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Contribution of Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms to U(VI) Immobilization Bin Cao,†,§ Bulbul Ahmed,† David W. Kennedy,‡ Zheming Wang,‡ Liang Shi,‡ Matthew J. Marshall,‡ Jim K. Fredrickson,‡ Nancy G. Isern,‡ Paul D. Majors,‡ and Haluk Beyenal*,† †
The Gene and Linda Voiland School of Chemical Engineering and Bioengineering and the Center for Environmental, Sediment and Aquatic Research (CESAR), Washington State University, Pullman, Washington, United States ‡ Pacific Northwest National Laboratory, Richland, Washington, United States
bS Supporting Information ABSTRACT: The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.
’ INTRODUCTION Shallow subsurface environments are inhabited by diverse populations of microorganisms that either form, or are capable of forming, biofilms.13 Biofilms are complex microenvironments containing microbial cells enclosed within a matrix of extracellular polymeric substances (EPS). Cells within biofilms conduct extracellular activities, including nutrient acquisition, chemical communication, and coordination of gene activities, more efficiently than their planktonic counterparts.4 In geologic systems, EPS can aid in the adhesion of microbes to mineral surfaces, bind ions, and in general contribute to geochemical properties in the microenvironment that may be similar to or distinct from those of the bulk environment.5 The presence of EPS in the subsurface environment can influence cellular metabolism as well as the migration and fate of heavy metal and radionuclide contaminants.68 EPS from most bacterial species are polyanionic at circumneutral pH and are able to form organometallic complexes with multivalent cations through electrostatic interactions.5,9 Because EPS contain a variety of functional groups such as carboxyl, phosphoryl, amide, and hydroxyl groups,10,11 they have also been widely studied for metal complexation.5,7,8,12,13 Subsurface uranium contamination at U.S. Department of Energy sites is one of r 2011 American Chemical Society
the most complex and intractable problems in environmental biogeochemistry.2 In natural subsurface environments, where nutrients are usually limited, resulting in relatively low cell density and extensive EPS production,14 the presence of EPS can influence cellular metabolism and the migration and fate of certain contaminants such as heavy metals and radionuclides in the subsurface environment.68 Several studies have reported interactions between EPS and U(VI). Macaskie et al.15 showed U(VI) could be immobilized in extracellular extracts of Citrobacter sp. via enzymatically released phosphate. Uranium sequestration in the EPS of Synechococcus elongatus16 and Acidithiobacillus ferrooxidans17 has also been reported. In addition, EPS of Myxococcus xanthus18 and Pseudomonas stutzeri DSM 5190 may also be involved in U(VI) biomineralization.11 These studies, however, mainly focused on U(VI) sorption by EPS. In addition to sorption, dissimilatory metal-reducing bacteria (DMRB) are also able to Received: January 10, 2011 Accepted: May 2, 2011 Revised: April 25, 2011 Published: May 31, 2011 5483
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Figure 1. Schematic illustration of cell preparation and EPS extraction for comparative analyses of U(VI) sorption and reduction: (A) cell with bEPS, (B) laEPS, (C) cell with minimal EPS, and (D) bEPS.
biotransform U(VI), making the interactions between U(VI) and EPS-producing DMRB difficult to predict. Many metal-reducing bacteria can reduce U(VI) to either uraninite (UO2(s))1921 or nonuraninite products such as U(IV)-orthophosphates.22,23 Specifically, Shewanella species can reduce U(VI) under anoxic conditions, resulting in the formation of uraninite (UO2(s)) or U(IV)-orthophosphates within the periplasm and external to the cell outer membrane (OM).20,2325 The EPS of S. oneidensis MR-1 have been reported to colocalize with uraninite nanoparticles (UO2-EPS),25 and immuno-localization studies have revealed that this UO2-EPS contains OM c-type cytochromes (OMCs) MtrC and OmcA in which MtrC possesses uranyl reductase activity. These results suggest that EPS may play a yet unrecognized role in U(VI) reduction.25 We have recently characterized the proteome of EPS from anaerobic Shewanella sp. HRCR-1 biofilms to reveal at least 20 redox active proteins, including homologues of MtrC and OmcA.26 The presence of electron transfer proteins within EPS suggests that Shewanella biofilms are redox active and may play important role(s) in the extracellular reduction of U(VI). In biofilms, EPS can be either tightly associated with the cells (i.e., bound EPS, bEPS) or indirectly attached to the cell surface (i.e., loosely associated EPS, laEPS).26 To the best of our knowledge, the relative contributions of bEPS, laEPS, and EPSfree cells to U(VI) immobilization have not been investigated, most likely because of a lack of appropriate methods to grow biofilms with high cell viability and minimal cell lysis for the preparation of bEPS, laEPS, and cells with minimal EPS. In our previous study we developed and optimized such methods.26 The goal of this study was to quantify the contribution of EPS to U(VI) immobilization using Shewanella sp. HRCR-1, an isolate from the Hanford Reach of the Columbia River, as a model organism. We prepared Shewanella sp. HRCR-1 biofilms in a hollow fiber membrane reactor (HfMBR) and quantitatively
analyzed the contribution of EPS to U(VI) immobilization (i.e., sorption and reduction) through (i) comparing U(VI) removal from the aqueous phase by cells with bEPS and cells with minimal EPS and (ii) directly testing the reactivity of isolated laEPS and bEPS with U(VI).
