Pseudomonas aeruginosa as well as preparation and application thereof

Abstract

The invention discloses pseudomonas aeruginosa as well as a preparation and application thereof, and belongs to the technical field of microbial strains, the pseudomonas aeruginosa is separated from a soil sample in a uranium mine area and is named as P. aeruginosa LXZ1, the P. aeruginosa LXZ1 and shewanella (S. oneidensis MR-1) can be constructed into a specific synthetic microbial flora, and the specific synthetic microbial flora can be used for preparing the microbial flora. When the synthesized microbial flora is applied to uranium-polluted water body remediation, P.aeruginosa LXZ1 can actively secrete phenazine PYO and release eDNA into a biological membrane matrix to generate a flavin protein compound and form a stable conductive biological membrane network, which can enhance the EET process, thereby accelerating the reduction of U (VI) and improving the remediation efficiency of the uranium-polluted water body. Meanwhile, the problem that in the prior art, due to the fact that the content of flavin accumulated in the S.oneidensis MR-1 biological membrane inhibits the efficient biological reduction process of U (VI), the remediation efficiency of the uranium-polluted water body is reduced is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial strains, in particular to a pseudomonas aeruginosa and a preparation and application thereof. BACKGROUND

[0002] Uranium (U) as a natural radioactive element, its pollution hazards have both radioactive hazards and chemical toxicity hazards, and the pollution has the characteristics of concealment, long-term and accumulation. After the weathering erosion of uranium-containing minerals, uranium will be released into the ecosystem, especially uranium mining, which will cause serious pollution to the environment. In uranium pollution, uranium usually exists in the form of hexavalent state (VI), therefore, if U (VI) in the pollutant can be reduced to insoluble U (IV), the pollution degree of uranium can be significantly reduced.

[0003] At present, U (VI) is generally reduced to U (IV) by dissimilatory metal-reducing bacteria (DMRB), such as shewanella (Shewanella oneidensis MR-1 (S. oneidensis MR-1) and Geobacter sulfurreducens (G. sulfurreducens). Shewanella oneidensis MR-1 (S. oneidensis MR-1) and Geobacter sulfurreducens (G. sulfurreducens). S. oneidensis MR-1) and Geobacter sulfurreducens (G. sulfurreducens). S. oneidensis However, the accumulated flavin content in the biofilm of MR-1 is extremely low, which cannot meet the efficient biological reduction process of U (VI), and will lead to the reduction of the efficiency of water body repair of uranium pollution. SUMMARY

[0004] The main purpose of the present application is to provide a pseudomonas aeruginosa and a preparation and application thereof, which aims to solve the problems of insufficient accumulated flavin content in the biofilm of MR-1, which cannot meet the efficient biological reduction process of U (VI), and will lead to the reduction of the efficiency of water body repair of uranium pollution in the prior art. S. oneidensis The main purpose of the present application is to provide a pseudomonas aeruginosa and a preparation and application thereof, which aims to solve the problems of insufficient accumulated flavin content in the biofilm of MR-1, which cannot meet the efficient biological reduction process of U (VI), and will lead to the reduction of the efficiency of water body repair of uranium pollution in the prior art.

[0005] In order to achieve the above purpose, the present application provides a pseudomonas aeruginosa, the preservation unit of the pseudomonas aeruginosa is Guangdong Microbial Culture Collection Center, the address of the preservation unit is No. 100, Er'lie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, the preservation date is September 15, 2025, the preservation number is GDMCC No: 66960, and the name is pseudomonas aeruginosa LXZ1. P. aeruginosa LXZ1.

[0006] The number of the pseudomonas aeruginosa in the BioSample database of NCBI is SAMN39097935.

[0007] The genome size of the *Pseudomonas aeruginosa* is 6,446,933 bp, the number of genes is 6,077, the total length of the genes is 5,740,371 bp, the average length of the genes is 944.61 bp, the proportion of gene length to the total genome length is 89.04%, and the GC content is 67.06%.

[0008] To achieve the above objectives, the present invention also provides a water remediation formulation comprising the aforementioned Pseudomonas aeruginosa.

[0009] Optionally, the preparation may also include Shewanella.

[0010] Optionally, the concentration of Pseudomonas aeruginosa in the formulation is 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 8 CUF / mL.

[0011] Optionally, when the formulation further includes Shewanella, the concentration of Shewanella is 1.0 × 10⁻⁶. 5 CUF / mL ~1.0×10 9 CUF / mL.

[0012] To achieve the above objectives, the present invention also provides an application of a water remediation agent, which is applied to the remediation of uranium-contaminated water bodies.

[0013] Optionally, when the preparation includes *Pseudomonas aeruginosa* and *Shewanella*, and when the uranium content in the uranium-contaminated water is >100 mg / L, the concentration of *Pseudomonas aeruginosa* is 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 8 CUF / mL, the concentration of Shewanella was 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 9 CUF / mL.

[0014] Optionally, the remediation time for the uranium-contaminated water body is 1 to 3 days.

[0015] Compared with the prior art, the beneficial effects that the present invention can achieve are as follows: 1. This invention isolates a strain of *Pseudomonas aeruginosa* from soil samples from a uranium mining area and names it... Pseudomonas aeruginosa LXZ1 ( P. aeruginosa LXZ1), obtained through uploading sequencing results to NCBI (National Center for Biotechnology Information), has a BioSample database ID: SAMN39097935. This ID is... P. aeruginosa LXZ1's identification code, through which one can obtain... P. aeruginosaThe complete genome sequence of LXZ1. Whole-genome sequencing revealed that... P. aeruginosa The LXZ1 genome is 6,446,933 bp in size, contains 6,077 genes, has a total length of 5,740,371 bp, an average gene length of 944.61 bp, a gene length as a percentage of the total genome length of 89.04%, and a GC content of 67.06%.

