Electrode material properties for designing effective microbial electrosynthesis systems

Abstract

The electrode material is one of the key components of a bioelectrochemical system (BES) as the biocatalyst or the electroactive biofilm will develop on this base material and hence plays a pivotal role in regulating the type and rate of electron transfer processes and bioelectrochemical conversions. Microbial electrosynthesis (MES) requires biocompatible electrode materials with a high surface area that can support the effective development of microbial biomass at high current densities to provide high product titers. Carbon-based materials are the best available practice today; yet, resolving which of their properties are truly relevant to achieve these goals remains elusive. The present study shows that biofilm coverage and the relative abundance of cell-bound polymeric filaments are directly proportional to the total charge consumed over time. The combination of high biofilm coverages and high relative abundance of the cell-bound polymeric filaments resulted in low charge transfer resistances, as determined by electrochemical impedance spectroscopy. Although a wide variety of the physicochemical parameters of the supporting carbon electrode materials (electric conductivity, specific surface area, porosity, roughness, thermogravimetric mass spectrometry, etc.), were characterized in this study, the only one that showed a consistent correlation with the total charge consumed by the electroactive biofilms was the contact angle. This suggests that the hydrophilic moieties and surface tension are two fundamental parameters to consider for an effective design of microbial electrosynthesis biocathodes. Among the carbon electrode materials used in the present study, the activated carbon-based VITO CORE™ electrode was considered the most suitable electrode material to provide such desired features while other electrodes exhibited roughnesses much higher than this biologically-amenable range. Thus, the superior wetting characteristics of the VITO CORE™ electrode seem highly reliant on the roughness provided by the manufacturing method (i.e., cold-rolling).

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Journal of
Materials Chemistry A
Materials for energy and sustainability
rsc.li/materials-a
ISSN 2050-7488
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Zhenhai Wen et al.
An electrochemically neutralized energy-assisted low-cost
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Volume 6
Number 12
28 March 2018
Pages 4883-5230
Journal of
Materials Chemistry A
Materials for energy and sustainability
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Alvarez-Gallego, W. Achouak, D. Pant, P. Sharma and X. Dominguez-Benetton, J. Mater. Chem. A, 2019,
DOI: 10.1039/C9TA04886C.

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Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Electrode material properties for designing effective microbial
electrosynthesis systems
Mohita Sharmaabc
†
Yolanda Alvarez-Gallegoa Wafa Achouakd Deepak Panta Priyangshu M Sarmabc‡
Xochitl Dominguez-Benettona*
The electrode material is one of the key components of a bioelectrochemical system (BES) as the biocatalyst or the
electroactive biofilm develops on this base material and hence plays a pivotal role in regulating the type and rate of electron
transfer processes and bioelectrochemical conversions. Microbial electrosynthesis (MES) requires biocompatible electrode
materials with a large surface area, that can support the effective development of microbial biomass at high current densities
to provide significant product titers. Carbon-based materials are the best available practice today; yet, resolving which of
their properties are truly relevant to achieve these goals remains elusive. The present study shows that biofilm coverage
and the relative abundance of cell-bound polymeric filaments are directly proportional to the total charge consumed over
time. The combination of high biofilm coverages and high relative abundance of the cell-bound polymeric filaments resulted
in low charge transfer resistances, as determined by electrochemical impedance spectroscopy. Although a wide variety of
the physicochemical parameters of the supporting carbon electrode materials (electric conductivity, specific surface area,
porosity, roughness, thermogravimetric mass spectrometry, etc.) were characterized in this study, the only one that showed
a consistent correlation with the total charge consumed by the electroactive biofilms was the contact angle. This suggests
that the hydrophilic moieties and surface tension are two fundamental parameters to consider for an effective design of
microbial electrosynthesis biocathodes. Among the carbon electrode materials used in the present study, the activated
carbon-based VITO CORE™ electrode was considered the most suitable electrode material to provide such desired features,
while other electrodes exhibited roughnesses much higher than those amenable for the microbiological range. The superior
wetting characteristics of the VITO CORE™ electrode seem highly reliant on the roughness provided by the manufacturing
method (i.e., cold-rolling).
.
Introduction
Microbial electrosynthesis involves microbes that exchange
electrons with solid-state electrodes to transform organic or
inorganic compounds, yielding more valuable chemicals in
relevant amounts (Schröder et al., 2015; Sharma et al., 2013,
2015). At present, microbial electrosynthesis is often
demonstrated by the reduction of CO2 to short-chain organic
chemicals, but the phenomenology is not limited to this specific
conversion. For instance, carbon chain elongation,
solventogenesis, and nanoparticle synthesis, among others, are
variants to CO2-based microbial electrosynthesis.
Operation of microbial electrosynthesis cells fundamentally
requires two key groups of components. The first one can be
simplified as the biological component, consisting of the
electrochemically-active microbes acting as electrocatalyst and
the culture medium required for their propagation–which
includes the electrolytes required to conduct ionic charge. The
second one can be generalized as the material components,
which mainly comprise the reactor constituents, such as the
electrode required for the development of the biocatalyst and
its electrochemical interactions, membranes, current collectors,
and the cell configuration. The microbe-electrode interactions
play the central role in determining the performance of the
bioelectrochemical cell (Zhang et al., 2013).
Since the electrode materials used in these
bioelectrochemical systems have a predominant effect on their
performance, it is paramount to unveil the physicochemical
features that favor their improved performance, i.e., selective
electrosynthesis, at high current densities, for high product
titers (Sharma et al., 2014a).
a Separation& Conversion Technologies, VITO - Flemish Institute for Technological Research,
Boeretang 200, 2400 Mol, Belgium
b TERI University, Plot No. 10, Institutional Area, Vasant Kunj, New Delhi, 110070, India
c The Energy and Resource Institute (TERI), IHC, Lodhi Road, New Delhi, 110003, India
d Laboratory of Microbial Ecology of the Rhizosphere (LEMiRE), BIAM, UMR 7265, CNRS-CEA-Aix-
Marseille, University, 13108 Saint Paul lez Durance, France
† Present address:Petroleum Microbiology Research Group, Department of Biological Sciences,
University of Calgary, Calgary – AB, T2N1N4, Canada
‡ For correspondence on the microbial consortium contact: priyangshu.sarma@innotechin.com
*Corresponding author: xoch@vito.be
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Microbial electrodes should have a base material with
efficient electrical conduction, good chemical stability, high
mechanical strength, low cost and efficient electron transfer
capacity at the microbe-electrode interface (Wei et al., 2011).
The physicochemical properties of the electrode material, in
turn, determine the extent of bacterial adhesion, growth
capabilities, and performance (Guo et al., 2015; Pham et al.,
2009; Sun et al., 2012; Logan et al., 2006).
In microbial fuel cell (MFC) studies, the ratio between the
surface area and the volume of an electrode is directly
proportional to the current density (You et al., 2009; Nevin et
al., 2008). Highly porous structures provide better microbial
growth as they promote efficient substrate delivery; resulting in
high current density, better effluent flow and minimal clogging
(Liu et al., 2010; Minteer et al., 2007). Geometric shape,
composition, conductivity, and quantity of the electrode also
play an important role, as these features determine its internal
resistance and play a significant role in current distribution. The
ratio between the electrode surface area and electrolyte
volume also impacts on the system performance. For instance,
Oh et al., (2004) reported an increase in power performance by
24% when the cathode surface area was increased from 22.5
cm2 to 67.5 cm2, converse to its reduction by 56% when the area
was reduced down to 5.8 cm2, for the same operational volume.
