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On the removal of sulfonamides using microbial bioelectrochemical systems
Electrochemistry Communications 26 (2013) 77–80
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
On the removal of sulfonamides using microbial bioelectrochemical systems Falk Harnisch ⁎, Carla Gimkiewicz, Birthe Bogunovic, Robert Kreuzig ⁎⁎, Uwe Schröder Institute of Environmental and Sustainable Chemistry, TU Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
a r t i c l e
i n f o
Article history: Received 3 September 2012 Received in revised form 5 October 2012 Accepted 8 October 2012 Available online 16 October 2012 Keywords: Microbial fuel cell Microbial biosensor Bioelectrochemical system Electroactive biofilms Wastewater treatment Sulfonamides
a b s t r a c t On the example of selected sulfonamides and N4-acteyl-analogues we demonstrate that antibiotic substances can be removed from artificial wastewater using anodic microbial biofilms. Particularly sulfamethoxazole and sulfadiazine are completely or partly removed, respectively, by biofilms within 7 days of batch operation. Thereby, the removal process is shown not to affect the anodic microbial biofilm performance. As further shown for N4-acetyl-sulfamethoxazole and N4-acetyl-sulfadiazine the microbial bioelectrochemical removal of these compounds does not proceed via the retransformation to the sulfonamides and is thus not leading to the environmentally relevant pharmaceutical reactivation, as often observed for aerobic degradation processes within wastewater treatment plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Human pharmaceuticals belong to the emerging group of micropollutants in wastewater that can be hardly removed in municipal wastewater treatment plant (WWTP). Hence, pharmaceutical residues may occur in the WWTP effluents up to the μg L −1 range [1,2]. Although these concentrations are below the ecotoxicologically effective mg L −1 levels [3], chronic environmental toxic effects cannot be excluded, as there is very little known about chronic ecotoxicity of single substances and almost nothing about potentially synergistic effects. Consequently, new methods for the removal of these micropollutants within WWTPs are investigated, comprising high-sophisticated technologies that often require a considerable consumption of energy and/ or chemicals, e.g., chlorination [4], UV irradiation [5], nanofiltration [6], ozonation, activated carbon filtration, biofiltration [7,8] and electrochemical decomposition using boron-doped diamond electrodes [9]. At the same time microbial bioelectrochemical systems (BES), most prominently microbial fuel cells (MFCs), are proposed as sustainable alternatives for wastewater treatment. It has been demonstrated that BES can be used not only for the oxidation of wastewater constituents, i.e., COD removal, but also for the removal of pollutants. These include, e.g., nitrogen [10], sulfide, nitrobenzene [11] as well as the dehalogenation of X-ray contrast media [12]. Furthermore, BES were shown to be efficient in the dehalogenation of halogen containing hydrocarbons, e.g., in soils [13]. ⁎ Corresponding author. Tel.: +49 5313918425; fax: +49 5319318428. ⁎⁎ Corresponding author. Tel.: +49 5313915962; fax: +49 5313915799. E-mail addresses: [email protected] (F. Harnisch), [email protected] (R. Kreuzig). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.10.015
Based on a previously demonstrated persistence of anodic microbial biofilms in BES against heavy metals and antibiotics at high concentrations, that are toxic to planktonic cells [14], we now investigated the potential removal of selected sulfonamides and corresponding N 4-acetyl-sulfonamides by wastewater derived anodic microbial biofilms using acetate as substrate. 2. Experimental 2.1. General conditions All microbiological and electrochemical experiments were conducted under strictly anoxic conditions at a temperature of 30 °C. All chemicals were of analytical or biochemical grade and were purchased from Sigma-Aldrich and Merck except the sulfonamides, which were purchased from Dr. Ehrenstorfer GmbH, Germany. If not stated otherwise, all potentials provided in this article refer to the Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE). 2.2. Microbial inoculums, growth medium and sulfonamides The source for the microbial inoculum was primary wastewater, which was collected from the WWTP Braunschweig/Steinhoff, Germany, and stored in sealed containers at 4 °C. The bacterial growth medium was prepared as reported by Kim et al. [15]. It contained NH4Cl (0.31 g L−1), KCl (0.13 g L−1), NaH2PO4·H2O (2.69 g L−1), Na2HPO4 (4.33 g L−1), trace metal (12.5 mL) and vitamin (12.5 mL) solutions [16] and was adjusted to pH 6.8. Acetate (10 mM) served as substrate in the growth medium. In order to ensure anaerobic conditions the substrate and buffer solutions were purged with nitrogen before use.
