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Enhanced removal of 1,2-dichloroethane by anodophilicmicrobial consortia
Hai Pham, Nico Boon, Massimo Marzorati, Willy Verstraete*
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B 9000 Gent, Belgium
a r t i c l e i n f o
Article history:
Received 16 January 2009
Received in revised form
2 April 2009
Accepted 2 April 2009
Published online 17 April 2009
Keywords:
Microbial fuel cell
Anodophilic bacteria
1,2-Dichloroethane
* Corresponding author. Tel.: þ32 (0)9 264 59E-mail addresses: willy.verstraete@ugentURL: http://labmet.ugent.be
0043-1354/$ – see front matter ª 2009 Elsevidoi:10.1016/j.watres.2009.04.004
a b s t r a c t
1,2-Dichloroethane (1,2-DCA) is a well-known recalcitrant groundwater contaminant. New
environment-friendly approaches for the removal of 1,2-DCA that does not bring about
volatilization of the compound are required. In this study, different anodophilic consortia
enriched in microbial fuel cells (MFCs) operated under airtight conditions were shown to
effectively degrade 1,2-DCA (up to 102 mg per liter reactor volume per day), while
concomitantly generating a current. An anodophilic consortium previously enriched with
acetate as the electron donor changed its composition at the rate of 48% per week and
increased its richness (Rr) 3-fold, upon adapting to 1,2-DCA as the new electron donor.
After being stable, during 1 month of operation, it removed up to 95% of the 1,2-DCA
amount in the medium in the first 2 weeks, while converting 43 � 4% of electrons available
from the removal to electricity. A natural consortium from a 1,2-DCA contaminated site
changed its composition at the rate of 9% per week and increased its Rr 2-fold, upon
adapting to the MFC anode conditions with 1,2-DCA as the electron donor. After being
stable, during 1 month of operation, it removed up to 85% of the 1,2-DCA amount in the
medium in the first 2 weeks and the coulombic efficiency was 25 � 4%. The operation of the
MFCs under closed circuit conditions resulted in higher 1,2-DCA removal rates than the
operation under open circuit conditions, indicating that bioelectrochemical activities
enhanced the removal of 1,2-DCA in the MFC anode. The production of ethylene glycol,
acetate and carbon dioxide indicated that the anodophilic bacteria oxidatively metabolized
1,2-DCA, probably by means of a hydrolysis-based pathway. The results show that MFCs
can be potentially used as a practically convenient technology for the biological removal of
1,2-DCA.
ª 2009 Elsevier Ltd. All rights reserved.
1. Introduction Dinglasan-Panlilio et al., 2006). Poor disposals practices resul-
1,2-Dichloroethane (1,2-DCA), or ethylene dichloride, is the
most abundant chlorinated industrial product (Bhatt et al.,
2007; De Wildeman et al., 2003). It is also an intermediate in
some other industrial processes (such as the synthesis of
fluorocarbon and 1,1,1-trichloroethane) (Bhatt et al., 2007;
76; fax: þ32 (0)9 264 6248.be, haithe.pham@ugent.
er Ltd. All rights reserved
ted in a wide spreading of this compound in groundwater
(Janssen et al., 1984, 1994). This is of major concern since the
compound may cause damages to kidney, liver and nerve
systems in humans and is a suspected carcinogen (Hughes
et al., 1994). Moreover, the possibility of human exposure to it
through groundwater is considerable (Hughes et al., 1994).
.be (W. Verstraete).
.
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 6 2937
Approaches to degrade 1,2-DCA have been investigated
intensively. Together with physical and chemical methods,
biodegradation of 1,2-DCA using microorganisms deserves
attention as it offers environment-friendly and low cost reme-
diation (De Wildeman and Verstraete, 2003; Marzorati et al.,
2006). 1,2-DCA can be microbially degraded both under aerobic
and anaerobic conditions (Dinglasan-Panlilio et al., 2006). A
number of pure cultures capable of aerobically degrading 1,2-
DCA have been isolated; most of them belonging to the bacterial
genera of Pseudomonas, Xanthobacter and Ancylobacter (Hage and
Hartmans, 1999; Janssen et al., 1985; Stucki et al., 1983;
Vandenwijngaard et al., 1992). These bacteria use 1,2-DCA as the
electron donor and the degradation occurs either through
a hydrolysis dehalogenation pathway or through an oxidation
pathway both leading to the formation of intermediates such as
2-chloroethanol and monochloroacetate and finally to the
formation of CO2 (Dinglasan-Panlilio et al., 2006; Nobre and
Nobre, 2004). Anaerobic degradation of 1,2-DCA is mainly based
on reductive dechlorination, in which the compound plays
the role of an electron acceptor (Bhatt et al., 2007). Bacteria
capable of doing this are Dehalococoides ethenogenes (He et al.,
2003; Maymo-Gatell et al., 1999) and some methanogens such as
Methanobacterium thermoautotrophicum, Methanosarcina barkeri
and Methylosinus trichosporium that can cometabolically dechlo-
rinate 1,2-DCA to ethene (Egli et al., 1987; Oldenhuis et al., 1989).
Notably, a pure culture, Desulfitobacterium dichloroeliminans DCA
1, was reported to be capable of complete reductive dehaloge-
nationof1,2-DCAtoethene(DeWildemanetal., 2003;Maeset al.,
2006; Marzorati et al., 2007).Onlyrecently, anaerobic oxidation of
1,2-DCA with nitrate as the electron acceptor has been reported
for the first time (Dinglasan-Panlilio et al., 2006).
