Methods and microbial agents for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste

Abstract

This invention discloses a method and microbial agents for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste, belonging to the field of chromium pollution control technology. The method includes: uniformly mixing livestock and poultry manure and agricultural and forestry waste, adding a No. 1 microbial agent for aerobic composting; after composting, mixing the compost product with the chromium-contaminated soil, adding a No. 2 microbial agent and sodium lactate for remediation, and then proceeding until remediation is complete. Both the No. 1 and No. 2 microbial agents used in this invention are composed of multiple microorganisms, and the No. 2 agent loads suitable microbial strains onto biochar to form small spherical microbial agents, which can effectively reduce and fix chromium, ensuring the long-term safety of the remediated soil. The microbial agents used have advantages such as high efficiency, environmental friendliness, and readily available materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chromium pollution treatment, and particularly relates to a method for repairing chromium-polluted soil by using livestock and poultry manure and agricultural and forestry waste and a microbial agent. BACKGROUND

[0002] At present, the repair methods for chromium-polluted sites mainly include physical repair, biological repair and chemical repair. The physical repair includes soil replacement, and the more soil to be replaced, the more obvious the repair effect is. However, this method needs to spend a large amount of manpower and financial resources in the actual application process, and is only suitable for small areas with serious pollution. The chemical repair includes solidification / stabilization, chemical reduction, chemical leaching and electrokinetic repair. The chemical method has the characteristics of wide application range, low repair cost and short repair time, but the repaired site is prone to secondary pollution risk of re-oxidation with the extension of time. The biological repair includes plant repair, microbial repair and microbial-plant combined repair method, which mainly uses microorganisms and plants to remove hexavalent chromium in the environment through processes such as adsorption, extraction, degradation and fixation of heavy metals. However, the repair period is relatively long.

[0003] The existing repair technologies mainly include chemical-biological combined use, such as CN202410316068.4 which discloses a method for repairing high-concentration hexavalent chromium-polluted soil by chemical-biological combination; electrokinetic-microbial combined use, such as CN202311142100.3 which discloses a method for repairing chromium-polluted soil by fixed Pseudomonas veronii combined with electro-osmosis; and plant-microbial combined use, such as CN202311047257.8 which discloses a method for in-situ repair of heavy metal-polluted soil by improved agent-plant-microbial combination. Although the combined use technology can reduce the defects of single technology to some extent, the defects still cannot be completely eliminated. The microbial repair technology has the advantages of environmental protection and economy, but it also has the disadvantages of long repair period and unstable microbial activity. How to shorten the microbial repair period and improve the stability of microorganisms in the soil system at low cost and in an environmentally friendly manner is of great significance to the wide use of microbial repair technology, and also attracts widespread attention in the repair of hexavalent chromium-polluted soil.

[0004] The existing technologies for improving the stability of microorganisms in the soil system by using biochar to load microorganisms and the repair technology for adding organic wastewater containing organic reducing substances to the soil can reduce hexavalent chromium while providing nutrients for microorganisms to accelerate microbial growth and shorten the repair period. For example, CN202310223746.8 discloses a repair agent for hexavalent chromium-polluted soil treatment by using organic wastewater, and CN202310819400.4 discloses a method for repairing heavy metal chromium-polluted soil in a mining area by using microbial-biochar combination. However, the existing biochar loading microbial technology mainly loads single functional microorganisms, and the single strain has weak anti-interference ability in the complex soil system. SUMMARY

[0005] The application adopts biochar loaded with various chromium-reducing functional microorganisms, which is easy to obtain, and has stronger adaptability, and uses the product obtained by composting livestock and poultry manure and agricultural and forestry waste to replace organic wastewater, thereby avoiding the use of organic wastewater with complex components and the risk of permeating underground water, and achieving the resource utilization of solid waste.

[0006] The application first provides a method for repairing chromium-contaminated soil by using livestock and poultry manure and agricultural and forestry waste, which comprises the following steps:

[0007] A, preparation of No. 1 microbial agent: Bacillus subtilis BNCC188080, Bacillus licheniformis BNCC221464, Phanerochaete chrysosporium BNCC190652, Cellulomonas umida BNCC336467 and Bacillus amyloliquefaciens BNCC132483 are respectively cultured, and the optical density value of each bacterial solution at 600 nm wavelength is 0.8-1.2, five kinds of bacterial solutions are obtained, and the volume ratio of Bacillus subtilis: Bacillus licheniformis: Cellulomonas umida: Bacillus amyloliquefaciens: Phanerochaete chrysosporium is 3±0.1: 3±0.1: 2±0.1: 1±0.05: 1±0.05, and the mixture is uniformly mixed to obtain No. 1 microbial agent, and then the sterile physiological saline is diluted by 8-10 times for standby;

[0008] B, preparation of compost product: livestock and poultry manure and agricultural and forestry waste are fully mixed at a mass ratio of 1.8-2.2:1, the diluted No. 1 microbial agent is added during the mixing process, and water is uniformly sprayed, a pile is prepared (the size of the pile can be determined according to the required amount of compost product), and the water content of the pile is controlled at 55%-60%; then, aerobic fermentation is carried out, the pile is turned over at an appropriate frequency (generally once every 7 days) during the fermentation process, and water is appropriately sprayed to ensure that the water content is 55%-60%, after the composting is completed, the pile is crushed to obtain the compost product;

[0009] C. Preparation of Microbial Agent No. 2: Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, and Pseudomonas stutzeri LS-2 were cultured separately until the absorbance of each bacterial solution at a wavelength of 600 nm was 1.0–1.2, resulting in three bacterial solutions. These solutions were then mixed thoroughly at a volume ratio of 2±0.1:1±0.05:1±0.05 to obtain a mixed bacterial solution. The mixed bacterial solution and biochar were then used to prepare microbial pellets, thus obtaining Microbial Agent No. 2.

