Appl Microbiol Biotechnol (2009) 82:829–836

DOI 10.1007/s00253-008-1796-4

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Methanogenesis in membraneless microbial electrolysis cells

Peter Clauwaert & Willy Verstraete

Received: 25 August 2008 / Revised: 12 November 2008 / Accepted: 13 November 2008 / Published online: 3 December 2008
# Springer-Verlag 2008

Abstract Operation of microbial electrolysis cells (MECs) Keywords Phosphate buffer . Biocatalyzed electrolysis .
without an ion exchange membrane could help to lower the Overpotentials . Biocatalysts . Ohmic resistance .
construction costs while lowering the ohmic cell resistance Microbial fuel cell
and improving MEC conversion rates by minimizing the
pH gradient between anode and cathode. In this research,
we demonstrate that membraneless MECs with plain Introduction
graphite can be operated for methane production without
pH adjustment and that the ohmic cell resistance could be Microbial electrolysis cells are bio-electrochemical systems
lowered with approximately 50% by removing the cation (BESs) where a voltage is applied to the cell to drive the
exchange membrane. As a result, the current production (bio)electrochemical reactions (Clauwaert et al. 2008a). In a
increased from 66±2 to 156±1 A m−3 MEC by removing microbial electrolysis cell (MEC), biocatalyzed oxidation of
the membrane with an applied voltage of −0.8 V. Methane organic compounds in the anode chamber is typically
was the main energetic product despite continuous opera- combined with chemical evolution of hydrogen in the
tion under carbonate-limited and slightly acidified condi- cathode chamber (Liu et al. 2005; Rozendal et al. 2006b).
tions (pH 6.1–6.2). Our results suggest that continuous So, the current production is directly proportional to the
production of hydrogen in membraneless MECs will be hydrogen gas produced in anaerobic conditions (Rozendal
challenging since methane production might not be avoided et al. 2006b). Recently, the possibility of biological
easily. The electrical energy invested was not always hydrogen production in a poised cathode has been
completely recovered under the form of an energy-rich described after an anodic acclimatization period with
biogas; however, our results indicate that membraneless acetate and hydrogen gas (Rozendal et al. 2008).
MECs might be a viable polishing step for the treatment of Separation between the anode and the cathode chambers
the effluent of anaerobic digesters as methane was of a MEC is accomplished with an ion-selective membrane.
produced under low organic loading conditions and at Ion-selective membranes are used to obtain cathodic
room temperature. hydrogen gas that is as pure as possible. However, ion-
selective membranes give rise to a higher ohmic voltage
loss in the cell and a pH gradient over the membrane,
Electronic supplementary material The online version of this article resulting in a lower current production for a given applied
(doi:10.1007/s00253-008-1796-4) contains supplementary material, cell voltage (Call and Logan 2008). Removing a part of the
which is available to authorized users. membrane helped to minimize the pH gradient between
P. Clauwaert : W. Verstraete (*) anode and cathode (Clauwaert et al. 2008b). Though the
Laboratory of Microbial Ecology and Technology (LabMET), goal of membraneless MECs is to produce hydrogen gas,
Ghent University, Call and Logan (2008) reported the production of methane
Coupure Links 653,
as an undesired side product. Methane production persisted
9000 Ghent, Belgium
e-mail: [email protected] in a membraneless MEC (28 mL volume) despite exposure
URL: http://labmet.UGent.be to the air for 30 to 45 min between batch-feeding cycles
830 Appl Microbiol Biotechnol (2009) 82:829–836

