Preparation method and application of compound microorganism sodium humate water-soluble fertilizer

Abstract

The invention discloses a preparation method and application of a compound microorganism sodium humate water-soluble fertilizer, and belongs to the technical field of agricultural water-soluble fertilizer preparation. According to the invention, a functional microbial flora composed of clostridium butyricum, bacillus coagulans, bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus, lactobacillus plantarum and lactobacillus casei is compounded, and high-purity sodium humate is used as a core matrix and is matched with nutrient components such as glucose, molasses and a seaweed extract; a special process of'matrix activation-step fermentation-micro-aerobic and anaerobic regulation 'is adopted to replace a traditional simple compounding mode, and the high-activity sodium humate water-soluble fertilizer is prepared. The water-soluble fertilizer can effectively solve the problems of nutrient imbalance, hardening, micro-ecological imbalance and the like of continuous cropping soil, the content of humic acid in the soil and the effectiveness of elements such as calcium, magnesium, iron, zinc and the like are remarkably improved, meanwhile, the rhizosphere environment of crops is improved, the stress resistance of the crops is enhanced, and finally, the quality optimization and the yield improvement of the crops are realized.

Description

Technical Field

[0001] This invention belongs to the field of agricultural water-soluble fertilizer preparation technology, and more specifically relates to a method for preparing compound microbial sodium humate water-soluble fertilizer and its application. Background Technology

[0002] Water-soluble fertilizers (WSF), a core fertilizer category in modern agriculture, are multi-component nutrient preparations with complete water solubility. Their key characteristics include thorough dissolution, no solid residue, and rapid integration with irrigation water to form a homogeneous nutrient system. Compared to traditional fertilizers such as superphosphate and granular compound fertilizers, water-soluble fertilizers exhibit significant advantages in crop absorption efficiency and application scenarios. They can be rapidly absorbed by crops through roots and leaves, achieving an effective nutrient utilization rate of 80-90%, far exceeding that of ordinary chemical fertilizers. Furthermore, they are precisely compatible with modern water-saving irrigation systems such as sprinkler and drip irrigation, enabling an integrated management model of "simultaneous water and fertilizer supply and water-borne fertilizer infiltration." This characteristic allows for a significant reduction in water and fertilizer consumption in agricultural production, while also reducing labor input. Especially against the backdrop of increasingly scarce agricultural water resources and rising environmental protection requirements, water-soluble fertilizers play an irreplaceable role in improving crop quality, reducing non-point source pollution, and promoting cost reduction and efficiency in agriculture.

[0003] Sodium humate, as a natural organic active substance, has excellent soil improvement and crop growth-promoting functions. It can effectively activate nutrients fixed in the soil, improve soil physicochemical properties, promote the reproduction of beneficial microorganisms, and increase soil organic matter content. Currently, there are some water-soluble fertilizer products containing sodium humate on the market, but most are mainly chemically compounded or simply physically mixed, with relatively limited functions and limited ability to systematically restore the soil microecology, especially in the face of complex soil problems such as nutrient preservation, soil compaction, and microbial imbalance.

[0004] Furthermore, while existing technologies include products that combine single or a few microbial agents with humic acid substances, these often employ simple compounding processes, resulting in poor maintenance of microbial activity, a lack of synergistic effects among the microbial communities, and difficulty in forming a stable microbial-humic acid linkage system. Especially when facing complex soil environments, these products still exhibit significant shortcomings in terms of microbial colonization, long-lasting effects, and nutrient cycling efficiency.

[0005] Therefore, there is an urgent need to develop a compound water-soluble fertilizer product that can not only efficiently improve soil structure and enhance nutrient utilization efficiency, but also systematically repair soil micro-ecology and enhance crop stress resistance, in order to address the current problems in agricultural production such as soil degradation, continuous cropping obstacles, and low fertilizer utilization. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a compound microbial sodium humate water-soluble fertilizer and its application, thereby solving the problems existing in the prior art. This invention utilizes a functional microbial community composed of Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Lactobacillus plantarum, and Lactobacillus casei, with high-purity sodium humate as the core matrix, combined with nutrients such as glucose, molasses, and seaweed extract. A proprietary process of "matrix activation - stepwise fermentation - micro-aerobic-anaerobic regulation" is employed to replace the traditional simple compounding method, producing a highly active sodium humate water-soluble fertilizer. This water-soluble fertilizer can effectively repair problems such as nutrient imbalance, compaction, and microecological imbalance in continuously cropped soils, significantly increasing the humic acid content and the availability of elements such as calcium, magnesium, iron, and zinc in the soil. Simultaneously, it improves the rhizosphere environment of crops, enhances crop resistance, and ultimately optimizes crop quality and increases yield.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is to provide a compound microbial sodium humate water-soluble fertilizer, which is prepared by co-fermentation of the following raw materials in parts by weight: 45-55 parts sodium humate, 3-7 parts carbon source, 1-3 parts nitrogen source, 1-2 parts phosphorus and potassium source, 0.5-1 part magnesium sulfate, 0.2-0.3 parts EDTA chelated trace elements, 1-2 parts seaweed extract, 200-300 parts water, and 0.46-0.77 parts of compound microbial agent based on dry weight of bacterial cells; The carbon source includes glucose and molasses, wherein the mass ratio of glucose to molasses is 2~4:1~3; The nitrogen source includes urea; The phosphorus and potassium source includes potassium dihydrogen phosphate; The compound microbial agent, based on the dry weight of the bacterial cells, comprises the following components: 0.05-0.1 parts of Clostridium butyricum fermentation broth, 0.05-0.1 parts of Bacillus coagulans fermentation broth, 0.15-0.2 parts of Bacillus subtilis fermentation broth, 0.07-0.1 parts of Bacillus amyloliquefaciens fermentation broth, 0.04-0.07 parts of Bacillus pumilus fermentation broth, 0.05-0.1 parts of Lactobacillus plantarum fermentation broth, and 0.05-0.1 parts of Lactobacillus casei fermentation broth.

