Science of the Total Environment 856 (2023) 159105

Contents lists available at ScienceDirect

Science of the Total Environment

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

Tailoring a highly conductive and super-hydrophilic electrode for

biocatalytic performance of microbial electrolysis cells
Sung-Gwan Park a,b, Chaeyoung Rhee c, Dipak A. Jadhav a, Tasnim Eisa a,b, Riyam B. Al-Mayyahi a,b, Seung Gu Shin c,
⁎ ⁎⁎
Mohammad Ali Abdelkareem d,e,f, , Kyu-Jung Chae a,b,
a
Department of Environmental Engineering, College of Ocean Science and Engineering, Korea Maritime and Ocean University, 727 Taejong-ro, Yeongdo-gu, Busan 49112, Republic of Korea
b
Interdisciplinary Major of Ocean Renewable Energy Engineering, Korea Maritime and Ocean University, 727 Taejong-ro, Yeongdo-gu, Busan 49112, Republic of Korea
c
Department of Energy Engineering, Future Convergence Technology Research Institute, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 52828, Republic of Korea
d
Chemical Engineering Department, Faculty of Engineering, Minia University, Minia, Egypt
e
Center of Advanced Materials Research, Research Institute of Science and Engineering, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates
f
Department of Sustainable and Renewable Energy Engineering, University of Sharjah, PO Box 27272, Sharjah, United Arab Emirates

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• The highly conductive and hydrophilic

properties were applied to carbon mate-
rial.
• The Coulombic efficiency was increased
by two-fold after functionalization of
anode.
• Increased internal mass transfer promoted
microbial growth on the anode surface.
• The bifunctional anode plays a role as an
electron-hive for efficient H2 production.
• Biofilm and microbial dynamics in 3D
structural anodes were first profiled in
depth.

A R T I C L E I N F O A B S T R A C T

Editor: Huu Hao Ngo Bioelectrochemical hydrogen production via microbial electrolysis cells (MECs) has attracted attention as the next gen-
eration of technology for the hydrogen economy. MECs work by electrochemically active bacteria reducing organic
Keywords: compounds at the anode. However, the hydrophobic nature of carbon-based anodes suppresses the release of the pro-
Bioelectrochemical system duced gas and water penetration, which significantly reduces the possibility of microbial attachment. Consequently, a
Hydrogen
limited surface area of the anode is used, which decreases hydrogen production efficiency. In this study, the bifunc-
Biocompatibility
Polymerized anode material
tional material poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was applied to the surface of a
Electrochemically active bacteria three-dimensional carbon felt anode to enhance the hydrogen production efficiency of an MEC owing to the high con-
Microbial electrolysis cells ductivity of PEDOT and super-hydrophilicity of PSS. In experiments, the PEDOT:PSS-modified anode almost doubled
the hydrogen production efficiency of the MEC compared with the control anode owing to the increased capacitance
current (239.3 %) and biofilm formation (220.7 %). The modified anode reduced the time required for the MEC to
reach a steady state of hydrogen production by 14 days compared to the control anode. Microbial community profiles
demonstrated that the modified anode had a greater abundance of electrochemically active bacteria than the control
anode. This simple method could be widely applied to various bioelectrochemical systems (e.g., microbial fuel cells
and solar cells) and to scaling up MECs.

Abbreviations: CE, Coulombic efficiency; CF, carbon felt; COD, chemical oxygen demand; CV, cyclic voltammetry; EAB, electrochemically active bacteria; EDLC, electric double-layer capaci-
tance; EDX, energy-dispersive X-ray; EIS, electrochemical impedance spectroscopy; GF, graphite felt.
⁎ Correspondence to: M.A. Abdelkareem, Chemical Engineering Department, Faculty of Engineering, Minia University, Minia, Egypt.
⁎⁎ Correspondence to: K.J. Chae, Department of Environmental Engineering, College of Ocean Science and Engineering, Korea Maritime and Ocean University, 727 Taejong-ro, Yeongdo-gu,
Busan 49112, Republic of Korea.
E-mail addresses: mabdulkareem@sharjah.ac.ae (M.A. Abdelkareem), ckjdream@kmou.ac.kr (K.-J. Chae).