’ MATERIALS AND METHODS Shewanella sp. HRCR-1 Biofilms. Biofilms of Shewanella sp. HRCR-1 were prepared in a HfMBR following previously reported protocols.26 Briefly, the sterilized HfMBR was filled with 2 L of modified M1 medium containing 10 mM Na-lactate and 10 mM Fe(III)-NTA as the sole electron donor and acceptor for biofilm growth, respectively.26 After 1% v/v inoculation with a seed culture (OD600 0.80), the culture was purged with O2-free N2 to maintain an anaerobic growth condition for biofilm formation. The flow rate of the recirculation was initially set at 3 mL/min; however, this flow rate slowed with time because of the attachment of cells and the maturation of the biofilm on the hollow fibers. Compared to biofilms prepared in other types of reactors, such as flat plate and fixed bed column reactors, biofilms grown in our HfMBR have several advantages including relatively high cell viability and minimal cell lysis, meeting crucial requirements for EPS studies.26 Cell Preparation and EPS Isolation. Previously we developed and optimized a protocol for the isolation of EPS fractions from Shewanella sp. HRCR-1 biofilms with negligible contamination from cellular materials.26 Briefly, biofilms grown on hollow fibers were dispersed by reversing the recirculation flow and the cells were harvested by centrifugation (5000g, 15 min). Using centrifugation followed by treatment with ethylenediaminetetraacetate (EDTA), two fractions of biofilm EPS (i.e., laEPS and bEPS) were obtained from the supernatants, and cells with bEPS as well as cells with minimal EPS were recovered from the pellets 5484
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Environmental Science & Technology (Figure 1). The details of the procedures are shown in Supporting Information (SI). The cells were kept at 4 °C for further use in U(VI) immobilization. Both laEPS and bEPS were dialyzed using a dialysis membrane with a molecular cutoff of 3 kDa to remove ethanol and EDTA. Protein and polysaccharide contents were measured using previously described protocols.26 Although the effectiveness of such EPS removal has been previously evaluated,26 in this study Shewanella sp. HRCR-1 cells with bEPS and with minimal EPS were imaged using FEI 200F SEM (FEI Company, Hillsboro, OR) to observe the morphological changes before and after bEPS removal. Low-vacuum SEM was used, and the images were taken directly using wet samples without dehydration or staining at an acceleration voltage of 10 kV, a vacuum pressure of 120 Pa, and a spot size of 3.5 nm. The viability of cells with bEPS and cells with minimal EPS was evaluated using a confocal laser scanning microscope (CLSM) (Zeiss LSM 510 Meta) and a BacLight LIVE/DEAD staining kit (Invitrogen, Carlsbad, CA). The green and red fluorescing cells were counted at five different locations within each sample, and viability was expressed as the percentage of green cells averaged over the five locations. Nuclear Magnetic Resonance (NMR). After dialysis, a portion of each EPS fraction was adjusted to pH 7.0 using 1.0 M NaOH or HCl and analyzed using a 600 MHz NMR to ensure the efficient removal of ethanol and EDTA. DSS (4,4-dimethyl-4-silapentane1-sulfonic acid) was added as a chemical shift standard. A sample (540 μL) was mixed with 10 mM DSS (60 μL) and then loaded into the NMR tube for analysis. The 1H spectra were acquired on a 600 MHz NMR spectrometer with a HCN cold probe (Varian, Walnut Creek, CA). The specific chemical shifts for EDTA were predicted to be at 2.96, 3.43, and 3.53 ppm (www.nmrdb.org). For ethanol, the typical peaks were at 3.60 ppm (quartet) and 1.20 ppm (triplet), which are available in the database of Chenomx NMR Suite (Edmonton, Alberta, Canada). U(VI) Immobilization by Cells With bEPS and Cells With Minimal EPS. Quantification of U(VI) immobilization was conducted anaerobically (90% N2/5% CO2/5% H2) in 20-mL serum bottles containing 10 mL of 10 mM PIPES buffer (pH 7.2) supplemented with 10 mM lactate as an electron donor. Appropriate amounts of a stock solution of uranyl chloride (UO2Cl2) (International Bio-Analytical Industries, Boca Raton, FL) in water were added to obtain different initial U(VI) concentrations. The nominal U(VI) concentrations we used were 100, 500, and 1000 μM; the exact initial concentrations were determined using a Kinetic Phosphorescence Analyzer (KPA) (Chemchek Instruments, Richland, WA) to be 102.4, 490.8, and 990.4 μM. Cells with bEPS and cells with minimal EPS were suspended and transferred into the serum bottles to a final cell density of 1.00 ( 0.02 mg wet-cell-biomass/mL (∼4.0 107 cells/mL). All experiments were conducted in triplicate. Samples (500 μL each) were taken periodically and centrifuged at 20 000g for 5 min at 4 °C, and U(VI) concentrations in the cell-free supernatants were analyzed27 on a KPA Analyzer. To estimate U(VI) reduction in the immobilization process, O2-catalyzed oxidatively solubilized U, presumably U(VI), was quantified: 200 μL of each sample taken at 5 h was reoxidized by exposure to the air for 2 h and intermittent aeration by pipetting. The samples were then centrifuged at 20 000g for 5 min at 4 °C, and the difference between the aqueous U(VI) concentrations before and after 2 h of air exposure was used to estimate the amount of U(VI) that had been reduced during U(VI) immobilization.19 Wilcoxon signed-rank test 25,28 was performed online to compare U(VI)
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immobilization curves (www.fon.hum.uva.nl/Service/Statistics/ Signed_Rank_Test.html) and the comparison was considered significant at p < 0.01. U(VI) Immobilization by laEPS and bEPS. Oxidized laEPS and bEPS samples were prepared by gently pipetting the samples in ambient air. Reduced laEPS and bEPS samples were prepared by mixing 1.5 mL of EPS sample with several grains (∼10 mg) of sodium dithionite (Sigma) in the anaerobic chamber (90% N2/ 5% CO2/5% H2) for 5 min and dialyzing against anoxic water. Both oxidized and reduced EPS samples were assayed for U(VI) immobilization (i.e., removal from solution) in triplicate. The difference in aqueous U(VI) removal by oxidized and reduced forms of laEPS or bEPS was attributed to U(VI) reduction by laEPS or bEPS, respectively. For each experiment, 1.0 mL of EPS sample (approximately 90 μg for bEPS and 115 μg for laEPS) was mixed with 1.0 mL of 5.0 μM UO2Cl2 aqueous solution in a 20-mL serum bottle and 300-μL samples were taken periodically for aqueous U(VI) concentration determination after centrifugation at 20 000g for 5 min. A mixture of 1.0 mL of distilled water and 1.0 mL of the diluted UO2Cl2 stock was prepared as an abiotic control. To rule out the possibility that residual dithionite in the dialyzed EPS samples reduced U(VI), a sodium dithionite control solution was prepared following the protocol used to reduce EPS. After dialysis, this solution was reacted with 1.0 mL of U(VI) solution and the extent of U reduction was measured. Visible spectra (600400 nm) of the oxidized (by air) and reduced (by sodium dithionite) EPS samples were obtained. Absorption spectra of the EPS samples were taken again after mixing reduced EPS with U(VI) and incubating anaerobically for 24 h. U(VI) fluorescence spectroscopic measurements were performed in a cryostat at liquid He-temperature (LHeT) as described previously.29,30 The fluorescence emission spectra were obtained by excitation at 415 nm.
