Environmental Research 209 (2022) 112801

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Soil microorganisms facilitated the electrode-driven trichloroethene

dechlorination to ethene by Dehalococcoides species in a
bioelectrochemical system
Lingyu Meng a, *, Naoko Yoshida a, Zhiling Li b
a
Department of Civil Engineering, Nagoya Institute of Technology (Nitech), Nagoya, 466–8555, Japan
b
State Key Laboratory of Urban Water Resources and Environment, School of Environment, Harbin Institute of Technology, Harbin, 150090, China

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

Keywords: Bioelectrochemical dechlorination using organohalide-respiring bacteria (ORBs) is a promising technique for
Syntrophic interaction remediating contaminated groundwater. Generally, a longer enrichment period is required for selecting the ORB
Bioelectrochemical dechlorination consortia to achieve bioelectrochemical dechlorination. However, the full dechloriantion is difficult to be ach­
Exocellular electron transfer
ieved due to the absence of functional species (e.g. Dehalococcoides) in previously used enrich cultures. To
Dehalococcoides
overcome these challenges, bioelectrochemical dechlorination using a culture enriched with the pre-augmented
Dehalococcoides was performed for the first time in this study. A two-chamber bioelectrochemical system (BES)
inoculated with a pure Dehalococcoides culture and paddy soil with an applied voltage of − 0.3 V (versus a
standard hydrogen electrode) as the sole electron donor was used to achieve dechlorination. The ethene for­
mation rate was 10–100 times higher than that in previous studies, indicating that inoculating the system with a
pure Dehalococcoides culture and soil microorganisms gave effective full dechlorination performance. Microbial
community analysis and bioelectrochemical analysis indicated that Desulfosporosinus species may have facilitated
dechlorination through syntrophic interactions with Dehalococcoides. The results indicated that adding Dehalo­
coccoides cells before operating a bioelectrochemical system is an effective way of achieving full dechlorination.

1. Introduction hydrocarbons and groundwater quality deterioration caused by large

Improper handling and disposal of chloroethenes (CEs), such as
tetrachloroethene (PCE) and trichloroethene (TCE) used as solvents and
metal degreasing agents, can lead to groundwater pollution (Binbin
et al., 2014; Hamonts et al., 2012). Anaerobic reductive dechlorination
of CEs and chloroethanes using organohalide-respiring bacteria (ORBs)
has been performed in various batch bioreactors and continuous bio­
reactors and in situ at remediation sites. In these systems, H2-releasing
organic compounds were the main electron donors involved in dechlo­
rination of CEs through the respiration by ORBs (Aulenta et al., 2006;
Morse et al., 1998; Palma et al., 2018). Bioelectrochemical dechlorina­
tion has been used to remediate water contaminated with chlorinated
solvents in the last decade. In bioelectrochemical dechlorination, ORBs
are used and an electrode acts as an electron donor. Different to the
conventional bioremediation, this approach can avoid the competition
for H2 caused by the proliferation of unwanted bacteria (e.g., metha­
nogens, nitrate- and sulfate-reducing bacteria) that consume excess
amounts of fermentation products being formed (Aulenta et al., 2007b,
2009; Fennell and Gossett, 2005; Palma et al., 2018; Wang et al., 2015).
In previous bioelectrochemical systems (BESs) studies, it was re­
ported that cathode-utilizing bacteria (e.g., Geobacter, Meth­
anobacterium, and Shewanella), which are found widely in the natural
environment, were involved in bioelectrochemical nitrite reduction,
methanogenesis, CO2 capture, and dechlorination (Chen et al., 2019a;
Leitão et al., 2015; Lu et al., 2018; Noori et al., 2020). However, no
clear-cut electron transfer (ET) mechanism between the electrode and
these bacteria is known. The mechanisms assumed to be involved in ET
from the electrode to ORBs during bioelectrochemical dechlorination
are described next. First, electrochemically generated H2 acts as the
ultimate electron donor at applied cathode potentials lower than − 0.41
V versus (vs.) a standard hydrogen electrode (SHE) (Chen et al., 2019a;
Leitão et al., 2015). Second, direct ET (DET) occurs via redox proteins in
ORBs, e.g., c-type cytochromes, or electrically conductive pili (Lovley,
2017; Strycharz et al., 2008). Third, indirect ET (IET) occurs through

* Corresponding author.
E-mail address: meng.lingyu@nitech.ac.jp (L. Meng).

https://doi.org/10.1016/j.envres.2022.112801
Received 11 November 2021; Received in revised form 17 December 2021; Accepted 20 January 2022
Available online 29 January 2022
0013-9351/© 2022 Elsevier Inc. All rights reserved.
