Elucidating the Role of Electron Shuttles in Reductive Transformations

In microbial systems, iron-reducing bacteria are thought to mediate the reduction ...... Lovley , D. R. Quinone moieties act as electron acceptors in ...
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Environ. Sci. Technol. 2009, 43, 1042–1048

Elucidating the Role of Electron Shuttles in Reductive Transformations in Anaerobic Sediments H U I C H U N Z H A N G †,‡ A N D E R I C J . W E B E R * National Exposure Research Laboratory, U.S. Environmental Protection Agency, 960 College Station Road, Athens, Georgia 30605

Received February 13, 2008. Revised manuscript received December 3, 2008. Accepted December 7, 2008.

Model studies have demonstrated that electron shuttles (ES) such as dissolved organic matter (DOM) can participate in the reduction of organic contaminants; however, much uncertainty exists concerning the significance of this solution phase pathway for contaminant reduction in natural systems. To compare the identity and reactivity of ES in anaerobic sediments with those in model systems, two chemical probes (4-cyano-4‘aminoazobenzene (CNAAzB) either free or covalently bound to glass beads) were synthesized that allowed for differentiation between surface-associated and solution-phase electron-transfer processes. The feasibility of these chemical probes were demonstrated in abiotic model systems (Fe(II)/Fe(III) oxide) and biotic model systems (Fe(II)/Fe(III) oxide or river sediment amended with S. putrefaciens strain cells). Experiments in the abiotic systems revealed that the addition of model hydroquinones and chemically reduced DOM increased reduction rates of free CNAAzB, whereas no enhancement in reactivity was observed with the addition of model quinones or DOM. Bound CNAAzB was also reduced by model hydroquinones and reduced DOMsbut not by model quinones and untreated DOMsin the abiotic model systems, indicating that Fe(II)/Fe(III) oxides do not function as a bulk reductant for the reduction of ES. Addition of model quinones or untreated DOM to the biotic models systems with sediment increased reduction rates of bound CNAAzB, which correlated well with the dissolved organic carbon content. In natural sediment slurries, reduction rates of bound CNAAzB correlated well with parameters for organic carbon (OC) content of both sediments and supernatants. Our results support a scenario in which reducible organic contaminants will compete with iron oxides for the electron flow generated by the microbially mediated oxidation of organic carbon and subsequent reduction of quinone functional groups associated with DOM.

Introduction Studies in anaerobic sediments and aquifers have demonstrated the significant role that surface-associated ferrous iron plays as an electron source for the reductive transfor* Corresponding author e-mail: [email protected]. † National Research Counsel Research Associate. ‡ Current Address: Department of Chemistry and Environmental Sciences; Southern Illinois University Edwardsville; Edwardsville, IL 62026-1652; Email: [email protected]. 1042

