Cometabolic degradation of chloramphenicol via a meta-cleavage pathway in a microbial fuel cell and its microbial community

Bioresource Technology 229 (2017) 104–110

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Cometabolic degradation of chloramphenicol via a meta-cleavage pathway in a microbial fuel cell and its microbial community Qinghua Zhang a,b,c, Yanyan Zhang a,c, Daping Li a,c,⇑ a Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Science, Chengdu 610041, China b College of Life Sciences, Sichuan University, Chengdu 610064, China c University of Chinese Academy of Sciences, Beijing 100049, China

h i g h l i g h t s
The MFC with acetate as electrons donor increased the removal rate of CAP.
The CAP degradation was optimized using Box-Behnken model.
Antibacterial activity of CAP was eliminated after treatment by MFC.
The electrogenic bacteria enriched in MFC under the closed-circuit mode.

a r t i c l e

i n f o

Article history: Received 1 December 2016 Received in revised form 10 January 2017 Accepted 11 January 2017 Available online 13 January 2017 Keywords: Chloramphenicol Microbial fuel cell Degradation pathway Microbial community

a b s t r a c t The performance of a microbial fuel cell (MFC) in terms of degradation of chloramphenicol (CAP) was investigated. Approximately 84% of 50 mg/L CAP was degraded within 12 h in the MFC. A significant interaction of pH, temperature, and initial CAP concentration was found on removal of CAP, and a maximum degradation rate of 96.53% could theoretically be achieved at 31.48 °C, a pH of 7.12, and an initial CAP concentration of 106.37 mg/L. Moreover, CAP was further degraded through a ring-cleavage pathway. The antibacterial activity of CAP towards Escherichia coli ATCC 25922 and Shewanella oneidensis MR-1 was largely eliminated by MFC treatment. High-throughput sequencing analysis indicated that Azonexus, Comamonas, Nitrososphaera, Chryseobacterium, Azoarcus, Rhodococcus, and Dysgonomonas were the predominant genera in the MFC anode biofilm. In conclusion, the MFC shows potential for the treatment of antibiotic residue-containing wastewater due to its high rates of CAP removal and energy recovery. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Since the discovery of the first natural antibiotic, penicillin, by Fleming, many natural antibiotics or their derivatives have been applied in medicine, animal feed, and agriculture (Andersson and Hughes, 2014). However, their widespread use has resulted in antibiotic residues becoming a serious problem. At present, medical facilities and pharmaceutical factories, particularly the animalbreeding industry, discharge large quantities of various antibiotics (Naquin et al., 2015), but few water-treatment plants have strictly implemented current standards, resulting in discharge of residual antibiotics into the environment (Wang et al., 2016). Residues of ⇑ Corresponding author at: Chengdu Institute of Biology, Chinese Academy of Science, Chengdu 610041, China. E-mail address: [email protected] (D. Li). http://dx.doi.org/10.1016/j.biortech.2017.01.026 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.

antibiotics in the environment are not only considered emerging contaminants but also pose a potential threat to human and animal health worldwide (Berendonk et al., 2015). The long-term effects of antibiotic residues include their bioaccumulation through the food chain and stimulation of the growth and spread of drug-resistant bacteria (Sarmah et al., 2006). The broad-spectrum nitroaromatic antibiotic, chloramphenicol (CAP), was the first synthetic antibiotic introduced into clinical practice on a large scale (Henry et al., 1981). Due to its costeffectiveness, it has been extensively used in the animal breeding industry globally since its discovery. Due to its serious toxicity (bone-marrow depression and aplastic anemia) in humans and animals (Martelli et al., 1991), CAP has been banned for use in foodproducing animals in many developed countries; however, it is still extensively used in many developing countries because of its low cost. CAP is frequently present in the effluent of breeding farms

