Effects of Electron Transfer Mediators on the Bioreduction of

Aug 20, 2008 - This study examines the effects of a series of compounds representing ... Environmental Science & Technology 2017 51 (21), 12573-12582...
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Environ. Sci. Technol. 2008, 42, 6876–6882

Effects of Electron Transfer Mediators on the Bioreduction of Lepidocrocite (γ-FeOOH) by Shewanella putrefaciens CN32 EDWARD J. O’LOUGHLIN* Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439-4843

Received March 07, 2008. Revised manuscript received June 09, 2008. Accepted July 01, 2008.

Electron transfer mediators (ETMs) such as low-molecularmass quinones (e.g., juglone and lawsone) and humic substances are believed to play a role in many redox reactions involved in contaminant transformations and the biogeochemical cycling of many redox-active elements (e.g., Fe and Mn) in aquatic and terrestrial environments. This study examines the effects of a series of compounds representing major classes of natural and synthetic organic ETMs, including low-molecular-mass quinones, humic substances, phenazines, phenoxazines, phenothiazines, and indigo derivatives, on the bioreduction of lepidocrocite (γ-FeOOH) by the dissimilatory Fe(III)-reducing bacterium Shewanella putrefaciens CN32. Although S. putrefaciens CN32 was able to reduce lepidocrocite in the absence of exogenous ETMs, the addition of exogenous ETMs enhanced the bioreduction of lepidocrocite. In general, the rate of Fe(II) production correlated well with the reduction potentials of the ETMs. The addition of humic acids or unfractionated natural organic matter at concentrations of 10 mg organic C L-1 resulted in, at best, a minimal enhancement of lepidocrocite bioreduction. This observation suggests that electron shuttling by humic substances is not likely to play a major role in Fe(III) bioreduction in oligotrophic environments such as subsurface sediments with low organic C contents.

Introduction The biogeochemistry of Fe in many aquatic and terrestrial environments is driven largely by microbial activity, particularly in Fe-rich soils and sediments where Fe redox cycling by microorganisms is a significant component of C cycling and energy flux (1, 2). Indeed, the presence of Fe(II) in suboxic and anoxic environments is commonly attributed to the action of dissimilatory iron(III)-reducing bacteria (DIRB) and archaea, phylogenetically diverse microorganisms that are able to obtain energy by coupling the oxidation of organic compounds or molecular hydrogen to the reduction of Fe(III) to Fe(II). In addition to their role in C and Fe redox cycling, the activity of Fe(III)-reducing microorganisms and the Fe(II) they produce can play a major role in many processes in suboxic aquatic and terrestrial systems, including the dissolution and precipitation of minerals (3, 4), the availability of nutrients such as phosphate (5), and the fate and transport of organic and inorganic contaminants (6). * Corresponding author telephone: 630-252-9902; fax: 630-2529793; [email protected]. 6876

