Low-temperature-resistant urease-producing bacterium as well as isolated culture method and application thereof

Abstract

The invention provides a low-temperature-resistant urease-producing bacterium as well as an isolated culture method and application thereof, and belongs to the technical field of microorganisms. The low-temperature-resistant urease-producing bacterium disclosed by the invention is named as Vidibacillus sp. DXA-9, and is preserved in Guangdong Microbiological Culture Collection Center on July 16, 2025, the address is Guangdong Institute of Microbiology on 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No. 66692. The strain disclosed by the invention can survive in an environment with the temperature as low as 10 DEG C and is applied to microbial remediation of low-temperature tailings and waste dump soil, the pH of the mining wasteland soil subjected to microbial remediation is remarkably increased, and the concentration of heavy metals in the soil is remarkably reduced, so that the heavy metals in the mining wasteland are effectively fixed.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a low-temperature resistant urease-producing bacterium and its isolation, culture, and application. Background Technology

[0002] Tailings, a major solid waste generated during mineral resource development, not only occupy valuable land resources but also pose a serious threat to the ecological environment due to their large-scale accumulation. Under long-term oxidation and environmental factors, tailings are highly susceptible to acidification, producing acidic wastewater with extremely low pH and rich in heavy metals. This wastewater pollutes surrounding soil and water bodies through infiltration and runoff. Because heavy metals are non-degradable and bioaccumulative, they accumulate in organisms through the food chain, and even at extremely low doses, they can cause cytotoxicity, gene mutations, and even cancer. This makes heavy metal pollution remediation one of the most pressing environmental problems today.

[0003] In recent years, heavy metal pollution control and environmental bioremediation technologies based on functional microorganisms have attracted widespread attention due to their environmental friendliness and sustainability. Addressing key issues such as the high mobility, bioavailability, and ecotoxicity of heavy metals in mining areas, as well as soil nitrogen deficiency, scholars both domestically and internationally have explored various biological immobilization methods. Among these, urease-producing microorganisms (UPMs) have become a research hotspot due to their unique heavy metal passivation function. Urease-producing bacteria are a class of microorganisms capable of secreting urease, including bacteria, fungi, and actinomycetes. They catalyze the hydrolysis of urea (CO(NH2)2 + H2O → 2NH3 + CO2) to produce ammonia and carbonates. This process not only increases soil pH but also produces NH3. 4+ It can provide nitrogen source for plants, and in addition, the carbonate ions and heavy metal ions (such as Cu) produced are also present. 2+ Cd 2+ Pb 2+ Zn 2+ (etc.) combine to transform into an insoluble carbonate form. Compared with traditional chemical passivating agents and physical covering methods, the microbial immobilization technology using urease-producing bacteria has advantages such as long-lasting effect, low cost, and ecological compatibility, and has shown important application value in the remediation of mining areas with high heavy metal activity.

[0004] While microbially induced carbonate precipitation (MICP) technology offers numerous advantages, its practical application still faces several limitations. Temperature is one of the most prominent limiting factors. Most ureases involved in the MMICP process exhibit peak activity within the 20–28°C range, and seasonal temperature variations (especially the low temperatures of winter in temperate regions) severely inhibit the growth and metabolic activity of native microorganisms, rendering MICP unsuitable for cold environments. Compared to ambient temperature remediation, research on low-temperature biomineralization for heavy metal remediation is insufficient. Therefore, further research into the characteristics of cryogenic bacteria and their mechanisms of heavy metal mineralization at low temperatures is essential. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a low-temperature resistant urease-producing bacterium, its isolation and cultivation method, and its application. The strain of this invention can grow at temperatures as low as 10°C and efficiently catalyzes urea hydrolysis, increasing soil nitrogen content and pH, reducing soil heavy metal concentration, and promoting vegetation restoration in mining areas.

