Biological organic fertilizer fermentation inoculant as well as preparation method and application thereof

Abstract

The invention discloses a bio-organic fertilizer fermentation inoculant as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The microbial agent comprises active microorganisms and a synergistic component, and the mass ratio of the active microorganisms to the synergistic component is 1: (0.3-0.6). By optimizing the active microorganism composition and synergistic components, the fermentation efficiency and the product quality are remarkably improved. The microbial inoculum not only can quickly decompose lignocellulose structures in straws, but also can effectively reduce nutrient loss and improve the decomposition degree and fertilizer efficiency stability of organic fertilizers. Aspergillus fungal spore powder and a cellulose degrading bacterium viable bacterial preparation are scientifically matched, and specific synergistic components are combined, so that the bacterial agent shows an excellent synergistic effect in a composting treatment process. According to the technical scheme, the problems of slow fermentation, incomplete decomposition and the like in traditional organic fertilizer production are solved, and a new solution path is provided for resource utilization of agricultural wastes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a bio-organic fertilizer fermentation agent and its preparation method. Background Technology

[0002] With the development of modern agriculture, the amount of chemical fertilizer used is increasing day by day. The amount applied far exceeds the needs of crops, and unreasonable fertilization methods have led to problems such as low fertilizer utilization, soil degradation and environmental pollution.

[0003] Organic fertilizer is a type of fertilizer produced from organic waste through technological processing. It is rich in nutrients, provides stable fertilization, and improves soil quality. Organic fertilizer can significantly mitigate the environmental pollution caused by excessive use of chemical fertilizers and effectively increase crop yield and quality. However, traditional commercial organic fertilizers have high raw material costs and may introduce certain chemicals during the manufacturing process, which could potentially impact the soil's ecological environment.

[0004] With the rapid development of agriculture and the economy in my country, the improper disposal of large quantities of crop straw and livestock manure has placed enormous pressure on the environment. Composting agricultural waste to produce organic fertilizer is an effective means of resource utilization and an important way to improve economic efficiency. However, composting suffers from problems such as slow natural fermentation, severe nutrient loss, and incomplete decomposition due to the presence of large amounts of cellulose, lignin, and other recalcitrant substances in the compost material. Utilizing compound microbial agents to promote the degradation of lignocellulose in the compost pile and improve fermentation efficiency is key to solving these problems.

[0005] For example, Chinese patent application CN201710676656.9 discloses an organic fertilizer fermentation agent, which is composed of bacterial preparations, mold preparations, yeast preparations, and actinomycete preparations mixed in the following weight ratio: bacterial preparation: mold preparation: yeast preparation: actinomycete preparation = 2-7: 2-8: 2: 1-3. However, existing bacterial agents often suffer from drawbacks such as single bacterial species, poor activity, and high cost, especially for wastes with high cellulose content such as straw, cow manure, and sheep manure, where the fermentation effect is unsatisfactory. Straw waste, in particular, faces the biggest obstacle due to its lignocellulose structure. The physical barrier formed by lignin encapsulates cellulose and hemicellulose, making it difficult for microorganisms to contact the substrate. Although physical crushing (such as steam explosion) can destroy the structure, the equipment investment is large, making it unaffordable for small and medium-sized enterprises. Biological pretreatment relies on cellulase, but enzyme preparations account for more than 30% of the cost, and enzyme activity is easily inhibited. This severely restricts the efficiency of agricultural waste resource utilization and the development of the organic fertilizer industry.

