Characterization of Pyoverdins Secreted by a Subsurface Strain of Pseudomonas fluorescens and Their Interactions with Uranium(VI)

Abstract

Fluorescent Pseudomonas species secrete pyoverdin-type siderophores with a high potential to dissolve, bind, and thus transport uranium in the environment. The formation of complexes of UO2 2 + with pyoverdins released by the groundwater bacterium Pseudomonas fluorescens (CCUG 32456) isolated at a depth of 70 m in the Äspö Hard Rock Laboratory, Sweden, was studied. Mass spectrometry indicated that the cells produce a pyoverdin–mixture with four main components: pyoverdin with a succinamide side chain, pyoverdin with a succinic acid side chain, ferribactin with a succinamide side chain, and ferribactin with a glutamic acid side chain. Three pK values could be determined from the pH-dependent changes in the absorption spectra of the pyoverdin mixture: log ß012 = 22.67 ± 0.15 (pK1 = 4.40), log ß013 = 29.15 ± 0.05 (pK2 = 6.48), and log ß014 = 33.55 ± 0.05 (pK3 = 10.47). The fluorescence properties of the pyoverdin mixture were pH-dependent. The emission maximum changed from 448 nm at pH = 2.1 to 466 nm in the pH 3.8–8.9 range. At pH > 4 a mono-exponential fluorescence decay dominates with a decay time of 5865 ± 640 ps. A drastic change in the intrinsic fluorescence properties, e.g., static fluorescence quenching, occurred due to the complex formation with UO2 2 +. Species containing UO2 2+ of the type M p L q H r were identified from the dependencies observed in the ultraviolet visible and time-resolved laser-induced fluorescence spectroscopy spectra at pyoverdin concentrations below 0.1 mM. The following average formation constants were determined: log ß112 = 30.00 ± 0.64 and log ß111 = 26.00 ± 0.85 at ionic strength I = 0.1 M (NaClO4). The determined stability constants can be used directly in safety calculations of the mobilizing effect of released pyoverdins on uranium, in uranium-contaminated environments such as mine waste disposal sites.