Biotechnology Advances 73 (2024) 108369

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Relevance of extracellular electron uptake mechanisms for

electromethanogenesis applications
Paola Andrea Palacios a, *, Jo Philips a, Anders Bentien b, Michael Vedel Wegener Kofoed a
a
Department of Biological and Chemical Engineering, Aarhus University, Gustav Wieds Vej 10C, 8200 Aarhus, Denmark
b
Department of Biological and Chemical Engineering, Aarhus University, Aabogade 40, Aarhus N, 8200 Aarhus, Denmark

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

Keywords: Electromethanogenesis has emerged as a biological branch of Power-to-X technologies that implements meth­
Electromethanogenesis anogenic microorganisms, as an alternative to chemical Power-to-X, to convert electrical power from renewable
Extracellular electron uptake sources, and CO2 into methane. Unlike biomethanation processes where CO2 is converted via exogenously added
Power-to-X
hydrogen, electromethanogenesis occurs in a bioelectrochemical set-up that combines electrodes and microor­
Hydrogenotrophic methanogens
ganisms. Thereby, mixed, or pure methanogenic cultures catalyze the reduction of CO2 to methane via reducing
Microbial induced corrosion (MIC)
equivalents supplied by a cathode. Recent advances in electromethanogenesis have been driven by interdisci­
plinary research at the intersection of microbiology, electrochemistry, and engineering. Integrating the knowl­
edge acquired from these areas is essential to address the specific challenges presented by this relatively young
biotechnology, which include electron transfer limitations, low energy and product efficiencies, and reactor
design to enable upscaling. This review approaches electromethanogenesis from a multidisciplinary perspective,
putting emphasis on the extracellular electron uptake mechanisms that methanogens use to obtain energy from
cathodes, since understanding these mechanisms is key to optimize the electrochemical conditions for the
development of these systems. This work summarizes the direct and indirect extracellular electron uptake
mechanisms that have been elucidated to date in methanogens, along with the ones that remain unsolved. As the
study of microbial corrosion, a similar bioelectrochemical process with Fe0 as electron source, has contributed to
elucidate different mechanisms on how methanogens use solid electron donors, insights from both fields, bio­
corrosion and electromethanogenesis, are combined. Based on the repertoire of mechanisms and their potential
to convert CO2 to methane, we conclude that for future applications, electromethanogenesis should focus on the
indirect mechanism with H2 as intermediary. By summarizing and linking the general aspects and challenges of
this process, we hope that this review serves as a guide for researchers working on electromethanogenesis in
different areas of expertise to overcome the current limitations and continue with the optimization of this
promising interdisciplinary technology.

1. Introduction
Carbon dioxide (CO2) emissions released from the burning of fossil
fuels for energy acquisition account for up to three-quarters of global
greenhouse gas emissions (Ritchie and Roser, 2017). Due to their
negative impact, energy production strategies are currently under a
transition towards sustainable and clean energy sources to help mitigate
climate change. Moreover, the continuous growth of society's energy
demand versus the inevitable depletion of fossil fuels, i.e., estimated in
remaining years of global coal, oil, and natural gas (114, 50.7, 52.8,
respectively) (Ritchie and Roser, 2017), have helped to introduce
renewable energy sources such as wind-, solar-, hydropower, and bio­
methane as suitable alternatives to fossil-based energy production.
One crucial factor to consider in the integration of renewable sources
is energy conversion and storage, as it plays a significant role in main­
taining a robust and reliable modern power grid (Ould Amrouche et al.,
2016). In other words, renewable energy storage strategies are required to
efficiently manage the fluctuating energy production from wind turbines
and solar panels to ensure a stable supply of electricity. Yet, compact and
cheap devices like batteries for large-scale storage of electric energy are
still under development (Debabov, 2017). A suitable alternative that has
emerged to help overcome this challenge are the Power-to-X technologies

* Corresponding author.
E-mail address: paola@bce.au.dk (P.A. Palacios).