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and water-treatment plants (Fan et al., 2010; Resende et al., 2014). One or more functional groups is essential for the antibacterial activity of most antibiotics; the 1,3-propanediol formation and nitro group are the functional groups of CAP (Miller and Halpert, 1986). However, most studies have focused on the change in CAP concentration rather than the degradation pathway. Furthermore, acetylate and amine metabolites of CAP are produced by its microbial degradation (Yun et al., 2016), but whether they are further degraded is unclear. These aromatic metabolites pose a potential risk to human health (Kadlubar et al., 1992). Various physicochemical methods have been used to investigate CAP degradation, such as photocatalytic reduction, zerovalent bimetallic nanoparticles, and the Fenton process (Sun et al., 2013). However, most of these methods consume energy and/or chemicals. Bioelectrochemical systems (BESs), including microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) with biocatalyzed electrodes, can be used to degrade various pollutants (e.g., transforming nitroaromatics) into amino-aromatic compounds and to remove antibiotics such as sulfamethoxazole, ceftriaxone and ampicillin (Körbahti and Tasßyürek, 2015; Liang et al., 2014; Wang et al., 2016; Wen et al., 2011). Biocatalytic electrochemical cells have also been used to degrade CAP using an external voltage and glucose as the electron donor (Sun et al., 2013). Unlike MECs, MFCs do not require an external input voltage and consume less energy. CAP degradation by MFCs with acetate as the sole electron donor has not been extensively investigated; therefore, CAP removal using MFC reactors requires further research. This study investigated the removal of CAP by an MFC with acetate as the sole electron donor. A Box-Behnken experimental design (BBD) combined with response surface methodology (RSM) was applied to optimize the CAP degradation conditions. CAP metabolites were identified by mass spectrometry, their antibacterial activity was evaluated, and the degradation pathway was deduced. In addition, high-throughput sequencing was employed to analyze the microbial composition of MFC biofilm. 2. Materials and methods 2.1. Chemicals and reagents CAP (>98% purity) was purchased from Solarbio (Beijing, China). High-performance liquid chromatography (HPLC)-grade methanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrapure water used in this experiment was generated by a MilliQ system (Bedford, MA, USA). All of the other chemicals were of analytical reagent grade and were obtained from commercial sources. 2.2. Reactor setup Dual-chamber MFC reactors separated by cationic exchange membranes (CMI-7000s, Membrane International Inc., Ringwood, NJ, USA) were assembled. Each chamber of the MFC reactor had a working volume of 120 mL. Active-carbon felt (3 cm diameter, 3 cm length) was used for the cathode and anode. The two electrodes were connected via a titanium wire (1 mm in diameter) with an external load of 1000 X. The MFC reactor output voltages were recorded using a digital multimeter (Keithley 2700, Cleveland, OH, USA).

sludge obtained from a wastewater treatment plant (Mianyang, China). The anaerobic sludge was fed with medium containing acetate in 50 mM phosphate buffer solution (PBS; 2.45 g/L NaH2PO4H2O, 4.57 g/L Na2HPO4, 0.13 g/L KCl, 0.31 g/L NH4Cl) supplemented with 5 mL/L vitamins and 12.5 mL/L mineral solutions in the presence of 50 mg/L CAP (Lovley and Phillips, 1988; Zhang et al., 2015). To start the MFC reactors, the anode chambers were inoculated with the anaerobic-acclimated sludge. Then CAP was injected into the anodes of the MFC reactors (final concentration, 50 mg/L). The reactors were randomly divided into three groups and operated in closed-circuit mode, open-circuit (OC) mode, and abiotic control (anodes were autoclaved). The OC test without a circuit load was regarded as the traditional anaerobic digestion test. The medium was purged with N2 gas (99.9%). After 6 months, CAP was rapidly degraded in the anode chamber and the anolyte was changed weekly thereafter. The catholyte was potassium ferricyanide solution (50 mM K3[Fe(CN)6] in 50 mM PBS) and was replaced after each cycle. For sampling, the anolyte was withdrawn from the anode chamber and filtered through 0.22 lm porosity polytetrafluoroethylene (PTFE) membranes prior to chemical analysis. Experiments were performed at room temperature (28 ± 2 °C) and the anode chambers were covered with silver paper to exclude light. 2.4. Analytic method The CAP concentration was determined using an Agilent 1260 HPLC system (Agilent, Santa Clara, CA, USA) with UV detection at 275 nm. CAP separation was achieved using an Agilent C18 column (4.6  250 mm, 5 lm) with a methanol/water mobile phase at a flow rate of 0.6 mL/min. CAP removal was calculated using the following equation:

