Chlorinated phenol treatment and in situ hydrogen peroxide production in a sulfate-reducing bacteria enriched bioelectrochemical system

Water Research 117 (2017) 198e206

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Chlorinated phenol treatment and in situ hydrogen peroxide production in a sulfate-reducing bacteria enriched bioelectrochemical system Waheed Miran, Mohsin Nawaz, Jiseon Jang, Dae Sung Lee* Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 January 2017 Received in revised form 16 March 2017 Accepted 3 April 2017 Available online 5 April 2017

Wastewaters are increasingly being considered as renewable resources for the sustainable production of electricity, fuels, and chemicals. In recent years, bioelectrochemical treatment has come to light as a prospective technology for the production of energy from wastewaters. In this study, a bioelectrochemical system (BES) enriched with sulfate-reducing bacteria (SRB) in the anodic chamber was proposed and evaluated for the biodegradation of recalcitrant chlorinated phenol, electricity generation (in the microbial fuel cell (MFC)), and production of hydrogen peroxide (H2O2) (in the microbial electrolysis cell (MEC)), which is a very strong oxidizing agent and often used for the degradation of complex organics. Maximum power generation of 253.5 mW/m2, corresponding to a current density of 712.0 mA/ m2, was achieved in the presence of a chlorinated phenol pollutant (4-chlorophenol (4-CP) at 100 mg/L (0.78 mM)) and lactate (COD of 500 mg/L). In the anodic chamber, biodegradation of 4-CP was not limited to dechlorination, and further degradation of one of its metabolic products (phenol) was observed. In MEC operation mode, external voltage (0.2, 0.4, or 0.6 V) was added via a power supply, with 0.4 V producing the highest concentration of H2O2 (13.3 g/L-m2 or 974 mM) in the cathodic chamber after 6 h of operation. Consequently, SRB-based bioelectrochemical technology can be applied for chlorinated pollutant biodegradation in the anodic chamber and either net current or H2O2 production in the cathodic chamber by applying an optimum external voltage. © 2017 Elsevier Ltd. All rights reserved.

Keywords: 4-Chlorophenol Sulfate-reducing bacteria Bioelectricity BES Wastewater treatment

1. Introduction Environmental contamination by toxic, recalcitrant, and xenobiotic compounds is a very serious global problem. Among these pollutants, chlorophenols (CPs) have garnered much attention because of their ubiquity in the environment and biota due to their extensive use in many industrial processes (Tobajas et al., 2012). CPs are the precursors or intermediates in many process industries, such as the dye, resin and plastics, pharmaceutical, and pulp and paper industries (Basak et al., 2013; Lim et al., 2013). They are widely used as paints, disinfectants, explosives, herbicides, pesticides, and fiber and leather preservatives (Gomez et al., 2009; Sahoo et al., 2010). CPs are irritants at low levels and have a very negative impact on the respiratory and central nervous systems at higher doses. They are regulated among the 65 priority pollutants

* Corresponding author. E-mail address: [email protected] (D.S. Lee). http://dx.doi.org/10.1016/j.watres.2017.04.008 0043-1354/© 2017 Elsevier Ltd. All rights reserved.

by the United States Environmental Protection Agency (USEPA) because of their acute toxicity and carcinogenic properties (Zhao et al., 2015). Toxicity and bioaccumulation of CPs are largely linked to their lipophilicity. An increase in the chlorination of CPs increases their lipophilicity, which leads to greater potential for uptake into the organism. Moreover, the CPs with meta- and parasubstituted chlorine generally are more toxic than orthosubstituted ones, as ortho-substituted chlorine shields the OH group that interacts with the active sites in aquatic organisms (Markovi c et al., 2015). Because the removal of CPs from wastewater is a contemporary and very important issue, physical, chemical, and biological methods, including adsorption on activated carbon or sludge, chemical or enzymatic oxidation, catalytic degradation, solvent extraction, and microbial degradation, have been proposed for removing or degrading several CPs from wastewaters (Monsalvo et al., 2012; Munoz et al., 2016; Wang et al., 2016). Despite this extensive research, suitable, vigorous, and low-cost treatment of these pollutants has yet to be implemented. Therefore, efficient