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F. Harnisch et al. / Electrochemistry Communications 26 (2013) 77–80
The sulfonamides sulfamethoxazole, sulfathiazole, sulfadimidine, sulfadiazine (mix-SA) and their corresponding N 4-acetyl metabolites (mix-AcSA) were studied at respective concentrations of 6 μg L −1. Structures of the test substances are listed in Fig. 2. 2.3. Electrochemical set-up All electrochemical experiments were carried out under potentiostatic control, using a three electrode arrangement consisting of the working electrode, an Ag/AgCl reference electrode (sat. KCl, Sensortechnik Meinsberg, Germany, 0.195 V vs. SHE) and a counter electrode. The working and counter electrodes used throughout this study were graphite rods (CP-Graphite GmbH, Germany). The experiments were conducted with a Potentiostat/Galvanostat Model VMP3 (BioLogic Science Instruments, France), equipped with 12 independent potentiostat channels. Cyclic voltammetry (CV) was performed during turnover and non-turnover conditions at a scan rate of 1 mV s−1 in accordance with previous studies, e.g., [17,18]. Current density data in this article are reported per footprint surface area and are denominated as “geometric current density”. 2.4. Biofilm growth in semi-batch experiments and sampling As described by Liu et al. [19] for the formation of primary biofilms, 1 mL of wastewater per 30 mL of the substrate solution was inoculated into the sealed 250-mL electrochemical cells. A constant potential of
0.2 V was applied to the working electrode and the substrate solution was replenished regularly. The biofilm growth was monitored by measuring the bioelectrocatalytic oxidation current. After usually 2 to 3 fed-batch cycles (which are usually required to achieve a sufficient biofilm thickness and thus biocatalytic performance), the substrate solution was replenished by fresh substrate solution additionally containing a mix of sulfonamides (Section 2.2. and Fig. 1A). This point of exposure is further referred to as t0. During the further potentiostatic biofilm operation (as described above) liquid samples were taken after 3 days and 7 days of continuous incubation as follows: after 3 days 1 × 1-mL and 2 × 25-mL samples were taken from each electrochemical cell for metabolite (Section 2.5.) and residue (Section 2.6) analysis, respectively, and 1 mL of 1 M sodium acetate solution was added (thus the total batch volume decreased, but all electrodes remained immersed). After 7 days this sampling procedure was repeated—without the subsequent addition of acetate. The t0 samples were taken from the bulk solutions. All presented data are based on, at least, two independent biological replicates and thus, at least, on 4 analytical replicates plus 4 blanks. All removal rates are provided in percentage to the initially used concentrations. Abiotic control experiments at 0.2 V vs. Ag/AgCl as well as at −0.7 V vs. Ag/AgCl (being a typical potential of the counter electrode) did show no removal of the substances. 2.5. Metabolite analysis The metabolic substrate consumption and non-gaseous fermentation product formation were followed by HPLC analysis. The HPLC (Spectrasystem P400, FINNIGAN Surveyor RI Plus detector, Fisher Scientific, Germany) was equipped with a Rezex HyperREZ XP Carbohydrate H + 8 μm column. The chromatograms were recorded at room temperature with 0.005 N sulfuric acid as the eluent.
A
2.6. Residue analysis
B
For residue analysis, 25-mL samples were diluted with 200 mL deionized water and were adjusted to pH 4. The test substances, i.e., sulfonamides and corresponding N4-acetyl metabolites, were then enriched by solid phase extraction (Oasis HLB; Waters, Germany). After methanol elution, the analytical solutions were analyzed using LC/MS/MS (4000 QTRAP; AB SCIEX, Germany) operating in ESI+ and MRM mode [20]. For quantitative analysis, the external calibration curves (5–100 pg μL −1) were recorded. The analytical quality assurance based on spiking before and spiking after extraction tests and the determination of quantifier and qualifier ions of each test substance at signal noise ratio of 10:1.
