Uranium pollution remediation method based on microorganism-clay mineral synergistic effect

Abstract

The invention discloses a uranium pollution remediation method based on a microorganism-clay mineral synergistic effect, belongs to the field of environmental engineering, and carries out uranium pollution remediation on a strain WG2-6 screened from a uranium tailing area and a montmorillonite binary body under the optimal condition. Experiments show that the uranium removal rate of the system reaches 90% or above, and the fixed product is kept stable in the environment. The binary system fixes uranium through multiple mechanisms mediated by surface coordination, ion exchange and extracellular polymeric substances, microorganisms participate in uranium fixation through phosphorus-containing groups, protein components and the like, montmorillonite serves as a microorganism protection carrier and promotes increase of the content of protein in the strain WG2-6, and then uranium fixation of the strain WG2-6 is strengthened. The microorganism-mineral synergistic remediation system is constructed by using natural components in the soil, has the advantages of low cost, environment friendliness, high remediation efficiency and the like, and is suitable for remediation of uranium contaminated sites.

Description

Technical Field

[0001] The present invention belongs to the field of environmental engineering, and in particular relates to a uranium pollution remediation method based on the synergistic effect of microorganisms and clay minerals. Background Art

[0002] Uranium, a key radioactive element, is widely used in nuclear energy, military industry, and scientific research. However, with the continued expansion of uranium mining, processing, and use, large amounts of uranium contaminants are released into the environment, causing severe contamination of soil, water, and ecosystems. Its migration and accumulation in the environment pose a serious threat to ecosystem stability and human health. Therefore, effectively limiting the migration of uranium in the environment is a key step in the remediation of uranium-contaminated sites.

[0003] Soil is the main carrier of uranium pollution. The migration and transformation behavior of uranium within it is affected by many factors, among which microorganisms play a particularly critical role in this process. Microorganisms, as tiny organisms with a large number and diverse metabolism in soil ecosystems, are widely distributed in various radioactive and non-radioactive environments due to their unique advantages of small size, large specific surface area and high biotransformation efficiency. Microorganisms can directly or indirectly interact with uranium through bioadsorption, bioreduction and biomineralization, changing the chemical form of uranium, thereby achieving the regulation of uranium conversion mechanisms and migration pathways. In addition, clay minerals, as important members of soil mineral composition, can adsorb and fix uranium through mechanisms such as ion exchange, surface complexation and co-precipitation due to their unique layered structure, high specific surface area and abundant surface active sites, showing great potential in the field of uranium pollution remediation.

[0004] In summary, it can be found that microorganisms and clay minerals have shown certain potential in the remediation of uranium contamination, but existing technologies still have obvious shortcomings. In terms of microbial remediation, the mechanism by which environmental factors (such as pH and temperature) affect microbial uranium fixation is still unclear, and the interaction mechanism between microorganisms and uranium still needs in-depth study. In terms of clay mineral remediation, existing research has mostly focused on the fixation of uranium by a single mineral, while there is a lack of systematic exploration of the synergistic mechanism between clay minerals and microorganisms under natural conditions. In addition, in actual soil environments, clay minerals and microorganisms do not exist independently, but form complex microaggregate structures through cementation. The research results of a single component are difficult to truly reflect the migration and transformation laws of uranium under natural conditions.

[0005] Therefore, developing a uranium contamination remediation technology based on the synergistic effect of microorganisms and clay minerals and deeply analyzing the interaction between them will help to enhance the uranium fixation effect and improve the remediation plan. Summary of the Invention

[0006] The technical problem to be solved by the present application is how to provide a uranium pollution remediation method based on the synergistic effect of microorganisms and clay minerals.

[0007] The technical solution of the present application is a Pseudomonas brassicacearum WG2-6 strain, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20251214.

[0008] A microorganism-clay mineral binary body, characterized in that the binary body is composed of Pseudomonas brassicacearum WG2-6 bacteria and montmorillonite powder (MMT).

[0009] Further, the weight ratio of the Pseudomonas brassicacearum WG2-6 bacteria and the montmorillonite powder is 2:1.

