Evidence of uranium uptake in Acidovorax facilis cells by TRLFS and EF-TEM/EELS

For bioremediation of uranium contaminated environments from activities such as uranium mining and uranium processing, microorganisms could be important due to their ability to immobilize radionuclides and heavy met-als. Since the main public concern is the possibility of radionuclide escaping and migrating into groundwater, there is an intense interest in the development of effective remediation methods. The aim is to improve biore-mediation strategies, based on a better understanding of binding mechanisms on the molecular level.

For our studies we used Acidovorax facilis (formerly Pseudomonas facilis), an aerobic Gram-negative Betapro-teobacteria, which is commonly found in soil. Experiments were performed in batch cultures under aerobic con-ditions at 25 °C using nutrient broth. The cells were grown to an optical density (OD600) of around 1.5. For U(VI) biosorption experiments the cultures were washed 2 times with tap water and then resuspended in tap water. After that UO2(NO3)2 was added to the solution to achieve an initial uranium concentration of 0.05 and 0.1 M, respectively, at a neutral pH range. The duration of the sorption experiments were limited to 48 h. As a re-sponse to uranium stress Acidovorax f. were forming extracellular polymeric substances (EPS) resulting in the formation of cell agglomerates. For separating the EPS from the bacteria, the cell agglomerates were ultra-centrifuged (40.000 x g) for 2 h at 10 °C. The cell pellet was used for time-resolved laser fluorescence spectros-copy (TRLFS). The U(VI) luminescence at 274 K was measured after excitation with laser pulses at 266 nm and with an average pulse energy of 300 μJ. The emitted fluorescence light of the cell pellet was recorded using an iHR550 spectrograph and an ICCD camera in the 370 – 670-nm wavelength range by averaging 100 laser pulses and using a gate of 2000 µs. The measured emission spectrum of the pellet is characterized by five emission bands. Their peak maxima were observed at 481.2, 497.8, 519.5, 544.1 and 569.3 nm ± 0.5 nm. In addition, the spectra of the Uranyl-complexes of lipopolysaccharide (R−O−PO3−UO2) and peptidoglycan (R−COO−UO2) were used for comparison. The reference spectra display band positions at 481.5, 498.1, 519.6, 542.9 and 567.5 nm for pH 4 [1] as well as 481.6, 498.1, 518.0, 539.0 and 566.0 nm for pH 4 [2], respectively. They show only a small deviation from those observed in our studies with the best agreement by those of the Uranyl-lipopolysaccharide-complex. Hence, it can be concluded that phosphoryl groups may be the main binding sites for uranyl, located in the lipopolysaccharide Acidovorax f. cells. But, at this moment it is not clear if it is bound on carboxylic functionality groups either.

Acidovorax f. cells were prepared for Energy-filtered transmission electron microscopy (EF-TEM) and electron energy-loss spectroscopy (EELS) by following the routine embedding protocol as described in [3]. The results provide microscopically and spectroscopically evidence of Uranium sorbed at the outer membrane of Acidovorax f. cells by showing high electron density and U ionization intensity peaks. The results support the TRLFS measurements and contribute to a better understanding of the binding mechanisms of U(VI) on Acidovorax f. cells.

[1] Barkleit, A. et al. (2008) Dalton Transaction, 2879–2886.
[2] Barkleit, A. et al. (2009) Dalton Transaction, 5379–5385.
[3] Lünsdorf, H. et al. (2001) Methods in Enzymology 331, 317–331.