0.50
0.40
3. Results and discussion 3.1. Influence of the sulfonamides on the biofilm performance / μA cm-2 V-1
j / μA cm-2
0.30
0.20
ΔjΔE
-1
0.10
0.00
30 25 20 15 10 5 0
-0.4
-0.3
-0.2
E / V (vs. Ag/ AgCl, sat. KCl.)
-0.10 -0.4
-0.2
0.0
0.2
0.4
E / V (vs. Ag/ AgCl, sat. KCl.) Fig. 1. A) Plot of the chronoamperometric fed-batch biofilm formation and sampling procedure on the example of exposure to mix-AcSA (details see 2.4). B) Turn-over CV of biofilm incubated with mix-AcSA after 3 days of operation. The scan rate was 1 mV s−1. Inset: 1st derivative of the CV.
During all applications of the sulfonamide (mix-SA) and acetylsulfonamide (mix-AcSA) mixtures the performance of the electroactive microbial biofilms was assessed in terms of maximum geometric current density, jmax, and coulombic efficiency, CE. Here the coulombic efficiency refers to the oxidation of the primary substrate: acetate. Both parameters, jmax =237±26 μA cm−2 and CE=72±16%, were within the usual range of primary anodic biofilms grown from wastewater inoculi [19,21]. The exposure of the biofilms to the sulfonamides did not decrease jmax. In contrast, the current densities further increased after t0 by an average of 11%. This increase has to be attributed to the fact that the biofilms had not reached steady state within the 2–3 growth cycles before exposure [19]. The coulombic efficiencies remained constant within a window of ±16% relative to CE before exposure to the sulfonamides. Thus, it is evident that in accordance with our previous study [14], the
F. Harnisch et al. / Electrochemistry Communications 26 (2013) 77–80
exposure to the micropollutants did not affect the electrochemical biofilm performance. Cyclic voltammetry was performed for turn-over, i.e., presence of the electron donor acetate, as well as non turn-over, i.e., depletion of the electron donor acetate conditions. Fig. 1 showing a turn-over CV clearly demonstrates that the formal potential of the active site, Ef, of 325± 25 mV (vs. Ag/AgCl) is similar to wastewater derived Geobacteraceae dominated biofilms [21,22]. This was confirmed by non turn-over CVs possessing a similar, characteristic CV shape—despite that DGGE analysis showed a multitude of signals, pointing to the presence of several (not necessarily bioelectrochemically active) microbial species. These were not analyzed in detail.
79
(decrease to 69%), whereas both other compounds were not removed from the solution. Importantly, the acetyl-analogues were removed from the solution by the bacterial activity, but not retransformed to the sulfonamides, as often found for aerobic wastewater treatments [24]. These differences of the substance specific removability of the sulfonamides and their N 4-acetyl-metabolites are under current research. Control experiments for sterile conditions did not show any removal. Furthermore, experiments using 14C-labeled acetyl-sulfamethoxazole, analyzed as described elsewhere [25], did not reveal any accumulation of this test substance in the biofilms.
4. Conclusions 3.2. Removal of the sulfonamides and N 4-acetyl-sulfonamides Fig. 2 illustrates the substance specific removal of test substances under study by the electroactive microbial biofilms. Sulfamethoxazole was completely removed from the solution facing the anodic biofilm, whereas the concentration of sulfathiazole remained constant. Similar tendencies were observed for the partly removed sulfadiazine and the unaffected sulfadimidine. These results indicate substance-specific biotransformation processes by biofilms. However, additional tests are necessary to identify the respective microbial transformation pathways and the formed transformation products. Furthermore, electroactive biofilms harboring a different (or more diverse) microbial community might possess an even higher degradation potential. The potential of the environmentally relevant regeneration of sulfonamides from N 4-acetyl-metabolites within wastewater treatment processes is well understood and represents a major risk, as this regeneration does particularly mean the reactivation of active compounds from the pharmacological viewpoint [23]. Therefore, the corresponding N4-acetyl-analogues of the sulfonamides were additionally studied for their microbial bioelectrochemical removal. As illustrated in Fig. 2B for mix-AcSA, the profiles of the N4-acetyl-metabolites qualitatively resembled the removal behavior of the corresponding sulfonamides. Thus, the concentration of N4-acetyl-sulfamethoxazole dropped to 41% of the initial amount within 7 days, followed by N4-acetyl-sulfadiazine
The authors acknowledge the financial support from the Fonds der Chemischen Industrie, the German Federal Ministry of Food, Agriculture and Consumer Protection through the Federal Office for Agriculture and Food, Bonn, Germany, (grant number 2810HS032), the Volkswagen AG and the Verband der Deutschen Biokraftstoffindustrie e.V. for funding the professorship for Sustainable Chemistry and Energy Research.
