Interactions of natural occurring eukaryotic microorganisms isolated from the uranium mine Königstein (Saxony, Germany) with U(VI)

Despite high uranium concentrations (up to 14 mg L-1) and low pH (2.5 - 3.0) a high microbial diversity was detected by culture independent methods in the flooding water of the former uranium mine Königstein (Saxony, Germany). In this study we used culture dependent techniques for the isolation of eukaryotic microorganisms from the flooding water. It was possible to isolate different eukaryotic fungi with a glucose riche medium. The microbial isolates identified by 16S rDNA and 18S rDNA analysis were tested for their uranium tolerance abilities by the determination of the minimal inhibitory concentration (MIC) on solid media. The results showed high tolerances of uranium (up to 6 mM) on solid agar plates. Based on these results isolate KS5 (Rhodosporidium toruloides) and one reference organism DSM 10134 (Rhodosporidium toruloides) were selected for further uranium interaction experiments. Uranium biosorption tests indicated that the cells of the strain KS5 remove high amounts of uranium (120 mg uranium/ 1 g dry biomass). Temperature dependent biosorption tests with a U(VI) concentration of 100 µM showed significant differences: KS5 revealed twice as much uranium removal at 30°C compared to at 4°C (s. Fig. 1). Since active processes, e.g., bioaccumulation do not occur at low temperature, only minor amounts of U(VI) are taking up in the cytoplasm of the cells. Hence, U(VI) is preferentially sorbed on the cell membrane by the passive process of biosorption.
In order to test the uranium tolerance quantitatively in liquid media flow cytometry experiments with the strains KS5 and DSM 10134 were performed. For this purpose live-dead staining were done to test the cell viability. The cells were stained with Propidium Iodid (PI - non viable cells) and Fluorescein Diacetate (FDA - viable cells). Furthermore the oxidative stress response was measured with the fluorescent dye 3,3'-Dihexyloxacarbocyanine Iodide (DiOC6 - cell membrane of living cells). The results showed that the isolate KS5 are able to tolerate higher U(VI) concentrations compared to the reference culture DSM 10134. More than 50 % of the KS5 cells are viable at an initial U(VI) concentration of 100 µM. In contrast less than 10 % of the reference cells are viable at the same uranium concentration. The results of the oxidative stress response showed a slight difference to the cell viability test. The isolate KS5 showed that nearly 50 % of the cells are active, like the cell viability test. Whereas the results for the reference DSM 10134 revealed that more than 30 % of the cells are active and exhibit an active membrane potential, in contrast to the results of the cell viability test. This can be explained by the stress response in the presence of heavy metals. Some cells produce mechanic-sensitive receptors which are permeable for Propidium Iodide, resulting in a wrong fluorescent staining signal. Thus, it would be an asset to combine both methods, like cell viability test and oxidative stress response tests.
Summarizing the results of this study, we were able to prove that eukaryotic microorganisms within a uranium-contaminated environment exhibit adaption mechanisms against high U(VI) concentrations. The expensive chemical treatment of the flooding water in Königstein could take a long time probably for the next 100 years. For that reason, these isolated eukaryotic microorganisms might play an important role in the bioremediation of radionuclides within the waste water treatment in Königstein.