Bioresource Technology 241 (2017) 879–886

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Efficient 2,4-dichloronitrobenzene removal in the coupled BES-UASB

reactor: Effect of external voltage mode
Hui Chen a, Xinyi Gao a, Caiqin Wang a, Junjie Shao a, Xiangyang Xu a,b, Liang Zhu a,b,⇑
a
Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China
b
Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

BES-UASB (10 L) was stable at a short

HRT of 4 h for DClNB degradation at
25 mg L1.

Sludge characteristics were enhanced
in the presence of external voltage.

Reductive dechlorination related
microbes were enriched in the
coupled system.

Intermittent mode reduced 50%
energy input and achieved
satisfactory performance.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, bioelectrochemical-upflow anaerobic sludge blanket (BES-UASB) system was developed for
Received 1 May 2017 treatment of 2,4-dichloronitrobenzen (DClNB) containing wastewater to investigate the effect of external
Received in revised form 30 May 2017 voltage with different supplying modes. Results showed that 2,4-dichloroaniline (DClAN) was under
Accepted 3 June 2017
detection limit in R1 (applied with intermittent voltage) and R2 (applied with continuous voltage) when
Available online 15 June 2017
the DClNB loading increased from 25 to 100 g m3 d1 (hydraulic retention time (HRT) was decreased from
24 to 6 h) while sudden accumulation of DClAN (1.7 mg L1) was observed in R0 (control). Dechlorination
Keywords:
efficiency (DE) decreased to 32.7%, 45.0% and 45.3% in R0, R1 and R2 when HRT was further shortened to
Chloronitrobenzene
Bioelectrochemical system (BES)
4 h. Microbial community analysis indicated the significant enrichment of dechlorination-related species
Upflow anaerobic sludge blanket (UASB) in R1 and R2 compared with R0, e.g., Dehalobacter and Dehalococcoides. In summary, the BES-UASB system
External voltage mode with intermittent voltage is an alternative process for efficient treatment of DClNB containing wastewa-
Microbial community ter, and the energy input was reduced markedly.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Chloronitrobenzenes (ClNBs), widely applied as a synthetic
intermediate in the production of dyes, herbicides, rubber, phar-
maceuticals and other industrial chemicals, are toxic to human
⇑ Corresponding author at: Department of Environmental Engineering, Zhejiang
University, No. 866 Yuhangtang Road, Hangzhou 310058, China.
E-mail address: felix79cn@hotmail.com (L. Zhu).
beings and other organisms (Wang et al., 2015; Wu et al., 2006;
Yuan et al., 2012). Due to the electron affinity of chloro- and
nitro-groups to benzene ring, ClNBs have stable chemical structure
and are difficult to be removed from wastewater (Yuan et al.,
2015). Physical adsorption, chemical reduction, zero-valent iron
(ZVI) reduction and advanced oxidization are typical physicochem-
ical treatment process for ClNBs removal (Xu et al., 2006; Ye et al.,
2009). However, high cost and the risk of secondary pollution limit
the industrial application of these processes. Biological treatment

http://dx.doi.org/10.1016/j.biortech.2017.06.010
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
880 H. Chen et al. / Bioresource Technology 241 (2017) 879–886
is considered as an alternative due to the highly efficient and cost
effective properties. Lin et al. (2011) found ClNBs were difficult to
be degraded under aerobic conditions. Owning to the electron-
deficient characteristic of ClNBs which protects the benzene ring
from the attack of oxygenase, anaerobic process is more appropri-
ate for the reduction of ClNBs. ClNBs was easily to be transformed
to less harmful compounds under anaerobic environment (Zhu
et al., 2012). Microbial reductive dechlorination has been proven
as an alternative technology for the treatment of chlorinated com-
pounds containing wastewater. However, it requires enough elec-
tron donors and reductive niche for reductive dechlorination, i.e.,
the addition of organic carbon sources which increases the opera-
tional cost (Shen et al., 2014).
Recently, bioelectrochemical system (BES) as a promising tech-
nology has been widely applied to recover energy from wastewa-
ter, i.e., producing valuable resources (H2, CH4, H2O2) (Cheng and
Logan, 2011; Jadhav et al., 2017; Logan et al., 2006; Zhang and
Angelidaki, 2016). BES make some thermodynamic unfavorable
reactions happen and accomplish the purification of wastewater.