[0016] 2. In the technical solution of the present invention, P. aeruginosa LXZ1 and / or Shewanella ( Shewanella oneidensis MR-1 ( S. oneidensis MR-1 is a key microbial species in water remediation formulations. When this formulation simultaneously includes... P. aeruginosa LXZ1 and S. oneidensis In MR-1, then P. aeruginosa LXZ1 and S. oneidensis MR-1 microorganisms can form specific synthetic microbial communities, which can enhance the remediation of uranium-contaminated water systems. S. oneidensis The extracellular electron transfer (EET) process of MR-1 accelerates the reduction rate of U(VI) to U(IV) in uranium-contaminated water, enabling the rapid reduction and fixation of U(VI), thereby removing U(VI) and achieving water remediation. This also solves the problems of existing technologies. S. oneidensis The flavin content accumulated in the MR-1 biofilm is insufficient to meet the efficient bioreduction process of U(VI), which leads to a decrease in the efficiency of uranium-contaminated water remediation.

[0017] 3. In the technical solution of the present invention, the [material] containing P. aeruginosa LXZ1 and S. oneidensis MR-1 formulations are used in the remediation of uranium-contaminated water bodies. P. aeruginosa LXZ1 can actively secrete phenazine PYO and release eDNA into the biofilm matrix, generating flavoprotein complexes and forming a stable conductive biofilm network, thereby enhancing the EET process and accelerating U(VI) reduction; this provides a powerful strategy for developing robust, scalable and sustainable bioremediation systems for uranium contamination. Attached Figure Description

[0018] Figure 1 To separate from soil samples from uranium mining areas P. aeruginosa A schematic diagram of the LXZ1 method; Figure 2 Subgraph a is P. aeruginosa Phylogenetic tree analysis diagram of LXZ1, subgraph b is P. aeruginosa LXZ1 (right) and P. aeruginosaThe growth curve of PAO1 (left), subgraph c is P. aeruginosa The UV-Vis spectrum of the bacterial suspension of LXZ1 and P. aeruginosa The growth curve of PAO1; Figure 3 The UV-Vis spectrum of the bacterial suspension of PAO1 and P. aeruginosa LXZ1 and P. aeruginosa The UV-Vis spectrum of the bacterial suspension of LXZ1; Figure 4 The UV-Vis spectrum of the bacterial suspension of LXZ1, P. aeruginosa LXZ1, S. oneidensis MR-1 and P. aeruginosa LXZ1 and S. oneidensis The fluorescence labeling diagram of extracellular eDNA and whole cell DNA of the double bacterial system of LXZ1 and MR-1; Figure 5 Subgraph a is the reduction rate curve of U(VI) to U(IV); subgraph b is P. aeruginosa The deposition distribution of U(IV) on the cell surface in the bacterial suspension of LXZ1 and S. oneidensis MR-1 cell suspension; subgraph c is P. aeruginosa The deposition distribution of U(IV) on the cell surface in the double bacterial cell mixed suspension of LXZ1 and S. oneidensis MR-1; Figure 6 The change of extracellular electron transfer current; Figure 7 The current (μA) response diagram under different potentials (V vs. SHE). DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0020] In order to solve the problem in the prior art that the accumulated flavin content in the MR-1 biofilm does not meet the efficient biological reduction process of U(VI), which leads to the reduction of the efficiency of the remediation of the water body polluted by uranium, S. oneidensis The present application provides a Pseudomonas aeruginosa which is isolated from a uranium mine soil sample, and the similarity with DSM50071 is 92% through phylogenetic tree analysis, so it can be confirmed that Pseudomonas aeruginosa LXZ1 belongs to the Pseudomonas aeruginosa (Pseudomonas aeruginosa) species, and is named as LXZ1. P. aeruginosa Pseudomonas aeruginosa P. aeruginosa ​​​

[0021] Regarding the above P. aeruginosa LXZ1 was sequenced, and the sequencing results were uploaded to the NCBI website, obtaining the BioSample database ID: SAMN39097935. This ID is... P. aeruginosa The LXZ1 identification code can be used to obtain... P. aeruginosa The complete genome sequence of LXZ1.

[0022] By analyzing the above P. aeruginosa Whole-genome sequencing of LXZ1 revealed a genome size of 6,446,933 bp, 6,077 genes, a total length of 5,740,371 bp, an average gene length of 944.61 bp, a gene length as a percentage of the total genome length of 89.04%, and a GC content of 67.06%. Phylogenetic analysis and... Pseudomonas aeruginosa The similarity to DSM50071 is 92%.

[0023] The above-mentioned soil samples from the uranium mining area were isolated P. aeruginosa A schematic diagram of the LXZ1 method is shown below. Figure 1 As shown, the details are as follows: S10. Collect contaminated soil from the uranium mining area, mix 5 g with 50 mL of ultrapure water, shake for 24 h, then centrifuge at 6000 rpm for 10 min to obtain the supernatant; S. oneidensis MR-1 was placed in 50 mL centrifuge tubes containing LB (Luria-Bertani Medium) and incubated at 180 rpm and 30°C for 24 h; then washed twice with DM medium supplemented with 10 mM sodium lactate to obtain... S. oneidensis MR-1 cells.

[0024] Optionally, the LB medium mentioned above is the LB agar medium commonly used in the prior art.

[0025] Optionally, the composition of the above-mentioned DM culture medium is as follows: NaHCO3 2.5 g / L, CaCl2·2H2O 0.08 g / L, NH4Cl 1.0 g / L, NaCl 10 g / L, MgCl2·6H2O 0.2 g / L, yeast extract 0.5 g / L and HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) 7.2 g / L.

