Accelerated microbial reductive dechlorination of 2,4,6-trichlorophenol by weak electrical stimulation

Water Research 162 (2019) 236e245

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Accelerated microbial reductive dechlorination of 2,4,6trichlorophenol by weak electrical stimulation Xiao-Qiu Lin a, Zhi-Ling Li a, *, Bin Liang b, Hong-Liang Zhai a, Wei-Wei Cai a, Jun Nan a, Ai-Jie Wang a, b, ** a b

State Key Laboratory of Urban Water Resources and Environment, School of Environment, Harbin Institute of Technology, Harbin, 150090, China Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 March 2019 Received in revised form 7 June 2019 Accepted 26 June 2019 Available online 27 June 2019

Microbial reductive dechlorination of chlorinated aromatics frequently suffers from the long dechlorination period and the generation of toxic metabolites. Biocathode bioelectrochemical systems were verified to be effective in the degradation of various refractory pollutants. However, the electrochemical and microbial related working mechanisms for bio-dechlorination by electro-stimulation remain poorly understood. In this study, we reported the significantly improved 2,4,6-trichlorophenol dechlorination activity through the weak electro-stimulation (cathode potential of
0.36 V vs. SHE), as evidenced by the 3.1 times higher dechlorination rate and the complete dechlorination ability with phenol as the end dechlorination product. The high reductive dechlorination rate (20.8 mM/d) could be maintained by utilizing electrode as an effective electron donor (coulombic efficiency of 82.3 ± 4.8%). Cyclic voltammetry analysis of the cathodic biofilm gave the direct evidences of the cathodic respiration with the improved and positive-shifted reduction peaks of 2,4,6-TCP, 2,4-DCP and 4-CP. The optimal 2,4,6-TCP reductive dechlorination rate (24.2 mM/d) was obtained when a small amount of lactate (2 mM) was added, and the generation of H2 and CH4 were accompanied due to the biological fermentation and methanogenesis. The electrical stimulation significantly altered the cathodic biofilm structure and composition with some potential dechlorinators (like Acetobacterium) predominated. The microbial interactions in the ecological network of cathodic biofilm were more simplified than the planktonic community. However, some potential dechlorinators (Acetobacterium, Desulfovibrio, etc.) shared more positive interactions. The co-existence and possible cooperative relationships between potential dechlorinators and fermenters (Sedimentibacter, etc.) were revealed. Meanwhile, the competitive interrelations between potential dechlorinators and methanogens (Methanomassiliicoccus) were found. In the network of plankton, the fermenters and methanogens possessed the more positive interrelations. Electro-stimulation at the cathodic potential of
0.36 V selectively enhanced the dechlorination function, while it showed little influence on either fermentation or methanogenesis process. The study gave suggestions for the enhanced bioremediation of chlorinated aromatics, in views of the electrostimulation capacity, efficiency and microbial interrelations related microbial mechanism. © 2019 Elsevier Ltd. All rights reserved.

Keywords: 2,4,6-Trichlorophenol Reductive dechlorination Electrical stimulation Electro-active microorganism Biocathode Microbial interaction

1. Introduction Halogenated aromatic compounds (HACs) constitute an important class of organic pollutants in the environment because of their

* Corresponding author. ** Co-corresponding author. State Key Laboratory of Urban Water Resources and Environment, School of Environment, Harbin Institute of Technology, Harbin, 150090, China. E-mail addresses: [email protected] (Z.-L. Li), [email protected] (A.-J. Wang). https://doi.org/10.1016/j.watres.2019.06.068 0043-1354/© 2019 Elsevier Ltd. All rights reserved.

€ggblom and Bossert, toxicity, persistence and widespread use (Ha 2003). Twenty-three types of halogenated benzenes or phenols have been recorded as priority pollutants by the US Environmental Protection Agency, which occupied 18% of the total list (EPA, 1981). 2,4,6-trichlorophenol (2,4,6-TCP) is one of the notorious representatives and has been extensively applied as a pesticide precursor or formed during drinking water disinfection (Tan et al., 2009). Reductive dehalogenation of HACs via organohalide-respiring bacteria has been considered as one of the most crucial bioremediation routes for HAC detoxification in anoxic or anaerobic

