HCH contaminated soil remediation agent and remediation method

Abstract

The invention relates to an HCH contaminated soil remediation agent and a remediation method. The repairing agent is composite zoogloea loaded biochar-nano zero-valent iron microspheres with the particle size of 20 to 40 meshes; the composite zoogloea loaded biochar-nano zero-valent iron microspheres are prepared from gel microspheres embedded with a composite zoogloea-biochar compound and a biochar-nano zero-valent iron composite material according to the weight ratio of 1 to (1 to 3); the remediation method comprises the following steps: S1, soil pretreatment; s2, applying a repairing agent; according to the method, firstly, nanoscale zero-valent iron is used for reductive dechlorination, high-chlorinated HCH is converted into a low-chlorinated benzene compound, then microorganisms such as sphingomonas are used for degrading an intermediate product, the repairing efficiency is greatly improved, meanwhile, the sphingomonas in the composite zoogloea is used for treating an HCH molecular ring firstly, and therefore the repairing efficiency is greatly improved. And then, the strain of the pseudomonas putida opens a benzene ring, compounds such as benzene, methylbenzene and phenols are used as carbon sources and energy sources, and finally, the compounds are thoroughly mineralized, so that the remediation effect of HCH pollution is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of HCH pollution remediation technology, specifically to an HCH contaminated soil remediation agent and remediation method. Background Technology

[0002] Pesticides are an indispensable production material in agricultural development. However, with the large-scale application of pesticides, the soil environment has been damaged, and the soil quality is declining. The characteristics of soil pesticide pollution are: large contaminated area, high risk to human health, and significant economic losses. Although my country banned the production and use of organochlorine pesticides in 1983, due to large-scale use in the past and the difficulty in degrading these substances, a large amount of organochlorine pesticide residues still remain in the soil. According to incomplete statistics in 2006, approximately 870,000 to 1,070,000 hectares nationwide still have such residues. 2 The soil was contaminated with pesticides. In the 1950s and 60s, hexachlorocyclohexane (HCH) was widely produced and used worldwide due to its wide range of applications, ease of manufacture, and low price. It was also once the pesticide with the largest production and usage volume in my country, but it is now banned. As a persistent organic pollutant (POP), HCH is highly toxic, difficult to degrade, and bioaccumulates. Among the controlled POPs listed in the Stockholm Convention, there are three isomers of HCH: α-HCH, β-HCH, and γ-HCH (also known as lindane), which are well-known for their toxicity, environmental persistence, and global presence.

[0003] Methods for remediating HCH in soil include: solidification and stabilization, thermal desorption, soil leaching, redox remediation, and bioremediation. Among these, microbial remediation within bioremediation can be combined with other remediation materials to effectively remediate HCH-contaminated soil. Biochar-supported nano-zero-valent iron (BC / nZVI) is a novel environmental functional material that leverages the adsorption properties of biochar and the reducing properties of nano-zero-valent iron to effectively remediate HCH-contaminated soil. Combining it with microbial remediation to degrade and remediate HCH in soil is feasible.

[0004] Sphingosomalidobacterium UT26 is one of the most thoroughly studied HCH-degrading bacteria. It possesses a classic γ-HCH (lindane) ascending degradation pathway, the main step of which is dechlorination and hydrogenation, ultimately producing chlorobenzene intermediates such as γ-pentachlorocyclohexene (γ-PCCH) and downstream chlorobenzenes and chlorophenols. Although Sphingosomalidobacterium is adept at "disassembling" HCH molecular rings, its ability to completely mineralize (ultimately turning into CO2 and water) these stubborn chloroaromatic intermediates is limited. This leads to the accumulation of these chlorobenzenes in the environment, resulting in incomplete remediation. Summary of the Invention

[0005] To address the above problems, this invention provides an HCH contaminated soil remediation agent and a remediation method.

[0006] The technical solution of the present invention is: an HCH contaminated soil remediation agent, wherein the remediation agent is a composite bacterial floc loaded with biochar-nano zero-valent iron microspheres with a particle size of 20-40 mesh; the composite bacterial floc loaded with biochar-nano zero-valent iron microspheres is composed of gel microspheres encapsulated with a composite bacterial floc-biochar composite and a biochar-nano zero-valent iron composite material in a weight ratio of 1:1-3. The method for preparing the composite bacterial micelles is as follows: Sphingomonas and Pseudomonas putida are inoculated into LB liquid medium and cultured with shaking at 28-30℃ and 150-180 rpm until the OD600 is 0.6-0.8. Then, the bacterial cells are collected by centrifugation at 7500-8000 rpm for 8-12 min. Finally, the cells are washed 2-4 times with sterile basal salt medium to obtain pure Sphingomonas bacterial suspension and pure Pseudomonas putida bacterial suspension. Then, according to the volume ratio of pure Sphingomonas sphingosine monocytogenes bacterial suspension and pure Pseudomonas putida bacterial suspension, the bacterial suspensions were inoculated into sterile basal salt medium at 1:1. Then, the carrier loaded with HCH was added to the sterile basal salt medium and cultured with shaking at 120-150 rpm for 3-5 days. When tight bacterial aggregates were observed, 5-10% of the total volume of bacterial aggregates and free bacteria was transferred to fresh sterile basal salt medium containing 50-100 mg / L HCH for subculture. This is the first generation. Repeat the subculture steps for 5-10 generations. Let the culture medium of the last generation stand for 10-30 minutes, then centrifuge at low speed of 2000-3000 rpm for 3-5 minutes and collect the composite bacterial flocs that settle at the bottom. The total inoculation amount of pure sphingomonas bacterial suspension and pure pseudomonas putrefactive bacteria suspension accounted for 1-5% of the volume of sterile basal salt medium; the ratio of HCH-loaded carrier to sterile basal salt medium was 50-100 mg / L. Explanation: Synergistic degradation of HCH: Nano-sized zero-valent iron first reduces and dechlorinates, converting highly chlorinated HCH into low-chlorinated benzene compounds. These intermediate products are generally more easily degraded by microorganisms such as Sphingomonas. This relay mode of "chemical destruction + biomineralization" greatly improves the remediation efficiency. Within the complex bacterial floc, strains can form metabolic specialization, working together to completely degrade HCH. Strains of *Pseudomonas putida* possess a strong ability to degrade aromatic compounds, exhibiting a complete metabolic pathway, especially the ortho-cleavage pathway, which can open the benzene ring, using benzene, toluene, phenols, and other compounds as carbon and energy sources, ultimately achieving complete mineralization. The remediation agent prepared in the above manner can reshape the soil biochemical environment and ultimately, by relieving pollution stress, stimulate plants to restore the activity of antioxidant enzymes (SOD, POD), helping plants resist oxidative damage caused by HCH, thereby increasing plant biomass and facilitating ecological restoration. The LB liquid culture medium formula is as described in commercially available media; the basic salt culture medium formula is: K₂HPO₄: 1.5 g / L; KH₂PO₄: 0.5 g / L; NH₄Cl: 0.5 g / L; NaCl: 0.5 g / L; MgSO₄·7H₂O: 0.2 g / L; pH 7.0-7.2; trace element solution: (disodium ethylenediaminetetraacetate: 500 mg / L, FeSO₄·7H₂O: 500 mg / L). The trace element solution is a 1000x concentrated stock solution, added at a ratio of 1 mL / L. (ZnSO4·7H2O: 100 mg / L; MnCl2·4H2O: 30 mg / L; CoCl2·6H2O: 20 mg / L; CuCl2·2H2O: 10 mg / L; NiCl2·6H2O: 10 mg / L; Na2MoO4·2H2O: 10 mg / L; H3BO3: 10 mg / L).