[0006] Therefore, developing a fermentation agent that combines efficient decomposition promotion and nutrient loss reduction to produce high-quality organic fertilizer with comprehensive nutrients and long-lasting fertilization is a technical problem that urgently needs to be solved. Summary of the Invention

[0007] This invention provides a bio-organic fertilizer fermentation agent, whose innovation lies in significantly improving fermentation efficiency and product quality through optimized ratio of active microorganisms and synergistic components. This agent not only rapidly decomposes the lignocellulose structure in straw but also effectively reduces nutrient loss, improving the maturity and stability of organic fertilizer. Furthermore, this invention provides a specific preparation method for the agent, ensuring its ease of operation and low cost in practical applications. By scientifically combining Aspergillus spore powder and live cellulose-degrading bacteria with specific synergistic components, this agent exhibits excellent synergistic effects during composting. This technical solution not only solves the problems of slow fermentation and incomplete maturity in traditional organic fertilizer production but also provides a new solution for the resource utilization of agricultural waste.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:(0.3-0.6).

[0009] Furthermore, the active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:1.

[0010] Furthermore, the method for preparing the Aspergillus spore powder is as follows: (1) The strain numbered CGMCC No.3.15548 of Aspergillus violaceus ( Purple monkfish Inoculate the culture into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant culture. (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

[0011] Aspergillus violaceus strain numbered CGMCC No. 3.15548 Purple monkfish This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of February 19, 2016. It is available for purchase through commercial channels.

[0012] Step (1) The composition of activation medium A is: 200g / L potato, 20g / L glucose, 5g / L peptone, 20g / L agar, pH natural.

[0013] Step (2) The liquid seed culture medium consists of: 6-8 g / L corn steep liquor powder, 15 g / L glucose, 3 g / L yeast extract, 0.5 g / L MgSO4・7H2O, and pH 6.0-6.5.

[0014] Step (3) The solid sporulation substrate consists of: 70% wheat bran, 20% corn cob powder, 5% soybean meal, 3% CaCO3, 2% KH2PO4, and water is added to adjust the moisture content to 60%-65%.

[0015] Furthermore, the preparation method of the cellulose-degrading live bacteria preparation is as follows: (1) Strain screening and activation: Guangxi high-temperature actinomycetes with strain number CGMCC No4.7156 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies; (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

[0016] The strain numbered CGMCC No. 4.7156 is *Geothermal Actinobacter guangxiensis* ( Thermoactinomyces Guangxi This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of January 22, 2014. It is available for purchase through commercial channels.

[0017] Furthermore, the composition of the activation medium B in step (1) is as follows: soluble starch 20g / L, KNO3 1g / L, K2HPO4 0.5g / L, MgSO4・7H2O 0.5g / L, NaCl 0.5g / L, FeSO4・7H2O 0.01g / L, pH 7.2-7.4.

[0018] Furthermore, in step (2), the liquid culture medium is composed of activated culture medium B with agar removed and 5 g / L sodium carboxymethyl cellulose added.

[0019] Further, the composition of the fermentation substrate in step (3) is: 50% straw powder (crushed to ≤2mm), 30% wheat bran, 15% soybean meal, 35% CaCO3, with water added to adjust the moisture content to 60%-65%, and sterilized at 121℃ for 30 minutes.

[0020] Furthermore, the synergistic component comprises the following raw materials in parts by weight: 30-40 parts of wood ash, 5-10 parts of bone meal, and 10-15 parts of brown sugar.

[0021] All raw materials used in this invention are commercially available.

[0022] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two in a mass ratio of 1:1; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0023] This application describes a bio-organic fertilizer fermentation agent used for the fermentation of straw to prepare organic fertilizer.

[0024] The method of using the microbial agent in this application is as follows: Crush crop straw into small pieces of 3-6cm in length, add urea to adjust the ratio to about 25:1, add the microbial agent to water and disperse evenly, spray evenly on the pile using a water sprayer, and finally spray clean water while turning the pile to adjust the moisture content to about 60%. The mass ratio of microbial agent to straw is 50g:10kg. Ferment naturally for 15-20 days, turning the pile every 3-4 days. After fermentation, dry the pile until the moisture content is no higher than 30% to obtain straw bio-organic fertilizer.

