A microbial composition, a gel material, a preparation method and application thereof in saline-alkali land afforestation

Abstract

This invention discloses a microbial composition, a gel material, a preparation method, and its application in afforestation of saline-alkali land, belonging to the field of microbial technology. It is composed of *Bacillus curvaturetus* (…). Bacillus bent E1, Bacillus argentea ( Bacillus aryabhattai Composed of GPR018 and lactic acid bacteria; Bacillus curvatureis ( Bacillus flexus The accession number for E1 is CGMCC No. 31832. The surface charge of these three microorganisms can synergistically interact with the gel network of the gel material, effectively inhibiting the excessive adsorption of sodium ions from the soil by the polymer lattice of the gel material. This reduces the risk of secondary soil salinization caused by the gel material lattice's "only adsorption and no release" of sodium ions, fundamentally solving the problem of soil salt accumulation caused by the retention of sodium ions during the repeated water absorption and release process of traditional water-retaining agents.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial composition, a gel material, a preparation method, and its application in afforestation in saline-alkali land. Background Technology

[0002] Soil salinization is a major challenge hindering sustainable agricultural development. High salinity, particularly sodium ion accumulation, leads to soil structure damage, low water use efficiency, and plant salt stress, which are the core difficulties in saline-alkali land management. Existing technologies for improving saline-alkali soils mainly revolve around physical engineering (such as underground salt drainage), chemical modification (such as adding gypsum and organic fertilizers), and biological modification (such as planting salt-tolerant plants and applying microbial agents). Among these, superabsorbent polymers (water-retaining agents) have shown application potential in improving soil moisture due to their excellent water retention capacity. Meanwhile, microbial modification technologies, especially those utilizing the properties of specific functional microorganisms (such as Bacillus and Pseudomonas) to secrete organic acids and produce extracellular polymers to neutralize alkalinity, integrate salt ions, and promote plant growth, have become a green and promising improvement strategy. However, these technologies tend to be applied independently, failing to fully realize the potential for synergistic effects between materials and organisms.

[0003] Plant rhizosphere growth-promoting bacteria (PGPR) are beneficial bacteria that live in the rhizosphere of plants, promoting plant growth or antagonizing pathogens, playing a crucial role in plant growth and disease control. PGPR significantly promotes the improvement of the rhizosphere soil ecological environment and plant growth, especially root growth. However, soil improvement effects from PGPR inoculation in saline-alkali environments are poor, failing to effectively enhance plant salt tolerance. This is because different environmental conditions, such as temperature, salinity, moisture, and the activity of other indigenous microorganisms, all affect microbial colonization and survival. While most microorganisms can reproduce and function normally under suitable conditions, they cannot colonize and survive long-term in saline-alkali environments, thus losing their unique functions.

[0004] Although water-retaining agents can theoretically improve drought problems in saline-alkali land, conventional water-retaining agents, when applied in high-salt-alkali environments, tend to selectively adsorb and fix sodium ions in the soil solution due to their polymer network structure, leading to a sodium ion lattice blockade phenomenon of "adsorption without release." This not only drastically reduces the water absorption and retention capacity of the water-retaining agent itself, failing to effectively improve its function, but also causes secondary soil salinization, exacerbating salt stress on plant roots. Therefore, developing a composite material that can effectively inhibit the excessive adsorption and fixation of sodium ions by water-retaining agents, while also possessing multiple functions such as improving soil structure and promoting plant growth, is crucial for the efficient management of saline-alkali land, especially for improving the survival rate of afforestation. Although some studies have attempted to develop functionalized hydrogels (e.g., introducing temperature-sensitive or pH-responsive properties) or utilize gels to encapsulate slow-release soil conditioners, a technical solution that precisely regulates and inhibits the adsorption of sodium ions by water-retaining agents through specific microbial compositions and their surface charge characteristics, thereby fundamentally solving the problem of salt accumulation, has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a microbial composition, a gel material, a preparation method, and its application in afforestation in saline-alkali land. The microbial composition provided by the present invention has strong targeting and effectiveness against arid environments and exhibits a clustering and growth-promoting effect in arid environments.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A first aspect of the present invention provides a microbial composition comprising Bacillus curvatureis ( Bacillus bent E1, Bacillus argentea ( Bacillus aryabhattai Composed of GPR018 and lactic acid bacteria;

[0008] The Bacillus curvatureis ( Bacillus flexus E1 was deposited on September 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31832.

[0009] In some embodiments of the present invention, the Bacillus curvatureus ( Bacillus flexus E1, Bacillus argentea ( Bacillus aryabhattai The ratio of GPR018 to lactic acid bacteria is 10:(1~2):(2~3).

[0010] In some embodiments of the present invention, the lactic acid bacteria is Lactobacillus casei subsp. ( Lactobacillus casei ).

[0011] A second aspect of the present invention provides a gel material comprising an adsorption carrier and a bacterial solution containing the microbial composition described in the first aspect.

[0012] In some embodiments of the present invention, the adsorption carrier is a grafted copolymer of humic acid and bentonite superabsorbent composite material.

[0013] In some embodiments of the present invention, the bacterial solution contains a viable count > 6.0 × 10⁻⁶. 8 CFU / mL.

[0014] A third aspect of the present invention provides a method for preparing the gel material described in the second aspect, comprising the following steps:

[0015] The bacterial culture was diluted with a diluent containing carbon and nitrogen sources to obtain a microbial adsorption solution.

[0016] Microbial adsorption solution is mixed with adsorption carrier to obtain gel material.

