A method for preparing a heavy metal contaminated soil activator using agricultural and rural waste

Abstract

The present application relates to a method for preparing a heavy metal contaminated soil activator by using agricultural and rural wastes, which comprises the following steps: composting agricultural and rural wastes (sludge, livestock and poultry manure, plant residues, kitchen waste, etc.) and soluble inorganic salt containing heavy metals as raw materials, then further domesticating and culturing microorganisms in the compost product by using non-selective liquid medium containing heavy metals, and then applying N-acetylglucosamine hydrolase to destroy the cells of the aforementioned microorganisms by ultrasonic crushing, freeze-thaw cycle and other methods, so as to obtain a biological polymer with heavy metal adsorption capacity. The water-soluble biological polymer prepared by the method can complex with cadmium, lead, arsenic, chromium and other heavy metals in the soil to increase the activity of the heavy metals in the soil, and can be used as an aid for chemical leaching or plant extraction remediation technology to enhance the removal efficiency of heavy metals in the soil.

Description

Technical Field

[0001] This invention belongs to the following fields: resource utilization of agricultural and rural waste, preparation of biopolymer-type heavy metal contaminated soil activators (remediation aids), and application in ecological environment restoration. Background Technology

[0002] Among various remediation technologies for heavy metal-contaminated soils, chemical leaching and phytoextraction are commonly used. These techniques typically require the use of activators as remediation adjuvants to enhance remediation effectiveness and efficiency. Sequential extraction methods can classify heavy metals in soil into five existence forms: water-soluble, exchangeable, carbonate-bound, iron-manganese oxide-bound, and residual. Water-soluble and exchangeable forms exhibit high activity and mobility, and are collectively referred to as the active forms of heavy metals in the soil. The proportion of these active forms in the total heavy metal content of the soil is called the activity level of the heavy metals. The purpose of applying activators is to increase the activity level of heavy metals in the soil. However, most commercially available chemically synthesized activators (remediation adjuvants), such as EDTA and EDDS, suffer from high costs and secondary pollution due to their residues in the soil, limiting their widespread and safe application. The demand for high-performance, cost-effective, and safe soil heavy metal activators (remediation adjuvants) remains strong.

[0003] The treatment and disposal of waste such as sludge, livestock and poultry manure, plant residues, and kitchen waste in rural areas has received increasing attention in recent years. Composting technology utilizes these wastes containing fertilizer components, mixing them with soil and minerals, and allowing them to ferment and decompose under high temperature and humidity conditions. This process involves various microorganisms, including bacteria, actinomycetes, fungi, and yeasts, releasing nutrients from the waste. However, the subsequent treatment of compost products also requires attention. Currently, returning them to the fields as supplementary "nutrient soil" for landscaping or agroforestry is a common treatment method, although relatively low-value, it is simple and easy to implement.

[0004] This invention uses rural agricultural waste (sludge, livestock and poultry manure, plant residues, kitchen waste, etc.) as raw materials. By adding heavy metals to domesticate the microorganisms within, it further extracts and prepares a highly efficient and environmentally friendly biopolymer-type soil heavy metal activator. This activator, used as a remediation aid in chemical leaching and phytoextraction technologies, will make a positive contribution to the fields of waste resource utilization and ecological environment restoration, and has broad market prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a heavy metal contaminated soil activator using agricultural and rural waste. This invention discovers that agricultural and rural waste (sludge, livestock and poultry manure, plant residues, kitchen waste, etc.) and soluble inorganic salts containing heavy metals are used as composting materials. Then, microorganisms selected from the compost products are further domesticated and cultured using a non-selective liquid culture medium containing heavy metals. Subsequently, N-acetylmuramic polysaccharide hydrolase is applied to destroy the aforementioned microbial cells by means of ultrasonic disruption, freeze-thaw cycles, etc., thereby obtaining a biopolymer with heavy metal adsorption capacity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a soil activator for heavy metal contaminated soil using agricultural and rural waste includes the following steps:

[0008] 1. Compost

[0009] Agricultural and rural waste (sludge, livestock and poultry manure, plant residues, kitchen waste, etc.) is composted using traditional aerobic methods (at room temperature of 25℃). On the fifth day of composting, a 10 mg / L solution of soluble inorganic salts containing heavy metals is evenly sprayed onto the material to achieve an overall moist state. 500 ml of the 10 mg / L inorganic salt solution is added per kilogram of fresh waste. The compost is turned over every three days until the microbial community stabilizes around the 20th day, at which point composting is stopped.