[0025] Beneficial effects: (1) The present invention screened two functional strains of highly efficient cellulose degradation, namely, Guangxi high-temperature actinomycete and purple red mold. In the slightly acidic environment at the beginning of fermentation, purple red mold can efficiently secrete highly active laccase, which can effectively break down the lignin structure of straw. Subsequently, Guangxi high-temperature actinomycete can secrete highly active heat-resistant cellulase to rapidly degrade exposed cellulose. When the two strains are mixed in equal proportion, they can produce a significant synergistic effect. The combined use of the two strains greatly improves the cellulose degradation rate. (2) An synergist composed of wood ash, bone meal, and brown sugar was added. The wood ash in the synergist can neutralize the organic acids produced in the early stage of composting and maintain a suitable pH value, thereby enhancing the activity of laccase secreted by Monascus purpureus. Bone meal, as a long-lasting phosphorus source, can not only promote the growth and reproduction of microorganisms, but also replenish the mineral elements lost during fermentation. Brown sugar, as a fast-acting carbon source, can quickly activate the metabolic activities of microorganisms and enhance the initial colonization ability of the inoculant. Through the scientific proportioning of each component, the inoculant exhibits excellent fermentation performance in composting treatment, significantly shortens the fermentation cycle, and improves the degree and efficiency of organic fertilizer decomposition. In addition, the inoculant has a simple production process, low raw material cost, and is easy to scale up, with significant economic and environmental benefits. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the antagonistic experiment of the strains of this invention; Figure 2 The images show the antagonistic experimental results of *Aspergillus violaceus* and *Guangxi high-temperature actinomycete*, where A represents *Guangxi high-temperature actinomycete* and B represents *Aspergillus violaceus*. Figure 3 This is a photograph of the transparent zone of Aspergillus purpureus and Guangxi high-temperature actinomycetes on Congo red agar medium. Figure 4 Scanning electron microscope images of the surface of rice straw before and after degradation in compost. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0028] Example 1 A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:0.3.

[0029] The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:1.

[0030] The method for preparing the Aspergillus spore powder is as follows: (1) The strain numbered CGMCC No.3.15548 of Aspergillus violaceus ( Purple monkfish Inoculate the culture into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant culture. (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

[0031] Aspergillus violaceus strain numbered CGMCC No. 3.15548 Purple monkfish This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of February 19, 2016. It is available for purchase through commercial channels.

[0032] Step (1) The composition of activation medium A is: 200g / L potato, 20g / L glucose, 5g / L peptone, 20g / L agar, pH natural.

[0033] Step (2) The liquid seed culture medium consists of: 6-8 g / L corn steep liquor powder, 15 g / L glucose, 3 g / L yeast extract, 0.5 g / L MgSO4・7H2O, and pH 6.0-6.5.

[0034] Step (3) The solid sporulation substrate consists of: 70% wheat bran, 20% corn cob powder, 5% soybean meal, 3% CaCO3, 2% KH2PO4, and water is added to adjust the moisture content to 60%-65%.

[0035] The preparation method of the cellulose-degrading bacteria live bacterial preparation is as follows: (1) Strain screening and activation: Guangxi high-temperature actinomycetes with strain number CGMCC No4.7156 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies; (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

[0036] The strain numbered CGMCC No. 4.7156 is *Geothermal Actinobacter guangxiensis* ( Thermoactinomyces Guangxi This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of January 22, 2014. It is available for purchase through commercial channels.

[0037] Step (1) The composition of activation medium B is: soluble starch 20g / L, KNO3 1g / L, K2HPO4 0.5g / L, MgSO4・7H2O 0.5g / L, NaCl 0.5g / L, FeSO4・7H2O 0.01g / L, pH 7.2-7.4.

[0038] Step (2) The liquid culture medium is composed of activated culture medium B with agar removed and 5 g / L sodium carboxymethyl cellulose added.

[0039] Step (3) The fermentation substrate consists of: 50% straw powder (crushed to ≤2mm), 30% wheat bran, 15% soybean meal, and 35% CaCO3. Water is added to adjust the moisture content to 60%-65%, and the substrate is sterilized at 121℃ for 30 minutes.

