A plant-microorganism combined uranium pollution control method

Abstract

This invention discloses a plant-microorganism combined method for uranium contamination remediation. The method involves planting sterilized uranium-tolerant plant seeds into the soil to be remediated. After applying a suspension of uranium-tolerant strains (such as Rhodotorula rubrum M1, Aspergillus M2, and Bacillus M3) to the soil, the uranium accumulation capacity of the uranium-tolerant plants is significantly enhanced, achieving good remediation results for uranium-contaminated soil in temperate climate zones. The uranium-tolerant plants can be harvested manually or mechanically and transported to a safe location for treatment, thus effectively remediating uranium-contaminated soil in temperate climate zones.

Description

Technical Field

[0001] This invention belongs to the field of uranium contaminated soil phytoremediation technology, specifically relating to a plant-microorganism combined uranium contamination remediation method. Background Technology

[0002] Nuclear energy is a low-carbon and clean energy source, and with industrial development, it has become one of my country's important energy sources. However, the development of nuclear energy depends on the mining and smelting of uranium. The rapid development of the nuclear industry has increased the demand for nuclear fuel, accelerating uranium mining and smelting. However, the mining and smelting process generates large amounts of waste and tailings, the uranium content of which is far higher than that of natural soil. The uranium in these wastes, when dispersed into the soil, air, or groundwater, can have a significant impact on the ecological environment. Compared to uranium pollution in the atmosphere and water, uranium pollution in soil is characterized by its insidious nature, cumulative effect, long remediation period, and high toxicity, attracting increasing attention from scholars and the public.

[0003] Remediation methods for uranium-contaminated soil can be mainly divided into physical remediation, chemical remediation, bioremediation, and integrated remediation technologies. In recent years, phytoremediation technology has attracted attention due to its advantages such as in-situ remediation, economy, and environmental friendliness, and has become an effective means of treating soil contaminated with heavy metals and radionuclides. Microbial-phytoremediation technology combines microbial and phytoremediation, fully leveraging the advantages of both materials. It can improve the rhizosphere microenvironment of plants, exhibiting synergistic effects and promoting plant growth, thereby enhancing the remediation efficiency of plants in radionuclide-contaminated soil. However, current methods for uranium contamination in soil mostly employ single-method microbial or phytoremediation approaches, both of which have limitations. Therefore, utilizing microorganisms to enhance phytoremediation is a mutually beneficial approach that does not cause secondary pollution to the environment and represents a future direction for sustainable development. Current uranium-accumulating plants and radiation-resistant microorganisms are mainly targeted at radioactive contaminated soil environments in subtropical regions of South China (such as East and South China). Hard-rock uranium deposits in Northwest my country (such as the Longshoushan and Helanshan mining areas in Gansu and Ningxia) generally exhibit typical continental arid climate characteristics: low annual precipitation, large diurnal temperature range, and low organic matter content. These extreme environments present multiple challenges to traditional bioremediation technologies. First, microorganisms and plants have poor adaptability: due to temperature differences, radiation-resistant strains and plants screened in East China have low survival rates in Northwest soils. Second, uranium has complex occurrence forms: weathering of hard-rock uranium deposits produces uranyl carbonate complexes (UO2(CO3)3). 4(⁻) High proportion of uranium, resulting in poor fixation efficiency of conventional microorganisms for soluble uranium; finally, weak ecological restoration capacity: the soil microbial diversity index in the Northwest mining area is significantly lower than that in the southern uranium mining area, and the indigenous microbial community is unable to support the niche competition of exogenous remediation bacteria. Therefore, finding a combined remediation method of radiation-resistant microorganisms and plants suitable for the mid-temperate climate zone is crucial for the remediation of radioactive contamination in the soil surrounding uranium mines in Northwest my country. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a plant-microorganism combined uranium contamination treatment method.

[0005] The objective of this invention is achieved by at least one of the following technical solutions.

[0006] A plant-microorganism combined method for uranium contamination remediation includes the following steps:

[0007] (1) Plant the disinfected uranium-tolerant plant seeds into the soil to be remediated;

[0008] (2) Apply a suspension of uranium-resistant bacterial strains to the soil to be remediated; the uranium-resistant bacterial strains include one or more of Rhodotorula rubra M1, Aspergillus M2 and Bacillus M3;

[0009] (3) Harvest uranium-tolerant plants and process them centrally.