http://dx.doi.org/10.1016/j.scitotenv.2022.159105
Received 20 July 2022; Received in revised form 14 September 2022; Accepted 24 September 2022
Available online 29 September 2022
0048-9697/© 2022 Elsevier B.V. All rights reserved.
S.-G. Park et al.
1. Introduction
As the energy sector moves toward the era of the hydrogen (H2) econ-
omy, sustainable and efficient H2 production has become a critical issue.
Microbial electrolysis cells (MECs) represent an ecofriendly technology
that recovers clean energy such as H2 from organic waste via
bioelectrochemical activity (Yang et al., 2021). In MECs, the organic com-
pounds in wastewater are initially degraded into CO2 and biomass, while
the electrochemically active bacteria (EAB) generate protons and electrons
at the anode (Jadhav et al., 2020; Santoro et al., 2022). Then, protons are
reduced by electrons at the cathode to produce H2. However, this reduction
at the cathode may be hindered by insufficient generation of protons and
electrons at the anode. Therefore, improving the bioelectrocatalytic activity
at the anode is crucial for the commercialization of MECs and realizing sus-
tainable and efficient H2 production (Jadhav et al., 2022). To increase the
concentrations of protons and electrons at the cathode, two essential pro-
cesses must be achieved: the rapid degradation of organic compounds
and efficient electron transfer to the anode with minimal electron loss. In
particular, EAB, which are also known as exoelectrogens, play a crucial
role in both processes (Pawar et al., 2022). The anode should have high
conductivity and biocompatibility to allow for vigorous EAB proliferation.
In this context, several types of carbon-based materials such as carbon felt
(CF), carbon nanotubes (CNTs), carbon cloth, and carbon paper have
been widely used as anodes because they are cheaper than other conductive
materials and have a three-dimensional (3D) structure comprising multiple
pores that is suitable for microbial community growth (Park et al., 2022).
However, the hydrophobicity of carbon-based materials hinders the mass
transfer of the solution and substrates, which forms dead space in the elec-
trode. In other words, only the outer surface of the electrode is available for
microbial growth, so only a 2D surface is used despite the electrode having
a 3D structure.
Acid treatment is typically used to increase the surface area and polarity
of carbon-based materials by damaging the carbon fibers (Zhang et al.,
2009). However, physically pitted and fragmented carbon fibers become
extremely fragile and have been shown to lose 28.4 % of their weight
when treated with 70 % nitric acid for 240 min (Wu et al., 1995). Hydro-
philic conductive polymers are a sustainable alternative because they
enable super-hydrophilicity without any damage or weight loss of the car-
bon fibers. This enables a rapid exchange of solution across carbon fibers
for active microbial growth even inside the anode. In addition, the electrical
conductivity of the polymer increases the conductivity of the treated carbon
fibers while acid treatment cannot enhance the conductivity beyond the
original level.
Most of the recent attempts to modify carbon-based anodes have used
conductive polymers to enhance the electron transfer efficiency. For exam-
ple, Cui et al. (2015) electropolymerized polyaniline (PANI) on the surface
of large porous graphite felt (GF) followed by the electrophoretic deposi-
tion of CNTs to improve the performance of microbial fuel cells (MFCs).
MFCs are similar to MECs within the framework of bioelectrochemical
systems except that the former outputs electricity while the latter outputs
hydrogen (Sunghoon et al., 2021). With the CNT-125/PANI/GF anode,
they realized a maximum power density (308 mW/m2) that was 6.2- and
3.9-fold higher than those with the original GF (49 mW/m2) and PANI/GF
(80 mW/m2) anodes, respectively. Feng et al. (2010) modified an anode
by using polypyrrole/anthraquinone-2,6-disulphonic disodium salt (PPy/
AQDS) and demonstrated a dual-chamber MFC with a maximum power den-
sity of 1303 mW/m2, which was 13-fold higher than that with an unmodi-
fied anode. Li et al. (2014) prepared PANI/multiwalled carbon nanotube
(MWCNT) and PPy/MWCNT composite anodes and revealed that the
PPy/MWCNT anode (34.95 Ω) exhibited better electrical conductivity
than the PANI/MWCNT (87.93 Ω) and unmodified (226.20 Ω) anodes. Fur-
thermore, some researchers fabricated magnetic and conductive anodes by
encapsulating uniformly dispersed SrFe12O19 nanoparticles into poly(vinyl
alcohol-co-ethylene) nanofibers to form a 3D PPy network and realized a
maximum power density of 3317 mW/m2, which was significantly greater
than that of the control anode (2471 mW/m2) (Li et al., 2019).
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Science of the Total Environment 856 (2023) 159105
Thus, most studies on polymer-modified anodes mainly focused on in-
creasing electron transfer efficiency by enhancing conductivity. However,
few studies have considered improving the hydrophilicity. The uniform
formation of microbial communities in 3D anodes is crucial for the rapid
decomposition of organic compounds and the promotion of extracellular elec-
tron transfer. In this context, poly(3, 4-ethylenedioxythiophene) (PEDOT)
and polystyrene sulfonate (PSS) can be combined into a dual-functional ma-
terial as a unique potential candidate for improving the MEC performance.
PEDOT is a positively charged ionic polymer that is highly conductive but
is naturally hydrophobic. However, when it is electropolymerized with PSS,
which is a negatively charged ionic polymer with SO− 3 groups and thus hy-
drophilic, PSS surrounds the outside of PEDOT, so PEDOT:PSS is hydrophilic
(Lu et al., 2019). Furthermore, the strong interaction between PEDOT and
PSS can result in anodes with high stability and easy modification. Zajdel
et al. (2018) developed a bioanode covered with a multilayer conductive bac-
terial composite film using PEDOT:PSS that improved the MFC performance.
Despite its effectiveness, the enrichment of specific microbial cultures for
industrial-scale plants is technically challenging. PEDOT:PSS increases both
the hydrophilicity and electron transfer of carbon-based anodes and allows
for easy mass transfer within. The hydrophilicity of the anode is important be-
cause it accelerates the biofilm formation. The hydrophobic nature of CF can
increase the resistance of the interface between the solution and anode by
preventing surface wetting. Additionally, this hydrophobicity can induce
the capture of produced gases, which interferes with internal mass transfer.
Therefore, the hydrophilic surface caused by the SO− 3 groups of PEDOT:PSS
smoothens the flow of the internal solution, hence increasing the microorgan-
ism attachment possibility and facilitating the natural release of produced
gases. Fig. 1 shows the concept of functionalizing the surface of a CF anode
with PEDOT:PSS to improve the MEC performance.
In this study, a simple method was used to apply a highly conductive
and super-hydrophilic electrode to an MEC for enhanced H2 production.
The optimal electropolymerization conditions of the PEDOT:PSS anode
were determined based on the physical and chemical characteristics. The
long-term effects of PEDOT:PSS application on the MEC performance
were investigated over a period of >4 months. The biofilm formation and
microbial dynamics in 3D-structure anodes were profiled in depth to pro-
vide insight into the relationship between the microbial community struc-
ture and MEC performance. The findings of this study may contribute to
the potential application of PEDOT:PSS in biocatalytic materials, particu-
larly for bioelectrochemical systems.
2. Materials and methods
2.1. Preparation of functionalized anodes
Anodes were manufactured in accordance with the design of the MEC
reactor used in a previous study (Park et al., 2017, 2021). Rinsed CF (5 ×
5 cm2, thickness 5 mm) was attached to stainless steel as an electron collec-
tor, which was used as a control anode for the experiment (Fig. 2).
Electropolymerization solutions (EPSs) were prepared by mixing 3,4-
ethylenedioxythiophene (EDOT, Sigma–Aldrich), poly(sodium 4-styrene
sulfonate) (NaPSS, Sigma–Aldrich), lithium chloride (LiClO4, Samchun
Chemicals), and deionized (DI) water at five concentrations. LiClO4 was
used as the electrolyte in a mixed solution. Concentration 8 (CONC.8)
was obtained by referring to Benoudjit et al. (2018), who successfully
electropolymerized PEDOT:PSS on electrochemical sensors, and four addi-
tional concentrations (i.e., CONC.1, 2, 4, and 16) were added to identify the
effects of different PEDOT:PSS concentrations (Table 1).
Electropolymerization was conducted to functionalize the CF surface by
using a three-electrode system comprising a control anode as the working
electrode, a graphite counter electrode, and an Ag/AgCl reference electrode
(Fig. S1a). A galvanostat (ZIVE SP1, WonATech, Korea) was operated in
galvanic mode at 100 mA for 12 h with each EPS concentration to fabricate
the PEDOT:PSS-functionalized CFs (PEDOT:PSS@CF). The three-electrode
cell was composed of quartz instead of the commonly used acrylic material
to prevent undesired reactions such as deformation of the surface into deep
S.-G. Park et al. Science of the Total Environment 856 (2023) 159105

