Soil remediation agent and application thereof

Abstract

The invention discloses a soil remediation agent and application thereof, and belongs to the technical field of soil remediation. The soil remediation agent comprises micro-nano bone black and a penicillium fistulosum solid fungicide. Penicillium fimbriatum can convert insoluble phosphorus in soil into available phosphorus and increase phosphorus nutrition of the soil, micro-nano bone black can improve cation exchange capacity of the soil and enhance water and fertilizer retention capacity of the soil, and soil fertility can be comprehensively improved by combining the two. The phosphorus-solubilizing fungus and micro-nano bone black combined remediation technology is adopted, dissolution of insoluble phosphate can be effectively promoted, and the content of available phosphorus in soil is increased by 2.69 times; the soil heavy metal cadmium (Cd) can be fixed, the effective state Cd of the soil is reduced by about 25.91%, and the soil heavy metal cadmium (Cd) fixing agent has high Cd fixing capacity and has good application value in farmland Cd pollution remediation and environmental protection. The soil remediation agent provided by the invention not only can realize efficient remediation of cadmium-contaminated soil, but also is green and environment-friendly, and is suitable for large-scale popularization and application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of soil remediation, and particularly relates to a soil remediation agent and application thereof. BACKGROUND

[0002] Farmland soil is facing the dual crisis of low-efficiency utilization of phosphorus and aggravation of cadmium pollution. Among them, phosphorus, as an essential element for crops, more than 95% exists in the form of insoluble calcium phosphate, resulting in a utilization rate of less than 30% of chemical phosphorus fertilizer; and cadmium pollution enters the food chain through crops, threatening food safety, both of which restrict the sustainable development of agriculture.

[0003] In traditional management, separate application of chemical phosphorus fertilizer and cadmium passivator not only has high cost, but also easily causes soil compaction and secondary pollution. Biological remediation has become a new direction due to its environmental protection and cost advantages. Some fungi can transform insoluble phosphorus by secreting organic acids, and reduce the availability of cadmium by cell wall adsorption and chelation, achieving the synergies of "dissolving phosphorus and reducing cadmium". Pure soil cultivation experiments can accurately simulate the field microenvironment, avoiding the disconnection problem between shake flask experiments and actual application. At the same time, as a natural carrier, bone charcoal is rich in insoluble phosphorus such as calcium phosphate for fungi to act on, and contains carbon and nitrogen nutrients to support its colonization. Therefore, a multifunctional and efficient microbial agent is developed to improve the transformation ability of insoluble phosphorus in bone charcoal in soil environment, improve the utilization rate of soil phosphorus, break the soil phosphorus-cadmium synergy barrier, and provide theoretical and experimental support for promoting the safe use of contaminated farmland. SUMMARY

[0004] To solve the above problems in the prior art, the application provides a soil remediation agent and application thereof.

[0005] To achieve the above-mentioned purpose, the application provides the following technical scheme: The application provides a soil remediation agent, which comprises micro-nano bone charcoal and a solid Penicillium crustosum microbial agent.

[0006] As a preferred technical scheme of the application, the preparation method of the micro-nano bone charcoal comprises the following steps: grinding the pyrolyzed pig bone.

[0007] As a preferred technical scheme of the application, the pyrolysis is carried out under an inert atmosphere, the pyrolysis temperature is 400-500 DEG C, the time is 100-140 min, and the grinding is to a particle size of 30 nm-100 mu m.

[0008] As a preferred technical scheme of the application, the preparation method of the solid Penicillium crustosum microbial agent comprises the following sterile operation steps: coating Penicillium crustosum on NBRIP solid culture medium for culture, washing to obtain spore solution after spore maturation; mixing a solid carrier with sterile water, then adding a solid microbial agent protective agent, and then adding the spore solution to obtain the solid Penicillium crustosum microbial agent.

[0009] As a preferred embodiment of the present invention, the culture temperature is 30℃ and the time is 3-4 days; the number of spores in the spore solution is ≥1×10⁻⁶. 8 cells / mL; As a preferred embodiment of the present invention, the solid carrier includes bacterial bran, the ratio of solid carrier to sterile water is 1 g: (2.5-3.5) mL, the solid bacterial agent protectant includes sorbitol, and the amount of solid bacterial agent protectant added is 0.8-1.2 wt% of the total amount of solid carrier and sterile water; the amount of spore liquid added is 8-10 wt% of the total amount of solid carrier and sterile water.

