Uranium(VI) reduction by a Desulfitobacterium species in pure culture and in artificial multispecies bio-aggregates

The reduction of highly mobile and water soluble U(VI) to less mobile U(IV) represents a key process influencing the migration of this radionuclide in the environment. Microorganisms such as for example iron and sulfate-reducers are capable of reducing U(VI) under various conditions. This interaction mechanism between microbes and U could play an important role in a final disposal site for high-level radioactive waste deposited in deep geological layers. Different host rocks are suitable for the long-term storage of nuclear waste, e.g. clay formations, crystalline rock and rock salt. Besides the geochemical, geophysical and geological properties of such a repository, little is known about the influence of naturally occurring microorganism on the safety of such a site. In a worst-case scenario, if water enters the repository, radionuclides can get distributed in the surrounding host rock and thus interact with the native microorganisms, potentially leading to an immobilization of radionuclides via bioreduction. Furthermore, a reduction of U(VI) could also play an important role in the development of different bioremediation approaches for radionuclide-contaminated environments. As a potential component of new remediation strategies, we introduce the use of artificial bio-aggregates of different bacterial genera. By the use of these artificial biofilms insights into the complex interactions in a multi-species environment can be obtained. In this study, we used derivatized polyelectrolytes to form aggregates of two different microorganisms to connect advantageous properties of the microorganisms in a complementary way and to investigate the reduction of U(VI) under different conditions.
Desulfitobacterium sp. G1-2 was chosen as an important representative of iron-reducing bacteria in anaerobic environments. This bacterial strain was isolated from bentonite samples of the Full-scale Engineered Barrier Experiment – Dismantling Project (FEBEX-DP) at the Helmholtz Center Dresden-Rossendorf. Bentonites are supposed to serve as a possible backfill material, not only for a final disposal site in clay formations but also in crystalline rock. Furthermore, Desulfitobacterium species were detected in other clay formations as well, for example in Opalinus Clay.[1] These were used to form artificial bio-aggregates with different bacterial strains (among others Desulfitobacterium sp. G1-2 and aerobic marine inhabitant Cobetia marina DSM 50416) using electrostatic modifications of surface charge of bacterial cells.
Time-dependent experiments of Desulfitobacterium sp. G1-2 alone in 30 mM bicarbonate buffer (100 µM U(VI), 10 mM lactate) showed a decrease in U concentrations in the supernatants. Moreover, artificial Opalinus Clay pore water[2] (100 µM U(VI), 10 mM lactate, pH 5.5) was used as background electrolyte, as well, to create more environment-related conditions. In both cases, approximately 80% of the uranium was removed from the supernatants after one week. In order to be able to exclude abiotic influences on the uranium(VI) reduction, experiments using heat-killed cells were carried out, as well. Thermodynamic calculations of the U(VI) speciation in both solutions revealed the predominance of different U(VI) complexes in the used media. UV/Vis studies of the dissolved cell pellets verified the formation of U(IV) by an almost complete reduction of U(VI) in bicarbonate buffer and artificial Opalinus Clay pore water. In contrast, experiments with heat-killed cells did not show any reduction of U(VI) in the samples. STEM investigations coupled with EDX analysis of U-incubated cells showed the presence of two different U-containing aggregates inside the cells of Desulfitobacterium sp. G1-2. On the one hand, spherical nanoparticles are present, which are probably containing organic uranium phosphate compounds, as shown by EDX mapping of the samples. On the other hand, rod-shaped particles consisting of inorganic uranium phosphate compounds occur inside the cells as well.
First experiments with artificial bio-aggregates that were formed from different bacterial strains (e.g. Desulfitobacterium sp. G1-2 and Cobetia marina DSM 50416) using electrostatic modification of surface charge of bacterial cells by different polyelectrolytes showed a promising U reduction capacity in bicarbonate buffer (30 mM, 100 µM U(VI), 10 mM lactate). Future investigation will focus on the elucidation of the complex interaction mechanisms in multi-species environments.
This study helps to close existing gaps in a comprehensive safeguard concept for a repository for high-level radioactive waste in clay rock. Moreover, the results of these investigations provide new insights into the U(VI) reduction by iron-reducing microorganisms and thus, contribute to new knowledge on the migration of uranium in the environment. In addition, it may help to establish new bioremediation approaches of contaminated environments, because beneficial microbes can be used for the artificial bio-aggregates, even if they do not form biofilms themselves.

References
[1] A. Bagnoud et al. (2016). Reconstructing a hydrogen-driven microbial metabolic network in Opalinus Clay rock. Nat. Commun. 2016, 7, 1–10
[2] P. Wersin et al. (2011). Biogeochemical processes in a clay formation in situ experiment: Part A - Overview, experimental design and water data of an experiment in the Opalinus Clay at the Mont Terri Underground Research Laboratory, Switzerland. Appl. Geochemistry 2011, 26, 931–953