Electrochemical Stimulation of Microbial Roxarsone Degradation

Jun 17, 2014 - Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been commonly used in animal feed as an organoarsenic additive, most of which is ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/est

Electrochemical Stimulation of Microbial Roxarsone Degradation under Anaerobic Conditions Lin Shi,† Wei Wang,†,‡ Shou-Jun Yuan,†,‡ and Zhen-Hu Hu*,†,‡ †

School of Civil Engineering, Hefei University of Technology, Hefei 230009, China Institute of Water Treatment and Wastes Reutilization, School of Civil Engineering, Hefei University of Technology, Hefei 230009, China

‡

S Supporting Information *

ABSTRACT: Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been commonly used in animal feed as an organoarsenic additive, most of which is excreted in manure. Roxarsone is easily biodegraded to 4-hydroxy-3-aminophenylarsonic acid (HAPA) under anaerobic conditions, but HAPA persists for long periods in the environment, increasing the risk of arsenic contamination through diffusion. We investigated the electrochemical stimulation of the microbial degradation of roxarsone under anaerobic conditions. After the carbon sources in the substrate were depleted, HAPA was slowly degraded to form arsenite under anaerobic conditions. The degradation rate of HAPA was significantly increased when 0.5 V was applied without adding a carbon source. The two-cell membrane reactor assays reveal that the HAPA was degraded in the anode chambers, confirming that the anode enhanced the electron transfer process by acting as an electron acceptor. The degradation product formed with electrochemical stimulation was arsenate, which facilitates the removal of arsenic from wastewater. Based on the high performance liquid chromatographyultraviolet-hydride generation-atomic fluorescence spectrometry (HPLC-UV-HG-AFS) and gas chromatography−mass spectrometry (GC−MS) data, the pathway for the biodegradation of roxarsone and the mechanisms for the electrochemically stimulated degradation are proposed. This method provides a potential solution for the removal of arsenic from organoarseniccontaminated wastewater.

■

INTRODUCTION Roxarsone (4-hydroxy-3-nitrophenylarsonic acid), which is an organoarsenic additive, has been widely used in animal feed to promote growth and to control coccidial intestinal parasites.1−3 However, most of the roxarsone added to the feed is excreted unchanged in manure.1−6 Reports indicate the presence of 21.6 and 89.3 mg/kg arsenic in chicken and pig manure in China, respectively.4 Although roxarsone has a low toxicity, the inorganic arsenic generated after roxarsone degradation is very toxic.2,6 Most of the arsenic compounds leached from animal manure are water-soluble,1,5 inevitably releasing inorganic arsenic into the surface and ground waters and causing arsenic contamination. The uptake and accumulation of arsenic by crops, fish and other organisms have been reported in recent years.7−9 Various microbial communities, such as bacteria, archaea, and eukaryotic microbes, play important roles in environmental arsenic cycles,10 including oxidation, reduction, methylation, and demethylation.11−13 Previous studies using chicken litter slurries or sewage sludge proved that biological processes transform roxarsone under anaerobic conditions.1,2 Roxarsone in the environment can be transformed into arsenite [As(III)] and arsenate [As(V)],1,2,5,6 which can be subsequently biotransformed to monomethylarsenite [MMA(III)], mono© 2014 American Chemical Society

methylarsenate [MMA(V)], dimethylarsenite [DMA(III)], and dimethylarsenate [DMA(V)].7,14 Roxarsone, as well as its degradation and transformation products, inhibited methanogenic processes.3 Many investigations have shown that roxarsone can be biotransformed under both aerobic and anaerobic conditions.15 Under anaerobic methanogenic and sulfate-reducing conditions, roxarsone was rapidly reduced to form 4-hydroxy-3aminophenylarsonic acid (HAPA), which persisted for a long time in the environment.1,2 Cortinas et al. reported that HAPA was slowly broken down to form inorganic As(III) and As(V) after incubation over 229 days.1 HAPA is highly water-soluble and resistant to degradation; therefore, if it reaches the surface water, its toxicity toward animals and humans will be significantly increased after generating inorganic arsenic. Electrochemistry has been used to stimulate microbial metabolism for more than 50 years.16 Although the exact genetic and biochemical mechanisms of the interaction between organisms and working electrodes remains unknown,16 this Received: Revised: Accepted: Published: 7951