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mation of organic contaminants (1-3). Uncertainty remains, however, concerning the potential contribution of aqueous phase chemical reductants to the rate of reduction in these systems. A plausible pathway for reduction in the aqueous phase is electron transfer mediated by electron shuttles (ES), which are defined as chemical substances that function to shuttle electrons from a bulk reductant to the oxidant of interest (e.g., iron oxides or chemical contaminants). Although numerous studies in model systems have demonstrated the plausibility of ES to function as electron carriers in the reduction of iron oxides and chemical contaminants (4-6), direct evidence for this pathway for electron transfer in natural systems has not been provided. Dissolved organic matter (DOM) is thought to be the most viable ES in natural systems because (1) DOM is ubiquitous in sediment and aquatic ecosystems (2); DOM contains redox active functional groups and metal chelating moieties; and (3), has been demonstrated to undergo microbially mediated reduction (i.e., is an electron acceptor) under a variety of redox conditions including fermentation, halo respiration, humicreducing, iron-reducing, methanogenesis, and sulfatereducing (7-11). Although DOM has been demonstrated to be reduced by chemical methods in the laboratory including eleoctrochemical reduction (12), metal-catalyzed hydrogenation (13), and reaction with sulfide (10), the contribution of surface-associated abiotic pathways for the reduction of DOM in natural systems remains an open question. Comparative studies with model compounds have provided critical information concerning the characterization and redox cycling of the predominant redox active functional groups in DOM (10, 12, 14-20). These studies have attributed the redox activity of DOM primarily to quinone functional groups that can be reduced to hydroquinones and the formation of soluble Fe(II) complexes (21, 22). More recently, chemical reduction studies of DOM have provided further evidence for the occurrence and chemical structure of the quinone functional groups in DOM (13). A similar reducing capacity for chemically reduced DOM was reported for microbially reduced analogs. A limited number of studies of biotic model systems containing pure cultures using organic carbon (e.g., acetate, glucose, and NADH) as the bulk electron source have provided evidence of reduced quinones and humic acids functioning as ES in contaminant degradation (4, 23, 24). In microbial systems, iron-reducing bacteria are thought to mediate the reduction of iron minerals through the coupling of the oxidation of organic carbon to the reduction of DOM (5-9); soluble chemical reductants (e.g., sulfide, humic-metal complexes) are typically used as the bulk electron donor (i.e., chemical reductant) in abiotic model systems (25, 26). To understand the role of ES in natural sediments, it is imperative to identify the predomiant bulk reductants as well (i.e., what are the sources of electrons for reduction of the ES). For this reason, the goals of this study were (1) to understand the relative contributions of surfaceassociated abiotic vs biotic pathways for the reduction of DOM in natural systems and (2) to compare the identity and reactivity of ES in natural sediments to those of model systems because significant difference may exist for both systems (27). To this end, a chemical probe [4-cyano-4′-aminoazobenzene (CNAAzB) covalently bound to a glass bead] was synthesized that allowed for differentiation between surfaceassociated and solution-phase electron-transfer processes. A similar chemical probe was used to study electron transfer proceses in zerovalent iron systems (28). Free CNAAzB was also studied for comparison. Upon reduction, 10.1021/es8017072 CCC: $40.75

 2009 American Chemical Society

Published on Web 01/22/2009

both chemical probes yield p-cyanoaniline (CNNA) as the reduction product. Reduction of the probes was carried out in abiotic model systems (aqueous suspensions of Fe(II)/ iron oxide dosed with ES), biotic model systems (addition of dissimilatory iron-reducing bacteria (DIRB) Shewanella putrefaciens strain CN32 to the abiotic systems), and in anaerobic sediments. Model ES (i.e., substituted quinones and hydroquinones and metal complexes) and DOM were studied. For sediment systems, the reactivity of sediments of different origins and their corresponding supernatants were studied and correlated with their physiochemical properties.

Materials and Methods Materials. A complete listing of chemicals used in this study and the procedure for preparing the reduced DOM (redDOM) is provided in Text S1 in the Supporting Information (SI). Bacteria and Media. S. putrefaciens strain CN32 (S. CN32, the American type Culture Collection) was provided courtesy of Dr. Edward O‘Loughlin (Argonne National Laboratory). Cells were cultured in tryptic soy broth (TSB) at ambient temperature (24-25 °C), harvested at late log phase by centrifugation (10 000 × g at 20 °C for 20 min), and washed twice with a sterilized defined mineral medium (DMM, Text S4). Bound CNAAzB reduction experiments were conducted in DMM (29). Analytical Procedures. Analytical procedures are provided in Text S1 in SI. Kinetic Experiments. Batch experiments were performed in 65-mL serum bottles sealed with butyl rubber stoppers in a COY anaerobic chamber (2 to 5% H2). For experiments in abiotic model systems, suspensions of 25 mM pH 7 MOPS buffer, 0.1 M NaCl, and 5 g/L goethite were prepared. After stirring for 16 to 18 h, Fe(II) was added to each serum bottle followed by dosage of the electron shuttle after 2-4 h. For experiments in biotic model systems, the experimental setup is similar to that described previously (30-32). Briefly, serum bottles preloaded with goethite or sediment were charged with 50-mL of DMM, formate, and filter sterilized ES. The mixture was stirred overnight, and a known amount of inoculum was spiked into each reactor to achieve a cell density of 2.7 × 108 to 7.7 × 109 cells/mL. For experiments with anaerobic sediments, the sediment incubation is similar to that described previously (2) and is reported in detail in Text S3 of the SI. Briefly, a typical batch experiment consisted of a 65-mL serum bottle charged with 10-mM sodium acetate, 25-mM pH 7.0 MOPS buffer and 0.05 to 2.5 g of sediment. The bottles were sealed, moved outside of the chamber, placed upside down on a tabletop shaker at room temperature for 5 w and then at the end of the 5-w incubation, ironreducing/methanogenic conditions have developed and the

