Biochar-microorganism composite soil conditioner as well as preparation method and application thereof

Abstract

The invention provides a biochar-microorganism composite soil conditioner as well as a preparation method and application thereof, and belongs to the crossing field of environmental functional materials and agricultural microorganism technologies. The biochar-microorganism composite soil conditioner comprises modified biochar and plant growth-promoting rhizobacteria loaded on the modified biochar, and further comprises an embedding agent, and the embedding agent wraps the outer surface of the modified biochar loaded with the plant growth-promoting rhizobacteria; the modified biochar is selected from any one of ball-milling modified biochar, kaolinite modified biochar or chitosan modified biochar. The biochar-microorganism composite soil conditioner disclosed by the invention can effectively reduce the content of available cadmium (Cd) in soil, and the Cd is converted into a stable residue state from a biological effective state; meanwhile, the growth of crops is remarkably promoted by releasing nutrients and secreting growth-promoting substances.

Description

Technical Field

[0001] This invention relates to the intersection of environmental functional materials and agricultural microbial technology, and particularly to a biochar-microbial composite soil conditioner, its preparation method, and its application. Background Technology

[0002] Heavy metal pollution in soil, particularly cadmium (Cd) pollution, has become an environmental problem threatening the quality and safety of agricultural products, the health of ecosystems, and human health. Cd is highly toxic, easily migrates, and bioaccumulates, and can be transferred and accumulated through the food chain, causing a range of diseases. Currently, the environmental quality of arable land soil is a prominent issue, and the need for remediation of Cd and other heavy metal pollution is urgent. However, traditional methods such as physical adsorption and biodegradation suffer from low removal efficiency, high costs, and the potential for secondary pollution. Summary of the Invention

[0003] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides a biochar-microorganism composite soil conditioner, its preparation method, and its application.

[0004] According to one embodiment of the present invention, a biochar-microbial composite soil conditioner is provided, comprising modified biochar and plant rhizosphere growth-promoting bacteria loaded on the modified biochar, and further comprising an encapsulating agent, the encapsulating agent being wrapped around the outer surface of the modified biochar loaded with plant rhizosphere growth-promoting bacteria; the modified biochar is selected from any one of ball-milled modified biochar, kaolinite modified biochar, or chitosan modified biochar.

[0005] According to another aspect of the present invention, a method for preparing a biochar-microorganism composite soil conditioner is provided, comprising: mixing modified biochar in a suspension of plant rhizosphere growth-promoting bacteria to obtain a first mixture; mixing the first mixture with a polyvinyl alcohol-sodium alginate gel-like mixed solution to obtain a second mixture; adding the second mixture dropwise to a crosslinking agent solution, crosslinking and fixing the mixture, and then freeze-drying it to obtain the biochar-microorganism composite soil conditioner.

[0006] According to another aspect of the present invention, an application of a biochar-microorganism composite soil conditioner is provided, including using the biochar-microorganism composite soil conditioner for cadmium pollution remediation and crop growth promotion.

[0007] This invention utilizes modified biochar, which increases the specific surface area and surface functional group abundance of biochar, enhancing the adsorption and complexation capacity of the biochar-microbial composite soil conditioner. Furthermore, it enables high-density loading of plant rhizosphere growth-promoting bacteria (PGPR) through electrostatic and hydrogen bonding interactions, thereby constructing an integrated micro-ecosystem of "biochar-functional bacteria," which facilitates the role of PGPR in promoting crop growth. Using environmentally responsive biodegradable biopolymers as encapsulating agents, the modified biochar is thin-layered, imparting slow-release and high mechanical strength properties while preventing the desorption of adsorbed pollutants and avoiding secondary pollution. Ultimately, this results in a biochar-microbial composite soil conditioner that combines the biological effects of microorganisms with the physicochemical advantages of modified biochar, simultaneously achieving the dual functions of crop growth promotion and Cd pollution remediation. Attached Figure Description

[0008] Figure 1 This is a schematic flowchart of the preparation method of the composite pollution remediation agent in an embodiment of the present invention;

[0009] Figure 2 The images shown are microscopic morphology diagrams of biochar in the embodiments of the present invention, wherein (a) is a microscopic morphology diagram of original biochar (BC), (b) is a microscopic morphology diagram of kaolinite-modified biochar (KLBC), (c) is a microscopic morphology diagram of chitosan-modified biochar (CSBC), and (d) is a microscopic morphology diagram of ball-milled modified biochar (BLBC).

[0010] Figure 3 The following are elemental distribution diagrams of biochar in the embodiments of the present invention, wherein (a) is the elemental distribution diagram of BC, (b) is the elemental distribution diagram of KLBC, (c) is the elemental distribution diagram of CSBC, and (d) is the elemental distribution diagram of BLBC.

[0011] Figure 4 The N2 adsorption / desorption curves of the original biochar and modified biochar in the embodiments of the present invention are shown in the figure.

[0012] Figure 5 This is a diagram showing the total pore size distribution of the original biochar and modified biochar in the embodiments of the present invention;

[0013] Figure 6 The Fourier transform infrared spectra of the original biochar and modified biochar in the embodiments of the present invention are shown.

[0014] Figure 7 This is a scanning electron microscope image of the biochar-microorganism composite soil conditioner in an embodiment of the present invention;

[0015] Figure 8 This is the Fourier transform infrared spectrum of the biochar-microorganism composite soil conditioner in this embodiment of the invention;

[0016] Figure 9 This is a graph showing the Bacillus subtilis loading rate of biochar in an embodiment of the present invention;

[0017] Figure 10 The graph shows the Cd removal rate results of each biochar and biochar-microorganism composite soil conditioner in the embodiments of the present invention.

[0018] Figure 11 This is a graph showing the results of the available Cd content in the soil after applying different remediation agents in Test Example 2 of this invention;

[0019] Figure 12 This is a graph showing the percentage of different Cd components in the soil after applying different remediation agents in Test Example 2 of this invention;

[0020] Figure 13 The figures show the plant height and root length results after applying different repair agents in Test Example 2 of this invention.

[0021] Figure 14 This is a graph showing the plant biomass results after applying different repair agents in Test Example 2 of the present invention. Detailed Implementation

[0022] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0024] In realizing the concept of this invention, it was discovered that among current remediation technologies for farmland soil with moderate to mild Cd contamination, in-situ passivation and stabilization remediation is widely used due to its low cost, ease of operation, and minimal environmental disturbance. The core of this technology is the addition of passivating materials, such as biochar, to the soil. Through adsorption, precipitation, and complexation, the form in which heavy metals exist is altered, reducing their bioavailability and mobility.

[0025] PGPR, as a functional microorganism, enhances crop resistance to stress and reduces its absorption of heavy metals. Although both biochar and PGPR exhibit good performance in soil remediation, simply mixing and applying them has significant limitations: free PGPR, when directly applied to the soil, is susceptible to external environmental stresses (such as pH, temperature, moisture, and competition from indigenous microorganisms), resulting in low colonization and survival rates, unstable remediation effects, and poor persistence. Microbial-carrier immobilization technology can provide functional microorganisms with a "micro-refuge" protected from harsh environments, improving their survival rate and activity. Biochar, due to its porous structure, good water retention, and biocompatibility, is considered an excellent microbial carrier. Targeted modification of biochar (ball milling, kaolinite modification, or chitosan modification) to increase its specific surface area and surface functional group abundance can enhance its adsorption and passivation capacity for target pollutants (Cd) and create a better colonization environment for functional microorganisms. Finally, encapsulation agents are used to improve the mechanical and slow-release properties of the biochar-microorganism composite soil conditioner, achieving long-term remediation of water and soil. This invention couples materials science methods with biological processes to synergistically achieve the dual effects of crop growth promotion and heavy metal pollution remediation.