L. Meng et al.
interspecies ET via electroactive microorganisms and biologically syn­
thesized mediators, e.g., cobalamin and flavins (Holmes et al., 2016; Yan
et al., 2012), or physiochemically produced mediators, e.g.,
anthraquinone-2,6-disulfonate, humin, and methyl viologen (Aulenta
et al., 2007a, 2010; Zhang et al., 2014). Generally, lower applied po­
tential is required for electrolytic H2 involved dechlorination than DET
or IET involved dechlorination. However, in the previous study of
dechlorination involving electrolytic H2, it was found that the dechlo­
rination efficiency was strongly affected by the presence of
hydrogen-utilizing methanogens because of competition for H2 (Aulenta
et al., 2011). Hence, accelerating DET and IET with a relative higher
potential could be an alternative way of achieving effective electro­
chemical dechlorination, which can avoid methane emissions and
reduce energy input.
To date, limited types of pure ORBs cultures can perform respiration
through gaining electrons from electrodes via DET. This form of respi­
ration has only been reported in Geobacter species, while Geobacter could
only dechlorinate TCE to give cis-Dichloroethene (cis-DCE) but not to the
nontoxic ethene (Strycharz et al., 2008). It is well known that only
Dehalococcoides and Dehalogenimonas species capable of reductive
dichlorination of vinyl chloride (VC) to nontoxic ethene (Puentes
Jácome et al., 2019). However, as one of representative
VC-dechlorinating species, Dehalococcoides has not previously been re­
ported that can use an electrode as the sole electron donor in a BES. To
promote the electrode-driven dechlorination, attempts have been made
to supply external dissolved electron mediators to BESs. The IET
employed external dissolved redox mediators, such as methyl viologen,
anthraquinone-2,6-disulfonate, and humin, have been well studied and
found to accelerate electrode-driven dechlorination of TCE, cis-DCE, and
phencyclidine, respectively (Aulenta et al., 2007a, 2010; Zhang et al.,
2014). Also, the IET is likely to be enhanced by the self-produced redox
mediators. Nevertheless, the microbial interactions involved in this
mechanism are poorly understood. Notably, an unknown produce­
d/excreted metabolite was likely to mediate the extracellular electron
transfer from the electrode to the dechlorinating microorganisms in the
Dehalococcoides contained mix culture (Aulenta et al., 2009). Addtion­
ally, it has been recently found that Geobacter, an anodic- and
cathodic-active bacteria, can transfer a lower ligand such as cobalamin
to Dehalococcoides, which improved the reductive activity of Dehalo­
coccoides (Yan et al., 2012). These studies implied that the IET between
Dehalococcoides and electrode could be enhanced probably by the sym­
biotic metabolism between Dehalococcoides and other cathode-utilizing
functional species. Moreover, a longer enrichment period more than
two months was required for constructing the syntrophic relation in
previous bioelectrochemically dechlorination studies (Leitão et al.,
2016; Lin et al., 2019). However, due to the low growth rate of Deha­
lococcoides, full dechlorination to nontoxic ethene was difficult to be
achieved (Chen et al., 2019a). Thus, if the functional species is presence
in soil environment, full electrode-driven dechlorination can be ach­
ieved by pre-augmenting Dehalococcoides to the contaminated soil and
groundwater. However, could this symbiotic relation be constructed by
pre-augmenting Dehalococcoides or not is remain poorly understand.
Therefore, in this study, the paddy soil was co-inoculated with pure
Dehalococcoides maccartyi NIT01 culture to the cathode with an applied
potential of − 0.3 V vs. a SHE. Also, the open circuit for the co-inoculated
cathode and the polarized cathode inoculating with only pure Dehalo­
coccoides culture or paddy soil was performed. The objectives of the
study presented here were (i) to confirm that if the effectively full
dechlorination can be achieved or not with the proposed strategy and to
assess the dechlorination performance of a BES under different condi­
tions; (ii) to explore the microorganisms which can facilitate the full
bioelectrochemical dechlorination employing Dehalococcoides and (iii)
to understand the interaction of the soil microorganisms with Dehalo­
coccoides related to the electrode-driven dechlorination.
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Environmental Research 209 (2022) 112801
2. Materials and methods
2.1. Dechlorination culture and paddy soil
A pure culture of Dehalococcoides maccartyi NIT01 isolated from an
enriched culture labeled YN3 was used in the bioelectrochemical ex­
periments (Asai et al., 2021). The original inoculum used to prepare the
YN3 culture was sediment from the Arako River, Nagoya, Japan (Ismaeil
et al., 2017). The pure culture was maintained in a 0.5 L batch culture
system (serum bottle, liquid volume 0.3 L), which was fed once a month
with TCE (1 mM), acetate (5 mM), and H2 (80% of the headspace).
Before each feed, 15 mL of the culture was transferred to a new serum
bottle filled with 300 mL of fresh anaerobic DHB–CO3–Br basal medium,
which was a modified version of DHB–CO3 medium with Cl− replaced
with Br− . The basal medium contained 1 g of NaBr, 2.5 g of NaHCO3, 0.5
g of KBr, 0.5 g of NH4Br, 0.1 g of MgBr2⋅6H2O, 0.2 g of KH2PO4, 1 mL of
trace element solution SL10, 10 mL of vitamin solution, and 1 mL of
Se/W solution per liter (Ismaeil et al., 2018). The culture was main­
tained for >5 y and could dechlorinate TCE to give ethene at 28 ± 1 ◦ C in
30 d. The paddy soil used in the experiments was collected in Nisshin
City, Japan (137◦ 04′ 51.3′′ E, 35◦ 08′ 22.3′′ N) and was stored at room
temperature until use.
