Pseudomonas aeruginosa capable of producing urease at low temperature and application of pseudomonas aeruginosa in heavy metal contaminated soil

Abstract

The invention belongs to the field of microorganisms, and provides a pseudomonas capable of producing urease at low temperature and application of the pseudomonas in heavy metal contaminated soil. The Pseudomonas sp. J-6 capable of producing urease at low temperature is preserved in the China General Microbiological Culture Collection Center on January 7, 2025, and the preservation number of the Pseudomonas sp. J-6 is 33317. The Pseudomonas sp. J-6 capable of producing urease at low temperature, provided by the invention, is applied to heavy metal pollutants, is applied to heavy metal contaminated soil and is applied to improvement of a physical structure of the heavy metal contaminated soil.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to a cold-loving urease-producing Pseudomonas and its application in heavy metal-contaminated soil. Background Art

[0002] High-altitude regions, due to their unique geological conditions, are rich in mineral resources. However, large-scale mining has led to the accumulation of massive amounts of tailings waste, which has also become a significant source of heavy metal pollution. Heavy metals are environmentally persistent, resistant to microbial degradation, and irreversible, causing significant damage to ecological systems. Furthermore, their toxicity can enter the human body through ingestion, skin contact, and inhalation, posing serious carcinogenic and non-carcinogenic risks to humans.

[0003] As a green and sustainable pollution control method, microbial remediation technology has become one of the main research directions for heavy metal pollution remediation due to its high efficiency, environmental friendliness and economy. Among them, microbial induced carbonate precipitation (MICP) technology has been widely used in recent years. The core mechanism of MICP is to decompose urea into ammonium salt (NH4 + ) and carbonate ions (CO3 2- ), thereby promoting the combination of heavy metal ions and carbonate ions to form stable heavy metal carbonate precipitates. This process effectively reduces the bioavailability of heavy metals in the soil, while achieving long-term stabilization of heavy metals. However, the climate in high-altitude areas is cold, and the low-temperature environment will significantly inhibit the activity of urease-producing bacteria, thereby reducing the heavy metal remediation effect of MICP technology. Current related research mainly focuses on normal temperature environments, and there are no reports on research on high-altitude low-temperature environments. There are no urease-producing bacteria that can tolerate low-temperature environments and their remediation effects in high-altitude mines. Existing urease-producing strains are difficult to meet the remediation needs in this special environment.

[0004] Patent document CN 115709217 A describes a biochar-immobilized urease-producing bacteria composite soil heavy metal remediation agent and its application. The urease-producing Pseudomonas aeruginosa is cultured at 28°C. The Pseudomonas aeruginosa disclosed in the document is not psychrophilic. The psychrophilic urease-producing bacteria described in the paper "A psychrotolerant Ni-resistant Bacillus cereus D2 induces carbonate precipitation of nickel at low temperature" exhibits poor growth at 10°C. Although a few studies have reported on psychrophilic urease-producing bacteria, their heavy metal resistance is still limited. For example, the strain Glutamicibacter DC1 described in the paper "The Isolation and Characterization of Glutamicibacter DC1 to Induce Carbonate Precipitation of Some Heavy Metals at Low-Temperature" exhibits a minimum inhibitory concentration of only 20 mg / L for cadmium (Cd) at 10°C, indicating low tolerance to heavy metals. The microbial remediation technology mentioned in the paper "A Brief Discussion on Heavy Metal Pollution in Soil Around Alpine and High-Altitude Mining Areas and Its Integrated Prevention and Control" and the biomineralization technology mentioned in "Arsenic biomineralization and selenium nanoparticles biosynthesis by Halomonas boliviensis strain H-10 isolated from the high-altitude Salar de Huasco salt flat (Chile)" can remove heavy metals from the soil, but cannot improve the physical structure of the soil. Summary of the Invention

[0005] The purpose of the present invention is to provide a cold-loving urease-producing Pseudomonas and its application in heavy metal contaminated soil. The cold-loving urease-producing Pseudomonas of the present invention has good low-temperature adaptability and heavy metal resistance, can remove heavy metals in the soil, and improve the physical structure of the soil.