[0007] Further, the preparation method of the HCH-loaded carrier is as follows: first, HCH is dissolved in acetone at a ratio of 8-10 mg: 1 mL to obtain an HCH-acetone solution; then, the HCH-acetone solution is added dropwise to sterile diatomaceous earth at a ratio of 1-1.5 mL: 1 g, stirred and mixed, and finally, the mixture is evaporated by ventilation at a temperature of 23-27℃ for 2-4 hours to obtain the HCH-loaded carrier. Note: Through this "carrier loading" method, HCH exists in a highly dispersed microcrystalline form on the huge surface of diatomaceous earth, which greatly increases its contact area with microorganisms and significantly improves the bioavailability of HCH.

[0008] Furthermore, the preparation method of the composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres is as follows: Step 1: Biochar Pretreatment The biochar material was pulverized and passed through an 80-200 mesh sieve. The pulverized biochar material was then soaked in a 1-1.2 mol / L acid solution at a solid-liquid ratio of 1 g: 8-10 mL, and shaken at 23-27℃ and 120-150 rpm for 2-4 hours. The acid solution was discarded, and the biochar material was washed with deionized water until the pH of the washing solution reached 6.8-7.2. Finally, the washed biochar material was dried in an oven at 100-105℃ and treated with nitrogen gas for 25-30 minutes to obtain pretreated biochar material. Pretreatment optimizes its surface functional groups and pore structure, facilitating microbial attachment. Step 2: Preparation of gel microspheres The composite mycelium flocs were mixed with the pretreated biochar material at a ratio of 1 / 4 to 3 / 4 wet weight of mycelium flocs to dry weight of biochar material of 1:1-3. The mixture was placed in a shaker and incubated at 28-30°C and 80-100 rpm for 6-8 hours. Finally, the mixture was placed in a refrigerator at 3-4°C and allowed to stand for 12-24 hours to fix the biochar with composite mycelium flocs loaded on the pores and surface. Biochar loaded with composite bacterial micelles was mixed with 2.0-4.0 wt% sodium alginate solution at a ratio of 1 g: 3-5 mL to obtain a mixed slurry. Then, 2-5 wt% calcium chloride solution was added dropwise at a volume ratio of 1:3-5 between the mixed slurry and calcium chloride solution, and the mixture was cross-linked and cured for 15-30 min to form gel microspheres embedded with the composite bacterial micelle-biochar composite. Due to their larger volume and viscosity, the composite bacterial micelles can more effectively adsorb and fix themselves in the pores of the biochar. Step 3: Preparation of composite materials A nano-zero-valent iron suspension was prepared, and then the remaining pretreated biochar material was mixed with the nano-zero-valent iron suspension at a ratio of 8-12g:30mL. The mixture was then shaken for 4-6h under nitrogen protection and at 40-50rpm, and finally dried in a vacuum drying oven at 55-60℃ for 12-24h to obtain the biochar-nano-zero-valent iron composite material. Step 4: Preparation of composite bacterial micelles loaded with biochar-nano zero-valent iron microspheres The wet weight of the gel microspheres prepared in step 2 and the dry weight of the biochar-nano zero-valent iron composite material prepared in step 3 were mixed at a ratio of 1:1-3 to obtain composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres; dehydrated and granulated for later use. Biochar, as a carrier, can prevent the agglomeration of nano-zero ferric iron particles and improve their dispersibility and reactivity. Nano-zero ferric iron first decomposes HCH through chemical reduction and then is degraded by microorganisms.

[0009] Furthermore, in step 1, the raw material for the biochar material is any one of peanut shells, coconut shells, walnut shells, and sawdust; Note: Peanut shells, coconut shells, and walnut shells have good pore structure, moderate ash content, and balanced performance, and can be used to prepare biochar with the highest specific surface area, providing the most adsorption sites for HCH molecules; the large and medium pore structure formed by sawdust is conducive to microbial loading, with sufficient space, which is beneficial to material exchange and microbial community reproduction.

[0010] Further, in step 1, the acid solution is obtained by mixing hydrochloric acid solution and nitric acid solution in a volume ratio of 1:1; Note: Hydrochloric acid can efficiently remove carbonates and some metal oxides from ash and increase porosity, but it is less effective at introducing oxygen-containing functional groups. In addition to removing ash, nitric acid's strong oxidizing properties can significantly create a large number of oxygen-containing functional groups on the surface of biochar, resulting in a more comprehensive modification effect.