https://doi.org/10.1016/j.biotechadv.2024.108369
Received 1 September 2023; Received in revised form 21 February 2024; Accepted 24 April 2024
Available online 27 April 2024
0734-9750/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P.A. Palacios et al.
(P2X or PtX), in which renewable energy can be converted into chemicals
and green fuels like H2, methane, NH3, methanol, ethanol, CO/syngas/
formic acid, H2O2 (Daiyan et al., 2020; Gong et al., 2021; Sternberg and
Bardow, 2015). Power-to-X offers an attractive approach for converting
CO2 into carbon-neutral fuels that can be easily transported and stored,
such as methane, methanol, and ethanol, using CO2 as carbon-based
feedstock (Bushuyev et al., 2018; Gong et al., 2021). One of the most
important parameters concerning the development of PtX technologies
and CO2 utilization processes is the use of cost-effective catalysts with
long-term stability, selectivity, and good performance (Bushuyev et al.,
2018; Gong et al., 2021; Tufa et al., 2020).
The use of microorganisms or enzymes as catalysts offers an
environmental-friendly option that can be suitable for different PtX
approaches in bioelectrochemical systems (BES) (de Vasconcelos and
Lavoie, 2019; Logroño et al., 2023; Ramamurthy et al., 2021). In BES,
biocatalysts can be used in two different modus operandi: As microbial
fuel cells (MFC), where certain microbes generate electricity by using an
anode as electron acceptor for their microbial metabolism (exoelec­
trogens), or as biological PtX technology through microbial electrosyn­
thesis systems (MES), where certain microbes use the reductive electron
equivalents or electrons provided by a cathode for reduction processes
that lead to the synthesis of valuable products such as methane and
acetate (Logan et al., 2008; Logan and Rabaey, 2012).
Application of MES for electromethanogenesis enables the conver­
sion of electrical energy into methane via CO2 reduction using metha­
nogens as biocatalysts (Beese-Vasbender et al., 2015; Nevin et al., 2010;
Rabaey and Rozendal, 2010). Methanogens are a phylogenetically
diverse group of anaerobic microorganisms within the domain Archaea,
responsible for producing about 70% of the global atmospheric methane
(Lyu et al., 2018). However, methanogens are also used in different
technical applications to produce methane that can be used as a drop-in
fuel to directly substitute our current use of fossil-derived natural gas.
Biologically produced methane can therefore be used in applications
where its fossil counterpart is predominantly used today: as fuel for
combined heat and power production, in industrial processes, as trans­
port fuel, or stored and transported in the natural gas grid in geographic
regions where such infrastructure is present (de Vasconcelos and Lavoie,
2019; Götz et al., 2016; Pasini et al., 2019; Sun et al., 2022).
Biological methane production coupled with Power-to-gas technolo­
gies can be obtained in a one-step process, where the renewable electricity
gets supplied directly to the methanogens (electromethanogenesis), or in
a two-step process, where H2 is synthesized via water electrolysis (using
renewable electricity), and then injected into a downstream gas fermen­
tation reactor (Angelidaki et al., 2018; Aryal et al., 2021; Cheng et al.,
2009). While the two-step approach is already implemented at industrial
scale by companies such as Electrochaea, MicrobEnergy, and Krajete,
along with other large-scale projects, the one-step approach is still under
investigation at laboratory-scale. As an alternative to the two-step
approach, where electrochemical H2 production and biological conver­
sion is separated into two units, the one-step approach presents a com­
bined solution. Some of the challenges encountered in the two-step
process include low H2 gas-to-liquid mass transfer, buffer capacity, and
excessive H2 supply or partial pressure (Sun et al., 2022). On the other
hand, the one-step approach encounters major challenges, including low
current densities, intricate system design, and low methane production
rates, which need to be addressed before electromethanogenesis can be
scaled up (Blasco-Gómez et al., 2017; Zakaria and Dhar, 2019).
A crucial step to achieve this goal is to comprehend the interaction
between the biological and electrochemical components of the system.
This review aims to summarize how methanogens make this one-step
process work (either in pure culture or in mixed communities), it
highlights the importance of H2 as intermediary, and it gives insights
into the electrochemical concepts that should be optimized according to
the microbial extracellular electron uptake mechanisms of the inoculum.
Furthermore, future perspectives for the development of bio­
electrochemical systems for electromethanogenesis are also considered.
2
Biotechnology Advances 73 (2024) 108369
2. Electromethanogenesis lab-scale set-up
The electrochemical set-up implemented for electromethanogenesis
generally consists of an anode and a cathode placed in a two-chamber
system (e.g., H-cell reactor), separated by a membrane (Fig. 1). The
cathode and anode are coupled by the external circuit so electrical en­
ergy can be supplied using a power source. A negative potential is
applied to the cathode, so methanogens, present in pure culture, co-
culture, or a mixed community, can use electrons and protons, or/and
reduced intermediaries to convert CO2 to methane. Meanwhile, in the
anode, water-splitting occurs generating O2 and protons, so protons
diffuse from the anode to the cathode chamber through a membrane,
whereas the O2 remains at the anode side (Mayer et al., 2019; Nevin
et al., 2010; Rabaey and Rozendal, 2010) (Fig. 1).
The magnitude of the negative potential applied to the cathode in­
fluences whether H2 (important intermediate for electromethano­
genesis, as will be discussed below) will be available for
methanogenesis, or other autotrophic metabolisms in mixed commu­
nities (e.g., acetogenesis by acetogenic bacteria). The cathode potential
at which H2 formation becomes feasible, i.e. the H2 evolution onset
potential, depends on the H2 partial pressure and the pH at the cathode
surface (May et al., 2016; Philips, 2020; Vincent et al., 2007):
( )
R⋅T pH2
EH /H2 = EH+ /H2 − (1)
◦
⋅ln
2⋅F [H+ ]2
+
With R the ideal gas constant (8.314 10− 3 kJ ⋅ mol− 1 ⋅ K− 1), T the
temperature (K), F the Faraday constant (96.485 kJ ⋅ V− 1), pH2 the H2
partial pressure (atm), and [H+] is the proton concentration (M).
EH+ /H2 is the standard potential (pH 0, 1 atm H2) for H2 evolution, which
◦
is 0 V (i.e., the potential of the SHE). Quite often, H2 evolution is not
considered to happen at cathode potentials less negative than − 414 mV
vs SHE (Ragab et al., 2020), which is the H2 evolution onset potential at
standard conditions (pH 7, all concentrations 1 M and gasses 1 atm).
However, actual conditions in MES are most often far from standard
conditions. For instance, at a H2 partial pressure of 50 Pa (5 × 10− 4 atm),
the H2 evolution onset potential becomes – 0.316 V (pH 7), while at pH
5.5, the H2 evolution onset potential is − 0.325 V (1 atm H2). Conse­
quently, H2 evolution can thermodynamically be favorable at cathode
potentials less negative than − 0.4 V vs. SHE (Philips, 2020). Further­
more, the thermodynamic minimum for electrotrophic methanogens to
utilize extracellular electrons from cathodes for CO2 reduction (Eqs.(4))
is E = − 244 mV vs. SHE at pH = 7 (Blasco-Gómez et al., 2017; Cheng
et al., 2009; Villano et al., 2010).
Fig. 1. Example of an H-cell reactor implemented for methanogenesis via mi­
crobial electrosynthesis for preliminary lab-scale studies.
P.A. Palacios et al. Biotechnology Advances 73 (2024) 108369

3. Electrosynthesis processes by methanogenic cultures
Methanogens are phylogenetically diverse, and the current taxon­
omy classifies them into seven different orders: Methanobacteriales,
Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyr­
ales, Methanocellales, and Methanomassiliicoccales (Lyu and Liu, 2019).
So far, electron uptake via electrotrophy or via extracellular enzymes is a
rare and an exclusive trait described in only a few methanogenic strains
or species, as presented in section 5. Therefore, H2 generated electro­
chemically on the cathode is thought to be an essential mediator for the
microbial reduction of CO2 in electromethanogenesis (Blanchet et al.,
2015).
Different screening approaches have been employed to obtain the
most suitable methanogens for electromethanogenesis. Some studies
have focused on enrichment cultures, isolates, or commercially available
strains, depending on the objectives of the study. Methanogenic pure
cultures capable of electromethanogenesis have been key to elucidate
extracellular electron transfer (EET) mechanisms involved in this pro­
cess (Fig. 2). This knowledge has been crucial to understand the dif­
ference in electron uptake efficiencies of different methanogenic strains.
Until now, only a few pure methanogenic cultures have been tested in
MES as described in Table 1 (Logan et al., 2019; Mayer et al., 2019).
The different experimental conditions (e.g., cathodic potential,
temperature, presence of electron shuttles, electrolyte composition) and
data reporting methods, make a comparison between the methanogenic
strains from Table 1 difficult. Still, the highest current densities have
been reported for Methanococcus maripaludis strain KA1 (800 ± 115 mA
m− 2) and Mic1c10 (487 ± 27 mA m− 2) (Mayer et al., 2022). Interest­
ingly, only hydrogenotrophic species (Table 1) have been found capable
of performing electromethanogenesis, while obligate acetoclastic and
methylotrophic methanogens such as Methanosarcina horonobensis have
failed to perform electromethanogenesis, even though some of them are
capable of other electroactive processes such as DIET (as described in
detail in section 4) (Yee et al., 2019). These findings thus suggest that H2
acts as an important intermediate in the uptake of extracellular electrons
from the cathode by methanogens as further discussed in section 5. It is
important to note that pure culture studies often reported low current
densities, likely because many of these studies were testing for direct
EET mechanisms and thus used cathode potentials around − 400 mV to
exclude H2 evolution. Using more negative cathode potentials, however,
usually leads to higher current densities (Table 1).
4. Electrosynthesis by methanogenic mixed communities
Mixed cultures, enrichment cultures, or defined co-cultures and
consortia have also been implemented in electromethanogenesis pro­
cesses (Table 2). The diversity of microbial metabolisms and interactions
that occur among the different microbial groups will be influenced by
the species or strains present in the cathodic chamber. As an example, a
complex microbial metabolic network involving microbial-cathode in­
teractions is expected in electromethanogenesis systems when digestate
from biogas reactors, one of the most common sources of mixed cultures
among the studies, is utilized as biocatalyst. Similar to the findings with
pure methanogenic cultures, in the studies using methanogenic mixed
communities, higher CH4 production rates are being reported at more
negative cathodic potentials (Cheng et al., 2009; Deutzmann and Spor­
mann, 2017; Jiang et al., 2013; Ragab et al., 2020; Villano et al., 2010;
Zhen et al., 2015) (Table 2). However, differences in inocula complexity
and operational parameters hinder direct comparison of CH4 rates for
each type of inoculum evaluated in the studies.
Despite the complexity of mixed communities in electrosynthesis
systems, which hampers elucidation of extracellular electron transfer
mechanisms, their implementation facilitates the production of a higher
variety of electron donors by different microbial groups interacting with
the cathode for methanogenesis. In general, the conventional meth­
anogenic substrates include H2, CO2, formate, acetate, methanol, and
methylated amines. Thereby, methanogens can be identified as hydro­
genotrophic, acetoclastic, and methylotrophic based on their substrate
utilization (Lyu et al., 2018). Considering that methanol and methylated
amines are uncommon substrates in electromethanogenesis, this
pathway will not be discussed further. Hence, in electrosynthesis, CH4
production can take place via two different metabolic pathways:

