A carbonate mineralizing bacterium and its application in the remediation of heavy metal polluted environments

Abstract

The present invention relates to the field of microorganisms and the technology of solidifying heavy metals, and specifically, to a carbonate mineralizing bacterium and its application in the remediation of heavy metal polluted environments. The carbonate mineralizing bacterium is Rummeliibacillus sp. ANY-1, and the Rummeliibacillus sp. ANY-1 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 24, 2024, with a deposit number of CGMCC No.31431. The carbonate mineralizing bacteria of the present invention have good cadmium and arsenic removal and passivation effects. The present invention lays a theoretical foundation for the application of the strain in the remediation of soil contaminated by cadmium and / or arsenic.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and heavy metal solidification, and in particular to a carbonate mineralizing bacterium and its application in the restoration of a heavy metal polluted environment. Background Art

[0002] Cadmium is a highly toxic heavy metal element, mainly derived from lead and zinc mines, as well as non-ferrous metal smelting, electroplating and factories using cadmium compounds as raw materials or catalysts. After entering the environment, cadmium will accumulate in the soil and enter the human body through the food chain, causing serious impacts on human health. Long-term inhalation of cadmium-containing smoke may cause diseases such as lung inflammation, bronchitis, and emphysema. Long-term, low-dose exposure to cadmium pollution may also damage the renal tubules, leading to symptoms such as diabetes and proteinuria. Cadmium can also replace zinc in plants, affecting plant growth and even causing death.

[0003] Arsenic is widely distributed in nature, mostly in the form of heavy metal arsenic compounds and arsenic sulfide in cadmium ores. Arsenic that pollutes the environment is mostly trivalent inorganic compounds, which are more toxic than pentavalent arsenic. Arsenic pollution is extremely harmful to the human body. Arsenic compounds are highly toxic and easily accumulate in the human body, causing chronic arsenic poisoning. The World Health Organization lists arsenic pollution as a major public health issue. The sources of arsenic pollution include arsenic-containing smoke, wastewater, waste gas, and waste residue emitted during the production of arsenic-containing metal ores, such as mining, roasting, smelting, chemicals, coking, thermal power, papermaking, and leather.

[0004] Cadmium and arsenic pollution are serious environmental problems that pose a threat to human health and ecosystems. At present, remediation technologies mainly include physical remediation, chemical remediation, microbial remediation and other methods. Physical remediation mainly repairs cadmium and arsenic pollution by excavating and transferring contaminated soil. However, this method is costly and may cause secondary pollution during operation. Chemical remediation mainly converts cadmium and arsenic into low-toxic or non-toxic forms through the action of chemical reagents. However, this method may cause other environmental pollution problems, such as chemical reagent residues and secondary pollution. Bioremediation refers to the use of biological or microbial action to degrade or transform cadmium and arsenic. This method is environmentally friendly and sustainable, but there are currently few microorganisms that can repair arsenic-cadmium complex pollution. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a carbonate mineralizing bacterium and its application in the remediation of heavy metal polluted environments.

[0006] One of the purposes of the present invention is to provide a carbonate mineralizing bacterium, wherein the carbonate mineralizing bacterium is Rummeliibacillus sp. ANY-1, and the Rummeliibacillus sp. ANY-1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 24, 2024, with a deposit number of CGMCC No.31431.

[0007] The carbonate mineralizing bacteria of the present invention have good mineralization ability and can better remove arsenic and cadmium at the same time compared with the widely used mineralizing bacteria S. pasteurii under the same conditions. The carbonate mineralizing bacteria of the present invention have good removal effects under the conditions of single arsenic and cadmium and arsenic-cadmium combined pollution. The present invention further achieves the passivation effect of the above-mentioned carbonate mineralizing bacteria on arsenic-cadmium combined pollution in farmland soil.

[0008] The second object of the present invention is to provide a bacterial agent containing the carbonate mineralizing bacteria.

[0009] The third object of the present invention is to provide the use of the carbonate mineralizing bacteria or the bacterial agent in the remediation of a heavy metal polluted environment, wherein the heavy metal is cadmium and / or arsenic.