[0017] In some embodiments of the present invention, a diluent containing a carbon source and a nitrogen source is used to dilute the bacterial solution, thereby diluting the bacterial solution by 8 to 10 times;

[0018] In the diluent, the concentration of carbon source is 0.5%~1%, and the concentration of nitrogen source is 0.1%~0.2%.

[0019] In some embodiments of the present invention, the carbon source includes any one or more of glucose, sucrose, fructose, and lactose; the nitrogen source includes any one or more of urea, beef extract, corn steep liquor, peptone, ammonium nitrate, ammonium chloride, ammonium bicarbonate, potassium nitrate, sodium nitrate, and calcium nitrate.

[0020] A fourth aspect of the present invention provides the application of the gel material described in the second aspect in afforestation of saline-alkali land.

[0021] In some embodiments of the present invention, the afforestation of saline-alkali land includes the following steps:

[0022] When planting seedlings in containers, after digging the tree pits, sprinkle the gel material at the bottom of the pits, plant the trees, and then evenly sprinkle the gel material around the seedling containers.

[0023] When planting bare-root seedlings, after digging the planting pit, mix the gel material with the soil and then evenly spread it around the roots of the seedlings.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention provides a microbial composition consisting of *Bacillus curvatureis* E1, *Bacillus aspergerii* GPR018, and lactic acid bacteria. The surface charge of these three microorganisms creates a synergistic effect with the gel network of the gel material. This effect effectively inhibits the excessive adsorption of sodium ions from the soil by the polymer lattice of the gel material, reducing the risk of secondary soil salinization caused by the gel material's "adsorption without release" of sodium ions. This fundamentally solves the problem of soil salt accumulation caused by the retention of sodium ions during repeated water absorption and release processes in traditional water-retaining agents. This provides key technical support for the safe and effective use of water-retaining agents in arid and semi-arid regions and in soils experiencing secondary salinization. Furthermore, these three microorganisms form a functionally complementary synthetic microbial community in the plant rhizosphere. The mutually beneficial relationship between them enhances the stability and functional durability of the entire microbial community in complex soil environments. This microbial-based improvement strategy effectively reduces dependence on chemical fertilizers, aligns with the development direction of green agriculture, and provides effective technical support for the remediation of soil degradation and sustainable agricultural development.

[0026] This invention also provides a gel material whose three-dimensional network structure effectively encapsulates and protects microorganisms, helping them resist stresses such as drought and pH fluctuations in the soil environment. The gel carrier can combine with nutrients such as carbon and nitrogen sources, providing "starting energy" for the colonization and initial proliferation of microorganisms in the soil, ensuring they can quickly establish a dominant population and thus exert lasting efficacy. This dual role of protection and nutrient supply overcomes the industry pain points of low survival rates and unstable effects when microorganisms are directly applied to the soil. The synergistic effect of *Bacillus curvatureii* E1, *Bacillus aspergillus* GPR018, and lactic acid bacteria effectively interferes with or inhibits the specific adsorption and fixation of sodium ions by the polymer lattice of the gel material. This allows the gel to reduce the "lock-in" of sodium ions during repeated water absorption-release cycles, preventing secondary soil salinization and providing key technical support for the safe use of water-retaining agents in arid and semi-arid regions.

[0027] The gel material provided by this invention can systematically improve the soil ecosystem. It helps promote the formation of soil aggregates, improves soil compaction caused by salinization, and enhances soil aeration and water retention capacity. Simultaneously, functional microorganisms can secrete organic acids and other substances, activating fixed nutrients such as phosphorus and potassium in the soil and increasing nitrogen sources through nitrogen fixation, thereby comprehensively improving soil fertility. Ultimately, these effects collectively create a healthy microenvironment for plant roots, significantly promoting plant growth and increasing the survival rate of afforestation on saline-alkali land. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 This shows the growth of Bacillus curvatureis E1 on PDA culture media with different salt concentrations in Example 1 of the present invention.

[0030] Figure 2 This is the growth curve of Bacillus curvatureis E1 in Example 1 of the present invention under different salt concentrations of PDB medium.

[0031] Figure 3 This is a standard curve for glucose concentration. Detailed Implementation

[0032] This invention provides a microbial composition, a gel material, a preparation method, and its application in afforestation of saline-alkali land. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0033] The Bacillus curvatureus described in this invention ( Bacillus flexus E1 was deposited on September 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31832.

[0034] The Bacillus argentea described in this invention ( Bacillus aryabhattai GPR018 refers to the *Bacillus argentea* species disclosed in patent "CN108102958A A *Bacillus argentea* strain for promoting rhizosphere growth in saline-alkali soil and its application" (…). Bacillus aryabhattai GPR018 was deposited on January 7, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 11973.

[0035] The lactic acid bacteria described in this invention are Lactobacillus casei subsp. ( Lactobacillus casei The strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with strain number 1.0580.

[0036] The grafted copolymer humic acid and bentonite superabsorbent composite material of the present invention is the grafted copolymer humic acid and bentonite superabsorbent composite material disclosed in the patent "CN101230181A Grafted copolymer humic acid and bentonite superabsorbent composite material and manufacturing method thereof". By uniformly mixing humic acid and bentonite together as a grafting skeleton, a graft copolymerization reaction is carried out with the skeleton composed of humic acid and bentonite using vinyl monomers. In the presence of a crosslinking agent, crosslinks are formed between the branches generated by graft copolymerization, and finally a grafted copolymer humic acid and bentonite superabsorbent composite material with several crosslinks in molecular structure is obtained.