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treatment methods of industrial wastewaters should be developed to prevent the discharge of CPs into downstream waterbodies, so that this environmental concern can be effectively addressed. In the last decade or so, bioelectrochemical systems (BESs), such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs), have been widely investigated for their novel aspects and potential environmental advantages (Rozendal et al., 2008; Zhang and Angelidaki, 2014). The basic principal of such systems is the oxidation of organics from wastewater by electrochemically active microorganisms, and, consequently, the microorganisms transport electrons resulting from this oxidation to the anode via extracellular electron transfer. Then, the electrons are transported to the cathode through an external circuit, where they are used for oxygen reduction and electricity generation (in MFCs) or other useful product formation, such as hydrogen (H2), caustics (NaOH/KOH), and hydrogen peroxide (H2O2) (with additional power supply in MECs). Overpotential can be substantially brought down with electrochemically active bacteria (EAB) at the anode/cathode, hence inexpensive materials (such as graphite and carbon) can be employed as electrodes in BESs (Mu et al., 2009). A number of valuable oxidation or reduction reactions demonstrating the versatility of BESs have been described. Bioelectricity generation can be achieved by using a large range of biodegradable fuels, including substrates such as acetate (Min et al., 2005) and organics in wastewater (Min and Logan, 2004; Shimoyama et al., 2008). Meanwhile, some studies have reported that BESs could greatly promote the removal of refractory organics such as pyridine, quinoline, indole, furfural, and phenol (Hu et al., 2011; Luo et al., 2010; Song et al., 2014). At the BES's anode, co-substrates (e.g. glucose for phenol and pyridine (Luo et al., 2009; Zhang et al., 2009), acetate and phenol for pentachlorophenol (Huang et al., 2011), brewery waste for azo dye (Miran et al., 2015)) provide electrons for the degradation of biorefractory compounds at higher rates, along with electricity production (in MFC) or useful product formation (in MEC); therefore, both electricity production/useful product formation and the degradation of biorefractory compounds are major focuses of BESs. Earlier studies have mainly investigated the reduction of CPs, such as 4-CP, at the abiotic cathode of an MFC; during this process, electricity was simultaneously generated (Gu et al., 2007; Wen et al., 2013). The limitation in these studies was that only dechlorination took place and no further mineralization occurred. Only a few researchers have given attention to aromatic chlorides in MFCs with microbial anodes. The microbial cultures in such anodes play a significant role in the overall performance of the BESs. Sulfatereducing bacteria (SRB) have been exploited successfully for the biodegradation of phenolic and other persistent organic pollutants in anaerobic processes (Haggblom and Young, 1995; Meckenstock et al., 2000). Lately, some researchers have used SRB for current generation with simultaneous organics oxidation and sulfate reduction, because of their electroactive nature in BESs (Cordas et al., 2008; Kang et al., 2014). Using an enriched SRB culture in a BES's anodic chamber for the treatment of toxic pollutants, such as 4-CP, may be an attractive option that will help in the efficient degradation of the pollutant, along with bioelectricity generation or other useful products formation. On the other hand, as stated earlier, a number of useful products can be produced at the cathode in MECs. Among these products, H2O2 is seen as a viable option based on life cycle assessment (LCA) testing, with significant environmental benefits through the displacement of chemicals produced by conventional means (Foley et al., 2010). H2O2 is environmentally friendly, as its degradation products are only water and oxygen, with no hazardous residues (Li et al., 2016). H2O2 has been applied (as a part of Fenton's reagent) to numerous industrial areas (including where CPs are involved) such