[0010] 2. Domestication and Cultivation

[0011] Prepare a non-selective liquid culture medium containing heavy metals, comprising glucose (4675 mg / L), peptone (1000 mg / L), K₂HPO₄ (3000 mg / L), KH₂PO₄ (1500 mg / L), NH₄Cl (1125 mg / L), and MgSO₄. 4· 7H2O (562.5 mg / L), FeSO4 4· 7H2O (18.75 mg / L), ZnSO 4· 7H2O (18.75 mg / L), MnSO4 4· 7H2O (18.75 mg / L), CaCl2 (75 mg / L), NaHCO3 (900 mg / L) and heavy metals Cd / Pb / As / Cr (10 mg / L) were autoclaved at 121°C for 20 minutes. The heavy metals were one or more of Cd, Pb, As and Cr, and the concentration of each heavy metal was 10 mg / L.

[0012] Microorganisms selected from the compost products obtained in step 1 were added to the aforementioned liquid culture medium at a ratio of 1g:100ml and incubated at 25°C for 24 hours. Then, a sample was taken from the liquid culture medium after incubation and added to freshly prepared liquid culture medium at a ratio of 1ml:100ml, and incubated at 25°C for 24 hours. To ensure the stability of the bacterial flora, this subculture step was repeated multiple times.

[0013] Collect the liquid culture medium from the last subculture (usually at least the sixth, preferably the sixth to the tenth) and centrifuge at 9000 rpm and 4°C for 5 minutes.

[0014] 3. Preparation of biopolymers

[0015] Discard the supernatant after centrifugation, rinse twice with UP water, and then suspend the remaining particles in 20 mmol / L Tris-HCl buffer (pH=8). Add N-acetylmuramic glycan hydrolase to a final concentration of 0.4 mg / mL and wait for 20 minutes.

[0016] Three freeze-thaw cycles were performed at -80°C and 30°C, respectively. Afterward, the suspension was sonicated for 15 minutes at 170W, 20kHz, and a 50% duty cycle pulse. During the sonication, the container holding the sample was placed in a low-temperature environment such as crushed ice.

[0017] Finally, the suspension was centrifuged at 9000 rpm and 4°C for 30 minutes. The supernatant was collected and filtered through a 0.45 μm membrane. The resulting filtrate was a biopolymer capable of activating heavy metals in contaminated soil (i.e., a heavy metal contaminated soil activator).

[0018] Furthermore, the heavy metals in the soluble inorganic salts containing heavy metals in step 1 specifically refer to one or more of Cd, Pb, As, and Cr. Taking Cd as an example, it includes Cd(NO3)2 or CdCl2, etc., and the concentration of each heavy metal is 10 mg / L.

[0019] Furthermore, the heavy metals in the soluble inorganic salts containing heavy metals added in step 1, as well as the heavy metals in the non-selective liquid culture medium, are consistent with the heavy metals referred to in the biopolymer of the target product that has the ability to activate heavy metals in contaminated soil. In reality, the preparation of this non-selective liquid culture medium needs to be based on the types of heavy metals contained in the target contaminated soil to be remediated, hence it is an "and / or" relationship. If a soil contains only Pb, then the culture medium will contain only Pb, with a Pb concentration of 10 mg / L; if a soil is contaminated with both Pb and Cd, then the culture medium must contain both, with each heavy metal at a concentration of 10 mg / L.

[0020] Furthermore, the microorganisms selected from the compost products in step 2 specifically include Bacillus, Flavobacterium, Escherichia coli, etc.

[0021] Furthermore, the specific selection process for microorganisms selected from the compost products in step 2 includes: During the composting process, a thin layer of microbial plaques will appear on the surface of the material, and in the later stages of composting, microbial moss may even form. The microbial plaques and microbial moss are selected (scraped) using an inoculation loop sterilized by high-pressure steam.

[0022] Furthermore, the main components of the prepared heavy metal contaminated soil activator include high molecular weight substances such as polysaccharides, proteins, and nucleic acids, with polysaccharide content of 0.1-10 wt%, protein content of 0.1-10 wt%, and nucleic acid content of 0.1-10 wt%.