[0010] Further, the Pseudomonas brassicacearum WG2-6 bacteria are prepared by the following method: inoculating Pseudomonas brassicacearum WG2-6 into sterile LB liquid medium and culturing for 24 hours, centrifuging the wet bacteria at 8000 r / min for 10 minutes, and washing the bacteria twice with sterile ultrapure water.

[0011] A uranium pollution remediation method based on the synergistic effect of a microorganism-clay mineral binary body, wherein the binary body is added to wastewater or soil containing uranium, the pH value of the system is controlled to be 3-9, and the temperature is controlled to be 25-40 DEG C, so as to complete the adsorption and solidification of uranium.

[0012] Further, the pH value is 5.

[0013] Further, the temperature is 35 DEG C.

[0014] Compared with the prior art, the present application has the following beneficial effects:

[0015] The present invention combines the synergistic properties of WG2-6 and montmorillonite minerals, resulting in a remediation system with excellent uranium fixation capabilities, effectively removing uranium contaminants from soil. Through its bioremediation mechanisms, such as surface coordination, ion exchange, and chelation mediated by extracellular polymers, WG2-6 collaborates with montmorillonite to convert uranium ions into a stable chemically bound state, facilitating long-term fixation. Montmorillonite provides a good growth substrate for WG2-6, promoting bacterial metabolic activity. Its layered structure and abundant surface sites significantly enhance uranium fixation. Experiments have shown that under the optimal ratio (WG2-6:MMT = 2:1) and suitable environmental conditions (pH = 5.0, 35°C), the binary formed by WG2-6 and montmorillonite achieves an adsorption rate of over 90% for uranium through the abundant amide II bonds, carboxyl groups, siloxane-aluminum bonds, siloxane-silicon bonds, and phosphate groups on its surface and in the extracellular polymers, and the immobilized product remains stable. The remediation system of the present invention not only efficiently fixes uranium but also improves the soil microenvironment. The introduction of montmorillonite promotes the secretion of extracellular polymers in WG2-6, enhancing its uranium complexing capacity. The formation of mineral-microbial aggregates also helps maintain soil structure and promotes the ecological function of the microbial community. Furthermore, both WG2-6 and montmorillonite used in the present invention are environmentally friendly materials that do not introduce secondary pollution, are low-cost, and are easy to operate. Experimental verification has demonstrated that this technology has a significant remediation effect on uranium-contaminated soil, while balancing environmental safety and economic feasibility.

[0016] Collection information:

[0017] Pseudomonas brassicacearum WG2-6 was deposited in the China Center for Type Culture Collection on May 28, 2025, with the deposit number: CCTCC NO: M 20251214, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology and cell morphology of WG2-6 are shown in the figure: a is the colony morphology of WG2-6 on LB plate culture medium; b is the cell morphology of WG2-6 at 100×.

[0019] Figure 2 This is the 16S rRNA phylogenetic tree of WG2-6.

[0020] Figure 3 The growth curves of WG2-6 at different ratios of WG2-6 to montmorillonite (WG2-6:MMT=0:1, 2:1, 1:1, 1:2, 1:0).

[0021] Figure 4Figure 3 shows the effects of different factors on the immobilization of uranium by WG2-6, MMT and WG2-6+MMT. In the figure: a shows the effect of pH on the immobilization of uranium by WG2-6, MMT and WG2-6+MMT; b shows the effect of temperature on the immobilization of uranium by WG2-6, MMT and WG2-6+MMT.

[0022] Figure 5 These are the SEM images of WG2-6, MMT and WG2-6+MMT before and after the reaction with uranium. In the figure: a is the SEM image of WG2-6; b is the SEM image of MMT; c is the SEM image of WG2-6+MMT; d is the SEM image of WG2-6+U; e is the SEM image of MMT+U; f is the SEM image of WG2-6+MMT+U.

[0023] Figure 6 FTIR analysis of WG2-6, MMT and WG2-6+MMT before and after reaction with uranium.

