Journal of Hazardous Materials 283 (2015) 617–622
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Anaerobic arsenite oxidation with an electrode serving as the sole electron acceptor: A novel approach to the bioremediation of arsenic-polluted groundwater Narcis Pous a , Barbara Casentini b , Simona Rossetti b , Stefano Fazi b , Sebastià Puig a , Federico Aulenta b,∗ a Laboratory of Chemical and Environmental Engineering (LEQUiA), Institute of the Environment, University of Girona, C/Maria Aurèlia Capmany, 69 E-17071 Girona, Spain b Water Research Institute (IRSA-CNR), National Research Council, Via Salaria Km 29.300, 00015 Monterotondo, Italy
h i g h l i g h t s • As(III) was oxidized to As(V) in a bioelectrochemical system. • A polarized graphite electrode served as electron acceptor. • Gammaproteobacteria were the dominating organisms at the electrode.
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 2 October 2014 Accepted 4 October 2014 Available online 20 October 2014 Keywords: Arsenite oxidation Arsenic bioremediation Bioelectrochemical systems
a b s t r a c t Arsenic contamination of soil and groundwater is a serious problem worldwide. Here we show that anaerobic oxidation of As(III) to As(V), a form which is more extensively and stably adsorbed onto metaloxides, can be achieved by using a polarized (+497 mV vs. SHE) graphite anode serving as terminal electron acceptor in the microbial metabolism. The characterization of the microbial populations at the electrode, by using in situ detection methods, revealed the predominance of gammaproteobacteria. In principle, the proposed bioelectrochemical oxidation process would make it possible to provide As(III)-oxidizing microorganisms with a virtually unlimited, low-cost and low-maintenance electron acceptor as well as with a physical support for microbial attachment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Groundwater contamination by arsenic poses important health risks and has prompted continued research efforts for the development of effective remediation technologies [1,2]. Although arsenic can exist in four oxidation states, the predominant forms typically found in water are inorganic As(III) (arsenite) and As(V) (arsenate). In aerobic oxidizing environments, arsenate is preeminent, while in anoxic reducing environments, such as subsurface waters, arsenite is the predominant form [3]. Arsenite is more harmful for human health than arsenate, being more toxic, soluble, and mobile [4]. In spite of that, established remediation technologies are typically more effective in removing As(V) than As(III) [5]. Indeed, due to its higher mobility, arsenite removal by precipitation or
∗ Corresponding author. Tel.: +39 06 90672751; fax: +39 06 90672787. E-mail address: [email protected] (F. Aulenta). http://dx.doi.org/10.1016/j.jhazmat.2014.10.014 0304-3894/© 2014 Elsevier B.V. All rights reserved.
adsorption is typically less effective than arsenate removal. For this reason, arsenic-treatment technologies should rely on a two-step approach, involving an initial oxidation of As(III) to As(V) followed by the adsorption of As(V). Traditionally, physicochemical techniques have been used to oxidize arsenite to arsenate [6,7]. However, these technologies typically suffer from high costs and generation of toxic by-products. Therefore, research on new sustainable and cost-effective arsenic removal technologies for water has recently become an area of intense research activity. Microbially catalysed arsenite oxidation is recently attracting considerable attention as an environmentally friendly pre-treatment for arsenic removal, being based on cheap and self-regenerating catalysts. Both heterotrophic and autotrophic As(III)-oxidizing microorganisms were described so far [8]. In heterotrophic bacteria, As(III) oxidation is typically regarded as a detoxification mechanism, whereby As(III) is enzymatically transformed into the less toxic As(V) [9]. By contrast, autotrophic As(III)-oxidizing bacteria typically use arsenite as electron donor
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N. Pous et al. / Journal of Hazardous Materials 283 (2015) 617–622