One challenge to the current 1,2-DCA degradation tech-
nologies is that the compound is volatile (Gordon et al., 2002)
and thus can escape into the air from groundwater, causing
a secondary pollution. Therefore, a technology that can cope
with this challenge is needed. As both aerobic and anaerobic
oxidation of 1,2-DCA is feasible, the bioanode of a microbial
fuel cell could be used to degrade 1,2-DCA under gastight
conditions. In bioanodes, microorganisms catalyze the
oxidation of substrates, converting part of the chemical
energy available in substrates into electrical energy (Allen and
Bennetto, 1993; Clauwaert et al., 2008). Reductive dehaloge-
nation of chlorinated compounds using MFCs has been
reported (Aulenta et al., 2007, 2008; Strycharz et al., 2008).
However, in those studies, the process was mainly based on
the activity of microorganisms at the cathode. In addition, no
MFC studies have addressed 1,2-DCA removal. In this article,
we reported for the first time the use of a microbial fuel cell for
an accelerated removal of 1,2-DCA, based on the anode
oxidation of this compound.
Fig. 1 – Descriptive plot of the MFC reactor (A) and
simplified scheme (B) of the setup to treat 1,2-DCA used in
this study. R: external resistor; CEM: cation exchange
membrane. Not shown in the figure is the cathode loop
recirculating the catholyte (hexacyanoferrate). The unit of
the dimensions of the anode working space indicated in (A)
is cm.
2. Experimental section
2.1. MFC reactors
Each microbial fuel cell reactor used in this study was con-
structed with Perspex frames and contained an anode
compartment and a cathode compartment (Fig. 1A). A cation-
specific membrane (Ultrex CMI7000, Membranes International
Inc., US) separated the two compartments. Each compartment
included an endplate (10 � 10 � 1.5 cm3) and a square-holed
subframe (8 � 8 � 0.9 cm3), with 0.9 cm being the distance
from the endplate to the membrane (Fig. 1A). There were two
side holes on each frame for the influent and effluent. The
working space of each compartment had a 7 � 7 � 0.9 cm3
dimension. The anodic electrode was a graphite plate
(5 � 4 � 0.3 cm3) (Le Carbone, Belgium) in contact with
a graphite rod (0.5 cm diameter) (Le Carbone, Belgium) that
penetrated the anode endplate through a hole (0.5 cm diam-
eter). Hence, the net anodic capacity (NAC) was 38 mL (cm3)
or 38 � 10�6 m3 (¼the anode working space � the inserting
electrode volume ¼ 7 � 7 � 0.9 cm3 � 5 � 4 � 0.3 cm3). The
cathode working space was filled with graphite granules
(diameters between 1.5 and 5 mm, Le Carbone, Belgium) in
contact with a graphite rod (0.5 cm diameter) (Le Carbone,
Belgium) that penetrated the cathode endplate through a hole
(0.5 cm diameter). The catholyte was an aqueous solution of
50 mM K3Fe(CN)6 and 100 mM KH2PO4 buffer (Merck, Belgium)
adjusted to pH 7 with 1 M NaOH. It was recirculated through
the cathode matrix and replenished before its decoloration
(Aelterman et al., 2006). The anode and cathode graphite rods
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 62938
were connected to an external resistor of 20 ohm and to a data
acquisition system (Fig. 1A). After assembling, the whole
reactor was sealed using superglue (SUPERCOL, BricoBi,
Belgium) to make it airtight.
2.2. Operation
Two types of MFCs were investigated for their capability of
removing 1,2-DCA. The first type (AceMFCs) included two
MFCs that already had acquired well-performing anodic
microbial consortia because they were previously enriched
with acetate, fed continuously, as the substrate. With an
acetate loading rate equal to 19 g COD L�1 NAC d�1, the
AceMFCs had generated a stable current of 6.8 � 0.2 mA for
2 months. This corresponds to the power output of
24.3 � 0.1 W m�3 NAC or 462 � 2 mW m�2 anodic electrode
surface, which are comparable with other working MFC
systems (Clauwaert et al., 2008). When the experiments
started, the acetate feeding was stopped and the entire
anodic content of each of the AceMFCs was replaced by a 1,2-
DCA containing medium before its anode compartment was
connected to a medium circulating system. The 1,2-DCA
containing medium was a modified M9 medium (with 6 g L�1
Na2HPO4.2H2O, 3 g L�1 KH2PO4.2H2O, 0.1 g L�1 NH4Cl, 0.05 g
L�1 NaCl, 0.24 g L�1 MgSO4.7H2O and trace elements as
previously described (Rabaey et al., 2005)) supplemented with
2.5 mL L�1 of a solution of 0.4 M 1,2-DCA in isopropanol.
(Isopropanol was used to lower the volatilization of 1,2-DCA).
Thus the final concentration of 1,2-DCA in the medium was
1 mM (or 99 mg L�1). Five hundreds mLs of the 1,2-DCA con-
taining medium was circulated between an airtight reservoir
that had a headspace volume of 100 mL and the anode
compartment of each MFC at the flow rate of ca. 0.5 mL min�1
by a pump and through an airtight tubing system (Fig. 1B). To
investigate the removal of 1,2-DCA in relation to bio-
electrochemistry, one AceMFC was operated under closed
(electrical) circuit conditions and the other under open
(electrical) circuit conditions while other conditions were the
same for both.
