Microbial source humic acid fertilizer for increasing relative abundance of arbuscular mycorrhizal fungi in soybean rhizosphere soil
By preparing and applying corn-based microbial humic acid fertilizer, the problem of low abundance of Arthrobacter spp. in the rhizosphere soil of cowpeas was solved, resulting in increased root length and nutrient absorption, and improved yield and quality of cowpeas.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-19
- Publication Date
- 2026-03-24
AI Technical Summary
In traditional green bean cultivation, the relative abundance of Arthrobacter spp. in the rhizosphere soil is low, which affects the yield and quality of green beans.
Using corn steep liquor as raw material, artificially synthesized microbial humic acid fertilizer was prepared by pre-decomposition with cellulase, alkaline protease and amylase, combined with Auricularia auricula species and catalyst. The fertilizer was then applied to the roots of cowpea seedlings to increase the abundance of Arthrobacter spp. in the rhizosphere soil.
It significantly increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, promotes root length growth to 23-24 cm, enhances nutrient absorption capacity, and improves the yield and quality of cowpea.
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Figure CN120329133B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to microbial fertilizer manufacturing technology, specifically to a synthetically produced microbial humic acid that can increase the relative abundance of Arthrobacter genus in the rhizosphere soil of cowpea. Background Technology
[0002] Beneficial rhizosphere bacteria play an irreplaceable role in plant growth, soil improvement, and ecosystem stability. These beneficial bacteria support healthy plant growth, optimize the soil environment, and maintain ecosystem balance through multiple pathways. First, rhizosphere bacteria significantly promote nutrient absorption by plants. They can convert atmospheric nitrogen into usable ammonium nitrogen through nitrogen fixation, thus improving nitrogen nutrition levels. Simultaneously, by secreting organic acids and specific enzymes, beneficial bacteria can convert insoluble phosphates, potassium salts, and certain trace elements (such as iron, zinc, and manganese) in the soil into forms that can be absorbed and utilized by plants, significantly improving nutrient absorption efficiency. Furthermore, through close interaction with plant roots, beneficial bacteria secrete plant hormones such as indoleacetic acid and gibberellins, directly stimulating root growth and development, thereby enhancing the plant's ability to utilize soil resources.
[0003] Exogenous additives can significantly increase the abundance of beneficial bacteria in plant roots through multiple mechanisms, thereby optimizing the rhizosphere microbial community and promoting plant growth and health. These additives include organic carbon sources, microorganisms (such as Trichoderma, Arthrobacter, and Bacillus), and chemical signaling substances (such as quercetin and flavonoids). They not only directly provide abundant nutrients for beneficial bacteria and improve the rhizosphere microenvironment, but also stimulate the increase of plant root exudates, further attracting and colonizing beneficial bacteria. In addition, soil conditioners such as biochar and humates can enhance the survival ability of beneficial bacteria by optimizing soil physicochemical properties and providing a stable microenvironment. Studies have shown that exogenous additives can increase the relative abundance of beneficial rhizosphere bacteria by 20%-50%, enhance their disease resistance, nitrogen fixation, and phosphorus solubilization capabilities, while improving plant nutrient absorption, inhibiting pathogens, and increasing crop yield and quality, showing broad application prospects in green agriculture and soil remediation. Summary of the Invention
[0004] The purpose of this invention is to provide a microbial humic acid fertilizer that increases the relative abundance of Arthrobacter in the rhizosphere soil of cowpea. This artificially synthesized microbial humic acid, which increases the relative abundance of Arthrobacter in the rhizosphere soil of cowpea, is used to solve the problem of low relative abundance of Arthrobacter in the rhizosphere soil of traditional cowpea cultivation, which affects the yield and quality of cowpea.
[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: This artificially synthesized microbial humic acid, which increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, is prepared by the following method:
[0006] First, corn steep liquor pre-decomposition is performed, which consists of two stages:
[0007] The first stage involves pre-decomposing corn steep liquor using cellulase, alkaline protease, and amylase. The second stage involves pre-decomposing the product from the first stage using Auricularia auricula-judae fungi, and then diluting the pre-decomposed corn steep liquor containing microbial fungal residues with water.
[0008] Diluted corn steep liquor containing microbial and fungal residues is processed into synthetic microbial-derived humic acid through a three-stage reaction process:
[0009] The first stage reaction: the diluted corn steep liquor containing microbial fungal residues was reacted at pH 7.0 and the temperature was controlled at 140℃ for 2 hours; potassium hydroxide and sodium hydroxide were added in a mass ratio of 10:1, and the temperature was controlled at 160℃ for the second stage reaction, which lasted for 2 hours; sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride were then added in a mass ratio of 3:1:1, and the temperature was controlled at 180℃ for the third stage reaction, which lasted for 2 hours. The resulting artificially synthesized microbial humic acid liquid was dried to obtain artificially synthesized microbial humic acid fertilizer.