[0010] A fourth object of the present invention is to provide a method for remediating heavy metal contaminated soil, comprising the following steps:

[0011] (1) culturing the carbonate mineralizing bacteria in a liquid culture medium to obtain a bacterial suspension;

[0012] (2) applying the bacterial suspension and nutrients to heavy metal contaminated soil;

[0013] The heavy metal is cadmium and / or arsenic.

[0014] Furthermore, in step (1), the carbonate mineralizing bacteria were inoculated into a liquid culture medium and activated overnight to the logarithmic phase, the cells were collected by centrifugation, and resuspended to an OD of 600nm is 0.8~1 to obtain bacterial suspension.

[0015] Furthermore, the liquid culture medium consists of:

[0016] 2.5-3.5g beef extract, 4-6g peptone, 4-6g sodium chloride, 28-20g urea, and water to make up to 1L.

[0017] Furthermore, in step (2), the amount of bacterial suspension applied is 1% to 10% of the mass of the heavy metal contaminated soil.

[0018] Furthermore, in step (2), the nutrients are urea and CaCl2, and the applied amounts of urea and CaCl2 are the same, both of which are 0.8% to 1.2% of the mass of the heavy metal contaminated soil.

[0019] Furthermore, in step (2), the moisture content of the heavy metal contaminated soil is 65 g / 100 g to 70 g / 100 g.

[0020] The present invention has the following beneficial effects:

[0021] Additional aspects and advantages of the present invention will be presented in the detailed description.

[0022] Storage Instructions:

[0023] Bacteria name: Bacillus rameliensis;

[0024] Latin name: Rummeliibacillus sp.;

[0025] Strain number: ANY-1;

[0026] Depository: China General Microbiology Center of Microbiological Culture Collection Administration (CGMCC);

[0027] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0028] Deposit date: July 24, 2024;

[0029] Deposit number: CGMCC No.31431. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The colony morphology of the strain on NBU plate.

[0031] Figure 2 Gram staining of the strain.

[0032] Figure 3 middle:

[0033] A is the growth curve of ANY-1.

[0034] B is the urease activity of ANY-1.

[0035] C is the pH change diagram of ANY-1.

[0036] Figure 4 middle:

[0037] A is a comparison of the arsenic removal effects of ANY-1 and S. pasteurii at a single arsenic concentration (0.5 mg / L). B is a comparison of the arsenic removal effects of ANY-1 and S. pasteurii at a single arsenic concentration (1 mg / L). C is a comparison of the arsenic removal effects of ANY-1 and S. pasteurii at a single arsenic concentration (5 mg / L). D is a comparison of the arsenic removal effects of ANY-1 and S. pasteurii at a single arsenic concentration (10 mg / L). Figure 5 middle:

[0038] A is a comparison of the cadmium removal effects of ANY-1 and S. pasteurii at a single cadmium concentration (0.5 mg / L). B is a comparison of the cadmium removal effects of ANY-1 and S. pasteurii at a single cadmium concentration (1 mg / L). C is a comparison of the cadmium removal effects of ANY-1 and S. pasteurii at a single cadmium concentration (5 mg / L). D is a comparison of the cadmium removal effects of ANY-1 and S. pasteurii at a single cadmium concentration (10 mg / L). Figure 6 middle:

[0039] A is the SEM image of the mineralization product of ANY-1.

[0040] B is the EDS image of the mineralization product of ANY-1.

[0041] Figure 7 middle:

[0042] A is the XRD pattern of ANY-1 mineralized cadmium product.

[0043] B is the XRD pattern of the ANY-1 mineralized arsenic product.

[0044] Figure 8 middle:

[0045] A is the arsenic removal effect of ANY-1 in the presence of different cadmium concentrations when the arsenic concentration is fixed at 5.0 mg / L.

[0046] B is the cadmium removal effect of ANY-1 in the presence of different concentrations of arsenic when the cadmium concentration is fixed at 10.0 mg / L.

