Bioelectrochemistry 109 (2016) 5762

Contents lists available at ScienceDirect

Bioelectrochemistry

journal homepage: www.elsevier.com/locate/bioelechem

Increased performance of hydrogen production in microbial electrolysis

cells under alkaline conditions
Laura Rago, Juan A. Baeza , Albert Guisasola
GENOCOV, Departament d'Enginyeria Qumica, Biolgica i Ambiental, Escola d'Enginyeria, Universitat Autnoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

a r t i c l e i n f o a b s t r a c t

Article history: This work reports the rst successful enrichment and operation of alkaline bioelectrochemical systems (microbial
Received 4 November 2015 fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH = 9.3) bioelectrochemical hydrogen pro-
Received in revised form 27 December 2015 duction presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres
Accepted 24 January 2016
of hydrogen gas per litre of reactor per day, LH2L1REACTORd1). Pyrosequencing results of the anodic bio-
Available online 27 January 2016
lm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter
Keywords:
sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the rst report of a high enrichment
Alkalibacter of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover,
Alkaline Alkalibacter sp. was highly present in the anodic biolm of the alkaline MFC (37%), which would indicate
Geoalkalibacter its potentiality as a new exoelectrogen.
Microbial electrolysis cell (MEC) 2016 Elsevier B.V. All rights reserved.
Microbial fuel cell (MFC)
Pyrosequencing

1. Introduction range to include wastewater treatment and energy generation under

Bioelectrochemical systems represent a promising strategy to har-
vest energy and to obtain added value products from renewable sources
with high organic content (e.g. wastewaters). Bioelectrochemistry com-
bines electrochemistry with the metabolism of anode respiring bacteria
(ARB), also known as exoelectrogenic bacteria. These bacteria are able
to transfer the electrons obtained in their metabolism to an external
solid anode which is, thus, the nal electron acceptor. These electrons
ow through an electrical circuit to a cathode where a reductive
reaction takes place [1].
In a microbial fuel cell (MFC), oxygen reduction occurs on the
cathode and the overall process is spontaneous, leading to electricity
generation simultaneous to substrate oxidation. On the other hand,
added-value compounds (such as hydrogen) are produced in the cath-
ode of a microbial electrolysis cell (MEC) through a reduction reaction.
These processes are not, in general, thermodynamically spontaneous
and additional energy supply is required [2,3]. Geobacteraceae and
Shewanaellaceae families are the most studied genera of ARB [4],
with Geobacter being the commonly found genus in acetate-fed high
bioelectrochemical systems [58].
Most of current studies are based on moderate pH conditions. How-
ever, bioelectrochemical systems should extend their applicability
Corresponding author.
E-mail addresses: Laura.Rago@live.com (L. Rago), JuanAntonio.Baeza@uab.cat
(J.A. Baeza), Albert.Guisasola@uab.cat (A. Guisasola).
different pH scenarios. Alkaline exoelectrogenesis is, a priori, very stim-
ulating since: i) the highest current densities ever achieved by pure cul-
tures were found under alkaline conditions with the Geoalkalibacter
genus [9]; ii) Geoalkalibacter are, in turn, very attractive since they are
also halophilic and give successful results under high-salt conditions
[912]; iii) alkaliphilic environments may also be favorable to prevent
acidity buildup [13,14]; iv) an alkaline environment could create
a more selective and favorable environment for ARB when competing
with methanogens for the electron donor and v) alkaline
bioelectrochemical systems can be a suitable technology for the direct
treatment of alkaline wastes (e.g. beamhouse wastewaters from leather
tannery or wastewaters with glycerol produced in alkaline biodiesel
production).
Thus, the aim of this study is the rst experimental evaluation of the
long-term performance of mixed-culture bioelectrochemical systems
under alkaline conditions. Electrochemical and advanced microbiologi-
cal tools are used to gain insight into the process performance.
2. Materials and methods
2.1. Reactor description, inoculation and operation
An air-cathode MFC, designed and built as previously described [15],
was used for enrichment of exoelectrogenic bacteria under alkaline con-
ditions (inoculum microbial fuel cell, i-MFC). It consisted of a 400 mL
glass vessel with a lateral aperture for the cathode assembling, which
was a graphite ber cloth that had a PTFE diffusion layer and a catalytic