[0010] The artificially synthesized microbial humic acid fertilizer was diluted with water and applied to the roots of the cowpea seedlings. Fertilizer was applied every 7 days for a total of 3 times. After the fourth week, the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea reached 5%-6%, and the length of the cowpea root system reached 23-24 cm.
[0011] The method for corn steep liquor pre-decomposition in the above scheme:
[0012] In the first stage, corn steep liquor is pre-decomposed using cellulase and alkaline protease at a ratio of 3:1, at a temperature of 50°C, and for 3 hours. Then, amylase is used for further pre-decomposition at a temperature of 45°C for 1 hour.
[0013] In the second stage, the products from the first stage were pre-decomposed using Auricularia auricula-judae fungi. The pre-decomposition culture temperature was 25℃, and the culture time was 10 days.
[0014] In the above scheme, when the corn steep liquor containing microbial and fungal residues is diluted with water, it is then treated by a water processor at a frequency of 20000Hz for 5-20 minutes after dilution with water.
[0015] In the above scheme, the total mass ratio of potassium hydroxide and sodium hydroxide to the volume ratio of the diluted corn steep liquor solution containing microbial and fungal residues is 0.5-1.5%.
[0016] In the above scheme, the total mass ratio of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume ratio of the diluted corn steep liquor solution containing microbial and fungal residues is 0.2-2%.
[0017] In the above scheme, when the artificially synthesized microbial humic acid fertilizer is diluted with water, the amount of water added should be 1000 to 3000 times the amount of the artificially synthesized microbial humic acid fertilizer. When applying fertilizer, the amount applied is 20 to 100 mL per plant. Beneficial effects
[0018] 1. This invention increases the content of *Arthrobacter* spp. (rhizosphere growth-promoting bacteria) in the rhizosphere of green beans by applying artificially synthesized microbial humic acid from corn steep liquor. It also provides a method for pre-decomposing corn steep liquor and artificially synthesizing microbial humic acid. *Arthrobacter* spp., as a type of plant rhizosphere growth-promoting bacteria, has the function of promoting plant growth and its absorption and utilization of mineral nutrients.
[0019] 2. The artificially synthesized microbial humic acid molecules prepared by this invention contain abundant active functional groups such as carboxyl, hydroxyl and ketone groups, which have strong complexing ability and environmental stability. They can promote the growth of cowpea roots, making the length of cowpea roots reach 23-24 cm, while making the relative abundance of Arthrobacter genus in the rhizosphere soil of cowpea reach 5%-6%.
[0020] 3. This invention decomposes macromolecules (such as proteins and polysaccharides) in corn steep liquor into smaller molecules, such as short peptides, free amino acids and monosaccharides, by adding cellulase, alkaline protease and amylase, and then generates a large amount of artificially synthesized microbial humic acid substances through subsequent reactions. Attached Figure Description
[0021] Figure 1 A comparison diagram showing the relative abundance of Arthrobacter spp. in the rhizosphere soil of *Gnaphalium* when the present invention is applied to *Gnaphalium*.
[0022] Figure 2 A comparison diagram showing the length of the root system of a green bean (cowpea) after applying the present invention. Detailed Implementation
[0023] The invention will be further described below with reference to the accompanying drawings: Example
[0024] This artificially synthesized microbial humic acid, which increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, was prepared by the following method:
[0025] The raw corn steep liquor was pre-decomposed in the first stage using cellulase, alkaline protease, and amylase. The product of the first stage was then pre-decomposed in the second stage using Auricularia auricula-judae fungi. The pre-decomposed corn steep liquor containing microbial fungal residues was diluted by treating it with a water processor at 20,000 Hz for 10 minutes. Then, the corn steep liquor containing microbial fungal residues was processed into artificially synthesized microbial humic acid in three stages. The first stage reaction temperature was controlled at 140℃, and the reaction was carried out at pH=7.0 for 2 hours. The second stage reaction temperature was controlled at 160℃, with the addition of potassium hydroxide and sodium hydroxide in a ratio of 10:1, and the total mass of potassium hydroxide and sodium hydroxide to the volume ratio of the corn steep liquor solution being 0.5%, and the reaction was carried out for 2 hours. The third stage reaction temperature was controlled at 180℃, and the reaction was carried out for 2 hours. The catalysts sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride were in a mass ratio of 3:1:1, and the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume ratio of the corn steep liquor solution was 0.2%. The reacted liquid is dried to obtain artificially synthesized microbial humic acid fertilizer.