[0008] Preferably, the proportion of live bacteria in the compound microbial sodium humate water-soluble fertilizer meets the following condition: live lactic acid bacteria count ≥ 5.0 × 10⁻⁶. 8 CFU / g, total viable bacteria count ≥2×10 9 CFU / g.

[0009] Preferably, the humic acid content in the compound microbial sodium humate water-soluble fertilizer is ≥30% by mass.

[0010] The second technical solution of this invention: provides a method for preparing the above-mentioned compound microbial sodium humate water-soluble fertilizer, comprising the following steps: Fermentation broths of Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Lactobacillus plantarum, and Lactobacillus casei were prepared separately. Mix sodium humate, water, glucose, and urea according to the formula, heat to 50-60℃ and keep warm for 20-40 minutes for activation, then cool to 30-35℃, add Clostridium butyricum fermentation broth and Bacillus coagulans fermentation broth, and carry out the first fermentation. After the first fermentation, the system temperature is lowered to 26-30℃, and molasses, potassium dihydrogen phosphate, magnesium sulfate, EDTA chelated trace elements, seaweed extract, and Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Bacillus pumilus fermentation broth, Lactobacillus plantarum fermentation broth and Lactobacillus casei fermentation broth are added for the second fermentation. The second fermentation includes an aerobic stage in the early stage and an anaerobic stage in the later stage. After the second fermentation, the liquid fertilizer is obtained by solid-liquid separation and concentration; or by further spray drying to obtain powdered water-soluble fertilizer, thus obtaining the compound microbial sodium humate water-soluble fertilizer.

[0011] Preferably, the conditions for the first fermentation are: temperature 30~32℃, stirring speed 140~160r / min, aeration rate 1:0.7~0.9V / V·min, fermentation time 44~52h, and pH maintained at 6.5~7.0.

[0012] Preferably, the aerobic stage of the second fermentation lasts for 14-18 hours with an aeration rate of 1:0.6-0.8 V / V·min; the anaerobic stage lasts for 6-10 hours with nitrogen introduced to replace the air and a stirring speed of 70-90 r / min; the temperature of the second fermentation is maintained at 26-30℃ throughout the process.

[0013] Preferably, the pH of the aerobic stage is maintained at 6.5 to 7.0; and the pH of the anaerobic stage is maintained at 5.5 to 6.0.

[0014] Preferably, the spray drying conditions are: inlet air temperature 170~190℃ and outlet air temperature 75~85℃.

[0015] The third technical solution of the present invention provides the application of the above-mentioned compound microbial sodium humate water-soluble fertilizer in agriculture. The compound microbial sodium humate water-soluble fertilizer is used to improve degraded soil, increase the content of soil organic matter and trace elements, enhance crop stress resistance, and promote crop yield and quality improvement.

[0016] Preferably, the compound microbial sodium humate water-soluble fertilizer is applied by foliar spraying, fertigation or drip irrigation, with a dilution ratio of 300 to 800 times.