Table 1
Methanogenic strains in electrosynthesis systems.
Methanogenic strain Type of Cathode Cathode Membrane Product rates Coulombic Current Reference
reactor potential (V) material efficiency (%) density

Methanococcus maripaludis S2 H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 8.81 (±0.51) 58.9 (±0.8) 219 (±21) (Mayer et al.,
DSM-14266 (37 ◦ C) rods mmol m− 2 d− 1 mA m − 2 2019)
Methanococcus maripaludis H-cell − 0.6 vs SHE graphite Nafion 117 formate (60 μg Hdr- 90 Unknown (Lienemann
MM1264 (purified Hdr-SC) rods SC): 0.12 μmol h− 1 et al., 2018)
(30 ◦ C) cm− 2
Methanococcus maripaludis H-cell − 0.7 vs SHE graphite Nafion 117 CH4: 1 μmol h− 1 70–80 Unknown (Lohner et al.,
MM901 rods 2014)
Methanococcus maripaludis KA1 H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 10.8 (±0.51) 35.1 (±11.8) 800 (±115) (Mayer et al.,
rods mmol m− 2 d− 1 mA m− 2 2022)
Methanococcus maripaludis H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 16.2 (±0.45) 61.3 (±1.5) 487 (±27) (Mayer et al.,
Mic1c10 rods mmol m− 2 d− 2 mA m− 2 2022)
Methanococcus vannielii H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 8.76 (±0.26) 55.4 (±0.5) 184 (±13) (Mayer et al.,
DSM-1224 (37 ◦ C) rods mmol m− 2 d− 1 mA m − 2 2019)
Methanobacterium congolense H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 4.12 (±0.36) 51.3 (±1.3) 140 (±20) (Mayer et al.,
DSM-7095 (37 ◦ C) rods mmol m− 2 d− 1 mA m − 2 2019)
1
Methanobacterium-like archaeon Two − 0.4 vs. SHE graphite n.a. salt CH4: 0.35 μmol d− 80 9.69 × 10− 5 (Beese-
strain IM1 (21 ◦ C) chambers* rods bridge cm− 2 mA Vasbender et al.,
m− 2 2015)
Methanoculleus submarinus H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 7.90 (±0.05) 45.3 (±10.9) 259 (±59) (Mayer et al.,
DSM-15122 (37 ◦ C) rods mmol m− 2 d− 1 mA m − 2 2019)
Methanolacinia petrolearia H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 7.49 (±0.25) 56.6 (±1) 189 (±4.9) (Mayer et al.,
DSM-11571 (37 ◦ C) rods mmol m− 2 d− 1 mA m − 2 2019)
Methanosarcina mazei Gö1 H-cell − 0.7 vs. SHE graphite Nafion 117 CH4: 0.14 (±0.01) n.a. n.a. (Mayer et al.,
DSM-3647 (37 ◦ C) rods mmol m− 2 d− 1 2019)
Methanosarcina barkeri DSM- H-cell − 0.45 vs. carbon Nafion 117 CH4: 104.0 ± 15.7 86 ± 28 120 (±35) (Rowe et al.,
804 (30 ◦ C) SHE cloth eeq total after 48 h eeq total 2019)
Methanosarcina barkeri DSM- H-cell − 0.4 vs. SHE graphite Nafion 117 CH4: 3.1 ± 0.34 Unknown ≈ 2.5 mA m (Yee et al., 2019)
− 2
800 (37 ◦ C) rods mM, after 28 days

(n.a. = not applicable; *Dual compartment connected via a salt bridge)

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P.A. Palacios et al. Biotechnology Advances 73 (2024) 108369

Table 2
Methanogenic mixed cultures and co-cultures performing methane electrosynthesis from cathodes.
Methanogens involved Bacteria involved Source Type of Cathode Cathode Observations Reference
reactor potential material
(V vs. SHE)

Methanococcus maripaludis Desulfopila corrodens Defined co- Two- − 0.4 V − Graphite CH4 rates at − 0.4 V: (Deutzmann
(MM901) (IS4) culture chamber 0.5 V 0.1–0.14 μmol cm− 2 h− 1. At – and
0.5 V: 0.6–0.9 μmol cm− 2 h− 1 Spormann,
2017)
Methanobacterium sp. (34×) Desulfovibrio and various Wastewater Two- − 0.77 V Carbon cloth CH4 production was higher at (Ragab et al.,
Methanobrevibacter sp. fermentative bacteria sludge chamber − 0.47 V − 0.77 V 2020)
Methanosarcina sp. enrichment
Methanobacterium sp. Desulfovibrionaceae Rice paddy Two- − 0.5 V Carbon cloth Highest rates of CH4 with (Bretschger
Methanobacterium bryantii Bacteroidetes and soil chamber multiple methanogenic et al., 2015)
Methanosarcina mazei Bacillaceae phylotypes
Hydrogenotrophic Not determined Anaerobic Two- From Carbon Higher CH4 production at more (Villano et al.,
methanogens sludge chamber − 0.65 to paper negative potentials: up to 0.32 2010)
− 0.9 V ± 0.01 mmol d− 1 at − 0.9 V (CE:
85 ± 2%)
Mainly Methanobacterium 62.8 ± 11.8% bacteria Anaerobic Two- From Carbon Considerable methane yield at (Zhen et al.,
(non-characterized) sludge chamber ** − 0.37 V to sticks − 0.67 V vs. SHE 2015)
− 0.77 V
Uncharacterized electrochemically active mixed culture Activated Two- − 0.62 V − Carbon-felt From − 0.62 to − 0.72 V CH4 and (Jiang et al.,
sludge chamber * 0.72 V H2 were produced. At lower 2013)
− 0.82 V potentials CH4, H2 and
CH3COOH. (CE: 60.9% at − 0.62
V; 95.8% at − 0.72 V; 56.7% at
− 0.82 V)
Predominated by Primarily Anaerobic Two- − 0.6 V Carbon The only reactors that were (Siegert et al.,
Methanobacterium except δ-Proteobacteria digester chamber brush, always stable in terms of CH4 2015)
for platinum cathodes that sludge graphite production and CE below 100%
were predominated by block with were those with platinum
Methanobrevibacter different cathodes
coatings
Methanoculleus thermophilus, Firmicutes, Thickened Cylindrical − 0.57 V 8 carbon Gas production increased with (Sasaki et al.,
Methanobacterium Bacteroidetes, sewage sludge (2-chamber) plates carbon fiber fabric on cathodes. 2013)
formicicum, Methanosarcina Synergistetes. A reactor for scale-up
thermophila,
Methanothermobacter
crinale
Methanobacterium palustre Sedimentibacter Solution from Single- − 0.47 V − Several Biofilm from cathode produced (Cheng et al.,
(most abundant), hongkongensis, an anode chamber 0.77 V carbon cloth CH4 at less than − 0.47 V but at 2009)
Methanoregula boonei, Clostridium sticklandii, chamber slower rates. (CE: 96%; − 0.77 V)
Methanospirillum hungatei Clostridium
aminobutyricum
Methanothermobacter sp (most Not determined Sludge from Two- − 0.57 V Carbon sheet Possibility of electron transfer (Sasaki et al.,
abundant) CH4 chamber − 0.37 V with AQDS from the cathodic 2011)
fermentor working electrode

