A bio-organic fertilizer of co-fermentation of auricularia auricula residue, tobacco dust and biomass charcoal, and a preparation method and application thereof

Abstract

This invention relates to the field of fertilizer manufacturing technology, specifically disclosing a bio-organic fertilizer prepared by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar, along with its preparation method and application. The bio-organic fertilizer is prepared by co-fermentation of 60-80 parts of *Auricularia auricula-judae* residue, 15-25 parts of tobacco dust, 8-15 parts of biochar, 2-5 parts of compound microbial inoculant, and 5-10 parts of auxiliary conditioner. The lignin depolymerization aromatic fragments and nicotine nitrogen are linked by C-N bonds and then anchored in the pores of the biochar by C-O-C bonds, forming a ternary covalent complex. During preparation, the inoculant is first activated, followed by free radical-initiated pulping, hot alkaline-oxygen prepolymerization, aerobic fermentation with biochar loading, and low-temperature drying. This fertilizer can simultaneously increase soil pH, available phosphorus, and organic matter content, reduce exchangeable aluminum, and significantly improve the yield and quality of crops such as tea, pickled mustard greens, and mustard greens, exhibiting long-term soil improvement and anti-degradation properties.

Description

Technical Field

[0001] This invention relates to the field of fertilizer manufacturing technology, and more specifically to a bio-organic fertilizer produced by co-fermentation of yellow-backed wood ear fungus residue, tobacco dust and biochar, as well as its preparation method and application. Background Technology

[0002] Yellow-backed wood ear (hairy wood ear) is a widely cultivated edible fungus in my country, generating a large amount of waste mushroom residue after harvesting. Currently, the residue is mostly disposed of by incineration or discarding, causing serious environmental pollution and resource waste. Yellow-backed wood ear residue is rich in nutrients such as crude protein, cellulose, nitrogen, phosphorus, potassium, amino acids, and vitamins. It also contains small amounts of lime, gypsum, and superphosphate, making it valuable for fertilizer use.

[0003] Tobacco dust is a waste product generated during tobacco processing. It contains nicotine, organic matter, and various mineral elements, but directly returning it to the field poses a risk that nicotine will inhibit crop growth.

[0004] Existing technologies, such as CN109400367A, disclose a bio-organic fertilizer prepared by aerobic fermentation of *Auricularia auricula-judae* residue and its preparation method. However, this technology uses conventional aerobic fermentation, which fails to fully utilize the synergistic effect of functional components in the residue, resulting in limited improvement on soil acidification and phosphorus deficiency. CN121970560A discloses a method for improving tea garden soil based on the combined application of biochar and organic fertilizer. However, the biochar and organic fertilizer are only physically mixed, and no chemical bonds are formed between them, resulting in a short-lasting synergistic effect.

[0005] Therefore, developing a bio-organic fertilizer and its preparation method that can achieve molecular-level chemical fusion of yellow-backed wood ear fungus residue, tobacco dust and biochar to synergistically solve the problems of soil acidification, phosphorus fixation and organic matter depletion is of great practical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-organic fertilizer made from the co-fermentation of yellow-backed wood ear fungus residue, tobacco dust, and biochar, as well as its preparation method and application, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, on the one hand, the present invention provides a bio-organic fertilizer co-fermented with *Auricularia auricula-judae* residue, tobacco dust, and biochar, comprising, by mass parts: 60-80 parts of *Auricularia auricula-judae* residue, 15-25 parts of tobacco dust, 8-15 parts of biochar, 2-5 parts of compound microbial agent, and 5-10 parts of auxiliary conditioner; the aromatic fragments generated by the depolymerization of lignin in the *Auricularia auricula-judae* residue are connected to the nitrogen atoms of nicotine in the tobacco dust through CN bonds, and then anchored to the pore structure of the biochar through COC bonds with the active functional groups of the biochar, forming a ternary covalent complex of lignin-nicotine-biochar. The formation of this complex enables the bio-organic fertilizer to have a triple synergistic function: (1) the phenolic hydroxyl groups and pyridine nitrogen functional groups on the surface of the complex can specifically complex the active Al in the soil. 3+ (1) Alleviate aluminum toxicity; (2) The lignin quinone structure can act as an electron shuttle to drive the reduction reaction of dissimilar iron and release Fe-P bound phosphorus into effective phosphorus; (3) The covalently linked ternary structure has strong resistance to microbial degradation and can achieve long-term carbon sequestration and soil structure improvement.

[0008] Preferably, the dry matter composition of the *Auricularia auricula-judae* residue, by mass percentage, includes: 35-45% sawdust, 10-20% cottonseed hulls, 15-25% rice bran, 8-12% wheat bran, 1-3% corn flour, 0.5-1.5% lime, 0.5-1.5% gypsum, and 0.5-1.5% superphosphate; the nicotine content of the tobacco powder is 0.8-2.5%. Nicotine's pyridine ring possesses a lone pair of electrons, and its nucleophilicity increases under alkaline conditions, making it a key catalyst for the Michael addition reaction of lignin quinone groups.

