X-Ray Fluorescence in Biological Sciences. Группа авторов

obvious effects on microbial growth [7, 8]. At the same time, the production of reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide dismutase (O₂ ⁻) and hydroxyl radicals (OH⁻) may alter the intracellular redox status and ultimately cause cell death when the ROS exceeds the antioxidant capacity of the micro‐organism [9]. Low concentration of AuCl₄ ⁻ (<130 μmol/l) could stimulate Rhizopusoryzae to secrete reductive proteins for biological detoxification, but when the concentration of AuCl₄ ⁻ was higher than 130 μmol/l, the ultrastructure of the cells would be destroyed, and growth was significantly inhibited.

5.3 Application of μ‐XRF and XAS in Understanding the Cycling of Elements Driven by Micro‐organism

      Micro‐organisms are key‐drivers of carbon‐, nitrogen‐, sulfur‐, and metal‐cycling on Earth, and they directly and indirectly drive element cycling [10]. Micro‐organisms can influence the cycling process of heavy metal elements through dissolution, adsorption, transformation, and other processes. XRF and XAS analysis techniques play important roles in revealing the mechanism of heavy metals migration between water, soils, plants, and gases. The toxicity of heavy metals is mainly influenced by its chemical species and mobility.

      XANES analysis, used to detect the species of As, can help to understand the redox mechanism of arsenic, the circulation of arsenic between solid and liquid phases, and the release mechanism of arsenic from sediments into water. Under reducing conditions, 74–85% of the arsenic exists as As (III), which has a high ecological risk. After switching to a toxic condition, the native micro‐organism can oxidize As (III) to As (V) and adsorb it to the solid phase, indicating that oxidation conditions can reduce the solubility and toxicity of As [11].

      Iron‐bearing minerals have strong adsorption capacity for heavy metals. The interaction between micro‐organisms and iron minerals is of great significance to the cycle of heavy metals in the environment. Arsenate is strongly associated with soil minerals, particularly iron(hydr)oxides [12]. The catalytic oxidation of iron has an important effect on the adsorption and chemical species of arsenate. Analysis of XRD and XANES showed that Shewanella putrefaciens could oxidize Fe (II) to feroxyhyte (δ^′‐FeOOH) and goethite (α‐FeOOH), while Sb(V) was reduced to Sb (III) and adsorbed on the surface of these secondary Fe(III) oxides, which can effectively reduce the toxic effect of Sb in aqueous solutions [13]. The mine water contained high concentrations of iron and other heavy metals, and the formation of iron‐containing biofilms may affect the cycling of heavy metals. 16S rDNA, TEM, XRD, XANES, and scanning‐particle induced X‐ray emission analysis (S‐PIXE) results revealed that under the action of Gallionella sp., the concentration of Fe and As decreased significantly, accompanied by the formation of a yellowish biofilm precipitate. The precipitate was confirmed to be schwertmannite containing Fe, As, and S, and consequently As was deposited in the mineral. These results indicated that Gallionella Sp. could promote the formation of iron‐bearing minerals, which improved the adsorption capacity to arsenic [14].

      In the reduction condition, some micro‐organisms may promote the release of arsenic. In anoxic sediments, Geobacter sp. one can use Fe (III) as the electron acceptor to reduce Fe (III) to Fe (II) to dissolve iron‐bearing minerals, accompanied by reduction of As (V) to As (III), so the As originally adsorbed on the surface of iron‐bearing minerals was dissolved and released into the aquatic environment, increasing the ecological risk of As entering the food chain [15].

5.4 Application of μ‐XRF and XAS in Understanding the Transformation of Elements Driven by Micro‐organisms

      In the heavy metal enriched environment, the chemical speciation of heavy metal elements is one of the important factors affecting the growth of micro‐organisms. Toxic heavy metal ions can easily replace essential metal ions that are necessary in many enzymes [4, 5]. Micro‐organisms can detoxify through biological processes and survive by changing the chemical speciation, bioavailability, and toxicity of heavy metals.

Redox of toxic elements by micro‐organisms is an important detoxification mechanism. Au (I/III)‐complexes are highly toxic to organisms, while Au nanoparticles (AuNPs) are relatively non‐toxic [16, 17]. Once Au (I/III)‐complexes enter the cells, they easily bind with glutathione and react with oxygen molecules to form oxidized glutathione and hydrogen peroxide, causing high oxidative stress and inhibiting catalase activity [18, 19]. Some micro‐organisms can detoxify through reductive precipitation of Au (III)‐complexes to less toxic Au (0) nanoparticles. Different micro‐organisms have different reduction and detoxification mechanisms for Au (I/III)‐complexes. Micro‐organisms can reduce Au (I/III) to Au (0) through extracellular secretions, reduced proteins on cell membranes, or intracellular reductase (Figure 5.1) [20]. Delftia acidovorans can survive in a solution containing 100 μmol/l AuCl₃, but was significantly inhibited when they could not secrete secondary metabolites, suggesting that secondary metabolites can help them to reduce Au (III) to Au(0) [21]. The membrane vesicles produced by many gram‐negative bacteria can be used as a protective shield against the toxic environment [22]. When cyanobacteria were exposed to an Au (III) solution, the cyanobacteria would release the membrane vesicles to wrap the cells and prevent the entrance of Au (III) into the cells, while the reduction of Au (III) to metallic Au involves the formation of Au (I)‐sulfide, as confirmed by XANES [23]. Analysis of XANES and μ‐XRF revealed that Cupriavidus metallidurans accumulated Au(III)‐complexes from the solution, and the cellular Au was coupled to the formation of Au(I)‐S species intermediates, finally leading to the formation of Au(0) in both intra‐ and extracellular reductive precipitation, accompanied by a efflux outside the cell [19].

Figure 5.1 Reduction and detoxification mechanism of gold ions by Rhizopous oryzae cells.

      Source: Reproduced from Das et al. [20] with the permission from American Chemical Society.

5.5 Application of μ‐XRF and XAS in Understanding the Mechanism of Using Micro‐organisms in Bioremediation

      In the environment, heavy metal pollution is a serious problem. However, it is well known that microbial remediation is highly efficient and indispensable in repairing heavy metal pollution. Thus, understanding the mechanism of using micro‐organisms in bioremediation may help to improve remediation efficiency.

      The biomineralization of heavy metals inside and outside the micro‐organism cells can precipitate heavy metals, which then reduces their bioavailability. Biomineralization is an important bioremediation method that includes phosphate mineralization, carbonate mineralization, and sulfide mineralization. XAS results showed that Shewanella oneidensis MR‐1 could reduce dissolved U(VI) to UO₂ minerals with poor solubility to achieve the purpose of bioremediation. The addition of S, Si, and P elements in the environment could promote the formation of EPS (Extracellular Polymeric Substances), and the proportion of UO₂ increases by 55–95%, which improved the efficiency of bioremediation significantly [24]. Uranium oxides were mineralized to secondary minerals by fungus, and the main processes were as follows: (1) When the fungus cells were exposed to UO₃ and U₃O₈, the cells secreted large amounts of oxalic acid, which caused UO₃ and U₃O₈ to dissolve and form (UO₂)²⁺ complexes; (2) U was adsorbed by cells, and the adsorption capacity could reach 80 mg/g; (3) The dissolved (UO₂)²⁺ further complexed with P to form UO₂HPO₄ and [UO₂(HPO₄)₂]²⁻, and finally mineralized to form the uranyl phosphate mineral NH₄(UO₂)(PO₄).3H₂O and H₂(UO₂)₂(PO₄)₂.8H₂O