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Application of Flow Field Flow Fractionation-ICPMS for the Study of Uranium Binding in Bacterial Cell Suspensions Brian P. Jackson,† James F. Ranville,*,‡ and Andrew L. Neal†,§
Savannah River Ecology Laboratory, University of Georgia, Aiken, South Carolina 29802, Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401, and Department of Microbiology, University of Georgia, Athens, Georgia 30602
Field flow fractionation (FFF) is a size-based separation technique applicable to biomolecules, colloids, and bacteria in solution. When interfaced with ICPMS on-line, elemental data can be collected concurrent with size distribution. We employed hyperlayer flow FFF (Fl FFF) methodology to separate cells of Shewanella oneidensis strain MR-1 from exopolymers present in washed cell suspensions. With a channel flow of 4 mL min-1 and a cross-flow of 0.4 mL min-1 cells eluted with a retention time of 4.7 min corresponding to an approximate equivalent spherical cell diameter of 0.8 µm. Cell suspensions were amended with increasing concentrations of U to establish an adsorption isotherm and with fixed U concentrations at varying pH to establish the pH dependence of sorption. A linear sorption isotherm was determined for U solution concentrations of 0.2-16 µM, maximum U sorption occurred at pH 5. A high molecular weight compound, presumably a cell exudate, was identified by Fl FFF-ICPMS. This cell exudate complexed U, and at elevated pH, the exudate appeared to have a greater affinity for U than cell surfaces. Thus, Fl FFF interfaced with ICPMS detection is a powerful analytical technique for metal sorption studies with bacteria; analysis can be carried out on small sample volumes (25 µL) and additional speciation information can be gained because of the versatile Fl FFF separation range and multielement detection capabilities of ICPMS. The extent to which bacteria interact with contaminants in the subsurface is important both in terms of the kinetics of remediation and in assessing the potential for bacteria to act as a vector for contaminant transport. Bioremediation methods can be broadly divided into active and passive approaches. In active bioremediation, bacteria effect a change in speciation of a contaminant that subsequently lowers its availability,1-3 while in passive remediation * Corresponding author. Phone: 303-273-3004. E-mail:
[email protected]. † Savannah River Ecology Laboratory, University of Georgia. ‡ Colorado School of Mines. § Department of Microbiology, University of Georgia. (1) Sani, R. K.; Peyton, B. M.; Smith, W. A.; Apel, W. A.; Petersen, J. N. Appl. Microbiol. Biot. 2002, 60, 192-199. (2) Finneran, K. T.; Anderson, R. T.; Nevin, K. P.; Lovley, D. R. Soil Sediment Contam. 2002, 11, 339-357. 10.1021/ac049278q CCC: $30.25 Published on Web 01/22/2005
© 2005 American Chemical Society
biomass sorbs the contaminant, which is subsequently removed from the system.4 For U remediation, both approaches have potential application.5 Uranium contamination at many DOE sites occurs in the subsurface, a complex system in which to model the transport of contaminants. Components frequently considered in transport models are mineral surfaces of mobile inorganic colloids and dissolved organic carbon (DOC). Bacteria may also significantly impact contaminant transport providing sorption sites and by mediating chemical transformations.6 Processes such as advection and size exclusion may result in contaminants sorbed to cells being transported at greater rates than either DOC or mineralassociated contaminants. Increasingly accurate models of contaminant transport require understanding of partitioning between multiple phases in complex systems. Clearly, analytical methods are required to investigate such complex mixed systems. A number of mechanisms have been employed for cell separation;7 however, these methods are geared to the separation of polydisperse cell suspensions rather than investigations of metal binding to bacteria. The simplest approach to metal-bacteria binding studies is a batch sorption experiment. In this approach, cell suspensions are equilibrated in the presence of known concentrations of metal ions; after an appropriate reaction time, the cells are separated from the supernatant by centrifugation or filtration and the concentration of metal ion remaining in the supernatant is then quantified. This method has been applied to study the thermodynamics of U binding to Shewanella putrefaciens8 and Bacillus subtilis9 and Cd binding to B. subtilis in a ternary system with humic acid.10 A potential drawback to this approach is that the speciation of the metal ion in solution is not directly determined. In ternary systems, increases in metal ion solubility in the presence of humic acid might (3) Lloyd, J. R.; Chesnes, J.; Glasauer, S.; Bunker, D. J.; Livens, F. R.; Lovley, D. R. Geomicrobiol. J. 2002, 19, 103-120. (4) Malik, A. Environ. Int. 2004, 30, 261-278. (5) Bender, J.; Duff, M. C.; Phillips, P.; Hill, M. Environ. Sci. Technol. 2000, 34, 3235-3241. (6) Neal, A. L.; Amonette, J. E.; Peyton, B. M.; Geesey, G. G. Environ. Sci. Technol. 2004, 38, 3019-3027. (7) Chianea, T.; Assidjo, N. E.; Cardot, P. J. P. Talanta 2000, 51, 835-847. (8) Haas, J. R.; Dichristina, T. J.; Wade, R. Chem. Geol. 2001, 180, 33-54. (9) Fowle, D. A.; Fein, J. B.; Martin, A. M. Environ. Sci. Technol. 2000, 34, 3737-3741. (10) Wightman, P. G.; Fein, J. B. Chem. Geol. 2001, 180, 55-65.