[0026] Specifically, according to one embodiment of the present invention, a biochar-microbial composite soil conditioner is provided, comprising modified biochar and plant rhizosphere growth-promoting bacteria loaded on the modified biochar, including an encapsulating agent that encapsulates the outer surface of the modified biochar loaded with plant rhizosphere growth-promoting bacteria; the modified biochar is selected from any one of ball-milled modified biochar, kaolinite modified biochar, or chitosan modified biochar.

[0027] According to embodiments of the present invention, biochar is produced by the pyrolysis of biomass under anaerobic conditions. Due to its rich porous structure, large specific surface area, abundant surface functional groups (such as carboxyl and hydroxyl groups), and alkaline properties, it exhibits excellent adsorption performance for heavy metal ions. Simultaneously, it can improve soil structure and enhance fertility, making it a promising environmental material. On the other hand, microbial remediation technology utilizes the metabolic activities (such as biosorption, extracellular precipitation, and biomineralization) of specific functional microorganisms (such as bacteria and fungi) to fix or transform heavy metals, also showing great potential. PGPR, as a common type of functional bacteria, not only has good environmental adaptability and rapid reproduction, but can also complex and fix Cd by secreting metabolites such as siderophores, organic acids, and polysaccharides. 2+ Meanwhile, its own microbial cells also have a strong biosorption capacity. In addition, PGPR can produce antibacterial substances and plant growth-promoting hormones (such as indoleacetic acid), effectively inhibiting soil-borne diseases and promoting crop root development, thereby indirectly enhancing crop stress resistance and reducing its absorption of heavy metals.

[0028] While raw biochar (BC) possesses basic adsorption properties, its limited surface functional groups, unoptimized pore structure, and low microbial loading capacity result in weak selective adsorption of Cd, insufficient cell survival and metabolic activity, making it difficult to achieve efficient and durable remediation in complex soil environments. Chemical modification can directionally introduce functional groups, optimize pore structure, and improve cell compatibility, thereby constructing an integrated synergistic remediation system of "adsorption-biotransformation-plant growth promotion," achieving efficient passivation of Cd-contaminated soil and safe crop production. Ball milling modification can increase the surface roughness of biochar, introduce abundant oxygen-containing functional groups, and enhance Cd adsorption. 2+ The physical adsorption and surface complexation of kaolin, along with its porous structure, provide a microenvironment for PGPR colonization, jointly achieving the fixation and speciation of Cd. Kaolin modification introduces a layered aluminosilicate structure, enhancing cation exchange capacity and strengthening Cd adsorption and co-precipitation capabilities. Its interlayer structure provides a protective habitat for PGPR, and PGPR metabolism further promotes the formation of stable complexes between Cd and minerals. Chitosan modification introduces -NH2 and hydroxyl-OH groups, significantly enhancing Cd chelation capacity and PGPR loading rate, systematically improving soil health and plant stress resistance. The modified biochar encapsulation agent forms a barrier, improving the mechanical properties of the biochar-microbial composite soil conditioner and achieving long-term slow-release remediation effects; it also prevents the desorption of adsorbed pollutants, avoiding secondary pollution.

[0029] According to embodiments of the present invention, the biochar-microbial composite soil conditioner comprises, by mass fraction: 30-45% modified biochar, 30-40% encapsulating agent, and 20-35% plant rhizosphere growth-promoting bacteria.

[0030] According to embodiments of the present invention, modified biochar with a mass fraction of 30-45% ensures sufficient specific surface area and pore structure for effective adsorption of pollutants in the soil, without crowding out the space of the encapsulating agent and microbial agent due to an excessively high proportion, thus avoiding re-adsorption of the remediation agent and neglecting its activation function. The encapsulating agent with a mass fraction of 30-40% has a narrower range, which can better balance the fixation and release processes, stably encapsulating the microbial agent and protecting it from damage by harsh soil environments (such as acids, alkalis, and toxins), while also slowly releasing the microbial agent to ensure its long-term effectiveness. Plant rhizosphere growth-promoting bacteria with a mass fraction of 20-35% are sufficient to support soil microecological improvement (such as nitrogen fixation, phosphorus solubilization, and pathogen inhibition), assisting modified biochar in enhancing the remediation effect, while avoiding reduced survival rates due to excessive competition for nutrients by the microbial agent.

[0031] According to embodiments of the present invention, the biochar-microbial composite soil conditioner comprises, by mass fraction: 30-45% modified biochar, for example, 30%, 35%, 38%, 40%, 45%; 30-40% encapsulating agent, for example, 30%, 32%, 35%, 38%, 40%; and 20-35% plant rhizosphere growth-promoting bacteria, for example, 20%, 22%, 25%, 30%, 35%.

[0032] According to embodiments of the present invention, the plant rhizosphere growth-promoting bacteria are selected from any one or more of the genera *Bacillus*, *Streptomyces*, *Pseudomonas*, and *Burkholderia*. Specifically, *Bacillus* can be *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus megaterium*, etc., preferably *Bacillus subtilis*; *Streptomyces* can be *Streptomyces griseus*, *Streptomyces hygroscopicus*, etc.; *Pseudomonas* can be *Pseudomonas fluorescens*, *Pseudomonas putida*, *Pseudomonas aeruginosa*, etc.; and *Burkholderia* can be *Burkholderia cepacia*, etc. Plant rhizosphere growth-promoting bacteria can directly provide nutrients and promote plant growth: *Bacillus subtilis* can decompose insoluble phosphorus and potassium elements in the soil, converting them into forms that plants can absorb, thus supplementing nutrients; it can also synthesize auxins (such as indoleacetic acid) required for plant growth, directly stimulating root development and plant growth. Plant rhizosphere growth-promoting bacteria can inhibit harmful organisms and protect plant health: *Bacillus subtilis* can produce a variety of antibiotics, effectively inhibiting fungi, bacteria, and other pathogens in the soil, reducing soil-borne diseases. Rhizosphere growth-promoting bacteria can also improve the soil environment and enhance plant stress resistance: the above-mentioned bacteria can regulate the pH value of rhizosphere soil, improve soil aggregate structure, enhance soil water and fertilizer retention capacity, and help plants resist abiotic stresses such as drought and salinity. For example, they can enhance plant stress resistance by synthesizing osmotic regulators such as proline.

