Compound bacteria for sheep manure fermentation and method for preparing organic fertilizer through fermentation

Abstract

The invention discloses compound bacteria for sheep manure fermentation and a method for preparing an organic fertilizer through fermentation, and belongs to the technical field of microbial materials. The compound bacteria comprise functional bacteria I, functional bacteria II, a compound porous carrier and a microbial activity accelerant. The compound bacteria comprise specific functional bacteria, a compound porous carrier and a microbial activity accelerant, and all the components have a synergistic effect, so that the fermentation of the sheep manure can be effectively promoted, and harmful substances such as antibiotics possibly existing in the sheep manure can be better treated. The compound bacteria are used for fermenting sheep manure to prepare the organic fertilizer, so that the composting time is expected to be shortened, the degradation degree of harmful substances in the manure is improved, the pollution to the environment is reduced, meanwhile, high-quality organic fertilizer is provided for agricultural production, and the sustainable development of livestock breeding industry and agriculture is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial materials, and particularly relates to a composite bacteria for sheep manure fermentation and a method for preparing organic fertilizer by fermentation. Background Art

[0002] The booming livestock and poultry industry has impacted the modern environment, placing immense pressure on environmental governance. Among these impacts, livestock and poultry manure is a major contributing factor. Pollution from livestock farming, particularly the various environmental pollution phenomena caused by improper manure disposal, such as water and soil contamination, harmful gas pollution, and high-volume greenhouse gas emissions, has become a new focus of environmental pollution control.

[0003] Composting is an effective method for treating and utilizing livestock and poultry manure. It effectively removes harmful substances remaining in manure and converts it into organic fertilizer. This not only solves the waste problem in livestock and poultry production, but also provides a highly efficient fertilizer resource and reduces environmental pollution from manure. However, traditional composting methods have some drawbacks, such as long composting times and incomplete degradation of harmful substances in manure.

[0004] In the prior art, one or more microbial combinations are usually used to treat livestock manure. For example, Chinese patent application CN201711146838.1 discloses a sheep manure fermented fertilizer specifically for leguminous forage and a preparation method thereof. The fertilizer is prepared from 90-120 parts of sheep manure, 10-12 parts of peanut seedlings, 8-20 parts of sweet potato vines, 5-20 parts of paper mulberry wood chips, 8-10 parts of mushroom chaff, 2-5 parts of charcoal, 3-5 parts of fermentation agents, and 4-6 parts of mixed additives, and the effective agent is EM bacteria.

[0005] For example, Chinese patent application CN201711211483.X relates to a method for rapid fermentation of sheep manure, which belongs to the field of organic fertilizer technology. The method comprises the following steps: 1) mixing pond mud and crushed crop straw to obtain a fermentation base; 2) mixing sheep manure with an auxiliary mixture to obtain a fermentation dry material; 3) mixing a microbial fermentation agent with brown sugar and distilled water to obtain a fermentation liquid, wherein the microbial fermentation agent contains Proteus morganii, Aspergillus niger and Bacillus subtilis; 4) using the fermentation liquid to moisten wood ash to obtain a fermentation wet material; 5) spreading the fermentation base material on the bottom of the fermentation tank, and then stacking the fermentation base material in the form of a layer of fermentation dry material on top of a layer of fermentation wet material, inserting 3-5 ventilation pipes into the stack, the ventilation pipes protruding from the stack and having ventilation holes on the pipe wall, and the stack is fermented for 5-10 days.

[0006] Current existing technologies mostly use common microorganisms to simply ferment animal manure. However, in the livestock farming industry, antibiotics are often used. Most of the antibiotics used in the farming process cannot be fully absorbed by livestock and poultry. A large part of them will be excreted in the form of feces and urine, spreading to various environments such as soil, water and atmosphere, posing a serious threat to the environment and human health.

[0007] Therefore, how to develop a truly efficient method for resource utilization of poultry and livestock manure, effectively remove antibiotics in manure, and improve composting effects is a technical problem that needs to be solved urgently. Summary of the Invention

[0008] The present invention aims to solve the problems existing in the prior art and provides a composite bacteria for sheep manure fermentation and a method for preparing organic fertilizer by fermentation, aiming to solve the problems existing in the existing composting methods, such as long composting time, incomplete degradation of harmful substances, and antibiotic residues in livestock and poultry feces, so as to achieve efficient resource utilization of sheep manure. The composite bacteria comprises specific functional bacteria, a composite porous carrier, and a microbial activity promoter. The synergistic effect of each component can not only effectively promote the fermentation of sheep manure, but also better deal with harmful substances such as antibiotics that may be present in sheep manure. By fermenting sheep manure to prepare organic fertilizer with this composite bacteria, it is expected to shorten the composting time, improve the degradation degree of harmful substances in feces, reduce pollution to the environment, and provide high-quality organic fertilizer for agricultural production, thereby promoting the sustainable development of animal husbandry and agriculture.

