Environ. Sci. Technol. 2003, 37, 2166-2172
Speciation of Pb(II) Sorbed by Burkholderia cepacia/Goethite Composites A L E X I S S . T E M P L E T O N , * ,† ALFRED M. SPORMANN,‡ AND G O R D O N E . B R O W N , J R . †,§ Surface & Aqueous Geochemistry Group, Department of Geological & Environmental Sciences, Stanford University, Stanford, California 94035-2115, Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305, and Stanford Synchrotron Radiation Laboratory, SLAC, MS 99, 2575 Sand Hill Road, Menlo Park, California 94025
Bacterial-mineral composites are important in the retention of heavy metals such as Pb due to their large sorption capacity under a wide range of environmental conditions. However, the partitioning of heavy metals between components in such composites is not probed directly. Using Burkholderia cepacia biofilms coated with goethite (RFeOOH) particles, the partitioning of Pb(II) between the biological and iron-(oxyhydr)oxide surfaces has been measured using an X-ray spectroscopic approach. EXAFS spectra were fit to quantitatively determine the fraction of Pb(II) associated with each component as a function of pH and [Pb]. At pH 70% Pb/goethite) above pH 6. Direct comparison can be made between the amount of Pb(II) bound to each component in the composite vs separate binary systems (i.e., Pb/biofilm or Pb/goethite). At high pH, Pb(II) uptake on the biofilm is dramatically decreased due to competition with the goethite surface. In contrast, Pb uptake on goethite is significantly enhanced at low pH (2-fold increase at pH 5) compared to systems with no complexing ligands. The mode of Pb(II)-binding to the goethite component changes from low to high [Pb]. Structural fitting of the EXAFS spectra collected from 10-5.6 to 10-3.6 M [Pb]eq at pH 6 shows that the Pb-goethite surface complexes at low [Pb] are dominated by inner-sphere bidentate, binuclear complexes bridging two adjacent singly coordinated surface oxygens, giving rise to Pb-Fe distances of ∼3.9 Å. At high [Pb], the dominant Pb(II) inner-sphere complexes on the goethite surface shift to bidentate edge-sharing complexes with Pb-Fe distances of ∼3.3 Å.
* Corresponding author phone: (858)822-1426; fax: (858)534-7313; e-mail:
[email protected]. Present address: Scripps Institution of Oceanography, Marine Biology Research Division, 9500 Gilman Drive, La Jolla, CA 92093-0202. † Surface & Aqueous Geochemistry Group, Department of Geological & Environmental Sciences, Stanford University. ‡ Department of Civil and Environmental Engineering, Stanford University. § Stanford Synchrotron Radiation Laboratory. 2166
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Introduction Pb is a widespread toxic environmental pollutant that poses varying levels of risk for human health depending upon its speciation. Several decades of experimental work have been dedicated to developing a molecular-level framework for describing metal sorption and precipitation processes at the mineral-water interface (e.g., ref 1 and references therein), in particular for Pb(II) (e.g., refs 2-9). There is also widespread recognition that natural organic matter (e.g., refs 10-15) and biological surfaces (16-22), especially high-surface area bacterial, algal, and fungal surfaces, are also potent sorbents of Pb. Fortunately, many of the surface complexation models developed to describe reactions at mineral surfaces can be modified to describe reactions of aqueous metal ions occurring at bacterial surfaces (e.g., refs 23 and 24). However, microorganisms are often intimately intermixed with mineral phases to form complex, highly hydrated, high surface-area composite materials. The surface properties of these composite materials can differ dramatically from pure bacterial or mineral surfaces, particularly in terms of surface charge, double-layer properties, numbers of reactive sites, and potential metal-binding affinity (13, 21, 25, 26). Strong electrostatic interactions between the wide array of acidic surface functional groups found on bacterial surfaces or exopolysaccharides lead to a common association between bacteria and many classes of minerals, especially iron- and aluminum- (oxy)hydroxides, which have a relatively high point-of-zero charge. Therefore, adsorbed organic compounds, which can include bacteria, form ubiquitous aggregates with minerals in aquatic and soil environments. In turn, colloidal mineral phases, particularly nanoscale oxides and aluminosilicate gels, form common coatings on bacterial surfaces. Often these mineral coatings form in-situ after association of dissolved metal ions on the bacterial surfaces leads to the precipitation of crystalline phases (27). Ultimately, these composite materials will exhibit heterogeneous adsorption behavior. It is critical to determine the relative importance of biological vs inorganic sorption processes as controls on trace metal concentrations over a large range in pH and metal-ion activity. Recent sorption studies have addressed aspects of this problem by testing whether additivity of metal uptake is achieved when uptake on the composite materials is compared to the mineral and bacterial surfaces alone (e.g., ref 28). Recent studies have also quantified the metal retention behavior of naturally occurring biofilm-mineral composites (e.g., refs 29 and 30). However, it is difficult to isolate the individual effects on the sorptive properties of each component or to determine the mode of metal binding within the composites. Our study was designed to investigate the partitioning behavior of Pb between crystalline goethite (R-FeOOH), a common iron-oxyhydroxide mineral, and Burkholderia cepacia biofilms that have formed in the presence of goethite. Extended X-ray absorption fine-structure (EXAFS) spectroscopy was used to determine the fraction of Pb(II) associated with the goethite surface and the biological material as a function of pH and [Pb]. This approach enables us to quantify the contribution of goethite vs the biofilm component for Pb sorption across the range of system variables. Moreover, the total Pb uptake data can be coupled to the partitioning data to directly compare the surface coverage of Pb(II) on each component with the surface-coverage measured under similar conditions in single-material studies. In addition, structural fitting of the Pb LIII-EXAFS spectra is used to determine how the association of the B. cepacia biofilm alters 10.1021/es026081b CCC: $25.00
2003 American Chemical Society Published on Web 04/08/2003
the molecular mechanisms of Pb(II) binding to the R-FeOOH surface.
