A method for remediating a phosphogypsum-contaminated site based on urease-loving bacteria

Abstract

The application discloses a method for repairing a phosphogypsum contaminated site based on urease, and the method comprises the following steps: subjecting urease producing bacteria to acid resistance, phosphorus resistance and fluorine resistance domestication, then carrying out fermentation culture, mixing obtained fermentation liquor to obtain mixed domesticated bacteria liquor, then mixing the mixed domesticated bacteria liquor with a protective agent and a solvent to obtain a mixed solution, mixing the mixed solution with a urea solution, using the mixed solution and the urea solution to repair the phosphogypsum contaminated site in real time, then covering the site with a guest soil, and carrying out normal maintenance to complete ecological repair. The urease strain subjected to acid resistance, phosphorus resistance and fluorine resistance domestication is combined with the EICP technology, high-efficiency and deep solidification and pollutant storage are directly carried out on the phosphogypsum stockyard, and high-efficiency ecological repair is completed.

Description

Technical Field

[0001] This invention belongs to the field of environmental geotechnical engineering and ecological restoration technology, specifically relating to a method for remediating phosphogypsum-contaminated sites based on acidophilus urease. Background Technology

[0002] Phosphogypsum is a major byproduct of wet-process phosphoric acid production and the production of phosphate fertilizers such as ammonium phosphate and superphosphate. Its main component is calcium sulfate dihydrate (CaSO4·2H2O), and its composition is complex, often containing undecomposed phosphate rock, fluorides, heavy metals, radioactive elements, and residual acidic substances. Currently, phosphogypsum treatment methods are mainly divided into two categories: comprehensive utilization and harmless stockpiling. Although the comprehensive utilization of phosphogypsum for ecological restoration and backfilling, and the preparation of building gypsum products and cement retarders is developing rapidly, the current rate of comprehensive utilization cannot fully absorb the annual increase in production due to the huge amount of phosphogypsum produced. Therefore, stockpiles remain a necessary facility for regulating the balance between production and consumption and ensuring the normal production of phosphate chemical enterprises, mainly playing a "safety net" and "buffer" role. However, long-term stockpiling poses multiple environmental risks: phosphogypsum stockpiles occupy large areas, are stored in the open, and rainwater from the stockpile area needs to be collected into open-air storage ponds. Its leachate is highly acidic (pH typically 2-4), easily leading to acidification of surrounding soil and water bodies. The soluble phosphorus (P), fluorine (F), and oxides it contains are washed into reservoirs by rainwater, further seeping into the ground or flowing into surface water bodies, causing water pollution and damage to the aquatic ecosystem. The acidic leachate also dissolves heavy metals from the soil, exacerbating land pollution. Furthermore, the fine particles of phosphogypsum easily generate dust, spreading pollutants such as fluorides and heavy metals, impacting the regional atmospheric environment.

[0003] Existing remediation technologies for stockpiled phosphogypsum mainly include physical, chemical, and biological methods, but all have significant limitations and are difficult to address complex stockpiles with strong acidity and multiple pollutants. Physical methods often use HDPE geomembranes combined with soil / vegetation cover to block rainwater infiltration and dust, but geomembranes are prone to aging, costly, and can easily lead to the accumulation of acidic liquids. Chemical methods mostly rely on silicate cement to neutralize acidity or the addition of stabilizers to solidify pollutants, but often require secondary landfilling or repeated chemical application, which can easily lead to soil salinization and pollutant migration. Furthermore, in a strong acid environment, heavy metal chelates are unstable, and radioactive nuclides are difficult to effectively fix. Biological methods rely on acid-resistant or enriching plants to absorb and transform pollutants, but the remediation cycle is long, the depth of action is limited, and the strong acid environment inhibits plant growth, usually requiring prior chemical neutralization.

[0004] Enzyme-induced calcium carbonate precipitation (EICP) is an emerging green reinforcement / remediation technology. It reacts at room temperature, consumes very little energy, causes minimal environmental pollution, and can penetrate into tiny pores. It is easy to apply and has strong bonding power, effectively removing impurities such as phosphorus and fluorine from phosphogypsum and fixing heavy metals. Therefore, it has great application potential in treating pollutants such as phosphogypsum. Chinese patent CN121426392A discloses an ecological remediation method for improving Yellow River sediment based on the combined use of EICP, phosphogypsum, and cement. This method extracts soluble calcium and removes heavy metals through acid hydrolysis of phosphogypsum, and then combines EICP and cement to synergistically improve the Yellow River sediment. Spatially, cement constructs the macroscopic framework, while EICP strengthens the microscopic bonding. Temporally, it provides early, mid, and late-stage strength enhancement, achieving multi-dimensional and stable improvement. Simultaneously, various agricultural wastes and industrial byproducts are introduced to improve nutrient content and environmental safety. Chinese patent CN117999896A discloses a method for in-situ biological soil remediation of phosphogypsum stockpiles. This method involves preparing a composite microbial agent by screening native acid-tolerant functional microorganisms from the phosphogypsum stockpile or its surrounding area, then spraying the agent, planting native plants, and introducing soil animals to complete the in-situ soil remediation of phosphogypsum stockpile derivatives. The native acid-tolerant functional microorganisms include phosphate-solubilizing bacteria, free-living nitrogen-fixing bacteria, and potassium-solubilizing bacteria. However, current methods for remediating phosphogypsum stockpiles only consider the impact of pH on microorganisms, neglecting the effects of high phosphorus and fluoride content in phosphogypsum on microbial life activities, urease activity, and even the remediation effect. Therefore, developing an EICP technology that can adapt to acidic, high-phosphorus, and high-fluoride environments, requires no pre-neutralization, and enables the resource utilization of pollutants is urgently needed and of great significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for remediating phosphogypsum-contaminated sites based on acidophilic urease. It employs urease strains acclimated to acid, phosphorus, and fluorine tolerance, combined with EICP technology, to directly and efficiently solidify and seal pollutants in phosphogypsum stockpiles, achieving highly efficient ecological restoration.