Fig. 1. Conceptual diagram of the advantage from functionalizing the MEC anode surface with PEDOT:PSS.

white during electropolymerization. The quartz reactor allowed identical
conditions to be maintained during each electropolymerization, such as
the distances between the three electrodes. Finally, the anodes were heat-
treated at 110 °C for 12 h to minimize the release of PEDOT:PSS into the
MEC anolyte. The prepared anodes were then equipped in the anode cham-
ber of the H2-producing MEC for performance evaluation (Fig. S1b).
2.2. Characterization of functionalized anodes
The surface morphology and elemental composition of the anodes were
characterized by field emission scanning electron microscopy (FE-SEM,
TESCAN-MIRA 3 LMU, Czech) coupled with energy-dispersive X-ray
spectroscopy (EDX) to confirm that PEDOT:PSS covered the CF. The ele-
mental structure and functional groups of the anodes were analyzed by
using Raman spectroscopy (JASCO, NRS-5100, Japan) with laser excitation
at 532 nm (range of 700–4200 cm−1) and Fourier-transform infrared spec-
troscopy (FT-IR; Spectrum GX, Perkin Elmer, USA) in attenuated total re-
flectance mode (650–4000 cm−1).
The water contact angle (WCA) test (DSA25, Kruss, Germany) was con-
ducted to evaluate the effect of different PEDOT:PSS concentrations on the
hydrophilicity of the anode. During the WCA analysis, 6 μL of DI water was
dropped on the surface and inside of the anodes, and the angle between the
droplet and sample was measured at 21° ± 1°. The immediate water ab-
sorption performance was filmed and can be seen in Video S1.

Fig. 2. Preparation of control and PEDOT:PSS anodes.

3
S.-G. Park et al.
Table 1
EPS concentrations for PEDOT:PSS synthesis.
EPS concentrations DI water (mL) LiClO4 (g) EDOT (mL) NaPSS (g)
CONC.1 20 0.0112 0.06 0.1
CONC.2 20 0.0112 0.12 0.2
CONC.4 20 0.0112 0.25 0.4
CONC.8 20 0.0112 0.5 0.8
CONC.16 20 0.0112 1 1.6
The electrocatalytic activity of the samples was examined by using cy-
clic voltammetry (CV) and electrochemical impedance spectroscopy (EIS;
ZIVE SP1, WonATech, South Korea) on a three-electrode cell (Fig. S1a).
The prepared anodes were vertically sliced into three layers to obtain the
inside (first and third layers) and outside (second layer) layers of the elec-
trode. Additionally, the electrochemical behaviors of the first and second
layers were investigated. Each sample was connected to ZIVE SP1 to act
as the working electrode, and Pt wire and Ag/AgCl were used as the counter
and reference electrodes, respectively. CV tests were conducted at −1 to
1 V at 50 mV/s in a phosphate buffer solution (pH 7) under anoxic condi-
tions, which were achieved by nitrogen gas purging. EIS measurements
were performed under the same conditions as CV, but the frequency
range, potential, and amplitude were fixed at 0.01–100 kHz, 0 V (vs.
open-circuit potentiometry), and 10 mV, respectively.
2.3. Microbial electrolysis cell setup and operation
H2-producing MECs were operated with two different anodes (CF and
PEDOT:PSS@CF) to compare their performances (Fig. S1c). MEC opera-
tions with each anode were conducted in triplicate: CF-MEC1, CF-MEC2,
and CF-MEC3 for CF and PEDOT:PSS-MEC1, PEDOT:PSS-MEC2, and
PEDOT:PSS-MEC3 for PEDOT:PSS@CF. The MEC reactors comprised an-
odic and cathodic chambers (200 mL; working volume = 160 mL in each
chamber) separated by a Nafion-211 membrane (5 × 5 cm2, DuPont,
USA). Identical Pt-coated perforated Ti plates (0.5 mg Pt/cm2) were used
as cathodes. The anodic chamber was filled with a mixture of nutrient min-
eral buffer solution and anaerobic digestion sludge (Soo-young Wastewater
Treatment Plant, South Korea) to inoculate microorganisms on the anode
surface while the cathodic chamber was filled with phosphate buffer
(electrolyte, pH 7). The substrate (0.5 mL of 500 mM acetic acid) was
injected into the anodic chamber for every batch after purging with nitro-
gen gas. In total, 30 batches were tested for 4 months. To supply an energy
deficit, an external voltage of 0.5 V was applied to the MEC reactors by
using a DC power supply (OPM\\303D, ODA, South Korea). A multimeter
(Keithley 2700, USA) was used to monitor the voltage and calculate the cur-
rent through the external wire. All experiments were performed at 30 °C
while using an incubator with a magnetic stirrer (350 rpm).
2.4. Electron transfer and gas production evaluation
The substrate-to-electron conversion efficiency can be expressed
in terms of the Coulombic efficiency (CE) from the measured current
flowing from the anode to the cathode through an external wire using a
fixed resistance:
Rt
M 0 I dt
CE ð%Þ ¼ (1)
FbV an ΔCOD
where M is the molecular weight of O2 (32 g/mol), F is Faraday's constant
(96,485 C/mol), b is the number of moles of electrons produced per mole
of substrate (4), and Van is the volume of the reactor. The current (I) flowed
through a fixed resistance (10 Ω) on the cathode side and was automatically
monitored by a Keithley multimeter. The chemical oxygen demand (COD)
was measured before and after each batch by using an HS COD-LR kit
(Humas, HS 2300 Plus, Korea).
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Science of the Total Environment 856 (2023) 159105
The gas production rate was automatically logged in real-time by using
a respirometer (BRS-110, EETech, South Korea), and its composition was
analyzed by gas chromatography (Clarus 580, Perkin Elmer, USA)
equipped with a thermal conductivity detector and Elite-PLOT Q Column
(6 f. × 1/8″, SS) with nitrogen gas (99.999 %) as the carrier gas. The gas
for gas chromatography analysis was sampled by using a gastight syringe
(2.5 mL, Hamilton SampleLock Syringe # 1002, USA) from the headspace
of each chamber. The amount of H2 production was calculated based on
the gas production rate and gas composition results. The overall H2 effi-
ciency (OHE) from the substrate to H2 was calculated as the ratio of the ac-
tual amount of H2 produced and theoretical amount of H2 in the substrate.
To compare the characteristics of H2 production in the MECs, the maximum
gas production rate, saturation constant, and reaction immediacy after sub-
strate injection were determined by using the modified Gompertz model,
which Ware and Power applied to the degradation of simple organic sub-
strates such as acetate (Ware and Power, 2017):
  