The second type of MFCs (InoMFCs) included two newly
started reactors, each of which was inoculated with 10 mL of
a 1,2-DCA degrading sample from a natural site contaminated
with 1,2-DCA (Maes et al., 2006) in the anode compartment
when the experiments started. When the experiments star-
ted, the InoMFCs were operated with the 1,2-DCA containing
medium in a mode similar to that applied to the AceMFCs, as
described above. Also, one InoMFC was operated under closed
circuit conditions and the other under open circuit conditions
while other conditions were the same for both.
To investigate whether the current production was due to
the degradation of 1,2-DCA or not, a medium with similar
composition, including 2.5 mL L�1 of isopropanol, but not
containing 1,2-DCA, was used to feed the reactors at times of
interest.
A system that circulated the 1,2-DCA containing medium
between a reservoir and an autoclaved and uninoculated MFC
type reactor was set up and monitored as an additional
control to investigate if 1,2-DCA could escape from the
system, adsorb to the reactor materials or could be abiotically
degraded.
2.3. Bacterial community analysis and isolation
In the first month of operation with 1,2-DCA as the
electron donor, after the first, the second and the forth
week, bacterial cells on 1 cm2 of the electrode surface of
each of the MFCs were sampled and suspended in 2 mL of
100 mM phosphate buffer saline (PBS). For the extraction
of total DNA of the original acetate-fed consortium, the
corresponding bacterial suspension as such was used.
For the extraction of total DNA of an anodic community at a
certain experiment time, the mixture of 2.5 mL of
the medium in the reservoir and 1/10 of the corresponding
bacterial suspension from 1 cm2 electrode surface
was used.(The liquid medium volume to electrode surface
ratio was 500 mL/20 cm2 ¼ 2.5 mL/0.1 cm2). The latter was
done because the system was operated in a recirculative
mode, thus the planktonic cells growing in the medium
should also be taken into account. The original 1,2-
DCA degrading inoculum was used as such for DNA
extraction.
Total DNA of the aforementioned sample mixtures were
extracted using standard methods (Boon et al., 2000). 16S rRNA
gene fragments were amplified with the primers PRBA338fGC
and P518 (Muyzer et al., 1993) and analyzed using denaturing
gradient gel electrophoresis (DGGE) with a denaturing
gradient ranging from 45 to 60% (Boon et al., 2002). Based on
the DGGE patterns, bacterial communities of the MFCs were
analyzed in terms of dynamics (rate of composition change),
range-weighted richness and functional organization (Pareto–
Lorenz curves) using the methods proposed previously (Mar-
zorati et al., 2008). For more details about these methods, see
Supporting Material.
2.4. Analysis
1,2-DCA in the medium was measured by headspace gas
chromatography with a flame ionization detector (Chrompack
9002) as previously described (De Wildeman et al., 2003). The
headspace gas was sampled using a needle by injecting it
through the gastight rubber stopper of the medium container.
The same procedure was applied to detect and measure
ethene, ethane, vinyl chloride, 2-chloroethanol, ethylene
glycol, which are the potential intermediates and products
produced during the removal of 1,2-DCA. The concentration of
ethylene glycol was calculated using calibration curves
established from the measurement of standards made from
the pure compounds (purchased from Sigma–Aldrich). CO2
and CH4 produced in the headspace of a medium reservoir
was measured with an Intersmat IGC 120MB gas chromato-
graph connected to a Hewlett–Packard 3390A. Chloride (Cl�)
was determined using an ion chromatography (Compact IC
761 with conductivity detector, Metrohm, Switzerland) with
a metrosep A supp 5 column and a metrosep A 4/5 guard
column. Acetate was measured by gas chromatography
(Rabaey et al., 2003). Electrical parameters were measured and
calculated as previously described (Logan et al., 2006). All
analyses or measurements, unless otherwise explained, were
repeated three times. Data reported were taken from three
repetitions of each experiment (n ¼ 3).
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 6 2939
3. Results
3.1. Current generation corresponding to 1,2-DCAremoval by the MFCs
In the first month of operation with 1,2-DCA as the main
substrate, the current generation by the closed circuit MFCs
started to increase after the first week (Fig. 2A), together with
the decrease of 1,2-DCA concentration in the medium (data
not shown). The current production reached a maximum level
in the third week (Fig. 2A). The maximum level of the current
generated by the AceMFC (containing a consortium previously
enriched with acetate) was 0.17 � 0.02 mA while that by the
InoMFC (inoculated with a sample from a natural site
contaminated with 1,2-DCA) were 0.10 � 0.03 mA (Fig. 2A).
The coulombic efficiency of the former was also higher than
that of the latter: 43 � 4% vs. 25 � 4%. After reaching the
Fig. 2 – (A): Typical patterns of current generation by the
MFCs during the first month of operation with 1,2-DCA as
the substrate (i.e. the adaptation period).(B): The changes of
the current generated by the AceMFC (after the anodic
consortium was stable) in response to the absence of 1,2-
DCA in the feeding medium. Dashed arrows indicate the
time points when the medium containing no 1,2-DCA was
fed to the systems. Solid arrows indicate the time points
when the medium containing 1,2-DCA was again fed to the
systems.
maximum level, if the AceMFC was operated with the medium
that had a similar composition, including 2.5 mL L�1 of iso-
propanol, but not containing 1,2-DCA, the current signifi-
cantly decreased to the background level of about 0.05 mA
(Fig. 2B). The InoMFC showed similar responses (data not
shown). This indicated that the current generation was due to
the presence of 1,2-DCA in the medium and that isopropanol
in the medium was not used by the anode bacteria as the main
electron donor.