Compared with the traditional process, the superiority of BES has
been demonstrated by the increase of reductive degradation rate
of toxic pollutants and BES requires much lower input energy than
the typical electrochemical process (Jiang et al., 2016).
Several researchers have investigated the feasibility of the cou-
pled BES-anaerobic process for the degradation of halogenated aro-
matic compounds (Table 1), e.g., p-ClNBs (Xu et al., 2016), 2,4-
dinitrochlorobenzene (Jiang et al., 2016), p-nitrophenol (Shen
et al., 2014) and 4-chlorophenol (Kong et al., 2015). The results
indicated that the coupled system could not only remarkably
improve the dechlorination efficiency but also reduce organic
cosubstrate consumption (Shen et al., 2014). More recently,
Rózsenberszki et al. (2017) found that one-step technology alone
was not able to achieve efficient organics removal and energy
recovery simultaneously from municipal waste liquor, e.g., dark
fermentative H2 production, biogas fermentation and microbial
fuel cells (MFC). A combined three-stage technology was an alter-
native to achieve the goals. Zhen et al. (2017) addressed that cou-
pled MEC system was capable of improving the overall
performance of UASB reactor for methanolic wastewater treat-
ment. The methane production in the coupled system was 10.1%
higher than the control UASB. Jia et al. (2016) developed a novel
monitoring system for the MFC-UASB process, which consisted of
a single-chamber MFC where the UASB reactor worked as anode
chamber and the carbon cloth worked as an air-cathode. The
research provided valuable information which is useful in the
future development of COD monitoring.
Although many studies have been undertaken to investigate the
coupled BES-anaerobic process, few studies have focused on the
reduction process of running cost. As is known, energy input, i.e.,
external electricity, is the main cost in the BES-anaerobic biological
process, and it’s of great importance to reduce the energy input to
make the process more cost-effective. However, few studies
reported the control strategy of running cost. Intermittent mode
Table 1
Summary of the studies for ClNBs treatment in BES-UASB system.
Pollutants HRT (h) Reactor Reference
volume (L)
p-chloronitrobenzene 24 4.8 Zhu et al. (2014)
4-chloronitrobenzene – 0.2 Guo et al. (2015)
p-chloronitrobenzene 36 4.8 Xu et al. (2016)
2,4-dinitrochlorobenzene 120–48 7.5 Jiang et al. (2016)
Aniline – 0.2 Li et al. (2017)
Aniline – 0.9 Wu et al. (2017)
2,4-dichloronitrobenzene 24–4 10.0 This study
of external voltage may be a substitute for continuous mode with
which energy input could be effectively reduced by decreasing the
duration of voltage supply. Therefore, it’s important to compare
the differences between continuous and intermittent modes. It is
necessary to be mentioned that previous studies on the dechlorina-
tion of ClNBs based on coupled system were undertaken with long
hydraulic retention time (HRT) (usually longer than 1 day) (Jiang
et al., 2016; Xu et al., 2016) and small volumes which was usually
below 1 L (Guo et al., 2015; Wu et al., 2017), leading to a relatively
low pollutant loading. Hence, the effect of short HRT and reactors
with bigger volume should be concerned and studied to provide
data foundations for larger-scale usage.
In the present study, 2,4-dichloronitrobenzene (DClNB) was
chosen as the target pollutant and we investigate (1) the perfor-
mance of DClNB degradation in the 10 L upflow anaerobic sludge
blanket (UASB) reactors under relatively short HRT (from 24 to
4 h), and (2) the difference between the supply modes, i.e., contin-
uous and intermittent voltage during DClNB degradation.
2. Materials and methods
2.1. Chemicals and electrodes
DClNB, 2,4-dichloroaniline (DClAN), 2-chloroaniline (2-ClAN),
4-chloroaniline (4-ClAN) and aniline (AN) were purchased from
Aladding Chemistry Co. Ltd (Shanghai, China). Sucrose was pur-
chased from Sinopharm Chemical Reagent Co. Ltd (Shanghai,
China). Methylalcohol (high performance liquid chromatography
(HPLC) grade) was purchased from Tianjin Shield Co. Ltd (Tianjin,
China).