P ¼ ðA  BÞ=A  100%

ð1Þ

where P is the CAP removal percentage, A is the initial concentration of CAP in the anolyte, and B is the CAP concentration in samples. Analysis of CAP degradation products was performed by electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS; Bruker, Germany). Positive and negative ESI modes with a capillary voltage of 4.5 kV were used to detect samples. The nebulizer was set at a 0.8 bar and the flow of dry gas was set at 6.0 mL/min (dry heater at 180 °C). High-purity nitrogen (N2) (99.999%) was used as the collision gas. 2.5. Optimization of the degrading conditions Based on the results of a single-factor experiment, the temperature, pH, and initial CAP concentration were selected as three independent variables. The dependent variable was the CAP degradation rate over 3 days in the MFC reactors. A three-factor and three-level BBD of RSM was applied to evaluate the combined effects of three independent variables on the CAP degradation rate using the Design Expert (DX) 8.0.5 software (Stat-Ease Inc., Minneapolis, MN, USA). The design matrix and levels of each variable are shown in Table 1. Seventeen experimental sets were conducted to assess the interactive effects of the three independent variables,

Table 1 Range and levels of independent variables and code values in BBD. Independent variables

Symbols

pH Temperature (°C) CAP concentration (mg/L)

X1 X2 X3

2.3. Reactor operation Before starting the MFC reactors, the microbial consortium used as the anode inoculum was pre-enriched using activated anaerobic

Range and levels 1

0

1

6.0 20 100

7.0 30 150

8.0 40 200

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Table 2 Box-Behnken design for CAP degradation. Run

Code

Response

pH

Temperature (°C)

CAP concentration (mg/L)

CAP degradation rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0 1 1 0 1 0 0 0 0 0 0 1 1 1 1 1 0

0 0 1 1 0 1 1 0 0 0 1 1 0 1 1 0 0

0 1 0 1 1 1 1 0 0 0 1 0 1 0 0 1 0

89.36 83.13 68.11 83.21 58.42 47.41 89.19 89.59 90.21 88.25 59.64 75.13 89.46 62.16 71.52 48.31 87.93

each at low (1), medium (0), and high (+1) levels, on the CAP degradation rate (Table 2). A reactor without microorganisms was used as the control. Each experiment was performed in triplicate. 2.6. Antibacterial activity test After a reaction for 120 h, most of the CAP (50 mg/L) was consumed. Then the reactor effluent (80 mL) was removed, centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was filtered through a 0.22 lm Millipore filter. The antibacterial activity of reactor effluent was evaluated against Escherichia coli ATCC 25922 (Gram-negative bacterium) and Shewanella oneidensis strain MR-1 (electrogenic bacterium). Three test groups were designated. The reactors in the CAP group contained 20 mL Luria–Bertani (LB) medium, 80 mL 20 mM PBS (pH 7.0), and 50 mg/L CAP. Those in the reactor effluent group contained 20 mL LB medium and 80 mL sterile anolyte. Reactors in the control group contained 20 mL LB medium and 80 mL 20 mM PBS. Each group comprised triplicate reactors. Bacteria were harvested during the exponential growth phase and were washed three times with sterile PBS. Following inoculation, bacteria were incubated on a shaker at 150 rpm and 37 °C. Cell concentration was determined by measuring the optical density at 600 nm (OD600). 2.7. Microbial community analysis and sequencing data processing The microbial community composition of the MFC anode biofilm was analyzed at the end of the test. The total genomic DNA of the samples was extracted using a TIANamp Bacteria DNA Kit (Tiangen Biotech Co., Ltd., China) according to the manufacturer’s instructions (Yan et al., 2015). For sequencing, the V4 region of the 16S rRNA gene was amplified using the universal primer pair 515F (50 -GTGYCAGCMGCCGCGGTA-30 ) and 909R (50 CCCCGYCAAT TCMTTTRAGT-3) to which the barcodes were attached. The conditions used for PCR and related procedures were previously described (Li et al., 2013). Amplicons were sequenced on the Illumina Miseq platform (San Diego, CA, USA). Based on the unique sample barcodes, the raw sequences were sorted and denoised using QIIME Pipeline (Caporaso et al., 2010). Raw sequences of low quality, a read length of <200 bp or an average base quality score of <20 were filtered out. Chimeric sequences were removed by the UCHIME algorithm (Edgar et al., 2011). Because the number of the sequences varied among the samples, a second resampling