[0011] Furthermore, nano-zero valent iron was prepared by liquid-phase reduction method, with the following steps: 4.5-5.5 g of FeSO4·7H2O was weighed and dissolved in 150-200 mL of a mixed solution obtained by mixing anhydrous ethanol and ultrapure water in a volume ratio of 25-30:70-75 (water was added first to dissolve the solution before adding the ethanol solution). The mixture was stirred evenly with a glass rod, and the solution was transferred to a three-necked flask and stirred continuously at 300-400 rpm. Then, nitrogen gas was introduced into the three-necked flask. Next, weigh 0.9-1.1 g of NaBH4 and dissolve it in 45-55 mL of deoxygenated ultrapure water to obtain a NaBH4 solution. Add the NaBH4 solution dropwise to a three-necked flask at a rate of 1-2 drops / s using a separatory funnel until no obvious bubbles are generated. After the addition is complete, continue the reaction under nitrogen protection and stirring for 50-70 min, and let it stand for 15-20 min to obtain a suspension. The obtained suspension was vacuum filtered through a constant pressure filter, then washed 1-2 times with an 85-90% ethanol solution, and then washed 2-4 times with deoxygenated ultrapure water. The filter cake was then dried in an oven at 60-80℃ for 1.5-2.5 hours. The dried powder was taken out and ground in an anaerobic glove box and passed through a 200-300 mesh sieve. Finally, it was dispersed in deoxygenated ultrapure water under nitrogen protection and ultrasonically treated for 10-15 minutes at a power of 250-300W to obtain a nano zero-valent iron suspension. Note: The ethanol-water mixed solvent and anaerobic operation ensured that the generated nZVI particles were smaller, more uniformly distributed, and more reactive, avoiding rapid oxidation and passivation in the air. Low-temperature vacuum drying and anaerobic operation maximized the preservation of the zero-valent state and reactivity of nZVI, allowing it to maintain high activity when applied to the soil. When this highly active nZVI is combined with biochar and composite bacterial micelles, it forms a better synergistic degradation effect on HCH.

[0012] Furthermore, the nitrogen flow rate is 0.5-1.0 L / min, and the introduction time is 15-20 min; Note: The introduction of nitrogen gas, under the conditions described above, can effectively remove dissolved oxygen from the solution and create an anaerobic environment.

[0013] This invention also provides a method for remediating HCH contaminated soil, based on the above-mentioned remediation agent, comprising the following steps: S1, Soil Pretreatment Take HCH-contaminated soil after removing stones and plant debris, air dry or air dry naturally, and crush it through a 1-2 mm sieve; take 250-300g of the sieved soil sample and place it in a 500 mL black culture bottle, spray it with deionized water until the soil moisture content reaches 58-62% of the field saturation moisture content; place the culture bottle in a constant temperature incubator at 23-25℃ and culture it in the dark for 1 week. S2, Application of Repair Agent Add 0.5-2g of the HCH contaminated soil remediation agent to every 100g of soil, stir well, and place the treated culture bottle back into a constant temperature incubator at 20-25℃ for two months of light-protected culture to complete the remediation.

[0014] Furthermore, in S2, during the two-month light-protected cultivation process, the soil was weighed every 2-3 days, and sterile water was added to maintain the soil moisture content at 55-65% of the field saturation moisture content; the soil was aerated every 3-5 days. Note: Stable moisture content is a prerequisite for ensuring microbial activity, and aeration treatment can ensure the oxygen supply for aerobic microorganisms.

[0015] Compared with existing technologies, the beneficial effects of this invention are: (1) This invention first uses nano-zero-valent iron to reduce and dechlorinate, converting highly chlorinated HCH into low-chlorinated benzene compounds, and then uses microorganisms such as Sphingomonas to degrade the intermediate products, which greatly improves the remediation efficiency. At the same time, the Sphingomonas inside the compound bacterial floc first "disassembles" the HCH molecular ring, and then the strain of Pseudomonas putida opens the benzene ring, using benzene, toluene, phenols and other compounds as carbon and energy sources, and finally completely mineralizes them. The remediation agent prepared in the above manner can relieve pollution stress, stimulate plants to restore the activity of antioxidant enzymes, enable plants to better resist oxidative damage caused by HCH, thereby increasing plant biomass and facilitating ecological restoration.

[0016] (2) The composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres prepared by the present invention first anchor the composite bacterial flocs in the deeper pores of biochar, and fix the nano zero-valent iron on the surface of the gel microspheres containing the composite bacterial flocs-biochar complex. The biochar acts as a "scaffold" to disperse and fix the nZVI particles, prevent their aggregation, and maximize the exposure of active sites, thereby achieving long-term and targeted nZVI activity, enabling it to continuously treat pollutants. The process of HCH remediation is that it is first adsorbed and enriched by biochar, then reduced and dechlorinated by the nano zero-valent iron on the surface, and converted into easily degradable intermediate products, which are then captured by the internal composite bacterial flocs and completely mineralized. This effectively reduces the risk of toxicity of intermediate products to the soil ecosystem and the degrading bacteria themselves. Attached Figure Description

[0017] Figure 1 This is a comparison chart of the total HCH degradation rate and catalase activity in the soil of Examples 1-11, Control Group 1-2, and Blank Group of the present invention; Figure 2 This is a comparison chart of the total HCH degradation rate and catalase activity in the soil of Examples 1, 12-27 and Control Groups 3-4 of the present invention; Figure 3 This is a comparison chart of the total HCH degradation rate and catalase activity in the soil of Examples 1, 28-29 and Control Group 5 of the present invention. Detailed Implementation