[0026] S20. Take the contents of S10... S. oneidensis MR-1 cells were formulated as OD 600 = 0.1 S. oneidensis MR-1 cell suspension; take 250 μL of supernatant from S10 and mix with 6 mL OD 600 = 0.1S. oneidensis The MR-1 cell culture was thoroughly mixed and then pre-cultured in a three-electrode system to selectively enrich electroactive microorganisms. After 48 hours of culture, the maximum current obtainable by the three-electrode system was 100 μA. The cell-containing supernatant from the three-electrode system was then inoculated into LB agar dishes. After overnight culture, a strain of *Pseudomonas aeruginosa* was obtained by monoclonal plate isolation and named... Pseudomonas aeruginosa LXZ1 ( P. aeruginosa LXZ1).

[0027] Optionally, the voltage of the above three-electrode system is 0.2V, the reference electrode is saturated Ag / AgCl, the working electrode is indium tin oxide, and the counter electrode is platinum wire.

[0028] It should be noted that the above OD 600 = 0.1 S. oneidensis MR-1 refers to the wavelength of this [unclear - possibly a specific material or indicator]. S. oneidensis The optical density of the MR-1 bacterial culture was 0.1, corresponding to a low cell concentration.

[0029] The above-mentioned results obtained through S10~S20 P. aeruginosa Genome analysis of LXZ1 was performed, and the colonies were plated using a single-clone plating method, comparing them with the type strain. Pseudomonas aeruginosa A comparative analysis of PAO1 was performed, and the results are as follows: Figure 2 As shown.

[0030] It should be noted that the above-mentioned model strains Pseudomonas aeruginosa PAO1 ( P. aeruginosa PAO1 belongs to Pseudomonas aeruginosa (PAO1) Pseudomonas aeruginosa Representative strains of the species.

[0031] like Figure 2 As shown, subgraph a is P. aeruginosa Phylogenetic tree analysis results of LXZ1, subgraph b is... P. aeruginosa LXZ1 (right) and P. aeruginosa The coating plate of PAO1 (left), sub-image c is... P. aeruginosa LXZ1 and P. aeruginosa Growth curve of PAO1.

[0032] Depend on Figure 2 As can be seen from neutron diagram a, P. aeruginosa LXZ1 and Pseudomonas aeruginosa The similarity to DSM50071 is 92%, therefore, it can be confirmed. P. aeruginosa LXZ1 belongs to Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Species.

[0033] Through observation Figure 2 From the colony growth in the left and right swabs of neutron diagram b, we can see that... P. aeruginosa The fact that single-clone colonies of LXZ1 can turn green in a short time proves that... P. aeruginosa LXZ1 can produce a number of secondary metabolites, including pigments, in a short period of time, indicating active secondary metabolism.

[0034] Analysis and comparison P. aeruginosa LXZ1 and P. aeruginosa The colony growth of PAO1 within 36 hours was as follows: Figure 2 As shown in neutron diagram c, after approximately 30 hours of culture, the OD values ​​of the two strains... 600 The values ​​are all close to 1.75, indicating that their bacterial reproduction rate and final bacterial density are basically the same. Therefore, P. aeruginosa LXZ1 and P. aeruginosa There was no significant difference in the basic growth performance of PAO1.

[0035] The above analysis shows that, compared with the model strain P. aeruginosa Compared to PAO1, P. aeruginosa OD of LXZ1 bacterial suspension 600 The value can also increase to 1.75 within 36 hours, indicating that... P. aeruginosa LXZ1 and the model strain P. aeruginosa The growth rate of PAO1 was almost uniform, but its pigment secretion capacity was significantly increased.

[0036] In the above analysis, by plating single-clone colonies and incubating them for 36 hours, the following results could be obtained: P. aeruginosa PAO1 and P. aeruginosa The LXZ1 colonies, after being dissolved separately, can be obtained separately. P. aeruginosa PAO1 and P. aeruginosa LXZ1 bacterial suspension.

[0037] The above-obtained values ​​were measured respectively. P. aeruginosa PAO1 and P. aeruginosa The UV-Vis spectrum of the bacterial suspension of LXZ1 was obtained as follows: Figure 3 As shown.

[0038] like Figure 3 The main image is P. aeruginosa PAO1 and P. aeruginosa The UV-Vis spectrum of LXZ1 bacterial suspension is shown in the inset, which is a photograph of the bacterial suspension. The portion on the left, appearing as a yellow liquid, represents... P. aeruginosa The bacterial suspension of PAO1, the one on the right showing a green liquid state is... P. aeruginosa LXZ1 bacterial suspension.

[0039] analyze Figure 3 As can be seen from the main image, P. aeruginosa PAO1 and P. aeruginosa The bacterial suspension of LXZ1 showed characteristic absorption peaks of the key pigment, pyocyanin (PYO), at approximately 310 nm, 370 nm, and 690 nm. P. aeruginosa The characteristic peak of LXZ1 is significantly higher than that of LXZ1. P. aeruginosa PAO1, this indicates, P. aeruginosa LXZ1 produces more PYO; and PYO is a key “electron shuttle” that can obtain electrons from the bacterial cell and then transfer them to U(VI) or Shewanella cytochrome c in the water, solving the problem of the large spatial distance between bacteria and U(VI) and the obstruction of electron transfer. Therefore, the higher the PYO content, the stronger the ability of the strain to remove U(VI).

[0040] Using fluorescent labeling methods, respectively for P. aeruginosa LXZ1, S. oneidensis MR-1 and P. aeruginosa LXZ1 and S. oneidensis The distribution of extracellular eDNA (green fluorescent label) and whole-cell DNA (red fluorescent label) in the MR-1 dual-strain system was determined, and the results are as follows: Figure 4 As shown.