[0033] According to embodiments of the present invention, the encapsulating agent is a cross-linked dual network formed by polyvinyl alcohol and calcium alginate. The cross-linked dual network formed by polyvinyl alcohol and calcium alginate is a dense and elastic gel structure with its own adsorption capacity. Its porous structure can further accommodate modified biochar, and the two form a synergistic adsorption effect. That is, biochar is mainly responsible for adsorbing pollutants, while the cross-linked dual network can intercept some small molecule pollutants and slow down the diffusion rate of pollutants into the biochar, allowing for more complete adsorption. The cross-linked dual network can encapsulate loose modified biochar particles, preventing the modified biochar from migrating or being lost in water or soil due to water flow or wind. The encapsulation structure also protects the adsorption active sites of the modified biochar, preventing them from being directly contaminated or blocked by external impurities, thus maintaining the long-term adsorption efficiency of the modified biochar. Polyvinyl alcohol enhances the mechanical strength of the cross-linked structure of the dual network, reducing breakage even under complex environments (such as repeated wetting and drying, acid and alkali fluctuations), and extending the service life of the remediation agent. For biochar that has adsorbed pollutants (such as heavy metal Cd), cross-linked double-network encapsulation can form a barrier to prevent the adsorbed pollutants from re-desorbing due to changes in environmental conditions (such as increased pH), thus avoiding secondary pollution. The encapsulated modified biochar has a more regular morphology (such as being made into granules or membranes), which facilitates subsequent recycling and regeneration, solving the problems of difficult separation and high recycling costs of traditional powdered biochar.

[0034] Figure 1 This is a schematic flowchart of the preparation method of the composite pollution remediation agent in an embodiment of the present invention.

[0035] According to another embodiment of the present invention, a method for preparing the above-mentioned biochar-microorganism composite soil conditioner is provided, such as... Figure 1 As shown, it includes steps S1-S3.

[0036] Step S1: Mix the modified biochar with a suspension of plant rhizosphere growth-promoting bacteria to obtain the first mixture.

[0037] Step S2: Mix the first mixture with the polyvinyl alcohol-sodium alginate gel solution to obtain the second mixture.

[0038] Step S3: Add the second mixture dropwise to the crosslinking agent solution, fix the crosslinking, and freeze-dry to obtain the biochar-microorganism composite soil conditioner.

[0039] According to embodiments of the present invention, the modified biochar is first mixed with plant rhizosphere growth-promoting bacteria, which allows the bacterial suspension to fully contact the modified biochar. This enables the bacterial agent to uniformly penetrate the porous structure of the modified biochar, preventing bacterial aggregation due to uneven local concentrations during subsequent encapsulation and ensuring stable bacterial activity in each part of the material. The step-by-step operation allows control over the amount of cross-linking agent and reaction conditions (such as temperature and time). Allowing the bacterial agent to adsorb into the pores of the modified biochar before gentle cross-linking reduces activity damage caused by direct contact between the cross-linking agent and the bacterial cells, thus improving the retention rate of viable bacteria.

[0040] According to an embodiment of the present invention, before step S1, unmodified raw biochar needs to be prepared. The preparation method includes: selecting agricultural waste straw as the raw material for biochar preparation, such as rice husks, wheat straw, cotton straw, corn straw, etc. The straw is repeatedly washed with deionized water to remove surface dust and soluble impurities. After drying at 105°C to constant weight, the dried straw is crushed using a crusher and passed through a 0.25mm sieve to obtain straw powder with uniform particle size. The straw powder is placed in an anaerobic pyrolysis furnace and heated to the target pyrolysis temperature of 600°C at a programmed heating rate of 10°C / min under the protection of inert gas N2, and then pyrolyzed at this temperature for 4 hours. After pyrolysis, it is naturally cooled to room temperature under continuous inert gas circulation, ground, and passed through a 100-mesh sieve to obtain raw biochar (denoted as BC), which is then sealed and stored in a desiccator for later use.

[0041] According to embodiments of the present invention, agricultural waste straw can be utilized as a raw material, resulting in low raw material and application costs. Ecologically, it reduces pollution from straw burning, and the prepared biochar-microbial composite soil conditioner produces no toxic emissions during the remediation process, avoiding secondary pollution. It is a safe in-situ remediation technology that can be widely used in Cd-contaminated farmland, possessing both significant ecological benefits and promising market application prospects.

[0042] According to an embodiment of the present invention, before step S1, the raw biochar needs to be modified to obtain modified biochar. The modified biochar is selected from any one of ball-milled modified biochar, kaolinite modified biochar, or chitosan modified biochar. The preparation method of ball-milled modified biochar includes: placing the raw biochar and grinding media into the grinding jar of a planetary ball mill. The grinding media used for ball milling is zirconia balls, placed in the grinding jar of the planetary ball mill at a ball-to-material mass ratio of 1:20 (i.e., 1 part zirconia balls: 20 parts raw biochar). The ball milling speed is 200-600 rpm / min, and the ball milling time is 24 hours. Anhydrous ethanol (60% of the biochar mass) can be added as a process control agent. After ball milling, the product is washed repeatedly with deionized water until the supernatant is neutral, and then vacuum dried at 80°C for 24 hours to obtain ball-milled modified biochar (denoted as BLBC).

[0043] According to embodiments of the present invention, in the preparation process of ball-milled modified biochar, adding anhydrous ethanol as a process control agent can inhibit the agglomeration of biochar caused by electrostatic adsorption and improve grinding efficiency; it can also reduce the temperature during the grinding process, reducing the thermal decomposition of components such as hemicellulose and lignin in the biochar. During ball milling, the grinding jar and grinding media will experience minor wear due to impact, which may introduce metal ions (such as Fe). 2+ Al 3+ Impurities such as solid debris or fragments, if left behind, will adhere to the surface of the biochar, occupying adsorption sites for the microbial agent or contaminants, and may even become toxic to the microbial agent, affecting its survival. After ball milling, washing with deionized water can remove residual impurities and bring the ball-milled modified biochar to neutral, reducing the adverse effects of unsuitable pH on the microbial agent.

[0044] According to an embodiment of the present invention, the preparation method of kaolinite-modified biochar includes: adding raw biochar and kaolinite powder to 1L of deionized water and mixing to obtain a suspension of raw biochar and kaolinite, then shaking at 150 r / min for 12 hours, centrifuging, filtering, drying at 60°C for 12 hours, and then sieving through a 0.25mm sieve to obtain kaolinite-modified biochar (denoted as KLBC).

[0045] According to embodiments of the present invention, in the preparation process of kaolinite-modified biochar, the mass ratio of raw biochar to kaolinite powder is 15-25:1. At this ratio, kaolinite can be uniformly dispersed in the pores of the biochar, neither clogging the large pores of the biochar (ensuring the adsorption of large molecular pollutants) nor hindering the addition of specific adsorption sites, thus improving adsorption capacity. Furthermore, biochar is mostly alkaline, and its use alone may alter the pH balance of soil or water bodies, while kaolinite is neutral or weakly acidic and has good pH buffering capacity. The 15-25:1 ratio can adjust the pH of the adsorbent to a neutral range of 6.5-7.5, avoiding the stimulation of soil microorganisms or aquatic organisms by excessively alkaline biochar, and providing suitable conditions for subsequent loading of bacterial agents or adsorption of acid- and alkali-sensitive pollutants.

[0046] According to an embodiment of the present invention, in the preparation process of kaolinite-modified biochar, the mass ratio of raw biochar to kaolinite powder is 15-25:1, for example, it can be 15:1, 17:1, 19:1, 21:1, 23:1, 25:1, preferably 20:1.

