Shewanella putrefaciens Strain CN-32 Cells and Extracellular

Mar 4, 2011 - stepwise fashion with As(V) or As(III) to achieve final concen- trations in the range 2-10 ..... 1300-1550 cm. -1 range of this ... phos...
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Impacts of Shewanella putrefaciens Strain CN-32 Cells and Extracellular Polymeric Substances on the Sorption of As(V) and As(III) on Fe(III)-(Hydr)oxides Jen-How Huang,*,† Evert J. Elzinga,‡ Yves Brechbuehl,† Andreas Voegelin,§ and Ruben Kretzschmar† †

Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, CHN, CH-8092 Zurich, Switzerland Department of Earth & Environmental Sciences, Rutgers University, Newark, New Jersey 07102, United States § Eawag, Swiss Federal Institute of Aquatic Science and Technology, CH-8600 D€ubendorf, Switzerland ‡

bS Supporting Information ABSTRACT:

We investigated the effects of Shewanella putrefaciens cells and extracellular polymeric substances on the sorption of As(III) and As(V) to goethite, ferrihydrite, and hematite at pH 7.0. Adsorption of As(III) and As(V) at solution concentrations between 0.001 and 20 μM decreased by 10 to 45% in the presence of 0.3 g L-1 EPS, with As(III) being affected more strongly than As(V). Also, inactivated Shewanella cells induced desorption of As(V) from the Fe(III)-(hydr)oxide mineral surfaces. ATR-FTIR studies of ternary As(V)-Shewanella-hematite systems indicated As(V) desorption concurrent with attachment of bacterial cells at the hematite surface, and showed evidence of inner-sphere coordination of bacterial phosphate and carboxylate groups at hematite surface sites. Competition between As(V) and bacterial phosphate and carboxylate groups for Fe(III)-(oxyhydr)oxide surface sites is proposed as an important factor leading to increased solubility of As(V). The results from this study have implications for the solubility of As(V) in the soil rhizosphere and in geochemical systems undergoing microbially mediated reduction and indicate that the presence of sorbed oxyanions may affect Fe-reduction and biofilm development at mineral surfaces.

’ INTRODUCTION Adsorption and desorption processes regulate, to a large extent, the partitioning of arsenic (As) between the aqueous and solid phases in soils and sediments and therefore play an important role in determining the mobility and bioavailability of As in geochemical systems. Much previous research has been dedicated to these processes (e.g., refs 1-10), and these studies have indicated that arsenic has a high affinity for adsorption to metal oxide surfaces, with the overall sorption behavior determined by numerous variables, including mineral surface structure, pH, surface coverage, redox potential, and the concentration of competing ions, including organic ligands. Both arsenate (As(V)) and arsenite (As(III)) have been shown to coordinate to metal oxide surfaces as inner-sphere complexes, most commonly in a bidentate binuclear (i.e., bridging) r 2011 American Chemical Society

configuration.1-4,7 In addition, outer-sphere and monodentate inner-sphere As(V) complexation have been reported.1,3,8-10 In many surface and near-surface geochemical environments, microbes are abundant, and their presence and activity may exert an important influence on trace element cycling. In soils, the microbial cell density may be up to several 1010 cells cm-3,11 which makes microbes an important reactive constituent of many soil systems. Due to the high affinity of As for Fe(III)-(hydr)oxides, arsenic cycling in soils and sediments is intimately linked to the Fe cycle, which is driven to a significant Received: November 28, 2010 Accepted: February 14, 2011 Revised: February 10, 2011 Published: March 04, 2011 2804