and oxygen (or nitrate) as terminal electron acceptor in their respiratory metabolism [10–12]. As far as in situ bioremediation of As-contaminated groundwater is concerned, the injection and distribution of oxygen remains a challenging aspect, primarily due to the low solubility of oxygen in water and the reduction of aquifer porosity caused by gas bubbles, among the others [13]. Bubble-less aeration using membranes was proposed as an effective method to transfer oxygen into groundwater but its application is still largely limited by the high installation and operational costs [14,15]. In recent years, the use of bioelectrochemical systems (BES), whereby microorganisms use solid-state electrodes as electron donors or acceptors in their metabolism, has raised considerable attention for bioremediation applications [16,17]. Until now, BES have been successfully applied (at least at the laboratory scale) for the bioremediation of several subsurface contaminants including perchlorate [18], trichloroethene [19–21], hexavalent chromium [22], benzene [23], nitrate [24], and sulphate [25]. So far, the potential of BES for treatment of As-contaminated (ground)water has been marginally explored. Recently, researchers [26] applied a microbial desalination cell (i.e. a device which exploits the electrochemical potential generated from microbial oxidation of organics to accomplish desalination) to separate hardness from water samples. It was found that the cell separated 89% of the arsenic contained in synthetic solutions. In another study [27], a hybrid bioelectrochemical system coupling a microbial fuel cell (MFC) to a zero-valent iron (ZVI) technology was proposed for arsenic removal from aqueous solutions. However, from the authors’ knowledge, the anaerobic bioelectrochemical oxidation of As(III) to As(V) with an electrode serving as direct electron acceptor in the metabolism of As(III)-oxidizing bacteria has not been so far investigated. In principle, with this approach, it could be possible to provide As(III)-oxidizing microorganisms with a virtually unlimited, lowcost and low-maintenance electron acceptor as well as with a physical support for attachment. Indeed, cheap carbon-based materials, such as graphite (approx. $1500 per ton), can be used as anodes and cathodes, which require minimal maintenance during operation not being prone to corrosion in aqueous environments. Furthermore, solar panels can possibly be used to drive the bioelectrochemical oxidation process, since it involves only low electric currents and/or applied voltages. Furthermore, the bioelectrochemical approach would eliminate the need for adding chemicals (i.e., oxygen) to groundwater to stimulate microbial activity and would minimize the generation of toxic by-products. Based on these considerations, the aim of this study was to demonstrate, for the first time, the possibility to stimulate the anaerobic oxidation of As(III) to As(V) using a polarized graphite electrode as the sole electron.
2. Experimental 2.1. Experimental set-up The bioelectrochemical experiments were conducted in a single-chamber cell having a total volume of 250 mL. The cell was equipped with two graphite rods (Sigma-Aldrich, Italy) serving as working- and counter-electrode, respectively, and an Ag/AgCl reference electrode (+0.197 V vs. standard hydrogen electrode, SHE) (Amel, Italy). The graphite rods were previously treated with HCl 1 M and NaOH 1 M, to removal metals and residual organics, as described previously [28]. The cell was filled with 150 mL of anaerobic medium. As a consequence, the resulting wet surface area of the working electrode was 5 cm2 . The medium used for the tests was prepared with deionized water and it contained 15 mg/L of As(III), 19.5 mg/L KH2 PO4 , 19.5 mg/L K2 HPO4 , 487 mg/L NaCl, 158 mg/L