Two-chamber reactors were separated by a Nafion 117 proton exchange membrane except for: * Cation exchange membrane (AMI7001), ** CSO monovalent-cation-
selective exchange membrane.

(i) CO2 reduction: in co-cultures or methanogenic mixed cultures in a process defined as

ʹ /
4H2 + CO2 →CH4 + 2H2 O ΔG0 = − 135 kJ mol
ʹ /
4HCOOH→3CO2 + CH4 + 2H2 O ΔG0 = − 130 kJ mol
8H+ + 8e− + CO2 →CH4 + 2H2 O
(ii) Acetoclastic pathway
' /
CH3 COOH→CH4 + CO2 ΔG0 = − 33kJ mol
0 cussed in section 5, but has been demonstrated in direct interspecies
ΔG ′ source: (Hedderich and Whitman, 2013; Liu and Whitman,
2008).
For instance, the CO2 reduction pathway (Eqs.(2)–(3)-(4)) is
considered to play a major role during electromethanogenesis, and thus
it determines the overall performance of the system (Blasco-Gómez
et al., 2017). Accordingly, H2 and formate as electron donors (Eqs.(2)–
(3)) (Liu and Whitman, 2008) can be electrochemically generated on the
cathode. However, they can also be obtained from syntrophic in­
teractions with H2 and formate-producing bacteria (Morris et al., 2013)
interspecies hydrogen/formic acid transfer (Bryant et al., 1967; Zhang
(2) et al., 2023), or hydrogen-, formate-mediated interspecies electron
transfer (MIET) (Storck et al., 2015). Opportunistic interactions with
autotrophic bacteria can also represent a H2 source for methanogens. In
(3)
this case, the microorganisms with lower H2 thresholds have the
(4) advantage (competitive microbial interaction) to scavenge any available
H2 produced or released from the lysis of H2-consuming bacteria (Pal­
acios et al., 2021). Alternatively, methanogens could in theory also use
electrons directly from the cathode to catalyze the reduction of CO2 via
Eq.4. However, such a direct mechanism has not been experimentally
(5)
characterized or elucidated for electromethanogenesis, as further dis­
electron transfer (DIET). For DIET, the syntrophic interaction has been
shown to involve different methanogenic genera (e.g., Methanosarcina,
Methanotrix, Methanobacterium strain YSL) that act as e- acceptors for
specific electroactive bacteria such as Geobacter (the e- donor) (Rotaru
et al., 2014b; Rotaru et al., 2014a; Yee et al., 2019; Zheng et al., 2020b).
This relationship is found to be mediated through the direct contact
between cells, which allows electron transfer from the bacteria to the
methanogens via nanowires and outer-membrane multi-heme c-type