[0006] To achieve the above objectives, the present invention provides a low-temperature resistant urease-producing bacterium, named *Bacillus aeruginosa* (…). Viridibacillus Bacillus sp.) DXA-9 was deposited on July 16, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at the Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66692. The inventors of this application isolated Bacillus aeruginosa (spe.) from the soil of the Dexing Copper Mine in Dexing City, Shangrao City, Jiangxi Province. Viridibacillus (spe.) DXA-9, this strain can grow under environmental temperatures as low as about 10℃, effectively reducing the concentration of heavy metals in the soil and increasing the soil pH.

[0007] As a preferred embodiment of the low-temperature resistant urease-producing bacterium described in this invention, the 16S rDNA gene sequence of Bacillus aeruginosa DXA-9 is shown in SEQ ID No. 3.

[0008] The present invention also provides the application of the aforementioned low-temperature resistant urease-producing bacteria and / or their fermentation products in the remediation of heavy metal contaminated soil.

[0009] In a preferred embodiment of the application described in this invention, the heavy metal includes at least one of Cu, Cd, Pb, and Zn.

[0010] In a preferred embodiment of the application described in this invention, the soil includes tailings and spoil heap soil.

[0011] As a preferred embodiment of the application described in this invention, the remediation includes at least one of reducing the concentration of available heavy metals in the soil, neutralizing the soil pH, and increasing the activity of soil urease.

[0012] The present invention also provides a biological agent comprising the aforementioned low-temperature resistant urease-producing bacteria and / or their fermentation products.

[0013] Preferably, the biological agent contains a bacterial suspension of the pyrothermic urease-producing bacteria.

[0014] More preferably, the OD of the pyrothermic urease-producing bacteria in the bacterial solution is... 600 The value is 0.5~1.0, with 1.0 being preferred.

[0015] More preferably, the bacterial solution also contains an enrichment culture medium.

[0016] More preferably, the enrichment culture medium contains at least one of NaCl, KH2PO4, urea, and glucose.

[0017] More preferably, the enrichment medium contains 1.99~2.01 g / L NaCl, 1.99~2.01 g / L KH2PO4, 0.011~0.013 g / L phenol red, 19.99~20.01 g / L urea, and 1.99~2.01 g / L glucose.

[0018] More preferably, the enrichment medium is prepared by weighing NaCl, KH2PO4, and phenol red, mixing them to obtain a mixed solution, adjusting the pH of the mixed solution to 6.8-7.0 with HCl before bringing the volume to 1 L, sterilizing at 121℃ for 20 min, and then adding urea and glucose to obtain the enrichment medium.

[0019] The present invention also provides the application of the aforementioned biological agent in the remediation of heavy metal contaminated soil.

[0020] Preferably, the remediation of heavy metal contaminated soil includes at least one of reducing heavy metal pollution in the soil and neutralizing soil pH.

[0021] Preferably, the heavy metal includes at least one of Cu, Cd, Pb, and Zn.

[0022] Preferably, the soil includes tailings soil or spoil heap soil.

[0023] The present invention also provides a method for microbial remediation of heavy metal contaminated soil, wherein the low-temperature resistant urease-producing bacteria and / or their fermentation products or the biological agents are applied to the heavy metal contaminated soil.

[0024] In a preferred embodiment of the method described in this invention, the amount of the low-temperature resistant urease-producing bacteria applied is 1 to 10% (v / v) of the volume of the heavy metal contaminated soil.

[0025] Preferably, the amount of the low-temperature resistant urease-producing bacteria applied is 10% of the volume of the heavy metal contaminated soil.

[0026] More preferably, the refractory urease-producing bacteria and / or their fermentation products or the biological agent are mixed with the soil in the form of a bacterial solution.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a low-temperature resistant urease-producing bacterium and its isolation and cultivation method and application. The present invention isolated *Bacillus aeruginosa* (*B. aeruginosa*) from the soil of the Dexing Copper Mine in Dexing City, Shangrao City, Jiangxi Province. Viridibacillus The strain DXA-9 can survive in environments with temperatures as low as 10°C and has been applied to the microbial remediation of soils in low-temperature tailings and spoil heaps. After microbial remediation, the pH of the soil in the mining waste site increased significantly and the concentration of heavy metals in the soil decreased significantly, thus achieving effective fixation of heavy metals in the mining waste site. Attached Figure Description

[0028] Figure 1 Low-temperature resistant urease-producing bacteria Viridibacillus sp. The isolation status of DXA-9 strains.