[0009] Preferably, the compound microbial agent includes *Phanerochaete chrysosporium*, *Bacillus subtilis*, *Thermophilic bacteria*, and *Bacillus amyloliquefaciens*, with the effective viable count of each of the four agents not less than 1 × 10⁻⁶. 7 CFU / mL. *Phanerochaete chrysosporium* is a highly efficient lignin-degrading bacterium, secreting laccase and manganese peroxidase to depolymerize lignin macromolecules into phenolic acid monomers, providing precursors for ternary assembly. *Bacillus subtilis* secretes organic acids to assist in phosphorus solubilization and forms spores during aging, improving product shelf life. *Thermophilic bacterium* is a thermophilic bacterium that maintains metabolic activity at high temperatures of 55-65℃, accelerating organic matter decomposition and promoting nicotine release. *Bacillus amyloliquefaciens* produces antibacterial substances to inhibit the growth of other bacteria and secretes extracellular polymers to assist in ternary stabilization. A 2:2:1:1 mass ratio of *Phanerochaete chrysosporium*, *Bacillus subtilis*, *Thermophilic bacterium*, and *Bacillus amyloliquefaciens* allows for the formation of dominant bacterial communities at different fermentation temperature stages, ensuring the coordinated progress of lignin degradation, ternary assembly, and functional bacterial colonization. The effective viable count of each strain is not less than 1×10⁻⁶. 7 CFU / mL ensures rapid establishment of the bacterial community after inoculation.

[0010] Preferably, the auxiliary conditioner is selected from one or more of dried chicken manure, oil cake, and peat moss. Its main function is to adjust the carbon-nitrogen (C / N) ratio of the fermentation material to a suitable range of (25-35):1. Yellow-backed wood ear mushroom residue has a relatively high C / N ratio (approximately 40-60:1), while tobacco dust has a relatively low C / N ratio (approximately 15-20:1). The addition of dried chicken manure (C / N approximately 7-10:1) or oil cake (C / N approximately 6-8:1) can adjust the C / N ratio of the mixture to the optimal range, avoiding slow fermentation and incomplete decomposition due to an excessively high C / N ratio, or significant nitrogen loss through volatilization in the form of ammonia due to an excessively low C / N ratio. Simultaneously, the auxiliary conditioner itself is rich in organic matter and trace elements, providing microorganisms with a readily available carbon source and growth factors, accelerating the fermentation process.

[0011] Preferably, the biochar is sawdust biochar or straw biochar, prepared at a temperature of 400-600℃, and has a specific surface area of ​​80-150 m². 2 / g.

[0012] On the other hand, the present invention also discloses a method for preparing a bio-organic fertilizer by co-fermentation of the above-mentioned yellow-backed wood ear fungus residue, tobacco dust and biochar, comprising the following steps: Yellow-backed wood ear fungus residue is mixed with tobacco dust and pre-treated by pulping in the presence of a free radical initiator (to initially destroy the rigid structure of lignin and generate quinone intermediates with higher reactivity). Then, a prepolymerization reaction is carried out under alkaline conditions and under heating and pressure (to induce Michael addition of lignin quinone groups with nicotine to generate water-soluble CN-bonded oligomers), thus generating water-soluble prepolymers. The prepolymer is mixed with acid-activated biochar and inoculated with the composite microbial agent for aerobic fermentation (under the catalysis of laccase / peroxidase secreted by the microorganisms, further polymerization and cross-linking occur, and the oligomers are covalently anchored to the pore surface of the biochar) to form a ternary covalent complex. Finally, the bio-organic fertilizer is obtained by low-temperature drying.

[0013] Preferably, the preparation method of the compound microbial agent is as follows: *Phanerochaete chrysosporium*, *Bacillus subtilis*, *Thermophilus*, and *Bacillus amyloliquefaciens* are inoculated into their respective liquid culture media and cultured with shaking at 28-65°C until the logarithmic growth phase. Then, they are mixed to obtain the compound microbial agent. Specifically, *Phanerochaete chrysosporium* is cultured in PDA liquid medium at 28-30°C with shaking for 48-72 hours to allow it to secrete sufficient lignin-degrading enzymes; *Bacillus subtilis* and *Bacillus amyloliquefaciens* are cultured in LB liquid medium at 35-37°C with shaking for 18-24 hours to allow them to rapidly multiply to the logarithmic phase; *Thermophilus* is cultured in modified Thermus medium at 60-65°C with shaking for 24-36 hours to maintain its high-temperature activity. The activated bacterial solution can have a viable bacterial count of up to 10-1.8 -10 9 CFU / mL, after inoculation, it can rapidly colonize and function in the material. Preferably, the aerobic fermentation includes two stages: a high-temperature period and an aging period. During the high-temperature period, the temperature is maintained to facilitate the metabolism of thermophilic bacteria, and forced ventilation is carried out to make the material mature quickly and promote the formation of covalent bonds. After the material temperature drops naturally, the aging period begins, forced ventilation is stopped, and anaerobic or facultative aging is carried out to stabilize the complex structure and promote the colonization of functional bacteria.

[0014] During the high-temperature stage of 55-65℃, thermophilic bacteria dominate the covalent cross-linking reaction between lignin-nicotine prepolymer and biochar, completing the in-situ assembly of the ternary complex. At this temperature, Bacillus subtilis and Bacillus amyloliquefaciens form spores and enter a dormant state, remaining in the fermentation system as spores. *Phanerochaete chrysosporium*, having completed the initial depolymerization of lignin during the heating phase, is naturally eliminated during the high-temperature stage. After the high-temperature fermentation phase, the material is allowed to cool naturally (e.g., to around 42℃) and enters the aging period. Forced ventilation is stopped. During the aging period, the spores of Bacillus subtilis and Bacillus amyloliquefaciens re-germinate and regain metabolic activity, further stabilizing the formed ternary structure.