[0024] Figure 7 This is the XPS analysis of WG2-6, MMT and WG2-6+MMT before and after the reaction with uranium. In the figure: a is the full spectrum of WG2-6, MMT and WG2-6+MMT before the reaction with uranium; b is the full spectrum of WG2-6, MMT and WG2-6+MMT after the reaction with uranium; c is the C1s fine spectrum of WG2-6, MMT and WG2-6+MMT before the reaction with uranium; d is the C1s fine spectrum after the reaction of WG2-6, MMT and WG2-6+MMT with uranium; e is the P 2p fine spectrum of WG2-6, MMT and WG2-6+MMT before the reaction with uranium; f is the P 2p fine spectrum after the reaction of WG2-6, MMT and WG2-6+MMT with uranium; g is the U 4f fine spectrum of WG2-6, MMT and WG2-6+MMT before the reaction with uranium; h is the U 4f fine spectrum after the reaction of WG2-6, MMT and WG2-6+MMT with uranium.

[0025] Figure 8 This is the 3D-EEM analysis, in the figure: a is the 3D-EEM image of WG2-6; b is the 3D-EEM image of WG2-6+U; c is the 3D-EEM image of WG2-6+MMT+U.

[0026] Figure 9 It is a mechanism diagram of the present invention. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0028] Example 1 Isolation, identification and preservation of WG2-6

[0029] (1) Separation of WG2-6

[0030] Rhizosphere soil samples at a depth of 10-20 cm were collected from a uranium tailings mine in southwestern China for the isolation of phosphate-solubilizing bacteria. Soil samples were collected in sterile sampling bags and stored in the laboratory at 4°C. The phosphate-solubilizing bacteria were isolated using the clearing zone method. Briefly, 20 g of soil sample was placed in a sterile Erlenmeyer flask containing 90 ml of sterile saline and several glass beads. The suspension was shaken at 180 rpm and 30°C for 30 minutes to prepare a soil suspension. The supernatant of the static soil suspension was diluted 100-fold and spread onto a phosphate-solubilizing agar plate. The plate was then incubated at 35°C for 3 days. The culture medium was observed for the presence of colonies and the formation of hydrolysis zones. Colonies that produced clear clearing zones were selected, streaked onto LB solid medium, numbered, and incubated at 35°C for 24 hours to obtain single colonies. Single colonies were then streaked onto LB solid medium. This process was repeated three times to obtain a pure culture.

[0031] The LB medium was prepared as follows: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1000 mL distilled water. The phosphate-solubilizing medium was prepared as follows: 10.0 g glucose, 0.5 g (NH₄)₂SO₄, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO₄·7H₂O, 0.03 g FeSO₄·7H₂O, 0.03 g MnSO₄·4H₂O, 5.0 g tricalcium phosphate, and 1000 mL distilled water. After the above medium was prepared, the pH was adjusted to 7.0 ± 0.5 using 1 M NaOH and 1 M HCl. For solid medium, 1.5% agar powder was added. Finally, the medium was autoclaved at 121°C for 20 min.

[0032] (2) Morphological identification of WG2-6

[0033] Use an inoculating loop to pick up a small amount of WG2-6 and streak it on an LB plate to isolate a single colony. Take a photo of the colony growing on the plate and record it. Then observe its cell morphology under a Gram staining microscope. WG2-6 forms colonies on the LB plate with a diameter of 2-5 mm. The colonies are round, convex, with a moist and smooth surface and neat edges. Its cell morphology: Gram staining is G-, rod-shaped, as shown in Figure 2. Figure 1 shown.

[0034] (3) 16S rRNA sequencing and preservation of WG2-6

[0035] The genome of WG2-6 was extracted using a kit, and 16S rDNA sequencing and strain identification were performed using the following primers: upstream: 5′-AGAGTTTGATCMTGGCTCAG-3′, downstream: 5′-TACGGYTACCTTGTTACGACTT-3′. After obtaining the sequencing results, they were imported into the NCBI database using BLAST software for homology comparison. The results showed that WG2-6 was Pseudomonas brassicae. A phylogenetic tree was constructed (see Figure 5). Figure 2 It was deposited in China Center for Type Culture Collection with the accession number CCTCC M 20251214.