4
P.A. Palacios et al.
cytochromes (MHC) (Lovley, 2017; Shi et al., 2016; Shrestha et al.,
2013b; Summers et al., 2010). The use of undefined mixed cultures not
only promotes syntrophic interactions but can also lead to competitive
interactions between methanogens and homoacetogens over electrons
and H2 in cathodes, although this interaction is more commonly
addressed for the cathodic production of acetate (Marshall et al., 2012;
Molenaar et al., 2017).
Although CO2 reduction pathways are the main focus of electro­
methanogenesis, the importance of the acetoclastic pathway should not
be underrated when working with mixed cultures (Blasco-Gómez et al.,
2017). Acetoclastic methanogenesis (Eq. (5)) (Liu and Whitman, 2008)
can be supported through syntrophic interactions with acetate-
producing bacteria (acetogens), which are very common in these sys­
tems. For instance, in complex microbial communities such as anaerobic
digestate, which is commonly used as inoculum in MES, cooperative
relationships can be formed when methanogens consume the accumu­
lated acetate generated via homoacetogenesis from H2 and CO2, or the
acetate and H2 accumulated from the fermentation of organic com­
pounds (Stams and Plugge, 2009). Methanogenesis with acetate as
electron donor yields little energy under standard conditions compared
to methane formation from CO2 reduction (Eqs. (2)–(3)-(5)) (Stams
et al., 2019). All acetoclastic methanogens belong to the order Meth­
anosarcinales, but acetate can also help to stimulate growth in other non-
acetoclastic methanogens (not as an energy source but as a carbon
source) (Fuchs and Stupperich, 1986).
Furthermore, different microbial interactions and products in addi­
tion to methane can also be expected, but this depends on the microbial
species and strains present in the MES. In addition to C1 compounds,
cathodic microorganisms have been shown to enable MES production of
C2-C6 compounds including short carboxylic acids like acetic and
butyric acid (Aryal et al., 2017b; Christodoulou and Velasquez-Orta,
2016; Nevin et al., 2010), medium-chain carboxylic acids like caproic
acid (Van Eerten-Jansen et al., 2013; Vassilev et al., 2018), and alcohols
like ethanol and butanol (Blanchet et al., 2015; Romans-Casas et al.,
2024). Furthermore, the diversity of metabolic pathways performed by
mixed methanogenic communities can help to develop flexible and
resilient biofilms on cathodes. These communities can then be adapted
to synthesize a wide array of products and tolerate stressful conditions
such as oxygen leaks and addition of contaminants (Mills et al., 2022).
5. Mechanisms employed by methanogens to use cathodes and
Fe0 as electron donors
Recently, different studies using methanogenic pure cultures eluci­
dated some of the mechanisms involved in extracellular electron transfer
from reduced solid surfaces such as cathodes and zero-valent iron (mi­
crobial corrosion). The economic and environmental problems caused
by microbial-induced corrosion processes under anoxic conditions have
led to the investigation of how different microbial groups induce severe
corrosion by harvesting electrons effectively from iron (Fe0) and thus
oxidizing it. Methanogens may be the second most common cause of
microbial-induced corrosion after sulfate-reducing bacteria in anoxic
environments (Dinh et al., 2004; Ibrahim et al., 2018; Tsurumaru et al.,
2018; Uchiyama et al., 2010), and therefore Fe0 corrosion assays along
with electrochemical systems are commonly implemented to evaluate
not only the corrosive potential of methanogens but also their ability to
uptake electrons from cathodes, which is relevant for electromethano­
genesis processes.
Both electromethanogenesis and microbial corrosion processes
involve electrochemical reactions. The mechanism engaged in capture
of electrons from the Fe0 surface also involves electron flow from an
anodic to a cathodic site on the Fe0 surface. At the anodic site, Fe0 gets
oxidized releasing electrons and ferrous iron (Fe2+) (indicative of
corrosion). Then, the electrons generated at the anode are simulta­
neously accepted at the cathodic surface, where some of them can be
used as electron donors for microorganisms via electrochemical H2
5
Biotechnology Advances 73 (2024) 108369
generation (Hamilton, 2003), or via other extracellular electron uptake
mechanisms (Holmes et al., 2022a; Lienemann et al., 2018; Tsurumaru
et al., 2018). The thermodynamic favorability of this process has been
expressed by the equation: 8H+ + 4Fe0 + CO2 → CH4 + 4Fe2+ + 2H2O,
ΔG0 = − 150.5 kJ/moL CH4 (Boopathy and Daniels, 1991; Wang et al.,
2021). Moreover, the use of metallic iron (Fe0) as sole electron donor in
anaerobic cultures, along with the quantification of metabolic products
and Fe2+, offers a suitable alternative to screen for microorganisms with
ability to uptake electrons from cathodes without assembling an elec­
trochemical set-up. For instance, pure Fe0, Fe0 with a 99.98% purity, and
stainless steel can be used for this purpose (Deutzmann et al., 2015;
Holmes et al., 2022a; Tsurumaru et al., 2018; Uchiyama et al., 2010).
Here, we describe several mechanisms that methanogens use during
electromethanogenesis processes, including the ones discovered in
highly corrosive species. From this section it can also be inferred that
non-electrotrophic methanogens, which utilize H2 as intermediary
instead of electrons, play a major role as biocatalysts in electro­
methanogenic systems.
5.1. Hydrogen uptake from cathodes and Fe0 (indirect)
As previously mentioned, the H2 generated from cathodes that are
poised at sufficient negative potentials, and from Fe0 (Hamilton, 2003)
represents an available electron donor for hydrogenotrophic metha­
nogens (Fig. 2). In microbial-induced corrosion studies, cathodic depo­
larization was the first proposed mechanism for corrosive sulfate-
reducing bacteria, and it remains one of the most frequently discussed
methods by which some bacteria and methanogens can stimulate
corrosion by consuming the abiotically produced H2 generated at the
cathodic zone of the metal surface (Uchiyama et al., 2010) (Fig. 2). In
more detail, this concept states that the presence of H2 at cathodic sites
causes ‘polarization’ of the cathodes, which limits the rate of the overall
corrosion reaction. Microorganisms stimulate the corrosion reaction by
removing the H2 (Costello, 1974), as this H2 utilization removes elec­
trons and hereby depolarizes the cathodic site, allowing more iron to be
corroded at the anode side (Iverson, 1966). Nevertheless, as reported for
some hydrogenotrophic methanogens such as Methanogenium, Meth­
anoplanus, Methanocalculus, and Methanobacterium, H2 utilization alone
is not sufficient to explain the observed significant corrosion rates
(Skovhus et al., 2017).
A better understanding on how some methanogenic strains were able
to induce severe Fe0 corrosion was acquired after specific extracellular
enzymes were discovered to catalyze cathodic H2 and formate genera­
tion coupled with CO2 reduction, as described in the next section. At low
enough potentials, electrochemically generated H2 acts as an interme­
diary, enhancing methanogenesis and growth rates in biocathodes
(Villano et al., 2010), which also corresponds with the enrichment of
hydrogenotrophic methanogens often observed in electromethano­
genesis (Table 2). H2 is considered to be the main electron transfer
molecule for electromethanogenesis (Fu et al., 2015), and this goes
along with the fact that some methanogens can effectively use H2, even
when it is only available at very low concentrations (Table 3). It is hy­
pothesized that strains with a low H2 threshold and a high affinity for H2
have an advantage on electrodes, where usually low H2 partial pressures
prevail. Moreover, the maintenance of low H2 levels on cathodes is
thought to thermodynamically stimulate the cathodic H2 evolution re­
action (Philips, 2020). Different H2-threshold values have been previ­
ously reported for different methanogenic strains, which in theory
should help to screen for methanogens that can use the electrochemi­
cally generated H2 from cathodes and Fe0. However, these values are to
some level condition dependent (e.g., different medium composition,
mixing intensity, temperature, pH) (Karadagli et al., 2019; Philips,
2020).
P.A. Palacios et al.
Table 3
H2- utilizing methanogens and their corresponding H2 thresholds.
Hydrogenotrophic methanogens H2 threshold (Pa)
Methanobacteriales order
Methanobacterium bryantii strain M.o.H. 6.9 ± 1.5
0.056 (0.028–0.14)
Methanobacterium formicicum strain JF-1 6.5 ± 0.6
Methanobacterium formicicum DSM 1535 2.8
Methanobacterium bryantii DSM 10113 2.5 ± 4.4
Methanobrevibacter arboriphilus DSM 744 9 and ~8
Methanobrevibacter smithii DSM 816 10
Methanobrevibacter strain AMG-1 5.7 ± 0.7
Methanothermobacter marburgensis DSM 2133 ~8
Methanomicrobiales order
Methanospirillum hungatei strain JF-1 9.5 ± 1.3
Methanospirillum hungatei DSM 864 3 (Cord-Ruwisch et al., 1988)
Methanoculleus bourgensis strain MAB1 0.15 ± 0.13
Methanogenium marinum strain AK-1 ~0.57
Methanococcales order
Methanococcus vannielii DSM 1224 7.5
Methanosarcinales order
Methanosarcina barkeri DSM 800 18.6 ± 10.0
Methanosarcina barkeri DSM 804 150
5.2. Electroactive extracellular enzymes (indirect)
Recently, a mechanism for extracellular electron uptake from solid
surfaces (Fe0 and electrodes) was proposed and reported in a few
methanogenic strains. The ability of Methanococcus maripaludis KA1/
Mic1c10/MM1264/OS7, Methanobacterium sp., and Methanobacterium-
like archaeon IM1 to efficiently uptake electrons from Fe0 and cathodes
was associated with excreted extracellular hydrogenases and formate
dehydrogenases, which were proven to be involved in severe corrosion,
and enhanced methane production in electrochemical reactors, due to
the generation of H2 or formate (Eqs. (2)–(3)) (Dinh et al., 2004; Lie­
nemann et al., 2018; Mori et al., 2010; Perona-Vico et al., 2019; Tsur­
umaru et al., 2018; Uchiyama et al., 2010).
Two publications have studied some of the enzymes involved in this
mechanism. Lienemann and co-authors reported that the multi-enzyme
heterodisulfide reductase supercomplex (Hdr-SC) from Methanococcus
maripaludis MM1264 could facilitate electron uptake through the rapid
generation of formate on Fe0 and cathodes (− 600 mV vs SHE) (Fig. 2).
This complex involves a heterodisulfide reductase (HdrABC), formate
dehydrogenase (FdhAB), and NiFe‑hydrogenase (VhuABDGU) (Liene­
mann et al., 2018), where VhuD is thought to facilitate electron flow
from the hydrogenase and formate dehydrogenase to HdrA (Costa et al.,
2013). The purified Hdr-SC did not show high catalytic activity for H2
formation under Fe0 corroding conditions, suggesting that H2 formation
from cell lysate is catalyzed by a non-Hdr-SC-associated hydrogenase
activity. This enzymatic complex acts as a highly selective, electroactive
formate evolution catalyst, and is capable of sustaining enzymatic
electrosynthesis of formate in the absence of an external mediator
(Lienemann et al., 2018).
Tsurumaru and co-authors furthermore discovered a novel family