Materials and Methods Sample Preparation. Biofilms of Burkholderia cepacia (ATCC 17616) were grown aerobically in 0.5 L flasks in the presence of laboratory synthesized goethite (acicular R-FeOOH needles ∼ 100 nm × 1 µm, 90 m2/g by BET/N2; prepared as described by (8)) using a minimal medium (200 µM CaCl2, 150 µM MgSO4, 90 µM (NH4)2SO4, 150 µM KNO3, 10 µM NaHCO3, 50 µM KH2PO4, and 1 mM sodium acetate as a carbon and energy source) at pH 6. The flasks were constantly shaken at 150 rpm at 30 °C. After the cultures reached late-exponential growth phase (∼4 days), the suspensions were left to settle, and the excess supernatant was decanted. The B. cepacia/ goethite suspensions (which include associated exopolysaccharides) were resuspended in 200 mL of 0.01 M NaNO3 (pH 6) three times to remove excess media. Subsequently, the B. cepacia/goethite composites were split into aliquots of approximately 150 mg and resuspended in 50 mL of variable concentration Pb(NO3)2 solutions at an initial pH of 4.5 (final solids concentration was 3 g/L; no surface-area measurement of the composite). The pH was adjusted to the desired value by addition of appropriate amounts of 0.01 M HNO3 and 0.01 M NaOH. Prior to and after the washing procedure, 1 mL of the B. cepacia/goethite suspensions was incubated with either Syto-9 or Live/Dead nucleic-acid stains (Molecular Probes, Inc.) and visualized using a Nikon E600 epifluorescent microscope to enumerate the cells and monitor the cell viability. Approximately 95% of the bacterial cells or greater were green (live) in these assays. The organic C and N content of the B. cepacia/goethite composites was analyzed using a CE Elantech NA1500 Series II Carlo Erba coupled to a Finnegan Delta Plus mass spectrometer. Elemental analysis was determined by comparison of the mass 44 peak intensity derived from the biofilm/goethite composites and a series of standards. The composites comprised ∼30% ((5%) biofilm material and 70% ((5%) goethite, assuming that the biofilm material was 50% carbon on a dry-weight basis. Therefore, for each slurry containing 150 mg of composite material, approximately 45 mg is the biofilm component, while 105 mg is R-FeOOH (9.45 m2 total mineral surface area). In fixed [Pb] experiments, B. cepacia/goethite suspensions were incubated with 10-3.7 M Pb(NO3)2 solutions at pH 2.75 to 7.5. In fixed pH experiments, B. cepacia/goethite suspensions were incubated with 10-4.7-10-2 M Pb(NO3)2 solutions at pH 5 and 6. During the incubation of the B. cepacia/goethite composites with Pb2+, the solutions were bubbled with N2 gas to prevent the saturation and precipitation of Pbcarbonate species. Samples were equilibrated for 24 h with minor additions of 0.01 M NaOH or 0.01 M HNO3 to adjust pH. After equilibration, the suspensions were centrifuged at 18 000 rpm, and the supernatant was decanted and acidified to measure the residual [Pb] by ICP-AES analysis (TJA IRIS Advantage). The cell-paste was transferred to a 2 mm × 20 mm Teflon holder sealed with Kapton tape for X-ray absorption spectroscopy (XAS) measurements (analysis occurred within 8 h). X-ray Absorption Spectroscopy. XAS measurements were performed at the Stanford Synchrotron Radiation Laboratory on wiggler beamline IV-3 (3 GeV, 30-100 mA, detuned 3060% at 13 600 eV). The incident X-ray beam was monochromatized using Si(111) crystals with 2 mm × 20 mm slits prior to the first nitrogen-filled ionization chamber (I0). The Teflon sample holder was placed in the beam path between the first and second ionization chambers (I0 and I1) at 45 degrees to a Xenon-filled Stearn-Heald type Lytle-detector with an As (6T) filter to attenuate elastic scatter and background fluorescence. Additional Al filters were placed between the sample and the Lytle-detector to reduce the fluorescence