process was performed using 17,329 reads per sample. The operational taxonomic units (OTUs) were classified using a 97% identity to the 16S rRNA gene sequence as a cutoff. The Chao1 estimator and Shannon diversity index were calculated at a 97% sequence similarity in the Ribosomal Database Project (RDP) pipeline (http://pyro.cme.msu.edu/). The RDP classifier was used to assess the phylogenetic affiliation of each sequence at a confidence level of 80%. To ensure the accuracy of the RDP classifier, representative sequences of dominant archaea and bacteria were subjected to BLAST homology search against non-metagenomes and nonenvironmental sequences in the NCBI nucleotide database (http://blast.ncbi.nlm.nih.gov/). 2.8. Statistical analysis The CAP concentration and OD600 values are expressed as the geometric means of triplicate measurements. Differences in mean values were examined by one-way analysis of variance (ANOVA). Statistical significance was assumed at a level of P < 0.05. Statistical analyses were performed using the Statistical Analysis System (SAS) 9.2 (SAS Institute Inc., Cary, USA). 3. Results and discussion 3.1. CAP concentration All of the reactors were operated in batch mode. The anolyte and catholyte were replaced when the output voltage dropped to less than 50 mV, which was considered a ‘‘cycle”. The maximum stable output voltage of MFC was 0.6 V (Fig. S1). And the corresponding maximum current was 0.6 mA, which indicated that the enrichment of exoelectrogenic bacteria in MFC anode. The changes in CAP concentration after a 6-month acclimation period are shown in Fig. 1. The order of CAP removal rate (PCAP) was MFC > open circuit > abiotic control. In the abiotic control group, the CAP concentration decreased slightly. However, after a 12 h incubation, approximately 84% of 50 mg/L CAP was rapidly removed in the MFC group, and the CAP concentration was less than the detection limit (0.1 mg/L) after 48 h. In the OC control group, 48% of added CAP was removed within 12 h, and 83% was removed after 72 h. Therefore, CAP removal in the MFC group was more rapid than that in the OC control group. CAP may have been directly degraded by bacteria or through physical adsorption by the anode. However, the concentration change in the abiotic control group was slight, suggesting that removal of CAP in the

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formation of acetylated-CAP (CAP-acetyl), which is a common mechanism of resistance to CAP (Cao et al., 1999). Then AMCl2 and CAP-acetyl were further catabolized by the microbes. AMCl2 was transformed via a meta-cleavage pathway (Bajaj et al., 2014; Ju and Parales, 2010). In addition, the nitro group of CAP-acetyl was further reduced to an amino group. Subsequently, AMCl2acetyl (amine product of CAP-acetyl) was converted into AMCl2. After 96 h incubation, only one metabolite, Mc-AMCl2 (the metacleavage product), was detected. Therefore, AMCl2-acetyl was completely further transformed to Mc-AMCl2 (Fig. S5). Moreover, the aromatic structure of CAP was destroyed via a meta-cleavage pathway. Therefore, CAP is degraded in MFC through diverse microbial metabolic pathways and can be transformed further if it is present as the sole carbon and nitrogen source (Dantas et al., 2008). 3.4. Antibacterial activity of CAP degradation products Fig. 1. CAP concentration change in MFCs, open-circuit controls (without current) and abiotic controls (without current and microbes) after 6 months of acclimation. The CAP removal rate in three groups (PCAP) are: MFC > open circuit > abiotic control. Test conditions: ambient temperature (28 ± 2 °C); initial CAP concentration of 50 mg/L.