Rmax  e
M ¼ Pmax  exp  exp  ðλ  tÞ þ 1 (2)
Pmax
where M is the gas production (mL), Pmax is the gas production constant,
Rmax is the maximum production rate constant, λ is the lag-phase time
(h), and t is the operating time (h). The software program MATLAB
R2015a was used to fit the experimental gas production data to the model-
ing algorithm.
2.5. Microbial attachment and community analysis according to anode depth
2.5.1. Sample preparation
Biofilm formation on the anodes of all experimental reactors was ob-
served after four months of MEC operation (Fig. S2). To identify the micro-
bial growth distribution and differences in microbial diversity depending
on the depth of the anode, a 1 × 1 cm2 piece was obtained from the anodes
and sliced into five layers, each with a thickness of 1 mm (Fig. S3). FE-SEM,
next generation sequencing (NGS), and quantitative polymerase chain reac-
tion (qPCR) were conducted as follows.
2.5.2. Field emission scanning electron microscopy
To observe the shape of microbes on both the surface and inside of the
anode using FE-SEM, the first and third anode layers were carefully
dehydrated, which was followed by the fixation process. The samples
were soaked in 2.5 % glutaraldehyde (primary fix, Sigma–Aldrich, USA)
and 1 % osmium tetroxide (secondary fix, Sigma–Aldrich, USA) for 1 h
and were washed three times with DI water after each step. Then, the dehy-
dration process was performed by using increasing concentrations (50 %,
70 %, 90 %, 95 %, and twice 100 %) of ethanol, and the solution was
changed twice to 99.9 % hexamethyldisilazane (Sigma–Aldrich, USA) for
10 min per step. Before observation, the samples were dried overnight at
25 °C ± 1 °C, and then the sample surfaces were coated with Pt by ion
sputtering (E-1030, Hitachi Ltd., Japan).
2.5.3. 16S rRNA gene sequencing and bacterial taxonomic assignment
The five layers of each anode were sonicated with distilled water and
centrifuged to obtain cell pellets. DNA was extracted by using the AccuPrep
Genomic DNA Extraction Kit (Bioneer, South Korea). To normalize the DNA
extraction yield, a self-ligated pGEM-T easy vector (3015 base pairs,
Promega, USA) was added during the cell lysis step. In total, 108 copies
of the internal standard were co-extracted with sample DNA and purified
in 100 μL of elution buffer. The amount of eluted internal standard was
quantified by real-time PCR.
Amplicon sequencing was performed according to the manufacturer's
instructions (Illumina, CA, USA). Oligomers containing the specific se-
quence of the V3–4 region of bacterial 16S rDNA (518F: 5′-CCAGCAGCC
GCGGTAATACG-3′ and 805R: 5′-GACTACCAGGGTATCTAATCC-3′)
(Lane, 1991; Yu et al., 2005) and the Illumina overhang adapter sequence
were used as amplicon primers. Sample DNA was PCR-amplified through
S.-G. Park et al.
28 cycles at an annealing temperature of 58 °C. The purified amplicons were
PCR-indexed by using the Illumina Nextera XT index kit. A TaqMan DNA po-
lymerase kit (Solgent, South Korea) was used for library construction. The pu-
rified library was quantified, pooled, and combined with PhiX control
(Illumina). The amplicon sequencing was paired-end (150 bp × 2) sequenced
by using the iSeq 100 platform. Sequence reads were first merged and then
processed to remove short-or low-quality sequences and potential chimeras.
More than 43,000 filtered reads (average: 76,000 reads) were obtained
from each sample. Operational taxonomic units (OTUs) were defined at a
97 % sequence identity cutoff by using the VSEARCH algorithm (Rognes
et al., 2016). Taxonomic assignment was conducted by using the online
RDP classifier (https://rdp.cme.msu.edu/classifier/).
2.5.4. Real-time quantitative polymerase chain reaction and quantification of
bacterial population
To measure the population size of bacteria, real-time qPCR was
performed by using the StepOnePlus Real-Time PCR system (Applied
Fig. 3. FE-SEM and EDX results for the surfaces of (a) CF and (b) PEDOT:PSS@CF with heat treatment. (c) Chemical structure of PEDOT:PSS. (d) Raman spectra and (e) FT-IR
results for CF and PEDOT:PSS@CF, where the rhombuses and stars indicate the thiophene ring and SO−
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Science of the Total Environment 856 (2023) 159105
Biosystems, USA). The qPCR assay was conducted by using the PowerUp
SYBR Green Master Mix (Applied Biosystems) with the bacteria-specific
primers and probe set (338F; 5′-ACTCCTACGGGAGGCAG-3′, 516F; 5′-
TGCCAGCAGCCGCGGTAATAC-3′, and 805R; 5′-GACTACCAGGGTATCT
AATCC-3′) (Shin et al., 2010). The cycling conditions were as follows: ini-
tial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation
at 95 °C for 6 s and primer annealing and elongation at 60 °C for 45 s.
The DNA standard was previously constructed as a purified plasmid derived
from the bacterial species Escherichia coli k12 (Shin et al., 2010). To normal-
ize the DNA extraction yield, the internal standard (pGEM-T easy plasmid)
was amplified with T7 (5′-TAATACGACTCACTATAGGGCGA-3′) and SP6
(5′-ATTTAGGTGACACTATAGAATACTCAAGCT-3′) primers by using the
PowerUp SYBR Green Master Mix. The cycling conditions were as follows:
initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation
at 95 °C for 5 s and primer annealing and elongation at 60 °C for 30 s. The
DNA extraction yield (%) was determined by dividing the plasmid copy
number in the extracted DNA by the input copy number of the plasmid.
3 group, respectively.
S.-G. Park et al.
3. Results and discussion
3.1. Confirmation of the functionalized anode surface
The surface morphology of the prepared anodes was confirmed by FE-
SEM (Fig. 3a and b). The CF anode had a clean surface, whereas the surface