A convoluted geometric shape of the electrode material also
improves the surface area per unit volume of electrode, which
has shown to have a positive impact on the performance of
anodic microbial electrocatalysis in MFCs (Ieropoulos et al.,
2008). Besides, biocompatibility is necessary. Although some of
these favorable properties are fairly well characterized for MFC
bioanodes, they are elusive for electrodes supporting cathodic
microbial electrosynthesis. Cathodic microbial electrosynthesis
is greatly favored over anodic microbial electrosynthesis, as
electrons are used as a green reagent in the process of product
formation.
Materials commonly used for the production of microbial
electrodes include carbon paper, carbon cloth (Cheng et al.,
2009), carbon felt, carbon mesh, carbon fiber, graphite
granules, graphite fiber brush, reticulated vitreous carbon (You
et al., 2009), and carbon fiber veil (Liu et al., 2010; Logan, 2010).
Zhao et al., reported activated carbon fabric as a superior
material over graphite foil and carbon fiber veil, due to
improved sulfide adsorption and oxidation which link to the
potential for harvesting energy from sulfate-rich solutions in the
form of electricity (Zhao et al., 2008). Activated carbon fabric
(ACF) electrodes have also been reported to provide higher
specific surface area and thus better performance (Logan,
2010).
Indeed, carbon-based materials are the most widely used
for microbial electrodes, because of their large specific surface
area, strong corrosion resistance, low price, high-temperature
stability, high electrical conductivity, and various machining and
manufacturing possibilities (Rosenbaum and Henrich 2014).
Current collectors, e.g., like stainless steel (SS) mesh (with high
chrome content to avoid corrosion), can be combined with
carbon to make composite materials, for achieving a higher
electric conductivity, and excellent biocompatibility (Zhang et
al., 2009).
Despite the extended use of carbon and its numerous
characterization studies available, the rational choice of a
particular type is still somewhat arbitrary for MFCs and even
more unfounded for the less studied microbial electrosynthesis
cells. In fact, it is not even certain that different carbon supports
would ensure the preservation of the electrochemical
functionality of the electrochemically-active (EA) biofilms, the
even progression of the bioelectrochemically-active surface
area, and an enhanced exchange current density, among
others.
A holistic and comprehensive investigation of materials for
microbial electrodes and evaluation of their performance is not
straightforward. From the materials science and engineering
point of view, the most common characterizations include the
electrode morphology assessed by different microscopies (SEM,
TEM, AFM, CLSM), energy dispersive X-ray spectroscopy (EDS)
to estimate the elemental composition, and Raman
spectroscopy to investigate additional characteristics of the
electrode material (Chen et al., 2012; Zhang et al., 2013). From
the electrochemical point of view, current density per projected
surface area (PSA) is the most commonly used parameter, but
due to the uneven porous three-dimensional structure of
different materials, a comparison with such parameter alone
becomes ineffective and often precedes misleading
interpretations (Sharma et al., 2014). A thorough
characterization of the microbial diversity and biofilm formation
are also necessary. Yet, the majority of studies available
investigate these aspects separately and do not correlate how
do they ultimately control the desired performance. Research
in this direction is thus required to progress beyond the current
state of the art in this field.
This work investigated the physicochemical and
bioelectrochemical response of sulfate-reducing bacteria (SRB)-
based biocathodes supported on commonly-employed carbon-
based electrode materials, aiming to understand and establish
correlations between their corresponding features. Multiple
carbon-based electrode materials were studied simultaneously
in a tubular reactor configuration. By the end of the 32nd day of
experimentation, the electrochemically-active biofilms reached
a pseudo-steady state situation over all the electrodes. The
microbial electrodes were then extensively characterized by
different electrochemical, analytical, physicochemical,
microbial and material characterization methods, to achieve
our intended aim.
Experimental
Reagents and materials
The experiments here described were conducted in a Plexiglas
reactor with tubular configuration, under potentiostatic control
at -0.85 V vs. Ag/AgCl (3.5 M KCl). A common counter electrode
and Ag/AgCl (3.5 M KCl) reference electrode were employed for
all the working electrodes, using a Bio-Logic N’Stat Box for
multi-electrode cell connections. A functional pre-colonized
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biocathode taken from a previously-running electrochemical
reactor in the same lab at VITO (Sharma et al., 2013) served as
the source of inoculum (TERI-MS-003) for the multi-electrode
electrochemical reactor operating in batch mode with
recirculation. The high-strength substrate mix used was
composed of 0.1 M of each acetic and butyric acids,
supplemented to a synthetic feed as described previously in
Sharma et al., 2014b. In addition, seven different clean working
electrodes were used for this study. The VITO CORE™ electrode
consisted of an 80-20% carbon-polytetrafluoroethylene (PTFE)
mix, used for the assembly of activated carbon layers from both
sides of a 316-grade stainless steel (SS) mesh. The electrode
manufacturing process has been reported previously (Alvarez-
Gallego et al., 2012; Zhang et al., 2011; Zhang et al., 2014). This
component, together with other six different electrode
materials used in the experiment, have been designated with
numbers in the rest of the paper. These are namely: E1 -
graphite felt (Morgan, Australia), E2 - VITO CORE™ electrode
(VITO, Belgium), E3 - pre-colonized cathode prepared using
VITO CORE™ electrode, E4 - activated carbon fleece (Pure
Nature, Spain), E5 - activated carbon cloth (HEG Limited, India),
E6 - carbon paper (Quintech, Germany), E7 - carbon felt (MAST
Carbon, U.K.), and E8 - graphitized carbon felt (PaxiTech,
France). These materials are amongst the most commonly
employed in microbial-electrochemical investigations for the
past two decades. The projected (geometric) surface area of
each working electrode was 4 cm2. The counter electrode was a
dimensionally stable anode (DSA) grid (Magneto, NL) of 57.6
cm2 projected (geometric) surface area; it was given a pre-
treatment as described in Sharma et al., 2013. The aqueous
solutions were prepared in deionized water, and all the
chemical reagents used for the experiments were purchased
from Sigma-Aldrich.
Physicochemical measurements of electrode surfaces
Through-plane electrical resistance
For the electrical resistance measurements, an in-house set-up
was employed (previously described in Alvarez-Gallego et al.,
2012). This set-up consisted of two smooth Pt/Pd flat plate
electrodes, which could be adjusted with the help of a screw. In
order to determine the contribution of the two contact
resistances occurring between the surface of the electrode
layer and the Pt/Pd electrodes, a single electrode layer to be
tested (sample diameter: 24 mm) was placed between the two
electrodes, which were screwed to each other to ensure electric
contact with the layer. At the time of measurement, when these
three layers touched each other, the resistance between the
Pt/Pd electrodes was determined (AC S HiTester 3560, Hioki).
All measurements were repeated with double and triple pieces
of electrode layers sandwiched between the two Pt/Pd
electrodes. The values of the total resistance measured were
then plotted against the total thickness of the respective stacks
and the slope obtained was hence considered equivalent to the
through-plane electrical resistance (Alvarez-Gallego et al.,
2012).
Hydrophilic porosity
For the determination of the hydrophilic porosity, each
electrode was immersed in deionised water for 24 hours. The
total volume of the hydrophilic pores was calculated from the
total amount of water that gets absorbed in the electrode layer.
Compared with the volume of the sample, the relative volume
of these hydrophilic pores was calculated. This is expressed as
the hydrophilic porosity:
Eq. 1
hydrophilic porosity
(
%
)
=


,

2
0





2
0
×
100
where Awet, H2O is the density of the water-wetted electrode
sample, Adry is the density of dry sample, and A52O is the density
of water (Alvarez-Gallego et al., 2012).
Contact angle measurements
In the present study, the contact angle was measured using the
sessile drop technique coupled with digital image analysis, as
described elsewhere (Yuan and Lee, 2013). The measurements
were performed using a contact angle system OCA-15EC
(DataPhysics Instruments GmbH, Germany) and the contact
angles were calculated using the software SCA 20.
