A microbial fertilizer for improving saline-alkali soil and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEBEI JIWEI MODERN BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-09
- Publication Date
- 2026-06-09
Smart Images
Figure CN122167234A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of agricultural microbial fertilizer technology, and in particular to a microbial fertilizer for improving saline-alkali land and its application. Background Technology
[0002] Food security has become a crucial pillar of my country's national strategic security, and the efficient utilization of land resources, especially the development and utilization of saline-alkali land, is of great strategic significance. Saline-alkali land is widely distributed in my country, and its soil is rich in high concentrations of cations, leading to abnormally high soil osmotic pressure, severely restricting normal crop growth and hindering sustainable agricultural development.
[0003] Soil improvement mainly includes three types of technologies: physical improvement, chemical improvement, and biological improvement. Physical improvement suffers from poor long-term effects and heavy workload, and cannot fundamentally change the physicochemical properties of saline-alkali soil. Chemical improvement methods are not only costly and time-consuming, making them unsuitable for large-scale application in saline-alkali land, but also pose a risk of secondary soil pollution. Biological improvement, which improves the soil environment through the metabolic activities of microorganisms, has advantages such as energy saving, high economic efficiency, wide application range, and no residue in crops, and has become the mainstream direction for saline-alkali land management.
[0004] Compound microbial fertilizers, as the core carrier of biological improvement, combine the characteristics of both microbial and organic fertilizers through the synergistic effect of functional microorganisms. However, some existing compound microbial fertilizers suffer from problems such as unscientific strain ratios, weak functional targeting, limited effects on saline-alkali land improvement, and insignificant growth-promoting effects on specific crops, making it difficult to simultaneously achieve the dual goals of efficient saline-alkali land improvement and the promotion of growth in multiple crops. Therefore, developing a saline-alkali land improvement microbial fertilizer with a reasonable ratio, stable improvement effect, and suitability for the growth needs of crops such as corn, cotton, and alfalfa is of great significance for promoting the sustainable development of agriculture in saline-alkali land. Summary of the Invention
[0005] This application provides a microbial fertilizer for improving saline-alkali land and its application, in order to solve the problems of short-lived physical effects of traditional microbial fertilizers, high cost and easy pollution of chemical improvement, and poor growth promotion effect.
[0006] This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial culture: 5-15%; *Pseudomonas saponin* bacterial suspension: 5-15%; Organic fertilizer: 70-90%.
[0007] Optionally, the organic fertilizer has an organic matter content of ≥30%, a C / N ratio of 15-25:1, and a decomposition degree of ≥85%.
[0008] Optionally, the ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension is 1:1 based on the effective viable count, and the effective viable count of each is not less than 2.0 × 10⁻⁶. 9 CFU / g.
[0009] Optionally, both Bacillus subtilis and Pseudomonas saponinophilus are prepared by culturing on NB medium, which consists of: 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.0-7.2.
[0010] Optionally, the culture conditions for Bacillus subtilis and Pseudomonas saponinophilus are: 30℃, 180 rpm shaking culture for 24 h, and after the culture is completed, the bacterial solution is confirmed by microscopic examination to be free of contamination (contamination rate ≤0.1%).
[0011] Optionally, the organic fertilizer is a mixture of well-rotted animal manure (chicken manure and sheep manure in a 3:2 ratio) and plant residues (corn stalks and wheat stalks in a 2:1 ratio). The well-rotted fertilizer needs to undergo high-temperature fermentation (55-65℃) for 7-10 days to ensure that there are no pathogens.
[0012] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, comprising the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. S3: Compounding and Formulation of Microbial Fertilizer: The compound microbial agent obtained in S2 is diluted 100 times with sterile water and mixed with the pretreated organic fertilizer in proportion. The mixture is stirred for 30 minutes using a double helix mixer at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, thus obtaining the microbial fertilizer for improving saline-alkali land.
[0013] Optionally, the organic fertilizer pretreatment in S3 includes the following steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%. After measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 25-30%, with a deviation of ≤±1%.
[0014] Optionally, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the pulverized organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature, and then the moisture content is adjusted to ensure that the residual pathogens in the organic fertilizer are ≤10 CFU / g. This application also proposes the application of a microbial fertilizer for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0015] Therefore, this application has at least the following beneficial effects: (1) In the embodiments of this application, Bacillus subtilis and Pseudomonas saponophila synergistically produce polybasic organic acids, which neutralize soil OH⁻ by ionizing H⁺, break the hydrolysis balance of CO3²⁻ / HCO3⁻ and convert it into CO2 volatilization, thereby reducing the soil pH from 8.14 to 7.32, with a reduction rate of 10.1%; the carboxyl and hydroxyl groups of organic acids complex with cations such as Na⁺ and Ca²⁺ in the soil to form water-soluble chelate salts, which are leached away from the rhizosphere, and the salt content is reduced by up to 71.3%; the extracellular polysaccharides (EPS) secreted by the strain synergistically bind soil particles with organic fertilizer humus to form a water-stable aggregate structure, which significantly improves soil aeration and permeability, reduces bulk density and reduces compaction.
[0016] (2) In the embodiments of this application, Bacillus subtilis secretes hydrolytic enzymes such as cellulase and protease, and Pseudomonas saponophila secretes lignin-degrading enzymes. Together, they decompose the large molecular organic matter in organic fertilizer into small molecular nutrients, which not only provides carbon source for the strain to promote reproduction, but also provides direct absorption by crops, resulting in a maximum increase of 24.4% in soil organic matter. The two strains activate nitrogen through nitrogenase, activate phosphorus through organic acid and phytase, and activate potassium through organic acid complexation. Combined with the fertilizer retention function of humus, they increase the soil alkaline nitrogen, available phosphorus and available potassium by a maximum of 18.3%, 21.8% and 9.88%, respectively, which significantly improves the soil nutrient supply capacity and nutrient utilization rate, and provides sufficient and balanced nutritional support for crop growth.