of the PEDOT:PSS@CF anodes was overlaid by a thin layer with a rough
texture. The heat treatment, which was intended to increase the durability
of the PEDOT:PSS coating, also seemed to increase the surface roughness.
The functionalization of the CF surface with PEDOT:PSS was confirmed
by EDX. The original CF comprised 100 % elemental C, whereas the surface
of the PEDOT:PSS@CF showed a lower C content of 77.05 wt% with S and
O contents of 17.09 and 5.86 wt%, respectively. This supports the presence
of PEDOT:PSS, whose structure comprises elemental S and O from PEDOT
and the SO− 3 groups of PSS on its surface (Fig. 3c).
The chemical structure of the prepared anodes was examined by using
Raman spectroscopy (Fig. 3d). The presence of the precursors EDOT and
NaPSS was observed in the Raman peaks of PEDOT:PSS@CF, which indi-
cates that the CF surface was successfully functionalized by PEDOT:PSS.
The Raman spectrum of CF showed only two peaks (D band: 1358 cm−1
and G band: 1589 cm−1), which are typical of carbon-based materials.
The peaks at 1003, 1269, 1361, and 1436 cm−1 in PEDOT:PSS@CF repre-
sent Cβ–Cβ′ asymmetric, Cα–Cα′ inter-ring stretching, Cβ–Cβ′ stretching, and
Cα = Cβ symmetric stretching vibrations, respectively (Chang et al., 2014;
Xiong et al., 2013). The peak at 1507 cm−1 corresponds to the Cα = Cβ
asymmetric elongation vibration associated with the thiophene ring
in the PEDOT chain (Lindfors et al., 2014). In addition, a peak at
1565 cm−1 was observed indicating an obvious quinoid structure (Cα–Cβ)
(Funda et al., 2016). These peaks have also been identified in commercial
PEDOT:PSS polymers, which indicates that PEDOT:PSS thoroughly coated
the CF surface.
FT-IR analysis was used to evaluate the surface compounds of the pre-
pared anodes with IR absorption properties that could not be identified
by Raman analysis (Fig. 3e). PEDOT:PSS@CF demonstrated the asymmetric
stretching modes of Cα = Cβ, symmetric Cα = Cβ, and asymmetric Cα–Cβ
indicating the quinoid structure of the thiophene ring by peaks at 1716,
1654, and 1427 cm−1, respectively (Han et al., 2014; Xu et al., 2017).
The bands at 1334 cm−1 correspond to ring strains in the stretching
mode of ethylenedioxy (C–O–C) group (Susanti et al., 2018). A band ap-
peared at approximately 1137 cm−1 because of the stretching vibrations
of the SO− 3 groups of PSS (Xu et al., 2017). The observed peak at
998 cm−1 (C\\S) can be attributed to the stretching vibrations of the thio-
phene ring (Olivares et al., 2019). The peak at 2875 cm−1 indicates a C\\H
Fig. 4. (a) WCA of prepared samples according to the PEDOT:PSS concentrations given in Table 1. Inside and outside WCAs of (b) CF and (c) PEDOT:PSS@CF.
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Science of the Total Environment 856 (2023) 159105
stretching bend (Mengistie et al., 2013). Therefore, these results indicate
that the CF surface was successfully functionalized by PEDOT:PSS.
3.2. Characterization of the anodes
3.2.1. Wettability
Fig. 4a shows the wettability, represented by the WCA, of the samples
according to the PEDOT:PSS concentration. Pristine CF had the largest
WCA at 136.5° ± 12.3°, and the hydrophilicity increased with the
PEDOT:PSS concentration. Super-hydrophilicity (i.e., WCA = 0°) was ob-
tained at PEDOT:PSS@CF_CONC.8. For further investigation, the prepared
anodes were cut in half, and the wettability inside and outside the anodes
was measured (Fig. 4b and c). Pristine CF was hydrophobic both outside
(136.5° ± 12.3°) and inside (143.2° ± 4.3°). PEDOT:PSS@CF_CONC.8
was super-hydrophilic both outside (0°) and inside (0°), which indicates
that the coating was uniform across the entire anode (Video S1).
Sharma et al. (2019a) studied the physicochemical properties of carbon-
based materials. These materials showed >100° of WCA regardless of sur-
face area and porosity, causing low mass transfer into the electrode. How-
ever, the superhydrophilic property allowed the solution to penetrate
deeply into the electrode and completely utilize the 3D porous structure
of CF. This should increase the mass transfer, electron transfer efficiency,
and biocompatibility of the anode. Based on the previous results, any fur-
ther discussion of the PEDOT:PSS@CF results is referring to PEDOT:PSS@
CF_CONC.8.
Guselnikova et al. (2017) also performed wettability studies using
PEDOT:PSS. They observed that pristine PEDOT:PSS films exhibited high
hydrophilicity owing to the presence of polar groups in the polymer
blend. Moreover, when PEDOT:PSS was modified by different substituents
(NO2, NH2, and CF3), the WCA increased because the hydrophilic SO− 3
groups turned into nonpolar esters. This implies that the SO− 3 groups of
PSS clearly contribute to the hydrophilicity of PEDOT:PSS. Lu et al.
(2019) developed pure PEDOT:PSS hydrogels that are highly desirable for
bioelectronic applications. They noted that a hydrophobic PEDOT-rich
core covered by a hydrophilic PSS-rich shell can improve the intercon-
nected networks of PEDOT:PSS due to the water-swelling characteristics
of the PSS-rich domain, which agrees with the results of the present
study. Biessmann et al. (2018) investigated the swelling behavior of
PEDOT:PSS electrodes under humid conditions and observed the highest
swelling ratio and water content in pristine PEDOT:PSS thin films. They
attempted to decrease the swelling of PEDOT:PSS by treatment with
Zonyl and post-treatment with ethylene glycol to stabilize the PEDOT:PSS
thin films. In the present study, however, the swelling of PEDOT:PSS was
S.-G. Park et al.
used to allow the solution to penetrate the anode, which increased the mass
transfer and fully exploited the 3D structure.