[0026] When artificially synthesized microbial humic acid fertilizer is applied to the roots of cowpeas, it needs to be diluted 3000 times, and the application amount after dilution is 80mL / plant. Example
[0027] This artificially synthesized microbial humic acid, which increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, was prepared by the following method:
[0028] The raw corn steep liquor was pre-decomposed in the first stage using cellulase, alkaline protease, and amylase. The product of the first stage was then pre-decomposed in the second stage using Auricularia auricula-judae fungi. The pre-decomposed corn steep liquor containing microbial fungal residues was diluted by treating it with a water processor at 20,000 Hz for 5 minutes. Then, the corn steep liquor containing microbial fungal residues was processed into artificially synthesized microbial humic acid in three stages. The first stage reaction was controlled at 140℃ and pH=7.0 for 2 hours. The second stage reaction was controlled at 160℃, with potassium hydroxide and sodium hydroxide added in a ratio of 10:1, and the total mass of potassium hydroxide and sodium hydroxide to the volume ratio of the corn steep liquor solution was 1%, for 2 hours. The third stage reaction was controlled at 180℃ for 2 hours, with the catalyst sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride in a mass ratio of 3:1:1, and the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume ratio of the corn steep liquor solution was 2%. The reacted liquid is dried to obtain a synthetically produced microbial humic acid fertilizer. When applying the synthetically produced microbial humic acid fertilizer to the roots of cowpeas, it needs to be diluted 3000 times, and the application amount after dilution is 100 mL per plant. Example
[0029] This artificially synthesized microbial humic acid, which increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, was prepared by the following method:
[0030] The raw corn steep liquor was pre-decomposed in the first stage using cellulase, alkaline protease, and amylase. The product of the first stage was then pre-decomposed in the second stage using Auricularia auricula-judae fungi. The pre-decomposed corn steep liquor containing microbial fungal residues was diluted by treating it with a water processor at 20,000 Hz for 5 minutes. Then, the corn steep liquor containing microbial fungal residues was processed into artificially synthesized microbial humic acid in three stages. The first stage reaction was controlled at 140℃ and pH=7.0 for 2 hours. The second stage reaction was controlled at 160℃, with potassium hydroxide and sodium hydroxide added in a ratio of 10:1, and the total mass of potassium hydroxide and sodium hydroxide to the volume ratio of the corn steep liquor solution was 1.5%, and the reaction was carried out for 2 hours. The third stage reaction was controlled at 180℃ for 2 hours, with the catalyst sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride in a mass ratio of 3:1:1, and the total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume ratio of the corn steep liquor solution was 2%. The reacted liquid is dried to obtain a synthetically produced microbial humic acid fertilizer. When applying the synthetically produced microbial humic acid fertilizer to the roots of cowpeas, it needs to be diluted 1000 times, and the application amount after dilution is 20 mL per plant.
[0031] To verify the effectiveness of the present invention, a confidentiality experiment was conducted, as follows:
[0032] This experiment aims to prepare artificially synthesized microbial humic acid fertilizer according to the method of the invention patent. First, corn steep liquor was selected as the raw material and pretreated. The specific steps were as follows: In the first stage, cellulase and alkaline protease were added to the corn steep liquor in a 3:1 ratio and reacted at 50℃ for 3 hours. Subsequently, amylase was added, and a second pre-decomposition was carried out at 45℃ for 1 hour. In the second stage, the product of the first stage was pre-decomposed with *Auricularia auricula-judae* strains at 25℃ for 10 days. The pre-decomposed corn steep liquor containing microbial fungal residues was then treated by a water processor at a frequency of 20000Hz for 5–20 minutes to complete dilution and further activation. Next, the treated corn steep liquor containing microbial fungal residues was processed through a three-stage reaction to produce artificially synthesized microbial humic acid: In the first stage, the temperature was controlled at 140℃ for 2 hours, and the pH was maintained at neutral. In the second stage, the temperature was controlled at 160℃ for 2 hours, and potassium hydroxide and sodium hydroxide were added in a mass ratio of 10:1, with the total mass of potassium hydroxide and sodium hydroxide being 0.5% of the volume of the corn steep liquor solution. The third stage: The temperature was controlled at 180℃, and the reaction was carried out for 2 hours. Sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride were added in a mass ratio of 3:1:1. The total mass of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride was 0.2% of the volume of the corn steep liquor solution. After the reaction was completed, the artificially synthesized microbial humic acid liquid was dried to produce artificially synthesized microbial humic acid fertilizer for subsequent experiments.