[0006] In order to solve the above technical problems, the present invention provides a psychrophilic urease-producing Pseudomonas and its application in heavy metal contaminated soil as follows:

[0007] A cold-loving, urease-producing Pseudomonas sp. J-6 was deposited in the China Microorganism Collection Center on January 7, 2025, with the accession number 33317.

[0008] Application of psychrophilic urease-producing Pseudomonas sp. J-6 in heavy metal pollutants.

[0009] Application of psychrophilic urease-producing Pseudomonas sp. J-6 in heavy metal contaminated soil.

[0010] Application of psychrophilic urease-producing Pseudomonas sp. J-6 in improving the physical structure of heavy metal contaminated soil.

[0011] Optionally, the Pseudomonas sp. J-6 removes heavy metals from heavy metal contaminated soil through microbial-induced carbonate precipitation technology.

[0012] Optionally, the Pseudomonas sp. J-6 improves the physical structure of heavy metal contaminated soil through microbial-induced carbonate precipitation technology.

[0013] Optionally, the urea concentration of the microbial induced carbonate precipitation technology is 30 g / L, the calcium ion concentration is 20 mM, the initial pH value is 9, and the initial addition amount of Pseudomonas (Pseudomonas sp.) J-6 bacterial liquid is 4%; the reaction temperature of the microbial induced carbonate precipitation technology is 5-20°C.

[0014] A heavy metal soil conditioner comprises: 30 g / L urea, 20 mM calcium ions, and 4% by mass of Pseudomonas sp. J-6 bacterial solution.

[0015] Optionally, the initial pH value of the heavy metal soil conditioner is 9.

[0016] Optionally, the heavy metal soil conditioner is used at a temperature of 5°C to 20°C.

[0017] The cold-loving urease-producing Pseudomonas sp. J-6 provided by the present invention has been shown to be cold-loving and exhibits strong resistance to heavy metals and growth effect at 10°C to 20°C; in particular, the growth and heavy metal resistance of the strain are best at 10°C.

[0018] Compared with the prior art, the strain of the present invention has stronger heavy metal tolerance. The strain of the present invention can still grow normally in an environment with a Cd concentration of 0.8 mM (i.e. 89.6 mg / L), and the strain of the present invention shows strong heavy metal tolerance.

[0019] The Pseudomonas sp. J-6 strain of the present invention has been shown to not only remove heavy metals from heavy metal-contaminated soil but also improve the soil's physical structure. Soil structure measurements conducted by the present invention showed increases in small aggregates (SMA), large aggregates (LMA), water retention, shear strength, cohesion, and internal friction angle in the experimental group containing the strain of the present invention. Compared to the control group, under 50 kPa and 100 kPa stresses, the SMA ratio of the optimal combination of strains of the present invention, T8, increased from 26.63% to 49.63%, water retention increased by 21.65%, and shear strength increased from 58 kPa and 88 kPa to 99 kPa and 141 kPa, respectively. The cohesion angle and internal friction angle increased from 28 kPa and 30.96° to 57 kPa and 40.03°, respectively.

[0020] The Pseudomonas sp. J-6 of the present invention is cryogenic and can better adapt to the low-temperature environment of high-altitude areas or severely cold areas. In a low-temperature environment, the strain of the present invention has a high growth efficiency, ensures the decomposition rate of urea in the MICP technology, can promote the generation of carbonate ions, and can effectively drive the precipitation of heavy metal ions.

[0021] Moreover, the strain of the present invention is not only cryogenic, but also can improve the physical structure of the soil while removing heavy metals in the soil. Therefore, it can be more applied in complex environments of high altitude areas or severely cold areas, such as low temperature and poor soil quality, to meet market application needs.