Profilometry of electrode materials
A profilometric analysis was performed to determine the
surface roughness (Ra, estimated by the arithmetic average
height calculated over the entire measured array) and the
profile height (Rt, calculated as the distance between the mean
Rs and the highest point over the evaluation length). These
measurements were done using a Wyko (Veeco, USA) surface
optical profiler, as per the instructions of the manufacturer.
Thermogravimetric analysis (TGA)
Thermogravimetric analyses (TGA) were conducted on a
thermobalance NETZSCH STA 499C at a heating rate of 10 K min-
1, from 100 C to 800 C under argon flow. Each sample (10–20
mg) was pre-heated at 130 C for 120 min to eliminate
physisorbed water and volatile compounds.
N2 adsorption measurement
N2 adsorption experiments were performed (Autosorb iQ
Station, Quantachrome) for the determination of the BET
surface area of the electrode materials, using Quantachrome®
ASiQwin (Version 3.01) for data acquisition and processing. The
typical measuring protocol included degassing for 20 h at 120
°C, followed by the subsequent measurement of the adsorption
isotherm at -196 °C.
Scanning electron microscopy (SEM) and energy dispersive x-ray
spectroscopy (EDS) measurements
The surface morphologies of the electrode samples and EDS
spectra were investigated using field-emission scanning
electron microscopy (FESEM; JSM-6304F, JEOL, Japan at an
acceleration voltage of 20 kV). The biocathodes were treated
for SEM at the termination of the experiment (after 32 days of
operation). The samples were dipped in a 2.5% glutaraldehyde
solution, overnight, at 4 °C. The samples were subsequently
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washed with 0.1 M cold phosphate buffer, thrice, for 15 minutes
each. The samples were then dehydrated, by using a series of
ethanol gradient solutions (30%, 50%, 70%, 80%, 90%, 95% and
100%) for 15 minutes each. They were critical point-dried with
liquid CO2, as per the instructions of the manufacturer (Bal-Tec
CPD 030). The samples were further mounted on aluminum
stubs; sputter-coated with platinum-iridium and visualized
using the scanning electron microscope (Sharma et al., 2015).
Reactor and inoculum source
A tubular reactor placed in horizontal position was used as the
electrochemical cell, with a working volume of 500 mL. This cell
was further connected with tubing to a feed tank in
recirculation mode (Fig. 1). The electrodes were positioned
sequentially next to each other from E1 to E8, with a separation
of 2 cm between each other. The first electrode, at the inlet of
the flow to the reactor, was the reference electrode, followed
by the eight working electrodes. The reference electrode was
placed outside of the current path, which has been proven to
enable accurate multi-electrode electrochemical
characterizations (Zhang et al., 2014). An anaerobic
environment was maintained in the headspace of the feed-tank,
by continuous sparging of N2 gas. The synthetic feed was at pH
5 throughout the operation of the reactor, and the conductivity
was 30.3 mS cm-1. The experiment was operated at room
temperature (18–22 °C). The counter electrode was positioned
below all the working electrodes so that it would project over
the full length and width of these (Fig. 1), and hence avoid
misleading effects and biocathode underperformance due to
position-altered current distributions and uneven
electromagnetic fields (Lanas & Logan, 2013; Zhang et al., 2014).
Given that the reference electrode was placed at the entrance
of the reactor (before electrode E1), the corresponding ohmic
drop corrections (determined by the current interrupt method,
using the standard settings provided by the potentiostat
employed) were carried out for all electrochemical
measurements reported in this study.
Microbial community analysis
The biofilms, which matured up to a pseudo-steady state (i.e.,
di/dt  0), were scraped from the electrode surface, and
bacterial DNA was extracted and stored at -80°C before analysis,
as previously described (Erable et al., 2009). SSU rRNA gene
amplification was performed with barcoded primers for the V1-
V3 regions. The 16S universal Eubacterial primers 27Fmod (5’-
AGRGTTTGATCMTGGCTCAG) and 519R (5’-
GTNTTACNGCGGCKGCTG) were used for the amplification of
the 500 bp region of the 16S rRNA genes. The 454 Titanium
sequencing run was performed on a 70675 GS PicoTiterPlate by
using a Genome Sequencer FLX System (Roche, Nutley, NJ).
Each individual sequence was trimmed to a Q25 average and
data derived from the sequencing process were processed using
a proprietary analysis pipeline (www.mrdnalab.com).
Processing of pyrosequencing data
The 16S pyrosequencing data (PD) were processed using the
Quantitative Insights into Microbial Ecology (QIIME)
pipeline (Caporaso et al., 2010). Demultiplex and quality filter
reads were used to perform quality filtering based on the
characteristics of each sequence. Sequences were trimmed of
barcodes and primers. Then short sequences (< 200bp),
sequences with ambiguous base calls, and sequences with
homopolymer runs exceeding 6bp were removed. Sequences
were then denoised and chimeras removed (Dowd et al., 2008).
The resulting sequences were then evaluated using the
classify.seqs algorithm (Bayesian method) in MOTHUR against a
database derived from the Greengenes set using a bootstrap
cutoff of 65%. Based upon sequence identity, each bacterium
was identified to its closest relative and taxonomic level.
Sequences were clustered into different OTUs, and the resulting
clusters were assessed at 3% and 5% dissimilarity to provide the
data needed for diversity analysis. The remaining 16S rRNA
sequences from pyrosequencing in this study were clustered
into OTUs using UCLUST, with setting 0.03 distance limit
(equivalent to 97% similarity, Edgar et al., 2010 ).
Final OTUs were taxonomically classified using RDP (Wang et al.,
2007), against a curated database derived from
GreenGenes. Representative sequences were aligned to the
Greengenes core set with PyNAST. All sequences that failed to
align were discarded. Alpha (or the within-sample) diversity for
samples or groups of samples was examined using Chao1 and
Shannon index and phylogeny-based measure as Faith’s PD.
Beta diversity analysis (between-samples) was based on Bray-
Curtis, weighted and unweighted UniFrac distance metrics,
PCoA analysis (Lozupone et al., 2006).
Potentiostat Box/
FRA interface
Electrochemical
cell
N’stat Box
Pump
Pump
Feed Tank
E1 E2 E3 E4 E5 E6 E7 E8
RE: Ag/AgCl/3.5M KCl
N2sparged
atmosphere
DSA: Counter electrode
Fig. 1: Schematic arrangement of electrodes in the electrochemical cell and recirculation
loop for the electrolyte. The electrodes were monitored simultaneously with the help of
N’ stat box, further connected to the potentiostat. RE and E denote reference electrode
and each independent working electrode, respectively.
Ex-situ confocal studies of electroactive biofilms
At the end of the experiment, the biofilm-embedded
biocathodes were also used for conducting confocal laser
scanning microscopy (CLSM) studies. CLSM images of biofilms
were obtained by staining bacteria with SYTO-9. Excitation
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wavelengths of 488 nm and 647 nm, and emission wavelengths
in the range of 560-590 nm for green channel and 585-640 nm
for the red channel, were employed.
Electrochemical measurements
Electrochemical measurements were recorded using a Bio-Logic
VMP-3 multi-potentiostat and frequency response analyser
(software EC-Lab v10.23). The working potential of each
individual electrode was constantly maintained by
chronoamperometry, at -0.85 V vs. Ag/AgCl/ 3.5 M KCl (+199
mV against SHE), throughout the operation of the cell.
Subsequently, upon attaining steady state at this polarization
condition, single-sine EIS was recorded at an AC amplitude of 10
mV (~7.07 mVrms), in the frequency range of 10 kHz to 1 mHz.