[0015] Preferably, the acid activation treatment of the biochar involves soaking in a dilute acid solution to increase the content of active functional groups such as carboxyl groups on the surface of the biochar, thereby improving the covalent bonding efficiency with the prepolymer. Soaking in a dilute acid solution (such as 0.5-1.0% dilute nitric acid) can increase the carboxyl content on the surface of the biochar by 20-40%. Carboxyl groups can not only directly react with the hydroxyl groups in lignin-nicotine oligomers to form covalent bonds through esterification, but also complex Fe... 3+ Metal ions provide trace elements for microorganisms.

[0016] Furthermore, this invention also discloses the application of the above-mentioned bio-organic fertilizer, which is co-fermented with the residue of yellow-backed wood ear fungus, tobacco dust and biochar, in the improvement of acidic soil and the cultivation of pickled mustard greens or mustard greens.

[0017] The beneficial effects of this invention are as follows: Lignin in *Auricularia auricula-judae* residue undergoes oxidative depolymerization under the action of free radical initiators and heat-alkali-oxygen treatment, generating active aromatic fragments containing quinone and phenolic hydroxyl groups. Nicotine in tobacco dust exhibits enhanced nucleophilicity of its pyrrolidine nitrogen atoms under alkaline conditions, undergoing Michael addition with lignin quinone groups to form water-soluble oligomers (lignin-nicotine prepolymers) linked by CN covalent bonds. This prepolymer further undergoes esterification with carboxyl groups on the surface of acid-pretreated biochar, achieving covalent anchoring under the synergistic effect of the fermentation system's chemothermodynamics and microbial metabolic activities, ultimately forming a ternary covalent complex of lignin-nicotine-biochar. When this complex is applied to acidic soil: ① its alkaline groups (derived from calcium in biochar and fungal residue) neutralize soil H₂. + Its phenolic hydroxyl and pyrrolidine nitrogen functional groups complex with Al 3+ A dual mechanism reduces aluminum toxicity; ② The quinone structure in the complex acts as an electron shuttle, driving dissimilar iron-reducing bacteria in the soil to reduce Fe. 3+ Reduced to Fe 2+ ① It releases phosphate ions fixed by iron; ② The covalently linked complex resists microbial degradation, forms stable large-particle organic-inorganic complexes in the soil, improves the aggregation structure, and realizes long-term carbon sequestration. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0019] It should be noted that all reagents and raw materials used in this invention are commercially available, and the reagents are of analytical grade.

[0020] *Phanerochaete chrysosporium* was obtained from the China Industrial Microbial Culture Collection Center, strain number CICC40719. *Bacillus subtilis* was obtained from Shandong Weilan Luyuan Biotechnology Co., Ltd., product code Shengzhuan 120. *Thermophilic bacterium* was obtained from Wuhan Gray Algae Biotechnology Co., Ltd., product code HZB218806. *Bacillus amyloliquefaciens* was obtained from Wuhan Kenuo Biotechnology Co., Ltd., product code KN-527. Wood chip biochar was obtained from Henan Lanri Environmental Protection Co., Ltd., product code r1168.

[0021] Tobacco dust refers to the broken by-products collected after tobacco leaves are threshed and re-dried and then screened, as well as the waste tobacco dust discharged from the cigarette manufacturing workshop.

[0022] Example 1 This embodiment provides a bio-organic fertilizer co-fermented from *Auricularia auricula-judae* residue, tobacco dust, and biochar. By weight, the raw material composition is: 70 parts *Auricularia auricula-judae* residue, 20 parts tobacco dust, 12 parts sawdust biochar, 3.5 parts compound microbial inoculant, and 8 parts dried chicken manure. The dry matter of the *Auricularia auricula-judae* residue, by weight percentage, includes: 40% birch sawdust, 20% rice bran, 15% cottonseed hulls, 10% corn cob, 2% corn flour, 10% wheat bran, 1% lime, 1% gypsum, and 1% superphosphate.

[0023] The preparation method is as follows: I. Preparation of Compound Microbial Agents Prepare the following liquid culture media respectively: (1) PDA liquid culture medium: Take 200g of peeled potatoes, cut them into pieces, add 1000mL of deionized water and boil for 30min. Filter with eight layers of gauze, add 20g of glucose, 3g of KH2PO4, 1.5g of MgSO4·7H2O and 0.01g of vitamin B1 to the filtrate, add deionized water to make up to 1000mL, and sterilize at 115℃ for 20min; (2) LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, add deionized water to make up to 1000mL, adjust pH to 7.0, sterilize at 121℃ for 20min; (3) Modified Thermus medium: 5g peptone, 3g yeast extract, 2g NaCl, 0.5g MgSO4·7H2O, 0.2g CaCl2, 0.01g FeSO4·7H2O, add deionized water to make up to 1000mL, adjust pH to 7.5, and sterilize at 121℃ for 20min.

[0024] Activation culture conditions: Phanerochaete chrysosporium: 1 g of lyophilized powder was inoculated into 100 mL of PDA liquid medium and cultured at 28 °C and 180 rpm for 60 h with shaking. Bacillus subtilis: Take 1g of powder and inoculate it into 100mL LB liquid medium, and culture at 36℃ and 200rpm for 20h. Thermophilic bacteria: 1g of lyophilized powder was inoculated into 100mL of modified Thermus medium and cultured at 63℃ and 150rpm for 30h. Bacillus amyloliquefaciens: Take 1g of powder and inoculate it into 100mL LB liquid medium, and culture at 32℃ and 200rpm for 20h with shaking. After cultivation, the viable cell count of each bacterial suspension was determined (dilution plating method). The required volume of each bacterial suspension was calculated based on the viable cell ratio of *Phanerochaete chrysosporium*: *Bacillus subtilis*: *Thermophilic bacteria*: *Bacillus amyloliquefaciens* = 2:2:1:1, and the two suspensions were mixed to obtain a compound microbial agent with a total effective viable cell count of approximately 8.5 × 10⁻⁶.8 CFU / mL. The compound microbial agent should be prepared and used immediately, and stored at 4℃ for no more than 24 hours.