2.2. Bioelectrochemical apparatus
The two-chamber BES used in the study consisted of two gastight
glass cells (total volume ~65 mL per cell) separated by a proton ex­
change membrane (N117; Fuel Cell Earth, Woburn, MA, USA) with a
cross-sectional area of 6 cm2. The BES is shown in Fig. S1. A piece of
GFA10 graphite felt (5 cm long, 1 cm wide, 0.3 cm thick; SGL, Wies­
baden, Germany) and a platinum wire (0.3 cm diameter, 50 cm long)
were used as the cathode and anode, respectively. The electrodes were
repeatedly soaked in deionized water before being used. A RE-1B satu­
rated Ag/AgCl electrode (+199 mV vs. a SHE) (BAS, Tokyo, Japan) in
the cathode compartment was used as a reference electrode. Polariza­
tion was measured using a potentiostat (Goto and Yoshida, 2017) and
the current was recorded using a digital data logger.
2.3. Bioelectrochemical experiments
Studies of dechlorination using the electrode as an electron donor in
the two-chamber BES were performed at 28 ◦ C. The open circuit voltage
(OCV) tests were performed using a series of gastight 60 mL borosilicate
glass bottles without setting the electrodes. A 20 mL aliquot of the
D. maccartyi NIT01 culture and 40 mL of DHB–CO3 medium were
transferred under anaerobic conditions into the cathode compartment of
the BES, and 65 mL of 100 mM ferrocyanide was added to the anode
compartment. Paddy soil was added to cathode cells to give 2.5% w/w of
the mass present to act as a source of potentially syntrophic microor­
ganisms to facilitate interspecies ET. Acetate, Na2S, and CaBr2 were also
added to the test cultures to give concentrations of 10 mM, 0.3 mM, and
0.01 g/L, respectively. Anaerobic gas (80:20 N2/CO2) was supplied to
replace the headspace above each culture for 15 min to ensure the sys­
tem was anaerobic. The reactors were left unconnected for 30 min until
they were stable, then TCE (to give a concentration of 1 mM) was added
to each reactor. Moreover, 2 μL of 1,1,2–trichloroethane (1,1,2-TCA)
was added to each reactor as the inhibitors of methanogens (Oremland
and Capone, 1988). The working electrode was kept at − 0.3 V (vs. a
SHE) in the BES (in tests called P-Dhc+S later), representing a condition
that dechlorination was almost driven by the ET from electrode without
abiotic H2-generation (Strycharz et al., 2008), while the electrodes were
not placed in the OCV tests (called OCV-Dhc+S later). Control tests using
the BES were performed using the mixture described above but without
paddy soil added, i.e., only the pure D. maccartyi NIT01 culture was
added, and these tests are called P-Dhc later. Also, the H2-fed OCV was
performed by repalcing the headspace of OCV-Dhc+S tests with the H2
L. Meng et al.
contained anaerobic gas (80:20 H2/CO2) (called OCV-Dhc+S+H2 later).
Each test was performed in duplicate.
Replicate batch experiments were performed to investigate the
microbe population structures and to conduct electrochemical analyses
after the stable dechlorination performance was observed. Moreover, to
understand the effect of the physical and chemical properties of paddy
soil on the dechlorination performance, the cathode inoculated with
only paddy soil (called P–S later), and co-inoculated with pure Dehalo­
coccoides culture and autoclaved paddy soil (called P-Dhc+AS later) was
also performed. Based on the second batch test, cyclic voltammetry (CV)
analyses were performed using an EC-lab SP-150 electrochemical
workstation (BioLogic, Seyssinet-Pariset, France) equipped with a three-
electrode system. The cathode was used as the working electrode and a
saturated Ag/AgCl electrode was used as the reference electrode. The
tests were performed using DHB–CO3–Br as the catholyte at pH 7. The
reductive dechlorination capacities of the cathodes in the P-Dhc+S and
P-Dhc tests and of the abiotic cathode were determined in the presence
and absence of TCE. CV curves were acquired over the voltage range
− 0.8 to 0.2 V vs. Ag/AgCl at a scanning rate of 2 mV/s. Each reactor was
allowed to stabilize unconnected for 1 h after catholyte exchange before
a CV test was performed. The TCE concentration used in the tests was 1
mM.
2.4. Analytical methods
The acetate, CEs, hydrogen, methane, and 1,1,2-TCA concentrations
were determined using previously described methods (Yoshida et al.,
2007, 2019). Briefly, 100 μL of a headspace sample taken from a reac­
tion cell was analyzed using a GC-2014 gas chromatograph (Shimadzu,
Kyoto, Japan) equipped with a Porapak Q column (GL Sciences, Tokyo,
Japan) and a flame ionization detector. The hydrogen concentration was
determined by analyzing 100 μL of a headspace sample using a GC-2014
gas chromatograph equipped with a stainless-steel column packed with
a 5A molecular sieve (GL Sciences) and a thermal conductivity detector.
