Environ. Sci. Technol. 2010, 44, 4685–4691
Bioelectrochemical Perchlorate Reduction in a Microbial Fuel Cell C A I T L Y N S . B U T L E R , †,| P E T E R C L A U W A E R T , ‡,⊥ STEFAN J. GREEN,§ WILLY VERSTRAETE,‡ A N D R O B E R T N E R E N B E R G * ,† Department of Civil Engineering and Geological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium, and Department of Oceanography, Florida State University, Tallahassee, Florida 32311
Received June 15, 2009. Revised manuscript received April 27, 2010. Accepted April 28, 2010.
Perchlorate is an emerging surface water and groundwater contaminant, and it is of concern because of its mobility in the environment and its inhibitory effect on thyroid function. Microbial fuel cells (MFCs) may be a suitable method for its treatment. We investigated a MFC with a denitrifying biocathode for perchlorate reduction and utilized the system to identify putativebiocathode-utilizingperchlorate-reducingbacteria(PCRB). Perchlorate reduction in the MFC was established by increasing the perchlorate loading to the biocathode, while decreasing nitrate loading. Perchlorate reduction was obtained without the need for exogenous electron shuttles or fixed electrode potentials, achieving a maximum perchlorate removal of 24 mg/ L-d and cathodic conversion efficiency of 84%. The perchloratereducing biocathode bacterial community, which contained putative denitrifying Betaproteobacteria, shared little overlap with a purely denitrifying biocathode community, and was composed primarily of putative iron-oxidizing genera. Despite differences in cathodic function, the anode communities from the perchlorate-reducing MFC and the denitrifying MFC were similar to each other but different than their corresponding biocathode community. These data indicate that PCRB can utilize a cathode as an electron donor, and that this process can be harnessed to treat perchlorate while producing usable electrical power.
Introduction Perchlorate is an emerging drinking water contaminant, and it is of environmental concern because of its inhibitory effect on the thyroid gland (1). There is a need to remove low-level perchlorate concentrations from contaminated drinking water supplies, as well as high-level perchlorate concentrations from superfund sites and industrial wastewaters (2). A potentially cost-effective approach is bioreduction to chloride by dissimilatory perchlorate-reducing bacteria (PCRB) (3). While a number of bioreactors have been developed for perchlorate reduction (4), reduction using a biologically active * Corresponding author phone: 574-631-4098; fax: 574-631-9236; e-mail:
[email protected]. † University of Notre Dame. ‡ Ghent University. § Florida State University. | Currently at Arizona State University. ⊥ Currently with Aquafin NV, Belgium. 10.1021/es901758z
2010 American Chemical Society
Published on Web 05/17/2010
cathode (biocathode) within a microbial fuel cell (MFC) is a novel and potentially cost-effective approach (5, 6). Many PCRB are facultative anaerobes and denitrifiers (3). The ClO4-/Cl- pair (+0.806 vs SHE; pH 7) has a redox potential similar to that of oxygen (+0.816 vs SHE; pH 7), and theoretical per-electron yields on perchlorate are similar to those of aerobic and nitrate-dependent respiration. However, growth on perchlorate is typically slower than growth on oxygen or nitrate, possibly because perchlorate and its reduction intermediate, chlorate, are reduced by the same enzyme, the (per)chlorate reductase (7-10). Biocathodes harness the capacity of specific microorganisms to accept electrons from a solid surface (cathode) (11). High-rate oxygen reduction without a platinum catalyst and denitrification have been accomplished using biocathodes (11, 12), as well as reduction of chlorinated groundwater pollutants and uranium(VI) (13, 14). An advantage of this process is the opportunity for electrical energy recovery from the treatment process. Since the cathode compartment of an MFC contains no organic carbon, biomass formation is low because of the autotrophic growth of the cathode biofilm organisms (15). Our research explores the remediation potential of a perchlorate-reducing MFC. Specifically, we developed a highly active perchlorate-reducing microbial community capable of obtaining electrons for reduction from a cathode. We further sought to determine if nitrate is necessary for perchlorate reduction activity, and we examined the electrode microbial community structure in perchlorate- and nitratereducing MFCs for insight into the key taxa involved in reduction processes.