3.2. Changes in bacterial communities of the MFCs
The most significant shifts of the bacterial communities in the
anodes of the MFCs occurred already after 1 week of operation
with 1,2-DCA as the substrate, as shown by the DGGE patterns
(Fig. 3), the changes of range-weighted richness (Rr) and
functional organization (Fo) values (Table 1, Fig. S1). During
the following weeks, smaller shifts continued to occur for
most of the communities, except for that of the open circuit
InoMFC. After 2 weeks, the changes slowed down for the
AceMFCs. The dynamic values (rates of change) of the AceMFC
communities were 5–6-fold higher than those of the InoMFC
ones: 48.3 vs. 8.9%/week (closed circuit conditions) and 47.3 vs.
9.3%/week (open circuit conditions).
Interestingly, when stability was achieved after the first
4 weeks, the community of the AceMFC operated under closed
circuits conditions was not significantly different from that
operated under open circuit conditions (Fig. 3). The commu-
nities indeed had changed at approximately equal rates (Table
1). However, in terms of richness and functional organization,
it could be observed that after the first week, the community
Fig. 3 – DGGE patterns of the 16S rRNA gene fragments of
the bacterial communities from the AceMFCs and the
InoMFCs at different time points during the first month of
operation with the 1,2-DCA containing medium (i.e. the
adaptation period). Meanings of sample abbreviations: A:
AceMFC; I: InoMFC; C: closed circuit; O: open circuit; to: at
the starting time of the experiment (1,2-DCA feeding
started); 1w: after 1 week; 2w: after 2 weeks; 4w: after
4 weeks.(Each sample was taken and analyzed in triplicate
and similar patterns were observed. Thus only one
triplicate of each sample was selected for this gel analysis).
Table 1 – Analyses of bacterial communities of the MFCs in terms of the rate of change, range-weighted richness andfunctional organization (after Marzorati et al. (2008)) during the first month of operation with 1,2-DCA containing medium(i.e. the adaptation period).
Community Rate of change (Dy) (%/week) Range-weighted richness (Rr) Functionality organization (Fo)(Gini index value*)
To 1w 2w 4w To 1w 2w 4w
AceMFC (closed ) 48.3 15.5 45.9 27.8 31.6 0.65 0.39 0.52 0.49
AceMFC (open) 47.3 15.5 38.5 21.6 25.3 0.65 0.49 0.52 0.51
InoMFC (closed ) 8.9 6.1 13.2 10.9 15.7 0.67 0.51 0.48 0.39
InoMFC (open) 9.3 6.1 6.0 2.4 1.6 0.67 0.63 0.68 0.65
*: the Gini index value reflects the degree of evenness of species distribution in a microbial community and is a value representing the area
between a given Pareto–Lorenz curve and the line of the perfect evenness. A perfectly even community corresponds to a Gini ¼ 0 (Lee, 1997;
Marzorati et al., 2008). Note: To: at the beginning of the experiments (1,2-DCA feeding started); 1w: after 1 week; 2w: after 2 weeks; 4w: after
4 weeks; closed: operated under closed circuit conditions; open: operated under open circuit conditions.
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 62940
of the closed circuit AceMFC changed more profoundly than
that of the open circuit AceMFC, becoming richer with more
even species distribution (Table 1, Fig. S1A). Specifically, the Rr
of the former increased from 15.5 to 45.9 while that of the
latter increased to 38.5; the Fo value of the former decreased
from 0.65 to 0.39 (i.e. the species within the community
became more evenly-distributed), while that of the latter only
decreased to 0.49. In these communities, dominant species of
the previous acetate-fed community became less common
while several previously less prominent species became
dominant (Fig. 3, Fig. S1A).
The anode bacterial community of the closed circuit
InoMFC also changed after the first week, becoming different
from that of the original inoculum and from those of the
AceMFCs (Fig. 3). However, no species tended to be dominant
after 4 weeks (Figs. 3 and S1B). The anode bacterial commu-
nity of the open circuit InoMFC remained unchanged and died
away. This can be observed from the decrease in intensity of
the DGGE bands (Fig. 3) and of the Rr values (Table 1).
3.3. 1,2-DCA removal by the MFCs
Hereafter, unless otherwise indicated, results are reported for
a batch of 1 month operation of the reactors after the micro-
bial consortia were stable (i.e. after the first month, which was
the adaptation period). Three replicates in each case repre-
sented 3 months of operation, during which the 1,2-DCA
containing medium was renewed after each month. The
AceMFC operated under closed circuit conditions already
removed more than 55% of 1,2-DCA after 1 week of operation
(Fig. 4B). Its highest removal rate of 102 mg 1,2-DCA L�1 NAC
d�1 was thus achieved in the first week. After 2 weeks, almost
95% of 1,2-DCA was removed by this closed circuit AceMFC.
After 4 weeks, the removal was 98%. The average 1,2-DCA
removal rate of this MFC in 1 month was 45.6 � 0.5 mg L�1
NAC d�1. The AceMFC operated under open circuit conditions
(after 1 month of enrichment) only removed about 25, 53 and
73% of 1,2-DCA after 1, 2 and 4 weeks of operation, respec-
tively. The average 1,2-DCA removal rate of the open circuit
AceMFC in 1 month was 34.0 � 1.5 mg L�1 NAC d�1.