[0047] Fig. 9 middle:

[0048] A is the passivation effect of ANY-1 on arsenic in arsenic-cadmium co-contaminated soil.

[0049] B is the passivation effect of ANY-1 on cadmium in arsenic-cadmium co-contaminated soil. DETAILED DESCRIPTION

[0050] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0051] Example 1: Screening and identification of carbonate mineralizing bacteria

[0052] 1 Materials and methods

[0053] 1.1 Test soil

[0054] The soil used for passivation remediation was collected from the arsenic-cadmium complex-contaminated farmland in Dongzhi County, Anhui Province. The soil samples were air-dried and passed through a 5 mm sieve for later use.

[0055] 1.2 Test culture medium

[0056] Enrichment medium (g / L): 20 g glucose, 2 g sodium acetate, 2 g potassium dihydrogen phosphate, 5 g sodium chloride, 20 g urea, and the balance is distilled water.

[0057] Screening medium (g / L): 3 g beef extract, 5 g peptone, 5 g sodium chloride, 20 g urea, 10 mL 0.2% phenol red solution, and the balance is distilled water.

[0058] NBU medium (g / L): 3 g beef extract, 5 g peptone, 5 g sodium chloride, 20 g urea, and the balance is distilled water.

[0059] Autoclave at 121°C for 25 min. Add urea, calcium chloride, glucose and phenol red solution after filtering through a 0.22 μm filter membrane. If a solid culture medium is required, add 2% agar powder.

[0060] 2 Test methods

[0061] (1) Screening

[0062] Take 1g of soil sample and inoculate it into 100mL of enrichment medium (100mL / 250mL conical flask), shake and culture at 30℃ and 160r / min for 24h, take 1mL of the upper suspension and add it into a centrifuge tube containing 9mL of sterile water, and mix it by pipetting (get 10 -1 Diluent), follow the same steps to dilute to 10 -7 , take 10 respectively -5 , 10 -6 , 10 -7 0.2 mL of the three dilutions were applied to the screening medium and incubated at 30°C for 72 h. The strains with obviously different colony characteristics and red surrounding the culture medium were selected for streaking separation. This step was repeated more than 3 times for purification until a pure single bacterium was obtained.

[0063] (2) Identification

[0064] Morphological identification: Inoculate a single bacterium into NBU medium and culture at 30℃ for 24h before observing the colony morphology.

[0065] Gram staining: Take a clean slide, drop a drop of sterile distilled water in the center of the slide, use a sterile inoculation loop to pick up a small amount of bacteria and apply it on the slide, break the colony, mix it with sterile water and spread it into a uniform thin layer; after air drying naturally in the air, quickly pass it over the flame three times to fix the bacteria on the slide; add a drop of crystal violet staining solution to stain for 1-2 minutes and then wash with water; add a drop of iodine solution to stain for 1-2 minutes and wash with water; then add 95% ethanol and wash with water after 20-30 seconds; then add safranin, stain for 2 minutes, wash with water and air dry naturally. After air drying, the slide preparation is completed and can be examined under a microscope.

[0066] 16S rDNA sequence identification: PCR amplification using bacterial 16s DNA universal primers:

[0067] 27F (SEQ ID NO. 1): 5'-AGAGTTTGATCMTGGCTCAG-3'.

[0068] 1492R (SEQ ID NO. 2): 5′-GGTTACCTTGTTACGACTT-3′.

[0069] The reaction system was: 10×Ex Taq Buffer 5μL, dNTP Mixture 5μL, upstream primer 2μL, downstream primer 2μL, template DNA 5μL, Ex Taq DNA polymerase 1μL, sterilized dd H2O 30μL. PCR reaction program: 94℃5min; 94℃30s; 55℃30s; 72℃90s, 35 cycles; 72℃10min, stored at 4℃. After the PCR reaction, all the products were spotted on 1.2% macroporous agarose gel, electrophoresed at 100V constant voltage for 45min, observed by UV gel imaging system, and the products were purified.