[0006] To achieve the above objectives, the present invention provides a method for remediating phosphogypsum-contaminated sites based on acidophilic urease-producing bacteria, comprising the following steps: (1) The urease-producing bacteria were acclimated to acid, phosphorus and fluorine tolerance respectively; (2) Ferment and culture acid-resistant strains, phosphorus-resistant strains and fluoride-resistant strains separately, and mix the fermentation broth to obtain a mixed acclimatization bacterial solution; (3) The mixed domesticated bacterial culture was separated and purified to obtain an acid urease solution; (4) Prepare urea solution; dissolve the protective agent in the solvent, let it stand, and then add acidophilus solution to obtain a mixture; (5) Mix the urea solution and the mixed solution to obtain the remediation solution, spray it onto the phosphogypsum contaminated site, and plant acid-resistant plants to complete the remediation.

[0007] Preferably, the acidophilic urease producing bacteria in step (1) is one or more of Alcaligenes faecalis, Bacillus spheroidae, Bacillus pasteurellii, and Bacillus licheniformis.

[0008] Preferably, the acid tolerance acclimatization in step (1) is carried out in a gradient manner at sulfuric acid concentrations of 10-20, 45-60, and 70-90 g / L; the phosphorus tolerance acclimatization is carried out in HPO4. 2- Gradual acclimation was performed at concentrations of 0.1-0.3, 0.4-0.6, and 0.8-1.0 g / L; the fluoride tolerance acclimation was carried out at F... - Gradual acclimatization was carried out at concentrations of 0.1-0.3, 0.4-0.6, and 0.8-1.0 g / L.

[0009] Preferably, the acid-resistant strain, phosphorus-resistant strain and fluoride-resistant strain described in step (2) are mixed in a live count of (1~3):(1~3):(1~3).

[0010] Preferably, the concentration of the acidophilus urease solution in step (3) is 2.0-4.0 g / L.

[0011] Preferably, the concentration of the urea solution in step (4) is 1-2 mol / L.

[0012] Preferably, the volume ratio of the protective agent, solvent and acid urease solution in step (4) is 1:(0.5~3):(1~5).

[0013] More preferably, the protective agent is any one of urea, cysteine, bovine serum albumin, or sucrose-glycerol-NaCl; and the solvent is any one of sodium citrate-hydrochloric acid buffer, acetate-sodium acetate buffer, or HEPES buffer.

[0014] Preferably, the separation and purification in step (4) consists of centrifugation to collect bacterial cells, lysozyme grinding and crushing with liquid nitrogen, and ethanol fractionation and precipitation.

[0015] Preferably, the volume ratio of urea solution to mixed solution in step (5) is 1:(0.5~3); the amount of repair solution used is 3.0-5.0 L / m³. 2 .

[0016] Preferably, the pH of the phosphogypsum contaminated site described in step (5) is 1.0-4.5, and the F... -The content is 30-50 g / L, the phosphorus content is 50-80 g / L, and the calcium mainly exists in the form of calcium sulfate dihydrate and calcium oxide. It also contains trace amounts of heavy metals such as lead (Pb), arsenic (As), and cadmium (Cd).

[0017] The beneficial effects of this invention are as follows: 1. Acid-resistant, phosphorus-resistant, and fluoride-resistant bacterial strains were obtained through three acclimation methods: acid-resistant, phosphorus-resistant, and fluoride-resistant. Their combined use maintained high activity even in acidic environments. The expressed acidophilic urease catalyzed the hydrolysis of urea at pH 2.0-4.5, generating carbonate ions, which combined with calcium ions in phosphogypsum to form calcium carbonate precipitate. This process not only immobilizes heavy metals in situ but also converts soluble phosphorus into insoluble calcium phosphate mineral phases through co-precipitation or adsorption, and converts fluorides into fluorapatite or encapsulates them with calcium carbonate, significantly reducing their leaching concentration and ultimately achieving the remediation of phosphogypsum stockpiles.

[0018] 2. The acclimatized microorganisms can gradually increase the pH value of the micro-area by releasing ammonia through urease hydrolysis of urea without large-scale chemical neutralization. During the remediation process, the pH can rise from 2.8 to 6.5-7.2, creating a suitable soil environment for subsequent microbial community reconstruction and plant colonization. Furthermore, the acclimatized microorganisms exhibit tolerance to high concentrations of phosphorus and fluorine (e.g., tolerance to HPO4). 2- Up to 0.9g / L, resistant to F - With a concentration of up to 0.9 g / L, it can function stably in stockpiles where pollutant concentrations fluctuate greatly, avoiding remediation interruptions caused by drastic environmental changes. This biologically driven pH regulation and pollutant stabilization process is characterized by its mildness, controllability, and durability, and is the core mechanism for realizing the transformation of ecological restoration from "passive storage" to "active recovery."