[0023] The water-soluble biopolymer prepared by this method can complex with heavy metals such as cadmium, lead, arsenic, and chromium in the soil, increasing their activity in the soil. It can be used as an adjuvant for chemical leaching or phytoremediation technologies to enhance the removal efficiency of heavy metals in the soil.

[0024] The purpose of this invention is to use the prepared biopolymer to activate heavy metals in contaminated soil and enhance their activity, thereby helping to improve the remediation efficiency of chemical leaching or phytoextraction techniques.

[0025] The significant advantages of this invention are:

[0026] Composting involves a diverse range of microorganisms, which produce abundant biopolymers both inside and outside the body. These biopolymers are primarily composed of high-molecular-weight substances such as polysaccharides, proteins, and nucleic acids, containing numerous phosphate, thiol, carboxyl, and hydroxyl groups, as well as a significant number of charged groups. When heavy metal stress occurs in the environment, these biopolymers exhibit excellent adsorption and binding capacity for heavy metal ions, thereby ensuring the stability of the microbial community structure. This invention uses rural agricultural waste (sludge, livestock and poultry manure, plant residues, kitchen waste, etc.) as raw materials. By adding heavy metals to acclimate the microorganisms within, a highly efficient and environmentally friendly biopolymer-based soil heavy metal activator is further extracted and prepared. This activator, used as a remediation aid in chemical leaching and phytoextraction techniques, can significantly increase the activity of heavy metals in the soil. It will make a positive contribution to waste resource utilization and ecological environment restoration, and has broad market prospects. Attached Figure Description

[0027] Figure 1 This is a photograph of the activator for heavy metal contaminated soil prepared according to the present invention. Detailed Implementation

[0028] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0030] Example 1

[0031] A method for preparing a soil activator for heavy metal contaminated soil using agricultural and rural waste includes the following steps:

[0032] 1. Compost

[0033] Agricultural and rural waste, including livestock and poultry manure, is composted using traditional aerobic methods (at room temperature of 25℃). On the fifth day of composting, a 10 mg / L solution of soluble inorganic salts containing heavy metals is evenly sprayed onto the material to achieve a generally moist state. 500 ml of the 10 mg / L inorganic salt solution is added per kilogram of fresh waste. The compost is turned over every three days until the microbial community stabilizes around the 20th day, at which point composting is stopped.

[0034] 2. Domestication and Cultivation

[0035] Prepare a non-selective liquid culture medium containing heavy metals, comprising glucose (4675 mg / L), peptone (1000 mg / L), K₂HPO₄ (3000 mg / L), KH₂PO₄ (1500 mg / L), NH₄Cl (1125 mg / L), and MgSO₄. 4· 7H2O (562.5 mg / L), FeSO4 4· 7H2O (18.75 mg / L), ZnSO 4· 7H2O (18.75 mg / L), MnSO4 4· The 7H2O (18.75 mg / L), CaCl2 (75 mg / L), NaHCO3 (900 mg / L), and heavy metals (10 mg / L) were autoclaved at 121°C for 20 minutes.

[0036] Microorganisms selected from the compost products obtained in step 1 were added to the aforementioned liquid culture medium at a ratio of 1g:100ml and incubated at 25°C for 24 hours. Then, a sample was taken from the liquid culture medium after incubation and added to freshly prepared liquid culture medium at a ratio of 1ml:100ml, and incubated at 25°C for 24 hours. To ensure the stability of the bacterial flora, this subculture step was repeated multiple times.

[0037] Collect the liquid culture medium from the sixth subculture and centrifuge at 9000 rpm and 4°C for 5 minutes.

[0038] 3. Preparation of biopolymers

[0039] Discard the supernatant after centrifugation, rinse twice with UP water, and then suspend the remaining particles in 20 mmol / L Tris-HCl buffer (pH=8). Add N-acetylmuramic glycan hydrolase to a final concentration of 0.4 mg / mL and wait for 20 minutes.

[0040] Three freeze-thaw cycles were performed at -80°C and 30°C, respectively. Afterward, the suspension was sonicated for 15 minutes at 170W, 20kHz, and a 50% duty cycle pulse. During the sonication, the container holding the sample was placed in a low-temperature environment such as crushed ice.