The acetate was measured using an HPLC system (Shim-pack IC-C4
column, Shimadzu, Kyoto, Japan).
2.5. Microbial community analysis
The structures and compositions of the microbial communities in the
cathode biofilms and suspensions in the P-Dhc+S and P–S systems as
well as in the suspensions in the OCV-Dhc+S systems were analyzed by
performing 16S rRNA gene sequencing using a MiSeq system (Illumina,
San Diego, CA, USA). Each cathode graphite felt sample was washed
with 2 mL of sterile water, then the solution was centrifuged at
12,000×g. DNA was then extracted from the cells and polymerase chain
reactions were performed targeting the V3–V4 region of the 16S rRNA
genes using the forward primer 341F (5′ -CCTACGGGNGGCWGCAG-3′ )
and the reverse primer 805R (5′ -GACTACHVGGGTATCTAATCC-3′ )
(Yoshida et al., 2016). PCR products were purified using an AMPure XP
PCR purification kit (Beckman Coulter, Inc., CA, USA). After which they
were sequenced using a MiSeq Reagent Kit v3 on a MiSeq DNA
sequencer (Illumina, USA) in paired-end sequencing mode (2 × 300 bp).
The PliX, low-quality (Q < 20) were removed (Sickle ver. 1.33) (Joshi
and Fass, 2011), and the pair-end sequences were assembled using
FLASH (ver.1.2.11) (Magoč and Salzberg, 2011). The chimeric se­
quences were removed using the data2 plugin on Qiime2 (ver. 2020.8)
(Bolyen et al., 2019) through the alignment with the Greengene data­
base (DeSantis et al., 2006). Taxonomic classification of each phylotype
was determined using the Greengene (ver. 13_8) with over 97% of
sequence similarity. The 16S rRNA gene sequence data were deposited
in the DNA Data Bank of Japan under the accession number of
DRA012421.
General differences in the microbial community structures were
identified by performing the principal component analysis (PCA) using
Canoco version 5.0 software. Network analysis was used to assess the
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Environmental Research 209 (2022) 112801
operational taxonomic units (OTUs) found under the different experi­
mental conditions, and the data were plotted using Cytoscape 3.8.2
software using the detected OTUs to indicate node connectivity between
groups and inter-group relationships (Cline et al., 2007; Herrgård et al.,
2008).
3. Results and discussion
3.1. Bioelectrochemical dechlorination
The data for dechlorination of TCE in ~70 d in the P-Dhc+S, OCV-
Dhc+S, and P-Dhc systems are shown in Fig. 1. TCE was added to each
system at a concentration of 1 mM, and the initial TCE concentrations of
0.9–1.2 mM on day 0 decreased gradually in all four systems (Fig. 1).
However, marked amounts of dechlorination products were only found
in the P-Dhc+S and OCV-Dhc+S+H2 system, in which the total amounts
of dechlorination products were comparable with the initial amounts of
TCE present, respectively (Fig. 1A and D). The OCV-Dhc+S system
produced the minor dechlorination products cis-DCE and VC, and hardly
any dechlorination products were found in the P-Dhc system (Fig. 1B
and C). At the end of the incubation, the total dechlorination product
concentrations found in the OCV-Dhc+S and P-Dhc systems were 26%
and 10% of the initial TCE concentrations, respectively. Moreover, since
the TCE (0.305 mM on days15) was detected in anode chamber in P-Dhc
in the preliminary test (data not shown), the decrease in OCV-Dhc+S
and P-Dhc were mainly caused by movement into the anode chamber or
volatilization rather than reductive dichlorination. As the inhibitor,
1,1,2-TCA show a slight decrease but the obvious dechlorination was not
found to have occurred in all systems (Fig. S2). Thus, the differences
between the amounts of TCE and total CEs present indicated that the
production of non-toxic ETH was mainly caused by bioelectrochemical
dechlorination of TCE in P-Dhc+S.
The maximum ETH formation rate with the BES system was 30 μM/
d, which was found in the P-Dhc+S system and was comparable with
that in OCV-Dhc+S+H2 (38 μM/d). The ETH formation rate achieved in
P-Dhc+S was 103 and 17 times higher than that observed in the OCV-
Dhc+S and P-Dhc systems, respectively (Fig. 1A, B and 1C). The ETH
concentration in the P-Dhc+S system (0.98 mM) was comparable to the
initial TCE concentration, but only trace amounts of ETH were observed
in the OCV-Dhc+S system (<0.3 mM) and P-Dhc system (<0.1 mM).
Effective TCE dechlorination was only achieved in the system containing
both Dehalococcoides and paddy soil under polarized conditions (P-
Dhc+S) and the H2-fed OCV conditions (OCV-Dhc+S+H2). The results
indicated that CEs can be dechlorinated by Dehalococcoides under
polarized conditions, but other microbes are required to facilitate
effective bioelectrochemical dechlorination.