Six points per logarithmic decade were taken. The previously
referred N’Stat box (Bio-logic) was used to simultaneously
connect, polarize, and monitor all the eight working electrodes
in the electrochemical cell (Fig. 2), including the frequency
response analysis. The electrochemical system was confirmed
for linearity, causality, finiteness, and stability, as described in
Dominguez-Benetton et al. (2012). Stability was defined as per
variations in current lower than ±10 [ in a period of 1 h,
immediately before the EIS run. Each EIS run took about 53
minutes. Causality was ensured, as spurious (noisy) data were
not regularly observed while recording EIS. Finally, the
measured frequency range met the constraints of the dynamic
response of the electrolysis system under study. Furthermore,
the validity of EIS data was assessed using the Kramers-Krönig
transforms (EC-Lab software).
Total charge
The total charge transferred during the selected polarization
condition (Qp) was calculated using the equation described
below (Gimkiewicz and Harnisch, 2012):
Eq. 2
!
=
"

0
#
Exchange current density
The exchange current density (jex) is an indicator of the
electrokinetic activity of a (bio)electrode. A (bio)electrode with
a high exchange current density has fast kinetics and can
respond rapidly to a change in potential (Srikanth and
Venkatamohan, 2012). This parameter reflects the intrinsic rate
of electron transfer between the bioelectrode and the
production generated thereby. The magnitude of jex also
denotes desirable electrocatalytic properties; for instance, the
degree of reversibility. A higher jex indicates lower
overpotential, which implies that the reaction would tend
towards reversibility. It is desirable to have the reduction
reaction occurring at potentials as close as possible to the
reversible electrode potential (thermodynamic electrode
potential) with satisfactory reaction rates. Thus, to obtain high
currents at low overpotential, jex should be large (Zhang, 2008).
The exchange current density can be expressed as per Eq. 3:
Eq. 3
$
%
=
R
'
(
F
*
+
where jex is the exchange current density, n represents number
of electrons transferred (20 for butyrate and 8 for acetate, thus
n = 28 in the context of Eq. 3), F is Faraday’s constant (96485 C
mole-1), R is the ideal gas constant (8.314 J K-1 mol-1). The
polarization resistance also known as charge transfer resistance
Rct as shown in the Eq. 3, can be obtained from EIS plots, and T
is the temperature (K) (Liu et al., 2011).
Electrochemically-active area
Neither the projected surface area or the BET surface area of
the electrodes truly represent the effective area utilized to
complete the electrochemical transformations attributed to the
charge consumed by the independent electrodes. Hence it
becomes necessary to calculate the electrochemically-active
surface area (A) responsible for such processes, in order to
ensure an objective benchmark on the underlying performance
of the electrodes.
The integrated Cotrell equation, referred sometimes as Anson
equation (Eq. 4), defines the charge-time dependence for linear
diffusion control (Fragkou et al. 2012), as described by Fragkou
et.al. 2012:
Eq. 4
=
2
(
F
,-
.
1/2
0
1/2

1/2
For the case of more than one electrochemically-active
chemical species, this equation can be rewritten as:
Eq. 4a
=
2F
,
0
1/2

1/2
(
1
(
#
=
1
(
#
-
#
.
#
1/2
)
And for the specific case of acetate and butyrate, as:
Eq. 4b
=
2F
,
0
1/2

1/2
(
(
2+
-
2+
.
2+
1/2
+
(
45
-
45
.
45
1/2
)
The linear addition implicitly assumes that the diffusion
coefficients of each chemical species is independent of their
concentration, which is valid for dilute solutions (as in the case
of this study).
Q refers to the experimental charge transferred (C), n
represents the number of electrons transferred (20 for butyrate
and 8 for acetate), F is Faraday’s constant (96485 C mole-1), A is
the real electrochemical surface area (cm2), C the concentration
of the substrate (in this case measured at the sampling points
referred in the results section), D the diffusion coefficient of the
substrate (in cm2 s-1, i.e., 12.1E-10 cm2 s-1 for acetate and 8.7E-10
cm2 s-1 for butyrate), and t the time (s).
The rationale for the choice of this equation is the following.
There are simultaneous phenomena taking place within the
system of study, which are relevant for determining the
electrochemically-active surface area. First, there are
phenomena related to the butyric and acetic acids supplied for
the bioelectrochemical transformations. The diffusion of acetic
and butyric acid towards the bioelectrochemically-active
surface, along with charge transfer limitations at the electrode-
biofilm interface (i.e., extracellular electron transfer), constitute
the most important occurrences in this regard.
The bacteria employed constitued the sole (bioelectro-)catalyst
involved on the conversions of both butyric and acetic acid. In
control experiments without the microbial biomass, the
conversions cannot be achieved by the sole effect of any of the
carbon electrodes supplied—as referred in our previous work
(Sharma et al., 2013). Thus, the bacteria are indeed key to the
charge transfer steps observed in the system. Although in the
beginning the bacteria are not attached to all electrodes, they
are issued from a highly electrochemically-active system, and
therefore they can convert the substrates provided right away.
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Upon development, they act mostly attached to the porous
macro-electrodes, following their structure. As it can be seen in
Figure 6 ouf our manuscript, the bacterial biofilms formed do
follow the structure of the porous electrodes.
On the other hand, the flowing electrolyte in the system,
follows a laminar flow pattern. This implies that there is a quasi-
still environment towards the electrodes and that a
concentration gradient of the substrates is developed from the
bulk to the electrode surface, especially due to the activity of
the microbes that deplete the substrate in the immediacy of the
electrodes. In our previous work regarding the colonizing
electrode presented in this work (Sharma et al., 2013), this
biocathode was reaching current densities far superior than the
ones presented in this study (e.g., up to >200 A m-2) which
shows its high electrocatalytic capabilities. It is only in the
reactor and flowing configuration investigated in our present
study that the biocathode reached more moderate current
densities, which are attributed to a different limiting
phenomenon.
Thus, although there are different phenomena contributing to
the transitions observed in the faradaic current (mainly charge-
transfer by the bacteria and diffusional limitations to the porous
electrodes), the overall phenomenology is considered to be
overall controlled by the substrate concentration gradient.
It should also be noted that in the experimental conditions of
the system investigated, abiotic hydrogen electrosynthesis is
also thermodynamically feasible, yet kinetically hampered in
carbon-based electrodes. The adsorption of electrochemically-
active species and the capacitive charge due to charging of the
double layer may also contribute to the total charge consumed.
However, we consider that these three phenomena are
negligible in comparison with the other phenomena taking
place in the system so they are not considered relevant for the
account of the total charge exchanged.
Analysis of impedance parameters
The impedance response can be explained by the use of
deterministic models, which offer information about
physiochemical processes occurring at the electrode-electrolyte
interface of an electrochemical system. Such processes may
explain not only kinetic rates, but also mass transfer reaction
mechanisms, as well as material properties, such as permittivity
and conductivity (Hirschorn et al., 2010a). An assumption that
is frequently used to model EIS data of heterogeneous
interfaces, such as those developed in porous electrodes
including electrochemically-active biofilms is the constant
phase element (CPE) (Dominguez-Benetton et al., 2012). The
impedance response of this CPE (ZCPE) is based on a
mathematical representation similar to the electrical reactance
of a pure capacitor, but it considers some deviation in the
complex plane, given by an angle of rotation E. The impedance
response of a CPE is represented by Eq. 5:
Eq.5
6
-!7
=
1
(
8
9
:
The CPE parameters Q and E are frequency-independent
constants; where Q represents the differential capacitance of
the interface. j is the imaginary unit (j2 = -1), and F is the angular
frequency (F = _f, f being the ordinary frequency, in Hz)
(Jorcin et al., 2006). The parameter E can be estimated from the
graphical representation of impedance concerning the negative
imaginary measurement (-ZIm) against the frequency (F), both
parameters in logarithmic scale (Dominguez-Benettton et al.,
2012; Devos et al., 2006). The first derivative d(-ZIm)/dF can be
obtained to determine the relative maxima of the function. The
slopes of the curves above the relaxation frequency are
estimated, which directly correspond to the magnitude of E.