[0017] (3) In this embodiment, by reducing soil pH and salt content to reduce stress sources, and combined with ACC deaminase and osmotic regulators produced by the strain, the crop’s salt and alkali resistance is significantly improved and the damage of reactive oxygen species to cell membranes is reduced. The IAA produced by metabolism activates crop cell signaling pathways, promotes cell elongation and division, and increases the maximum growth rate of cotton seedling stem length, plant height and root length by 66.52%, 66.02% and 54.29% respectively, and alfalfa seedling plant height and root length by 65.3% and 55.4% respectively. Soil nutrient optimization and vitamin B complex secreted by the strain ensure chlorophyll synthesis and photosynthetic enzyme activity. The chlorophyll content of cotton seedlings increases by a maximum of 8.38%, and the maximum increase rate of chlorophyll a and b in alfalfa seedlings reaches 80.5% and 113.4% respectively, improving photosynthetic efficiency. Finally, it coordinates the nutritional and reproductive growth of corn, resulting in a maximum increase of 28.1% in corn yield, and a maximum increase of 27% and 17% in ear grain number and ear grain weight respectively.
[0018] (4) In the embodiments of this application, the microbial fertilizer components are all environmentally friendly and have no chemical additives. The two strains are beneficial soil microorganisms. They inhibit pathogens such as Fusarium through nutrient competition and secretion of antibacterial substances, significantly improving the soil microecological balance and reducing the occurrence of soil-borne diseases of crops. The organic fertilizer is derived from decomposed animal and plant waste, realizing resource recycling, reducing the risk of environmental pollution from waste, and improving the utilization rate of agricultural resources. After the strains complete metabolism, they will be assimilated and decomposed by the soil microbial community, with no residual risk. The environmental safety of soil and crops is guaranteed throughout the process, improving the sustainable utilization capacity of saline-alkali land agriculture, meeting the requirements of green agricultural development, and having no secondary pollution risks.
[0019] This solves the problems of traditional microbial fertilizers, such as short-lived physical effects, high cost and easy pollution from chemical modification, and poor growth promotion effect.
[0020] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description
[0021] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a flowchart of a method for preparing microbial fertilizer for improving saline-alkali land according to an embodiment of this application; Figure 2 This is a graph showing maize yield and related trait indicators provided according to embodiments of this application; Figure 3 This is a diagram showing the results of the physicochemical property determination of maize rhizosphere soil according to the embodiments of this application; Figure 4 This is a graph showing the comprehensive measurement results of cotton seedling growth indicators according to the embodiments of this application; Figure 5 This is a graph showing the comprehensive measurement results of alfalfa seedling growth indicators provided in the embodiments of this application. Detailed Implementation
[0022] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.
[0023] In the embodiments of this application, unless otherwise specified, the raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.
[0024] The present application will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present application in any way.
[0025] In this application embodiment, the drug source includes: *Pseudomonas saponophilus*, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit date of January 7, 2026, and classified as *Pseudomonas saponophilus*. Pseudomonas saponiphila The registration number is: CGMCC No. 37314; Bacillus subtilis Bacillus subtilis The registration number is: CGMCC No.37313.
[0026] Example 1 This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial culture: 5%; *Pseudomonas saponin* bacterial suspension: 5%; Organic fertilizer: 90%.
[0027] Among them, the organic matter content of organic fertilizer is ≥30%, the C / N ratio is 15:1, and the degree of decomposition is ≥85%.
[0028] The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension, based on the effective viable count, was 1:1, and the effective viable count of each was not less than 2.0 × 10⁻⁶. 9 CFU / g.
[0029] Bacillus subtilis and Pseudomonas saponinophilus were both cultured on NB medium, which consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.0.
[0030] The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
[0031] The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to be fermented at a high temperature of 55℃ for 7 days to ensure that there are no pathogens.
[0032] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, such as... Figure 1 As shown, it includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. It is understood that in the embodiments of this application, the activity of the strain is first activated to ensure its metabolic capacity through gradient activation and large-scale culture, and then the cell volume is increased through large-scale culture in a fermenter. Strict control of culture parameters can ensure the purity and activity of single strain bacterial solution, avoid contamination by other bacteria, and lay the foundation for the precise formulation and function of subsequent compound bacterial agents.
[0033] S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. It is understood that the precise control of the mixing ratio of the two strains in this application embodiment ensures the basis for their synergistic effect. The gentle stirring rate and temperature conditions can reduce bacterial cell damage, and avoiding direct sunlight can prevent ultraviolet rays from damaging the bacterial cell structure, thus ensuring the overall activity of the compound bacterial agent and improving the improvement efficiency of subsequent bacterial fertilizers.
[0034] S3: Compounding and Formulation of Microbial Fertilizer: Dilute the compound microbial agent obtained in S2 with sterile water by 100 times, mix it with the pretreated organic fertilizer in proportion, and stir it with a double helix mixer for 30 minutes at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, so as to obtain microbial fertilizer for improving saline-alkali land.
[0035] It is understood that diluting the compound microbial agent before mixing it with organic fertilizer in this embodiment can improve the uniformity of mixing; the double-helix stirring method can ensure that the microbial agent and organic fertilizer are fully integrated, and the appropriate stirring rate and time can avoid damage to the activity of the microbial agent, while ensuring the overall quality of the microbial fertilizer is uniform, thus providing a guarantee for the stable improvement effect during subsequent field application.
[0036] Among them, the organic fertilizer pretreatment in S3 includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%; after measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 25%, with a deviation of ≤±1%.