member of [NiFe] hydrogenases. This enzyme denominated MIC‑hy­
drogenase (from microbiologically influenced corrosion, MIC) is pro­
duced by specific strains of Methanococcus maripaludis (KA1, OS7, and
Mic1c10), and can make direct contact with Fe0 and oxidize it through
the generation of H2 gas (Fig. 2). It is encoded in an unstable genomic
region named “MIC island”, which explains why corrosive strains
growing on H2 after several transfers for culture maintenance, lose their
ability to induce severe corrosion along with loss of this specific genomic
6
Biotechnology Advances 73 (2024) 108369
Reference
(Lovley, 1985), (Karadagli and Rittmann, 2007)
(Lovley, 1985)
(Cord-Ruwisch et al., 1988)
(Neubeck et al., 2016)
(Cord-Ruwisch et al., 1988), (Kaster et al., 2011)
(Cord-Ruwisch et al., 1988)
(Feldewert et al., 2020)
(Kaster et al., 2011)
(Lovley, 1985)
(Neubeck et al., 2016)
(Chong et al., 2002)
(Cord-Ruwisch et al., 1988)
(Neubeck et al., 2016)
(Kaster et al., 2011)
region via spontaneous deletion (Tsurumaru et al., 2018). Interestingly,
the MIC island can spread among methanogens beyond the genus level,
and its presence was also confirmed in Methanobacterium congolense
(Tsurumaru et al., 2018). Currently, the large subunit of this hydroge­
nase (labeled micH) has been used as a biomarker for microbial corro­
sion and has being detected in multiple oil fields and corrosive biofilms,
suggesting that this enzyme is geographically widespread (Lahme et al.,
2021). Moreover, in 2019, Perona-Vico and co-authors found that
[NiFe]‑hydrogenases, heterodisulfide reductase, F420-reducing
[NiFe]‑hydrogenase, and the hydrogenase maturation protein: HypD
were constitutively expressed during electromethanogenesis in an
enriched Methanobacterium population (Perona-Vico et al., 2019). Hy­
drogenases from Methanosarcina barkeri have also been reported to
catalyze the H2 production on electrodes in electrochemical reactors.
Cathodes inoculated with this methanogen at a set potential of − 0.6 V
vs. SHE, produced H2 at a rate of 120 ± 18 nmol d− 1 ml− 1, and about
half of this rate was detected with the non-viable cells of M. barkeri
(Yates et al., 2014). It has been reported that hydrogenases in combi­
nation with ferredoxin (Fd) and F420 from M. barkeri can attach to the
electrode surface to catalyze H2 formation (Rowe et al., 2019).
Furthermore, only those strains capable of reaching high methane pro­
duction rates, and higher electron transfer rates should be considered for
upscaling and for industrial applications. Thereby, hydrogenotrophic
methanogens and H2 as an intermediary offer a suitable alternative to
further develop these systems.
The purification of electroactive hydrogenases and formate de­
hydrogenases has helped to demonstrate their ability to generate H2 or
formate on cathodes and Fe0. In a recent study, it was shown that three
different hydrogenases from Clostridium pasteurianum and Meth­
anococcus maripaludis, when immobilized on a cathode in a cobaltocene-
functionalized polyallylamine (Cc-PAA) redox polymer, mediated rapid
and efficient H2 evolution. The Cc-PAA-mediated hydrogenases oper­
ated at high faradaic efficiency (80–100%) and low apparent over­
potential (− 0.578 to − 0.593 V vs. SHE) (Ruth et al., 2020). Since one of
the main challenges of H2 production on an industrial scale is to stabilize
the hydrogenases on the electrodes, this study on immobilization of
enzymes on a cathode illustrates an interesting approach to work with
purified enzymes. Still, whole cells of mixed or pure cultures of actively
P.A. Palacios et al.
growing microorganisms represent a cost-effective alternative to the
elaborate enzyme purification procedures (Sané et al., 2014; Sané et al.,
2013; Yates et al., 2014), which is critical for large-scale applications.
5.3. Electron shuttles (potential indirect mechanism)
Another way of enhancing the transfer of electrons between cathode
and microorganism is through the use of redox mediators. Redox me­
diators are stable and electrochemically active molecules that can be
reduced and oxidized reversibly, thereby acting as electron shuttles
between electron donating cathodes and electron accepting microor­
ganisms (Van Der Zee and Cervantes, 2009). The use of electron shuttles
is a common mechanism that electroactive microbes use to achieve
extracellular electron transfer. In methanogens, electron shuttles are
well known as intermediary electron acceptors (available in their
oxidized state), and some examples of methanogens reducing artificial
and natural extracellular electron shuttles are available in literature (e.
g., AQDS, Fe3+, U6+, and V4+) (Bond and Lovley, 2002; Holmes et al.,
2019; Holmes et al., 2018; Palacios et al., 2023; Zhang et al., 2014).
However, in terms of electromethanogenesis, electron shuttles must act
as intermediary electron donors switching between reduced and
oxidized form when transferring electrons from the electron donating
cathode to the electron accepting methanogen to facilitate extracellular
electron uptake by the methanogens. To date, only electrically reduced
neutral red has been reported as electron donor for growth and pro­
duction of methane from CO2 in a methanogenic mixed culture from
anaerobic sludge (Park et al., 1999). From this study, the authors
affirmed that much remains to be learned about the exact biochemical
mechanisms involved in this process. In a more recent study involving
pure cultures of Methanosarcina mazei, methane production was also
enhanced by neutral red (no electrodes present), but only when acetate
was used as substrate in the absence of H2 (Beckmann et al., 2016). The
lack of activity in the absence of acetate demonstrated that neutral red
was not used as an electron donor for methanogenesis via CO2 reduction.
Instead, this mediator could drive H2 production via the Vho hydroge­
nase by delivering reducing equivalents to the membrane bound HdrED,
which enabled the reduction of the carbonyl group from acetate to
methane as shown in in vitro electron transport assays on isolated
membrane fractions of the methanogen (Beckmann et al., 2016).
Mediated extracellular electron uptake by methanogens via artificial
electron shuttles is still largely unexplored. Future use of such redox
mediators should consider cellular toxicity of such shuttles, along with
their stability and redox potential. The screening and selection of an
adequate electron mediator could help alleviate the low electron
transfer rates from cathode to redox-active proteins and hereby improve
methane production rates and efficiencies.
5.4. Multiheme c-type cytochromes (direct mechanism)
The important role of outer membrane c-type cytochromes (OMCs)
as electron carrier proteins in electroactive bacteria like Shewanella
oneidensis MR-1 and Geobacter sulfurreducens (Paquete et al., 2022; Shi
et al., 2009; Tang et al., 2019) leads to question if similar membrane-
bound redox-active proteins are involved in direct extracellular elec­
tron uptake mechanisms in methanogens. The only methanogens with
known membrane-bound cytochromes are Methanotrix, and Meth­
anosarcina (Kletzin et al., 2015; Kühn et al., 1983; Rotaru et al., 2014a;
Thauer et al., 2008). Even though, a study in direct interspecies electron
transfer (DIET) suggested that extracellular electron uptake in
7
Biotechnology Advances 73 (2024) 108369
Methanosarcinales is independent of multiheme c-type cytochromes (Yee
et al., 2018), the presence of OMCs in the model organism Meth­
anosarcina acetivorans (denominated as MmcA) has given some insight
on the crucial role that these redox-active proteins can have in the EET
processes carried by this Type II Methanosarcina. The multiheme, outer-
surface c-type cytochrome MmcA can act not only as a terminal reduc­
tase capable of transferring electrons to the humic analog, anthraquione-
2,6-disulfonate (AQDS) (Holmes et al., 2019) but also as a conduit for
direct electron uptake from Fe0 (metal-to-microbe), when acetate is
available as an additional energy source. Moreover, during DIET,
Methanosarcina also receive acetate from its electron-donating partner as
an additional source of electrons (Holmes et al., 2022b; Holmes et al.,
2022a; Rotaru et al., 2014a; Shrestha et al., 2013a, 2013b). For the
fundamental understanding of the microbial physiology in MES, it is
important to elucidate the mechanisms behind the extracellular electron
uptake processes. However, in terms of applicability direct mechanisms
such as the outer-surface c-type cytochromes have not been proven to be
suitable candidates to develop efficient electromethanogenesis pro­
cesses on an industrial scale, especially with pure cultures. For example,
it has been suggested that Methanosarcina barkeri uses both a
hydrogenase-mediated and a putatively direct mechanism to feed from
cathodes (Rowe et al., 2019). The methane production values obtained
from whole cultures (including extracellular enzymes) were fivefold
higher (104.0 ± 15.7 eeq total) compared to washed cells cultures (18.9
± 3.3 eeq total), in which extracellular enzymes such as hydrogenases
have previously been washed out (Rowe et al., 2019). This indicates that
the direct mechanism only played a minor role in comparison to the
hydrogenase-mediated mechanism with H2 as intermediary.
5.5. Archaella (potential direct mechanism)
Extracellular electron transfer (EET) mechanisms have been widely
studied in bacteria, such as Geobacter and Shewanella species (Shi et al.,
2016), compared to Archaea. This can give us some hints on potential
and similar EET mechanisms that might be present in methanogens
growing on cathodes and Fe0 surfaces (e.g., Archaellum, c-type cyto­
chromes, electron shuttles). The archaeal flagellum, now defined as
archaellum (Albers and Jarrell, 2015; Jarrell and Albers, 2012), is a
particularly interesting structure to consider in EET studies. For
example, in terms of conductivity it has been reported that the arch­
aellum of Menthanospirillum hungatei has a conductance 4-fold higher
compared to G. sulfurreducens nanowires (Walker et al., 2019). Even
though, the structure of this conductive protein filament is unknown, the
authors identified a core of tightly packed phenylalanines as a potential
the route for its electron conductance (Walker et al., 2019). Since
archaella are also involved in cellular motility, biofilm formation, and
cellular adhesion (Jarrell et al., 2011; Jarrell and Albers, 2012; Schopf
et al., 2008), this structure could also play an important role in the
formation of methanogenic biofilms on cathodes. However, archaella
from studied methanogens such as Methanococcus maripaludis are not
constitutively expressed, and several nutritional factors may increase (e.
g., H2 and phosphate limitation) or decrease (e.g., nitrogen limitation)
their expression (Albers and Jarrell, 2015; Hendrickson et al., 2008; Xia
et al., 2009). Further studies could clarify if conductive archaella, like
the one from Menthanospirillum hungatei (Walker et al., 2019), or the
archaellum of Methanococcus maripaludis are capable of performing EET
from cathodes to cells.
P.A. Palacios et al. Biotechnology Advances 73 (2024) 108369