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as chemical synthesis, pulp paper and textile bleaching, medical disinfection, treatment of wastewater, and destruction of hazardous organic wastes (Khataee et al., 2011; Siedlecka and Stepnowski, 2006). Therefore, CPs removal and in situ H2O2 production could be a viable option in BESs. Recently, a few researchers tried to produce H2O2 in situ in BESs (a bioelectro-Fenton system) or ex situ along with treatment of pollutants (such as dye) (Ling et al., 2016; Liu et al., 2012). The in situ production of H2O2 in comparison to the commonly used anthraquinone oxidation method (which requires the use of hydrogen and non-aqueous solvents with significant energy input and generates substantial waste along with possible hazards due to transportation (Campos-Martin et al., 2006)) will likely be more secure and efficient, as there are no transports or handling issues. Therefore, the overall objectives of this study were to enrich the SRB-dominated microbial community and to assess its capacity to biotransform 4-CP and produce current/H2O2 in batch-fed BESs. The effect of the initial 4-CP concentration on the performance of the BESs in terms of bioelectricity generation and organics and sulfate removal were evaluated, and optimum externally added voltage conditions were determined for maximizing H2O2 production. 2. Materials and methods 2.1. MFC and MEC reactor setups The MFC was developed with two equally sized rectangular Plexiglass chambers. Each chamber had a volume of 200 mL. The anode was carbon felt (3.18 mm thick, 5 cm
5 cm; Alfa Aesar, Haverhill, USA). The cathode was carbon cloth, purchased from Fuel Cell Earth (Wakefield, USA), with a surface area of 25 cm2, containing 1.0 mg/cm2 (20 wt.%) Pt on conductive specialty carbon black (XC-72), and Nafion treated to avoid any damage of the Pt catalyst from the catholyte. The anode and cathode were separated by a cation exchange membrane (Nafion 117, Dupont Co., USA), which was treated with H2O2, H2SO4, and deionized water under boiling condition prior to application between electrodes to enhance performance (Miran et al., 2016a). The system was sealed to ensure an anaerobic environment in the anode. A pure nitrogen gas bag was attached to the anodic chamber to prevent ingress of atmospheric oxygen into the chamber. For the MEC only, the cathode was replaced with graphite felt having the same dimensions as that of the carbon felt. During MEC operation, external voltage was provided by a power supply (Model 2231A-30-3, Keithley Instruments Inc., USA). 2.2. Inoculum, anolyte, and catholyte An SRB culture was enriched with anaerobic sludge collected from a domestic wastewater treatment plant (Sincheon wastewater treatment plant) in Daegu, South Korea. The sludge was inoculated into modified Postgate's B medium (contained the following (in mM): KH2PO4 3.7, NH4Cl 18.7, FeSO4$7H2O 0.4, MgSO4$7H2O 0.2, yeast extract 3.6, sodium citrate 1.2, ascorbic acid 0.6, and thioglycollic acid 1.7 with sodium lactate and sodium sulfate) as the main carbon and sulfate sources, respectively (lactate COD/SO2 4 mass ratio of 2.0). Lactate COD/SO2 4 mass ratio was kept constant in all experiments for comparisons. Sodium 2-bromoethanesulfonate (Na-BES) (2.5 g/L (11.8 mM)) was added at the enrichment stage to suppress acetoclastic methanogenic activity, which can consume lactate in the medium and adversely affect a BES's performance. A trace element solution (1.0 mL) was also added. Prior to culture enrichment, the medium was autoclaved at 15 psi and 120  C for 20 min and deoxygenated by bubbling with high purity nitrogen to