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contaminated sites (sediments, groundwater, etc.) (Jugder et al., 2016). However, because of the physiological toxicity of HACs and the external electron donor deficiency, the currently reported dehalogenation populations possess either low dehalogenation rate or incomplete dehalogenation capacity (Richardson, 2016; €ggblom et al., 2000). The reported HACs dehalogenation period Ha would usually take from a few days to weeks (Richardson, 2016; €ggblom et al., 2000). To date, some obligate organohalideHa respiring isolates like Dehalococcoides and Dehalobacter were found that could efficiently dechlorinate 2,4,6-TCP (Wang and He, 2013), but no isolate was able to dechlorinate 2,4,6-TCP completely to phenol (Wang et al., 2014; Villemur et al., 2006). The lack of electron donor (short-chain organics, H2) in dechlorination systems was majorly caused by the low H2 solubility or electron scramble of competitive metabolic processes (methanogenesis, denitrification, etc). (Chambon et al., 2013; Li et al., 2010). The enhanced reduction of various refractory pollutants has been observed in bioelectrochemical cathodes by introducing a constant negative potential (Aulenta et al., 2008; Zhang et al., 2014). The application of electrode as a potential electron donor is a favorable means because of its continuous, direct and sustainable characteristics (Chen et al., 2018). Previously, Anaeromyxobacter dehalogenans and Geobacter lovelyi were found to utilize electrons from the electrode surface and reductively dechlorinate 2-chlorophenol or tetrachloroethylene (Strycharz et al. 2008, 2010). The reductive dechlorination of chlorophenols have been investigated through adding co-substrates or electron mediators in constructed bioelectrochemical systems (Zhang et al., 2011, 2014; Liu et al., 2013; Wang et al., 2019). However, the electrochemical and microbial mechanisms of the electrostimulated bio-dechlorination require further study. The electrochemical evidence by utilizing electrode as electron donor for stimulating the dechlorination activity is still lacking. Besides, studies on the cathodic microbiome were majorly focused on the bacterial community composition and structure succession by high-throughput sequencing of 16S rRNA gene (Chen et al., 2019; Zhang et al., 2011), while the microbial interactions of different populations in the biocathode systems are poorly understood. Notably, in most of the bio-dehalogenation systems, dehalogenation was always accompanied with fermentation and methanogenesis because of the electron scramble by adding organic acids as carbon and electron source (Chambon et al., 2013; Li et al., 2010; Farai et al., 2012). In natural environment, dehalogenators and fermenters are symbiotic, while dehalogenators and methanogens are competitive (Chambon et al., 2013; Farai et al., 2012). However, how those functional populations were influenced under the electro-stimulated environments including not only the microbial community succession but also the microbial ecological interrelations remain poorly understood. In this study, microbial dechlorination activities for 2,4,6-TCP were investigated by stimulation with a negatively polarized graphite electrode (
0.36 V vs. SHE). This study aims to reveal whether cathode could serve as an alternative electron donor to stimulate bio-dechlorination, and how the potential dechlorinators, methanogens, fermenters or electroactive populations associate to achieve the high dechlorination efficiency when double electron donors (hydrosoluble lactate and insoluble solid-state electrode) co-existed. Microbiome structure and ecological network analyses aimed to identify the potential dechlorinators, fermenters and methanogens and their collaborative or competitive interrelations. The obviously improved dechlorination performance and the microbiome analyses highlighted the function and mechanism of the accelerated electro-stimulated dechlorination system for potential bioremediation applications.

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2. Materials and methods 2.1. Microbial consortium enrichment The microbial source was obtained from chlorophenol-polluted soil collected at around a pesticide factory in Shandong Province, China. The detail information of the microbial consortium enrichment process was described before (Li et al., 2013b). After acclimatization for more than one year, the culture could stably dechlorinate 100 mM of 2,4,6-TCP to 4-CP within 14 d, by utilizing 20 mM of lactate as the electron donor. The 2,4,6-TCP dechlorination results were shown in Fig. S1. 2.2. Biocathode bioelectrochemial system (BES) setup Biocathode BESs were set up in traditional cathode batch-type chamber by assembling two equal-size Lexan-made cubes (7  7  4 cm3) separated with a proton exchange membrane (N115, Membranes International Inc., NJ, USA). A graphite fiber brush (diameter 2.5 cm and length 2.5 cm, Toho Tenax Co. Ltd., Tokyo, Japan) and a carbon cloth (6 cm in diameter, YB-20, YiBang Technology Co. Ltd., Shenzhen, China) were used as anode and cathode, respectively. A saturated calomel reference electrode (SCE, 0.247 V vs. standard hydrogen electrode, SHE) was inserted into the cathode chamber for cathode potential detection. Before commencing, both cathodes and anodes were pretreated by immersing in 1 M hydrochloric acid (24 h) and then in deionized water (24 h). The effective volume of cathode and anode were 58 and 55 mL, respectively. Data acquisition system (Model 2700, Keithley Instruments Inc., Ohio, USA) was applied to detect the cathode potential with a high-precision resistor (10 U) connected in the circuit. The applied external voltage was 0.6 V and the cathode potential was measured at
0.36 V (Fig. S2). All the potential without special statement was against SHE. 2.3. Biocathode BESs acclimation and start up Catholyte composed of 5 mM phosphate buffered saline (PBS), 1 mL/L vitamin stock solution, l mL/L SL-10 trace element solution, 100 mM 2,4,6-TCP, and/or 2 mM sodium lactate (Li et al., 2013a). The anaerobic anolyte contained 100 mM of potassium ferrocyanide (99% purity). During preparation, both catholyte and anolyte were bubbled with N2 (99.99% of purity) for 15 min to remove oxygen. For solution exchange, the catholyte and anolyte were all discharged and meanwhile, the chambers were refilled with N2. Then the prepared catholyte and anolyte were injected immediately into cathode and anode chambers. The 2,4,6-TCP dechlorination consortium (5 mL, about 10% of inoculation percentage) was inoculated into cathode to allow the cathodic biofilm development. Once 2,4,6-TCP dechlorination activity was observed in the cathode, the catholyte exchange and repeated inoculation were conducted. After repeated inoculation for about four times (nearly 20 d), the microbial inoculation terminated and catholyte exchange was started every 4 d when 2,4,6-TCP dechlorination was observed. The stable dechlorination performance (rate, efficiency and metabolites generation) was observed after acclimation for about 40 d (about 20 d of acclimation and 20 d of medium exchange). After that, acclimation was continued for another 20 d to confirm the stable dechlorination activity. The whole acclimation procedure lasted for approximately 60 d to achieve the stable dechlorination activity. Separate reactors were simultaneously started up with four different operating conditions including (a) lactate-fed biocathode (L-BC), (b) non-lactate-fed biocathode (nL-BC), (c) open circuit and (d) abiotic cathode. The L-BC indicated the reactors fed with 2 mM sodium lactate and the cathodic potential of
0.36V throughout the