[0041] Depend on Figure 4 It can be seen that, in S. oneidensis In the fluorescence images of MR-1, the green fluorescence is densely distributed, and the red fluorescence is also clearly distributed, indicating that this bacterium can secrete a certain amount of extracellular eDNA into the surrounding environment. When the images are superimposed, the green and red fluorescence show good overlap, indicating that there is a spatial correlation between the extracellular eDNA and whole-cell DNA. This may be because the extracellular eDNA is released from the cell's secretion. P. aeruginosa In the fluorescence images of LXZ1, the green fluorescence is densely distributed, while the red fluorescence distribution is less characteristic, indicating that this bacterium can secrete a large amount of extracellular eDNA into the surrounding environment; when the images are superimposed, it reflects... P. aeruginosa LXZ1 exhibits highly characteristic extracellular eDNA secretion; eDNA not only adsorbs U(VI) but also acts as an "electron conduction bridge" because its backbone contains numerous conjugated double bonds that mediate electron transfer. Simultaneously, its surface functional groups (such as hydroxyl and amino groups) can immobilize U(VI), enriching it around the bacteria and increasing the probability of bacterial contact with U(VI). P. aeruginosa LXZ1 and S. oneidensisIn the fluorescence images of the MR-1 dual-strain system, the distribution of extracellular eDNA and whole-cell DNA showed different characteristics from the single-strain system, with a bright green color indicating that it produced more eDNA. Its biofilm evenly covered the entire field of view, showing a strong biofilm-forming ability compared to the single-strain system. The biofilm thickness and eDNA content were significantly higher than those of the single-strain system. Under the action of the electron mediator PYO, the extracellular electron transfer efficiency of microorganisms can be significantly improved.

[0042] To address the aforementioned problems, the present invention also provides a water remediation formulation comprising the aforementioned *Pseudomonas aeruginosa* (…). P. aeruginosa LXZ1).

[0043] In one possible implementation, the above-mentioned preparation also includes Shewanella (… Shewanella oneidensis MR-1 ( S. oneidensis MR-1).

[0044] In one possible implementation, in the above-described formulation, P. aeruginosa The concentration of LXZ1 can be 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 8 CUF / mL.

[0045] In one possible implementation, when the above-mentioned formulation further includes S. oneidensis During MR-1, S. oneidensis The concentration of MR-1 can be 1.0 × 10⁻⁶. 5 CUF / mL ~1.0×10 9 CUF / mL.

[0046] It should be noted that when the above-mentioned water remediation agents only include P. aeruginosa When LXZ1, P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 8 At CUF / mL, during the remediation of water bodies contaminated with heavy metals, P. aeruginosa LXZ1 can secrete large amounts of PYO and eDNA. PYO is a phenazine pigment. PYO and eDNA may be able to remove heavy metal ions through complexation or other mechanisms. For example, in the remediation of uranium-contaminated water, PYO and eDNA may complex with uranium ions or alter their valence state through redox reactions, reducing and fixing them, thereby decreasing the mobility and toxicity of uranium ions. Furthermore, P. aeruginosaThe abundant extracellular polymeric substances of LXZ1 may flocculate suspended particles and organic pollutants in water, improving water transparency and apparent quality. In a possible implementation, if the concentration of heavy metals in the polluted water is low, for example ≤50 mg / L, [the following method can be selected]. P. aeruginosa LXZ1 formulations in the low concentration range, for example, can be selected P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 7 A formulation with CUF / mL; if the heavy metal concentration is high, then a formulation with CUF / mL can be selected. P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 8 A formulation with CUF / mL.

[0047] Furthermore, when the above-mentioned formulation simultaneously includes P. aeruginosa LXZ1 and S. oneidensis MR-1, the two can form a specific microbial colony, and P. aeruginosa LXZ1 possesses the ability to secrete phenazine PYO and eDNA, which can enhance... S. oneidensis MR-1's extracellular electron transport capability, and S. oneidensis MR-1's cytochrome c can directly accept electrons transferred from phenazine PYO or eDNA, thereby efficiently reducing U(VI) to U(IV), effectively degrading pollutants in water and achieving the remediation of polluted water bodies. Furthermore, P. aeruginosa LXZ1's phenazine PYO and eDNA may also complex with heavy metal ions such as uranium, while S. oneidensis MR-1 can also reduce other heavy metals, and the synergy between the two can expand the range and efficiency of heavy metal removal. When P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 8 CUF / mL S. oneidensis The concentration of MR-1 was 1.0 × 10⁻⁶. 5 CUF / mL ~1.0×10 9 The concentrations of CUF / mL and CUF work synergistically to enhance the removal capacity of heavy metals.

[0048] To address the aforementioned problems, the present invention also provides an application of a water remediation preparation, which is applied to the remediation of uranium-contaminated water bodies.

[0049] In one possible implementation, when the above-mentioned water remediation agent is applied to the remediation of uranium-contaminated water, the water remediation agent may include... P. aeruginosa LXZ1 orS. oneidensis MR-1.

[0050] In one possible implementation, the above-mentioned water remediation agent includes P. aeruginosa LXZ1 and S. oneidensis MR-1.

[0051] In one possible implementation, when the uranium concentration in the uranium-contaminated water body is >100 mg / L, P. aeruginosa The concentration of LXZ1 can be 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 8 CUF / mL; S. oneidensis The concentration of MR-1 can be 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 9 CUF / mL.

[0052] In one possible implementation, when the uranium content in the uranium-contaminated water body is in the range of 100 mg / L to 200 mg / L, the above-mentioned water body remediation agent may be used for water body remediation, which may also achieve rapid water body remediation.

[0053] In one possible implementation, the remediation of the aforementioned uranium-contaminated water body takes 1 to 3 days.

