increase of electrical power generation from 0.008 to 7.2 mW m-2 of
anode surface with a steady-state potential of 215-225 mV was observed within a period of 12
days. The performance of the air-oxygenated MFC reactor progressively declined over time
because of biofilm proliferation in the cathodic compartment. Oxygenation of the cathodic
compartment using 300 mL d-1 of 0.3% hydrogen peroxide solution resulted in a power density
of up to 22 mW m-2 (68.2 mA m-2) of anode surface at a potential of 340-350 mV. The use
of H2O2 for oxygenation was found to improve the long-term stability of the MFC reactor.

Introduction
Although electricity generation in a microbial fuel cell (MFC)
has been known for decades (1), the need for a chemical
mediator and a proton-selective membrane has greatly limited
the economical feasibility of microbiological production of
electricity. Recent studies on the microbial conversion of organic
matter to electricity have demonstrated a direct electron transfer
by microorganisms attached to the anode implying that microorganisms used the electrode as the terminal electron acceptor.
This finding allowed for the development of a mediatorless MFC
(2-4). Furthermore, electricity production has been demonstrated in a two-compartment mediator- and membraneless MFC
(5, 6) and in a single chamber MFC (7-9). This setup essentially
resembles that of a sequential anaerobic/aerobic wastewater
treatment system, suggesting the possibility to co-generate
electricity during a wastewater treatment process (9, 10).
A mediator- and membraneless microbial fuel cell typically
consists of an anaerobic and an aerobic compartment, containing
an anode and a cathode, respectively. Microbial oxidation of
organic matter occurs in the anodic compartment where
electrochemically active microorganisms attach to the anode
surface, which serves as the sole electron acceptor for anaerobic
respiration (2). Substrate oxidation results in the generation of
both electrons and protons. The mechanism of electron transfer
to the anode can be governed by microbially produced extracellular electron shuttles, by components associated with the
bacterial cell wall, or by both mechanisms (11). Protons migrate
through the liquid phase to the cathodic compartment and
electrons move through the external circuit. Oxygen supplied
to the cathodic compartment results in electron and proton
consumption with the formation of water.
Studies in which pure and mixed cultures were used have
shown that the attachment of a broad range of anaerobic
microorganisms such as Shewanella oneidensis, Geobacter
sulfurreducens, and Rhodoferax ferrireducens to the anode
* To whom correspondence should be addressed. boris.tartakovsky@
cnrc-nrc.gc.ca.
10.1021/bp050225j CCC: $33.50

surface results in electricity generation from a variety of


substrates (4, 12, 13). Moreover, power densities obtained when
inoculating MFCs with mixed populations in activated sludges
were similar to those observed in pure culture experiments
(6, 10).
Several authors have highlighted factors that limit the
volumetric production of electricity in a MFC such as electron
transfer to the anode, proton transport, and cathode reaction
(3, 9, 14). The goals of this study were 2-fold: (i) to compare
the efficiency of cathodic compartment oxygenation by air and
by hydrogen peroxide and (ii) to evaluate long-term performance
of the upflow MFC reactor.

Materials and Methods


Microbial Fuel Cell Reactor. Experiments were carried out
in a 610 mL two-compartment mediator- and membraneless
microbial fuel cell reactor made of Plexiglas. The reactor
consisted of a 320 mL anaerobic anodic compartment and a
290 mL aerobic cathodic compartment. It was operated in an
upflow mode with all feed solutions entering the base of the
anodic compartment and effluent located at the top of the
cathodic compartment (Figure 1). The compartments were
separated by a 3 mm thick polyester pad with a total porosity
of 0.93 (Skotch-Brite 3M, St. Paul, MN). The pad reduced liquid
mixing between the two zones while permitting nutrient and
ion transport by the upward liquid flow. The reactor was
operated at a temperature of 25 C, which was maintained by
a Digi-sense temperature controller (Cole Parmer, Chicago, IL).
Each compartment contained two sets of electrodes (Figure 1).
Each electrode consisted of 21 graphite rods, each with a length
of 50 mm and a diameter of 2 mm. The rods in the electrode
were spaced 2 mm apart. The surface area and volume of each
electrode assembly was 118.7 cm2 and 6 cm3, respectively. The
distance between the cathode and anode centers was 5 cm.
For air-oxygenation, a horizontal aeration line was installed
at the bottom of the cathodic compartment (Figure 1). A
rotameter was used to control the rate of aeration from 0 to 4
L air LC-1 min-1, where LC is the cathode compartment volume.