’ RESULTS AND DISCUSSION Cell Preparation and EPS Isolation. The preparation of cells and isolation of EPS fractions for U(VI) sorption and reduction studies are shown schematically in Figure 1. EPS removal from representative Shewanella sp. HRCR-1 biofilms is shown in SEM images (Figure SI-1). The cells were completely covered by amorphous materials, indicating extensive EPS production in the biofilms grown in the HfMBR.26 The laEPS were readily separated from the cells with bEPS through centrifugation. There was little difference in the appearance of the cells after the removal of laEPS; in contrast, after bEPS removal individual cells became more apparent.26 With a radial flow delivering nutrients throughout the biofilm and minimizing diffusion limitations, biofilms prepared in our HfMBR maintain a significantly higher proportion of viable cells26 than those grown in other types of reactors (e.g., flat plate and fixed bed reactors). The removal of bEPS did not significantly affect cell viability (91 ( 6% for untreated cells vs 86 ( 7% for EPS-removed cells; p = 0.625). U(VI) Immobilization by Cells With bEPS and Cells With Minimal EPS. To determine the effects of EPS removal on U(VI) immobilization, we added U(VI) at three different concentrations to the suspensions of cells with bEPS and cells with minimal EPS. The results are shown in Figure 2. Although both cells with bEPS and cells with minimal EPS effectively removed U(VI) from the aqueous phase, there were significant differences (p < 0.01) between their U(VI) 5485
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Figure 3. U(VI) reduction efficiency (expressed as % of total U immobilized) during immobilization by the Shewanella sp. HRCR-1 cells with bEPS and by those with minimal EPS at different initial U(VI) concentrations. U released into the solution following air oxidation relative to the total amount of immobilized U was assumed to represent microbially reduced U(IV). Error bars represent SD (n = 3).
Figure 2. Immobilization of U(VI) at different initial concentrations by cells with bEPS and cells with minimal EPS: (A) 100 μM, (B) 500 μM, and (C) 1000 μM. The final U(VI) removal efficiencies (expressed as % of initial U(VI) removed from the aqueous phase) are provided. Abiotic controls at the different initial U(VI) concentrations are also shown. Error bars represent standard deviation (SD) (n = 3).
immobilization profiles. The abiotic controls with the three different initial concentrations showed U(VI)(aq) concentrations remaining constant over time. The differences could be attributed to the variation in viability (91% for cells with bEPS vs 86% for cells with minimal EPS). However, we normalized the data shown in Figure 2 using viable cell counts and found that the differences remained significant (Figure SI-2), suggesting that bEPS account for the differences in U(VI) immobilization. The U(VI) immobilization efficiencies and initial immobilization rates of cells with bEPS and cells with minimal EPS at different initial U(VI) concentrations were calculated from Figure 2 and are shown in Figure SI-3. Although the initial immobilization rates differed by relatively little, a higher U(VI) immobilization efficiency was observed for cells with bEPS at different initial U(VI) concentrations. These results show that EPS removal
significantly lowers the ability of Shewanella cells to immobilize U(VI). Furthermore, the difference in U(VI) immobilization between cells with bEPS and with minimal EPS was proportionally more significant at low U(VI) concentrations (19.0%, 5.8%, and 3.4% at initial U(VI) concentrations of 100, 500, and 1000 μM, respectively), suggesting that EPS play more important roles at lower U(VI) concentrations. In the presence of aqueous U(VI), EPS are likely the first reactive barrier to U(VI) immobilization, before U(VI) can diffuse through the EPS and react with the cells. Thus, EPS likely contribute more significantly to U(VI) immobilization at low U(VI) concentrations; at high U(VI) concentrations, we speculate that the EPS are probably saturated in terms of binding sites for U(VI) sorption so that cells might play a more dominant role. Similar saturation effects in metal sorption by EPS or cell biomass have been reported for other organisms. For example, Zn(II) and Pb(II) adsorption on S. oneidensis;10 binding of Pu(IV) to EPS from S. putrefaciens;31 binding of Np(V) to cells and EPS of S. alga;32 and U(VI) sorption onto cells and EPS of Pseudomonas sp.33 With an initial U(VI) concentration of 1000 μM, the aqueous U(VI) concentration decreased to 755.9 ( 3.0 and 722.1 ( 4.2 μM within 5 h in the presence of cells with minimal EPS and cells with bEPS, respectively (Figure 2). Similarly, with an initial