(Call and Logan 2008). Other strategies for inhibition of Materials and methods
methanogenesis in membraneless MECs have been sug-
gested: (1) operation under low pH conditions, (2) washout Microbial electrolysis cell construction and operation
of methanogens by a short hydraulic retention time, or (3)
low carbonate levels (Call and Logan 2008; Rozendal et al. The MECs were made of two plexiglass frames (8×8×
2008). Since BES favor biofilm-associated biomass, 2 cm3 per frame; 0.256 L MEC). The anodic and cathodic
preventing methanogenesis is challenging as biofilms frames were filled with graphite granules (type 00514,
might protect the methanogens from high oxygen concen- diameter between 1.5 and 5 mm, Le Carbone, Belgium) and
trations and washout. At the cathode, where local connected to the external circuitry with a graphite rod
alkalinization occurs, creating low pH conditions will be (5 mm diameter, Morgan, Belgium; Figure S1 of the
also difficult. Additionally, carbon dioxide, continuously Electronic Supplementary Material). A cation exchange
produced as a result of anodic oxidation of organic membrane (CEM; Ultrex CMI7000, Membranes Interna-
compounds, may cross over to the cathode, making tional) was inserted between the anodic and cathodic
operation under carbon-limited conditions in a membrane- frames for the tests indicated by an asterisk (*). In the
less MEC rather unlikely. MEC tests completed without a membrane, a nonwoven
Methane is an energetic compound, and as a product of cloth (Liplisse 3, Libeltex, Belgium) was used to prevent
anaerobic digestion, it is widely used to produce electricity contact between anodic and cathodic graphite granules.
in wastewater treatment facilities (Pham et al. 2006). During the experiments where sodium acetate (1 g per
Though methane contains less energy than hydrogen per feeding cycle) was batch fed to the membrane-containing
unit of mass, a MEC without ion exchange membrane will MECs (R1*, R2*, RA*, and RB*), the anode and the
have lower capital costs. Methane production in a MEC cathode had a separate recirculation vessel (2 L) from
may also be more robust than hydrogen production. Hence, which the liquid was pumped through the anode or cathode
a MEC installed after a conventional anaerobic digester to at a rate of 0.35 L h−1 (Figure S2 of the Electronic
remove the residual organics present in the effluent of the Supplementary Material). During the experiments where
digester could be a practical polishing step. Indeed, sodium acetate or acetic acid were batch fed to the
effluents from completely stirred tank digesters treating membraneless MECs (RA and RB), a recirculation vessel
wastes such as biosolids or manure typically have effluents (0.25 L) was placed in the recirculation loop (0.35 L h−1) so
still containing 0.5 to a few grams of residual volatile fatty that the cathode effluent was mixed in the recirculation
acids (van Lier et al. 2001). Interestingly, BES are typically vessel before entering the anode (Figure S3 of the
effective at such low substrate concentrations and can also Electronic Supplementary Material). In the other tests,
work at ambient temperatures (Pham et al. 2006). the MEC (RBc) was operated continuously and fed fresh
Sulfide is a threat to the long-term performance of a medium (0.64 L day−1; hydraulic retention time, 5.3 h)
membraneless MEC intended for hydrogen production. that was flushed for 30 min with nitrogen gas prior to use.
Sulfate is often reduced in the anode compartment, and A concentrated solution of acetic acid, dissolved in
sulfide can end up in the cathode compartment (Rabaey et medium used at that time, was continuously added
al. 2006). In the cathode, sulfide can lower hydrogen gas (8.5 mL day−1) to the recirculation loop (0.35 L h−1) to
purity as well as the activity of the platinum catalyst often obtain the desired volumetric chemical oxygen demand
used in MECs (Niessen et al. 2004). (COD) loading rate (Bv, kg COD m−3 MEC day−1; Figure
The goal of this research was to determine the S4 of the Electronic Supplementary Material). The
effectiveness of a membraneless MEC with high specific medium always contained 6 g Na2HPO4·2H2O L−1, 3 g
surface area granular graphite electrodes (in the order of KH2PO4 L−1, 1 g NaHCO3 L−1, 0.2 g MgSO4·7H2O L−1,
6 × 10 6 m 2 m −3 ; Freguia et al. 2007) for methane 0.1 g NH4Cl L−1, 0.0146 g CaCl2 L−1, and trace elements
production. As has been suggested for microbial fuel as previously described (Clauwaert et al. 2007b), unless
cells (MFCs), activation overpotentials may be lowered stated differently. All tests were performed at room
by increasing the specific surface area of the cathode temperature (22±2 °C).
with granular graphite (Freguia et al. 2007), making
chemical catalysts that are fouled by sulfide not necessary. Electrochemical measurements
We constructed MECs with and without a cation exchange
membrane. The viability of these configurations was The cell voltage of each MEC was applied by a direct
evaluated based on the operational performances and current (DC) power supply or by a potentiostat (PAR Bi-
polarization behavior of a MEC. The methane production Stat Potentiostat, Princeton Applied Research, France).
was monitored in the presence and the absence of an When a DC power supply was used, the current was
applied cell voltage. measured by the voltage difference over a 1-Ω resistor in
Appl Microbiol Biotechnol (2009) 82:829–836 831

the electrical circuit between the DC power supply and the over acetate dosed (ηAc-CH4 and hTh:MAX
Ac
H2 , dimensionless)
cathode. The cell voltage, current production, and the is calculated as follows:
cathode potential were recorded every minute with a data-  
rCH4  8 mol e
mol
1 CH4  32 g COD mol
1 COD
acquisition unit (HP 34970A, Agilent). The hourly aver- 24:2 L mol
1  4 mol e
mol
1 COD
hAc
CH4 ¼  100% ð4Þ
ages were then used for further calculations. Polarization Bv
EAc
curves were obtained with a potentiostat by increasing the
applied voltage by −0.050 V increments every hour  
rHTh:MAX  2 mol e
mol
1 H2  32 g COD mol
1 COD
between −0.100 and −0.800 V. The polarization curves 2
24:2 L mol
1  4 mol e
mol
1 COD
were used to evaluate the electrode potential in function of hAc
HTh:MAX ¼  100%
the current production. The ohmic cell resistance was
2 Bv
EAc
determined with the current interrupt method based on cell ð5Þ
voltage change 0.2 ms after disconnecting the cell during a
with Bv, the volumetric COD loading rate, EAc, the specific
potentiostatic program. The potential of the cathodic elec-
COD flux leaving the microbial electrolysis cell (kg COD m−3
trode was monitored with a Ag/AgCl reference electrode
MEC day−1), and AcCOD the acetate concentration expressed
(assumed to be +0.197 V vs Standard Hydrogen Electrode
in COD equivalents (g COD L−1).
(SHE); model RE-5B, BASi), and the anode potential was