Non-waterproof graphite felt was used as electrodes and pur-
chased from Beijing Sanye Carbon Co. (Beijing, China). The elec-
trodes were degreased ultrasonically and cleaned in acetone for
20 min, and washed by deionized water for three times. Then,
the electrodes were dried at 105 °C.
2.2. Synthetic wastewater and inoculation
Synthetic wastewater was used during the experiment, includ-
ing organic component, buffer solution and trace elements. The
composition of the synthetic wastewater is listed in Table S1.
Sucrose was used as the carbon source and the chemical oxygen
demand (COD) in the wastewater was 468.5–549.2 mg L1, while
the concentration of DClNB was at 23.5–25.3 mg L1. pH of the
wastewater was not controlled and ranged from 7.3 to 7.6.
Seeding sludge was taken from a wastewater treatment plant
(WWTP) in Hangzhou, China. The suspended solid (SS) and volatile
suspended solid (VSS) of the seeding sludge were 42.6 and
27.6 g L1, respectively. The initial SS of the three UASB reactors
was 23.5 g L1 after inoculation.
2.3. Reactor configuration and operation conditions
Three UASB reactor was designed according to our previous
study with some modifications (Zhu et al., 2015) (Fig. S1), i.e., R0,
R1 and R2. R1 was intermittently supplied with 1.2 V external volt-
age, R2 was continuously supplied with 1.2 V external voltage,
while R0 was operated as the control. The plexiglass-based UASB
reactors were constructed with an internal diameter of 14 cm
and height of 48 cm. The effective volume of the UASB reactor
reached 10 L. The reactors were placed in the laboratory with a
temperature of 30 ± 2 °C. A pair of graphite felt electrodes
(100  200  3 mm) were set in R1 and R2 and supplied with a
voltage of 1.2 V utilizing a direct current power source (Victo-
ry3003D, China). Intermittent supply of voltage with 12 h ON:
 H. Chen et al. / Bioresource Technology 241 (2017) 879–886
12 h OFF was applied in R1 while the supply of voltage in R2 was
continuous. The reactors were operated in parallel with an initial
HRT of 24 h.
The detailed procedure of the experiment was shown in Fig. S1.
The experiment was conducted with a duration of 172 days.
According to the supply of DClNB in the wastewater, the experi-
ment was divided into two phases, i.e., acclimation and DClNB
treatment phases. The duration of phase one was 38 days while
phase two lasted for 134 days. DClNB concentration in phase two
was constant in the wastewater and HRT of the reactors was grad-
ually decreased from 24 h to 4 h simultaneously.
2.4. Analytical methods
DClNB, DClAN, 2-ClAN, 4-ClAN and AN were identified by the
gas chromatography-mass spectrometer (GC–MS) (Agilent
7890B/7000C, USA) and the column temperature was programmed
as follows. The temperature was held at 50 °C for 2 min and was
then increased to 150 °C (10 °C min1) and held for 2 min. The
temperature was increased to 270 °C with 20 °C min1 afterwards,
where the temperature lasted for 5 min. The main reductive prod-
ucts, i.e., DClAN, 4-ClAN and AN were quantified by the HPLC
(Waters 2487, USA). The mobile phases were methanol-water
(6/4, v/v) with the flowing rate of 1.0 mL min1. Cl was monitored
by the ion chromatography (IC) (Dionex 1100, USA) equipped with
electron capture detector (ECD) detector, a IonPacÒ AS01-HC
4  250 mm separation column and a IonPacÒ AG11-HC
4  50 mm guard column.
The granular characteristics were measured according to Zhu
et al. (2014). The morphology of the sludge was observed by scan-
ning electron microscope (SEM) (HitachiS-3700N, Japan). Size of
the sludge was measured by a mastersizer (Malvern 2000, UK).
Extracellular polymeric substance (EPS) was extracted by heating
method (Morgan et al., 1990). The pH, SS, VSS and COD were mea-
sured according to the standard methods (APHA, 2005).
2.5. Illumina high-throughput sequencing
The DNA of the sludge samples collected from the reactors on
day 172 along with the seeding sludge were extracted with MoBio
PowerSoilÒ DNA Isolation Kit (12888) according to the manufac-
turer’s instructions.