When E = 1, Eq. 5 becomes equal to that of the reactive
capacitance. Thus, the CPE represents a pseudo-circuit element
with limiting behaviour as a capacitor for E = 1.
Correspondingly, the CPE behaves as a resistor when E = 0.
However, many electrochemical systems behave differently,
and when 0 < E < 1, Q cannot represent either a capacitance or
resistance (Hirschorn et al., 2010b). In this way, the CPE is used
as a flexible parameter for fitting impedance data.
The existence of a CPE in the model of EIS response of a
microbially-electrocatalyzed interface can be clearly justified
from the magnitude of E (Dominguez-Benetton et al., 2012;
Devos et al., 2006). However, the physical meaning of the
process underlying such response cannot be clarified from just
the purely CPE mathematical description (Dominguez-Benetton
et al., 2012; Hirschorn et al., 2010a). As explained by Hirschorn
et al., the physical origins of the CPE are polemic and may arise
from a distribution of time-constants which cannot always be
elucidated with ease. For this reason, here the analysis of E is
solely taken as an indicative of the distributed properties of the
microbial-electrochemical interface (i.e., its electrochemical
heterogeneity, as evolving along experimentation).
Results and discussion
Designing bioelectrochemical systems essentially requires
working electrodes that have the expected physiochemical
properties supporting the growth of a biocathode with a
sustained functionality. These should also provide the suitable
surface properties that further enhance its performance, e.g.,
by overcoming mass transfer limitations and other resistances
intrinsic to the system. In the following sections, a comparative
evaluation of selected electrode materials is provided, from
both the physicochemical and bioelectrochemical perspectives.
Physicochemical properties of selected electrode materials
Electrical resistance
The electrical resistance of a carbon-based electrode material is
directly dependent on its structure and contributes to the total
resistance of both the system and the interface of study during
operation. Hence, as desirable in an efficiently-performing set-
up, the electrode resistance should be as minimal as possible.
From the range of electrodes that were shortlisted for this
operation, E1 exhibited the least electrical resistance of 1.6 ×
10-3 S (Table 1). This was followed by E8 and E6, with electrical
resistances of 3.2 × 10-3 S and 1.1 × 10-2 S respectively. The
remaining electrodes, in increasing order of electrical
resistance, were E5 (4.02 × 10-2 S< E7 (4.69 × 10-2 S< E2 (2.61 ×
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10-1 S< and E4 (372.5 S< correspondingly. Summarizing, the
magnitude of the electrical resistances behaved as follows:
E1<E8<E6<E5,E7<E2<E4. E3 was not considered for this and
other subsequent physicochemical characterizations due to its
completely different state of development, which would lack of
objectivity for our purposes at this point. Different commercial
electrode materials exhibit a wide range of electrical resistances
which in turn depend on other properties as described below.
Hydrophilic porosity
As can be seen in Table 1, it was not possible to measure the
hydrophilic porosity of all the selected bare materials, as
electrodes E1, E2, and E7, swell considerably when dipped in
water. Electrode E4 exhibited the highest hydrophilic porosity
(48.21 %) amongst the other measurable electrode materials,
followed by E5 (32.27 %), E6 (20.12 %), and E8 (5.27 %), i.e.,
E8<E6<E5<E4. Among the measured electrodes, the hydrophilic
porosity was found to be directly proportional to the electrical
resistance.
Contact angle
The wetting characteristics of a solid depend on the roughness
of its surface, this being characteristic of the fundamental
theory of wetting action (Wenzel, 1936). This is immediately
apparent when analyses of wetting phenomena take into
account the actual area of the several interfaces involved, as
well as their respective specific energy values. This method of
analysis helps in the quantitative evaluation of the effects of
surface heterogeneity and surface porosity (Cassie and Baxter,
1944). Small contact angles (<90°) correspond to high
wettability, while large contact angles (>90°) correspond to low
wettability.
Electrode surfaces like E1, E2, E4, E6, E7 and E8, rendered
contact angles ranging in between 101° to 138°
(E4<E7<E1<E6<E8<E2), indicating that wetting is unfavorable
(i.e., tending to hydrophobic), as the liquid droplet tends to
minimize its contact with the surface of the solid sample. For
electrode E5, the contact angle measurement was not possible
due to the immediate drain of the water drop, which
qualitatively characterizes it as highly hydrophilic. The contact
angle is a property of the material and should depend
exclusively on the characteristics of the solid surface under
investigation; yet, in practice, external factors can affect the
measurement, such as geometric factors and the characteristics
of the solid-liquid interface formed (Snoeijer et al., 2008; Yuan
& Lee, 2013). This is probably the case for the E5 electrode,
whose surface has a relatively open structure (cf. Figure 4). One
must note that the exposed porosity of these materials
contributes to the surface water contact angle. In the case of
porous materials, contact angle alone cannot differentiate the
surface chemistry of the electrodes.
Although hydrophilic porosity and contact angle
measurements give an indication of electrode properties, the
electrolyte reachability will majorly depend on the carbon
structure and surface properties, viscosity, dielectric constant,
dipole moment and size of the organic constituents of the
electrolyte (Koresh and Soffer, 1977; Salitra et al., 2000;
Pandolfo and Hollenkamp, 2006).
Here, a specific association between the electrical
resistance, the hydrophilic porosity, and the contact angle
behaviors could not be established. However, in general, those
electrodes with a lower electrical resistance rendered higher
contact angles, and vice versa.
Thermogravimetric Analysis (TGA)
An isothermic pre-treatment at 130 °C was carried out prior to
the TGA essay, to desorb loosely bounded physisorbed
molecules of water and volatile compounds. Except for E2 and
E5, all electrode materials examined lost less than 3% of the
original mass during the isothermic pretreatment (Table 2).
Dynamic TGA was subsequently carried out (100 – 800 °C) in
order to monitor the general thermal resistance of the materials
as a function of temperature. The thermal resistance of the
electrode materials showed different trends as a function of the
presence of functional groups on their surface. E1, E5, E6, E7
and E8 showed one single major degradation, with a maximum
in the rate of mass loss (peak in DTG) at relatively high
temperatures: 617 C for E5, accounting for a mass loss of 72%;
656 C for E6, accounting for a mass loss of 99%; 696C for E8,
accounting for a mass loss of 98%; 600 C for E7, accounting for
a mass loss of 72%; and >800 C for E8 (the degradation step
was not completed when the upper temperature limit of 800 C
had been reached), accounting for a mass loss of 52% (Figure 2,
Table 2).
The EDS spectra of E1, E6, and E8 (Figures SI S1.1, S1.5, and
D1.7) show one single peak corresponding to carbon, i.e., no
oxygen or other functional groups are present on the surface of
these electrode materials according to EDS analysis. The mass
loss observed in E1, E6, and E8 would be thus ascribable to a
general degradation of the carbon skeleton of the material. In
the case of E5 and E7, the EDS revealed the presence of more
elements than C, namely O, Na, and P, in the case of E5 (Figures
SI S1.4); O and S, as well as Na, Ti, and Pt, in the case of E7
(Figure SI S1.6). The thermal degradation step in these two
samples should be related to both the general degradation of
the carbon structure and the loss of oxygenated functional
groups, possibly carboxylic anhydrides or lactone groups, given
the temperature range at which the process is observed
(Figueiredo et al., 1999; Szymanski et al., 2002).