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As a group, DIRB can use a wide range of Fe(III) forms as terminal electron acceptors for anaerobic respiration, including soluble Fe(III) complexes and Fe(III) oxides such as ferrihydrite, hematite (R-Fe2O3), goethite (R-FeOOH), akagane´ite (β-FeOOH), and lepidocrocite (γ-FeOOH) (7-10). Iron(III) oxides are often among the most abundant forms of Fe(III) in near-surface aquatic and terrestrial systems; however, they are relatively insoluble at circumneutral pH. Therefore, their use by DIRB as terminal electron acceptors for anaerobic respiration requires different mechanisms for electron transfer compared to soluble electron acceptors that are easily transported into the cell (e.g., molecular oxygen and nitrate). The transfer of electrons from microbes to sparingly soluble extracellular electron acceptors such as Fe(III) oxides can occur (i) via direct microbial contact with the mineral surface; e.g., reductases located on the outer membrane of Gram-negative bacteria such as Geobacter and Shewanella (11) or by means of cellular appendages such as electrically conductive pili or “nanowires” (12, 13); (ii) by the enhanced dissolution of the mineral by exogenous or endogenous ligands and subsequent reduction of the dissolved Fe(III) complex (14, 15); and (iii) by facilitated electron transfer involving endogenous or exogenous electron transfer mediators (ETMs), also commonly referred to as electron shuttles (14, 16-26). The ETMs are reduced by the microbes and then subsequently diffuse away from the cell. On encountering a suitable electron acceptor (e.g., Fe(III) oxide), the ETMs are converted back to the oxidized form, completing the cycle. Several ETMs have been shown to facilitate the bioreduction of Fe(III) oxides. Many microorganisms excrete soluble ETMs, and of these, melanin (16), phenazine derivatives (17), flavins (18), and uncharacterized quinone(s) (14) have been shown to enhance the microbial reduction of Fe(III) oxides. Exogenous ETMs such as humic substances (HS) and 9,10-anthraquinone-2,6-disulfonate (AQDS), a synthetic quinone that has been used extensively as a model for the redox properties of quinone groups in HS, enhance the reduction of iron oxides by DIRB, as well as a phylogenetically diverse range of bacteria and archaea not primarily classified as dissimilatory Fe(III)-reducing microorganisms (27). Despite the interest in the role of ETMs in microbial metal reduction in natural and engineered systems, there has not been an extensive investigation of the relative effectiveness of a broad range of natural and synthetic ETMs for enhancing the bioreduction of Fe(III)-bearing minerals. The vast majority of studies examining ETM effects on Fe(III) bioreduction have focused on the use of AQDS as a surrogate for quinone groups in HS. A few studies have compared the ability of multiple compounds to enhance Fe(III) bioreduction by electron shuttling, but they have been limited in terms of the number of potential ETMs examined. For example, Nevin and Lovley (24) compared the ability of AQDS, humics, U(VI), cystine, and S(0) to enhance Fe(III) bioreduction by electron shuttling; Royer et al. (28) compared AQDS, NOM, methyl viologen, methylene blue, and benzoquinone; Hernandez et al. (17) compared AQDS and phenazine derivatives; and von Canstein et al. (18) examined AQDS and flavins. In total, these four studies examined at least 14 different potential ETMs. However, because the experimental conditions in these studies were significantly different (e.g., different forms and concentrations of Fe(III), different types and numbers if DIRB, different concentrations of ETMs, etc.), it is not possible to make determinative comparisons between the various ETMs examined in these studies. Therefore, the main 10.1021/es800686d CCC: $40.75

 2008 American Chemical Society

Published on Web 08/20/2008

TABLE 1. Total Fe(II) Production, Formate Consumption, Ratio of Formate Consumption to Fe(II) Production, and Time Needed to Reach One Half the Final Fe(II) Concentration for Each of the Experimental Systems Examining the Bioreduction of Lepidocrocite by S. putrefaciens CN32 system

Fe(II) produceda (mM)

formate consumeda (mM)

Synthetic ETMs 53.1 ( 0.9 30.7 ( 0.1

Fe(II)/formatea

time to 1/2 Fe(II)max (d)

reduction potentialb Em7 (V)

1.73 ( 0.05

62

59.9 ( 0.5

31.5 ( 0.6

1.90 ( 0.07

25

61.5 ( 0.3 60.0 ( 2.7 62.1 ( 0.5 59.9 ( 2.2 59.4 ( 1.4 53.4 ( 0.2 58.8 ( 1.1 60.2 ( 0.7 62.6 ( 0.6

31.8 ( 0.5 31.7 ( 0.1 32.2 ( 0.1 31.7 ( 1.1 31.7 ( 0.5 30.1 ( 0.1 32.1 ( 0.8 30.3 ( 0.5 32.6 ( 0.2

1.94 ( 0.06 1.89 ( 0.12 1.93 ( 0.03 1.89 ( 0.19 1.88 ( 0.10 1.77 ( 0.02 1.83 ( 0.11 1.98 ( 0.08 1.92 ( 0.04

10 20 18 38 37 48 34 40 37

-0.247c -0.184d -0.225d

54.4 ( 3.1

31.5 ( 0.5

1.73 ( 0.18

56

+0.011e

55.8 ( 2.0 54.2 ( 2.4

31.1 ( 0.4 31.6 ( 0.2

1.79 ( 0.12 1.71 ( 0.13

39 36

-0.003e -0.137e

61.6 ( 1.0

32.4 ( 0.3

1.90 ( 0.07

30

-0.325d

57.8 ( 3.0

31.1 ( 0.2

1.86 ( 0.16

51

-0.051d

Natural Organic Matter no mediator 52.2 ( 0.1 30.3 ( 0.5 Suwannee River fulvic acid (SRFA) 56.2 ( 0.8 33.0 ( 0.5 Suwannee River humic acid (SRHA) 58.0 ( 0.6 33.1 ( 0.7 Suwannee River natural organic matter (SRNOM) 56.5 ( 1.2 33.2 ( 0.7