Methods Reactor Configuration. Two well-mixed, continuously fed MFCs were used concurrently in this research: a control MFC, where the cathode was fed with nitrate only, and an experimental MFC, which was initially operated with nitrate, but then supplied with perchlorate (Supporting Information, Figure S1). The MFCs were constructed from rectangular Plexiglas frames and filled with graphite granules, as described in Clauwaert et al. 2007 (16). Each electrode compartment had a 400-mL total volume and a 200-mL liquid volume after the granular electrode material was added, and contained a 200-mL external vessel within the recirculation loop to remove nitrogen gas produced in the cathode. External contact to the electrodes was made via graphite rods (McMaster-Carr) inserted into the anode and cathode chambers. The anode and cathode compartments were separated by an Ultrex cation exchange membrane (CMI700, Membranes International, Glen Rock, NJ). The influent flow rate was 0.23 mL/min, and recirculation rate was 100 mL/min, providing well-mixed conditions. Media and Inocula. The MFC anodes were inoculated with bacteria from activated sludge and existing MFC anodes. The cathodes were inoculated with effluent from the biocathode of a denitrifying MFC, activated sludge, a chloratereducing enrichment from lake sediment, and a pure culture of Dechloromonas sp. PC1 (GenBank accession No. AY126452). Potassium acetate (200 mg-acetate/L) was used as the organic substrate in the anodes. The nitrate and perchlorate concentrations within the cathode of the MFCs are presented in Table 1. The medium for the anode was a 16-mM phosphatebuffered minimal growth medium (Minimal Media #1, Supporting Information, Table S1). The anode medium was purged with nitrogen for 15-20 min and maintained under a nitrogen atmosphere when fed to the anode chambers of VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Influent Concentration and MFC Performance duration stage
days
Ib
0–58
II
59–84
III
85–101
IV
influent perchlorate concentration mg/L
influent nitrate concentration mg N/L
102–122
1
10
V
123–145
10
5
VI
146–179
1
5
VII
180–221
10
5
VIII
222–253
5
10
IX
254–302
20
1
X
303–337
20
338–388
10
389–421
20
XII
0–421
average nitrate removal
average current
CCE
mg/L-d (%)
mA
%
5.6 (24) 24 (100) 25 (100) 12 (100) 6.2 (100) 5.6 (100) 5.6 (100) 11 (100) 1.2 (100)
0.28
16–28
3.1
64–84
3.1
68–78
1.5
78–82
0.76
21–78
0.54
38–88
0.77
51–85
0.97
47–80
0.28
47–91
0.21
67–97
0.24
38–51
0.28
26–96
3.3
79–88
a
mg/L-d (%)
20 20
d
a
Perchlorate-Reducing Biocathode 20
0.1
XId
maximum perchlorate removal
0.096 (81) 0.62 (52) 5.0 (44) 1.1 (94) 11 (96) 5.3 (88) 24 (99c) 9.1 (39) 16 (99) 22 (97)
Control Denitrifying Biocathode 20
26 (100)
Based on the total liquid volume of the cathode compartment. b Rext ) 100 Ω. For all other conditions, Rext ) 10 Ω. Maximum occurred when the pH of the solution was increased to 8.5. Perchlorate removal was not sustained when the pH fell below 7.5. d The pH of the influent was increased to 8.5. a
c
the MFCs. The growth medium supplied to the biocathodes (Stages I-II, Table 1) contained 20 mg-N/L of nitrate and a slight variation of the previously described medium to compensate for the alkalinity produced during nitrate reduction (Minimal Media #2, Supporting Information, Table S1). From Stages III-X, the medium in the experimental biocathode was Minimal Media #1, and in the final two stages of the experimental MFC operation, the experimental biocathode medium was adjusted from neutral pH to a pH of 8.5 with 1 M NaOH. Analytical Techniques. Ion chromatography (ICS-2500, DIONEX Corporation, Sunnyvale, CA) was used to measure the acetate, nitrate, nitrite, chlorate, chloride, and perchlorate for the influent and effluent of the anode and cathode compartments of the MFCs. Samples were collected one to three times a week, filtered with 0.1 µm syringe filters, and stored at 4 °C for no more than 4 days before they were prepared for analysis on the ion chromatograph. Dilutions were made in 20:1, 50:1, and 100:1 depending on the initial concentrations and run against a standard calibration curve for each species on an AS-11 column (DIONEX Coporation, Sunnyvale, CA) with a 4 mM NaOH eluent at 1.5 mL/min for 9 min and 50 mM NaOH eluent at 1.5 mL/min for 11 min at 30 °C. The conductivity of the eluted species was measured at 190 mA. The detection limit for acetate was 0.5 mg/L, for nitrate 0.1 mg/L, and for perchlorate 0.01 mg/L. A pH meter (Fisher Scientific, Pittsburgh, PA) was used to monitor the pH within each electrode compartment during sampling. Electrochemical Performance. Each MFC was operated initially with a 100-Ω external resistance and, once the voltage drop was sustained, the external resistance was reduced to 10 Ω, following Clauwaert et al. (16). The voltage output across the external resistance (v) was recorded every 7 min using a digital multimeter (Keithley Instruments, Inc., Cleveland, 4686