http://dx.doi.org/10.1016/j.bioelechem.2016.01.003
1567-5394/ 2016 Elsevier B.V. All rights reserved.
58 L. Rago et al. / Bioelectrochemistry 109 (2016) 5762
layer coated with platinum (5 mg Pt/cm2, ElectroChem Inc.). The anode
(area 0.8 m2) was a graphite brush made with graphite bers (diameter
7.2 m, PANEX33 160K, ZOLTEK) and titanium wire, which was pierced
on a cap provided with a silicone septum. The anode and the cathode
were connected with an external resistance using titanium wire. The al-
kaline i-MFC was inoculated with sludge from the anaerobic digester of
a local WWTP (Manresa, Barcelona) and operated for three months in
fed-batch mode under moderate alkaline conditions (i.e. pH was
daily adjusted to 8.5). Acetate pulses were added when intensity
dropped down.
A cube microbial fuel cell (c-MFC) was designed and built [16] using
28 mL methacrylate vessels with an air-cathode and an anode connect-
ed through a 220 external resistance. The cathode consisted of
a graphite ber cloth (3.8 cm diameter, 7 cm2 total exposed area)
coated with platinum (5 mg Pt/cm2, ElectroChem Inc.) in the catalytic
layer and a PTFE diffusion layer which permitted oxygen diffusion into
the cell while preventing water leakage [17]. It was placed 2.5 cm
apart from the anode. The anode was a graphite ber brush (20 mm
diameter 30 mm length; 0.18 m2) made with bers (diameter
7.2 m, PANEX33 160K, ZOLTEK) connected with a titanium wire core.
The alkaline c-MFC was inoculated by mixing (1:1) used media from
the alkaline i-MFC and fresh media. The alkaline c-MFC anode was
moved to an alkaline cube microbial electrolysis cell (c-MEC) and a con-
stant potential of 0.8 V was applied. The c-MEC was analogous to the c-
MFC, but the cathode was not exposed to the air. The complete c-MEC
characteristics are detailed in Montpart et al. [16].
The medium used was 100 mM NaH2PO4 with 30 mM of acetate and
the following additional components in 1 L of deionized water: NH4Cl
(0.41 g), mineral media (10 mL), 1 mL of 4 gL1 FeCl2 stock
solution, and 0.5 mL of 37.2 gL 1 Na2S9H2O stock solution. The
mineral media composition was as described in [18]. 50 mM of
2-bromoethanesulfonate was added to prevent methanogenesis. A
parallel set of experiments was conducted under neutral pH condi-
tions (pH = 7.3) also with i-MFC, c-MFC and c-MEC.
pH was monitored on-line with pH electrodes (Crison pH 5233) con-
nected to a pH meter (Crison MultiMeter 44) as previously described [2]
and controlled with base dosage (NaOH 3 M) using an automatic bu-
rette (Crison MultiBurette 2S). pH control experiments were performed
on an orbital agitator at 100 rpm (DOS-20L ELMY Sky Line digital orbital
shaker).
2.2. Electrochemical, chemical and microbiological analyses
Coulombic Efciency (CE) was calculated using Eq. (1).
t
Coulombs recovered as current intensity t 0F I dt
CE
Coulombs in substrate F  bAc  V L  c  M1
1 An alkaline MFC was obtained from a conventional anaerobic sludge
where t0 and tF are the initial and nal time of an experiment, I is the
current intensity, F is the Faraday's constant (96,485 C/mol e), bAc is
the number of e transferred per mole of acetate (8 mol e/mol ace-
tate), VL is the volume of liquid in the reactor, c is the change in acetate
concentration during the experiment (g acetate/L cell) and M is acetate
molecular weight (59 g/mol).
The energy recovery of the cell was calculated as the amount of
energy produced as hydrogen with respect to the electrical input (rE,
Eq. (2)) and the electrical input and the energy content of the substrate
(rE + S, Eq. (3)) [19].
nH2  H H2
rE  
tF
t0 I  Eap  I2  Rext dt
where nH2 are the moles of produced hydrogen, HH2 is the heat of
combustion of hydrogen (285.83 kJmol1), Eap is the applied voltage
(V) and Rext is the external resistance used for monitoring ().
nH2  HH2
r ES   3
tF
nS  H S t0 I  Eap  I2  Rext dt
where nS are the moles of consumed substrate and HS is the heat of
combustion of the substrate (870.28 kJmol1 for acetate).
Cathodic gas recovery (rCAT) was calculated as in Eq. (4).
Coulombs in H2 V F;H2  2  F  V m 1
r CAT t
Coulombs recovered as current intensity t F Idt
0
4
where VF,H2 is the volume of H2 at the end of the cycle and Vm is the
molar gas volume (24.03 Lmol1) at 20 C.
Acetate was analyzed by gas chromatography (Agilent Technologies,
7820-A) using a ame ionization detector (FID) with helium as carrier
gas. H2 and CH4 production were analyzed with the same chromato-
graph but using a thermal conductivity detector (TCD) with argon as
carrier gas [19].
The anode graphite bers for high-throughput 16S rRNA gene pyro-
sequencing were rinsed with 1 mL of sterile MilliQ water to remove any
residue and then they were cut and combined for DNA extraction. Total
DNA was extracted from approximately 0.2 g of samples using a
PowerBiolm DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,
CA) according to the manufacturer's instructions. Quality and quantity
of the DNA were evaluated as detailed in Rago et al. [20]. High-
throughput 16S rRNA gene pyrosequencing was performed in a 454
Titanium FLX system by the Research and Testing Laboratory (Lubbock,
TX), based upon their protocols for anode DNA samples. Each DNA
sample (20 ng/L, quality ratio of 1.8) was analyzed with an average of
3000 reads/assay and using 338F907F and 338F907R primers couple,
comprising the V3V5 regions of the bacterial 16S rRNA gene [21].
Sequences were checked using Uchime [22], sorted and trimmed
using the Pipeline Initial Process at the Ribosomal Database Project
(RDP) Pyrosequencing Pipeline (http://rdp.cme.msu.edu/index.jsp;
[23]) with the default settings. The RDP Classier was used to assign
16S rRNA gene sequences to a taxonomical hierarchy with a condence
threshold of 95%, since the DNA sequences were b 250 bp [24]. The
relative abundance of a given phylogenetic group was calculated as
the number of sequences associated with that group divided by the
total number of sequences per sample.
3. Results and discussion
through a two-step procedure to avoid a rough pH change: a rst en-
richment period in an alkaline i-MFC operated at pH = 8.5 and, then,
this enriched alkaline microbial community was inoculated in a c-MFC
(day 0 in Fig. 1) and operated for three months with a controlled pH
of 9.3. The cell showed initial exoelectrogenic activity with low current
density values (~1 mAm2) during the rst batch cycles. However, a
high increase of activity was observed in the fourth cycle because of
the anode adaptation to the new operational alkaline conditions
(i.e. pH = 9.3). The system showed a reliable performance for more
than two months and stabilized current intensity around 9.5 mAm2
under non-limiting substrate concentrations. A parallel c-MFC at neutral
pH was operated for three months and the current intensity achieved
was around 9 mAm2 (Fig. 1 shows the rst 30 days). The CE for the
alkaline c-MFC was 60 5% (n = 3), which is higher than that obtained
with the neutral c-MFC (43 10%, n = 3). Similar MFC congurations
at neutral pH in the literature also reported lower CE values [2527]
2 suggesting less competition for substrate or, in other words, a more se-
lective environment for exoelectrogens at alkaline pH.
 L. Rago et al. / Bioelectrochemistry 109 (2016) 5762 59