[0041] Finally, the suspension was centrifuged at 9000 rpm and 4°C for 30 minutes. The supernatant was collected and filtered through a 0.45 μm membrane. The filtrate was the biopolymer with the ability to activate heavy metals in contaminated soil (i.e., heavy metal contaminated soil activator).

[0042] Application Example 1

[0043] Specifically, the heavy metal-containing soluble inorganic salt solution in step 1 of Example 1 is defined as a solution containing Na2HAsO4 and Cd(NO3)2, wherein the concentration of As is 10 mg / L and the concentration of Cd is 10 mg / L.

[0044] In step 2, the non-selective liquid culture medium containing heavy metals is specifically limited to Na2HAsO4 and Cd(NO3)2, with As concentration of 10 mg / L and Cd concentration of 10 mg / L.

[0045] The biopolymer-type heavy metal activator prepared in this way is used in the following applications.

[0046] a. Collect arsenic- and cadmium-contaminated red soil from Fuzhou, air-dry, grind, and pass through a 10-mesh sieve.

[0047] b. The biopolymer-type heavy metal activator is prepared according to a 1m... 3 Add 500L of soil to the contaminated soil and allow them to mix thoroughly for 24 hours.

[0048] c. Discard the liquid phase and air dry the remaining soil.

[0049] d. The Tessier continuous extraction method was used to determine the occurrence forms of heavy metals in pristine red soil and red soil after the application of activators, especially their active forms (water-soluble + exchangeable forms). The results showed that the activity of soil arsenic increased from 9.06% to 34.69%; the activity of soil cadmium increased from 10.02% to 41.16%.

[0050] Application Example 2

[0051] Specifically, the heavy metal-containing soluble inorganic salt solution in step 1 of Example 1 is defined as a solution containing Pb(NO3)2 and Cd(NO3)2, wherein the concentration of Pb is 10 mg / L and the concentration of Cd is 10 mg / L.

[0052] In step 2, the non-selective liquid culture medium containing heavy metals is specifically limited to Pb(NO3)2 and Cd(NO3)2, with Pb concentration of 10 mg / L and Cd concentration of 10 mg / L.

[0053] The biopolymer-type heavy metal activator prepared in this way is used in the following applications.

[0054] a. Collect lead- and cadmium-contaminated yellow soil from Kunming, air-dry, grind, and pass through a 10-mesh sieve.

[0055] b. The biopolymer-type heavy metal activator is prepared according to a 1m... 3 Add 500L of soil to the contaminated soil and allow them to mix thoroughly for 24 hours.

[0056] c. Discard the liquid phase and air dry the remaining soil.

[0057] d. The Tessier continuous extraction method was used to determine the occurrence forms of heavy metals in the original yellow soil and the yellow soil after the application of activator, especially their active forms (water-soluble + exchangeable forms). The results showed that the active form of lead in the soil increased from 9.94% to 37.33%; the active form of cadmium in the soil increased from 11.21% to 50.65% before and after remediation.

[0058] Application Example 3

[0059] Specifically, the heavy metal-containing soluble inorganic salt solution in step 1 of Example 1 is defined as a solution containing Na2HAsO4 and Pb(NO3)2, wherein the concentration of As is 10 mg / L and the concentration of Pb is 10 mg / L.

[0060] In step 2, the non-selective liquid culture medium containing heavy metals is specifically limited to Na2HAsO4 and Pb(NO3)2, with As concentration of 10 mg / L and Pb concentration of 10 mg / L.

[0061] The biopolymer-type heavy metal activator prepared in this way is used in the following applications.

[0062] a. Collect black soil from Heilongjiang Province that is contaminated with arsenic and lead, air-dry, grind, and pass through a 10-mesh sieve.

[0063] b. The biopolymer-type heavy metal activator is prepared according to a 1m... 3 Add 500L of soil to the contaminated soil and allow them to mix thoroughly for 24 hours.

[0064] c. Discard the liquid phase and air dry the remaining soil.

[0065] d. The Tessier continuous extraction method was used to determine the occurrence forms of heavy metals in the original black soil and the black soil after the application of activators, especially their active forms (water-soluble + exchangeable forms). The results showed that the active form of arsenic in the soil increased from 16.95% to 44.22%; the active form of lead in the soil increased from 15.01% to 50.43% before and after remediation.