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OH). The current density (i ) v/Rext/estimated cathode surface area) was calculated based on the maximum sustained voltage drop across the external resistance. The theoretical current was calculated based on the total electrons released in the reduction of each species in the cathode compartment: Ith )
FbNO3-q∆NO3 MNO3-
+
FbClO4-q∆ClO4 MClO4-
(1)
where Ith is the theoretical current, MNO3- is the molecular weight of nitrate (14 g-N/mol), MClO4- is the molecular weight of perchlorate (99.5 g/mol), F is Faraday’s constant (96485 C/mol), bNO3- is the number of electrons exchanged per mole of nitrate (5 e- eq.-N/mol), bClO4- is the number of electrons exchanged per mole of perchlorate when reduced to Cl- (8 e- eq./mol), q is the influent flow rate, ∆[NO3-] is the difference in the influent and effluent nitrate concentration measured as nitrogen and ∆[ClO4-] is the difference in the influent and effluent perchlorate concentration. The Cathodic Conversion Efficiency (CCE) was calculated as the ratio of actual current (Iactual) to theoretical current. Polarization curves were obtained by varying the external resistance of the cell from 10 to 5000 Ω and measuring the resulting cell voltage. The cell was allowed 1 hydraulic retention time (HRT; 22 h within this system) to achieve a steady voltage. After voltage output stabilized at each external resistance step, samples were collected from the anode and cathode compartments for ion analysis, as described above. Current and power were calculated as described above. The cathode potentials of each MFC were measured with a handheld digital multimeter (Fluke 12 Multimeter) versus an Ag/ AgCl reference electrode connected to the cathode chamber
by a 4 M KCl salt bridge, and anode potentials were calculated as the difference between the cell potential and the cathode potential. Analysis of Electrode Bacterial Community Structure. After an operating period of 420 days, total genomic DNA was extracted from microbial biomass associated with anodic and cathodic granules within the control and experimental MFCs. Genomic DNA was independently extracted in triplicate from granules taken from throughout each electrode compartment, using the Ultraclean soil DNA extraction kit (Mo Bio Laboratories, Inc., Carlsbad, CA) according to the manufacturer’s instructions. PCR amplification of portions of bacterial 16S rRNA genes was performed as described previously, with a working concentration of 4 mM Mg2+ and an annealing temperature of 62 °C (17). Initially, individual PCR products from each independent DNA extract were compared using denaturing gradient gel electrophoresis (DGGE) analyses (18). Because of the high reproducibility of replicate samples from each electrode, replicate PCR amplicons from each electrode were pooled to create a composite sample. The PCR amplicons were purified using the Mo Bio PCR Clean-up kit, ligated into into the pCR2.1 TOPO TA cloning vector (Invitrogen, Carlsbad, CA), and sent to the Washington University Genome Center for sequencing. Sequences were analyzed using a multipronged approach, including analysis with the basic local alignment search tool (BLAST), rapid classification using the classifier tool at the Ribosomal Database Project, diversity analysis using the software package DOTUR and phylogenetic analysis using the software package ARB. Details and references regarding the processing of sequence data can be found in the Supporting Information. To compare the communities on each electrode, a phylogenetic tree was generated from the sequence data (Supporting Information, Figure S4), and coupled with information about the electrode from which each sequence was derived, was used to compute a phylogenetic diversity metric for each pair of electrodes using the online software package Unifrac (19).
