Slow-release bacterial fertilizer, preparation method and application

Abstract

The invention discloses a slow-release bacterial fertilizer as well as a preparation method and application thereof, and relates to the technical field of biotechnology. Comprising the following steps: step 1, preparing a bacterial liquid of bacillus cereus, a bacterial liquid of lysinibacillus slender, a bacterial liquid of acinetobacter schiffei, a bacterial liquid of stenotrophomonas maltophilia, a bacterial liquid of sphingobacterium alcohovorans and a bacterial liquid of bacillus licheniformis into a mixed bacterial suspension according to a volume ratio of 1: (1-2): (1-2): (1-2): (1-2): (1-2); 2, mixing the mixed bacterium suspension with an embedding agent and a cross-linking agent to obtain a first mixed colloid; step 3, embedding the first mixed colloid into the second mixed layer to obtain first compound fertilizer particles; and step 4, embedding the first compound fertilizer particles into a fermented third mixed layer to obtain the slow-release bacterial fertilizer. The prepared slow-release bacterial fertilizer improves the remediation effect of organic phosphorus contaminated soil. The soil fertility is improved, and a good growth promoting effect on plants is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a slow-release bacterial fertilizer, a preparation method and an application thereof. Background Art

[0002] In agricultural production, soil mainly provides a living environment and nutrients for the growth of crops, so soil conditions are an important factor affecting the quality and yield of crops. However, over the years, with the rapid development of agriculture, land is facing more and more problems. Among them, a large amount of old pesticide residues in the soil seriously hinder the replanting of crops in the land, causing great harm to the ecological environment around the polluted land.

[0003] Microorganisms are decomposers in the natural ecosystem. They can continuously degrade various pollutants entering the environment and eventually convert them into inorganic substances such as CO2 and H2O, so that the polluted environment can be purified. There are many challenges in directly applying beneficial microorganisms to agriculture. Therefore, immobilization technology can be used, that is, a new technology that fixes specific microorganisms on carriers, locates or restricts them to a certain space with high density and maintains biological activity. It is an important technical means in the field of bioengineering. This technology can not only play a good protective role for specific microorganisms applied to the soil, but also provide certain nutrients for the growth and reproduction of microorganisms, improve their adaptability to the external environment and colonization rate. Microbial treatment technology is not only suitable for large-scale organophosphorus pesticide pollutant treatment sites, but also can be used in farmland soil, thereby greatly saving treatment costs.

[0004] However, the existing immobilization technology only fixes the bioactive substances on a specific carrier to prepare an immobilized material with only one layer of embedding, which has a short duration. Therefore, frequent application of pesticides is required to achieve the ideal pesticide degradation effect, and there is a problem of low soil remediation efficiency. Summary of the invention

[0005] To solve the above problems, the present invention provides a slow-release bacterial fertilizer, a preparation method and an application thereof, so as to solve the problem of low soil remediation efficiency.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for preparing a slow-release bacterial fertilizer comprises the following steps:

[0008] Step 1: prepare a mixed bacterial suspension of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Sphingobacillus alcoholivorans, and Bacillus licheniformis in a volume ratio of 1:1-2:1-2:1-2:1-2:1-2:1-2; the number of viable bacteria in the mixed bacterial suspension is 10 6 CFU / mL~108 CFU / mL.

[0009] Step 2: Mix the mixed bacterial suspension with an embedding agent and a cross-linking agent to obtain a first mixed colloid; the volume ratio of the mixed bacterial suspension, the embedding agent and the cross-linking agent is 1:2-4:9-11.

[0010] Step 3: embedding the first mixed colloid into the second mixed layer to obtain first compound fertilizer particles; the volume ratio of the first mixed colloid to the second mixed layer is 8-9:1-2.

[0011] The second mixed layer is obtained by mixing adsorbent and nutrients, sterilizing, and cooling to room temperature; the mass of the adsorbent accounts for 1% to 6% of the mass of the slow-release fertilizer, and the mass of the nutrients accounts for 5% to 25% of the mass of the slow-release fertilizer.

[0012] Step 4: embedding the first compound fertilizer particles into the fermented third mixed layer to obtain the slow-release bacterial fertilizer; the volume ratio of the first compound fertilizer particles to the third mixed layer is 7-8:2-3.

[0013] The third mixed layer is prepared by composting 19 to 21 parts of crop straw, 4 to 6 parts of sawdust or rapeseed cake, 20 to 30 parts of bean dregs or peanut bran, 20 to 30 parts of livestock and poultry manure, 10 to 15 parts of fish pond sludge and 40 to 60 parts of water. After fermentation, 4 to 6 parts of wood ash and 18 to 20 parts of mixed bacterial suspension are added for secondary fermentation.

[0014] Preferably, the embedding agent is a mixture of polyvinyl alcohol and sodium alginate; the mass ratio of the sodium alginate to polyvinyl alcohol is 3:1.5-2.

[0015] Preferably, the cross-linking agent is an aqueous solution of CaCl2 and H3BO3 with a pH of 6 to 7; the mass ratio of CaCl2 to H3BO3 is 1:1 to 3.

[0016] Preferably, the adsorbent is composed of biochar and diatomaceous earth; the mass ratio of the biochar to diatomaceous earth is 1:1-2.

[0017] Preferably, the nutrient substance consists of γ-polyglutamic acid, humic acid and glucose; the mass ratio of γ-polyglutamic acid, humic acid and glucose is 2-3:3-4:1.

[0018] The slow-release bacterial fertilizer is prepared by the slow-release bacterial fertilizer preparation method.

[0019] The application of the slow-release bacterial fertilizer includes at least one of the following:

[0020] 1) Degradation of organic phosphorus.

[0021] 2) Degradation of inorganic phosphorus.

[0022] 3) Degradation of cellulose.

[0023] 4) Promote plant growth.