[0037] Among them, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
[0038] This application also proposes the application of microbial fertilizers for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0039] For example, before planting corn, cotton and alfalfa in saline-alkali land, the preparation of compound microbial fertilizer for improving saline-alkali land is completed first, and then it is applied to the experimental plot in an appropriate manner and dosage. The specific process and effect are as follows: Bacillus subtilis B from a laboratory of an agricultural university is selected. subtilis HZ-1 and Pseudomonas saponinophilus P. saponiphilaGZ-2 was used as a functional microbial strain. Two strains were separately inoculated into NB medium and cultured for 24 h. They were then mixed at a 1:1 volume ratio to obtain a compound microbial agent. Organic fertilizer with an organic matter content of 30% (purchased from Liaoning Kuningwang Agricultural Co., Ltd.) was used as a carrier. The compound microbial agent was diluted 100 times and mixed evenly with the organic fertilizer to prepare a compound microbial fertilizer. Subsequently, one control group (CK) and three experimental groups were set up for the experiment. The control group received no microbial agent, only an equal volume of water. Experimental group 1 (T1) received only the aforementioned organic fertilizer, experimental group 2 (T2) received only the aforementioned compound microbial agent, and experimental group 3 (T3) received the prepared compound microbial fertilizer. All groups were treated according to the standard per 100 m²: T3 received a mixture of 10 L of the 100-fold diluted compound microbial agent and 250 kg of organic fertilizer; T1 received 250 kg of organic fertilizer; and T2 received a mixture of 10 L of the 100-fold diluted compound microbial agent and 250 kg of organic fertilizer. L-compound microbial agent was applied, and the control group was treated with an equal amount of water. All agents were evenly spread and then tilled. Corn, cotton, and alfalfa were then planted in the treated saline-alkali plots and corresponding pot experiments, respectively, with consistent planting conditions to ensure experimental validity. A five-point sampling method was used to collect rhizosphere soil samples. A clean, alcohol-sterilized small shovel was used to collect samples, digging 5-10 cm around the target crop to a depth of 20 cm. After removing sand and debris, each sample was replicated three times, placed in 50 mL centrifuge tubes, and labeled for later use. Specific measurement indicators and methods were established. For corn, after maturity, ear length, ear weight, ear diameter, number of rows per ear, number of kernels per row, number of kernels per ear, ear kernel weight, 100-kernel weight, and actual yield were measured and recorded. For cotton seedlings, after 45 days of growth, stem length, root length, plant height, and chlorophyll content were measured. For alfalfa seedlings, after 45 days of growth, stem length, root length, plant height, and chlorophyll content were measured. After d, root length, plant height, aboveground fresh weight, underground fresh weight, and chlorophyll a and chlorophyll b contents were measured. For soil physicochemical properties, pH was measured using the potentiometric method, conductivity was measured using a magnetic conductivity meter and converted to salt content, organic matter content was measured using the potassium dichromate volumetric dilution pyrometric method, available nitrogen content was measured using the alkaline hydrolysis diffusion method, available phosphorus content was measured using the sodium bicarbonate extraction molybdenum-antimony colorimetric method, and available potassium content was measured using the NaNO3 extraction sodium tetraphenylborate turbidimetric method. The final experimental results were significant. Compound microbial fertilizer can improve saline-alkali land and promote crop growth through the synergistic effect of microbial agents and organic fertilizers: for maize, the yields of groups T1, T2, and T3 increased by 13.4%, 19.8%, and 28.1% respectively compared to the control group (group T3 showed the largest increase). Figure 2 A) both ear length and ear weight increased, ear grain number increased by 13%, 20%, and 27%, respectively, and ear grain weight increased by 7%, 13%, and 17%, respectively. Both ear grain number and ear weight showed significant differences compared to the CK group. Figure 2 B, Figure 2(See the graph showing maize yield and related trait indicators). Regarding soil, the pH of groups T1, T2, and T3 decreased by 4.1%, 6.1%, and 10.1% respectively compared to the control group (CK), and the salt content decreased by 52.9%, 58.9%, and 71.3% respectively (group T3 showed the most significant salt reduction effect). Furthermore, the contents of soil organic matter, available nitrogen, available phosphorus, and available potassium were all higher than in the control group, with the largest increase in group T3, increasing by 24.4%, 18.3%, 21.8%, and 9.88% respectively. Figure 3 , Figure 3 (Figure showing the results of the physicochemical properties determination of maize rhizosphere soil); For cotton seedlings, after 45 days of growth, the stem length of groups T1, T2, and T3 increased by 22.17%, 10.86%, and 66.52% respectively compared with the CK group. Figure 4 A), the plant height of group T3 increased by 66.02%, 34.92%, and 45.05% compared with groups CK, T1, and T2, respectively. Figure 4 B), the root length increased by 54.29% compared to the CK group ( Figure 4 C), chlorophyll content increased by 8.38%, 4.86%, and 4.58% respectively compared to CK, T1, and T2 groups. Figure 4 D, Figure 4 E is a graph showing the growth of cotton seedlings. Figure 4 (See the comprehensive measurement results of cotton seedling growth indicators); For alfalfa seedlings, the plant height of group T3 increased by 65.3% compared with group CK 45 days after harvest in the pot experiment. Figure 5 A), the root lengths of groups T1, T2, and T3 increased by 11.8%, 8%, and 55.4% respectively compared to the CK group. Figure 5 B), the aboveground fresh weight of group T3 increased by approximately 184.1% ( Figure 5 C) The increase in underground fresh weight was approximately 144.9% ( Figure 5 D), the contents of chlorophyll a and b increased by 80.5% compared with the CK group (D). Figure 5 E), 113.4% Figure 5 F, significantly higher than CK group (P<0.05), all three experimental groups showed significant growth-promoting effects, with T3 group showing the most prominent effect. Figure 5 G is a graph showing the growth of alfalfa seedlings. Figure 5 (Graph showing the comprehensive measurement results of alfalfa seedling growth indicators).
[0040] Example 2 This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial suspension: 7.5%; *Pseudomonas saponinophilus* bacterial suspension: 7.5%; Organic fertilizer: 85%.
[0041] Among them, the organic matter content of organic fertilizer is ≥30%, the C / N ratio is 17.5:1, and the degree of decomposition is ≥85%.
[0042] The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension, based on the effective viable count, was 1:1, and the effective viable count of each was not less than 2.0 × 10⁻⁶. 9 CFU / g.
[0043] Bacillus subtilis and Pseudomonas saponinophilus were both cultured on NB medium, which consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.05.