[0017] The present invention discloses the following technical effects: (1) Synergistic effect of microbial strains to enhance soil microecological activity. The water-soluble fertilizer of this invention is compounded with Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Lactobacillus plantarum, and Lactobacillus casei, forming a multi-level symbiotic and complementary system of seven functional microorganisms. The short-chain fatty acids secreted by Clostridium butyricum can regulate the soil acid-base environment, creating suitable conditions for lactic acid bacteria that require an acidic environment; Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus pumilus constitute a "Bacillus antibacterial alliance", which respectively secretes antimicrobial peptides, chitinase, and lipopeptides to target and inhibit soil-borne fungal and bacterial diseases, forming a "bacteria + fungi" broad-spectrum antibacterial combination with the bacteriocins secreted by Lactobacillus plantarum and Lactobacillus casei, thus broadening the scope of disease control; Bacillus amyloliquefaciens has the ability to secrete highly active amylase and cellulase, which can rapidly degrade insoluble starch and cellulose organic matter in the soil, providing easy access for other microbial strains. They absorb carbon sources; Bacillus pumilus secretes extracellular polysaccharides, which synergistically work with the adhesive substances of Bacillus coagulans to promote the colonization and proliferation of all functional microorganisms in soil particles and crop roots, strengthening the rhizosphere colonization capacity of the microbial community; Bacillus coagulans enhances the stress resistance of the microbial community by regulating the osmotic pressure of the fermentation system and soil microenvironment; Lactobacillus plantarum and Lactobacillus casei, as dominant lactic acid bacteria, can secrete organic acids such as lactic acid and acetic acid to further optimize the pH value of the rhizosphere microenvironment, activate elements such as phosphorus and calcium fixed in the soil, and the organic acids produced by their metabolism can also promote the germination of Bacillus spores, forming a "proliferation-activation" synergistic effect. The seven microorganisms each perform their own functions and support each other, synergistically increasing the abundance of beneficial microorganisms in the soil, while activating nutrients such as calcium, phosphorus, and potassium fixed in the soil by secreting hydrolytic enzymes, significantly increasing the content of available calcium, available phosphorus, and available potassium in the soil, optimizing the soil nutrient structure, and providing sufficient mineral nutrition for crop growth.

[0018] (2) Sodium humate interacts with microorganisms to enhance nutrient conversion efficiency. As a natural organic carrier, sodium humate provides carbon sources and growth substrates for functional microorganisms, promoting rapid reproduction and metabolism. On the other hand, its rich humic acid functional groups can chelate nitrogen, phosphorus, potassium, and trace elements in the soil, reducing nutrient fixation and loss. Microbial metabolites can also degrade sodium humate, releasing small-molecule organic nutrients, realizing a closed-loop effect of "microbial activation - humic acid chelation - nutrient slow release", which greatly improves the crop's absorption and utilization rate of nutrients.

[0019] (3) Enhances crop resistance to stress in multiple dimensions, resulting in significant improvement in quality and yield. The synergistic effect of various microorganisms in the formula can induce crops to produce disease-resistant enzymes (such as peroxidase and polyphenol oxidase), enhancing the crop's resistance to cold, drought, and pests and diseases; sodium humate can promote crop root development and increase root absorption area and vitality. The combination of the two can significantly improve crop photosynthetic efficiency, promote dry matter accumulation, and achieve crop yield improvement and quality optimization (such as increased sugar and vitamin content in fruits), without chemical residues. Detailed Implementation

[0020] Currently, most existing soil remediation and fertility enhancement products are single humic acid fertilizers or conventional compound microbial agents. However, there are few reports on the use of a specific ratio of seven functional microorganisms (Clostridium butyricum, Bacillus coagulans, etc.) in synergistic fermentation with high-purity sodium humate to construct a "microorganism-humic acid" linkage system for soil remediation and crop quality and yield improvement. This invention is the first to use a compound microbial agent composed of Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Lactobacillus plantarum, and Lactobacillus casei to prepare a water-soluble fertilizer through targeted fermentation with high-purity sodium humate. This fertilizer is used to remediate degraded soil and promote crop quality and yield improvement. Field trials have demonstrated for the first time that the compound microbial sodium humate water-soluble fertilizer provided by this invention has significant advantages in regulating soil microecological balance, breaking through continuous cropping barriers, and enhancing nutrient utilization efficiency. It provides a new and efficient solution for soil remediation and green agricultural production, and has significant application value.

[0021] The preparation steps of the composite microbial sodium humate water-soluble fertilizer provided by the present invention are as follows: Preparation of fermentation broth: (1) After activating the preserved Clostridium butyricum on a plate culture medium, the activated bacteria are inoculated into the modified CM medium and cultured at 32-38℃ for 12-18h to obtain the seed liquid; then the seed liquid is transferred to the modified CM medium at an inoculation rate of 1-3% (v / v) and fermented at 30-38℃ for 35-40h. The obtained fermentation broth is centrifuged to obtain the Clostridium butyricum fermentation broth.

[0022] (2) Inoculate the Bacillus coagulans strain into MRS liquid medium and activate it by culturing at 37°C for 18 h. Repeat the activation twice to obtain the activated solution. Inoculate the activated solution into MRS liquid medium at an inoculation rate of 2% (v / v) and culture at 37°C for 24 h to obtain the Bacillus coagulans fermentation broth.

[0023] (3) The activation medium (compound nutrient medium, 100mL) for Bacillus amyloliquefaciens and Bacillus pumilus consisted of: 1.2g tryptone, 0.6g yeast extract, 0.2g beef extract, 0.3g soluble starch, 0.8g sodium chloride, 0.15g dipotassium hydrogen phosphate, 1.5g agar, and 100mL purified water, with a final pH of 7.1. The expansion culture medium (agar-free liquid, 100mL) consisted of: 1.8g glucose, 1.5g peptone, 0.8g yeast extract, 0.5g corn steep liquor powder, 0.02g calcium chloride, 0.03g magnesium sulfate, and 100mL purified water, with a final pH of 7.1. The slant cultures of Bacillus amyloliquefaciens and Bacillus pumilus were inoculated into 100mL of activation medium and cultured at 37℃ and 180r / min for 24h. Then, 10 mL of seed culture was added to 200 mL of expansion medium, and cultured at 37 °C and 180 r / min for 24 h to obtain Bacillus amyloliquefaciens fermentation broth and Bacillus pumilus fermentation broth.