[0047] According to an embodiment of the present invention, the preparation method of chitosan-modified biochar includes: slowly adding raw biochar and chitosan to 1L of 2% acetic acid solution, continuously heating and stirring in a 50℃ water bath until a uniform and viscous mixed solution is formed. After the mixed solution cools, the pH of the mixed solution is adjusted to 7.0 by adding NaOH solution dropwise, stirring for 1 hour and letting stand for 7 hours. After washing several times with deionized water and drying in an oven at 50℃, the product is ground and passed through a 0.25mm sieve to obtain chitosan-modified biochar (denoted as CSBC).

[0048] According to an embodiment of the present invention, in the preparation process of chitosan-modified biochar, heating in a water bath at 50 °C is mainly to promote the dissolution of chitosan and facilitate its reaction with the original biochar; stirring for 1 hour after adding NaOH solution is to fully adjust the pH; and letting it stand for 7 hours is to allow the chitosan-modified biochar to precipitate, making it easier to separate and collect.

[0049] According to an embodiment of the present invention, in the preparation process of chitosan-modified biochar, the mass ratio of original biochar to chitosan is 20-30:1. At a ratio of 20-30:1, a small amount of chitosan can uniformly cover the surface of biochar and the inner wall of pores, without clogging the large pores of biochar, and can supplement specific adsorption sites, thereby increasing the adsorption capacity of the adsorbent for heavy metals such as cadmium.

[0050] According to an embodiment of the present invention, in the preparation process of chitosan-modified biochar, the mass ratio of the original biochar to chitosan is 20-30:1, for example, it can be 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, preferably 25:1.

[0051] According to an embodiment of the present invention, before step S1, a suspension of plant rhizosphere growth-promoting bacteria needs to be prepared, including: inoculating the plant rhizosphere growth-promoting bacteria onto LB solid medium plates and activating them at 35°C for 24 hours; picking single colonies and inoculating them into LB liquid seed medium, and shaking and culturing them at 35°C and 175 rpm until the late logarithmic growth phase (OD600=1.0); transferring them to fermentation medium at an inoculation rate of 5% to obtain a high-concentration bacterial fermentation broth; collecting the bacterial cells by aseptic centrifugation at 4°C and 8000 rpm for 10 min; resuspending and washing twice with sterile 0.85% physiological saline, and finally resuspending with physiological saline to the required concentration (viable cell count not less than 1×10⁻⁶). 9 (CFU / mL) was used to prepare a suspension of plant rhizosphere growth-promoting bacteria for later use.

[0052] According to an embodiment of the present invention, in step S1, modified biochar is mixed in a suspension of plant rhizosphere growth-promoting bacteria to obtain a first mixture. The plant rhizosphere growth-promoting bacteria are connected to the modified biochar through adsorption and electrostatic interactions, with an addition ratio of 1g:5-30mL. This addition ratio allows the plant rhizosphere growth-promoting bacteria to maintain a good crop growth-promoting effect without excessively occupying the pores of the modified biochar and reducing the adsorption effect.

[0053] According to an embodiment of the present invention, in step S1, the addition ratio of modified biochar to plant rhizosphere growth-promoting bacteria suspension is 1g:5-30mL, for example, it can be: 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL, 1g:30mL, preferably 1g:10mL.

[0054] According to an embodiment of the present invention, in step S2, polyvinyl alcohol and sodium alginate are first weighed and soaked overnight in cold water at about 20°C. After being removed, they are slowly stirred to allow the polyvinyl alcohol and sodium alginate particles to fully swell and for the volatile substances in the polyvinyl alcohol to escape. Then, the water bath temperature is raised to about 95°C, and the mixture is stirred at 70-100 r / min to accelerate dissolution. The mixture is kept at this temperature for 2-2.5 hours to obtain a polyvinyl alcohol-sodium alginate gel-like mixed solution. After the temperature of the polyvinyl alcohol-sodium alginate gel-like mixed solution drops below 40°C, which will not affect the bacterial agent, the first mixture is then mixed with the polyvinyl alcohol-sodium alginate gel-like mixed solution and stirred thoroughly to obtain a second mixture.

[0055] According to an embodiment of the present invention, in step S2, the volume / mass ratio of polyvinyl alcohol to sodium alginate in the polyvinyl alcohol-sodium alginate gel-mixed solution is 8:1.0-1.4. This allows the formed cross-linked double network to have high mechanical strength, while also controlling the swelling properties and preserving biocompatibility.

[0056] According to an embodiment of the present invention, in step S2, the volume / mass ratio of polyvinyl alcohol to sodium alginate in the polyvinyl alcohol-sodium alginate gel mixed solution is 8:1.0-1.4, for example, it can be 8:1.0, 8:1.2, 8:1.4, preferably 8:1.2.

[0057] According to an embodiment of the present invention, in step S3, the second mixture is added dropwise to the crosslinking agent solution, and after crosslinking fixation, it is freeze-dried to obtain a biochar-microorganism composite soil conditioner. The crosslinking agent is selected from a mixed solution of calcium chloride and boric acid, wherein the mass fraction of calcium chloride is 4% and the mass fraction of boric acid is 6%. Step S3 specifically includes: adding the second mixture dropwise to the crosslinking agent solution using a peristaltic pump at a dropping rate controlled at 7.0 mL / s; the modified biochar forms small spheres with a particle size of 3-5 mm under the encapsulation effect of the encapsulating agent; and the small spheres are soaked in the crosslinking agent solution at 4°C for 4-24 hours for full crosslinking fixation. Next, the pellets were washed three times with 0.9% sterile saline to remove residual CaCl2 on the surface, and then placed in a vacuum freeze dryer (cold trap temperature -50℃, vacuum degree ≤10 Pa) for 2 hours to dry them until the moisture content was ≤10%, thus obtaining biochar-microorganism composite soil conditioner (among which, ball milled modified biochar-microorganism composite soil conditioner is denoted as BLBC@BS.sp, kaolinite modified biochar-microorganism composite soil conditioner is denoted as KLBC@BS.sp, and chitosan modified biochar-microorganism composite soil conditioner is denoted as CSBC@BS.sp).

[0058] According to another aspect of the present invention, an application of a biochar-microorganism composite soil conditioner is provided, including using the biochar-microorganism composite soil conditioner for cadmium pollution remediation and crop growth promotion.

[0059] According to embodiments of the present invention, modified biochar has a larger specific surface area and more uniform pore size than original biochar, resulting in higher Cd ion removal efficiency; furthermore, the micro-nano structure and encapsulating agent of modified biochar provide a protective barrier for the microbial agent. Modified biochar and microbial agent form a synergistic effect through mechanisms such as bioadsorption, biomineralization, and biotransformation (converting exchangeable Cd into residual state), effectively enabling cadmium pollution remediation and crop growth promotion.

[0060] According to embodiments of the present invention, cadmium pollution remediation includes cadmium pollution remediation in water bodies and cadmium pollution remediation in soil; wherein, in the cadmium pollution remediation in water bodies, the dosage of biochar-microorganism composite soil conditioner is 0.1~2 g / L, the reaction time is 0.5-35 hours, and the cadmium concentration is 10-100 mg / L; in the cadmium pollution remediation in soil bodies, the dosage of biochar-microorganism composite soil conditioner is 2-5%.