[0044] The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
[0045] The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to undergo high-temperature fermentation at 57.5℃ for 7.75 days to ensure that there are no pathogens.
[0046] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, such as... Figure 1 As shown, it includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. It is understood that in the embodiments of this application, the activity of the strain is first activated to ensure its metabolic capacity through gradient activation and large-scale culture, and then the cell volume is increased through large-scale culture in a fermenter. Strict control of culture parameters can ensure the purity and activity of single strain bacterial solution, avoid contamination by other bacteria, and lay the foundation for the precise formulation and function of subsequent compound bacterial agents.
[0047] S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. It is understood that the precise control of the mixing ratio of the two strains in this application embodiment ensures the basis for their synergistic effect. The gentle stirring rate and temperature conditions can reduce bacterial cell damage, and avoiding direct sunlight can prevent ultraviolet rays from damaging the bacterial cell structure, thus ensuring the overall activity of the compound bacterial agent and improving the improvement efficiency of subsequent bacterial fertilizers.
[0048] S3: Compounding and Formulation of Microbial Fertilizer: Dilute the compound microbial agent obtained in S2 with sterile water by 100 times, mix it with the pretreated organic fertilizer in proportion, and stir it with a double helix mixer for 30 minutes at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, so as to obtain microbial fertilizer for improving saline-alkali land.
[0049] It is understood that diluting the compound microbial agent before mixing it with organic fertilizer in this embodiment can improve the uniformity of mixing; the double-helix stirring method can ensure that the microbial agent and organic fertilizer are fully integrated, and the appropriate stirring rate and time can avoid damage to the activity of the microbial agent, while ensuring the overall quality of the microbial fertilizer is uniform, thus providing a guarantee for the stable improvement effect during subsequent field application.
[0050] Among them, the organic fertilizer pretreatment in S3 includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%; after measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 26.25%, with a deviation of ≤±1%.
[0051] Among them, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
[0052] This application also proposes the application of microbial fertilizers for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0053] Example 3 This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial suspension: 10%; Pseudomonas saponin bacterial suspension: 10%; Organic fertilizer: 80%.
[0054] Among them, the organic matter content of organic fertilizer is ≥30%, the C / N ratio is 20:1, and the degree of decomposition is ≥85%.
[0055] The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension, based on the effective viable count, was 1:1, and the effective viable count of each was not less than 2.0 × 10⁻⁶. 9CFU / g.
[0056] Bacillus subtilis and Pseudomonas saponinophilus were both cultured on NB medium, which consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.1.
[0057] The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
[0058] The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to be fermented at a high temperature of 60℃ for 8.5 days to ensure that there are no pathogens.
[0059] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, such as... Figure 1 As shown, it includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. It is understood that in the embodiments of this application, the activity of the strain is first activated to ensure its metabolic capacity through gradient activation and large-scale culture, and then the cell volume is increased through large-scale culture in a fermenter. Strict control of culture parameters can ensure the purity and activity of single strain bacterial solution, avoid contamination by other bacteria, and lay the foundation for the precise formulation and function of subsequent compound bacterial agents.
[0060] S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. It is understood that the precise control of the mixing ratio of the two strains in this application embodiment ensures the basis for their synergistic effect. The gentle stirring rate and temperature conditions can reduce bacterial cell damage, and avoiding direct sunlight can prevent ultraviolet rays from damaging the bacterial cell structure, thus ensuring the overall activity of the compound bacterial agent and improving the improvement efficiency of subsequent bacterial fertilizers.
[0061] S3: Compounding and Formulation of Microbial Fertilizer: Dilute the compound microbial agent obtained in S2 with sterile water by 100 times, mix it with the pretreated organic fertilizer in proportion, and stir it with a double helix mixer for 30 minutes at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, so as to obtain microbial fertilizer for improving saline-alkali land.
[0062] It is understood that diluting the compound microbial agent before mixing it with organic fertilizer in this embodiment can improve the uniformity of mixing; the double-helix stirring method can ensure that the microbial agent and organic fertilizer are fully integrated, and the appropriate stirring rate and time can avoid damage to the activity of the microbial agent, while ensuring the overall quality of the microbial fertilizer is uniform, thus providing a guarantee for the stable improvement effect during subsequent field application.
[0063] Among them, the organic fertilizer pretreatment in S3 includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%; after measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 27.5%, with a deviation of ≤±1%.
[0064] Among them, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
[0065] This application also proposes the application of microbial fertilizers for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0066] Example 4 This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial suspension: 12.5%; *Pseudomonas saponinophilus* bacterial suspension: 12.5%; Organic fertilizer: 75%.
[0067] Among them, the organic matter content of organic fertilizer is ≥30%, the C / N ratio is 22.5:1, and the degree of decomposition is ≥85%.
[0068] The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension, based on the effective viable count, was 1:1, and the effective viable count of each was not less than 2.0 × 10⁻⁶. 9 CFU / g.
[0069] Bacillus subtilis and Pseudomonas saponinophilus were both cultured on NB medium, which consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.15.
[0070] The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
[0071] The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to undergo high-temperature fermentation at 62.5℃ for 9.25 days to ensure that there are no pathogens.
[0072] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, such as... Figure 1 As shown, it includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. It is understood that in the embodiments of this application, the activity of the strain is first activated to ensure its metabolic capacity through gradient activation and large-scale culture, and then the cell volume is increased through large-scale culture in a fermenter. Strict control of culture parameters can ensure the purity and activity of single strain bacterial solution, avoid contamination by other bacteria, and lay the foundation for the precise formulation and function of subsequent compound bacterial agents.
[0073] S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. It is understood that the precise control of the mixing ratio of the two strains in this application embodiment ensures the basis for their synergistic effect. The gentle stirring rate and temperature conditions can reduce bacterial cell damage, and avoiding direct sunlight can prevent ultraviolet rays from damaging the bacterial cell structure, thus ensuring the overall activity of the compound bacterial agent and improving the improvement efficiency of subsequent bacterial fertilizers.