Overall, the closed circuit InoMFC had a poorer perfor-
mance, compared to the closed circuit AceMFC. It removed 32,
85 and 95% of 1,2-DCA after 1, 2 and 4 weeks of operation,
respectively (Fig. 4B). The highest removal rate of 98 mg 1,2-
DCA L�1 NAC d�1 was achieved in the second week. The
average 1,2-DCA removal rate of this closed circuit InoMFC in
1 month was 44.1 � 0.2 mg L�1 NAC d�1. The open circuit
InoMFC almost could not remove 1,2-DCA (Fig. 4B).
The 1,2-DCA concentrations in the autoclaved and unin-
oculated MFC (control) did not change significantly (Fig. 4B),
indicating that 1,2-DCA did not escape from the system or
adsorb onto the materials and was not abiotically degraded.
It should be noticed that the production of current by the
MFCs corresponded to the 1,2-DCA removing capability of
the MFCs (Figs. 4A,B). Usually after 4 weeks of operation, the
currents decreased, probably due to the shortage of the main
electron donor (1,2-DCA).
3.4. Changes of the pH, chloride and CO2 concentrations
The amount of chloride produced was proportional to the
amount of 1,2-DCA removed (Fig. 4C, Table 2). After 4 weeks of
operation, the concentration of chloride produced in the
medium of the AceMFC reached 2 mM, which is the theoret-
ical maximum concentration of chloride that can be produced
from 1 mM 1,2-DCA. Chloride was produced at the highest
rates and levels in the closed circuit AceMFC (Fig. 4C).
CO2 was produced in the systems together with the
removal of 1,2-DCA (Table 2). Interestingly, the CO2 concen-
trations were the highest after 1 week of operation. CO2 was
produced the most in the closed circuit AceMFC (up to
0.22 mmol L�1 medium) and the second most in the closed
circuit InoMFC. Much less CO2 was produced in the reactors
operated under open circuit condition and no CO2 was
detected in the open circuit InoMFC. CO2 was not produced
when the MFCs were fed with the medium containing no 1,2-
DCA. Also, in the control (abiotic MFC), no CO2 was detected
(Table 2). These observations indicate that the production of
CO2 was a biological process related to the removal of 1,2-DCA.
The pH of the medium decreased in the closed circuit
AceMFC and closed circuit InoMFC (Table 2) although the
medium was buffered. For the AceMFC, the pH decrease was
the most pronounced, from 7.1 � 0.1 to 6.6 � 0.1 after 1 week
of operation, and to 6.5 � 0.1 after 2 weeks. The pH changes in
the InoMFC were lower but had a similar trend. Finally, the pH
changes were also less prominent in the AceMFC operated
Fig. 4 – The changes of average current (A), 1,2-DCA concentration in the medium (B) and chloride concentration in the
medium (C) during a month of operation of the MFCs with their stable anodic consortia. Control: the autoclaved and
uninoculated MFC operated under closed circuit conditions; closed circuit: operated under closed circuit conditions; open
circuit: operated under open circuit conditions.(Error bars were calculated based on three replicates representing 3 months
of operation; the 1,2-DCA containing medium was renewed after each month).
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 6 2941
under open circuit conditions and null in the InoMFC operated
under open circuit conditions.
3.5. The production of potential intermediates andproducts
2-Chloroethanol, a typical intermediate in aerobic oxidation of
1,2-DCA (Nobre and Nobre, 2004), was not detected in the
circulated medium in any the experimental case. Ethene,
ethane and vinyl chloride, which are potential products and
intermediates of reductive dechlorination of 1,2-DCA (Nobre
and Nobre, 2004), were also not detected. Meanwhile, ethylene
glycol, an intermediate in anaerobic hydrolysis-based degra-
dation of 1,2-DCA (Nobre and Nobre, 2004), was found to
accumulate in the medium of the AceMFCs, both under closed
circuit and open circuit conditions (Table 2). Ethylene glycol
was less prevalent in the medium of the closed circuit AceMFC
after 1 and 2 weeks but reached 0.25 � 0.05 mM after 4 weeks,
when the currents significantly decreased. In the medium of
the open circuit AceMFC, ethylene glycol was already present
at a concentration of 0.2 � 0.06 mM after 2 weeks. Acetate was
also detected in the medium of the AceMFCs, both under
closed circuit and open circuit conditions, e.g. at 0.15 � 0.02
and 0.08 � 0.03 mM, respectively, after the first week (Table 2).
Table 2 – The values of the parameters monitored at different times during a month of operating the MFCs with the stable anodic consortia.
Reactors Time [1,2-DCA](mM)
[Cl� produced] (mM) CO2 produced (mmol L�1 medium) pH [EthylGly](mM)
[Acetate](mM)
CH4 produced(mmol L�1 medium)
Aver. I(mA)
CE (%)
Theo Real Theo Real
AceMFC (closed ) To 1.00 – – – – 7.1 � 0.1 0.015 � 0.005 – – –
1w 0.44 � 0.07 1.12 1.20 � 0.33 1.11 0.22 � 0.02 6.6 � 0.1 0.08 � 0.005 0.15 � 0.02 0.02 � 0.007 0.22 � 0.04
2w 0.05 � 0.004 1.90 1.77 � 0.27 1.90 0.14 � 0.02 6.5 � 0.1 0.12 � 0.02 0.22 � 0.03 0.03 � 0.01 0.17 � 0.02
4w 0.03 � 0.005 1.95 2.03 � 0.06 1.95 0.10 � 0.02 6.8 � 0.1 0.25 � 0.05 0.05 � 0.01 0.06 � 0.01 0.10 � 0.02 43 � 4
AceMFC (open) To 1.00 – – – – 7.1 � 0.1 0.02 � 0.004 – – n.a.
