Biochar-based nitrogen-fixing bacterium agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial agents, and particularly relates to a charcoal-based nitrogen-fixing bacterium agent as well as a preparation method and application thereof. According to the invention, a biochar-humic acid-sodium alginate embedding mode is adopted, so that a porous protection cabin can be provided to adsorb plant rhizosphere nutrients. Meanwhile, a low-oxygen microcell is created to protect nitrogenase, physical protection is provided for thalli, and external environmental stress is buffered. Wherein humic acid is used as a high-quality organic carbon source and is beneficial to stimulating thalli to rapidly proliferate in embedded particles and rhizosphere, and a large number of active cells can be obtained in a short time. The biochar-based nitrogen-fixing bacterium agent (namely biochar-humic acid-sodium alginate microspheres) prepared by the invention has the advantages of simple and convenient preparation method, stable storage performance and the like, and compared with other biological agents, the biomass of the dried microbial agent reaches 2.2 * 10 < 10 > CFU / g, so that the biochar-based nitrogen-fixing bacterium agent has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, and specifically relates to a biochar-based nitrogen-fixing bacterium inoculant based on diazotrophic Gluconacetobacter diazotrophicus ZJB-2025351, its preparation method, and its application. Background Technology

[0002] The improper use of chemical fertilizers has led to numerous problems, such as soil acidification, atmospheric nitrogen deposition, water quality deterioration, declining agricultural product quality, and nitrogen-phosphorus imbalance. In recent decades, the widespread application of plant growth-promoting microorganisms (PGPBs) has been accepted as an effective alternative to chemical agricultural products. PGPBs are promising green agricultural biopharmaceuticals with effects such as promoting plant growth and reducing environmental stress, improving soil microbiology, and enhancing soil fertility. However, a major challenge in using PGPBs is the loss of cell viability during storage and under harsh field conditions. When PGPBs are directly injected into the soil, their effectiveness is significantly affected due to the loss of their biological activity and efficacy. Therefore, it is necessary to improve the survival rate of soil-introduced microorganisms and enhance their growth-promoting effects on cultivated crops.

[0003] Currently, there are two main research hotspots in microbial immobilization technology: the implementation techniques for immobilized bacterial agents and the selection of immobilization carrier materials. The implementation techniques for immobilized bacterial agents can be mainly divided into adsorption, encapsulation, cross-linking, and covalent bonding methods, depending on the interaction between the immobilization carrier and cells. Adsorption methods are widely used due to their simplicity and high bacterial survival rate. Commonly used immobilization carrier materials include alginate, agar, carrageenan, gelatin, sponge, polyvinyl alcohol, diatomaceous earth, kaolin, biochar, wheat bran, straw, rice husks, and coconut shells, but reports on endophytic nitrogen-fixing microorganisms are still relatively few.

[0004] Biochar not only serves as a slow-release carrier for bacterial strains, but its rich porous structure and adsorption properties can also improve the physical and chemical properties of soil, regulate soil pH, enhance nutrient and water use efficiency, increase soil microbial abundance, and increase soil element content. Humic acid (HA), as a high-quality organic carbon source, can stimulate rapid proliferation of bacteria within the encapsulated particles and in the rhizosphere, and can also synergistically improve the rhizosphere microecology with biochar. Sodium alginate (SA) is a commonly used material for preparing gel beads; it is non-toxic, easily degradable, and low-cost, and can interact with calcium and other divalent metal cations to prepare gel beads for protecting microorganisms adsorbed on biochar.

[0005] Therefore, this invention uses biochar as a carrier and employs an adsorption-embedding immobilization method, with plant endophytic bacteria—diazotrophic glucose acetic acid bacillus—as the embedding target, to prepare a biochar-based composite immobilized bacterial agent, in order to ensure the long-term survival of beneficial microorganisms and improve their application performance. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of existing PGPB in terms of instability and poor environmental adaptability under biotic and abiotic stresses, and provides a biochar-based nitrogen-fixing bacterium inoculant and its preparation method, and applies it to plant growth promotion.