[0010] D. Soil remediation for hexavalent chromium contaminated soil: Mix the compost product obtained in step B with the soil to be remediated at a ratio of 8% to 12% by mass. Add 0.5% to 1% of the No. 2 microbial agent and 1% to 2% of sodium lactate by mass of the soil to be remediated. Soil remediation can be carried out after the soil remediation is completed.

[0011] In the above-mentioned method for remediating chromium-contaminated soil, in step A, the culture medium for Bacillus subtilis, Bacillus licheniformis, Moistened Fibromonas hydrophila, and Bacillus amyloliquefaciens is: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH value: 7.3 ± 0.1.

[0012] In the above-mentioned method for remediating chromium-contaminated soil, in step A, the culture conditions for Bacillus subtilis, Bacillus licheniformis, and Moist Cellomonas are as follows: cultured at 30±1℃ on a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 0.8-1.2 (generally cultured for 18-24 hours).

[0013] In the above-mentioned method for remediating chromium-contaminated soil, in step A, the culture conditions for Bacillus amyloliquefaciens are as follows: cultured at 37±1℃ on a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 0.8-1.2 (generally cultured for 18-24 hours).

[0014] In the above-mentioned method for remediating chromium-contaminated soil, in step A, the culture medium for *Phanerochaete chrysosporium* is as follows: potato extract powder: 10.0 g / L, glucose: 20.0 g / L, KH2PO4: 3.0 g / L, MgSO4·7H2O: 1.5 g / L, thiamine: 0.008 g / L, pH: 6.0 ± 0.2.

[0015] In the above-mentioned method for remediating chromium-contaminated soil, in step A, the culture conditions for *Phanerochaete chrysosporium* are as follows: cultured at 30±1℃ on a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 0.8-1.2 (generally cultured for 3-5 days).

[0016] In the above-mentioned method for remediating chromium-contaminated soil, in step B, the livestock and poultry manure is fresh pig manure or fresh cow manure (the moisture content of fresh livestock and poultry manure is generally 60% to 70%).

[0017] In the above-mentioned method for remediating chromium-contaminated soil, in step B, the agricultural and forestry waste is corn stalks, wheat stalks, or rice stalks (the agricultural and forestry waste is naturally air-dried and crushed stalks, with a moisture content of generally 5% to 12%, crushed into uniform small segments of 10 to 50 mm).

[0018] In the above-mentioned method for remediating chromium-contaminated soil, in step B, the amount of diluted No. 1 microbial agent added is 0.2-0.3 L / kg of the total mass of livestock and poultry manure and agricultural and forestry waste.

[0019] In the above-mentioned method for remediating chromium-contaminated soil, step B involves aerobic fermentation lasting 30–35 days.

[0020] In the above-mentioned method for remediating chromium-contaminated soil, step B yields a compost product with a particle size of 1–5 mm.

[0021] In the above-mentioned method for remediating chromium-contaminated soil, in step C, the culture medium for Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, and Pseudomonas stutzeri LS-2 is: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH value: 7.3±0.1.

[0022] In the above-mentioned method for remediating chromium-contaminated soil, in step C, the culture conditions for Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, and Pseudomonas stutzeri LS-2 are as follows: cultured at 25-28℃ on a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 1.0-1.2 (generally cultured for 8-14 hours).

[0023] In the above-mentioned method for remediating chromium-contaminated soil, step C, the specific steps for preparing microbial pellets from the mixed bacterial solution and biochar are as follows: after centrifuging the mixed bacterial solution and discarding the supernatant, resuspend it in sterile physiological saline and centrifuge again, repeating this process 2-3 times (the total number of operations after resuspending in sterile physiological saline and centrifuging is 3-4 times); then, the bacterial solution concentration is adjusted to OD using sterile physiological saline. 600 Add 1.0–1.2 g / L of biochar, shake, then add 0.2–0.3 g / L of sterile sodium alginate and 0.5–0.8 g / L of sterile polyvinyl alcohol. After stirring and standing, drop the bacterial solution into a sterile saturated calcium borate solution to form a stable microsphere structure. The microspheres are then subjected to freezing, filtration, washing, and vacuum freeze-drying to obtain microbial spheres (which can be stored at 4°C for later use).

[0024] In the above-mentioned method for remediating chromium-contaminated soil, when preparing microbial microspheres, the mixed bacterial solution is centrifuged at 6000-8000 rpm for 5-10 minutes.

[0025] In the above-mentioned method for remediating chromium-contaminated soil, when preparing microbial microspheres, the volume ratio of sterile physiological saline to mixed bacterial solution is 2-5:8 each time the microspheres are resuspended.

[0026] In the above-mentioned method for remediating chromium-contaminated soil, the shaking conditions for preparing microbial pellets are 150-180 rpm at 28-30℃ for 1-2 hours.

[0027] In the above-mentioned methods for remediating chromium-contaminated soil, the settling time for preparing microbial pellets is 30–60 min;

[0028] In the above-mentioned method for remediating chromium-contaminated soil, when preparing microbial microspheres, the freezing and settling conditions are 0-5℃ for 24-36 hours.

[0029] In the above-mentioned method for remediating chromium-contaminated soil, in step C, the biochar is prepared by the following method: after drying and pulverizing biomass, it is heated to 400-700°C at a heating rate of 10-30°C / min under an inert atmosphere and held at that temperature for 1-4 hours to obtain carbonized material; the carbonized material and activator are mixed evenly at a mass ratio of 1:1-3, and then activated at 400-700°C at a heating rate of 10-30°C / min under an inert atmosphere and held at that temperature for 1-4 hours to obtain biochar.

[0030] In the above-mentioned method for remediating chromium-contaminated soil, step D involves a soil remediation period of 30 to 40 days, during which the soil is watered daily to maintain a moisture content of 55% to 60%, and the soil is tilled every two days.