1
calculated from the cathode potential, the cell voltage COD
ACCOD  0:64 L day
AcIN OUT

applied, and the ohmic cell voltage (Clauwaert et al. 2008a). EAc ¼
ð6Þ
ð256  10
6 m3 MECÞ  1; 000 g kg
1
The coulombic efficiency (CE) was determined as previ-
ously described (Clauwaert et al. 2007b): In the case of batch-operated tests, rbiogas, rCH4, rH2
Th:MAX
, and
  Ac
E need to be replaced in Eqs. 2 to 5 by V biogas
(L), VCH4
(L), and the acetate concentration (g COD L−1)
I Th:MAX
139:59A kg
1 COD d (L), VH2
CE ¼  100% ð1Þ at the end of the batch tests, respectively. The equations to
Bv
calculate the percentage of the substrate dosed that could be
lost as soluble methane and hydrogen gas in the effluent
Analytical measurements (FCH4dis and FH2dis, %) and the specific COD removal rate
due to sulfate reduction to sulfide are given in the Electronic
Acetate, sulfate, and ammonium concentrations were Supplementary Material.
determined as previously described (Clauwaert et al.
2007a). For the batch-fed tests, a gas bag was connected
to the headspace of the recirculation vessel. When operated Results
continuously, a gas trap was used to separate the biogas
produced from the effluent. Methane (CH4biogas, (vol.) %) Batch-fed microbial electrolysis cells
and carbon dioxide (CO2biogas, (vol.) %) were measured as
previously described (Clauwaert et al. 2008b). The specific MECs R1* and R2*, both with a CEM between anode and
methane production rate (rCH4, L CH4 L−1 MEC day−1) and cathode, were constructed and operated in a batch fed mode
the theoretical maximum hydrogen production rate (Figure S2 of the Electronic Supplementary Material) with

1
1

an applied cell voltage of −0.601±0.013 V. The anode
Th:MAX
rH2 ; L H2 L MEC day were calculated from the
chambers were inoculated with 5 mL of anode effluent
from a MFC, and the cathode chambers were inoculated
biogas production rate (rbiogas, L biogas L−1 MEC day−1) with 5 mL nongranular anaerobic sludge obtained from a
and volumetric fractions of methane and carbon dioxide. mesophilic winery digester. After 24 to 48 h of lag phase,
the current production remained between 8 and 30 A m−3
CHbiogas MEC for both reactors for 28 days (Eanode, −0.269 V vs
rCH4 ¼ 4
 rbiogas ð2Þ
100% SHE, Ecathode, −0.844±0.014 V vs SHE). There was no gas
  production detected in the cathode.
100%
CHbiogas
4
CO biogas
2 To verify microbial involvement in the anode and
rHTh:MAX ¼ rbiogas  ð3Þ cathode conversion processes, the graphite granules of the
2
100%
anode and the cathode of one of the two MECs were
This means that the actual hydrogen production rate could autoclaved. The autoclaved anode was combined with the
even be lower if traces of nitrogen gas, hydrogen sulfide, not autoclaved cathode of (RA*), and the autoclaved
etc. would be present in the biogas. The percentage of cathode was combined with the not autoclaved anode
methane or (theoretical maximum) hydrogen recovered (RB*). Re-inoculation occurred from the recirculation
832 Appl Microbiol Biotechnol (2009) 82:829–836