The primer pairs 520F (50 -AYTGGGYDTAAAGNG-30 ) and 802R (
50 -TACNVGGGTATCTAATCC-30 ) targeting the V4 region of the bac-
terial 16S rRNA were used for amplification and sequencing. For
16S rRNA gene amplification, each polymerase chain reaction
(PCR) mixture (25 mL) was composed of 11.25 mL sterilized ultra-
pure water, 1 mL forward primer (10 mM), 1 mL reverse primer
(10 mM), 1 mL template DNA, 5 mL reaction buffer, 5 mL high GC buf-
fer, 0.5 mL dNTP (10 mM) and 0.25 mL Q5 high-fidelity DNA poly-
merase. PCR conditions for the sequencing process were as
follows: initial denaturation at 98 °C for 30 s, followed by 25–27
cycles, each circle was consist of denaturation at 98 °C for 15 s,
annealing at 50 °C for 30 s, an extension at 72 °C for 30 s and the
a final extension at 72 °C for 5 min. After that, the temperature
was maintained at 4 °C.
2.6. Statistical analysis and calculations
Statistical comparison between variables was conducted by t-
test with SPSS software (SPSS 18.0). A p-value of 0.05 or lower indi-
cates that the difference between the variables under comparison
is statistically significant. In this study, the operational condition
of reactors and experimental conditions were identical as
described above. Therefore, errors in the study mainly due to sam-
881
ple and instrument analysis which are inevitable during the biolog-
ical experiment.
DClNB and 4-ClAN removal efficiency (REDClNB and RE4-ClNB), AN
formation efficiency (FE) and dechlorination efficiency (DE) are
defined as:
DClNBinf  DClNBeff
REDClNB ¼  100% ð1Þ
DClNBinf
where DClNBinf and DClNBeff are the concentrations of DClNB in the
influent and effluent, respectively.
4  ClANtheo  4  ClANeff
RE4ClAN ¼  100% ð2Þ
4  ClANtheo
where 4-ClANtheo is the theoretical concentration of 4-ClAN when
influent DClNB is completely transformed to 4-ClAN according to
stoichiometric ratio, 4-ClANeff is the concentration of 4-ClAN in
the effluent.
ANeff
FE ¼  100% ð3Þ
ANtheo
where ANeff is the concentration of AN in the effluent and ANtheo is
the theoretical concentration of AN when DClNB is completely con-
verted to AN.
 
Cleff  Clinf
DE ¼   100% ð4Þ
Cltheo
where Cl  
eff and Clinf are the concentration of Cl in the effluent and
influent, respectively, Cl is the theoretical Cl
formation when
theo
DClNB is completely dechlorinated.
3. Results and discussion
3.1. Pollutants removal performance of reactors
In phase one, DClNB was absent and effluent COD was moni-
tored. Fig. 1 showed that the COD removal when the influent
COD was 510.7 ± 21.5 mg L1. During phase one, the effluent COD
of R0 ranged from 401.5 to 40.5 mg L1 while the corresponding
COD of R1 and R2 were 382.1–18.5 mg L1 and 381.5–10.0 mg L1,
respectively. The COD concentrations in the effluent of R1 and R2
were slightly lower than that in R0 during phase one (p > 0.05),
while the differences in R0 and the other two reactors were signif-
icant (p < 0.05). When the effluent COD of the reactors were all
constantly below 50 mg L1, DClNB was added in the wastewater.
DClNB was added with a dosage of 25 mg L1 during phase two.
The intermediates during DClNB degradation were identified by
GC–MS (Fig. S2). DClAN was observed rapidly as DClNB was
reduced in the BES-UASB and then DClAN was converted to 4-
ClAN and AN. Additionally, 2-ClAN was not detected. The results
indicated that DClNB degradation consisted of three steps: (1)
Nitro group was reduced to produce DClAN, (2) ortho-position
chlorine group was reduced with 4-ClAN production and (3)
para-position chlorine group was 4-ClNB was further dechlori-
nated to AN.
When the HRT was gradually shortened from 24 h to 4 h, COD
removal efficiency kept stable which were >90.0%in all reactors.