1.72 ( 0.04 1.70 ( 0.07 1.76 ( 0.08 1.70 ( 0.10

38 37 36 39

mo mediator 3,4-dihydroxy-9,10-anthraquinone-2-sulfonate (alizarin red S-ALZRS) 9,10-anthraquinone-2-carboxylic acid (AQC) 9,10-anthraquinone-2,6-disulfonate (AQDS) 9,10-anthraquinone-2-sulfonate (AQS) 1,4-dihydroxy-2-naphthoic acid (DHNA) 5,8-dihydroxy-1,4-naphthoquinone (DHNQ) isatin-5-sulfonate (ISAT) 5,5′-indigodisulfonate (indigo carmine-I2S) 5,5′,7-indigotrisulfonate (I3S) 5,5′,7,7′-indigotetrasulfonate (I4S) 3,7-bis(dimethylamino)phenazathionium chloride (methylene blue-MB) 5-hydroxy-1,4-naphthoquinone (juglone-NQJ) 2-hydroxy-1,4-naphthoquinone (lawsone-NQL) 3-amino-7-dimethylamino-2-methylphenazine hydrochloride (neutral red-NR) 7-hydroxy-3H-phenoxazine-3-one (resorufin-RESR)

-0.05e -0.125d -0.081d -0.046d

The values (average ( one standard deviation) reported for each of these parameters reflect conditions at the final sampling event; 180 d for the experiments with synthetic ETMs or 134 d for the experiments with natural organic matter. b The potential at pH 7 and 50% reduction. c The reduction potential for AQC was estimated from Figure 4 in Rau et al. (35). d From Clark (47) and references therein. e From Fultz (48) and references therein. a

objective of this study is to compare the effects of a diverse range of compounds from several major classes of organic ETMs, including low-molecular-mass quinones, humic substances, phenazines, phenoxazines, phenothiazines, and indigo derivatives, on the bioreduction of lepidocrocite by Shewanella putrefaciens CN32.

Experimental Section Chemicals. Lepidocrocite was obtained from LANXESS Corp., Leverkusen, Germany/Pittsburgh, PA. NQJ (see abbreviations for ETMs in Table 1), NQL, DHNQ, DHNA, AQC, AQS, MB, NR, RESR, I2S, I3S, I4S, and Pd catalyst (0.5 wt % Pd on 3.2-mm alumina pellets) were purchased from Sigma-Aldrich. ISAT, AQDS, and ALZRS were purchased from Fluka. SRFA, SRHA, and SRNOM were purchased from the International Humic Substance Society (http://www.ihss.gatech.edu/). Individual stock solutions containing 10 mM AQS, AQDS, ALZRS, NR, RESR, MB, ISAT, I2S, I3S, or I4S, or SRFA, SRHA, or SRNOM at an organic carbon (OC) of 1 g L-1, were prepared in water and filter-sterilized (0.22 µm). Because of their lower aqueous solubility, stock solutions of DHNA (100 mM), NQJ (33 mM), and NQL (50 mM) were prepared in methanol, and 10 mM stock solutions of ALZRS, AQC, and DHNQ were prepared in acetone. Experimental Setup. The experimental systems were sterile 160-mL serum vials containing 100 mL of sterile defined mineral medium (DMM) consisting of 80 mM lepidocrocite, 75 mM formate, 22 mM (NH4)Cl, 1.5 mM NaCl, 1.2 mM KCl, 1.1 mM MgSO4, 680 µM Na3NTA, 670 µM CaCl2, 270 µM MnSO4, and 10 mL L-1 of Wolfe’s mineral solution (29), modified to include 50 mg L-1 NiCl2 · 6H2O, 263 mg L-1 Na2SeO3 · 5H2O, and 25 mg L-1 Na2WO4 · 2H2O and omitting MgSO4 and NaCl. The DMM was prepared by combining all