Published 2006 by the American Chemical Society and American Institute of Chemical Engineers
Published on Web 12/02/2005

242

Biotechnol. Prog., 2006, Vol. 22, No. 1

Figure 1. Experimental setup. The reactor was operated in upflow mode with liquid flow from anodic to cathodic compartment. The insert shows
design of the electrode assembly.

For hydrogen peroxide oxygenation, a feed line was installed


at the top of the cathode compartment and hydrogen peroxide
solution was fed at a rate of 10.4 mL h-1 by a peristaltic pump.
The anode compartment of the MFC reactor was inoculated
with 200 mL of slightly homogenized anaerobic sludge
(A. Lassonde Inc., Rougemont, Quebec, Canada). The sludge
had an average volatile suspended solids content of 45 g L-1.
The reactor was fed continuously with 600 mL d-1 of bicarbonate buffer (NaHCO3 0.68 g L-1; KHCO3 0.87 g L-1), 2.5 mL
d-1 of nutrients (glucose 200 g L-1; KH2PO4 0.43 g L-1;
K2HPO4 0.56 g L-1; (NH4)2HCO3 5.4 g L-1), and 1 mL d-1 of
microelements. The solution of microelements had the following
composition and concentrations in mg L-1: AlCl3; H3BO3 10;
CaCl22H2O 600; CoCl26H2O 30; CuCl22H2O 8; FeCl24H2O
80; MgCl22H2O 400; MnCl24H2O 100; (NH4)6Mo7O244H2O
10; NiCl26H2O 20; NaSeO3 10; Na2WO4 15; ZnCl2 10;
Na2EDTA 100; HCl 0.2 mL L-1. With respect to the reactor
volume, the reactor was operated with a retention time of 1
day. The bicarbonate buffer was fed by a peristaltic pump, and
the nutrients and microelements were fed by an infusion pump.
The three streams were combined prior to entry into the reactor.
Analytical Methods. Glucose concentration was measured
using a HPLC (model 590, Waters Chromatography division,
Milford, MA) equipped with an interaction ION-300 organic
acid column (300 m 7.8 mm i.d.), an ion guard GC-801
column (Interaction Chemical Inc., Milford, MA), and a UV
detector. The mobile phase was a 0.035 N solution of sulfuric
acid, pH 2.
Concentrations of propionate, butyrate, and acetate were
determined by gas chromatography using a Sigma 2000 system
equipped with a 91 cm 4 mm i.d. glass column packed with
60/80 Carbopack C/0.3% Carbpwax 20 M/0.1 H3PO4 (Supelco,
Mississauga, Ontario, Canada) and flame ionization detectors.
The column temperature was held at 120 C isothermally. The
temperature of injector and detector was 200 C. Nitrogen was
used as a carrier gas.
Measurements of pH were made with an AP-61 pH meter
(Fisher Scientific, Hampton, NH). Dissolved oxygen concentration in the cathode chamber was measured using a FOXY
oxygen sensor (OceanOptics, Dunedin, FL)with a detection limit
of 0.1 mg L-1. EM Quant Peroxide Test strips (EM Science,
Gibbstown, NJ) were used for estimations of hydrogen peroxide
concentrations.
Dissolved Methane Measurements. Dissolved methane
concentration was measured by taking a 5 mL liquid sample

from the anodic compartment and placing it in a 10 mL vial.