March 22, 2014 June 12, 2014 June 17, 2014 June 17, 2014 dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

mm in diameter) was clamped in the middle of the cells to prevent the diffusion of roxarsone and HAPA. Both electrode chambers had the equal working volumes (100 mL) and one graphite rod. An external power source (0.5 V) was applied using a constant-voltage power supply to maintain the electrochemical stimulation. Saturated calomel reference electrodes (SCE, 241 mV vs standard hydrogen electrode) were inserted into the anode chambers to measure the real-time electrode potentials. Experimental Design. The experiments composed of three parts. Experiment I was carried out in single cell reactors to investigate the effects of electrochemical stimulation on microbial roxarsone degradation, as listed in assays 1−7 in Table 1. Roxarsone degradation without electrochemical

method is effective for promoting the biotransformation of organic contaminants, such as chlorinated compounds,17−21 aromatic hydrocarbons,22−26 and antibiotics.27,28 A promising alternative would be to use electrodes as anodic electron acceptors or cathodic electron donors for the microbial communities responsible for degradation processes.19 This method has also successfully produced electrodes that provide continuous and fine stimulation to microbes in situ when treating contaminants.21,24 The removal of inorganic arsenic during wastewater treatment has been widely investigated. Various physicochemical treatment methods, such as adsorption, precipitation, and coagulation, have been developed to remove inorganic arsenic from water and wastewater.29 While most of the techniques were effective for As(V) removal, they failed for As(III).30 Effective removal processes for organoarsenics have not been reported. The purpose of this study was to investigate the following: (1) the use of electrochemical stimulation during the degradation of roxarsone into inorganic arsenic, (2) the speciation of arsenic after the electrochemically stimulated degradation under anaerobic conditions, and (3) the possible pathway for roxarsone degradation and biotransformation, as well as the mechanisms through which electrochemical stimulation enhanced the degradation. To prevent side reactions on electrodes, a low voltage (0.5 V) was applied. Hypothetically, electrochemical stimulation enhances the biodegradation of roxarsone to inorganic arsenic, and through controlling the redox potential, the soluble inorganic arsenic exists as As(V), facilitating the arsenic removal through the physicochemical treatment of wastewater.

Table 1. Operation Conditions of the Reactors part I

II

■

MATERIALS AND METHODS Microbes and Chemicals. Anaerobic methanogenic sludge was used as the inoculum and was collected from the Zhu Zhuanjing wastewater treatment plant (Hefei, China). The volatile solid (VS) content was 2.03%. Roxarsone (4-hydroxy-3-nitrophenylarsonic acid, purity >99%; CAS No., 121−19−7, molecular formula, C6H6AsNO6; molecular weight, 263.04) was purchased from Sigma-Aldrich. HAPA (4-hydroxy-3-aminophenylarsonic acid, purity >97%; CAS No., 2163−77−1, molecular formula, C6H8AsNO4; molecular weight, 233.04) was purchased from International Laboratory. As(III), As(V), MMA(V), and DMA(V) were reagent grade and were purchased from CRM/RM Information Center of China. The methanol, dichloromethane and hexane were chromatographic grade and were purchased from Tedia Company. The other chemicals used in this study were analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. All of the solutions were prepared with ultrapure water obtained from a thermocompression water distiller. Reactor Setup. Two types of reactors were used during the experiment. One was a single cell reactor with a close, cylindrical, undivided 250 mL glass electrolytic cell 7 cm in diameter for investigating the effects of electrochemical stimulation on the degradation of roxarsone. Three holes were punched in the top of the electrolytic cell: the middle one was used to introduce the sample, and the other two contained graphite rods (6 mm in diameter and 150 mm in length) as electrodes. The working volume of the electrolytic cell was 200 mL. To investigate the possible mechanisms for the electrochemical stimulation, membrane reactors with two separated electrolytic cells were used. An anion-exchange membrane (20

III

assay

sludge (g/L)

1 2 3 4 5 6 7

4 4 4 4 (heat killed)

4

8 9 10 11

4 4 4

12 13

4 4

roxarsone (mg/L)

acetate (g/L)

voltage (V)

20 20 20 20 20 20 20

3.81 3.81

0.5

20 20

3.81

3.81 3.81

3.81 20 20

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

stimulation was investigated in assays 1, 3, and 4 as the control. The function of microbes and acetate on the degradation was explored. Electrochemically stimulated roxarsone degradation was investigated in assays 2 and 5−7, in which the function of microbes, acetate and their combination on the degradation was identified. Experiment II was carried out in membrane reactors to investigate the possible mechanisms for the electrochemical stimulation during the degradation of HAPA and the influence of the anode potentials. The operation conditions were set as listed in assays 8−11 in Table 1. Experiment III (assays 12 and 13) was performed in membrane reactors to investigate the intermediate degradation products, as listed in Table 1, with eight replicates in each; assay 13 was the control. After 7 days of cultivation, a 500 mL liquid sample was collected from the anode chambers for analysis by gas chromatography−mass spectrometry (GC−MS). Except assays 6 and 11, the solution used for all of the assays was prepared using tap water and nutrients. The tap water was exposed to air for 2 days before use to remove any residual chlorine. The added nutrients were composed of NH4Cl (0.287 g/L) and KH2PO4 (0.066 g/L). The pH was adjusted to approximately 7.5 with 1.0 mol/L NaHCO3 solution. Assays 6 and 11 were conducted in ultrapure water without added nutrient or pH adjustments. All of the reactors were purged with N2 gas for 3 min to maintain anaerobic conditions and sealed before incubation, and placed in an incubator at 35 °C. Liquid samples were collected from the reactors using 5 mL syringes, centrifuged for 10 min at 10 000 rpm, and filtered 7952