bottles were moved back into the chamber. The experimental systems were allowed to equilibrate for 2 d prior to addition of probe compounds, which marked time zero for the kinetic studies, at concentrations to achieve maximum CNNA formation of 0.65-1.60 µM for bound CNAAzB (i.e., the initial loading of CNAAzB to the glass bead ) [bound-CNAAzB]0) or 25-30 µM for free CNAAzB. When conducting experiments in sediment supernatants, sediments were removed by centrifugation (8000 × g at 20 °C for 20 min). All reactors were prepared in duplicate and placed on a rotator inside the chamber (49 rpm/min). Samples (0.5 mL) were periodically taken and filtered by Acrodisc 0.2 µm syringe filters with PTFE membrane, and the filtrates were collected for CNNA analysis. Kinetic Data Analysis. A kinetic model for the degradation of bound CNAAzB in reducing environments (similar to the models for other surface-mediated systems) is presented in detail in Text S5 to allow comparison of the reactivity of ES in various systems (33). Briefly, k1 k2

bound-CNAAzB + ESS If P

(1)

k-1

where I ) reaction intermediate; P ) reaction product (i.e., CNNA). Assuming that steady-state conditions apply to [I] and [ES] is a constant because ES are regenerated by bulk reductants, we can solve for [P]: [P] ) [P]max(1 - ebt)

(2)

[P]max ) [bound-CNAAzB]0

(5)

where

b)

k1k2 [ES] k-1 + k2

(3)

Parameter b is associated with the initial generation rate of P. Knowing [P]max, the value of b can be obtained by nonlinear fitting eq 2 to the observed reaction kinetics. Because k1, k-1, and k2 are all constants for a given system, we expect to see a linear correlation between parameter b and [ES] (eq 3).