1w 0.74 � 0.13 0.52 0.42 � 0.07 0.52 0.08 � 0.01 6.8 � 0.1 0.05 � 0.005 0.08 � 0.03 0.11 � 0.02 n.a.
2w 0.47 � 0.05 1.06 1.05 � 0.32 1.06 0.07 � 0.01 6.9 � 0.1 0.25 � 0.06 0.13 � 0.01 0.23 � 0.04 n.a.
4w 0.27 � 0.03 1.46 1.15 � 0.34 1.46 0.02 � 0.01 6.9 � 0.1 0.41 � 0.07 0.02 � 0.01 0.31 � 0.05 n.a.
InoMFC (closed ) To 1.00 – – – – 7.1 � 0.1 0.015 � 0.005 – – –
1w 0.68 � 0.05 0.64 0.51 � 0.14 0.64 0.18 � 0.02 6.8 � 0.1 0.01 � 0.002 0.18 � 0.01 0.01 � 0.001 0.13 � 0.03
2w 0.15 � 0.007 1.70 1.58 � 0.18 1.70 0.10 � 0.02 6.8 � 0.1 0.01 � 0.001 0.03 � 0.01 0.17 � 0.07 0.12 � 0.02
4w 0.05 � 0.004 1.90 1.70 � 0.14 1.90 0.02 � 0.01 6.9 � 0.1 0.01 � 0.001 0.02 � 0.01 0.20 � 0.03 0.07 � 0.02 25 � 4
InoMFC (open) To 1.00 – – – – 7.1 � 0.1 0.01 � 0.001 – – n.a.
1w 0.99 � 0.03 0.02 w0 0.02 w0 7.0 � 0.2 0.01 � 0.002 w0 0.01 � 0.001 n.a.
2w 0.97 � 0.03 0.06 w0 0.06 w0 7.2 � 0.1 0.01 � 0.003 w0 0.01 � 0.001 n.a.
4w 0.84 � 0.04 0.32 w0 0.32 w0 7.1 � 0.1 0.01 � 0.002 w0 0.10 � 0.05 n.a.
Control To 1.00 – – – – 7.1 � 0.1 w0 – – w0
1w 0.99 � 0.03 w0 w0 w0 w0 7.1 � 0.2 w0 w0 w0 w0
2w 1.02 � 0.03 w0 w0 w0 w0 7.1 � 0.1 w0 w0 w0 w0
4w 0.98 � 0.04 w0 w0 w0 w0 7.1 � 0.1 w0 w0 w0 w0
n.a.: not applicable. Abbreviations: EthylGyl: ethylene glycol; Aver. I: average current; CE: coulombic efficiency (calculated for the whole period of operation: 4 weeks). Other notes: Real: real values;
Theo: theoretical values, calculated from the concentrations of 1,2-DCA removed; To: at the beginning of the operation; 1w: after 1 week; 2w: after 2 weeks; 4w: after 4 weeks; closed: operated under
closed circuit conditions; open: operated under open circuit conditions; Control: the autoclaved and uninoculated MFC operated under closed circuit conditions. The theoretical values and coulombic
efficiencies were calculated based on the assumption that 1,2-DCA is oxidized completely to CO2 (1 mole of 1,2-DCA produces 10 moles of electrons). The concentration of produced chloride was
calculated as the concentration of chloride in the medium at the time of interest subtracted by the chloride concentration in the original medium (110 mg L�1).
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w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 6 2943
The concentration of acetate was low in both cases after
4 weeks of operation.
In the cases of the InoMFCs, ethylene glycol was only
detected at the basic level (equal to that of the initial medium).
Acetate was detected only in the medium of the closed circuit
InoMFC, but at lower concentrations (Table 2). The acetate
concentration also tended to decrease during a course of a test
run (lowest after 4 weeks, when the currents significantly
decreased).
4. Discussion
4.1. Closed circuit operation vs. open circuit operation
The results showed that the closed circuit MFCs performed
better than their open circuit counterparts. For instance, the
closed circuit operation led to a 1.34-fold higher 1,2-DCA
removal rate in the AceMFCs. This indicated that the anode
activities accelerated the removal of 1,2-DCA. Being operated
under open circuit conditions means that the processes
occurring in the anodes are not electrochemical but plainly
aerobic oxidation or anaerobic fermentation/oxidation, as the
medium was anoxic (gastight). Possibly, due to the fact that
1,2-DCA is a recalcitrant compound (Bhatt et al., 2007), the
spontaneous metabolism of the bacteria on 1,2-DCA occurs at
a low rate. When an active electrode (anode) based with-
drawal of electrons was introduced, more pathways might be
activated and the metabolic rate increased.