[0007] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A biochar-based nitrogen-fixing bacteria agent includes a diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 adsorbed on biochar. The diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 is embedded in a mixed solution of humic acid and sodium alginate and is cross-linked and fixed by divalent metal ions.

[0008] This invention, through in-depth analysis of the complementary relationship between strain characteristics and carrier function, organically combines biochar, humic acid, and sodium alginate to develop a novel immobilization system that synergistically enhances the overall performance of nitrogen-fixing bacterial agents. Regarding the carrier selection, biochar not only serves as a physical adsorption matrix, but its rich porous structure also provides a habitat for the bacteria (i.e., the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351), while simultaneously adsorbing rhizosphere nutrients, creating a suitable microenvironment for the microorganisms. Humic acid, as a high-quality organic carbon source, can be directly utilized by microorganisms, stimulating rapid bacterial proliferation within the particles and in the rhizosphere; while sodium alginate, under the cross-linking of divalent metal ions, forms a stable gel network, effectively encapsulating the biochar containing the adsorbed bacteria, constructing a microcapsule system that combines physical protection and nutrient slow-release functions.

[0009] This invention employs a step-by-step immobilization process of "adsorption-embedding-crosslinking." First, the high adsorption capacity of biochar enriches the bacterial cells within a porous carrier. Then, a secondary embedding is performed using a humic acid-sodium alginate mixed solution. Finally, a stable three-dimensional network structure is formed through crosslinking with divalent metal ions. This step-by-step immobilization method not only effectively avoids bacterial damage that might occur with direct embedding but also fully utilizes different carrier materials: biochar provides habitat and soil amendment, humic acid provides nutrients, and sodium alginate acts as a physical barrier. Furthermore, this invention significantly improves the rhizosphere microecological environment through the combination of humic acid and biochar, while the sodium alginate gel network maintains structural stability and its moderate permeability ensures nutrient transport and metabolite diffusion, thereby promoting a significant synergistic effect among the three components.

[0010] Preferably, the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 has the accession number CCTCC NO: M 20252292 and is deposited at the China Center for Type Culture Collection.

[0011] Preferably, the divalent metal ion is a calcium ion.

[0012] Preferably, the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 is at least one of bacterial powder, wet bacterial cells, and culture.

[0013] A method for preparing a biochar-based nitrogen-fixing bacterial inoculant as described above includes the following steps: S1: The culture of diazotrophic glucose acetic acid bacteria mutant was mixed with biochar for adsorption, and centrifuged to obtain biochar loaded with diazotrophic glucose acetic acid bacteria mutant. S2: The biochar loaded with the diazotrophic glucose acetic acid bacteria mutant obtained in step S1 is added to a mixed solution of humic acid and sodium alginate for encapsulation to obtain encapsulated biochar. S3: The encapsulated biochar obtained in step S2 is added to a divalent metal ion solution for cross-linking and solidification. After filtration and washing, biochar-humic acid-sodium alginate microspheres are obtained, which are biochar-based nitrogen-fixing bacteria agents.

[0014] In the biochar-humic acid-sodium alginate microspheres prepared in this invention, the diazotrophic glucose acetic acid bacterium mutant strain ZJB-2025351 serves as the active ingredient, and biochar acts as an adsorbent and fixative. It not only acts as a slow-release carrier for the strain but also, due to its rich pore structure and adsorption properties, improves the physical and chemical properties of the soil, regulates soil pH, enhances nutrient and water use efficiency, increases the abundance of soil microorganisms, and increases the content of soil elements. Simultaneously, it creates a low-oxygen microzone to protect nitrogenase, providing physical protection for the bacteria and buffering external environmental stresses. Humic acid serves as a high-quality organic carbon source, helping to stimulate rapid bacterial proliferation within the embedded particles and rhizosphere, enabling the rapid acquisition of a large number of active cells. It also synergistically works with biochar to significantly improve the rhizosphere microecology. The cross-linking and calcification of sodium alginate with calcium chloride significantly improves the embedding rate, survival rate, stability, and environmental adaptability of the biochar-humic acid-sodium alginate microspheres, thereby effectively enhancing their application performance. The biochar-based nitrogen-fixing bacteria inoculant prepared by this invention (i.e., biochar-humic acid-sodium alginate microspheres) has advantages such as simple preparation method and stable storage performance. Compared with other biological agents, the biomass of this inoculant reaches 2.2 × 10⁻⁶ after drying. 10 CFU / g has good application prospects.