[0019] 3. During the EICP catalysis process, the ammonia produced by urea hydrolysis by the acclimated microorganisms provides a nitrogen source for plants, while the phosphorus and sulfur originally present in the phosphogypsum are retained in the solidified layer, becoming usable nutrients for plants. The presence of the acclimated microorganisms ensures the continuous release and transformation of nutrients in the remediation layer, avoiding soil salinization or nutrient loss caused by traditional chemical neutralization, truly achieving the ecological restoration goal of "treating waste with waste and turning waste into resources." The significance of acclimated microorganisms in the remediation of phosphogypsum stockpiles lies not only in their high tolerance and activity to acids, phosphorus, and fluorine, but also in their ability to drive the EICP reaction in extreme environments, achieving a three-pronged ecological restoration pathway of in-situ pollutant stabilization, soil pH regulation, and nutrient regeneration. Compared to traditional methods, the use of acclimated microorganisms significantly reduces engineering costs and environmental risks, providing key technical support for the green and sustainable remediation of phosphogypsum stockpiles.

[0020] 4. The three domesticated bacterial strains exhibited synergistic dynamic functional transformations during the ecological restoration process. In the early stages of restoration, acid-tolerant strains drove urease hydrolysis, gradually neutralizing acidity and increasing pH. As pH rose, phosphorus-tolerant and fluoride-tolerant bacteria were gradually activated, participating in the fixation and stabilization of phosphorus and fluoride. The three strains played their primary functions in different pH ranges throughout the entire restoration cycle, ensuring the continuous and efficient operation of the EICP reaction at different stages. This mechanism overcomes the bottleneck of traditional microbial remediation technologies, which are often interrupted by drastic environmental changes, enabling this invention to complete the entire process of ecological reconstruction from strongly acidic environments to near-neutral soils without pre-neutralization.

[0021] 5. Compared with traditional neutralization methods, this invention saves over 90% of the costs associated with the procurement, transportation, and application of neutralizing agents. It overcomes the application limitations of EICP technology regarding pH, phosphorus content, and fluorine content, achieving a technological leap from "neutralization followed by solidification" to "direct in-situ solidification," significantly simplifying the process. Furthermore, the reaction process is mild, without introducing strongly alkaline substances, fundamentally avoiding soil salinization and compaction. The enzymatic reaction rate is controllable, and pollutants are gradually stabilized and fixed through calcium carbonate crystallization, avoiding the risk of secondary pollution and exhibiting high environmental compatibility. The calcium carbonate precipitate generated by this invention not only binds particles but also firmly encapsulates or fixes soluble phosphorus, fluorides, and heavy metals within the mineral lattice through adsorption, co-precipitation, and ion exchange mechanisms, transforming them into chemically stable, leaching-resistant mineral phases for long-term safe storage. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the repair mechanism of the present invention.

[0023] Figure 2 The image shows the repair result of Example 2. In the image, A is an overall view of the repair block, and B is a magnified view of a portion of A.

[0024] Figure 3 The images show the repair results of Example 2 and Comparative Example 2. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0026] In the following embodiments: The phosphogypsum stockpile is a legacy site of a former phosphate fertilizer plant, covering an area of ​​approximately 5000 m². 2The stockpiled material was approximately 2.0m high. Long-term open-air storage resulted in a highly acidic environment, with the overall pH of the stockpile ranging from 1.9 to 5.2. The leachate contained fluoride at a concentration of 120 mg / L and soluble phosphorus at 80 mg / L. Regarding heavy metals, tests were conducted according to the "Identification Standard for Hazardous Waste: Leaching Toxicity Identification" (GB5085.3-2007). The lead leaching concentration was 15.0 mg / L, exceeding the standard limit by 3 times (5.0 mg / L); the cadmium leaching concentration was 2.0 mg / L, exceeding the standard limit by 2 times (1.0 mg / L).

[0027] Bacillus licheniformis strain: purchased from Shanghai Preservation Microbiology Co., Ltd., accession number SHMCCD10742; LB (Luria-Bertani) medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being deionized water; adjust the pH to 7.0±0.2 (25℃) with 1 mol / L NaOH, and autoclave at 121℃ for 20 min. MSM (Minimal Salts Medium) medium: Na2HPO4·2H2O 3.5 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 0.5 g / L, MgCl2·6H2O 0.1 g / L, calcium salt 0.05 g / L, adjust pH to 7.0±0.2 (25℃), sterilize at 121℃ for 15 min; 9K medium: (NH4)2SO4 3.0 g / L, KCl 0.1 g / L, K2HPO4 0.5 g / L, Ca(NO3)2 0.01 g / L, MgSO4·H2O 0.5 g / L, FeSO4·7H2O 50.0 g / L, adjust pH to 3.0~3.5 with 10 N H2SO4, sterilize at 121℃ for 15 min.

[0028] Example 1 (1) Take a strain of Bacillus licheniformis and place it in an activation medium. Incubate at 37°C for 24.0 h to obtain an activated strain; (2) The activated strain was inoculated into LB (Luria-Bertani) medium (pH 7.0±0.2) containing 15.0 g / L H2SO4 and cultured at 30℃ for 72 h to obtain the first-stage acclimatized strain; (3) The first-acclimatized strain was inoculated into LB (Luria-Bertani) medium (pH 4.0) containing 50.0 g / L H2SO4 and cultured at 30±5 ℃ for 72 h to obtain the second-acclimatized strain; (4) The secondary acclimatized strain was inoculated into LB (Luria-Bertani) medium (pH 2.0) containing 80.0 g / L H2SO4 and cultured at 30±5 ℃ for 72 h to obtain an acid-resistant strain; (5) The activated strain was inoculated sequentially into MSM (Minimal Salts Medium) medium containing 0.2 g / L, 0.5 g / L, and 0.9 g / L KH2PO4, and cultured at 30±5 ℃ for 72 h to obtain phosphorus-resistant strains; (6) The activated strain was inoculated sequentially into 9K medium containing 0.2 g / L, 0.5 g / L, and 0.9 g / L NH4F, and cultured at 30±5℃ for 72 h to obtain fluoride-resistant strain; (7) The acid-resistant strain was inoculated into LB medium and cultured at 30 °C for 48 h to obtain the acid-resistant bacterial fermentation broth (OD600=2.0). The phosphorus-resistant strain was inoculated into MSM medium and cultured at 30 °C for 48 h to obtain the phosphorus-resistant bacterial fermentation broth (OD600=1.5). The fluoride-resistant strain was inoculated into 9K medium and cultured at 30 °C for 72 h to obtain the fluoride-resistant bacterial fermentation broth (OD600=1.0).