[0010] Furthermore, in step (1), the uranium-tolerant plants include one or more of the following: corn, mustard greens, asparagus, yellow privet, rosemary, spruce, gentian, fescue, caragana, pine wormwood, Paris polyphylla, thistle, sedge, and white sheep grass.

[0011] Furthermore, in step (1), the uranium-tolerant plants include one or more of corn, mustard greens, and sow thistle.

[0012] Furthermore, in step (1), the seed spacing of the uranium-tolerant plant is 20-50cm.

[0013] Furthermore, in step (1), the seed spacing of the uranium-tolerant plant is 30cm.

[0014] Further, in step (1), the method for disinfecting the seeds of the uranium-tolerant plant is as follows: the seeds of the uranium-tolerant plant are first washed with 75% ethanol for 3-10 min, then washed 3 times with sterile distilled water, then soaked in 10% H2O2 for 10-30 min, and finally washed with 30-100 mL of Hogland nutrient solution.

[0015] Furthermore, in step (1), the method for disinfecting the seeds of the uranium-tolerant plant is as follows: the seeds of the uranium-tolerant plant are first washed with 75% ethanol for 4 min, then washed 3 times with sterile distilled water, then soaked in 10% H2O2 for 20 min, and finally washed with 50 mL of Hogland nutrient solution.

[0016] Furthermore, in step (2), the method of applying the bacterial suspension of the uranium-resistant strain is to apply it to the soil above the seeds of the uranium-resistant plant or to the rhizosphere soil of the uranium-resistant plant.

[0017] Furthermore, in step (2), the amount of the uranium-resistant bacterial suspension applied is 30-100 mL per plant per application.

[0018] Furthermore, in step (2), the amount of the uranium-resistant bacterial suspension applied is 50 mL per plant per application.

[0019] Furthermore, in step (2), the uranium-resistant strain in the bacterial suspension is in the logarithmic growth phase.

[0020] Furthermore, the concentration of Rhodotorula rubra M1 in logarithmic growth phase was OD600 = 2.5-3.6; the concentration of suspended solids in the mixed solution of Aspergillus M2 in logarithmic growth phase was 3.0-4.5 mg / L; and the concentration of Bacillus M3 in logarithmic growth phase was OD600 = 0.6-1.2.

[0021] Furthermore, in step (2), the bacterial suspension of the uranium-resistant strain is repeatedly applied to the soil to be remediated every 3-10 days.

[0022] Furthermore, in step (2), the bacterial suspension of the uranium-resistant strain is repeatedly applied to the soil to be remediated every 5 days.

[0023] Furthermore, in step (3), the specific method for harvesting uranium-resistant plants and carrying out centralized processing is as follows: the uranium-resistant plants are harvested manually or mechanically, and then the harvested uranium-resistant plants are transferred to a safe place for centralized drying, crushing, and incineration, and finally landfilling or leaching to recover uranium.

[0024] Furthermore, in step (3), the uranium-tolerant plants are harvested once every 30 days or 60 days.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) After applying uranium-tolerant strains M1, M2 or M3 to the rhizosphere of uranium-tolerant plants, their uranium enrichment capacity is significantly enhanced, achieving good results in the remediation of uranium-contaminated soil in temperate climate areas. Uranium-tolerant plants can be harvested manually or mechanically and transferred to a safe place for treatment.

[0027] (2) Uranium-tolerant strains M1, M2 and M3 can enhance the enrichment of uranium in the roots of uranium-tolerant plants and increase the translocation of uranium to the aboveground parts of the plants, thus promoting the enrichment of uranium in plants.

[0028] (3) Under the combined action of plants and microorganisms, the overall remediation effect of adding M1 and M3 bacterial agents is better than that of M2. The soil uranium loss in cornfields and mustard fields with added M1 is more obvious. After adding M1, the transfer coefficients of corn, mustard and sow thistle all exceeded 1, and the transfer of uranium from underground to surface increased. Among them, the transfer coefficient of mustard was the highest, reaching 2.818. Attached Figure Description

[0029] Figure 1 This is a graph showing the content of typical nuclide U in each sampled plant from Example 1.

[0030] Figure 2 This is a graph showing the changes in soil uranium concentration during a pot experiment involving combined plant-microbe uranium contamination treatment in Example 2.