[0009] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0010] Furthermore, the functional bacteria I is Methylobacterium marinum ( Methylobacterium salsuginis ), whose strain number is CGMCC No.1.6474; the functional bacteria II is Bacillus cellulolyticus ( Bacillus cellulosilyticus ), whose strain number is CGMCC No.1.15312.

[0011] Furthermore, the Methylobacterium marinum strain was purchased from the China General Microorganism Collection Center, with an original deposit date of November 1, 2006. The Bacillus cellulolyticus strain was purchased from the China General Microorganism Collection Center, with an original deposit date of June 12, 2015. Both functional microorganisms are commercially available through the China General Microorganism Collection Center, and do not require biological deposit.

[0012] Furthermore, the preparation method of the composite porous carrier is: (1) Palm leaves are dried and crushed, and then mixed with diatomaceous earth in a mass ratio of 10:1, and then polylactic acid (PLA) microspheres (30% by mass of the mixture) are added, and after mixing, they are hot-pressed at 180-200°C; in an N2 atmosphere, the temperature is raised to 600°C at a rate of 2°C / min, PLA decomposes and forms pores while biomass is carbonized, and then the temperature is raised to 700°C and CO2 is activated for 1 hour to increase the specific surface area, and then the temperature is naturally lowered to obtain biochar; (2) The biochar was then immersed in nano-silica sol at a solid-liquid ratio of 1 g:10 mL, and vacuum impregnated at -0.1 MPa for 1-2 h. After impregnation, it was filtered and dried at 70-80 ° C for 6-10 h to obtain activated biochar; (3) The activated biochar obtained in step (2) was immersed in an ethanol solution of 3-aminopropyltriethoxysilane (APTES) with a mass concentration of 5 wt% at a solid-liquid ratio of 1 g:15 mL, refluxed at 70 ° C for 3 h, filtered, washed with ethanol three times, and dried at 55-60 ° C to obtain a composite porous carrier.

[0013] Furthermore, the average particle size of the polylactic acid (PLA) microspheres is 10-20 μm, the solid content of the nano-silica sol is 15-20%, and the average particle size is 100-200 nm.

[0014] Furthermore, the microbial activity promoter is sodium alginate, mannitol and sucrose, which are mixed in a mass ratio of 1:1:10.

[0015] Furthermore, the preparation method of the composite bacteria for sheep manure fermentation includes the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 1:1 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3-0.5 to obtain a mixed solution, and the composite porous carrier is added. The mixed solution and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3-5 hours so that the carrier can fully absorb the mixed solution. Finally, the product is obtained by granulation and drying.

[0016] Furthermore, the composition of the seed culture medium is: 20g glucose, 10g peptone, 5g beef extract, 5g sodium chloride, 1000mL distilled water, and the pH value is 7.0-7.2; the composition of the fermentation culture medium is: 30g corn flour, 20g soybean meal powder, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate, 1000mL distilled water, and the pH value is 7.0-7.2.

[0017] The method for preparing organic fertilizer by fermenting with the composite bacteria of the present invention comprises the following steps: crushing sheep manure and screening the crushed sheep manure to obtain a sieved particle size of ≤2 cm, adding straw powder, and uniformly mixing the crushed sheep manure and straw at a mass ratio of sheep manure to straw of 7:1, adjusting the water content of the system to 55-58%, adding composite bacteria in an amount of 5% by mass of the sheep manure to carry out composting fermentation, and fermenting the compost for 15-20 days, turning the compost every 2 days during the heating / cooling period, and turning the compost every 3 days during the high temperature period, until there is no odor and the mixture is dark brown and loose, and the fermentation is terminated.

[0018] During the fermentation process, the pile is in the form of strips with a length, width and height of 2m×1m×0.6m. Water can be added when the pile is turned over to ensure that the moisture content of the pile is 50%-60%.

[0019] Beneficial effects: (1) The present invention selects two functional microorganisms to form the core strain, of which Methylobacterium marinum can effectively degrade antibiotic residues in sheep manure, and Bacillus cellulolyticus can decompose cellulose and other macromolecular organic matter in sheep manure. The two strains work synergistically, greatly improving the degradation efficiency of harmful substances in sheep manure, improving fermentation efficiency, and reducing environmental pollution. (2) The preparation of the composite porous carrier adopts a unique process. Through PLA decomposition and biomass carbonization, and then CO2 activation, the carrier has a large specific surface area and rich pore structure. The multi-layer petal-like structure formed provides a good living and reproduction space for functional microorganisms, which is conducive to the microorganisms to perform their functions. At the same time, it has a strong adsorption capacity, adsorbs and fixes harmful substances, and has the effect of deodorization and purification. (3) The addition of microbial activity promoters can significantly improve the activity and metabolic capacity of functional microorganisms, further promote the fermentation process of sheep manure, and shorten the composting time. (4) The composting time is significantly shortened compared with the traditional method by using the composite bacteria fermentation method of the present invention. The organic fertilizer after fermentation is of high quality and rich in nutrients. It can provide high-quality fertilizer resources for agricultural production, reduce environmental pollution, and promote the sustainable development of animal husbandry and agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the co-cultivation effect of Methylobacterium marinum and Bacillus cellulolyticus of the present invention; Figure 2 The electron microscope image of the composite porous carrier of the present invention is as follows Figure 2 As shown, (ab) are internal structure diagrams and (cd) are surface structure diagrams.