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Environmental Science & Technology extent by microbial activity, e.g., through dissolution of Fe(III)oxides by siderophore-producing or Fe-reducing bacteria. Whereas indirect effects of microbial activity on As cycling (through effects on the Fe cycle) have been addressed in previous studies (e.g., refs 12-14), little is known about possible direct effects on As retention in geochemical systems that may be associated with the presence of reactive microbial surfaces. The surfaces of microorganisms contain functional groups that act as sorption sites for a variety of aqueous chemical species, particularly trace metals.15-18 In addition, bacteria exude extracellular polymeric substances (EPS) into their environment, which are a heterogeneous mixture composed primarily of polysaccharides and proteins and with nucleic acids and lipids as minor constituents.19 The exterior surface of bacterial cells is composed of EPS, teichoic acids (Gram-positive bacteria), lipopolysaccharides (Gram-negative bacteria), and membranebound proteins.20 Reactive groups in bacterial cell walls and EPS include peptidoglycan carboxyl groups, which are important cation complexation sites on Gram-positive cell walls, and phosphate groups, which contribute significantly in Gramnegative species.21 Besides facilitating biosorption, the functional groups on microbial surfaces also mediate adhesion between microbial cells and mineral surfaces through formation of Coulombic, van der Waals, and covalent bonds between C-, N-, and P-containing cell wall moieties and mineral surfaces sites.20-25 Studies using ATR-FTIR spectroscopy have indicated that phosphate and phosphodiester groups may play an important role in the attachment of bacterial cells and EPS to Fe(III)oxide minerals through the formation of covalent bonds.19,20,26,27 The possible involvement of these groups in bacterial cell and EPS adhesion implies that the presence of bacteria may impact As sorption to Fe(III)-oxide substrates because of competition between phosphate and As for coordination at surface sites. On the other hand, bacterial cell walls may provide sites for As complexation, which may promote As sorption in mineralbacteria suspensions. Additional considerations are that microbes may change As oxidation state and consume organic ligands that may compete with As for coordination at surface sites. These potential influences of microbial cells on As sorption have not been addressed in previous studies. The objectives of this study were to (1) characterize and quantify the adsorption of As(V) and As(III) on goethite, hematite, and ferrihydrite (Fh) as affected by the presence of EPS and microbial cells and (2) obtain spectroscopic information on the interaction of As(V) and microbial cells with hematite substrate in ternary As(V)-bacteria-hematite systems.

’ MATERIALS AND METHODS Iron Hydroxides. Two-line ferrihydrite (Fe5HO8 3 4H2O) was synthesized by neutralizing 500 mL of 0.2 M FeCl3 solution under constant stirring to pH 7 by dropwise addition of 1 M NaOH. The resulting suspension was centrifuged (30 min, 2100g, 20 °C) and the precipitate washed several times with autoclaved, deionized water (DI water, 18.2 MΩ cm-1; Milli-Q, Millipore, Milford, MA). To prevent possible changes in aggregate structure and porosity by freeze-drying,28 the material was kept in aqueous suspension and used for experiments shortly after synthesis. Goethite (R-FeOOH) was synthesized by adding 180 mL of 5.0 M NaOH to 100 mL of 1.0 M FeCl3 solution. The resulting suspension was diluted to 2 L with DI water and then transferred to a polypropylene flask that was sealed and placed in