[0024] (4) The culture medium for *Lactobacillus plantarum* and *Lactobacillus casei* consisted of: 10.0 g peptone, 4.0 g yeast extract, 1.0 g Tween 80, 2.0 g dipotassium hydrogen phosphate heptahydrate, 5.0 g sodium acetate trihydrate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate tetrahydrate, 10.0 g maltose, 15.0 g agar, 0.01 g vancomycin, and 0.0048 g bromocresol purple, with a final pH of 6.2 ± 0.2. *Lactobacillus plantarum* and *Lactobacillus casei* slant cultures were inoculated into 100 mL of culture medium and cultured at 30 °C and 150 r / min for 48 h. Then, 8 mL of seed culture was added to 200 mL of expansion culture medium and cultured at 30 °C and 150 r / min for 48 h to obtain *Lactobacillus plantarum* fermentation broth and *Lactobacillus casei* fermentation broth.

[0025] (5) The composition of the Bacillus subtilis activation medium (broth medium) is: 2g tryptone, 2g NaCl, 1g yeast extract, 100mL purified water, pH 6.8. The composition of the expansion culture medium is: 2g glucose, 2g peptone, 1g yeast extract, 100mL purified water, pH 6.8. Pick one loop of fresh Bacillus subtilis slant culture and inoculate it into a shake flask containing 100mL activation medium. Shake culture at 30℃ and 150r / min for 48h. Then take 8mL of the above seed culture and inoculate it into a shake flask containing 200mL expansion culture medium. Shake culture at 30℃ and 150r / min for 48h to obtain the Bacillus subtilis fermentation broth.

[0026] Preparation of compound microbial sodium humate water-soluble fertilizer: Add 45-55 kg of high-purity sodium humate, 200-300 L of deionized water, 2-4 kg of glucose, and 1-3 kg of urea to a fermenter. After stirring evenly, raise the temperature to 50-60℃ and maintain this temperature for 20-40 min to activate the active groups of humic acid. Then cool to 30-35℃ and add 50-100 g (based on dry weight of bacteria) of Clostridium butyricum fermentation broth and 50-100 g (based on dry weight of bacteria) of Bacillus coagulans fermentation broth from the compound bacterial agent to carry out the first fermentation. Control the fermentation temperature at 30-32℃, keep the agitator running throughout the process at a speed of 140-160 r / min, and an aeration rate of 1:0.7-0.9 (V / V·min) to ensure thorough agitation of the materials. The fermentation time is 44-52 h. During this period, monitor the pH value every 12 h. If it is lower than 6.0, adjust it to the range of 6.5-7.0 with 0.1 mol / L sodium hydroxide solution.

[0027] After the first fermentation is complete, there is no need to let it stand. Directly lower the temperature to 26~30℃, adjust the stirring speed to 120r / min, and add 1~3kg of molasses, 1~2kg of potassium dihydrogen phosphate, 0.5~1kg of magnesium sulfate, 0.2~0.3kg of EDTA chelated trace elements, and 1~2kg of seaweed extract in sequence. Add 150~200g of Bacillus subtilis fermentation broth (based on cell dry weight), 70~100g of Bacillus amyloliquefaciens fermentation broth (based on cell dry weight), 40~70g of Bacillus pumilus fermentation broth (based on cell dry weight), 50~100g of Lactobacillus plantarum fermentation broth (based on cell dry weight), and 50~100g of Lactobacillus casei fermentation broth (based on cell dry weight). Stir until the materials are completely dissolved and uniform. The second fermentation was then carried out under phased control of the fermentation conditions: for the first 14-18 hours, an aerobic environment was maintained with an aeration rate of 1:0.6-0.8 (V / V·min) to meet the growth requirements of Bacillus; for the next 6-10 hours, the aeration valve was closed, and anaerobic fermentation was initiated. Nitrogen gas (purity ≥99.9%) was introduced into the top space of the fermenter for 3 minutes to replace the air and promote lactic acid bacteria growth. The temperature was controlled at 26-30℃ throughout the process. During the anaerobic stage, the stirring speed was reduced to 70-90 r / min to minimize air introduction. The pH value was monitored every 12 hours during fermentation. If the pH value was below 6.0 during the aerobic stage, it was adjusted to 6.5-7.0 with 0.1 mol / L sodium hydroxide solution; if the pH value was below 5.5 during the anaerobic stage, it was adjusted to the range of 5.5-6.0.