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legitimately be explained by the formation of metal-humate solution complexes, however, it is also possible that bacterial extracellular polymeric species could complex metal ions. The recent application of pore exclusion chromatography-ICPMS presents a technique that could be used for the separation and quantitation of metal-cell and metal-complex binding on-line in one analytical run.11 In this technique, an aliquot of a bacterial suspension is injected onto a size exclusion column (SEC), and bacteria are excluded from the pore space, thus eluting in the excluded volume. The dissolved constituents that are within the size separation range of the column elute with a retention time inversely related to their molecular weight. Interfacing SEC to ICPMS allows element-specific detection so that metal ion binding by bacteria or dissolved constituents can be determined. However, this analytical approach is somewhat compromised in that bacteria are not retained on the column, and a metal signal in the excluded volume will correspond to metal binding by any excluded species, including dissolved compounds above the molecular weight cutoff of the size exclusion column. Field flow fractionation (FFF) is a size-based separation technique easily interfaced with ICPMS,12,13 which has previously has been applied to the separation of bacteria.7,14,15 The theory of FFF has been well described elsewhere.16 Briefly, a polydisperse sample is injected into a carrier flow in a thin channel and an external force is applied perpendicular to the channel flow. This forces molecules, colloids, and particles toward one of the channel walls (termed the accumulation wall). Counter forces operate against the applied perpendicular force causing the molecules or particles to occupy different equilibrium distances from the accumulation wall. For molecules and colloids of 1 µm (including bacterial cells), diffusion is insignificant. Instead, hydrodynamic lift forces act to move larger particles further away from the accumulation wall, termed hyperlayer mode FFF. Channel flow in FFF has a laminar profile, flow velocity at the center of the channel is greater than at the walls, and the equilibrium height above the channel wall determines the elution time. Thus, a sized-based separation occurs along the length of the channel. However, there is an important difference between normal mode and hyperlayer mode FFF that must be recognized, namely, that for normal mode separations small molecules elute from the channel first, while for hyperlayer mode the order of separation is from large to small. A number of different FFF techniques exist and are differentiated by the type of applied force. Sedimentation FFF, where a centrifugal force is the external applied force, has been most commonly used for bacterial separations15,17,18 and has been successfully used to separate flagellated and nonflagellated Escherichia coli strains.16,19
Flow FFF (Fl FFF), where the field is a perpendicular fluid flow applied to a thin channel via porous frits, has also been used to perform bacterial separations.14,20 In this paper, we report on the use of Fl FFF-ICPMS to study U binding to Shewanella oneidensis. This hyphenated technique has great potential for the study of metal binding in bacterial systems; because bacteria are retained from the void volume, it is possible to alter their retention time by varying the separation conditions. Therefore, it should be possible to study competitive binding in binary and ternary systems. In this preliminary study, we demonstrate the applicability of the technique for determining metal-bacteria sorption and pH isotherms.