Results Reactor Optimization for Perchlorate Reduction. The experimental and control MFCs were inoculated and started under identical conditions. The start-up period lasted 1 month, during which the MFCs were operated with a 100-Ω external resistance (Stage I, Table 1). Acetate was supplied to the anode as the sole electron donor, and nitrate was supplied to the cathode as the sole electron acceptor. At the end of the startup period (Stage 1, Table 1), both MFCs began to partially remove nitrate from the cathode compartment, producing a sustained current. The external resistance was decreased to 10 Ω, yielding more current and higher denitrification rates (Stage II). The current and denitrification rates increased over a month until a sustained current of 3-3.5 mA (17-18 mA/m2 cathode, 3 mW/m2 cathode) was achieved, and nitrate was completely removed. The control MFC displayed similar performance throughout the 420-day experimental period (Table 1). The effluent pH in both cathodes during Stage I and II averaged 7.1. Effluent acetate concentrations were typically below detection (0.50 mg/L). At day 87, perchlorate was supplied to the experimental MFC at 0.1 mg/L (Stage III). The perchlorate removal (influent concentration- effluent concentration) was as high as 0.096 mg/L (Figure 1). A control verified that perchlorate did not adsorb to the graphite granules used in this experiment. The influent perchlorate concentration was then increased to 1 mg/L, and the nitrate was decreased to 10 mg-N/L (Stage IV). The current decreased to 1.5 mA (9 mA/m2-cathode, 1.5 mW/m2-cathode), but a progressive increase in perchlorate removal was observed (Figure 1). After 15 days, perchlorate
FIGURE 1. Influent nitrate (gray diamonds) and perchlorate (black triangles) concentrations and the effluent nitrate (gray squares) and perchlorate (×) concentrations in the experimental biocathode. removal reached 0.52 mg/L. On day 127, the influent nitrate concentration to the experimental reactor was decreased to 5 mg-N/L, and the perchlorate concentration increased to 10 mg/L. The current decreased to 0.76 mA (4.8 mA/m2 cathode, 0.75 mW/m2 cathode). The perchlorate removal reached 4.4 mg/L, subsequently decreasing to 3.5 mg/L (Stage V). The effluent cathode pH in this stage began at 7, but then decreased to 6.5 because of proton production via perchlorate reduction (Supporting Information, Figure S2). The influent perchlorate concentration was decreased to 1 mg/L, while maintaining nitrate at 5 mg N/L (Stage VI). The current decreased from 0.76 to 0.54 mA (4.8 to 3.4 mA/ m2-cathode), and greater than 96% removal of perchlorate was achieved after 20 days (Figure 1, Stage VI). The average effluent pH was 7.0. In Stage VII, the influent concentrations were returned to 10 mg/L of perchlorate and 5 mg-N/L nitrate. Initial perchlorate removal was 9.6 mg/L, but subsequently decreased to 2 mg/L, at which point the effluent pH was 6.5. The influent perchlorate concentration was decreased to 5 mg/L and the nitrate increased to 10 mg N/L (Stage VIII). The current increased to roughly 1 mA (6.3 mA/m2-cathode, 1 mW/m2-cathode). Approximately 2 mg/L perchlorate was removed (Figure 1), and the average effluent pH was 6.7. Subsequently, the influent perchlorate concentration was increased to 20 mg/L and the nitrate concentration decreased to 1 mg N/L (Stage IX). The maximum perchlorate removal was 8 mg/L (Figure 1) and the cathode pH was 6.5. However, perchlorate removal increased to 19.8 mg/L when the pH of medium was increased to 8.5 for 1 day (equal to 1 HRT). The effluent pH was 7.3. During Stage X, the influent pH was returned to 7, and no nitrate was added to the cathode influent. The influent perchlorate remained at 20 mg/L. Perchlorate removal was less than 8 mg/L. During Stage XI, the influent pH again was adjusted to 8.5, and the influent perchlorate concentration decreased to 10 mg/L, and perchlorate removal increased to 100%. When the influent perchlorate was increased to 20 mg/L (Stage XII), the removal was nearly complete. The average effluent pH was 7.8, and the current production was 0.28 mA (0.17 mA/m2-cathode, 0.3 mW/m2-cathode). Under all influent conditions, the nitrate was removed to below 0.1 mg-N/L, and no chlorate or nitrite was detected. Chloride was produced stoichiometrically from perchlorate. The CCE under all conditions was relatively high (Table 1), and the theoretical current correlated well with the measured current. The CCE was less than 100%, suggesting that acetate may have crossed over from the anode (20) or electrons were consumed in microbial growth. While the inocula may have been a source of substrates, any organic carbon contained in the inocula was either consumed during the first days of operation or otherwise washed out of this flow-through system. VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Perchlorate removal as a function of effluent pH for Stages IX and XI. Perchlorate removal as a function of effluent pH for Stages X and XII. For both stages, the influent perchlorate concentration was 20 mg/L and there was no nitrate in the medium. To confirm that the current resulted from perchlorate reduction and not from