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Samples taken from the anolyte at regular intervals were immediately filtered through a 0.22 mm filter. Soluble COD was measured in a Hach COD reactor (DRB200, Hach Co., Loveland, CO) using Hach vials and a spectrophotometer (1412 V, Optizen, Mecasys Co., Korea). Total organic carbon (TOC) was measured using a TOC analyzer (TOC-VCPH, Shimadzu, Kyoto, Japan). Sulfates were analyzed using a DX ICS-1000 ion chromatography unit (Dionex, USA) equipped with a conductivity detector and selfregenerating suppressor. A DX Ionpac AS14-HC analytical column (4
250 mm) connected to a DX Ionpac AG14-HC guard column (4
50 mm) was used. The unit was operated in autosuppression recycle mode with the eluent (3.5 mM Na2CO3 þ 1 mM NaHCO3) at a flow rate of 1.2 mL/min 4-CP and phenol were analyzed by high performance liquid chromatography (HPLC) using an XDB-C18 column (4.6 mm
150 mm). A methanol/water mixture (60:40, v/v) was used as the mobile phase, where water contains 1% acetic acid. The mobile phase flow rate of 1 mL/min was used during the analysis. The detection was performed at 280 nm. The residence times for phenol and 4-CP were 2.6 and 4.3 min, respectively. The concentrations of volatile fatty acids (VFAs) were analyzed by HPLC (Model 1200, Agilent Inc., USA) using a refractive index detector and an Aminex HPX-87H column (300 mm
7.8 mm) with 4 mM H2SO4 as the mobile phase. H2O2 was measured using the iodometric titration method and reported in g/L-m2 based on the cathode surface area as power density is reported in units of W/m2 based on the anode surface area in case of MFCs. Cell voltage (V) was measured (every 8 min) by using a multimeter with a digital data acquisition system (Model 2700, Keithley Instruments Inc., USA) connected to a personal computer. Current density (I) and power density (P) normalized to the anode area (25 cm2) were calculated according to I ¼ V/R and P ¼ V2/R, respectively, where R is the external resistance. Power and current density curves were monitored to evaluate the maximum power using a single cycle method. This method is based on the change in external loads during a single batch-fed cycle operating at its stable potential, where external resistance was varied from 10 to 10,000 U (Miran et al., 2016b). Cyclic voltammetry (CV) was used to electrochemically characterize the anode biofilm activities with an Ivium Compact Stat potentiostat (Ivium Technologies, Netherlands). X-ray photoelectron spectroscopy (XPS) using a Quantera SXM (ULVACPHI, Japan) was employed to study the anaerobic sludge composition by recording mainly the S2p XPS spectra. Experiments were repeated for at least two successive cycles (n ¼ 2) in single reactor. Error bars with standard deviations were provided where required.

3.1. Electricity generation in the MFC After operation of the MFC stabilized, repetitive 48 h feeding cycles were conducted for analysis of the SRB-acclimated MFC performance. The results (Fig. 1a) demonstrated that an average voltage of 0.464 V (330 U) over the period of 48 h was generated in the cycles with lactate (COD of 500 mg/L) as the sole carbon source (no 4-CP). Metabolites formed after the oxidation of lactate were acetate and propionate (Fig. S1), which were also the electron

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2.3. Analytical methods and calculations

3. Results and discussion

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ensure anaerobic conditions. The initial pH of the medium was adjusted to 7.5 using both 1 N NaOH and 1 N HCl solution. The mixture was cultured and subclultured weekly in 1 L bottles at 30  C on a shaking incubator at 130 rpm until the SRB (indicated by blackening) from the sludge were enriched. The MFC anode was inoculated with the enriched SRB, at a starting external resistance of 1000 U, which was gradually decreased to 500 U, and finally 330 U, to promote the growth of exoelectrogenic bacteria. The medium used in the anode was the same as that used for the SRB enrichment, minus all carbon sources except lactate. 4-CP was added to the anode after stable operation of the MFC at different initial concentrations of 10, 25, 50, and 100 mg/L (0.078, 0.19, 0.39, and 0.78 mM). The medium used in the cathode was 0.1 M potassium phosphate buffer solution (pH 7.0; 10.71 g/L K2HPO4þ5.24 g/L KH2PO4), and oxygen (air) was continuously added by using an air pump for reduction at the cathode. Temperature of the setup was maintained at 30 ± 1  C via a water bath.

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Current density (mA/m ) Fig. 1. (a) Voltage generation, (b) power curves, and (c) polarization curves for batches fed with lactate alone or lactate with 4-CP.

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Intensity (a.u.)

