The immobilization of uranium in multispecies biofilms studied by microsensors and confocal microscopy

Multispecies biofilms were cultured in standard culture media (Sifin; TN 1171) with a pH of approximately 7.2, and in air atmosphere condition at room temperature (20 °C). The culture media were pumped through three annular reactors for two months with a flow-rate of 15.2 ml/min, and an inner cylinder rotation speed of 14 rpm. Inside the reactors biofilms were grown on glass slides to a thickness of approximately 600 µm. Two reactors were fed with UO2(ClO4)2 to adjust the total uranium concentration in the culture medium in ecological relevant concentration (5×10-5 M and 5×10-6 M). After three weeks the cultured multispecies biofilms were used for microscopical and spectroscopical studies (CLSM) as well as for O2 microsensor studies.
Fluorescent uranium(V) and uranium(VI) particles were observed for the first time in vivo by a combined laser fluorescence spectroscopy and confocal laser scanning microscopy approach in a living multispecies biofilm grown on biotite plates. These particles ranged between 1 and 7 µm in width and up to 20 µm in length and were located at the bottom and at the edges of biofilms colonies. Anaysis of amplified 16S rRNA fragments and fluorescence in situ hybridization were used to characterize the biofilm communities. Laser fluorescence spectroscopy was used to identify these particles. The particles showed either a characteristic fluorescence spectrum in the wavelength range of 415-475 nm, indicative for uranium(V), or in the range of 480-560 nm, which is typical for uranium(VI). Particles of uranium(V) as well as uranium(VI) were simultaneously observed in the biofilms. These uranium particles were attributed for uranium(VI) to biologically mediated precipitation and for uranium(V) to redox processes taking place within the biofilms. The detection of uranium(V) in a multispecies biofilm was interpreted as a short-lived intermediate of the uranium(VI) to uranium(IV) redox reaction. Its presence clearly documents that the uranium(VI) reduction is not a two electron step but that only one electron is involved.
Concentration profiles of oxygen versus biofilm depths were measured in the biofilms by electrochemical microsensors with a tip diameter of 10µm. A motor-driven micromanipulator was used for moving downwards through the biofilm in 20 or 50µm steps. The microsensor results clearly showed that the vertical profiles of the O2 concentration within the biofilms are affected by the presence as well as by the concentration of U(VI) in the culture media. In the absence of uranium the O2 concentration in the well aerated biofilm decreased slightly. In contrast, O2 concentrations in the biofilms, which were exposed to different concentrations of uranium, decreased with increasing uranium concentration.