[0036] Example 2 Cultivation of WG2-6 and Montmorillonite Pretreatment

[0037] Streak WG2-6 onto LB agar medium and incubate at 35°C for 12-24 hours. Pick a single colony and transfer it to fresh sterile LB liquid medium for 24 hours. Centrifuge at 8000 rpm for 10 minutes to collect the wet cells and wash twice with sterile ultrapure water.

[0038] Montmorillonite was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd. A slurry suspension was prepared by mixing montmorillonite with ultrapure water at a ratio of 1:10. The suspension was stirred at 500 rpm in a magnetic stirrer at 40°C for 60 minutes. The suspension was then sonicated for 5 minutes and centrifuged at 4000 rpm for 9 minutes. The supernatant was removed, and the precipitate was dried in an 80°C drying oven. Finally, the suspension was ground through a 100-mesh sieve to obtain montmorillonite powder.

[0039] Example 3 Effect of montmorillonite on the growth of WG2-6

[0040] Weigh the corresponding mass of bacteria and minerals according to different ratios (WG2-6:MMT=0:1, 2:1, 1:1, 1:2, 1:0), place them in 5 centrifuge tubes, add LB medium to 40mL, and mix thoroughly. Then, take 10mL of the mixture and transfer it to a sterile growth curve reaction tube, with 3 replicates for each group. Place the reaction tube in a constant temperature shaker (35℃, 180rpm) and culture it. Take samples every 2 hours to measure the OD 600 The values ​​were continuously monitored for 68 hours, and bacterial growth was recorded. A single-mineral control group (montmorillonite only) and a single-bacteria control group (WG2-6 only) were also set up to eliminate background interference. After the experiment, the bacterial growth curves under different ratios were analyzed to determine the optimal ratio.

[0041] Experiments show that the optimal ratio of WG2-6 to montmorillonite is 2:1. At this time, montmorillonite provides a good growth environment for bacteria and is conducive to the exchange of substances between cells. When the bacterial ratio drops to 1:1, the relative lack of WG2-6 leads to less contact between WG2-6 and montmorillonite, and montmorillonite is not fully utilized. When montmorillonite is not added at all, bacteria grow poorly due to lack of mineral protection. On the contrary, when the montmorillonite ratio is increased to 1:2, its dense structure will hinder the transmission of oxygen and nutrients, which also limits bacterial growth ( Figure 3 ).

[0042] Example 4 Effect of different conditions on uranium fixation in WG2-6 and montmorillonite binary

[0043] Batch adsorption experiments were conducted in sterile 50ml polypropylene centrifuge tubes with a Pseudomonas concentration of 2g / L, montmorillonite concentration of 1g / L, and uranium concentration of 100mg / L. The reaction volume was set at 40ml and the pH range was 3.0-9.0. Adsorption experiments were performed in a thermostatic shaker (35°C, 180 rpm).

[0044] (1) Effect of pH on uranium fixation in WG2-6 and montmorillonite binary

[0045] Adjust the pH of the reaction solution to 3.0-9.0 (in intervals of 1.0), react at 35°C until adsorption equilibrium is reached, and then measure the residual uranium concentration. Uranium concentration determination steps: Centrifuge the reaction suspension at 8000 rpm for 10 minutes. Transfer 0.5 ml of the supernatant to a 50 ml colorimetric tube, add 1 ml of arsenazo III and 5 ml of sodium chloroacetic acid buffer solution, and fill the tube to the 50 ml mark with ultrapure water. Mix by repeated inversion. After reacting for 10 minutes, measure the absorbance of uranium at a wavelength of 652 nm.

[0046] The uranium removal rate R (%) is calculated by the following formula:

[0047] R=(C0-C e ) / C0×100%

[0048] Where R (%) represents the removal rate of uranium, C0 (mol / L) is UO2 2+ The initial concentration of the solution, C e (mol / L) is UO2 2+ The equilibrium concentration of the solution after adsorption.

[0049] The results of uranium removal efficiency of WG2-6 and montmorillonite binary immobilized uranium under different pH conditions are shown in Figure 4 The results show that the uranium removal rate of this binary system increases first and then decreases with increasing pH, reaching a peak at pH 5.0. Therefore, the optimal pH for uranium fixation in the WG2-6 and montmorillonite binary system is 5.0.