donors in the MFC after further degradation. Sulfate reducers have the tendency to either completely degrade organic compounds to carbon dioxide or incompletely to acetate (Muyzer and Stams, 2008). This disadvantage of incomplete degradation was eliminated by using the MFC, because organic acids can be further degraded by exoelectrogens in the presence of an anode (Cao et al., 2010; Liu et al., 2005). It is well known that members of the SRB have these exoelectrogenic properties (tendency to transport electrons to the anode via the external membrane) (Angelov et al., 2013; Cordas et al., 2008). The experimental results illustrated that the voltage and power density both varied with increasing 4-CP concentration. After adding 4-CP, there was a drop, although not a very significant one, in voltage, i.e., the average voltage decreased by 5%e14%. The average voltages generated at the initial 4-CP concentrations of 10, 25, 50, and 100 mg/L (0.078, 0.19, 0.39, and 0.78 mM) in 48 h were 0.440, 0.432, 0.420, and 0.396 V, respectively. The voltage evolution over time is also shown (Fig. S2). Similar trends were observed with the power density curves when drawn at the stable stages of the different initial 4-CP-fed MFC batches (Fig. 1b). The maximum power density achieved with lactate alone was 323.2 mW/m2, which decreased to 253.5 mW/m2 when 4-CP (100 mg/L (0.78 mM)) was added; whereas the internal resistances of the MFC system obtained from polarization curves (Fig. 1c) were approximately 168 and 189 U, respectively. The reduction in voltage and maximum power density during 4-CP degradation was most likely attributable to electron loss or activity repression of electrogens due to the reduction in 4-CP. The part of electrons generated in the anodic chamber of MFC was consumed by sulfate (and reduced to sulfide) for maintaining the growth of the SRB. Biological reduction of sulfate to sulfide theoretically requires eight reducing equivalents i.e., a minimum COD/ sulfate mass ratio of 0.67 is (theoretically) required to achieve complete removal of sulfate (Archilha et al., 2010; Lens et al., 1998). This sulfide formation is considered toxic and harmful to microbes, reactors, and pipelines, if there are no other electron acceptors available to ensure its conversion to S0, i.e., elemental sulfur, as there are in denitrifying sulfide removal (where nitrate is used as the electron acceptor) (Lee et al., 2014). However, BESs have the advantage of using an anode as the electron acceptor, which helps convert the HS to S0 abiotically (Rabaey et al., 2006; Sun et al., 2009b). This was confirmed in the current study. XPS wide spectra (Fig. S3) and data for sulfur-related peaks were obtained. In Fig. 2, anode sludge analyzed for XPS showed that there were two broad characteristic peaks present on the XPS S2p spectra. The

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observed peaks were interpreted using the National Institute of Standards and Technology (NIST) XPS database (https://srdata.nist. gov/xps/). The first broad peak, found between 158 and 167 eV, was divided into two subpeaks, at 162.5 eV represented by S(II) i.e., sulfide, and 163.6 eV by S0. The second broad peak, at 168.5 eV, was attributed to S(þVI) i.e., sulfate. From these results, it can be inferred that sulfate was microbially reduced to sulfide and further abiotically oxidize to S0 on the anode (because of the oxidative effect of the anode). The possibility of elemental sulfur to be further oxidized to sulfate cannot be ruled out in such systems. However, elemental sulfur can be precipitated out and ultimately removed (dependent on biological oxidation, or electrode potential) (Rabaey et al., 2006). Based on sulfate's final conversion to elemental sulfur, along with lactate and 4-CP degradation, the probable mechanism for the anode was proposed (Fig. 3). The proposed mechanism can be summarized as; acetate, propionate and sulfide were electron donors, whereas 4-CP, sulfate, and anode electrode were electron acceptors. Microbial activity was involved in 4-CP, phenol, lactate, acetate, propionate and sulfate reactions, and extracellular electron transfer to anode for current generation. Abiotic reaction involved the sulfide to sulfur oxidation on anode and electrons transfer to anode. Major theoretical electron balance involved the 8-electron reduction (Sulfate to sulfide), 2-electron reduction (4-CP to phenol), 8-electron oxidation (acetate to carbon dioxide) and 2electron oxidation (sulfide to sulfur). Sulfate was most likely to be reduced at the top of anodic biofilm and the produced sulfide migrates into matrix where it was abiotically oxidized on the electrode (Rabaey et al., 2006). 4-CP, phenol, and lactate degradation is possible in bulk phase or on the anode biofilm, whereas acetate and propionate was most like to be oxidized at anode biofilm from where electron transferred to the anode through microbial extracellular electron transfer involving typical c-type cytochromes. It has been reported that when SRBs were used as a microbial catalyst in a mediator less MFC, electrons were transferred to the anode via contact between microbes and anode through a c-type cytochrome in the outer cell membrane (Cordas et al., 2008). CV, which assists in understanding the bioelectrochemical behavior of the anode/cathode biofilm in BESs, was conducted to compare the different batches. The performance was evaluated by setting the anode, the cathode, and Ag/AgCl (s) as the working, counter, and reference electrodes, respectively. The anode potentials were scanned from 0.5e1.5 V (vs. Ag/AgCl) at a scan rate of 10 mV/s. Voltammograms determined in situ in the reactor showed significant variation in the electrochemical properties (Fig. S4). Reactors fed with lactate only had higher current densities in the CV tests than those fed with lactate þ4-CP. It can be inferred that the lactate fed batches had better anodic activity than those with the lactate þ4-CP did. Reduction peaks were observed at 0.42 (lactate) and 0.48 V (lactate þ 4-CP) whereas two set of oxidation peaks were found at 0.30 V (both for lactate and lactate þ 4-CP) and 0.11(lactate) and 0.16 V (lactate þ 4-CP), respectively. No significant redox peak was observed without matured biofilm presence on the anode.