[0031] Figure 3 This is a graph showing the change in uranium concentration in the soil under single-plant conditions before the addition of microbial agents in the field experiment of Example 3. Detailed Implementation

[0032] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0033] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0034] In this invention, the terms "red yeast M1" and "M1" both refer to the strain with accession number CGMCC No. 34591, and are classified and named accordingly. Rhodotorula toruloides This strain was deposited on May 19, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0035] In this invention, the terms "Aspergillus M2" and "M2" both refer to the strain with accession number CGMCC No. 41942, and are classified and named as follows: Aspergillus welwitschiaeThis strain was deposited on May 19, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] In this invention, the terms "Bacillus M3" and "M3" both refer to the strain with accession number CGMCC No. 34592. Classification and naming. Bacillus wiedmannii This strain was deposited on May 19, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] In this invention, the mixed liquor suspended solids concentration (MLSS) refers to the mass of activated sludge suspended solids per unit volume of mixed liquor, expressed in milligrams per liter (mg / L).

[0038] In this invention, red yeast M1 and Aspergillus M2 are cultured in potato dextrose liquid medium to the logarithmic growth phase.

[0039] In this invention, Bacillus M3 is cultured in beef extract peptone liquid medium to the logarithmic growth phase.

[0040] In this invention, the method for preparing potato glucose liquid culture medium is as follows:

[0041] Cut peeled and washed potatoes into evenly sized small cubes. Weigh out 200 g of the cubes and add them to an appropriate amount of boiling water. Simmer over low heat for about 15-20 minutes (stop heating when a glass rod can just pierce the potato cubes). Then filter the solution through eight layers of gauze and collect the filtrate. Add 20 g of glucose to the filtrate while stirring constantly with a glass rod to accelerate its dissolution. Finally, bring the volume to 1000 mL with distilled water. Dispense the solution, autoclave at 121 °C for 30 minutes, cool, and store for later use.

[0042] In this invention, the method for preparing beef extract peptone liquid culture medium is as follows:

[0043] All solutes were dissolved in deionized water at a ratio of 3 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl, and then sterilized in an autoclave at 121°C for 20 min.

[0044] In this invention, the radionuclide content of soil samples is determined using the following method:

[0045] Soil samples were air-dried naturally, impurities were removed, and then dried in an 80 ℃ oven until constant weight was achieved. The samples were then ground, passed through a 200-mesh sieve, and collected in a desiccator for later use. Soil samples for pH determination by potentiometric method were dried to constant weight and then directly passed through a 20-mesh sieve. Soil pH was measured using a pH meter (water to soil mass ratio m(water):m(soil) = (2.5:1)).

[0046] Weigh 0.1 g of soil sample into a beaker and digest it using aqua regia and perchloric acid (2:1, v / v). After digestion, dilute to a volumetric flask of 100 mL and determine the radionuclide content of the sample using inductively coupled plasma atomic emission spectrometry.

[0047] In this invention, the nuclide content of plant samples is determined using the following method:

[0048] The plant was divided into above-ground and underground parts. After washing with tap water, it was rinsed three times with deionized water. The roots were then soaked in 10 mmol·L⁻¹ solution. -1 Remove adsorbed metal ions from the root surface by soaking in EDTA solution for 10 min, then wash with deionized water, drain off the water, place in an oven at 105 ℃ for 30 min to blanch, then place in an oven at 75 ℃ to constant weight, then place the above-ground and underground parts into porcelain crucibles respectively, gradually raise the temperature in a muffle furnace to 600 ℃, ashed for 6 h, collect the samples and place them in a desiccator for later use.

[0049] Weigh 0.1 g of plant sample into a beaker and digest it using aqua regia and perchloric acid (2:1, v / v). After digestion, dilute to a 100 mL volumetric flask and determine the radionuclide content of the sample using inductively coupled plasma atomic emission spectrometry (ICP-AES). The uranium content in the plant is expressed in mg·kg⁻¹. -1 (As ash content).

[0050] In a specific embodiment of the present invention, the disinfection of plant seeds is carried out using the following method:

[0051] The plant seeds were first washed with 75% ethanol for 4 min, then washed three times with sterile distilled water, soaked in 10% H2O2 for 20 min, and finally washed with 50 mL of Hogland nutrient solution.

[0052] In this invention, the formula for Hogland nutrient solution is as follows:

[0053] Calcium nitrate 945 mg / L

[0054] Potassium nitrate 607 mg / L

[0055] Ammonium phosphate 115 mg / L

[0056] Magnesium sulfate 493 mg / L

[0057] Iron salt solution 2.5 ml / L

[0058] Trace elements 5ml / L

[0059] pH=6.0. The trace element solution formula is as follows:

[0060] Potassium iodide 0.83 mg / L

[0061] Boric acid 6.2 mg / L

[0062] Manganese sulfate 22.3 mg / L

[0063] Zinc sulfate 8.6 mg / L

[0064] Sodium molybdate 0.25 mg / L

[0065] Copper sulfate 0.025 mg / L

[0066] Cobalt chloride 0.025 mg / L.