[0022] The Pseudomonas sp. J-6 provided by the present invention is a low-temperature strain that can grow efficiently in low-temperature environments at high altitudes or in severely cold areas, and can achieve heavy metal fixation and repair through MICP technology. It can achieve heavy metal repair in high-altitude areas in an efficient, low-cost, and green and environmentally friendly manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the screening process and technical mechanism of urease-producing Pseudomonas of the present invention;

[0024] Figure 2 Graphs showing the test results of Example 2 of the present invention, FIG. a shows the urease activity of ten bacterial strains, FIG. b shows the calcium carbonate precipitation ability of ten bacterial strains, FIG. c shows the heavy metal removal ability of ten bacterial strains, and FIG. d shows the phylogenetic tree;

[0025] Figure 3: These are test results from Example 3 of the present invention, FIG. a is a growth curve of Pseudomonassp. J-6 of the present invention in LB culture medium at different Cd concentrations, FIG. b is a growth curve of Pseudomonassp. J-6 of the present invention in LB culture medium at different temperatures, FIG. c is a growth curve of Pseudomonassp. J-6 of the present invention in UNB culture medium at 5°C, 10°C, and 20°C, and FIG. d is a Cd removal rate of Pseudomonas sp. J-6 of the present invention in UNB culture medium;

[0026] Figure 4 Graphs showing the test results of Example 4 of the present invention are shown. Graph a shows the heavy metal removal rates at different urea concentrations, Graph b shows the heavy metal removal rates at different calcium ion concentrations, Graph c shows the heavy metal removal rates at different initial pH values, and Graph d shows the heavy metal removal rates at different bacterial solution addition amounts.

[0027] Figure 5 The experimental results of Example 5 of the present invention are shown in Figure a. Figure a is the SEM-EDS diagram of the precipitate, Figure b is the XRD diagram of the precipitate under different conditions, and Figure c is the intracellular structure of the Pseudomonassp. J-6 bacteria of the present invention.

[0028] , SEM-EDS images of extracellular;

[0029] Figure 6 1 and 2 are test results of Example 6 of the present invention, wherein Figure a is a bar graph of the Cd content of the slag of the T1-T8 group, Figure b is a bar graph of the aggregate composition of the T1-T8 group, Figure c is a bar graph of the water retention rate of the T1-T8 group, Figure d is a bar graph of the shear stress of the T1-T8 group, and Figure e is a line graph of the internal friction angle and cohesion of the T1-T8 group. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail in the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] The urease-producing Pseudomonas strains of the present invention were isolated and purified from tailings soil collected from the high-altitude region of Tibet at latitude and longitude (92°1'E, 30°19'N). Ten strains of urease-producing bacteria were isolated and purified at a low temperature of 10°C. The strains were compared for their calcium carbonate precipitation ability, urease activity, and heavy metal removal ability. The strain with the best overall performance was selected as the dominant strain and identified. The mineralization ability of the strains was further studied by optimizing colony growth conditions through a one-way experiment, adjusting variables such as urea concentration, calcium concentration, pH, and bacterial inoculum size to achieve optimal MICP mineralization. The mineralization products and bacterial characteristics were characterized, and Cd distribution was analyzed to further explore the mineralization mechanism under low-temperature conditions. Simulating the actual environment of high-altitude areas, different treatment methods were designed to verify the heavy metal remediation effect of high-altitude soil. Experiments have demonstrated that the urease-producing bacteria selected by the present invention have excellent psychrophilicity and heavy metal removal efficiency, effectively remediating heavy metals in high-altitude mining soil while also improving the physical structure of the soil. Figure 1 It is a diagram of the screening process and technical mechanism of urease-producing Pseudomonas of the present invention.

[0032] As follows, the present invention introduces in detail the screening of bacterial strains and the mechanism of action of the bacterial strains.