of the components except formate and ETM. The pH was adjusted to 7.5, and the medium was autoclaved. After the medium cooled to ambient temperature, formate was added from a filter-sterilized stock solution to achieve a concentration of 75 mM. The DDM was then portioned into sterile serum bottles and spiked with ETM from the stock solution to provide 100 µM ETM (10 mg OC L-1 for SRFA, SRHA, and SRNOM), with the exception of the ETMs in acetone or methanol, which were added to the sterile serum bottles, and the solvents were evaporated under a stream of sterile Ar before the addition of DDM. Experiments examining the effects of ETM concentration contained 0-1 mM AQDS. The vials were sealed with Teflon-lined rubber septa and aluminum crimp caps. Molecular oxygen was removed from the vials by sparging with sterile Ar. All systems were prepared in duplicate. The inoculum was prepared from late log-phase cultures of S. putrefaciens CN32 (American type Culture Collection BAA-453) as described by O’Loughlin et al. (30). The experiments were initiated by spiking each vial with the volume of inoculum needed to achieve a cell density of 5 × 109 cells mL-1 (the experiments with synthetic ETMs and those with humics were initiated on separate occasions using freshly prepared inoculum). Experiments examining the effects of cell density on AQDS-enhanced bioreduction of lepidocrocite were inoculated to achieve initial cell densities ranging from 1 × 109 to 1 × 1010 cells mL-1. Sterile controls consisted of uninoculated replicate systems with and without lepidocrocite. The suspensions were placed on a roller drum and incubated at 30 °C in the dark. Samples of the suspensions, for monitoring pH, Fe(II), and formate, as well as for identification of biomineralization products by X-ray diffraction (XRD), were collected with sterile syringes. Unless VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Production of total 0.5 M HCl extractable Fe(II) during the bioreduction of 80 mM lepidocrocite by S. putrefaciens CN32 provided with 75 mM formate as the electron donor and in the presence and absence of 100 µM synthetic exogenous electron transfer mediators (abbreviations are defined in Table 1). Inset shows an expanded view of Fe(II) production during the first 30 d; a larger version of the inset is provided in Figure S1. Lines are a visual aid only. Error bars indicate one standard deviation. otherwise indicated, sample collection and processing were conducted in a glovebox containing an anoxic atmosphere (95% N2 with 5% H2). The reduction of lepidocrocite by abiotically reduced AQDS was examined using an approach for reducing quinones adapted from Tratynek and Macalady (31), modified to simulate the redox cycling of AQDS in the bioreduction experiments. Serum bottles (500 mL) containing 50 mL of DDM (without formate or ETM) and 0.5 g of Pd catalyst (0.5 wt % Pd on 3.2-mm alumina pellets) were sealed with rubber septa and aluminum crimp caps and then autoclaved. After the bottles were cooled to ambient temperature, AQDS was added to achieve a concentration of 100 µM, and the bottles were sparged with sterile H2 (assuming a headspace of pure H2, the bottles each contained ∼19 mmol H2). AQDS was not added to the control. After sparging with H2, 20 mM NaHCO3 was added to account for the formation of carbonate resulting from the oxidation of formate in the microbial reduction systems. Analytical Methods. The reduction of lepidocrocite was monitored by measuring the Fe(II) content of 0.5 M HCl extracts of the suspensions; extraction with 0.5 M HCl has been shown to readily solubilize sorbed Fe(II) and to effectively dissolve green rust, siderite, and vivianite (7). The suspensions were extracted for at least 24 h and then centrifuged at 25 000g for 10 min. The Fe(II) concentrations in the supernatants were determined by the ferrozine assay (32). Briefly, 1 mL of HEPES-buffered ferrozine reagent (33) was added to 50 µL of supernatant, and the absorbance at 562 nm was measured. The secondary mineralization products were analyzed by XRD with a Rigaku MiniFlex X-ray diffractometer with Ni-filtered Cu KR radiation. Samples for XRD analysis were collected by filtration on 25-mm, 0.22-µm nylon filters and covered with 8.4-µm-thick Kapton film under anoxic conditions; the filtrate was saved for analysis of dissolved Fe(II), formate, and pH. Samples prepared in this manner showed no evidence of oxidation over the course of data collection. The samples were scanned between 5° and 80° 2θ at a speed of 2.5° 2θ min-1. The XRD patterns were analyzed with the JADE 6 software package (MDI, Livermore, CA). Formate concentrations were measured with an Agilent 1100 series HPLC equipped with an ultraviolet absorbance detector. Each sample was diluted with an equal volume of 10 mM H2SO4, 6878