SDZ
SMX
STZ
AcSDM
AcSDZ
AcSMX
AcSTZ
B
100
concentration/ %
100
concentration/ %
Acknowledgment
SDM
A
80 60 40
80 60 40 20
20 0
This study demonstrates on the example of the selected sulfonamides and corresponding N4-acetyl-analogues that antibiotic micropollutants can be principally removed from wastewater using acetate grown anodic microbial biofilms. This removal does not affect the microbial bioelectrocatalytic performance, however, shows a substance specific behavior. Particularly, the removal of N4-acetyl-sulfamethoxazole proceeds not via the retransformation to the pharmaceutically active parent compound, proved microbial bioelectrochemical systems to be a promising alternative for the removal of those micropollutants. Certainly, further investigations are inevitable for revealing the underlying bacterial mechanisms and the transformation products and metabolic pathways as well as studying the removal potential of other (more diverse) anodic and cathodic microbial biofilms.
SDM
SDZ
SMX
STZ
AcSDM
AcSDZ AcSMX AcSTZ
Fig. 2. Removal using the primary anodic microbial biofilms at chronoamperometric operation 0.2 V (vs. Ag/AgCl) with black: day 0, shaded: day 3 and white: day 7, error bars indicate the standard deviation of 2 biological and 4 analytical replicates. A) sulfonamides (SDM: sulfadimidine, SDZ: sulfadiazine, SMX: sulfamethoxazole, STZ: sulfathiazole), and B) N4-acetyl-sulfonamides (AcSDM: acetyl-sulfadimidine, AcSDZ: acetyl-sulfadiazine, AcSMX: acetyl-sulfamethoxazole, AcSTZ: acetyl-sulfathiazole).
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F. Harnisch et al. / Electrochemistry Communications 26 (2013) 77–80
References [1] C.G. Daughton, T.A. Ternes, Environmental Health Perspectives 107 (1999) 907. [2] D. Fatta-Kassinos, S. Meric, A. Nikolaou, Analytical and Bioanalytical Chemistry 399 (2011) 251. [3] M.D. Hernando, A.R. Fernández-Alba, R. Tauler, D. Barceló, Talanta 65 (2005) 358. [4] E. Chamberlain, C. Adams, Water Research 40 (2006) 2517. [5] C. Adams, M. Asce, Y. Wang, K. Loftin, M. Meyer, Journal of Environmental Engineering 128 (2002). [6] M. Röhricht, J. Krisam, U. Weise, U.R. Kraus, R.-A. Düring, Clean 37 (2009) 638. [7] J. Reungoat, M. Macova, B.I. Escher, S. Carswell, J.F. Mueller, J. Keller, Water Research 44 (2010) 625. [8] J. Reungoat, B.I. Escher, M. Macova, J. Keller, Water Research 45 (2011) 2751. [9] J. Radjenović, M.J. Farre, Y. Mu, W. Gernjak, J. Keller, Water Research 46 (2012) 1706. [10] B. Virdis, K. Rabaey, Z. Yuan, J. Keller, Water Research 42 (2008) 3013. [11] A.J. Wang, H.Y. Cheng, B. Liang, N.Q. Ren, D. Cui, N. Lin, B.H. Kim, K. Rabaey, Environmental Science & Technology 45 (2011) 10186. [12] Y. Mu, J. Radjenović, S. Sinyou, R.A. Rozendal, K. Rabaey, J. Keller, Environmental Science & Technology 45 (2010) 782. [13] F. Aulenta, A. Canosa, L. De Roma, P. Reale, S. Panero, S. Rossetti, M. Majone, Journal of Chemical Technology and Biotechnology 84 (2009) 864.