[0037] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0038] The embodiments and comparative examples of this invention describe some cases. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these cases. In fact, any concentration of each component between the two endpoints shown in the embodiments can achieve a good effect on improving sleep quality. The comparative examples only list some cases where the results were unsatisfactory in the experiments. In addition, many other attempts were made during the research and development process, such as using different components, different ratios, different culture media, or different addition times.

[0039] This invention provides a microbial composition composed of Bacillus curvaturei (Bacillus) Bacillus flexus E1, Bacillus argentea ( Bacillus aryabhattai Composed of GPR018 and lactic acid bacteria;

[0040] The Bacillus curvatureis ( Bacillus flexus E1 was deposited on September 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31832.

[0041] Upon testing, the *Bacillus curvatureis* E1 used in this invention is a Gram-negative bacterium. The colonies are round and flat, relatively small, with a moist, raised surface, grayish-white in color, and opaque. They typically spread along the inoculation line. The bacteria are small, rod-shaped, and lack spores and capsules. The physiological and biochemical characteristics of *Bacillus curvatureis* E1 are shown in Table 1. Based on the results of 16S rDNA sequencing and comparison with the NCBI database, *Bacillus curvatureis* E1 is similar to... Bacillus fecusLocated on the same branch with 97.00% homology, it was identified as Bacillus curvatureis ( Bacillus fecus ).

[0042] Table 1. Physiological and biochemical characteristics of Bacillus curvatureis E1

[0043]

[0044] Note: In Table 1, "+" indicates a positive result, meaning that Bacillus curvatureis E1 possesses this physiological and biochemical characteristic; "-" indicates a negative result, meaning that Bacillus curvatureis E1 does not possess this physiological and biochemical characteristic.

[0045] 16S rDNA sequence of Bacillus curvatureis E1

[0046]

[0047] The Bacillus curvatureis E1 used in this invention, such as Figure 1 As shown, it has strong salt tolerance and can grow normally in potato dextrose agar (PDA) medium with a salt concentration of 12% (mass percentage). The extracellular polysaccharide yield in potato dextrose broth (PDB) medium with a salt concentration of 9% can reach 154.28 μg / mL. The specific experimental process is as follows.

[0048] I. Determination of Salt Tolerance of Bacillus Curvularia E1

[0049] 1. Plate Experiment

[0050] 1.1 Experimental Methods

[0051] Activated *Bacillus curvatureis* E1 was inoculated into PDB medium and cultured at 180 rpm and 37°C with shaking for 12 h to obtain activated bacterial suspension of *Bacillus curvatureis* E1. PDB solid medium with NaCl concentrations of 0%, 2%, 5%, 7%, 10%, 11%, 12%, 13%, 14%, 15%, 17%, and 20% (mass percentage) was prepared. 100 mL of the bacterial suspension was spread onto PDB plates with different NaCl concentrations and incubated at 37°C for 24–48 h. The growth status of *Bacillus curvatureis* E1 under different NaCl concentrations was observed.

[0052] 1.2 Experimental Results

[0053] Depend on Figure 1 It is known that Bacillus curvatureis E1 stops growing on PDA plates with a salt concentration of 14%, therefore the highest salt tolerance of Bacillus curvatureis E1 in solid PDA medium is 13%.

[0054] 2. Shaking Bottle Experiment

[0055] 2.1 Experimental Methods

[0056] The activated Bacillus curvatureis E1 bacterial suspension was inoculated at a rate of 2% into 50 mL PDB medium containing different NaCl concentrations (0%, 3%, 6%, 9%, and 12%). The culture was incubated at 37°C, and the growth curves of Bacillus curvatureis E1 at different salt concentrations were measured (OD values ​​of the bacterial suspension were measured using a microplate reader at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 30 h, 36 h, 48 h, and 60 h after inoculation). 600 Values, with three parallel sets for each salt gradient.

[0057] After 24 hours of bacterial culture, the viable count of *Bacillus curvaturelis* E1 suspensions at different NaCl concentrations was determined using the dilution plating method. Specifically:

[0058] Take 100 μL of bacterial suspension from 0% salt PDB medium and add it to a centrifuge tube containing 900 μL of sterile water. Vortex to mix. Repeat this process to dilute the bacterial suspension to 10. -5 100 μL of bacterial suspension from each gradient was taken and spread onto PDA plates with 0% salt concentration. Three plates were spread for each gradient and incubated at 37°C for 24 h. The colonies were then counted.

[0059] Take 100 μL of bacterial suspension from 3% PDB medium and add it to a centrifuge tube containing 900 μL of sterile water. Vortex to mix. Repeat this process to dilute the bacterial suspension to 10 μL. -5 100 μL of bacterial suspension from each gradient was taken and spread onto PDA plates with a 3% salt concentration. Three plates were spread for each gradient and incubated at 37°C for 24 h. The colonies were then counted.

[0060] Take 100 μL of bacterial suspension from 6% PDB medium and add it to a centrifuge tube containing 900 μL of sterile water. Vortex to mix. Repeat this process to dilute the bacterial suspension to a final concentration of 10. -4 100 μL of bacterial suspension from each gradient was taken and spread onto PDA plates with a 6% salt concentration. Three plates were spread for each gradient and incubated at 37°C for 24 h. The colonies were then counted.

[0061] Take 100 μL of bacterial suspension from 9% PDB medium and add it to a centrifuge tube containing 900 μL of sterile water. Vortex to mix. Repeat this process to dilute the bacterial suspension to 10. -4 100 μL of bacterial suspension from each gradient was taken and spread onto PDA plates with a 9% salt concentration. Three plates were spread for each gradient and incubated at 37°C for at least 48 h. The colonies were then counted.