[0040] The synergistic component comprises the following raw materials in parts by weight: 30 parts wood ash, 5 parts bone meal, and 10 parts brown sugar.

[0041] All raw materials used in this embodiment are commercially available.

[0042] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two in a mass ratio of 1:1; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0043] Example 2 A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:0.6.

[0044] The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:1.

[0045] The method for preparing the Aspergillus spore powder is as follows: (1) The strain numbered CGMCC No.3.15548 of Aspergillus violaceus ( Purple monkfish Inoculate the culture into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant culture. (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

[0046] Aspergillus violaceus strain numbered CGMCC No. 3.15548 Purple monkfish This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of February 19, 2016. It is available for purchase through commercial channels.

[0047] Step (1) The composition of activation medium A is: 200g / L potato, 20g / L glucose, 5g / L peptone, 20g / L agar, pH natural.

[0048] Step (2) The liquid seed culture medium consists of: 6-8 g / L corn steep liquor powder, 15 g / L glucose, 3 g / L yeast extract, 0.5 g / L MgSO4・7H2O, and pH 6.0-6.5.

[0049] Step (3) The solid sporulation substrate consists of: 70% wheat bran, 20% corn cob powder, 5% soybean meal, 3% CaCO3, 2% KH2PO4, and water is added to adjust the moisture content to 60%-65%.

[0050] The preparation method of the cellulose-degrading bacteria live bacterial preparation is as follows: (1) Strain screening and activation: Guangxi high-temperature actinomycetes with strain number CGMCC No4.7156 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies; (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

[0051] The strain numbered CGMCC No. 4.7156 is *Geothermal Actinobacter guangxiensis* ( Thermoactinomyces Guangxi This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of January 22, 2014. It is available for purchase through commercial channels.

[0052] Step (1) The composition of activation medium B is: soluble starch 20g / L, KNO3 1g / L, K2HPO4 0.5g / L, MgSO4・7H2O 0.5g / L, NaCl 0.5g / L, FeSO4・7H2O 0.01g / L, pH 7.2-7.4.

[0053] Step (2) The liquid culture medium is composed of activated culture medium B with agar removed and 5 g / L sodium carboxymethyl cellulose added.

[0054] Step (3) The fermentation substrate consists of: 50% straw powder (crushed to ≤2mm), 30% wheat bran, 15% soybean meal, and 35% CaCO3. Water is added to adjust the moisture content to 60%-65%, and the substrate is sterilized at 121℃ for 30 minutes.

[0055] The synergistic component comprises the following raw materials in parts by weight: 40 parts wood ash, 10 parts bone meal, and 15 parts brown sugar.

[0056] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two in a mass ratio of 1:1; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0057] Comparative Example 1 In this comparative example, except that only a single microorganism, *Guangxi thermophilic actinomycete*, was used in the active microorganisms, all other raw materials and process steps were the same as in Example 1. That is: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:0.3.

[0058] The active microorganisms are live cellulose-degrading bacteria.

[0059] The preparation method of the cellulose-degrading bacteria live bacterial preparation is as follows: (1) Strain screening and activation: Guangxi high-temperature actinomycetes with strain number CGMCC No4.7156 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies; (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

[0060] The strain numbered CGMCC No. 4.7156 is *Geothermal Actinobacter guangxiensis* ( Thermoactinomyces Guangxi This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of January 22, 2014. It is available for purchase through commercial channels.

[0061] Step (1) The composition of activation medium B is: soluble starch 20g / L, KNO3 1g / L, K2HPO4 0.5g / L, MgSO4・7H2O 0.5g / L, NaCl 0.5g / L, FeSO4・7H2O 0.01g / L, pH 7.2-7.4.

[0062] Step (2) The liquid culture medium is composed of activated culture medium B with agar removed and 5 g / L sodium carboxymethyl cellulose added.