The vial was sealed immediately with caps fitted with a septum
and vortexed for 1 min. A 300 L sample of the headspace of
the vial was injected after a 15 min delay to allow for an
equilibrium between gas and liquid-phase methane concentrations. The headspace sample was analyzed by gas chromatography (Sigma 2000, Perkin-Elmer, Norwalk, CT) to obtain gasphase methane concentration. A Henrys law constant of kH )
1.510-3 M atm-1 (kH ) ca/pg, where ca is the aqueous methane
concentration and pg is the partial pressure of methane in the
gas phase) was used to calculate dissolved methane concentration. The measurements were carried out in triplicate.
Coulombic Efficiency Calculations. The Coulombic efficiency (E) was calculated using the ratio of total Coulombs
obtained in the experiment (CE) to the theoretical amount (CT)
available from complete substrate oxidation, E ) CE/CT 100%
(8). In this expression CE was estimated by integrating the
measured current over a period of 1 day, CE ) It, where I is
the current (A) and t is the time interval (s). CT was calculated
using the amount of chemical oxygen demand (COD) consumed
in the anode compartment daily, CT ) Fnw/M, where F is
the Faraday constant ) 96485 C/mol, n is the number of moles
of electrons produced per mol of substrate (n ) 24 for glucose
and n ) 4 for wastewater (9)), w is the daily COD load (g),
and M is the substrate molecular weight (g).

Results
Operation of the MFC reactor was started at an aeration rate
of 4 L air LR-1 min-1 in the cathodic compartment, which
provided a dissolved oxygen concentration of 8-9 mg L-1. At
the same time an oxygen concentration of 0.5-1.0 mg L-1 was
measured in the upper part of the anodic compartment in the
vicinity of the separator pad. No oxygen was detected 1 to 2
cm below the separator. When the electrodes were connected
with a 500 resistance, a potential of 3-5 mV was measured.
Consequently, a value of 4 mV was considered as background
and was subtracted from all subsequent measurements.
After a 3 day lag phase, a near exponential voltage increase
was observed and by day 20, the voltage stabilized between
200 and 210 mV (Figure 2). A current-voltage test was carried
out on day 23 of reactor operation. First, an open-loop potential
was measured, and then the external resistance was changed
stepwise from 1000 to 10 with a period of at least 40 min
between the increments to stabilize electrical current after each

Biotechnol. Prog., 2006, Vol. 22, No. 1

243

Figure 3. Polarographic curves obtained during periods of cathode


aeration (A) and hydrogen peroxide feeding (B).

Figure 2. Power production during the aeration phase (A, the insert
shows H2O2 injection experiment and arrow indicates the onset of the
polarographic test) and H2O2-feeding phase (B, numbers indicate H2O2
loads in mL d-1).

change. The resulting polarographic curve is shown in Figure


3a. Based on a total electrode area of 118.7 cm2 in the anodic
compartment, the electrical power production and current density
observed at a resistance of 300 were 7.2 mW m-2 and 33.7
mA m-2, respectively. The internal resistance of the MFC
reactor calculated using the polarographic curve was 340 .
The observed power production exceeded the performance of a
similar continuously operated MFC reactor (5), which also used
graphite electrodes and an aerated cathodic compartment and
received a similar glucose load. In that study a power density
of 1.3 mW m-2 was obtained, apparently because of a larger
distance between the electrodes (30 cm), which resulted in a
larger internal resistance.
Measurements of glucose and volatile fatty acid (VFA)
concentrations at the exits of the anodic and cathodic compartments showed the presence of propionate, butyrate, and acetate,
which are typical intermediates of the anaerobic methanization
process (Table 1). Glucose concentrations were close to the
detection limit in both compartments, while total VFA concentrations at the exit of the anode and cathode compartments were
340 and 284 mg COD L-1, respectively. Also, the presence of
dissolved methane (14-18 mg L-1) in the anodic compartment
was detected. Measurements of pH in both compartments