[0050] (2) Temperature effect on uranium adsorption by WG2-6 and montmorillonite binary system

[0051] The adsorption performance of WG2-6 and montmorillonite binary system for uranium was investigated at 25℃, 30℃, 35℃ and 40℃ under the optimum pH condition.

[0052] The results showed that temperature had a certain influence on biosorption Figure 4 In the temperature range of 25-40℃, increasing the experimental temperature helped to speed up the adsorption rate of uranium on WG2-6 and montmorillonite binary system. This finding was consistent with existing research, indicating that the adsorption of uranium on microorganisms and montmorillonite was an endothermic process, and higher temperature promoted the Brownian motion of uranium ions, thereby enhancing the adsorption effect. However, some studies have shown that excessively high temperature may cause bacteria to die, so the experimental temperature was set at around 35℃, which was more appropriate.

[0053] Example 5: Systematic characterization of uranium immobilized by WG2-6 and montmorillonite binary system

[0054] The experiment was carried out in a 50mL sterile polypropylene centrifuge tube, and the reaction system was 40mL, with WG2-6 concentration of 2g / L, montmorillonite concentration of 1g / L, and uranium concentration of 100mg / L. The pH was adjusted to 5.0, and the experiment was uniformly carried out in a constant temperature shaker (35℃, 180rpm). After the reaction was completed, the sample was centrifuged at 8000rpm for 10min, the supernatant was discarded, and the precipitate was washed with ultrapure water three times and used for systematic characterization to analyze its composition.

[0055] (1) Scanning electron microscopy (SEM, sample morphology analysis)

[0056] SEM (Germany, ZEISS Sigma 300) was used to analyze the sample morphology and elemental composition: The collected precipitate was washed with ultrapure water three times, fixed in 2.5% glutaraldehyde at 4℃ for 12h, and gradient dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol for 10min each time. Then the sample was dried with a vacuum freeze dryer, and finally a small amount of sample powder was placed on the surface of conductive glue and vacuum coated with gold. The sample was observed by SEM. The acceleration voltage was 0-20kV, and the excitation electron beam was 30-40 ·· A.

[0057] Figure 5 The SEM characterization results showed that WG2-6 presented a typical smooth and complete surface morphology Figure 5 In the temperature range of 25-40℃, increasing the experimental temperature helped to speed up the adsorption rate of uranium on WG2-6 and montmorillonite binary system. This finding was consistent with existing research, indicating that the adsorption of uranium on microorganisms and montmorillonite was an endothermic process, and higher temperature promoted the Brownian motion of uranium ions, thereby enhancing the adsorption effect. However, some studies have shown that excessively high temperature may cause bacteria to die, so the experimental temperature was set at around 35℃, which was more appropriate. Figure 5Middle c); After uranium treatment, the surface of WG2-6 becomes significantly roughened ( Figure 5 b), montmorillonite crystals partially dissolved ( Figure 5 In the binary system of WG2-6 and montmorillonite, montmorillonite can effectively adsorb WG2-6 through its surface and layered structure, and white particles can be seen on its surface ( Figure 5 After the binary reacted with uranium, the surface of WG2-6 showed significant roughening and wrinkling, and the crystal structure of montmorillonite was partially dissolved ( Figure 5 f).

[0058] (2) Fourier transform infrared spectroscopy (FT-IR, functional group characteristics and molecular structure analysis) and X-ray photoelectron spectroscopy (XPS, elemental composition and chemical valence state analysis)

[0059] FT-IR (Thermo Scientific Nicolet 6700, USA) was used to analyze the changes in the characteristic peaks of the main functional groups on the sample surface: the collected reaction precipitate was dried in a freeze dryer. The dried sample powder was thoroughly mixed with KBr at a mass ratio of 1:100, ground, and then pressed into a tablet for measurement. The scanning range was 400-4000 cm -1 , resolution 4cm -1 , the sample was scanned 32 times.

[0060] XPS (Thermo Scientific K-Alpha, USA) was used to analyze the main elemental composition and valence state changes of the three sample groups. Sample powder was evenly spread on the XPS sample stage and scanned using an Al Kα excitation source at an energy of 1486.6 eV and a voltage of 12 kV to determine the elements present.