The first aim of this study was to confirm that if the effectively full
electrode-driven dechlorination employing Dehalococcoides can be ach­
ieved or not by the co-relation with soil microorganisms. The perfor­
mance observed through this study was compared to that of previously
reported TCE dechlorinating BES with ORB-enriched cultures or with
electron mediators added (Aulenta et al., 2007a, 2008, 2009; Chen et al.,
2018a; Verdini et al., 2015). The highest ETH formation rate was close to
30 μM/d, which was found for the P-Dhc+S system. This was 10–100
times higher than ETH formation rates previously found for most BESs
using Dehalococcoides-enriched microbial consortia with or without
electron mediators added (Table S1). A higher ETH formation rate (74
μM/d) than this study was reported in a BES using an enriched dech­
lorinating culture without electron mediators added (Chen et al.,
2018a). However, the amount of ETH produced was only 7.6% of the
amount of dechlorination products produced, whereas the amount of
ETH produced was 77.4% of the amount of dechlorination products
produced in the present study (Table S1 and Fig. 1A). In the previous
study, cis-DCE contributed ~80% of the dechlorination products
because the enriched dechlorinating culture lacked Dehalococcoides
species (Chen et al., 2018a). The authors concluded that inoculating
L. Meng et al. Environmental Research 209 (2022) 112801

Fig. 1. Reductive dechlorination of trichloroethene (TCE) and formation of metabolites (ethylene (ETH), vinyl chloride (VC), and cis-dichloroethene (cis-DCE)) in the
inoculated bioelectrochemical system (A) with polarization (P-Dhc+S), (B) with an open circuit (OCV-Dhc+S), (C) with Dehalococcoides maccartyi NIT01 only (P-
Dhc), and (D) with an open circuit and hydrogen fed in headspace (OCV-Dhc+S+H2).

Dehalococcoides into a BES is an effective way of fully dechlorinating
TCE to give ETH. Effective electrode-driven dechlorination of Dehalo­
coccoides can be facilitated by microbes that are found widely in soil.
This would decrease the dependency of the dechlorination process on
enriched microbial consortia or electron mediators by applying low
energy consumed potential. These results indicated that a pure Dehalo­
coccoides culture can be used in a BES to effectively and sustainably
achieve dechlorination.
3.2. Hydrogen and methane production in the BES
The H2 concentration in the BES was measured during incubation to
determine whether hydrogen or electrons were provided to Dehalo­
coccoides (Fig. 2A). As expected, only trace amounts of methane (<0.02
mM) were found in all three systems (Fig. 2B), due to the addition of
1,1,2-TCA as an inhibitor (Oremland and Capone, 1988). The highest H2
concentration (2.4 mM) was found in the P-Dhc system, while H2 con­
centrations in the P-Dhc+S and OCV-Dhc+S systems were 0.1 and 0.4
mM, respectively, which were much lower than the concentration in the
P-Dhc system. Only slight dechlorination was found (Fig. 1C), meaning
electrolytically produced H2 probably contributed to dechlorination in
the P-Dhc system (which contained the pure Dehalococcoides culture).
However, effective dechlorination was not achieved, probably because
of the relatively low H2 production rate at the voltage that was used
(− 0.3 V vs. a SHE) (Cheng et al., 2009). Due to the lack of a certain
metabolite later produced by other soil microorganisms in P-Dhc,
Dehalococcoides had lost their activity or dead before the hydrogen
reached the preferred level. H2 was detected in the P-Dhc+S and
OCV-Dhc+S systems only on day 6 (Fig. 2A), suggesting that the H2 that
was potentially consumed by the interaction of Dehalococcoides and
microorganisms added in the paddy soil. The dechlorination and H2
production results for the P-Dhc+S and P-Dhc systems (Fig. 1A, C, and
2A) suggested that effective dechlorination in the P-Dhc+S system was
principally achieved via interaction of Dehalococcoides and the soil
microorganism, such as the interspecies ET as well as metabolite transfer
(cobamides) as reported in previous studies (Aulenta et al., 2009; Yan
et al., 2012).
3.3. Electrochemical analysis
The replicated batch BES study was repeated following the same
procedure to confirm the dechlorination performance results. Stable

Fig. 2. Temporal changes in the (A) H2 concentration, and (B) CH4 concentration in the inoculated bioelectrochemical system. (P = polarization, OCV = open circuit,
Dhc = Dehaloccoides, S = soil).

4
L. Meng et al.
dechlorination performance and similar trends to those described above
were confirmed (see section 3.1) (Fig. 1A–C and S3A–S3C). Effective
dechlorination was not found in P–S, because the dechlorination product
concentrations were <0.01 mM (Fig. S3D). Moreover, the total con­
centration (<0.2 mM) of the dechlorination product in P-Dhc+S was
much lower than that (0.97 mM) observed in P-Dhc+S (Fig.S3A and
S3E), indicated the physical and chemical properties (e.g., redox medi­
ators, conductivity) of paddy soil maybe contribute to the dechlorina­
tion, but not the main factor for the effective dechlorination in P-Dhc +
S.