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an oven for 72 h at 70 °C. The resulting goethite was then centrifuged (30 min, 2100 g, 20 °C), washed several times with DI water, freeze-dried, and stored at 4 °C until use. Hematite was synthesized following a method reported by Sugimoto et al.:29 500 mL of a 2 M FeCl3 solution was continuously added during 5 min to 500 mL of a stirred 5.4 M NaOH solution. The resulting Fe(III)-hydroxide gel was aged in a tightly sealed Pyrex glass bottle for 8 days at 101 °C. 29 The oxide sediment was allowed to cool to room temperature and was subsequently washed several times with DI water in order to remove excess salt until a conductivity of less than 5 μS cm-1 was achieved in the supernatant. The N2-BET (Sorptomatic, Thermo Scientific, Waltham, MA) surface areas of the goethite and hematite were 18.1 m2 g-1 and 24.0 m2 g-1, respectively; the specific surface area of the ferrihydrite was assumed to be 300 m2 g-1.30 Sterilization of the working assays with goethite was done by autoclaving goethite suspensions; X-ray diffraction (Bruker Model AXS D4 Endeavor, Karlsruhe, Germany) analyses of autoclaved goethite indicated no changes in mineral structure resulting from the autoclaving procedure. Extraction and Characterization of Extracellular Polymeric Substances. Shewanella putrefaciens strain CN-32 obtained as ATCC BAA-1097 was grown for 24 h to lateexponential phase under aerobic conditions in tryptic soy broth at 30 °C. Cells were harvested by centrifugation (2100g, 15 min at 4 °C) and washed twice with a solution containing 1 M NaCl. The viscous pellet obtained from 500 mL cell suspensions was resuspended in 15 mL of 50 mM EDTA to chemically separate the capsular EPS from the cells. The mixture was incubated for 4 h at 4 °C with frequent gentle agitation and then centrifuged at 12300g for 30 min at 4 °C. The extracellular polymeric substances (EPS) were precipitated from the supernatant liquid by addition of two volumes of cold ethanol followed by overnight incubation at 4 °C. The precipitates were then dialyzed (molecular weight cutoff: 6000-8000 Da, Spectra/Por1, Breda, The Netherlands) against continuously flowing DI water for two days and then freeze-dried. The amount of EPS was determined with a digital analytical balance (AE 163, Mettler, Switzerland) with 0.1 mg precision. Elemental analysis of the EPS material indicated C, N, S (CHNS-analyzer, CHNS-932, LECO, Germany) and P contents (dissolved in DI water and measured with ICPOES, Vista-Pro radial, Varian, Germany) of 308 ( 7, 74.5 ( 1.4, 0.60 ( 0.30, and 0.98 ( 0.06 g kg-1, respectively. Binary As Batch Adsorption Experiments. Binary As(V) and As(III) adsorption experiments were performed in pHbuffered electrolyte solutions at pH 7 containing 5 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 25 μM sodium lactate, and 10 mM PIPES (piperazine-N,N0 -bis(2-ethanesulfonic acid)) buffer. This composition was chosen because it corresponds to solutions we used in a related study on microbial reduction of As(V) in the presence of mineral sorbents.31 To test for a possible influence of lactate and PIPES on As sorption, we conducted additional sorption experiments in (i) 8 mM NaNO3 and 10 mM PIPES, and (ii) 20 mM NaNO3 (pH 7; checked hourly), respectively. Arsenic(III) and As(V) adsorption isotherms were determined at solids concentrations of 0.2 g L-1 goethite and hematite, and 0.02 g L-1 ferrihydrite. Mineral suspensions (1 L) in PIPES-buffered solutions were allowed to equilibrate while stirring for 24 h and were then spiked in a stepwise fashion with As(V) or As(III) to achieve final concentrations in the range 2-10 μM and 0.4-4 μM for As(V) and As(III), respectively. After each step, a 10 mL subsample was 2805

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Environmental Science & Technology removed from the main vessel under vigorous stirring and pipetted into a 10 mL polyethylene vial, which was equilibrated on an end-overend shaker (50 rpm). Experiments using 20 mM NaNO3 as background solute involved reacting a series of stirred 200 mL mineral suspensions with As(V) concentrations in the range 0.5-40 μM, and As(III) concentrations in the range 0.430 μM. All As(V)-spiked suspensions were equilibrated at room temperature for 24 h; the equilibration time for As(III) was limited to 2 h to minimize oxidation.5 Following equilibration, the solutions were syringe-filtered through 0.2 μm nitrocellulose filter membranes, and analyzed for total As using ICP-MS (Agilent 7500ce, Japan) with Rh as internal standard. The adsorbed amounts of As were calculated from the difference between the initial and final As concentrations. Ternary As-EPS Batch Adsorption Experiments. The effects of EPS on As sorption were studied by preparing suspensions identical to the samples of the binary experiments, except that EPS was added to the suspensions just prior to As spiking at concentrations of 0.03 or 0.3 g L-1. The samples were reacted for 24 h (As(V)) or 2 h (As(III)), and were then filtered and analyzed for dissolved As following the methods described above. As(V) Desorption Induced by Shewanella putrefaciens Bacterial Cells. To investigate the effects of Shewanella cells on As(V) sorption to goethite, ferrihydrite, and hematite, Shewanella cells were first deactivated by treatment with 4% paraformaldehyde to avoid microbial As(V) reduction or As(V) uptake. The deactivated cells were then added to goethite, hematite, or ferrihydrite suspensions with solids concentrations of 0.2, 2.0, and 20 g L-1 that had been equilibrated for 24 h with 10 μM As(V) at pH 7.0 under N2 atmosphere. The final microbial cell density in the samples was estimated at 5  109 cells mL-1, based on DAPI staining and optical density measurements at 600 nm wavelength (OD600). Sampling was performed with syringes under N2 atmosphere at regular time intervals following Shewanella cells introduction. The mineralmicrobe suspensions were filtered (0.2 μm) for subsequent analysis of aqueous As speciation with HPLC-ICP-MS. Control experiments indicated no reduction of As(V) by deactivated Shewanella cells after treatment with paraformaldehyde. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. ATR-FTIR experiments were performed for direct monitoring of the effect of bacterial cells on As(V) adsorption to hematite. Hematite was used as sorbent because it lacks IR absorption bands in the spectral range of interest (1700-700 cm-1) that may interfere with the IR bands of bacterial cell groups and As(V) oxyanions. EXAFS studies have indicated that As(V) sorption to hematite occurs through similar mechanisms as for goethite and ferrihydrite.1,7,32,33 We therefore assume that the results obtained in the IR experiments using hematite can be expected to occur at the surfaces of goethite and ferrihydrite as well. The ATR-FTIR spectra were recorded on a Perkin-Elmer Spectrum 100 IR spectrometer equipped with a liquid N2-cooled MCT/A detector and an optics compartment that was continuously purged at 10 L min-1 with CO2- and H2O-free air delivered by a Balston Parker dry air purger. The ATR-FTIR experiments were performed using the flow cell technique described in previous studies.34,35 Briefly, a horizontal 45° ZnSe ATR crystal with nine internal reflections was coated with a thin layer of colloidal hematite at pH 5 and sealed in a flow cell. The flow cell was placed on the ATR stage of the spectrometer and connected through polypropylene tubing to a reaction vessel containing