[0031] The present invention also provides a microbial agent for remediating chromium-contaminated soil, which is composed of microbial agent No. 1 and microbial agent No. 2 in a volume-to-mass ratio of 4-6 mL: 5 g;

[0032] The No. 1 microbial inoculant was prepared by the following method: *Bacillus subtilis* BNCC188080, *Bacillus licheniformis* BNCC221464, *Phanerochaete chrysosporium* BNCC190652, *Cellulosum humicum* BNCC336467, and *Bacillus amyloliquefaciens* BNCC132483 were cultured until the absorbance of each bacterial solution at a wavelength of 600 nm was 0.8–1.2, resulting in five bacterial solutions. These solutions were then mixed evenly in a volume ratio of *Bacillus subtilis*: *Bacillus licheniformis*: *Cellulosum humicum*: *Bacillus amyloliquefaciens*: *Phanerochaete chrysosporium* of 3±0.1:3±0.1:2±0.1:1±0.05:1±0.05.

[0033] The No. 2 microbial inoculant was prepared by the following method: Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, and Pseudomonas stutzeri LS-2 were cultured separately until the absorbance of each bacterial solution at a wavelength of 600 nm was 1.0 to 1.2, resulting in three bacterial solutions. These solutions were then mixed thoroughly at a volume ratio of 2±0.1:1±0.05:1±0.05 to obtain a mixed bacterial solution. The mixed bacterial solution and biochar were then used to prepare microbial pellets.

[0034] Among the microbial agents used for remediating chromium-contaminated soil, the culture medium for Bacillus subtilis, Bacillus licheniformis, Moistened Fibromonas hydrophila, and Bacillus amyloliquefaciens in the preparation of microbial agent No. 1 is: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH value: 7.3±0.1.

[0035] Among the microbial agents used for remediating chromium-contaminated soil, the culture conditions for Bacillus subtilis, Bacillus licheniformis, and Moist Cellomonas hydrophila in the preparation of microbial agent No. 1 are as follows: cultured at 30±1℃ in a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600nm is 0.8-1.2 (generally cultured for 18-24h).

[0036] Among the microbial agents used for remediating chromium-contaminated soil, the culture conditions for Bacillus amyloliquefaciens in the preparation of agent No. 1 are as follows: cultured at 37±1℃ in a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600nm is 0.8-1.2 (generally cultured for 18-24 hours).

[0037] Among the microbial agents used for remediating chromium-contaminated soil, the culture medium for preparing Microbial Agent No. 1 was as follows: potato extract powder: 10.0 g / L, glucose: 20.0 g / L, KH2PO4: 3.0 g / L, MgSO4·7H2O: 1.5 g / L, thiamine: 0.008 g / L, pH: 6.0 ± 0.2.

[0038] Among the microbial agents used for remediating chromium-contaminated soil, the culture conditions for preparing Microbial Agent No. 1 are as follows: cultured at 30±1℃ in a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600nm is 0.8-1.2 (generally cultured for 3-5 days).

[0039] Among the microbial agents used for remediating chromium-contaminated soil, the culture medium for preparing microbial agent No. 2 for Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, and Pseudomonasstutzeri LS-2 was: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.3 ± 0.1;

[0040] Among the microbial agents used for remediating chromium-contaminated soil, the culture conditions for preparing microbial agent No. 2, namely Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32 and Pseudomonasstutzeri LS-2, are as follows: cultured at 25-28℃ in a shaker at 150-180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 1.0-1.2 (generally cultured for 8-14 hours);

[0041] Among the aforementioned microbial agents for remediating chromium-contaminated soil, the specific steps for preparing microbial agents No. 2, specifically the preparation of microbial microspheres from the mixed bacterial solution and biochar, are as follows: After centrifuging the mixed bacterial solution and discarding the supernatant, resuspend it in sterile physiological saline and centrifuge again, repeating this process 2-3 times (the total number of resuspending and centrifuging operations is 3-4 times); then, adjust the bacterial solution concentration to OD using sterile physiological saline. 600Add 1.0–1.2 g / L of biochar, shake, then add 0.2–0.3 g / L of sterile sodium alginate and 0.5–0.8 g / L of sterile polyvinyl alcohol. After stirring and standing, drop the bacterial solution into a sterile saturated calcium borate solution to form a stable microsphere structure. The microspheres are then subjected to freezing, filtration, washing, and vacuum freeze-drying to obtain microbial spheres (which can be stored at 4°C for later use).

[0042] In this invention, the microbial strains used in Agent No. 1 are all commercially available, including Bacillus subtilis BNCC188080, Bacillus licheniformis BNCC221464, Phanerochaete chrysospora BNCC190652, Moistened Fibromonas hydrophila BNCC336467, and Bacillus amyloliquefaciens BNCC132483, all purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0043] In this invention, the microbial strains used in inoculant No. 2 were all commercially available. *Shewanella oneidensis* MR-1 and *Shewanella putrefaciens* CN-32 were purchased from the China Center for Type Culture Collection (CCTCC), with numbers CCTCC AB 2013238 and CCTCC AB 2013239, respectively. *Pseudomonas stutzeri* LS-2 was purchased from the China General Microbiological Culture Collection Center, with number CGMCC No. 11556. *Pseudomonas stutzeri* LS-2 can also be referenced in the following paper: Li,S.,Li,X.&Li,F.Fe(II)oxidation and nitrate reduction by adenitrifying bacterium, *Pseudomonas stutzeri* LS-2, isolated from paddy soil. *JSoils Sediments* 18, 1668–1678 (2018).

[0044] In this invention, biochar can be a commercially available product or homemade. When homemade, common biomass in the field can be used, such as mango kernels, peanut shells, coconut shells, walnut shells, etc. Considering that Panzhihua is a major producer of mangoes, this embodiment of the invention uses mango kernels as raw material to prepare biochar.