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operation. For the nL-BC, the initial operation mode was the same with the L-BC. After the mature biofilm was formed (about 40 d), sodium lactate was replaced with NaHCO3 for at least 3 cycles to testify the biofilm cathode-respiring ability during dechlorination. Open circuit reactors were the ones with the disconnected circuits throughout the operation. Abiotic cathode reactors indicated the ones without microbial inoculation during acclimation. Sextuplicate batch experiments were conducted in parallel under each identical condition. All experiments were conducted at a constant ambient temperature (25 ± 2  C). 2.4. Chemicals and analytical methods 2,4,6-trichlorophenol (2,4,6-TCP, 99% purity), 2,4dichlorophenol (2,4-DCP) (>97% purity), 4-chlorophenol (4-CP, 98% purity) and phenol ( 99% purity) were obtained from SigmaAldrich, USA. Sodium lactate (98% purity) were purchased from Xiya Reagent, China. The concentration of 2,4,6-TCP and its dechlorination metabolites, including 2,4-DCP, 4-CP and phenol, were determined by GC-MS (Shimadzu, Kyoto, Japan) (Li et al., 2016). Before performing GC-MS analysis, a liquid sample (1 mL) was taken from the cathode chamber, pretreated by acidifying, extracted with acetonitrile (1 mL) and ethyl acetate (1 mL), and finally dehydrated with sodium sulfate anhydrous. For each test, standard samples were pretreated and determined together with samples to be tested to determine the recovery rate. Lactate and organic metabolites (acetate and propionate) were determined by high-performance liquid chromatography (CTO-10A; Shimadzu) equipped with an L-column ODS (inner diameter 4.6 mm, length 250 mm, CRRI, Saitama, Japan) and UV detection (210 nm) (Li et al., 2010). Gas concentration of H2, CO2 and CH4 were determined by gas chromatography (GC-7890A, Agilent Technologies, CA, USA) and Oswald's equation conversion (Li et al., 2013a). Current (I) was calculated with I ¼ V/R according to Ohm's law, where the voltage was obtained from the resister through the data acquisition system. The coulombic efficiency (CE, %) was calculated according to equation CE ¼ fðCT0
CT Þ  V  6  F  100%g= Q . In this equation, Q indicates the generated electricity quantity (C), calculated by multiplying the accumulated area of current (A) and time (s). CT0 and CT indicate the determined concentration of 2,4,6TCP at time 0 and t (d). V represents the effective cathodic volume (L). The “6” in the formula is the theoretical number of electrons the cathode gets, when 1 mole of 2,4,6-TCP is completely dechlorinated to phenol. F is the Faraday constant (96485 C/mol). 2,4,6-TCP dechlorination efficiency (%) was calculated by dividing the decreased 2,4,6-TCP concentration with the initial one. The dechlorination rate constant was simulated by fitting with the firstorder kinetic model C ¼ C0 expð
kT tÞ, where C represents 2,4,6TCP concentration (mM) at time t (d) and C0 is the initial 2,4,6-TCP concentration. The rate constant kT (d
1) was calculated using OriginPro 2016. Cyclic voltammetry (CV) of the established systems were performed using a CHI 660E electrochemical workstation (CH Instruments, Chenhua Co., Ltd., Shanghai, China) equipped with a three-electrode system. The cathode was set as a working electrode and a saturated calomel electrode (SCE) was applied as the reference electrode. All the tests were operated with PBS as catholyte under medium condition (pH ¼ 7.2). The reductive dechlorination capacity of cathodes in acclimated systems of L-BC, nL-BC and abiotic cathode were testified fed with or without one kind of chlorophenols (2,4,6-TCP, 2,4-DCP or 4-CP). The condition without adding in any chlorophenol was blank control. CV curves were recorded from
1 to 0.2 V vs. SCE at the scanning rate of 5 mV/s. All the reactors needed to keep stable and unconnected for 1 h after catholyte exchange before CV test. The applied concentration of

