Study of Microbial Chromium(VI) Reduction by Electron Energy

T.L. Daulton,¹ B.J. Little,² and J.M. Jones-Meehan³
¹Marine Geosciences Division
²Oceanography Division
³Chemistry Division

Introduction: The geochemistry and toxicity of chromium (Cr) are controlled by valence state. Chromium is a redox active 3d transition metal with a wide range (-2 to +6) of possible oxidation states, of which only two are stable. Thermodynamic calculations predict that soluble Cr(VI) is energetically favored for oxic conditions, while insoluble Cr(III) is favored under anoxic or suboxic conditions. Hexavalent chromium species are strong oxidants that act as carcinogens, mutagens, and teratogens in biological systems. Therefore, microbial Cr(VI) reduction is of particular technological and biological importance because it converts a toxic, mobile element into a less toxic, immobile form.

Study of microbial Cr(VI) reduction, such as identification of reduction intermediates, has been hindered by the lack of analytical techniques that can identify oxidation state with subcellular spatial resolution. The most common method for measuring Cr(VI) reduction in bacterial cultures is the diphenylcarbazide colorimetric assay in which Cr(VI) concentration is determined from absorbance at 540 nm by the stoichiometric oxidation products of diphenylcarbazide reagent. However, this bulk technique cannot provide the submicron-scale information necessary for understanding microbial reduction processes. One technique with sufficient spatial resolution is electron energy loss spectroscopy (EELS). EELS directly measures the energy loss of incident electrons that inelastically scatter from atoms in the specimen and is a direct probe of the electron configuration around atoms. Consequently, EELS can identify the oxidation state of 3d and 4d transition metals.¹ Despite the detailed, submicron-scale information EELS techniques can provide on oxidation state, they have never been applied in microbial reduction studies. This article demonstrates the application of EELS for the determination of metal oxidation state in studies of microbial reduction. Specifically, we examined reduction of Cr(VI) in anaerobic cultures of Shewanella oneidensis containing Cr(VI)O₄^2-. S. oneidensis is a gram-negative, facultative bacterium, capable of respiring aerobically and anaerobically by using a variety of terminal electron acceptors.² It is a member of the g-subclass of Proteobacteria, and has been isolated from lascustrine and marine environments.

Methodology: Determination of oxidation state by EELS is accomplished by analyzing valence-induced differences in fine structure of L₂ and L₃ (or collectively L_2,3) absorption edges. The L_2,3 absorption edges arise from transitions to unoccupied d levels from two spin-orbit split levels: the 2p_1/2 level (producing the L₂ edge) and the 2p_3/2 level (producing the L₃ edge). The valence of a transition metal is related to the number of holes in the d level, i.e., the 3d (or 4d) configuration. For example, tetrahedral Cr(VI) has an empty d orbital (3d⁰ configuration) and octahedral Cr(III) has a 3d³ configuration. Since L_2,3 absorption edges are inherently dependent on the number of unoccupied d levels in 3d and 4d transition metals, they are sensitive to valence state. Bacterial cultures were examined directly by environmental cell (EC)-transmission electron microscopy (TEM) at 100 Torr, under a circulation of air saturated with water vapor. The EC-TEM system is of the closed-cell type. A pressurized environment is maintained by two electron-transparent, amorphouscarbon windows with the specimen supported on the lower window. Bacteria were also examined in cross section by conventional TEM after embedding and thin sectioning.

FIGURE 11
Shewanella oneidensis imaged by ECTEM at 100 Torr: bacteria (a) exhibiting low contrast and (b) encrusted with electron dense precipitates. Arrowhead in (b) points to a low contrast bacterium, illustrating the dramatic contrast difference with respect to encrusted bacteria.

Results: Examination by EC-TEM shows the typical rod-shaped morphology of S. oneidensis. In particular, the bacterial membranes are intact and do not show evidence of rupture by partial decompression. Cells remain plump/hydrated, while extracellular polymeric substances encapsulating the cells retain moisture. Electron microscopy reveals two distinct populations of S. oneidensis in incubated cultures containing Cr(VI): cells that exhibit low image contrast (Fig. 11(a)) and heavily precipitate-encrusted cells that exhibit high image contrast (Figs. 11(b)).

Several EELS techniques were applied to determine the oxidation state of Cr associated with the encrusted cells. Oxidation state was determined by measuring the chemical shift and intensity ratios of Cr-L_2,3 adsorption peaks.³ Figure 12 compares the EELS spectra of encrusted, hydrated S. oneidensis collected by EC-TEM to that of Cr oxidation-state standards collected by conventional TEM. The correlation between measured L₃/L₂ integrated-peak intensity ratios and L₃ peak positions for standards demonstrates that different oxidation states fall within well-separated regions (Fig. 13). Within a given oxidation state, spectra of individual standards fall within separate groupings, reflecting possible differences in atom coordination, spin-orbit interactions, and crystal field splitting. Comparison with the standards demonstrates that precipitate-encrusted bacteria contain Cr in oxidation state +3 or lower (Fig. 13). Precipitates encrusting bacteria were also examined in cross section. EELS measurements by conventional TEM of cross sections (Fig. 13) are consistent with measurements of encrusted, hydrated bacteria by ECTEM, demonstrating that EELS provides accurate data, even under the more onerous experimental conditions of the EC.

FIGURE 12
Comparison of EELS spectra of encrusted Shewanella oneidensis in the EC and Cr oxidation-state standards. Spectra were normalized to the intensity of the L3 peak and offset from one another.

Fig13 Image

FIGURE 13
Correlation between measured L3/L2 integrated-peak ratios and L3 peak positions (a) Cr oxidation-state standards, (b) bacteria and precipitates (solid data points represent the mean of the data for a particular Cr standard).

Summary: Chemical and oxidation state information for the microbial reduction of Cr(VI) by the facultative anaerobe Shewanella oneidensis was acquired with high spatial resolution using EELS. We demonstrate that quantitative measurements of oxidation state can be performed on hydrated specimens by EC-TEM. This is the first time the oxidation state of microbial metal-reduction products, localized with bacteria, has been measured. Such information is vital for identifying microbial electron transfer sites and transfer mechanisms.

[Sponsored by ONR]References
¹ R.F. Egerton, Electron Energy-loss Spectroscopy in the Electron Microscope (Plenum, New York, 1996).
² K. Venkateswaran, D.P. Moser, M.E. Dollhopf, D.P. Lies, D.A. Saffarini, B.J. MacGregor, D.B. Ringelberg, D.C. White, M. Nishijima, H. Sano, J. Burghardt, E. Stackebrandt, and K.H. Nealson, "Polyphasic Taxonomy of the Genus Shewanella and Description of Shewanella oneidensis sp. nov.," Int. J. Syst. Bacteriol. 49, 705-724 (1999).
³ T.L. Daulton, B.J. Little, K. Lowe, and J. Jones-Meehan, "Insitu Environmental Cell -Transmission Electron Microscopy Study of Microbial Reduction of Chromium(VI) using Electron Energy Loss Spectroscopy," Microscopy Microanal., in press.