[0062] Take 100 μL of bacterial suspension from 12% salt PDB medium and add it to a centrifuge tube containing 900 μL of sterile water. Vortex to mix. Repeat this process to dilute the bacterial suspension to 10. -4 100 μL of bacterial suspension from each gradient was taken and spread onto PDA plates with a 12% salt concentration. Three plates were spread for each gradient and incubated at 37°C for at least 48 h. The colonies were then counted.

[0063] 2.2 Experimental Results

[0064] 1. Growth curves of Bacillus curvatureis E1 under different salt concentrations in PDB medium.

[0065] Depend on Figure 2It was found that under 0% salt concentration PDB medium, Bacillus curvatureis E1 entered the logarithmic phase at 4 h, the stationary phase at 24 h, and the apoptosis phase at 36 h; under 3% salt concentration PDB medium, Bacillus curvatureis E1 entered the logarithmic phase at 4 h, the stationary phase at 24 h, and the apoptosis phase at 36 h; under 6% salt concentration PDB medium, Bacillus curvatureis E1 entered the logarithmic phase at 12 h, the stationary phase at 24 h, and the apoptosis phase at 36 h; while under high salt concentration (9%, 12%) PDB medium, the logarithmic phase of Bacillus curvatureis E1 was inhibited, its growth was slow, and its overall biomass decreased.

[0066] 2. Viable cell count of Bacillus curvatureis E1 after 24 h of culture in PDB medium with different salt concentrations

[0067] Under 0% salt concentration PDB medium conditions, the viable count of 1 mL of bacterial suspension was 1.96 × 10⁻⁶. 7 Under 3% salt concentration PDB medium conditions, the viable count of 1 mL of bacterial suspension was 4.25 × 10⁻⁶. 6 Under 6% salt concentration PDB medium conditions, the viable count of 1 mL of bacterial suspension was 3.03 × 10⁻⁶. 5 Under 9% salt concentration PDB medium conditions, the viable bacterial count in 1 mL of bacterial suspension was 1.816 × 10⁻⁶. 4 Under 12% salt concentration PDB medium conditions, the viable count of 1 mL of bacterial suspension was 1.19 × 10⁻⁶. 3 Among them, Bacillus curvaturee E1 bacterial suspensions coated in PDA with 9% and 12% salt concentrations require more than 48 hours of incubation to grow, while other salt concentrations require 24 hours of incubation.

[0068] II. Determination of the extracellular polysaccharide production capacity of Bacillus curvatureis E1

[0069] 1. Isolation and extraction of extracellular polysaccharides

[0070] Bacillus curvatureis E1 was inoculated into normal PDB medium and shaken at 37°C and 180 rpm for 12 h. The resulting seed culture was then transferred at a 2% inoculation rate to PDB and LB liquid media with salt concentrations of 0%, 3%, 6%, 9%, and 12%, respectively, and cultured under the same conditions for 48 h, with two replicates for each salt concentration. The shaken extracellular polysaccharide fermentation broth was centrifuged at 4°C and 10,000 rpm for 10 min to remove the precipitate, and the supernatant was transferred to a new centrifuge tube. The supernatant was placed in a 100°C water bath for 10 min to denature the proteins. After natural cooling, it was centrifuged at 4°C and 10,000 rpm for 15 min to remove the proteins, retaining the supernatant (10 ml of supernatant was obtained from each treatment).

[0071] Take 10 mL of the supernatant, add 30 mL of pre-cooled anhydrous ethanol, and extract at 4℃ for 24 h to fully extract the extracellular polysaccharides. Centrifuge the extracted extracellular polysaccharide supernatant at 10000 rpm / min at 4℃ for 15 min. Remove the supernatant, retain the extracellular polysaccharide precipitate, and dry the centrifuge tube containing the precipitate at 50℃ in an oven. Store at -20℃ for later use.

[0072] 2. Plotting the standard curve for glucose concentration

[0073] Glucose concentration standard curve: Accurately weigh 0.25 g of glucose dried to constant weight at 50℃, dissolve it in 50 mL of distilled water to obtain a 5 mg / mL glucose standard solution A. Pipette 1 mL of glucose standard solution A into a 100 mL volumetric flask, and dilute to 100 mL with distilled water to obtain a 50 ug / mL glucose solution B. Add 0, 0.5, 1, 1.5, 2, and 2.5 mL of glucose solution B to clean test tubes, respectively, and add distilled water to test tubes with a volume less than 2.5 mL to a final volume of 2.5 mL.

[0074] Add 5 mL of sulfuric acid-anthrone reagent to each of the above test tubes and immediately stopper them with rubber to prevent water evaporation. Heat the test tubes in a boiling water bath for 10 min, then remove and allow them to cool naturally to room temperature. Using the reaction solution in a test tube with 2.5 mL of distilled water as a blank, measure the absorbance at 620 nm. Plot a glucose concentration standard curve with glucose concentration on the x-axis and OD value on the y-axis, as shown below. Figure 3 .

[0075] 3. Determination and Calculation of Extracellular Polysaccharide Yield

[0076] Extracellular polysaccharide samples extracted from PDB and LB liquid media with different salt concentrations were dissolved in 5 mL of distilled water and thoroughly mixed by shaking. Two replicates were performed for each gradient. 0.2 mL of the dissolved extracellular polysaccharide solution was pipetted into clean test tubes, and distilled water was added to a total volume of 2.5 mL. After mixing, 5 mL of sulfuric acid-anthrone reagent was added to each tube, and the tubes were immediately sealed with rubber stoppers to prevent evaporation. The test tubes were then placed in a boiling water bath for 10 min and allowed to cool naturally to room temperature. A test tube containing 2.5 mL of distilled water was used as a blank control. The OD values ​​of each test tube solution were measured. 620 Absorbance at that location.