[0045] When preparing biochar, the specific surface area can be effectively increased and the repair effect can be improved by adding an activator. Common activators in the field, such as KOH, NaOH, water vapor, hydrochloric acid, sulfuric acid, etc., are all suitable for this invention.

[0046] In this invention, the mass concentration of sterile saline is generally 0.85%.

[0047] In this invention, when preparing microbial agent No. 2, in the amounts of 1.0–1.5 g / L biochar, 0.2–0.3 g / L sterile sodium alginate, and 0.5–0.8 g / L sterile polyvinyl alcohol, "L" represents the amount used to adjust OD. 600 The bacterial culture volume is 1.0 to 1.2.

[0048] The beneficial effects of this invention are:

[0049] This invention utilizes readily available biochar loaded with various chromium-reducing microorganisms. The resulting biochar-loaded functional microbial pellets are convenient to use and exhibit stronger adaptability in soil systems. This invention adds the product obtained from composting No. 1 microbial agent, livestock and poultry manure, and agricultural and forestry waste to contaminated soil. The humus in the compost product acts as a shuttle, accelerating the chromium reduction by the functional microorganisms in No. 2 microbial agent. Simultaneously, it provides a carbon source for the growth and reproduction of microorganisms. The use of compost is less likely to cause secondary pollution and achieves resource utilization of solid waste.

[0050] The No. 1 and No. 2 microbial agents used in this invention are both composed of multiple microorganisms. The No. 2 agent is used after solidification treatment, that is, it is loaded onto biochar prepared from mango kernels and immobilized with sodium alginate gel to form small spherical microbial agents, which can more effectively reduce and fix chromium, ensuring long-term safety after soil remediation. The microbial agents used have the advantages of high efficiency, green environmental protection and readily available materials.

[0051] The *Pseudomonas stutzeri* LS-2 of this invention has the accession number CGMCC No. 11556. The accession date was November 2, 2015. The accession center is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is *Pseudomonas stutzeri* LS-2. Attached Figure Description

[0052] Figure 1 The graph shows the results of Cr(VI) reduction by different bacterial concentrations of Shewanella oneidensis MR-1.

[0053] Figure 2 The graph shows the results of Cr(VI) reduction by different bacterial concentrations of Shewanella putrefaciens CN-32.

[0054] Figure 3 The graph shows the results of Cr(VI) reduction by different bacterial concentrations of Pseudomonas stutzeri LS-2.

[0055] Figure 4 The microbial spheres prepared in Example 4 of this invention. Detailed Implementation

[0056] Specifically, the No. 2 microbial agent of this invention is first prepared by mixing various bacterial solutions in the following volume ratio: the volume ratio of Shewanella oneidensis MR-1: Shewanella putrefaciens CN-32: Pseudomonas stutzeri LS-2 is 2±0.1:1±0.05:1±0.05; the culture medium for the three bacteria is: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH value: 7.3±0.1; and cultured under aerobic conditions at 25-28℃ and 150-180 rpm on a shaker until the absorbance of the bacterial solution at a wavelength of 600 nm is 1.0-1.2 (generally cultured for 8-14 h), and then mixed according to the aforementioned volume ratio;

[0057] After mixing, aliquot the bacterial suspension into sterile centrifuge tubes (800 mL / tube), centrifuge at 6000–8000 rpm for 5–10 min, discard the supernatant, add 200–500 mL of 0.85% sterile physiological saline to each tube to resuspend, and centrifuge again. Repeat 2–3 times. Then adjust the concentration of the mixed bacterial suspension to OD using 0.85% sterile physiological saline. 600 The concentration of the solution was 1.0–1.2. 1.0–1.5 g / L of biochar was added, and the mixture was shaken at 150–180 rpm for 1–2 hours at 28–30°C. Then, 0.2–0.3 g / L of sterile sodium alginate and 0.5–0.8 g / L of sterile polyvinyl alcohol were added, and the mixture was stirred thoroughly. After standing for 30–60 minutes, the mixture was slowly dripped into a pre-prepared sterile saturated calcium borate solution using a sterile syringe to form stable microspheres. These microspheres were then allowed to stand in a 0–5°C refrigerator for 24–36 hours before filtration. The microspheres were then washed 2–3 times with 0.85% physiological saline and finally dried in a vacuum freeze dryer for 18–24 hours to obtain microbial spheres, which became microbial agent No. 2. This agent was stored at 4°C for later use.

[0058] Specifically, the biochar of this invention can be prepared using the following methods:

[0059] Mango pits are rinsed with deionized water to remove surface impurities and then dried at 80–105°C for 12–24 hours. Next, they are pulverized and sieved through a 30–200 mesh screen. 10.0g of mango pit powder is weighed and placed in a quartz reactor. To remove residual air from the quartz tube, nitrogen (N2) is continuously purged at a stable flow rate of 200–400 mL / min for 10 minutes (the exact purging time depends on the size of the pyrolysis tube). (To ensure the exhaust of residual air); the termination temperature of the tubular furnace pyrolysis reaction is set at 400-700℃, the heating rate is 10-30℃ / min, and after reaching the final decomposition temperature, it is kept constant for 1-4 hours. During the carbonization process, the gas containing tar is purified by the tail gas treatment device; then, after cooling to room temperature, it is washed with deionized water to remove residual tar, ash and other impurities on the carbon surface. Then, after vacuum filtration, the material is placed in an oven at 80-105℃ for drying to obtain carbonized material.

[0060] Using KOH as an activator, the above-mentioned biochar and KOH were mixed at a mass ratio of 1:1 to 3, and an appropriate amount of deionized water was added and stirred evenly. After being naturally dried, it was placed in a quartz reactor. To remove residual air in the quartz tube, nitrogen (N2) was continuously introduced into it, and the flow rate was kept stable at 200 to 400 mL / min. The purging process lasted for 10 minutes (the specific purging time was determined according to the size of the pyrolysis tube to ensure that the remaining air was removed). The termination temperature of the tubular furnace pyrolysis reaction was set at 400 to 700℃, and the heating rate was 10 to 30℃ / min. After reaching the final decomposition temperature, the temperature was kept constant for 1 to 4 hours, and then cooled to room temperature. The activated material was washed with 10% (V / V) hydrochloric acid and deionized water until neutral, and then dried at 80 to 105℃ to obtain biochar.