[0029] Example 2 (1) Take 5.0 L each of the acid-resistant bacteria fermentation broth, phosphorus-resistant bacteria fermentation broth, and fluoride-resistant bacteria fermentation broth prepared in Example 1, and centrifuge them at 4℃ and 8000 rpm for 15 min respectively. Discard the supernatant and collect the bacterial precipitate. Resuspend the bacterial cells in sterile physiological saline and adjust the viable cell concentration of the three bacterial suspensions to 1.0 × 10⁻⁶ using the plate count method. 9 CFU / mL; then the acid-resistant bacteria suspension, phosphorus-resistant bacteria suspension and fluoride-resistant bacteria suspension were mixed at a volume ratio of 1:1:1 to obtain 1.5 L of mixed acclimatization bacterial solution; (2) Centrifuge the above 1.5 L mixed acclimatization bacterial culture at 8000 rpm for 15 min at 4℃, collect the bacterial precipitate, and discard the supernatant; wash the bacterial cells twice with pre-cooled (4℃) sterile physiological saline, and resuspend them each time by adding 1.0 L of physiological saline, and centrifuge at 8000 rpm for 15 min at 4℃ to collect the bacterial cells; add lysozyme buffer at a ratio of 5 mL per gram of wet bacterial cells (lysozyme concentration of 1 mg / mL) to resuspend the bacterial cells to obtain a bacterial suspension; place the bacterial suspension in a 37℃ water bath for 30 min to lyse, then use liquid nitrogen grinding to break the cells, repeat freeze-thaw three times, and finally centrifuge at 12000 rpm for 20 min at 4℃ to collect the supernatant; dilute the obtained supernatant with pre-cooled sodium citrate-hydrochloric acid buffer to adjust the protein concentration to 2.8 g / L to obtain an acidurase solution; the urease activity of the acidurase solution was determined by the indophenol blue method to be 13.5 mM / min; (3) Place urea in deionized water and stir at 50 rpm until completely dissolved to obtain a urea solution with a concentration of 1.5 mol / L; (4) Add the urea solution from step (3) to the protective agent (2% trehalose solution by mass) and part of the solvent pre-cooled to 4°C (sodium citrate-hydrochloric acid buffer, pH 3.0), stir at 50 rpm and let stand for 8 min; then add the acid urease solution and stir at 50 rpm for 25 min; finally, use the solvent pre-cooled to 4°C to make up the volume so that the final concentration of urea in the mixed solution is 0.5 mol / L, the acid urease activity is 13.5 mM / min, and the volume ratio of protective agent, solvent and acid urease solution is kept at 1:2:2 to obtain the mixed solution; (5) The 1.5 mol / L urea solution obtained in step (3) and the mixed solution obtained in step (4) are mixed at a volume ratio of 2:1 to obtain the repair solution. The repair solution is prepared fresh for use. (6) Level the phosphogypsum stockpile and spray the remediation solution prepared in step (5) evenly onto the surface of the phosphogypsum stockpile. Spray for 30 minutes every 4 hours until the total amount of remediation solution used reaches 4.0 L / m². 2 Control the spraying intensity during the spraying process to avoid surface runoff. (7) After spraying, let it stand for 72 hours, then cover it with 20 cm of topsoil (made of sludge, loess and humus in a mass ratio of 2:3:3); then plant Miscanthus sinensis and Centipede Grass, with a planting density of 1500 plants / mu for Miscanthus sinensis and 3000 plants / mu for Centipede Grass; water immediately after planting, with a watering volume of 8 L / m 2 Subsequently, during the 24-day curing period, watering was carried out on days 3, 7, 14, 18, and 22, with each watering amount being 5 L / m². 2 The soil moisture content should be maintained at 60%-70%. If the daily rainfall is ≥5 mm, watering should be cancelled. No additional lime, chemical neutralizers, or compound fertilizers should be applied during the maintenance period. Plant survival rate should only be checked on the 7th day. If the survival rate is below 85%, the same species should be replanted. After 24 days of maintenance, if the pH of the restored site reaches 6.0-7.5, the fluoride leaching concentration is not higher than 10 mg / L, the total phosphorus leaching concentration is not higher than 5 mg / L, the Pb leaching concentration is not higher than 0.10 mg / L, the Cd leaching concentration is not higher than 0.05 mg / L, and the vegetation coverage is not lower than 85%, then the ecological restoration is considered complete. Figure 2 ); (8) On the 15th day of maintenance, test the pH value, leachate concentration, vegetation coverage, and average plant height of the phosphogypsum layer. When the pH is 5.5-6.0, the fluoride leachate concentration is 10-20 mg / L, the total phosphorus leachate concentration is 5-10 mg / L, the Pb leachate concentration is 0.05-0.10 mg / L, the Cd leachate concentration is 0.02-0.05 mg / L, the vegetation coverage is 70%-85%, and the average plant height is 20-30 cm, spray the repair solution once more, with a spraying volume of 1.2 L / m². 2 When pH < 5.5, fluoride leaching concentration > 20 mg / L, total phosphorus leaching concentration > 10 mg / L, Pb leaching concentration > 0.10 mg / L, Cd leaching concentration > 0.05 mg / L, vegetation coverage < 70%, and average plant height < 20 cm, apply the remediation solution twice. The first application should be 2.0 L / m². 2 The second supplementary spraying dosage was 1.2 L / m². 2 There should be a 7-day interval between the two supplementary sprays.