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and 50 µL of the diluted sample was injected onto a Bio-Rad Aminex HPX-87H ion-exchange column (7.8 × 300 mm). The column was eluted with 5 mM H2SO4 at a flow rate of 0.6 mL min-1 at 50 °C, with analyte detection at 210 nm.

Results and Discussion ETM Effects on Fe(II) Production. The bioreduction of lepidocrocite by S. putrefaciens CN32 in the absence of exogenous ETMs was characterized by the initial production of ∼3 mM Fe(II) within the first 24 h (Figure S1, Supporting Information), followed by a lag period of nearly 22 d during which the concentration of Fe(II) increased by only 2 mM. The lag period was followed by sustained Fe(II) production until the Fe(II) concentration leveled off at ∼53 mM after 180 d (Figure 1). Although no exogenous ETMs were added to this system, several Shewanella spp. are known to produce ETMs that might facilitate the reduction of solid-phase oxidants such as Fe(III) oxides (16, 19-21), and thus the presence of endogenous ETMs in this system cannot be discounted. All of the synthetic ETMs examined in this study, which include analogues for the redox active moieties in NOM (ALZRS, AQC, AQDS, AQS, DGNA, DHNA, NQJ, and NQL), analogues for microbially produced phenazines (NR) and phenoxazines (RESR), and commonly used redox indicators (I2S, I3S, I4S, and MB), were reduced by S. putrefaciens CN32, as indicated by the characteristic changes in color between the oxidized and reduced forms of the ETMs (Figure S2, Supporting Information). Compared to the control, all of the synthetic ETMs enhanced the bioreduction of lepidocrocite by S. putrefaciens CN32, as indicated by the production of Fe(II) (Figure 1). As with the control, the initial production of Fe(II) was followed by a lag period (except for AQC and AQS), the length of which varied from 3 d with NR to 18 d with MB (Figure S1, Supporting Information). After the lag period, Fe(II) production increased substantially. No lag period was observed in the abiotic system containing Pd catalyst as a surrogate for microbial reduction of AQDS (Figure S3, Supporting Information), suggesting that the lag period observed in the biotic AQDS system is the result of physiological factors and not some inherent chemical or mineralogical constraint on Fe(III) reduction. The various ETMs differed substantially in the time to onset of sustained Fe(III) reduction, as well as the rates of

FIGURE 3. Relationship between the formal redox potential at pH 7 (E7M) of the ETM and the time to reach half the maximum Fe(II) concentration. Data from experimental systems containing ETMs for which published reduction potential are unavailable are not included. Neutral red (NR) was not included in the correlation. Dashed lines indicate 95% confidence intervals. Abbreviations are defined in Table 1. FIGURE 2. Effects of cell density and AQDS concentration on the production of total 0.5 M HCl-extractable Fe(II) during the bioreduction of 80 mM lepidocrocite by S. putrefaciens CN32 provided with 75 mM formate as the electron donor. Lines are a visual aid only. Error bars indicate one standard deviation. Fe(II) production; however, by 180 d all of the systems attained steady-state Fe(II) concentrations ranging from ∼53 to 63 mM Fe(II) (Table 1), with an average of 58.7 ( 3.2. Shewanella putrefaciens CN32 can effectively utilize formate as an electron donor for the dissimilatory reduction of lepidocrocite (30), and therefore the production of Fe(II) should be coupled to the oxidation of formate, with 1 mol of formate oxidized for every 2 mol of Fe(III) reduced. The ratios of Fe(II) produced to formate consumed (1.71-1.98) (Table 1) are marginally lower than expected on the basis of the stoichiometry of the above reaction (2:1), but they are within reasonably good agreement. Factors influencing the effectiveness of ETMs for enhancing lepidocrocite bioreduction included inoculum density, ETM concentration, and the reduction potential of the ETMs. The rate of Fe(II) production increased substantially in the presence of 100 µM AQDS, with increases in cell numbers from 1 × 109 to 3 × 109 to 5 × 109 cells mL-1 (Figure 2). However, no increase in lepidocrocite bioreduction occurred when cell density was increased from 5 × 109 to 1 × 1010 cells mL-1. A similar response was observed with changes in AQDS concentration (Figure 2). The addition of 10 µM AQDS substantially enhanced Fe(II) production compared with the system without AQDS. Increasing the AQDS concentration to 100 µM further enhanced lepidocrocite bioreduction, but no additional increase in the rate of Fe(II) production was observed with the addition of 1 mM AQDS (which may indicate a sensitivity to AQDS at higher concentrations (34)). In general, the rate of enhanced Fe(II) production (as indicated by the time required to reach one-half the maximum Fe(II) concentration in each system) correlated with the formal potentials (E7M) of the ETMs (Figure 3), such that ETMs with lower E7M values tended to have faster rates. These results are consistent with previous studies, which indicated a correlation between the rates of mediated redox reactions and the reduction potentials of ETMs (35, 36). Neutral red was an outlier and was not included in the correlation shown in Figure 4. The reduced form of NR is colorless; however, a soluble, yellow, fluorescent pigment formed in both the biotic control without lepidocrocite (Figure S2, Supporting Information) and the complete experimental system (data not shown). A yellow fluorescent