vessels. It took the reactor with the autoclaved anode 39 h The applied cell voltage of the membraneless MEC RA
to regain the production of 8 A m−3 MEC, whereas the was increased to approximately −0.8 V. After an acclimatiza-
current production in the reactor with the autoclaved tion period of 10 days, cell RA was fed with four subsequent
cathode was not hampered (12–20 A m−3 MEC) during spike doses of 12.2 mmol sodium acetate (Na-Ac) upon
the next 14 days. Polarization curves were obtained before depletion (Table 1, test 1), four spike doses of 12.2 mmol
and 3 days after autoclaving the respective graphite acetic acid (H-Ac; Table 1, test 2), and then, as a control
electrode granules (Fig. 1), indicating that mainly the anode experiment with no applied voltage, two times with
was affected due to the autoclaving and the disruption by 12.2 mmol acetic acid (Table 1, test 3). The medium was
replacing the graphite electrode granules. renewed and sparged for 30 min with nitrogen gas when the
Subsequently, the CEMs were removed, and a nonwoven substrate was changed from sodium acetate to acetic acid and
cloth was placed between the anode and the cathode to before the control experiment. An intermediate feeding cycle
create the membraneless MEC configuration (Figure S3 of (data not shown) was applied each time to remove most of
the Electronic Supplementary Material). The reactors (RA the nitrogen gas from the headspace after sparging. The
and RB) were inoculated with 5 mL of the same maximum current density was higher in the case of sodium
nongranular anaerobic sludge as used for R1* and R2*. acetate, and the average time per cycle was lower. The
Removing the CEM lowered the ohmic cell resistance from addition of HCl to compensate the pH increase, resulting in a
2.2±0.6 Ω (N=14) to 1.1±0.3 Ω (N=14). The MECs were higher conductivity, could have accounted for the higher
operated for 22 days with an applied cell voltage of −0.610± current production. However, there was a higher rate of
0.006 V. The current production immediately increased to carbon dioxide production for the feeding cycles with acetic
35 A m−3 MEC after removing the CEM and further acid (Table 1). When sodium acetate was supplied to the
increased up to 55±6 A m−3 MEC in the following days MEC, the pH needed to be corrected from 7.4–7.6 to 7.0–7.2
(Eanode, −0.225 V vs SHE, Ecathode, −0.818±0.006 V vs with 1 M HCl after every feeding cycle. For acetic acid,
SHE). A polarization curve (Fig. 1) was performed, and there was also an increase in pH during every cycle, which
higher current densities could be achieved compared to the compensated for the initial decrease in pH to 6.4±0.2 after
membrane-containing MEC. Especially the cathode over- the spiking with acetic acid. This lower pH might explain the
potentials in the high current range did not increase as fast as higher CO2 content in the case of acetic acid.
in the case of a membrane-containing MEC. An over-
potential at an electrode is defined as the voltage difference Continuous operated microbial electrolysis cell
between the electrode potential and the equilibrium poten-
tials, which is the equilibrated electrode potential in the Cell RBc was first operated at a volumetric COD loading
absence of current production. rate of 1.38 kg COD m−3 MEC day−1 with continuous
recirculation, and various parameters were monitored
Current production (A m-3 MEC) (Figure S4 of the Electronic Supplementary Material and
0.100 Table 2, test 1). When there was no recirculation, the
0.000 current production, coulombic efficiency, and methane
0 25 50 75 100 125 150
production decreased (Table 2, test 2). An increase in the
Electrode potential (V vs SHE)

-0.100

-0.200
loading rate resulted in a higher current and methane
Anode R1*/R2*
production, while the acetate removal efficiency was much
-0.300
Cathode R1*/R2* lower (Table 2, tests 3 and 4). As a control, no voltage was
-0.400 Anode RA*
applied in a subsequent test. Methane production continued
Cathode RA*
-0.500 but at a lower rate (Table 2, test 5). In test 6, the carbonate
Anode RB*
-0.600 Cathode RB* buffer was omitted from the influent media, resulting in an
Anode RA/RB
-0.700 Cathode RA/RB influent pH decrease from 7.2 to 7.0. This had no clear
-0.800
impact on the current production nor methane production
rate compared with test 1. The phosphate buffer strength
-0.900
was lowered by a factor of 10 in test 7. As a result, the
-1.000
effluent pH decreased to 6.1–6.2. This lower pH lowered
Fig. 1 The anode and cathode potential in function of the current the cathodic overpotentials, but the anodic overpotentials
density in the presence (asterisk) or absence of a cation exchange increased even stronger (Fig. 2). In test 8, the pH of the
membrane during polarization. In the case of RA*, the polarization was concentrated solution of acetic acid was adjusted from 2.4
performed 3 days after autoclaving the anodic graphite granules, while
to 6.9 with 1 N NaOH, which lowered the anodic
in the case of RB*, the polarization was performed 3 days after
autoclaving the cathodic graphite granules. The error bars indicate the overpotentials compared to test 7. The production of current
minimum and maximum of a duplicate scan for R1*, R2*, RA, and RB remained lower than in test 1 (Table 1), which was likely
Appl Microbiol Biotechnol (2009) 82:829–836 833

Table 1 Summary of three membraneless microbial electrolysis tests in cell RA, where every feeding cycle, 12.2 mmol of sodium acetate or
acetic acid was batch-fed as a substrate

Test number 1 2 3

Test duration (h cycle−1) 49±2 66±8 96±1

Applied cell voltage (V) −0.807±0.005 −0.816±0.002 –
Substrate (−) Na-Ac H-Ac H-Ac
Total number of feeding cycles (−) 4 4 2
VCH4 (L CH4 cycle−1) 0.23±0.02 0.26±0.02 0.20±0.04
CH4biogas (% vol.) 71±3 58±2 37±15
CO2biogas (% vol.) 8±3 24±11 13±9
Acetate removal (%) 100 100 100
ηAc-CH4 (%) 79±7 87±7 69±13
ηAc-H2Th.MAX (%) 6±3 6±3 23±3
Imax (A m−3 MEC) 223 190 –
CE (%) 87±5 83±2 –
Eanode (V vs SHE) −0.148 −0.052 −0.316
Ecathode (V vs SHE) −0.896±0.012 −0.820±0.021 −0.308±0.018
ΔpH day−1 (−) 0.2±0.1 0.2±0.1 0.2±0.1