2,4,6-TCP, 2,4-DCP or 4-CP were 100 mM. 2.5. Microbial community analysis The microbial community structure and composition in both cathodic biofilm and suspension of L-BC, nL-BC and open circuit were analyzed by 16S rRNA gene-based Illumina MiSeq sequencing. When lactate was removed for at least six cycles, samples were taken from nL-BC. Samples in cathode carbon brush were pretreated by cutting into pieces, immerging into 10 mL PBS and washing out by an oscillator. And then the washed-out solution was collected and concentrated to 1.5 mL by centrifugation at 12000 rps. The detail methods for DNA extraction, amplification, sequencing and taxonomic classification have been described previously (Chen et al., 2018). Principal component analysis (PCA) was performed to evaluate the topological variation of microbial community composition among the different operation conditions by Canoco 4.5. Pearson correlation between microbes and three crucial environmental variables (2,4,6-TCP dechlorination efficiency, acetate generation and CH4 generation) were calculated by Majorbio ISanger Cloud Platform (www.i-sanger.com). The visualization of both PCA and Pearson correlation were using OriginPro 2016. The 16S rRNA gene sequence data were deposited in NCBI Sequence Read Archive under the accession number of SRP151178. 2.6. Construction of molecular ecological network To evaluate the microbial interactions of potential dechlorination populations, fermentation populations and methanogens in cathodic biofilm and suspension, two phylogenetic molecular ecological networks (MENs) were constructed with the random matrix theory-based (RMT-based) interface approach (Zhou et al., 2010; Deng et al., 2012; Liang et al., 2019). All identified operational taxonomic units (OTUs) for bacteria and archaea were applied for the network construction. The topological properties of the network including number of nodes and links, averaged connectivity, path length, clustering coefficient and modularity were processed by network analysis pipeline (http://ieg2.ou.edu/MENA/ ). The constructed networks for biofilm and plankton community were visualized by Cytoscape 3.6.1 software. 3. Results 3.1. Reductive dechlorination of 2,4,6-TCP After inoculating for approximately 60 d, the similar current variation trend, stable cathode potential (Fig. S2) and 2,4,6-TCP dechlorination efficiency were observed under each batch cycle, suggesting the constructed systems have become stable. 2,4,6-TCP dechlorination efficiencies and kinetics measured in a cycle were shown in Fig. 1 and Table S1. The significantly enhanced 2,4,6-TCP dechlorination activity was observed in L-BC, with the removal efficiency (Er) achieving 83.7 ± 2.1% and 99.5 ± 3.2% at 2 d and 4 d, respectively (Fig. 1A, Table S1). Phenol was the major determined dechlorination product in L-BC. A trace amount of 2,4-DCP and 4-CP once appeared at 2 d, and they were further dechlorinated completely to phenol after 4 d (Fig. 1A). The further degradation of phenol was not observed after the following 10 d (Fig. S3). In comparison, little 2,4,6-TCP was removed in abiotic cathode at 4 d (Fig. 1D), reflecting the restrictive dechlorination capacity at potential of
0.36 V. The result was coincident with a previous study finding that the pure electrochemical process contributed little to chlorophenol decomposition when cathode potential was higher than
0.44 V (Cheng et al., 1997). Meanwhile, the dechlorination process in open circuit was similar with that by the

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Fig. 1. 2,4,6-TCP reductive dechlorination and metabolites formation in lactate-fed biocathode (L-BC) (A), non-lactate-fed biocathode (nL-BC) (B), open circuit (C) and abiotic cathode (D)

enriched microbial consortium (Fig. S1, S3B). Only 36.4% of Er was observed at 4 d and the end of dechlorination appeared after 12 d, following with 2,4-DCP and 4-CP as the intermediates or end dechlorination products. In either the enriched microbial consortium or open circuit, 4-CP was the final dechlorination product. After lactate was removed (nL-BC), the dechlorination activity could still be maintained with phenol as the end dechlorination product. 2,4,6-TCP dechlorination efficiency was reduced to 88.2 ± 3.1% at 4 d with the generation of some metabolites (2,4-DCP, 4-CP and phenol) (Fig. 1B). The complete dechlorination of 2,4,6TCP to phenol was observed at 8 d (Fig. S3). Under all the tested conditions, the rate constants (k) for 2,4,6TCP reductive dechlorination was fitted well with the first-order kinetics (r2 > 0.970, Table S1). The distinctly higher k was observed in L-BC (k ¼ 1.37 ± 0.02, r2 ¼ 0.970) than in nL-BC (k ¼ 0.53 ± 0.02, r2 ¼ 0.990) or open circuit (k ¼ 0.11 ± 0.01, r2 ¼ 0.995). The coulombic efficiencies in L-BC and nL-BC achieved 43.8 ± 3.2% and 82.3 ± 4.8%, respectively, revealed 2,4,6-TCP reductive dechlorination relied more on electrode after lactate removal. 3.2. Performance of lactate fermentation and gas generation In L-BC, lactate was completely consumed within 2 d and the gradual generation of short chain acid and gas were observed as the fermentation byproducts (Fig. 2a). At 4 d, acetate and propionate approached 1.1 mM and 0.5 mM, respectively, accompanied with some amount of H2 (0.35 mM), CH4 (1.7 mM) and CO2 (0.15 mM) (Fig. 2b). The gradual increase in the concentration of CH4 indicated the increased activity of methanogens. Neither lactate fermentation nor gas generation was observed in the abiotic cathode, confirming their reliance on microbial participation. Lactate fermentation performance in open circuit was similar with L-BC, and both lactate

fermentation and gas formation were observed (Fig. 2a and b). This indicated that electro-stimulation had little impact on both lactate fermentation and gas generation. As lactate was removed (nL-BC), only negligible acetate or CH4 were detected, while no H2 or CO2 generation was observed, indicating their restrictive ability for biocathode gas generation under cathode potential of
0.36 V. Besides, it also illustrated that the generation of CH4 and H2 majorly relied on the biological fermentation and methanogenesis. Previously, some studies found that the generation of H2 or CH4 in BESs could only proceed below
0.41 V and
0.5 V, respectively (Cheng et al., 2009). Combing the lactate decomposition results in L-BC, nL-BC and abiotic cathode, it is inferred that the generation of H2 and CH4 would probably be derived from biological effect but not by the cathodic potential. 3.3. Electrochemical evidences for the enhanced dechlorination by CV analysis After the stable dechlorination activity was obtained, CV analysis was performed to testify the electrochemical characteristics of cathodic biofilm. In abiotic cathode added with 2,4,6-TCP, CV curve presented a small peak (peak a) arising at
0.27 V (Fig. 3A), while no recognizable peak was observed without 2,4,6-TCP (Fig. 3B). Two obvious reduction peaks were found at
0.057 V (peak b, nLBC) and
0.005 V (peak c, L-BC) with the onset potential ranged between 0.1 and 0.2 V, respectively (Fig. 3A). Since no remarkable peak was observed in the absence of 2,4,6-TCP, peaks b (nL-BC) and c (L-BC) would probably derive from 2,4,6-TCP reduction. Both peak b and c were positively shifted compared with peak a. Also, the 13.8 times (peak c/a, nL-BC) and 3.6 times (peak b/a, L-BC) higher peak currents were observed. The much smaller peak area of b than c demonstrated the distinctly affected electrochemical characteristics after lactate removal.