Results and Discussion Free CNAAzB Reduction in Abiotic Model Systems. For a compound to serve as an electron shuttle, a source of electrons (i.e., bulk reductant) must be present and capable of reducing the ES to initiate electron transfer. Given that surface-bound Fe(II) (i.e., Fe(II)/iron oxides) are one of the predominant reductants for contaminant reduction in natural sediments (1), it was of interest to determine if Fe(II)/iron oxides function as a bulk reductant for the reduction of ES, thus, providing an abiotic pathway for the direct reduction of electron shuttles. To test this hypothesis, degradation of free CNAAzB was carried out in abiotic model systems containing Fe(II)/goethite as the reducing surface, and model quinones or DOM as ES. Due to the strong adsorption of CNAAzB to goethite surfaces (>80% CNAAzB adsorbed), reduction of CNAAzB was measured by the formation of CNNA. As shown in Figure 1a, CNAAzB was stable in control experiments with pH buffer and NaCl. Reduced juglone (JUGH2Q) reduced CNAAzB at a fairly fast rate as demonstrated by the formation of CNNA; however, reduced Suwannee river natural organic matter (red-SRNOM) reduced CNAAzB at a much slower rate. Similarly, we found that hydroquinones with standard reduction potentials (E0) lower than that of naphthahydroquinone (NH2Q)sincluding anthrahydroquinone-2,6-disulfonic acid (AH2QDS), reduced lawsone (LAWH2Q), and NH2Qsreduced CNAAzB at fairly VOL. 43, NO. 4, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Free CNAAzB reduction in abiotic model systems: typical CNNA formation time course with (a) red-ES and/or soluble Fe(II) (the slow reactions with SRNOM are shown in the inset), and (b) Fe(II)/goethite (Fe(II)/G) with red-ES (the open symbols and dotted lines correspond to the additive results for two models systems containing either Fe(II)/G or red-ES). Error bars are the standard deviation of duplicate results. Reaction conditions unless otherwise stated: 5 g/L goethite, 400 µM Fe(II), 200 µM hydroquinones or 5 mg/L red-DOM, 25-30 µM CNAAzB, pH 7 buffer, 0.1 M NaCl. Points are experimental data and lines are model fits. DOM studied includes HA, FA, SRNOM, and NRNOM; each showed similar reactivity to SRNOM (results not shown). estimated the range for DOM E0 values to be between 0.5 fast rate; red-ES with E0 higher than that of NH2Q - including 2,6-Dimethoxyhydroquinone (DMOH2Q), 2,6-Dimethylhyand 0.7 V, which is in agreement with the previously estimated droquinone, hydroquinone (H2Q) and catechol - and DOM ranges in E0 values from < -0.48 V to > + 0.83 V (14). (including humic acid (HA), fulvic acid (FA), SRNOM, and Bound CNAAzB Reduction in Abiotic Model Systems. Nordic Reservoir natural organic matter (NRNOM), either The feasibility of the glass bead-azo compound complex (i.e., untreated or chemically reduced) did not reduce CNAAzB to bound CNAAzB) to differentiate between surface-associated any appreciable extent (see Table S2 of the SI for E0 values). and solution-phase electron-transfer processes was initially Also shown in Figure 1a, soluble Fe(II) only reduced tested in abiotic model systems. Control experiments conCNAAzB at a slow rate; however, addition of soluble Fe(II) taining bound CNAAzB with pH buffer demonstrated that to systems containing JUGH2Q or red-SRNOM increased the bound CNAAzB was stable through the study period (up to reduction rate. This increase in reactivity indicates that red3 weeks). Reduction of bound CNAAzB by either aqueous ES are capable of forming soluble complexes with Fe(II) that Fe(II) or Fe(II)/Fe oxide was very slow. Negligible bound have greater reactivity than red-ES themselves. CNAAzB reduction was also observed over a 2-w period with Significant reduction of CNAAzB occurred in the presence the addition of 150-200 µM AQDS, LAW, benzoquinone, of Fe(II)/goethite (Figure 1b). Furthermore, the addition of H2Q, catechol, ubiquinone-5, hematin (3.3 mg/L), chlorophyll (26.7 µM), HA or FA (5 mg/L, either untreated or chemically red-ES to Fe(II)/goethite systems increased the formation of reduced) to systems with or without Fe(II)/goethite. In CNNA, with a rate greater than the simple addition of the contrast, reduction of bound CNAAzB by 200 µM AH2QDS, rates for the two model systems containing either red-ES or JUGH2Q, LAWH2Q, or 62.5 mg/L red-DOM was significant Fe(II)/goethite. This increase in reactivity indicates that there as shown by the formation of CNNA (Figure 2). These results is a synergistic effect between Fe(II)/goethite and red-ES demonstrate that hydroquinones with low reduction poresulting in greater reducing capacity. Further experiments tentials (i.e., E0 < 0.428 V) and high concentrations of redwith LAWH2Q, lawsone (LAW), AH2QDS, or anthrabenzoquinone-2,6-disulfonic acid (AQDS), and Fe(II)/goethite DOM can reduce bound CNAAzB. The fact that only high suspensions demonstrated that either LAWH2Q or AH2QDS concentrations of red-DOM effect reduction of bound can reduce goethite, and that the reverse reaction (i.e., CNAAzB at substantial rates indicates that the concentrations reduction of LAW or AQDS by Fe(II)/goethite) does not occur of quinone functional groups in the DOM are quite low and/ (see Text S6 and Figure S2 of the SI for details). These results or not all of the quinone functional groups present are suggest that the observed synergistic effect is due to the susceptible to reduction. The addition of Fe(II)/FeOOH to reduction of the goethite surface by red-ES resulting in an the systems containing AH2QDS, JUGH2Q, or LAWH2Q decreased the rate and extent of bound CNAAzB reduction increase in the formation of surface complexed Fe(II), which (Figures 2 and S4 of the SI), most likely due to oxidation of leads to an increased rate of reduction of CNAAzB. The the hydroquinones by goethite resulting in a decrease in estimated lower reduction potentials for the quinones than hydroquinone concentration (Text S6 of the SI) (34-36). The that of goethite indicate that reduction of the goethite surface inhibitory effect of Fe(II)/goethite was not as significant for by the hydroquinones is indeed thermodynamically favorable JUGH2Q, likely due to the higher E0 of JUGH2Q (Table S2 of (calculations shown in Text S7 of the SI). The inability of the SI), and thus, less oxidation by goethite. The similar Fe(II)/goethite to reduce oxidized ES indicates that Fe(II)/ reactivity observed for JUGH2Q and red-DOM also provides goethite does not function as a bulk reductant (i.e., does not evidence that quinone functional groups in DOM play an function as an electron source for the reduction of the important role in the redox activity of DOM. Overall, these quinones or DOM) in the abiotic model system. results demonstrate that CNAAzB covalently bound to glass The reaction kinetics of CNAAzB in the abiotic model beads is a suitable chemical probe for distinguishing between systems containing Fe(II)/goethite and red-ES as shown in solution phase and surface-mediated electron-transfer Figure 1b can all be fitted well by a similar kinetic model as processes. eq 2 (Text S5 of the SI). As the fitting parameters show in Bound CNAAzB Reduction in Biotic Model Systems. On Figure S3 and Table S2, CNNA formation rate decreases with the basis of the results of numerous studies demonstrating an increase in E0 of the ES. On the basis of values for parameter b (Table S2 of the SI), JUG seems to be a better model the ability of iron-reducing bacteria to reduce model quinones compound than AQDS for simulating electron shuttling by and DOM (5-7, 23, 35, 36), it is most likely that anaerobic DOM (12). Measured E0 values for DOM are not available. bacteria function as a source of electrons through the On the basis of dependence of parameter b on E0, we oxidation of organic carbon (i.e., the bulk electron donor) 1044