[0028] After the second fermentation, the mixture was allowed to stand for 1 hour. Impurities were removed by plate and frame filtration. The filtrate was then concentrated under vacuum at 60℃ and 0.08MPa to a solid content of 40%, yielding a sodium humate water-soluble fertilizer (liquid type) containing Clostridium butyricum and compound lactic acid bacteria. For powder preparation, the concentrated solution was spray-dried under conditions of 170-190℃ inlet air and 75-85℃ outlet air, with an effective viable count ≥2.0×10⁻⁶. 9 CFU / g (of which the number of live lactic acid bacteria is ≥5.0×10⁻⁶) 8Products with a CFU / g content and a humic acid content ≥30wt% are considered qualified.

[0029] Among them, the EDTA chelated trace elements include 0.06~0.09 parts of EDTA chelated calcium, 0.04~0.06 parts of EDTA chelated magnesium, 0.03~0.045 parts of EDTA chelated iron, 0.03~0.045 parts of EDTA chelated zinc, 0.02~0.03 parts of EDTA chelated boron, and 0.02~0.03 parts of EDTA chelated manganese.

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0036] The bacterial strains involved in this invention are all existing bacterial strains that have been publicly disclosed and used in the prior art, wherein: Clostridium butyricum (accession number: CCTCC NO: M2022151), Bacillus coagulans (accession number: CGMCC NO. 32216), and Bacillus subtilis (accession number: CCTCC NO: M20232018) have been bio-deposited at designated international depositary units for biological materials and have been published in invention patents with publication numbers CN116103200A, CN111500508A, and CN120888468A. Bacillus pumilus (accession number: CGMCC No. 31078) and Bacillus amyloliquefaciens (accession number: CGMCC No. 31076) have also been bio-deposited at designated international depositary units for biological materials and have been published in invention patents with publication numbers CN118879548A and CN119020194A. Lactobacillus casei (accession number: CICC 6114) and Lactobacillus plantarum (accession number: CICC 21809) were purchased from CICC (China Industrial Microbial Culture Collection Center).

[0037] The EDTA chelated trace elements used in the following examples (containing 0.06-0.09 parts of EDTA chelated calcium, 0.04-0.06 parts of EDTA chelated magnesium, 0.03-0.045 parts of EDTA chelated iron, 0.03-0.045 parts of EDTA chelated zinc, 0.02-0.03 parts of EDTA chelated boron and 0.02-0.03 parts of EDTA chelated manganese) were purchased from Tianjin Huazhen Agricultural Technology Co., Ltd.

[0038] Example 1 Preparation of fermentation broth: (1) After activating the preserved Clostridium butyricum on a plate culture medium, the activated bacteria were inoculated into the modified CM medium and cultured at 37℃ for 15h to obtain the seed liquid; then the seed liquid was transferred to the modified CM medium at an inoculation rate of 2~3% (v / v) and fermented at 35℃ for 38h. The resulting fermentation broth was centrifuged to obtain the Clostridium butyricum fermentation broth.

[0039] (2) Inoculate the Bacillus coagulans strain into MRS liquid medium and activate it by culturing at 37°C for 18 h. Repeat the activation twice to obtain the activated solution. Inoculate the activated solution into MRS liquid medium at an inoculation rate of 2% (v / v) and culture at 37°C for 24 h to obtain the Bacillus coagulans fermentation broth.

[0040] (3) The activation medium (compound nutrient medium, 100mL) for Bacillus amyloliquefaciens and Bacillus pumilus consisted of: 1.2g tryptone, 0.6g yeast extract, 0.2g beef extract, 0.3g soluble starch, 0.8g sodium chloride, 0.15g dipotassium hydrogen phosphate, 1.5g agar, and 100mL purified water, with a final pH of 7.1. The expansion culture medium (agar-free liquid, 100mL) consisted of: 1.8g glucose, 1.5g peptone, 0.8g yeast extract, 0.5g corn steep liquor powder, 0.02g calcium chloride, 0.03g magnesium sulfate, and 100mL purified water, with a final pH of 7.1. The slant cultures of Bacillus amyloliquefaciens and Bacillus pumilus were inoculated into 100mL of activation medium and cultured at 37℃ and 180r / min for 24h. Then, 10 mL of seed culture was added to 200 mL of expansion medium, and cultured at 37 °C and 180 r / min for 24 h to obtain Bacillus amyloliquefaciens fermentation broth and Bacillus pumilus fermentation broth.

[0041] (4) The culture medium for *Lactobacillus plantarum* and *Lactobacillus casei* consisted of: 10.0 g peptone, 4.0 g yeast extract, 1.0 g Tween 80, 2.0 g dipotassium hydrogen phosphate heptahydrate, 5.0 g sodium acetate trihydrate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate tetrahydrate, 10.0 g maltose, 15.0 g agar, 0.01 g vancomycin, and 0.0048 g bromocresol purple, with a final pH of 6.2 ± 0.2. *Lactobacillus plantarum* and *Lactobacillus casei* slant cultures were inoculated into 100 mL of culture medium and cultured at 30 °C and 150 r / min for 48 h. Then, 8 mL of seed culture was added to 200 mL of expansion culture medium and cultured at 30 °C and 150 r / min for 48 h to obtain *Lactobacillus plantarum* fermentation broth and *Lactobacillus casei* fermentation broth.