The cathode current density for the P-Dhc+S system during TCE
dechlorination was markedly different from the cathode current den­
sities for the P-Dhc and P–S systems, indicating that electrons supplied
by the cathode accelerated effective TCE dechlorination in the P-Dhc+S
system (Fig. S4). Random changes in the cathode current were probably
caused by the dechlorination reaction at the reference electrode, as was
found in a previous study of bioelectrochemical dechlorination using a
Ag/AgCl reference electrode (Fernández-Verdejo et al., 2021).
Once the dechlorination activity had stabilized, CV analysis was
performed to assess the electrochemical characteristics of the cathode
biofilm and catholyte (Fig. 3). The abiotic cathode in the P-Dhc system
and the biocathode in the P–S system with TCE added gave two small
peaks at 0.002 V (peak a) and − 0.03 or − 0.01 V (peaks b and c), but
similar peak areas were found for the P-Dhc+S, OCV-Dhc+S, and P-Dhc
systems (Fig. 3A). The biocathode in the P-Dhc+S system with TCE
added gave two pairs of bigger peaks at − 0.11 V (peak d) and − 0.14 V
(peak e) that were larger than the peaks given by the abiotic cathode
(Fig. 3B). The midpoint between peaks d and e was − 0.13 V, which
indicated that adding TCE affected the electrochemical characteristics of
the system. The P-Dhc+S–C system gave much larger areas for peaks f
(0.27 V) and g (− 0.006 V) than for peaks d (which was a fifth of the area
of peak g) and e (which was one 28th of the area of peak h) (Fig. 3B).
This indicated that the peaks were markedly different from the peaks
given by the abiotic cathode in the P-Dhc+S–B system. The midpoint
potential of the redox pair was +0.138 V (vs. a SHE), which was similar
to the potential for ubiquinone (+0.113 V vs. a SHE) (Du et al., 2007).
Since the reverse electron transfer from several redox molecules to
the functional dechlorination protein, reductive dehalogenase (RDase),
driven by the proton motive force has been reported in a previous study
(Wang et al., 2018). Thus, these unknown redox molecules involved
were probably produced or excreted during metabolism related to bio­
electrochemical dechlorination and may have played an important role
in the mediated interspecies ET or interspecies metabolite transfer in the
mixed culture. The results described above (Fig. 3, S3, and S4) suggested
that bioelectrochemical dechlorination involving the pure Dehalo­
coccoides culture was potentially enhanced by the microbes supplied by
the paddy soil. The CV results and the good dechlorination performance
suggested that redox mediators produced by the microbes effectively
caused electrode-driven dechlorination.
Fig. 3. (A) Cyclic voltammograms of the abiotic cathode and biocathodes (P-Dhc-B and P–S–B). (B) Cyclic voltammograms of the abiotic cathode, biocathode (P-
D+S–B), and catholyte (P-D+S–C). Each test was performed in the presence of trichloroethene. The potential range was 0.4 to − 0.6 V versus a standard hydrogen
electrode. Each experiment was performed using neutral conditions (DHB–CO3–Br basal medium, pH 7).
5
Environmental Research 209 (2022) 112801
3.4. Microbial community structures and compositions
The secondary BES test results (Fig. S3A–4D) led us to perform 16s
rRNA gene-based amplicon analyses of the samples collected under
different operating conditions to gain insights into the dynamics of the
microbial community. The dominant phyla and classes are shown in
Fig. S6.
A light grey biofilm was observed on the biocathode of P-Dhc+S
(Fig. 6). The dominant genera are shown in Fig. 4. Desulfosporosinus,
Pseudomonas, and Clostridium were the dominant genera in the biofilm in
the P-Dhc+S system, with relative abundances of 23.2%, 8.7%, and
6.9%, respectively. Oscillospira, Caloramator, Desulfovibrio, and five
other genera were also detected but at relative abundances <5%. In
contrast, Clostridium was the dominant genus and Cellulomonas was the
second most dominant genus in the P–S–B system biofilm, with relative
abundances of 41.6% and 25.9%, respectively. The Desulfosporosinus
relative abundance was only 5.4%. Desulfosporosinus was the dominant
genus in the P-Dhc+S and P–S system catholytes, the relative abun­
dances being 70.6% and 27.9%, respectively. Most of the other genera
found in the P-Dhc+S–C system catholyte had relative abundances <5%,
but Massilia had a relative abundance of 7.9%. Clostridium, Cellulomonas,
and Massilia were found in the P–S–C system catholyte at relative
abundances of 21.8%, 18.2%, and 11.9%, respectively (i.e., >10%). The
dominant genera in the OCV-Dhc+S system catholyte were Thermoa­
naerosceptrum and Sedimentibacter, which had relative abundances of
46.9% and 5.7%, respectively, and the other genera had relative abun­
dances <1%. The microbial communities in the biofilms and catholytes
were clearly different. Very different microbial profiles were found for
the cultures acquired after polarization and in the OCV tests. The results
indicated that Desulfosporosinus was the most enriched genus in the P-
Dhc+S system. This may have been related to the higher
Fig. 4. Taxonomic classification of the MiSeq sequenced bacterial communities
from the biofilm and catholyte from the P-Dhc+S, P–S, and OCV-Dhc+S sys­
tems at the genera level (relative abundances >1%).