[0063] Step (3) The fermentation substrate consists of: 50% straw powder (crushed to ≤2mm), 30% wheat bran, 15% soybean meal, and 35% CaCO3. Water is added to adjust the moisture content to 60%-65%, and the substrate is sterilized at 121℃ for 30 minutes.

[0064] The synergistic component comprises the following raw materials in parts by weight: 30 parts wood ash, 5 parts bone meal, and 10 parts brown sugar.

[0065] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Preparation of live cellulose-degrading bacteria preparation; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0066] Comparative Example 2 In this comparative example, except that only a single microorganism, *Aspergillus violaceus*, was used in the active microorganisms, all other raw materials and process steps were the same as in Example 1. That is: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:0.3.

[0067] The active microorganism is Aspergillus spore powder.

[0068] The method for preparing the Aspergillus spore powder is as follows: (1) The strain numbered CGMCC No.3.15548 of Aspergillus violaceus ( Purple monkfish Inoculate the culture into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant culture. (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

[0069] Aspergillus violaceus strain numbered CGMCC No. 3.15548 Purple monkfish This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of February 19, 2016. It is available for purchase through commercial channels.

[0070] Step (1) The composition of activation medium A is: 200g / L potato, 20g / L glucose, 5g / L peptone, 20g / L agar, pH natural.

[0071] Step (2) The liquid seed culture medium consists of: 6-8 g / L corn steep liquor powder, 15 g / L glucose, 3 g / L yeast extract, 0.5 g / L MgSO4・7H2O, and pH 6.0-6.5.

[0072] Step (3) The solid sporulation substrate consists of: 70% wheat bran, 20% corn cob powder, 5% soybean meal, 3% CaCO3, 2% KH2PO4, and water is added to adjust the moisture content to 60%-65%.

[0073] The synergistic component comprises the following raw materials in parts by weight: 30 parts wood ash, 5 parts bone meal, and 10 parts brown sugar.

[0074] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Preparation of Aspergillus spore powder; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0075] Comparative Example 3 In this comparative example, except for changing the mass ratio of Aspergillus spore powder to cellulose-degrading live bacteria preparation in the active microorganisms to 2:1, all other raw materials and process steps are the same as in Example 1. That is: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:3.

[0076] The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 2:1.

[0077] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two at a mass ratio of 2:1; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0078] Comparative Example 4 In this comparative example, except for changing the mass ratio of Aspergillus spore powder to cellulose-degrading live bacteria preparation in the active microorganisms to 1:2, all other raw materials and process steps are the same as in Example 1. That is: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:3.

[0079] The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:2.

[0080] A method for preparing a bio-organic fertilizer fermentation agent includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two in a mass ratio of 1:2; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

[0081] Comparative Example 5 In this comparative example, except for the strain used to prepare the Aspergillus spore powder, the raw materials and process steps are the same as in Example 1. That is: A bio-organic fertilizer fermentation agent, wherein the agent comprises active microorganisms and synergistic components in a mass ratio of 1:0.3.

[0082] The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:1.

[0083] The method for preparing the Aspergillus spore powder is as follows: (1) The strain numbered CGMCC No. 3.15543 of Aspergillus violaceus ( Purple monkfish Inoculate the culture into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant culture. (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

[0084] Aspergillus violaceus strain numbered CGMCC No. 3.15543 Purple monkfish This strain was purchased from the China General Microbiological Culture Collection Center, with an original deposit date of February 19, 2016. It is available for purchase through commercial channels.

[0085] Comparative Example 6 In this comparative example, except for changing the strain type of thermotolerant actinomycetes in the cellulose-degrading bacteria live bacterial preparation, the raw materials and process steps are the same as in Example 1. That is: The cultivation method is the same as in Example 1. The preparation method of the cellulose-degrading bacteria live bacterial preparation is as follows: (1) Strain screening and activation: The thermophilic actinomycetes with strain number GDMCC No4.160 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies. (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

[0086] Thermostable actinomycetes with strain number GDMCC No. 4.160 ( Thermoactinomyces guangxiensis This strain was purchased from the Guangdong Provincial Microbial Culture Collection Center, with an original preservation date of June 13, 2019. It is available for purchase through commercial channels.