4.2. Acetate feeding enriched anodophilic consortia vs.1,2-DCA feeding enriched anodophilic consortia
The consortia previously enriched with acetate functioned
better than the ones enriched with 1,2-DCA from a 1,2-DCA
removing inoculum, in terms of both current generation and
1,2-DCA removal. Even under open circuit conditions, the
former removed 75% of 1,2-DCA after 4 weeks while the latter
almost could not remove 1,2-DCA. The reason for this might be
the higher flexibility of the former, as they were richer in
species (Table 1). It is known that not only stability but also
flexibility are essential for a stable performance and swift
adaptation of a microbial community (Loreau et al., 2001;
Torsvik and Ovreas, 2002). The results showed that the
communities of the AceMFCs changed faster than those of
the InoMFCs in response to the feeding with 1,2-DCA (Table 1).
The higher metabolic versatility probably allows the AceMFC
communities to more easily shift to new compositions and
adapt to new conditions. The InoMFC communities might be
rather specific for their original bio-niche (as the species
evenness of the inoculum was relatively low – Fig. S1) and
appeared less adaptable to new conditions. The site from
which the inoculum of the InoMFCs was taken was treated
with the strain Desulfitobacterium dichloroeliminans DCA 1 and
subjected to reductive dechlorination (Maes et al., 2006). This is
probably another reason why the InoMFC communities were
less adaptable to the oxidative conditions in the MFC anode
than the AceMFC communities. However, the profound
changes of the community of the closed circuit InoMFC showed
that the MFC anode could stimulate this consortium to shift its
composition, survive and be able to metabolize 1,2-DCA.
4.3. Possible metabolism of 1,2-DCA by the anodophilicbacteria
As shown in Table 2, the measured concentration values of
chloride produced in the medium of the MFCs corresponded to
the theoretical values. This indicates complete dechlorination
of 1,2-DCA. The decreases of pH and the production of CO2
strongly suggested that the degradation of 1,2-DCA was an
oxidative process. The fact that potential products of reduc-
tive dechlorination of 1,2-DCA such as ethene, ethane and
vinyl chloride were not detected while acetate was produced
in the medium of the MFCs also corroborated this. Indeed, the
possibility for oxidative dechlorination in the anode is
eminent as the redox potential of the cathode oxidant, which
is hexacyanoferrate, is high (Eh� ¼ þ 436 mV vs. SHE as
mentioned above). The current generation also indicated that
an oxidative process must have occurred in the anodes of the
closed circuit MFCs.
This raises the question about the pathway employed by
the anodophilic bacteria for the oxidative dechlorination of
1,2-DCA. In this study, ethylene glycol and acetate, and not
chloroethanol, were detected in the medium of the MFCs,
showing that the anode bacteria might not employ an oxida-
tive pathway similar to that in aerobic oxidation of 1,2-DCA.
Indeed, a pathway that involves ethylene glycol and acetate
was previously reported for the degradation of 1,2-DCA under
anaerobic conditions (Nobre and Nobre, 2004; Stucki and
Thuer, 1995). In this process, 1,2-DCA is first hydrolyzed to
ethylene glycol, which is subsequently converted to acetate
and CO2 by acetogenic bacteria. This pathway fits the case of
the anodophilic bacteria, which are used to utilizing acetate as
their only substrate. Interestingly, the conversion of 1,2-DCA
to glycol was described as an abiotic process that occurs very
slowly (half life: 5.8 years) (Nobre and Nobre, 2004). In this
study, ethylene glycol kept accumulating in the medium of
the AceMFCs during a month of operation (Table 2). Therefore,
in our MFCs, probably this process was biotically accelerated
by the anodophilic bacteria. Another possibility is that the
bacteria kept consuming ethylene glycol to produce acetate
and consuming acetate to produce electrons; which might
cause the equilibrium of the abiotic hydrolytic reaction to
keep moving towards the production of ethylene glycol.
The fact that the current generation of the AceMFC
decreased in response to the decrease of acetate concentra-
tion (after 4 weeks) also strongly corroborates the hypothesis
that the oxidation of acetate (produced from ethylene glycol)
was a cause of the current generation. Moreover, ethylene
glycol concentrations were low in the medium of the closed
circuit AceMFC during the first and the second weeks (in an
1 month test run), when the currents were high, indicating
that this compound was also used for current generation. The
accumulation of ethylene glycol indicates that there might be
an ethylene glycol concentration limit for the conversion by
the anodophilic bacteria. Also, this was probably the reason
why the coulombic efficiency could not be higher.
Based on the discussion above, it can be concluded that the
hydrolysis pathway is the most possible explanation for the
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 62944
degradation of 1,2-DCA by the anodophilic bacteria in the
AceMFCs. It can be summarized as follows:
Step 1: Cl–CH2–CH2–Cl (1,2-DCA) þ 2H2O / CH2OH–CH2OH
(ethylene glycol) þ 2HCl
Step 2: 2CH2OH–CH2OH (ethylene glycol) þ 2H2O�������!Acetogenesis
CH3COOH (acetate) þ 2CO2 þ 12Hþ þ 12e�
Step 3: CH3COOH (acetate) þ 2H2O / 2CO2 þ 8Hþ þ 8e�
Overall, the electron balances at the beginning and at the
end of an 1 month test run of the closed circuit AceMFC
appeared to be relatively equal (Table S1). This suggests that
the above proposed pathway is probably the sole process in
the anode of the AceMFC under closed circuit conditions.