[0077]

[0078] The extracellular polysaccharide (EPS) yield of Bacillus curvaturee E1 was 419.03 μg / mL in 0% PDB; 295.44 μg / mL in 3% PDB; 233.64 μg / mL in 6% PDB; 154.28 μg / mL in 9% PDB; 55.97 μg / mL in 12% PDB; and 47.54 μg / mL in LB liquid medium.

[0079] The Bacillus argentea used in this invention ( Bacillus aryabhattai GPR018 can promote plant growth, increase the water content of plant leaves, and reduce the peroxidation damage of plant membranes in a heavily saline-alkali environment, thus improving the salt and alkali tolerance of Tamarix chinensis in such environments.

[0080] The lactic acid bacteria used in this invention are Lactobacillus casei subsp. ( Lactobacillus casei It can regulate the microecological environment of plant rhizosphere and promote the colonization ability of Bacillus curvatureis E1 in the rhizosphere environment.

[0081] This invention provides a microbial composition with Bacillus curvatureis E1 as the core component and optimized combination with other functional bacteria, achieving a clustering and growth-promoting effect of multiple strains in arid environments, exhibiting stronger targeting and effectiveness in arid conditions. These three microorganisms demonstrate multi-level synergistic effects of complementary environmental colonization, mutual assistance in nutrient metabolism, and synergistic pathogen antagonism, forming a functionally complementary micro-ecosystem in the soil. Furthermore, they cleverly utilize their surface charge characteristics to synergistically inhibit the excessive fixation of sodium ions by water-retaining agents.

[0082] In some specific embodiments, the Bacillus curvatureis ( Bacillus flexus E1, Bacillus argentea ( Bacillus aryabhattai The ratio of GPR018 to lactic acid bacteria is 10:(1~2):(2~3).

[0083] At this ratio, the resulting microbial composition exhibits high stability.

[0084] The present invention also provides a gel material comprising an adsorption carrier and a bacterial solution containing the above-described microbial composition.

[0085] The gel material provided by this invention can prolong the survival time of microorganisms and increase the number of effective microorganisms in a saline-alkali environment; it can also reduce the adsorption of sodium ions by the adsorption carrier in a saline-alkali environment, thereby improving the soil, enhancing the plant's ability to adapt to saline-alkali conditions, and increasing the survival rate of afforestation in saline-alkali land.

[0086] The saline-alkali environment described in this invention is mainly soda-alkali soil composed primarily of NaHCO3 and Na2CO3. Plants growing in this type of soil are susceptible to Na+ damage. + Multiple stresses, including high pH and low water potential, reduce soil permeability and aeration, increase soil solution osmotic pressure, and decrease soil nutrient availability, severely limiting normal plant growth.

[0087] The cell walls of these three microorganisms are rich in teichoic acid, giving them a generally negatively charged surface. When *Bacillus curvatureis* E1, *Bacillus aspergeria* GPR018, and lactic acid bacteria coexist in the network structure of a gel material, they create a dynamic, specifically charged "micro-electric field" or "ion cloud" around the polymer chains of the gel material through their own surface charges. This charged environment established by the microbial community can, to some extent, shield or interfere with the electrostatic attraction of the polymer lattice in the gel material to sodium ions in the solution.

[0088] These factors work together to help prevent the rigid accumulation of sodium ions inside the gel material, which is "only in and not out," and promote a healthier dynamic exchange between sodium ions and other cations, thereby effectively reducing the risk of salt accumulation caused by the repeated absorption and inability to release sodium ions by the gel material.

[0089] In some specific embodiments, the adsorption carrier may be a natural polymer material, a functional inorganic mineral, a synthetic polymer gel, or an organic-inorganic complex that is compatible with microorganisms.

[0090] Natural polymer materials include, but are not limited to, any one of gelatin, carboxymethyl chitosan, sodium alginate, and agarose. Natural polymer materials exhibit excellent biocompatibility; being derived from nature, they provide a more natural living environment for microorganisms; their three-dimensional network structure typically possesses good hydrophilicity and water absorption / retention capabilities. For example, the gelatin-carboxymethyl chitosan composite system has been studied as an excellent carrier matrix.

[0091] Functional inorganic minerals typically possess unique pore structures and cation exchange capacities, which facilitate the adsorption of substances such as ammonia nitrogen, regulate the pH of the microenvironment, and may immobilize microorganisms through physical adsorption. These include, but are not limited to, any one of zeolite, diatomaceous earth, porous ceramics, and kaolin.

[0092] Synthetic polymeric gels exhibit high water absorption capacity, precisely controllable gel strength and water structure, good stability, and long service life. For example, by introducing micro-nano fibrillated cellulose (MNFC) to reinforce polyacrylic acid-based hydrogels, their water absorption capacity and mechanical strength can be significantly improved simultaneously. This includes, but is not limited to, any one of polyacrylamide (PAAm), polyacrylic acid (PAA), and polyvinyl alcohol (PVA) hydrogels.

[0093] Organic-inorganic composites can combine the advantages of both organic and inorganic phases, achieving complementary and synergistic performance. These include, but are not limited to, grafted copolymerized humic acid and bentonite composites, cellulose / clay composites, and protein-mineral composite gels.

[0094] In some specific embodiments, the adsorption carrier is a grafted copolymer of humic acid and bentonite superabsorbent composite material.