3.2.2. Electrochemical properties
The cyclic voltammograms showed mixed capacitive behavior along the
tested potential for both CF and PEDOT:PSS@CF (Fig. 5a). The current in-
creased positively with the charging forward scan, followed by a similar
smooth increase in negative current during the reverse scan. In both for-
ward and reverse scans, the change in current showed no peaks or a sudden
onset of rapid current increase. Therefore, the response was inferred to ex-
hibit capacitive behavior. Electric double-layer capacitance (EDLC) domi-
nated the response, whereas pseudocapacitance was closer to the
maximum and minimum in the tested potential window (−1 and 1 V).
The noticeable deviation from the rectangular response of the EDLC can
Fig. 5. (a) CV and (b) EIS results of CF and PEDOT:PSS@CF; (c) magnified view of
the red box in (b).
7
Science of the Total Environment 856 (2023) 159105
be attributed to the high scan rate (50 mV/s), which exceeded the charging
speed of the electrochemical capacitor, as well as the pore resistance for
mass transfer (Lee et al., 2015). Neither electrode showed any irreversible
or faradic response, which indicated their inactivity in phosphate buffer
and consequently their potential to promote stable and direct contact for in-
terspecies electron transfer. However, the PEDOT:PSS@CF electrode exhib-
ited a considerably higher capacitance current (9.90 mA) than the CF
electrode (4.17 mA) at the same specific potential (0.37 V) owing to its in-
herent capacitive properties (Volkov et al., 2017). This increase in the redox
current indicates that the modified anode demonstrated better electrocata-
lytic behavior than the unmodified anode. The enhanced EDLC of the mod-
ified anode may also be a result of the larger electrochemically active
surface area and more accessible pores because of the higher hydrophilicity
(Barbieri et al., 2005; Chmiola et al., 2006; Hryniewicz et al., 2018). More-
over, the current densities were similar inside and outside PEDOT:PSS@CF.
Thus, PEDOT:PSS thoroughly coated even inside the CF, which increased
the active surface area and promoted electrical conductivity.
EIS was performed to obtain a better understanding of the electrochem-
ical behavior of the electrodes in the phosphate buffer. The EIS data before
and after electropolymerization showed a clear difference between CF and
PEDOT:PSS@CF. Overall, the Nyquist plot showed a decrease in the imped-
ance magnitude of the modified electrode (Fig. 5b and c). The impedance
spectrum showed three distinct regions depending on the dominant
limiting process in each frequency range: physical, electrochemical, or
mass transfer.
At high frequencies, the physical impedance had a close encounter with
the x-axis (i.e., real impedance), which represented the electrode resistance,
electrode–current collector contact resistance, and bulk electrolyte resis-
tance (Mei et al., 2018). The first minimum at the starting point for
PEDOT:PSS@CF ranged between 63 and 66 Ω, which is considerably
lower than that of CF, ranging between 829 and 895 Ω. The lower intercept
indicates that the introduction of PEDOT:PSS improved the conductivity of
the original electrode material. As the frequency decreased, the impedance
manifests as a distorted arc. The impedance varied between CF and PEDOT:
PSS. This represents a unique interaction at the electrode surface and is not
a result of a common feature such as electrolyte resistance. Consequently,
the phenomena were attributed to electrochemical interfacial surface inter-
actions between the electrolyte and electrode as well as the pore resistance
(Bastidas, 2007; Lee et al., 2015). Such impedance was high for CF because
the hydrophobic pores promoted the charging resistance at a lower capac-
itance (Choi, 2010). In contrast, the resistance of PEDOT:PSS@CF was
only ~16.2 Ω (Jones et al., 2020; Nuramdhani et al., 2018; Stöcker et al.,
2012). The distortion in the arc indicates non-ideal capacitance for the
EDLC (constant-phase element) (Chen et al., 2015; Mei et al., 2018;
Zajdel et al., 2018).
Finally, at lower frequencies, ions started to move a considerable dis-
tance demonstrating the effects of diffusion, as evident by the ascending
line following the arc. The cumulative diffusive behavior of both electrodes
can be better understood via anomalous diffusion owing to their complex
structures (Bisquert et al., 1999). For CF, higher frequencies prompted a
low-angle transition state best described by Warburg diffusion, which is
caused by a short diffusion length compared to the pore length at relatively
high AC signal frequencies (Bastidas, 2007; Lasia, 1995). Thereafter, ions
diffused further through the CF pore matrix against the 3D structure,
while the response further deviated from the Warburg ideal diffusion ac-
cording to the pore capacitance assuming a higher WCA (~78°).
Similarly, PEDOT:PSS exhibited complex behavior as a result of ion dif-
fusion through the CF pores because PSS opposed the charge transfer
through the PEDOT layer. The ionic diffusion through the conductive film
was slower than that through the CF pores as evidenced by the wider War-
burg diffusion response (Hernández-Labrado et al., 2011). The noticeable
diffusion effect proves the deposition and formation of PEDOT:PSS. There-
after, the accumulation of charges and ions in the electrode pores and con-
ductive film EDLC added a capacitive aspect to the diffusion at lower
frequencies by increasing the slope. This behavior is common in ion-
conductive polymers (Hernández-Labrado et al., 2011; Sharifi-Viand
S.-G. Park et al. Science of the Total Environment 856 (2023) 159105