Fig. 1. Current density proles for alkaline cube microbial fuel cell (c-MFC) and cube microbial electrolysis cell (c-MEC) during all the operational periods and for neutral c-MFC (rst
30 days) and c-MEC (last 25 days).

The performance of the alkaline c-MFC was further evaluated on day
70 through polarization and power curves (Fig. 2). The maximum
power (2.8 mWm2) for the alkaline c-MFC (Fig. 2A) was obtained
with an external resistance of around 100 , which was in the same
order of magnitude as the resistance used under normal operation
(220 ). This power was slightly lower than that observed for the neu-
tral c-MFC (3.4 mWm2) despite that the proles were very similar.
DNA was extracted from the anode of the alkaline c-MFC (day 90)
and the microbial community was analyzed using pyrosequencing
(Fig. 3). The dominant bacterial genus was Alkalibacter (37% of all the
sequences) which, to the best of our knowledge, has not been described
as an ARB yet. Nevertheless, we consider Alkalibacter sp. as a possible
ARB because of its high abundance in this c-MFC. Alkalibacter genus
has never been reported before in any bioelectrochemical system [16,
20,2830] under neutral conditions. Alkalibacter genus is strictly anaer-
obic and an alkaliphilic genus of Firmicutes phylum [31]. Many genera
and species from Firmicutes phylum, often from Clostridiales order,
have shown exoelectrogenic activity [4,32]. A high percentage of
Geoalkalibacter genus was also detected in the alkaline c-MFC (21% of
sequences). Geoalkalibacter is a genus of the Geobacteraceae family,
and an exoelectrogenic genus. Badalamenti et al. [9] operated a MEC
with a pure culture of Geolkalibacter ferrihydriticus demonstrating its
high capacity for current generation. The results obtained in the alkaline
c-MFC are promising and represent a high enrichment for this genus in
an alkaline mixed-culture bioelectrochemical system. On the other
hand, Geobacter was not detected in the alkaline c-MFC, while high
values of Geobacter (around 70%) are normally present in neutral
MFCs [28]. The Acetobacterium sp. presence (3%) agrees with previous
results in other bioelectrochemical systems [20,28,29]. It is a
homoacetogenic genus able to use H2 as electron donor and CO2 as
electron acceptor to produce acetic acid [33]. Other classied genera
of Clostridiales order detected were Proteiniclasticum sp. (2%) and
Anoxynatronum sp. (1%). Species of the Anoxynatronum genus (1%) are
known to be true alkaliphilic with a growth range from pH 7.1 to
pH 10.1 and optimal pH for growth at pH 9.1 [31]. Nitrincola sp.,
which represents 2% of total reads, is also recognized as an alkaliphilic
genus. Unclassied family of Clostridiales order represents 2% of the
bacteria in the anode. Unclassied microorganisms of the phylum
Bacteroidetes were also found (6%). Their presence is frequent in natural
environments and has been reported in anaerobic digestion sludge [34],
which was our initial inoculum. Almost 9% of the sequences could not
be ascribed to particular phyla. 10% of the sequences were included in