[0015] Preferably, in step S1, adsorption is carried out under water bath oscillation conditions, with an adsorption temperature of 20~30℃, an adsorption time of 20~40min, and an oscillation rate of 30~180r / min.

[0016] Preferably, the amount of biochar added in step S1 is 3-8%; the concentration of the diazotrophic glucose acetic acid bacteria mutant culture is 1-9 × 10⁻⁶. 9 CFU / mL.

[0017] Preferably, the method for preparing the diazotrophic glucose acetic acid bacterium mutant culture in step S1 includes the following steps: The diazotrophic glucose acetic acid bacterium mutant strain ZJB-2025351 was cultured in a liquid culture medium containing carbon source, nitrogen source and inorganic salts for 24-72 h.

[0018] Preferably, the final concentration of humic acid in the mixed solution in step S2 is 3~6 g / L, and the final concentration of sodium alginate is 2~8 g / L.

[0019] Preferably, in step S3, the concentration of the divalent metal ion solution is 0.03~0.11 mol / L, and the cross-linking curing time is 20~60 min.

[0020] Preferably, the divalent metal ion solution in step S3 is a calcium chloride solution.

[0021] The application of biochar-based nitrogen-fixing bacteria inoculants in promoting plant growth, as described above.

[0022] A method for using the biochar-based nitrogen-fixing bacteria agent as described above in promoting plant growth is to apply it directly or freeze-dry it to the soil around the roots of the plant after transplanting or sowing, at a dosage of 1-5g per plant, wherein the plant is any one or more of tomato, corn, soybean, pepper, rice, and wheat.

[0023] Therefore, the present invention has the following beneficial effects: Compared with existing technologies, the biochar-based nitrogen-fixing bacteria agent of this invention has fewer raw materials, a simpler preparation method, stable strain function, and a long-lasting effect, making it easy to carry out industrial production and widespread application. Moreover, in plant pot experiments, compared with free diazotrophic glucose acetic acid bacteria agents, this immobilized agent can effectively enhance its growth-promoting effect and improve soil physicochemical properties. Especially after 180 days of storage at room temperature, the immobilized agent still maintains high activity, meeting the requirements of green agricultural development and laying the foundation for the further field application of environmentally friendly microbial agents and sustainable agricultural development. Attached Figure Description

[0024] Figure 1A schematic diagram of the preparation process for biochar-based nitrogen-fixing bacteria inoculants.

[0025] Figure 2 This is a schematic diagram of the dry and wet states of biochar-humic acid-sodium alginate microspheres.

[0026] Figure 3 The graph shows the test results of the heat resistance of biochar-humic acid-sodium alginate microspheres.

[0027] Figure 4 The figure shows the test results of the sustained-release performance of biochar-humic acid-sodium alginate microspheres.

[0028] Figure 5 Figure showing the survival rate test results of diazotrophic glucose acetic acid bacteria mutants stored at different temperatures.

[0029] Figure 6 The figure shows the effect of biochar-based nitrogen-fixing bacteria inoculant on tomato quality. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0031] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

[0032] Example 1: Preparation of biochar-humic acid-sodium alginate microspheres (i.e., biochar-based nitrogen-fixing bacteria inoculant) The immobilized strain in this embodiment is the diazotrophic glutamate mutant ZJB-2025351, with accession number CCTCC NO: M 20252292, accession date October 22, 2025, and depositary institution China Center for Type Culture Collection, Wuhan University, China, 430072, China.