For DClNB and its intermediates, no DClAN was observed in the
reactor effluent with an HRT above 6 h. The average concentration
of 4-ClAN during these days (days 39–140) in R0, R1 and R2 were
11.4, 11.0 and 11.0 mg L1 (p = 0.058) while the corresponding val-
ues for AN were 2.1, 2.4 and 2.5 mg L1 (p = 0.062). In R0, R2 and
R3, DE gradually increased from 16.5% to 59.2%, 22.6% to 63.2%
and 24.3% to 63.9%, respectively (Fig. 2). When the HRT was further
shortened to 6 h, significant differences were observed between R0
and the coupled reactors. Sudden accumulation of DClAN was
882 H. Chen et al. / Bioresource Technology 241 (2017) 879–886
Fig. 1. Evolution of the indices in different reactors (a, b and c for R0; d, e and f for R1; g, h and i for R2).
Fig. 2. Dechlorination efficiency of the reactors. * stands for statistical difference; **
stands for significant statistical difference.
observed in the effluent of R0 while DClAN was under detection
limit in the effluent of R1 and R2 (HRT = 6 h). Corresponding DE
in R0, R1 and R2 decreased from 59.2% to 50.1%, 63.2% to 56.9%
and 63.9% to 58.4% (p < 0.05). It could be concluded that the cou-
pled BES-UASB reactors were slightly influenced by high hydraulic
loadings while sever influence was observed in the control UASB.
Finally, the HRT was decreased to 4 h with which DClAN accumu-
lated in all reactors (6.9, 2.0 and 1.8 mg L1 in R0, R1 and R2,
respectively) (p < 0.01).The results of DClAN accumulation demon-
strated that the supply of external voltage accelerated the transfor-
mation of DClAN and the difference between the UASB and BES-
UASB was significant. There were also a significant difference in
AN production (0.7, 2.1 and 2.0 mg L1 in R0, R1 and R2, respec-
tively) (p < 0.01). Additionally, pH is an important index in the
anaerobic system (Zhang et al., 2016) and pH of the three reactors
was similar (p > 0.05) in this study which were around 7.0, indicat-
ing that no volatile fatty acid (VFA) accumulated during the
experiment.
The performance of the reactors were similar during phase one.
It might be that the anaerobic microbes had low growth rate and
the stimulation of external voltage can’t be observed immediately.
During phase two, especially when the HRT was shorten to 4 h, the
superiority of the coupled system could be achieved. In R1 and R2,
DClNB was converted to 4-ClAN and AN more efficiently with
much less DClAN accumulation compared with R0 (p < 0.01). To
be added, the coupled reactors R1 and R2 had higher DE than R0
(p < 0.01). It is necessary to be mentioned that although the perfor-
mance of R2 was a bit better than that of R1 (p > 0.05), R1 was
more satisfactory due to the decrease of energy input by 50%.
In this study, the performance of coupled BES-UASB reactor is
influenced by various operational factors such as external voltage
and HRT. Thereinto, the working voltage which is higher than the
value of water electrolysis (1.23 V) leads to the production of oxy-
gen and hydrogen around the electrodes with which active
microorganisms may be severely inhibited (Shen et al., 2014).
HRT is another key parameter in the anaerobic system. Results
showed that biocatalytic reduction performance of 2,4-
dinitrochlorobenzene declined along with the decrease of reactor
HRT (Cui et al., 2014). It has been reported that the pH value is
another factor affecting the reactor performance (Li and Zhu,
2014; Wu et al., 2017). Wu et al. (2017) found that p-
chloronitrobenzene can be degraded in a faster rate with more
final product (AN) under acidic condition. As described above, it
is of great significance to enhance the mineralization of AN by opti-
mizing the operational parameters, e.g., alternative electrodes.
 H. Chen et al. / Bioresource Technology 241 (2017) 879–886
3.2. Characteristics of granular sludge
Sludge characteristics are important which is related with the
reactor performance. Size distribution was observed during the
experiment (Fig. 3). The mean diameter of the seeding sludge
was around 130 mm. The diameter in the reactors increased slowly
during the first 72 day when the HRT was maintained at 24 h. Slow
up-flow rate and low COD in the influent might be two possible
reasons for the slow granulation. When HRT was gradually
decreased to 4 h, it can be concluded from Fig. 3 that the mean
diameter of the sludge from R2 was greater than that from R0
and R1 (p < 0.01), while the diameters of R0 and R1 were similar.