other potential acceptors, such as oxygen entering the system by diffusion or leaks, the cathode medium was maintained for 5 HRTs without nitrate or perchlorate. The current decreased from 0.3 mA (1.9 mA/ m2-cathode) to 0.01 mA (0.06-mA/m2 cathode) within 1 HRT. pH and Perchlorate Reduction. When the pH of the influent was briefly raised to 8.5 (day 297 to day 300), complete perchlorate removal was achieved (Figure 1). To explore the effect of pH on perchlorate removal, the percent of perchlorate removed was plotted as a function of effluent pH for Stages X and XII. In both stages, the influent perchlorate concentration was 20 mg/L, and perchlorate served as the sole electron acceptor. The effluent cathode pH correlated well with the perchlorate removal in the system (Figure 2). With an increase in pH from 6.2 to 8.1, the perchlorate reduction nearly tripled, increasing from 19% to 57%. Effect of Cathode Potential on Perchlorate Reduction. Polarization curves were obtained for the experimental reactor during Stage XI (10 mg/L ClO4-, pH 8.5). The changes in anode and cell potential versus current are reported in the Supporting Information, Figure S3. The polarization curve shows that an increase in cathode potential corresponds to lower current production and perchlorate reduction (Figure 3). Establishing a correlation between cathode potential and perchlorate reduction is further evidence the cathode serves as a donor for perchlorate-reducing microorganisms. Had the microbial community used organic matter (i.e., from dead biomass or the inoculum) as the primary electron donor, changing the cathode potential would have had little effect on perchlorate reduction. On the reverse sweep of the polarization curve, the current and perchlorate removal did not return to the level achieved in the forward sweep, particularly at the most negative cathode potentials. At 10-Ω external resistance, on the forward sweep the cathode potential was -0.375 mV versus Ag/AgCl and perchlorate removal was 10 mg/L. On the reverse sweep, at 10 Ω, the cathode potential was -0.350 mV versus Ag/AgCl and perchlorate removal was less than 1 mg/L. The microbial community may have been adversely affected by the cathodic condition. The observed decrease in perchlorate removal could be the result of sustained MFC operation (up to 9 days) at a cathode potential that the perchlorate-reducing bacteria are not able to use efficiently. One week after the polarization curve was obtained, the MFC perchlorate removals returned to the levels achieved prior to the polarization curve measurement. The polarization curve for the control denitrifying MFC (data not shown) revealed 4688
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FIGURE 3. Perchlorate removal (gray triangles) and cathode potential (gray circles) as a function of current. The external resistance of the cell was varied in the following order: 10, 50, 100, 500, 1000, 500, 100, 50, and 10 Ω. similar cathode potentials to the experimental MFC. However, unlike the experimental MFC, nitrate removal was restored on the reverse sweep. Analysis of Electrode-Associated Bacterial Communities. For each electrode and MFC, small clone libraries of 72-76 sequences were generated, and sequence data were used to generate diversity statistics (Supporting Information, Table S2), and a phylogenetic tree (Supporting Information, Figure S4). A UniFrac-based principal coordinates analysis (PCoA) revealed that the bacterial community structure of the anodes was highly similar, as both clone libraries were dominated by sequences from the genus Geobacter (Figure 4 and Supporting Information, Figure S4). Both anode clone libraries were additionally populated with sequences from the order Bacteroidales (phylum Bacteroidetes) and the phyla Acidobacteria and Chlorobi. Conversely, the denitrifying and perchlorate-reducing cathode bacterial communities were highly distinct from each other and from the anode communities (Figure 4), and shared few bacterial lineages (Supporting Information, Figure S4). At a broad phylogenetic scale, the biocathode communities were dominated by Betaproteobacteria, including established denitrifying lineages. The denitrifying biocathode clone library had the lowest diversity (Supporting Information, Table S2), and was predominantly composed of sequences affiliated with ironoxidizing bacteria (FeOB) from the Betaproteobacterial genera Ferritrophicum and Sideroxydans (21, 22). Sequences from these organisms were almost entirely absent from the perchlorate-fed biocathode clone library. The microbial community within the perchlorate-fed biocathode was significantly more diverse than the denitrifying cathode community (Supporting Information, Table S2), and was primarily composed of bacterial sequences most similar to a clone from a dioxin-dechlorinating microcosm (23), Denitratisoma oestradiolicum, a cholesterol-oxidizing denitrifier (24), and to sequences from bacteria of the genera Chryseobacterium and Kaistella (phylum Bacteroidetes) (Figure 4 and Supporting Information, Figure S4). Sequences from bacteria of the genus Dechloromonas were not abundant in the perchlorate-reducing biocathode clone library.