The test duration was defined as the time between substrate feeding and a complete removal of the substrate.
ηAc-CH4 percentage of methane recovered in the biogas over acetate removed on COD basis ×100%, ηAc-H2Th.MAX percentage of theoretical
maximum hydrogen recovered in the biogas over moles acetate removed on COD basis ×100%, Imax the maximum hourly averaged current
production, CE the coulombic efficiency, Eanode and Ecathode the electrode potential of anode and cathode, ΔpH day−1 the daily increase in pH in
the recirculation vessel

Table 2 Summary of the continuous membraneless microbial electrolysis tests in RBc

Test number T1 T2a T3 T4 T5 T6b T7b,c T8b,c,d

Test duration (h) 165 167 166 102 147 169 166 164
Applied cell (V) −0.812± −0.831± −0.818± −0.813± – −0.827± −0.844± −0.833±
voltage 0.004 0.009 0.009 0.014 0.007 0.004 0.002
Bv (kg COD m−3 1.38 1.38 2.41 4.13 1.38 1.38 1.38 1.38
MEC day−1)
rCH4 (L CH4 L−1 MEC 0.33± 0.28± 0.35± 0.75± 0.17± 0.30± 0.29± 0.34±
day−1) 0.07 0.02 0.06 0.12 0.06 0.03 0.02 0.03
CH4biogas (% vol.) 80±5 77±5 78±5 79±8 69±17 79±7 72±3 90±8
CO2biogas (% vol.) 5±1 4±1 8±5 11±1 6±1 4±0 9±1 2±1
Acetate (%) 98±1 93±3 58±23 56±2 69±5 99±1 96±1 96±2
removal
ηAc-CH4 (%) 65±13 57±4 66±11 86±14 47±17 58±7 59±4 67±6
I (A m−3 MEC) 121±5 72±6 198±6 211±10 – 116±3 56±3 74±2
CE (%) 63±3 37±3 59±2 36±2 – 60±1 29±2 39±1
Eanode (V vs SHE) −0.094 −0.065 −0.100 −0.096 −0.358 −0.085 +0.016 −0.143
Ecathode (V vs SHE) −0.859± −0.856± −0.842± −0.828± −0.363± −0.840± −0.694± −0.886±
0.006 0.008 0.004 0.004 0.012 0.012 0.010 0.025
Ammonium (%) 6±4 20±11 8±7 17±2 4±4 20±4 6±3 18±7
removal
Sulfate (%) 66±5 52±12 56±8 68±10 28±14 63±4 70±6 82±7
removal
pHin−pHout (−) 0.2±0.0 0.2±0.0 0.3±0.1 0.5±0.0 0.2±0.0 0.2±0.0 0.8±0.1 −0.5±0.1

Bv volumetric COD loading rate, rCH4 the specific methane production rate, ηAc-CH4 percentage of methane recovered over acetate removed on
COD basis ×100%, I the average current production, CE the coulombic efficiency, Eanode and Ecathode the electrode potential of anode and
cathode
a
No recirculation
b
No carbonate buffer used
c
5.6 mM phosphate buffer used instead of 56 mM
d
pH in syringe adjusted to 6.9 with 1 N NaOH
834 Appl Microbiol Biotechnol (2009) 82:829–836

Current production (A m-3 MEC) 0.04±0.1 kg COD m−3 MEC day−1 (Table 2). If sulfide
0.100
was oxidized at the anode to elemental sulfur, the fraction
0.000 of the COD loading rate that was not recovered as current
0 25 50 75 100 125 150
Electrode potential (V vs SHE)