[0042] (5) The composition of the Bacillus subtilis activation medium (broth medium) is: 2g tryptone, 2g NaCl, 1g yeast extract, 100mL purified water, pH 6.8. The composition of the expansion culture medium is: 2g glucose, 2g peptone, 1g yeast extract, 100mL purified water, pH 6.8. Pick one loop of fresh Bacillus subtilis slant culture and inoculate it into a shake flask containing 100mL activation medium. Shake culture at 30℃ and 150r / min for 48h. Then take 8mL of the above seed culture and inoculate it into a shake flask containing 200mL expansion culture medium. Shake culture at 30℃ and 150r / min for 48h to obtain the Bacillus subtilis fermentation broth.

[0043] Preparation of compound microbial sodium humate water-soluble fertilizer: 45 kg of high-purity sodium humate, 250 L of deionized water, 3 kg of glucose, and 2 kg of urea were added to a 500 L fermenter. After stirring evenly, the mixture was heated to 55 °C and kept at this temperature for 30 min to activate the active groups of humic acid. Then, it was cooled to 32 °C, and 54.2 g of Clostridium butyricum fermentation broth (based on cell dry weight) and 54.2 g of Bacillus coagulans fermentation broth (based on cell dry weight) from the compound bacterial agent were added to initiate the first fermentation. The fermentation temperature was controlled at 30–32 °C, with stirring continuously at a speed of 150 r / min and an aeration rate of 1:0.8 (V / V·min) to ensure thorough agitation of the materials. The fermentation time was 48 h, during which the pH value was monitored every 12 h. If the pH value was below 6.0, it was adjusted to the range of 6.5–7.0 using 0.1 mol / L sodium hydroxide solution.

[0044] After the first fermentation is complete, there is no need to let it stand. Directly lower the temperature to 28℃, adjust the stirring speed to 120r / min, and add 2kg of molasses, 1.5kg of potassium dihydrogen phosphate, 0.8kg of magnesium sulfate, 0.25kg of EDTA chelated trace elements, and 1.5kg of seaweed extract in sequence. Add 162.5g of Bacillus subtilis fermentation broth (based on cell dry weight), 82g of Bacillus amyloliquefaciens fermentation broth (based on cell dry weight), 54g of Bacillus pumilus fermentation broth (based on cell dry weight), 54.2g of Lactobacillus plantarum fermentation broth (based on cell dry weight), and 54.2g of Lactobacillus casei fermentation broth (based on cell dry weight). Stir for 1.5h until the materials are completely dissolved and uniform. The second fermentation was then carried out under phased control of the fermentation conditions: for the first 16 hours, an aerobic environment with an aeration rate of 1:0.7 (V / V·min) was maintained to meet the growth requirements of Bacillus; for the next 8 hours, the aeration valve was closed, and the fermentation was switched to anaerobic fermentation. Nitrogen gas (purity ≥99.9%) was introduced into the top space of the fermenter for 3 minutes to replace the air and adapt to the growth of lactic acid bacteria. The temperature was controlled at 28~30℃ throughout the process. During the anaerobic stage, the stirring speed was reduced to 80 r / min to reduce the introduction of air. The pH value was monitored every 12 hours during fermentation. If the pH value was lower than 6.0 during the aerobic stage, it was adjusted to 6.5~7.0 with 0.1mol / L sodium hydroxide solution. If the pH value was lower than 5.5 during the anaerobic stage, it was adjusted to the range of 5.5~6.0.

[0045] After the second fermentation, the mixture was allowed to stand for 1 hour. Impurities were removed by plate and frame filtration. The filtrate was then concentrated under vacuum at 60℃ and 0.08MPa to a solid content of 40%, yielding a compound microbial sodium humate water-soluble fertilizer (liquid type). For powder preparation, the concentrated solution was spray-dried under conditions of 180℃ inlet air and 80℃ outlet air to obtain the compound microbial sodium humate water-soluble fertilizer (powder type). The effective viable count was ≥2.0×10⁻⁶. 9 CFU / g (of which the number of live lactic acid bacteria is ≥5.0×10⁻⁶) 8 CFU / g), humic acid content ≥30wt%.

[0046] The components of the compound microbial sodium humate water-soluble fertilizer (powder type) obtained in this embodiment were analyzed. The comparison results of the components of the compound microbial sodium humate water-soluble fertilizer (powder type) obtained in this embodiment and the commercially available ordinary humic acid water-soluble fertilizer (humic acid water-soluble fertilizer purchased from Shandong Shengjiu Agricultural Chemical Co., Ltd.) are shown in Table 1.