[0024] Preferably, the organic phosphorus is at least one of ethoprophos and lecithin.

[0025] Preferably, the increasing plant growth refers to promoting plant leaf length, plant height and stem thickness.

[0026] Preferably, the plant includes Lepidium dimorphum, Chinese cabbage, tomato, cantaloupe, melon or loofah.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a slow-release bacterial fertilizer, which is prepared into a mixed bacterial suspension by preparing bacterial liquids of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Bacillus alcoholicus and Bacillus licheniformis. First, the mixed bacterial suspension is mixed with an embedding agent and a cross-linking agent to form a first mixed colloid. Then, the first mixed colloid is embedded in a second mixed layer to obtain a first composite fertilizer particle. The second mixed layer is composed of an adsorbent and a nutrient, which can provide necessary nutrition when the mixed bacterial suspension is released, enhance the survival rate of the strain, enable it to reproduce rapidly in the soil, have strong adaptability, thereby improving degradation efficiency and repairing the soil.

[0029] Subsequently, the first compound fertilizer particles are embedded in the fermented third mixed layer to finally obtain the slow-release bacterial fertilizer. The third mixed layer is made by mixing and composting the following ingredients in proportion: 19 to 21 parts of crop straw, 4 to 6 parts of sawdust or rapeseed cake, 20 to 30 parts of bean dregs or peanut bran, 20 to 30 parts of livestock and poultry manure, 10 to 15 parts of fish pond sludge and 40 to 60 parts of water. After fermentation, 4 to 6 parts of wood ash and 18 to 20 parts of mixed bacterial suspension are added for secondary fermentation. The mixed bacterial suspension can accelerate the decomposition of cellulose of other substances in the third mixed layer, making it more fertile.

[0030] When applying the slow-release bacterial fertilizer of the present invention, first, the outermost third mixed layer begins to decompose and play a role, degrading pesticide residues and improving soil quality. As time goes by, the effect of existing bacterial fertilizers begins to weaken, and the second layer of the slow-release bacterial fertilizer of the present invention begins to decompose, releasing the embedded mixed bacterial suspension, and starts to degrade pesticide residues again to improve soil quality. Compared with the existing immobilization technology, the present invention has three layers, which decompose and play a role at different times, and the action time is long, which greatly improves the degradation efficiency of residual pesticides, and converts them into absorbable phosphorus required by plants, improves soil fertility, and effectively solves the problem of poor soil remediation effect. The slow-release bacterial fertilizer of the present invention has significant growth-promoting and yield-increasing effects on wheat, corn, rice, vegetables, fruits, wetland plants, etc.

[0031] The invention can improve the activity environment of bacterial strains when stored or used, can provide nutrients to microorganisms in the bacterial fertilizer, can promote the formation of bacterial flora, and improve the activity of bacterial strains and the application effect of microbial slow-release bacterial fertilizer.

[0032] The invention has a simple formula, a long effective fertilizer time, and is convenient and fast to apply fertilizer. The fertilizer has a large diameter and is condensed into a mass, which greatly increases the sustained-release time. Compared with the previous small granular fertilizer, the fertilizer effect is prolonged and the fertilization speed is increased. This also greatly reduces the fertilization cost of farmers and the manufacturing cost of manufacturers.

[0033] The present invention utilizes crop straw, sawdust, rapeseed cake, peanut bran residue and the like as starting raw materials, and can effectively utilize the plant fiber and plant protein therein as raw materials for slow-release fertilizer after hydrolysis and fermentation, and the decomposed substances, such as lignin, cellulose, chitin and the like, have a degradation effect and are easily absorbed by animals and plants, can fertilize the soil, promote the growth of animals and plants, and enhance the resistance and immunity of animals and plants to various diseases.

[0034] In addition, Bacillus cereus and Bacillus cereus can produce antibacterial substances, inhibit the reproduction of harmful microorganisms, degrade nutrients in the soil, and improve the ecological environment. Oligotrophomonas maltophilia has multiple growth-promoting properties such as decomposing organic phosphorus, decomposing potassium, and fixing nitrogen. The microbial agent prepared using this bacterium can increase biomass, improve seed germination rate, promote the growth of tomatoes and peppers, improve plant salt tolerance, and reduce the use of chemical fertilizers. Based on its excellent comprehensive biological characteristics, the agent has a wide range of application prospects. Bacillus licheniformis has the effects of disease resistance and killing harmful bacteria. The microbial agent prepared using this bacterium can be used as a soil remediation agent for organic pollution.

[0035] A variety of beneficial microorganisms multiply in the rhizosphere of crops, forming a dominant bacterial community, and secrete plant growth-promoting substances and antibiotics to inhibit the growth of pathogenic microorganisms. They are resistant to repeated cropping diseases, soil-borne diseases, cold and frost resistance, and premature aging of plants, and extend the growth period. They improve quality and mature earlier. By appropriately reducing the amount of chemical fertilizers, alleviating soil pollution, and degrading nitrates and pesticide residues, the protein, sugar, and vitamin content of agricultural products can be increased, making vegetables, fruits, and melons fresh, delicious, and of excellent quality. They also mature earlier and have significant effects in increasing production and income. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is the first mixed colloid diagram of the present invention;

[0038] Figure 2 This is a second mixed layer diagram of the present invention;

[0039] Figure 3 This is a third mixed layer diagram of the present invention;

[0040] Figure 4 This is the first mixed colloid diagram of the present invention;

[0041] Figure 5 This is a second mixed layer diagram of the present invention;

[0042] Figure 6 This is a third mixed layer diagram of the present invention;

[0043] Figure 7 This is a diagram showing the plant height measurement results of Carex dimorphus of the present invention;

[0044] Figure 8 This is a diagram showing the diameter measurement results of the dimorphic sedge of the present invention;

[0045] Fig. 9 The degradation efficiency of ethoxyprolin in the present invention is 200 μg / kg when the concentration of ethoxyprolin is only treated with the plant Lepidium dimorphum, 6 mixed bacteria, and Lepidium dimorphum + mixed bacteria;

[0046] Fig.10 This is a photo of the growth of Chinese cabbage of the present invention;

[0047] Fig.11 The results of the three treatments of the present invention on the plant height of Chinese cabbage are shown in FIG.