-0.100 was 25% lower. Polarization curves were performed

-0.200 during tests 1, 2, 6, 7, and 8 (Fig. 2).
-0.300 Anode T1
Cathode T1
-0.400 Anode T2
Cathode T2 Discussion
-0.500
Anode T6
-0.600 Cathode T6
Anode T7 Ion exchange membranes in BESs are generally considered
-0.700 Cathode T7 necessary to avoid short-circuit conversion of the reagents.
Anode T8
-0.800 Cathode T8
They however cause higher ohmic cell resistance and the
-0.900 buildup of a pH gradient across the membrane (Clauwaert
-1.000
et al. 2008a; Rozendal et al. 2006a). Here, we have found
that removing the cation exchange membrane lowered the
Fig. 2 The anode and cathode potential in function of the current ohmic cell resistance by approximately 50% (2.2 vs 1.1 Ω
density of cell RBC during polarization. The cells were fed in a or 0.56 vs 0.28 mΩ.m3 MEC), while alleviating the need to
continuous way, and there was no recirculation during test T2, no
carbonate buffer during tests T6, T7, and T8, and a 5.6 mM phosphate adjust the pH with acid or base. As previously described for
buffer instead of 56 mM during tests T7 and T8. The pH in the MFCs, the membrane avoids the direct crossover conver-
feeding syringe during T7 was 2.4 and was adjusted to 6.9 with 1 N sion of the organic substrate by aerobic microorganisms,
NaOH during T8 (more details in Table 2) which lowers the coulombic efficiency (Liu and Logan
2004). MECs with an ion exchange membrane resulted in
due to the lower pH buffer capacity. The average ohmic cell production of relative pure hydrogen gas in the cathode
resistance was 1.6±0.3 Ω (N=3) during tests 1, 3, and 4. (Rozendal et al. 2007). However, when this membrane is
During test 2, the ohmic cell resistance was 2.2 Ω, and omitted from the MEC, hydrogenotrophic methanogens can
removing the carbonate buffer (test 6) resulted in an combine anodically produced carbon dioxide with cathod-
increase of the ohmic cell resistance to 2.5 Ω. Lowering ically produced hydrogen gas (Call and Logan 2008;
the phosphate buffer strength resulted in an increase of the Clauwaert et al. 2008b). In this research, we have
ohmic cell resistance to 8.9 Ω (test 7), and during test 8, the demonstrated that methanogenesis can easily become
ohmic cell resistance decreased to 4.8 Ω, most likely due to dominant in membraneless MECs that are not exposed to
the continuous addition of NaOH that increased the ionic air. Of an acetate-containing solution (0.554 g COD L−1),
strength. 98±1% was removed in a membraneless MEC. Of the ace-
The acetate that was removed in the reactor and that was tate removed, 65±13% could be recovered as methane at a
not recovered as methane in the biogas (100%−η
Ac-CH4) rate of 0.33±0.07 L CH4 L−1 MEC day−1 (Table 2, test 1).
was either converted into hydrogen gas hTh:maxAc
H2 , soluble Methanogenesis was persistent throughout the experiments
methane, and hydrogen in the effluent, reduced sulfur despite short hydraulic retention times of 5.3 h and slightly
species, or biomass. The theoretical maximal hydrogen acidic conditions (pH 6.1–6.2). Operation under more
fraction in the biogas was calculated by excluding the acidified conditions is not desired as it would result in a
measured volume of methane and carbon dioxide (Eq. 3). further increase of anodic overpotentials during anodic
The theoretical percentage of acetate

removed that was substrate oxidation, especially since the pH in the proximity
recovered as hydrogen hAc
H2 was calculated to be
Th:max
of the anode is even lower than in the bulk solution
between 2% and 5% for tests 1 to 8. The dissolved fraction (Clauwaert et al. 2008a). Also, the operation without
of the biogas produced in the effluent could not be carbonate in the medium (Table 2, test 6) did not result in
recovered. For a volumetric loading rate of 1.38 kg COD a lower methane production rate. Further research is
m−3 MEC day−1, a maximum percentage of 14% and 2% of necessary to reveal the proportion, localization, and activity
the COD could be lost to dissolved methane and hydrogen, of hydrogenotrophic and acetoclastic methanogens in the
respectively, in the effluent (assumed solubility at 22 °C, anode and cathode compartment. Moreover, other organic
20 mg CH4 L−1 and 1.6 mg H2 L−1). Sulfate reduction, substrates and their effect on the microbial community are
when assumed to sulfide (8 mol electrons per mol of specific interest for further research.
sulfate), could on average have contributed to a COD Our findings suggest that methane production is most
removal rate of 0.08±0.02 kg COD m−3 MEC day−1 for likely to occur in membraneless MECs, even in acidified,
all tests, except for test 5 where the calculated COD carbonate-limited continuous systems. If, however, hydro-
removal rate based on the sulfate removal was only gen production would be preferred over methane produc-
Appl Microbiol Biotechnol (2009) 82:829–836 835