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50 mL of a 0.01 M NaCl solution adjusted to pH 7.0. A peristaltic pump was used to circulate solute from this vessel through the flow cell at a rate of 100 mL h-1 for 2 h in order to equilibrate the hematite deposit with the background electrolyte. Following preequilibration, a background scan consisting of the combined absorbances of the ZnSe crystal, the hematite film, and the 0.01 M NaCl background solute was collected as the average of 500 scans at a 4 cm-1 resolution; all subsequent spectra were ratioed to this background spectrum. Next, the reaction solute was spiked with 25 μM As(V) by addition of the appropriate amount of a 0.01 M NaH2AsO4 stock solution. The adsorption of As(V) to the hematite deposit was monitored by observing the υ3 vibrations of adsorbed As(V) species in the spectral range 7001000 cm-1. After 3 h, no further increases in As(V) adsorption were seen, and the final spectrum of adsorbed As(V) was collected. Next, 50 mL of a pH 7.0 stock suspension of S. putrefaciens with a cell density of 1010 cells mL-1 and spiked with 25 μM As(V) was added to the reaction vessel in order to obtain a final 0.01 M NaCl solution of pH 7.0 with an As(V) concentration of 25 μM and a cell density of 5  109 cells mL-1. This solution was pumped through the flow cell for equilibration with the hematite deposit for 2 h. Characteristic absorbance of adsorbed As(V) species and of functional groups associated with bacterial cells were recorded in the 1700-700 cm-1 range throughout this time period by collecting spectra as the average of 250 scans at 4 cm-1 resolution (4 min per spectrum) every 10 min. Simultaneous recording of the IR frequencies of adsorbed As(V) and bacterial cells permitted direct assessment of the effect of cells on As(V) adsorption at the hematite surface in this system. Following reaction, the spectrum of aqueous Shewanella cells deposited directly onto the ZnSe ATR crystal was collected as follows: (1) the ATR-FTIR spectrum of the “supernatant” (i. e., the reacting solute flowing over the flow cell hematite deposit) used in the adhesion experiment was collected with a bare (i.e., without hematite film) ZnSe crystal; (2) the spectrum of a 0.1 M NaCl solution (pH 7) was collected with the same bare ZnSe crystal; (3) the spectrum of the 0.1 M NaCl solution was subtracted from the spectrum of the flow cell supernatant to isolate the spectrum of Shewanella cells adhered to the ZnSe crystal surface.

’ RESULTS AND DISCUSSION As(III) and As(V) Adsorption to Goethite and Ferrihydrite in the Absence of EPS and Bacterial Cells. The adsorption

isotherms of As(III) and As(V) on goethite, hematite, and ferrihydrite measured in the absence of EPS and Shewanella cells are presented in Figure 1. We observed no noticeable effects of the presence of 10 mM PIPES, 25 μM lactate, or the background electrolytes used on As(III) and As(V) sorption. The isotherm data show that As(V) has a higher affinity than As(III) for goethite, hematite, and ferrihydrite at the experimental pH (pH 7.0) over the entire concentration range studied (Figure 1a-c). However, the sorption isotherms of As(V) and As(III) converged at the high end of the concentration range, suggesting that As(V) adsorption approached saturation while the adsorption maximum of As(III) was not reached. These findings are consistent with the results from previous studies indicating stronger sorption of As(III) than As(V) to goethite and ferrihydrite at high As concentrations and pH values in the near neutral range and higher.2,6,36 The data further show that lactate and PIPES do not effectively compete with As(V) for surface sites under the 2806