[0029] Figure 2 Low-temperature resistant urease-producing bacteria Viridibacillus sp. Phylogenetic tree of DXA-9.

[0030] Figure 3 Low-temperature resistant urease-producing bacteria Viridibacillus sp. Results of the low-temperature urease production capacity test of DXA-9.

[0031] Figure 4 Low-temperature resistant urease-producing bacteria Viridibacillus sp. Results of heavy metal tolerance test for DXA-9.

[0032] Figure 5 Low-temperature resistant urease-producing bacteria Viridibacillus sp. Test results of the heavy metal removal capacity of DXA-9.

[0033] Figure 6 The total content of heavy metals Cu, Cd, Zn and Pb in soil samples from the spoil heap.

[0034] Figure 7 A is a low-temperature resistant urease-producing bacterium. Viridibacillus sp. pH changes in the spoil heap before and after DXA-9 remediation; B is a low-temperature resistant urease-producing bacterium. Viridibacillus sp. Changes in soil urease activity before and after DXA-9 remediation.

[0035] Figure 8 Low-temperature resistant urease-producing bacteria Viridibacillus sp. DXA-9 microbial remediation of Zn in spoil heaps 2+ Pb 2+ Cd 2+ Cu 2+ Changes in the effective state content. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention is further described through the following embodiments. Obviously, the following embodiments are only a part of the embodiments of the present invention, and not all of them. It should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the methods and experimental conditions used in the embodiments of the present invention are all methods and experimental conditions conventionally used in the art, and the reagents, equipment, and culture media used are all reagents, equipment, and culture media conventionally used in the art, and can all be prepared by existing methods or purchased commercially.

[0037] The formulations and preparation methods of the culture medium and solution involved in the embodiments of the present invention are as follows: Enrichment medium: Weigh 2.0 g NaCl, 2.0 g KH2PO4 and 0.012 g phenol red, mix them to obtain a mixture, adjust the pH of the mixture to 6.8-7.0 with HCl before bringing the volume to 1 L, sterilize at 121℃ for 20 min, and then add 20 g urea and 2.0 g glucose to obtain the enrichment medium.

[0038] Culture medium for strain purification: Weigh 2.0 g peptone, 10.0 g beef extract, 2.0 g NaCl, 2.0 g KH2PO4, and 0.012 g phenol red, dissolve them in 700 mL distilled water, and adjust the pH to 6.8-7.0 with HCl to obtain culture medium a; Separately weigh 15 g agar and dissolve it in 300 mL distilled water, sterilize at 121℃ for 20 min, and after cooling to 50℃, mix culture medium a and agar solution evenly, then add 20 g urea and 2.0 g glucose (filtered and sterilized) to obtain solid culture medium. Pour the medium into plates at a volume of approximately 15 mL per plate, and after the plates solidify, obtain the culture medium for strain purification. Seal the plates with sealing film and store them upside down at 4℃ until use.

[0039] Heavy metal tolerance medium: Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride, dissolve in 1 L distilled water, sterilize at 121℃ for 15 min, cool, filter to remove bacteria, and add 20 g urea and 27.75 g / L CaCl2 to obtain medium b. Separately prepare medium containing 1 g / L Cu. 2+ Pb2+ Zn 2+ Cd 2+ The mother liquor (prepared using CuCl2, PbCl2, ZnCl2, CdCl2·2.5H2O) was used to obtain culture medium c. Finally, culture medium c and culture medium b were used to prepare a solution containing Cu... 2+ Pb 2+ Zn 2+ Cd 2+ Heavy metal tolerance culture media with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, 600 mg / L, and 800 mg / L were prepared, with three replicates for each gradient.