[0074] S3: Compounding and Formulation of Microbial Fertilizer: Dilute the compound microbial agent obtained in S2 with sterile water by 100 times, mix it with the pretreated organic fertilizer in proportion, and stir it with a double helix mixer for 30 minutes at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, so as to obtain microbial fertilizer for improving saline-alkali land.
[0075] It is understood that diluting the compound microbial agent before mixing it with organic fertilizer in this embodiment can improve the uniformity of mixing; the double-helix stirring method can ensure that the microbial agent and organic fertilizer are fully integrated, and the appropriate stirring rate and time can avoid damage to the activity of the microbial agent, while ensuring the overall quality of the microbial fertilizer is uniform, thus providing a guarantee for the stable improvement effect during subsequent field application.
[0076] Among them, the organic fertilizer pretreatment in S3 includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%; after measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 28.75%, with a deviation of ≤±1%.
[0077] Among them, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
[0078] This application also proposes the application of microbial fertilizers for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0079] Example 5 This application provides a microbial fertilizer for improving saline-alkali land, which, by weight percentage, comprises: Bacillus subtilis bacterial culture: 15%; *Pseudomonas saponin* bacterial suspension: 15%; Organic fertilizer: 70%.
[0080] Among them, the organic matter content of organic fertilizer is ≥30%, the C / N ratio is 25:1, and the degree of decomposition is ≥85%.
[0081] The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension, based on the effective viable count, was 1:1, and the effective viable count of each was not less than 2.0 × 10⁻⁶. 9 CFU / g.
[0082] Bacillus subtilis and Pseudomonas saponinophilus were both cultured on NB medium, which consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.2.
[0083] The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
[0084] The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to be fermented at a high temperature of 65℃ for 10 days to ensure that there are no pathogens.
[0085] This application also proposes a method for preparing microbial fertilizer for improving saline-alkali land, such as... Figure 1 As shown, it includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL of NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. It is understood that in the embodiments of this application, the activity of the strain is first activated to ensure its metabolic capacity through gradient activation and large-scale culture, and then the cell volume is increased through large-scale culture in a fermenter. Strict control of culture parameters can ensure the purity and activity of single strain bacterial solution, avoid contamination by other bacteria, and lay the foundation for the precise formulation and function of subsequent compound bacterial agents.
[0086] S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and the mixture is stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 30℃ and direct sunlight is avoided. It is understood that the precise control of the mixing ratio of the two strains in this application embodiment ensures the basis for their synergistic effect. The gentle stirring rate and temperature conditions can reduce bacterial cell damage, and avoiding direct sunlight can prevent ultraviolet rays from damaging the bacterial cell structure, thus ensuring the overall activity of the compound bacterial agent and improving the improvement efficiency of subsequent bacterial fertilizers.
[0087] S3: Compounding and Formulation of Microbial Fertilizer: Dilute the compound microbial agent obtained in S2 with sterile water by 100 times, mix it with the pretreated organic fertilizer in proportion, and stir it with a double helix mixer for 30 minutes at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, so as to obtain microbial fertilizer for improving saline-alkali land.
[0088] It is understood that diluting the compound microbial agent before mixing it with organic fertilizer in this embodiment can improve the uniformity of mixing; the double-helix stirring method can ensure that the microbial agent and organic fertilizer are fully integrated, and the appropriate stirring rate and time can avoid damage to the activity of the microbial agent, while ensuring the overall quality of the microbial fertilizer is uniform, thus providing a guarantee for the stable improvement effect during subsequent field application.
[0089] Among them, the organic fertilizer pretreatment in S3 includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%; after measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 30%, with a deviation of ≤±1%.
[0090] Among them, the organic fertilizer pretreatment in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
[0091] This application also proposes the application of microbial fertilizers for improving saline-alkali land, which can be used to promote the growth of corn, cotton, and alfalfa seedlings and increase corn yield.
[0092] Comparative Example 1 This comparative example provides a compound microbial fertilizer for improving saline-alkali land and its application. The only difference between this example and Example 1 is that it does not contain Pseudomonas saponophila bacterial solution. The reduced amount of Pseudomonas saponophila bacterial solution is distributed to Bacillus subtilis bacterial solution. The other components, component contents, and application process are the same as in Example 1.
[0093] Comparative Example 2 This comparative example provides a compound microbial fertilizer for improving saline-alkali land and its application. The only difference between this example and Example 1 is that it does not contain Bacillus subtilis bacterial solution. Instead, the reduced amount of Bacillus subtilis bacterial solution is distributed to Pseudomonas saponinophilus bacterial solution. The other components, component contents, and application process are the same as in Example 1.
[0094] Comparative Example 3 This comparative example provides a compound microbial fertilizer for improving saline-alkali land and its application. The only difference between this example and Example 1 is that it does not contain organic fertilizer. The amount of organic fertilizer is reduced and distributed to the Pseudomonas saponinophilus bacterial solution and Bacillus subtilis bacterial solution. The other components, component contents, and application process are the same as in Example 1.
[0095] Performance testing The pot experiment was used to simulate saline-alkali soil and analyze the effect of the microbial fertilizers prepared in Examples 1-5 and Comparative Examples 1-3 on improving saline-alkali soil. Typical saline-alkali soil with pH 8.14 and salt content 2.5 g / kg was selected. Each pot contained 5 kg of soil and applied the corresponding microbial fertilizer (20 g / kg soil). The control group without microbial fertilizer was used. The soil was placed under normal temperature and natural light for 30 days, and the soil moisture content was maintained at 25%. After the incubation, the soil pH, salt content and bulk density were measured. The pH reduction rate ((initial pH - post-treatment pH) / initial pH × 100%) and the salt content reduction rate ((initial salt content - post-treatment salt content) / initial salt content × 100%) were calculated. The performance test data are shown in Table 1.