[0033] The preparation method of biochar-humic acid-sodium alginate microspheres (i.e., biochar-based nitrogen-fixing bacteria inoculant) in this embodiment includes the following steps: S1: The diazotrophic glycocalycemic mutant *Glucosamine acetate* ZJB-2025351, stored in glycerol tubes at -80℃, was removed and activated by streaking on MA solid medium. It was then incubated overnight at 28℃. The activated strain was transferred to test tubes containing MA liquid medium and incubated at 28℃ and 200 rpm for 24 h to obtain a seed culture. The seed culture was then transferred to shake flasks containing MA liquid medium and incubated at 28℃ and 200 rpm for 48 h to obtain the diazotrophic glycocalycemic mutant *Glucosamine acetate* ZJB-2025351 culture. The viable count was calculated using the dilution plating method. The MA medium consisted of 3.0 g / L peptone, 5.0 g / L yeast extract, and 25.0 g / L mannitol. S2: Add 5g of sterilized dry biochar to 100mL of diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 culture, place in a water bath and shake to adsorb, set the adsorption temperature to 28℃, the shaking rate to 90r / min, and the adsorption time to 30min, so that ZJB-2025351 is fully adsorbed onto the biochar, and a biochar-bacterial solution is obtained, which is recorded as the first product; S3: Centrifuge the first product at 8000 rpm for 10 min to obtain biochar loaded with the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351, which is denoted as the second product. S4: Add 0.5g of humic acid and 0.7g of sodium alginate to 100mL of water, mix well, sterilize and cool to obtain a mixed solution of humic acid and sodium alginate (i.e., embedding solution). Add the second product obtained in step S3 to the mixed solution of humic acid and sodium alginate at a mass ratio of 3:7, shake well to coat the second product with the mixed solution, and obtain the embedded biochar, which is denoted as the third product. S5: The third product obtained in step S4 is added dropwise to a 0.07 mol / L calcium chloride solution using a sterile syringe. The mixture is cross-linked and fixed for 30 min. Microspheres are collected using a sterile sieve and gently washed 2-3 times with sterile 0.85% NaCl solution to remove residual Ca2+ from the surface. 2+ The unencapsulated bacterial cells were combined to obtain biochar-humic acid-sodium alginate microspheres, i.e., biochar-based nitrogen-fixing bacteria inoculum. A schematic diagram of the preparation process of the biochar-based nitrogen-fixing bacteria inoculum is shown below. Figure 1 As shown.

[0034] Example 2: Determination of the physicochemical properties of biochar-humic acid-sodium alginate microspheres Biochar-humic acid-sodium alginate microspheres were prepared according to the method in Example 1. A schematic diagram of the dry and wet states of the biochar-humic acid-sodium alginate microspheres is shown below. Figure 2 As shown. Among them, Figure 2In the figure, A represents biochar-humic acid-sodium alginate microspheres in a moist state (i.e., wet microspheres). Figure 2 In this context, B represents the dried biochar-humic acid-sodium alginate microspheres (i.e., dry microspheres). The biochar-humic acid-sodium alginate microspheres prepared in Example 1 were subjected to physicochemical property testing according to the following method: (1) Morphology of biochar-humic acid-sodium alginate microspheres: Observe the morphology and uniformity of biochar-humic acid-sodium alginate microspheres. Randomly select 50 prepared biochar-humic acid-sodium alginate microspheres and measure their particle size with vernier calipers. Take the average value. (2) Mechanical strength: 50 biochar-humic acid-sodium alginate microspheres were placed in a 250mL conical flask, 100mL of deionized water was added, and the microspheres were shaken on a constant temperature shaker for 12h. The ratio of intact microspheres to the original total number of microspheres was used to represent the strength coefficient of the microspheres. (3) Moisture content: 30 wet microspheres were randomly selected and weighed, recorded as m1. They were then dried in an oven at 60℃ for 36 hours and weighed again, recorded as m2. The moisture content was calculated using the following formula: Moisture content (%) = (m1 - m2) / m1 × 100%; (4) Swelling performance: Take 30 wet microspheres and measure their diameter (d1) with vernier calipers. Then place them in a 250mL conical flask, add 100mL of deionized water, and refrigerate at 4℃ for 6h. Then measure their diameter (d2) again. The ratio of the total diameter of the expanded microspheres (D2) to the total diameter of the original microspheres (D1) represents the swelling performance of the microspheres. The physicochemical properties of the biochar-humic acid-sodium alginate microspheres are shown in Table 1 below.