In the case of E2, a carbon-PTFE composite, two different
degradation steps can be distinguished in the DTG traces (Figure
2b, Table 2). The first degradation step, with a maximum in the
rate of mass loss at ca. 540 C, would be mainly related to the
decomposition of the PTFE component (through unzipping to
tetrafluoroethylene monomer) and to the degradation of
functional groups, most likely carboxylic acid groups (Schild,
1993; Figueiredo et al., 1999; Szymanski et al., 2002) and other
acid moieties. Although not immediately identifiable in the EDS
spectra (Figure SI S1.2), S and Cl are present in this electrode,
as it pre-treated with acid-washed steam by the manufacturer
(i.e., HCl and H2SO4 are likely involved in this process).
E4 also showed two different degradation steps (Figure 2,
Table 2). The first step, with a peak in DTG at 356 C, may be
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assigned to the decomposition of carboxylic acid functional
groups. The second degradation step would be then due to the
thermal degradation of the carbon backbone of the material,
along with the thermal decomposition of functional groups such
as carboxylic anhydrides or lactones, which are thermally more
stable than carboxylic acids (Figueiredo et al., 1999; Szymanski
et al., 2002). Moreover, the EDS spectra of this electrode (Figure
SI S1.3) confirmed a high presence of metals in the electrode.
Table 1: Physicochemical properties of the selected electrode materials
Name of the electrode
Surface
area
(m2g-1)
Pore
volume
(ccg-1)
Pore
diameter
;[<
Electric
resistanc
e ;S<
Thickness
(mm)
Contact
angle
(°)
Hydrophilic
porosity
(%)
Profilometry Ra
(surface
&.[<
Profilometry
Rt (maximum
profile
&.[<
E
1
Glass wool filter
3.94
0.424
0.002
1.6E-3
2.85
118.7
NA
76.56
569.41
E
2
VITO CORE TM
620.6
0.424
0.004
2.6E-1
1.24
137.9
NA
1.33
12.52
E
4
Activated carbon
fleece
125.7
0.041
0.002
372.5
1.51
100.97
48.21
18.42
353.32
E
5
Activated carbon
fabric
1118.5
0.144
0.001
4.02E-2
0.63
n.m.
32.27
20.92
184.37
E
6
Carbon paper
4.5
0.001
0.087
1.1E-2
0.25
122.6
20.12
14.07
99.06
E
7
Carbon felt
847.9
0.056
0.002
4.69E-2
2.59
117.03
NA
44.51
416.45
E
8
Paxitech
1.37
0.003
0.005
3.21E-3
0.32
130.4
5.27
15.28
201.24
Table 2: TGA features of the selected electrode materials
Name of the electrode
Mass change pre-treatment
130C (%)
T DTG peak
step 1
(C)
Mass change step
1 (%)
T DTG peak
step 2
(C)
Mass change step
2 (%)
Char yield
800C (%)
E
1
Glass wool filter
-0.28
>800*
-52
--
--
47
E
2
VITO CORE TM
-8.1
537
-29
607
-57
4.2
E
4
Activated carbon
fleece
-2.9
369
-42
449
-46
9.7
E
5
Activated carbon
fabric
-21
617
-72
--
--
7.4
E
6
Carbon paper
-0.15
656
-99
--
--
1.1
E
7
Carbon felt
-1.62
600
-79
--
--
1.0
E
8
Paxitech
-0.40
694
-98
--
--
19
* The degradation step was not completed when the upper temperature limit of 800 C had been reached.
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Fig. 2: (a) Thermogravimetric (TG) profiles of various electrodes studied and (b) the corresponding derivative thermogravimetric curves (DTG).
N2 adsorption measurements
The characteristic BET surface area, pore volume, and pore
diameter of each electrode sample are shown in Table 1. A
decreasing surface area was found for E5 (1118.5 m2g-1), E7
(881.6 m2g-1), E2 (585.5 m2g-1), E4 (116.8 m2g-1), E6 (6.9 m2g-1),
and E8 (1.37 m2g-1), correspondingly. Based on the pore
diameter size determination, as described by Sing et al. (1985),
it was here found that all electrodes, with exception of E6,
contained mesopores (2 – 50 nm pore diameter). E6 was found
to have macropores (>50 nm).
Though the BET surface analysis method is widely used for
measuring the apparent surface area of carbon-based porous
electrodes, it has some limitations, which cannot be ruled out.
This includes the assumption that the surface area accessed by
nitrogen gas is similar to that of the electrolyte while
performing experiments. Though nitrogen gas can indeed
penetrate open pores down to the size that approaches the
molecular size of the adsorbate, this is not necessarily the case
with the whole electrolyte components.
No evident relationship was found between the BET surface
area; however, those electrodes with a small pore diameter
coincided with those within the lower range of contact angle
and vice versa.
Profilometry and SEM of sterile electrode surfaces
Structural studies are also essential to understand electrode
materials, i.e., the geometric surface irregularities. A surface
can be curvy, wavy, rough, or smooth, depending on the
magnitude of the spacing between peaks and valleys and how
the surface is produced (Wyko surface Profilers). A high
roughness of the electrode surface is generally preferred for
biofilm development, as it increases the reactive surface area
available for the microbes to interact with the electrode. This
eventually results in higher current densities due to lower
ohmic resistance and lesser instances of bioclogging (Jourdin et
al., 2014).
To determine the roughness of the electrode surfaces
investigated here, profilometric analyses were done. All
profilometric calculations were done in triplicate. The average
surface roughness is reported in Table 1. E1 exhibited the
highest surface roughness of 76.56 [ followed by E7, which
exhibited surface roughness of 44.51 [ A common feature
between E1 and E7 is that both these are felt-based materials.
E5 presented a surface roughness of 20.92 [ followed by E8,
E4, E6 and E2 with surface roughnesses of 15.28 [ 18.42 [
14.07 [ and 1.33 [ respectively.
The profilometric surface images and the roughness values are
in good agreement with the corresponding SEM micrographs
(50x magnification), as shown in Fig. 4. As the packing of carbon
fibers increases, the roughness of the corresponding electrodes
decreases (Fig. 4.). Electrode E1, with the highest asymmetry,
also showed the highest surface roughness. In contrast, E2, with
the highest packed surface, showed a surface roughness of 1.33
[ It is commonly believed that a surface roughness close to
the size of microbial cells promotes more biofilm coverage,
ultimately contributing to higher current densities in the case of
MFC bioanodes. It has also been reported that a roughness
higher than 4µm does not show any enhanced effect on biofilm
development (Pons et al., 2011). From all the electrode
materials selected here, only electrode E2 falls in this category.
The remaining electrode materials exhibited roughnesses much
higher than this biologically-amenable range. Thus, the superior
wetting characteristics of the VITO CORE™ electrode seem
highly reliant on the roughness provided by the manufacturing
method (i.e., cold-rolling).
It has been reported that besides the surface area and porosity
of the electrode material, variations in carbon precursor,
thermal treatment method applied during the manufacture of
electrode, the presence or absence of surface functionalities,
and associated changes in the mode of electronic conduction,
influence together the performance of any electrode material
(Biniak et al., 2001). Yet, these assertions may be valid only for
a few electrochemical systems and do not necessarily correlate
to the suitable establishment of bioelectrodes with superior
performance on the parameters that define an effective
microbial electrosynthesis (e.g., effective charge transfer,
sustained high current density, preservation of the targeted
microbial-electrochemical activity).
Electrochemical response of the biocathodes
The SRB-biocathodes were simultaneously polarized at -0.85 V
vs. Ag/AgCl within the multi-working-electrode tubular
electrochemical cell. The current was monitored for 32 days in
chronoamperometric experiments (Fig. 5).