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Figure 1. Adsorption isotherms of arsenate (As(V)) and arsenite (As(III)) in the absence of bacteria on (a) ferrihydrite, (b) goethite, and (c) hematite at pH 7.0 and room temperature (23 ( 2 °C). Full medium: 5 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 25 μM sodium lactate, and 10 mM PIPES. The legend in panel c applies for all panels.

conditions of the isotherm experiments, as indicated by the similarity in As(V) adsorption in the full medium and the solutions free of lactate and PIPES, respectively (Figure 1a-c). Therefore, changes in the concentrations of lactate and acetate (and presumably of similar organic ligands) resulting from microbial activity are expected to have no direct impact on As(V) retention in aqueous Fe(III)-hydroxide systems under the current conditions. Ternary As-EPS Adsorption Experiments. The effects of EPS on the adsorption of As to goethite, hematite, and ferrihydrite are presented in Figure 2a-c. The adsorption of As(V) and As(III) strongly decreased with increasing EPS additions for all three sorbents, with 10-45% lower sorption of As(V) in the presence of 0.3 g L-1 of EPS. Previous studies have indicated that phosphate groups in EPS form inner-sphere surface complexes on Fe(III)-oxide surfaces,19,20,26,27 and therefore the current results can be explained by competition between As(V) and EPS functional groups for sorption sites on the Fe(III)-(oxyhydr)oxide

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Figure 2. Adsorption isotherms of arsenate (As(V)) and arsenite (As(III)) on (a) ferrihydrite, (b) goethite, and (c) hematite in the absence or presence of 0.03 or 0.3 g/L microbial exopolysaccharide (EPS) isolated from Shewanella putrefaciens CN-32. Background solutions: 5 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 25 μM lactate, PIPES 10 mM, pH 7.0, room temperature (23 ( 2 °C). The legend in panel c applies for all panels.

surfaces. The formation of stable aqueous As(V)-EPS complexes may also contribute to the observed decrease in As sorption in EPS-containing systems but would likely be less important than direct displacement of sorbed As(V) by competitive coordination of EPS phosphate groups. The microbial EPS used in the experiments had a P content of 0.98 g P kg-1. Accordingly, addition of 0.3 g L-1 EPS (the highest EPS concentration used for the experiments presented in Figure 2) corresponds to introduction of phosphoryl groups at a concentration of 9.5 μM. The resulting decrease in As(V) sorption on the Fe(III)-(hydr)oxides is approximately 10-40% at [As(V)] = 1 μM (i.e., at a 10-fold excess of added biophosphate), and approximately 15-45% at [As(V)] = 5 μM (2-fold excess of biophosphate) (Figure 2a-c). These results compare to 50-60% As(V) desorbed from goethite at pH 4 and 6 induced by orthophosphate addition at a 5-fold excess reported by O’Reilly et al.4 Dixit and Hering6 2807

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Figure 3. Release of dissolved arsenate (As(V)) after spiking suspensions of (a) ferrihydrite, (b) goethite, and (c) hematite containing 10 μM As(V) with deactivated cells (5  109 mL-1) of Shewanella putrefaciens CN-32. All suspensions were prepared in 5 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 25 μM lactate, 10 mM PIPES, at pH 7.0 and 25 °C. The solids concentrations were 0.2, 2, or 20 g L-1 (legend in panel a applies for all panels).

reported decreases in adsorption of As(V) and As(III) to ferrihydrite and goethite by 40-60% in the presence of 4-10 fold excess orthophosphate at pH 7.0, similar to the effects observed in other studies.37,38 Assuming that the observed decrease in As sorption in the presence of EPS was predominantly due to competition with EPS phosphate groups, the results presented in Figure 2 suggest that these biological phosphates have comparable affinity toward Fe(III)-oxide surfaces as orthophosphate and inhibit As sorption substantially. Influence of Shewanella putrefaciens Cells on As(V) Sorption. Figure 3 presents the results of the ternary As(V)Shewanella desorption experiments, demonstrating that introduction of Shewanella cells to Fe(III)-(hydr)oxide suspensions with sorbed As(V) results in As(V) release to solution. The pore size of the filter membranes used to extract the reaction solutes in these experiments (0.2 μm) was sufficiently small to remove