[0070] The purified samples were sequenced. A phylogenetic tree was constructed for the 16S rRNA gene of the strain finally measured to determine the position of its species in the classification system. The specific steps of phylogenetic tree construction are: using the BLAST function in the website of the National Center for Biotechnology Information (NCBI) of the United States, the 16S rRNA sequence obtained by sequencing was subjected to homology comparison analysis. The corresponding gene sequences of the model strains with a close relationship to the measured gene sequence were selected, and these sequences were used as the analysis objects. The Clustal W function of MEGA7 was used to compare the target gene sequence with the related sequence. Then, the phylogenetic tree was constructed using the Neighbor-joining method using the MEGA7 software to compare the compared sequences.

[0071] 3 Test results

[0072] The carbonate mineralizing bacteria screened by the present invention can grow in large quantities when cultured in NBU solid medium at 30°C for 24 hours, and the colonies are round, with neat edges and opaque ( Figure 1 ). Optical microscope observation revealed that bacterial cells were short rod-shaped.

[0073] Physiological and biochemical characteristics are:

[0074] ①Suitable culture temperature: 25~40℃.

[0075] ②Suitable culture pH: 6-8.

[0076] ③ Gram staining: positive, Figure 2 shown.

[0077] The sequences were aligned by 16S rDNA sequencing to construct a phylogenetic tree. The results showed that the strain was 99% homologous to Bacillus Rummelii bacillus sp. and was finally identified as Bacillus Rummelii bacillus sp. and named ANY-1.

[0078] Example 2: Study on the properties of ANY-1

[0079] 1 Test method

[0080] (1) Growth characteristics of strains:

[0081] Preparation of bacterial suspension: The purified ANY-1 single bacteria were inoculated into NBU liquid medium and activated overnight to the logarithmic phase. The bacteria were collected by centrifugation at 5000 rpm for 10 min, washed three times with PBS and resuspended. The OD was adjusted. 600nm =0.8 as bacterial suspension.

[0082] Prepare 50 mL of NBU liquid culture medium, adjust the initial pH value to 4, 5, 6, 7, 8, and 9, inoculate 2% (v:v) bacterial suspension, and culture at 30°C, 160 r / min for 24 h. Measure the OD of the bacterial solution under different treatments. 600 Each treatment was set up in triplicate.

[0083] Prepare 50 mL of NBU liquid culture medium, inoculate 2% (v:v) bacterial suspension, place in a shaker at 20℃, 25℃, 30℃, 35℃, and 40℃, shake at 160r / min for 24h, and measure the OD of the bacterial solution under different treatments 600 Each treatment was set up in triplicate.

[0084] (2) Growth curve of strain:

[0085] Prepare 200 mL of NBU liquid medium, inoculate with 2% (v:v) bacterial suspension, and culture at 30°C, 160 r / min shaking. During the culture process, take samples continuously at 0, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 144 h to measure OD 600 , which is used to characterize the growth of bacteria. The experiment was set up in three parallel groups.

[0086] (3) Urease activity and alkali production ability of the strain:

[0087] Prepare 200mL of NBU liquid medium, inoculate 2% (v:v) bacterial suspension, and culture at 30°C and 160r / min shaking. During the culture process, continuous sampling is performed at 0, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 144h to determine the urease activity and pH of the strain. The experiment is set up in three parallel groups.

[0088] The conductivity method was used to determine the urease activity of microorganisms. The measurement method was as follows: 1 mL of bacterial solution was added to 9 mL of 1.11 mol / L urea solution and mixed. The conductivity change of the mixed system at 25°C for 5 min was measured using a conductivity meter. The average conductivity change within 5 min (ms / (cm·min)) was recorded and multiplied by the dilution factor to obtain the urease activity of the bacterial solution (mol / (L·min)).

[0089] 2 Test results

[0090] The strain has obvious stagnation period, logarithmic growth period, stable period and decay period. It grows rapidly in 4-24h, which is the logarithmic growth period, and grows slowly after 24h and enters the stable period. Figure 3 A). The urease activity of the strain gradually increased during growth, reaching a maximum at 96h, and then began to decrease ( Figure 3 B). The pH value during the growth of the strain gradually increased with the increase of culture time, reaching a maximum of 9.07 ( Figure 3 C).