[0025] II. Preparation of Bio-organic Fertilizer Step 1: Raw material pretreatment and pulping activation: Take the yellow-backed wood ear fungus residue and tobacco dust, crush them separately and pass them through a 60-mesh sieve. Then weigh 70 kg of the sieved yellow-backed wood ear fungus residue and 20 kg of tobacco dust and mix them together. Weigh 0.68 kg of ferrous sulfate, add it to 20 kg of water and stir to dissolve it. Spray it evenly into the mixture while stirring it to raise the moisture content of the material to 68%. Pile the material in the pulping tank, cover it with plastic film and let it stand for 24 hours. Step 2, Heat-Alkali-Oxygen Three-Field Coupled Prepolymerization: The slurry material is transferred to a closed reaction vessel with a stirring and heating jacket. Hot water vapor is introduced into the heating jacket for heating, and the temperature is raised to 70°C while stirring. Lime and water are mixed at a weight ratio of 2:10 to obtain lime slurry, which is added to the reaction vessel. The final pH of the material is adjusted to 7.5. The exhaust valve is closed, and the pressure inside the vessel is maintained at 0.08 MPa. The vessel is kept at 70±2°C for 9 hours, and stirred for 5 minutes every 1 hour during this period. Step 3, Biochar Loading and Deep Microbial Assembly: Allow the material treated in Step 2 to cool naturally to 58℃; take 12kg of sawdust biochar and pretreat it as follows: soak the biochar in 0.8wt% dilute nitric acid solution for 3 hours, drain, wash with water until neutral, and dry at 95℃ for later use; add the pretreated biochar to the reaction vessel and stir for 15 minutes to ensure uniform mixing; take 3.5kg of the composite microbial agent prepared in Step 1 and add 20kg of warm water (… Dilute in 30℃ and spray evenly onto the material. Transfer the material to a windrow fermentation tank (8m long, 2m wide, and 0.8m high), cover with non-woven fabric for heat and moisture retention, and maintain an ambient temperature of 28±2℃. Carry out high-temperature aerobic fermentation for 8 days at 60±2℃, using forced ventilation (ventilation volume 0.45L / (min·kg dry material)). Turn the pile once a day. After 8 days, the material temperature will naturally drop to 42℃ and enter the aging period. Stop forced ventilation and change to turning the pile once every 2 days for 4 days. Step 4: Low-temperature drying: Spread the fermented material in the drying workshop to a thickness of 5cm, and dry it at 55±5℃ using a heat pump dryer until the moisture content is 13%. Then crush it through an 80-mesh sieve to obtain the bio-organic fertilizer.

[0026] Example 2 This embodiment provides a bio-organic fertilizer co-fermented with yellow-backed wood ear fungus residue, tobacco dust and biochar, which includes, by mass parts: 60 parts yellow-backed wood ear fungus residue, 25 parts tobacco dust, 11 parts sawdust biochar, 4 parts compound microbial agent and 10 parts oil cake.

[0027] The preparation method is the same as in Example 1.

[0028] Example 3 This embodiment provides a bio-organic fertilizer made by co-fermentation of yellow-backed wood ear fungus residue, tobacco dust and biochar. By weight, it includes: 80 parts yellow-backed wood ear fungus residue, 15 parts tobacco dust, 15 parts straw biochar, 2 parts compound microbial agent, 5 parts dried chicken manure, 3 parts peat moss (8 parts auxiliary conditioning agent).

[0029] The preparation method is the same as in Example 1.

[0030] Example 4 This embodiment provides a bio-organic fertilizer co-fermented with yellow-backed wood ear fungus residue, tobacco dust and biochar, which includes, by mass parts: 65 parts yellow-backed wood ear fungus residue, 22 parts tobacco dust, 10 parts sawdust biochar, 3.0 parts compound microbial agent and 8 parts peat.

[0031] The preparation method is the same as in Example 1.

[0032] Comparative Example 1 (without chemical prepolymerization treatment) Compared with Example 1, the difference is that step two is omitted, that is, biochar and microbial agents are directly added after pulping for aerobic fermentation.

[0033] Comparative Example 2 (without chemical prepolymerization and biochar, using conventional composting) Compared with Example 1, the differences are as follows: Step 2 is omitted, biochar is not added in Step 3, and the process adopts a conventional aerobic composting method: Take 3.5 kg of the compound microbial agent prepared according to the same method as Example 1 (preparation method is the same as Example 1), dilute it in 20 kg of warm water (30℃), and spray it evenly onto the material (the material in Step 1). Stack the material into windrows and ventilate and ferment for 20 days at 50±5℃ with a forced ventilation rate of 0.3 L / (min·kg). Turn the pile once every 2 days, for a total of 8 times. There are no separate high-temperature period and aging period segments.

[0034] Comparative Example 3 (Biochar was physically mixed after fermentation) Compared to Example 1, the difference lies in the following: Organic fertilizer (without biochar) was first prepared according to the method of Comparative Example 2. After fermentation and drying, it was physically mixed with sawdust biochar: 12 kg of sawdust biochar was taken and pretreated using the same method as in Example 1 (soaked in 0.8 wt% dilute nitric acid solution for 3 hours, washed until neutral, and dried at 95°C). The pretreated biochar and the basic organic fertilizer obtained in step four were then mixed in a three-dimensional mixer for 30 minutes to ensure uniform physical mixing of the biochar and organic fertilizer.