[0048] Fig.12The diagram is a result diagram of the effects of the three treatments of the present invention on the leaf width of Chinese cabbage;

[0049] Fig.13 The results of the three treatments of the present invention on the effect of tomato plant height are shown in FIG.

[0050] Fig.14 The results of the three treatments of the present invention on the diameter of tomatoes are shown in FIG.

[0051] Fig.15 This is a photo of the growth of the tomato plant + clean water group of the present invention;

[0052] Fig.16 This is a photo of the growth of the tomato plant + organic fertilizer group of the present invention;

[0053] Fig.17 This is a photo of the growth of the tomato plant + slow-release bacterial fertilizer group of the present invention;

[0054] Fig.18 These are the effect diagrams of the mixed bacteria on the first and third days of the present invention;

[0055] Fig.18 In the figure, A is the effect picture of the mixed bacteria on the first day; B is the effect picture of the mixed bacteria on the third day;

[0056] Fig.19 This is a graph showing the effect of the mixed microorganisms of the present invention on the decomposition of straw for one day;

[0057] Fig. 20 This is a graph showing the effect of the mixed microorganisms of the present invention on the decomposition of straw for 6 days;

[0058] Fig.21 This is a diagram showing the effect of the mixed microorganisms of the present invention on straw decomposition after one month; a total of eight parallel experiments were set up.

[0059] Fig. 22 This is a diagram showing the first day effect of the present invention on the decomposition of organic phosphorus;

[0060] Fig.23 This is a diagram showing the effect of the present invention on the decomposition of organic phosphorus on the third day;

[0061] Fig.24 This is a diagram showing the first day effect of the inorganic phosphorus solution of the present invention;

[0062] Fig.25 This is a diagram showing the effect of the inorganic phosphorus solution on the third day of the present invention;

[0063] Fig.26 This is a photo of the growth of cantaloupe after adding bacterial fertilizer to the present invention;

[0064] Fig. 27 This is a photo of the growth of melons after adding bacterial fertilizer to the present invention;

[0065] Fig.28This is a photo of the growth of loofah after adding bacterial fertilizer to the present invention. DETAILED DESCRIPTION

[0066] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in the present invention. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0067] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0068] The identification of the strain genome was carried out by 16S rDNA sequence analysis. The strain genes were extracted using a bacterial genomic DNA extraction kit. Using this as a template, PCR was used to amplify the 16S rDNA sequences of the two bacteria. The forward primer for PCR amplification was B341F: 5′-CCTACGGGNGGCWGCAG-3′; the reverse primer was B785R: 5′-GACTACHVGGGTATCTAAT-3′. The PCR reaction procedure was: 95°C pre-denaturation for 3min, 95°C denaturation for 30s, 25 cycles, 54°C annealing for 30s, 72°C extension for 30s, and 72°C final extension for 5min. The PCR product was tested by 2% agarose gel electrophoresis and sent to Jiangsu Sanshu Biotechnology Co., Ltd. for sequencing.

[0069] In order to better illustrate the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0070] The materials used in this invention are purchased from the following sources:

[0071] The pesticide chlorpyrifos was purchased from Tanmo Quality Inspection Technology Co., Ltd.; the crop straw was corn straw powder, purchased from Taobao Su Rui Agricultural Products Deep Processing; the sawdust was fir pine sawdust, purchased from Carpenter Family Gardening Museum; the rapeseed cake was rapeseed cake, purchased from Taobao Leipin Home Furnishing Store; bean dregs were purchased from Taobao Suizhou Jia Ren Local Products; peanut bran was purchased from Taobao Chunzhen Tu You Fang; pig manure was purchased from Taobao Happy Countryside Agricultural Products Sales; horse manure was purchased from Shen Diao Daily Chemicals; sheep manure was purchased from Mengmin Gardening Products Flagship Store; fish pond sludge was purchased from Taobao Feng Ming Jing Enterprise Factory; and wood ash was purchased from Kunning Wang Home Furnishing Official Flagship Store.

[0072] Example 1

[0073] A method for preparing a slow-release bacterial fertilizer:

[0074] Step 1: prepare a mixed bacterial suspension of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Sphingobacillus alcoholivorans, and Bacillus licheniformis in a volume ratio of 1:1:1:1:1:1, wherein the number of viable bacteria in the mixed bacterial suspension is 10 6 CFU / mL.

[0075] Step 2. Weigh 3g of polyvinyl alcohol and 1.5g of sodium alginate and put them into a 50mL conical flask containing 20mL of distilled water. Heat and stir in a magnetic stirring water bath at 70°C and a speed of 15000r / min to dissolve them and obtain an embedding agent. Add 10mL of mixed bacterial suspension to the embedding agent to obtain a colloid, place the colloid on a sterile operating table, and wait for it to cool to room temperature to obtain an embedding agent. Weigh 2g of CaCl2 and 2g of H3BO3 and add them to 100mL of deionized water, stir to dissolve them, and use sodium carbonate to adjust the pH to 6 to obtain a cross-linking agent. Use a 50mL syringe with a caliber of 2mm to take 50mL of the embedding agent, add it dropwise to 90mL of the cross-linking agent liquid from a height of 10cm, fix it for 1h, and obtain the first mixed colloid. After taking it out, wash it three times with sterile deionized water. Dry it in a 35°C oven, put it into a self-sealing bag, and put it in a refrigerator at 4°C for use. The results are as follows. Figure 1 and Figure 4 shown.