[0054] It should be noted that the above technical solution will include... P. aeruginosa LXZ1 and / or S. oneidensis MR-1, a water remediation agent, is used in the remediation of uranium-contaminated water. During the remediation process, the system temperature significantly affects the remediation rate. If the system temperature is too low or too high, it inhibits the proliferation rate of bacterial cells, making it difficult to maintain the bacterial concentration within the set range, thus reducing the remediation rate. In other words, temperature affects the water remediation rate by influencing the proliferation rate of bacterial cells. Therefore, in the technical solution of this invention, the suitable water remediation temperature is 15℃~35℃, for example, 25℃~30℃. This temperature range matches the optimal growth temperature of the two bacteria, promoting active bacterial proliferation and stably maintaining the concentration set in the solution.

[0055] It should be noted that when the above-mentioned formulations simultaneously include P. aeruginosa LXZ1 and S. oneidensisWhen MR-1 is applied to the remediation of uranium-contaminated water, the two bacterial colonies can work together through different mechanisms to act on uranium ions, significantly reducing the amount of mobile, biotoxic soluble uranium in the water. They not only convert free uranium in the water into a solid form, such as reducing U(VI) to U(IV), but also enhance the stability of solid uranium in the water sediment through bacterial metabolites or cellular characteristics, preventing uranium re-leaching due to subsequent changes in environmental conditions. Furthermore, while remediating uranium contamination, the metabolic activities of the two bacterial strains can indirectly improve the aquatic microenvironment, such as… P. aeruginosa During its growth, LXZ1 decomposes organic matter, producing a small amount of organic acids, which can lower the pH in alkaline water, preventing excessively high pH from causing U(VI) to form soluble anionic complexes. Meanwhile, Shewanella, when reducing U(VI), produces H+. + The consumption of uranium can raise the pH in acidic water, preventing the pH from being too low and inhibiting bacterial activity; the two work together to form a pH buffering mechanism, stabilizing the water pH in the optimal range of 5-7, providing a suitable environment for uranium reduction and adsorption.

[0056] Furthermore, when the formulation contains P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 8 CUF / mL S. oneidensis The concentration of MR-1 was 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 9 At CUF / mL, numerous functional groups such as carboxyl, hydroxyl, and amino groups on the surface of bacteria can directly adsorb U(VI), increasing the uranium adsorption capacity per unit volume of water. Simultaneously, high-concentration bacteria exhibit stronger metabolic activity, generating electron carriers more quickly and accelerating the reduction reaction of U(VI) to U(IV), shortening the remediation cycle. In environments with uranium concentrations >100 mg / L, this combined concentration can reduce uranium levels in water below the limit in a relatively short time. Furthermore, high-concentration bacteria can resist the toxic inhibitory effect of high U(VI) on individual bacteria through a "group synergistic effect." On one hand, some bacteria can preferentially adsorb uranium, reducing the toxicity of U(VI) to other bacteria in the surrounding environment; on the other hand... S. oneidensis MR-1 has strong metal resistance and can be combined with... P. aeruginosa LXZ1 synergistically maintains the overall activity of the microbial community, avoiding a sharp drop in remediation efficiency due to high contamination load.

[0057] Furthermore, when the above-mentioned formulation simultaneously includes P. aeruginosa LXZ1 and S. oneidensis During MR-1, P. aeruginosa The phenazine PYO secreted by LXZ1 can act as an electron shuttle, transporting substances from its own respiratory chain or... S. oneidensisMR-1 gains electrons in the respiratory chain and is reduced to "reduced PYO", such as PYO. - Or PYO 2- Restore U(VI) to U(IV); at the same time, S. oneidensis MR-1 possesses unique "outer membrane cytochromes," such as MtrC and OmcA, which efficiently transfer electrons generated by intracellular metabolism to extracellular PYO, reducing U(VI) to U(IV). The presence of phenazine PYO breaks the limitation that bacteria need direct contact with U(VI) for reduction, allowing electrons to be transported long distances in water, covering more U(VI) molecules. Therefore, P. aeruginosa LXZ1 and S. oneidensis The two bacteria in MR-1 can work synergistically to maintain the "oxidation-reduction cycle" of phenazine PYO, significantly improving electron transfer efficiency.

[0058] Furthermore, P. aeruginosa LXZ1 and S. oneidensis The synergistic effect of MR-1 is also manifested in the following ways: First, P. aeruginosa The phenazine PYO and eDNA secreted by LXZ1 can not only mediate electron transport, but may also form complexes with U(VI), enhancing the adsorption affinity of U(VI) on the bacterial cell surface; simultaneously, the reduced U(IV) can interact with... P. aeruginosa U(VI) adsorbed on the LXZ1 surface can form co-precipitates, further enhancing the stability of uranium retention. Secondly, S. oneidensis MR-1 is a typical "metal-reducing bacterium." Its cytochrome c cells form an efficient electron transport chain, transferring electrons produced during metabolism to U(VI), reducing it to U(IV). U(IV) likely exists primarily as UO2. U(IV) has extremely low solubility and rapidly precipitates from water, directly reducing the concentration of soluble uranium in the water. Furthermore, the biotoxicity of U(IV) is only 1 / 100 to 1 / 1000 that of U(VI), significantly reducing the harm of uranium to aquatic organisms. In addition, S. oneidensis MR-1, while reducing U(VI), also produces bicarbonate (HCO3-) through metabolism. - ), phosphate (PO4) 3- These ions can react with reduced U(IV) or unreduced U(VI) to form uranium minerals, such as UO₂CO₃ and CaUO₂(PO₄)₂. These minerals have stable crystal structures, are not easily dissolved, and can further enhance the fixation of uranium in the sediment of water bodies. Therefore, P. aeruginosa LXZ1 and S. oneidensisMR-1 complements each other in terms of adsorption capacity and reduction efficiency, forming a closed loop of uranium adsorption-reduction-immobilization, reducing uranium loss in the intermediate process, and ultimately achieving a synergistic effect of "1+1>2".

[0059] Example 1 S10. *Pseudomonas aeruginosa* was isolated from the soil samples from the uranium mining area as described above. P. aeruginosa After culturing LXZ1, a bacterial suspension was prepared. The bacterial suspension contained... P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 7 CUF / mL.