[0033] Example 1 Screening of the psychrophilic urease-producing Pseudomonas strain of the present invention

[0034] Tailings soil was collected from a high-altitude area in Tibet, specifically the Mengyaa lead-zinc mine in Jiali County, Nagqu City, Tibet Autonomous Region, at 92°1'E, 30°19'N. The collected tailings soil sample was mixed with water and shaken for 2 hours. After standing for 30 minutes, 1 mL of the supernatant was added to 50 mL of LB culture medium and incubated at 10°C, 150 rpm for 2 days. Subsequently, 100 μL of the turbid culture medium was taken and the sample was precipitated according to the following method:

[0035] Dilute the solution in a gradient of 10-1 to 10-6 times and spread evenly on a solid screening plate. Incubate at 10°C until the color of the culture medium gradually changes from yellow to red. Pick 10 well-growing colonies and perform four streak purifications on each. Compare the urease activity, calcium carbonate precipitation capacity, and heavy metal removal capacity of the ten initially screened strains at 10°C to select the strain with the best overall performance for strain identification.

[0036] Example 2 The urease activity, calcite yield and heavy metal removal rate of the psychrophilic urease-producing Pseudomonas sp. of the present invention were determined to screen the dominant strains.

[0037] 2.1. The urease activity of 10 strains was determined by the hypochlorous acid-phenol method.

[0038] The bacterial suspension was mixed with phosphate buffer and urea solution and incubated at 10°C for 2 hours. Subsequently, sodium phenolate and sodium hypochlorite were added. After color development at room temperature for 20 minutes, the absorbance was measured at 578 nm using a UV-visible spectrophotometer, with ammonium chloride (0-10 μM) as the standard curve. The urease activity unit (IU) is the production of 1 μg of NH4 per minute. + The amount of bacterial suspension required. Figure 2 As shown in a.

[0039] 2.2. Determination of the ability to precipitate calcite (CaCO3) at low temperatures

[0040] To evaluate the ability of the strain to precipitate calcite at low temperatures, the strain was incubated at 10°C, washed with phosphate buffered saline (PBS), and resuspended in PBS. 1 mL of bacterial suspension was added to a solution of urea (3 M) and calcium chloride (0.35 M), with deionized water as a control. All samples were incubated at 10°C until a white precipitate formed. The precipitate was collected by centrifugation, dried at 50°C for 48 hours, and weighed; the results are shown in Table 1. Figure 2 b.

[0041] 2.3 Determination of heavy metal removal rate

[0042] In order to determine the ability of the strain to remove heavy metals, the strain was added to UNB medium containing 5 mg / L Cd and cultured at 10°C and 150 rpm. Samples were taken regularly and the supernatant obtained by centrifugation was used to determine the heavy metal content by flame atomic absorption spectrophotometry (FAAS). The removal rate of the strain for heavy metals was calculated. The results are shown in Figure 2. Figure 2 c.

[0043] Dominant strains with high urease activity, strong CaCO3 precipitation ability and high Cd removal ability were screened for biological identification.

[0044] 2.4. 16S rDNA Identification of Pseudomonas Strain of the Present Invention

[0045] DNA extraction was performed using a DNA extraction kit (universal). The extracted DNA was used as a template for polymerase chain reaction amplification using bacterial primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCT ACCTTGT TACGAC-3'). The PCR conditions were 98°C amplification for 3 minutes, followed by 39 cycles (98°C amplification for 10 seconds, 55°C amplification for 15 seconds, and 72°C amplification for 15 seconds / kb), and finally amplification at 72°C for 5 minutes. The PCR product was sequenced by Sanger sequencing and compared with the NCBI database (blast.ncbi.nlm.nih.gov). The similarity with yamanorum SFB8.6 (GenBank number CP143576) in the NCBI database was 99%, and it belonged to the genus Pseudomonas. The strain was named YY4BVEUV013Pseudomonas J-6. The phylogenetic tree constructed using MEGA-X software is shown in the figure. Figure 2 As shown in d.

[0046] The selected strains showed higher urease activity ( Figure 2 a) and CaCO3 precipitation capacity ( Figure 2 b), which were 192.84 IU and 1.67 mg / mL respectively. The Cd removal rate was the highest on the 7th day ( Figure 2 c) is 44.56%. Generally, the higher the urease activity and precipitation capacity of urea-producing bacteria, the better the removal effect of heavy metals.