[0076] Step 3: embed the first mixed colloid into the second mixed layer to obtain the first compound fertilizer particles. The volume ratio of the first mixed colloid to the second mixed layer is 8:1. Figure 2 and Figure 5 As shown. The second mixed layer is obtained by mixing an adsorbent and a nutrient and sterilizing them under high pressure at 100°C and 15psi, and then cooling them to room temperature. The mass concentration of the adsorbent is 1%, and the mass concentration of the nutrient is 5%. The adsorbent is composed of biochar and diatomaceous earth, and the mass ratio of biochar to diatomaceous earth is 1:1; the nutrient is composed of γ-polyglutamic acid, humic acid and glucose, and the mass ratio of γ-polyglutamic acid, humic acid and glucose is 2:3:1.

[0077] Step 4: embed the first compound fertilizer particles into the fermented third mixed layer to obtain a slow-release bacterial fertilizer. The mass ratio of the first compound fertilizer particles to the third mixed layer is 7:2. The obtained slow-release bacterial fertilizer is placed in an organic fertilizer preparation machine for extrusion molding. The result is as follows: Figure 3 and Figure 6 shown.

[0078] The preparation method of the third mixed layer is as follows: 19 parts of crop straw, 4 parts of sawdust, 20 parts of bean dregs, 20 parts of livestock and poultry manure, and 10 parts of fish pond sludge are mixed evenly one month in advance according to the weight ratio, and the drying temperature is 60°C for 2 hours; after crushing, pass through a 20-mesh sieve to screen out large impurities of stones, add 40 parts of water to mix the compost evenly, and ferment it at room temperature for 5 days at a fermentation temperature of 55°C. After the fermentation product is fully ventilated, 4 parts of plant ash are added, and after adjusting the pH to 6, an effective viable bacteria count of 0.4×10 9 18 portions of mixed bacterial suspension with a concentration of 0.1 cfu / ml were continuously stirred for secondary fermentation, and water was added until the humidity of the pile reached 50%. The fertilizer was turned over every 2 days, and the fermentation was completed after 14 days to obtain the third mixed layer.

[0079] The raw materials are placed in a mold and then demolded after cooling to become cylindrical, spherical and other shaped fertilizers. The diameter of this fertilizer is 5mm. When applying fertilizer, just place one within 30 meters. Moreover, because the fertilizer has a large diameter and is condensed into a mass, the sustained-release time is greatly increased. Compared with the previous small granular fertilizers, the fertilizer effect is prolonged and the fertilization speed is increased. This also greatly reduces the fertilizer cost of farmers and the manufacturing cost of manufacturers.

[0080] Example 2

[0081] A method for preparing a slow-release bacterial fertilizer:

[0082] Step 1: prepare a mixed bacterial suspension of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Sphingobacillus alcoholivorans, and Bacillus licheniformis in a volume ratio of 1:1:1:1:1:1, wherein the number of viable bacteria in the mixed bacterial suspension is 10 7 CFU / mL.

[0083] Step 2. Weigh 3g of polyvinyl alcohol and 1.5g of sodium alginate and put them into a 50mL conical flask containing 30mL of distilled water. Heat and stir in a magnetic stirring water bath at 70°C and a speed of 15000r / min to dissolve them and obtain an embedding agent. Add 10mL of mixed bacterial suspension to the embedding agent to obtain a colloid, and place the colloid on a sterile operating table until it cools to room temperature. Weigh 2gCaCl2 and 3g H3BO3 and add them to 100mL of deionized water, stir to dissolve them, and use sodium carbonate to adjust the pH to 6 to obtain a cross-linking agent. Use a 50mL syringe with a caliber of 2mm to take 50mL of colloid, and add it dropwise to 100mL of cross-linking agent liquid from a height of 10cm. Fix it for 1h to obtain the first mixed colloid. After taking it out, wash it three times with sterile deionized water. Dry it in a 35°C oven, put it into a self-sealing bag, and put it in a refrigerator at 4°C for use. The results are as follows. Figure 1 and Figure 4shown.

[0084] Step 3: embed the first mixed colloid into the second mixed layer to obtain the first compound fertilizer particles. The volume ratio of the first mixed colloid to the second mixed layer is 9:2. Figure 2 and Figure 5 As shown. The second mixed layer is obtained by mixing an adsorbent and a nutrient and sterilizing them under high pressure at 100°C and 15psi, and then cooling them to room temperature. The mass concentration of the adsorbent is 3%, and the mass concentration of the nutrient is 15%. The adsorbent is composed of biochar and diatomaceous earth, and the mass ratio of biochar to diatomaceous earth is 1:1; the nutrient is composed of γ-polyglutamic acid, humic acid and glucose, and the mass ratio of γ-polyglutamic acid, humic acid and glucose is 2:3:1.

[0085] Step 4: The first compound fertilizer particles are embedded in the fermented third mixed layer to obtain a slow-release bacterial fertilizer. The mass ratio of the first compound fertilizer particles to the third mixed layer is 8:3. The obtained slow-release bacterial fertilizer is placed in an organic fertilizer preparation machine for extrusion molding. The result is as follows: Figure 3 and Figure 6 shown.

[0086] The preparation method of the third mixed layer is as follows: one month in advance, 20 parts of crop straw, 5 parts of sawdust, 25 parts of bean dregs, 25 parts of livestock and poultry manure, and 12 parts of fish pond sludge are mixed evenly by weight, and the drying temperature is 60°C for 2 hours; after crushing, pass through a 20-mesh sieve to screen out large impurities of stones, add 50 parts of water and mix the compost evenly, and ferment at room temperature for 5 days at a fermentation temperature of 55°C. After the fermentation product is fully ventilated, 5 parts of plant ash are added, and after adjusting the pH to 6, an effective viable bacteria count of 0.5×10 9 19 portions of mixed bacterial suspension with a concentration of 0.1 cfu / ml were continuously stirred for secondary fermentation, water was added until the humidity of the pile reached 50%, the fertilizer was turned over every 2 days, and the fermentation was completed after 14 days to obtain the third mixed layer.