[0061] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described herein.

[0062] Experimental Example 1: Investigation into the ability of a single bacterial strain to remove Cr(VI) from water

[0063] Prepare 750 mL of beef meal culture medium: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH: 7.3 ± 0.1. Dispense into 3 500 mL Erlenmeyer flasks (250 mL / flask), autoclave, and set aside for use. Inoculate one flask each of *Shewanella oneidensis* MR-1, *Shewanella putrefaciens* CN-32, and *Pseudomonas stutzeri* LS-2. Incubate the inoculated bacterial suspension at 25°C and 150 rpm on a shaker until the absorbance at 600 nm is approximately 1.0, about 8–14 hours. Aliquot the bacterial suspension into sterile centrifuge tubes (50 mL / tube), centrifuge at 8000 rpm for 5 min, discard the supernatant, resuspend in 40 mL of 0.85% sterile physiological saline in each tube, and centrifuge again. Repeat twice. Finally, the concentrations of the three different bacterial solutions were adjusted to OD using 0.85% sterile saline. 600 The value is 1.0, which will be used as the mother culture for future use.

[0064] Prepare a 20 mM HEPES buffer solution and add 100 mg / L sodium lactate. Add 12 mg / L Cr(VI) to Shewanella oneidensis MR-1 and Shewanella putrefaciens CN-32, respectively, and add 3 mg / L Cr(VI) to Pseudomonas stutzeriLS-2. Construct 100 mL reaction systems for each. Samples were taken at different time points to test the Cr(VI) concentration in the solution. The results are as follows: Figures 1-3 As shown.

[0065] Depend on Figures 1-3It is known that the three bacterial species *Shewanella oneidensis* MR-1, *Shewanella putrefaciens* CN-32, and *Pseudomonas stutzeri* LS-2 all have the ability to reduce Cr(VI) in water. However, single bacterial species have weak resistance to interference in complex soil systems and their Cr(VI) remediation effect is poor. Therefore, this invention uses a composite bacterial species approach. MR-1 and CN-32 individually reduce Cr(VI) more quickly and utilize sodium lactate better, while LS-2 tends to utilize sodium acetate (an intermediate product of sodium lactate oxidation). By controlling the proportion of each bacterial species, the synergistic effect of each species can be fully utilized. This not only improves the adaptability of the bacteria in the natural environment but also ensures long-term effectiveness through division of labor, thereby enhancing the Cr(VI) remediation effect in the soil. Meanwhile, different functional bacteria such as Bacillus subtilis BNCC188080, Bacillus licheniformis BNCC221464, Phanerochaete chrysospora BNCC190652, Moistened Fibromonas hydrophila BNCC336467, and Bacillus amyloliquefaciens BNCC132483 are mixed in proportion to work together to complete composting. The humus in the compost product can act as a shuttle, which can accelerate the reduction of chromium by functional microorganisms in the bacterial agent. At the same time, it can also provide a carbon source for the growth and reproduction of microorganisms. The use of compost is not likely to cause secondary pollution and can realize the resource utilization of solid waste.

[0066] Example 1: Preparation of Inoculum Agent No. 1

[0067] Prepare 9L beef meal culture medium: beef meal 3.0g / L, peptone 10.0g / L, NaCl 5.0g / L, pH: 7.3±0.1. Dispense into 36 500mL Erlenmeyer flasks (250mL / flask), autoclave, and set aside for use. Inoculate 12 flasks with *Bacillus subtilis*, 12 flasks with *Bacillus licheniformis*, 8 flasks with *C. humicola*, and 4 flasks with *Bacillus amyloliquefaciens*. Then, incubate *Bacillus subtilis*, *Bacillus licheniformis*, and *C. humicolaciformis* at 30℃ and 150rpm on a shaker until the absorbance at 600nm is approximately 0.8, which takes approximately 18–24 hours. Incubate *B. amyloliquefaciens* at 37℃ and 150rpm on a shaker until the absorbance at 600nm is approximately 0.8, which also takes approximately 18–24 hours.

[0068] Prepare 1L potato culture medium with the following components: potato extract powder: 10.0 g / L, glucose: 20.0 g / L, KH₂PO₄: 3.0 g / L, MgSO₄·7H₂O: 1.5 g / L, thiamine: 0.008 g / L, pH: 6.0 ± 0.2. Dispense the medium into 4 500 mL Erlenmeyer flasks (250 mL each). Inoculate with *Phanerochaete chrysosporium* and incubate at 30°C and 150 rpm on a shaker until the absorbance at 600 nm is approximately 0.8, which takes about 3–5 days.

[0069] Mix the cultured bacterial solution according to the volume ratio of Bacillus subtilis: Bacillus licheniformis: Moistened cellulomonas: Bacillus amyloliquefaciens: Phanerochaete chrysosporium = 3:3:2:1:1 and set aside for later use.

[0070] Example 2: Preparation of compost products

[0071] Pig manure and crushed corn stalks (corn stalks ≤15cm in length, naturally air-dried and crushed into uniform pieces of 10-50mm) are thoroughly mixed at a mass ratio of 2:1. During mixing, 0.2L / kg of a microbial agent (diluted 10 times with 0.85% sterile saline solution) is added, and water is sprayed evenly. The mixture is then piled into a heap 0.5m wide, 1m long, and 0.2m high, with a moisture content of 55%-60%. After construction, the heap is covered with plastic film. Every 7 days, the film is removed, the heap is turned over, and water is sprayed appropriately to maintain a moisture content of 55%-60%. Composting is complete after 30-35 days. The resulting compost product is then crushed and sieved through a 2mm sieve for later use.