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Fig. 2. Lactate fermentation and the generation of organic metabolites (acetate and propionate) and gas (CH4, CO2 and H2) accompanied with 2,4,6-TCP reductive dechlorination in lactate-fed biocathode (L-BC) and non-lactate-fed biocathode (nL-BC).

Fig. 3. (A): Cyclic voltammograms (CVs) of the abiotic cathode, non-lactate-fed biocathode (nL-BC) and lactate-fed biocathode (L-BC) in the presence of 2,4,6-TCP. (B): CVs of the abiotic cathode, nL-BC and L-BC in the absence of 2,4,6-TCP. (C): CVs of the abiotic cathode with and without 2,4-DCP or 4-CP. Inset: enlarging for comparison of the different operating conditions with the potential ranged between 0.2V and
0.8V. (D): CVs of L-BC with and without 2,4-DCP or 4-CP. The applied concentration of 2,4,6-TCP, 2,4-DCP and 4CP were 100 mM. All experiments were conducted in neutral conditions (PBS, pH ¼ 7.2).

The reduction capacity of 4-CP or 2,4-DCP by cathodic biofilm were also testified (Fig. 3 C, D). Two small reduction peaks d (
0.19 V) or f (
0.25 V) were observed with the addition of 4-CP or 2,4-DCP in the abiotic cathode. Similarly, in biocathode with the addition of 4-CP or 2,4-DCP, two positively shifted peaks were observed (peak D, 0.016 V; peak F, 0.003 V). Meanwhile, the peak currents of D and F were 2.7 and 1.4 times higher than d and f,

respectively. The obvious positive shift of reduction peak and the higher peak current indicated the enhanced reduction activity of 2,4-DCP or 4-CP in L-BC. 3.4. Microbial community structure and composition The detailed information on the high-throughput sequencing,

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the diversity and dominant phyla and classes for bacteria and archaea were described in Supplementary Materials (Tables S2 and S3 and Fig. S4). Principal component analysis (PCA) (Fig. S5A) showed the similar bacterial OTU composition in biofilm and plankton of L-BC and biofilm of nL-BC, while distinctly different bacterial communities were obtained in the enriched culture, open circuit or the plankton of nL-BC. For the archaea, the similar OTUs were observed in the biofilms of L-BC, nL-BC and open circuit. However, the considerable different archaeal community composition was found between clusters of electro-stimulated samples with the original enrichment (Fig. S5B). The PCA analysis showed that electro-stimulation obviously altered the microbial structure. Bacterial structure and composition (relative abundance over 0.1%) at genus level were described in Fig. 4A. Electro-stimulation distinctly promoted the enrichment of potential electro-active, dechlorinating and fermenting genera, like Acetobacterium, Clostridium, Pseudomonas and Desulfovibrio. Acetobacterium was the most abundant in all electro-stimulated systems (46.5% in biofilm and 44.3% in the plankton of L-BC, 47.1% in biofilm and 25.8% in the plankton of nL-BC), except for the open circuit or the original enrichment. Acetobacterium is homoacetogenic and halogenrespiring which utilizes halogenated organic compounds, like

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tetrachloromethane and pentabrominated diphenyl ether (Michal et al., 2012; Ding et al., 2013). Clostridium (2.1e9.1%) and Pseudomonas (4.6e7.9%) which hold the diverse physiologies, were predominant in all electro-stimulated systems. Both Clostridium and Pseudomonas were reported as lactate-utilizing fermenters (Akao et al., 2007; Ma et al., 2007) and reductive dechlorinators (Xu et al., 2014; Garg et al., 2013). Besides, Clostridium and Pseudomonas were reported as electro-active which could respire with both anode and cathode (Choi et al., 2014; Logan, 2009; Su et al., 2012). Desulfovibrio, responsible for 2-chlorophenol reductive dechlorination (Sun et al., 2000), was more abundant in biofilms of L-BC (5.8 %e6.7%) and nL-BC (4.3 %e4.9%). And Desulfovibrio was also reported that could respire with either anode (Logan, 2009) or cathode (Croese et al., 2011). Some fermentative genera, like Anaerocella, Sedimentibacter or Hydrogenoanaerobacterium (Yang et al., 2009; Imachi et al., 2016; Song and Dong, 2009), took higher proportion in L-BC (4.6%, 3.4% and 3.0%, respectively) than nL-BC (1.5%, 1.2% and 1.2%, respectively), indicating their close correlation with lactate. As for open circuit, the proportions of Acetobacterium (16.3%) and Desulfovibrio (0.2%) were obviously lower than the electro-stimulation system (L-BC, nL-BC). However, the abundance of other potential functional genera like Anaerocella

Fig. 4. Taxonomic classification of the obtained 16S rRNA gene sequences at the genus level in the kingdom of bacteria (A) and archaea (B). L-BC: Lactate-fed biocathode; nL-BC: Non-lactate-fed biocathode; OP: Open circuit; Culture: the enriched 2,4,6-TCP dechlorination culture; E: OTUs in cathodic biofilm; S: OTUs of plankton in suspension.

