The effect of bacterial growth phase and culture concentration on U(VI) removal from aqueous solution

Bacteria play a key role in controlling the mobility of contaminants, such as uranium (U), in the environment. Uranium could be sourced from disposed radioactive waste, derived either from surface disposal trenches for Low Level Waste (LLW) that, because of the waste type and disposal concept, would typically present acidic conditions or from the geological disposal of LLW or Intermediate Level Waste (ILW) that, because of the waste type and the disposal concept, would typically present alkaline conditions. In disposed radioactive waste, there could be variable amounts of cellulosic material. Bacterial cells may be living in a range of different growth phases, depending on the growth conditions and nutrients available at the time any waste-derived U migrated to the cells. A key knowledge gap to date has been the lack of a mechanistic understanding of how bacterial growth phases (exponential, stationary, and death phase) affect the ability of bacteria to remove U(VI) from solution. To address this, we first characterised the cells using potentiometric titrations to detect any differences in proton binding to proton active sites on Pseudomonas putida cells at each growth phase under aerobic conditions, or under anaerobic conditions favourable to U(IV) reoxidation. We then conducted batch U(VI) removal experiments with bacteria at each phase suspended in 1 and 10 ppm U aqueous solutions with the pH adjusted from 2 to 12 as well as with culture concentrations from 0.01 to 10 g/L, to identify the minimal concentration of bacteria in solution necessary to affect U removal. We found that, in death phase, P. putida cells exhibited double the concentration of proton active sites than bacteria grown to exponential and stationary phase. However, we did not see a difference in the extent of U(VI) removal, from a 10 ppm U solution, between the different growth phases as a function of pH (2 to 12). Culture concentration affected U removal between pH 2–8, where U removal decreased with a decreasing concentration of cells in solution. When the pH was 10–12, ≤55% of U precipitated abiotically. The presence of bacteria in solution (0.01–10 g/L), regardless of growth phase, increased the precipitation of U from ≤55% up to 70–90%, accumulating inside the cells and on the cell walls as ~0.2 μm uranyl phosphate precipitates. These precipitates were also found at low pH with the exception of cells at exponential growth phase. This study demonstrates that growth phase affects the proton-active site concentration but not the extent of U bound to P. putida cells and that growth phase dictates the form of U removed from solution. Since the pH of trench-disposed LLW is controlled by the degradation of cellulosic waste, leading to acidic conditions (pH 4–6), bacterial concentrations would be expected to highly affect the extent of U removed from solution. The cement in grouted ILW and LLW, for geologic disposal, will allow for the development of extremely high pH values in solution (pH 9–13), where even the smallest concentrations of bacteria were able to significantly increase the removal of U from solution under aerobic conditions, or under anaerobic conditions favourable to U(IV) reoxidation