[0060] S20. For Shewanella S. oneidensis MR-1 was cultured and then prepared into a bacterial suspension. The bacterial suspension contained... S. oneidensis The concentration of MR-1 was 1.0 × 10⁻⁶. 7 CUF / mL.

[0061] S30. Take the contents of S10... P. aeruginosa LXZ1 bacterial suspension and S20 S. oneidensis The MR-1 bacterial suspension was mixed evenly at a volume ratio of 1:1 to obtain a dual-bacterial system, which was used as a preparation for the remediation of uranium-contaminated water bodies.

[0062] S40. The dual-bacterial system of S30 was added to uranium-contaminated water with a U(VI) concentration of 50 mg / L for water remediation. The reduction of U(VI) to U(IV) at different time periods was observed and detected.

[0063] Comparative Example 1 is set up under Example 1. The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not include S20, and the bacterial suspension in S30 has a concentration of 1.0 × 10⁻⁶. 7 CUF / mL P. aeruginosa A single bacterial suspension of LXZ1; in S40, using P. aeruginosa LXZ1 single bacterial suspension for uranium contaminated water remediation.

[0064] Comparative Example 2 is set up under Example 1. The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not include S10, and the bacterial suspension in S30 has a concentration of 1.0 × 10⁻⁶. 7 CUF / mL S. oneidensis A single bacterial suspension of MR-1; in S40, using S. oneidensis MR-1 single bacterial suspension for uranium contaminated water remediation.

[0065] The remediation of uranium-contaminated water in Example 1, Comparative Example 1, and Comparative Example 2 was monitored at different time periods. The reduction rate of U(VI) to U(IV) was determined through detection and calculation, and the results are as follows: Figure 5 Subgraph a is shown in the figure.

[0066] After the uranium-contaminated water bodies were remediated, samples were taken sequentially from the remediated water bodies of Example 1, Comparative Example 1, and Comparative Example 2, obtaining three types of water samples. These three water samples were then separated and extracted to obtain three types of bacterial cell suspensions, as follows: P. aeruginosa LXZ1 and S. oneidensis MR-1 dual-strain cell suspension P. aeruginosa LXZ1 cell suspension and S. oneidensis MR-1 cell suspension.

[0067] Observed using transmission electron microscopy P. aeruginosa LXZ1 and S. oneidensis MR-1 dual-strain cell suspension P. aeruginosa LXZ1 cell suspension and S. oneidensis The deposition and distribution of U(IV) on the cell surface in MR-1 cell suspension are shown in the following results. Figure 5 Subgraphs b and c are shown. Subgraph b is... P. aeruginosa LXZ1 cell suspension and S. oneidensis The deposition and distribution of U(IV) on the cell surface in MR-1 cell suspension; subfigure c shows... P. aeruginosa LXZ1 and S. oneidensis The deposition and distribution of U(IV) on the cell surface in a two-cell mixed suspension of MR-1 bacteria.

[0068] First, by Figure 5 As shown in neutron diagram a, when the U(VI) concentration in uranium-contaminated water is 50 mg / L, when using P. aeruginosa LXZ1 and S. oneidensis When the MR-1-constructed dual-bacterial system was used for the reduction and fixation of U(VI) to U(IV), 75% of U(VI) could be reduced and fixed to U(IV) during 12 hours of remedy; after 48 hours of remedy, almost all of U(VI) could be reduced. In contrast, the ability to reduce and fix U(VI) was weaker when using a single bacterial culture. This is because, in the dual-bacterial system, P. aeruginosa The phenazine PYO and eDNA secreted by LXZ1 can act as electron shuttles, accelerating extracellular electron transfer and thus enhancing… S. oneidensis MR-1's ability to reduce U(VI) allows U(VI) to be rapidly reduced to U(IV) and then fixed, enabling rapid remediation of uranium-contaminated water bodies.

[0069] Secondly, by Figure 5 As can be seen from neutron diagram b, P. aeruginosa LXZ1 cells showed no obvious uranium particles on their surface, while S. oneidensis Uranium particles were clearly visible on the surface of MR-1 cells, but the deposition amount was low, indicating that the reduction and fixation ability of a single bacterial cell for U(VI) was weak. In contrast, as shown in sub-figure c, where DF is a bright-field image and O and U represent the elemental surface distribution of uranium (U) and oxygen (O), a large number of uranium particles were visible in the bright-field image (DF). The elemental surface distribution showed that the distribution of uranium (U) and oxygen (O) highly overlapped, indicating that after U(VI) was reduced, the resulting U(IV) was mainly fixed in the form of oxygen-containing compounds.

[0070] according to Figure 5 Analysis shows that, P. aeruginosa LXZ1 and S. oneidensis The MR-1 cells enhanced the reduction and fixation efficiency of U(VI) to U(IV) through synergistic effects, indicating that in a dual-strain system, the reduction rate of U(VI) is not increased through simple adsorption or synergistic effects, but rather... P. aeruginosa LXZ1 and S. oneidensis The MR-1 molecules synergistically enhanced the reduction and fixation rate of U(VI) to U(IV) in uranium-contaminated water because, P. aeruginosa The metabolites of LXZ1 are S. oneidensis The reduction reaction of MR-1 provides electron transfer assistance, which accelerates the rate of U(VI) reduction to U(IV). Furthermore, in the dual-bacterial system, the bacterial species can jointly regulate the microenvironment of the water, such as pH and redox potential, avoiding the stringent requirements of artificially adjusting the acid-base environment of the water in water remediation. This is also more conducive to the reduction of U(VI), and the secreted extracellular polymers and other substances can also enhance the adsorption of uranium, ultimately achieving a remediation effect of 1+1>2.