[0095] The grafted copolymerized humic acid and bentonite superabsorbent composite material possesses high water absorption and retention capabilities, is pressure-resistant, salt-alkali resistant, and has a long service life. It can also improve desertified soil, promote plant growth, and enhance crop drought resistance. By mixing humic acid and bentonite as a grafting skeleton, a graft copolymerization reaction is carried out using vinyl monomers with humic acid and bentonite. Under the action of a crosslinking agent, crosslinked structures are formed between the branches generated by the graft copolymerization, resulting in a crosslinked grafted copolymerized humic acid and bentonite superabsorbent composite material with a crosslinked molecular structure. The structure and preparation method of this material have been disclosed in patent "CN101230181A Grafted Copolymerized Humic Acid and Bentonite Superabsorbent Composite Material and its Manufacturing Method".

[0096] In some specific embodiments, the bacterial solution contains a viable count > 6.0 × 10⁻⁶. 8 CFU / mL.

[0097] In some specific embodiments, the gel material contains a viable bacteria count > 1.8 × 10⁻⁶. 8 CFU / g.

[0098] A high number of viable bacteria ensures that the amount of functional microorganisms applied to the gel material and its final soil environment is sufficient, which can quickly form a dominant microbial community and effectively play its role in improving soil, inhibiting sodium ion adsorption and promoting plant growth.

[0099] The present invention also provides a method for preparing the above-mentioned gel material, comprising the following steps:

[0100] The bacterial culture was diluted with a diluent containing carbon and nitrogen sources to obtain a microbial adsorption solution.

[0101] Microbial adsorption solution is mixed with adsorption carrier to obtain gel material.

[0102] The preparation method of this invention achieves a synergistic effect (1+1>2) through precise step design, ultimately yielding not just a simple mixture, but a highly functional microbial preparation. This gel material effectively protects the functional bacteria and provides initial nutrition, ensuring rapid colonization and sustained function of the microbial community upon arrival in the soil. High-concentration bacterial solutions are diluted with a specialized diluent containing carbon and nitrogen sources, providing the microorganisms with an "on-the-go nutrient package," maintaining bacterial activity, effectively preventing activity reduction due to "starvation" during processing and drying, buffering environmental shocks, and ensuring high activity of the microorganisms during mixing, storage, and initial application, laying the foundation for rapid colonization. The microbial adsorption solution is mixed with the adsorption carrier in a specific ratio to promote uniform adsorption, forming a stable three-dimensional gel network structure that provides physical protection for the microorganisms, creating a "micro-ecosystem" that both protects the microorganisms and slowly releases nutrients and bacterial cells.

[0103] In some specific embodiments, the bacterial culture is diluted with a diluent containing carbon and nitrogen sources, and the bacterial culture is diluted 8 to 10 times.

[0104] In the diluent, the concentration of carbon source is 0.5%~1%, and the concentration of nitrogen source is 0.1%~0.2%.

[0105] In some specific embodiments, the carbon source includes, but is not limited to, any one or more of glucose, sucrose, fructose, and lactose, preferably glucose; the nitrogen source includes, but is not limited to, any one or more of urea, beef extract, corn steep liquor, peptone, ammonium nitrate, ammonium chloride, ammonium bicarbonate, potassium nitrate, sodium nitrate, and calcium nitrate, preferably urea.

[0106] Directly adsorbing bacterial suspension using an adsorption carrier is too costly, and high bacterial concentrations reduce bacterial activity and the water absorption capacity of the adsorption carrier. Therefore, this invention dilutes the bacterial suspension before adsorption. This invention uses a diluent containing carbon and nitrogen sources to treat the bacterial suspension before mixing it with the adsorption carrier. This maximizes the "start-up energy" for the organisms, optimizes their colonization environment, and significantly improves the performance and stability of the gel material.

[0107] The present invention also provides an application of the above-mentioned gel material in afforestation in saline-alkali land.

[0108] In some specific embodiments, the afforestation of saline-alkali land includes the following steps:

[0109] When planting seedlings in containers, after digging the planting pit, sprinkle the gel material at the bottom of the pit, plant the tree, and then sprinkle the gel material evenly around the seedling container.

[0110] Spreading gel material at the bottom of the planting pit creates a "bottom-layer water-retaining and moisture-retaining layer" for the root ball of the seedling. This effectively utilizes the trace moisture in the deeper soil layers, preventing excessive water loss after planting and providing guidance and a moist environment for new root growth. Evenly spreading the gel material around the seedling container creates a buffer zone and resource enrichment zone between the original root ball and the existing soil in the pit. After absorbing irrigation water, the gel material keeps the root zone moist, and its functional microorganisms can quickly colonize around the seedling roots, forming a beneficial rhizosphere microenvironment, inhibiting soil-borne diseases, and effectively blocking the direct stress of saline-alkali soil on the roots. This method avoids damaging the original root ball structure during transplanting and significantly shortens the recovery period.

[0111] This invention does not limit the amount of gel material applied to the bottom of the pit or around the seedling container; those skilled in the art can choose an appropriate amount based on the specific circumstances. For example, 200-300 g of gel material can be applied to the bottom of the pit, and 600-800 g of gel material can be applied around the seedling container.

[0112] In some specific embodiments, the afforestation of saline-alkali land includes the following steps:

[0113] When planting bare-root seedlings, after digging the planting pit, mix the gel material with the soil and then evenly spread it around the roots of the seedlings.