Fig. 2. General schematic of extracellular electron uptake mechanisms performed by methanogens in electromethanogenesis processes using cathodes as energy
source. Indirect mechanisms (left panel) include the utilization of electrochemically generated H2, and the ability of specific methanogenic strains to perform
enzyme-dependent electron uptake via the MIC-hydrogenase ([NiFe]-hydrogenase), and the heterodisulfide reductase supercomplex (Hdr-SC) (both can interact with
the cathode to generate H2 and formate, respectively). On the other hand, a direct electron uptake mechanism (right panel) involves the use of multiheme c-type
cytochromes by the model organism Methanosarcina acetivorans to perform direct metal-to-microbe electron uptake during acetoclastic methanogenesis. Moreover, if
conductive archaella are involved as a direct mechanism, or if redox mediators can facilitate an indirect electron uptake mechanism require further research.

6. Optimization of electromethanogenesis processes
The idea of displaying all elucidated mechanisms to date, is to
recognize the major role that H2 plays as an intermediary generated
electrochemically, enzymatically, or microbially. Still, the mechanisms
represent just a part of the interdisciplinary MES technology, which is
why the optimization of other parameters that can enhance electron and
H2 transfer rates from cathodes to microbes and their corresponding
redox enzymes is largely encouraged.
From a microbiological point of view, it is critical to understand how
the selected pure or mixed cultures of methanogens interact with the
cathode at specific voltages. Knowledge and selection of specific meth­
anogenic strains and their ability to either take up electrons directly
(electrotrophic methanogens) or via reduced intermediaries such as H2
(non-electrotrophic methanogens) will be key for pairing the optimal
electrochemical conditions of the set-up, with the physiology of the
methanogenic strains. Finding the right combination between the
methanogenic inoculum of interest and its optimal cathodic potential
can result in higher energy efficiencies and production rates, which are
some of the main limitations encountered in this system (Su and Ajo-
Franklin, 2019; Zheng et al., 2020a).
An efficient MES is, however, not dependent on the microorganisms
alone, but also on their interaction with the cathode. Some of the factors
limiting electron transfer are related to the design of cathodes to opti­
mize their surface area, conductivity, and biocompatibility. Efforts are
therefore being made to improve the cathodes that are used in electro­
methanogenesis by combining new and existing materials, and shapes,
or applying different treatments to the surface of the cathodes (Blasco-
Gómez et al., 2017). Several research studies have contributed to the
design, evaluation, and comparison of different cathodes for electro­
synthesis processes (Aryal et al., 2017a; Bajracharya et al., 2022; Siegert
et al., 2014; Zhang et al., 2013; Zhen et al., 2018). The most commonly
used electrode materials in electrosynthesis lab-scale studies include
carbon-based materials such as carbon felt, carbon cloth, and graphite,
but also stainless steel, nickel, and titanium (Park et al., 2022).
Furthermore, the utilization of conductive particles or molecules could
potentially facilitate electron transfer. However, their effect on
microbial stability (e.g., toxicity tests) and activity must be determined
to assure biocompatibility and higher efficiencies. From the electro­
chemical perspective, it would also be convenient to apply more nega­
tive potentials to the cathode to increase the electron flow delivery and
transfer. One important factor to consider when applying more negative
potentials is the increase of pH at the cathode as protons are continously
removed to produce H2 (Lim et al., 2018). pH control is key in this case,
and the addition of CO2 or acidic solutions (e.g., HCl) would be rec­
ommended to maintain the pH at neutral or slightly acidic to sustain the
biocathode performance.
The H-cell system (Fig. 1) is ideal for investigations on mechanisms
and optimization studies involving different membranes, cathode ma­
terials, and well-defined methanogenic cultures as sterile conditions can
be achieved by autoclaving most parts of the cell (Table 1) (Liu et al.,
2023). However, the H-cell system presents some limitations such as
non-optimal gassing, stirring geometry, large internal resistance as a
result of the long distance between electrodes (Liang et al., 2007), and
challenging scale-up (Krieg et al., 2019). The reactor design and
configuration are therefore important engineering aspects to consider to
obtain more efficient electron and gas (CO2 and H2) transfer processes.
MES design includes both single-chamber systems, which are
membrane-less and contain the cathode and anode in the same chamber,
or dual- or multi-chamber systems, where electrodes are separated by a
membrane. Even though the single-chamber system has a simpler design
compared to dual-chamber systems that can reduce the cost and
complexity of the system, the main disadvantage of the single-chamber
system is the electrochemical production of O2 at the anode, also known
as oxygen evolution reaction (OER), which can affect the obligate
anaerobic methanogens also present in the single-chamber MES. The
membrane included in dual and multi-chamber systems can limit the O2
diffusion from anode to cathode (Aryal et al., 2021) and reduce its
negative impact on the obligate anaerobic methanogens. Considering
that nearly all BES require the spatial separation of anode and cathode to
maintain redox gradients for energy conversion and to prevent crossover
processes (Krieg et al., 2014), one of the most important up-scaling
challenges involves highly efficient and low-cost electrochemical re­
actors. Here many different designs can be considered, but some of the