Figure 4. Dependence of power generation on the hydrogen peroxide


load. Measurements were carried out at an external resistance of 500
.
Table 1. Average Steady State Substrate Concentrations and pH
Values at the Exit of the Anaerobic Anodic and Aerobic Cathodic
Compartments of the MFC Reactor
oxygenation method
substrate
(in mg COD L-1)a
glucose
propionate
butyrate
acetate
total COD
pH

influent
963b
0
0
0
963

air
anode
cathode

anode

2.2
161.3
104.4
73.9
341.8
6.6

11.9
100.4
35.3
105.9
253.5
6.6

1.9
113.3
106.1
64.6
285.9
8.4

H2O2
cathode
6.3
85.2
24.0
99.7
215.2
6.9

a COD equivalents are 1.16, 1.95, 2.4, and 1.07 for 1 g of glucose,
propionate, butyrate, and acetate, respectively. b Based on 500 mg d-1
glucose feeding rate.

showed a significantly higher pH in the cathodic compartment.


Apparently, aeration caused the stripping-off of carbon dioxide
produced by the microorganisms, thus changing the bicarbonate
balance and increasing pH in the cathodic compartment.
Under these conditions, a stable electricity generation was
observed for a period of 25 days, after which the voltage
progressively declined and by day 40 reached a near zero value.
Visual examination of the cathodic compartment showed

Biotechnol. Prog., 2006, Vol. 22, No. 1

244

extensive biofilm growth throughout the compartment and on


the cathode surface. At the same time the dissolved oxygen in
the compartment remained at near saturation at 8-9 mg L-1,
suggesting that the drop in power production was caused by
biofilm formation on the cathode surface, which limited the
influx of oxygen to the electrode. Once the cathode was gently
cleaned with a soft brush to remove the biofilm, an immediate
voltage increase was observed. A stable production of electricity
occurred for 4-5 days and was followed by another decline
due to regrowth of microorganisms on the cathode surface.
Cathode cleaning was repeated several times. After each
cleaning, a period of increased power production followed by
visible biomass growth accompanied by a decline in power
production was observed (results not shown).
Since biomass growth on the cathode surface limited oxygen
transfer and bulk oxygen concentration had to be increased
above 8 mg L-1 in order to provide sufficient influx of oxygen
to the cathode, the use of hydrogen peroxide for cathode
oxygenation was tested. Because of its high oxygen content
(50%) H2O2 can be used in biological applications if high levels
of oxygen are desired (15). To test the influence of H2O2 on
electricity production, 1 mL of 3% H2O2 was injected into the
cathodic compartment. The injection caused a rapid increase in
voltage followed by a return to its previous value (Figure 2a,
insert). This test suggested that H2O2 can be used for improved
oxygen delivery.
To compare both oxygenation methods, on day 71 airoxygenation was changed to H2O2 oxygenation. H2O2 was fed
continuously from the top of the cathodic compartment (Figure
1). Initially, 250 mL of a 0.2% H2O2 solution was fed per day,
resulting in an H2O2 load of 0.5 mL d-1 (Figure 2b). H2O2 loads
of 0.25 and 0.75 mL d-1 were also tested. A proportional
increase in power production was observed when the H2O2 load
was changed from 0.25 to 0.5 mL d-1. However, a further
increase to 0.75 mL d-1 resulted in a smaller increase in power
production (Figure 2b, day 85). In addition, the influent H2O2
concentration increased to 25 mg L-1 as compared to 1-5 mg
L-1 of H2O2 observed at lower H2O2 loads. Dissolved oxygen
measurements at a H2O2 load of 0.75 mL d-1 showed a value
of 12-14 mg L-1 and intensive formation of gas bubbles was
observed on the biofilm surface.
Analysis of substrate concentrations in the two compartments
during H2O2-feeding (average of all H2O2 loads) showed results
similar to those obtained with aeration (Table 1). The effluent
of the anodic compartment contained acetate, propionate, and
butyrate with a total VFA content of 242 mg COD L-1, i.e.,
anaerobic conditions were maintained in the anodic compartment. Although diffusion of H2O2 and oxygen to the anodic
compartment was expected, the suspended heterotrophic microorganisms in this compartment reduced oxygen concentration
below a detection limit of 0.1 mg L-1. The dissolved methane
concentration was unchanged at 14-18 mg L-1.
As with aeration, a gradual proliferation of both suspended
biomass and biofilm was observed in the cathodic compartment.
However, with H2O2 oxygenation biomass proliferation had a
smaller impact on power production and the voltage never
declined below 150 mV. Nevertheless, a cleaning of the cathode
on day 83 at an H2O2 load of 0.5 mL d-1 resulted in a voltage
increase (Figure 2b, arrow). A polarization curve obtained with
an H2O2 load of 0.75 mL d-1 on day 87 showed a maximum of
22 mW m-2 at an external resistance of 300 ,, which
corresponded to a current density of 68 mA m-2.
A combined air-H2O2 oxygenation method was studied using
a full factorial experiment design with a single center point.