[0035] Comparative Example 4 (The bacterial agent did not contain thermophilic bacteria, and there was no forced ventilation during high-temperature periods) Compared to Example 1, the difference lies in that the compound microbial agent contains only *Phanerochaete chrysosporium*, *Bacillus subtilis*, and *Bacillus amyloliquefaciens* (mass ratio 2:2:1), without the addition of thermophilic bacteria. Fermentation conditions: No forced ventilation is used; ventilation relies solely on natural turning of the pile. The maximum fermentation temperature is 48°C, and the total fermentation time is 20 days, with the pile turned once daily. There are no separate high-temperature and aging periods.

[0036] Comparative Example 5 (Smokeless Powder) Compared with Example 1, the difference is that no tobacco powder is added to the raw materials, and the amount of yellow-backed wood ear fungus residue is increased to 90 parts.

[0037] The physicochemical properties of the fertilizers prepared in Examples 1-4 and Comparative Examples 1-5 were tested. The test indicators and methods are as follows: Organic matter: Refer to HJ761-2015 "Determination of Organic Matter in Solid Waste - Loss on Ignition Method". Place the dried fertilizer sample in a muffle furnace at 600℃ and ignite for 3-4 hours. The organic matter is completely burned at high temperature, and the organic matter content is calculated based on the mass difference before and after ignition.

[0038] Total nitrogen: Referring to NY / T2542-2014 "Determination of total nitrogen content in fertilizers", the sample is digested with sulfuric acid-hydrogen peroxide to convert nitrogen-containing organic matter into ammonium sulfate. Ammonia gas is released by distillation under alkaline conditions, absorbed by boric acid solution, and titrated with standard acid. The nitrogen content is calculated based on the amount of acid consumed.

[0039] Total phosphorus: Referring to NY / T2541-2014 "Determination of phosphorus content in fertilizers", the sample was digested with sulfuric acid-hydrogen peroxide to convert phosphorus into orthophosphate, which reacted with ammonium vanadate in an acidic medium to form a yellow complex. The absorbance was measured at a wavelength of 440 nm, and the phosphorus content was calculated according to the standard curve.

[0040] Total potassium: Referring to NY / T2540-2014 "Determination of potassium content in fertilizers", the sample was digested with sulfuric acid-hydrogen peroxide to convert potassium into ionic state. The emission spectrum intensity of potassium element (approximately 767nm) was measured using a flame photometer, and the potassium content was calculated based on the standard curve.

[0041] pH value: Refer to Appendix E of NY / T525-2021 "Organic Fertilizer" for determination. Weigh 5.00g of air-dried sample that has passed through a Φ1mm sieve into a 100mL beaker, add 50mL of water without carbon dioxide (after boiling to remove carbon dioxide), stir manually or with a magnetic stirrer for 3min, let stand for 30min, and measure with a pH meter.

[0042] The test results are shown in Table 1.

[0043] Table 1. Results of Phytochemical Properties Testing of Fertilizer

[0044] As shown in Table 1, the organic matter content of Examples 1-4 (44.8%-47.1%) was significantly higher than that of Comparative Example 2 (without biochar, 38.5%) and Comparative Example 4 (without thermophilic bacteria, 39.7%), indicating that the addition of biochar and the participation of thermophilic bacteria promoted the full decomposition of organic materials and the retention of organic matter. The total nutrient content of the Examples (6.87%-7.33%) was higher than that of Comparative Examples 1-5 (5.41%-6.33%), with Comparative Example 2 (without biochar) having the lowest total nutrient content (5.41%). Although the total nutrient content of Comparative Example 5 (without tobacco dust) (6.33%) was higher than that of the other comparative examples, it was lower than that of all Examples, indicating that nicotine in tobacco dust has a catalytic effect on nutrient conversion.

[0045] Field trial conditions: The test soil was paddy soil (acidic purple soil), with an initial pH of 4.93, organic matter of 15.2 g / kg, available phosphorus of 8.16 mg / kg, and exchangeable Al content of [missing information]. 3+ 2.83 cmol / kg. The test crop was mustard (variety Daoguanqing). 80 kg of the tested fertilizer was applied per mu as basal fertilizer. The fertilizer was harvested 90 days after transplanting. Soil samples were collected after two consecutive seasons of application for each treatment.

[0046] The physicochemical properties of the soil after fertilization with the fertilizers prepared in Examples 1-4 and Comparative Examples 1-5 were tested. The test indicators and methods are as follows: Soil pH value: NY / T1377-2007 "Determination of pH value in soil", potentiometric method was used. The air-dried soil sample was passed through a 2 mm sieve, and water was added at a soil-to-water ratio of 1:2.5 and shaken to extract. After standing, the suspension was measured with a pH meter. The electromotive force was measured using a glass electrode as the indicator electrode and a calomel electrode as the reference electrode, and then converted to pH value.

[0047] Soil organic matter: NY / T1121.6-2006 "Soil Testing Part 6: Determination of Soil Organic Matter", using the potassium dichromate oxidation-external heating method. Weigh an air-dried soil sample into a test tube, add potassium dichromate-sulfuric acid solution, and heat in an oil bath at 185℃ for 5 minutes to digest the organic carbon, ensuring complete oxidation. The remaining potassium dichromate is back-titrated with ferrous sulfate standard solution, using o-phenanthroline as an indicator; the endpoint color changes from orange-yellow through blue-green to brown-red. The organic carbon content is calculated based on the potassium dichromate consumption using an oxidation correction factor, multiplied by 1.724 to convert to organic matter content, and the result is expressed in g / kg.