FIGURE 4. Ordination of MFC samples with UniFrac. Principal coordinates analysis (PCoA) of UniFrac values calculated from a phylogenetic tree of 16S rRNA gene sequences recovered from anode and cathode communities (Supporting Information, Figure S4). The figure shows a plot of the first 2 principal coordinate axes, representing 44.54 and 31.62% of the variation, respectively. Each point represents a single environment from this study; squares represent samples from the nitrate control MFC and circles represent samples from the perchlorate MFC. Filled symbols represent anodes and empty symbols represent cathodes. The composition of the bacterial community in each sample, as derived from the phylogenetic tree, is indicated below the PCoA.
Discussion A MFC capable of perchlorate-reduction was developed from a mixed denitrifying consortium from environmental and activated sludge sources. A gradual improvement of perchlorate reduction was observed, consistent with previous findings with denitrifying and perchlorate-reducing bioreactors, suggesting an enrichment for a perchlorate-reducing microbial community (7). Our system demonstrated that 24 mg/L-d could be removed in a full MFC configuration with perchlorate as the sole electron acceptor in the cathode compartment and acetate oxidation in the anode compartment. Perchlorate-reduction in the biocathode of MFC was dependent on both cathode potential and pH. The maximum perchlorate reduction was observed at a cathode potential of -375 mV versus Ag/AgCl and pH of 8.5. A previous study reported higher perchlorate removal rates, 60 mg/L-d, at an imposed electrode potential of -500 mV versus Ag/AgCl (6). However, the half cell system described by Thrash et al. (7) had an influent perchlorate concentration of 90 mg/L and exogenous electron shuttles. Additionally, the highly negative operating potential may have caused formation of hydrogen at the electrode surface, supplying the PCRB with a soluble electron donor for perchlorate reduction. At the potentials reported in this study, the theoretical hydrogen evolution is negligible (calculated via Nernst equation using experimental conditions), and even lower at a pH of 8.5, when the maximum perchlorate reduction was observed. Thus, this study demonstrated significant perchlorate reduction in a bioelectrochemical system without a fixed cathode potential, exogenous electron shuttles, or other soluble electron donor. Bioelectrochemical perchlorate reduction was complete when the pH was above 7.5. Nerenberg et al. (7) showed that maximum perchlorate reduction rates in a denitrifying membrane-biofilm reactor (MBfR) occurred at a pH of 8, but dropped sharply at pH values below 7, and fell to zero at a pH of 6. In our experimental reactor, the amount of nitrate reduced correlated with perchlorate reduction rates. For example, when the influent nitrate was greater than 5 mgN/L, perchlorate was completely removed (Figure 1, Stages III-IV). However, when influent nitrate was 1 mg-N/L or less, perchlorate removal was incomplete (Figure 1, Stage VIII). Increases in pH that would create favorable conditions for PCRB have been observed in other denitrifying biocathodes (25). The accumulation of OH- ions in the cathode is the result of 6 mols of protons consumed per mole of nitrate completely reduced to N2 and inefficient transport of protons from the anode to the cathode. Proton exchange membranes,
like the Ultrex membrane used in these experiments, preferentially transport other cations over protons between anode and cathode, creating a pH decrease in the anode and increase in the cathode (26). Analysis of the bacterial communities developing on each of the electrodes revealed that while the biocathode microbial communities were sensitive to the available electron acceptor, the anodes from both MFCs were highly similar despite slightly different anode potentials of -340 mV for the denitrifying MFC and -376 mV (vs Ag/AgCl). The abundance of bacteria from the genus Geobacter is consistent with other anode communities described in the literature, and these organisms are capable of oxidizing acetate and using an electrode as an electron acceptor (27). For the other abundant constituents of the anode communities less is known, though environmental sequences from dechlorinating and denitrifying enrichments or reactors were frequently the most similar sequences to those recovered (data not shown). A clear effect of the terminal electron acceptor on the electrode microbial community was observed in the cathodes. Although sequences from the class Betaproteobacteria and the genera Chryseobacterium and Kaistella comprised 72-91% of all the sequences recovered from the cathodes (and less than 2% of the anodes), distinct microbial communities with limited overlap developed, indicating a strong selection within each cathode. The denitrifying