Fig. 2. Power (P, solid line with circles) and polarization curves (E, dashed line with triangles) for the alkaline and neutral cube microbial fuel cells (c-MFCs).
60 L. Rago et al. / Bioelectrochemistry 109 (2016) 5762
Fig. 3. 16S rRNA gene pyrosequencing results of anodic alkaline bacterial communities in a cube microbial fuel cell (c-MFC) and in a cube microbial electrolysis cell (c-MEC).
the others category as its representation was below 1% of the total
reads.
After 90 days of MFC operation, both cells (alkaline and neutral)
were moved to a MEC conguration. Current intensity, CE and H2 pro-
duction were monitored for both alkaline and neutral c-MEC (Fig. 1).
The exoelectrogenic activity in the alkaline c-MEC increased after
35 days of MEC operation and was maintained stable for one month
with high current intensity around 50 mAm 2 for all batch cycles.
On the other hand, the maximum current intensity obtained in the
neutral c-MEC was only slightly higher than 30 mAm 2. The best
alkaline c-MEC performance was obtained after around 140 days with
2.6 LH2L1REACTORd 1, while H2 production in neutral c-MEC was
only 1.2 LH2L1REACTORd1, resulting in an increased alkaline produc-
tion rate of +117%. These results present, for the rst time, the experi-
mental evidence of mixed-culture alkaline bioelectrochemical systems
having better performance than those under neutral conditions. Energy
recovery was also improved under alkaline operation: rE increased from
139 13% (n = 2) in the neutral c-MEC to 213 20% (n = 2) in the
alkaline c-MEC, while rE + S was improved from 55 9 (n = 2) to
87 6 (n = 2) in the alkaline c-MEC. In addition, rCAT in both cells
was close to the maximum (around 100%). CE was similar for both
cells: 110 5% (n = 2) for alkaline c-MEC and 102 12% (n = 2) for
neutral c-MEC. CE values higher than 100% are often observed in
single-chamber MECs and attributed to the presence of hydrogen
scavengers which would use part of the hydrogen produced (for
example, homoacetogens using hydrogen as electron donor for acetate
production). The presence of homoacetogens was conrmed in the py-
rosequencing analysis described below. This hydrogen recycling biases
the calculation of conventional MEC indicators as CE and a thorough
electron balance is required to fully understand the fate of electrons
[19].
Table 1 summarizes the major differences found among neutral and
alkaline cells in this work. The operation at alkaline pH seems to be a
stable process, since the higher performance obtained was maintained
stably during all the operational period tested. Hydrogen production
with MEC at alkaline pH could be implemented in full-scale systems
 L. Rago et al. / Bioelectrochemistry 109 (2016) 5762
Table 1
Summary of the main results obtained in this work. All the cells were operated with acetate as sole carbon source.
MFC
Neutral Alkaline
Voltage obtained (mV) 360 380
1 1
CE (%) 43 10 60 5
Maximum power obtained (mWm2) 3.4 2.8
% Geobacter 79 b1
% Geoalkalibacter b1 21
% Alkalibacter b1 37
1
CE: Coulombic efciency calculated as percentage of coulombs recovered as intensity with respect to coulombs in substrate (Eq. 1).
2
rE+S: percentage of energy recovered as hydrogen with respect to the electrical input plus the energy content in the substrate (Eq. 3).
as a suitable operation for wastewaters with high pH, which could make
a sustainable treatment with higher performance than neutral pH.
DNA was extracted from the anode of the alkaline c-MEC at day 145
for a pyrosequencing analysis (Fig. 3). The dominant bacterial genus
identied was Geoalkalibacter sp. (43%). When compared to the alkaline
MFC data, a high increase of Geoalkalibacter genus (from 23 to 43%) and
a high decrease of the Alkalibacter genus (from 37 to 8%) were observed.
The Geoalkalibacter increase may be linked to the high current density
increase observed between both scenarios (up to six-fold higher). The
applied potential may have created a positive selection force for
Geoalkalibacter. Further research is required at this stage to understand