[0040] Heavy metal removal medium: Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride, dissolve them in 1000 mL distilled water, sterilize at 121℃ for 15 min, and after cooling, filter to remove bacteria. Add 20 g urea, 27.75 g / L CaCl2, and 10 mg each of CuCl2, ZnCl2, PbCl2, and CdCl2 to obtain the heavy metal removal medium.

[0041] In the embodiments of the present invention: strain Corynebacterium glutamicum It is a known strain that is resistant to low temperatures of 10℃ and is positive for urease. It was obtained from the China Industrial Microbial Culture Collection Center, with strain number CICC 1355.

[0042] Example 1: Isolation and Screening of Low-Temperature Urease-Producing Strains 1. Enrichment culture: Take 0.5 g of soil sample from the spoil heap of Dexing Copper Mine in Jiangxi Province into an Erlenmeyer flask, add 2 / 3 volume of enrichment culture medium to the Erlenmeyer flask, and incubate at 10℃ and 150 rpm on a shaker until the culture medium turns from yellow to red for bacterial isolation.

[0043] 2. Strain strain isolation: such as Figure 1 As shown, the bacterial culture obtained in step 1 was serially diluted to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Then, 100 μL of the culture medium was spread onto a plate containing solid culture medium and incubated at 10°C until single colonies grew. Single colonies were picked and streaked onto a plate, and purified for 3-4 generations until stable, single-morphological single colonies grew.

[0044] Example 2: Identification of pyrothermic urease-producing strains Genomic DNA was extracted from the strain isolated in Example 1. Primers 27F (5'-3': AGAGTTTGATCMTGGCTCAG, SEQ ID NO: 1) and 1492R (5'-3': GGTTACCTTGTTACGACTT, SEQ ID NO: 2) were used to amplify the 16S rRNA gene sequence. The PCR amplification system was 25 μL, specifically containing 12.5 μL Taq mix, 0.5 μL 27F primers, 0.5 μL 1492R primers, 1 μL strain DNA, and 10.5 μL ddH2O. The PCR amplification program was as follows: pre-denaturation temperature 94℃, 5 min; denaturation temperature 94℃, 30 s; annealing temperature 55℃, 30 s; extension temperature 72℃, 90 s; 30 cycles; supplemental extension temperature 72℃, 10 min. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the sequence information. The 16S rRNA gene sequence of the strain obtained by sequencing was compared with the database sequence using NCBI BLAST.

[0045] The results showed that this strain was related to Bacillus spp. ( Viridibacillus sp. The *Viridibacillusarvi* sequence showed 100% homology. Twelve 16S rRNA gene sequences from the same species, genus, or family of microorganisms were downloaded from the NCBI database as reference sequences and used together with the strain's sequence to construct a phylogenetic tree, confirming that the strain belongs to the genus *Bacillus*. Viridibacillus sp. () Figure 2 ).

[0046]

[0047] After the above screening, isolation, and identification, the obtained strain was named Bacillus aeruginosa (B. aeruginosa). Viridibacillus sp. DXA-9 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 16, 2025, with accession number GDMCC No: 66692. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0048] Example 3: Low-temperature resistant urease-producing bacteria Viridibacillus sp. DXA-9 Performance Evaluation This embodiment focuses on Bacillus aeruginosa ( Viridibacillus sp. The low-temperature urease-producing ability of DXA-9 was evaluated. The specific experimental method is as follows: The *Bacillus aeruginosa* (…) isolated in Example 1… Viridibacillus sp. DXA-9 was inoculated at a ratio of 3% into enrichment medium, with three replicates. The culture was carried out at a constant temperature of 10℃ to obtain bacterial suspension. The absorbance of the bacterial suspension was measured at a wavelength of 600 nm using a spectrophotometer every 8 h.

[0049] The results are as follows Figure 3 As shown, the logarithmic mid-growth stage will be... Viridibacillus sp. The urease activity of DXA-9 was 116.69 μM / min and 1861.03 μM / min at 10℃ and 30℃, respectively, while that of the control strain was lower. Corynebacterium glutamicum The enzyme activities were only 29.11 μM / min and 1015.79 μM / min, respectively (e.g., Figure 3 B), indicating that the *Bacillus aeruginosa* isolated in this invention ( Viridibacillus sp. DXA-9 has a stronger ability to produce urease under low temperature conditions compared to other known low-temperature resistant urease-producing strains.