[0066] Application Example 4

[0067] Specifically, the heavy metal-containing soluble inorganic salt solution in step 1 of Example 1 is defined as a solution containing K2Cr2O7 and Cd(NO3)2, wherein the concentration of Cr is 10 mg / L and the concentration of Cd is 10 mg / L.

[0068] In step 2, the non-selective liquid culture medium containing heavy metals is specifically limited to K2Cr2O7 and Cd(NO3)2, with Cr concentration of 10 mg / L and Cd concentration of 10 mg / L.

[0069] The biopolymer-type heavy metal activator prepared in this way is used in the following applications.

[0070] a. Collect chromium- and cadmium-contaminated brown soil from Shijiazhuang, air-dry, grind, and pass through a 10-mesh sieve.

[0071] b. The biopolymer-type heavy metal activator is prepared according to a 1m... 3 Add 500L of soil to the contaminated soil and allow them to mix thoroughly for 24 hours.

[0072] c. Discard the liquid phase and air dry the remaining soil.

[0073] d. The Tessier continuous extraction method was used to determine the occurrence forms of heavy metals in the original brown soil and the brown soil after the application of activator, especially their active forms (water-soluble + exchangeable forms). The results showed that the active form of chromium in the soil increased from 13.98% to 42.11%; the active form of cadmium in the soil increased from 10.06% to 49.52% before and after remediation.

[0074] Matters not covered in this invention are common knowledge.

[0075] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a heavy metal contaminated soil activator using agricultural rural waste, characterized by: The method comprises the following specific steps: (1) during the aerobic composting of agricultural and rural waste, 500 ml of inorganic salt solution with a heavy metal concentration of 10 mg / L is added per kilogram of fresh weight of waste until the composting is completed; (2) a non-selective liquid medium with a heavy metal content of 10 mg / L is prepared; (3) the microorganisms in the compost product obtained in step (1) are further domesticated and cultured in the non-selective liquid medium, and the domestication and culture are carried out at 25 DEG C for 24 hours; (4) the liquid is taken from the liquid medium after the domestication and culture is completed, and is added into the newly prepared non-selective liquid medium, and sub-cultivation is carried out at 25 DEG C for 24 hours, and the sub-cultivation is repeated for multiple times with a cycle of 24 hours; (5) the liquid medium after the final sub-cultivation is collected, and the solid particles are resuspended in 20 mM Tris-HCl buffer after centrifugation; (6) after N-acetyl muramidase is added for 20 minutes, the microbial cells are destroyed by ultrasonic crushing and freeze-thaw cycle, and the supernatant after membrane filtration is collected, so that a biological polymer with heavy metal adsorption capacity is obtained; The agricultural and rural waste comprises sludge, livestock and poultry manure, plant residues and kitchen waste; The heavy metal in the inorganic salt solution containing heavy metal is one or more of Cd, Pb, As and Cr, and the concentration of each heavy metal is 10 mg / L; The non-selective liquid medium containing heavy metal is prepared and comprises the following components: glucose 4675 mg / L, peptone 1000 mg / L, K2HPO4 3000 mg / L, KH2PO4 1500 mg / L, NH4Cl 1125 mg / L, MgSO4·7H2O 562.5 mg / L, FeSO4·7H2O 18.75 mg / L, ZnSO4·7H2O 18.75 mg / L, MnSO4·7H2O 18.75 mg / L, CaCl2 75 mg / L, NaHCO3 900 mg / L and heavy metal 10 mg / L, and the high-pressure steam sterilization is carried out at 121 DEG C for 20 minutes; wherein the heavy metal is one or more of Cd, Pb, As and Cr, and the concentration of each heavy metal is 10 mg / L; The pH of the Tris-HCl buffer is 8.

2. The method of claim 1, wherein, The dosage ratio of the microorganisms selected from the compost product to the non-selective liquid medium in step (3) is 1 g:100 ml.

3. The method of claim 1, wherein, The final concentration of N-acetyl muramidase added in step (6) is 0.4 mg / mL.

4. The method of claim 1, wherein, The freeze-thaw cycle in step (6) specifically refers to 3 times of freeze-thaw cycles at-80 DEG C and 30 DEG C respectively.

5. The method of claim 1, wherein, The ultrasonic crushing in step (6) specifically refers to ultrasonic treatment for 15 minutes under the condition of 170 W, 20 kHz and 50% duty cycle pulse.

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