[0047] Then, the present invention conducts further experiments and identification on the screened strains.

[0048] Example 3: Determination of heavy metal resistance and cold tolerance of selected strains

[0049] 3.1. Growth in LB medium with different Cd concentrations

[0050] The selected strains were cultured in LB medium with Cd concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM and 0.8 mM, and their Cd resistance was determined. Figure 3 a.

[0051] like Figure 3 As shown in Figure a, the strain growth curve decreases with increasing Cd concentration, indicating that Cd is toxic and can inhibit microbial growth. Despite this, the strain of the present invention continued to grow at a Cd concentration of 0.8 mM. This demonstrates that, despite the toxicity of Cd to J-6, the strain exhibits significant resistance, highlighting its potential for application in Cd remediation.

[0052] 3.2. Inoculate the selected strains into LB medium at different temperatures (5°C, 10°C, 20°C, 30°C, 40°C) to analyze whether they are psychrophilic strains. Measure the OD every 12 hours. 600 , the results are as follows Figure 3 b.

[0053] We from Figure 3 b It was observed that the strain showed better growth performance under low temperature conditions, while at higher temperature (40℃), its growth rate slowed down significantly and OD 600 The peak value is significantly lower than that under low temperature conditions, indicating that the bacteria is a psychrophilic bacterium.

[0054] 3.3. Inoculate the activated strain into UNB medium containing 0.4 mM Cd to create a MICP mineralization system environment; UNB medium components: 10 g / L peptone, 3 g / L beef powder, 5 g / L calcium chloride, and 30 g / L urea. Incubate at 5°C, 10°C, and 20°C at 150 rpm, and measure the strain's OD regularly. 600 and the concentration of Cd in the culture medium. The results are shown in Figure 3 c and Figure 3 d.

[0055] Figure 3 In c, the growth curve at 5°C was significantly lower than that at 10°C and 20°C. Figure 3 After 14 days, the Cd removal rate was only 25.72% at 10°C. Compared with 20°C, early growth was inhibited at 10°C, but by the 14th day, the Cd removal rate still reached 65.85%, close to the 67.48% at 20°C. Microbial growth and urea hydrolysis were inhibited at low temperatures, resulting in reduced heavy metal removal. However, at lower temperatures, urease activity was maintained for a longer period of time, which led to more carbonate production, thereby enhancing the MICP treatment effect under these conditions. In summary, the Pseudomonas sp. J-6 strain has the ability to grow and remove heavy metals at 5°C and has excellent growth and removal effects at 10°C. In summary, the Pseudomonas sp. J-6 strain of the present invention exhibits strong heavy metal resistance and strong cold tolerance.

[0056] Example 4 Optimization of conditions for the participation of Pseudomonas sp. J-6 in the MICP process

[0057] 4.1. Optimization of the strains of the present invention at low temperatures

[0058] The MICP process requires both nutrients for microbial growth and substrates for microbial hydrolysis and carbonate precipitation to ensure the continued progress of the reaction. In this example, a Pseudomonas sp. J-6 bacterial solution was inoculated into NB medium containing 0.4 mM Cd and urea. NB medium components include 10 g / L peptone, 3 g / L beef meal, and 5 g / L calcium chloride.

[0059] Then the urea concentration, calcium ion concentration, initial pH value and initial bacterial solution addition amount were changed by single factor.

[0060] , and then determine the heavy metal removal rate and select the one with the best conditions.

[0061] 4.2 Experimental Procedure

[0062] Keeping other conditions unchanged, only the urea concentration was changed (0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L); Figure 4 a shows that the optimal urea concentration is 30 g / L.

[0063] Keeping other conditions unchanged, only the calcium ion concentration was changed (0mM, 10mM, 20mM, 30mM, 40mM). Figure 4 b shows that the optimal calcium ion concentration is 20 mM.