[0061] According to embodiments of the present invention, a biochar-microbial composite soil conditioner at a concentration of 0.1–2 g / L can provide sufficient adsorption sites and exhibit high adsorption efficiency. The reaction time of 0.5–35 hours covers both the rapid and equilibrium phases of adsorption. A short time (0.5 hours) allows for the rapid adsorption of most free cadmium ions in water, suitable for emergency treatment; a longer time (35 hours) allows the biochar-microbial composite soil conditioner to fully react with the difficult-to-adsorb cadmium ions, reaching adsorption equilibrium and ensuring stable remediation results. The cadmium concentration is 10–100 mg / L, a relatively wide range, indicating the broad adaptability of the biochar-microbial composite soil conditioner, applicable to diverse polluted environments. In the remediation of cadmium pollution in soil, the dosage of the biochar-microbial composite soil conditioner is 2–5%. This range balances fixation efficiency with soil impact, both initially fixing highly reactive cadmium in the soil through the pores and functional groups of modified biochar and enhancing the fixation effect by converting cadmium into a stable form (such as residual form), reducing plant absorption. At the same time, this dosage will not cause a decrease in soil permeability or drastic changes in pH due to excessive material, thus avoiding damage to the soil micro-ecology.

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] Preparation Example 1

[0065] Agricultural waste straw such as rice husks was selected and repeatedly washed with deionized water to remove surface dust and soluble impurities. It was then dried at 105℃ to constant weight. The dried straw was pulverized using a crusher and passed through a 0.25mm sieve to obtain straw powder with uniform particle size. 500g of the straw powder was placed in a vacuum tube furnace or muffle furnace. Under the protection of high-purity inert gas (N2, purity ≥99.99%), the temperature was increased to 600℃ at a rate of 10℃ / min and pyrolyzed at this temperature for 4 hours. After pyrolysis, the mixture was naturally cooled to room temperature under continuous N2 purging. The resulting raw biochar (denoted as BC) was then removed, ground, passed through a 100-mesh sieve, and sealed for storage in a desiccator for later use.

[0066] Preparation Example 2

[0067] Bacillus subtilis was inoculated onto LB solid agar plates and activated at 35°C for 24 hours. Single colonies were picked and inoculated into LB liquid seed medium and cultured with shaking at 35°C and 175 rpm until the late logarithmic growth phase (OD600=1.0). A 5% inoculum was then transferred to fermentation medium to obtain a high-concentration fermentation broth. The high-concentration fermentation broth was aseptically centrifuged at 4°C and 8000 rpm for 10 min to collect the bacterial cells. The cells were resuspended twice in sterile 0.85% physiological saline and finally resuspended in physiological saline to the desired concentration (viable cell count not less than 1×10⁻⁶). 9 (CFU / mL) was used to prepare a Bacillus subtilis suspension for later use.

[0068] Example 1

[0069] Ball milling modification of raw biochar: 100 g of raw biochar and 5 g of zirconia balls (ball-to-material mass ratio 1:20, zirconia ball particle sizes including 5 mm, 10 mm, and 15 mm, with a mass ratio of 1:2:2) were placed in a planetary ball mill and dry-milled at 600 rpm / min for 24 hours. 76.05 mL of anhydrous ethanol (C2H6O) was added as a process control agent during milling. After milling, the product was washed repeatedly with deionized water until the supernatant was neutral. It was then vacuum-dried at 80℃ for 24 hours and passed through a 0.25 mm sieve to obtain ball-milled modified biochar (denoted as BLBC).

[0070] Subsequently, 100g of BLBC was slowly added to 1000mL of Bacillus subtilis suspension activated and cultured in LB liquid medium (viable count concentration 1.0×10⁻⁶). 9 The sample was placed in a constant temperature shaker and subjected to low-speed shaking at 35°C and 175 rpm for 4 hours for adsorption.

[0071] 80g of polyvinyl alcohol (PVA) and 12g of sodium alginate (SA) were added to 1L of deionized water and stirred in a 95℃ water bath until completely dissolved, yielding a polyvinyl alcohol-sodium alginate gel-like mixed solution. After cooling to 40℃, the first mixture of BC and Bacillus subtilis was slowly added to the polyvinyl alcohol-sodium alginate gel-like mixed solution under aseptic conditions, and stirred for 30 minutes to allow the bacteria and biochar to be initially encapsulated, forming a second mixed solution.

[0072] Subsequently, the second mixture was added dropwise to a 3L solution of 4% calcium chloride (CaCl2) and 6% boric acid (H3BO3) by a peristaltic pump at a rate of 7.0 mL / second. After the addition was completed, the mixture was allowed to stand and solidify at 4°C for 4 hours to form a ball-milled modified biochar-microorganism composite soil conditioner (denoted as BLBC@BS.sp) with a particle size of 3-5 mm. The conditioner was then stored in a refrigerator at 4°C for later use.

[0073] Example 2

[0074] The preparation process of Example 2 is the same as that of Example 1, except that the original biochar is modified with kaolinite:

[0075] 100 g of raw biochar and 5 g of kaolinite powder were weighed and added to 1 L of deionized water to form a suspension of raw biochar and kaolinite. The suspension was then placed on a magnetic stirrer and continuously shaken at 150 r / min for 12 hours to ensure that the kaolinite sheets were fully exfoliated and in full contact with the active sites on the biochar surface. During this period, the walls of the container were manually scraped with a glass rod every 2 hours to prevent particle deposition. The resulting suspension was transferred to a centrifuge tube and centrifuged at 4000 r / min for 15 minutes. After discarding the supernatant, the sample was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane. The filtered sample was then dried in a 60℃ forced-air oven for 12 hours. The dried sample was immediately transferred to a desiccator to cool to room temperature and then sieved through a 0.25 mm stainless steel sieve to obtain kaolinite-modified biochar (denoted as KLBC).

[0076] Example 2 prepared a kaolinite-modified biochar-microorganism composite soil conditioner (denoted as KLBC@BS.sp).

[0077] Example 3

[0078] The preparation process of Example 3 is the same as that of Example 1, except that the original biochar is modified with chitosan:

[0079] 100 g of raw biochar and 4 g of chitosan powder were slowly added to 1 L of a 2% acetic acid solution. The mixture was heated continuously and stirred at a constant temperature in a 50°C water bath until a homogeneous and viscous solution was formed. After the solution cooled to room temperature, NaOH solution was slowly added dropwise while stirring to precisely adjust the pH of the mixture to 7.0. Stirring was continued for 1 hour to ensure complete reaction, followed by aging for 7 hours. After the reaction was complete, the product was repeatedly washed with deionized water until neutral and dried in a 50°C oven for 8 hours. The resulting product was ground and passed through a 0.25 mm sieve to obtain chitosan-modified biochar (denoted as CSBC).

[0080] Example 3 prepared a chitosan-modified biochar-microorganism composite soil conditioner (denoted as CSBC@BS.sp).

[0081] The mass fractions of each component in BC, BLBC, KLBC, CSBC, BC@BS.sp, BLBC@BS.sp, KLBC@BS.sp, and CSBC@BS.sp obtained in Preparation Example 1 and Examples 1-3 are shown in Table 1.

[0082] Table 1

[0083]

[0084] Scanning electron microscopy (SEM) was used to analyze BC, BLBC, KLBC, and CSBC, obtaining their surface microstructure images, such as... Figure 2 As shown.