S(-II)

3.2. MFC treatment efficiency and 4-CP biodegradation

S(VI)

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Binding energy (eV) Fig. 2. XPS S2p spectra of the sludge scraped from the carbon-felt anode.

Removal of organics along with current generation is one of the important objectives of wastewater treatment in MFCs. The anodic chamber in MFCs also acts as an anaerobic treatment reactor and thus is considered a substitute for the conventional processes of biological wastewater treatment (Mansoorian et al., 2013; Miran et al., 2015). Therefore, this simultaneous treatment of wastewater(s) in the anode chamber helps in making MFCs environmentally friendly and sustainable. Effects of the initial 4-CP concentration on TOC and sulfate removal efficiencies were evaluated (Fig. 4). The

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Fig. 3. Proposed mechanism of current generation and 4-CP degradation in the MFC.

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mixed SRB enriched anodic chamber showed notable organics treatment along with current generation. TOC removal of 79% was achieved with lactate as the sole carbon source. This removal decreased with an increase in 4-CP concentration. TOC removal efficiencies were 74.2, 65.8, 61.8, and 50.1% at 4-CP initial concentrations of 10, 25, 50, and 100 mg/L (0.078, 0.19, 0.39, and 0.78 mM), respectively, in 48 h of operation. The decrease in TOC removal can be attributed to the toxicity of recalcitrant pollutants, such as 4-CP, to cell growth and therefore degradation ability of organics (Tobajas et al., 2012). TOC removal in MFCs is considered a complex function of reactor design, operational conditions, and wastewater composition. Oxygen ingress from cathode to anode can positively affect TOC removal but resulted in lower power generation and sulfate removal tendency because of electron uptake for its reduction. Sulfate removal efficiency of the SRB enriched MFC demonstrated that more than 90% of the total sulfate removal was achieved in the first 24 h of all batch operations. Total sulfate removal was 72 ± 3.0% in all MFC batches after 48 h operation, showing a less significant effect of 4-CP addition on sulfate removal. This was most likely due to a higher reduction potential for sulfate than for 4-CP. The standard redox potential of the 4-CP and sulfate reactions approximates 430 mV (Shaikh et al., 2002) and 220 mV (Jones and Ingle, 2005), respectively. Thus, under standard conditions, the reduction of 4-CP to phenol is thermodynamically less favorable than the reduction of sulfate to sulfide. 4-CP and its main degradation product i.e., phenol's concentration profiles obtained during MFC batch studies, are shown in Fig. 5. It can be seen that, unlike in earlier studies where only dechlorination of 4-CP was achieved in abiotic cathodes, a bioanode can lead to further biotransformation of degraded products of 4-CP, which is the ultimate goal of treatment involving such recalcitrant pollutants. Phenol was most likely degraded to acetate and then to CO2 and H2O as earlier reported (Zeng et al., 2015). Due

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based/platinum coated), because inexpensive carbon materials are considered preferable catalysts for this reaction (Rozendal et al., 2009). Theoretically, H2O2 can be produced bioelectrochemically by the MFCs, as the cathode potential for two-electron ORR (~260 mV vs. normal hydrogen electrode (NHE)) is higher than the anode potential (~340 mV vs. NHE) (Chen et al., 2015), based on the degradation of lactate, as stated in reactions (1) and (2). These are the general reactions at pH 7 in the MFC with their corresponding apparent standard potentials.