[0072] Example 3: Preparation of biochar powder

[0073] Mango pits were rinsed with deionized water to remove surface impurities and then dried at 105°C for 24 hours. They were then pulverized and sieved through a 50-mesh sieve for later use. 10.0g of mango pit powder was weighed and placed in a quartz reactor. To remove residual air from the quartz tube, nitrogen (N2) was continuously introduced at a stable flow rate of 400mL / min for 10 minutes. The pyrolysis reaction in the tubular furnace was set to terminate at 700°C with a heating rate of 10°C / min. After reaching the termination temperature, the temperature was maintained at a constant level for 4 hours until cooling. During carbonization, tar-containing gases were purified using a tail gas treatment device. After cooling to room temperature, the biochar was rinsed with deionized water to remove residual tar, ash, and other impurities. The biochar was then vacuum filtered and dried in a 105°C oven to obtain the final biochar.

[0074] Using KOH as an activator, biochar and KOH were mixed at a ratio of 1g / 3g carbon-alkali, and an appropriate amount of deionized water was added and stirred until homogeneous. After natural air drying, the mixture was placed in a quartz reactor. To remove residual air from the quartz tube, nitrogen (N2) was continuously introduced into it, ensuring a stable flow rate of 400 mL / min, and the purging process lasted for 10 minutes. The termination temperature of the tubular furnace pyrolysis reaction was set at 700℃, with a heating rate of 10℃ / min. After reaching the final decomposition temperature, the temperature was maintained constant for 4 hours until cooling. The resulting activated biochar was washed with 10% (v / v) hydrochloric acid and deionized water until neutral, and then dried at 105℃ to obtain biochar powder for later use.

[0075] Example 4: Preparation of Inoculum Agent No. 2

[0076] Prepare 4L beef meal culture medium: beef meal 3.0g / L, peptone 10.0g / L, NaCl 5.0g / L, pH: 7.3±0.1. Dispense into 16 500mL Erlenmeyer flasks (250mL / flask), autoclave, and set aside for use. Inoculate 8 flasks with *Shewanella oneidensis* MR-1, and 4 flasks each with *Shewanella putrefaciens* CN-32 and *Pseudomonas stutzeri* LS-2. Incubate the inoculated cultures at 25℃ and 150rpm on a shaker until the absorbance at 600nm is approximately 1.0, about 8–14 hours.

[0077] The cultured bacterial suspension was mixed at a volume ratio of Shewanella oneidensis MR-1: Shewanella putrefaciens CN-32: Pseudomonas stutzeri LS-2 = 2:1:1. The mixed bacterial suspension was dispensed into sterile centrifuge tubes (800 mL / tube). After centrifugation at 8000 rpm for 5 min, the supernatant was discarded. 200 mL of 0.85% sterile physiological saline was added to each centrifuge tube to resuspend the suspension, and then centrifuged again. This process was repeated twice (for a total of 3 times).

[0078] Finally, the concentration of the mixed bacterial solution was adjusted to OD using 0.85% sterile physiological saline. 600The concentration was 1.0, and then 1.0 g / L of biochar was added. The mixture was shaken at 180 rpm for 1 hour at 30°C. Then, 0.2 g / L of sterile sodium alginate and 0.5 g / L of sterile polyvinyl alcohol were added, and the mixture was stirred thoroughly and allowed to stand for 30 minutes. The mixture was then slowly dripped into a pre-prepared sterile saturated calcium borate solution using a sterile syringe to form stable microspheres. These microspheres were allowed to stand at 4°C for 24 hours, filtered, washed three times with 0.85% physiological saline, and finally dried in a vacuum freeze dryer for 18 hours to obtain microbial spheres, which were stored at 4°C for later use.

[0079] Example 5: Soil Remediation Experiment

[0080] Soil samples were collected from the top layer (0-20cm depth) of a city park in the Dongqu District of Panzhihua City, Sichuan Province (27°21'N, 102°15'E). After removing plant debris and pebbles, the soil was dried in a cool, ventilated place and then ground through a 2mm sieve. The Cr(VI) contamination concentration in the soil was set at 400mg / kg. 10kg of the soil was weighed and mixed with 2L of a 2.0g / L potassium dichromate solution to obtain chromium-contaminated soil (400mg / kg). After air-drying, the soil was again ground and sieved through a 2mm sieve. Three portions of the soil (3.0kg each) were weighed and mixed with 0.3kg of compost product, followed by 15.0g of No. 2 microbial agent and 30.0g of sodium lactate. The soil was watered daily and tilled every two days for 30 days for remediation. The distribution of Cr(VI) in the above soil samples was determined using a modified BCR continuous extraction method, with specific procedures following the national standard CB / T 25282-2010. Table 1 shows the distribution of different forms of Cr(VI) in the soil before and after remediation.

[0081] Table 1. Changes in the speciation of Cr(VI) in the soil before and after remediation.

[0082] Before repair After repair Weak acid extractable (%) 16.5 1.2 Reducible (%) 17.5 28.2 Oxidisable (%) 41.2 26.5 Residual (%) 24.8 44.1

[0083] The national standard CB / T 25282-2010 defines the weakly acid-extractable state as the elemental form electrostatically adsorbed on the surface of soil and sediment particles, which can be released through ion exchange, as well as the elemental form bound in carbonates. The reducible state refers to the elemental form held by iron oxide, manganese oxide, etc. The oxidizable state mainly refers to the elemental form bound to the active groups of organic matter, as well as the elemental form of sulfides oxidized to soluble sulfates. The residual state mainly refers to the elemental form existing in the silicate lattice. Among these, the weakly acid-extractable state of heavy metals is relatively reactive and considered a bioavailable component that can be absorbed and utilized by plants. The reducible and oxidizable states are relatively stable and are often considered as the potential bioavailability of heavy metals. The residual state is the stable state of heavy metals, with poor mobility, not easily utilized by plants, and poses less harm to organisms.