[0010] As a preferred embodiment of the present invention, each liter of NBRIP solid culture medium contains: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 5.0 g Ca3(PO4)2, 10 g sorbitol protectant, 20 g agar, and pH 5.0.

[0011] The present invention also provides an application of the soil remediation agent described above in reducing cadmium content and increasing phosphorus content in soil.

[0012] As a preferred technical solution of the present invention, the application amount of the soil remediation agent is as follows: the content of micro-nano bone char in the soil is 0.4-0.6 wt%, and the content of Penicillium chrysogenum solid inoculant in the soil is 2-4 wt%.

[0013] Existing remediation technologies often focus on a single mechanism, while the combined remediation technology of this invention utilizes micro / nano bone char, which, due to its abundant porous structure and large specific surface area, rapidly adsorbs cadmium ions from the soil. Meanwhile, *Penicillium fimbriatum* secretes organic acids and other substances, lowering the soil pH and transforming cadmium ions from a poorly soluble form into a more readily adsorbable one. The synergistic effect of these two technologies significantly improves the removal and fixation efficiency of cadmium. Furthermore, the metabolites produced by *Penicillium fimbriatum* during phosphorus dissolution can form stable complexes or precipitates with cadmium ions, further reducing the bioavailability of cadmium. This is more effective than using physical or chemical adsorption methods alone in reducing cadmium bioavailability.

[0014] Traditional chemical remediation methods use large amounts of chemical agents, which can easily leave residues and cause secondary pollution. In contrast, the combined remediation technology of this invention uses *Penicillium chrysogenum* and micro / nano bone char, both of which are naturally derived or biodegradable materials. These materials do not introduce harmful chemicals into the soil, making them more environmentally friendly to the soil and surrounding ecosystem. Furthermore, *Penicillium chrysogenum* can improve the soil microbial community structure, promoting the growth and reproduction of beneficial microorganisms, while micro / nano bone char provides a favorable habitat for soil microorganisms, jointly maintaining the soil ecological balance—something many traditional remediation technologies struggle to achieve.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) In the soil remediation agent provided by this invention, *Penicillium fimbriatum* can convert insoluble phosphorus in the soil into available phosphorus, increasing soil phosphorus nutrition. Micro-nano bone char can improve the cation exchange capacity of the soil and enhance the soil's water and fertilizer retention capacity. The combination of the two can comprehensively improve soil fertility and create good nutritional conditions for crop growth. This invention adopts a combined remediation technology of phosphate-solubilizing fungi and micro-nano bone char, which can effectively promote the dissolution of insoluble phosphate, increasing the available phosphorus content in the soil by 2.69 times (168.87%); and can also fix the soil heavy metal Cd, reducing the available Cd in the soil by about 25.91%, showing strong Cd fixation capacity. It has good application value for the remediation of Cd pollution in farmland and environmental protection.

[0016] (2) The soil remediation agent provided by the present invention can not only achieve efficient remediation of cadmium-contaminated soil, but is also green and environmentally friendly, and is suitable for large-scale promotion and application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A graph showing the dynamic changes in soil pH under the influence of Penicillium chrysogenum and bone char at different sampling times; Figure 2 A dynamic change diagram of soil cation exchange capacity under the interaction of Penicillium frenulum and bone char at different sampling times; Figure 3 A graph showing the dynamic changes in available phosphorus content in soil under the influence of Penicillium chrysogenum and bone carbon at different sampling times. Figure 4 This figure shows the dynamic changes in available cadmium in the soil under the influence of Penicillium chrysogenum and bone carbon at different sampling times. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The following examples use Penicillium fimbriae ( Penicillium steckii The sample was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC NO: 41749, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. All other raw materials were commercially available and conventional, with no special restrictions. These will not be repeated below.