[14] S.A. Patil, F. Harnisch, U. Schröder, Chemie, Physik und Physikalischen Chemie 11 (2010) 2834. [15] J.R. Kim, B. Min, B.E. Logan, Applied Microbiology and Biotechnology 68 (2005) 23. [16] W.E. Balch, G.E. Fox, L.J. Magrum, C.R. Woese, R.S. Wolfe, Microbiological Reviews 43 (1979) 260. [17] K. Fricke, F. Harnisch, U. Schröder, Energy & Environmental Science 1 (2008) 144. [18] S. Srikanth, E. Marsili, M.C. Flickinger, D.R. Bond, Biotechnology and Bioengineering 99 (2008) 1065. [19] Y. Liu, F. Harnisch, K. Fricke, R. Sietmann, U. Schröder, Biosensors & Bioelectronics 24 (2008) 1012. [20] M. El Dosoky, in, vol. PhD TU Braunschweig, 2012. [21] S.A. Patil, F. Harnisch, C. Koch, T. Hübschmann, I. Fetzer, A.-A. Carmona-Martinez, S. Müller, U. Schröder, Bioresource Technology 102 (2011) 9683. [22] F. Harnisch, C. Koch, S.A. Patil, T. Hübschmann, S. Müller, U. Schröder, Energy & Environmental Science 4 (2011) 1265. [23] A. Göbel, C.S. McArdell, M.J.-F. Suter, W. Giger, Analytical Chemistry 76 (2004) 4756. [24] M. Clara, B. Strenn, O. Gans, E. Martinez, N. Kreuzinger, H. Kroiss, Water Research 39 (2005) 4797. [25] S. Höltge, R. Kreuzig, Clean 35 (2007) 104.
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
On the removal of sulfonamides using microbial bioelectrochemical systems Falk Harnisch ⁎, Carla Gimkiewicz, Birthe Bogunovic, Robert Kreuzig ⁎⁎, Uwe Schröder Institute of Environmental and Sustainable Chemistry, TU Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
a r t i c l e
i n f o
Article history: Received 3 September 2012 Received in revised form 5 October 2012 Accepted 8 October 2012 Available online 16 October 2012 Keywords: Microbial fuel cell Microbial biosensor Bioelectrochemical system Electroactive biofilms Wastewater treatment Sulfonamides
a b s t r a c t On the example of selected sulfonamides and N4-acteyl-analogues we demonstrate that antibiotic substances can be removed from artificial wastewater using anodic microbial biofilms. Particularly sulfamethoxazole and sulfadiazine are completely or partly removed, respectively, by biofilms within 7 days of batch operation. Thereby, the removal process is shown not to affect the anodic microbial biofilm performance. As further shown for N4-acetyl-sulfamethoxazole and N4-acetyl-sulfadiazine the microbial bioelectrochemical removal of these compounds does not proceed via the retransformation to the sulfonamides and is thus not leading to the environmentally relevant pharmaceutical reactivation, as often observed for aerobic degradation processes within wastewater treatment plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Human pharmaceuticals belong to the emerging group of micropollutants in wastewater that can be hardly removed in municipal wastewater treatment plant (WWTP). Hence, pharmaceutical residues may occur in the WWTP effluents up to the μg L −1 range [1,2]. Although these concentrations are below the ecotoxicologically effective mg L −1 levels [3], chronic environmental toxic effects cannot be excluded, as there is very little known about chronic ecotoxicity of single substances and almost nothing about potentially synergistic effects. Consequently, new methods for the removal of these micropollutants within WWTPs are investigated, comprising high-sophisticated technologies that often require a considerable consumption of energy and/ or chemicals, e.g., chlorination [4], UV irradiation [5], nanofiltration [6], ozonation, activated carbon filtration, biofiltration [7,8] and electrochemical decomposition using boron-doped diamond electrodes [9]. At the same time microbial bioelectrochemical systems (BES), most prominently microbial fuel cells (MFCs), are proposed as sustainable alternatives for wastewater treatment. It has been demonstrated that BES can be used not only for the oxidation of wastewater constituents, i.e., COD removal, but also for the removal of pollutants. These include, e.g., nitrogen [10], sulfide, nitrobenzene [11] as well as the dehalogenation of X-ray contrast media [12]. Furthermore, BES were shown to be efficient in the dehalogenation of halogen containing hydrocarbons, e.g., in soils [13]. ⁎ Corresponding author. Tel.: +49 5313918425; fax: +49 5319318428. ⁎⁎ Corresponding author. Tel.: +49 5313915962; fax: +49 5313915799. E-mail addresses: [email protected] (F. Harnisch), [email protected] (R. Kreuzig). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.10.015