[0114] Bare-root seedlings have exposed roots during lifting and transportation, making them prone to water loss and lacking the protection of their original soil. Applying a gel material mixed with soil around the roots creates a customized "micro-zone for establishment and growth" for the seedlings. This mixing maximizes the contact area between the roots and the moisturizing material, providing immediate and usable moisture to the exposed roots and preventing death from physiological dehydration. Simultaneously, the humid environment effectively stimulates new root germination and growth. Furthermore, direct contact between the bare-root seedling roots and the gel material containing highly active microbial agents allows beneficial microorganisms to adhere to the root surface more quickly and exert their effects. These microorganisms not only produce growth-promoting substances, but their metabolic activities also improve the soil structure of the rhizosphere microzone, alleviate salt and alkali stress, and create favorable conditions for seedling survival.

[0115] In this invention, the mass of the gel material mixed with the soil is not limited, and those skilled in the art can select an appropriate mass according to the actual situation. For example, 1200-1500 g of gel material can be mixed with the soil.

[0116] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0117] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0118] Example 1

[0119] This embodiment provides a microbial composition comprising Bacillus curvatureis E1, Bacillus argentea GPR018, and Lactobacillus casei subsp.

[0120] The preparation method of the microbial composition bacterial solution is as follows:

[0121] Bacillus curvatureis E1, Bacillus aspergillus GPR018, and Lactobacillus casei subsp. were mixed in a CFU ratio of 10:1.5:2. The resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0122] Example 2

[0123] This embodiment provides a microbial composition comprising Bacillus curvatureis E1, Bacillus argentea GPR018, and Lactobacillus casei subsp.

[0124] The preparation method of the microbial composition bacterial solution is as follows:

[0125] Bacillus curvatureis E1, Bacillus aspergillus GPR018, and Lactobacillus casei subsp. were mixed in a CFU ratio of 10:1:2. The resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0126] Example 3

[0127] This embodiment provides a microbial composition comprising Bacillus curvatureis E1, Bacillus argentea GPR018, and Lactobacillus casei subsp.

[0128] The preparation method of the microbial composition bacterial solution is as follows:

[0129] Bacillus curvatureis E1, Bacillus aspergillus GPR018, and Lactobacillus casei subsp. were mixed in a CFU ratio of 10:2:3. The resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0130] Comparative Example 1

[0131] This comparative example provides a microbial composition comprising Bacillus curvatureis E1, Bacillus argentea GPR018, and Lactobacillus casei subsp.

[0132] The preparation method of the microbial composition bacterial solution is as follows:

[0133] Bacillus curvatureis E1, Bacillus aspergillus GPR018, and Lactobacillus casei subsp. were mixed in a CFU ratio of 10:0.5:2. The resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0134] Comparative Example 2

[0135] This comparative example provides a microbial composition comprising Bacillus curvatureis E1, Bacillus argentea GPR018, and Lactobacillus casei subsp.

[0136] The preparation method of the microbial composition bacterial solution is as follows:

[0137] Bacillus curvatureis E1, Bacillus aspergillus GPR018, and Lactobacillus casei subsp. were mixed in a CFU ratio of 10:2.5:2. The resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0138] Comparative Example 3

[0139] This comparative example provides a microbial composition comprising Bacillus curvatureis E1 and Bacillus argentis GPR018.

[0140] The preparation method of the microbial composition bacterial solution is as follows:

[0141] Bacillus curvatureis E1 and Bacillus aspergillus GPR018 were mixed at a CFU ratio of 10:1.8, and the resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0142] Comparative Example 4

[0143] This comparative example provides a microbial composition comprising Bacillus curvatureis E1 and Lactobacillus casei subsp.

[0144] The preparation method of the microbial composition bacterial solution is as follows:

[0145] Bacillus curvatureis E1 and Lactobacillus casei subsp. were mixed at a CFU ratio of 10:2, and the resulting microbial composition solution contained 7.25 × 10⁻⁶ viable bacteria. 8 CFU / mL.

[0146] Efficacy verification: Potted experiment of bare-root ash seedlings.

[0147] When transplanting ash trees, 10 mL of the bacterial solutions prepared in Examples 1-3 and Comparative Examples 1-4 were diluted to 100 mL and evenly poured around the roots of the ash trees. Then, water was applied to 80% of the field capacity. Simultaneously, equal amounts of *Bacillus aspergerii* GPR018 (control group 1), *Bacillus curvatureis* E1 (control group 2), and *Lactobacillus casei* subsp. *casei* (control group 3) were applied to maintain a consistent total viable count across treatments, and normal management was maintained. Forty-five days after transplanting, salt stress treatment was initiated. Every 7 days, potted plants from different treatments were immersed in an equal volume of the corresponding concentration of 0.60% NaCl solution for 4 hours, with the solution submerging 3 / 4 to 4 / 5 of the pot. Afterward, all plants were replenished with clean water once a week, 300 mL per pot each time, to prevent salt loss. After 30 days, the relative water content, relative conductivity, and total chlorophyll content of the ash leaves were measured.

[0148] The results are shown in Table 2.

[0149] Table 2. Relative water content, relative electrical conductivity, and soil nutrients of *Fraxinus chinensis* leaves under different treatments.

[0150]

[0151] Note: Different letters in Table 2 indicate statistically significant differences between groups (P<0.05).

[0152] As shown in Table 2, compared with the control group, Examples 1-3 exhibited the highest relative leaf water content and total chlorophyll content, and the lowest relative leaf electrical conductivity. Therefore, based on the principle that the strains do not antagonize each other, the combined bacterial solution can fully leverage the synergistic effect of the bacterial community and improve the adaptability of ash in saline-alkali environments.

[0153] Example 4

[0154] This embodiment provides a gel material comprising a microbial composition bacterial solution and a grafted copolymer of humic acid and bentonite superabsorbent composite material.