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Figure 4. (a) Spectra showing desorption of arsenate from the hematite surface following introduction of Shewanella putrefaciens CN-32. The first spectrum (red) was collected after initial equilibration of the hematite deposit with 50 μM arsenate in the absence of Shewanella at pH 7.0. The following spectra (arrows to blue) were collected during the 2 h following introduction of Shewanella (5  109 cells mL-1) immediately after the first spectrum had been collected. (b) Comparison of the IR spectra of hematite-adsorbed and aqueous Shewanella cells. The inset zooms in on the phosphoryl region and indicates a feature characteristic of inner-sphere P-O-Fe bonds.

bacterial cells from solution, and therefore the increase in solution As(V) concentrations seen in Figure 3 represents an increase in dissolved As(V). The bacterial cells used in the experiments had been deactivated and washed, and therefore interference by the release of EPS from inactivated Shewanella cells was likely to be minimal.39 Instead, the results in Figure 3 suggest displacement of adsorbed As(V) through competitive complexation of Shewanella cells at goethite, hematite, and ferrihydrite surface sites. ATR-FTIR experiments reported by Chorover and co-workers19,20,26,27 point to the importance of microbial phosphate and phosphodiester groups in the attachment of intact bacterial cells to Fe(III)-(hydr)oxide minerals. Since phosphate ions effectively compete with As(V) for coordination at Fe(III)-oxide surface sites,8,11 the release of As(V) from the surface of goethite following Shewanella introduction is likely due to competition between As(V) and microbial phosphate groups resulting in As(V) desorption. ATR-FTIR Experiments. Figure 4a presents the results of the ATR-FTIR experiments investigating the effect of Shewanella 2808

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Environmental Science & Technology cells on the retention of As(V) by hematite. The first spectrum collected following equilibration of As(V) with hematite in the absence of bacterial cells shows the As-O υ3 stretching bands of adsorbed As(V) in the 925-750 cm-1 spectral range, consistent with results from previous ATR-FTIR studies characterizing As(V) surface complexes.40,41 Negative absorbances in the 1300-1550 cm-1 range of this spectrum indicate desorption of carbonate surface species as a result of competitive adsorption of As(V).41 Immediately after this spectrum was collected, deactivated Shewanella cells were introduced into the system to give a concentration of 5  109 cells mL-1, and additional spectra were collected during the following 2 h. The spectra show pronounced increases in IR absorbances of functional groups associated with bacterial cells coordinating to the hematite surface (frequencies in the 1700-900 cm-1 range20, 27) as time progressed. A close-up of the frequencies of adsorbed As(V) in these spectra is shown in the inset of Figure 4a and shows a progressive decrease in the intensity of As(V) absorbances with time after Shewanella addition. These results indicate that increasing bacterial coordination at the hematite surface causes the competitive desorption of complexed As(V). To identify the functional groups involved in Shewanella cell attachment to the hematite surface, the IR spectrum of adsorbed bacteria in the presence of As(V) (after 2 h; Figure 4a) was compared to the spectrum of Shewanella cells deposited directly onto the ZnSe ATR crystal (Figure 4b). Comparison of these spectra indicates differences in the vibrational frequencies of the phosphate (1200-1000 cm-1) and carboxylate (14501360 cm-1) regions, consistent with interactions between bacterial phosphate and carboxylate groups with the hematite surface. A close-up of the spectral region containing the phosphate P-O υ3 stretching bands is shown in the inset of Figure 4b and shows frequencies in the spectrum of adsorbed Shewanella that have been assigned to P-O-Fe vibrational bands19,20,26,27 and thus suggest inner-sphere coordination of bacterial phosphate groups at the hematite surface. The appearance of the IR band near 1320 cm-1 in the spectrum of adsorbed Shewanella suggests additional surface interactions with bacterial carboxylate groups, with the 1320 cm-1 band representing the symmetric stretching frequency of the COO- group, while the corresponding asymmetric stretch is masked by overlap with the strong amine bands in the 1500-1650 cm-1 region.42 Inner-sphere complexation of carboxylate groups with Fe was observed during adhesion of Cryptosporidium oocysts and Pseudomonas putida to hematite.42,43 The inner-sphere coordination of bacterial phosphate and carboxylate functional groups and the concurrent desorption of As(V) evident from the ATR-FTIR results are consistent with displacement of As(V) from surface sites by cell wall functional groups through competitive interactions. An additional factor that may have contributed to As(V) desorption from the hematite surface in the IR experiments is adsorption of As(V) at the bacterial cell surfaces. Chubar et al.44 report substantial adsorption of phosphate anions on Shewanella cells but note that a preconditioning step is required to obtain significant phosphate adsorption. Further studies on the direct interaction of As ions with bacterial surfaces and EPS molecules are required to assess the importance of As retention at bacterial cell surfaces. Implications for Arsenic Mobilization. The primary finding from this study is that microbial cells and EPS can mobilize As from Fe(III)-hydroxides because of competitive interactions between adsorbed As and functional moieties (phosphoryl and carboxylate groups) of cell surface molecules and EPS