signal derived from Fe in the samples. An elemental Pb calibration foil was placed between the second and third ionization chambers, and the first inflection of the Pb-foil absorption edge was taken at 13 055 eV. Extended X-ray absorption fine-structure (EXAFS) spectra were collected from 12 800 to 13 600 eV at room temperature. Typically 14-20 successive scans were averaged together, and the data were background subtracted and normalized to a k3-weighted spline. E0 for the EXAFS was taken at 13 070 eV. To obtain partitioning data, the EXAFS spectra were fit using the linear-combination fitting procedure employing the DATFIT module of the EXAFSPAK program (31). Initially, test fits of the spectra were conducted using a library of more than 30 Pb model compounds, which includes Pb sorbed to B. cepacia, Pb sorbed to goethite, and several crystalline phases such as Pb-carbonates, Pb-sulfates, and Pb-phosphates (32), to determine whether biomineralization or abiotic precipitation of Pb occurred under any conditions within the composites. Ultimately, empirical model spectra of Pb(II) sorbed to the surface of heat-treated B. cepacia (sample C056) equilibrated with 10-5 M Pb, pH 6 and R-FeOOH equilibrated with 10-4.3 M Pb, pH 6 (without bacteria present, 2 µm/m2 surface coverage) were used as end-members for two-component fits, since all the crystalline phases were rejected in the initial fits (lower limit of detection is 6) where strong Pb(II) adsorption on R-FeOOH typically occurs, we observe a large shift in the partitioning of Pb(II) between the biofilm and goethite fractions, leading to a dramatic reduction in the total amount of Pb(II) bound to the biofilm component. Structural fits of the EXAFS spectra provide more detailed information on changes in the types of Pb(II) complexes formed at the goethite surfaces in the B. cepacia/goethite composites. Although neither Ostergren et al. (7, 8) nor Bargar et al. (4, 36) observed differences in the Pb LIII-EXAFS as a function of surface coverage from 0.1 to 5 µmol/m2, a marked change in the mode of Pb(II) binding to the R-FeOOH surface at pH 6 at low vs high [Pb] was observed in the present study. In particular, in the EXAFS fit for the low Pb coverage sample at pH 6 (spectrum 4A), the dominance of the long Pb-Fe distance (3.85 Å) and lack of the shorter second-shell Fe (∼3.3 Å) that is typically observed even in the presence of sulfate (7), indicates that the biofilm may enhance the stability of bidentate, binuclear Pb(II) sorption complexes to a greater degree than observed for small anions. Conceptually, the generation of such “new”, high-affinity sites or ternary complexes is often suggested in metal-humic-mineral studies (e.g., refs 11, 13, and 40). At high [Pb], Pb(II) binding to the goethite fraction is again dominated by edge-sharing complexes, and little effect can be attributed to the presence of a microbial biofilm. Therefore, the structural fitting of the EXAFS spectra provides molecular-level insights on how changes in the mode of Pb binding to the goethite surfaces over several orders of magnitude [Pb] drives changes in the partitioning data derived for the Pb isotherms.
Acknowledgments This work was supported by the National Science Foundation (NSF-EAR-9905755 and NSF-CHE-0089215) and the Eugene Holman Stanford Graduate Fellowship. We thank Tom Trainor, Scott Fendorf, Craig Criddle, and four anonymous reviewers for constructive criticism of this manuscript. We also thank Alice Dohnalkova for TEM assistance provided at the W.R. Wiley Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory. The Stanford Synchrotron Radiation Laboratory and PNNL are funded by the Department of Energy (Offices of Basic Energy Sciences and Biological and Environmental Research) and by the National Institutes of Health.
Supporting Information Available Epifluorescent images of B. cepacia/goethite composites (S1) and Pb(II) uptake, biofilm-Pb(II), and R-FeOOH-Pb(II) for B.cepacia/goethite composites reacted with 50 mM PbT (10-3 M [Pb]i) (S2). This material is available free of charge via the Internet at http://pubs.acs.org. VOL. 37, NO. 10, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Received for review August 22, 2002. Revised manuscript received February 4, 2003. Accepted February 7, 2003. ES026081B