[0087] Performance testing Antagonism experiment: *Aspergillus violaceus* strain CGMCC No. 3.15548 and *Actinomyces guangxiensis* strain CGMCC No. 4.7156 were compared and contrasted. Figure 1 (Schematic diagram) As shown, the bacteria were inoculated onto CMC-Na solid medium and cultured at 25-30℃ for 3 days. When the strains grew into bands, observe whether inhibition zones appeared at the intersections of different strains. If no inhibition zones appeared, it indicates that there was no antagonistic effect between the strains, and they could be used in combination; otherwise, they could not be used in combination. The culture diagram is shown below. Figure 2 As shown, the two bacteria do not inhibit each other and can be used in combination.

[0088] Cellulose degradation ability test of strains: The target strains were inoculated onto sodium carboxymethyl cellulose (CMC-Na) solid medium, with 3 spots per plate. The plates were then inverted and incubated at 25-30℃. After 3 days, colonies grew. Staining was performed by carefully pouring 1.0 g / L Congo red dye into the plates, allowing them to stand for 30 min, then removing the Congo red dye. The plates were then destained with 1.0 mol / L NaCl solution for 20 min. The clear zones appearing on the plates were observed, and the diameters of the clear zones (D) and colonies (d) were measured. Strains capable of degrading cellulose showed obvious clear zones after treatment with Congo red dye. The ratio of the clear zone (D) to the colony diameter (d) reflects their degradation ability. The culture results are as follows: Figure 3 As shown in Table 1.

[0089] Table 1. Ratio of the diameter of the transparent zone D to the diameter of the colony d for each strain. c Straw fermentation composting experiment: CMC-Na enzyme, xylanase, and laccase are biocatalysts that directly reflect the intensity of microbial metabolism during the degradation of lignocellulose. Therefore, the activities of these three enzymes are measured during the mid-fermentation stage to determine the fermentation effect.

[0090] Fermentation agents were prepared according to the methods described in the examples and comparative examples.

[0091] Rice straw was selected, dried, and pulverized into 3-6cm pieces. Urea was added to adjust the ratio to approximately 25:1. The microbial agent to be tested was added to water and dispersed evenly. The mixture was then sprayed evenly onto the pile using a water sprayer. Finally, the pile was turned over while spraying clean water until the moisture content was adjusted to approximately 60%. The mass ratio of microbial agent to straw was 50g:10kg. Fermentation was carried out naturally for 20 days, with the pile turned over every 3-4 days. After fermentation, the straw was dried until the moisture content did not exceed 30% to obtain straw bio-organic fertilizer. Samples were taken for testing at 3, 10, and 20 days. The microstructure of the straw surface was observed at 3 days, key enzyme activity was tested at 10 days, and compost indicators were tested at 20 days.

[0092] Test method: (1) Cellulase activity assay Cellulase activity was determined using the carboxymethyl cellulose saccharification power method (Li Lanxiao, Du Jinhua, Li Junxun, et al. Study on the conditions for determining cellulase activity by CMC saccharification power method, Feed Industry, 2006, 27(24): 49-52). Cellulase activity was defined as the amount of enzyme that hydrolyzes carboxymethyl cellulose to produce 1.0 μg of glucose in 1 min at 40 ℃ and pH 4.6, defined as one enzyme activity unit (U / g).

[0093] (2) Xylanase activity assay The determination of xylanase activity follows the method in the national standard GB / T 23874—2009. Xylanase activity is defined as the amount of enzyme required per minute to degrade and release 1 mol of reducing sugar from a xylan solution with a mass concentration of 5 mg / mL at 37℃ and pH 5.5. This amount is defined as 1 unit of enzyme activity (U / g).