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

through 0.45 μm filters under N2 for analysis. The gas samples from the headspace were collected using a pressure-lock gas syringe. Analytical Procedures. The concentrations of roxarsone and HAPA were measured using high performance liquid chromatography (HPLC, 1260 Infinity, Agilent Technologies) with UV detection at 260 nm. The roxarsone and HAPA were separated on a C18 column (ZORBAX SB-C18, 150 × 4.6 mm, 5 μm, Agilent Technologies) with a mobile phase (KH2PO4 50 mmol/L, formic acid 0.1%, methanol 10%, pH 3.2) flowing at 1.0 mL/min and 30 °C. Atomic fluorescence spectrometry (AFS-8220, Beijing Titan, China) was used to determine the concentration of the total inorganic arsenic. The arsenic species were analyzed using high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) combined with a species analysis instrument (SAP-10, Beijing Titan, China). The arsenic species were separated with a 250 × 4.1 mm Hamilton PRP X-100 anion-exchange column at 30 °C with an isocratic elution of 10 mmol/L (NH4)2HPO4 solution (adjusted to pH 6.0 with 10% formic acid) at 1.0 mL/min. A peristaltic pump was used to pump the current-carrying solution (7% HCl) and reductant (20 mg/L KBH4), and the flow rates were 7 mL/min and 4 mL/min, respectively. Because roxarsone, HAPA and the intermediate products from the roxarsone degradation cannot be directly hydrogenated or quantitatively measured by AFS, they must be degraded to form inorganic arsenic before the AFS analysis. Therefore, high performance liquid chromatography-ultraviolet-hydride generation-atomic fluorescence spectrometry (HPLC-UV-HG-AFS) was used to assess the intermediate products of roxarsone degradation. An oxidant (20 mg/L K2S2O8) and UV irradiation were combined to oxidize the organoarsenic to inorganic arsenic between the separation and detection system,31 and the chromatographic column equipped for the separation was a C18 column. The methane content of the gas samples was determined using a gas chromatography (SP-6800A, Lu’nan Co., China) equipped with a thermal conductivity detector and a 1.5 m stainless steel column packed with 5 Å molecular sieves. The temperatures of the column, injector port and detector were 90, 100, and 100 °C, respectively. The degradation products without arsenic were analyzed using GC−MS (Clarus SQ8 T, PerkinElmer) with an Elite-5 capillary column (30 m × 0.25 mm i.d., 0.25 μm df). Before the GC−MS determination, a 500 mL sample collected from the anode chambers was extracted using a solid-phase extraction column (C18). The products were eluted from the column using 10 mL dichloromethane and flushed with nitrogen to dryness, and the dry residue was dissolved in 1.0 mL of hexane. The sample was mixed with 50 μL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) and stored at 35 °C for 12 h before the GC−MS analysis. The GC column was operated in temperature-programmed mode at 40 °C for 3 min, raised at 10 °C/min to 280 °C, and held for 5 min. The mass range was 20−500 m/z.

activity by 84% when 263 mg/L of roxarsone is present in the substrate.3 In this case, 20 mg/L of roxarsone decreased the total methane production by 27%. When 0.5 V was applied for the first time, an obvious decrease in methane production was observed (SI Figure S1), regardless of whether roxarsone was added. The impact of applying current on the performance of the bioelectrochemical system has been reported previously, and these effects were mitigated during the cultivation cycles before eventually disappearing.32 In this study, all of the anaerobic sludge was acclimated before formal experiments to mitigate the impact of the electric current. Reduction of Roxarsone. Figure 1 shows the degradation of roxarsone under anaerobic conditions at 35 °C. The

Figure 1. Degradation of roxarsone under anoxic conditions. Note: 20 mg/L of initial roxarsone was added into the single cells in assay 1 with acetate addition but without 0.5 V voltage application, in assay 2 with acetate addition and 0.5 V voltage application, in assay 3 without acetate addition or 0.5 V voltage application, in assay 4 with heat-killed sludge and acetate addition but without 0.5 V voltage application, in assay 5 with acetate addition and 0.5 V voltage application but without anaerobic sludge, and in assay 6 with 0.5 V voltage application but without acetate addition or anaerobic sludge in pure water. The roxarsone was measured by HPLC.

anaerobic microbial consortium in the reactors exhibited good methanogenic activity at 0.5 V. The concentration of roxarsone in the reactors of assays 1−3 with acclimated anaerobic sludge decreased more than 90% after 2 days of cultivation, while in assay 4, which added heat-killed sludge, or 5, which contained only acetate, the decrease was less than 50%. The slow removal rate of roxarsone in heat-killed sludge was reported previously,1 confirming that some mechanisms for roxarsone removal were abiotic. The degradation of roxarsone in assay 5 was also attributed to the minerals and microelements contained in the water and nutrients. However, roxarsone was not reduced in pure water, as demonstrated by assay 6 (Figure 1). The reductive abilities of the anaerobic microbial system transformed roxarsone to HAPA, remaining consistent with the results of Cortinas et al.; in their study, 263 mg/L of roxarsone was completely reduced after 15 days of fermentation.1 Similarly, 210 mg/L of roxarsone disappeared after 200 h of incubation in chicken litter enrichments.2 The rapid and facile reduction in methanogenic sludge was attributed to the added or endogenous substrates in the sludge, which supplied enough electrons for the reductive biotransformation of a nitro group (−NO2) to an amino group (−NH2).33,34

