Biological Reduction of a U(V)-Organic Ligand Complex

Abstract

Metal-reducing microorganisms such as Shewanella oneidensis MR-1 reduce highly soluble species of hexavalent uranyl (U(VI)) to less mobile tetravalent uranium (U(IV)) compounds. The biologically mediated immobilization of U(VI) is being considered for the remediation of U contamination. However, the mechanistic underpinnings of biological U(VI) reduction remain unresolved. It has become clear that a first electron transfer occurs to form pentavalent (U(V)) intermediates, but it has not been definitively established whether a second one-electron transfer can occur or if disproportionation of U(V) is required. Here, we utilize the unusual properties of dpaea^2- ((dpaeaH₂═bis(pyridyl-6-methyl-2-carboxylate)-ethylamine)), a ligand forming a stable soluble aqueous complex with U(V), and investigate the reduction of U(VI)-dpaea and U(V)-dpaea by S. oneidensis MR-1. We establish U speciation through time by separating U(VI) from U(IV) by ion exchange chromatography and characterize the reaction end-products using U M₄-edge high resolution X-ray absorption near-edge structure (HR-XANES) spectroscopy. We document the reduction of solid phase U(VI)-dpaea to aqueous U(V)-dpaea but, most importantly, demonstrate that of U(V)-dpaea to U(IV). This work establishes the potential for biological reduction of U(V) bound to a stabilizing ligand. Thus, further work is warranted to investigate the possible persistence of U(V)-organic complexes followed by their bioreduction in environmental systems.

Keywords: HERFD-XANES; Shewanella; bioremediation; disproportionation; electron transfer; pentavalent uranium; scanning transmission electron microscopy (STEM); uranium reduction.

Figure 1

A. U concentration in the incubation supernatants (pink dots) and in no-cell controls (yellow dots), measured from 0 to 96 h of incubation with U(VI)–dpaea (in duplicates). B. U speciation in the solid phase and in the aqueous phase of the incubations (in μg). The U(VI) and U(IV) fractions were obtained by ion exchange chromatography.

Figure 2

U M₄-edge HR-XANES spectra demonstrating reduction of (A) U(VI)–dpaea and (B) biologically produced U(V)–dpaea. The references U(VI)–dpaea (in red), U(V)–dpaea (in pink) and U(IV)–dpaea₂ (in blue) are presented in both panels. The spectra obtained for UO₂ is shown as a dashed line. U(IV)–dpaea₂ was used as a reference and likely does not fully characterize the U(IV) phases present. In addition to the references, the spectra obtained for (A) the aqueous phase after 72h and 96h of incubation, and for (B) the solid phase (cell pellet) collected after 85 h of incubation of S. oneidensis MR-1 with U(V)–dpaea are presented.

Figure 3

Aqueous and solid phase U concentration through time in incubations with S. oneidensis MR-1 and no-cell controls with (A) biologically produced U(V)–dpaea (B) synthetic U(V)–dpaea.

Figure 4

U oxidation state in the solid phase (cell pellet) and in the aqueous phase (supernatant) of (A) incubations with S. oneidensis MR-1 (B) and no-cell controls, in the presence of “biological” U(V)–dpaea. The U(VI) and U(IV) fractions were obtained by ion exchange chromatography. The ion exchange chromatography separation cannot directly identify U(V), because the samples are acidified prior to loading onto the column. Acid treatment is known to disproportionate uranyl(V) and to produce equal proportions of U(VI) and U(IV). Therefore, here the equal proportions observed for U(VI) and U(IV) in the supernatant are a proxy for U(V) (result demonstrated by U M₄-edge HR-XANES).

Figure 5

STEM images acquired on S. oneidensis MR-1 incubated with U(V)–dpaea for 3 months. We mainly observed two types of uranium morphologies. A, B, and C describe morphology type 1 corresponding to U aggregates smaller than 200 nm dispersed at the surface of the bacteria. D, E, and F show morphology type 2, in which U forms larger (>1 μm or μm) clusters in association with the bacterial cells.

Figure 6

Aqueous U(V)–dpaea concentration (A) and cell viability (B) through time in incubations with S. oneidensis MR-1, the deletion mutant ΔccmG and a no-cell control.