U(VI) concentration at 500 μM, U(VI) concentration decreased to 361.5 ( 4.1 and 333.4 ( 7.4 μM. We expected that all the U(VI) would be immobilized by the cells when the initial concentration of U(VI) was 100 μM. Interestingly, the final aqueous U(VI) concentration remained at 67.9 ( 4.1 and 48.4 ( 2.5 μM. A wide range of sorption sites with different binding affinities may have contributed to U(VI) removal from the aqueous phase. The sites with weaker affinity, which play a more significant role at high U(VI) concentrations, likely result in a decrease in efficiency at low U(VI) concentrations. The presence or absence of EPS significantly affected U(VI) immobilization by Shewanella sp. HRCR-1 cells, demonstrating directly that EPS can play an important role in controlling U(VI) solubility, especially at low initial U(VI) concentrations. Previous 5486
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studies have shown that EPS have important roles in metal ion immobilization by Shewanella species. The effect of the EPS of S. oneidensis MR-1 on Zn(II) and Pb(II) sorption have been studied using a combination of experimental and thermodynamic modeling approaches.10 By comparing S. oneidensis MR-1 with a genetically modified strain with inhibited EPS production (ΔEPS strain), it was concluded that the greater amount of EPS in wild type MR-1 enhanced metal ion uptake and hindered the diffusion of Zn(II) to the cell walls relative to the ΔEPS strain. Harper et al.31 demonstrated the role of EPS from S. putrefaciens CN32 in binding Pu(IV). Their results implied that carboxylic groups in EPS were the primary binding sites for Pu(IV). Here, we have quantitatively shown the difference in U(VI) removal from the aqueous phase by Shewanella sp. HRCR-1 cells with bEPS and with minimal EPS. Since EPS have been shown to be able to remove metals and radionuclides by sorption,10,31 the difference in U(VI) removal from solution between cells with bEPS and cells with minimal EPS in our study could be, at least partially, due to the sorption of U(VI) to bEPS. U(VI) Reduction by Cells With bEPS and Cells With Minimal EPS. In addition to the sorption of U(VI), Shewanella species are also capable of reducing U(VI) into U(IV).20,2325 To understand the role of EPS in U(VI) reduction, we compared the U(VI) reduction efficiency of the cells with bEPS and those with minimal EPS (Figure 3). The efficiency of U(VI) reduction depended on the initial U(VI) concentration (Figure 3). With an initial U(VI) concentration of 500 or 1000 μM, approximately 60% of the total U was reduced, compared to ∼30% at an initial U(VI) concentration of 100 μM, indicating that proportionally less U(VI) was reduced at lower concentrations. This finding is consistent with previous reports. For example, 73.9% of the U(VI) at an initial concentration of 30 mg/L was removed by sulfate reducing biofilms, while only 30.4% was removed when the initial U(VI) concentration was 3 mg/L.35 This suggests that, at low U concentrations, U(VI) sorption to the biomass (e.g., cells and EPS) rather than reduction, presumably by redox active proteins such as OMCs, might become the dominant factor controlling U(VI) immobilization. The presence or absence of EPS did not affect U(VI) reduction efficiency at the three different initial U(VI) concentrations (Figure 3). Assuming that the amounts of U(VI) reduced and adsorbed by treated cells or untreated cells are Rt and At or Ri and Ai, respectively, we have the following approximate correlation: R t =ðR t þ At Þ R i =ðR i þ Ai Þ
ð1Þ
It is possible that the removal of EPS changes not only the quantity but also the types of functional groups associated with the biomass that are available for metal binding.8 For approximation, we assumed that the types of functional groups did not change significantly after EPS removal from Shewanella sp. HRCR-1. In fact, it has been reported that S. oneidensis MR-1 and a mutant that is deficient in EPS production have the same three major types of functional groups (carboxyl, phosphoryl, and amide groups).10 Therefore, the difference in U(VI) reduction and sorption between cells with bEPS and cells with minimal EPS can be attributed to reduction and sorption by bEPS, i.e., Re and Ae. Thus, Re Ri Rt
ð2Þ
Ae Ai At
ð3Þ
From eqs 13, we can derive Re/(Re þ Ae) ≈ Ri/(Ri þ Ai), which suggests that the ratio of U(VI) reduction to U(VI)
Table 1. Removal of U(VI) from Aqueous Solution Using Oxidized or Reduced EPS bEPS EPS time (h) % removala
oxidized
laEPS reduced
oxidized
reduced
13
13
20
20
11.1 ( 4.9
38.9 ( 3.4
28.3 ( 2.3
37.7 ( 2.0
a Percent removal was calculated from the ratio of U(VI)(aq) to the initial U(VI)(aq) concentration in the reaction mixture. Data are reported as mean ( SD (n = 3).