[0067] In this invention, the formulas for calculating the transfer coefficient and enrichment coefficient of plants are as follows:

[0068] (1)

[0069] (2)

[0070] BCF: Plant enrichment coefficient; Cplant: Concentration of pollutants in plants, mg·kg-1; Csoil: Concentration of pollutants in plant rhizosphere soil, mg·kg-1.

[0071] TF: Plant transfer coefficient; Cabove: Concentration of pollutants in the aboveground parts of plants, mg·kg-1; Cbelow: Concentration of pollutants in the underground parts of plants, mg·kg-1.

[0072] The transfer coefficient reflects a plant's ability to transfer specific substances from its roots to its stems and leaves. A higher transfer coefficient indicates a higher efficiency in the translocation and distribution of specific substances. Plants with high transfer coefficients can remove specific pollutants by harvesting their above-ground parts, making them preferred plants for phytoremediation applications.

[0073] This invention provides a plant-microorganism combined uranium contamination remediation method, specifically including the following steps:

[0074] (1) Plant the disinfected uranium-tolerant plant seeds into the soil to be remediated;

[0075] (2) Apply a suspension of uranium-resistant bacterial strains to the soil to be remediated; the uranium-resistant bacterial strains include one or more of Rhodotorula rubra M1, Aspergillus M2 and Bacillus M3;

[0076] (3) Harvest uranium-tolerant plants and process them centrally.

[0077] In some embodiments of the present invention, in step (1), the uranium-tolerant plants include one or more of the following: corn, mustard greens, asparagus, yellow privet, rosemary, spruce, gentian, fescue, caragana, pine wormwood, Paris polyphylla, thistle, *Smilax china*, and white sheep grass. Preferably, in step (1), the uranium-tolerant plants include one or more of the following: corn, mustard greens, and thistle.

[0078] In some embodiments of the present invention, in step (1), the seed spacing of the uranium-tolerant plant is 20-50 cm. In a preferred embodiment of the present invention, in step (1), the seed spacing of the uranium-tolerant plant is 30 cm.

[0079] In some embodiments of the present invention, in step (1), the method for disinfecting the seeds of the uranium-tolerant plant is as follows: the seeds of the uranium-tolerant plant are first washed with 75% ethanol for 3-10 min, then washed three times with sterile distilled water, then soaked in 10% H2O2 for 10-30 min, and finally washed with 30-100 mL of Hoagland nutrient solution. Preferably, in step (1), the method for disinfecting the seeds of the uranium-tolerant plant is as follows: the seeds of the uranium-tolerant plant are first washed with 75% ethanol for 4 min, then washed three times with sterile distilled water, then soaked in 10% H2O2 for 20 min, and finally washed with 50 mL of Hoagland nutrient solution.

[0080] In some embodiments of the present invention, in step (2), the method of applying the bacterial suspension of the uranium-resistant strain is to apply it to the soil above the seeds of the uranium-resistant plant or to the rhizosphere soil of the uranium-resistant plant.

[0081] In some embodiments of the present invention, in step (2), the amount of the uranium-resistant bacterial suspension applied is 30-100 mL per plant per application. Preferably, in step (2), the amount of the uranium-resistant bacterial suspension applied is 50 mL per plant per application.

[0082] In some embodiments of the present invention, in step (2), the uranium-resistant strain in the bacterial suspension is in the logarithmic growth phase. Preferably, the concentration of the Rhodotorula rubra M1 bacterial suspension in the logarithmic growth phase is OD600 = 2.5-3.6; the concentration of the suspended solids in the Aspergillus M2 bacterial suspension mixture in the logarithmic growth phase is 3.0-4.5 mg / L; and the concentration of the Bacillus M3 bacterial suspension in the logarithmic growth phase is OD600 = 0.6-1.2.

[0083] In some embodiments of the present invention, in step (2), the bacterial suspension of the uranium-resistant strain is repeatedly applied to the soil to be remediated every 3-10 days. Preferably, in step (2), the bacterial suspension of the uranium-resistant strain is repeatedly applied to the soil to be remediated every 5 days.