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FIGURE 2. Typical reduction kinetics of bound CNAAzB in abiotic model systems containing (a) LAWH2Q or (b) DOM (untreated or chemically reduced) (slow reactions are shown in inset). Equal volumes of 62.5 mg/L reduced HA, FA, SRNOM, and NRNOM were mixed to form the red-DOM mixture. Reaction conditions: 5 g/L goethite or FeOOH, 400 µM Fe(II), 200 µM model quinone or 5-62.5 mg/L DOM, 25 mg bound CNAAzB (max CNNA formation ) 1.6 µM), pH 7 buffer, 0.1 M NaCl. Points are experimental data and lines are model fits. Results with AH2QDS are similar to those of LAWH2Q, and thus not shown.

FIGURE 3. Typical reduction kinetics of bound CNAAzB in: (a) biotic model systems with goethite as the solid phase; (b) biotic model systems with OR as the solid phase; (c) anaerobic OR sediments amended with DOM; and (d) the corresponding supernatants. In parts a-b, DOM studied included HA, FA, SRNOM, and NRNOM. Results are shown for SRNOM and similar kinetics were observed for the other DOM. Reaction conditions: (a) 2.7 × 108 cells/mL, 5 g/L goethite, 200 µM JUG or 5 mg/L DOM, 50 mg bound CNAAzB; (b) 7.7 × 109 cells/mL, 10 g/L OR sediment, 250 mg/L DOM or 2 g/L soil, 25 mg bound CNAAzB; c-d) 10 g/L OR sediment + 250 mg/L DOM or 2 g/L soil incubated for 5 w before the addition of bound CNAAzB 25 mg. Points are experimental data and lines are model fits. for the reduction of ES in natural systems. Accordingly, biotic model systems were developed by the addition of a pure culture of S. CN32 to the abiotic model systems. Cells at a low density of 2.7 × 108 cells/mL did not mediate bound CNAAzB reduction (Figure 3a). At this low density of cells, however, significant reduction of bound CNAAzB with the addition of JUG was observed either with or without goethite; on the other hand, 200 µM DMBQ, 5 mg/L HA, or 5 mg/L SRNOM (concentration based on mass) did not mediate reduction of bound CNAAzB either with or without goethite (Figure 3a and Table S3 of the SI). The fact that goethite had no effect on reactivity agrees well with the observations from