L. Meng et al.
bioelectrochemical dechlorination performance of the P-Dhc + S system
than the P–S and OCV-Dhc+S systems (Fig. 4 and S3).
Desulfosporosinus have not been found to be dominant in other BESs.
The dominant genera in a BES in which activated sludge were the
original inoculum were Geobacter, Lactococcus, and Pseudomonas (Chen
et al., 2018b, 2019b, 2018b). Desulfosporosinus is a sulfate-reducing and
halogen-respiring bacteria (e.g., it weakly dechlorinates tetra­
chloroethene to VC but cannot respire using VC) (Ramamoorthy et al.,
2006). Some species of Desulfosporosinus also show cathode activity
(Rodrigues and Rosenbaum, 2014). Representative Dehalococcoides
species are capable of dechlorinating VC to give ETH (Cheng and He,
2009), however had a quite low abundance (0.02%, data not shown),
which could explain why the dechlorination reaction in the P-Dhc+S
system almost stopped on day 20 (Fig. S3A). The low abundance prob­
ably due to the nutrients or electron donor competition between Deha­
lococcoides with other soil microorganisms. The enrichment of such
competitors during the incubation reduced the electron uptake effi­
ciency of Dehalococcoides. Further study is needed for understanding this
mechanism and clarify the operating condition for conducting a more
stable syntrophic relationship.
Cellulomonas, Clostridium, Oscillospira, Massilia, and Pseudomonas
have been found in various BESs used to produce electricity or dechlo­
rinate pollutants (Awate et al., 2017; Choi et al., 2014; Lu et al., 2019;
Sotres Fernández, 2015). Most of these genera were found in the
P-Dhc+S and P–S systems but not in the OCV-Dhc+S system (Fig. 4),
indicating that they were closely associated with polarization. Some
fermentative genera (including Bacillus, Caloramator, Macellibacterioides,
and Ruminococcus) had higher abundances in the P-Dhc+S and P–S
systems (1.4%–3.7%) than in the OCV-Dhc+S system (<0.1%) (Ai et al.,
2018; Jabari et al., 2012; Latham and Wolin, 1977; Ogg and Patel,
2009). Thermoanaerosceptrum and Sedimentibacter, which are also
fermentative genera, were only detected in the OCV-Dhc+S system and
had abundances of 46.9% and 5.7%, respectively (Imachi et al., 2016;
Yamagishi et al., 2019). This indicated that different fermentative
pathways were followed under different operating conditions and with
and without polarization. We concluded that Desulfosporosinus species
were the main microbes that facilitated bioelectrochemical dechlorina­
tion by the pure Dehalococcoides culture.
3.5. PCA and network analysis
The PCA results indicated that the genera found in the systems
operated under different conditions (with and without polarization)
were very different, which indicated that the functional bacterial com­
munity compositions were different (Fig. 5A). The abundances of the
particularly enriched genus Desulfosporosinus and the other genera
(Caloramator, Cellulomonas, Clostridium, Massilia, Oscillospira, and
Fig. 5. (A) Principal component analysis results for the identified operational taxonomic units for the OCV, P-Dhc+S (biofilm and catholyte), and P-S (biofilm and
catholyte) systems. (B) Operational taxonomic unit sharing network for the bacterial communities in the bioelectrochemical system used to dechlorinate tri­
chloroethene with and without polarization.
6
Environmental Research 209 (2022) 112801
Pseudomonas) found in other BESs were positively associated with the
degree of polarization. The PCA profiles for the P-Dhc+S and P–S sys­
tems were different, indicating that functional genera such as Desulfo­
sporosinus were associated with dechlorination. Fermentative genera
(Bacillus, Sedimentibacter, and Thermoanaerosceptrum) were found in the
OCV-Dhc+S system. The PCA results indicated that there were clear
relationships between the bacterial consortium structures and certain
environmental variables.
Microbiome network analysis was used to visualize the associations
between the OTUs and conditions (Fig. 5B). A total of 392 OTUs were
distributed among all operating conditions, and exclusive OTUs
belonged to the OCV–Dhc+S and P-Dhc+S–B were 58 and 53, which
were more abundant than in the other cultures (Fig. 4B). The genera that
were found in more than one system (including Cellulomonas, Clos­
tridium, Desulfosporosinus, Massilia, and Oscillospira) were clustered
together in the center of the network. Desulfosporosinus are
organohalide-consuming bacteria (Robertson et al., 2001) and some of
the genera (Cellulomonas, Clostridium, Massilia, and Oscillospira) are
electroactive or have been found to be highly enriched in BESs (Awate
et al., 2017; Choi et al., 2014; Lu et al., 2019; Sotres Fernández, 2015).
The network analysis results indicated that there were more unique
OTUs in the P-Dhc+S and OCV-Dhc+S systems than the other systems
and that almost all of the OTUs in the P–S system were also found in the
other cultures. This may have been because the P–S system operating
conditions were between the P-Dhc+S and OCV-Dhc+S system oper­
ating conditions, i.e., polarization was used but TCE was not dechlori­
nated because Dehalococcoides was not added. This indicated that the
relationship between the Dehalococcoides abundance and the functional
OTUs was strong in the P-Dhc+S system and that this facilitated
biochemical dechlorination by the pure Dehalococcoides culture.