[0064] Keeping other conditions unchanged, only by changing the initial pH value (5, 6, 7, 8, 9), Figure 4 cThe optimal initial pH value is 9.

[0065] Keeping other conditions unchanged, only by changing the initial bacterial solution addition amount (1%, 2%, 3%, 4%), Figure 4 d shows that the optimal bacterial solution addition amount is 4%.

[0066] According to the experiment of Example 3, 10° C. was selected as the optimal temperature for mineralization in the above experimental process.

[0067] Finally, it was determined that the preferred MICP conditions of the present invention are 0.4 mM Cd concentration, 30 g / L urea concentration, 20 mM Ca 2+ , NB culture medium with an initial pH of 9, and an addition amount of the bacterial solution of the present invention of 4%.

[0068] Example 5 MICP mineralization test and product analysis

[0069] 5.1 Distribution of Cd and Collection of Bacteria and Sediment

[0070] The strain was inoculated into 100 mL of UNB medium containing Cd and Ca and cultured at 10°C and 150 rpm for 14 days. The culture was then allowed to settle for 24 hours to allow the precipitate to settle. The bacterial solution was removed, centrifuged, and washed twice with PBS buffer. The precipitate was collected and freeze-dried for 24 hours to obtain pure, intact bacterial cells. The above steps were repeated, followed by two washes with PBS buffer and the addition of EDTA to elute extracellular Cd. The mixture was centrifuged, and the eluate (S1) was collected (S1: extracellular Cd). The remaining precipitate was digested on a hot plate to obtain solution S2 (S2: intracellular Cd). Furthermore, the EDTA-eluted bacterial cells were further disrupted using ultrasonic technology. The precipitate was collected, washed with distilled water, and freeze-dried for 24 hours to obtain disrupted cells. For the mineralized precipitate, after standing for 24 hours, the supernatant was removed, and the culture system was washed with sterile water and anhydrous ethanol. The precipitate was collected after centrifugation and dried at 50°C for 24 hours. The Cd content in the precipitate (S3) was determined by digestion with concentrated HCl and HNO3. The heavy metal Cd content of S1, S2, and S3 was determined to determine the distribution of Cd in the mineralization system. The bacterial bodies were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and Fourier transform infrared spectroscopy (FTIR), and the sediments were analyzed by X-ray diffraction (XRD) and SEM-EDS.

[0071] 5.2 Analysis of mineralized products

[0072] The generated precipitate was characterized by SEM-EDS. Figure 5 As shown in a, the surface of the calcite precipitate was found to be broken and rough. This is because Cd 2+ The urease-producing bacteria were inhibited, resulting in incomplete mineralization, thus affecting the complete crystallization of the crystals. At the same time, due to the similar ionic radius, Cd 2+ Replaces Ca in the lattice 2+ , forming a CdCO3 precipitate. This substitution resulted in an inhomogeneous crystal structure and overall fragmentation of the precipitate. EDS analysis revealed that C, O, Cd, and Ca were the main elements in the precipitate, confirming that Cd could be removed by precipitation.

[0073] In order to further analyze the composition of the precipitate, Figure 5 b XRD characterization was performed. In the diffraction angle range of 30° to 70°, the precipitate produced by the UNB medium without Cd had only 6 characteristic peaks of CaCO3, indicating that the carbonate compounds in the precipitate only existed in the form of CaCO3 crystals. In the UNB medium containing Cd, in addition to the characteristic peaks of CaCO3, CdCO3 and CaCO3 were also observed in the precipitate. 0.67 Cd 0.33The characteristic peak of CO3. The presence of CdCO3 crystals confirmed that the selected urease-producing strain removed Cd through MICP-mediated carbonate mineralization. 2+ In addition, this reaction generates Ca 0.67 Cd 0.33 The CO3 crystals demonstrate the strong adsorption of CaCO3 for heavy metal ions. Therefore, during the MICP process, heavy metal Cd is removed not only through biomineralization to produce CdCO3 precipitation but also through CaCO3 adsorption. Furthermore, a comparison of the crystal compositions generated at 10°C and 20°C revealed identical crystal compositions, demonstrating that the MICP reaction can proceed effectively at low temperatures (10°C).