[0061] Figure 6 It shows that WG2-6, montmorillonite and its binary adsorbed uranium at 915 cm -1 UO2 appears nearby 2+ The characteristic peaks indicate that uranium is successfully adsorbed. It is worth noting that after WG2-6 and montmorillonite adsorb uranium, the peak located at 1641.03 cm -1 、1532.05cm -1 、1049.32cm -1 、526.15cm -1 、469.75cm -1 The peak shifted to 1645.61 cm -1 、1048.81cm -1 、525.71cm -1 、469.92cm -1The results show that the functional groups on the surface of the binary, such as carboxyl, phosphate, silicon-oxygen-aluminum bond and silicon-oxygen-silicon bond, are the main participants in the interaction with uranium in the adsorption process.

[0062] Figure 7 The results show that the functional groups on the surface of the binary, such as carboxyl, phosphate, silicon-oxygen-aluminum bond and silicon-oxygen-silicon bond, are the main participants in the interaction with uranium in the adsorption process.

[0063] (3) Three-dimensional fluorescence excitation-emission matrix spectroscopy (3D-EEM, composition, source and characteristics of fluorescent components)

[0064] Three-dimensional fluorescence excitation-emission matrix spectroscopy (3D-EEM, composition, source and characteristics of fluorescent components) was used to qualitatively and semi-quantitatively determine the composition of EPS and its changes: EPS was extracted from three groups of samples by heat extraction method, and three-dimensional fluorescence spectroscopy data of EPS were obtained by 3D-EEM after extraction. The excitation wavelength and emission wavelength range were both 200 nm-550 nm, the increment was 5 nm, and the scanning rate was 2400 nm / min.

[0065] The heat extraction method steps are as follows: after the reaction is completed, the sample solution is placed in a 40℃ water bath for 30 min, and then centrifuged at 8000 rpm for 10 min in a centrifuge, and the supernatant is filtered through a 0.22 μm filter membrane to obtain the extracted EPS solution.

[0066] Figure 8 The 3D-EEM analysis of the EPS of WG2-6 shows that the EPS of WG2-6 presents characteristic fluorescence peaks at 280 / (330-340) nm (peak A, tryptophan protein) and 230 / (320-340) nm (peak B, tyrosine). After uranium treatment, the intensities of peaks A and B change significantly, confirming that the protein components in WG2-6 participate in the fixation of uranium. After the addition of montmorillonite, the intensity of peak B is significantly enhanced, indicating that montmorillonite can promote the secretion of tyrosine in WG2-6, which is consistent with the change of nitrogen-containing functional groups detected by XPS, indicating that montmorillonite enhances the uranium fixation capacity of WG2-6 by regulating the content of EPS components in WG2-6.

Claims

1. A strain of Pseudomonas brassicacearum WG2-6, deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20251214.

2. A microorganism-clay mineral binary system, characterized in that: The binary body is composed of the Pseudomonas brassicacearum WG2-6 bacterial cell according to claim 1 and montmorillonite powder.

3. The binary body according to claim 2, characterized in that: The weight ratio of the Pseudomonas brassicae WG2-6 bacterial cells to the montmorillonite powder is 2:

1.

4. The binary body according to claim 3, characterized in that: The Pseudomonas brassicacearum WG2-6 bacterial cells were prepared by the following method: inoculating Pseudomonas brassicacearum WG2-6 into a sterile LB liquid culture medium and culturing for 24 hours, collecting the wet bacterial cells by centrifugation at 8000 r / min for 10 minutes, and washing them twice with sterile ultrapure water.

5. A uranium contamination remediation method based on the synergistic effect of microorganisms and clay minerals, characterized in that: The binary according to any one of claims 2 to 4 is added to wastewater or soil containing uranium, and the pH value of the system is adjusted to 3-9 and the temperature to 25-40° C. to complete the adsorption and solidification of uranium.

6. The method according to claim 5, characterized in that The pH value is 5.

7. The method according to claim 5, characterized in that The temperature was 35°C.

Images