[0018] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0019] Example 1: A soil remediation agent for HCH contaminated soil, wherein the remediation agent is a composite microbial floc supported on biochar-nano zero-valent iron microspheres with a particle size of 20-40 mesh; the composite microbial floc supported on biochar-nano zero-valent iron microspheres are composed of gel microspheres embedded with a composite microbial floc-biochar composite and biochar-nano zero-valent iron composite material in a weight ratio of 1:2; The preparation method of the compound bacterial micelle is as follows: Sphingomonas and Pseudomonas putida are inoculated into LB liquid medium and cultured with shaking at 29℃ and 165 rpm until the OD600 is 0.7. Then, the bacterial cells are collected by centrifugation at 7750 rpm for 10 min. Finally, the cells are washed three times with sterile basal salt medium to obtain pure Sphingomonas bacterial suspension and pure Pseudomonas putida bacterial suspension. Then, the bacterial cultures were inoculated into sterile basal salt medium at a volume ratio of 1:1 between pure Sphingomonas sphingosine monocytogenes and pure Pseudomonas putida. The carrier loaded with HCH was then added to the sterile basal salt medium and cultured with shaking at 130 rpm for 4 days. When dense bacterial aggregates were observed, a culture accounting for 8% of the total volume of bacterial aggregates and free bacteria was transferred to fresh sterile basal salt medium containing 75 mg / L HCH for subculture. This was the first generation. The subculture steps were repeated for 8 generations. The culture medium of the last generation was allowed to stand for 20 minutes, then centrifuged at 2500 rpm for 4 minutes and the composite bacterial flocs settled at the bottom were collected. The total inoculation amount of pure sphingomonas and pure pseudomonas putrefactive bacteria was 3% of the volume of sterile basal salt medium; the ratio of HCH-loaded carrier to sterile basal salt medium was 75 mg / L. The preparation method of the HCH-loaded carrier is as follows: First, HCH is dissolved in acetone at a ratio of 9mg:1mL to obtain an HCH-acetone solution. Then, the HCH-acetone solution is added dropwise to sterile diatomaceous earth at a ratio of 1.3mL:1g, stirred and mixed, and finally evaporated by ventilation at a temperature of 25℃ for 3h to obtain the HCH-loaded carrier. The preparation method of biochar-nano zero-valent iron microspheres supported by composite bacterial micelles is as follows: Step 1: Biochar Pretreatment The biochar material was pulverized and passed through an 80-200 mesh sieve. The pulverized biochar material was then soaked in a 1.1 mol / L acid solution at a solid-liquid ratio of 1 g: 9 mL and shaken at 25 °C and 135 rpm for 3 h. The acid solution was then discarded, and the biochar material was washed with deionized water until the pH of the washing solution was 7. Finally, the washed biochar material was dried in an oven at 103 °C and treated with nitrogen gas for 28 min to obtain the pretreated biochar material. Step 2: Preparation of gel microspheres The composite fungal flocs were mixed with 1 / 2 of the pretreated biochar material at a ratio of 1:2 (wet weight of fungal flocs to dry weight of biochar material). The mixture was placed in a shaker and incubated at 29°C and 90 rpm for 7 hours. Finally, it was placed in a 4°C refrigerator and allowed to stand for 18 hours to fix the mixture, resulting in biochar with composite fungal flocs loaded on the pores and surface. Biochar loaded with composite bacterial micelles was mixed with 3wt% sodium alginate solution at a ratio of 1g:4mL to obtain a mixed slurry; then, 3wt% calcium chloride solution was added dropwise at a volume ratio of 1:4 between the mixed slurry and calcium chloride solution, and cross-linked and cured for 22min to form gel microspheres embedded with composite bacterial micelle-biochar complex. Step 3: Preparation of composite materials A nano-zero-valent iron suspension was prepared, and then the remaining pretreated biochar material was mixed with the nano-zero-valent iron suspension at a ratio of 10g:30mL. The mixture was then shaken for 5h under nitrogen protection and at 45rpm, and finally dried in a vacuum drying oven at 58℃ for 18h to obtain the biochar-nano-zero-valent iron composite material. Step 4: Preparation of composite bacterial micelles loaded with biochar-nano zero-valent iron microspheres The wet weight of the gel microspheres prepared in step 2 and the dry weight of the biochar-nano zero-valent iron composite material prepared in step 3 were mixed at a ratio of 1:2 to obtain composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres; dehydrated and granulated for later use. In step 1, the raw material for biochar is peanut shells; In step 1, the acid solution is obtained by mixing hydrochloric acid solution and nitric acid solution in a volume ratio of 1:1; Nano-sized zero-valent iron was prepared by liquid-phase reduction method, and the steps are as follows: 5.0 g of FeSO4·7H2O was weighed and dissolved in 175 mL of a mixed solution obtained by mixing anhydrous ethanol and ultrapure water at a volume ratio of 28:72 (water was added first to dissolve the iron before adding the ethanol solution). The mixture was stirred evenly with a glass rod, and the solution was transferred to a three-necked flask and stirred continuously at 350 rpm. Nitrogen gas was then introduced into the three-necked flask at a flow rate of 0.8 L / min for 18 min. Next, weigh 1.0 g of NaBH4 and dissolve it in 50 mL of deoxygenated ultrapure water to obtain a NaBH4 solution. Add the NaBH4 solution dropwise to a three-necked flask at a rate of 2 drops / s using a separatory funnel until no obvious bubbles are generated. After the addition is complete, continue to react under nitrogen protection and stirring for 60 min, and let it stand for 18 min to obtain a suspension. The obtained suspension was vacuum filtered through a constant pressure filter at -0.08 MPa until the surface of the filter cake dried. It was then washed twice with 88% ethanol solution and three times with deoxygenated ultrapure water. The filter cake was then dried in an oven at 70°C for 25 hours. The dried powder was taken out and ground in an anaerobic glove box and passed through a 200-300 mesh sieve. Finally, it was dispersed in deoxygenated ultrapure water at a ratio of 1 g: 75 mL under nitrogen protection and ultrasonically treated for 13 minutes at a power of 275 W to obtain a nano-zero valent iron suspension. This embodiment also provides a method for remediating HCH contaminated soil, based on the above-mentioned remediation agent, including the following steps: S1, Soil Pretreatment Take HCH-contaminated soil after removing stones and plant debris, air dry or air dry naturally, and crush it through a 1-2 mm sieve; take 275 g of the sieved soil sample and place it in a 500 mL black culture bottle, spray it with deionized water until the soil moisture content reaches 60% of the field saturation moisture content; place the culture bottle in a constant temperature incubator at 24℃ and culture it in the dark for 1 week. S2, Application of Repair Agent Add 1.2g of remediation agent to every 100g of soil to HCH contaminated soil remediation agent, stir well, and place the treated culture bottle back into a 23℃ constant temperature incubator for 2 months of light-protected culture to complete the remediation; in S2, during the 2 months of light-protected culture, weigh the soil every 2 days and add sterile water to maintain the soil moisture content at 60% of the field saturation moisture content; aerate the soil every 4 days; Soil samples were collected uniformly after mixing and taken on days 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63. The cultured soil samples were harvested periodically, air-dried, ground through a 60-mesh sieve, and 1.0 g of soil sample was weighed and added to a 40 mL brown headspace vial. 5 mL of acetone and 5 mL of n-hexane were added, and the mixture was sonicated for 45 min. After centrifugation at 2000 pm for 5 min, the supernatant was purified using a solid-phase extraction column (the column was activated with chromatographically pure n-hexane before purification). The peak area was determined using gas chromatography (GC), and the corresponding concentration was calculated using a standard curve. The degradation rate (%) of total HCH in the soil was calculated according to the formula, and the average value was taken.