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Fig. 4: Left panel represents the 50 X SEM micrograph of the seven electrodes namely E1
to E8 from top to bottom and right panel shows the corresponding profilometric images.
These pictures are also provided individually in the supplementary information.
The average and maximal current densities obtained with each
electrode material are reported in Table 3. The electrode E2
(VITO CORE™) showed a superior performance when compared
to the other electrodes, with a with total charge of 1012.37 C.
Electrode materials E1 (-16.78 A m-2), E6 (-14.40 A m-2), E8 (-
13.01 A m-2), and E3 (-12 A m-2) exhibited comparable
magnitudes of maximum current density with respect to E2 (-
17.14 A m-2). However, there was clear drop in the total electric
charge utilized by the biocathodes grown over such electrodes
materials, i.e., E1 (572.87 C), E6 (615.64 C), E8 (555.11 C) and E3
(368.44 C), to achieve the microbial-electroconversions
targeted. E4 (-0.95 A m-2), E5 (-2.61 A m-2), and E7 (-6.68 A m-2)
were the least performing biocathodes in terms of both
maximum current density and total charge transferred (E4:
35.03 C, E5: 135.21 C, and E7: 310.69 C).
The electrode E2 (VITO CORE™) transferred about double the
charge (1012.37 C) of the other electrode materials, as shown
in Table 3. The seven electrode materials were
electrochemically synchronized in the same electrochemical cell
setup (with the help of the N’Stat and potentiostat software),
sharing the same electrolyte via the recirculation set up, and
exposed to the same inoculum source, nutrients, and flow rate.
Therefore, the trigger for high current densities and total charge
consumed relates to the physicochemical properties of the
electrode material supports and the associated biofilms
individually formed on them.
It is to be noted that some electrodes showed an important
current from the beginning of the experiments. In a typical
experiment, in our previous works (Sharma et al., 2013) and in
most works published on microbial electrochemistry,
experiments start using an inoculum which has been cultivated
a priori in a non-electrochemical context, i.e., using soluble
electron donnors and acceptors.
Fig. 5: Chronoamperometric response of the selected electrodes during the experiment.
This graph represents a five point average value and hence maximum current density
values for individual electrodes may not be represented in this figure.
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In this case, microbes would require to change some of their
metabolic (and perhaps structural) machinery to be able to use the
electrode as electron donor or acceptor, and thus a delay time for
developing current is expected. In a few instances electrodes are
colonized starting from a pre-colonized electrode, which implies that
the microbes are readily active for the electrochemical processes of
interest at the solid state electrode. In our case, the precolonizing
electrode had displayed a remarkable electrochemical activity prior
to being placed in the setup object of the present study. Thus, as the
microbes are released towards the electrolyte they can effectively
interact right away with the different electrodes. We attribute such
starting currents to this phenomenon. Otherwise, a constant current
could be expected if the system would work in continuous mode, but
in this case the reactor was operating in batch mode with
recirculation. Thus, fluctuations are normal as substrate is added or
depleted.
Microscopic analysis of the selected electrode materials
To track the morphological characteristics of the electrodes
before and after a biofilm formed over their individual surfaces,
SEM measurements were done. EDS measurements were also
done on all the electrode materials, before the experiment was
initiated. An important observation made in the EDS
measurements (SI-1) of these carbon electrodes before the
experiments were performed was that within the EDS
detectable range no remarkable metal impurities were found in
electrodes E1, E2, E6, and E8, contributing to their good
performance. A very small fraction of some other elements,
besides carbon, were found in E5 and E7 contributing to their
medium performance, while electrode E4 contained many
impurities which contributed to its low performance, as
indicated in Table 3. Metal impurities can promote other
undesired side-reactions on the metal electrode interface,
hence compromising the overall performance of the electrode
or can even deter formation of electroactive biofilms as some
of these constituting metals may be toxic for biofilm growth.
On the other hand, three possible mechanisms have been
described in microbe-electrode interfaces for transfer of
electrons, including mediated electron transfer (via redox
mediators), direct electron transfer via cytochromes, and via
extrapolymeric or pili-like structures, often called bacterial
nanowires. As discussed in previous sections, the bacterial
settlement on the electrode surface is favored by a surface
roughness close to the size of the microbial cells. For instance,
electrode E2 shows more dense cell packing than the other
electrodes with a roughness factor Ra of 1.33 [ which roughly
corresponds to the microbial cell size, as can be observed from
the SEM results (Figure SI S2.3).
He et al. (2011) defined the “critical fiber diameter (CFD)
factor”, which is the fiber diameter of around the length of the
dominant microorganisms present in the electroactive biofilms;
this contributes to shift from porous to continuous solid
biofilms with a reduction in the CFD. The depth of biofilms was
reduced with a decrease in fiber diameter due to smaller pore
sizes that limit mass transport.
Table 3: Average current density, maximum current density and total charge calculated
for the working electrodes used in the reactor. Steady state was reached between 5th to
9th day and afterwards the behavior was constant.
Name of
the
electrode
Average
current
density per
projected
surface area
(A m-2)
Maximum
current
density per
projected
surface area
(A m-2)
Total
charge
(C)
Biofilm
Coverage
(%)
E2
-9.41
-17.14
1012.37
44.3
E6
-5.525
-14.40
615.64
27.7
E1
-5.24
-16.78
572.87
23.3
E8
-4.99
-13.01
555.11
46.5
E3
-4.62
-12.00
368.44
40.4
E7
-2.96
-6.68
310.69
23.4
E5
-1.22
-2.61
135.21
25.2
E4
-0.33
-0.95
35.03
17.5
In electrodes with fiber diameter less than the CFD, solid
continuous biofilms were observed. In the present work, the
highest current density was attributed to electrodes with
moderate roughness, like E2, around the size of microbes,
presumably due to the formation of proper size pores for
colonization. These ensured the formation of thick and
continuous solid biofilms as the fine inner pores of these
materials might not be accessible to the microorganisms (Kong
et al., 2014).
As can be deduced from the SEM images (Fig 6), filamentous
extrapolymeric structures with microorganisms were formed
over the electrode surfaces by the SRB consortium TERI-MS-
003. The coverage factor of the biofilms is summarized in Table
SI S2 (related to the processed micrographs shown in SI-2),
clearly demonstrating to be directly proportional to the amount
of current density generated over time (total charge), as shown
previously in the chronoamperograms of Figure 5. Electrodes
E2, E6, E1, and E8, which showed comparatively higher average
current densities, showed a higher prevalence of filamentous
extrapolymeric structures forming networks, as can be
onserved from the SEM images shown in Figure 6 and SI-2. Such
filaments appear to be cell-bound and extend into the
extracellular environment, besides being consistently found in
higher abundance in high performing electrodes, like E1 and E8.
Although their exact implication in the electrochemically-active
phenomena observed is not elucidated in this work, our
experimental results suggest that the current densities
achieved increase in proportion to their qualitative abundance.
These biofilms also displayed a lower resistance to electron
transfer across the biofilm-cathode interface, as per the
correlation observed with the average polarization resistance
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(Rp)—which was obtained by EIS and shown in data further
explained—and the coverage factor calculated from the SEM
images using Gwyddion software (version 2.42), as shown in
supplementary material SI-2. A quasi-linear correlation (R2=
0.87) was seen between the total charge transferred at the
bioelectrochemical interface and the percentage of biofilm
coverage, with E2 and E8 emerging as the best electrode
materials for biofilm formation.
The long filamentous structures found were seen both over
the bacterial surface and out of the surface, seemingly
connecting cells in a wire-like network. Wire-like structures
have been previously reported for long filamentous bacteria of
the Desulfobulbaceae family and have shown to transport
electrons generated from one end of the wire to the other end.