tion, alternative strategies have to be brought forward. been demonstrated yet in BESs (Kim et al. 2008),
Draining the reactor and exposure to air between every ammonium was most likely used for biomass buildup.
feeding cycle lowered the methane production rate (Call The use of pH buffers in BESs is popular because it masks
and Logan 2008). Another strategy could be the stimulation strong pH increases or decreases in cathodes or anodes,
of nitrifying microorganisms on internally aerated hollow respectively. When sodium acetate is the substrate and
fiber membranes inside the cathode compartment (Downing completely oxidized, only seven protons are delivered per
and Nerenberg 2007). The presence of low levels of eight electrons, and this alkalinization effect becomes more
oxygen, nitrate, and denitrification intermediates could dominant in poorly buffered solutions. The disadvantage of
inhibit the methane production (Roy and Conrad 1999). acetic acid as the substrate is that, especially in poorly
The combined nitrification and denitrification in the buffered solutions, pH control might be necessary in order
cathode can be considered as an environmental benefit in to avoid pH shocks.
the context of wastewater purification, however, rendering If the substrate would be completely converted into
the produced hydrogen gas impure with nitrogen gas, the hydrogen gas, the theoretical energy yield would be
end product of autotrophic or heterotrophic denitrification. 5 kWh kg−1 COD converted, while this would only be
The impure hydrogen gas produced might than be of 3.5 kWh kg−1 COD converted for methane (0.079 kWh
interest for the production of biopolymers like polyhydrox- mol−1 H2; 0.225 kWh mol−1 CH4). During the continuous
ybutyrate (Ishizaki et al. 1993). Another strategy to operation, the energy recovery, based on the measured
minimize methanogenesis in situ could be a regular short- methane production and calculated hydrogen production,
time polarity reversal at a higher applied cell voltage to for tests 1 to 8 was between 2.15 and 3.13 kWh kg−1 COD
produce oxygen and free radicals. substrate converted, except for test 5 where the recovery
The maximum reported current productions described was 1.87 kWh kg−1 COD substrate converted. The
for MECs where the anode and cathode were in the same efficiency from biogas combustion for electricity produc-
electrolyte were 292 A m−3 MEC for 28 mL MECs (Call tion is around 35% (Zeeman et al. 2008). The overall
and Logan 2008) and 145 A m−3 MEC for 225 mL MECs energetic efficiency can be much higher, since the heat
(Clauwaert et al. 2008b). In both cases, platinum was used that is liberated through combustion is not used for
as a catalyst in the cathode. In our research, no chemical heating the methane producing reactor but can be used
catalysts were used in the cathode, and the maximum for other processes. When no voltage was applied, the
current production was 223 A m−3 MEC (or 446 A m−3 methane production was not completely hampered
total cathodic compartment (TCC)) with graphite granules. (Tables 1 and 2), which indicates that also acetoclastic
The continuous removal of hydrogen by hydrogenotrophic methanogens were present and/or that syntrophic acetate
methanogens lowering the hydrogen partial pressure might oxidation coupled with hydrogenotrophic methanogenesis
have contributed to lower cathodic overpotentials. Current occurred in the absence of a voltage applied. The energy
densities from hydrogen producing cathodes were reported input under the form of electrical energy was between 2.72
in the order of 2 A m−2 (approximately 121 A m−3 TCC, and 2.83 kWh kg−1 COD converted into current for tests 1
calculated value for 413 mL TCC) for a noncatalyzed to 8, except for test 5 where no voltage was applied
graphite felt as a cathode, while a biological catalyzed (Table 2). This means that the energy invested was not
graphite felt as cathode could produce up to 3.7 A m−2 always completely recovered under the form of an energy-
(approximately 225 A m−3 TCC, calculated value) when the rich biogas. Improvement in the reactor configuration and
cathode potential was potentiostatically poised at −0.8 V vs biofilm structure might enhance the conversion efficiency
SHE (Rozendal et al. 2008). In our research, no biological at a lower applied cell voltage.
involvement in the cathodic reaction could be determined Further research might demonstrate that a hydrogen-
by autoclaving the cathodic graphite granules. This indi- producing cathode at the effluent of an anaerobic digester is
cates that it may be necessary to acclimate an anode with able to attract or retain the slow-growing, nongranular
hydrogen as the electron donor to establish an electrochem- methanogens that would otherwise be flushed out of the
ically active biofilm that can later be used for biocathodic reactor. Chemical reduction of polar compounds like humic
catalysis of hydrogen production. acids in the proximity of a hydrogen producing cathode
In this research, we demonstrated that no pH correction might also be used to make them apolar in order to remove
was needed when acetic acid was used as the electron them more easily by absorption or flotation (Satyawali et al.
donor. Besides acetate removal up to 99% during contin- 2007).
uous MEC operation, 52% to 82% of the 0.81 mM sulfate We have demonstrated that methane production can
and between 4% and 20% of the 1.87 mM ammonium in easily be established in a membraneless MEC at ambient
the influent were removed when a voltage was applied to temperatures. However, the energy balance remains less
the MEC. Since anaerobic ammonium oxidation has not favorable than anaerobic digestion where no energy
836 Appl Microbiol Biotechnol (2009) 82:829–836