[0025] Example 1 The preparation steps for soil remediation agents are as follows: (1) Preparation of micro / nano bone char Pig bones were pyrolyzed at 450 °C for 120 min in a nitrogen-filled tube furnace at a heating rate of 10 °C / min to produce bone biochar (BC). The BC was then further ground to a particle size of 30 nm to 100 μm using a planetary ball mill to obtain micro / nano bone biochar (MNBC).

[0026] (2) Preparation of solid inoculum of Penicillium fimbriae Penicillium brevicornu, preserved in glycerol at -80 °C, was spread onto NBRIP solid medium (1 L formulation: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 5.0 g Ca3(PO4)2, 10 g sorbitol protectant, 20 g agar, pH 5.0) and incubated at 30 °C for 4 days. After spore maturation, fungal spore suspension was prepared. The prepared solid plates were rinsed with sterile water, and spores were gently scraped off using a spreading stick during rinsing. Each plate was rinsed with 50 mL of sterile water to ensure a spore count of 1 × 10⁻⁶ spores in the spore suspension. 8 spores / mL (spore counting was performed using a hemocytometer). Fungal residue was selected as the solid carrier. After sterilization, a material-to-liquid ratio of fungal residue:sterile water = 1g:3mL was used. Under aseptic conditions, 1wt% sorbitol (based on the total amount of fungal residue and sterile water) was added as a solid inoculum protectant. Finally, 9wt% spore liquid (based on the total amount of fungal residue and sterile water) was added to prepare the *Penicillium fimbriatum* solid inoculum (hereinafter referred to as...). Ps ).

[0027] Example 2 The preparation steps for soil remediation agents are as follows: (1) Preparation of micro / nano bone char Pig bones were pyrolyzed at 400 °C for 140 min in a nitrogen-filled tube furnace at a heating rate of 10 °C / min to produce bone biochar (BC). The BC was then further ground to a particle size of 30 nm to 100 μm using a planetary ball mill to obtain micro / nano bone biochar (MNBC-2).

[0028] (2) Preparation of solid inoculum of Penicillium fimbriae Penicillium brevicornu, preserved in glycerol at -80 °C, was spread onto NBRIP solid medium (1 L formulation: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 5.0 g Ca3(PO4)2, 10 g sorbitol protectant, 20 g agar, pH 5.0) and incubated at 30 °C for 4 days. After spore maturation, fungal spore suspension was prepared. The prepared solid plates were rinsed with sterile water, and spores were gently scraped off using a spreading stick during rinsing. Each plate was rinsed with 50 mL of sterile water to ensure a spore count of 1 × 10⁻⁶ spores in the spore suspension. 8 Specimen count / mL (spore count was performed using a hemocytometer). Fungal residue was selected as the solid carrier and sterilized. After sterilization, a material-to-liquid ratio of fungal residue:sterile water = 1 g:2.5 mL was used. Under aseptic conditions, 1.2 wt% of sorbitol (based on the total amount of fungal residue and sterile water) was added as a solid inoculum protectant. Finally, 10 wt% of spore liquid (based on the total amount of fungal residue and sterile water) was added to prepare the *Penicillium fimbriatum* solid inoculum (abbreviated as...). Ps-2 ).

[0029] Example 3 The preparation steps for soil remediation agents are as follows: (1) Preparation of micro / nano bone char Pig bones were pyrolyzed at 500 °C for 100 min in a nitrogen-filled tube furnace at a heating rate of 10 °C / min to produce bone biochar (BC). The BC was then further ground to a particle size of 30 nm to 100 μm using a planetary ball mill to obtain micro / nano bone biochar (MNBC-3).

[0030] (2) Preparation of solid inoculum of Penicillium fimbriae Penicillium brevicornu, preserved in glycerol at -80 °C, was spread onto NBRIP solid medium (1 L formulation: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 5.0 g Ca3(PO4)2, 10 g sorbitol protectant, 20 g agar, pH 5.0) and incubated at 30 °C for 4 days. After spore maturation, fungal spore suspension was prepared. The prepared solid plates were rinsed with sterile water, and spores were gently scraped off using a spreading stick during rinsing. Each plate was rinsed with 50 mL of sterile water to ensure a spore count of 1 × 10⁻⁶ spores in the spore suspension. 8Specimen count / mL (spore count was performed using a hemocytometer). Fungal residue was selected as the solid carrier and sterilized. After sterilization, a material-to-liquid ratio of fungal residue:sterile water = 1 g:3.5 mL was used. Under aseptic conditions, 0.8 wt% of sorbitol (based on the total amount of fungal residue and sterile water) was added as a solid inoculum protectant. Finally, 8 wt% of spore liquid (based on the total amount of fungal residue and sterile water) was added to prepare the *Penicillium fimbriatum* solid inoculum (abbreviated as...). Ps-3 ).