[0020] D=(Q 0 -Q t ) / Q t In the formula, Q t : Pesticide residue, mg / kg; Q0: Initial pesticide content, mg / kg; D: Degradation rate; The soil was air-dried and ground through a 60-mesh sieve. The soil enzyme activity was measured using a soil catalase kit before and after the addition of materials, and the catalase activity decline rate was calculated and averaged.

[0021] Example 2: Unlike Example 1, in the preparation method of the composite bacterial micelle, Sphingomonas and Pseudomonas putida were inoculated into LB liquid medium and cultured with shaking at 28°C and 150 rpm until the OD600 reached 0.6. Then, the bacterial cells were collected by centrifugation at 7500 rpm for 8 min. Finally, the cells were washed twice with sterile basal salt medium to obtain pure Sphingomonas bacterial suspension and pure Pseudomonas putida bacterial suspension.

[0022] Example 3: Unlike Example 1, in the preparation method of the composite bacterial micelle, Sphingomonas and Pseudomonas putida were inoculated into LB liquid medium and cultured with shaking at 30°C and 180 rpm until the OD600 was 0.8. Then, the cells were collected by centrifugation at 8000 rpm for 12 min. Finally, the cells were washed 4 times with sterile basal salt medium to obtain pure Sphingomonas bacterial suspension and pure Pseudomonas putida bacterial suspension.

[0023] Example 4: Unlike Example 1, the culture was shaken at 120 rpm for 3 days. When dense bacterial aggregates were observed, a portion of the culture, accounting for 5% of the total volume of bacterial aggregates and free bacteria, was transferred to a fresh sterile basal salt medium containing 50 mg / L HCH for subculture. This was the first generation. The subculture process was repeated for 5 generations.

[0024] Example 5: Unlike Example 1, the culture was shaken at 150 rpm for 5 days. When dense bacterial aggregates were observed, a culture comprising 10% of the total volume of bacterial aggregates and free bacteria was transferred to a fresh sterile basal salt medium containing 100 mg / L HCH for subculture. This was the first generation. The subculture process was repeated for 10 generations.

[0025] Example 6: Unlike Example 1, the culture medium of the last generation was allowed to stand for 10 minutes, then centrifuged at 2000 rpm for 3 minutes and the composite bacterial flocs that settled at the bottom were collected.

[0026] Example 7: Unlike Example 1, the culture medium of the last generation was allowed to stand for 30 minutes, then centrifuged at 3000 rpm for 5 minutes and the composite bacterial flocs that settled at the bottom were collected.

[0027] Example 8: Unlike Example 1, the total inoculation amount of pure Sphingomonas sphingosine monocytogenes and pure Pseudomonas putida is 1% of the volume of sterile basal salt medium; the ratio of HCH-loaded carrier to sterile basal salt medium is 50 mg / L.

[0028] Example 9: Unlike Example 1, the total inoculation amount of pure sphingomonas bacterial suspension and pure pseudomonas putrefactive bacteria suspension accounted for 5% of the volume of sterile basal salt medium; the ratio of HCH-loaded carrier to sterile basal salt medium was 100 mg / L.

[0029] Example 10: Unlike Example 1, the method for preparing the HCH-loaded carrier is as follows: First, HCH is dissolved in acetone at a ratio of 10mg:1mL to obtain an HCH-acetone solution. Then, the HCH-acetone solution is added dropwise to sterile diatomaceous earth at a ratio of 1.5mL:1g, stirred and mixed, and finally evaporated under ventilation at a temperature of 23°C for 4 hours to obtain the HCH-loaded carrier.

[0030] Example 11: Unlike Example 1, the preparation method of the HCH-loaded carrier is as follows: First, HCH is dissolved in acetone at a ratio of 8mg:1mL to obtain an HCH-acetone solution. Then, the HCH-acetone solution is added dropwise to sterile diatomaceous earth at a ratio of 1mL:1g, stirred and mixed, and finally evaporated under ventilation at a temperature of 27°C for 2 hours to obtain the HCH-loaded carrier.

[0031] Example 12: Unlike Example 1, in the preparation method of composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres, the biochar material was pulverized through an 80-200 mesh sieve, and the pulverized biochar material was soaked in a 1 mol / L acid solution at a solid-liquid ratio of 1 g: 8 mL, and shaken at 23°C and 120 rpm for 2 h; the acid solution was discarded, and the biochar material was washed with deionized water until the pH of the washing solution was 6.8; finally, the washed biochar material was placed in an oven at 100°C to dry, and nitrogen gas was introduced for 25 min to obtain the pretreated biochar material.

[0032] Example 13: Unlike Example 1, in the preparation method of composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres, the biochar material was pulverized through an 80-200 mesh sieve, and the pulverized biochar material was soaked in a 1.2 mol / L acid solution at a solid-liquid ratio of 1 g: 10 mL, and shaken at 27°C and 150 rpm for 4 h; the acid solution was discarded, and the biochar material was washed with deionized water until the pH of the washing solution was 7.2; finally, the washed biochar material was placed in an oven at 105°C to dry, and nitrogen gas was introduced for 30 min to obtain the pretreated biochar material.

[0033] Example 14: Unlike Example 1, the composite bacterial flocs and 1 / 4 of the pretreated biochar material were mixed at a ratio of 1:1 (wet weight of bacterial flocs to dry weight of biochar material). The mixture was placed in a shaker and incubated at 28°C and 80 rpm for 6 hours. Finally, it was placed in a 3°C refrigerator and allowed to stand for 12 hours to fix, resulting in biochar with composite bacterial flocs loaded on its pores and surface.

[0034] Example 15: Unlike Example 1, the composite bacterial flocs and 3 / 4 of the pretreated biochar material were mixed at a ratio of 1:3 (wet weight of bacterial flocs to dry weight of biochar material). The mixture was placed in a shaker and incubated at 30°C and 100 rpm for 8 hours. Finally, it was placed in a 4°C refrigerator and allowed to stand for 24 hours to fix, resulting in biochar with composite bacterial flocs loaded on its pores and surface.