[0084] In some embodiments of the present invention, the specific method for harvesting uranium-resistant plants and centrally processing them in step (3) is as follows: the uranium-resistant plants are harvested manually or mechanically, and then the harvested uranium-resistant plants are transferred to a safe place for centralized drying, crushing, and incineration, and finally landfilling or leaching to recover uranium.

[0085] In some embodiments of the present invention, in step (3), the uranium-tolerant plants are harvested once every 30 days or 60 days.

[0086] In one specific embodiment of the present invention, the uranium-tolerant plant is mustard greens, and the uranium-tolerant strain is Rhodotorula rubra M1. The combination of the two can effectively promote the translocation of uranium to the aboveground parts of the plant, promote the enrichment of uranium by the plant, and demonstrates excellent performance in the remediation of uranium-contaminated soils in temperate climate zones.

[0087] Example 1: Screening of uranium-tolerant plants

[0088] This study selected soil samples from the area surrounding a uranium mine in the northwestern Liupanshan Basin as the research area. The Liupanshan Basin extends northwestward, wider in the north and narrower in the south, forming an inverted triangle. The basin occupies a unique tectonic location, bordered by the North Qilian-Qinling orogenic belt to the southwest, the Xiangshan fault uplift on the southern edge of the Alashan Block to the north, and the western rift zone of the Ordos Block to the east. Three sedimentary cover layers are present in the basin: Upper Paleozoic Devonian-Carboniferous-Permian, Mesozoic Triassic-Lower Cretaceous, and Cenozoic Paleogene-Quaternary. The study area belongs to the arid hilly region of the Loess Plateau, with a terrain that slopes from south to north, ranging in elevation from 1688 to 2633 m. It is 140 km east of Liupanshan, 28 km from the Xumishan Scenic Area, 63 km from Guyuan City, and 391 km north of Yinchuan City.

[0089] Twelve native plant species were collected from the surrounding contaminated soil in the study area, including annuals, perennials, flowering plants, and non-flowering plants. Different plant types showed varying enrichment capacities for radionuclides. These included *Asparagus officinalis*, *Gynostemma pentaphyllum*, *Rosemary's wort*, *Picea spp.*, *Gentiana scabra*, *Festuca pubescens*, *Caragana sinica*, *Artemisia argyi*, *Paris polyphylla*, *Cirsium japonicum*, *Smilax china*, and *Hemiberlesia sapiens*. Plant samples were collected at approximately 30 cm × 40 cm. Small and long, slender plants were collected whole and folded into V or N shapes for storage; larger plants were collected only from representative portions. The collected plant samples were placed in collection tubes lined with tissue paper and stored uniformly before being sent to the laboratory for processing and testing. To differentiate the enrichment characteristics of the aboveground and belowground parts of the plants, the collected plants were divided into aboveground and belowground samples for separate radionuclide content testing. The content of the typical radionuclide U in each sampled plant is shown below. Figure 1 As shown.

[0090] from Figure 1 It can be seen that different plants have different enrichment abilities for uranium. Among them, thistle, asparagus, and spruce have the best enrichment ability (above ground and below ground), Paris polyphylla, Podocarpus spp., fescue, Caragana korshinskii, and Gentiana have moderate enrichment abilities for uranium, while Smilax china and rosemary have poor enrichment effects for uranium.

[0091] Analysis of plant growth characteristics revealed that the three plant species with the best uranium accumulation capacity were all perennials, while those with moderate accumulation capacity were mainly annuals. The uranium accumulation capacity of the aboveground parts of plants was generally higher than that of the underground parts; among the 12 plant species, the uranium accumulation content in the aboveground parts ranged from 59.76 to 1267.07 mg·kg⁻¹. -1 The underground portion ranges from 53.66 to 370.73 mg / kg. -1 Among them, the above-ground parts of thistle had the highest uranium content, reaching 1267.07 mg·kg⁻¹. -1 The transfer coefficient was also the highest, at 3.79. In contrast to thistle, while asparagus generally exhibits strong uranium enrichment, its underground parts contain a higher uranium content, at 370.73 mg·kg⁻¹. -1 Conversely, the uranium content in the above-ground portion was relatively low, at 59.76 mg·kg⁻¹. -1 Therefore, its transfer coefficient is the lowest, at only 0.16.

[0092] In summary, the uranium enrichment capacity of the 12 representative native plants around the study area varied considerably. *Cirsium japonicum* showed the most significant uranium enrichment capacity among the 12 plants, mainly because it is a perennial plant, cold-resistant, drought-resistant, and highly adaptable. Its well-developed root system and high biomass are conducive to uranium absorption and translocation.