[0050] Example 4: Low-temperature resistant urease-producing bacteria Viridibacillus sp. DXA-9's tolerance to heavy metal ions This embodiment focuses on Bacillus aeruginosa ( Viridibacillus sp. The tolerance of DXA-9 to heavy metal ions was evaluated using the following experimental method: The *Bacillus aeruginosa* isolated in Example 1 (…) Viridibacillus sp. DXA-9 was inoculated at a ratio of 3% into the prepared heavy metal tolerance medium, with three replicates. The cultures were incubated at 10℃ and 150 rpm / min, and OD was measured every 12 h. 600 value.

[0051] The results are as follows Figure 4 As shown, Viridibacillus sp. DXA-9 is resistant to heavy metals, especially Cu. 2+ Good tolerance, in Cu 2+It still has a high OD at a concentration of 600 mg / L. 600 Value, for Zn 2+ Pb 2+ It also has a certain tolerance and can still grow at concentrations of 300 mg / L and 100 mg / L respectively.

[0052] Example 5: Low-temperature resistant urease-producing bacteria Viridibacillus sp. DXA-9's ability to fix heavy metals in liquid culture medium Specific experimental method: The *Bacillus aeruginosa* isolated in Example 1 (… Viridibacillus sp. DXA-9 was inoculated at a ratio of 3% in heavy metal removal medium, with three replicates, and cultured at 10℃, 30℃, and 150 rpm / min respectively.

[0053] The results are as follows Figure 5 As shown, 7 days later Viridibacillus sp. DXA-9 at 10℃ for Pb 2+ The removal rate reached 86.07%, and other elements Cu... 2+ Zn 2+ Cd 2+ The removal rates (also known as fixation rate or passivation rate) of Pb were 2.76%, 23.29%, and 14.39%, respectively; under conditions of 30℃, Pb 2+ Cu 2+ Zn 2+ Cd 2+ The removal rates were 75.56%, 91.23%, 29.73%, and 88.75%, respectively. This indicates that Bacillus aeruginosa (…) Viridibacillus sp. DXA-9 has a good fixation effect on heavy metals under both low and high temperature conditions.

[0054] Example 6: Low-temperature resistant urease-producing bacteria Viridibacillus sp. Applications of DXA-9 This embodiment investigates the *Bacillus aeruginosa* isolated in Example 1. Viridibacillus sp. The effect of DXA-9 on microbial remediation of low-temperature acidic spoil heaps is illustrated by the following experimental methods: 1. Determination of Total Heavy Metal Content in Acidic Dump Soil: After air-drying, the soil was passed through a 100-mesh sieve. 0.100 g (accurate to 0.001 g) of soil was weighed and added to a digestion tube. 9 mL of concentrated HCl, 3 mL of concentrated HNO3, and 3 drops of HClO4 were added sequentially. The mixture was heated at 120-150-185℃ for 2-5-25 min according to the microwave digestion protocol. After digestion, acid removal was carried out (at 175℃ for approximately 1 hour or more) until the solution in the digestion tube was reduced to approximately 1 ml. After cooling, the soil sample was diluted to 25 ml with ultrapure water, filtered through a membrane into a capped centrifuge tube, and the filtrate was analyzed using AAS / ICP-MS. The final results are shown in Table 1.

[0055] 2. Acidic tailings pond soil bottling: Use 100 mL bottles, each containing approximately 50 g of tailings pond soil sample. The soil samples used in the experiment came from the tailings pond of the Dexing Copper Mine in Dexing City, Shangrao City, Jiangxi Province.