[0033] This experiment, conducted in pots, investigated the effects of artificially synthesized microbial humic acid on the abundance of *Arthrobacter* spp. in the rhizosphere soil of *Caulis vinifera* seedlings. An experimental group and a control group were established. The experimental group received 50 mL of artificially synthesized microbial humic acid diluted 1000 times per plant, while the control group received an equal volume of water. The experiment was carried out in a greenhouse using standard nutrient soil as the culture medium, lasting for 4 weeks, with fertilization every 7 days for a total of 3 applications. Rhizosphere soil samples were collected in the fourth week of the experiment and sent to AVC Revo sequencing company for 16S rRNA high-throughput sequencing to analyze rhizosphere microbial community diversity and the relative abundance of *Arthrobacter* spp. Simultaneously, growth indicators of *Caulis vinifera* seedlings were statistically analyzed.
[0034] The experimental results show that, see Figure 1 Compared with applying water, applying the artificially synthesized microbial humic acid provided by this invention can increase the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea, with the relative abundance of Arthrobacter spp. reaching 5%-6%.
[0035] The experimental results show that, compared with applying water, applying the artificially synthesized microbial humic acid provided by this invention can increase the root length of the cowpea, which reaches 23-24 cm.
Claims
1. A microbial humic acid fertilizer that increases the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpeas, characterized in that: The artificially synthesized microbial humic acid that increases the relative abundance of Arthrobacter in the rhizosphere soil of cowpea is prepared by the following method: First, corn steep liquor pre-decomposition is performed, which consists of two stages: The first stage involves pre-decomposing corn steep liquor using cellulase, alkaline protease, and amylase. The second stage involves pre-decomposing the product from the first stage using Auricularia auricula-judae fungi, and then diluting the pre-decomposed corn steep liquor containing microbial fungal residues with water. Diluted corn steep liquor containing microbial and fungal residues is processed into synthetic microbial-derived humic acid through a three-stage reaction process: The first stage reaction: the diluted corn steep liquor containing microbial fungal residues was reacted at pH 7.0 and the temperature was controlled at 140℃ for 2 hours; potassium hydroxide and sodium hydroxide were added in a mass ratio of 10:1, and the temperature was controlled at 160℃ for the second stage reaction, which lasted for 2 hours; sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride were then added in a mass ratio of 3:1:1, and the temperature was controlled at 180℃ for the third stage reaction, which lasted for 2 hours. The resulting artificially synthesized microbial humic acid liquid was dried to obtain artificially synthesized microbial humic acid fertilizer. The total mass ratio of potassium hydroxide and sodium hydroxide to the volume ratio of the diluted corn steep liquor solution containing microbial and fungal residues is 0.5-1.5%. The total mass ratio of sodium dodecylbenzenesulfonate, sodium persulfate, and ferric chloride to the volume ratio of the diluted corn steep liquor solution containing microbial and fungal residues is 0.2-2%. The artificially synthesized microbial humic acid fertilizer was diluted with water and applied to the roots of the cowpea seedlings. Fertilizer was applied every 7 days for a total of 3 times. After the fourth week, the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea reached 5%-6%, and the length of the cowpea root system reached 23-24 cm.
2. The microbial humic acid fertilizer for increasing the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea according to claim 1, characterized in that: The method for pre-decomposing corn steep liquor: In the first stage, corn steep liquor is pre-decomposed using cellulase and alkaline protease at a ratio of 3:1, at a temperature of 50°C, and for 3 hours. Then, amylase is used for further pre-decomposition at a temperature of 45°C for 1 hour. In the second stage, the products from the first stage were pre-decomposed using Auricularia auricula-judae fungi. The pre-decomposition culture temperature was 25℃, and the culture time was 10 days.
3. The microbial humic acid fertilizer for increasing the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea according to claim 2, characterized in that: When the corn steep liquor containing microbial and fungal residues is diluted with water, it is then processed by a water processor at a frequency of 20000Hz for 5-20 minutes.
4. The microbial humic acid fertilizer for increasing the relative abundance of Arthrobacter spp. in the rhizosphere soil of cowpea according to claim 3, characterized in that: When diluting the artificially synthesized microbial humic acid fertilizer with water, the amount of water added should be 1000 to 3000 times the amount of the artificially synthesized microbial humic acid fertilizer. When applying the fertilizer, the amount applied should be 20 to 100 mL per plant.