Based on a previously demonstrated persistence of anodic microbial biofilms in BES against heavy metals and antibiotics at high concentrations, that are toxic to planktonic cells [14], we now investigated the potential removal of selected sulfonamides and corresponding N 4-acetyl-sulfonamides by wastewater derived anodic microbial biofilms using acetate as substrate. 2. Experimental 2.1. General conditions All microbiological and electrochemical experiments were conducted under strictly anoxic conditions at a temperature of 30 °C. All chemicals were of analytical or biochemical grade and were purchased from Sigma-Aldrich and Merck except the sulfonamides, which were purchased from Dr. Ehrenstorfer GmbH, Germany. If not stated otherwise, all potentials provided in this article refer to the Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE). 2.2. Microbial inoculums, growth medium and sulfonamides The source for the microbial inoculum was primary wastewater, which was collected from the WWTP Braunschweig/Steinhoff, Germany, and stored in sealed containers at 4 °C. The bacterial growth medium was prepared as reported by Kim et al. [15]. It contained NH4Cl (0.31 g L−1), KCl (0.13 g L−1), NaH2PO4·H2O (2.69 g L−1), Na2HPO4 (4.33 g L−1), trace metal (12.5 mL) and vitamin (12.5 mL) solutions [16] and was adjusted to pH 6.8. Acetate (10 mM) served as substrate in the growth medium. In order to ensure anaerobic conditions the substrate and buffer solutions were purged with nitrogen before use.
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F. Harnisch et al. / Electrochemistry Communications 26 (2013) 77–80
The sulfonamides sulfamethoxazole, sulfathiazole, sulfadimidine, sulfadiazine (mix-SA) and their corresponding N 4-acetyl metabolites (mix-AcSA) were studied at respective concentrations of 6 μg L −1. Structures of the test substances are listed in Fig. 2. 2.3. Electrochemical set-up All electrochemical experiments were carried out under potentiostatic control, using a three electrode arrangement consisting of the working electrode, an Ag/AgCl reference electrode (sat. KCl, Sensortechnik Meinsberg, Germany, 0.195 V vs. SHE) and a counter electrode. The working and counter electrodes used throughout this study were graphite rods (CP-Graphite GmbH, Germany). The experiments were conducted with a Potentiostat/Galvanostat Model VMP3 (BioLogic Science Instruments, France), equipped with 12 independent potentiostat channels. Cyclic voltammetry (CV) was performed during turnover and non-turnover conditions at a scan rate of 1 mV s−1 in accordance with previous studies, e.g., [17,18]. Current density data in this article are reported per footprint surface area and are denominated as “geometric current density”. 2.4. Biofilm growth in semi-batch experiments and sampling As described by Liu et al. [19] for the formation of primary biofilms, 1 mL of wastewater per 30 mL of the substrate solution was inoculated into the sealed 250-mL electrochemical cells. A constant potential of
0.2 V was applied to the working electrode and the substrate solution was replenished regularly. The biofilm growth was monitored by measuring the bioelectrocatalytic oxidation current. After usually 2 to 3 fed-batch cycles (which are usually required to achieve a sufficient biofilm thickness and thus biocatalytic performance), the substrate solution was replenished by fresh substrate solution additionally containing a mix of sulfonamides (Section 2.2. and Fig. 1A). This point of exposure is further referred to as t0. During the further potentiostatic biofilm operation (as described above) liquid samples were taken after 3 days and 7 days of continuous incubation as follows: after 3 days 1 × 1-mL and 2 × 25-mL samples were taken from each electrochemical cell for metabolite (Section 2.5.) and residue (Section 2.6) analysis, respectively, and 1 mL of 1 M sodium acetate solution was added (thus the total batch volume decreased, but all electrodes remained immersed). After 7 days this sampling procedure was repeated—without the subsequent addition of acetate. The t0 samples were taken from the bulk solutions. All presented data are based on, at least, two independent biological replicates and thus, at least, on 4 analytical replicates plus 4 blanks. All removal rates are provided in percentage to the initially used concentrations. Abiotic control experiments at 0.2 V vs. Ag/AgCl as well as at −0.7 V vs. Ag/AgCl (being a typical potential of the counter electrode) did show no removal of the substances. 2.5. Metabolite analysis The metabolic substrate consumption and non-gaseous fermentation product formation were followed by HPLC analysis. The HPLC (Spectrasystem P400, FINNIGAN Surveyor RI Plus detector, Fisher Scientific, Germany) was equipped with a Rezex HyperREZ XP Carbohydrate H + 8 μm column. The chromatograms were recorded at room temperature with 0.005 N sulfuric acid as the eluent.