■

RESULTS AND DISCUSSION Acclimation of the Anaerobic Microbial Consortium. The acclimation of the anaerobic microbial consortium toward the electric current and the inhibitory effect of roxarsone on the microbial consortium were evaluated using batch bioassays (SI Figure S1 and Table S1). Roxarsone is a severe inhibitor for acetate-utilizing methanogens, decreasing the methanogenic 7953

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

Electrochemically Stimulated Biodegradation of HAPA. As shown in Figure 2, the concentration of HAPA

mechanisms were further explored using two-chamber membrane reactors. Generation and Speciation of Inorganic Arsenic. Figure 3A shows the generation of the total inorganic arsenic

Figure 2. Degradation of HAPA under anaerobic conditions. Note: 20 mg/L of initial roxarsone was added into the single cells in assay 1 with acetate addition but without 0.5 V voltage application, in assay 2 with acetate addition and 0.5 V voltage application, in assay 3 without acetate addition or 0.5 V voltage application, and in assay 7 with 0.5 V voltage application but without acetate addition. The HAPA was measured by HPLC.

rapidly increased to a high level over the first 2 days as the roxarsone was reduced. Approximately 10−15% of the roxarsone disappeared during this stage because of the adsorption on sludge. With the added acetate in the assays 1 and 2, the HAPA concentration decreased only slightly after 24 days of incubation, regardless of whether 0.5 V voltage was applied. Therefore, HAPA resisted degradation under anaerobic conditions, and reports indicate that HAPA was converted over 229 days of anaerobic digestion.1 Due to its high solubility, the HAPA in animal wastewater will be transported to the surface water, releasing inorganic arsenic and causing arsenic contamination. Therefore, HAPA removal from wastewater is an important challenge. Interestingly, the HAPA degradation was accelerated without adding acetate. After 24 days of incubation, the HAPA in assay 3, which contained no added acetate, was more than 50% degraded, implying that HAPA could be degraded to form inorganic arsenic when the roxarsone contaminated wastewater is discharged into surface water, because the organic matter content of the surface water is very low. Reports indicate that many recognized toxicants can be utilized by microbes as carbon or energy sources, including benzoates,35 phenolic derivatives,36 and other refractory pollutants. During the initial phase of anaerobic digestion, acetate or endogenous substrates provided electrons for the microbes, and the roxarsone was reduced to HAPA because it is an electron acceptor. However, after the acetate was depleted, HAPA was utilized by the microbes as a carbon source, accelerating the degradation of HAPA. This result demonstrates the risk of releasing the roxarsone in animal wastes and wastewater into the environment. Therefore, removing the roxarsone and HAPA in animal wastewater before it is discharged with the effluent is important. Moreover, as shown in Figure 2, the HAPA was completely degraded after 8 days of incubation when 0.5 V was applied without added acetate (assay 7), indicating that applying 0.5 V significantly enhanced the degradation by enhancing the electron transfer during the degradation process. The possible

Figure 3. Generation of the total inorganic arsenic (A), As(III) (B) and As(V) (C) in solution. Note: 20 mg/L of initial roxarsone was added into the single cells in assay 1 with acetate addition but without 0.5 V voltage application, in assay 2 with acetate addition and 0.5 V voltage application, in assay 3 without acetate addition or 0.5 V voltage application, and in assay 7 with 0.5 V voltage application but without acetate addition. The total inorganic arsenic was measured by AFS, and the As(III) and As(V) were analyzed by HPLC-HG-AFS.

in solution during the incubation relative to Figure 2. In the assay with the added acetate, the HAPA persisted for 24 days (Figure 2), and the corresponding inorganic arsenic in the solution remained unchanged at a very low concentration. However, in the assays without acetate addition (assays 3 and 7), the degradation of HAPA was accompanied by the generation of inorganic arsenic. The sharp decrease in HAPA 7954

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

Figure 4. Degradation of HAPA and generation of As(III) and As(V) in the electrode chambers with 0.5 V voltage application. Degradation of HAPA (A), generation of As(III) (B), generation of As(V) (C), and real-time anode potentials vs standard hydrogen electrode (SHE) (D). Note: 20 mg/L of initial roxarsone was added into the anode chamber in assay 8 with acetate addition, cathode chamber in assay 8 with acetate addition, anode chamber in assay 9 without acetate addition, and cathode chamber in assay 9 without acetate addition. Anode potential was measured in assay 8 with acetate addition, in assay 9 without acetate addition, in assay 10 with acetate but without roxarsone addition, and in assay 11 in abiotic control. The HAPA was measured by HPLC, and the As(III) and As(V) were analyzed by HPLC-HG-AFS.