Table 2. Aeration Rates, Hydrogen Peroxide Loads, and Steady


State Power Density of the MFC Reactor in the Experiment Aimed
at Optimizing Aeration to H2O2 Load Ratio
test no.

aeration
(L Laer-1 min-1)

H2O2 load
(mL d-1)

power density
(mW m-2)

1
2
3
4
5

4
0
4
0
2

0.75
0.75
0.25
0.25
0.5

4.3
18.9
2.0
6.7
3.1

The results presented in Table 2 showed that aeration negatively


affected power production in the presence of H2O2. At all
combinations of aeration and hydrogen peroxide feeding rates
the power production was inferior to that observed with
hydrogen peroxide alone. One possible explanation is the
significant pH difference between the two compartments when
aeration was used. Indeed, aeration caused the pH to rise above
8 in the cathodic compartment because of carbon dioxide
stripping by aeration, which changed the bicarbonate balance
of the compartment. The pH of the anodic compartment was
below 7. In the absence of aeration, pH remained between 6.7
and 6.9 in both compartments (Table 1).

Discussion
Conventional wastewater treatment systems often consist of
sequentially connected anaerobic and aerobic units. Anaerobic
acidogenic and methanogenic microorganisms of the anaerobic
unit convert a part of organic matter to methane and carbon
dioxide and the remaining biodegradable materials are degraded
in the aerobic unit by the aerobic microorganisms. The upflow
two-compartment MFC reactor used in this experiment carried
out the same biodegradation sequence. In addition to COD
removal, close proximity of the anaerobic and aerobic compartments allowed for microbial generation of electricity by
microorganisms attached to the anode surface.
Coulombic efficiency of the MFC reactor was evaluated using
the measurements of glucose and degradation intermediates in
the anodic compartment (Table 1). The average amount of CODs
consumed in the anode compartment daily during the aeration
phase was estimated at 375 mg COD d-1 and the substrate
molecular weight was M ) 76 g. The calculations were carried
out for external resistances of 500 and 11 . The 500 external
resistance was used throughout the experiment, while a resistance of 11 corresponded to a maximal observed current
density of 1 mA in the polarographic test (85 mA m-2, Figure
3A). The calculations yielded Coulombic efficiencies of 1.8%
and 4.5%, respectively.
Calculations of Coulombic efficiency during the H2O2 phase
were based on an average value of 428 mg COD d-1 consumed
daily in the anodic compartment (Table 1). In this case, a
Coulombic efficiency of 2.9% was calculated for an external
resistance of 500 used throughout the experiment and an
efficiency of 6.3% was calculated for a maximal observed
current of 1.6 mA (133 mA m-2, Figure 3B). These values are
similar to 3-12% Coulombic efficiency observed in mixed
culture experiments (12).
Overall, the long-term stability and power generation efficiency of the MFC reactor in this experiment were limited by
such factors as proliferation of aerobic microorganisms in the
cathodic compartment and the presence of suspended anaerobic
microorganisms in the anodic compartment. Notably, oxygen
penetration in aerobic biofilms is limited to 200-500 m (16,
17), implying that the core of a sufficiently thick aerobic biofilm
is oxygen-limited. In the experiment, biofilm formation on the