[0096] Table 1 Test of Saline-Alkali Land Improvement Effect
[0097] As shown in Table 1, the microbial fertilizers prepared in Examples 1-5 of this application all exhibited excellent saline-alkali land improvement effects, and the improvement effect gradually increased with the increase of the proportion of microbial solution added: the pH reduction rate increased from 7.6% in Example 1 to 10.1% in Example 5, and the pH after treatment dropped to a minimum of 7.32, close to the neutral soil range; the salt content reduction rate increased simultaneously from 55.2% to 71.6%, and the salt content reduction rates of Examples 4 and 5 both exceeded 70%, achieving a significant desalination effect; the soil bulk density decreased from 1.35 g / cm³ in Example 1 to 1.22 g / cm³ in Example 5, and the soil looseness was significantly improved. This result fully confirms the effectiveness of the technical solution proposed in this application: the polybasic organic acids produced by the synergistic metabolism of Bacillus subtilis and Pseudomonas saponophilus can effectively ionize H⁺ to neutralize OH⁻ in the soil and disrupt the hydrolysis balance of CO₃²⁻ / HCO₃⁻. At the same time, they are removed by leaching through the formation of water-soluble chelate salts by complexing Na⁺, Ca²⁺ and other cations with carboxyl and hydroxyl groups. The EPS secreted by the strains synergistically binds soil particles with organic fertilizer humus to form a water-stable aggregate structure, thereby simultaneously achieving the improvement effects of reducing pH, reducing salinity and reducing bulk density. Moreover, the higher the proportion of bacterial solution, the stronger the synergistic metabolic efficiency and the more significant the improvement effect.
[0098] In comparison, the improvement effects of Comparative Examples 1-3 on saline-alkali land were significantly worse than those of the Example, and the improvement effect showed a stepwise decline as the core functional components were missing: Comparative Example 1 (missing Pseudomonas saponophila bacterial solution) only retained a single strain of Bacillus subtilis, which could not form efficient synergistic metabolism, with a pH reduction rate of only 3.6% and a salt content reduction rate of 27.6%; Comparative Example 2 (missing Bacillus subtilis bacterial solution) only retained Pseudomonas saponophila, and the improvement effect was further weakened due to the limited metabolic capacity of a single strain, with a pH reduction rate of 2.7% and a salt content reduction rate of 22.8%; Comparative Example 3 (missing organic fertilizer) lacked both the nutritional support of synergistic metabolism of the strain and the effect of humus cementing soil particles, resulting in the worst improvement effect, with a pH reduction rate of only 1.1%, a salt content reduction rate of less than 10%, and a soil bulk density as high as 1.55 g / cm³. The synergistic effect of two strains is the core prerequisite for the efficient production of multi-organic acids and EPS. Organic fertilizer provides a guarantee for the reproduction of strains and helps to improve soil structure. The lack of either core component will lead to a significant reduction in the improvement function, further highlighting the necessity of the synergistic effect of two strains and the combination of organic fertilizer in the embodiment.
[0099] The soil nutrient enhancement effect of the microbial fertilizers prepared in Examples 1-5 and Comparative Examples 1-3 was analyzed using a field plot experiment method. Typical saline-alkali land was selected and divided into 24 plots (each plot area 20 m²). 2The corresponding microbial fertilizers (300 kg / mu) were applied to randomly divided groups, with no microbial fertilizer applied as a blank control. Corn was planted as the indicator crop, and conventional field management was carried out during the growth cycle. Soil samples were collected before planting and after harvest to determine the contents of soil organic matter, available nitrogen, available phosphorus, and available potassium. The increase rate of each nutrient index was calculated as ((content after harvest - content before planting) / content before planting × 100%). The performance test data are shown in Table 2.
[0100] Table 2 Soil Nutrient Enhancement Effect Test
[0101] As shown in Table 2, the microbial fertilizers prepared in Examples 1-5 of this application can significantly improve and maintain soil nutrient levels, and the effect shows a steady increasing trend with the increase of the proportion of microbial solution added. Although corn absorbs a large amount of nutrients during growth, the organic matter, available nitrogen, available phosphorus, and available potassium contents of the soil in each treatment group after harvest showed a net increase compared with those before planting. The organic matter increase rate increased from 15.6% in Example 1 to 24.4% in Example 5, achieving a significant accumulation of soil organic matter; the increase rates of available nitrogen, available phosphorus, and available potassium increased simultaneously, with the increase rate of available nitrogen reaching 18.3%, available phosphorus reaching 21.8%, and available potassium reaching 9.9% in Example 5. This result fully confirms the effectiveness of the technical solution proposed in this application: the cellulase and protease secreted by Bacillus subtilis and the lignin-degrading enzyme secreted by Pseudomonas saponophila work synergistically to efficiently decompose organic fertilizer and large-molecule organic matter in the soil into small-molecule nutrients, and promote the activation of inherent soil nutrients; the two strains supplement nitrogen through nitrogen fixation, secrete organic acids and phytase to activate fixed phosphorus, and release slow-release potassium through ion exchange and complexation, which, together with the continuous provision of nutrient raw materials by organic fertilizer and the fertilizer retention function of humus, not only compensates for the consumption by crop absorption, but also achieves a net increase in the supply capacity of various readily available nutrients in the soil. The higher the proportion of bacterial solution, the stronger the enzyme secretion and activation efficiency, and the more significant the nutrient enhancement and maintenance effect.
[0102] In comparison, the soil nutrient maintenance and enhancement effects of Comparative Examples 1-3 were significantly weaker than those of the Example, and the lack of core components led to a significant reduction in their capabilities: Comparative Example 1 (lacking Pseudomonas saponophila bacterial solution) lacked lignin-degrading enzymes, resulting in insufficient decomposition of macromolecular organic matter, with an organic matter increase rate of only 8.2%, and a low increase rate for each available nutrient; Comparative Example 2 (lacking Bacillus subtilis bacterial solution) lacked cellulase, protease, and nitrogen fixation, weakening its ability to decompose organic matter and supplement nitrogen, further reducing its nutrient enhancement effect, with an organic matter increase rate of 7.5%; Comparative Example 3 (lacking organic fertilizer) lacked both exogenous nutrients and organic matter supplementation, and also lost the fertilizer retention function of humus, resulting in the worst nutrient enhancement effect, with an organic matter increase rate of only 3.2%, and an increase rate for each available nutrient of less than 5%. The synergistic degradation and activation by the two strains and the provision of material basis by organic fertilizer are the core mechanisms for enhancing and maintaining soil fertility. The lack of any core component will lead to a significant reduction in this function, further highlighting the necessity of the synergistic effect of the two strains and the combination of organic fertilizer in the Example.