(11) Zhang, B.; Li, F. M.; Houk, R. S.; Armstrong, D. W. Anal.Chem. 2003, 75, 6901-6905. (12) Ranville, J. F.; Chittleborough, D. J.; Shanks, F.; Morrison, R. J. S.; Harris, T.; Doss, F.; Beckett, R. Anal. Chim. Acta 1999, 381, 315-329. (13) Taylor, H. E.; Garbarino, J. R.; Murphy, D. M.; Beckett, R. Anal. Chem. 1992, 64, 2036-2041. (14) Saenton, S.; Lee, H.; Gao, Y. S.; Ranville, J. F.; Williams, S. K. R. Sep. Sci. Technol. 2000, 35, 1761-1775. (15) Sharma, R. V.; Edwards, R. T.; Beckett, R. Water Res. 1998, 32, 14971507. (16) Giddings, J. C. Science 1993, 260, 1456-1465.
(17) Sharma, R. V. Edwards, R. T.; Beckett, R. Water Res. 1998, 32, 15081514. (18) Battu, S.; Roux, A.; Delebasee, S.; Bosgiraud, C.; Cardot, P. J. P. J Chromatogr., B 2001, 751, 131-141. (19) Reschiglian, P.; Zattoni, A.; Roda, B.; Casolari, S.; Moon, M. H.; Lee, J.; Jung, J.; Rodmalm, K.; Cenacchi, G. Anal. Chem. 2002, 74, 4895-4904. (20) Lee, H.; Williams, S. K. R.; Wahl, K. L.; Valentine, N. B. Anal. Chem. 2003, 75, 2746-2752. (21) Kostka, J. E.; Nealson, K. H. In Techniques in Microbial Ecology; Burlage, R. S., Atlas, R., Stahl, D., Geesey, G. G., Sayler, G., Eds.; Oxford University Press: New York, 1998; pp 58-78.
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MATERIALS AND METHODS Bacterial Growth and Spiking Protocol. S. oneidensis strain MR-1, a Gram-negative facultative Fe-reducing bacterium was used in the experiments described here. The organism as used in our laboratory expresses green fluorescent protein (GFP) encoded on a p519nGFP plasmid. MR-1 was cultured aerobically for 24 h in M1 medium21 at pH 7.2 and 27 °C. In addition to 32 mM lactate, which served as carbon source, the medium contained 32 mM NaHCO3, 9 mM (NH4)2SO4, 1 mM MgSO4, 0.5 mM CaCl2, 67.2 µM Na2EDTA, 56.6 µM H3BO3, 10 µM NaCl, 5.4 µM FeSO4, 5 µM CoSO4, 5 µM Ni(NH4)2SO4, 3.9 µM Na2MoO4, 1.5 µM Na2SeO4, 1.3 µM MnSO4, 0.2 µM CuSO4, 20 mg L-1 arginine, 20 mg L-1 serine, and 20 mg L-1 glutamic acid. This medium commonly also contains a phosphate salt; however, this was not included in order to avoid the possibility of uranium phosphate mineral precipitation. The culture was continuously supplied with filter-sterilized (0.2 µm) air. Cells were harvested by centrifugation and washed 3 times in sterile defined medium lacking lactate. Washed cells were resuspended in 100 µL of sterile medium and used as a cell stock solution. The cell density of the stock solution, as estimated by optical density, was ∼107 cells mL-1. For sorption experiments, 10 µL of cell suspension was added to 100 µL of pH buffer and 10 µL of the appropriate U solution. The U amendment was prepared from a 1000 mg kg-1 primary U standard and serially diluted in the carrier solution buffer. Uranium solubility calculations using Visual MINTEQ predicted no U mineral precipitation under all the conditions of the experiments. Initial experimentation showed that 1 h was sufficient for sorption to reach completion. Cell integrity following exposure to U was checked using epifluorescent microscopy. GFP fluorescence was observed using a WIBA filter block (460-490-nm excitation; 505nm dichroic mirror; 515-550-nm emission, Olympus America Inc.) and 600× magnification. Images indicated that no cell lysis occurred during U exposure. Flow Field Flow Fractionation-ICPMS. A FFF1000 flow field flow fractionation system (Postnova Analytics, Salt Lake City, UT) was used to perform the cell separations. Hyperlayer separation
Figure 2. Separation of cells by Fl FFF with 210-nm detection.