A
2.6. Residue analysis
B
For residue analysis, 25-mL samples were diluted with 200 mL deionized water and were adjusted to pH 4. The test substances, i.e., sulfonamides and corresponding N4-acetyl metabolites, were then enriched by solid phase extraction (Oasis HLB; Waters, Germany). After methanol elution, the analytical solutions were analyzed using LC/MS/MS (4000 QTRAP; AB SCIEX, Germany) operating in ESI+ and MRM mode [20]. For quantitative analysis, the external calibration curves (5–100 pg μL −1) were recorded. The analytical quality assurance based on spiking before and spiking after extraction tests and the determination of quantifier and qualifier ions of each test substance at signal noise ratio of 10:1.
0.50
0.40
3. Results and discussion 3.1. Influence of the sulfonamides on the biofilm performance / μA cm-2 V-1
j / μA cm-2
0.30
0.20
ΔjΔE
-1
0.10
0.00
30 25 20 15 10 5 0
-0.4
-0.3
-0.2
E / V (vs. Ag/ AgCl, sat. KCl.)
-0.10 -0.4
-0.2
0.0
0.2
0.4
E / V (vs. Ag/ AgCl, sat. KCl.) Fig. 1. A) Plot of the chronoamperometric fed-batch biofilm formation and sampling procedure on the example of exposure to mix-AcSA (details see 2.4). B) Turn-over CV of biofilm incubated with mix-AcSA after 3 days of operation. The scan rate was 1 mV s−1. Inset: 1st derivative of the CV.
During all applications of the sulfonamide (mix-SA) and acetylsulfonamide (mix-AcSA) mixtures the performance of the electroactive microbial biofilms was assessed in terms of maximum geometric current density, jmax, and coulombic efficiency, CE. Here the coulombic efficiency refers to the oxidation of the primary substrate: acetate. Both parameters, jmax =237±26 μA cm−2 and CE=72±16%, were within the usual range of primary anodic biofilms grown from wastewater inoculi [19,21]. The exposure of the biofilms to the sulfonamides did not decrease jmax. In contrast, the current densities further increased after t0 by an average of 11%. This increase has to be attributed to the fact that the biofilms had not reached steady state within the 2–3 growth cycles before exposure [19]. The coulombic efficiencies remained constant within a window of ±16% relative to CE before exposure to the sulfonamides. Thus, it is evident that in accordance with our previous study [14], the
F. Harnisch et al. / Electrochemistry Communications 26 (2013) 77–80
exposure to the micropollutants did not affect the electrochemical biofilm performance. Cyclic voltammetry was performed for turn-over, i.e., presence of the electron donor acetate, as well as non turn-over, i.e., depletion of the electron donor acetate conditions. Fig. 1 showing a turn-over CV clearly demonstrates that the formal potential of the active site, Ef, of 325± 25 mV (vs. Ag/AgCl) is similar to wastewater derived Geobacteraceae dominated biofilms [21,22]. This was confirmed by non turn-over CVs possessing a similar, characteristic CV shape—despite that DGGE analysis showed a multitude of signals, pointing to the presence of several (not necessarily bioelectrochemically active) microbial species. These were not analyzed in detail.