[0094] (3) Laccase activity assay Laccase activity was determined using the ABTS oxidation method. Enzyme activity is defined as the amount of enzyme required to catalyze the generation of 1 μmol ABTS・⁺ (cationic free radical) per minute at 30℃ and pH 4.0 (refer to FAN Lili, JI Jiaming, XUN Yuefeng, et al. Isolation and characterization of laccase from Trichoderma asperellum Tasjk26[ J]. Food and Fermentation Industries, 2025, 51(13): 45-52.). Five samples were collected from different parts of the compost, and the average value was taken.

[0095] Table 2. Experimental results of fermentation effect After fermentation, fertilizer indicators, seed germination tests, and product physicochemical indicators were measured. The sample was mixed with sterile distilled water at a volume ratio of 1:10. 5 mL of the compost extract was pipetted into a sterile petri dish lined with filter paper, and 10 plump Chinese cabbage seeds were evenly placed on the surface. Sterile water was used as a control. Each treatment was performed in triplicate. The petri dishes were incubated at 25℃ for 48 hours, and the seed germination rate (GI) was measured. Generally, a seed germination rate of 50% or higher indicates that the organic fertilizer is basically decomposed. Other relevant indicators were tested according to the corresponding methods in the national quality standard for organic fertilizer NY525-2021. Five composting experiments were conducted for each experimental group, and the average results were taken.

[0096] Table 3 Composting Test Results As can be seen from the data in Tables 1-3, the fermentation agents of Examples 1-2 of this invention exhibited high enzyme activity during straw composting. This indicates that when the mass ratio of the selected Aspergillus spore powder to the live cellulose-degrading bacteria preparation is 1:1, they can work synergistically to promote the degradation of lignocellulose. Furthermore, as can be seen from the data in Table 2, in the key enzyme activity assays on day 10 of fermentation in Example 1, the activities of cellulase and xylanase also reached high levels, further verifying the superiority of this combination. Figure 4The straw degradation effect diagram also shows that before degradation, the rice straw surface was smooth, well-organized, and compact, with a tightly arranged, undamaged lignocellulose structure and intact thin-walled cell mesh structure. After 3 days of treatment with the fermentation agent in Example 1, the waxy layer on the surface of the rice straw was completely decomposed, and the surface showed obvious degradation and deformation. The lignocellulose structure was clearly broken, and severe surface shrinkage occurred, destroying the mesh integrity. This is highly beneficial for subsequent fermentation and decomposition.

[0097] Analysis of seed germination rates in different treatment groups revealed that the germination rate of bok choy seeds in Example 1 reached 85%, significantly higher than that of the treatment groups in Comparative Examples 3 to 6. Furthermore, the physicochemical indicators of the compost products met the requirements of the national quality standard for organic fertilizer, NY525-2021. This indicates that the fermentation agent not only effectively improves composting efficiency but also ensures the safety and fertilizer efficacy of the compost products. In Comparative Examples 1-6, where the strain composition was altered, the synergistic balance of key strains was disrupted, leading to a decrease in enzyme activity and consequently a weakening of the fermentation effect. Based on the above experimental results, the bio-organic fertilizer fermentation agent provided by this invention possesses significant technical advantages, particularly in improving enzyme activity, accelerating straw degradation, and enhancing the quality of compost products, demonstrating promising application prospects.

[0098] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A bio-organic fertilizer fermentation agent, characterized in that, The microbial agent contains active microorganisms and synergistic components in a mass ratio of 1:(0.3-0.6).

2. The bio-organic fertilizer fermentation agent according to claim 1, characterized in that, The active microorganisms include Aspergillus fungal spore powder and cellulose-degrading bacteria live bacterial preparation, with a mass ratio of 1:

1.