[0035] Table 1: Physicochemical properties of biochar-humic acid-sodium alginate microspheres form Particle size (mm) Mechanical strength Moisture content swelling properties Small spherical, black in appearance 3.2 100% 83.95% 1.088 .

[0036] Example 3: Encapsulation effect of biochar-humic acid-sodium alginate microspheres (1) Method for determining the microsphere encapsulation rate (EE%): According to the preparation method of biochar-humic acid-sodium alginate microspheres in Example 1, the total number of cells in the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 culture before encapsulation was first determined. 1 mL of the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 culture was taken, diluted, and cultured in MA medium for counting. The total number of cells in a certain volume of the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 culture was then calculated and recorded as N0. 1 mL of the encapsulated, cross-linked, and washed solution was taken, diluted, and cultured in MA medium for counting. The total number of unencapsulated cells was calculated and recorded as N1. The encapsulation rate (EE%) was calculated according to the following formula: Encapsulation rate (EE%) = (N0 - N1) / N0 × 100%; The encapsulation efficiency of biochar-humic acid-sodium alginate microspheres is shown in Table 2 below: Table 2: Encapsulation efficiency of biochar-humic acid-sodium alginate microspheres Numbering <![CDATA[N0(CFU)]]> <![CDATA[N1(CFU)]]> Encapsulation rate (EE%) 1 <![CDATA[4.20×10 10 ]]> <![CDATA[5.9×10 9 ]]> 85.95 2 <![CDATA[3.53×10 10 ]]> <![CDATA[3.8×10 9 ]]> 89.23 3 <![CDATA[5.22×10 10 ]]> <![CDATA[8.4×10 9 ]]> 83.91 average value <![CDATA[4.31×10 10 ]]> <![CDATA[6.0×10 9 ]]> 86.36 .

[0037] As shown in Table 2, after three repeated tests, the embedding effect was above 83% each time, with an average embedding effect of 86.36%. This indicates that biochar-humic acid-sodium alginate microspheres can effectively embed and fix the diazotrophic glucosinolate mutant ZJB-2025351.

[0038] (2) UV resistance test method: According to the preparation method of biochar-humic acid-sodium alginate microspheres in Example 1, microsphere products with the same bacterial count and untreated diazotrophic glucose acetic acid bacillus mutant cell culture were placed in sterile petri dishes and irradiated under a UV lamp for 30 min. The microsphere samples before and after irradiation were placed in an appropriate amount of sterilized sodium citrate solution (0.1 M, pH 7.0), and sonicated to rupture and dissolve the microspheres. They were then diluted and cultured in MA medium, and the survival rate was calculated. The UV resistance of biochar-humic acid-sodium alginate microspheres is shown in Table 3 below.

[0039] Table 3: UV resistance properties of biochar-humic acid-sodium alginate microspheres Unburied Embedding group Before irradiation (CFU / mL) <![CDATA[(6.33±0.17)×10 9 ]]> <![CDATA[(6.12±0.17)×10 9 ]]> Post-irradiation (CFU / mL) <![CDATA[(2.66±0.05)×10 9 ]]> <![CDATA[(3.93±0.02)×10 9 ]]> Survival rate (%) 42.02% 64.22% .