MFC was mainly due to microbial biodegradation. In addition, after a long-term acclimation, the growth of exoelectrogenic bacteria provided electrons to increase the metabolic reactions rates of microbes in the MFC anode (Xian et al., 2015). So the generation of electricity by exoelectrogenic bacteria made the MFC a more suitable niche for the CAP degradation, which was the main reason for the improvement of the CAP removal rate. 3.2. Optimization of CAP reduction BBD and RSM were used to investigate the interaction of pH, temperature, and initial concentration of CAP, as well as three variables, on CAP degradation during 17 runs of the experiment. The results in Table 2 were subjected to ANOVA and multiple regression analyses using the DX software. The polynomial model equation was used to predict the optimum variables and the corresponding maximum CAP-degradation rates. A quadratic model of CAP degradation exhibited the best fit (F-value 233.87; p < 0.001; Table S1), which indicates a significant effect on CAP degradation (Fig. 2). The p-value of the ‘‘lack of fit” was 0.36, suggesting that the prediction of the model fit was good. The equation (Eq. (2)) for the maximum CAP degradation rate was as follows:

Degradation ratio ¼ 88:85 þ 3:25  X1 þ 4:32  X2  16:40  X3  0:59  X1 X2 þ 0:95  X1 X3 þ 1:56  X2 X3  9:83  X21  9:79  X22  9:10  X23

ð2Þ

The results of the RSM analysis showed that the optimal conditions were a pH of 7.12, temperature of 31.48 °C, and initial CAP concentration of 106.37 mg/L, which yielded a predicted CAP degradation rate of 96.53% after 3 days, the theoretical maximum shown in Eq. (2). 3.3. Proposed CAP degradation pathways MFC anode samples were subjected to MS analysis, which resulted in identification of four probable metabolites (Figs. S2– 4). At early time points, CAP was transformed into two primary metabolic intermediates. The nitro group of CAP was transformed into an amine group with the formation of AMCl2 (amine product of CAP). The 3-hydroxy group CAP occurred acetylation with the

The biotoxicity of CAP degradation products is unclear. To investigate this, MFC anode effluent was inoculated with E. coli ATCC 25922 and S. oneidensis MR-1 and the antibacterial activity was assessed. E. coli ATCC 25922 is a Gram-negative bacterium used for antibiotic susceptibility assays, and S. oneidensis MR-1 is used to research microbial electrochemical behavior. CAP completely inhibited the growth of E. coli and S. oneidensis MR-1 (Fig. 3). However, after degradation by the reactor, the antibacterial activity of CAP towards E. coli and S. oneidensis MR-1 was significantly attenuated. For E. coli, its growth curve in the reactor effluent group was similar to that in the control group (Fig. 3a). For S. oneidensis MR-1, its growth curve in the reactor effluent group was almost the same to that in the control group. Therefore, the growth of E. coli and S. oneidensis was not markedly affected, which indicated that the antibacterial activity of CAP was largely eliminated by the MFC. CAP was first transformed to the CAPacetyl and AMCl2, which involved changes to its functional groups (Jiang et al., 2016; Wolter et al., 2005). Moreover, the aromatic structure of these two CAP metabolites was destroyed via a meta-cleavage pathway. Thus, after biodegradation in the MFC reactor, the reactor effluent exhibited little activity against E. coli and S. oneidensis. 3.5. Microbial community analysis of anode biofilm To assess the microbial community structure of the MFC anode biofilm, the total DNA of the activated sludge group, the MFC group, and the open-circuit control group was extracted and amplified. The amplification products were subjected to sequencing of bacterial and archaeal 16S rRNA genes. The samples yielded qualified sequencing reads in the range of 17,329–33,306 bp. The sequences were aligned and clustered to calculate OTUs using 97% identity as a cutoff. The sludge exhibited a diversity (Shannon = 8.17) greater than that of the MFC and controls (Shannon = 7.39 and 6.06, respectively) (Table S2). The Simpson index of the MFC (Simpson = 0.96) was higher than that of the opencircuit control group and activated sludge group (Simpson = 0.94 and 0.81, respectively). Qualified reads retrieved from the sludge, MFC, and control groups were assigned to known phyla, classes, and genera. The samples belonged to 10 phyla (Fig. 4a). At the class level (Fig. 4b), the biofilm community in MFC was dominated by the classes Betaproteobacteria (47.61%), Actinobacteria (15.40%), Alphaproteobacteria (9.35%), Flavobacteriia (8.00%), Sphingobacteriia (5.05%), and Gammaproteobacteria (3.52%), which was obviously different with that in open-circuit group and anaerobic sludge control. Microbial identification to the genus level was performed to determine the mechanism of CAP degradation in the MFC. At the

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Fig. 2. Effects of pH, temperature, and initial CAP concentration on CAP degradation rate. (a) Interactive effects of pH and temperature on CAP degradation while fixing the value of initial CAP concentration at 150 mg/L. (b) Interactive effects of pH and initial CAP concentration on CAP degradation while fixing the value of temperature at 30 °C. (c) Interactive effects of temperature and initial CAP concentration on CAP degradation while fixing the value of pH at 7.0.