sorption by cells with bEPS in the presence of cells as reducing equivalent sources was comparable with that by cells with minimal EPS. On a biomass basis, the EPS in a subsurface biofilm has, presumably, a higher reactive surface area than cells because it is mostly a highly hydrated open matrix. Hence, EPS could be much more reactive in terms of sorption than cells depending on the relative density of sorption sites. In the subsurface environment, where nutrients are usually limited, microorganisms tend to produce more EPS.14 In addition, during in situ stimulation in bioremediation processes, microorganisms can readily form sediment biofilms on mineral surfaces, producing up to 19 times more EPS than planktonic microorganisms.36,37 According to our results, EPS associated with cells in the subsurface environment are expected to be an important factor controlling U(VI) mobility through U(VI) sorption as well as reduction. U(VI) Immobilization by EPS. To further quantify the relative contributions of sorption and reduction in U(VI) immobilization by EPS, we directly tested the reactivity of the isolated laEPS and bEPS with U(VI). Table 1 shows the decreases of aqueous U(VI) concentration in the presence of oxidized or reduced EPS under anoxic conditions. The EPS fractions in both oxidized and reduced forms caused decreases in aqueous U(VI) concentration. The decreases in the presence of oxidized bEPS and laEPS can be attributed to U(VI) sorption by the EPS fractions. The U(VI) removed from the aqueous phase by reduced bEPS or laEPS was ∼250% or ∼33% more than that removed by oxidized bEPS or laEPS, respectively. The difference can be attributed to U(VI) reduction by the reduced EPS fractions. Only ∼30% of the total U(VI) removal by reduced bEPS could be attributed to sorption, while 70% was likely due to reduction. In contrast, U(VI) removal by reduced laEPS was mainly (∼75%) due to sorption, while only 25% could be attributed to reduction. Throughout the experiment, the abiotic controls showed no decrease in soluble U(VI) concentration, indicating that dithionite was not a significant contributor to U(VI) reduction by EPS. In addition, the EPS samples showed none of the typical peaks for ethanol or EDTA in NMR spectra (Figure SI-4), suggesting there was no detectable residual ethanol or EDTA from EPS that might have impacted the U(VI) immobilization in results. To confirm that a portion of the U immobilized by the EPS was reduced, one needs to elucidate the oxidation states of the U in the EPS or monitor the change of oxidation states of the EPS. However, to unambiguously analyze U oxidation states in the EPS sample using XANES, a significant amount of U accumulation is required, which is very challenging for the EPSU reaction system because of the relatively low concentration of redox active components in the EPS. Alternatively, since homologues of c-cytochromes including MtrC and OmcA have been identified in EPS from Shewanella sp. HRCR-1 biofilms,26 we 5487
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Figure 4. Absorption spectra of the bEPS-associated OMCs.