As(V). In the absence of carbon sources, the anaerobic sludge did not have enough electron donors, and the arsenite outside of the cytoplasm could provide electrons through arsenite oxidation.11 In the assay without acetate at 0.5 V voltage (assay 7), the conversion of As(III) to As(V) was accelerated, rapidly decreasing the As(III) and initially increasing the As(V). The acceleration might be attributed to the increased redox potential when 0.5 V was applied in the absence of carbon sources, generating a microcurrent stimulation for the oxidation of As(III). As(V) is favorable for arsenic removal from wastewater through physicochemical processes.30 Function of Electrodes on the Stimulation. Figure 2 shows that the applied voltage accelerated the degradation of HAPA. However, the purpose of the electrodes and the electrode potentials on the degradation of HAPA remained unclear. A short-term experiment with membrane reactors was designed to investigate the function of the electrodes and electrode potentials on the electrochemical stimulated HAPA degradation. In membrane reactors, the diffusion of HAPA was blocked by anion-exchange membrane, but As(III) and As(V) were both able to diffuse because of their anionic forms in the solution. Figure 4 shows the variations in the HAPA, As(III), As(V), and the anode potentials at 0.5 V voltage with 20 mg/L of added roxarsone (assays 8−11). The concentration of HAPA increased quickly in both two chambers over the first 2 days. The conversion rate of roxarsone to HAPA in the cathode chambers was universal higher than that in the anode chambers.

at 0.5 V (assay 7) immediately increased the inorganic arsenic production in solution. Figure 3B, C and SI Figure S2 show the speciation of As(III) and As(V). With acetate and 20 mg/L of initial roxarsone addition in assay 1, As(III) and As(V) were both generated, but the concentration of As(III) was higher than that of As(V); little As(V) was detected during the first 5 days of the incubation. In the assay without acetate addition (assay 3), As(III) was generated and increased steadily, peaking on the 20th day before decreasing (Figure 3B). Accompanied with the generation of As(III), As(V) was generated until the end of the incubation (Figure 3C). In the assay without acetate addition at 0.5 V (assay 7), As(III) appeared but decreased quickly during the early days of the incubation, while the As(V) concentration increased sharply, reaching a high level. Interestingly, approximately 35% of total arsenic contained in the roxarsone was released to form the inorganic arsenic in solution on the 10th days of the incubation in assay 7 after complete degradation of roxarsone, which was similar to the result reported by Cortinas et al. over 132 days of incubation.1 The distribution analysis of arsenic was carried out in SI Figure S3. As(V) would be the most likely species of inorganic arsenic released from the degradation of roxarsone or HAPA.1 However, As(III) was the predominant inorganic arsenic species detected in the early days of the incubation due to the subsequent biological reduction of arsenate to arsenite under anaerobic methanogenic conditions.33 In the assay without acetate (assay 3), As(III) was gradually converted to 7955

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

Figure 5. Degradation and biotransformation pathway for roxarsone under electrochemical stimulation.

the stimulation was related to the microbes. Meanwhile, a slight decrease in the roxarsone at the abiotic cathode was observed at −700 mV, while the HAPA remained unchanged (SI Figure S4B). Reports indicate that some organoarsenic acids could be reduced on the electrodes,41 but rapidly reducing roxarsone to HAPA was not easy under abiotic experimental conditions. The transformation of electrons by microbes is also essential for the HAPA degradation during the electrochemical stimulation process. Possible Mechanisms for the Accelerated HAPA Degradation and Biotransformation. Under anaerobic conditions, roxarsone was initially converted to HAPA after the nitro group (−NO2) was reduced to an amino group (−NH2); the electrons were donated by the added or endogenous substrates in solution. The reduction process could be slightly stimulated at a low reduction potential in the cathode chambers containing microbes. However, the anode chamber contributed to the degradation of HAPA: the anode acted as a stable electron acceptor for the degradation of HAPA. A similar action has been reported in microbial electrochemical systems.16,21,24,37 The degradation products were analyzed further using HPLC-UV-HG-AFS and GC−MS; no other arsenical compound peak appeared except HAPA and inorganic arsenic (SI Figure S5), and o-aminophenol was detected in the solution (SI Figure S6). O-aminophenol is a phenolic intermediate and can be degraded relatively easily by anaerobic microbes over several days.42 Combining the HPLCUV-HG-AFS and GC−MS, data revealed that the degradation of HAPA began with arsenic removal. As(III) was the predominant inorganic species initially (Figure 4B) due to the subsequent biological reduction of As(V) to As(III) under the reducing methanogenic conditions.1 As the carbon sources were depleted, the anode potential began to rise, oxidating of As(III) to As(V) and explaining the rapid increase in the As(V) concentration (Figure 4C). Therefore, the possible pathway for the roxarsone degradation and biotransformation under electrochemical stimulation are proposed, as illustrated in Figure 5. Environmental Significance. Organoarsenics, such as roxarsone and arsanilic acid, have been broadly applied as