[0035] Example 16: Unlike Example 1, biochar loaded with composite bacterial micelles was mixed with 2.0 wt% sodium alginate solution at a ratio of 1 g: 3 mL to obtain a mixed slurry; then, 2 wt% calcium chloride solution was added dropwise at a volume ratio of 1:3 between the mixed slurry and calcium chloride solution, and cross-linked and cured for 15 min to form gel microspheres embedded with composite bacterial micelle-biochar composite.

[0036] Example 17: Unlike Example 1, biochar loaded with composite bacterial micelles was mixed with 4.0 wt% sodium alginate solution at a ratio of 1 g: 5 mL to obtain a mixed slurry; then, 5 wt% calcium chloride solution was added dropwise at a volume ratio of 1:5 between the mixed slurry and calcium chloride solution, and cross-linked and cured for 30 min to form gel microspheres embedded with composite bacterial micelle-biochar composite.

[0037] Example 18: Unlike Example 1, a nano-zero-valent iron suspension was prepared, and then the remaining pretreated biochar material was mixed with the nano-zero-valent iron suspension at a ratio of 8g:30mL. The mixture was then shaken for 4h under nitrogen protection at 40rpm, and finally dried in a vacuum drying oven at 55℃ for 12h to obtain the biochar-nano-zero-valent iron composite material.

[0038] Example 19: Unlike Example 1, a nano-zero-valent iron suspension was prepared, and then the remaining pretreated biochar material was mixed with the nano-zero-valent iron suspension at a ratio of 12g:30mL. The mixture was then shaken for 6 hours under nitrogen protection at 50rpm, and finally dried in a vacuum drying oven at 60℃ for 24 hours to obtain the biochar-nano-zero-valent iron composite material.

[0039] Example 20: Unlike Example 1, the wet weight of the gel microspheres prepared in step 2 and the dry weight of the biochar-nano zero-valent iron composite material prepared in step 3 were mixed in a 1:1 ratio to obtain composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres.

[0040] Example 21: Unlike Example 1, the wet weight of the gel microspheres prepared in step 2 and the dry weight of the biochar-nano zero-valent iron composite material prepared in step 3 were mixed in a ratio of 1:3 to obtain composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres.

[0041] Example 22: Unlike Example 1, nano-zero valent iron was prepared by liquid-phase reduction method. The steps are as follows: 4.5 g of FeSO4·7H2O was weighed and dissolved in 150 mL of a mixed solution obtained by mixing anhydrous ethanol and ultrapure water in a volume ratio of 25:75. The mixture was stirred evenly with a glass rod, and the mixed solution was transferred to a three-necked flask and stirred continuously at 300 rpm. Then, nitrogen gas was introduced into the three-necked flask at a flow rate of 0.5 L / min for 15 min.

[0042] Example 23: Unlike Example 1, nano-zero valent iron was prepared by liquid-phase reduction method. The steps are as follows: 5.5 g of FeSO4·7H2O was weighed and dissolved in 200 mL of a mixed solution obtained by mixing anhydrous ethanol and ultrapure water in a volume ratio of 30:70. The mixture was stirred evenly with a glass rod and transferred to a three-necked flask. The mixture was stirred continuously at 400 rpm, and then nitrogen gas was introduced into the three-necked flask. The flow rate of nitrogen gas was 1.0 L / min and the introduction time was 20 min.

[0043] Example 24: Unlike Example 1, 0.9 g of NaBH4 was weighed and dissolved in 45 mL of deoxygenated ultrapure water to obtain a NaBH4 solution. The NaBH4 solution was added dropwise to a three-necked flask at a rate of 1 drop / s through a separatory funnel until no obvious bubbles were generated. After the addition was completed, the reaction was continued for 50 min under nitrogen protection and stirring, and then allowed to stand for 15 min to obtain a suspension.

[0044] Example 25: Unlike Example 1, 1.1 g of NaBH4 was weighed and dissolved in 55 mL of deoxygenated ultrapure water to obtain a NaBH4 solution. The NaBH4 solution was added dropwise to a three-necked flask at a rate of 2 drops / s through a separatory funnel until no obvious bubbles were generated. After the addition was completed, the reaction was continued for 70 min under nitrogen protection and stirring, and then allowed to stand for 20 min to obtain a suspension.

[0045] Example 26: Unlike Example 1, the filter cake was washed once with an 85% ethanol solution and then twice with deoxygenated ultrapure water. The filter cake was then dried in an oven at 60°C for 1.5 hours. The dried powder was then removed and ground in an anaerobic glove box and passed through a 200-300 mesh sieve. Finally, under nitrogen protection, the powder was dispersed in deoxygenated ultrapure water at a ratio of 1 g: 50 mL and ultrasonically treated for 10 minutes at a power of 250 W to obtain a nano-zero valent iron suspension.

[0046] Example 27: Unlike Example 1, the filter cake was washed twice with a 90% ethanol solution and then four times with deoxygenated ultrapure water. The filter cake was then dried in an oven at 80°C for 2.5 hours. The dried powder was then removed and ground in an anaerobic glove box and passed through a 200-300 mesh sieve. Finally, it was dispersed in deoxygenated ultrapure water at a ratio of 1 g: 100 mL under nitrogen protection and ultrasonically treated for 15 minutes at a power of 300 W to obtain a nano-zero valent iron suspension.

[0047] Example 28: Unlike Example 1, in the S2 of the remediation method, during the two-month light-protected culture, the soil was weighed every two days and sterile water was added to maintain the soil moisture content at 55% of the field saturation moisture content; the soil was aerated every three days.

[0048] Example 29: Unlike Example 1, in the S2 of the remediation method, during the two-month light-protected culture, the soil was weighed every three days and sterile water was added to maintain the soil moisture content at 65% of the field saturation moisture content; the soil was aerated every five days.

[0049] Experimental Example: The description of this experimental example is based on the scheme described in Example 1, and aims to illustrate the practical application effect of the present invention.

[0050] 1. Investigate the effects of remediation material composition on the degradation effect of HCH and catalase activity in soil: Blank group: Unlike Example 1, the repair agent was replaced with an equal amount of sterile water.