[0030] Example 3 (1) Take 5.0 L each of the acid-resistant bacteria fermentation broth, phosphorus-resistant bacteria fermentation broth, and fluoride-resistant bacteria fermentation broth prepared in Example 1, and centrifuge them at 4℃ and 8000 rpm for 15 min respectively. Discard the supernatant and collect the bacterial precipitate. Resuspend the bacterial cells in sterile physiological saline and adjust the viable cell concentration of the three bacterial suspensions to 1.0 × 10⁻⁶ using the plate count method. 9 CFU / mL; then the acid-resistant bacteria suspension, phosphorus-resistant bacteria suspension and fluoride-resistant bacteria suspension were mixed at a volume ratio of 1:2:2 to obtain 1.5 L of mixed acclimatization bacterial solution; (2) Centrifuge the above 1.5 L mixed acclimatization bacterial culture at 8000 rpm for 15 min at 4℃, collect the bacterial precipitate, and discard the supernatant; wash the bacterial cells twice with sterile physiological saline pre-cooled to 4℃, and resuspend them each time by adding 1.0 L of physiological saline, centrifuge at 8000 rpm for 15 min at 4℃, and collect the bacterial cells; add lysozyme buffer at a ratio of 5 mL per gram of wet bacterial cells (lysozyme concentration of 1 mg / mL); lyse the bacterial suspension in a 37℃ water bath for 30 min, then break the cells by liquid nitrogen grinding, repeat freeze-thaw three times, and finally centrifuge at 12000 rpm for 20 min at 4℃, and collect the supernatant; dilute the obtained supernatant with pre-cooled acetate-sodium acetate buffer to adjust the protein concentration to 2.5 g / L, and obtain the acidurase solution; the urease activity of the acidurase solution was determined by the indophenol blue method to be 13.0 mM / min; (3) Place cysteine ​​in deionized water and stir at 50 rpm until completely dissolved to obtain a cysteine ​​solution with a concentration of 1.0 mol / L; place urea in deionized water and stir at 50 rpm until completely dissolved to obtain a urea solution with a concentration of 1.0 mol / L. (4) Add the urea solution from step (3) to the protective agent (1.0 mol / L cysteine ​​solution) and part of the solvent pre-cooled to 4°C (acetic acid-sodium acetate buffer, pH 3.0), stir at 50 rpm and let stand for 8 min; then add the acid urease solution and stir at 50 rpm for 25 min; finally, make up the volume with the solvent pre-cooled to 4°C so that the concentration of cysteine ​​in the mixed solution is 0.2 mol / L, the activity of acid urease is 13.0 mM / min, and the volume ratio of protective agent, solvent and acid urease solution is kept at 1:2:2 to obtain the mixed solution; (5) The 1.0 mol / L urea solution obtained in step (3) and the mixed solution obtained in step (4) are mixed at a volume ratio of 3:1 to obtain the repair solution. The repair solution is prepared fresh for use. (6) Level the phosphogypsum stockpile and spray the remediation solution prepared in step (5) evenly onto the surface of the phosphogypsum stockpile. Spray for 30 minutes every 4 hours until the total amount of remediation solution used reaches 3.0 L / m². 2 Control the spraying intensity during the spraying process to ensure that the repair solution penetrates evenly into the phosphogypsum layer and avoid surface runoff. (7) After spraying, let stand for 72 hours, then cover with 20 cm of topsoil (mixed with sludge, loess and humus in a mass ratio of 2:3:3). After covering with topsoil, plant centipede grass and sorrel, with a planting density of 1500 plants / mu for centipede grass and 2500 plants / mu for sorrel. Water immediately after planting, with a watering rate of 8 L / m³. 2 Subsequently, during the 24-day curing period, watering was carried out on days 3, 7, 14, 18, and 22, with each watering amount being 5 L / m². 2 The soil moisture content should be maintained at 60%–70%. If the daily rainfall is ≥5 mm, watering should be cancelled. No additional lime, chemical neutralizers, or compound fertilizers should be applied during the maintenance period. Plant survival rate should only be checked on the 7th day. If the survival rate is below 85%, the same species of plants should be replanted. After 24 days of maintenance, if the pH of the restored site reaches 6.0–7.5, the fluoride leaching concentration is not higher than 10 mg / L, the total phosphorus leaching concentration is not higher than 5 mg / L, the Pb leaching concentration is not higher than 0.10 mg / L, the Cd leaching concentration is not higher than 0.05 mg / L, and the vegetation coverage is not lower than 85%, then the ecological restoration is considered complete. 8) On the 15th day of maintenance, monitor the pH value, leachate concentration, vegetation coverage, and average plant height of the phosphogypsum layer. When the pH is 5.5-6.0, the fluoride leachate concentration is 10-20 mg / L, the total phosphorus leachate concentration is 5-10 mg / L, the Pb leachate concentration is 0.05-0.10 mg / L, the Cd leachate concentration is 0.02-0.05 mg / L, the vegetation coverage is 70%-85%, and the average plant height is 20-30 cm, spray the repair solution once more at a rate of 0.9 L / m². 2 When pH < 5.5, fluoride leaching concentration > 20 mg / L, total phosphorus leaching concentration > 10 mg / L, Pb leaching concentration > 0.10 mg / L, Cd leaching concentration > 0.05 mg / L, vegetation coverage < 70%, and average plant height < 20 cm, apply the remediation solution twice. The first application should be at a rate of 1.5 L / m². 2 The second supplementary spraying dosage was 0.9 L / m². 2 There should be a 7-day interval between the two supplementary sprays.

