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 bac- teria 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 equiva- lent 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 con- centrations 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 ad- ditional 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 remedia- tion 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 bioremedia- tion, bacteria effect a change in speciation of a contaminant that subsequently lowers its availability, 1-3 while in passive remediation 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 mineral- associated contaminants. Increasingly accurate models of con- taminant 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 putrefaciens 8 and Bacillus subtilis 9 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 * Corresponding author. Phone: 303-273-3004. E-mail: jranvill@mines.edu. † 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. (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. Anal. Chem. 2005, 77, 1393-1397 10.1021/ac049278q CCC: $30.25 © 2005 American Chemical Society Analytical Chemistry, Vol. 77, No. 5, March 1, 2005 1393 Published on Web 01/22/2005