[0071] To further confirm the technical solution of the present invention, P. aeruginosa LXZ1 and S. oneidensis The MR-1 molecules enhance the reduction capacity and mechanism of U(VI) to U(IV) through synergistic effects. Using an electrolytic cell, the changes in extracellular electron transport current were monitored and recorded in Examples 1, 1, and 2 during the remediation of uranium-contaminated water. The results are as follows: Figure 6 As shown; and the current (μA) response under different potentials (V vs. SHE) was measured, the results are as follows. Figure 7 As shown.

[0072] Depend on Figure 6 It can be seen that, Example 1 ( P. aeruginosaLXZ1 and S. oneidensis MR-1 dual-strain system Syncom S+P The extracellular electron transport current curve rises the fastest and reaches its highest value over time, indicating that... P. aeruginosa LXZ1 and S. oneidensis The extracellular electron transfer activity of the MR-1 dual-strain system is much higher than that of a single-strain system; secondly, in Comparative Example 2... S. oneidensis The current in MR-1 is higher than that in Comparative Example 1. P. aeruginosa The presence of LXZ1 indicates that the former has a stronger extracellular electron transport capacity. Furthermore, during the remediation of uranium-contaminated water, visual observation of the system's color changes revealed that after 6 hours of remediation, both the electrolyte solutions of Example 1 and Comparative Example 1 exhibited a visible green color. This indicates that after 6 hours of remediation, P. aeruginosa LXZ1 and S. oneidensis The rapid increase in extracellular electron transport current in the MR-1 two-strain system may be due to P. aeruginosa LXZ1 produced large amounts of phenazine PYO and eDNA, accelerating the process. S. oneidensis The electron transport rate of MR-1 thus achieves the effect of current enhancement.

[0073] Figure 6 The mechanism by which extracellular electrons transfer current changes within the mesocell may be as follows: First, S. oneidensis MR-1 possesses a relatively sophisticated extracellular electron transport system, capable of directly transferring electrons generated during metabolism to uranium (U) (VI), thus exhibiting a high electron transport current; secondly, P. aeruginosa Electron transport in LXZ1 may depend on secreted metabolites, indirectly mediating electron transport and resulting in a lower current; in contrast, P. aeruginosa LXZ1 and S. oneidensis In the MR-1 two-strain system, P. aeruginosa LXZ1 can secrete the metabolite phenazine PYO and release eDNA into the biomembrane matrix, generating a flavoprotein complex and forming a stable conductive biomembrane network, thereby accelerating electron transfer. S. oneidensis The transfer between MR-1 and uranium; at the same time, S. oneidensis The MR-1's highly efficient electron transport system can provide... P. aeruginosa The LXZ1 provides a more active electron receiver, and the two form a synergistic closed loop of "electron generation-shuttle-transmission", which greatly enhances the overall electron transfer activity.

[0074] In Example 1 P. aeruginosa LXZ1 and S. oneidensisIn the MR-1 dual-strain system, deoxyribonuclease I (DNase I) was added. During the remediation of uranium-contaminated water, changes in extracellular electron transport current in this system were monitored. The results are as follows: Figure 6 As shown by the light blue line, the extracellular electron transport current decreased after the addition of DNase I. This may be because DNase I affects the extracellular environment. P. aeruginosa The eDNA released by LXZ1 was digested, resulting in P. aeruginosa LXZ1 and S. oneidensis The electron transport structures on the cell membrane of MR-1 cells are disrupted, preventing electrons from being smoothly transferred from inside the cell to the outside, thus weakening the extracellular electron transport current. Therefore, it can be concluded that... P. aeruginosa The eDNA released by LXZ1 significantly enhances the extracellular electron transport current, thereby strengthening the reduction of U(VI).

[0075] Depend on Figure 7 It can be seen that in Example 1 P. aeruginosa LXZ1 and S. oneidensis MR-1 dual-strain system ( Syncom S+P In the process of the potential changing from negative to positive, the current rises rapidly and reaches a high current value in the higher potential region, demonstrating that the dual-bacterial system has a stronger electron transport capability; and two pairs of reversible redox peaks appear at -80 mV and 120 mV, indicating that the electron shuttle in the dual-bacterial system is indeed phenazine PYO and has a strong electron transport capability; when DNase I is added to this system ( Syncom S+P+ DNase I digested the eDNA in the system, resulting in a significant suppression of the current intensity, indicating that eDNA has a significant impact on the electron transport process of uranium reduction. In contrast, the overall current values ​​were lower in the single-strain systems of Comparative Examples 1 and 2, suggesting that the electron transport capacity of a single strain is limited under different potentials.

[0076] According to Figure 7 Analysis shows that, P. aeruginosa LXZ1 and S. oneidensis The electron transfer performance of the MR-1 dual-strain system was significantly better than that of the single-strain system. S . oneidensis MR-1 or P . aeruginosa (LXZ1) This is because the two strains have a synergistic effect in electron transport, which enhances the efficiency of electron generation and transport; however, the electron transport performance of the mixed system decreases after the addition of DNase I because DNase I degrades the eDNA related to electron transport in the system, destroying the relevant structures or media of electron transport, thereby hindering the electron transport process.

[0077] Example 2 S10. *Pseudomonas aeruginosa* was isolated from the soil samples from the uranium mining area as described above. P. aeruginosa After culturing LXZ1, a bacterial suspension was prepared. The bacterial suspension contained... P. aeruginosa The concentration of LXZ1 was 1.0 × 10⁻⁶. 8 CUF / mL.

[0078] S20. For Shewanella S. oneidensis MR-1 was cultured and then prepared into a bacterial suspension. The bacterial suspension contained... S. S. oneidensis The concentration of MR-1 was 1.0 × 10⁻⁶. 7 CUF / mL.