The electrons donated by the cathode can accelerate the reduction, which has been reported previously.24−26 Afterward, a sharp decrease in HAPA only occurred in the anode chamber without acetate addition (assay 9), while a small decrease was observed in the cathode chamber (Figure 4A). As(III) was generated in the early days, and As(V) was formed later in both chambers in all assays (Figure 4B and C). Furthermore, the concentration of As(III) was higher than that of As(V) in both chambers during the early days of the incubation. In assay 9, the concentration of As(V) increased more in the anode chamber than that in the cathode chamber. The sharp decrease in the HAPA in the anode chamber proved that the HAPA was degraded on the anode. Reports indicate that electrochemical stimulation can accelerate the oxidation of organic contaminants by providing the anode as an electron acceptor.20,21,24,37 In this case, the graphite anode was a stable electron acceptor for the degradation of HAPA, and As(V) was the major degradation product. Function of the Anode Potential during Stimulation. Figure 4D shows the real-time variations in the anode potentials in the different assays. The initial anode potentials were all approximately +500 mV vs standard hydrogen electrode (SHE). The two assays with the added acetate showed a rapid decrease in the anode potentials and stabilized to a baseline (about −250 mV vs SHE) regardless of whether roxarsone or HAPA was present. Many investigations have also revealed low potentials for working electrodes in the acetate solutions because the electron source is stable.38,39 In the assay without acetate (assay 9), the anode potential initially decreased due to the residual carbon in the sludge. Afterward, the potential increased, obviously stimulating the degradation. As shown in Figure 4D, the anode potential should be at least +300 mV vs SHE to maintain the biological oxidation. Based on the Nernst Equation at a similar concentration of arsenic and pH 7.5, the high anode potential facilitated the oxidation of As(III) to As(V).40 However, in the pure water system (assay 11), a continuous high potential +650 mV vs SHE was observed, roxarsone and HAPA was not reduced and degraded in the anode chambers (SI Figure S4A), indicating that the roxarsone and HAPA was only degraded by microbes and that 7956

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

animal feed additives.1−3 The pollution caused by organoarsenic additives has generated attention. In China, high arsenic concentrations have been reported in the soil around the outlet of wastewater effluent from chicken and pig farms.4 Anaerobic digestion and aerobic treatments are often used to treat wastewater, but HAPA is highly water-soluble and is very difficult to degrade under aerobic or anaerobic conditions.1−3,15 Previous investigation also showed that most of roxarsone or HAPA still exists in the solution after sequencing batch reactor (SBR) treatment including nitrification and denitrification process.4 Therefore, discharging HAPA with wastewater contaminates the surface water or groundwater with arsenic. To the best of our knowledge, treating organoarsenic additivecontaminated wastewater has not been reported previously. Arsenic is very toxic, and the levels allowed in drinking water are limited to 10 μg/L by the WHO.8 The biochemical oxygen demand (BOD) value in the wastewater effluent is usually very low, making the application of electrochemical stimulation possible. Applying electrochemical stimulation significantly enhanced the biodegradation of HAPA to As(V), facilitating the final removal of arsenic from wastewater. Therefore, electrochemical stimulation provides a potential solution for treating organoarsenic additive-contaminated wastewater.

