Controls of uranyl species and metabolism on Bacillus subtilis-enhanced uranium bio-immobilization

Although uranium (U) bio-immobilization under aerobic conditions has been proven to be a viable bioremediation strategy, the roles of uranyl species and microbial metabolism related to specific hydrochemical components in U bio-immobilization are not well-understood. Groundwater samples were collected in a typical U-containing sandstone aquifer mined by acid in-situ leaching to investigate the specific hydrochemical components that potentially affected uranyl species and microbial metabolism and subsequently U bio-immobilization in the presence of Bacillus subtilis. Results showed that groundwater in the decommissioned acid in-situ leaching U mining area was characterized by low pH and high concentrations of U, SO₄^2-, Ca²⁺, Mg²⁺, K⁺, TOC, and F^-. According to the toxicity to microorganisms and the impacts on the elemental compositions of U immobilization products, H⁺, U, SO₄^2-, Ca²⁺, Mg²⁺, K⁺, F^-, HA, and FA were screened as the specific components potentially affecting U bio-immobilization. Uranium bio-immobilization experiments showed that Bacillus subtilis immobilized U by biosorption, biomineralization (forming sodium uranite), and bio-reduction of U(VI). The contribution of metabolic dependent U bio-immobilization to the total U bio-immobilization reached the maximum under neutral conditions (about 45 %) due to the non-acid resistance properties of Bacillus subtilis. Solid characterization showed that Bacillus subtilis-induced U extracellular bio-immobilization was preferred under neutral conditions, followed by cell surface bio-immobilization and intracellular bio-immobilization. The hydrochemical components (including HA, FA, H⁺, U, SO₄^2-, Ca²⁺, and F^-) changed the U bio-immobilization priority by regulating uranyl species and the microbial metabolism. HA and FA decreased the nucleation and growth rate of intracellular uranyl phosphate mineral by forming cell-HA/FA-UO₂²⁺ ternary complexes. As the H⁺ concentration increased, the metabolic activity gradually decreased, with U bio-immobilization location shifting from extracellular space to the cell membrane and intracellular space, and the gradually increased competitive adsorption of H⁺ with UO₂²⁺ reduced U bio-immobilization efficiency. SO₄^2-, Ca²⁺, and F^- affected U bio-immobilization by regulating microbial metabolic pathways. SO₄^2- promoted the bio-reduction of U(VI), and Ca²⁺ entered the cell to complex with U, both of which promoted the accumulation of intracellular uranyl phosphate minerals. The presence of F^- stimulated the P metabolism to reduce F^- toxicity to cells, weakening the complexation between U and P and ultimately inhibiting U bio-immobilization. This study provides new insights into the roles of hydrochemical components in U bio-immobilization by regulating uranyl species and the microbial metabolism.