Fig. 3. Growth curve of E. coli ATCC 25922 (a) and S. oneidensis MR-1 (b) in the control, MFC, and CAP groups. The growth of E. coli ATCC 25922 and S. oneidensis MR-1 was severely inhibited by CAP, whereas the inhibition effect of MFC effluent was not significant.

genus level (Fig. 4c), the dominant bacteria in MFC anode biofilm were Azonexus (19.94%), Comamonas (19.41%), Nitrososphaera (12.15%), Chryseobacterium (8.86%), Gaiella (5.45%), Azoarcus (3.10%), Azospirillum (2.46%), Rhodococcus (1.91%), Taibaiella (1.79%), Gp4 (1.41%), Dysgonomonas (1.31%), and Gp6 (1.28%). Azonexus and Comamonas dominate the communities on anodes of

MFCs used for electricity generation with acetate as the electron donor (Jangir et al., 2016; Xing et al., 2010). Moreover, members of the genus Comamonas can mineralize aniline (Peres et al., 1998). Nitrososphaera is an ammonia-oxidizing archaeal genus (Tourna et al., 2011). Many species of the genus Chryseobacterium have antibiotic-resistance genes, the products of which inactivate

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Fig. 4. Microbial community composition of sludge, MFC anode biofilm and the control at the phylum (a), class (b) and genus (c) levels. Only relative abundances >1% are shown.

CAP (Shaw et al., 1979). The dominant genera Azoarcus and Rhodococcus are degraders of various aromatic compounds, including some toxic and degradation-recalcitrant compounds, via a ringcleavage pathway under anaerobic conditions (Larkin et al., 2005; Qiu et al., 2013; Widdel and Rabus, 2001). The abundance of Azoarcus, Pseudomonas, and Dysgonomonas was higher in the control group than in the MFC group, but the community diversity of the MFC group was higher than that of the control group. Thus, the microorganisms in MFC anode biofilm can be functionally categorized into the following two groups: exoelectrogenic bacteria (Azonexus and Comamonas) and CAP-degradation-related bacteria (Azoarcus, Rhodococcus, Comamonas, Nitrososphaera, and Chryseobacterium). The complex syntrophic interactions and spatial distribution of these functional bacteria resulted in the rapid degradation of CAP. First, metabolism of acetate by exoelectrogenic bacteria was responsible for current generation in the MFC reactor. And the electrons provided by exoelectrogenic bacteria could increase the metabolic reaction rates of anaerobic bacteria in MFC (Xian et al., 2015). Second, bacteria with strong catabolic versatility were enriched in the anode biofilm. Therefore, the presence of various functional microbial communities was crucial for both CAP degradation via a ring-cleavage pathway and electricity generation.

4. Conclusions The MFC system with acetate as the electron donor can significantly improve CAP removal rate. Meanwhile, pH, temperature and

initial CAP concentration had a significant effects on the removal of CAP. Antibacterial activity of CAP was largely eliminated via metacleavage degradation pathway in MFCs. The microbial community composition analysis results showed that the MFC promoted the growth and multiplication of electrogenic bacteria. The electrons provided by electrogenic bacteria was significant for the improvement of CAP removal rate. The MFCs seemed to be promising as a potential approach to energy recovery and treatment of residual contaminants in the environment. Acknowledgements This work was supported by the National Natural Science Foundation of China (31270166). 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.2017.01. 026. References Andersson, D.I., Hughes, D., 2014. Microbiological effects of sublethal levels of antibiotics. Nat. Rev. Microbiol. 12, 465–478. Bajaj, A., Mayilraj, S., Mudiam, M.K.R., Patel, D.K., Manickam, N., 2014. Isolation and functional analysis of a glycolipid producing Rhodococcus sp. strain IITR03 with potential for degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). Bioresour. Technol. 167, 398–406.

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