FIGURE 4. Production of total 0.5 M HCl extractable Fe(II) during the bioreduction of 80 mM lepidocrocite by S. putrefaciens CN32 provided with 75 mM formate as the electron donor, in the presence and absence of 10 mg OC L-1 of Suwannee River fulvic acid (SRFA), humic acid (SRHA), or unfractionated natural organic matter (SRNOM). Lines are a visual aid only. Error bars indicate one standard deviation. compound has been observed to form in cultures of Escherichia coli (37) containing NR, and Clark and Perkins (38) reported that this compound has markedly different redox properties than NR. Effectiveness of Humic Substances as ETMs for Lepidocrocite Bioreduction. Humic substances are ubiquitous in aquatic and terrestrial environments and are often identified as ETMs for many biogeochemical processes, including dissimilatory Fe(III) reduction (20, 22-25). The ability of HS to act as ETMs in the bioreduction of Fe(III) minerals, which has largely been attributed to the presence of quinone groups within their structures (39-41), is the rationale for the common use of synthetic quinones such as AQDS as surrogates for the redoxactive moieties in humic substances. The addition of SRFA, SRHA, or SRNOM resulted in, at best, a minimal enhancement of lepidocrocite bioreduction relative to the control without any added exogenous ETM (Figure 4). Studies showing substantial enhancement of Fe(III) bioreduction in the presence of HS (20, 22-25) have typically examined HS derived from terrestrial sources (soils and coals), at loadings ranging from 50 mg to 20 g VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Time-resolved XRD patterns showing the disappearance of lepidocrocite and the formation of carbonate green rust (with the corresponding hkl values in parentheses) during the bioreduction of lepidocrocite by S. putrefaciens CN32 in the presence of 100 µM AQDS. L-1 (∼25 mg to 10 g OC L-1)—concentrations of OC are typical for high-OC environments (e.g., eutrophic sediments and OC-rich soils). Humic substances isolated from terrestrial materials tend to have higher electron-accepting capacities and, per unit mass, are generally more effective as ETMs for the bioreduction of Fe(III) than humics isolated from water (25, 39, 41). Thus, the nominal enhancement of Fe(III) bioreduction by the aquatic HS examined in this study is likely the result of relatively lower net electron transfer capacity (due to the lower overall mass of humics and lower electron transfer capacity per unit mass of humics) relative to the studies discussed above. Formation of Secondary Mineralization Products. The reduction of Fe(III) oxides by DIRB can result in the production of a suite of Fe(II) species including soluble Fe(II) complexes, Fe(II) complexes with organic and inorganic solid phases, and a host of mineral phases containing structural Fe(II) including magnetite (Fe3O4), siderite (FeCO3), vivianite [Fe3(PO4)2], and green rusts (3, 7, 9, 30). Analysis of the solids by XRD during Fe(II) production showed the gradual disappearance of lepidocrocite and concomitant formation of carbonate green rust (Figure 5). Green rusts are mixed ferrous/ferric hydroxides that have structures consisting of alternating positively charged hydroxide layers and hydrated anion layers with the general composition: [FeII(6-x)FeIIIx(OH)12]x+ [(A)x/nyH2O]x-