[0087] The raw materials are placed in a mold and then demolded after cooling to become cylindrical, spherical and other shaped fertilizers. The diameter of this fertilizer is 5mm. When applying fertilizer, just place one within 30 meters. Moreover, because the fertilizer has a large diameter and is condensed into a mass, the sustained-release time is greatly increased. Compared with the previous small granular fertilizers, the fertilizer effect is prolonged and the fertilization speed is increased. This also greatly reduces the fertilizer cost of farmers and the manufacturing cost of manufacturers.

[0088] Example 3

[0089] A method for preparing a slow-release bacterial fertilizer:

[0090] Step 1: prepare a mixed bacterial suspension of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Sphingobacillus alcoholivorans, and Bacillus licheniformis in a volume ratio of 1:2:2:2:2:2, wherein the number of viable bacteria in the mixed bacterial suspension is 10 8 CFU / mL.

[0091] Step 2. Weigh 3g of polyvinyl alcohol and 2g of sodium alginate and put them into a 50mL conical flask containing 40mL of distilled water. Heat and stir in a magnetic stirring water bath at 70°C and a speed of 15000r / min to dissolve them and obtain an embedding agent. Add 10mL of mixed bacterial suspension to the embedding agent to obtain a colloid, and place the colloid on a sterile operating table until it cools to room temperature. Weigh 2gCaCl2 and 4g H3BO3 and add them to 100mL of deionized water, stir to dissolve them, and use sodium carbonate to adjust the pH to 6 to obtain a cross-linking agent. Use a 50mL syringe with a caliber of 2mm to take 50mL of colloid, and add it dropwise to 110mL of cross-linker liquid from a height of 10cm. Fix it for 1h to obtain the first mixed colloid. After taking it out, wash it three times with sterile deionized water. Dry it in a 35°C oven, put it into a self-sealing bag, and put it in a refrigerator at 4°C for use. The results are as follows. Figure 1 and Figure 4 shown.

[0092] Step 3: embed the first mixed colloid into the second mixed layer to obtain the first compound fertilizer particles. The volume ratio of the first mixed colloid to the second mixed layer is 9:2. Figure 2 and Figure 5 As shown. The second mixed layer is obtained by mixing an adsorbent and a nutrient and sterilizing them under high pressure at 100°C and 15psi, and then cooling them to room temperature. The mass concentration of the adsorbent is 6%, and the mass concentration of the nutrient is 25%. The adsorbent is composed of biochar and diatomaceous earth, and the mass ratio of biochar to diatomaceous earth is 1:2; the nutrient is composed of γ-polyglutamic acid, humic acid and glucose, and the mass ratio of γ-polyglutamic acid, humic acid and glucose is 3:4:1.

[0093] Step 4: The first compound fertilizer particles are embedded in the fermented third mixed layer to obtain a slow-release bacterial fertilizer. The mass ratio of the first compound fertilizer particles to the third mixed layer is 8:3. The obtained slow-release bacterial fertilizer is placed in an organic fertilizer preparation machine for extrusion molding. The result is as follows: Figure 3 and Figure 6 shown.

[0094] The preparation method of the third mixed layer is as follows: 21 parts of crop straw, 6 parts of sawdust, 30 parts of bean dregs, 30 parts of livestock and poultry manure, and 15 parts of fish pond sludge are mixed evenly one month in advance according to the weight ratio, and the drying temperature is 60°C for 2 hours; after crushing, pass through a 20-mesh sieve to screen out large impurities of stones, add 60 parts of water to mix the compost evenly, and ferment it at room temperature for 5 days at a fermentation temperature of 55°C. After the fermentation product is fully ventilated, 6 parts of plant ash are added, and after adjusting the pH to 6, an effective viable bacteria count of 0.6×10 9 20 portions of mixed bacterial suspension with a concentration of 0.1 cfu / ml were continuously stirred for secondary fermentation, water was added until the humidity of the pile reached 50%, the fertilizer was turned over every 2 days, and the fermentation was completed after 14 days to obtain the third mixed layer.

[0095] The raw materials are placed in a mold and then demolded after cooling to become cylindrical, spherical and other shaped fertilizers. The diameter of this fertilizer is 5mm. When applying fertilizer, just place one within 30 meters. Moreover, because the fertilizer has a large diameter and is condensed into a mass, the sustained-release time is greatly increased. Compared with the previous small granular fertilizers, the fertilizer effect is prolonged and the fertilization speed is increased. This also greatly reduces the fertilizer cost of farmers and the manufacturing cost of manufacturers.

[0096] It should be noted that the sawdust in the above embodiment can be replaced by rapeseed cake, bean dregs can be replaced by peanut bran, and livestock and poultry manure can be pig manure, horse manure or sheep manure. The prepared slow-release bacterial fertilizer and the slow-release bacterial fertilizer prepared in Examples 1 to 3 all have the following effects: 1) degrading organic phosphorus, wherein the organic phosphorus is at least one of chloranthate and lecithin; 2) degrading inorganic phosphorus; 3) degrading cellulose; 4) promoting plant growth, wherein promoting plant growth refers to increasing plant leaf length, plant height and stem thickness.

[0097] Lysinibacillus elongatus is disclosed in "Coorevits A, Dinsdale AE, Heyrman J, Schumann P, Van Landschoot A, Logan NA, De Vos P. Lysinibacillus macroides sp. nov., nom. rev. Int J Syst Evol Microbiol. 2012 May; 62(Pt 5): 1121-1127. doi: 10.1099 / ijs.0.027995-0. Epub 2011 Jul 1. PMID: 21724959."