It might be the external voltage supply which stimulated sludge
granulation, especially the continuous mode.
EPS which consists of a rich polymer matrix of proteins (PN) and
polysaccharides (PS) plays an important role during sludge granu-
lation (Liu et al., 2004; Su and Yu, 2005). On day 10, EPS contents
didn’t increase obviously compared with the value on day-38
(Fig. 4). After the addition of DClNB in the influent on day 0, EPS
contents increased obviously on day 20. However, it was unex-
pected that EPS of R2 was less than that of R0 (119.6 vs.
126.9 mgEPS g1VSS). It has been reported that microorganisms
would secret more EPS to protect themselves from toxic sub-
stances (Aquino and Stuckey, 2004; Fang et al., 2002). EPS contents
continuously increased with the decrease of HRT in the following
Fig. 3. Size distribution of sludge in the reactors at different HRTs.
Fig. 4. Evolution of EPS contents during the experiment.
883
days. EPS contents in the sludge from R1 and R2 were significantly
greater than that from R0 (p < 0.01) (Fig. 4) and this was in accor-
dance with the results of DClNB degradation and granular size
distribution.
The seeding sludge and sludge samples from the reactors on day
132 were observed by SEM (Fig. S3). It can be observed that the
surface of the seeding sludge was irregular while after 172-day
operation the surface of the sludge changed. The sludge from R0
was close to the seeding sludge except the existence of some
cocci-shaped aggregates. In contrast, the sludge from R1 and R2
which were stimulated by external voltage changed obviously.
The surfaces were more regular than that of the seeding sludge
and R0. In Figs. S3c, d, bacillus were supposed to be the dominant
microbes. Fig. S3g was the image of the electrode before experi-
ment and the surface of the electrode was clean without the
attachment of microbes. After 172-day operation, a layer of various
microbes were observed on the electrode surface, forming a net-
work (Fig. S3e, f).
3.3. Microbial community analysis
In order to further investigate the microbial community diver-
sity, detailed analysis was carried out. A total number of 39563,
38735, 35525, 33715, 34711 and 38717 high-quality reads were
achieved for each sample using the high-throughput sequencing
(Table 2). The observed number of operational taxonomic units
(OTUs) at a 3% distance for the samples ranged from 1571 to
2453. The Shannon diversity index was often used as an index to
indicate the species diversity in the samples (Jiang et al., 2016).
The samples from the coupled reactors (R1 and R2) had a higher
diversity than that of the control (R0) (7.47 and 7.88 vs. 6.58), indi-
cating that the microbial community in R1 and R2 were richer due
to electrical stimulation. It has been reported that higher biodiver-
sity benefit to the ecological stability and make the system more
tolerant to environmental changes, e.g., pH, loadings, toxic com-
pounds (Tilman et al., 2006; Wrighton et al., 2008; Zhang et al.,
2013). In the present study, when the HRT of the reactor decreased
from 24 h to 4 h, DClNB degradation was severely influenced in R0,
including a sudden accumulation of DClAN and sharp decrease of
DE, while gentle fluctuation were observed in R1 and R2. The
higher biodiversity might be responsible for the better tolerance
to higher hydraulic loadings.
Phylogenetic analyses of the gene sequences were performed in
phylum and genus levels in order to investigate the microbial com-
munity distribution. Fig. 5 presented the top 20 abundant phyla
and genera of the samples. S1–S6 stands for seeding sludge, R0,
R1, R2, cathode (R1) and cathode (R2) sampled on day 132, respec-
tively. In Fig. 5a the relative bacterial community abundances were
identified at phylum level and Proteobacteria, Bacteroidetes, Planc-
tomycetes and Euryarchaeota were dominant in the samples. There
were significant differences between the samples from the coupled
reactors and that from the control. The relative abundances of the
phyla Proteobacteria, Bacteroidetes, Planctomycetes and Eur-
yarchaeota were 60.6%, 16.3%,7.2% and 5.2% in the seeding sludge.