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(10.0%), Sedimentibacter (9.7%), Clostridium (2.9%) and Pseudomonas (4.1%) were similar. In the original enrichment, the unclassified genera in family Anaerolineaceae were the most predominant (46.8%), while little was found in the other electro-stimulated systems. Some obligate organohalide-respiring bacteria were also detected in the original enriched culture like Dehalobacter (0.05%) and Dehalobacterium (0.5%) (Wang and He, 2013; Trueba-Santiso and Parlad, 2017). However, none of them was found in either cathodic biofilm or suspension, demonstrating their irrelevance with biocathode dechlorination. Archaeal composition and structure at the genus level were presented in Fig. 4B. Methanosarcina was a typical acetate-utilizing methanogen (Chae et al., 2010), which was the most predominant in all electro-stimulated systems or open circuit (64.6 %e93.4%). Methanomassiliicoccus, a hydrogen-utilizing methanogen (Dridi et al., 2012), was dominant in the enriched culture (85.7%), while its abundance became much lower after electro-acclimation (3.8e24.3%). Methanobacterium, a hydrogen-utilizing methanogen (Kotsyurbenko et al., 2007) was observed in all electro-stimulated systems (2.5 %e8.9%) but was barely found in the enriched culture.

3.5. Pearson correlation and molecular ecological networks Pearson correlation of the identified OTUs with environmental variables and the ones with which showed the significant correlations were displayed in Fig. 5, respectively. 2,4,6-TCP dechlorination

Fig. 5. Pearson correlation analysis of the identified OTUs in both biofilm and plankton which showed the significant difference (p < 0.05) with the environmental variables, including 2,4,6-TCP dechlorination efficiency (TCP), acetate (Ace) or CH4. (A): identified OTUs for bacterial genera; (B): identified OTUs for archaeal genera.

efficiency was positively correlated with some potential dechlorinators or fermenters, including Acetobacterium (Pearson correlation coefficient r > 0.85, significant correlation coefficient p < 0.01), Pseudomonas (r > 0.6, p < 0.05), Desulfovibrio (r > 0.7, p < 0.05), Anaerocella (r > 0.6, p < 0.05) and Hydrogenoanaerobacterium (r > 0.7, p < 0.01) (Fig. 5A). Among them, Hydrogenoanaerobacterium, the potential fermenter, displayed the significantly negative correlation with acetate and CH4. It should be noticed that Acetobacterium showed a negative correlation (without significant difference) with acetate (Fig. 5), so its homoacetogenic function was excluded in the system. Meanwhile, Sedimentibacter was significantly positivecorrelated with both acetate and CH4, and yet, significantly negative-correlated with the dechlorination efficiency. Some identified archaea, like Methanosarcina, Methanomassiliicoccus and Methanoplasma were significantly positive-correlated with CH4. Of which, Methanomassiliicoccus showed a positive correlation with acetate and a negative correlation with 2,4,6-TCP dechlorination efficiency. While Methanosarcina displayed a negative correlation with acetate (Fig. 5B). The overall RMT-based networks of cathodic biofilm and plankton were both divided into two distinct modules (with >32 nodes) (Fig. S6). The obviously different topological properties of biofilm and plankton networks indicated their great difference in microbial interaction (Table S4). The network of plankton possesses distinctly higher averaged connectivity, demonstrating a more complex network. Several Clostridium and Pseudomonas species spread over the major modules, consistent with their high abundance and diverse physiologies. Archaea like Methanosarcina and Methanomassiliicoccus shared numerous associations with bacteria, suggesting their tight relationships with bacteria. Subnetworks were constructed with the OTUs which represented the significant correlation with environmental variables (Fig. 6). The distinct different network interactions were found between biofilm and plankton. In the network of biofilm, Acetobacterium (OTU123) displayed a positive correlation with Desulfovibrio (OTU137), Pseudomonas (OTU143) and Sedimentibacter (OTU126) (Fig. 6A), demonstrating their similar preferred ecological niche or cooperative activities (Long and Azam, 2001). While both Acetobacterium (OTU123) and Pseudomonas (OTU143) showed a negative correlation with Methanomassiliicoccus (OTU256), which signified their potential competing relationships (Long and Azam, 2001). Pseudomonas (OTU234) was positively related with two typical methanogens, Methanosarcina (OTU250) and Methanobacterium (OTU257). While Desulfovibrio (OTU137) was negatively correlated with Methanobacterium (OTU257). However, in the plankton, Acetobacterium (OTU123) showed only a positive correlation with Pseudomonas (OTU143 and OTU234). OTU10, also from the genus of Acetobacterium, displayed positive correlations with Sedimentibacter (OTU74) and Pseudomonas (OTU35). Specifically, some nodes from the same genus (OTU125 and OTU137 from Desulfovibrio, OTU224 and OTU19 from Clostridium) shared diverse correlations, indicating somehow exclusion mechanisms or niche partitioning among the versatile genus. Both Clostridium (OTU224) and Pseudomonas (OTU139) showed positive correlations with Hydrogenoanaerobacterium (OTU116) and Methanosarcina (OTU250). While Methanomassiliicoccus (OTU256) displayed a negative correlation with Sedimentibacter (OTU74). Among of them, Acetobacterium (OTU123) and Pseudomonas (OTU143) displayed the consistent positive correlation with each other in either biofilm or plankton, implying the very similar phylogenetic relatedness between the two genera (Fuhrman and Steele, 2008). 4. Discussion In this study, the remarkably enhanced 2,4,6-TCP bio-