[0047] Table 1. Comparison of the components of the compound microbial sodium humate water-soluble fertilizer (powder type) obtained in Example 1 and commercially available ordinary humic acid water-soluble fertilizer. As shown in Table 1, the compound microbial sodium humate water-soluble fertilizer (powder type) prepared in Example 1 is rich in macronutrients such as phosphorus and potassium, as well as essential micronutrients such as calcium, magnesium, boron, iron, and zinc. The ratio of various elements is scientific and the activity is high, which can accurately replenish the soil nutrient gap and play an important role in maintaining the soil element balance. At the same time, the content of sodium humate and seaweed polysaccharide effective components is prominent, which can enhance the soil's fertilizer retention capacity and the efficiency of crop nutrient supply.

[0048] Example 2 Select the same plot of land and divide it into two plots, each 4m². 2 The plots were labeled A and B. Plot A was treated with the compound microbial sodium humate water-soluble fertilizer (powder type) prepared in Example 1, diluted 300 times, and sprayed evenly at a rate of 300 mL / mu. Plot B was treated with commercially available ordinary humic acid water-soluble fertilizer (purchased from Shandong Shengjiu Agricultural Chemical Co., Ltd.), sprayed evenly at a rate of 300 mL / mu. After 30 days, 100 g of soil samples were collected from the 0-20 cm topsoil layer at five random locations, and the soil element content was measured. The results are shown in Table 2.

[0049] Table 2. Soil element content As shown in Table 2, the compound microbial sodium humate water-soluble fertilizer (powder type) prepared in Example 1 can effectively improve the soil and increase the content of various effective elements in the soil.

[0050] Example 3 An experiment was conducted in a tomato continuous cropping greenhouse, divided into two plots, labeled A and B. Plot A was treated with the compound microbial sodium humate water-soluble fertilizer (powder type) prepared in Example 1, diluted 300 times and sprayed evenly at a rate of 300 mL / mu. Plot B was treated with commercially available ordinary humic acid water-soluble fertilizer (purchased from Shandong Shengjiu Agricultural Chemical Co., Ltd.), sprayed evenly at a rate of 300 mL / mu. The fertilizer was applied every 15 days after transplanting, for a total of 4 applications. Each group had an area of ​​16 m². 2 Forty-eight plants were planted in each plot, with all other management practices being the same. At harvest time, the incidence of Fusarium wilt, average number of fruits, and vitamin C content of the fruits were measured in plots A and B. The results are shown in Table 3.

[0051] Table 3 Comparison of Tomato Yield and Quality Table 3 shows that the application of the compound microbial sodium humate water-soluble fertilizer (powder type) prepared in Example 1 reduced the incidence of Fusarium wilt in tomatoes by 75% compared with the application of commercially available ordinary sodium humate water-soluble fertilizer, increased the number of fruits per plant by 15.6%, increased the vitamin C content of fruits by 21.4%, and increased the yield by 18.7%. These results demonstrate that the compound microbial sodium humate water-soluble fertilizer described in this invention plays an important role in increasing tomato yield and improving quality.

[0052] Example 4 This embodiment verifies the effect of different compound microbial agent compositions on the fertilizer efficacy of water-soluble fertilizer: The test crop was tomato (variety: Zhongza 105). Two groups of fertilizers were used in the experiment, with identical basic raw materials (sodium humate, glucose, urea, etc.) and dosages, differing only in preparation process, as follows: Group A (Exclusive Process Group): Prepared using the "substrate activation-stepwise fermentation-micro-anaerobic regulation" process of this invention. First, the substrate, including sodium humate, was activated at 55℃ for 30 minutes, then a compound microbial agent was added stepwise for aerobic-anaerobic staged fermentation, with temperature, aeration rate, and pH controlled throughout (see Example 1 for specific raw materials and preparation process). Group B (Traditional Compound Group): Prepared using a traditional simple compounding method, directly mixing all basic raw materials with the compound microbial agent dry powder (same composition and dosage as Group A), dissolving in water to produce a water-soluble fertilizer, without substrate activation or stepwise fermentation. Both products were liquid.

[0053] Experimental design: Both treatments were diluted 300 times and applied via root drenching and foliar spraying (the application time and dosage were consistent with the tomato experiment in Example 3); each group was replicated 3 times, with a plot area of ​​20m². 2 The planting density is 370 plants per mu, and other field management is completely consistent.

[0054] Testing indicators: At the end of the tomato harvest, the abundance of beneficial microorganisms in the soil, the incidence of tomato root rot, the average yield per mu, and the soluble solids content of the fruit were tested after the application of water-soluble fertilizer in each group. At the same time, the stability of the effective viable bacteria count of the two groups of water-soluble fertilizer products was tested (tested at 0 days and 30 days after preparation).

[0055] Results and analysis of process comparison test: The statistical results of each test index are shown in Table 4.