[0031] Example 4 (1) Take 5.0 L each of the acid-resistant, phosphorus-resistant, and fluoride-resistant bacterial fermentation broths prepared in Example 1, and centrifuge them at 4℃ and 8000 rpm for 15 min respectively. Discard the supernatant and collect the bacterial precipitate. Resuspend the bacterial cells in sterile physiological saline, and adjust the viable cell concentration of the three bacterial suspensions to 1.0 × 10⁻⁶ using the plate count method. 9 CFU / mL. Then, 900 mL of acid-resistant bacteria suspension, 300 mL of phosphorus-resistant bacteria suspension, and 300 mL of fluoride-resistant bacteria suspension were measured and mixed in a volume ratio of 3:1:1 to obtain 1.5 L of mixed acclimatization bacterial solution.

[0032] (2) Centrifuge the above 1.5 L mixed acclimatization bacterial culture at 8000 rpm for 15 min at 4℃, collect the bacterial precipitate, and discard the supernatant; wash the bacterial cells twice with sterile physiological saline pre-cooled to 4℃, and resuspend them each time by adding 1.0 L of physiological saline, centrifuge at 8000 rpm for 15 min at 4℃, and collect the bacterial cells; add lysozyme buffer at a ratio of 5 mL per gram of wet bacterial cells (lysozyme concentration of 1 mg / mL); lyse the bacterial suspension in a 37℃ water bath for 30 min, then break the cells by liquid nitrogen grinding, repeat freeze-thaw three times, and finally centrifuge at 12000 rpm for 20 min at 4℃, and collect the supernatant; dilute the obtained supernatant with pre-cooled HEPES buffer to adjust the protein concentration to 3.2 g / L, and obtain the acidurase solution; the urease activity of the acidurase solution was determined by the indophenol blue method to be 13.2 mM / min; (3) Bovine serum albumin was placed in deionized water and stirred at 50 rpm until completely dissolved to obtain a bovine serum albumin solution with a mass concentration of 20 g / L; urea was placed in deionized water and stirred at 50 rpm until completely dissolved to obtain a urea solution with a concentration of 2.0 mol / L. (4) Add the urea solution from step (3) to the protective agent (20 g / L bovine serum albumin solution) and part of the solvent pre-cooled to 4°C (HEPES buffer, pH 3.0), stir at 50 rpm and let stand for 5 min; then add the acid urease solution and stir at 50 rpm for 15 min; finally, use the solvent pre-cooled to 4°C to make up the volume so that the final concentration of bovine serum albumin in the mixed solution is 4 g / L, the acid urease activity is 13.5 mM / min, and the volume ratio of protective agent, solvent and acid urease solution is kept at 1:2:2 to obtain the mixed solution; (5) The 2.0 mol / L urea solution obtained in step (3) and the mixed solution obtained in step (4) are mixed at a volume ratio of 3:1 to obtain the repair solution. The repair solution is prepared fresh for use. (6) Level the phosphogypsum stockpile and drill vertical holes on the surface of the stockpile. The holes should be 200 cm deep, 8 cm in diameter, and 5 holes per m³. 2 The repair solution prepared in step (5) is injected into each well in several stages: 0.4 L is injected into each well for the first time, and the injection time is controlled within 15 min; 0.4 L is injected into each well after an 8 h interval; 0.2 L is injected into each well after another 8 h interval; after the three injections, a total of 1.0 L of repair solution is injected into each well, at a rate of 5 wells / m 2 The total volume of repair solution used is estimated to be 5.0 L / m³. 2 Control the injection speed during the injection process to avoid overflow from the orifice and surface runoff; (7) After injection, let stand for 72 hours, then cover with 20 cm of topsoil (mixed with sludge, loess and humus in a mass ratio of 2:3:3). After covering with topsoil, plant centipede grass, leucocephala and lupins. The planting density of centipede grass is 1500 plants / mu, the planting density of leucocephala is 3000 plants / mu, and the planting density of lupins is 2500 plants / mu. Water once immediately after planting, with a watering volume of 8 L / m 2 Subsequently, during the 24-day curing period, watering was carried out on days 3, 7, 14, 18, and 22, with each watering amount being 5 L / m². 2The soil moisture content should be maintained at 60%–70%. If the daily rainfall is ≥5 mm, watering should be cancelled. No additional lime, chemical neutralizers, or compound fertilizers should be applied during the maintenance period. Plant survival rate should only be checked on the 7th day. If the survival rate is below 85%, the same species of plants should be replanted. After 24 days of maintenance, if the pH of the restored site reaches 6.0–7.5, the fluoride leaching concentration is not higher than 10 mg / L, the total phosphorus leaching concentration is not higher than 5 mg / L, the Pb leaching concentration is not higher than 0.10 mg / L, the Cd leaching concentration is not higher than 0.05 mg / L, and the vegetation coverage is not lower than 85%, then the ecological restoration is considered complete. (8) On the 15th day of maintenance, the pH value, leachate concentration, vegetation coverage, and average plant height of the phosphogypsum layer should be measured. When the pH is 5.5-6.0, the fluoride leachate concentration is 10-20 mg / L, the total phosphorus leachate concentration is 5-10 mg / L, the Pb leachate concentration is 0.05-0.10 mg / L, the Cd leachate concentration is 0.02-0.05 mg / L, the vegetation coverage is 70%-85%, and the average plant height is 20-30 cm, a repair solution should be injected once, with a total injection volume of 1.5 L / m². 2 When pH < 5.5, fluoride leaching concentration > 20 mg / L, total phosphorus leaching concentration > 10 mg / L, Pb leaching concentration > 0.10 mg / L, Cd leaching concentration > 0.05 mg / L, vegetation coverage < 70%, and average plant height < 20 cm, two additional injections of remediation solution should be administered. The first injection should have a total volume of 2.5 L / m². 2 The total amount injected in the second injection was 1.5 L / m³. 2 There was a 7-day interval between the two supplementary injections.