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coordinating at Fe(III)-hydroxide surfaces. These competitive interactions may significantly contribute to generally increased solubility of As in Fe(III)-(hydr)oxide systems. Mobilization of anionic species by microbial attachment may enhance the availability of anionic nutrients and of oxyanions serving as electron acceptors for respiration of organic molecules under reducing conditions. The effects of EPS on As solubility may be of major significance in surface soils, which typically contain substantial bacterial populations. Moreover, exopolysaccharides are produced (as mucilage) in the soil rhizosphere by plant roots, and the current results imply that these compounds may influence As uptake by plants. Our results further suggest that microbial attachment to mineral surfaces may be impeded as a result of competition with oxyanions for surface sites. Consequently, the presence of adsorbed oxyanions such as phosphate and As(V) may affect biofilm development and possibly microbially mediated Fe-reduction.

’ ASSOCIATED CONTENT

bS

Supporting Information. Details on the method used for As(III) and As(V) quantification in solution and on the method used for deactivating Shewanella putrefaciens cells. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ41 44 632 88 19; fax: þ41 44 633 11 18; e-mail: [email protected].

’ ACKNOWLEDGMENT We gratefully acknowledge Kurt Barmettler (ETH Zurich) for technical support in the laboratory and Josef Zeyer (ETH Zurich) and Anna Lazzaro (ETH Zurich) for valuable discussions and microbiological advice. This research was financially supported by a Marie Curie Intra-European Fellowship for JHH (No. 039074, “Arsenic Reduction”) and an Ambizione research grant to JHH from the Swiss National Science Foundation (No. PZ00P2_122212). ’ REFERENCES (1) Fendorf, S.; Eick, M. J.; Grossl, P.; Sparks, D. L. As(V) and chromate retention mechanisms on goethite. 1. Surface structure. Environ. Sci. Technol. 1997, 31, 315–320. (2) Manning, B. A.; Fendorf, S. E.; Goldberg, S. Surface structures and stability of arsenic(III) on goethite: Spectroscopic evidence for inner-sphere complexes. Environ. Sci. Technol. 1998, 32, 2383–2388. (3) Arai, Y.; Elzinga, E. J.; Sparks, D. L. X-Ray absorption spectroscopic investigation of arsenite and As(V) adsorption at the aluminum oxide-water interface. J. Colloid Interface Sci. 2001, 235, 80–88. (4) O’Reilly, S. E.; Strawn, D. G.; Sparks, D. L. Residence time effects on As(V) adsorption/desorption mechanisms on goethite. Soil Sci. Soc. Am. J. 2001, 65, 67–77. (5) Goldberg, S. Competitive adsorption of As(V) and arsenite on oxides and clay minerals. Soil Sci. Soc. Am. J. 2002, 66, 413–421. (6) Dixit, S.; Hering, J. G. Comparison of arsenic (V) and arsenic (III) sorption onto iron oxide minerals: Implications for arsenic mobility. Environ. Sci. Technol. 2003, 37, 4182–4189. (7) Sherman, D. M.; Randall, S. R. Surface complexation of arsenic(V) to iron(III) (hydr)oxides: Structural mechanism from ab initio molecular geometries and EXAFS spectroscopy. Geochim. Cosmochim. Acta 2003, 67, 4223–4230. 2809

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