[0056] Table 4 Results of process comparison test Table 4 shows that the initial effective viable bacteria count of product group B decreased by 46.4% compared to group A, the effective viable bacteria count after 30 days decreased by 63.6%, the abundance of beneficial soil microorganisms decreased by 50.0%, the incidence of tomato root rot increased by 152%, and the average yield per mu (kg) decreased by 20.3%. In other words, group A, using the proprietary "substrate activation-step fermentation-micro-anaerobic regulation" process, significantly reduced the incidence of tomato root rot and significantly increased the average yield per mu compared to group B, which used a traditional compound formulation. These results demonstrate that the proprietary process of this invention significantly improves product stability and fertilizer efficiency compared to traditional compound formulations, which is crucial for increasing tomato yield and quality.

[0057] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound microbial sodium humate water-soluble fertilizer, characterized in that, It is prepared by co-fermentation of the following raw materials in parts by weight: 45-55 parts sodium humate, 3-7 parts carbon source, 1-3 parts nitrogen source, 1-2 parts phosphorus and potassium source, 0.5-1 part magnesium sulfate, 0.2-0.3 parts EDTA chelated trace elements, 1-2 parts seaweed extract, 200-300 parts water, and 0.46-0.77 parts of compound microbial agent based on dry weight of bacterial cells; The carbon source includes glucose and molasses, wherein the mass ratio of glucose to molasses is 2~4:1~3; The nitrogen source includes urea; The phosphorus and potassium source includes potassium dihydrogen phosphate; The compound microbial agent, based on the dry weight of the bacterial cells, comprises the following components: 0.05-0.1 parts of Clostridium butyricum fermentation broth, 0.05-0.1 parts of Bacillus coagulans fermentation broth, 0.15-0.2 parts of Bacillus subtilis fermentation broth, 0.07-0.1 parts of Bacillus amyloliquefaciens fermentation broth, 0.04-0.07 parts of Bacillus pumilus fermentation broth, 0.05-0.1 parts of Lactobacillus plantarum fermentation broth, and 0.05-0.1 parts of Lactobacillus casei fermentation broth.

2. The compound microbial sodium humate water-soluble fertilizer according to claim 1, characterized in that, The proportion of live bacteria in the compound microbial sodium humate water-soluble fertilizer meets the following condition: live lactic acid bacteria count ≥ 5.0 × 10⁻⁶. 8 CFU / g, total viable bacteria count ≥2×10 9 CFU / g.

3. The compound microbial sodium humate water-soluble fertilizer according to claim 1, characterized in that, The compound microbial sodium humate water-soluble fertilizer contains ≥30% humic acid by mass.

4. The method for preparing the compound microbial sodium humate water-soluble fertilizer according to any one of claims 1 to 3, characterized in that, Includes the following steps: Fermentation broths of Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Lactobacillus plantarum, and Lactobacillus casei were prepared separately. Mix sodium humate, water, glucose, and urea according to the formula, heat to 50-60℃ and keep warm for 20-40 minutes for activation, then cool to 30-35℃, add Clostridium butyricum fermentation broth and Bacillus coagulans fermentation broth, and carry out the first fermentation. After the first fermentation, the system temperature is lowered to 26-30℃, and molasses, potassium dihydrogen phosphate, magnesium sulfate, EDTA chelated trace elements, seaweed extract, and Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Bacillus pumilus fermentation broth, Lactobacillus plantarum fermentation broth and Lactobacillus casei fermentation broth are added for the second fermentation. The second fermentation includes an aerobic stage in the early stage and an anaerobic stage in the later stage. After the second fermentation, the liquid fertilizer is obtained by solid-liquid separation and concentration; or by further spray drying to obtain powdered water-soluble fertilizer, thus obtaining the compound microbial sodium humate water-soluble fertilizer.

5. The preparation method according to claim 4, characterized in that, The conditions for the first fermentation are: temperature 30~32℃, stirring speed 140~160r / min, aeration rate 1:0.7~0.9V / V·min, fermentation time 44~52h, and pH maintained at 6.5~7.

0.

6. The preparation method according to claim 4, characterized in that, The aerobic stage of the second fermentation lasts for 14-18 hours, with an aeration rate of 1:0.6-0.8 V / V·min; the anaerobic stage lasts for 6-10 hours, with nitrogen introduced to replace the air and a stirring speed of 70-90 r / min; the temperature of the second fermentation is maintained at 26-30℃ throughout the process.

7. The preparation method according to claim 4, characterized in that, The pH during the aerobic phase is maintained at 6.5-7.0; the pH during the anaerobic phase is maintained at 5.5-6.

0.

8. The preparation method according to claim 4, characterized in that, The conditions for spray drying are: inlet air temperature 170~190℃, outlet air temperature 75~85℃.

9. The application of the compound microbial sodium humate water-soluble fertilizer according to any one of claims 1 to 3 in agriculture, characterized in that, The compound microbial sodium humate water-soluble fertilizer is used to improve degraded soil, increase soil organic matter and trace element content, enhance crop stress resistance, and promote crop yield and quality improvement.

10. The application according to claim 9, characterized in that, The compound microbial sodium humate water-soluble fertilizer is applied by foliar spraying, fertigation or drip irrigation, with a dilution ratio of 300 to 800 times.

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