[0048] Available phosphorus in soil: NY / T1121.7-2014 "Soil Testing Part 7: Determination of Available Phosphorus in Soil". Weigh 2.5g of air-dried soil sample that has passed through a 2mm sieve into a 150mL Erlenmeyer flask, add 50mL of 0.5mol / L sodium bicarbonate extract, and shake at 20-25℃ for 30 minutes. Filter with phosphorus-free filter paper, and after the filtrate is developed with a molybdenum-antimony anti-chromic reagent, measure the absorbance with a spectrophotometer. Calculate the available phosphorus content according to the standard curve.

[0049] Soil exchangeable aluminum: LY / T1240-1999 "Determination of exchangeable acidity of forest soils". Weigh 5g of air-dried soil sample, add 50mL of 1mol / L potassium chloride solution, shake for 30 minutes, and filter. Take 25mL of the filtrate, add 0.1mol / L sodium hydroxide standard solution (neutralized to pH 8.0), heat to boiling for 5 minutes, and back-titrate with 0.02mol / L hydrochloric acid standard solution until the phenolphthalein endpoint disappears. Calculate the exchangeable aluminum based on the amount of hydrochloric acid consumed. 3+ content.

[0050] The test results are shown in Table 2.

[0051] Table 2 Soil Test Results

[0052] As shown in Table 2, all indicators of the embodiment are superior to those of the comparative example, as detailed below: (1) pH value: The soil pH after treatment in Examples 1-4 was 5.33-5.42, which was 0.37-0.46 higher than the initial soil pH (4.96) and 0.27-0.36 higher than that in Comparative Example 2 (without biochar, 5.06). This indicates that the biochar and residual lime in the product of this invention have a synergistic effect in neutralizing soil acidity, while the acid-adjusting ability of simple organic fertilizer (Comparative Example 2) is limited.

[0053] (2) Organic matter: The organic matter content of soil in Examples 1-4 (20.8-22.1 g / kg) was significantly higher than that of the initial soil value (15.5 g / kg) and Comparative Example 2 (17.8 g / kg). The highest organic matter content was found in Example 3 (15 biochar samples) (22.1 g / kg), while the lowest was found in Comparative Example 2 (no biochar) (17.8 g / kg), demonstrating that biochar not only contributes to stable carbon but also promotes the retention of exogenous organic matter.

[0054] (3) Available phosphorus: The available phosphorus content in the soil of Examples 1-4 (34.78-38.21 mg / kg) increased by 308-348% compared with the initial soil value (8.52 mg / kg), and by 112-133% compared with Comparative Example 2 (16.37 mg / kg). The available phosphorus content in Comparative Example 5 (no soot) was 24.86 mg / kg, significantly lower than that in Examples, indicating that nicotine has a catalytic effect on phosphorus activation. The available phosphorus content in Comparative Example 3 (physical mixing of biochar) was 19.68 mg / kg, lower than that in Comparative Example 5, proving that chemical covalent bonding is more effective than physical mixing in promoting phosphorus release.

[0055] (4) Exchangeable aluminum: Al from Examples 1-4 3+ The content (0.83-0.92 cmol / kg) was 66-70% lower than the initial soil value (2.76 cmol / kg), and 43-49% lower than Comparative Example 2 (1.62 cmol / kg). Comparative Example 5 (no smoke) Al 3+ The concentration was 1.19 cmol / kg, which is still higher than in the examples, verifying the key contribution of nicotine nitrogen atoms to aluminum complexation.

[0056] In summary, the bio-organic fertilizer of this invention can effectively improve soil acidification, significantly increase the available phosphorus content and organic matter content of the soil, and at the same time greatly reduce the exchangeable aluminum content of the soil, demonstrating multiple synergistic effects of "increasing yield, adjusting acidity, activating phosphorus, and reducing aluminum".

[0057] Application Example 1 (Application in Pickled Mustard Tuber Cultivation) The tested soil was paddy soil with a topsoil texture of clay and a moderate fertility level. The basic physicochemical properties of the soil before the experiment were: pH 4.96, organic matter 18.3 g / kg, available nitrogen 116.5 mg / kg, available phosphorus 8.2 mg / kg, and available potassium 122.6 mg / kg.

[0058] The crop tested was pickled mustard tuber, specifically the Huazha No. 1 variety.

[0059] The experiment consisted of four treatments, each with three replicates, arranged in a randomized block design. Each plot was 21.0 m² (7.0 m × 3.0 m) in size, with a planting size of 0.35 m × 0.40 m. 150 plants were transplanted per plot, resulting in 4762 plants per acre.

[0060] The following are the processing steps: Treatment ① (blank control): No fertilizer was applied; Treatment ② (conventional fertilization): Apply 40 kg of 48% compound fertilizer (16-16-16) per mu as base fertilizer, apply 10 kg / mu of urea as top dressing during the seedling stage, and apply 20 kg / mu of urea as top dressing during the fruit enlargement stage; Treatment ③ (conventional fertilization + ordinary organic fertilizer): Based on treatment ②, apply 100 kg of ordinary commercial organic fertilizer per mu as base fertilizer during land preparation; Treatment ④ (conventional fertilization + product of this invention): Based on treatment ②, apply 80 kg of the bio-organic fertilizer prepared in Example 1 of this invention per mu during land preparation.