79
(decrease to 69%), whereas both other compounds were not removed from the solution. Importantly, the acetyl-analogues were removed from the solution by the bacterial activity, but not retransformed to the sulfonamides, as often found for aerobic wastewater treatments [24]. These differences of the substance specific removability of the sulfonamides and their N 4-acetyl-metabolites are under current research. Control experiments for sterile conditions did not show any removal. Furthermore, experiments using 14C-labeled acetyl-sulfamethoxazole, analyzed as described elsewhere [25], did not reveal any accumulation of this test substance in the biofilms.
4. Conclusions 3.2. Removal of the sulfonamides and N 4-acetyl-sulfonamides Fig. 2 illustrates the substance specific removal of test substances under study by the electroactive microbial biofilms. Sulfamethoxazole was completely removed from the solution facing the anodic biofilm, whereas the concentration of sulfathiazole remained constant. Similar tendencies were observed for the partly removed sulfadiazine and the unaffected sulfadimidine. These results indicate substance-specific biotransformation processes by biofilms. However, additional tests are necessary to identify the respective microbial transformation pathways and the formed transformation products. Furthermore, electroactive biofilms harboring a different (or more diverse) microbial community might possess an even higher degradation potential. The potential of the environmentally relevant regeneration of sulfonamides from N 4-acetyl-metabolites within wastewater treatment processes is well understood and represents a major risk, as this regeneration does particularly mean the reactivation of active compounds from the pharmacological viewpoint [23]. Therefore, the corresponding N4-acetyl-analogues of the sulfonamides were additionally studied for their microbial bioelectrochemical removal. As illustrated in Fig. 2B for mix-AcSA, the profiles of the N4-acetyl-metabolites qualitatively resembled the removal behavior of the corresponding sulfonamides. Thus, the concentration of N4-acetyl-sulfamethoxazole dropped to 41% of the initial amount within 7 days, followed by N4-acetyl-sulfadiazine
The authors acknowledge the financial support from the Fonds der Chemischen Industrie, the German Federal Ministry of Food, Agriculture and Consumer Protection through the Federal Office for Agriculture and Food, Bonn, Germany, (grant number 2810HS032), the Volkswagen AG and the Verband der Deutschen Biokraftstoffindustrie e.V. for funding the professorship for Sustainable Chemistry and Energy Research.
SDZ
SMX
STZ
AcSDM
AcSDZ
AcSMX
AcSTZ
B
100
concentration/ %
100
concentration/ %
Acknowledgment
SDM
A
80 60 40
80 60 40 20
20 0
This study demonstrates on the example of the selected sulfonamides and corresponding N4-acetyl-analogues that antibiotic micropollutants can be principally removed from wastewater using acetate grown anodic microbial biofilms. This removal does not affect the microbial bioelectrocatalytic performance, however, shows a substance specific behavior. Particularly, the removal of N4-acetyl-sulfamethoxazole proceeds not via the retransformation to the pharmaceutically active parent compound, proved microbial bioelectrochemical systems to be a promising alternative for the removal of those micropollutants. Certainly, further investigations are inevitable for revealing the underlying bacterial mechanisms and the transformation products and metabolic pathways as well as studying the removal potential of other (more diverse) anodic and cathodic microbial biofilms.
SDM
SDZ
SMX
STZ
AcSDM
AcSDZ AcSMX AcSTZ
Fig. 2. Removal using the primary anodic microbial biofilms at chronoamperometric operation 0.2 V (vs. Ag/AgCl) with black: day 0, shaded: day 3 and white: day 7, error bars indicate the standard deviation of 2 biological and 4 analytical replicates. A) sulfonamides (SDM: sulfadimidine, SDZ: sulfadiazine, SMX: sulfamethoxazole, STZ: sulfathiazole), and B) N4-acetyl-sulfonamides (AcSDM: acetyl-sulfadimidine, AcSDZ: acetyl-sulfadiazine, AcSMX: acetyl-sulfamethoxazole, AcSTZ: acetyl-sulfathiazole).
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