[0074] Microorganisms have a strong ability to adsorb heavy metals. To explore this, the bacteria were characterized using SEM-EDS. The results are shown in Figure 2. Figure 5 c. SEM images of extracellular bacteria revealed intact, rod-shaped bacterial cells with smooth surfaces and orderly arrangement. Energy dispersive spectrometer (EDS) detected Cd and Ca on the cell surface. After cell disruption, the cells showed depressions, fractures, and fragments, but Cd and Ca remained. This indicates that the microorganisms adsorbed Cd extracellularly and accumulated it intracellularly. EDS analysis of the precipitate revealed 2.39% extracellular Cd and 1.55% intracellular Cd, indicating that Cd adsorption primarily occurred outside the cells.

[0075] Example 6 A remediation test of slag soil containing heavy metals was conducted using the Pseudomonas sp. J-6 bacteria of the present invention.

[0076] The slag soil was collected from the Mengyaa lead-zinc mine. In addition to soil components, the slag also contains other main components, namely SiO2, heavy metal content Pb135.73 mg / kg, Zn320.84 mg / kg, Cd1.02 mg / kg, Mn2719.54 mg / kg, Cu62.34 mg / kg.

[0077] The collected slag was subjected to eight different treatments at 10°C (see Table 1 for details). Each can contained 200g of slag soil, and the binder solution consisted of 1% bacterial solution, Ca 2+ 5.55 g / kg, 3 g / kg urea, total volume 100 ml. Three replicates per group. After 30 days, Cd concentrations and physical structural properties were measured.

[0078] Table 1. Eight different soil treatments

[0079] serial number Treatment T1 control group T2 Urease-producing bacteria T3 urea T4 Urease-producing bacteria + urea T5 calcium chloride T6 Urease-producing bacteria + calcium chloride T7 Urea + calcium chloride T8 Urease-producing bacteria + urea + calcium chloride

[0080] The concentration of available cadmium (Cd) in the slag was determined by diethylenetriaminepentaacetic acid (DTPA) extraction method. 10 g of air-dried soil sample was mixed with 20 mL of DTPA and shaken at 200 rpm for 2 h. The Cd concentration in the sample was determined by flame atomic absorption spectrophotometry (FAAS).

[0081] Soil aggregates were determined using the dry sieving method. 40 g of air-dried soil samples were sieved through sieves with different pore sizes (from top to bottom: 2 mm, 1 mm, 0.5 mm, 0.25 mm, 0.075 mm). Soil aggregates of different particle sizes were separated layer by layer, and the weight of each component was recorded. The samples were divided into three particle size groups: (1) >2 mm (LMA: large aggregates), (2) 2-0.25 mm (SMA: small aggregates), and (3) <0.25 mm (MA: microaggregates)

[20] .

[0082] Water retention was assessed gravimetrically. Air-dried soil was passed through a 2 mm sieve, and a 30 g sample was weighed and placed in a beaker. Water was added until the sample was completely submerged and allowed to soak for approximately 24 hours to ensure complete saturation. The saturated soil was then placed on filter paper and allowed to drain by gravity for 40 minutes. After drainage, the wet weight of the soil was recorded (M1). The sample was then dried at 105°C to constant weight, and the dry weight was recorded (M2).

[0083]

[0084] The unconfined compressive strength (UCS) test method was used to determine the shear strength of the soil. The soil was compacted into a cylindrical ring mold with a size of Φ61.80×20mm to make specimens for shear strength analysis. The load and displacement data were automatically recorded by a data recorder. τ is the shear stress, c is the soil cohesion, σ is the normal (vertical) stress, is the internal friction angle.

[0085] τ=c+σ·tanφ

[0086] After 30 days, the Cd content, aggregate composition, water retention, shear stress, internal friction angle and cohesion of slag after 8 different treatment methods were as follows: Figure 6 a, Figure 6 b, Figure 6 c. Figure 6 d and Figure 6 As shown in e.