NH4 Cl, 97.4 mg/L CaCl2 , 165 mg/L MgCl2 ·6H2 O, 7980 mg/L NaHCO3 , 0.974 mL/L of a metals solution [29], and 5 mL/L of a vitamins solution [30]. In order to ensure anaerobic conditions, the headspace of the cell was purged with N2 gas. When needed, N2 /CO2 (70:30) was purged to maintain the medium pH in the range 7.5–8.5. 2.2. BES operation At the start of the study, the bioelectrochemical cell was inoculated with 150 mL of an aerobic arsenite-oxidizing enrichment culture. The original inoculum from which the enrichment culture was derived was a groundwater sample from an arsenic-rich volcanic area in the Northern Latium (Italy). The groundwater was repeatedly spiked with As(III) to a final concentration of 100 mg/L and kept under aerobic conditions. Before each spike, cells were harvested by filtration through 0.2-m polycarbonate filters (Nuclepore) and resuspended in mineral medium lacking organic carbon sources, in order to promote the enrichment of autotrophic As(III)-oxidizing microorganisms. In order to promote an electrode-driven, anaerobic oxidation of As(III), after a period of about 2 weeks during which the enrichment culture was kept under anaerobic conditions in the presence of a polarized (+0.497 V vs. SHE) graphite electrode, the liquid medium was almost entirely removed in order to eliminate freeliving microorganisms and retain only those staying attached onto the electrode surface. The removed liquid was replaced with fresh anaerobic medium. Subsequently, potentiostatic batch tests were carried out to study the capability of microorganisms to anaerobically oxidize As(III), with the polarized (+0.497 V vs. SHE) graphite electrode serving as electron acceptor. The choice of the polarization potential was based on the formal redox potential of the arsenite oxidase of the autotrophic bacterium NT-26 [31]. In order to determine the As(III), As(V), and total arsenic (AsTOT ) concentrations, 2.5 mL of liquid samples were taken daily. To assess the effect of electrode polarization on microbial As(III) oxidation, batch tests were also carried whereby the inoculated cell was maintained at open circuit potential (OCP). Furthermore, in order to assess the contribution of purely electrochemical As(III) oxidation, abiotic control tests with the graphite electrode polarized at +0.497 V vs. SHE were carried out in a non-inoculated cell containing only anaerobic mineral medium. In the biotic and abiotic batch tests (four tests in total), As(III) was spiked to an initial concentration of approximately 15 mg/L and the headspace was continuously flushed with N2 in order to prevent oxygen intrusions. Throughout the study, the liquid content of the cells was continuously stirred with a stirring bar in order to prevent mass transport limitations; the cells were maintained in a water bath at 22–24 ◦ C. Low scan-rate (5 mV/s) cyclic voltammetry was performed at the end of each batch test on the working electrode of both inoculated and non-inoculated cells. All electrochemical tests and measurements were carried out using a multichannel potentiostat (Ivium Technologies, The Netherlands). Throughout the study, all redox potentials are reported with respect to the SHE. 2.3. Analytical methods and calculations Liquid samples taken from the cells during the batch tests were immediately filtered (0.2 m pore size syringe filters) and stored at −20 ◦ C until use. As(III) and total arsenic (AsTOT ) concentrations were determined by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) (Perkin-Elmer, USA). For the selective As(III) determination, samples were buffered to pH 5.5 by addition of acetate buffer 2 M. Determination of AsTOT was performed by treating the sample (1 mL) with reducing agents (i.e., 1 mL HCl 37% and 1 mL of 5% ascorbic acid and potassium iodide
N. Pous et al. / Journal of Hazardous Materials 283 (2015) 617–622
solution, reaction time 30 min). Before the analysis, the treated sample was diluted 1:20. As(V) was calculated as the difference between AsTOT and As(III). The Coulombic efficiency of the (bio)electrochemical oxidation process was calculated as the ratio between the produced amount of As(V) (considering that 1 mol of produced As(V) consumed two electron equivalents) and the electric charge transferred to the electrode over the period of electrode polarization; both parameters were expressed as electron equivalents.
