## 4 Results and Discussion
### Assessment of Biofilm Formation and the Role of biofilms in Uranium Immobilization  
In laboratory experiments using several methods, the formation of microbial biofilms by microorganisms on loam (sandy loam) from boreholes 1, 2, and 3 after single glucose-acetate activation was investigated. Microphotographs obtained using confocal microscopy (Figure 4) show maximum biofilm development on the 20 th day of the experiment. On the 40 th day, the biofilm matrices of microbial communities on the three rock samples deteriorated, and only in some areas of the rock did attached microorganisms remain. Based on visual analysis, the maximum accumulation of cellular nucleic acids (green colour) before glucose activation was observed in the loam of Sample 3. The maximum accumulation of polysaccharides on the 20 th day was observed for the rock of Sample 1, but on the 40 th day, the maximum polysaccharide amount was observed for the loam of Sample 3, indicating uneven biofilm development on rocks during interactions with solutions of different compositions. On the rock sample suffering from maximum technogenic impact, biofilm development was slower on the 20 th day, but the deterioration rate on the 40 th day was also not rapid. The confocal microscopy visualization data were confirmed by the MTT assay data (Table 6). The respiratory metabolism of microbial biofilms reached the maximum on the 20 th day, with the largest respiratory activity observed for the microbial community of Sample 1, which was in contact with groundwater and the minimum observed for the rock of Sample 3, which was mostly affected by nitrate sulphate water. The maximum area occupied by biofilms before microbial stimulation was largest on the sample with maximum contamination, and after stimulation, the area was largest on the uncontaminated sample. It should be noted that on the 40 th day, the biofilm area on Sample 3 was two times larger (15.5%) than the areas measured on the other samples. The formation of a more developed biofilm and the lower rate of biofilm deterioration on the most contaminated rock may indicate the important role of biofilms in microbial processes with a high nitrate background. It should be added that, based on the analysis of 16S rRNA genes in the biofilms, on both samples the dominance of representatives of the families _Commonadaceae_ (genus _Acidovorax_) and _Pseudomonadaceae_ (genus _Pseudomonas_) was observed. The formation of biofilms on rocks led to a change in the sorption capacity of the samples towards uranium. Samples after 20 days of the experiment were used as sorbents (Figure 4, Table 6). The \(\rm K_{d}\) values calculated from the experimental results are presented in Table 7. A notable increase in the \(\rm K_{d}\) value occurred for the rock of Sample 3, with increases of 24% and 30.5% (for distillate and groundwater solutions, respectively), and for Sample 1, with values of 33% and 37%, caused by the coating of particles with biofilms. We associated the higher sorption characteristics for Sample 1 with the higher degree of biofilm coating of the particles, explained by an increase in the number of sorption sites on the polysaccharide matrix on the rock surface, including carboxyl and hydroxyl groups [40]. \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \multirow{2}{*}{**Sample**} & \multicolumn{3}{c}{**0 Day**} & \multicolumn{3}{c}{**20 Days**} & \multicolumn{3}{c}{**40 Days**} \\ \cline{2-10}  & **NA** & **Bf** & **MTT** & **NA** & **Bf** & **MTT** & **NA** & **Bf** & **MTT** \\ \hline
1 & 1.03 \(\pm\) 0.3 & 1.7 \(\pm\) 0.4 & 0.13 & 10.3 \(\pm\) 2.6 & 41.8 \(\pm\) 3.4 & 13.5 & 2.6 \(\pm\) 1.2 & 7.8 \(\pm\) 1.8 & 1.7 \\
2 & 3.6 \(\pm\) 0.5 & 3.8 \(\pm\) 0.6 & 1.15 & 7.8 \(\pm\) 1.5 & 34.8 \(\pm\) 2.7 & 11.1 & 7.9 \(\pm\) 1.2 & 8.4 \(\pm\) 1.3 & 2.1 \\
3 & 4.05 \(\pm\) 0.5 & 4.7 \(\pm\) 0.5 & 1.12 & 4.2 \(\pm\) 0.5 & 28.9 \(\pm\) 1.2 & 5.2 & 6.6 \(\pm\) 1.2 & 15.5 \(\pm\) 0.7 & 1.8 \\ \hline \end{tabular}
\end{table}