the interactions between Geoalkalibacter and Alkalibacter genera.
Acetobacterium genus was more abundant in c-MEC (9%) than in
c-MFC (3%), in agreement with homoacetogenesis occurrence. The
rest of the variations of the bacterial community with minor representa-
tion between alkaline c-MFC and c-MEC scenarios were not relevant.
The same microbiological analysis was conducted in a similar MEC but
operated under neutral conditions [20] and the results were very differ-
ent. The neutral MEC mainly contained Geobacter (72%) together with
other genera with minor representation. Geoalkalibacter represented
1% of the total population and the number of reads of Alkalibacter was
less than 1%.
4. Conclusions
Alkaline (pH = 9.3) exoelectrogenesis had a better performance
than neutral, obtaining high current density and hydrogen production
under MEC conditions (50 mAm2 and 2.6 LH2L1REACTORd1). CE
for alkaline MFC was higher than that obtained with neutral MFC (60%
vs 43%). The anodic exoelectrogenic biolm was mainly enriched with
alkaliphilic bacteria such as Geoalkalibacter sp., with exoelectrogenic
activity detected in previous studies, and Alkalibacter sp., which was
identied as a highly abundant microorganism in alkaline MXCs for
the rst time. Future studies can conrm the exoelectrogenic activity
of species of the Alkalibacter genus.
Acknowledgments
The authors are members of the GENOCOV group (Grup de Recerca
Consolidat de la Generalitat de Catalunya, 2014 SGR 1255). Laura Rago
is grateful for the FPI grant BES-2011-051308 within the research
project CTM2010-20384 from the Spanish Ministerio de Economa y
Competitividad.
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[27] A.P. Borole, C.Y. Hamilton, T. Vishnivetskaya, D. Leak, C. Andras, Improving power Dr. Juan A. Baeza has been an associate professor in the Department of Chemical Engineering
production in acetate-fed microbial fuel cells via enrichment of exoelectrogenic at Universitat Autnoma de Barcelona since December 2004. His research is focused on
organisms in ow-through systems, Biochem. Eng. J. 48 (2009) 7180. Environmental Engineering: nutrient removal from wastewater (nitricationdenitrication,
[28] P. Parameswaran, C.I. Torres, D.-W. Kang, B.E. Rittmann, R. Krajmalnik-Brown, The enhanced biological phosphorus removal), bioelectrochemical systems (MFC, MEC), model-
role of homoacetogenic bacteria as efcient hydrogen scavengers in microbial ing and process control. He leads research on nutrient removal from urban wastewater in the
electrochemical cells (MXCs), Water Sci. Technol. 65 (2011) 1. "GENOCOV group on biological treatment of liquid and gas efuents" of the UAB, http://orcid.
[29] P. Parameswaran, C.I. Torres, H.-S. Lee, B.E. Rittmann, R. Krajmalnik-Brown, org/0000-0003-1290-1669.
Hydrogen consumption in microbial electrochemical systems (MXCs): the role of
homo-acetogenic bacteria, Bioresour. Technol. 102 (2011) 263271. Dr. Albert Guisasola is a chemical engineer and Ph.D. in environmental engineering. He is
[30] N. Montpart, L. Rago, J.A. Baeza, A. Guisasola, Hydrogen production in single associate professor in the Department of Chemical Engineering at Universitat Autnoma
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nov. from a soda lake in the Transbaikal region of Russia, Extremophiles 8 (2004) http://orcid.org/0000-0002-3012-7964.
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Harbin Gongye Daxue Xuebao 46 (2014) 3642.

Laura Rago earned her degree in Biotechnology and Master's degree in Pharmaceutical,
Veterinary and Medical Biotechnologies at Universit Magna Grcia di Catanzaro in
Italy, in 2008 and 2011 respectively. She became PhD in late 2015 at the Department of
Chemical, Biological and Environmental Engineering at Universitat Autnoma de Barcelona.
She is a member of the GENOCOV research group and focuses her research on the character-
ization of the microbiological community in bioelectrochemical systems, such as MFC
and MEC. In 2013, she participated in a research stay at the Swette Center for Environmental
Biotechnology at Arizona State University (Tempe, USA).