[0084] As shown in Table 1, the remediation method of this invention significantly reduced the extractable Cr(VI) in the weakly acidic state, while the sum of the reduced and oxidizable Cr(VI) remained unchanged, and the residual Cr(VI) significantly increased. This indicates that the bioavailability of chromium in the soil was significantly reduced after remediation, demonstrating a significant remediation effect.

[0085] Example 6: Pot Experiment

[0086] The chromium-contaminated soil before and after remediation was placed into pots (1 kg / pot), and then sown with 5 seeds per pot. Seedlings were selected based on their growth status and size. Watering was carried out regularly, maintaining soil moisture content at approximately 20% during seed germination, no less than 50% during the rosette stage, and approximately 80% before and during the heading stage. After growth, the cabbages were harvested, and their fresh and dry weights, as well as plant height, were recorded. The harvested cabbages were first rinsed with deionized water, dried, and weighed fresh. Then, the cabbages were placed in a 105℃ oven for 2 hours to blanch, and then dried in an 80℃ oven until constant weight; this weight was recorded as the dry weight. After weighing, the above-ground parts and roots of the cabbage were separated, ground through a 100-mesh sieve, and then the Cr(VI) concentration was tested using the HNO3-H2O2 method. The results are shown in Table 2.

[0087] Table 2. Cr(VI) concentration in the soil before remediation for Chinese cabbage planting

[0088]

[0089] As shown in Table 2, the concentration of Cr(VI) in the aboveground parts and roots of Chinese cabbage planted in unrestored soil was much higher than that in Chinese cabbage planted in restored soil. This indicates that Cr(VI) in the restored soil is more stable and not easily absorbed and utilized by plants. Therefore, the method of this invention can effectively reduce the bioavailability of Cr(VI) in the soil.

Claims

1. A method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste, characterized in that: Includes the following steps: A. Preparation of Microbial Agent No. 1: Bacillus subtilis BNCC188080, Bacillus licheniformis BNCC221464, Phanerochaete chrysosporium BNCC190652, Cellulosum humicum BNCC336467, and Bacillus amyloliquefaciens BNCC132483 were cultured separately until the absorbance of each bacterial solution at a wavelength of 600 nm was 0.8~1.2, resulting in five bacterial solutions. These solutions were then mixed evenly in a bacterial solution volume ratio of Bacillus subtilis: Bacillus licheniformis: Cellulosum humicum: Bacillus amyloliquefaciens: Phanerochaete chrysosporium of 3±0.1:3±0.1:2±0.1:1±0.05:1±0.05 to obtain Microbial Agent No.

1. The solution was then diluted 8~10 times with sterile physiological saline and set aside for use. B. Preparation of compost products: Livestock and poultry manure and agricultural and forestry waste are thoroughly mixed at a mass ratio of 1.8~2.2:

1. During the mixing process, diluted microbial agent No. 1 is added and water is sprayed evenly to form a pile. The moisture content of the pile is controlled at 55%~60%. Then, aerobic fermentation is carried out. During the fermentation process, the pile is turned at appropriate frequencies and water is sprayed appropriately to ensure the moisture content is 55%~60%. After composting is completed, the pile is crushed to obtain the compost product. In step B, the amount of diluted microbial agent No. 1 added is 0.2~0.3 L / kg of the total mass of livestock and poultry manure and agricultural and forestry waste. C. Preparation of Microbial Agent No. 2: Infecting Shewanella spp. (…) Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri LS-2 was cultured until the absorbance of each bacterial suspension at 600 nm was 1.0~1.2, resulting in three bacterial suspensions, which were then cultured according to the genus Shewanella (…). Shewanella oneidensis MR-1: Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32: Pseudomonas stearothermiae ( Pseudomonas stutzeri The bacterial solution of LS-2 was mixed evenly at a volume ratio of 2±0.1:1±0.05:1±0.05 to obtain a mixed bacterial solution; the mixed bacterial solution and biochar were then used to prepare microbial pellets, which yielded microbial agent No.

2. D. Soil remediation for hexavalent chromium contaminated soil: Mix the compost product obtained in step B with the soil to be remediated at a ratio of 8% to 12% by mass. Add 0.5% to 1% of the No. 2 microbial agent and 1% to 2% of sodium lactate by mass of the soil to be remediated. Soil remediation can be carried out after the soil remediation is completed.

2. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to claim 1, characterized in that: In step A, at least one of the following conditions must be met: The culture medium for Bacillus subtilis, Bacillus licheniformis, Moistened Fibromonas hydrophila and Bacillus amyloliquefaciens was: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.3±0.1; The culture conditions for Bacillus subtilis, Bacillus licheniformis, and Pseudomonas hygroscopicus were as follows: cultured at 30±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.

2. The culture conditions for Bacillus amyloliquefaciens were as follows: cultured at 37±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.2; The culture medium for *Phanerochaete chrysosporium* was as follows: potato extract: 10.0 g / L, glucose: 20.0 g / L, KH2PO4: 3.0 g / L, MgSO4·7H2O: 1.5 g / L, thiamine: 0.008 g / L, pH: 6.0±0.

2. The culture conditions for *Phanerochaete chrysosporium* were as follows: cultured at 30±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.

2.

3. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to claim 1, characterized in that: In step B, at least one of the following must be satisfied: The livestock and poultry manure is fresh pig manure or fresh cow manure; The agricultural and forestry waste mentioned is corn stalks, wheat stalks, or rice stalks; The aerobic fermentation time is 30-35 days; The resulting compost product has a particle size of 1-5 mm.

4. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to claim 1, characterized in that: In step C, at least one of the following must be satisfied: Shewanella ( Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri The culture medium for LS-2 was: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH: 7.3±0.1; Shewanella ( Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri The culture conditions for LS-2 are as follows: cultured at 25~28℃ in a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 1.0~1.

2.

5. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to claim 1, characterized in that: In step C, the specific steps for preparing microbial spheres from the mixed bacterial solution and biochar are as follows: after centrifuging the mixed bacterial solution and discarding the supernatant, resuspend it in sterile physiological saline and centrifuge again, repeating this process 2-3 times; then, adjust the bacterial solution concentration to OD using sterile physiological saline. 600 The concentration of the solution was 1.0-1.

2. 1.0-1.5 g / L of biochar was added. After shaking, 0.2-0.3 g / L of sterile sodium alginate and 0.5-0.8 g / L of sterile polyvinyl alcohol were added. After stirring and standing, the bacterial solution was dropped into a sterile saturated calcium borate solution to form a stable microsphere structure. The microspheres were then subjected to freezing, filtration, washing and vacuum freeze-drying to obtain microbial spheres.

6. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to claim 1, characterized in that: In step C, the biochar is prepared by the following method: after drying and pulverizing the biomass, it is heated to 400-700°C at a heating rate of 10-30°C / min under an inert atmosphere and held at that temperature for 1-4 hours to obtain carbonized material; the carbonized material and activator are mixed evenly at a mass ratio of 1:1-3, and then activated at 400-700°C at a heating rate of 10-30°C / min under an inert atmosphere and held at that temperature for 1-4 hours to obtain biochar.

7. The method for remediating chromium-contaminated soil using livestock and poultry manure and agricultural and forestry waste according to any one of claims 1 to 6, characterized in that: In step D, the soil remediation time is 30 to 40 days. During the remediation process, the soil is watered daily to ensure that the soil moisture content is 55% to 60%, and the soil is tilled every two days.

8. The microbial inoculant for remediating chromium-contaminated soil according to claim 1, characterized in that: It consists of microbial inoculant No. 1 and microbial inoculant No. 2 in a volume-to-mass ratio of 4-6 mL: 5 g; The No. 1 microbial inoculant was prepared by the following method: *Bacillus subtilis* BNCC188080, *Bacillus licheniformis* BNCC221464, *Phanerochaete chrysosporium* BNCC190652, *Cellulosum humicum* BNCC336467, and *Bacillus amyloliquefaciens* BNCC132483 were cultured until the absorbance of each bacterial solution at 600 nm was 0.8–1.2, resulting in five bacterial solutions. These solutions were then mixed thoroughly in a volume ratio of *Bacillus subtilis*: *Bacillus licheniformis*: *Cellulosum humicum*: *Bacillus amyloliquefaciens*: *Phanerochaete chrysosporium* of 3±0.1:3±0.1:2±0.1:1±0.05:1±0.

05. The No. 2 microbial inoculant was prepared by the following method: for Shewanella spp. (… Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri LS-2 was cultured until the absorbance of each bacterial suspension at 600 nm was 1.0~1.2, resulting in three bacterial suspensions, which were then cultured according to the genus Shewanella (…). Shewanella oneidensis MR-1: Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32: Pseudomonas stearothermiae ( Pseudomonas stutzeri The bacterial culture of LS-2 was mixed evenly with a volume ratio of 2±0.1:1±0.05:1±0.05 to obtain a mixed bacterial culture; the mixed bacterial culture and biochar were then used to prepare microbial spheres.

9. The microbial inoculant for remediating chromium-contaminated soil according to claim 8, characterized in that: When preparing microbial inoculant No. 1, at least one of the following conditions must be met: The culture medium for Bacillus subtilis, Bacillus licheniformis, Moistened Fibromonas hydrophila and Bacillus amyloliquefaciens was: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.3±0.1; The culture conditions for Bacillus subtilis, Bacillus licheniformis, and Pseudomonas hygroscopicus were as follows: cultured at 30±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.

2. The culture conditions for Bacillus amyloliquefaciens were as follows: cultured at 37±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.2; The culture medium for *Phanerochaete chrysosporium* was as follows: potato extract: 10.0 g / L, glucose: 20.0 g / L, KH2PO4: 3.0 g / L, MgSO4·7H2O: 1.5 g / L, thiamine: 0.008 g / L, pH: 6.0±0.

2. The culture conditions for *Phanerochaete chrysosporium* were as follows: cultured at 30±1℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8~1.

2.

10. The microbial inoculant for remediating chromium-contaminated soil according to claim 8, characterized in that: When preparing microbial inoculant No. 2, at least one of the following conditions must be met: Shewanella ( Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri The culture medium for LS-2 was: beef meal 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH: 7.3±0.1; Shewanella ( Shewanella oneidensis MR-1, Shewanella putrefactive bacteria ( Shewanella putrefaciens CN-32 and Pseudomonas stearothermia ( Pseudomonas stutzeri The culture conditions for LS-2 are as follows: cultured at 25~28℃ on a shaker at 150~180 rpm under aerobic conditions until the absorbance of the bacterial solution at a wavelength of 600 nm is 1.0~1.2; The specific steps for preparing microbial spheres from the mixed bacterial culture and biochar are as follows: After centrifuging the mixed bacterial culture and discarding the supernatant, resuspend it in sterile physiological saline and centrifuge again, repeating this process 2-3 times; then adjust the bacterial culture concentration to OD using sterile physiological saline. 600 The concentration of the solution was 1.0-1.

2. 1.0-1.5 g / L of biochar was added. After shaking, 0.2-0.3 g / L of sterile sodium alginate and 0.5-0.8 g / L of sterile polyvinyl alcohol were added. After stirring and standing, the bacterial solution was dropped into a sterile saturated calcium borate solution to form a stable microsphere structure. The microspheres were then subjected to freezing, filtration, washing and vacuum freeze-drying to obtain microbial spheres.

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