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Fig. 6. Subnetwork visualization of OTUs (from archaea and bacteria) which possessed the significant correlation with the aforementioned environmental variables. (A) OTUs in the cathodic biofilm; (B) OTUs in the plankton. A blue line indicated a positive interaction between two individual nodes, while a red line indicated a negative interaction. Different color of nodes represented OTUs from different phyla, and the node size was proportional to its connections with neighbors. The labels displayed names of the OTUs at the genus level from archaea, and bacteria which was reported as dechlorinators or fermenters (relative abundance over 0.1%). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

dechlorination activity was obtained by supplying a constant cathode potential of
0.36 V. The outstanding 2,4,6-TCP reductive dechlorination capacity was manifested in the accelerated dechlorination rate (24.2 mM/d, 3.0 times higher than open circuit) and the complete dechlorination ability with phenol as the sole final dechlorination product (no accumulation of toxic intermediate 4-CP) (Fig. 1). As reported, the longest distance from hydroxyl to para-chlorine made chlorine in para position stable and 4-CP the most difficult to be dechlorinated (Wang et al., 2014). To date, this is one of a very few that represented the complete dechlorination capacity during 2,4,6-TCP reductive dechlorination (Wang et al., 2014; Villemur et al., 2006). CV analysis for the first time gave the clear electrochemical evidence for the dechlorination of 2,4,6-TCP in biocathode and further confirmed the complete dechlorination ability by using electrode as the effective electron donor (Fig. 3). Fig. 2 showed electro-stimulation had little influence on either fermentation or methanogenesis. Microbiome structure and ecological network analyses suggested that under the cathodic potential of
0.36V, electro-stimulation selectively increased the abundance of some potential dechlorinators (Acetobacterium, Desulfovibrio, etc.), while no obvious impact on methanogens and fermenters was observed. The potential dechlorinators displayed collaborative interrelations with fermenters but somewhat

competitive interrelations with methanogens. So far, although Anaeromyxobacter or Geobacter have been confirmed to possess the cathode-respiring ability, the enhanced dechlorination capacity in the two strains-acclimated/electrostimulation systems had never been mentioned (Strycharz et al., 2008, 2010). Zhang et al. (2014) reported that pentachlorophenol was dechlorinated to 4-CP and phenol by immobilizing humin on cathode, however, the dechlorination activity was lost in the absence of formate. Liu et al. (2013) speculated the electron transfer between electrode and electro-active bacteria during pentachlorophenol dechlorination, but the direct bioelectrochemical evidence was lacking and the dechlorination metabolites were unclear. Acetobacterium was found as the most abundant potentialdechlorinating genus in biofilm (Figs. 4 and 5). However, its cathode respiring ability was never reported. Instead, some other dominant genera which was highly related with 2,4,6-TCP dechlorination were reported as cathode-respiring, like Pseudomonas and Desulfovibrio (Figs. 4 and 5). The widely existed positive correlations between Acetobacterium (OTU 123), Pseudomonas (OTU 143) and Desulfovibrio (OTU 137) in the network of biofilm (Fig.6), demonstrated their coexistence and cooperative interactions to achieve the high dechlorination rate by respiring electrons directly from electrode or indirectly via the mutual intraspecific or interspecies

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electron transfer processes (Nagarajan et al., 2013; Maphosa et al., 2012). On the other hand, the highest dechlorination rate was observed when lactate and electrode coexisted (Fig. 1). Lactate decomposition laws in different systems verified H2 and CH4 generation came solely from the microbial-participated lactate fermentation and H2/ acetate utilized methanogenesis, independent from electrode electrolysis (Fig. 2). Therefore, the high dechlorination rates would possibly due to the availability of sufficient electrons from both electrode and lactate fermentation. Otherwise, the low dechlorination performance in nL-BC could be possibly caused by the deficiency of either electrons or carbon sources. The microbial community analyses found that, compared with open circuit, electro-stimulation distinctly increased the relative abundance of some potential dechlorinators, while it did not show the obvious impact on fermenters and methanogens in both composition and abundance. In the network of biofilm, Acetobacterium (OTU 123) displayed sort of competitive relations with Methanomassiliicoccus (OTU 256), a potential hydrogen-utilizing methanogen, demonstrated their potential relations by scrambling for electrons (Chambon et al., 2013). Meanwhile, Acetobacterium (OTU 123) was positively correlated with a potential fermenter of Sedimentibacter (OTU126), demonstrating their potential syntrophic relationships (Farai et al., 2012). OTU 10, also from the genus of Acetobacterium, was highly related with other OTUs in the network of plankton. Acetobacterium OTU 10 showed the diversified interactions as compared with OTU 123, demonstrating their different physiologies. Clostridium represented close relations with methanogens (like Methanosarcina) and fermenters (like Hydrogenoanaerobacterium) in the network of plankton, demonstrating its potential fermentation or dechlorination function. Pseudomonas, also known as fermenters, displayed the positive correlations with methanogens in biofilm (such as OTU 234 with both OTU 257 and OTU 250) and plankton (such as OTU 110 with both OTU 257 and OTU 250), suggesting their possible syntrophic functions (Deng et al., 2012). In general, the ecological interrelations of potential dechlorinators, fermenters and methanogens in the electrostimulated systems followed the similar law with the microbial enrichment systems. The more complex network in plankton than in biofilm revealed the more diversified microbial interrelations. The possible reason would be that electro-stimulation had great impact on the biofilm community to attract the colonization of electro-active bacteria or the ones which shared the preferred environmental conditions with them. Although the microbial community in plankton was supposed to come majorly from biofilm detachment, the presence of lactate boosted the growth of microbial communities which utilized lactate and its fermentation metabolites (acetate, propionate, H2, etc). On the other hand, although less connectivity was observed in the network of biofilm, functional OTUs (dechlorinators and fermenters) gathered around and shared more interactions, while they dispersed with little interactions in the network of plankton (Fig. 6). Therefore, the electro-stimulation could optimize the microbial structure and interrelations which might corporately enhance 2,4,6-TCP dechlorination. 5. Conclusions This study reported the significantly enhanced 2,4,6-TCP reductive dechlorination activity by the weak electrical stimulation. Dechlorination performances and electrochemical evidence clearly revealed the biofilm community utilized electrode as an effective electron donor to maintain the high dechlorination ability. Electro-stimulation at the cathodic potential of
0.36 V selectively enhanced the dechlorination function, while, it showed little