Table 2
Biodiversity estimation based on high-throughput sequencing.
Sample Reads OTU Shannon
Seeding sludge 39563 1571 5.25
R0 38735 2112 6.58
R1 35525 2107 7.47
R2 33715 2240 7.88
Cathode biofilm (R1) 34711 2379 8.27
Cathode biofilm (R2) 38717 2453 8.22
884 H. Chen et al. / Bioresource Technology 241 (2017) 879–886

Fig. 5. Taxonomic classification of bacterial 16S rRNA genes reads (a, phylum level; b, genus level; S1–S6 were the samples from seeding sludge, R0, R1, R2, R1 cathode and R2
cathode).

After 172-day operation, Proteobacteria decreased in all reactors
significantly (27.2%, 45.7% and 46.6% for R0, R1 and R2). The abun-
dance of Bacteroidetes and Planctomycetes in R0 increased from
16.3% and 7.2% to 21.9% and 17.9%. However, the abundance of
these two phyla slightly decreased in the coupled reactors. The
result was opposite in the case of Euryarchaeota. The corresponding
abundance in R0, R1 and R2 were 9.1%, 15.3% and 14.5%. Similar
microbial community distribution was observed in the samples
of the electrodes (S5 and S6) compared with the samples of R1
and R2 (S3 and S4). Above results indicated that the microbial spe-
cies were selectively enriched with the supply of external voltage
(Proteobacteria and Euryarchaeota).
 H. Chen et al. / Bioresource Technology 241 (2017) 879–886
To get a better understand of the microbial community struc-
ture in the samples, the dominant communities were analyzed
on the genus level. As shown in Fig. 5b, some differences in micro-
bial abundance could be observed. The microbial structure was
homogenous in the seeding sludge (Planctomyces (7.1%), Prosthe-
cobacter (5.3%), Phemylobacterium (5.1%), Thauera (4.6%), Dehalo-
coccoides (3.3%) and Syntrophobacter (3.0%).After 172-day
operation, significant evolution of the microbial community was
observed. For S2, the dominant genera were Methanoregula
(19.4%), Clostridium (10.7%), Dehalococcoides (3.9%), Syntrophobac-
ter (3.6%), Syntrophomonas (3.4%), Methanolinea (3.0%) and Syntro-
phus (3.3%). In S3 and S4, the abundances of Methanoregula were
close to that in S1 (19.2% and 22.1% in S3 and S4, respectively).
However, the corresponding values in S5 and S6, i.e., the cathode
biofilm, were much greater than the one in the reactor bulk
(26.6% and 25.9% in S5 and S6). Similar trends were observed in
the evolution of genera Syntrophus. Fang et al. (2004) reported that
Syntrophus was involved in the phenol degradation under metha-
nogenic conditions and phenol was converted to benzoate by
Clostridium, and then to acetate, H2 and CO2 by Syntrophus. The
intermediate was finally transferred to CH4 by methanogens. Sim-
ilar results were obtained by Shin et al. (2010) that genus Syntro-
phus can participate in fatty acids degradation to produce
acetate, CO2 and H2, associating with methanogens. More recently,
Regueiro et al. (2015) also reported that Syntrophuswas present in
the anaerobic membrane bioreactor (AnMBR) which could degrade
butyrate and propionate to acetate, favoring CH4 production. Syn-
trophomonas was another microbial specie which could convert
fatty acid (butyric acid) to acetic acid with H2 production simulta-
neously (Zhao et al., 2016). In this study, the abundance of Syn-
trophomonas in R0, R1 and R2 were, 3.4%, 8.3% and 6.4%,
respectively which was in accordance with the trend of Syntrophus.
Dehalobacter and Dehalococcoides are microbes known to be
capable of complete reductive dechlorination, e.g., chloroethenes
and chlorobenzenes (Cheng and He, 2009; Li et al., 2015; Marco-
Urrea et al., 2011; Mortan et al., 2017; Wu et al., 2002). Compared
with S1, the abundance of the two genera in S2 were slightly
enriched (from 3.3% to 3.9% for Dehalococcoides while from 0.4%
to 1.7%Dehalobacter). In contrast, the corresponding values for
Dehalococcoides and Dehalobacter were 5.9% and 2.6%in S3, respec-
tively. In S4, the abundances of Dehalococcoides and Dehalobacter
were 7.9% and 3.0%, respectively. The results of microbial analysis
was consistent with the results of reactor performance. As dis-
cussed in Section 3.1, DE in the coupled reactor (R1 and R2) were
significantly greater than that in R0 (p < 0.01), especially when
the HRT of the reactors was shorten to 6 or 4 h (DE in R1 and R2
were about 10% greater than that in R0).