[0103] The effects of the microbial fertilizers prepared in Examples 1-5 and Comparative Examples 1-3 on crop growth promotion and yield increase were analyzed using a combination of pot and field experiments. Cotton seedlings, alfalfa seedlings, and corn were selected as test crops. Pot experiment: Each pot contained 5 kg of saline-alkali soil, applied the corresponding microbial fertilizer (20 g / kg soil), and sowed cotton and alfalfa seeds. The plants were cultured at room temperature and under natural light for 30 days. Seedling stem length, plant height, root length, and chlorophyll content were measured, and the growth rate of growth indicators was calculated as ((treatment group index - control group index) / control group index × 100%). Field experiment: Saline-alkali plots identical to those used in the pot experiment were selected, and the corresponding microbial fertilizer (300 kg / mu) was applied. Corn was planted and managed conventionally until harvest. Corn yield, number of grains per ear, and grain weight per ear were measured. The yield increase rate and the increase rate of yield components were calculated. Performance test data are shown in Table 3.
[0104] Table 3. Crop growth promotion and yield increase effects test
[0105] As shown in Table 3, the microbial fertilizers prepared in Examples 1-5 of this application can significantly promote crop growth and increase corn yield, and the effect gradually increases with the increase of the proportion of microbial solution added: In terms of seedling growth, the cotton seedling height growth rate of Example 5 reached 66.0% and the root length growth rate reached 54.3%, and the alfalfa seedling height growth rate reached 65.3% and the root length growth rate reached 55.4%, all of which achieved a significant improvement in seedling growth; In terms of yield, the corn yield increase rate of Example 5 reached 28.1% and the number of grains per ear increased by 27.0%, and the yield composition was significantly optimized. This result fully confirms the effectiveness of the technical solution proposed in this application: the microbial fertilizer creates a superior growth environment for crops by reducing soil pH and salinity, optimizing soil structure (Table 1), and enhancing nutrient supply capacity (Table 2); plant growth regulators (such as IAA) produced by the bacterial strains can activate crop cell signaling pathways and promote cell elongation and division; at the same time, the optimization of soil nutrients and the activity of the bacterial strains ensure chlorophyll synthesis and photosynthetic enzyme activity, improve photosynthetic efficiency, and ultimately coordinate crop vegetative and reproductive growth, achieving growth promotion and yield increase. The higher the proportion of bacterial solution, the more abundant the improvement of the growth environment and the more sufficient the supply of beneficial substances, and the more significant the effect.
[0106] In comparison, the crop growth promotion and yield increase effects of Comparative Examples 1-3 were significantly inferior to those of the Example, and the lack of core components led to a substantial reduction in the effect: Comparative Example 1 (lacking Pseudomonas saponophila bacterial solution) lacked some degrading enzymes and synergistic effects, resulting in limited soil improvement and nutrient activation effects, and restricted crop growth, with cotton seedling height growth rate of only 20.5% and corn yield increase rate of only 8.2%; Comparative Example 2 (lacking Bacillus subtilis bacterial solution) lacked IAA, cellulase, and nitrogen fixation effects, resulting in weakened growth promotion and nutrient activation capabilities, with alfalfa seedling root length growth rate of only 15.9% and corn yield increase rate of only 7.1%; Comparative Example 3 (lacking organic fertilizer) lacked nutrient base, strain nutrition, and humus protection, resulting in a harsh crop growth environment, with cotton seedling height growth rate of only 10.3% and corn yield increase rate of only 3.5%. The synergistic effect of dual strains combined with organic fertilizer, through comprehensive improvement of soil physicochemical properties, enhancement of fertility, and regulation of crop physiology, is key to achieving crop growth promotion and yield increase. The lack of any core component will lead to a significant reduction in this function.
[0107] In summary, the microbial fertilizers prepared in Examples 1-5 of this application demonstrate significant advantages in three dimensions: saline-alkali land improvement, soil nutrient enhancement, and crop growth promotion and yield increase, comprehensively outperforming comparative examples 1-3. Regarding saline-alkali land improvement, relying on the core mechanism of synergistic metabolism between Bacillus subtilis and Pseudomonas saponophilus, and the assistance of organic fertilizer, the pH reduction rate reaches 7.6%-10.1% (down to a minimum of 7.32, close to neutral), the salt content reduction rate reaches 55.2%-71.6% (over 70% in Examples 4-5), and the soil bulk density decreases to 1.22-1.35. The solution achieves synergistic improvement in soil quality by reducing pH, desalinizing, and loosening the soil. Regarding soil nutrient enhancement, the synergistic degradation and activation by the two bacterial strains, combined with the nutrient retention and replenishment effects of organic fertilizer, resulted in an increase in organic matter of 15.6%-24.4%, with simultaneous increases in available nitrogen, phosphorus, and potassium (18.3%, 21.8%, and 9.9% respectively in Example 5), achieving net growth and improved supply capacity of soil nutrients. In terms of crop growth and yield, by improving the soil environment, secreting growth regulators, and enhancing photosynthetic efficiency, the solution significantly promoted crop growth and yield, resulting in increases in plant height and root length of cotton and alfalfa seedlings of up to 66.0% and 55.4%, respectively, and an increase in corn yield of up to 28.1% and an increase in ear grain number of up to 27.0%. These effects gradually increased with the increase in the proportion of bacterial solution added, fully demonstrating the effectiveness and application value of the technical solution.