(1)

where x ) 0.9-4.2, A is an n-valent anion (typically Cl-, SO42-, or CO32-), and y denotes varying amounts of interlayer water (y ) 2-4 for most green rusts). Green rusts typically form in suboxic environments and have been reported as products of the bioreduction of ferrihydrite by S. putrefaciens CN32 ( 4, 7, 42) and lepidocrocite by S. putrefaciens 8071 (9) and S. putrefaciens CN32 (30). In the experimental systems examined in this study, carbonate green rust was the dominant solid phase formed from the reduction of lepidocrocite, as indicated by XRD analysis of the solids remaining after 180 d (Figures S4 and S5, Supporting Information), with or without the addition of exogenous ETMs and irrespective of whether lepidocrocite reduction was microbially driven or abiotic (i.e., the AQDS/Pd/H2 system). However, minor amounts of 6880

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other Fe(II)-bearing secondary mineralization products may be present; e.g., there are indications in several of the diffractograms of minor peaks consistent with the (104) reflection of siderite. The formation of green rust as the dominant biomineralization product is consistent with the incomplete reduction of Fe(III) observed in all of the experimental systems (only 53-63 mM of the initial 80 mM of Fe(III) was reduced to Fe(II)); i.e., the 17-27 mM Fe(III) remaining after Fe(II) production plateaued was likely incorporated into green rust and thus effectively sequestered from reduction over the time scale of these experiments. Environmental Relevance. Recent studies suggest that ETMs such as low molecular mass quinones (e.g., juglone and lawsone) and humic substances may play a role in many redox reactions involved in contaminant transformations and the biogeochemical cycling of redox active elements in aquatic and terrestrial environments. For example, ETMs have been shown to enhance the microbial degradation of a range of organic contaminants including azo dyes, chlorinated hydrocarbons, and triazine explosives (35, 43, 44). Similarly, ETMs, primarily AQDS, have also been shown to enhance the bioreduction of metals such as Fe(III) and U(VI) (7, 20, 22, 24, 45), although this is not always the case (46). The ubiquity of HS in soils and sediments has generated interest in determining their potential as ETMs for microbially mediated Fe(III) reduction. Although substantial evidence suggests that in OCrich environments, electron shuttling by HS can significantly contribute to the bioreduction of Fe(III) (20, 24, 26), the results of this study suggest that electron shuttling by HS is not likely to play a major role in Fe(III) bioreduction in oligotrophic environments, such as subsurface sediments with low OC contents, where the concentration of humics is often relatively low. Much of the work examining ETM-enhanced metal reduction has, for obvious reasons, focused on DIRB; however, the presence of exogenous ETMs has been shown to facilitate the reduction of Fe(III) oxides and other dissolved and solid-phase metals by bacteria that are not typically classified as dissimilatory metal-reducing microorganisms (27). This suggests that the presence of these compounds in natural environments, or their introduction into systems engineered for contaminant remediation, may increase the diversity of microbial populations participating in contaminant reduction. Moreover, soluble ETMs may increase the effectiveness of the remediation of contaminated environments by facilitating microbial transformation of contaminants that are physically/spatially unavailable to microbes (e.g., contaminants located in pore spaces too small to be accessed by bacteria but large enough to accommodate ETMs) (47) (48).

Acknowledgments I thank Karen Haugen and three anonymous reviewers for their thoughtful reviews of the manuscript. Funding was provided by the U.S. Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research, Environmental Remediation Science Program, under contract DE-AC02-06CH11357.

Supporting Information Available Photographs showing solutions of oxidized and bioreduced synthetic ETMs, plots of initial Fe(II) production in the systems with synthetic ETMs, the abiotic reduction of lepidocrocite in the presence of AQDS/Pd/H2, and the XRD patterns of the secondary mineralization products. This material is available free of charge via the Internet at http:// pubs.acs.org.

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