This phenomenon is balanced by the ionic charge transport by
ions in the surrounding environment, maintaining the overall
charge balance. It has also been also postulated that these wire-
like networks have several conductive filaments that are
isolated by outer membrane (Pfeffer et al., 2012). Although
individual or “frayed” filaments, popularely termed bacterial
nanowires, have been reported to have dimensions of 2-20 nm
in diamenter or thickness (Wang et al., 2019; Maruthupandy et
al., 2015; Gorby et al., 2006) , these usually appear bundled
together reaching thicknesses of up to >150 nm and
micrometers length (Gorby et al. 2006). The filamentous
structures found in our systems thus seem to correspond to
bundles of nanowires. In broad terms, bacterial nanowires have
been defined as protein filaments produced by microbes,
showing properties similar to supercapacitors and transistors
(Lovley, 2012). The exact nature of the structure and electron
transfer mechanism along such nanowires is still a debated
subject (Filman et al., 2018) and out of scope of the present
work. However, since the filamentous structures here observed
were in good correlation with the current density production
pattern in this study, we believe that these filament structures
are certainly implicated in long-range electron transfer. The
exact nature of the phenomenology implicated warrants
further investigations using more sophisticated methods like
conductive atomic force microscopy (AFM), among others.
Otherwise, such filaments can have additional functions,
beyond electron transfer, e.g., increase of biofilm ashesiveness,
establishing contact with insoluble electron acceptors, among
other unique cell-cell and cell-surface interactions (Reguera et
al., 2005), which may additionally enhance extracellular
electron transfer.
Frequency response of the SRB biocathode
Electrochemical impedance spectroscopy (EIS) is a non-
intrusive method that helps in the identification of the different
phenomenological contributions that impact the performance
of the bioelectrochemical reactor as a whole (Dominguez-
Benetton et al., 2012; Sevda et al., 2015). EIS also helps to
understand the phenomena taking place at the established
bioelectrochemical interface.
Preservation of the electrochemical features of the SRB
biocathodes using different carbon materials was established
by the EIS response. Figures 7 and 8 exhibit the Nyquist
diagrams with area correction (using the BET surface area) and
ohmic drop correction.
A characteristic response was observed with all the
electrode materials subject to new SRB-biofilm colonization,
which demonstrated that the EA-biofilm plays the central role
in accelerating the electron transfer processes and associated
microbe-electrode interface reactions (Fig. 7 and Fig. 8). This is
consistent with the observations of previous works (Yu et al.,
2013; Wang et al., 2013). In general, a unique pseudo-capacitive
loop developed as shown in the corresponding Nyquist
diagrams from high to middle frequencies, i.e., 10 kHz to 1 Hz
(Fig. 7 a,c,e,g and Fig. 8 a,c,e,g). In all instances, this loop
increased in magnitude through time, up to a final stable limit
where maximal biofilm growth is assumed. This pseudo-
capacitive loop was correlated to the charge transfer processes
occurring within the developing biofilm. As the electroactive
biofilms evolve over the electrode materials, there is evident
built-up charge at the interface, evidenced by such growing-
semicircle trend. An exception was noted in the initial stages of
development in E2, that had a differentiated impedance
response, but later on adopted a more consistent behavior with
the rest of the electrodes.
At lower frequencies (i.e., below 100 mHz), a pseudo-
inductive behaviour appeared. This has been previously
associated with an adsorption-limited mechanism during the
bioelectrochemical reduction of acetic and butyric acids
(Sharma et al., 2013), which is the conversion associated to the
present case. This pseudo-inductive loop is preserved in most
electrode materials, with a few exceptions. The first exception
is E2, the newly colonized VITO CORE™ electrode (Fig. 7c). Yet,
the behavior shifted towards this adsorption-like shape as time
evolved. However, the pseudo inductive loop on E2 is presented
at much higher frequencies than for the rest of the electrode
materials. This indicates the associated phenomenon, i.e., the
adsorption processes occur at higher rates as compared to
other electrodes, E5 (Fig. 8a) and E7 (Fig. 8e) also exhibit a
differing behaviour. The similarities and discrepancies on the
EIS response can also be observed in the Bode diagrams shown
in Figures 7 and 8. E2, E5, and E7 can be considered to maintain
the same limiting electrochemical activity but with a different
rate. Adsorption is present, but perhaps the distribution of the
adsorption sites in such electrodes is more scattered when
concerning the total electrode BET surface area.
Polarization behaviour and exchange current density
On the other hand, the polarization resistance (Rp) extracted
from the Nyquist diagrams (diameter of the semicircle) also
constitutes a performance criterion (Dominguez-Benetton et
al., 2012). Rp is correlated to the energy losses and kinetic
limitations of the electrode and is inversely proportional to ease
of electron transfer (He and Mansfeld, 2009; Aulenta et al.,
2010; Lepage et al., 2014). E1, E2, E6, and E8 presented the
lowest diameters from the beginning of the experiment and
through time up to day 32.
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Fig. 7: Nyquist and corresponding phase angle plots for electrodes E1-E4. (a), (c), (e), (g)
represent the Nyquist plots for Electrodes E1 to E4, respectively. (b), (d), (f), (h) represent
the phase angle plots for Electrodes E1 to E4, respectively. The Nyquist response is
calculated per unit of respective BET surface area and additionally ohmic drop corrected.
Fig. 8: Nyquist and corresponding phase angle plots for electrodes E5-E8. (a), (c), (e), (g)
represent the Nyquist plots for Electrodes E5 to E8. (b), (d), (f), (h) represent the phase
angle plots for Electrodes E5 to E8, respectively. The Nyquist response is calculated per
unit of respective BET surface area and additionally ohmic drop corrected.
Fig. 9: (a) Polarization resistance developed by the 8 working electrodes in the electrochemical set up. (b) Evolution of the exchange current density with respect to time for the 8
working electrodes in the electrochemical set up.
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M.S. acknowledges Indo-Belgian scholarship from the Flemish
Government (VlaamseGemeenssschap) and Department of Science and
technology (India). We thank C. Porto-Carrero (VITO) for technical help
and Raymond Kemps (VITO) for assisting during SEM visualizations. We
would also like to thank Pradeep from HEG Ltd; Eileen from Newcastle
group, UK; USC Spain and VITO for gifting us electrode samples for
testing. We thank Marie Huleux-Bertrand, Mohamed Barakat from CNRS-
CEA-Aix-Marseille for providing assistance with processing of microbial
community data. Finally, X.D.B. thanks Jan Fransaer (KU Leuven) for the
productive discussions regarding the equations concerning the
electrochemically-active surface area.
Notes
a Separation& Conversion Technologies, VITO - Flemish Institute for
Technological Research, Boeretang 200, 2400 Mol, Belgium
b TERI University, Plot No. 10, Institutional Area, Vasant Kunj, New
Delhi, 110070, India
c The Energy and Resource Institute (TERI), IHC, Lodhi Road, New Delhi,
110003, India
d Laboratory of Microbial Ecology of the Rhizosphere (LEMiRE), BIAM,
UMR 7265, CNRS-CEA-Aix-Marseille, University, 13108 Saint Paul lez
Durance, France
† Present address: Department of Biological Sciences, University of
Calgary, Calgary – AB, T2N1N4, Canada
‡ For correspondence on the microbial consortium contact:
priyangshumsarma@yahoo.co.in
* Corresponding author: xoch@vito.be
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Graphical abstract: (a) Pictograph and (b) schematic representation of placement of
multi-working-electrodes with single counter electrode and reference electrode using
the N’Stat setup and (b) represents the schematic of the potentiostat interface
connection with the electrochemical cell.
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