cathode had the lowest bacterial diversity, with most sequences similar to those recovered from Betaproteobacterial FeOB. Although inferring physiological capabilities from rRNA gene sequence similarity can be difficult, we note that that the detected rRNA gene sequences are most similar to sequences from bacteria of the genera Ferritrophicum and Sideroxydans, and are likely derived from lithoautotrophic bacteria with very limited metabolic capabilities (21). Nitrate-reduction for some of these organisms has been shown, though nitrate may sometimes be reduced only to nitrite (28-30). If the dominant nitrate biocathode organisms are neutrophilic Fe(II) oxidizers capable of utilizing the cathode as an electron donor, this could represent a significant limitation for operating a mixed perchlorate- and nitrate-reducing MFC, as many neutrophilic FeOB do not grow above pH 7 (21), while maximum perchlorate removal is observed above this pH (24). The dominance of these putative FeOB is striking, however, as they have not been reported in biocathodes without transition metal mediators. Previously, FeOB such as Leptothrix discophora (31) and Thiobacillus ferroxidans (32) have been VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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shown to mediate electron transfer from a cathode using iron and manganese compounds as shuttles. Putative denitrifying Betaproteobacteria were abundant on the perchlorate biocathode, coupled with an abundance of Bacteroidetes from the genera Kaistella and Chryseobacterium. Although there is precedence for denitrification activity with the genus Chryseobacterium (33), these organisms are generally considered aerobic heterotrophs (34). The high relative abundance of these organisms in the perchlorate biocathode is possibly a response to the dead microbial biomass associated with the transition from denitrifying to perchlorate-reducing conditions. However, the abundance of organisms affiliated with the genera Chryseobacterium and Kaistella on the perchlorate biocathode nearly 4 months after the removal of nitrate from the reactor could also indicate that these organisms are directly interacting with the electrode. Further work is required to determine the function of these organisms and that of the putative FeOB in the denitrifying and perchlorate-reducing biocathodes. We note that the seeding of the biocathodes with a complex mixture of microorganisms from activated sludge, an active denitrifying cathode, and a chlorate-reducing enrichment was effective in establishing a perchloratereducing biocathode community. However, the addition of Dechloromonas sp. PC1, a known perchlorate-reducing bacterium, was not essential as these species were not detected as significant members of either biocathode community. In this work, a perchlorate-reducing microbial community was developed in a MFC biocathode supplied with nitrate and perchlorate. Low-level perchlorate was reduced concurrently with nitrate, and high-level perchlorate was reduced as the sole electron acceptor, making this technology potentially suitable for treating water supplies, superfund sites, or industrial wastewaters. In contaminated water supplies, typically containing 10-100 µg/L perchlorate, little power can be obtained from perchlorate alone. However, nitrate is a typical co-contaminant that could help sustain the perchlorate-reducing community and increase power production.
Acknowledgments We would like to acknowledge support for Caitlyn Butler from the EC-US Task Force via the Transatlantic Environmental Biotechnology Fellowship, and the Center for Environmental Science and Technology at the University of Notre Dame via the Bayer Fellowship. Peter Clauwaert was funded by a Ph.D. grant (IWT Grant 53305) of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and by FWO Vlaanderen (K.2.050.07.N.01).
Supporting Information Available The contents of the Supporting Information includes (1) a table containing the constituents of the media used for each electrode compartment (Table S1), (2) a schematic of the experimental setup (Figure S1), (3) detailed methods for the raw sequence processing for the clone libraries created for each electrode compartment, (4) a figure containing pH data for the influent and effluent of the experimental perchloratereducing biocathode for the duration of operation in these experiments (Figure S2), (5) a figure containing anode and cell potentials obtained during the polarization curves (Figure S3), (6) a table containing the diversity and richness indices for clone libraries of each electrode compartment (Table S2), and (7) a phylogenetic tree of bacterial 16S rRNA gene sequences recovered in this study. This material is available free of charge via the Internet at http://pubs.acs.org. 4690
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