[0108] According to the embodiments of this application, a microbial fertilizer for improving saline-alkali land and its application are proposed. Through the synergistic metabolism of Bacillus subtilis and Pseudomonas saponophila, multi-component organic acids are produced, neutralizing soil OH⁻, disrupting carbonate hydrolysis balance to lower pH (maximum reduction rate 10.1%), and forming water-soluble chelate salts through leaching to desalinate (maximum reduction rate 71.3%). Furthermore, the EPS secreted by the strains synergistically forms water-stable aggregate structures with organic fertilizer humus to improve soil. The two strains respectively secrete hydrolytic enzymes and lignin-degrading enzymes to synergistically decompose macromolecular organic matter, while simultaneously activating nitrogen, phosphorus, and potassium, and enhancing soil nutrient retention (organic matter). The maximum increase was 24.4%, and the maximum increase in available phosphorus was 21.8%; it enhances crop resistance by reducing salt and alkali stress and producing ACC deaminase, promotes crop growth by secreting IAA (the maximum increase in cotton seedling height was 66.02%), and ensures chlorophyll synthesis and photosynthetic efficiency to increase yield (the maximum increase in corn yield was 28.1%); moreover, the microbial fertilizer components are environmentally friendly, the dual strains inhibit pathogens and maintain microecological balance, the organic fertilizer achieves resource recycling, and there is no residue after the strains metabolize. Ultimately, it solves the problems of limited effectiveness of traditional saline-alkali land improvement materials, insufficient soil nutrient supply, weak crop salt and alkali resistance, and potential agricultural non-point source pollution.
[0109] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.
[0110] The present application and its embodiments have been described above. This description is not restrictive, and the actual application is not limited thereto. In conclusion, if a person skilled in the art is inspired by this description and designs a similar structure and embodiment without departing from the spirit of this application, such design should fall within the protection scope of this application.
Claims
1. A microbial fertilizer for improving saline-alkali land, characterized in that, Its composition by weight percentage is as follows: Bacillus subtilis bacterial culture: 5-15%; *Pseudomonas saponin* bacterial suspension: 5-15%; Organic fertilizer: 70-90%.
2. The microbial fertilizer for improving saline-alkali land according to claim 1, characterized in that, The organic fertilizer has an organic matter content of ≥30%, a C / N ratio of 15-25:1, and a decomposition degree of ≥85%.
3. The microbial fertilizer for improving saline-alkali land according to claim 1, characterized in that, The ratio of Bacillus subtilis bacterial suspension to Pseudomonas saponophila bacterial suspension is 1:1, based on the effective viable count, and the effective viable count of each is not less than 2.0 × 10⁻⁶. 9 CFU / g.
4. The microbial fertilizer for improving saline-alkali land according to claim 1, characterized in that, Both Bacillus subtilis and Pseudomonas saponinophilus were prepared by culturing on NB medium, which consisted of: 10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 1000 mL distilled water, and pH 7.0-7.
2.
5. The microbial fertilizer for improving saline-alkali land according to claim 1, characterized in that, The culture conditions for Bacillus subtilis and Pseudomonas saponinophilus were: 30℃, 180 rpm shaking culture for 24 h. After the culture was completed, the bacterial solution was examined under a microscope to confirm that there was no contamination by other bacteria (the contamination rate was ≤0.1%).
6. The microbial fertilizer for improving saline-alkali land according to claim 1, characterized in that, The organic fertilizer is a mixture of well-rotted animal manure and plant residues. The well-rotted animal manure is a mixture of chicken manure and sheep manure in a 3:2 ratio, and the plant residues are a mixture of corn stalks and wheat stalks in a 2:1 ratio. The well-rotted fertilizer needs to undergo high-temperature fermentation at 55-65℃ for 7-10 days to ensure that there are no pathogens.
7. A method for preparing microbial fertilizer for improving saline-alkali land as described in any one of claims 1-6, characterized in that, Includes the following steps: S1: Activation and scale-up culture of strains: Bacillus subtilis and Pseudomonas saponophila were inoculated from slant culture medium into 50 mL NB seed culture and cultured at 30℃ and 180 rpm for 12 h for activation. Then, they were transferred to a 10 L fermenter (NB medium) at a 5% inoculation rate and cultured at 30℃, 180 rpm and 0.5 vvm for 24 h to obtain single strain bacterial culture. S2: Preparation of compound microbial agent: The two single-strain bacterial solutions obtained in S1 are mixed at a ratio of 1:1 for effective viable bacteria, and stirred at 50 rpm for 15 min to obtain the compound microbial agent. During the mixing process, the temperature is controlled at 28-30℃ and direct sunlight is avoided. S3: Compounding and Formulation of Microbial Fertilizer: The compound microbial agent obtained in S2 is diluted 100 times with sterile water and mixed with the pretreated organic fertilizer in proportion. The mixture is stirred for 30 minutes using a double helix mixer at a stirring speed of 30 rpm to ensure that the uniformity of the mixture between the microbial agent and the organic fertilizer is ≥90%, thus obtaining the microbial fertilizer for improving saline-alkali land.
8. The method for preparing microbial fertilizer for improving saline-alkali land according to claim 7, characterized in that, S3 organic fertilizer pretreatment includes crushing and moisture content adjustment steps: the well-rotted mixed organic fertilizer is crushed by a crusher and then passed through a 5 mm sieve. The material on the sieve is returned to the machine for re-crushing to ensure that the particle size is ≤5 mm and the qualified rate is ≥95%. After measuring the initial moisture content, sterile water is sprayed and stirred to adjust the moisture content to 25-30%, with a deviation of ≤±1%.
9. The method for preparing microbial fertilizer for improving saline-alkali land according to claim 7, characterized in that, The pretreatment of organic fertilizer in S3 also includes a high-temperature sterilization step: the crushed organic fertilizer is sterilized at 105℃ for 2 hours, cooled to room temperature, and then the moisture content is adjusted to ensure that the residual amount of pathogens in the organic fertilizer is ≤10 CFU / g.
10. A microbial fertilizer for improving saline-alkali land as described in any one of claims 1-6, applied to promote the growth of corn, cotton seedlings, alfalfa seedlings and increase corn yield.