[0155] The preparation method of the gel material is as follows:

[0156] The microbial composition solution obtained in Example 1 was diluted 9 times with a diluent (an aqueous solution containing 0.75% glucose and 0.15% urea) to obtain a microbial adsorption solution.

[0157] Microbial adsorption liquid was mixed with grafted copolymerized humic acid and bentonite superabsorbent composite material at a ratio of 40:1, mL / g to form a gel material.

[0158] In this embodiment, the gel material absorbs approximately 4 times the amount of saline solution (0.9%). After the grafted copolymerized humic acid and bentonite superabsorbent composite material absorbs the same volume of water as the microbial adsorption solution, the absorption capacity of the saline solution (0.9%) is approximately 7 times. Therefore, it can be concluded that after the grafted copolymerized humic acid and bentonite superabsorbent composite material adsorbs the microbial composition solution, its ability to adsorb saline solution decreases significantly, indicating that the microbial composition inhibits the adsorption of sodium ions by the grafted copolymerized humic acid and bentonite superabsorbent composite material.

[0159] Example 5

[0160] This embodiment provides a gel material comprising a microbial composition bacterial solution and a grafted copolymer of humic acid and bentonite superabsorbent composite material.

[0161] The preparation method of the gel material is as follows:

[0162] The bacterial culture of the microbial composition obtained in Example 1 was diluted 10 times with a diluent (an aqueous solution containing 0.5% glucose and 0.1% urea) to obtain a microbial adsorption solution;

[0163] Microbial adsorption liquid was mixed with grafted copolymerized humic acid and bentonite superabsorbent composite material at a ratio of 30:1, mL / g to form a gel material.

[0164] Example 6

[0165] This embodiment provides a gel material comprising a microbial composition bacterial solution and a grafted copolymer of humic acid and bentonite superabsorbent composite material.

[0166] The preparation method of the gel material is as follows:

[0167] The bacterial culture of the microbial composition obtained in Example 1 was diluted 8 times with a diluent (an aqueous solution containing 1% glucose and 0.2% urea) to obtain a microbial adsorption solution;

[0168] Microbial adsorption liquid was mixed with grafted copolymerized humic acid and bentonite superabsorbent composite material at a ratio of 50:1, mL / g to form a gel material.

[0169] Efficacy verification: The afforestation application effect of gel materials

[0170] A planting experiment of white elm was conducted in Kenli District, Dongying City. At the time of planting, 1200 g of gel material was mixed with some soil and evenly applied around the roots of the bare-root white elm seedlings. A control group (containing the same amount of microbial agent and grafted copolymerized humic acid and bentonite superabsorbent composite material as in Example 4) and a blank group (without gel material) were set up, maintaining the same number of microorganisms and amount of gel material in both groups. The survival rate was investigated in the year of planting and again in the following year. The results are shown in Table 3.

[0171] Table 3. Growth, survival rate and retention rate of *Ulmus pumila* under different treatments

[0172]

[0173] Note: Different letters in Table 3 indicate statistically significant differences between groups (P<0.05).

[0174] As shown in Table 3, the dry application of gel material (control group) has little effect on the survival rate of white elm, but significantly reduces the survival rate. In contrast, the gel material treatment (Examples 4-6) not only significantly improved the survival rate of white elm, but also improved the survival rate in the second year. The survival rate in the second year of Example 4 was significantly higher than that of the blank group by 12.11%.

[0175] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gel material, characterized in that, comprising an adsorption carrier and a bacterial solution containing a microbial composition consisting of Bacillus flexus (Bf) Bacillus flexus ), Bacillus aryabhattai (Ba) Bacillus aryabhattai ), GPR018 and lactic acid bacteria; The Bacillus curvatureis ( Bacillus flexus E1 was deposited on September 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31832. The Bacillus curvatureis ( Bacillus flexus E1, Bacillus argentea ( Bacillus aryabhattai The ratio of GPR018 to lactic acid bacteria is 10:(1~2):(2~3); The lactic acid bacteria is Lactobacillus casei subsp. ( Lactobacilluscasei The strain number is CGMCC 1.0580; The preservation number of the Bacillus argentea GPR018 is: CGMCC No. 11973; The adsorption carrier is a grafted copolymer of humic acid and bentonite superabsorbent composite material. The preparation method of the gel material includes the following steps: The bacterial culture was diluted with a diluent containing carbon and nitrogen sources to obtain a microbial adsorption solution. The microbial adsorption solution was mixed with an adsorption carrier to obtain a gel material. In the diluent, the concentration of carbon source was 0.5%~1% and the concentration of nitrogen source was 0.1%~0.2%. The carbon source was glucose and the nitrogen source was urea. The ratio of the microbial adsorption liquid to the adsorption carrier is 30:1, 40:1, or 50:1 mL / g.

2. The gel material as described in claim 1, characterized in that, The bacterial liquid contains a viable count > 6.0 x 10 8 CFU / mL.

3. The gel material as described in claim 1, characterized in that, In the preparation method of the gel material, the bacterial solution is diluted with a diluent containing carbon and nitrogen sources, and the bacterial solution is diluted 8 to 10 times to obtain a microbial adsorption solution.

4. The application of the gel material according to any one of claims 1-3 in afforestation of saline-alkali land.

5. The application as described in claim 4, characterized in that, The afforestation of saline-alkali land includes the following steps: When planting seedlings in containers, after digging the tree pit, sprinkle the gel material at the bottom of the pit, plant the tree, and then sprinkle the gel material evenly around the seedling container. When planting bare-root seedlings, after digging the planting pit, mix the gel material with the soil and then evenly spread it around the roots of the seedlings.

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