10 mg/L 25 mg/L 50 mg/L 100 mg/L

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3CO2 þ 10Hþ þ 10e / CH2CHOHCOO þ 3H2O; E ¼ 0.34 V (vs. NHE) (1)

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Time (h) Fig. 5. (a) 4-CP and (b) phenol concentration time profile for MFC batches fed with various initial 4-CP concentrations.

to the large background of chloride in the saline sulfidogenic medium (NH4Cl of 1 g/L used in medium, which corresponds to chloride concentration of 662.8 mg/L), the very small release of chloride (maximum upto 1.1e11.6 mg/L) from the little amount of 4-CP utilized could not be determined to meet exact chloride balance. It was also true for carbon from little amount of phenol degradation relative to total carbon. The MFC results showed higher 4-CP removal when compared to control experiments without external circuits i.e., converting the reactor to a traditional anaerobic reactor (Fig. S5). The 4-CP removal was 18.9% without external circuits. The higher performance of MFC systems for the degradation of refractory contaminants in comparison to traditional anaerobic systems is also well reported in earlier studies (Sun et al., 2009a). The abiotic control showed approximately 9% removal, which was most likely due to adsorption at the anode. Although, the complete degradation of 4-CP and its metabolic products was not reached in this study, further optimization in future studies by acclimating the SRB well to 4-CP (with higher degradation ability) prior to their addition to an MFC can be achieved. 3.3. MEC system performance for hydrogen peroxide production and 4-CP treatment An MEC system for the production of H2O2 was set up with inexpensive graphite felt as the cathode to favor the two-electron oxygen reduction reaction (ORR). Notably, the production of H2O2 in BES cathodes does not necessitate any high-cost catalysts (e.g., Pt

Moreover, it is important to discuss here that formal potential bring very theoretical hypothesis i.e., the lactate does not interact directly with the anode surface and therefore its formal potential does not give the true picture of thermodynamic of the electrochemical cell reaction. In this study, the redox compound oxidized at the anode is sulfide (redox potential S2/S0 ¼ ~160 mV), and in electroactive biofilm, the compounds performing the last electron transfer from the biofilm to the anode in case of SRBs are typically c-type cytochromes (redox potential ~200 to 400 mV) (Choi and Sang, 2016). However, no substantial amount of H2O2 can be obtained in such a spontaneous production process because of energy losses (overpotential), which is a usual feature of such systems (Modin and Fukushi, 2012; Rozendal et al., 2009). Therefore, to improve the yield and production rate, a small external voltage needs to be added to an MEC system. H2O2 production was investigated by applying the voltages of 0.2, 0.4, or 0.6 V, using a DC power supply. In the anodic chamber, the initial feed composition was kept the same, with a lactate COD of 500 mg/L and a 4-CP concentration of 100 mg/L (0.78 mM) Fig. 6 shows the maximum H2O2 concentrations after 6 h of operation in the MEC and found that by increasing the external voltage from 0.2 to 0.4 V, the H2O2 concentration increased from 8.84 ± 0.97 g/L-m2 (649 ± 71 mM) to 13.26 ± 1.44 g/L-m2 (974 ± 105 mM). By further increasing the voltage to 0.6 V, a drop in maximum H2O2 concentration to 5.74 ± 0.54 g/L-m2 (422 ± 39 mM) was observed. The increase in H2O2 concentration from 0.2 to 0.4 V was most likely due to the enhanced two-electron ORR pathway for H2O2 production from the increase in current density. Conversely, the decline in production rate of H2O2 at a higher added voltage resulted from the reduction of H2O2 to water, which competed with the two-electron ORR. It is also very important to set the optimum time for harvesting maximum concentration of H2O2, because accumulation of H2O2 can result in its conversion to H2O or disproportionation to O2, as per the following equations (Li et al., 2016): H2O2 þ 2Hþ þ 2e / 2H2O