It can be noted that the concentration of CO2 produced was
decreased and not well correlated with 1,2-DCA removal and
that the total carbon balances of the substrate (1,2-DCA) and
of the analyzed products (ethylene glycol, acetate, CO2) were
not equal (Table S2). Our hypothesis is that part of the carbon
in 1,2-DCA was possibly used for building biomass. This
aspect requires further examination.
A similar pathway is probably applied for the degradation
of 1,2-DCA in the anode of the open circuit AceMFC, because
the same intermediates were detected. Indeed, this is
corroborated by the insignificant difference in composition
between the stable microbial community of the closed circuit
AceMFC and that of the open circuit AceMFC. However, the
data indicated that the conversion rate under open circuit
conditions might be significantly lower than under closed
circuit conditions. In addition, instead of the generation of
electrons and protons, anaerobic digestion products might be
produced. Indeed, methane was detected from the open
circuit AceMFC, more than from the closed circuit AceMFC
(Table 2). The electron and carbon balance data for the open
circuit AceMFC (with methane also considered) (Tables S1 and
S2), showing high percentages of recovery (>90%), corroborate
this hypothesis.
For the closed circuit InoMFC, acetate and CO2 were also
detected in its medium, indicating that also in this case, the
degradation of 1,2-DCA might be an oxidative process.
However, due to the fact that no other intermediates could be
detected and that the electron and carbon balances at the
beginning and at the end of each operational batch were
significantly unequal (Tables S1 and S2), the possible mecha-
nisms for the degradation remain unclear. As the InoMFCs
performed more poorly than the AceMFCs, we decided not to
further investigate these mechanisms.
4.4. The potential use of MFCs for the removal of1,2-DCA
In this study, the maximum 1,2-DCA removal rate of 102 mg
L�1 reactor volume d�1 and the average removal rate of
45.6 mg L�1 reactor volume d�1 were achieved with the
AceMFC operated under closed circuit condition. These values
are comparable to those reported for a 1,2-DCA oxidizing
mixed culture using nitrate as the electron acceptor (Table S3).
Although this value is still lower than those reported for pure
cultures (Table S3), the removal efficiency reached >99% after
1 month for the AceMFC. One reason for the low removal rates
of the MFCs might be the limited mass transfer due to the
restricted contact surface and the low hydraulic mixing of the
medium in the system. Another reason might be the low
intrinsic rate of the hydrolytic conversion, as mentioned
above, which the anodophilic bacteria employ for the
metabolism of 1,2-DCA. Yet, as discussed above, the use of the
MFC anode significantly improved this rate. Pure cultures,
though reported to have high rates of 1,2-DCA removal (De
Wildeman et al., 2003; Hage and Hartmans, 1999; Janssen
et al., 1985), require specific environmental conditions.
Moreover, contamination is a significant problem decreasing
the performance of the reactors operating with pure cultures.
Anaerobic treatment (Dinglasan-Panlilio et al., 2006; Gupta
and Mali, 2008; Maes et al., 2006) for 1,2-DCA removal repre-
sent other options but these still require special care and
operation to maintain anaerobic conditions. Meanwhile, the
operation of MFCs is relatively simple, no special care is
required, and they can be used to recover part of the energy
derived from the oxidation of 1,2-DCA, transforming it into
electricity. Assuming that 1,2-DCA could be oxidized
completely to CO2, the coulombic efficiency of the AceMFCs
was about 43% (Table 2), which is indeed competitive with
respect to using MFCs for removing pollutants. In addition, in
this study, it was shown that using a gastight MFC system, 1,2-
DCA volatilization problems could be resolved. The fact that
the AceMFCs functioned better than the InoMFCs suggested
that there is no need of a special inoculum to enrich a 1,2-DCA
removing anodic consortium, which is convenient for prac-
tical application.
The remaining challenge for the use of MFCs might be how
to improve the removal rate of the anode consortia to the
levels comparable with those reported for practice (at gram
per liter reactor per day levels). Definitely, the mass transfer of
1,2-DCA to the anodophilic degraders will need optimizing.
Further studies of the 1,2-DCA metabolizing pathway(s)
employed by these bacteria in order to improve the removal
rate are also warranted.
5. Conclusion
In this study, we have shown that:
– 1,2-DCA, a well-known recalcitrant chlorinated ground-
water pollutant, was efficiently degraded by anodophilic
bacteria enriched in microbial fuel cells, at the rate of up to
102 mg per liter reactor volume per day. Concomitantly,
energy released from this degradation could be partially
recovered (up to 43%) as electricity.
– The flexibility and the anodic pre-acclimation of a microbial
community is probably more important than its substrate
pre-acclimation in determining its 1,2-DCA removing
performance in a MFC anode.
– A hydrolysis-based oxidative pathway is possibly employed
by anodophilic bacteria for the degradation of 1,2-DCA.
– The metabolic flexibility of anodophilic consortia, the high
1,2-DCA removal efficiency and the simple operation of
MFCs offer a new potential application of MFCs in the
domain of bioremediation.
w a t e r r e s e a r c h 4 3 ( 2 0 0 9 ) 2 9 3 6 – 2 9 4 6 2945
Acknowledgement
This research was supported by a grant from the Flanders
Research Foundation (FWO project G.0172.05) and an EU
Neptune project (Contract No 036845, SUSTDEV-2005-3.II.3.2).
The authors would like to deeply thank Peter Clauwaert for
critically reading the manuscript and giving useful comments.
Appendix A.Supplemental material
Supplementary information for this manuscript can be
downloaded at doi:10.1016/j.watres.2009.04.004.
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