influence on either fermentation or methanogenesis processes. In the electro-stimulated systems, the potential dechlorinators presented symbiotic or somewhat competitive relationships with fermenters or methanogens. The distinctly enhanced dechlorination performance and the reveal of microbial mechanism would be beneficial for the in situ bioremediation of chlorinated aromatics through the weak electrical stimulation. Acknowledgements This research was supported by the NSFC-EU joint program (No. 31861133001), National Natural Science Foundation of China (NSFC, No. 31400104), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. ES201806), and Natural Science Foundation of Heilongjiang Province China (No. C2018035). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2019.06.068. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Akao, S., Tsuno, H., Horie, T., Mori, S., 2007. Effects of pH and temperature on products and bacterial community in l-lactate batch fermentation of garbage under unsterile condition. Water Res. 41 (12), 2636e2642. Aulenta, F., Canosa, A., Majone, M., Panero, S., Reale, P., Rossetti, S., 2008. Trichloroethene dechlorination and H2 evolution are alternative biological pathways of electric charge utilization by a dechlorinating culture in a bioelectrochemical system. Environ. Sci. Technol. 42 (16), 6185e6190. Chae, K.J., Choi, M.J., Kim, K.Y., Ajayi, F.F., Chang, I.S., Kim, I.S., 2010. Selective inhibition of methanogens for the improvement of biohydrogen production in microbial electrolysis cells. Int. J. Hydrogen Energy 35 (24), 13379e13386. Chambon, J.C., Bjerg, P.L., Scheutz, C., Bælum, J., Jakobsen, R., Binning, P.J., 2013. Review of reactive kinetic models describing reductive dechlorination of chlorinated ethenes in soil and groundwater. Biotechnol. Bioeng. 110 (1), 1e23. Chen, F., Li, Z.-L., Liang, B., Yang, J.-Q., Cheng, H.-Y., Huang, C., Nan, J., Wang, A.-J., 2019. Electrostimulated bio-dechlorination of trichloroethene by potential regulation: kinetics, microbial community structure and function. Chem. Eng. J. 357, 633e640. Chen, F., Li, Z.-L., Yang, J.-q., Liang, B., Lin, X.-Q., Nan, J., Wang, A.-J., 2018. Effects of different carbon substrates on performance, microbiome community structure and function for bioelectrochemical-stimulated dechlorination of tetrachloroethylene. Chem. Eng. J. 352, 730e736. Cheng, I.F., Fernando, Q., Korte, N., 1997. Electrochemical dechlorination of 4chlorophenol to phenol. Environ. Sci. Technol. 31 (4), 1074e1078. Cheng, S., Xing, D., Call, D.F., Logan, B.E., 2009. Direct biological conversion of electrical current into methane by electromethanogenesis. Environ. Sci. Technol. 43 (10), 3953e3958. Choi, O., Kim, T., Woo, H.M., Um, Y., 2014. Electricity-driven metabolic shift through direct electron uptake by electroactive heterotroph Clostridium pasteurianum. Sci. Rep. 4, 6961. Croese, E., Pereira, M.A., Euverink, G.-J.W., Stams, A.J.M., Geelhoed, J.S., 2011. Analysis of the microbial community of the biocathode of a hydrogen-producing microbial electrolysis cell. Appl. Microbiol. Biotechnol. 92 (5), 1083e1093. Deng, Y., Jiang, Y.H., Yang, Y., He, Z., Luo, F., Zhou, J., 2012. Molecular ecological network analyses. BMC Bioinf. 13 (1). Ding, C., Chow, W.L., He, J., 2013. Isolation of Acetobacterium sp. strain AG, which reductively debrominates octa- and pentabrominated diphenyl ether technical mixtures. Appl. Environ. Microbiol. 79 (4), 1110e1117. Dridi, B., Fardeau, M.L., Ollivier, B., Raoult, D., Drancourt, M., 2012. Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int. J. Syst. Evol. Microbiol. 62 (8), 1902e1907. EPA, 1981. Priority Pollutant List [online], Office of Solid Waste and Emergency Response. US Environ- mental Protection Agency, Washington, D.C. 20460. EPA 530-R-10-002. https://www.epa.gov/eg/toxic-and-priority-pollutants-underclean-water-act#priority. Farai, M., JP, M.W., V, W.M., Hauke, S., 2012. Metagenome analysis reveals yet

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