[0098] Bacillus cereus is disclosed in "Senesi S, Ghelardi E. Production, secretion and biological activity of Bacillus cereus enterotoxins. Toxins (Basel). 2010 Jul; 2(7): 1690-703. doi: 10.3390 / toxins2071690. Epub 2010 Jun 29. PMID: 22069656; PMCID: PMC3153264."

[0099] Stenotrophomonas maltophilia was published in "Brooke JS. 2012. Stenotrophomonas maltophilia: an Emerging Global Opportunistic Pathogen. Clin Microbiol Rev 25:. https: / / doi.org / 10.1128 / cmr.00019-11"

[0100] Sphingobacterium alcoholicum is disclosed in “Sphingobacterium gen.nov., Sphingobacterium spiritivorum comb.nov., Sphingobacterium multivorum comb.nov., Sphingobacterium mizutae sp.nov., and Flavobacterium indologenes sp.nov.: Glucose-Nonfermenting Gram-Negative Rods in CDC Groups IIK-2andIIb”

[0101] Bacillus licheniformis is disclosed in “Muras, A., Romero, M., Mayer, C., & Otero, A. (2021). Biotechnological applications of Bacillus licheniformis. Critical Reviews in Biotechnology, 41(4), 609–627. https: / / doi.org / 10.1080 / 07388551.2021.1873239”.

[0102] The deposit number of Acinetobacter severii is CCTCC NO: M20241302.

[0103] Experimental Example 1: The ability of slow-release bacterial fertilizer to degrade organophosphorus pesticides and its effect on the growth of Carex dimorphus.

[0104] The mixed bacterial suspension described in Example 1 was taken and activated and fermented at 30°C and 170 r / min to culture the bacterial suspension for 12 hours in broth medium. After the expansion was completed, the concentration of the bacterial suspension was adjusted with sterile water, and the absorbance value of the bacterial suspension at a wavelength of 600 nm was measured to be about 1.0, which was used for subsequent experiments.

[0105] The test soil is composed of peat soil and loess in a volume ratio of 1:1. After high temperature and high pressure sterilization, it is placed in a plastic pot with a height of 9.8 cm and a width of 9.8 cm, 300 g per pot, and the soil moisture is 35%. Select the healthy and consistent growing plant Carex dimorphus, which is common in the watershed area, and rinse the roots with sterile water before planting to clean the attached soil and microorganisms.

[0106] In this experiment, a total of 6 treatment groups were set up, namely, plants + 50 mg / pot of chlorpyrifos pesticide, plants + 10 ml / pot of clean water, plants + 50 mg / pot of organic fertilizer, plants + 10 ml / pot of mixed bacterial suspension + 50 mg / pot of chlorpyrifos pesticide, plants + 10 ml / pot of mixed bacterial suspension, and plants + 50 mg / pot of slow-release bacterial fertilizer. Each group had 5 replicates, and the cultivation lasted for a total of 35 days. Water was replenished every day to maintain the soil moisture content. The organic fertilizer in the plant + organic fertilizer group was composed of 21 parts of crop straw, 6 parts of sawdust, 30 parts of bean dregs; 30 parts of livestock and poultry manure, 15 parts of fish pond sludge and 6 parts of wood ash. The height and diameter of the moss were measured every 7 days. Figure 7 and Figure 8 As shown in the figure, the results of the growth promotion experiment showed that there were significant differences between the treatment group and the control group. Among them, the plant + slow-release bacterial fertilizer group 50 mg / pot significantly increased the height and diameter of the sedge seedlings, and had the best growth promotion effect. After 35 days, the leaf length and stem diameter increased by 34.02 cm and 2.46 mm respectively.

[0107] Four treatment groups were set up in this experiment, namely: natural degradation group of chlorpyrifos pesticide, chlorpyrifos pesticide 50mg / pot, mixed bacterial suspension 10ml / pot + chlorpyrifos pesticide 50mg / pot, plant + chlorpyrifos pesticide group 50mg / pot and plant + mixed bacterial suspension 10ml / pot + chlorpyrifos pesticide group 50mg / pot. The concentration of chlorpyrifos pesticide is 200μg / kg, and the mixed bacterial suspension is added every 7d, 10ml each time. Each group of chlorpyrifos pesticide is 50g. The temperature of all treatment tests is 30℃, water is added once every 3d to maintain soil moisture, and a cycle of 30d is set, with 5 replicates in each group. After 30d, freeze-dry in a freeze dryer, sieved, and tested.

[0108] Take 5g of sieved soil in a 100mL plastic centrifuge tube, add 40mL of acetonitrile, shake vigorously several times, add 5g of anhydrous sodium chloride, shake again, and centrifuge at 8000r / min for 5min. Accurately pipette 10mL of supernatant into a 100mL eggplant-shaped bottle. Rotate and evaporate in a 40℃ water bath to about 1mL, wash the remaining sample with 3mL of acetonitrile-toluene solution into a solid phase extraction column, then wash with 2mL of acetonitrile-toluene solution, and transfer the washing liquid into the column, repeat twice, elute the small column with 25mL of acetonitrile-toluene solution, collect all the above effluent in a 100mL eggplant-shaped bottle, and rotate and concentrate in a 40℃ water bath to near dryness. Make up to 1mL with ethyl acetate, pass through an organic microporous filter membrane, and determine by gas chromatography-mass spectrometry. Microsoft Excel2016 software was used for data collation and drawing, and SPSS24.0 software was used for statistical analysis.

[0109] The results are as follows Fig. 9 As shown, when the concentration of fenthion was 200 μg / kg, the degradation rate of fenthion by the plant Lepidoptera dimorpha alone was 73.76%, the degradation efficiency of the mixed bacterial suspension was 84.14%, and the degradation efficiency of the plant Lepidoptera dimorpha + mixed bacterial suspension was as high as 93.27%, indicating that the mixed bacterial suspension had a significant degradation efficiency for organophosphorus pesticides.