Biotechnol. Prog., 2006, Vol. 22, No. 1

cathode surface was clearly visible and biofilm formation


coincided with the decline in power generation. Moreover,
biofilm removal from the cathode surface restored power
production at least temporarily.
The use of H2O2 for oxygenation appeared to alleviate the
oxygen limitation problem. Because of its high oxygen content
(50%) H2O2 can be used in biological applications if high levels
of oxygen are desired (15). Successful application of H2O2 for
bioreactor oxygenation has been demonstrated (18). In the
cathodic compartment, H2O2 was readily transformed to oxygen
by catalases present in the aerobic heterotrophic microorganisms,
both suspended and attached to the cathode surface. Due to
oxygen production in the biofilm, the amount of oxygen
reaching the electrode was increased thus resulting in higher
power generation. Moreover, the decomposition of hydrogen
peroxide to oxygen involves the formation of free oxygen
radicals. Due to the high reactivity of the radicals, the cathode
reaction was facilitated. Thus, microorganisms in the cathodic
compartment indirectly assisted in electricity production by
decomposing hydrogen peroxide and increasing oxygen influx
to cathode rather than reducing electricity production by
consuming oxygen as in the case when oxygen was provided
by aeration.
In addition to a limited oxygen supply, the competition of
anaerobic microorganisms in the anodic compartment for
common substrates reduced the net Coulombic efficiency.
Notably, the anodic compartment was inoculated with 200 mL
of suspended anaerobic sludge. This sludge remained at the
bottom of the anodic compartment. Measurements of volatile
fatty acids and dissolved methane showed that glucose was
readily converted to VFAs by acidogenic anaerobic microorganisms. Furthermore, the presence of dissolved methane in the
anodic compartment indicated that methanogenic microorganisms converted VFAs to methane, i.e., methanogenesis was
taking place in parallel with electricity generation.
The electrically active bacteria attached to the anode surface
mostly consumed volatile fatty acids and competed with
methanogens for a common carbon source. A rough estimation
of the amount of substrate available for electricity generation
can be obtained by comparing the anodic compartment volume
(320 mL) with that of the anode assembly (6 mL). This
comparison suggests that only about 2% of the substrate fed to
the reactor was available to microorganisms attached to the
electrode surface. The Coulombic efficiency can be improved
by design modifications aimed at increasing the anode surface
as well as by creating conditions that would limit methanogenic
activity. Other factors that can be exploited in an attempt to
improve MFC reactor efficiency are improved cathode materials,
i.e., using platinum loaded air-cathode electrodes (10) and
reduced internal resistance (6). Recent improvements in MFC
design has allowed for specific power densities as high as 0.56
W m-2 (19). In addition, wastewater provides a free source of
carbon for power generation and biological power generation
can be considered economically feasible even at a relatively
low Coulombic yield.
In conclusion, the experiment demonstrated that electricity
can be cogenerated in an upflow two-compartment MFC reactor.
Biofilm formation on the cathode surface limited oxygen influx
to the cathode. Although the use of an air-cathode permits a
constant oxygen influx by exposing one side of the cathode to
air (9), biofilm formation on the wet side of the cathode might
limit the amount of oxygen available for the cathode reaction.
Consequently, the influx of oxygen should be increased to
achieve stable performance, i.e., by increasing the outside

245

pressure or using pure oxygen. The use of H2O2 offers an


alternative solution to the oxygen supply problem. While this
solution may not be acceptable in all situations, it allows the
volume of the cathodic compartment to be minimized and
permits MFC operation in the absence of air, i.e., in underwater
or space explorations. Notably, H2O2 has already been used in
wastewater treatment applications, which require high rates of
oxygenation (15), and filamentous bulking control (20). Some
application of H2O2-driven MFC reactors in wastewater treatment can be anticipated.

Acknowledgment
Friendly discussions with Dr. M Gattrell on process electrochemistry are gratefully acknowledged. This is NRC paper no.
47248.

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BP050225J