[0085] Figure 2 These are microscopic morphology images of biochar in the embodiments of the present invention, wherein (a) is a microscopic morphology image of BC, (b) is a microscopic morphology image of KLBC, (c) is a microscopic morphology image of CSBC, and (d) is a microscopic morphology image of BLBC.

[0086] Depend on Figure 2 It can be seen that the surface of the original biochar is relatively rough and the pore structure is obvious. The surface of the kaolinite-modified biochar shows the deposition of kaolinite particles, which form a new microporous structure on the surface of the biochar.

[0087] Energy dispersive spectroscopy (EDS) was used to analyze BC, BLBC, KLBC, and CSBC, obtaining their surface elemental distribution maps, such as... Figure 3 As shown.

[0088] Figure 3 The following are elemental distribution diagrams of biochar in the embodiments of the present invention, wherein (a) is the elemental distribution diagram of BC, (b) is the elemental distribution diagram of KLBC, (c) is the elemental distribution diagram of CSBC, and (d) is the elemental distribution diagram of BLBC.

[0089] Depend on Figure 3 The EDS analysis results show that, in addition to C and O, kaolinite-modified biochar also contains high levels of Al and Si, which is consistent with the main components of kaolinite. A uniform chitosan coating forms on the surface of the chitosan-modified biochar, creating numerous pores of varying sizes, which provides more favorable conditions for efficient microbial loading onto the biochar surface. The ball-milled biochar surface shows more pronounced wear and fragmentation compared to the original biochar, with a more diverse and uniformly distributed pore structure.

[0090] N2 adsorption / desorption analysis was performed on BC, BLBC, KLBC, and CSBC, and the N2 adsorption / desorption curves were obtained, as shown in the figure. Figure 4 As shown.

[0091] Figure 4 The N2 adsorption / desorption curves of the original biochar and modified biochar in the embodiments of the present invention are shown in the figure.

[0092] Depend on Figure 4 It can be seen that in P / P 0 In the region greater than 0.8-1.0, the curve rises faster due to the adsorption and aggregation effect. Among them, the adsorption capacity of kaolinite-modified biochar increases the fastest, indicating that the pores of kaolinite-modified biochar are mainly composed of mesopores and micropores.

[0093] BET surface feature analysis was performed on BC, BLBC, KLBC, and CSBC to obtain the total pore size distribution map, as shown below. Figure 5 As shown.

[0094] Figure 5 This is a diagram showing the total pore size distribution of the original biochar and modified biochar in the embodiments of the present invention.

[0095] Depend on Figure 5 It can be seen that, compared with the original biochar, the number of small and mesopores in the structure of kaolinite-modified biochar is significantly increased. The pore size distribution of chitosan-modified biochar is mainly concentrated in the range of 2.5-10 nm, and the volume converges to 0 when the pore size is above 2 nm. The number of detected pores and the pore volume corresponding to each pore size are higher than those of the original biochar. By using the mechanical impact force of a planetary ball mill, the dense structure of the original biochar is broken down, forming ball-milled modified biochar with more micropores / mesopores and an increased number of pores.

[0096] Fourier transform infrared (FTIR) spectra of BC, BLBC, KLBC, and CSBC were analyzed to obtain their Fourier transform infrared spectra, such as... Figure 6 As shown.

[0097] Figure 6 The images show the Fourier transform infrared spectra of the original biochar and modified biochar in the embodiments of the present invention.

[0098] Depend on Figure 6 It can be seen that kaolinite-modified biochar at 1420 cm⁻¹ -1 and 600cm -1 A specific peak exists at 600 cm⁻¹, corresponding to functional groups such as -COOH and Si-O-Al. -1The broadband at these sites may be related to halogen or metal -O band stretching vibrations. These results further indicate that the specific functional groups of kaolinite form hydrogen bonds with the -OH groups of biochar, enhancing the bonding stability between the two. This strong interaction allows the layered structure of kaolinite to encapsulate the surface of biochar, forming a loose composite layer.

[0099] Compared to raw biochar, chitosan-modified biochar showed better performance at 3328 cm⁻¹. -1 2875cm -1 1650cm -1 and 1155cm -1 A specific peak appeared at this point, corresponding to ≡CH and -CH. n Functional groups such as -NO and CO ( Figure 4 This may be due to the protonation of chitosan in acetic acid solution to generate -NH3. + The chitosan forms a strong electrostatic attraction with the -COO- atoms dissociated from the biochar surface; simultaneously, the -OH groups of chitosan form hydrogen bonds with the -OH / -COOH groups of biochar; and some –NH3 groups... + It can also form weak coordination bonds with oxygen atoms on the surface of biochar, further enhancing the bonding stability.

[0100] The grinding action of zirconia balls removes impurities from the surface of biochar, exposing more surface active sites (such as -OH and -COOH), which can provide more attachment sites for Bacillus subtilis.

[0101] Scanning electron microscopy was used to analyze BC@BS.sp, BLBC@BS.sp, KLBC@BS.sp, and CSBC@BS.sp, obtaining their surface microstructure images, such as... Figure 7 As shown. Fourier transform infrared spectroscopy was used to analyze BC@BS.sp, BLBC@BS.sp, KLBC@BS.sp, and CSBC@BS.sp, obtaining their Fourier transform infrared spectra, as shown. Figure 8 As shown in the figure, the loading rate of Bacillus subtilis was tested on BC, BLBC, KLBC, and CSBC, and the results of the Bacillus subtilis loading rate of different biochar-microbial composite soil conditioners are shown in the figure. Figure 9 As shown.

[0102] Figure 7 This is a scanning electron microscope image of the biochar-microorganism composite soil conditioner in an embodiment of the present invention; Figure 8 This is the Fourier transform infrared spectrum of the biochar-microorganism composite soil conditioner in this embodiment of the invention; Figure 9The graph shows the Bacillus subtilis loading rate of biochar in this embodiment of the invention. The letters a, b, c, and d in the bar chart are statistical differences between groups. The same letter indicates that there is no significant difference between the corresponding groups at the p<0.05 level, and different letters indicate that there is a significant difference between the corresponding groups at the p<0.05 level (one-way ANOVA, Tukey HSD test).

[0103] Depend on Figures 7-9 It is known that the loose, composite interlayered pores formed by kaolinite-modified biochar can store moisture and nutrients, providing continuous nutrition for the bacteria and significantly increasing the bacterial loading rate compared to the original biochar. After deprotonation, chitosan forms hydrogen bonds with the phospholipid groups on the surface of Bacillus subtilis cell membranes. Figure 8 Furthermore, chitosan is biocompatible and can be used as a slow-release nutrient agent for Bacillus subtilis. The bacterial load rate of chitosan-modified biochar is 90.62%. Figure 9 The loading rate of the modified biochar was significantly higher than that of the original biochar (80.05%), demonstrating that chitosan can enhance the colonization capacity of bacteria through hydrogen bonding and nutrient supply. The microporous / mesoporous structure of the ball-milled modified biochar provides a micro-refuge for Bacillus subtilis, reducing the stress caused by the external environment and resulting in a significant increase in the loading rate of Bacillus subtilis compared to the original biochar.