[0040] The survival results in Table 3 show that microsphere encapsulation of the diazotrophic glucose acetic acid bacillus mutant can improve its UV resistance. After UV irradiation treatment, the survival rate of the diazotrophic glucose acetic acid bacillus mutant encapsulated in microspheres was 64.22%, which was 22.2% higher than that of the unencapsulated group.

[0041] (3) Method for determining heat resistance: Five portions of the microspheres prepared in Example 1 of the same mass were placed in sterile 10 mL centrifuge tubes, and an equal volume of sterilized sodium citrate solution (0.1 M, pH 7.0) was added. The tubes were heated in water baths at 40℃, 50℃, 60℃, 70℃, and 80℃ for 10 min, respectively. Another group was placed at room temperature for 10 min as a control. Finally, the microspheres were sonicated to rupture and dissolve, diluted and cultured in MA medium, and the survival rate was calculated. The heat resistance test results of the biochar-humic acid-sodium alginate microspheres are shown in […]. Figure 3 .

[0042] Depend on Figure 3Analysis shows that under the protection of biochar-humic acid-sodium alginate microspheres, even after treatment at 80℃ for 10 min, the survival rate of the diazotrophic glucose acetic acid bacteria mutant was still 73.7%, indicating that biochar-humic acid-sodium alginate microspheres can also improve the heat resistance of the diazotrophic glucose acetic acid bacteria mutant, which is beneficial for its application in the field.

[0043] Example 4: Nitrogen fixation performance of biochar-humic acid-sodium alginate microspheres The diazotrophic *Gluconobacterium acetate* mutant ZJB-2025351, as a strain with nitrogen-fixing capabilities, was used to determine the nitrogen-fixing performance of biochar-humic acid-sodium alginate microspheres obtained after encapsulation treatment. Using the acetylene reduction method, 0.2 g of the biochar-humic acid-sodium alginate microspheres prepared according to the method in Example 1 were placed in a sealed serum bottle containing nitrogen-free culture medium. 10% acetylene gas was injected, and the mixture was incubated at 28°C for 24 h. The amount of ethylene generated was measured by gas chromatography, and the nitrogenase activity of the microspheres was calculated. Empty microspheres without the strain were used as a control. The nitrogen-free culture medium was formulated as follows: mannitol 10g, KH₂PO₄ 0.2g, MgSO₄·7H₂O 0.2g, NaCl 0.2g, CaSO₄·2H₂O 0.1g, CaCO₃ 5g, agar powder 15-20g, and water was added to a final volume of 1000mL, adjusting the pH to 7.2-7.4. The nitrogen fixation results of the microspheres showed that the nitrogen fixation activity of the biochar-humic acid-sodium alginate microspheres reached 463.2 nmol·mg⁻¹. -1 ·h -1 The nitrogen fixation efficiency was 1.26 times higher than that of the unencapsulated strain, indicating that the encapsulation operation does not negatively affect the nitrogen fixation function of the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351.

[0044] Example 5: Determination of sustained release of biochar-humic acid-sodium alginate microspheres Take 2g of the biochar-humic acid-sodium alginate microspheres prepared in Example 1 and place them in 10mL of sterile PBS buffer. At room temperature, the number (CFU / mL) of the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 in the PBS buffer was measured every 12h using the dilution coating method. The results were recorded at 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, and 108h. Each time period was coated three times. The sustained-release performance test results of the biochar-humic acid-sodium alginate microspheres are shown in the figure. Figure 4 The sustained-release results of biochar-humic acid-sodium alginate microspheres showed that the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 encapsulated in biochar-based materials could reach its maximum release amount of 1.88 × 10⁻⁶ h at 72 h. 8The CFU / mL reading indicates that during application, the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 can be rapidly released from the immobilized material to perform its function within a short period of time.