Figure 1. Separation of four sized polystyrene beads by hyperlayer Fl FFF.
conditions were used with channel flow of 4 mL min-1, a crossflow of 0.4 mL min-1, and a relaxation time of 4 min. The carrier solution was 10 mM NH4Cl with 0.005% v/v Triton added to reduce interactions between the cells and the FFF membrane. The carrier solution was adjusted to the desired experimental pH with NH4OH or HCl. A 25-µL injection loop was employed with a 1 kDa MWCO regenerated cellulose membrane acting as the accumulation wall. The FFF separation conditions were first optimized with separation of a mixture of polystyrene beads (Duke Scientific) of 8, 5, 2, and 0.8 µm (UV detection only). Sorption isotherms were carried out with buffer and FFF carrier solution at pH 5. pH isotherm experiments were performed over the pH range 5-9, and in this case, the FFF carrier solution was also pH adjusted and allowed to equilibrate through the FFF channel for 30 min prior to the appropriate FFF analysis. The carrier solution from the channel was directed first through a UV detector (210 nm) and then to an ICPMS (Elan DRC, Perkin-Elmer, Shelton, CT.) The carrier flow was split after the UV detector, and the peristaltic pump of the ICPMS was adjusted to draw carrier flow to the ICPMS spray chamber at a flow rate of 2 mL min-1. A concentric nebulizer and cyclonic spray chamber were used for sample introduction to the ICPMS. Nebulizer gas flow and lens voltage were optimized daily for maximum signal intensity at m/z 238. ICPMS data were collected at m/z 238 using a data-only method, with a dwell time of 500 ms, 1 sweep per reading, and 1800 readings per replicate. This enabled ICPMS data to be collected at the same time frequency as UV data. RESULTS AND DISCUSSION Optimization of the Hyper Layer Fl FFF Separation. A full mathematical description of hyperlayer FFF is lacking, so polystyrene microspheres of known diameter were used to derive an empirical calibration (Figure 1). Smaller particles are retained longer than large particles, but complete separation of the two larger bead sizes is incomplete. The channel volume is 1.5 mL, and the channel flow rate is 4 mL min-1. The 8-µm bead is poorly
retained under these conditions. However, good baseline resolution was achieved over the 1-5-µm size range, expected for S. oneidensis. It should be noted that biomolecules and particles of 200 kDa in mass, while epifluorescent microscopy of the cell suspensions did not indicate any cell agglomeration, which could otherwise be responsible for an early-eluting peak under hyperlayer separation mode. Further investigations of the exopolymer, including purification and characterization studies, are ongoing. Increased sorption by the exopolymer and decreased sorption at the cell surface led to the expolymer being a stronger sportive phase for U at pH >8 (Figure 5). However, it should be noted that the amount of U retained by the FFF channel, i.e., U bound by the cell surface or the exopolymer, decreases as the pH of the system
increases, consistent with the formation of U carbonate and hydroxide species that would pass through the FFF membrane. CONCLUSIONS Flow FFF interfaced with ICPMS is a powerful technique for studies of metal binding to bacteria. Previous studies have shown that Fl FFF can separate different bacterial strains, and this study shows that by interfacing Fl FFF with ICPMS competitive metal binding between bacterial strains and other solid-phase and solution species could be studied. Similarly, competitive binding between multiple metals and bacteria, or studies of ternary systems containing humic acids or colloids, could also be analyzed by Fl FFF-ICPMS. Unexpected solution speciation, in this case the production of an exopolymer with binding affinity for U, which would not be observed by conventional batch analysis, is detected using this on-line sized-based, metal-specific analytical technique.
ACKNOWLEDGMENT This research was partially supported by the Environmental Remediation Sciences Division of the Office of Biological and Environmental Research, U.S. Department of Energy, through Financial Assistance Award DE-FC09-96-SR18546 to the University of Georgia Research Foundation. Funding was also provided by USEPA through a Star Grant (R-82-6651-01-0) and through the Center for the Study of Metals in the Environment (EPA Grant R-82-950001, Sub award 718).
Received for review May 17, 2004. Accepted November 19, 2004. AC049278Q
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