[0061] The entire growth period of pickled mustard tuber is 119 days. Field management measures were consistent across all treatments.

[0062] Pickled mustard tubers are harvested during their mature harvest period, and various data indicators of the pickled mustard tubers are tested.

[0063] Plant height: Ten representative plants were randomly selected from each plot, and the height from the ground to the highest point of the plant was measured with a ruler as the plant height (cm). Weight of a single tuberous stem: The average value of the fresh weight of each tuberous stem was taken as the weight of a single stem (g) after weighing it with an electronic balance. Actual yield of each plot: Harvest and weigh all the tuberous stems of all plants in each plot, and record the actual yield of the plot (kg / 21m). 2 ).

[0064] Yield per mu (unit of land area): After obtaining the actual yield of the plot, based on the plot area (21m²) 2 The yield per mu (kg / mu, based on 1 mu = 666.7 m³) is converted to yield per mu. 2 (Conversion).

[0065] The experimental results are shown in Table 3.

[0066] Table 3. Statistical Results of Various Data for Pickled Mustard Tuber

[0067] Statistical results show that applying the bio-organic fertilizer of Example 1 of this invention can improve the economic traits of pickled mustard tuber, increasing the weight of the tuberous stems by 524.44 g / stem compared to the blank control, 37.78 g / stem compared to conventional fertilization, and 35.51 g / stem compared to the control treated with ordinary organic fertilizer. The actual yield of pickled mustard tuber using the bio-organic fertilizer of Example 1 of this invention was 3514.32 kg / mu, an increase of 189.22 kg / mu (5.69%) compared to the control treated with ordinary organic fertilizer; an increase of 216.74 kg / mu (6.57%) compared to the conventional control; and an increase of 2513.05 kg / mu (250.98%) compared to the blank control. This demonstrates that the bio-organic fertilizer of this invention has a good yield-increasing effect on pickled mustard tuber.

[0068] Economic benefit analysis: Based on the market price of pickled mustard tuber (0.80 yuan / kg), the price of this invention's product (1.20 yuan / kg), and the price of ordinary organic fertilizer (0.90 yuan / kg): Processing ④ yield per mu: 3514.30 kg × 0.80 yuan / kg = 2811.44 yuan Processing method ③ Yield per mu: 3325.10 kg × 0.80 yuan / kg = 2660.08 yuan The additional output value of processing ④ compared to processing ③ was 151.36 yuan. The additional fertilizer cost for treatment ④ compared to treatment ③ is: 80kg × (1.20 - 0.90) yuan / kg = 24.00 yuan. Compared to treatment ③, treatment ④ resulted in an additional net income of 151.36 - 24.00 = 127.36 yuan / mu. The ratio of new output to input is 151.36:24.00 = 6.31:1 The above results show that when the product of the present invention is applied to pickled mustard tuber, it can significantly improve the biological characteristics of pickled mustard tuber (plant height increases by 2.11 cm, and the weight of the tuberous stem increases by 35.51 g), and has outstanding yield and income-increasing effects.

[0069] Application Example 2 (Application in Mustard Green Cultivation) The tested soil was paddy soil with a topsoil texture of clay and a moderate fertility level. The basic physicochemical properties of the soil before the experiment were: pH 4.93, organic matter 15.2 g / kg, available nitrogen 98.7 mg / kg, available phosphorus 8.5 mg / kg, and available potassium 115.3 mg / kg.

[0070] The test crop was mustard greens, specifically the variety "Daoguanqing".

[0071] The experiment consisted of three treatments, each replicated three times, in a randomized block design with a plot size of 21.0 m². 2 (7.0m×3.0m), cultivation specification 0.45m×0.50m, 90 plants per plot, equivalent to 2857 plants per mu.

[0072] The following are the processing steps: Treatment ① (conventional fertilization): Apply 40 kg of 48% ternary compound fertilizer (16-16-16) per mu as base fertilizer, and apply 10 kg / mu of urea as top dressing during the vigorous growth period; Treatment ② (conventional fertilization + ordinary organic fertilizer): Based on treatment ①, apply 100 kg of ordinary commercial organic fertilizer per mu as base fertilizer during land preparation; Treatment ③ (conventional fertilization + product of this invention): Based on treatment ①, apply 80 kg of the bio-organic fertilizer prepared in Example 1 of this invention per mu as a base fertilizer during land preparation.

[0073] The entire growth period of mustard greens is 120 days. Field management measures were consistent across all treatments.

[0074] Pickled mustard greens are harvested during the mature harvest period, and various data indicators of the mustard greens are tested.

[0075] Plant height: Ten representative plants were randomly selected from each plot, and the height from the ground to the highest point of the plant was measured with a ruler as the plant height (cm). Single plant product weight: The average value of the fresh weight of each plant was taken as the single plant weight (kg) after weighing each plant with an electronic balance. Actual yield of each plot: Harvest and weigh all the tuberous stems of all plants in each plot, and record the actual yield of the plot (kg / 21m). 2 ).

[0076] Yield per mu (unit of land area): After obtaining the actual yield of the plot, based on the plot area (21m²) 2 The yield per mu (kg / mu, based on 1 mu = 666.7 m³) is converted to yield per mu. 2 (Conversion).

[0077] The experimental results are shown in Table 4.

