Anaerobic of DIET in anaerobic cultures, Rotaru et al.,

Anaerobic digestion is a complex system involving
many redox reactions, and interspecies electron transfer process plays a key role
in the proper functioning of an AD system (Boone et al., 1989; De Bok et al., 2004, 2002; Sieber
et al., 2012).
Methanogenesis and Sulphur reduction reactions depend highly on the syntrophic
communities of bacteria and archaea. These syntrophic communities take
advantage of the metabolic abilities of corresponding syntrophic partner to
overcome energy barriers and breakdown compounds that are difficult to
metabolise by themselves (Stams and Plugge, 2009). As
discussed in the previous section, until early 2000s it was believed that IET
occurred only via electron shuttle components especially hydrogen and formate. The
other potential alternative for electron transfer was suggested to be direct
interspecies electron transfer (DIET) (Reguera
et al., 2005).

The process of DIET by methanogenic wastewater
digester aggregates is important to discuss because it decreases a number of
intermediary steps and intermediary products thereby decreasing process
dependency and increasing process stability. This paradigm shift in the
mechanisms of electron transfer significantly impact the “modelling and design
of anaerobic wastewater reactors and the understanding of how methanogenic
communities respond to environmental perturbations” (Morita et al., 2011).

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The first evidence of DIET through conductive pili which
are otherwise called as nanowires, was provided by Reguera and team (2005). Subsequently,
Gorby et al., (2006)
demonstrated that Shewanella oneidensis strain MR-1 developed
electrically conductive pili when it was deprived of electron acceptor
molecules. The study also confirmed that syntrophic methanogenic
micro-organisms, P. thermopropionicum and M. thermoautotrophicus are
connected by flagellum like appendages to establish not only IET but also other
energy exchange processes such as interspecies hydrogen transfer (IHT). Further
strengthening the idea of DIET in anaerobic cultures, Rotaru et al., (2014b) showed
carbon dioxide reduction through DIET between Geobacter metallireducens and
Methanosaeta harundinacea. The same team of researchers explored the
DIET capability of Methanosarcina barkeri when co-cultured with pilin-deficient
Geobacter metallireducens (Rotaru et al., 2014a).
The authors demonstrated that granular active carbon material can act as a
conductive material replacing pili to transport electrons.

Following this discovery, a correlation was obtained
between microbial community and granule conductivity by (Shrestha et al., 2014). A
correlation of about r=0.67 was observed between the abundance of Geobacter
species and granule conductivity proving that the supplement of granular
conductive material for the enhancement of DIET and thereby process efficiency.
Another study was conducted to compare the DIET efficiencies with different
conductive materials where carbon cloth demonstrated most effective syntrophic
electron transfer in comparison to graphite and biochar (Zhao et al., 2015b). The above
discovery was also remarkable in terms of electrochemical carbon dioxide
reduction, as dispersed and high surface area cathodes such as granular
activated carbon (Liu et al., 2012), biochar (Chen et al., 2014b), carbon
cloth (Chen et al., 2014a) and
magnetite (Liu et al., 2015) can, not
only donate electrons to microbes, but also transport electrons between
microorganisms. The microbes can just attach themselves to these conductive
materials (called as biocathodes, discussed in the next section) and transport
electron among them by saving the energy used in the generation of conductive
pili (Zhao et al., 2015b). 

This was immediately put into practice by (Zhao et al., 2015a) where an AD
system was attached with an electric circuit containing graphite rod cathode
and graphite brush anode. The authors observed 30% more electric conductivity
in the electric-AD in comparison to the control AD system. It was also observed
that rate of production of methane was higher with the electric-AD system by 3
times at the 33rd hour mark. In a study combining MEC-AD system for
carbon dioxide reduction, the authors compared reactor performances with and
without Geobacter species. It was observed that the carbon dioxide content
in total gas generated from the AD reactor with Geobacter was only half
of that generated from the same reactor without Geobacter, suggesting
that Methanosarcina may obtain the electron transferred from Geobacter
for the reduction of carbon dioxide to methane (Yin et al., 2016). One of the
most recent studies on MEC-AD system it was observed that the conductive
material carbon cloth, apart from enhancing DIET among the microbes, it was
able to stabilise the system for acid impacts and high hydrogen partial
pressures (Zhao et al., 2017).

Although it is a proven theory that direct
interspecies electron transfer improves AD process efficiency and in turn DIET
can be enhanced with the supplement of conductive material, there is no clear
explanation on the mechanism of these processes. Also, the details of
interspecies connecting networks are not entirely clear, however, Malvankar et al., (2011); Morita et al., (2011);
Reguera et al., (2005) have
reported metal like conductivity through the microbial nanowire network.  Another possible mechanism suggested for
electron transfer was through the c-type cytochromes present on the cell
surface of microorganisms (Lovley et al., 2011).


Biocathodes unlike bioanodes have
gained research interest only recently, especially in the field for
bioelectrochemical energy generation processes. These were developed as cheaper
alternative to the expensive metal cathode catalysts such as Platinum. Although
several ways have been implemented for the development of biocathodes (Morozov et al., 2002; Pershad et al., 1999; Rhoads et al., 2005; ter
Heijne et al., 2007), biofilm based
biocathodes hold the highest position in terms process efficiency due to the
possibility of direct electron transfer as mentioned in the previous section.
Several research papers have been published since its inception in the early
2010s and have been summarised in Table 1.