After 172-day operation the microbial community shifted com-
pared with the seeding sludge. The total abundance of the top 20
genera increased obviously, indicating oriented enrichment of the
microbes was achieved and this phenomenon was more obvious
in R1 and R2, especially in the cathodes from R1 and R2. Microbial
analysis based on genus level indicated that microbes related to
reductive dechlorination in R1 and R2 in the presence of external
voltage could be enriched more effectively than that in R0 without
external voltage. More specifically, compared with R0, higher
abundance of dominant microbe related to dechlorination in R1
and R2 was observed, e.g., Dehalobacter and Dehalococcoides, which
was in accordance with higher DE achieved in R1 and R2. More-
over, the biofilm kept direct contact with the electrodes, resulting
in the most abundant dominant microbes in the biofilm from the
cathodes. The SEM image found the surface of the electrodes were
attached by various of microbes to form a network with which
electron transfer might be promoted, leading to the increase in
DE. Similar phenomenon was observed by De Vireze et al. (2014)
885
who detected higher abundance of functional microbes on the
electrode in the BES-anaerobic digester compared with the control.
3.4. Potential mechanism for the enhancement of coupled system
As discussed above, the performance of R1 and R2 was better
than that of R0. It was reported that bioelectrodes could offer a
continuous electron supply for microorganisms on the surface of
electrodes (Chun et al., 2013). Moreover, reduction-related
microbes could obtain electrons from electrode with the assist of
electroactive microbes in the coupled BES-UASB system, catalyzing
the dechlorination and nitro reduction. This is also an alternative to
obtain electrons effectively compared with the control which
obtained electrons from fermentation.
3.5. Implication of this work
In the present study, we investigated the degradation of DClNB in
the 10 L UASB and coupled BES-UASB reactors and the results were
satisfactory. Here, the preliminary study indicated that both inter-
mittent and continuous supply of external voltage could improve
the performance of DClNB degradation compared with the control.
When the HRT was gradually decreased from 24 to 4 h, less fluctu-
ation was observed in coupled BES-UASB reactors, i.e., less DClAN
accumulation and stable DE. DClAN accumulation indicated that
an HRT as short as 4 h is inadequate for the microbes to transform
the intermediate and another possibility might be the carbon source
used in this study was relatively low (500 mgCOD L1 in the
influent).
The differences between intermittent and continuous modes
were insignificant (p > 0.05), i.e., intermittent mode (12 h ON:
12 h OFF) could be an alternative in this study which not only pro-
moted DClNB transformation, but also reduced 50% cost of energy
input. However, more work should be carried out to investigate the
influence of ON:OFF ratio, determining the optimum mode of the
supplied external voltage. Moreover, the experimental parameters,
e.g., material and site of the electrodes, concentration of carbon
source and pollutant, initial pH, etc., should be further investigated
to provide foundations for longer-term and scale-up stable opera-
tion of the coupled BES-UASB system.
4. Conclusions
External voltage supply enhanced the DClNB microbial degrada-
tion, and the coupled BES-UASB (R1 and R2) were more tolerant to
high hydraulic loadings compared with the control due to higher
microbial diversity in the coupled BES-UASB. According to micro-
bial community analysis, more microbes related to reductive chlo-
rination and syntrophism were enriched in MES-UASB system
which might be responsible for the superior performance for
DClNB degradation. The results demonstrated that the coupled
BES-UASB intermittently supplied with external voltage was able
to be utilized in treatment of DClNB containing wastewater even
when the HRT was as short as 6 h.
Acknowledgement
This work was funded by the National Natural Science Founda-
tion of China (No. 51378454).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.06.
010.
886 H. Chen et al. / Bioresource Technology 241 (2017) 879–886

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