[0110] Experimental Example 2: Experiment on the effect of slow-release bacterial fertilizer on Chinese cabbage

[0111] The test soil is composed of peat soil and loess in a volume ratio of 1:1. After being sterilized at 100℃ and 15psi, an equal volume of soil is placed in a flowerpot. Healthy pakchoy seeds are selected and rinsed with sterile water before planting to clean attached microorganisms. There are three treatment groups in this experiment, namely the blank group, plant + clean water group, plant + organic fertilizer group, and plant + slow-release bacterial fertilizer group. Each group has 5 replicates, and each pot has 8 pakchoy seedlings. The cultivation lasts for a total of 28 days, and water is added every time to maintain the soil moisture content. The organic fertilizer in the plant + organic fertilizer group is a mixture of 21 parts of crop straw, 6 parts of sawdust, 30 parts of bean dregs, 30 parts of livestock and poultry manure, 15 parts of fish pond sludge and 6 parts of wood ash. Plant height and leaf width are measured every 7 days. Photos of the growth of pakchoy are shown below. Fig.10 As shown in Figure 2, the effects of the three treatments on the plant height of Chinese cabbage are as follows: Fig.11 As shown in Figure 2, the effects of the three treatments on the leaf width of Chinese cabbage are as follows: Fig.12 As shown, the results of the growth promotion experiment showed that compared with the plant + organic fertilizer group and the blank group, the slow-release bacterial fertilizer group increased the leaf length and stem thickness of the small white grass seedlings, and the growth and development were obvious. The fertilizer had the best growth promotion effect. After 28 days, the leaf length and leaf width increased by 35.28cm and 57.92mm respectively.

[0112] Experimental Example 3: Determination of the ability of slow-release bacterial fertilizer to promote the growth of tomato seedlings.

[0113] Tomato seeds were sown on May 15, 2024, and experimental seedlings with consistent plant height and diameter were selected on May 29. This experiment will set up three treatment groups for tomato seedlings grown in a greenhouse, namely the blank group, plant + water group, plant + organic fertilizer group, and plant + slow-release bacterial fertilizer group. Each group has 5 replicates, and each replicate has 3 tomato seedlings. The cultivation lasts for a total of 28 days, and water is replenished every day to maintain the soil moisture content. Plant height and diameter are measured every 7 days. How do the three treatments affect tomato plant height? Fig.13 As shown in the figure, the effects of the three treatments on tomato diameter are as follows: Fig.14 As shown in the figure, the height of plants added with slow-release bacterial fertilizer increased by 84 cm and the diameter increased by 0.43 cm, which were significantly higher than the height increase of 62 cm and the diameter increase of 0.17 cm in the blank group and the height increase of 72 cm and the diameter increase of 0.32 cm in the plant + organic fertilizer group. The results of the fertilizer-promoting growth test showed that the height of plants added with slow-release bacterial fertilizer was significantly higher than that of the blank group and the plant + organic fertilizer group. For details, see Fig.15 The tomato seedlings in the medium plant + water group were weak. Fig.16 Medium plant + organic fertilizer group tomato seedlings medium height and thickness, Fig.17 The tomato seedlings in the plant + slow-release bacterial fertilizer group had a high rate of robustness and fruiting. The soil in the plant + slow-release bacterial fertilizer group was looser than that in the control group, which shows that the slow-release bacterial fertilizer of the present invention can effectively promote plant growth, improve soil environment, loosen soil, and enhance soil fertility.

[0114] Experimental Example 4: Effect of slow-release bacterial fertilizer on cellulose degradation in soil

[0115] Cellulose Congo red medium was used for cellulose determination.

[0116] Cellulose Congo red medium: sodium nitrate 1.0 g·L -1 , disodium hydrogen phosphate 1.2 g·L -1 , potassium dihydrogen phosphate 0.9 g·L -1 , magnesium sulfate 0.5 g·L -1 , Potassium chloride 0.5 g·L -1 , yeast extract powder 0.5g·L -1 , Acid hydrolyzed casein 0.5g·L -1 , Congo red 0.2 g·L -1 , cellulose powder 5.0g·L -1 , agar 15.0 g·L -1 , water to 1L, pH 7.0. The effect of mixed bacteria on the first and third days is shown in the figure Fig.18 shown.

[0117] Effect of mixed microorganisms on straw decomposition for 1 day Fig.19 As shown, the effect of decomposition for 6 days is as follows Fig. 20 As shown in Figure 1, after one month, the microbial mixture had a significant effect on straw decomposition. Fig.21 As shown, from the data of plants planted in the above-mentioned greenhouse, it can be seen that after the application of the bacterial fertilizer, the growth of the plants planted was significantly accelerated, and the fertility of the soil was significantly increased, which is beneficial to the remediation of pollution in the land and the re-planting of the soil.

[0118] Experimental Example 5: Determination of the degradation capacity of organic phosphorus and inorganic phosphorus.

[0119] 1. Qualitative and quantitative experiments on organic phosphorus

[0120] (1) Solution for qualitative test of organic phosphorus: Egg yolk agar test medium: 15 g peptone, 3 g beef powder, 5 g sodium chloride, 15 g agar, 10 g glucose, dilute to 1 L with water, pH 7.4, autoclave at 121°C for 15 minutes, cool to about 50°C, add 10 ml egg yolk emulsion, shake well, and pour into a sterile plate. Unit for qualitative test of organic phosphorus: mm.

[0121] Use a small hole puncher to punch three holes in the egg yolk agar medium and inoculate 30 μL of bacterial solution. For each bacteria, place three plates and measure the diameter of the turbid circle of bacterial growth on day 1 and day 3 and record it. The effect on day 1 is shown in the figure below. Fig. 22 As shown, the effect diagram on the third day is as follows Fig.23 shown.