[0045] Example 6: Storage stability of biochar-humic acid-sodium alginate microspheres The biochar-humic acid-sodium alginate microspheres prepared according to the method in Example 1 were freeze-dried for 48 h. The dried microspheres were stored at -20℃, 4℃, and 25℃, respectively. Periodically, 0.5 g of the dried microspheres were placed in 10 mL of sterile PBS buffer and sonicated to rupture and dissolve the microspheres. The microspheres were then diluted and cultured in MA medium, and the survival rate of the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 was determined after different storage times. The survival rate test results of the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 under different storage temperatures are shown below. Figure 5 After being stored at -20°C for 180 days, the dried biochar-humic acid-sodium alginate microsphere formulation still maintained a survival rate of approximately 80% (4.0 × 10⁻⁶ viable cells). 9 After being stored at 4℃ for 180 days, the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 still maintained a survival rate of approximately 71.2% (3.56 × 10⁻⁶ CFU / g). 9 After being stored at 25℃ for 180 days, the diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 still maintained a survival rate of approximately 53.83% (2.69 × 10⁻⁶ CFU / g). 9 CFU / g).

[0046] Example 7: Effects of biochar-based nitrogen-fixing bacteria inoculant on tomato growth Plump, uniformly sized tomato seeds were selected, disinfected with 6% sodium hypochlorite for 5 minutes, rinsed three times with distilled water, and then germinated in sterile vermiculite at room temperature. After 7 days of germination, seedlings with uniform growth were selected and placed in small pots (7cm in diameter and 10cm in height) containing 100g of sterilized substrate (vermiculite and soil volume ratio 1:1), with one seedling per pot. A total of three groups were set up: one control group and two experimental groups. Each group underwent five parallel experiments to examine the effects of biochar-humic acid-sodium alginate microspheres on tomato growth. The pot experiment group settings are shown in Table 4 below.

[0047] Table 4: Potted Plant Experiment Group Settings Serial Number deal with CK No bacteria T1 Inoculation of free bacterial solution (equal amount of bacterial cells) T2 Empty carrier microspheres T3 Immobilized microspheres .

[0048] The biochar-humic acid-sodium alginate microspheres prepared according to the method in Example 1 were applied at a rate of 3g per pot, once every 7 days, to the roots of the plants, for a total of 3 applications. The control group received no additional treatment. After applying the biochar-humic acid-sodium alginate microspheres, 20mL of sterile water was applied. The tomatoes were collected 30 days after sowing, and the growth indicators of the tomatoes were recorded. The growth of tomatoes in different groups is shown in Table 5 below.

[0049] Table 5: Growth indicators of tomato plants under different treatments deal with Plant height (cm) Stem diameter (mm) Fresh weight above ground (g / plant) Fresh weight underground (g / plant) Total dry weight (g / plant) CK 18.5±1.80 6.2±0.4 12.8±1.9 3.5±0.3 2.8±0.1 T1 21.3±2.11 6.6±0.3 15.9±2.8 4.4±0.6 3.5±0.1 T2 19.8±1.91 6.3±0.3 13.5±1.6 3.9±0.4 3.0±0.2 T3 25.7±2.33 7.5±0.5 25.8±2.6 6.8±0.2 4.6±0.2 .

[0050] Data from group T2 (empty carrier microspheres) showed that even without bacteria, biochar and humic acid could improve the rhizosphere environment and bring about a certain growth-promoting effect (compared to the CK group). In group T3 (immobilized microspheres, i.e., biochar-humic acid-sodium alginate microspheres), tomato plants showed the highest plant height and biomass, and their growth was better than that of groups T1 and T2, respectively. This indicates that the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 and the biochar-humic acid-sodium alginate carrier material jointly promoted plant growth, demonstrating the necessity and application potential of developing and using this immobilization and embedding technology.