8
P.A. Palacios et al.
main parameters for optimization are (i) reduction of the geometrical
distance between the anode and cathode to decrease the electrical
resistance and ohmic losses (ii) stacking of cells to obtain higher cell
voltages as power electronics is not (cost-) efficient at low voltages and
(iii) have OER catalysts that work efficiently at near neutral conditions
(Aryal et al., 2021; Krieg et al., 2014). Here it is natural to be inspired by
the technical advancements within fuel cells, electrolyzers, and flow
batteries that use the same overall design strategy for optimization and
upscaling. I.e. thin electrodes (typically <3 mm) stacked in cells from 40
to 100 and separated by bipolar plates (typically either carbon graphite
or nickel-based, depending on the specific technology) (Grigoriev et al.,
2020; Ke et al., 2018). Whereas H-cell current densities in general are
well below <1000 mA m− 2 (Table 1), the use of a redox-flow battery
design using a non-enriched inoculum from a biogas reactor resulted in
current densities of up to 35 A m− 2 (Geppert et al., 2019). Since elec­
tromethanogenesis is still being developed at lab-scale, the economic
implications can be difficult to predict, however, even at current den­
sities of 35 A m− 2, the optimization of charge transfer can still be seen as
a major challenge for the technology, which would result in large and
costly electrochemical cells (stacks) (Aryal et al., 2021), and effectively
hinder the practical application of such systems at large-scale. Future
development of electromethanogenesis as a relevant PtX technology
therefore requires continued efforts in microbiology, electrochemistry,
and engineering. Understanding the mechanisms employed by metha­
nogens to utilize reducing equivalents from cathodes represents one
piece of this puzzle.
7. Conclusions
Electromethanogenesis, as a branch of PtX technologies, has many
benefits to offer as a green approach that utilizes CO2 to produce
methane biofuel, using methanogens as the main catalyst and renewable
electricity as electron donor. However, the technology faces limitations
and challenges that require interdisciplinary research in microbiology,
electrochemistry, and engineering to facilitate its development. A key
aspect of electromethanogenesis is to understand how methanogens
interact with cathodes and utilize electrons or reduced products from
these systems. Considering all the discoveries made in recent years on
the mechanisms involved in the extracellular electron uptake from
cathodes and Fe0 (biocorrosion), particularly through the study of
methanogenic pure cultures, this review makes emphasis on the di­
versity of these mechanisms. This knowledge is essential for the devel­
opment of an efficient and productive system under the right
electrochemical conditions. Methanogens can use a cathode or Fe0 as
energy source, mainly by using different indirect mechanisms such as
H2-uptake, and extracellular electroactive enzymes (MIC‑hydrogenase,
heterodisulfide reductase supercomplex). So far, direct mechanisms
have been corroborated only via multi-heme c-type cytochromes in
Methanosarcina acetivorans, when acetate was supplemented as co-
substrate (Holmes et al., 2022a). Yet, novel extracellular electron up­
take mechanisms remain to be elucidated as suggested for conductive
archaella (direct), and redox molecules (indirect). This could be ach­
ieved by screening archaellated methanogens such as Methanospirillium
hungatei, and by adding redox molecules with as low redox potentials as
possible in lab-scale set-ups. Since electromethanogenesis processes also
use mixed methanogenic communities as biocatalysts, this review de­
scribes the microbial interactions that can have an impact in methano­
genesis. For instance, a complex methanogenic consortium, enriched or
not, will be normally dominated by non-electrotrophic methanogens.
Hydrogenotrophic methanogens can therefore use the H2 generated
from the cathode poised at very low potentials, or accomplish methane
production via syntrophic or direct interspecies electron transfer (DIET)
interactions. Thus, in electromethanogenesis processes, non-
electrotrophic/hydrogenotrophic methanogens can be utilized as
mixed or pure cultures. Each type of inoculum offers distinct advantages
in these processes where H2 plays a major role as intermediary.
9
Biotechnology Advances 73 (2024) 108369
CRediT authorship contribution statement
Paola Andrea Palacios: Conceptualization, Writing – review &
editing. Jo Philips: Writing – review & editing. Anders Bentien:
Writing – review & editing, Funding acquisition. Michael Vedel
Wegener Kofoed: Writing – review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by the Novo Nordisk Foundation (NNF),
Exploratory Interdisciplinary Synergy Programme 2020 (grant number
0064571).
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