[0051] Control Group 1: Unlike Example 1, the compound bacterial suspension was replaced with Sphingomonas bacterial suspension. (The Sphingomonas strain was obtained from the Microbial Culture Collection Center. The preparation process of the bacterial suspension was as follows: 10.0g of peptone, 10.0g of sodium chloride, and 5.0g of yeast extract were weighed and dissolved in 1L of deionized water to prepare LB liquid medium. The medium was autoclaved before use. Single colonies were picked and inoculated into LB liquid medium and cultured at 30℃ and 250r / min in a shaker for about 24 hours. The bacterial suspension was collected by centrifugation, washed twice with sterile water, and then resuspended in sterile water. The OD600 of the obtained bacterial suspension was measured to be 0.7. It was then stored at 4℃ for subsequent use.) Control group 2: Unlike Example 1, the compound bacterial solution was not prepared in the form of micelles.

[0052] Conclusion: From Figure 1 It can be seen that the HCH pollution in the blank group was remediated by the soil's own weak degradation capacity and possible volatilization, resulting in a very poor remediation effect. The activity of catalase in the soil also decreased at a low rate due to continuous inhibition by HCH.

[0053] In the comparison of Examples 1, 2-3, and Control Groups 1 and 2, we found that although the single high-efficiency bacterial strain has a significant degradation ability, the survival rate and stability of the strain are limited when directly exposed to the soil environment, and it lacks the ability to completely degrade intermediate products. Therefore, the degradation rate does not reach an extremely high level, and its metabolic activities also have a certain impact on soil enzyme activity. In Control Group 2, the compound bacterial solution has complementary functions, and theoretically the degradation rate should be higher than that of Control Group 1. However, the free strain has a significant competitive disadvantage in the soil and is easy to be lost and die, which may result in the actual effect being slightly lower than that of the well-fixed single strain, and causing greater disturbance to the soil microenvironment.

[0054] 2. To investigate the effects of the preparation method of biochar-zero-valent iron nanospheres supported on composite bacterial micelles on the degradation of HCH and catalase activity in soil. Control group 3: Unlike Example 1, the composite bacterial flocs and nano-zero valent iron were directly mixed and loaded onto the surface of the biochar material.

[0055] Control group 4: Unlike Example 1, the nano-zero ferric bacterial suspension was obtained by directly dispersing commercially available nZVI powder in deoxygenated ultrapure water under nitrogen protection and then ultrasonically dispersing it evenly.

[0056] Conclusion: From Figure 2 As can be seen from the comparison of Examples 1, Examples 12-27, and Control Group 3, the high reactivity of nZVI in the synchronous mixing in Control Group 3 directly toxicizes the composite bacterial micelles, inhibiting their activity and even causing their death. Furthermore, it cannot form a "chemical-biological" degradation pipeline, resulting in low reaction efficiency. Simultaneously, due to the lack of stable dispersion by biochar, nZVI rapidly aggregates and passivates, severely weakening both chemical and biological degradation capabilities, eliminating the synergistic effect, and significantly reducing overall efficiency. Therefore, it can be concluded that the "spatial hierarchical structure" of this application is essential for avoiding mutual inhibition of functional components and achieving efficient synergy. In Control Group 4, commercially available nZVI powder typically undergoes partial oxidation and aggregation, resulting in lower reactivity than the highly active nZVI suspension. Even after ultrasonication, it is difficult to achieve the dispersion and binding strength of nanoparticles prepared by the liquid-phase reduction method, thus reducing the efficiency of the chemical reduction initiation step. Although the biodegradation portion is unaffected, the overall repair efficiency is still lower than that of the examples using highly active nZVI.

[0057] 3. Investigate the effects of remediation methods on the degradation of HCH and catalase activity in soil. Control group 5: Unlike Example 1, no intervention was made on soil moisture content and aeration.

[0058] Conclusion: From Figure 3Comparing Examples 1, 28-29, and Control Group 5, it can be seen that the 60% saturated water content set in this application provides optimal conditions for microbial life activities; regular aeration ensures sufficient oxygen supply for the aerobic degradation process; while in Control Group 5, soil moisture evaporates naturally, leading to dryness, and microorganisms enter a dormant or dead state due to water loss, with a sharp decrease in activity. In the sealed bottle, aerobic microorganisms rapidly consume the limited oxygen, forming an anaerobic environment. The complex bacterial flocs, mainly composed of aerobic Sphingomonas and Pseudomonas putida, have extremely low degradation activity or even cannot survive under anaerobic conditions, causing the entire remediation system to fail. The chemical reduction of nZVI also cannot work efficiently due to the lack of subsequent biodegradation.

[0059] In summary, the remediation agent prepared in this application has a significant effect on HCH pollution.

Claims

1. A soil remediation agent for HCH contaminated soil, characterized in that, The repair agent is a composite fungal floc supported on biochar-nano zero-valent iron microspheres with a particle size of 20-40 mesh; the composite fungal floc supported on biochar-nano zero-valent iron microspheres are composed of gel microspheres embedded with composite fungal floc-biochar composite and biochar-nano zero-valent iron composite material in a weight ratio of 1:1-3. The method for preparing the composite bacterial micelles is as follows: Sphingomonas and Pseudomonas putida are inoculated into LB liquid medium and cultured with shaking at 28-30℃ and 150-180 rpm until the OD600 is 0.6-0.

8. Then, the bacterial cells are collected by centrifugation at 7500-8000 rpm for 8-12 min. Finally, the cells are washed 2-4 times with sterile basal salt medium to obtain pure Sphingomonas bacterial suspension and pure Pseudomonas putida bacterial suspension. Then, according to the volume ratio of pure Sphingomonas sphingosine monocytogenes bacterial suspension and pure Pseudomonas putida bacterial suspension, the bacterial suspensions were inoculated into sterile basal salt medium at 1:

1. Then, the carrier loaded with HCH was added to the sterile basal salt medium and cultured with shaking at 120-150 rpm for 3-5 days. When dense bacterial aggregates were observed, 5-10% of the total volume of bacterial aggregates and free bacteria was transferred to fresh sterile basal salt medium containing 50-100 mg / L HCH for subculture. This is the first generation. Repeat the subculture steps for 5-10 generations. Let the culture medium of the last generation stand for 10-30 minutes, then centrifuge at low speed of 2000-3000 rpm for 3-5 minutes and collect the composite bacterial flocs that settle at the bottom. The total inoculation amount of pure sphingomonas and pure pseudomonas putrefactive bacteria was 1-5% of the volume of sterile basal salt medium; the ratio of HCH-loaded carrier to sterile basal salt medium was 50-100 mg / L.