[0051] Example 8: Effects of biochar-based nitrogen-fixing bacteria inoculant on tomato quality The difference between this embodiment and Embodiment 7 is that this embodiment focuses on determining the effect of biochar-based nitrogen-fixing bacteria inoculant on tomato quality, mainly including the chlorophyll content and soluble sugar content of tomatoes. The chlorophyll content of tomato leaves was determined using the 95% ethanol extraction-spectrophotometric method according to national standards, and the soluble sugar content was determined using the anthrone colorimetric method. The results of the effect of the biochar-based nitrogen-fixing bacteria inoculant on tomato quality are shown below. Figure 6 .Depend on Figure 6 Analysis showed that the chlorophyll content in group T3 was 17.96% and 26.33% higher than that in groups T1 and T2, respectively; the soluble sugar content in group T3 was 24.66% and 32.48% higher than that in groups T1 and T2, respectively. This indicates that the diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 and the biochar-humic acid-sodium alginate carrier material can both improve tomato quality, and the two can work synergistically. The biochar-based nitrogen-fixing bacteria inoculant is more effective than applying free bacteria or empty carrier alone.

Claims

1. A biochar-based nitrogen-fixing bacterial agent, characterized in that, The product includes a diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 adsorbed on biochar. The diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 is embedded in a mixed solution of humic acid and sodium alginate and is cross-linked and fixed by divalent metal ions.

2. The biochar-based nitrogen-fixing bacterial agent according to claim 1, characterized in that, The diazotrophic glucose acetic acid bacterium mutant ZJB-2025351 has the accession number CCTCC NO: M 20252292 and is deposited at the China Center for Type Culture Collection.

3. The biochar-based nitrogen-fixing bacterial agent according to claim 1, characterized in that, The divalent metal ion is a calcium ion.

4. The biochar-based nitrogen-fixing bacterial agent according to claim 1, characterized in that, The diazotrophic glucose acetic acid bacillus mutant ZJB-2025351 is at least one of the following: bacterial powder, wet bacterial cells, and culture.

5. A method for preparing a biochar-based nitrogen-fixing bacterial inoculant as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1: The culture of diazotrophic glucose acetic acid bacteria mutant was mixed with biochar for adsorption, and centrifuged to obtain biochar loaded with diazotrophic glucose acetic acid bacteria mutant. S2: The biochar loaded with the diazotrophic glucose acetic acid bacteria mutant obtained in step S1 is added to a mixed solution of humic acid and sodium alginate for encapsulation to obtain encapsulated biochar. S3: The encapsulated biochar obtained in step S2 is added to a divalent metal ion solution for cross-linking and solidification. After filtration and washing, biochar-humic acid-sodium alginate microspheres are obtained, which are biochar-based nitrogen-fixing bacteria agents.

6. The method for preparing a biochar-based nitrogen-fixing bacterial agent according to claim 5, characterized in that, In step S1, adsorption is carried out under water bath oscillation conditions, with an adsorption temperature of 20~30℃, an adsorption time of 20~40min, and an oscillation rate of 30~180r / min.

7. The method for preparing a biochar-based nitrogen-fixing bacterial agent according to claim 5, characterized in that, In step S1, the amount of biochar added is 3-8%; the concentration of the diazotrophic glucose acetic acid bacteria mutant culture is 1-9 × 10⁻⁶. 9 CFU / mL.

8. The method for preparing a biochar-based nitrogen-fixing bacterial agent according to claim 5, characterized in that, In step S2, the final concentration of humic acid in the mixed solution is 3-6 g / L, and the final concentration of sodium alginate is 2-8 g / L.

9. The method for preparing a biochar-based nitrogen-fixing bacterial agent according to claim 5, characterized in that, In step S3, the concentration of the divalent metal ion solution is 0.03~0.11 mol / L, and the cross-linking curing time is 20~60 min.

10. The application of the biochar-based nitrogen-fixing bacteria inoculant as described in any one of claims 1 to 4 in promoting plant growth.

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