Identification of Fluorescent U(V) and U(VI) Microparticles in a Multispecies Biofilm by Confocal Laser Scanning Microscopy and Fluorescence Spectroscopy.

A combined approach of laser fluorescence spectroscopy and confocal laser scanning microscopy was used to study in vivo fluorescent particles in living multispecies biofilms which were exposed to 10-5 M and 5×10-6 M uranium, respectively. 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.
Analysis of the amplified 16S rRNA gene fragments have shown that the biofilm consisted of members of the alpha-, beta-, and, gamma-subgroup of Proteobacteria, as well as of representatives of Actinobacteria, Firmicutes, and Bacteroidetes. Restriction fragment length polymorphism (RFLP) screening and cloning of selected clones indicated a similar composition of the microbial communities in biofilm reactors with and without U addition. Both clone libraries were dominated by sequences affiliated to the alpha-subgroup of Proteobacteria. Fluorescence in situ hybridization (FISH) with group specific probes indicated a slightly higher coverage of the uranium contaminated biofilm by cells affiliated with the beta-Proteobacteria. Beta-Proteobacteria are recognized for their ability to reduce uranium in the presence of nitrate. However, an enrichment of additional bacteria known for their ability to reduce U(VI) did not become apparent, neither by 16S rRNA gene retrieval nor by the FISH technique. From both techniques it can be concluded that the multispecies biofilms grown without and with uranium addition were very similar regarding their microbial composition.
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 biofilm. 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.