619
a
2.4. Microbiological analysis At the end of the incubations, liquid samples (50 mL) were taken in triplicates from the bioelectrochemical cell and immediately fixed with formaldehyde (2%, v/v, final concentration). Moreover, the biofilm attached to the graphite electrode was gently scraped, suspended in ultrapure sterilized water, and fixed. Both water samples and biofilm suspensions (50 mL) were filtered through 0.2 m polycarbonate filters (Ø 47 mm, Millipore) by gentle vacuum (
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Anaerobic arsenite oxidation with an electrode serving as the sole electron acceptor: A novel approach to the bioremediation of arsenic-polluted groundwater Narcis Pous a , Barbara Casentini b , Simona Rossetti b , Stefano Fazi b , Sebastià Puig a , Federico Aulenta b,∗ a Laboratory of Chemical and Environmental Engineering (LEQUiA), Institute of the Environment, University of Girona, C/Maria Aurèlia Capmany, 69 E-17071 Girona, Spain b Water Research Institute (IRSA-CNR), National Research Council, Via Salaria Km 29.300, 00015 Monterotondo, Italy
h i g h l i g h t s • As(III) was oxidized to As(V) in a bioelectrochemical system. • A polarized graphite electrode served as electron acceptor. • Gammaproteobacteria were the dominating organisms at the electrode.
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 2 October 2014 Accepted 4 October 2014 Available online 20 October 2014 Keywords: Arsenite oxidation Arsenic bioremediation Bioelectrochemical systems
a b s t r a c t Arsenic contamination of soil and groundwater is a serious problem worldwide. Here we show that anaerobic oxidation of As(III) to As(V), a form which is more extensively and stably adsorbed onto metaloxides, can be achieved by using a polarized (+497 mV vs. SHE) graphite anode serving as terminal electron acceptor in the microbial metabolism. The characterization of the microbial populations at the electrode, by using in situ detection methods, revealed the predominance of gammaproteobacteria. In principle, the proposed bioelectrochemical oxidation process would make it possible to provide As(III)-oxidizing microorganisms with a virtually unlimited, low-cost and low-maintenance electron acceptor as well as with a physical support for microbial attachment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Groundwater contamination by arsenic poses important health risks and has prompted continued research efforts for the development of effective remediation technologies [1,2]. Although arsenic can exist in four oxidation states, the predominant forms typically found in water are inorganic As(III) (arsenite) and As(V) (arsenate). In aerobic oxidizing environments, arsenate is preeminent, while in anoxic reducing environments, such as subsurface waters, arsenite is the predominant form [3]. Arsenite is more harmful for human health than arsenate, being more toxic, soluble, and mobile [4]. In spite of that, established remediation technologies are typically more effective in removing As(V) than As(III) [5]. Indeed, due to its higher mobility, arsenite removal by precipitation or
∗ Corresponding author. Tel.: +39 06 90672751; fax: +39 06 90672787. E-mail address: [email protected] (F. Aulenta). http://dx.doi.org/10.1016/j.jhazmat.2014.10.014 0304-3894/© 2014 Elsevier B.V. All rights reserved.
adsorption is typically less effective than arsenate removal. For this reason, arsenic-treatment technologies should rely on a two-step approach, involving an initial oxidation of As(III) to As(V) followed by the adsorption of As(V). Traditionally, physicochemical techniques have been used to oxidize arsenite to arsenate [6,7]. However, these technologies typically suffer from high costs and generation of toxic by-products. Therefore, research on new sustainable and cost-effective arsenic removal technologies for water has recently become an area of intense research activity. Microbially catalysed arsenite oxidation is recently attracting considerable attention as an environmentally friendly pre-treatment for arsenic removal, being based on cheap and self-regenerating catalysts. Both heterotrophic and autotrophic As(III)-oxidizing microorganisms were described so far [8]. In heterotrophic bacteria, As(III) oxidation is typically regarded as a detoxification mechanism, whereby As(III) is enzymatically transformed into the less toxic As(V) [9]. By contrast, autotrophic As(III)-oxidizing bacteria typically use arsenite as electron donor