[0031] Effect verification 1. Soil collection The soil used in the experiment was collected from the top 0-20 cm layer of paddy fields in Huadu District, Guangzhou (23.415°N, 113.047°E). Before use, the soil was air-dried, ground, and passed through a 2 mm mesh sieve.

[0032] 2. Soil cultivation experiment Based on the presence or absence of phosphate-solubilizing fungi combined with micro / nano bone char, three treatment groups were set up: a 15 mg / Kg Cd control group, a 15 mg / Kg Cd + 0.5% 450 ℃ MNBC (i.e., the MNBC prepared in step (1) of Example 1), and a 15 mg / Kg Cd + 0.5% 450 ℃ MNBC + 3% MNBC. Ps (i.e., the product prepared in step (2) of Example 1) Ps Each treatment was performed in quadruplicate, with a total weight of 300 g, and cultured in brown wide-mouth bottles. The bottles were placed in a constant temperature environment at 25 ℃, and water was added daily to maintain a constant moisture content. Soil samples were collected on days 0, 10, and 30, cooled, ground, and sieved through a 100-mesh sieve for subsequent testing. Cd treatment was performed by artificially adding an analytical grade CdCl2·2.5H2O aqueous solution to the soil.

[0033] 3. Measurement Indicators Soil pH determination: According to the "Determination of Soil pH Value by Potential Method" (HJ 962-2018), in short: vortex ratio 1:2.5 ( g / mL The soil water ratio of the sample was 2 min, and after standing for 30 min, the electrode was inserted to read the pH value of the soil in all treatments.

[0034] Determination of soil cation exchange capacity: soil to water 1:2 ( g / mL Soil cation exchange capacity (CEC) was measured using a cobalt trichloride solution spectrophotometric method.

[0035] Determination of available phosphorus content in soil: Following the standard "Determination of Available Phosphorus in Soil - Sodium Bicarbonate Extraction-Molybdenum Antimony Spectrophotometric Method" (HJ 704-2014), the soil was mixed with 0.5 mol / L sodium bicarbonate extract at a ratio of 1:20. g / mLThe sample was shaken for 30 min at a constant temperature reciprocating shaker at 25 ℃ and 200 r / min. The supernatant was then filtered and tested.

[0036] Determination of available Cd in soil: According to the "Determination of 8 available elements in soil by diethylenetriaminepentaacetic acid extraction-inductively coupled plasma atomic emission spectrometry" (HJ 804-2016), 5.00 g of freeze-dried and sieved soil sample was weighed and placed in a 50 mL centrifuge tube. 20 mL of diethylenetriaminepentaacetic acid (DTPA)-calcium chloride (CaCl2)-triethanolamine (TEA) extraction reagent (pH 7.3) was added. The sample was extracted at 160 r / min for 2 h at room temperature. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane and then analyzed.

[0037] 4. Statistical Analysis Statistical analysis was performed using IBM SPSS 26.0 software. One-way ANOVA and Duncan's test were used to analyze the data. All samples were replicated four times. Data are expressed as mean ± standard error. p < 0.05 is considered significant. GraphPad Prism version 8 software was used for graphing.

[0038] Test Results Figures 1-4 In the middle, Cd, Cd MNBC and Cd MNBC Ps These represent the control group (15 mg / Kg Cd), the MNBC at 450 ℃ (15 mg / Kg Cd +0.5%), and the MNBC at 450 ℃ (15 mg / Kg Cd +0.5%) with a concentration of 3%, respectively. Ps Groups, which will not be described further below.