[0033] Comparative Example 1 The method and steps are the same as in Example 2, except that the mixed bacterial solution in step (1) is changed to a mixture of acid-resistant bacterial fermentation solution and phosphorus-resistant bacterial fermentation solution in equal volume ratio to complete the ecological restoration.

[0034] Comparative Example 2 The method and steps are the same as in Example 2, except that the mixed bacterial solution in step (1) is changed to a mixture of acid-resistant bacterial fermentation solution and fluoride-resistant bacterial fermentation solution in equal volume ratio to complete the ecological restoration.

[0035] Comparative Example 3 The method and steps are the same as in Example 2, except that the mixed bacterial solution in step (1) is changed to a mixture of phosphorus-resistant bacterial fermentation solution and fluoride-resistant bacterial fermentation solution in equal volume ratio to complete the ecological restoration.

[0036] Comparative Example 4 The method and steps are the same as in Example 2, except that the mixed bacterial solution in step (1) is changed to a fermentation liquid obtained entirely from undomesticated Bacillus licheniformis strains to complete the ecological restoration.

[0037] Comparative Example 5 The method and steps are the same as in Example 2, except that urea is not added as a protective agent to the mixed solution in step (3) to prepare the mixed solution, and then the remediation solution is prepared to complete the ecological restoration.

[0038] Comparative Example 6 The method and steps are the same as in Example 2, except that the sodium citrate-hydrochloric acid buffer solution in step (3) is replaced with water to prepare a mixed solution, and then a remediation solution is prepared to complete the ecological remediation.

[0039] Comparative Example 7 The method and steps are the same as in Example 2, except that in step (5), the amount of repair solution is changed to 2 L / m 2 Ecological restoration was completed.

[0040] Comparative Example 8 The method and steps are the same as in Example 2, except that the amount of repair solution used in step (5) is changed to 6 L / m 2 Ecological restoration was completed.

[0041] Example 5 Soil samples from the above examples and comparative examples were collected at different time points after spraying the remediation solution. pH was measured, and the leaching concentrations of soluble phosphorus, fluoride, and heavy metals Pb and Cd were determined using the "Solid Waste Leaching Toxicity Leaching Method: Sulfuric Acid and Nitric Acid Method" (HJ / T 299-2007). Vegetation growth was statistically analyzed, and the results are shown in Tables 1-3. Table 1. pH and leachate levels on day 7 post-remediation.

[0042] Table 2. pH and leachate levels on day 15 post-remediation.

[0043] Table 3 Plant growth status

[0044] Note: Plant growth was assessed 24 days after ecological restoration.

[0045] The results showed that in Examples 2-4, the pH significantly increased on days 7 and 15 after ecological restoration, and the leaching concentrations of fluoride, total phosphorus, and heavy metals Pb and Cd significantly decreased, with removal rates all exceeding 80% (over 95% after 15 days). Plant growth was vigorous, with vegetation coverage exceeding 90%. In contrast, Comparative Examples 1-3 showed significantly reduced removal efficiency for the corresponding pollutants due to the lack of certain acclimatized strains; Comparative Example 4, using unacclimatized strains, showed the worst remediation effect; Comparative Examples 5-6, lacking protective agents or buffer solutions, suffered from impaired enzyme activity, resulting in significantly reduced effectiveness; Comparative Example 7 showed insufficient remediation due to inadequate dosage, while Comparative Example 8, with excessive dosage, showed acceptable results but poor economic efficiency (Tables 1-2). These results demonstrate that the present invention, employing acid-resistant, phosphorus-resistant, and fluoride-resistant triple-acclimatized strains combined with EICP technology, can achieve efficient and stable remediation of phosphogypsum-contaminated sites.

[0046] Meanwhile, the average plant height, average root length, and aboveground biomass dry weight of the plants in Examples 2-4 were significantly higher than those in the control group, and the vegetation coverage reached over 93% (Table 3). Figure 3 This indicates that the remediation methods described in Examples 2-4 can significantly improve plant growth, thereby achieving rapid remediation of phosphogypsum-contaminated sites.