Fig. 6. Performances of CF-MEC and PEDOT:PSS-MEC: (a) CE, (b) H2 production and OHE, and (c) gas composition (CH4 in anodic chamber and H2 in cathodic chamber) of
CF-MEC and PEDOT:PSS-MEC.

et al., 2012). The transition from Warburg to capacitive diffusion occurred (Hernández-Labrado et al., 2011; Sharma et al., 2019b). Meanwhile,
at similar frequencies both inside (0.4 Hz) and outside (0.8 Hz) PEDOT:PSS, CF_outside (~1.6 Hz) exhibited a higher difference in transition frequency
which implies similar film thicknesses, ion conductivities, and pore sizes compared to CF_inside (~0.5 Hz), which indicates differences in the pore

8
S.-G. Park et al.
Fig. 7. Morphology of bacteria attached to the outside (first layer) and inside (third layer) of (a) CF-MEC and (b) PEDOT:PSS-MEC.
structure and hydrophilicity (Sharma et al., 2019b). The overall diffusion
impedance of CF declined >10 decades upon the introduction of PEDOT:
PSS owing to the enhanced hydrophilicity.
3.3. Performance of the microbial electrolysis cell
In Fig. 6, PEDOT:PSS-MEC showed a significantly higher average CE
(41.89 % overall, 56.48 % for the last 10 cycles in the steady state) than
CF-MEC (18.25 % overall, 33.38 % for the last 10 cycles in the steady
state, p < 0.001; Table S1). In addition, PEDOT:PSS-MEC required 7 batches
to reach 10 % CE as opposed to 12 batches for CF-MEC, and PEDOT:PSS-
MEC achieved a maximum CE of 73.3 % as opposed to 38.2 % for CF-
MEC (Fig. 6a). From the MFC's perspective, these results indicate that the
9
Science of the Total Environment 856 (2023) 159105
power production can be increased as much as CE increased because the
amount of electrons flowing from the anode to the cathode on the external
wire determines the performance of the MFC (Chae et al., 2010; Kim et al.,
2008). To demonstrate reproducibility, the voltage measurements of all re-
actors are shown in Fig. S4. Because of the current flowing along the exter-
nal wire, OHE began exceeding 10 % from batch 7 for PEDOT:PSS-MEC and
batch 11 for CF-MEC, and the maximum OHE was 73.4 % for PEDOT:PSS-
MEC and 32.7 % for CF-MEC (Fig. 6b). All batch cycle data of H2 production
were averaged by using the modified Gompertz model for further compar-
ison (Fig. S5). The maximum H2 production constant (Pmax) was twice as
high with PEDOT:PSS-MEC compared to with CF-MEC, and the H2 produc-
tion rate constant (Rmax) also increased from 0.68 for CF-MEC to 1.57
for PEDOT:PSS-MEC. The lag-phase time constant (λ) was 6.68th batch
S.-G. Park et al.
Fig. 8. (a) NMDS of bacterial communities with centroids and polygons (n = 3) (green: CF-MEC, red: PEDOT:PSS-MEC). (b) Rarefied richness of anode layers with the
annotation indicating statistical differences based on Duncan's test (n = 3) (C: CF-MEC, P: PEDOT:PSS-MEC). (c) 16S rRNA copy number of bacterial phyla present in
each anode layer.
(≒27 days) for CF-MEC and 3.25th batch (≒13 days) for PEDOT:PSS-MEC,
which indicates that the latter had a faster response to the substrate. More-
over, Pasupuleti et al. (2015) spent 70 days obtaining a maximum current
density from the enriched bioanode using CF. Similarly, in this study, CF-
MEC showed Pmax at the 16th batch (≒64 days), implying that progressively
enriched EAB produce stable performance. PEDOT:PSS-MEC required more
time to get Pmax (18th batch, ≒72 days). However, its Rmax was faster than
CF-MEC, resulting in a continuously increasing absolute amount of hydro-
gen production (mL). This implies that more EAB were enriched in the
PEDOT:PSS-MEC than in the CF-MEC (Jang et al., 2013). The PEDOT:
PSS-MEC began surpassing Pmax of CF-MEC from the 10th batch (≒40
days). These results indicate that the PEDOT:PSS coating reduced the initial
startup time and increased the amount of H2 available for maximum pro-
duction. The additional cost of coating the basic CF with PEDOT:PSS
is around 14 USD/12.5 cm3CF when calculated based on CONC.8
(EDOT: 5.8, NaPSS: 8.1, LiClO4: 0.1, and the power needed to conduct
electropolymerization). As the long-term effect of the PEDOT:PSS electrode
has been verified, the initial coating cost can be covered by a continuous hy-
drogen production.
Introducing the PEDOT:PSS coating also improved the maximum purity
of H2 in the cathodic chamber from 71.95 % to 82.56 % (Fig. 6c). In partic-
ular, the increase in H2 purity in the cathodic chamber reduced the CH4
composition in the anodic chamber because the CH4 and H2 production
processes competed for electrons, as demonstrated previously (Park et al.,
2019). In other words, the amount of EAB that could support H2 production
increased.
10
Science of the Total Environment 856 (2023) 159105
After batch 30 (i.e., the end of MEC operation), the biofilms formed
differently in each anode according to their biocompatibility (Fig. 7). CF
showed a distinct boundary between the inside and surface, which
implies that the anolyte did not sufficiently penetrate the anode. In
contrast, a well-developed biofilm was observed in PEDOT:PSS@CF
from the surface to the most internal area, which implies that
PEDOT:PSS increased the internal mass transfer within the anode. Fur-
thermore, a large number of rod-shaped potential EAB were observed
inside PEDOT:PSS@CF (Feng et al., 2014). They formed a rigid connec-
tion using their pilus to enable active electron transfer (Kumar et al.,
2016).
3.4. Microbial dynamics across the anode layers
Fig. 8a presents the bacterial community dynamics in terms of nonmetric
multidimensional scaling (NMDS) ordination. Each polygon and its centroid
are presented in triplicate. The CF-MEC community shifted greatly toward
the center of the anode (i.e., layer 3), whereas the outer layers (1 and
5) were close to each other, which indicate similar bacterial community
compositions. In contrast, PEDOT:PSS-MEC showed no significant difference
among the layers, although the outermost (layer 1) was slightly different
from the rest. Fig. 8b presents the microbial diversity within each sample
in terms of the rarefied richness. All layers of PEDOT:PSS-MEC showed no