[0078] Table 4. Statistical Results of Mustard Green Data

[0079] Observational results show that applying the bio-organic fertilizer of this invention can improve the economic traits of mustard greens, increasing their weight by 0.19 kg compared to conventional fertilization and 0.16 kg compared to the control group using ordinary organic fertilizer. The actual yield per mu (approximately 0.067 hectares) of mustard greens fertilized with the bio-organic fertilizer of this invention was 8468.08 kg, an increase of 484.98 kg per mu (6.08%) compared to the control group using ordinary organic fertilizer, and an increase of 574.39 kg per mu (7.28%) compared to the conventional control group. This indicates that the bio-organic fertilizer of this invention has a good yield-increasing effect on mustard greens.

[0080] Economic benefit analysis: Based on the market price of mustard greens (0.80 yuan / kg), the price of this invention's product (1.20 yuan / kg), and the price of ordinary organic fertilizer (0.90 yuan / kg): Processing method ③ Yield per mu: 8468.08 kg × 0.80 yuan / kg = 6774.46 yuan Processing method ② Yield per mu: 7983.11 kg × 0.80 yuan / kg = 6386.49 yuan The additional output value of treatment ③ compared to treatment ② was 387.97 yuan. The additional fertilizer cost for treatment ③ compared to treatment ② is: 80kg × (1.20 - 0.90) yuan / kg = 24.00 yuan. The net income increase from treatment ③ compared to treatment ② is: 387.97 - 24.00 = 363.97 yuan / mu The ratio of new production to input is 387.97:24.00 = 16.17:1.

[0081] The bio-economic traits and yield of mustard greens treated with bio-organic fertilizer as a base fertilizer were superior to those treated with ordinary organic fertilizer, with an increase of 484.98 kg per mu (approximately 0.067 hectares), a yield increase rate of 6.08%, and an increase in net income of 387.97 kg per mu. This demonstrates that the bio-organic fertilizer of this invention has significant yield and income-increasing benefits for mustard greens.

[0082] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations.

[0083] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A bio-organic fertilizer co-fermented with *Auricularia auricula-judae* residue, tobacco dust, and biochar, characterized in that... By mass fraction, it includes: 60-80 parts of yellow-backed wood ear fungus residue, 15-25 parts of tobacco dust, 8-15 parts of biochar, 2-5 parts of compound microbial inoculant, and 5-10 parts of auxiliary conditioning agent; the aromatic fragments generated by the depolymerization of lignin in the yellow-backed wood ear fungus residue are connected to the nitrogen atoms of nicotine in the tobacco dust through CN bonds, and then anchored to the pore structure of biochar through COC bonds with the active functional groups of biochar, forming a ternary covalent complex of lignin-nicotine-biochar.

2. The bio-organic fertilizer produced by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 1, characterized in that, The dry matter composition of the yellow-backed wood ear fungus residue, by mass percentage, includes: sawdust 35-45%, cottonseed hulls 10-20%, rice bran 15-25%, wheat bran 8-12%, corn flour 1-3%, lime 0.5-1.5%, gypsum 0.5-1.5%, and superphosphate 0.5-1.5%; the nicotine content of the tobacco dust is 0.8-2.5%.

3. The bio-organic fertilizer produced by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 1, characterized in that... The compound microbial agent includes *Phanerochaete chrysosporium*, *Bacillus subtilis*, *Thermophilus thermophilus*, and *Bacillus amyloliquefaciens*, with each of the four agents having an effective viable count of not less than 1 × 10⁻⁶. 7 CFU / mL.

4. The bio-organic fertilizer produced by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 1, characterized in that, The auxiliary conditioning agent is selected from one or more of dried chicken manure, oil cake, and peat moss.

5. The bio-organic fertilizer produced by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 2, characterized in that, The biochar is wood chip biochar or straw biochar, prepared at a temperature of 400-600℃, with a specific surface area of ​​80-150 m². 2 / g.

6. A method for preparing a bio-organic fertilizer by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar as described in any one of claims 1-5, characterized in that, Includes the following steps: Yellow-backed wood ear fungus residue was mixed with tobacco dust and pretreated by pulping in the presence of a free radical initiator. Then, a prepolymerization reaction was carried out under alkaline conditions and under heating and pressure to generate a water-soluble prepolymer. The prepolymer is mixed with acid-activated biochar and inoculated with the composite microbial agent for aerobic fermentation to form a ternary covalent complex. Finally, the bio-organic fertilizer is obtained by low-temperature drying.

7. The method for preparing bio-organic fertilizer by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 6, characterized in that, The preparation method of the compound microbial agent is as follows: Phanerochaete chrysosporium, Bacillus subtilis, Thermophilic bacteria and Bacillus amyloliquefaciens are inoculated into their respective liquid culture media, shaken and cultured at 28-65℃ until the logarithmic growth phase, and then mixed to obtain the compound microbial agent.

8. The method for preparing bio-organic fertilizer by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 6, characterized in that, The aerobic fermentation includes two stages: a high-temperature period and an aging period. During the high-temperature period, the temperature is maintained to be conducive to the metabolism of thermophilic bacteria and forced ventilation is carried out. After the material temperature drops naturally, the aging period begins, forced ventilation is stopped, and anaerobic or facultative aging is carried out.

9. The method for preparing bio-organic fertilizer by co-fermentation of *Auricularia auricula-judae* residue, tobacco dust, and biochar according to claim 6, characterized in that, The acid activation treatment of the biochar involves soaking in a dilute acid solution.

10. The application of a bio-organic fertilizer made by co-fermentation of yellow-backed wood ear residue, tobacco dust and biochar as described in any one of claims 1-5 in the improvement of acidic soil and in the cultivation of pickled mustard greens or mustard greens.

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