618
N. Pous et al. / Journal of Hazardous Materials 283 (2015) 617–622
and oxygen (or nitrate) as terminal electron acceptor in their respiratory metabolism [10–12]. As far as in situ bioremediation of As-contaminated groundwater is concerned, the injection and distribution of oxygen remains a challenging aspect, primarily due to the low solubility of oxygen in water and the reduction of aquifer porosity caused by gas bubbles, among the others [13]. Bubble-less aeration using membranes was proposed as an effective method to transfer oxygen into groundwater but its application is still largely limited by the high installation and operational costs [14,15]. In recent years, the use of bioelectrochemical systems (BES), whereby microorganisms use solid-state electrodes as electron donors or acceptors in their metabolism, has raised considerable attention for bioremediation applications [16,17]. Until now, BES have been successfully applied (at least at the laboratory scale) for the bioremediation of several subsurface contaminants including perchlorate [18], trichloroethene [19–21], hexavalent chromium [22], benzene [23], nitrate [24], and sulphate [25]. So far, the potential of BES for treatment of As-contaminated (ground)water has been marginally explored. Recently, researchers [26] applied a microbial desalination cell (i.e. a device which exploits the electrochemical potential generated from microbial oxidation of organics to accomplish desalination) to separate hardness from water samples. It was found that the cell separated 89% of the arsenic contained in synthetic solutions. In another study [27], a hybrid bioelectrochemical system coupling a microbial fuel cell (MFC) to a zero-valent iron (ZVI) technology was proposed for arsenic removal from aqueous solutions. However, from the authors’ knowledge, the anaerobic bioelectrochemical oxidation of As(III) to As(V) with an electrode serving as direct electron acceptor in the metabolism of As(III)-oxidizing bacteria has not been so far investigated. In principle, with this approach, it could be possible to provide As(III)-oxidizing microorganisms with a virtually unlimited, lowcost and low-maintenance electron acceptor as well as with a physical support for attachment. Indeed, cheap carbon-based materials, such as graphite (approx. $1500 per ton), can be used as anodes and cathodes, which require minimal maintenance during operation not being prone to corrosion in aqueous environments. Furthermore, solar panels can possibly be used to drive the bioelectrochemical oxidation process, since it involves only low electric currents and/or applied voltages. Furthermore, the bioelectrochemical approach would eliminate the need for adding chemicals (i.e., oxygen) to groundwater to stimulate microbial activity and would minimize the generation of toxic by-products. Based on these considerations, the aim of this study was to demonstrate, for the first time, the possibility to stimulate the anaerobic oxidation of As(III) to As(V) using a polarized graphite electrode as the sole electron.
2. Experimental 2.1. Experimental set-up The bioelectrochemical experiments were conducted in a single-chamber cell having a total volume of 250 mL. The cell was equipped with two graphite rods (Sigma-Aldrich, Italy) serving as working- and counter-electrode, respectively, and an Ag/AgCl reference electrode (+0.197 V vs. standard hydrogen electrode, SHE) (Amel, Italy). The graphite rods were previously treated with HCl 1 M and NaOH 1 M, to removal metals and residual organics, as described previously [28]. The cell was filled with 150 mL of anaerobic medium. As a consequence, the resulting wet surface area of the working electrode was 5 cm2 . The medium used for the tests was prepared with deionized water and it contained 15 mg/L of As(III), 19.5 mg/L KH2 PO4 , 19.5 mg/L K2 HPO4 , 487 mg/L NaCl, 158 mg/L