[0093] Example 2: Pot Experiment of Combined Plant-Microorganism Uranium Contamination Treatment

[0094] The experiment used corn and mustard, uranium-accumulating plants that had been previously verified. The seeds were first washed with 75% ethanol for 4 min, then washed three times with sterile distilled water, then soaked in 10% H2O2 for 20 min, and finally washed with 50 mL of Hogland nutrient solution.

[0095] Plant control group: Disinfected corn and mustard seeds were planted in an environment containing a certain concentration of uranium (244.88 mg·kg⁻¹). -1 In the potting soil, three parallel samples were set up for each plant. The plants were watered and observed regularly, and the plants were removed after 50 days of growth.

[0096] Control group of strains: Inoculated with a certain concentration of uranium (244.88 mg·kg⁻¹) -1 Every 5 days, 50 mL of bacterial suspension (50 mL per kg of soil) of either Rhodotorula rubra M1, Aspergillus M2, or Bacillus M3 in the logarithmic growth phase were added to the potting soil. Three replicates were set up for each strain, and the experiment lasted for 50 days. The concentration of Rhodotorula rubra M1 in the logarithmic growth phase was [OD value missing]. 600 =2.5-3.6; the suspended solids concentration of Aspergillus M2 bacterial suspension in logarithmic growth phase is 3.0-4.5 mg / L; the concentration of Bacillus M3 bacterial suspension in logarithmic growth phase is OD 600 =0.6-1.2.

[0097] Experimental group: Disinfected corn and mustard seeds were planted in an environment containing a certain concentration of uranium (244.88 mg·kg⁻¹). -1 In potting soil, bacterial solutions of Rhodotorula rubra M1, Aspergillus M2, or Bacillus M3 in the logarithmic growth phase were added every 5 days (50 mL of bacterial solution per kilogram of soil per application). Three replicates were prepared for each plant + bacterial strain combination. Plants were removed after 50 days of growth. The concentration of Rhodotorula rubra M1 in the logarithmic growth phase was [OD value missing]. 600 =2.5-3.6; the suspended solids concentration of Aspergillus M2 bacterial suspension in logarithmic growth phase is 3.0-4.5 mg / L; the concentration of Bacillus M3 bacterial suspension in logarithmic growth phase is OD 600 =0.6-1.2.

[0098] After the experiment, samples were taken from the potting soil and all the above-ground and underground parts of the plants for analysis, and the nuclide content was determined.

[0099] Table 1. Sample numbers from potted plants subjected to combined plant-microbe uranium contamination treatment

[0100]

[0101] The changes in soil uranium concentration in a pot experiment involving combined plant-microbe uranium contamination treatment are as follows: Figure 2 As shown in Table 2, both single-plant and combined plant-microbe remediation methods reduced the uranium concentration in the soil to varying degrees. However, combined plant-microbe remediation was superior to single-plant or single-strain remediation methods for uranium removal. To further analyze the transfer characteristics of uranium within the plant, enrichment and transfer coefficients of uranium in the aboveground and underground parts of the plant were calculated. The results are shown in Table 2.

[0102] Table 2. Plant uranium enrichment coefficient and transfer coefficient.

[0103]

[0104] As shown in Table 2, for the maize system, the combined use with microorganisms significantly improved the uranium enrichment capacity of maize. Specifically, in the maize-M3 co-remediation system, the uranium enrichment coefficient of plants was the highest both aboveground and belowground, followed by maize-M1, maize-M2, and maize alone. Figure 2 It is evident that the residual uranium concentration in the soil was lowest under the maize-M3 condition, further indicating that the combined remediation efficiency of maize-M3 is optimal. However, the change in the transfer coefficient reveals that the transfer coefficient of maize-M1 is the highest in the maize system, indicating that M1 promotes the transfer of uranium from underground to aboveground parts within the maize system.

[0105] For the mustard system, the trend of combined microbial use is consistent with that of the maize system, indicating that the combined use of plants and microorganisms is more effective in remediating uranium-contaminated soil. In the combined remediation of mustard-M1, the enrichment coefficient of uranium by mustard was the highest in both aboveground and belowground environments, followed by mustard-M2, and then mustard alone. Figure 2 It is evident that the residual uranium concentration in the soil was lowest under the mustard-M1 condition, further demonstrating that the combined remediation effect of mustard-M1 is better.