3. The bio-organic fertilizer fermentation agent according to claim 2, characterized in that, The method for preparing the Aspergillus spore powder is as follows: (1) Inoculate the purple-red Aspergillus strain with strain number CGMCC No3.15548 into activation medium A and incubate at 25±2℃ for 3-5 days to obtain activated slant strain; (2) Inoculate the activated bacterial strain into liquid seed culture medium and incubate for 48-72 hours at a shaking speed of 220-250 rpm and a temperature of 23-25°C, until the bacterial concentration in the culture is ≥10%. 8 CFU / mL was used to obtain the seed culture; (3) Inoculate the seed culture at a rate of 5%-8% into the solid sporulation substrate, place it in a breathable culture bag, and culture it in the dark at 23-25℃ and 75%-85% relative humidity for 7-10 days until a red spore layer covers the substrate surface. Simultaneously, observe the spore density under a microscope to ensure it is ≥10⁻⁶. 9 pcs / g; (4) Add sterile water containing 0.05% Tween-80 to the sporulation substrate, stir to wash away the spores, filter through a 300-mesh filter cloth to remove mycelial residues; collect the spore precipitate by centrifuging the filtrate at 4℃ and 3000rpm for 10 minutes, and freeze-dry it under vacuum to obtain Aspergillus spore powder with a moisture content ≤8% and a spore survival rate ≥90%.

4. The bio-organic fertilizer fermentation agent according to claim 2, characterized in that, The preparation method of the cellulose-degrading bacteria live bacterial preparation is as follows: (1) Strain screening and activation: Guangxi high-temperature actinomycetes with strain number CGMCC No4.7156 were inoculated into activation medium B and placed in a constant temperature incubator at 42±2℃ for 48-72 hours in the dark to obtain grayish-white radial colonies; (2) Seed culture expansion: Inoculate activated colonies into liquid culture medium at an inoculation rate of 6%-8%, and incubate at 40℃ and 220rpm for 36-48 hours. The viable bacterial concentration in the culture solution should be ≥10%. 9 CFU / mL terminates culture; (3) Solid-state fermentation for microbial production: Inoculate the seed liquid into the fermentation substrate at an inoculum rate of 10%-12%, place it in a breathable fermentation bag, and incubate it in an environment of 45±2℃ and 70%-80% relative humidity for 5-7 days. The characteristics of the fermentation endpoint are: the substrate is brownish-yellow and loose, and the mycelial density is ≥10 under a microscope. 5 strips / g, viable count ≥10 10 CFU / g, dried and pulverized to obtain a live cellulose-degrading bacteria preparation.

5. The bio-organic fertilizer fermentation agent according to claim 1, characterized in that, The synergistic component comprises the following raw materials in parts by weight: 30-40 parts wood ash, 5-10 parts bone meal, and 10-15 parts brown sugar.

6. A method for preparing the bio-organic fertilizer fermentation agent according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Prepare Aspergillus spore powder and cellulose-degrading bacteria live bacteria preparation by mixing the two in a mass ratio of 1:1; (2) Mix the active microorganisms and the synergistic components in a certain proportion. First, sieve the wood ash and bone meal in the synergistic components to 80-100 mesh to ensure uniform particle size. Then, add brown sugar and an appropriate amount of water to the mixing equipment to dissolve and form a sugar solution. Spray the sugar solution evenly on the surface of the mixture of wood ash and bone meal. Add the active microorganisms while stirring, and control the stirring speed to 60-80 rpm for 5-10 minutes to ensure that the components are fully mixed. After mixing, place the material in an environment with a temperature of 25-30℃ and a relative humidity of 50%-60% for 1-2 hours to promote the microorganisms to adapt to the environment and be initially activated. Finally, pack the mixed microbial agent into a sealed packaging bag and use vacuum packaging technology to avoid external contamination.

7. The application of the bio-organic fertilizer fermentation agent according to any one of claims 1-5, characterized in that, Used for fermenting straw to produce organic fertilizer.

Images