[0104] Test Example 1

[0105] Heavy metal Cd was selected as the characteristic pollutant for adsorption and degradation performance testing: First, a 100 mg·L⁻¹ solution was prepared. -1 Cd 2+ Transfer 100 mL of the solution to a 100 mL headspace bottle and add 2 g·L⁻¹ biochar-microbial composite soil conditioner. -1 The reaction was carried out in a horizontal shaking chamber (180 rpm) at 25±2℃, with the cap sealed with a PTFE-lined jaw cover.

[0106] Samples were taken from the reaction mixture at specific time intervals (0, 3, 4, 5, 10, 15, 20, 25, and 30 hours). Subsequently, 5 mL of the supernatant was collected, filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane, and the filtered sample was analyzed for residual Cd using inductively coupled plasma mass spectrometry (ICP-MS). 2+ The concentration was determined, and the removal efficiency of Cd was calculated according to Equation (1).

[0107] Removal efficiency = (C0 - C) t Equation (1) is: ) / C0×100%

[0108] Where C0 is the initial Cd 2+ Concentration, C t Cd during detection2+ concentration.

[0109] The removal rates of Cd by various biochars and biochar-microbial composite soil conditioners are as follows: Figure 10 As shown.

[0110] Figure 10 The graph shows the Cd removal rate results of various biochar and biochar-microorganism composite soil conditioners in the embodiments of the present invention.

[0111] Depend on Figure 10 It can be seen that BC@BS.sp, BLBC@BS.sp, KLBC@BS.sp, and CSBC@BS.sp all achieved removal rates of over 85% for the heavy metal Cd, significantly higher than the 73% of the original biochar. Among them, the CSBC@BS.sp treatment showed the highest removal rate of heavy metal Cd, reaching 98%. This indicates that the different biochar-microorganism composite soil conditioners of this invention all have good removal capabilities for heavy metal Cd.

[0112] The Cd removal rate results show a significant synergistic effect between modified biochar and Bacillus subtilis. While the original biochar possesses certain structural characteristics, its surface chemical properties and pore structure are insufficient for its ability to support bacterial loading and adapt to the environment. Modification methods such as kaolinite, chitosan, and ball milling optimize the pore structure of the biochar, increase its specific surface area and active sites, and introduce key functional groups such as -OH, -C=O, -COOH, and -NH2. This not only significantly improves its loading rate and colonization stability for Bacillus subtilis but also enhances its ability to synergize with Cd. 2+ Forming stable complexes, or reacting with Cd 2+ The formation of electrostatic attraction enhances the attraction of Cd 2+ Physical retention of Cd 2+ The removal rate is further improved compared to the original biochar. Simultaneously, the loaded microbial cells can further complex heavy metal Cd through mechanisms such as extracellular polymers, complementing the adsorption and complexation effects of the modified biochar to achieve highly efficient Cd removal. Therefore, the modification of the original biochar aims to fully leverage the synergistic function of the material and microorganisms through precise control of its structure and surface properties, thereby significantly improving its overall performance in heavy metal remediation.

[0113] To evaluate the passivation effect of the biochar-microorganism composite soil conditioner of this invention on available Cd (DTPA-Cd) in Cd-contaminated soil and its promoting effect on crop growth, this embodiment uses Cd-contaminated farmland soil as the research matrix. The available Cd content in the soil was determined by pot experiment and diethylenetriaminepentaacetic acid (DTPA) extraction method. At the same time, the plant height, root length, biomass and other indicators of maize were monitored. The effects of applying Bacillus subtilis suspension alone and applying different biochar / biochar-microorganism composite soil conditioners on reducing soil Cd bioavailability and regulating crop growth were analyzed.

[0114] Test Example 2

[0115] The soil used in the pot experiment was collected from a Cd-contaminated farmland. The basic physicochemical properties of the soil were: pH 7.15, organic matter content 4.52%, and total Cd content 8.69 mg / kg.

[0116] Add 3% (by dry weight) of Bacillus subtilis suspension / different types of biochar / different types of biochar-microbial composite soil conditioner to the original Cd-contaminated soil (CK), and mix thoroughly. Fill each pot (polyethylene pot, 13 cm top diameter, 15 cm height) with 1.5 kg of soil. Sow 5 corn seeds in each pot, and thin to 2 uniformly vigorous seedlings per pot after germination. Each treatment has 3 replicates. Culture in a greenhouse (day / night temperature 25 / 18℃, 12 hours light), watering regularly to maintain soil field capacity at 60%-70%. The culture period is 30 days.

[0117] After the 30-day culture period, relevant indicators were measured:

[0118] Determination of available Cd content in soil: Soil samples from the 0-15 cm soil layer were collected, air-dried, and passed through a 2 mm nylon sieve. 10.0 g of soil sample was weighed and added to DTPA extraction solution (0.005 mol / L DTPA - 0.01 mol / L CaCl2 - 0.1 mol / L triethanolamine (TEA), pH 7.3) at a soil-to-liquid ratio of 1:2. Extraction was carried out at 25℃ with shaking at 180 rpm / min for 2 hours. The extract was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane, and the Cd concentration in the filtrate was determined using inductively coupled plasma mass spectrometry (ICP-MS, NexION 350X, PerkinElmer). The determination process strictly followed the standard "Determination of Available Zinc, Manganese, Iron, and Copper Content in Soil - Diethylenetriaminepentaacetic Acid Extraction Method" (GB / T 23739-2009). Results are as follows: Figure 11 As shown.

[0119] Soil Cd speciation analysis: The same soil sample was sequentially extracted using acetic acid (for weakly acid-extractable Cd), hydroxylamine hydrochloride (for reducible Cd), and hydrogen peroxide + ammonium acetate (for oxidizable Cd). The final residue was digested and stored as a residue. The Cd content of each extract was determined by ICP-MS after centrifugation and filtration. The procedure strictly followed the "Sequential Extraction Procedure for 13 Trace Elements in Soil and Sediment" (GB / T25282—2010). Results are as follows: Figure 12 As shown.

[0120] Crop growth indicators: Carefully remove the entire corn plant and wash it with deionized water. Use a measuring tape to measure the root length (from the root-stem junction to the longest root tip) and plant height (from the root-stem junction to the longest leaf tip). Results are as follows: Figure 13 As shown. The plant was divided into root and aboveground parts, which were blanched at 105℃ for 30 minutes, then dried at 75℃ to constant weight. The dry weight was measured using a 0.01 g electronic balance, which is the biomass. The results are as follows. Figure 14 As shown.

[0121] Figure 11 The graph shows the results of soil available Cd content after applying different remediation agents according to the present invention. In the bar chart, the letters a, b, c, d, e, and f are statistical indicators of differences between groups. The same letter indicates that there is no significant difference between the corresponding groups at the p < 0.05 level, and different letters indicate that there is a significant difference between the corresponding groups at the p < 0.05 level (one-way ANOVA, Tukey HSD test). Figure 12 The graph shows the percentage of different Cd components in the soil after applying different remediation agents according to this invention. Figure 13 The graph shows the results of plant height and root length after applying different remedial agents according to the present invention. In the bar chart, the letters a, b, c, d, e, f, g, and h are statistical indicators of differences between groups. The same letter indicates that there is no significant difference between the corresponding groups at the p < 0.05 level, and different letters indicate that there is a significant difference between the corresponding groups at the p < 0.05 level (one-way ANOVA, Tukey HSD test). Figure 14 The graph shows the results of plant biomass after applying different remedial agents according to the present invention. In the bar chart, the letters a, b, c, d, e, f, g, h, i, and f are statistical differences between groups. The same letter indicates that there is no significant difference between the corresponding groups at the p<0.05 level, and different letters indicate that there is a significant difference between the corresponding groups at the p<0.05 level (one-way ANOVA, Tukey HSD test).