[0047] Example 6 In the ecological restoration of Example 2 and Comparative Example 4, samples were taken on days 3, 7, 15, and 24 after planting to detect pH changes in the stockpile. Simultaneously, the viable counts of acid-tolerant, phosphorus-tolerant, and fluoride-tolerant bacteria were tracked using plate counting. The results are shown in Table 4. Table 4. Changes in pH and viable cell count

[0048] The results showed that in Example 2, the number of viable acid-tolerant bacteria remained at a high level during the initial stage of ecological restoration (0-7 days), reaching 2.0 × 10⁻⁶ on day 7. 7 The CFU / g count indicates that acid-resistant strains can maintain high activity under strongly acidic conditions, driving urease hydrolysis and promoting a pH increase from 2.8 to 4.8. During the mid-stage of ecological restoration (7–15 days), as the pH further increased, phosphorus-resistant and fluoride-resistant bacteria gradually entered their optimal growth range, with their viable cell counts increasing to 2.1 × 10⁻⁶. 7 CFU / g and 1.9×10 7CFU / g, and participated in the fixation and stabilization of phosphorus and fluorine; in the later stage of ecological remediation (15-24 days), the pH of the stockpile was maintained at 6.8-7.2, and the three acclimatized strains maintained a high number, forming a relatively stable functional microbial community system. In contrast, Comparative Example 4 used unacclimatized strains, which were difficult to survive stably in strong acid, high phosphorus, and high fluorine environments. The number of detectable viable bacteria under the corresponding selective culture conditions decreased significantly with the remediation time. On day 24, the number of detectable viable bacteria for acid-resistant, phosphorus-resistant, and fluorine-resistant strains dropped to 8.0 × 10⁻⁶. 4 CFU / g, 2.0×10 4 CFU / g and 1.0×10 4 With CFU / g, the pH of the stockpile only increased to 3.4, indicating that the unacclimated strains could not adapt to the complex pollution environment of the phosphogypsum stockpile.

[0049] The results showed that in the early stage of ecological restoration (0-7 days), the viable count of acid-tolerant bacteria remained at a high level, indicating that acid-tolerant strains played a major role at this time, possessing the ability to maintain urease activity and urea hydrolysis in a strongly acidic environment, thus driving a slow increase in pH. In the middle stage of ecological restoration (7-15 days), as pH rose, phosphorus-tolerant and fluoride-tolerant bacteria gradually entered their suitable growth range, began to colonize in large numbers, and began to perform phosphorus and fluoride fixation functions. In the later stage of restoration (pH 6.0-7.2), the pH approached neutral, and all three strains were in a metabolically active state, forming a stable functional microbial ecosystem. During EICP, NH3 produced by urea hydrolysis creates a slightly alkaline microenvironment around the strains, and NH4+... + This acts as a pH buffer, ensuring that the local pH changes around the bacteria are less significant than changes in the overall soil environment, thus providing a certain survival buffer for the strains. The strains, after gradient acclimatization, have a wider pH tolerance range (stronger adaptability) and can maintain a certain level of urease activity within a pH range of 2.5-7.5. Therefore, when the pH of the phosphogypsum dump gradually rises and approaches neutrality as the ecological restoration process progresses, it will not significantly affect the life activities of the three strains, thus preventing their inactivation and demonstrating a good ecological restoration effect.

Claims

1. A method for remediating phosphogypsum-contaminated sites based on acidophilus urease, characterized in that: Includes the following steps: (1) The urease-producing bacteria were acclimated to acid, phosphorus and fluorine tolerance respectively; (2) Ferment and culture acid-resistant strains, phosphorus-resistant strains and fluoride-resistant strains separately, and mix the fermentation broth to obtain a mixed acclimatization bacterial solution; (3) The mixed domesticated bacterial culture was separated and purified to obtain an acid urease solution; (4) Prepare urea solution; dissolve the protective agent in the solvent, let it stand, and then add acidophilus solution to obtain a mixed solution; (5) Mix the urea solution and the mixed solution to obtain the remediation solution, spray it onto the phosphogypsum contaminated site, and plant acid-resistant plants to complete the remediation.

2. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The acidophilic urease producing bacteria in step (1) is one or more of Alcaligenes faecalis, Bacillus spheroidae, Bacillus pasteurellii, and Bacillus licheniformis.

3. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The acid tolerance acclimatization in step (1) is carried out in a gradient manner at sulfuric acid concentrations of 10-20, 45-60, and 70-90 g / L; the phosphorus tolerance acclimatization is carried out in HPO4. 2- Gradual acclimation was performed at concentrations of 0.1-0.3, 0.4-0.6, and 0.8-1.0 g / L; the fluoride tolerance acclimation was carried out at F... - Gradual acclimatization was carried out at concentrations of 0.1-0.3, 0.4-0.6, and 0.8-1.0 g / L.

4. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The acid-resistant strains, phosphorus-resistant strains and fluoride-resistant strains mentioned in step (2) are mixed in a viable count of (1-3):(1-3):(1-3).

5. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The concentration of the acidophilus solution in step (3) is 2.0-4.0 g / L.

6. A method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The concentration of the urea solution mentioned in step (4) is 1-2 mol / L.

7. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The volume ratio of the protective agent, solvent and acid urease solution in step (4) is 1:(0.5~3):(1~5).

8. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 7, characterized in that: The protective agent is any one of urea, cysteine, bovine serum albumin, and sucrose-glycerol-NaCl; the solvent is any one of sodium citrate-hydrochloric acid buffer, acetate-sodium acetate buffer, or HEPES buffer.

9. A method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The volume ratio of urea solution to mixed solution in step (5) is 1:(0.5-3); the amount of repair solution used is 3.0-5.0 L / m³. 2 .

10. The method for remediating phosphogypsum-contaminated sites based on acidophilus urease according to claim 1, characterized in that: The pH of the phosphogypsum contaminated site described in step (5) is 1.0-4.5, F - The content is 30-50g / L, the phosphorus content is 50-80g / L, and the calcium mainly exists in the form of calcium sulfate dihydrate and calcium oxide. It also contains heavy metals such as lead, arsenic and cadmium.

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