■

(5) Garbarino, J. R.; Bednar, A. J.; Rutherford, D. W.; Beyer, R. S.; Wershaw, R. L. Environmental fate of roxarsone in poultry litter. I. Degradation of roxarsone during composting. Environ. Sci. Technol. 2003, 37 (8), 1509−1514. (6) Bednar, A. J.; Garbarino, J. R.; Ferrer, I.; Rutherford, D. W.; Wershaw, R. L.; Ranville, J. F.; Wildeman, T. R. Photodegradation of roxarsone in poultry litter leachates. Sci. Total Environ. 2003, 302 (1− 3), 237−245. (7) Kim, K.-W.; Bang, S.; Zhu, Y.; Meharg, A. A.; Bhattacharya, P. Arsenic geochemistry, transport mechanism in the soil-plant system, human and animal health issues. Environ. Int. 2009, 35 (3), 453−454. (8) Abedin, M. J.; Cresser, M. S.; Meharg, A. A.; Feldmann, J.; Cotter-Howells, J. Arsenic accumulation and metabolism in rice (Oryza sativa L.). Environ. Sci. Technol. 2002, 36 (5), 962−968. (9) Itai, T.; Hayase, D.; Hyobu, Y.; Hirata, S. H.; Kumagai, M.; Tanabe, S. Hypoxia-induced exposure of isaza fish to manganese and arsenic at the bottom of lake Biwa, Japan: Experimental and geochemical verification. Environ. Sci. Technol. 2012, 46 (11), 5789− 5797. (10) Qin, J.; Lehr, C. R.; Yuan, C.; Le, X. C.; McDermott, T. R.; Rosen, B. P. Biotransformation of arsenic by a Yellowstone thermoacidophilic eukaryotic alga. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (13), 5213−5217. (11) Kruger, M. C.; Bertin, P. N.; Heipieper, H. J.; Arsene-Ploetze, F. Bacterial metabolism of environmental arsenic-mechanisms and biotechnological applications. Appl. Microbiol. Biotechnol. 2013, 97 (9), 3827−3841. (12) Mukhopadhyay, R.; Rosen, B. P.; Phung, L. T.; Silver, S. Microbial arsenic: From geocycles to genes and enzymes. FEMS Microbiol. Rev. 2002, 26 (3), 311−325. (13) Oremland, R. S.; Stolz, J. F. The ecology of arsenic. Science 2003, 300 (5621), 939−944. (14) Yin, X.-X.; Chen, J.; Qin, J.; Sun, G.-X.; Rosen, B. P.; Zhu, Y.-G. Biotransformation and volatilization of arsenic by three photosynthetic cyanobacteria. Plant Physiol. 2011, 156 (3), 1631−1638. (15) Neely, M. M.; Schreiber, M. E. Investigation of roxarsone biotransformation under aerobic conditions. 2008 NSF REU Proceedings of Research Opportunities in Interdisciplinary Watershed Sciences and Engineering, 2008; 14−16. (16) Thrash, J. C.; Coates, J. D. Review: Direct and indirect electrical stimulation of microbial metabolism. Environ. Sci. Technol. 2008, 42 (11), 3921−3931. (17) Strycharz, S. M.; Woodard, T. L.; Johnson, J. P.; Nevin, K. P.; Sanford, R. A.; Loeffler, F. E.; Lovley, D. R. Graphite electrode as a sole electron donor for reductive dechlorination of tetrachlorethene by Geobacter lovleyi. Appl. Environ. Microbiol. 2008, 74 (19), 5943−5947. (18) Aulenta, F.; Catervi, A.; Majone, M.; Panero, S.; Reale, P.; Rossetti, S. Electron transfer from a solid-state electrode assisted by methyl viologen sustains efficient microbial reductive dechlorination of TCE. Environ. Sci. Technol. 2007, 41 (7), 2554−2559. (19) Chun, C. L.; Payne, R. B.; Sowers, K. R.; May, H. D. Electrical stimulation of microbial PCB degradation in sediment. Water Res. 2013, 47 (1), 141−152. (20) Aulenta, F.; Verdini, R.; Zeppilli, M.; Zanaroli, G.; Fava, F.; Rossetti, S.; Majone, M. Electrochemical stimulation of microbial cisdichloroethene (cis-DCE) oxidation by an ethene-assimilating culture. New Biotechnology 2013, 30 (6), 749−755. (21) Lohner, S. T.; Becker, D.; Mangold, K.-M.; Tiehm, A. Sequential reductive and oxidative biodegradation of chloroethenes stimulated in a coupled bioelectro-process. Environ. Sci. Technol. 2011, 45 (15), 6491−6497. (22) Zhang, T.; Gannon, S. M.; Nevin, K. P.; Franks, A. E.; Lovley, D. R. Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor. Environ. Microbiol. 2010, 12 (4), 1011−1020. (23) Bond, D. R.; Holmes, D. E.; Tender, L. M.; Lovley, D. R. Electrode-reducing microorganisms that harvest energy from marine sediments. Science 2002, 295 (5554), 483−485.

ASSOCIATED CONTENT

S Supporting Information *

The results for the methane production, arsenic speciation during different phases, the distribution of arsenic, the control groups with the abiotic electrodes, and the HPLC-UV-HG-AFS and GC−MS analysis of the degradation products are included. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 86-551-62904144; fax: 86-551-62902066; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was partially supported by the NSFC (51078122, 51108149, 51208164), the Key Special Program on the S&T for Pollution Control (2012ZX07103-001), and the Program for New Century Excellent Talents in University (NCET-110624).

■

REFERENCES

(1) Cortinas, I.; Field, J. A.; Kopplin, M.; Garbarino, J. R.; Gandolfi, A. J.; Sierra-Alvarez, R. Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids. Environ. Sci. Technol. 2006, 40 (9), 2951−2957. (2) Stolz, J. F.; Perera, E.; Kilonzo, B.; Kail, B.; Crable, B.; Fisher, E.; Ranganathan, M.; Wormer, L.; Basu, P. Biotransformation of 3-nitro-4hydroxybenzene arsonic acid (roxarsone) and release of inorganic arsenic by Clostridium species. Environ. Sci. Technol. 2007, 41 (3), 818−823. (3) Sierra-Alvarez, R.; Cortinas, I.; Field, J. A. Methanogenic inhibition by roxarsone (4-hydroxy-3-nitrophenylarsonic acid) and related aromatic arsenic compounds. J. Hazard. Mater. 2010, 175 (1− 3), 352−358. (4) Guo, Q.; Liu, L.; Hu, Z.; Chen, G. Biological phosphorus removal inhibition by roxarsone in batch culture systems. Chemosphere 2013, 92 (1), 138−142. 7957