2. The HCH contaminated soil remediation agent as described in claim 1, characterized in that, The method for preparing the HCH-loaded carrier is as follows: First, HCH is dissolved in acetone at a ratio of 8-10 mg: 1 mL to obtain an HCH-acetone solution. Then, the HCH-acetone solution is added dropwise to sterile diatomaceous earth at a ratio of 1-1.5 mL: 1 g, stirred and mixed, and finally evaporated under ventilation at a temperature of 23-27℃ for 2-4 hours to obtain the HCH-loaded carrier.

3. The HCH contaminated soil remediation agent as described in claim 1, characterized in that, The preparation method of the composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres is as follows: Step 1: Biochar Pretreatment The biochar material was pulverized and passed through an 80-200 mesh sieve. The pulverized biochar material was then soaked in an acid solution of 1-1.2 mol / L at a solid-liquid ratio of 1 g: 8-10 mL and shaken at 23-27℃ and 120-150 rpm for 2-4 hours. The acid solution was then discarded, and the biochar material was washed with deionized water until the pH of the washing solution was 6.8-7.

2. Finally, the washed biochar material was dried in an oven at 100-105℃ and treated with nitrogen gas for 25-30 minutes to obtain the pretreated biochar material. Step 2: Preparation of gel microspheres The composite fungal flocs were mixed with 1 / 4 to 3 / 4 of the pretreated biochar material at a ratio of 1:1-3 (wet weight of fungal flocs to dry weight of biochar material). The mixture was placed in a shaker and incubated at 28-30°C and 80-100 rpm for 6-8 hours. Finally, it was placed in a refrigerator at 3-4°C and allowed to stand for 12-24 hours to fix the mixture, resulting in biochar with composite fungal flocs loaded on its pores and surface. The biochar loaded with composite bacterial micelles was mixed with 2.0-4.0 wt% sodium alginate solution at a ratio of 1 g: 3-5 mL to obtain a mixed slurry; then, 2-5 wt% calcium chloride solution was added dropwise at a volume ratio of 1:3-5 between the mixed slurry and calcium chloride solution, and the mixture was cross-linked and cured for 15-30 min to form gel microspheres embedded with the composite bacterial micelle-biochar complex. Step 3: Preparation of composite materials A nano-zero-valent iron suspension was prepared, and then the remaining pretreated biochar material was mixed with the nano-zero-valent iron suspension at a ratio of 8-12g:30mL. The mixture was then shaken for 4-6h under nitrogen protection and at 40-50rpm, and finally dried in a vacuum drying oven at 55-60℃ for 12-24h to obtain the biochar-nano-zero-valent iron composite material. Step 4: Preparation of composite bacterial micelles loaded with biochar-nano zero-valent iron microspheres The wet weight of the gel microspheres prepared in step 2 and the dry weight of the biochar-nano zero-valent iron composite material prepared in step 3 were mixed at a ratio of 1:1-3 to obtain composite bacterial flocs loaded with biochar-nano zero-valent iron microspheres.

4. The HCH contaminated soil remediation agent as described in claim 3, characterized in that, In step 1, the raw material for the biochar material is any one of peanut shells, coconut shells, walnut shells, and wood chips.

5. The HCH contaminated soil remediation agent as described in claim 3, characterized in that, In step 1, the acid solution is obtained by mixing hydrochloric acid solution and nitric acid solution in a volume ratio of 1:

1.

6. The HCH contaminated soil remediation agent as described in claim 3, characterized in that, Nano-zero valent iron was prepared by liquid-phase reduction method, and the steps are as follows: 4.5-5.5 g of FeSO4·7H2O was weighed and dissolved in 150-200 mL of a mixed solution obtained by mixing anhydrous ethanol and ultrapure water in a volume ratio of 25-30:70-75. The mixture was stirred evenly with a glass rod, and the mixed solution was transferred to a three-necked flask and stirred continuously at 300-400 rpm. Then, nitrogen gas was introduced into the three-necked flask. Next, weigh 0.9-1.1 g of NaBH4 and dissolve it in 45-55 mL of deoxygenated ultrapure water to obtain a NaBH4 solution. Add the NaBH4 solution dropwise to a three-necked flask at a rate of 1-2 drops / s using a separatory funnel until no obvious bubbles are generated. After the addition is complete, continue the reaction under nitrogen protection and stirring for 50-70 min, and let it stand for 15-20 min to obtain a suspension. The obtained suspension was vacuum filtered at -0.08 MPa, then washed 1-2 times with 85-90% ethanol solution, and then washed 2-4 times with deoxygenated ultrapure water. The filter cake was then dried in an oven at 60-80℃ for 1.5-2.5 hours. The dried powder was taken out and ground in an anaerobic glove box and passed through a 200-300 mesh sieve. Finally, under nitrogen protection, it was dispersed in deoxygenated ultrapure water at a ratio of 1g:50-100mL and ultrasonically treated for 10-15 minutes at a power of 250-300W to obtain a nano-zero valent iron suspension.

7. The HCH contaminated soil remediation agent as described in claim 6, characterized in that, Nitrogen gas is introduced into a three-necked flask at a flow rate of 0.5-1.0 L / min for 15-20 min.

8. A method for remediating HCH contaminated soil, using the remediation agent according to any one of claims 1-7, characterized in that, Includes the following steps: S1, Soil Pretreatment Take HCH-contaminated soil after removing stones and plant debris, air dry or air dry naturally, and crush it through a 1-2 mm sieve; take 250-300g of the sieved soil sample and place it in a 500 mL black culture bottle, spray it with deionized water until the soil moisture content reaches 58-62% of the field saturation moisture content; place the culture bottle in a constant temperature incubator at 23-25℃ and culture it in the dark for 1 week. S2, Application of Repair Agent Add 0.5-2g of the HCH contaminated soil remediation agent to every 100g of soil, stir well, and place the treated culture bottle back into a constant temperature incubator at 20-25℃ for two months of light-protected culture to complete the remediation.

9. A method for remediating HCH contaminated soil as described in claim 8, characterized in that, In S2, during the two-month light-protected cultivation process, the soil was weighed every 2-3 days, and sterile water was added to maintain the soil moisture content at 55-65% of the field saturation moisture content; the soil was aerated every 3-5 days.

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