**4. Biologically assisted synthesis**

Nanoparticles UO2 can be obtained also by mediation of living organisms. *Shewanella* genus, for example, belongs to a well-known group of U6+ reducing bacteria. Within this group, anaerobic *Shewanella oneidensis MR-1* and *Shewanella* *putrefaciens CN32* species have been widely used by several authors to produce biogenic uraninite nanoparticles [39–42]. Other species studied were: *Desulfovibrio vulgaris* [43, 44], *Geobacter sulfurreducens* [45, 46], and *Anaeromyxobacter dehalogenans 2CP-C* [47].

One of the main interests related to this topic is the possible use of bacteria in reducing the environmental mobility of the U6+ ions by transforming them into U4+ species. Thus, there is a big effort in determining the factors that affect the physiological state of the microorganisms, which mediate the U6+ reduction, as well as in determining which geochemical and environmental conditions modify the nanobiogenic UO2 surface reactivity [40] and redox potentials [42].

These experiments are conducted in a series of stages: the preparation of a background electrolyte, where the bacteria is allowed to live and growth, the cell cultivation, the U6+ bioreduction experiments and, finally, the determination of U4+ re-oxidation rates under different conditions. In natural environments, uranium might be present in different sites due to the geology of the area but also as a contaminant in soils, sediments, and groundwater [40]. So, on the one hand, the background electrolyte implies the preparation of buffered (6.8–8 pH range) artificial groundwater made of uranyl acetate in 1.2–4 mM concentrations and some organic additives as lactic acid and macronutrients for bacterial growth. On the other hand, cell suspensions are cultured aerobically at 30°C for 24 h; centrifuged, washed with an anaerobic buffer, and resuspended in an anaerobic solution. From this suspension, a portion is inoculated into the buffered, anaerobic uranyl-bearing solution to initiate uranium reduction. After a bioreduction essays, cell-uranium precipitates are pasteurized at 70°C to deactivate biological activity [40–42]. Burgos et al., for example, reported that it was challenging to determine what constitutes a single discrete particle in samples with thick uraninite coatings or large extracellular deposits but regardless of the bioreduction rate or the electrolyte used, identified a mean particle size structure of around 3 nm in TEM micrographs as well as with X-ray absorption fine structure spectroscopy (EXAFS) [39], while Singer et al. found stoichiometric uraninite with particle diameters of 5–10 nm by DRX [40].

Probably one of the most interesting results obtained by these authors was that the bioreduction rate is not the unique factor which controls the particle size of biogenic uraninite. Within the parameters that influence the obtaining of certain particle size, it can be include cell cultivation methods, metabolic state of cells, molecular-scale mechanisms of U6+ reduction, U4+ nucleation site, and cellular location of uraninite precipitates [39].