[0122] On the first day, the diameters of the three flat plate phosphorus dissolution circles were 17.4mm, 15.6mm, and 17.7mm respectively; on the third day, the diameters of the three flat plate phosphorus dissolution circles were 18.0mm, 18.6mm, and 24.1mm respectively, and the dissolution of organic phosphorus was significant.

[0123] 2. Qualitative and quantitative experiments on inorganic phosphorus

[0124] (1) Qualitative experiment of inorganic phosphorus: Inorganic phosphorus experimental medium: glucose 10g, ammonium sulfate 0.5g, yeast extract powder 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, tricalcium phosphate 5g, water to 1L, pH 7.0;

[0125] Use a small hole puncher to punch three holes on the inorganic phosphorus solid culture medium, and add 30 μL of mixed bacterial solution. Repeat on three plates, measure the diameter of the transparent circle of bacterial growth on day 1 and day 3, and record it. The effect on day 1 is shown in the figure below. Fig.24 As shown, the effect diagram on the third day is as follows Fig.25 shown.

[0126] On the first day, the diameters of the three flat plate phosphorus dissolution circles were 7.43mm, 8.91mm, and 8.70mm, respectively; on the third day, the diameters of the three flat plate phosphorus dissolution circles were 11.78mm, 13.88mm, and 13.59mm, respectively, and the dissolution of inorganic phosphorus was significant.

[0127] (2) Solution of Inorganic Phosphorus Quantification Experiment:

[0128] Take the activated bacterial liquid and inoculate it into inorganic phosphorus liquid culture medium at a rate of 1wt%. The shaking speed was 150r / min and the culture was carried out at 30°C for 1d and 3d. Take 6ml of inorganic phosphorus culture medium solution, digest it with 1ml of potassium persulfate, take 3ml and add it to a 50ml volumetric flask. The molybdenum antimony colorimetric method was used for colorimetry. Three shaking flasks were set for the same strain and the measurement was performed three times.

[0129]

[0130] The bacterial fertilizer was also used in the planting of cantaloupe, melon, loofah and other vegetables and fruits. The growth rate was higher than that of the control group. The results are as follows: Figure 26 to Figure 28 shown.

[0131] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a slow-release bacterial fertilizer, characterized in that: The following steps are involved: Step 1: prepare a mixed bacterial suspension of Bacillus cereus, Bacillus elongatus, Acinetobacter severii, Stenotrophomonas maltophilia, Sphingobacillus alcoholivorans, and Bacillus licheniformis in a volume ratio of 1:1-2:1-2:1-2:1-2:1-2:1-2; the number of viable bacteria in the mixed bacterial suspension is 10 6 CFU / mL~10 8 CFU / mL; Step 2: Mixing the mixed bacterial suspension with an embedding agent and a cross-linking agent to obtain a first mixed colloid; the volume ratio of the mixed bacterial suspension, the embedding agent and the cross-linking agent is 1:2-4:9-11; Step 3: embedding the first mixed colloid into the second mixed layer to obtain first compound fertilizer particles; the volume ratio of the first mixed colloid to the second mixed layer is 8-9:1-2; The second mixed layer is obtained by mixing an adsorbent and a nutrient, sterilizing the mixture, and cooling the mixture to room temperature; the mass of the adsorbent accounts for 1% to 6% of the mass of the slow-release fertilizer, and the mass of the nutrient accounts for 5% to 25% of the mass of the slow-release fertilizer; Step 4: embedding the first compound fertilizer particles into the fermented third mixed layer to obtain the slow-release bacterial fertilizer; the volume ratio of the first compound fertilizer particles to the third mixed layer is 7-8:2-3; The third mixed layer is prepared by composting 19 to 21 parts of crop straw, 4 to 6 parts of sawdust or rapeseed cake, 20 to 30 parts of bean dregs or peanut bran, 20 to 30 parts of livestock and poultry manure, 10 to 15 parts of fish pond sludge and 40 to 60 parts of water. After fermentation, 4 to 6 parts of wood ash and 18 to 20 parts of mixed bacterial suspension are added for secondary fermentation.

2. The method for preparing the slow-release bacterial fertilizer according to claim 1, characterized in that: The embedding agent is a mixture of polyvinyl alcohol and sodium alginate; the mass ratio of the sodium alginate to the polyvinyl alcohol is 3:1.5-2.

3. The method for preparing the slow-release bacterial fertilizer according to claim 1, characterized in that: The cross-linking agent is an aqueous solution of CaCl2 and H3BO3, with a pH of 6-7; the mass ratio of CaCl2 to H3BO3 is 1:1-3.

4. The method for preparing the slow-release bacterial fertilizer according to claim 1, characterized in that: The adsorbent consists of biochar and diatomaceous earth; the mass ratio of the biochar to the diatomaceous earth is 1:1-2.

5. The method for preparing the slow-release bacterial fertilizer according to claim 1, characterized in that: The nutrient substance consists of gamma-polyglutamic acid, humic acid and glucose; the mass ratio of the gamma-polyglutamic acid, humic acid and glucose is 2-3:3-4:

1.

6. The slow-release bacterial fertilizer prepared by the method for preparing the slow-release bacterial fertilizer according to any one of claims 1 to 5.

7. The use of the slow-release bacterial fertilizer as claimed in claim 6, characterized in that: Include at least one of the following: 1) Degradation of organic phosphorus; 2) Degradation of inorganic phosphorus; 3) Degradation of cellulose; 4) Promote plant growth.

8. The use of the slow-release bacterial fertilizer as claimed in claim 7, characterized in that: The organic phosphorus is at least one of ethoprophos and lecithin.

9. The use of the slow-release bacterial fertilizer as claimed in claim 7, characterized in that: The promoting plant growth refers to increasing the leaf length, plant height and stem thickness of the plant.

10. The use of the slow-release bacterial fertilizer as claimed in claim 9, characterized in that: The plants include Lepidium dimorphum, Chinese cabbage, tomato, cantaloupe, musk melon or loofah.

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