[0087] Compared with the control group (T1), the Cd contents in all groups decreased. Figure 6a shows that T8 had the highest immobilization effect. The groups with microorganisms (T2, T4, T6, and T8) were more effective at removing Cd than the groups without microorganisms (T1, T3, T5, and T7). In the groups without added microorganisms, Cd concentrations decreased due to the presence of small amounts of naturally occurring urease-producing bacteria and urea in the slag. However, due to the limited number of these bacteria and their low mineralization capacity, the cadmium removal effect remained minimal. Therefore, the addition of urease-producing bacteria significantly improved the MICP process.

[0088] Soil structure measurements show that Figure 6 The be analysis showed that compared with the CK (T1), all groups showed increases in small aggregates (SMA), large aggregates (LMA), water retention, shear strength, cohesion, and internal friction angle. This was most evident in the microbial groups (T2, T4, T6, and T8), especially T8. Compared with T1, under stresses of 50 kPa and 100 kPa, the SMA ratio of T8 increased from 26.63% to 49.63%, the water retention increased by 21.65%, and the shear strength increased from 58 kPa and 88 kPa to 99 kPa and 141 kPa, respectively. The cohesion angle and internal friction angle increased from 28 kPa and 30.96° to 57 kPa and 40.03°, respectively.

[0089] The combination of T8 urease-producing bacteria, urea, and calcium chloride is ideal because the key reactants are in sufficient supply, ensuring an efficient MICP process. The CaCO3 precipitate produced by MICP fills soil pores, promotes aggregate formation, improves soil water retention, and enhances the shear strength and cohesion of the soil structure. These structural improvements further enhance the stability and erosion resistance of the soil while enhancing its ability to adsorb and fix heavy metals. In summary, the J-6 strain of the present invention can not only effectively remove Cd from alpine slag at 10°C, but also improve soil structure. The addition of urea and calcium chloride promotes the mineralization process.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cold-loving, urease-producing Pseudomonas sp. J-6 was deposited in the China Center for Microbiological Collection on January 7, 2025, with the accession number 33317.

2. Use of the psychrophilic urease-producing Pseudomonas sp. J-6 according to claim 1 in treating heavy metal pollutants.

3. The psychrophilic urease-producing Pseudomonas sp. J-6 according to claim 2, characterized in that Application in heavy metal contaminated soil.

4. The psychrophilic urease-producing Pseudomonas sp. J-6 according to claim 3, characterized in that Application in improving the physical structure of heavy metal contaminated soil.

5. The psychrophilic urease-producing Pseudomonas sp. J-6 according to claim 3, characterized in that The Pseudomonas sp. J-6 removes heavy metals from heavy metal-contaminated soil through microbial-induced carbonate precipitation technology.

6. The psychrophilic urease-producing Pseudomonas sp. J-6 according to claim 4, characterized in that The Pseudomonas sp. J-6 improves the physical structure of heavy metal contaminated soil through microbial-induced carbonate precipitation technology.

7. The psychrophilic urease-producing Pseudomonas sp. J-6 according to claims 5 and 6, characterized in that The urea concentration of the microbial-induced carbonate precipitation technology is 30 g / L, the calcium ion concentration is 20 mM, the initial pH value is 9, and the initial addition amount of Pseudomonas sp. J-6 bacterial liquid is 4%; the reaction temperature of the microbial-induced carbonate precipitation technology is 5-20°C.

8. A heavy metal soil conditioner, characterized in that: include: The concentration of urea is 30 g / L, the concentration of calcium ions is 20 mM, and the mass percentage of Pseudomonas sp. J-6 bacterial liquid is 4%.

9. The heavy metal soil conditioner according to claim 8, characterized in that The initial pH value of the heavy metal soil conditioner is 9.

10. The heavy metal soil conditioner according to claim 8, characterized in that The heavy metal soil conditioner is used at a temperature of 5°C to 20°C.

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