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958

Environmental Science & Technology

Article

(24) Sun, M.; Reible, D. D.; Lowry, G. V.; Gregory, K. B. Effect of applied voltage, initial concentration, and natural organic matter on sequential reduction/oxidation of nitrobenzene by graphite electrodes. Environ. Sci. Technol. 2012, 46 (11), 6174−6181. (25) Wang, A.-J.; Cheng, H.-Y.; Liang, B.; Ren, N.-Q.; Cui, D.; Lin, N.; Kim, B. H.; Rabaey, K. Efficient reduction of nitrobenzene to aniline with a biocatalyzed cathode. Environ. Sci. Technol. 2011, 45 (23), 10186−10193. (26) Mu, Y.; Rozendal, R. A.; Rabaey, K.; Keller, J. Nitrobenzene removal in bioelectrochemical systems. Environ. Sci. Technol. 2009, 43 (22), 8690−8695. (27) Dirany, A.; Sires, I.; Oturan, N.; Ozcan, A.; Oturan, M. A. Electrochemical treatment of the antibiotic sulfachloropyridazine: Kinetics, reaction pathways, and toxicity evolution. Environ. Sci. Technol. 2012, 46 (7), 4074−4082. (28) Liang, B.; Cheng, H.-Y.; Kong, D.-Y.; Gao, S.-H.; Sun, F.; Cui, D.; Kong, F.-Y.; Zhou, A.-J.; Liu, W.-Z.; Ren, N.-Q.; Wu, W.-M.; Wang, A.-J.; Lee, D.-J. Accelerated reduction of chlorinated nitroaromatic antibiotic chloramphenicol by biocathode. Environ. Sci. Technol. 2013, 47 (10), 5353−5361. (29) Vaclavikova, M.; Gallios, G. P.; Hredzak, S.; Jakabsky, S. Removal of arsenic from water streams: An overview of available techniques. Clean Technol. Environ. Policy 2007, 10 (1), 89−95. (30) Wang, H.-J.; Gong, W.-X.; Liu, R.-P.; Liu, H.-J.; Qu, J.-H. Treatment of high arsenic content wastewater by a combined physicalchemical process. Colloids Surf., A 2011, 379 (1−3), 116−120. (31) Mazej, D.; Falnoga, I.; Veber, M.; Stibilj, V. Determination of selenium species in plant leaves by HPLC-UV-HG-AFS. Talanta 2006, 68 (3), 558−568. (32) Lin, H.; Wu, X.; Miller, C.; Zhu, J. Improved performance of microbial fuel cells enriched with natural microbial inocula and treated by electrical current. Biomass Bioenergy 2013, 54, 170−180. (33) Field, J. A.; Sierra-Alvarez, R.; Cortinas, I.; Feijoo, G.; Moreira, M. T.; Kopplin, M.; Gandolfi, A. J. Facile reduction of arsenate in methanogenic sludge. Biodegradation 2004, 15 (3), 185−196. (34) Razo-Flores, E.; Lettinga, G.; Field, J. A. Biotransformation and biodegradation of selected nitroaromatics under anaerobic conditions. Biotechnol. Prog. 1999, 15 (3), 358−365. (35) Kasai, Y.; Kodama, Y.; Takahata, Y.; Hoaki, T.; Watanabe, K. Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11. Environ. Sci. Technol. 2007, 41 (17), 6222−6227. (36) Dibenedetto, A.; Lo Noce, R. M.; Narracci, M.; Aresta, M. Structure-biodegradation correlation of polyphenols for Thauera aromatica in anaerobic conditions. Chem. Ecol. 2006, 22, 133−143. (37) Wagner, R. C.; Call, D. I.; Logan, B. E. Optimal set anode potentials vary in bioelectrochemical systems. Environ. Sci. Technol. 2010, 44 (16), 6036−6041. (38) Bond, D. R.; Lovley, D. R. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl. Environ. Microbiol. 2003, 69 (3), 1548−1555. (39) Liu, H.; Grot, S.; Logan, B. E. Electrochemically assisted microbial production of hydrogen from acetate. Environ. Sci. Technol. 2005, 39 (11), 4317−4320. (40) Bard, A. J.; Parsons, R.; Jordan, J. Standard Potentials in Aqueous Solutions; CRC press, 1985; Vol. 6. (41) Elton, R. K.; Geiger, W. E., Jr. Analytical and mechanistic studies of the electrochemical reduction of biologically active organoarsenic acids. Anal. Chem. 1978, 50 (6), 712−717. (42) Karim, K.; Gupta, S. K. Biotransformation of nitrophenols in upflow anaerobic sludge blanket reactors. Bioresour. Technol. 2001, 80 (3), 179−186.

7958

dx.doi.org/10.1021/es501398j | Environ. Sci. Technol. 2014, 48, 7951−7958