the abiotic model systems and is consistent with a pathway for electron transfer in which the cells directly reduced JUG, which in turn reduced bound CNAAzB. On the basis of the measured high RC values (Table S3 of the SI), DMBQ and DOM were also reduced by the cells (4, 5, 23), but did not mediate reduction of bound CNAAzB due to either the high reduction potential for DMBQ (Table S2 of the SI), or low concentration of DOM. To better mimic natural sediments, DOM at a much higher concentration of 250 mg/L, or soil at 2 g/L was added to the biotic model system. Also goethite was replaced by Oconee River sediment (OR), which has a low OC content and does VOL. 43, NO. 4, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Typical reduction kinetics of bound CNAAzB in (a) anaerobic sediment suspensions and (b) anaerobic sediment supernatants. Reaction conditions: 10 g/L anaerobic sediment, 25 mM pH 7 buffer, 25 mg bound CNAAzB. Points are experimental data and lines are model fits. not exhibit significant reactivity for bound CNAAzB reduction. As the kinetic results show in Figure 3b and Table S3, little reduction of bound CNAAzB was observed in cell-free controls containing DOM except for Gascoyne leonardite soil and Pahokee peat soil II (results for Pahokee peat soil II are similar to those of Gascoyne and thus not shown). In contrast, significant CNNA formation occurred in the presence of a high density of cells at 7.7 × 109 cells/mL. The addition of DOM to the high density of cells increased the reduction rate for bound CNAAzB to a rate comparable to that of the anaerobic sediment systems (see subsequent discussion), and the rate increase observed for the Gascoyne and Pahokee Soils was not as significant as for the other sources of DOM. This is likely due to the already reducing nature of the two soils that contain reduced functional groups prior to incubation (6, 37). The degradation kinetics can be fitted well by eq 2 (see Table S3 of the SI for fitting parameters). The reduction of bound CNAAzB by the high density of cells (versus no reduction in cell free controls) indicates that S. CN32 cells are capable of reducing the chemical probe (6). On the basis of the previous conclusion that iron reduction by S. oneidensis MR-1 occurred through both direct and indirect pathways for electron transfer, (38), we speculate that bound CNAAzB was reduced through the release of cell exudates (i.e., indirect pathway) or through direct contact. Model quinones and DOM enhanced the contribution of microbial reduction through the indirect pathway by functioning as ES. The pure cultures employed in the biotic model systems are far less complex than the bacterial communities found in sediment environments. To better simulate the latter, we studied model sediment systems in which DOM from various sources was incubated with anaerobic OR sediment to stimulate the growth of native bacteria. A significant increase in the reduction rate of bound CNAAzB was observed for both the sediment suspensions and the corresponding supernatants amended with DOM (Figure 3c-d). The reduction kinetics can be fitted well by eq 2 and the fitting parameters are shown in Tables S3 and Figures S5-S6 of the SI. A good correlation can be established between parameter b for model sediments (bmodel-sed) and DOC content (eq 4, Figure S5 of the SI), which agrees with eq 3 and thus provides quantitative evidence that DOM functions as ES in model anaerobic sediments to facilitate reductive degradation of bound CNAAzB. In addition, parameter b for supernatants (bmodel-sup) increases with an increase in DOC for model sediment supernatants (Figure S6 of the SI), indicating that DOM functions as the predominant reductant in the supernatants. We attribute the relatively poor model fitting of the reaction kinetics for CNNA formation in supernatants (Figure 3d) to the fact that the assumption made for the 1046