Table 1: Biocathodes with Biofilm


In 2008, Rozendal et al., for the first time,
demonstrated hydrogen gas production using biofilms that were developed on
graphite felt cathode in three phases. The authors were successfully able to
convert a bioanode which was oxidising acetate and hydrogen into a hydrogen
producing biocathode by through inversion of polarity and slowly adapting the
microorganisms and the electrodes to reducing environments. The authors were
able to achieve a current density of about -1.2 A/m2 to hydrogen at -0.7V and
suggested hydrogenotrophic methanogenic losses to be one of the reasons for low
hydrogen yield even under carbon limited conditions. This research laid
foundation for Bioelectrochemical hydrogen production, for instance, Croese et al., 2011 were able to culture Desulfovibrio spp. (an active hydrogen
consumer at anode) on graphite felt cathode to produce hydrogen at similar
rate, however, the group was yet to characterise two other dominant microbial
species on the cathode.

In 2008 Clauwaert and Verstraete showed that methane production can occur in a
membraneless MEC and suggested that MECs can be combined with AD systems for
efficient methane production. Infact they were able to verify a number of
advantages with membrane removed from the MEC: a) reduced ohmic resistance and
b) minimized pH gradient and higher (Clauwaert et al., 2008a). It was also shown that membraneless MECs are able to
combine anodically produced carbon dioxide with cathodically produced hydrogen
gas with the help of hydrogenotrophic methanogens (Call and Logan, 2008; Clauwaert et al., 2008b).

These observations were further strengthened by Cheng et al., 2009 with the demonstration of
methane production in a single chamber microbial electrochemical cell using a
biocathode at a potential of -0.7 V vs Ag/AgCl (~0.491 V vs SHE). Several
experimental strategies were applied (such as electrodes with and without
biofilm and electrolyte with and without organic and inorganic carbon source) to
verify whether the methane production was acetoclastic or hydrogenotrophic.
Direct electron transfer was evident as current flow with an abiotic cathode
reduced to 0 A at -0.95 V whereas for biocathode high currents were observed in
the range of -1 to -0.70 V. The authors were able to generate methane when the
sole source of carbon was CO2 at a set potential of -1.0 V. Methane
production rate of ?200mmol-CH4d-1m-2 with a CO2
consumption rate of ?210 mmol-CO2 d-1m-2 was achieved
using a two-chamber MEC.

            Following these research results, the focus of production
in MEC systems changed from Hydrogen to Methane which was earlier considered an
unavoidable by-product. Graphite based electrodes were used as cathodes
(Villano et al., 2011 and Mueller, 2012) so as to increase the geometric
surface area of the electrode where the biofilms can grow. Villano et al., 2011 were able to show that,
when anode potential was maintained constant at +0.50 V in a 2 chamber acetate
supplied MEC inoculated with G. sulfurreduccens, the acetate oxidation was
linearly related to the biomass (micro-organisms inoculated). These results
indicated that electron transport is directly proportional to the biomass
density on the biofilm. Carbon felt was also explored as a cathode material for
the first time in electromethanogenesis by Jiang et al., 2013. The authors showed how
CO2 is reduced to different products depending on the cathode potentials
applied on carbon felt cathode. Methane and hydrogen were produced in the range
-0.65 to -0.75 V (vs SHE), methane, hydrogen and acetic acid were produced
simultaneously at -0.75 V and only methane and acetic acid were produced at
-0.95 V.

            With such developments in methane production many
scientists approached electromethanogenesis as a biogas upgrading system. In
such systems, the electrochemical setup was combined with an existing anaerobic
digestion system such that the CO2 in biogas is reduced to methane and thereby
improving the biogas quality. Xu et al., 2014 compared two systems
where the AD system was attached externally to the MEC system and the other
where the MEC system was also an AD system. In the latter, the headspace of the
digester was connected to the MEC system which contained anaerobic granular
sludge without any organic carbon source. The authors observed that coulombic
efficiency of the ex-situ biogas upgrading system was more than the in-situ
system. Similar study was done by Zhao et al., 2014 and showed a 25% increase
in methane production and 19% increase in acetate consumption in an AD-MEC
system as compared to the an only AD system.

these studies with graphite rod cathodes revealed a large concentration of
hydrogenotrophic methanogens found on the electrode surface biofilm. Similarly,
Siegert et al., 2014a compared an acetoclastic culture rich AD sludge and
hydrogenotrophic culture rich anaerobic bog sediment for electromethanogenesis.
The results showed that bog samples as inoculum were most efficient attributing
it to the high concentration of Methanobacterium spp. as they significantly
reduced hydrogen gas recycling. They showed that Methanobacterium spp.
was the most abundant species in the biofilms followed by Methanosaeta spp.

the research area of MEC-AD systems, Fu et al., 2015 showed that thermophilic
microorganisms can be used as biocatalysts for electromethanogenesis. At 55°C
and an applied voltage of 0.8 V high methane production rates of about 1103 mmol
m-2 day-1 were obtained. Further, it was suggested that Synergistetes- and
Thermotogae-related bacteria established syntrophic associations with methanogens
for efficient methanogens. In a novel approach for integrating MEC and AD
systems, (Liu et al., 2016) were able to show that
the MEC systems not only improved methane production rate by 3 – fold but also
enhance substrate degradation.

the importance of hydrogenotrophic methanogens Dykstra and Pavlostathis, 2017, laid out a direct
comparison between mixed methanogenic (MM) and enriched hydrogenotrophic
methanogenic (EHM) culture for methane production. The EHM based system
produced 4 times more methane than MM based system. Many new bacterial species
such as Propionivibrio, Thiomonas, Citrobacter, Actinomyces etc., were
identified at the biocathode due to their exoelectrogenic properties.