[0056] 3. Functional microbial inoculation: Based on the soil volume, inoculate at a rate of 10% (v / v) with... Viridibacillus sp. DXA-9 uses bacterial culture (OD) 600 =1) The bacterial culture was mixed with soil in an Erlenmeyer flask, and the flask was sealed with a breathable sealing film. The flasks were incubated at 10℃ and 30℃ at 150 rpm / min, and the physicochemical properties of the soil were monitored and tested. The blank treatment group (Control) used sterile water instead of bacterial culture, the culture medium treatment group (LB+urea) used urea-containing culture medium instead of bacterial culture, and the experimental group (Bacterial culture) used urea-containing culture medium.

[0057] The results are shown in Tables 2-3. Figures 7 - 8 As shown, the low-temperature acidic spoil heap microcosm experiment lasted for 14 days. At a cultivation temperature of 10℃, the soil pH increased from 2.85 to 3.94, and the soil urease activity increased from 30.57 μg / g / d to 122.56 μg / g / d. The Cu content in the soil... 2+ The effective state decreased from 68.7.4 mg / kg to 53.53 mg / kg, Zn 2+ Pb 2+ The effective levels decreased from 14.77 mg / kg and 0.044 mg / kg to 3.65 mg / kg and 0.027 mg / kg, respectively. Cd 2+ The concentration of Cu in the soil decreased from 0.008 mg / kg to below the instrument detection limit; the soil pH increased from 2.85 to 5.92 at a culture temperature of 30℃, and the soil urease activity increased from 30.57 μg / g / d to 62.75 μg / g / d. 2+The effective state decreased from 68.7.4 mg / kg to 30.67 mg / kg, Zn 2+ Pb 2+ The effective doses decreased from 14.77 mg / kg and 0.044 mg / kg to 1.77 mg / kg and 0.024 mg / kg, respectively. (Cd) 2+ The decrease from 0.008 mg / kg to below the instrument's detection limit indicates a significant reduction in the bioavailability of pollutants in the soil. From the above, it can be understood that this invention... Viridibacillus sp. DXA-9 has the function of acid control under low temperature conditions and can be applied to the ecological reconstruction of mining waste sites.

[0058] Table 1. Total heavy metal content in soil samples from spoil heaps Table 2 Viridibacillus sp. Changes in physicochemical properties of spoil heaps after 14 days of treatment with DXA-9 at 10℃ Table 3 Viridibacillus sp. Changes in physicochemical properties of spoil heaps after DXA-9 treatment at 30℃ for 14 days *Note: ND indicates that the detection limit is below the detection limit and no detection was performed.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A low-temperature resistant urease-producing bacterium, characterized in that, The low-temperature resistant urease-producing bacterium was named Bacillus aeruginosa ( ). Viridibacillus sp.) DXA-9 was deposited on July 16, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66692.

2. The pyrothermic urease-producing bacterium according to claim 1, characterized in that, The 16S rDNA gene sequence of Bacillus aeruginosa DXA-9 is shown in SEQ ID No.

3.

3. The application of the low-temperature resistant urease-producing bacteria and / or their fermentation products as described in claim 1 in the remediation of heavy metal contaminated soil.

4. The application according to claim 3, characterized in that, The heavy metals include at least one of Cu, Cd, Pb, and Zn.

5. The application according to claim 3, characterized in that, The soil includes tailings soil or spoil heap soil.

6. The application according to claim 3, characterized in that, The remediation includes at least one of the following: reducing the concentration of available heavy metals in the soil, neutralizing soil pH, and increasing soil urease activity.

7. A biological agent, characterized in that, The biological agent includes the low-temperature resistant urease-producing bacteria of claim 1 and / or its fermentation products.

8. The application of the biological agent according to claim 7 in the remediation of heavy metal contaminated soil.

9. A method for microbial remediation of heavy metal contaminated soil, characterized in that, Apply the low-temperature resistant urease-producing bacteria of claim 1 and / or its fermentation products or the biological agent of claim 7 to heavy metal contaminated soil.

10. The method according to claim 9, characterized in that, The application rate of the low-temperature resistant urease-producing bacteria is 1-10% (v / v) of the volume of the heavy metal contaminated soil.

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