[0091] Example 3: Removal of arsenic and cadmium by ANY-1

[0092] 1. Removal of arsenic and cadmium by strains under single pollution conditions:

[0093] The bacterial suspension was inoculated into NBU+5g / L CaCl2 liquid culture medium containing 0.5, 1.0, 5.0, 10.0 mg / L cadmium chloride and 0.5, 1, 5, 10 mg / L sodium arsenite (urea and CaCl2 were sterilized by filtration). The control group was added with an equal amount of sterile water, cultured at 30°C, 160 r / min, and continuously sampled at 0, 6, 12, 24, 48, 96, and 144 h. Each sample was 5 mL, centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to determine the heavy metal concentration. The experiment was set up in three parallel groups. Figure 4 and Figure 5 It can be seen that ANY-1 is significantly better than the widely used mineralizing bacteria S. pasteurii in removing arsenic and cadmium.

[0094] Characterization of mineralization products of 2 strains:

[0095] (1) Scanning electron microscopy and energy spectrum analysis:

[0096] Take an appropriate amount of mineralized sample into a 50mL centrifuge tube, centrifuge at 4000r / min for 10min, discard the supernatant, fix with 2.5% glutaraldehyde precooled at 4℃ for 8-12h; then rinse and mix with pH 7.2 phosphate buffer, centrifuge again to precipitate the bacteria to the bottom of the centrifuge tube, repeat 2-3 times, 15min each time; then dehydrate with different concentrations of ethanol gradient (30%, 50%, 70%, 80%, 90%, 100%) in turn, each concentration treatment for 15min, after the last centrifugation, aspirate and discard the supernatant, put the sample in a CO2 critical point dryer to dry for 2h, and after coating treatment, observe the bacterial structure and mineralization product element analysis by scanning electron microscopy (SEM) and energy spectrum (EDS) analysis. The results are as follows: Figure 6 As shown, the SEM image shows a clear mineral structure, and the cells undergo deformations such as rupture and depression. The EDS image shows a clear Ca peak.

[0097] (2) Fourier infrared spectroscopy and x-ray diffraction:

[0098] The samples were centrifuged at 8000 r / min for 15 min, the supernatant was discarded, and the samples were washed 2-3 times. The collected precipitates were freeze-dried for 24 h, ground with an agate mortar and set aside for use. Fourier transform infrared spectroscopy (FTIR) was performed with a scanning range of 4000 cm -1 -400cm -1The precipitated phase was detected by X-ray diffractometer (XRD), with a scanning angle (2θ) of 10° to 90°, a step size of 0.02, and a count of 8°min -1 The data were analyzed using MDI JADE 6.0 software. Figure 7 As shown, according to the XRD of the mineralization product, it was verified that the main component of the generated mineral is CaCO3.

[0099] 2 Effects of strains on the removal of arsenic and cadmium under complex pollution conditions:

[0100] The concentration of arsenic was fixed at 5 mg / L, and the concentration of cadmium was 1.0, 5.0, 10.0, 20.0, and 40.0 mg / L, respectively. The concentration of cadmium was fixed at 10 mg / L, and the concentration of arsenic was 0.5, 1.0, 3.0, 5.0, and 10.0 mg / L, respectively. The bacterial suspension was inoculated into NBU+5g / L CaCl2 liquid culture medium containing the above concentrations of arsenic and cadmium (urea and CaCl2 were both filtered and sterilized), 30°C, 160r / min shaking culture, 0, 6, 12, 24, 48, 96, 144h continuous sampling, 5mL each time, 8000r / min centrifugation for 10min, and the supernatant was filtered through a 0.22μm filter membrane to determine the heavy metal concentration. The experiment was set up in three parallel groups.