(tropical climates) or low caloric energy is needed to obtain tion using gaseous substrate to guarantee safety from explosion. J
Chem Eng Jpn 26:225–227
methane production. The methane production rate in
Kim JR, Zuo Y, Regan JM, Logan BE (2008) Analysis of ammonia
conventional anaerobic reactors fed with concentrated loss mechanisms in microbial fuel cells treating animal waste-
organic substrates (> 1 g COD L−1) can be in the order of water. Biotechnol Bioeng 99:1120–1127
several liters methane per liter reactor per day, even at Liu H, Logan BE (2004) Electricity generation using an air-cathode
single chamber microbial fuel cell in the presence and absence of
moderate temperatures, while methane production rates in
a proton exchange membrane. Environ Sci Technol 38:4040–
our lower-loaded MECs (<1 g COD L−1) were below 0.7 L 4046
methane per liter reactor per day. Methane-producing Liu H, Grot S, Logan BE (2005) Electrochemically assisted microbial
MECs could be used in combination with conventional production of hydrogen from acetate. Environ Sci Technol
39:4317–4320
anaerobic digestion as a way to remove residual fatty acid Niessen J, Schroder U, Rosenbaum M, Scholz F (2004) Fluorinated
and sulfides, for instance from anaerobically, low-temper- polyanilines as superior materials for electrocatalytic anodes in
ature-treated domestic wastewater (Zeeman et al. 2008). bacterial fuel cells. Electrochem Commun 6:571–575
Pham TH, Rabaey K, Aelterman P, Clauwaert P, De Schamphelaire L,
Boon N, Verstraete W (2006) Microbial fuel cells in relation to
Acknowledgments The useful comments of Caitlyn Shea, Robert conventional anaerobic digestion technology. Eng Life Sci
Nerenberg, Pieter Van de Caveye, Liesje De Schamphelaire, and 6:285–292
Jingxing Ma are kindly acknowledged. This research was funded by a Rabaey K, Vandesompel K, Maignien L, Boon N, Aelterman P,
Ph.D grant (IWT grant 53305) of the Institute for the Promotion of Clauwaert P, De Schamphelaire L, Pham HT, Vermeulen J,
Innovation through Science and Technology in Flanders (IWT- Verhaege M, Lens P, Verstraete W (2006) Microbial fuel cells for
Vlaanderen) and by MIP-2007-04-Sewage Plus. sulfide removal. Environ Sci Technol 40:5218–5224
Roy R, Conrad R (1999) Effect of methanogenic precursors (acetate,
hydrogen, propionate) on the suppression of methane production
by nitrate in anoxic rice field soil. FEMS Microbiol Ecol 28:49–
References
61
Rozendal RA, Hamelers HVM, Buisman CJN (2006a) Effects of
Call D, Logan BE (2008) Hydrogen production in a single chamber membrane cation transport on pH and microbial fuel cell
microbial electrolysis cell lacking a membrane. Environ Sci performance. Environ Sci Technol 40:5206–5211
Technol 42:3401–3406 Rozendal RA, Hamelers HVM, Euverink GJW, Metz SJ, Buisman CJN
Clauwaert P, Rabaey K, Aelterman P, DeSchamphelaire L, Pham TH, (2006b) Principle and perspectives of hydrogen production
Boeckx P, Boon N, Verstraete W (2007a) Biological denitrifica- through biocatalyzed electrolysis. Int J Hydrogen Energy
tion in microbial fuel cells. Environ Sci Technol 41:3354–3360 31:1632–1640
Clauwaert P, Van der Ha D, Boon N, Verbeken K, Verhaege M, Rozendal RA, Hamelers HVM, Molenkmp RJ, Buisman JN (2007)
Rabaey K, Verstraete W (2007b) Open air biocathode enables Performance of single chamber biocatalyzed electrolysis with
effective electricity generation with microbial fuel cells. Environ different types of ion exchange membranes. Water Res 41:1984–
Sci Technol 41:7564–7569 1994
Clauwaert P, Aelterman P, Pham TH, De Schamphelaire L, Carballa M, Rozendal RA, Jeremiasse AW, Hamelers HVM, Buisman CJN (2008)
Rabaey K, Verstraete W (2008a) Minimizing losses in bio- Hydrogen production with a microbial biocathode. Environ Sci
electrochemical systems: the road to applications. Appl Microbiol Technol 42:629–634
Biotechnol 79:901–913 Satyawali Y, Van de Wiele T, Saveyn H, Van der Meeren P, Verstraete W
Clauwaert P, Toledo R, Van der Ha D, Crab R, Verstraete W, Hu H, (2007) Electrolytic reduction improves treatability of humic acids
Udert KM, Rabaey K (2008b) Combining biocatalyzed electrol- containing water streams. J Chem Technol Biotechnol 82:730–737
ysis with anaerobic digestion. Water Sci Technol 57:575–579 van Lier JB, Tilche A, Ahring BK, Macarie H, Moletta R, Dohanyos
Downing LS, Nerenberg R (2007) Performance and microbial ecology M, Pol LWH, Lens P, Verstraete W (2001) New perspectives in
of the hybrid membrane biofilm process for concurrent nitrification anaerobic digestion. Water Sci Technol 43:1–18
and denitrification of wastewater. Water Sci Technol 55:355–362 Zeeman G, Kujawa K, de Mes T, Hernandez L, de Graaff M, Abu-
Freguia S, Rabaey K, Yuan Z, Keller J (2007) Non-catalyzed cathodic Ghunmi L, Mels A, Meulman B, Temmink H, Buisman C, van
oxygen reduction at graphite granules in microbial fuel cells. Lier J, Lettinga G (2008) Anaerobic treatment as a core
Electrochim Acta 53:598–603 technology for energy, nutrients and water recovery from
Ishizaki A, Tanaka K, Takeshita T, Kanemaru T, Shimoji T, Kawano T source-separated domestic waste(water). Water Sci Technol
(1993) Equipment and operation for fermentative PHB produc- 57:1207–1212