[0122] according to Figures 11-14 The following calculations were performed on the rate of decrease in available Cd content, the conversion rate of residual Cd, the root length growth rate, the plant height growth rate, and the total biomass growth rate. The results are shown in Table 2 below.

[0123] Table 2

[0124]

[0125] Note: The data in Table 2 are obtained by comparing each group with the original Cd contaminated soil (CK) group data.

[0126] As shown in Table 2, compared with treatments involving Bacillus subtilis (BS.sp) or raw biochar (BC), the three modified biochars (BLBC, ball-milled modified biochar; KLBC, kaolinite-modified biochar; CSBC, chitosan-modified biochar) and their biochar-microbial composite soil conditioners (BLBC@BS.sp, ball-milled modified biochar-microbial composite soil conditioner; KLBC@BS.sp, kaolinite-modified biochar-microbial composite soil conditioner; CSBC@BS.sp, chitosan-modified biochar-microbial composite soil conditioner) all exhibited significant advantages in the remediation of Cd-contaminated soil and the promotion of maize growth. Compared with the control group (CK), the modified biochar and biochar-microbial composite soil conditioner treatment groups significantly reduced the available Cd content in the soil. Among them, the reduction rate of available Cd content in the biochar-microbial composite soil conditioner treatment group was 47.74%, 50.49%, and 57.50%, respectively, which was higher than 1.37% for BS.sp, 20.62% for BC, and 41.63% for BC@BS.sp.

[0127] Soil Cd speciation analysis showed that ( Figure 12 After adding biochar-microbial composite soil conditioner, the bioavailable, weakly acidic extractable Cd in the soil was converted into oxidizable and residual forms. Simultaneously, the root length, plant height, and biomass of maize plants also increased compared to the control group. Figure 13 These advantages stem from the synergistic mechanism between different modification methods and Bacillus subtilis.

[0128] In summary, the preparation of this biochar-microbial composite soil conditioner uses agricultural waste straw as raw material. Raw biochar is prepared through an oxygen-limited, temperature-controlled pyrolysis process. Modified biochar carriers with high specific surface area and rich functional groups are obtained through ball milling, kaolinite, or chitosan modification. Furthermore, plant rhizosphere growth-promoting bacteria are loaded onto the modified biochar using a microbial immobilization method, forming a "modified biochar-functional bacteria" composite micro-ecosystem. This simultaneously achieves straw nano-sizing, surface functionalization, and efficient bacterial loading, avoiding the loss of bacterial activity in multi-step processes. This biochar-microbial composite soil conditioner, through the synergistic effect of biochar's adsorption and passivation and the bio-immobilization and transformation mechanism of functional bacteria, significantly reduces the content of available Cd in the soil and transforms Cd from a bioavailable form into a stable residual state. Simultaneously, it significantly promotes crop growth by releasing nutrients and secreting growth-promoting substances. Experiments show that this biochar-microbial composite soil conditioner has a significant effect on Cd... 2+ The removal rate exceeded 70%, and the reduction rate of available Cd in the soil reached a maximum of 57.50%. Compared with the control group, the root length, plant height, and biomass of maize increased by up to 46.82%, 73.12%, and 92.11%, respectively. This invention combines the resource utilization of agricultural waste, high remediation efficiency, environmental friendliness, and operational economy, providing a practical solution for the safe in-situ remediation of Cd-contaminated farmland and safe crop production, demonstrating its practical application value.

[0129] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biochar-microorganism composite soil conditioner, characterized in that, include: Modified biochar, and plant rhizosphere growth-promoting bacteria supported on the modified biochar; An embedding agent is used to coat the outer surface of modified biochar loaded with plant rhizosphere growth-promoting bacteria. The modified biochar is selected from any one of ball-milled modified biochar, kaolinite modified biochar, or chitosan modified biochar.

2. The biochar-microorganism composite soil conditioner according to claim 1, characterized in that: The biochar-microorganism composite soil conditioner comprises, by mass fraction: 30-45% modified biochar, 30-40% encapsulating agent, and 20-35% plant rhizosphere growth-promoting bacteria.

3. The biochar-microorganism composite soil conditioner according to claim 1, characterized in that: The plant rhizosphere growth-promoting bacteria are selected from any one or more of the genera Bacillus, Streptomyces, Pseudomonas, and Burkholderia. The encapsulating agent is a cross-linked dual network formed by polyvinyl alcohol and calcium alginate.

4. A method for preparing a biochar-microorganism composite soil conditioner as described in any one of claims 1-3, characterized in that, include: The modified biochar was mixed in a suspension of plant rhizosphere growth-promoting bacteria to obtain the first mixture. The first mixture was mixed with the polyvinyl alcohol-sodium alginate gel solution to obtain the second mixture. The second mixture was added dropwise to the crosslinking agent solution, and after crosslinking and fixation, it was freeze-dried to obtain a biochar-microorganism composite soil conditioner.

5. The preparation method according to claim 4, characterized in that: The mass ratio of polyvinyl alcohol to sodium alginate in the polyvinyl alcohol-sodium alginate gel-like mixed solution is 8:1.0-1.

4. The crosslinking agent is selected from a mixed solution of calcium chloride and boric acid.

6. The preparation method according to claim 4, characterized in that: The ratio of modified biochar to plant rhizosphere growth-promoting bacteria suspension is 1g:5-30mL.

7. The preparation method according to claim 4, characterized in that: The modified biochar is selected from any one of ball-milled modified biochar, kaolinite modified biochar, or chitosan modified biochar. The preparation method of kaolinite-modified biochar includes: adding raw biochar and kaolinite powder into deionized water, stirring, centrifuging, filtering, drying, and then passing through a 0.25mm sieve to obtain kaolinite-modified biochar; The preparation method of chitosan-modified biochar includes: adding raw biochar and chitosan into an acetic acid solution, heating and stirring, adjusting the pH to 7.0 after cooling, drying, grinding and passing through a 0.25 mm sieve to obtain chitosan-modified biochar.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the original biochar to kaolinite powder is 15-25:1; The mass ratio of the original biochar to chitosan is 20-30:

1.

9. The application of the biochar-microorganism composite soil conditioner as described in claim 1, wherein: The applications include using the biochar-microorganism composite soil conditioner for cadmium pollution remediation and crop growth promotion.

10. The application according to claim 9, characterized in that: The cadmium pollution remediation includes the remediation of cadmium pollution in water bodies and the remediation of cadmium pollution in soil; In the remediation of cadmium pollution in the water body, the dosage of the biochar-microorganism composite soil conditioner is 0.1-2 g / L, the reaction time is 0.5-35 hours, and the cadmium concentration is 10-100 mg / L. In the remediation of cadmium pollution in the soil, the dosage of the biochar-microorganism composite soil conditioner is 2-5% of the dry weight of the soil.

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