[0039] 1. Dynamic changes in soil pH under the influence of Penicillium frenulum and bone carbon at different sampling times, as shown in the figure. Figure 1 As shown, Figure 2 This illustrates the dynamic changes in soil cation exchange capacity under the interaction of *Penicillium fimbriae* and bone char at different sampling times. Figure 1 and Figure 2 It was found that co-culturing fungi and MNBC significantly increased soil pH and CEC, enhancing soil nutrient retention capacity. Compared with the cadmium control treatment, Ps In synergy with fungi and MNBC, soil pH and CEC increased by 11.08% and 135.64% on day 10, respectively; and by 8.70% and 5.18% on day 30, respectively.

[0040] 2. Figure 3The graph shows the dynamic changes in available phosphorus content in soil under the influence of *Penicillium chrysogenum* and bone biochar at different sampling times. As can be seen from the graph, the application of bone biochar significantly increased the available phosphorus content in the soil. Compared with the Cd control group, the combined effect of bone biochar and bone biochar significantly improved the available phosphorus content. Ps On day 10, the available phosphorus content in the soil increased by 145.25% under fungal treatment, while the increase was 66.77% under MNBC treatment alone. This indicates that under cadmium pollution, the combined application of bone char and... Ps Fungi can further increase the available phosphorus content in the soil. After 30 days of combined application of bone char and... Ps The available phosphorus content in soil treated with fungi increased by 168.87%.

[0041] 3. Figure 4 The graph shows the dynamic changes in available cadmium in the soil under the influence of *Penicillium chrysogenum* and bone biochar at different sampling times. As can be seen from the graph, the application of bone biochar significantly reduced the available cadmium content in the soil. Compared with the Cd control group, the MNBC treatment alone and the combined bone biochar treatment on day 10... Ps The available cadmium content in the soil decreased by 15.06% and 17.22% in the fungal treatment group, respectively. After 30 days of combined application of bone char and... Ps In soil treated with fungi, the available Cd decreased by 25.91%.

[0042] The effects of the soil remediation agents prepared in Examples 2 and 3 were verified using the same verification method described above, and the results were basically the same as those of the soil remediation agent in Example 1.

[0043] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A soil remediation agent, characterized in that, Including micro / nano bone char and Penicillium fimbriae solid inoculum.

2. The soil remediation agent according to claim 1, characterized in that, The preparation method of the micro / nano bone char includes the following steps: pyrolyzing pig bones and then grinding them.

3. The soil remediation agent according to claim 2, characterized in that, The pyrolysis is carried out under an inert atmosphere at a temperature of 400–500 °C for 100–140 min, and the grinding is performed to a particle size of 30 nm–100 μm.

4. The soil remediation agent according to claim 1, characterized in that, The preparation method of the Penicillium chrysogenum solid inoculum includes the following steps: Penicillium chrysogenum is coated on NBRIP solid medium and cultured. After the spores mature, they are washed to obtain spore liquid. The solid carrier is mixed with sterile water, then a solid inoculum protectant is added, and then the spore liquid is added to obtain the Penicillium chrysogenum solid inoculum.

5. The soil remediation agent according to claim 4, characterized in that, The culture temperature was 30℃, and the time was 3–4 days; the number of spores in the spore solution was ≥1×10⁻⁶. 8 per mL.

6. The soil remediation agent according to claim 4, characterized in that, The solid carrier includes bacterial bran, and the ratio of solid carrier to sterile water is 1 g: (2.5-3.5) mL. The solid bacterial agent protectant includes sorbitol, and the amount of solid bacterial agent protectant added is 0.8-1.2 wt% of the total amount of solid carrier and sterile water. The amount of spore liquid added is 8-10 wt% of the total amount of solid carrier and sterile water.

7. The soil remediation agent according to claim 4, characterized in that, Each liter of NBRIP solid medium contains: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 5.0 g Ca3(PO4)2, 10 g sorbitol protectant, 20 g agar, and pH 5.

0.

8. The application of a soil remediation agent according to any one of claims 1 to 7 in reducing cadmium content and increasing phosphorus content in soil.

9. The application according to claim 8, characterized in that, The application rates of the soil remediation agents are as follows: the content of micro-nano bone char in the soil is 0.4-0.6 wt%, and the content of Penicillium chrysogenum solid inoculant in the soil is 2-4 wt%.

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