[0079] S30. Take the contents of S10... P. aeruginosa LXZ1 bacterial suspension and S20 S. oneidensis The MR-1 bacterial suspension was mixed evenly at a volume ratio of 1:1 to obtain a mixed bacterial solution, which was used as a preparation for the remediation of uranium-contaminated water bodies.

[0080] S40. Add the mixed bacterial solution of S30 to uranium-contaminated water with a U(VI) concentration of 100 mg / L, carry out water remediation at room temperature, and observe and detect the reduction of U(VI) to U(IV) at different time periods.

[0081] The kinetic simulation analysis of the reduction process of U(VI) in Example 2 is as follows.

[0082] (1) Model Formula Assuming the reaction rate depends only on the residual U(VI) concentration, the differential form of its first-order kinetic equation is:

[0083] in: C t : t The concentration of remaining U(VI) in the system at a given time, in mg / L; t Reaction time, in hours (h). k U(VI) is the reduction rate constant, in units of: h -1 .

[0084] Integral differential equation, initial conditions: t When =0, C t = C 0, C0 = 100 mg / L, the integral form is:

[0085] (2) Data monitoring and calculation During the monitoring of the reaction process in Example 2, the concentration of residual U(VI) in the system was observed as the reaction time increased. C t The value of kt was calculated using calculus equations, and the results are shown in Table 1.

[0086] Table 1

[0087] Analyzing the data in Table 1, it can be seen that within the range of 0~12h, Ct The rapid decrease is due to the high initial concentration of U(VI), and P. aeruginosa LXZ1 and S. oneidensis MR-1 has high reactivity, resulting in a fast reaction rate; in the later stage (24-48 hours), Ct The decrease slows down because the remaining U(VI) concentration is low, and the driving force of the reaction weakens. As shown in the table, for water with a uranium concentration of 100 mg / L, at the bacterial concentration corresponding to Example 2, almost 100% of U(VI) can be reduced and fixed to U(IV) within 48 hours.

[0088] Example 3 The difference between Example 3 and Example 2 is that S. oneidensis The concentration of MR-1 was 1.0 × 10⁻⁶. 8 CUF / mL. All other conditions were the same as in Example 2.

[0089] Example 4 The difference between Example 4 and Example 3 is that the concentration of U(VI) is 200 mg / L. All other conditions are the same as in Example 3.

[0090] Comparative Examples 3 and 4 were set up in Example 4.

[0091] Compared with Example 4, Comparative Example 3 differs in that it does not contain... S. oneidensis MR-1, the rest are the same as in Example 4.

[0092] The difference between Comparative Example 4 and Example 4 is that Comparative Example 4 does not contain... P. aeruginosa LXZ1, the rest are the same as in Example 4.

[0093] The reduction of U(VI) in uranium-contaminated water was monitored in Examples 3, 4, Comparative Examples 3 and 4, and the removal rate of U(VI) was calculated. The results are shown in Table 2.

[0094] The removal rate of U(VI) is the ratio of the concentration of U(VI) removed to the initial concentration of U(VI) in the system, expressed in units of %.

[0095] Table 2

[0096] As shown in Table 2, the removal rate of U(VI) in Example 3, i.e., the reduction of U(VI) to U(IV) and its fixation and removal, is higher than that in Example 4. This is because when the concentration of U(VI) is 200 mg / L, it may destroy... P. aeruginosa LXZ1 and S. S. oneidensis The cell membrane integrity of MR-1 slightly reduces the extracellular electron transport rate, resulting in a slight decrease in the removal rate. Secondly, in Comparative Examples 3 and 4, the presence of only a single bacterial species weakens the ability of extracellular electrons to reduce U(VI), leading to a significant decrease in the removal rate of U(VI). Furthermore, the data in the table show that... P. aeruginosa LXZ1 and S. oneidensis The synergistic effect between MR-1 molecules can significantly improve the removal rate of U(VI).

[0097] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A type of Pseudomonas aeruginosa, characterized in that, The *Pseudomonas aeruginosa* strain described was deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit date was September 15, 2025, and the accession number is GDMCC No: 66960. The strain was named *Pseudomonas aeruginosa*. P. aeruginosa LXZ1.

2. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The Pseudomonas aeruginosa strain in the NCBI BioSample database is identified as SAMN39097935.

3. The *Pseudomonas aeruginosa* according to claim 2, characterized in that, The genome size of the *Pseudomonas aeruginosa* is 6,446,933 bp, the number of genes is 6,077, the total length of the genes is 5,740,371 bp, the average length of the genes is 944.61 bp, the proportion of gene length to the total genome length is 89.04%, and the GC content is 67.06%.

4. A water remediation preparation, characterized in that, The preparation comprises Pseudomonas aeruginosa as described in any one of claims 1 to 3.

5. The formulation according to claim 4, characterized in that, The preparation also includes Shewanella.

6. The formulation according to claim 4, characterized in that, In the formulation, the concentration of *Pseudomonas aeruginosa* is 1.0 × 10⁻⁶. 6 CUF / mL ~1.0×10 8 CUF / mL.

7. The formulation according to claim 5, characterized in that, When the formulation further includes Shewanella, the concentration of Shewanella is 1.0 × 10⁻⁶. 5 CUF / mL ~1.0×10 9 CUF / mL.

8. The application of a water remediation preparation, characterized in that, The preparation according to any one of claims 4 to 7 is applied to the remediation of uranium-contaminated water bodies.

9. The application according to claim 8, characterized in that, When the preparation includes *Pseudomonas aeruginosa* and *Shewanella*, and when the uranium content in the uranium-contaminated water is >100 mg / L, the concentration of *Pseudomonas aeruginosa* is 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 8 CUF / mL, the concentration of Shewanella was 1.0 × 10⁻⁶. 7 CUF / mL ~1.0×10 9 CUF / mL.

10. The application according to claim 8 or 9, characterized in that, The remediation time for the uranium-contaminated water is 1 to 3 days.

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