significant difference in rarefied richness, and at least one annotation was
identical to another. In contrast, the innermost layer of CF-MEC (C3) showed
significantly less richness than those of C1, C2, and C5, which indicates poor
S.-G. Park et al.
microbial growth and distribution at the center of the anode. These results
further imply that the PEDOT:PSS treatment made the entire 3D structure
of CF a suitable environment for microbial growth. Fig. 8c presents the pop-
ulation sizes of the bacterial phyla. Overall, PEDOT:PSS-MEC had a larger
bacterial population than CF-MEC. For CF-MEC, the population size signifi-
cantly decreased toward the center of the anode. In contrast, PEDOT:PSS-
MEC showed no significant differences in population size among layers
except for layer 1, which had a significantly larger population owing to
the enhanced biocompatibility resulting from the positive effects of the
overlying PEDOT:PSS. In particular, CF-MEC showed a 98 % reduction
in the population size for layer 3 compared with layer 1, whereas
PEDOT:PSS-MEC showed an average reduction of only 63 %.
Proteobacteria was the most dominant bacterial phylum in all samples.
The largest contributor to the phylum Proteobacteria was the genus
Dechloromonas, which was assigned to OTU 7 (Table S2). Dechloromonas,
with Geobacter, is a known EAB with both nitrifying and electrogenic func-
tions, and it is commonly observed in the anodes of bioelectrochemical
systems such as MECs and MFCs (Hao et al., 2020; Li et al., 2021). Thus, the
increase in the Proteobacteria population, including Dechloromonas, most likely
enhanced the electron transfer and consequently improved H2 production.
3.5. Effect of bifunctional properties on the anode in bioelectrochemical systems
Compared with the control anode, the PEDOT:PSS-CF anode clearly
showed a heavier microbial load on both the surface and internal layers
(Fig. 7). Additionally, the total copy number of the bacterial population, es-
pecially that of EAB including Dechloromonas, was significantly higher for
the PEDOT:PSS-CF anode (Fig. 8c). According to these results, surface char-
acteristics, such as roughness and hydrophilicity, affect the formation of
biofilms in previous studies. Rough surfaces provide suitable conditions
for biofilm formation compared to smooth surfaces (Zhang et al., 2012).
As confirmed in Section 3.1, PEDOT:PSS@CF has a rougher surface than
CF. Moreover, as discussed in Section 3.2.1, the superhydrophilicity of
PEDOT:PSS boosted the internal mass transfer of the substrate and inocu-
lum, resulting in a well-established microbial community throughout the
3D structure of the anode. The increased microbial population further con-
tributed to H2 production by transferring more electrons. Microbial diver-
sity was more stable and greater across the layers with PEDOT:PSS, while
significantly lower diversity and more drastic microbial shifts were ob-
served in the control anode (Fig. 8a and b). Here, the higher EAB population
and stable microbial diversity across the PEDOT:PSS-CF anode layers likely
contributed to the reduced startup period in PEDOT:PSS-MEC. The electro-
chemical experiments (Section 3.2.2) also demonstrated that enhanced
electrical conductivity and reduced internal resistance were important fac-
tors in improving CE. These properties enabled the rapid transfer of elec-
trons generated by the densely formed EAB on the PEDOT:PSS@CF anode
to the cathode with minimal energy loss.
4. Conclusions
PEDOT:PSS successfully improved the CE of the anode; therefore, H2
yield was approximately two times higher than that of the original CF.
The major contributing factors were increased EAB population size
and density at the anode and enhanced electrical conductivity. The
superhydrophilicity of PEDOT:PSS allowed for the rapid exchange of the so-
lution across the carbon fibers, inducing a rich microbial distribution in the
anode. The FE-SEM results and NGS analysis confirmed the maximized oc-
cupancy of the EAB population in the 3D structural anode after the PEDOT:
PSS treatment. This first attempt to apply the bioelectrocatalytic material
(PEDOT:PSS) to modify the MEC anodes proved its potential applicability
in bioelectrochemical systems, including MFCs, microbial solar cells,
MEC-coupled anaerobic digestion, and dark fermentation. Additionally,
the importance of fully utilizing the anode for the superior performance
of MECs has been highlighted.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.159105.
11
Science of the Total Environment 856 (2023) 159105
CRediT authorship contribution statement
Sung-Gwan Park: Conceptualization, Methodology, Visualization,
Writing – original draft. Chaeyoung Rhee: Data curation, Software, Visual-
ization, Writing – original draft. Dipak A. Jadhav: Writing – original draft,
Writing – review & editing. Tasnim Eisa: Writing – original draft. Riyam
B. Al-Mayyahi: Writing – review & editing. Seung Gu Shin: Resources.
Mohammad Ali Abdelkareem: Supervision. Kyu-Jung Chae: Supervi-
sion, Writing – review & editing.
Data availability
Data will be made available on request.
Declaration of competing interest
The authors declare the following financial interests/personal relation-
ships which may be considered as potential competing interests: Kyu-Jung
Chae reports financial support was provided by National Research Founda-
tion of Korea (NRF). Sung-Gwan Park reports financial support was pro-
vided by National Research Foundation of Korea (NRF). Kyu-Jung Chae
reports financial support was provided by Rural Development Administra-
tion in Republic of Korea.
Acknowledgments
This work was supported by the National Research Foundation of Korea
(NRF) funded by the Korea government (No. 2019R1A2C1006356), Basic
Science Research Program through the NRF funded by the Ministry of
Education (2022R1I1A1A01072864) and “Cooperative Research Program
for Agriculture Science and Technology Development (Project No.
PJ016259022021)” funded by Rural Development Administration in
Republic of Korea.
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