NH4 Cl, 97.4 mg/L CaCl2 , 165 mg/L MgCl2 ·6H2 O, 7980 mg/L NaHCO3 , 0.974 mL/L of a metals solution [29], and 5 mL/L of a vitamins solution [30]. In order to ensure anaerobic conditions, the headspace of the cell was purged with N2 gas. When needed, N2 /CO2 (70:30) was purged to maintain the medium pH in the range 7.5–8.5. 2.2. BES operation At the start of the study, the bioelectrochemical cell was inoculated with 150 mL of an aerobic arsenite-oxidizing enrichment culture. The original inoculum from which the enrichment culture was derived was a groundwater sample from an arsenic-rich volcanic area in the Northern Latium (Italy). The groundwater was repeatedly spiked with As(III) to a final concentration of 100 mg/L and kept under aerobic conditions. Before each spike, cells were harvested by filtration through 0.2-m polycarbonate filters (Nuclepore) and resuspended in mineral medium lacking organic carbon sources, in order to promote the enrichment of autotrophic As(III)-oxidizing microorganisms. In order to promote an electrode-driven, anaerobic oxidation of As(III), after a period of about 2 weeks during which the enrichment culture was kept under anaerobic conditions in the presence of a polarized (+0.497 V vs. SHE) graphite electrode, the liquid medium was almost entirely removed in order to eliminate freeliving microorganisms and retain only those staying attached onto the electrode surface. The removed liquid was replaced with fresh anaerobic medium. Subsequently, potentiostatic batch tests were carried out to study the capability of microorganisms to anaerobically oxidize As(III), with the polarized (+0.497 V vs. SHE) graphite electrode serving as electron acceptor. The choice of the polarization potential was based on the formal redox potential of the arsenite oxidase of the autotrophic bacterium NT-26 [31]. In order to determine the As(III), As(V), and total arsenic (AsTOT ) concentrations, 2.5 mL of liquid samples were taken daily. To assess the effect of electrode polarization on microbial As(III) oxidation, batch tests were also carried whereby the inoculated cell was maintained at open circuit potential (OCP). Furthermore, in order to assess the contribution of purely electrochemical As(III) oxidation, abiotic control tests with the graphite electrode polarized at +0.497 V vs. SHE were carried out in a non-inoculated cell containing only anaerobic mineral medium. In the biotic and abiotic batch tests (four tests in total), As(III) was spiked to an initial concentration of approximately 15 mg/L and the headspace was continuously flushed with N2 in order to prevent oxygen intrusions. Throughout the study, the liquid content of the cells was continuously stirred with a stirring bar in order to prevent mass transport limitations; the cells were maintained in a water bath at 22–24 ◦ C. Low scan-rate (5 mV/s) cyclic voltammetry was performed at the end of each batch test on the working electrode of both inoculated and non-inoculated cells. All electrochemical tests and measurements were carried out using a multichannel potentiostat (Ivium Technologies, The Netherlands). Throughout the study, all redox potentials are reported with respect to the SHE. 2.3. Analytical methods and calculations Liquid samples taken from the cells during the batch tests were immediately filtered (0.2 m pore size syringe filters) and stored at −20 ◦ C until use. As(III) and total arsenic (AsTOT ) concentrations were determined by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) (Perkin-Elmer, USA). For the selective As(III) determination, samples were buffered to pH 5.5 by addition of acetate buffer 2 M. Determination of AsTOT was performed by treating the sample (1 mL) with reducing agents (i.e., 1 mL HCl 37% and 1 mL of 5% ascorbic acid and potassium iodide
N. Pous et al. / Journal of Hazardous Materials 283 (2015) 617–622
solution, reaction time 30 min). Before the analysis, the treated sample was diluted 1:20. As(V) was calculated as the difference between AsTOT and As(III). The Coulombic efficiency of the (bio)electrochemical oxidation process was calculated as the ratio between the produced amount of As(V) (considering that 1 mol of produced As(V) consumed two electron equivalents) and the electric charge transferred to the electrode over the period of electrode polarization; both parameters were expressed as electron equivalents.
619
a
2.4. Microbiological analysis At the end of the incubations, liquid samples (50 mL) were taken in triplicates from the bioelectrochemical cell and immediately fixed with formaldehyde (2%, v/v, final concentration). Moreover, the biofilm attached to the graphite electrode was gently scraped, suspended in ultrapure sterilized water, and fixed. Both water samples and biofilm suspensions (50 mL) were filtered through 0.2 m polycarbonate filters (Ø 47 mm, Millipore) by gentle vacuum (