3.6. Dechlorination of TCE during metabolic processes involving
polarization
The dechlorination products and pathways led us to develop the
conceptual model for enhanced TCE dechlorination in the biocathode
BESs shown in Fig. 6. Representative Dehalococcoides species dechlori­
nate VC to give ETH, but effective dechlorination did not occur in the P-
Dhc or P–S system (Fig. 1 and Fig. S3D). The authors therefore
concluded that the effective dechlorination was mainly achieved via the
interaction of Dehalococcoides and the soil microbes, such as
Desulfosporosinus.
The CV results indicated that two unknown redox molecules could
have facilitated reductive dechlorination. Since the effective dechlori­
nation was also not observed in the BES inoculated with autoclaved soil
(P-Dhc+AS, Fig. S3E), these functional redox molecules were not the
potential redox mediators (e.g., humic substances) from the original
L. Meng et al.
Fig. 6. Conceptual model of enhanced trichloroethene (TCE) dechlorination in
the bioelectrochemical system.
paddy soil, but should be produced/excreted by the enriched bacteria.
These redox molecules potentially mediated the interspecies ET from
soil microbes to Dehalococcoides and finally contributed to the effective
dechlorination in P-Dhc+S. Moreover, the interspecies metabolite
transfers also potentially occurred and contributed to the dechlorina­
tion. In a previous study, a PCE dechlorinator, Geobacter was found
could transfer a ligand (e.g., cobalamin) to Dehalococcoides and that this
could support reduction by Dehalococcoides (Yan et al., 2012). However,
Geobacter was not dominant in the P-Dhc+S system, but another
potentially electroactive species, Desulfosporosinus, was dominant in
both the P-Dhc+S system biofilm and suspension (Fig. 5). It has been
known that this species could weakly dechlorinate PCE to cis-DCE
(Robertson et al., 2001), which potentially produce and share cobamides
like Geobacter. Thus, the dominant species in the P-Dhc+S system (e.g.,
such as Desulfosporosinus species) could therefore have been involved in
interspecies metabolite transfer to Dehalococcoides and facilitated
dechlorination of TCE to ETH. However, since the homology species of
Desulfosporosinus can growth with H2 as the electron donor, the cathodic
activity of the dominant microbes could not be confirmed in the present
study. Also, the detail mechanisms of interaction between Dehalo­
coccoides and Desulfosporosinus need to be studied, such as by the
co-culture test.
The trace amount of hydrogen also contributed to the dechlorination
in P-Dhc+S, since it has been shown that ET from the electrode to
dechlorinators via intermediate electrolytically produced H2 is an
important pathway that is largely dependent on the applied cathode
potential (Aulenta et al., 2011; Lai et al., 2017). Theoretically, the
applied cathode potential (− 0.3 V vs. a SHE) was not negative enough
for sustainable electrolytic H2 production. The trace amounts of
hydrogen detected in the present study maybe because the introduced
biocatalysts changed the unfavorite energy balance. Similar results were
also found in a study performed by Leitão (Leitão et al., 2015). However,
although H2 were detected in all conditions, effective dechlorination
was only observed in P-Dhc+S suggesting H2 was not the main role for
the effective dechlorination in this study. These results suggested that
hydrogen contributed to the reductive dechlorination, nevertheless, the
functional redox molecules which promoted the cycle of hydrogen uti­
lization and dechlorination were more important for the effective
dechlorination in the present study.
4. Conclusions
Microorganisms capable of having symbiotic relationships with
Dehalococcoides in the BES to achieve effective dechlorination were
investigated. The ETH formation rate was 10–100 times higher than
7
Environmental Research 209 (2022) 112801
what has been found in previous studies using BESs using Dehalo­
coccoides-enriched microbial consortia with and without electron me­
diators added. The effective dechlorination performance did not appear
to involve electron transfer only via intermediate electrolytically pro­
duced H2 but was mainly attributed to microbial interactions through
interspecies electron transfer. The microbial structure analysis results
indicated that Desulfosporosinus species were potentially syntrophic with
Dehalococcoides and that the syntrophic relationships facilitated
electrode-driven dechlorination. The results suggested that inoculating a
BES with pure Dehalococcoides is an effective way of achieving full
dechlorination.
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.
Acknowledgments
This work was supported by the Japan Society for the Promotion of
Science through a Grant-in-Aid for Young Scientists (grant no.
21K17900), the JSPS Joint Research Program (JRP) with National
Natural Science Foundation of China (NSFC), and the Kato Memorial
Bioscience Foundation. The authors thank Mrs. Kyou Ikeru, Tomomi
Suzuki, and Asuka Akita (Nitech) for technical assistance. We thank
Gareth Thomas, PhD, from Edanz (https://jp.edanz.com/ac) for editing
a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envres.2022.112801.
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