[0106] In conclusion, the combined remediation of plants and microorganisms is more effective than that of a single plant or a single strain. Adding native uranium-tolerant strains to contaminated soil can promote the transfer of uranium from soil to plants. Among the three uranium-tolerant strains, M1 showed the most outstanding performance, exhibiting a significant enrichment-promoting function in various plants. The combined remediation of mustard greens and M1 showed the best removal effect on uranium contamination in soil.

[0107] Example 3: Field Experiment of Plant-Microorganism Combined Uranium Contamination Treatment

[0108] The experimental site was located in the study area, covering a total area of ​​100 m2. The site was divided into three zones: corn, mustard, and thistle, covering 45 m2, 33 m2, and 22 m2 respectively. The initial uranium concentration in the soil was 198.66 mg·kg⁻¹. -1 196.95 mg·kg -1192.38 mg·kg -1 .

[0109] Disinfected corn and mustard seeds, along with native transplanted *Cirsium japonicum* plants, were planted in the soil of the study area (8 rows, 6 rows, and 4 rows), maintaining a plant spacing of 30 cm to form a plant community. The first plant harvest and soil sampling were conducted 60 days after the community's establishment. Every 5 days, 50 mL of bacterial solution from *Rhodotorula rubra* M1, *Aspergillus* M2, or *Bacillus* M3 in the logarithmic growth phase was added to the soil, establishing a plant-microbe symbiotic system. The concentration of *Rhodotorula rubra* M1 in the logarithmic growth phase was [OD value missing]. 600 =2.5-3.6; the suspended solids concentration of Aspergillus M2 bacterial suspension in logarithmic growth phase is 3.0-4.5 mg / L; the concentration of Bacillus M3 bacterial suspension in logarithmic growth phase is OD 600 =0.6-1.2. After 30 days of growth, the plants were harvested and soil samples were collected for the second time. The sample numbers after harvesting are shown in Table 3. The field experiment lasted for 90 days, and the soil remediation depth was 20cm.

[0110] The control group was given an equal volume of culture medium without bacterial strain.

[0111] Table 3. Number of uranium-enriched samples from the remediation site

[0112]

[0113] Under field conditions, the changes in soil uranium concentration under single-plant conditions before the addition of inoculants are as follows: Figure 3 As shown. From Figure 3 The trends in soil uranium content show that all three plants have a certain removal effect on uranium in the soil. Compared with the indoor pot experiment, the soil planted with maize experienced greater uranium concentration loss, while the soil planted with mustard showed less loss. This may be related to the meteorological and hydrological environment of the field site. Maize adapts well to the local environment, while mustard prefers moist conditions and is intolerant of heat, growing slowly at temperatures above 25 ℃. The study area is located in Northwest my country, where the field experimental site has a dry and hot climate, which is unsuitable for mustard growth compared to southern regions. This may also lead to a decrease in the uranium enrichment effect of mustard.

[0114] To further analyze the transfer characteristics of uranium into plants under field conditions, the enrichment coefficient and transfer coefficient of uranium in the aboveground and underground parts of plants were calculated, and the results are shown in Table 4.

[0115] Table 4. Effects of plants on uranium enrichment

[0116]

[0117] Table 4 shows that there is still a certain difference in uranium accumulation capacity between the aboveground and underground parts of the plants, with the underground parts generally showing a higher accumulation capacity than the aboveground parts. This is consistent with the typical distribution pattern of heavy metal absorption by plants and is also consistent with the results of indoor potted plant experiments. Under single plant conditions, the aboveground parts of *Thistle* showed the best uranium accumulation capacity, reaching 84.15 mg·kg⁻¹. -1 It is higher than that of corn (53.66 mg·kg). -1 ) and mustard greens (59.76 mg·kg) -1 Regarding the underground parts, all three plants were enriched in uranium roots, with the highest uranium content (108.54 mg·kg) found in the underground parts of *Cirsium japonicum*. -1 ), followed by corn (71.95 mg·kg). -1 ) and mustard greens (65.85 mg·kg) -1 ).

[0118] In single-plant conditions, compared with indoor pot experiments, it can be seen that the uranium enrichment coefficients of the above-ground and below-ground parts of corn and mustard do not change much, and the enrichment coefficients of corn above-ground and below-ground parts increase slightly. This may be related to the fact that the concentration of uranium in the outdoor soil is lower than that in the indoor pot experiment soil, indicating that corn and mustard still have a certain enrichment effect on uranium in temperate climate regions.