suspect that EPS-associated c-cytochromes contribute, at least in part, to U(VI) reduction during interactions between EPS and U(VI). Marshall et al.25 reported that c-cytochrome MtrC in S. oneidensis MR-1 could be oxidized by U(VI). Hence, the oxidation of reduced c-cytochromes in EPS by U(VI) could be used as an indicator for U(VI) reduction. To verify that electron transfer occurred between EPS and U(VI), we used UVvis spectra to monitor the oxidation state of the EPS-associated OMCs before and after reactions with U(VI). Two prominent features can be observed in the UVvis absorption spectra of OMCs when they change from a reduced form to an oxidized form: (i) the Soret band shifts from 420 to 408 nm and (ii) a broad band at 530 nm replaces the β and R bands at 523 and 552 nm.38,39 Figure 4 shows the absorption spectra of the bEPS. The oxidized bEPS have two absorption peaks, at 408 and 530 nm, sharing the same features as oxidized OMCs. After reduction by dithionite, the absorption spectra showed features of reduced OMCs, suggesting the bEPS were in reduced form. Electron transfer between the bEPS and U(VI) was examined by adding U(VI) to the reduced bEPS. After 24 h of reaction with U(VI) at room temperature under an anaerobic condition, the absorption spectra showed that the EPS-associated OMCs had become oxidized, suggesting the U(VI) had accepted electrons from the bEPS. We were not able to measure the absorption spectra for laEPS. This was most likely due to the low abundance of OMCs in laEPS, which was also suggested in our previous study.26 In addition, highly sensitive LHeT laser fluorescence spectroscopy was used to obtain emission spectra of U(VI) in the EPSU systems containing oxidized or reduced EPS. Significant decreases in the U(VI) signals in the reduced EPS compared to the U(VI) peaks in the oxidized EPS fractions suggest the reduction of U(VI) (Figure SI-5). Upon reoxidation, U(VI) peaks became apparent in the spectra (Figure SI-5). The changes in U(VI) fluorescence spectra provide additional evidence supporting electron transfer from both EPS fractions to U(VI). The total concentrations of polysaccharides and proteins in the EPS fractions were determined, and it was revealed that there was a much higher carbohydrate/protein ratio in laEPS than in bEPS (3.64 vs 0.77) (Figure SI-6). Interestingly, the removal by laEPS of U(VI) from the aqueous phase was dominated by sorption (∼75%) while that by bEPS occurred mainly through
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reduction (∼70%) (Table 1). Taken together, these results suggest that (i) isolated EPS, especially bEPS, contain redox active components such as OMCs and possibly other redox active molecules that can be reduced by dithionite and subsequently reduce U(VI), and (ii) polysaccharides are most likely the dominant reactive component of laEPS, contributing to U(VI) sorption while redox active components (e.g., OMCs) in bEPS would have facilitated U(VI) reduction. In summary, we have determined the contribution of EPS from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization via (i) comparison of U(VI) sorption and reduction by cells with bEPS and cells with minimal EPS, and (ii) direct testing of the reactivity of bEPS and laEPS with U(VI). The comparison between cells with bEPS and cells with minimal EPS showed that bEPS from Shewanella sp. HRCR-1 biofilms significantly contributed to U(VI) immobilization through both sorption and reduction mechanisms. Furthermore, EPS likely play more important roles at lower initial U(VI) concentrations. The ratio of U(VI) reduction to U(VI) sorption by bEPS in the presence of cells as sources of reducing equivalents was comparable with that obtained for the cells, suggesting that bEPS could be considered a functional extension of the cells for U(VI) immobilization. In addition, the U(VI) reduction efficiency was found to be dependent upon the initial U(VI) concentration; the efficiency decreased at lower concentrations. By directly testing the reactivity of isolated EPS fractions with U(VI), we showed that, when in reduced form, isolated EPS from Shewanella sp. HRCR-1 biofilms can transfer electrons to U(VI). Polysaccharides likely contributed to U(VI) sorption by both EPS fractions, and they dominated the reactivity of the laEPS. In contrast, the higher abundance of redox proteins (e.g., OMCs) in the bEPS is expected to drive U(VI) immobilization by bEPS via a reductive mechanism.
’ ASSOCIATED CONTENT
bS
Supporting Information. Representative SEM images for evaluation of EPS removal (Figure SI-1), normalized U(VI) immobilization by viable cells (Figure SI-2), capacity and initial rate of U(VI) immobilization (Figure SI-3) and NMR spectra (Figure SI-4), LHeT fluorescence spectra (Figure SI-5), and a quantitative analysis of polysaccharide and protein contents (Figure SI-6). Detailed materials and methods for cell preparation, EPS isolation, and protein and polysaccharide assays are also provided (SI Materials and Methods). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Present Addresses §
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States.
’ ACKNOWLEDGMENT The research was supported by the U.S. DOE Office of Biological and Environmental Research under the Subsurface Biogeochemistry Research (SBR) Program (grant DE-FG9208ER64560) and the DOE-BER SBR Program’s Scientific Focus 5488
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Environmental Science & Technology Area (SFA) at the Pacific Northwest National Laboratory (PNNL). NMR and LHeT laser fluorescence spectroscopy were performed in the William R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at PNNL. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.
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