(3)

H2O2 / H2O þ 1/2O2

(4)

This issue needs to be addressed, and a suitable low cost stabilizer for enhancing H2O2 stability can be investigated in detail in future studies. The low concentration may be a concern if it does not meet the requirement for specific applications. Nonetheless, there are certain applications where a concentration of 0.1e0.5% is low enough to meet requirements, e.g., for membrane bioreactor treatment plants and disinfection of wastewater (Arends et al., lot et al., 2008). Moreover, a lower concentration has 2014; Gre the advantage in that it is less likely to affect the integrity of the proton exchange membrane. However, development of advanced cathode and anode designs can further improve the H2O2 yield and

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4-CP concentration (mg/L)

H2O2 concentraion (g/L-m2)

20

15

10

5

30 0.2 V 0.4 V 0.6 V

100

25

80

20

60

15

40

10 0.2 V 0.4 V 0.6 V

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5

0

0 0

0 0.2 V

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Phenol concentration (mg/L)

204

8

16

24

32

40

48

Time (h)

0.6 V

Voltage supplied (V)

Fig. 8. (a) 4-CP (open symbols) and (b) phenol (closed symbols) concentration time profiles for MEC batches fed at different externally added voltages.

Fig. 6. H2O2 concentration produced at different externally added voltages in the MEC.

lessen the extra energy required by decreasing the overpotential. Moreover, connecting multiple MFCs in series can draw higher voltages/useful products, which are suitable for practical applications (Aelterman et al., 2006). MEC system treatment efficiency is shown in Figs. 7 and 8. The results showed that TOC removal, sulfate reduction, and 4-CP degradation increased with the increase in added external voltage. At the highest added external voltage (0.6 V) in this study, TOC, sulfate, and 4-CP removal were 51.2, 76.0, and 42.9%, respectively, after 48 h of operation. Further degradation 100 a)

0.2 V 0.4 V 0.6 V

TOC removal (%)

75

4. Conclusions

50

25

0 0

8

16

24

32

40

48

Time (h) 100

Sulfate removal (%)

b)

0.2 V 0.4 V 0.6 V

75

of phenol was also more obvious. These results are consistent with earlier studies, where an increase in voltage favored organics degradation until there was no negative impact on the microbial communities of the anode (Ding et al., 2015). The lower 4-CP removal is a concern, which was most likely due to lower sulfate concentration used in this study. It has been reported earlier that low sulfate in anaerobic reduction process for 4-CP removal resulted in its lower degradation (Haggblom and Young, 1995). In future studies, 4-CP degradation can be enhanced by increasing the enrichment time of SRBs and optimum sulfate concentration. Moreover, the produced H2O2 in the cathode can be evaluated for further degradation of chlorinated pollutants that were not degraded in the anode chamber.

Environmentally friendly and economical removal methods are needed for pollutants such as 4-CP. Hence, this work was carried out to ascertain the role of SRB in the treatment of chlorinated pollutants using advanced BESs. SRB enriched BESs removed 4-CP well, and further degradation of dechlorinated products was achieved. Power and current densities were negatively affected by increasing 4-CP concentrations. In the MEC system, externally added voltage helped produce the very useful oxidizing agent, H2O2, and was optimum at 0.4 V. The overall advantages of SRB enriched BESs might lead to important methods for the treatment of recalcitrant waste, with bioenergy production, reduction of energy consumption, or production of other useful products as added benefits. Acknowledgements This work was supported by a grant provided by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education (ME) and National Research Foundation (NRF) of Korea (NRF-2014H1C1A1066929). This study was also supported by grants (NRF-2016R1A2B4010431, NRF-20090093819) through the ME and NRF of Korea. Additionally, this research was supported by an NRF grant from the Korean government (MSIP) (NRF-2015M2A7A1000194).

50

25

0 0

8

16

24

32

40

48

Time (h) Fig. 7. (a) TOC and (b) sulfate removal time profiles for MEC batches fed at different externally added voltages.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2017.04.008.

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