[0119] After the addition of the microbial agent, all types of crops showed stronger uranium accumulation capacity. In particular, the mustard field with added M1 had a conversion ratio of 2.818 between the aboveground and underground parts, which was significantly higher than other groups, indicating that M1 can effectively enhance the plant's absorption and translocation efficiency of uranium, consistent with the results of indoor pot experiments.

[0120] Table 5 shows the results of microbial-plant co-remediation of uranium-contaminated soil under field conditions.

[0121] Table 5. Changes in uranium concentration in soil at field sites

[0122]

[0123] Table 5 shows that the uranium concentration in the soil of the field sites decreased significantly after the addition of microbial agents. Specifically, the uranium concentration in the field sites treated with M1 and M3 microbial agents was significantly lower than that treated with M2, and the degree of reduction in soil uranium concentration was greater compared to the indoor pot experiment. This further demonstrates that strains M1 and M3 have significant advantages in enhancing the uranium accumulation capacity of plants and removing uranium from the soil, especially when applied to corn and mustard fields.

[0124] Indoor pot experiments and field experiments further demonstrated that corn, mustard greens, and thistle, as well as uranium-tolerant bacterial strains M1, M2, and M3, all possess a certain remediation capacity for uranium-contaminated soil. Furthermore, the addition of bacterial agents facilitated the accumulation of uranium in contaminated soil by plants. Strain M1 exhibited the best uranium accumulation effect and the best effect on promoting uranium accumulation in plants.

[0125] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A plant-microorganism combined method for uranium contamination remediation, characterized in that, Specifically, the following steps are included: (1) The seeds of uranium-tolerant plants that have been disinfected are planted in the soil to be remediated; the soil to be remediated is uranium-contaminated soil in the temperate climate zone; (2) Apply a suspension of uranium-resistant bacterial strains to the soil to be remediated; the uranium-resistant strains include one or more of Rhodotorula rubrum M1, Aspergillus M2, and Bacillus M3; the preservation number of Rhodotorula rubrum M1 is CGMCC No. 34591, and its classification name is Rhodotorula toruloides The Aspergillus M2 strain has the preservation number CGMCC No. 41942 and is classified as follows: Aspergillus welwitschiae The Bacillus M3 specimen has the accession number CGMCC No. 34592 and is classified as follows: Bacillus wiedmannii ; (3) Harvest uranium-tolerant plants and process them centrally.

2. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (1), the uranium-tolerant plants include one or more of the following: corn, mustard greens, asparagus, yellow privet, rosemary, spruce, gentian, fescue, caragana, pine wormwood, Paris polyphylla, thistle, sedge, and white sheep grass.

3. The plant-microorganism combined uranium contamination remediation method according to claim 2, characterized in that, In step (1), the uranium-tolerant plants include one or more of corn, mustard greens, and sow thistle.

4. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (1), the seed spacing of the uranium-tolerant plant is 20-50cm.

5. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (1), the method for disinfecting the seeds of uranium-tolerant plants is as follows: the seeds of uranium-tolerant plants are first washed with 75% ethanol for 3-10 min, then washed 3 times with sterile distilled water, then soaked in 10% H2O2 for 10-30 min, and finally washed with 30-100 mL of Hogland nutrient solution.

6. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (2), the amount of the uranium-resistant bacterial suspension applied is 30-100 mL per plant per application.

7. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (2), the uranium-resistant strain in the bacterial suspension is in the logarithmic growth phase.

8. The plant-microorganism combined uranium contamination remediation method according to claim 7, characterized in that, The concentration of Rhodotorula glutinis M1 culture in the logarithmic growth phase is OD. 600 =2.5-3.6; the suspended solids concentration of Aspergillus M2 bacterial suspension in logarithmic growth phase is 3.0-4.5 mg / L; the concentration of Bacillus M3 bacterial suspension in logarithmic growth phase is OD 600 =0.6-1.

2.

9. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (2), the bacterial suspension of the uranium-resistant strain is repeatedly applied to the soil to be remediated every 3-10 days.

10. The plant-microorganism combined uranium contamination remediation method according to claim 1, characterized in that, In step (3), the specific method for harvesting uranium-resistant plants and carrying out centralized processing is as follows: the uranium-resistant plants are harvested manually or mechanically, and then the harvested uranium-resistant plants are transferred to a safe place for centralized drying, crushing, and incineration, and finally landfilling or leaching to recover uranium.

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