[0101] Depend on Figure 8 It can be seen that when the fixed arsenic concentration is 5.0 mg / L, when the cadmium addition is low, the arsenic removal effect increases with the increase of cadmium concentration. When the cadmium addition is greater than 5.0 mg / L, the arsenic removal effect of the strain is inhibited. When the fixed cadmium concentration is 10.0 mg / L, the arsenic removal effect of the strain is inhibited when the arsenic concentration is greater than 1.0 mg / L.

[0102] The passivation effect of the three strains on farmland soil contaminated by arsenic and cadmium:

[0103] Weigh 100g of farmland soil contaminated with arsenic and cadmium into a beaker, and add 1%, 5%, and 10% of the soil mass of bacterial suspension, 1% urea, and 1% CaCl2, respectively. The moisture content was maintained at 100% of the maximum field water holding capacity (70g). An equal amount of ultrapure water was added to the control group. The soil was exposed to light at 25℃ day and night. Samples were collected at 7, 14, and 21 days, respectively, and the concentrations of available Cd and As in the soil samples were determined.

[0104] Extraction of available Cd in soil: 1.0 g soil sample was mixed with 10 mL 0.1 mol·L -1 The CaCl2 solution was mixed in a 15 mL centrifuge tube, shaken at 250 r / min for 2 hours at room temperature, and then centrifuged at 6000 r / min for 10 minutes. The supernatant was filtered through a 0.45 μm membrane and tested.

[0105] Extraction of available As in soil: 1.0 g dry soil was mixed with 25 mL extractant (0.05 M NH4H2PO4) in a 50 mL polypropylene centrifuge tube, and shaken at 250 r / min for 16 hours at room temperature. Centrifuged at 6000 r / min for 10 min, and the supernatant was filtered with a 0.45 μm membrane for testing.

[0106] Depend on Fig. 9 It can be seen that the addition of strains can effectively passivate arsenic and cadmium in the soil, providing a favorable approach for the remediation of complex-contaminated soil.

[0107] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A carbonate mineralizing bacterium, characterized in that: The carbonate mineralizing bacteria is Rummeliibacillus sp. ANY-1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 24, 2024, with a deposit number of CGMCC No.31431.

2. A bacterial agent containing the carbonate mineralizing bacteria according to claim 1.

3. Use of the carbonate mineralizing bacteria according to claim 1 or the bacterial agent according to claim 2 in the remediation of a heavy metal polluted environment, characterized in that: The heavy metal is cadmium and / or arsenic.

4. A method for remediating heavy metal contaminated soil, characterized in that: The following steps are involved: (1) culturing the carbonate mineralizing bacteria according to claim 1 in a liquid culture medium to obtain a bacterial suspension; (2) applying the bacterial suspension and nutrients to heavy metal contaminated soil; The heavy metal is cadmium and / or arsenic.

5. The method for remediating heavy metal contaminated soil according to claim 4, characterized in that: In step (1), the carbonate mineralizing bacteria were inoculated into a liquid culture medium and activated overnight to the logarithmic phase, the cells were collected by centrifugation, and resuspended to an OD of 600nm is 0.8~1 to obtain bacterial suspension.

6. The method for remediating heavy metal contaminated soil according to claim 4, characterized in that: In step (1), each 1L of the liquid culture medium is composed of: 2.5-3.5g beef extract, 4-6g peptone, 4-6g sodium chloride, 28-20g urea, and water to make up to 1L.

7. The method for remediating heavy metal contaminated soil according to claim 4, characterized in that: In step (2), the amount of bacterial suspension applied is 1% to 10% of the mass of the heavy metal contaminated soil.

8. The method for remediating heavy metal contaminated soil according to claim 4, characterized in that: In step (2), the nutrients are urea and CaCl2, and the applied amounts of urea and CaCl2 are the same, both of which are 0.8% to 1.2% of the mass of the heavy metal contaminated soil.

9. The method for remediating heavy metal contaminated soil according to claim 4, characterized in that: In step (2), the moisture content of the heavy metal contaminated soil is 65 g / 100 g to 70 g / 100 g.

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