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heterogeneous systems (i.e., ES are regenerated by a bulk reductant) does not hold for the solution phase systems. bmodel-sed ) (9 ( 2) × 10-5(DOC) - (5 ( 5) × 10-5 R 2 ) 0.80(n ) 6) (4) In addition to monitoring CNNA generation, soluble Fe(II) concentration and reducing capacity (RC), as measured by Fe(III) reduction in the supernatants, were measured for each biotic model system (Table S3 of the SI). Soluble Fe(II) concentration was stable over the entire reaction course for each of the model systems. Typically, a decrease in soluble Fe(II) concentration is concurrent with the reduction of organic compounds in Fe(II)/iron oxide systems (3, 39). Thus, no change in soluble Fe(II) concentration indicates that Fe(II)/iron minerals did not participate in bound CNAAzB reduction (i.e., do not function as a bulk reductant in reducing the ES). Typical RC values for original HAs and HAs in biotic model systems are in the range of 0-266 and 43-337 µM; the increase of 43-208 µM in RC values strongly indicate that HAs have been microbially reduced, but the extent of reduction varies among the HAs. No direct correlation between RC and the amount of bound CNAAzB reduced can be established (R2 < 0.46, Table S3 of the SI); thus RC as measured by Fe(III) reduction is not a good indicator for the electron shuttling capacity of DOM. Bound CNAAzB Reduction in Anaerobic Sediments. Significant CNNA formation was observed in the presence of all of the anaerobic sediments examined (examples of CNNA formation kinetics are shown in Figure 4a). CNNA formation rates (i.e., parameter bsed) were obtained by fitting the experimental data to eq 2 (Table S1of the SI). Linear regression analysis indicates that with the exception of Specific UV Absorbance at 254 nm (SUVA254nm) (Figures 5 and S7 of the SI), sediment OC-related properties (particularly SUVA350 nm) correlate reasonably well with parameter bsed, providing further evidence that DOM is the predominant ES in the anaerobic sediments. SUVA254 nm and SUVA350 nm can be used to indicate the aromatic carbon content and the fraction of quinone functional groups in DOM, respectively (15). The poor correlation with SUVA254 nm (R2 ) 0.25) and relatively good correlation with SUVA350 nm (R2 ) 0.61) is another indication that the electron shuttling ability of DOM is related primarily to quinone content. Anaerobic sediment supernatants also exhibited a range in reactivity for bound CNAAzB (Figure 4b). The formation kinetics for CNNA (bsup) could be fitted reasonably well by eq 2, likely due to their high DOC content that made the effect of ES regeneration negligible. As demonstrated by the fitting parameters in Figure S7c and Table S1 of the SI, a higher DOC corresponds to a higher value for parameter

FIGURE 5. Relationships between parameter b and sediment OC-related properties. (a) bsed vs DOC and (b) bsed vs SUVA350 nm. bsup. These results provide further evidence that DOM functions as the predominant reductant in the supernatants. Comparison of the reactivity of the sediments and the corresponding supernatants reveals that generally the sediments are more reactive. The lower reactivity of the supernatants versus that of the sediments can be reasonably explained by repeated reduction of the oxidized DOM through microbial processes in sediment suspensions. The unusually high reactivity of the BU, PL, and RA supernatants is likely in part related to their high DOC content, which minimizes the effect of regeneration of ES through microbial activity; however, it is not clear why the presence of these sediment suspensions actually impedes the reduction of bound CNAAzB. Environmental Significance. This study provides direct evidence for a solution phase pathway for electron transfer for contaminant reduction through a microbially mediated electron shuttling process. The study of model systems of increasing complexity and reactivity resembling that of natural sediments provided clear evidence for the role of DOM as the dominant ES in natural sediments. The similar reactivity observed for DOM and model quinones suggests that DOM functions as ES most likely through its quinone functional groups. However, it is possible the nonquinone structures in DOM also play a role (13). Our experimental results support a scenario in which reducible organic contaminants will compete with iron oxides for the electron flow generated by the microbially mediated oxidation of organic carbon and subsequent reduction of quinone functional groups associated with DOM.

The redox cycle continues with the reduction of the oxidized DOM by anaerobic bacteria. Chemical reduction of DOM by surface-associated Fe(II) does not appear to be a significant process. Further work is necessary to establish the relative contribution of this solution phase pathway for contaminant reduction versus surface-mediated processes such as reduction by surface-associated Fe(II). An important implication of this work is the role that ES may have in the reduction of organic contaminants associated

with soil and sediment surfaces (i.e., contaminants chemically or physically bound to surfaces), for which bound CNAAzB serves as a good model compound. For such contaminants, reduction by reducing surfaces such as Fe(II)/iron oxides are controlled by the availability of the soluble contaminants with desorption the rate-limiting step (40). The reduction of surface-associated contaminants by soluble ES, however, may provide a viable pathway for the natural attenuation of such contaminants.

Supporting Information Available Supporting Information including Text S1-S7, three tables, and seven figures is available.This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments The authors are thankful to Jack Jones for providing technical support on culturing the bacterial samples; John W. Washington, Y. P. Chin and Dharni Vasudevan for providing sediment and soil samples; B. T. Thomas for providing goethite, and Edward O’Loughlin for providing cell cultures.

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