[0021] Figure 3 The internal structure (a) and surface structure (b) of the biochar obtained in Comparative Example 5; Figure 4 This is a diagram showing the experimental effects of cellulose decomposition by Methylobacterium marinum and Bacillus cellulolyticus, where (A) is Methylobacterium marinum and (B) is Bacillus cellulolyticus. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0023] Example 1 A composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0024] The functional bacteria I is seawater methylbacterium ( Methylobacterium salsuginis ), whose strain number is CGMCC No.1.6474; the functional bacteria II is Bacillus cellulolyticus ( Bacillus cellulosilyticus ), whose strain number is CGMCC No.1.15312.

[0025] The Methylobacterium marinum strain was purchased from the China General Microorganism Collection Center, with an original deposit date of November 1, 2006. The Bacillus cellulolyticus strain was purchased from the China General Microorganism Collection Center, with an original deposit date of June 12, 2015. Both functional microorganisms are commercially available through the China General Microorganism Collection Center and do not require biological deposit.

[0026] The preparation method of the composite porous carrier is: (1) Palm leaves are dried and crushed, and then mixed with diatomaceous earth in a mass ratio of 10:1, and then polylactic acid (PLA) microspheres (30% by mass of the mixture) are added, and after mixing, they are hot-pressed at 180-200°C; in an N2 atmosphere, the temperature is raised to 600°C at a rate of 2°C / min, PLA decomposes and forms pores while biomass is carbonized, and then the temperature is raised to 700°C and CO2 is activated for 1 hour to increase the specific surface area, and then the temperature is naturally lowered to obtain biochar; (2) The biochar was then immersed in nano-silica sol at a solid-liquid ratio of 1 g:10 mL, and vacuum impregnated at -0.1 MPa for 1 h. After the impregnation was completed, it was filtered and dried at 70-80 ° C for 6 h to obtain activated biochar; (3) The activated biochar obtained in step (2) was immersed in an ethanol solution of 3-aminopropyltriethoxysilane (APTES) with a mass concentration of 5 wt% at a solid-liquid ratio of 1 g:15 mL, refluxed at 70 ° C for 3 h, filtered, washed with ethanol three times, and dried at 55-60 ° C to obtain a composite porous carrier. The scanning electron microscope image of the composite porous carrier is as follows Figure 2 As shown, (AB) is the internal structure diagram and (CD) is the surface structure diagram.

[0027] The average particle size of the polylactic acid (PLA) microspheres is 10-20 μm, the solid content of the nano-silica sol is 15-20%, and the average particle size is 100-200 nm.

[0028] The microbial activity promoter is sodium alginate, mannitol and sucrose, which are mixed in a mass ratio of 1:1:10.

[0029] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 1:1 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3 to obtain a mixed liquid, and the composite porous carrier is added. The mixed liquid and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3 hours so that the carrier can fully absorb the mixed liquid. Finally, the product is obtained by granulation and drying.

[0030] The seed culture medium is composed of: 20g glucose, 10g peptone, 5g beef extract, 5g sodium chloride, 1000mL distilled water, and a pH value of 7.0-7.2; the fermentation culture medium is composed of: 30g corn flour, 20g soybean meal powder, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate, 1000mL distilled water, and a pH value of 7.0-7.2.

[0031] The method for preparing organic fertilizer by fermenting the composite bacteria of the present embodiment comprises the following steps: crushing sheep manure and sieving it, the sieved particle size is ≤2 cm, adding straw powder, mixing uniformly according to the mass ratio of sheep manure: straw=7:1, adjusting the water content of the system to 55-58%, adding 5% of the composite bacteria of the sheep manure mass to carry out compost fermentation, fermenting for 15-20 days, heating / cooling period once every 2 days, high temperature period turning once every 3 days, until there is no odor, the mixture is dark brown and loose, and fermentation is terminated.

[0032] During the fermentation process, the pile is in the form of strips with a length, width and height of 2m×1m×0.6m. Water can be added when the pile is turned over to ensure that the moisture content of the pile is 50%-60%.

[0033] Strain compatibility analysis: Single colonies of Methylobacterium marinum and Bacillus cellulolyticus were cultured on LB solid medium and cultured in a constant temperature incubator at 30-37℃ for 24 hours to observe whether there was growth inhibition. Figure 1 As shown in Figure 2, Methylobacterium marinum and Bacillus cellulolyticus can co-grow and culture together without antagonism.

[0034] Example 2 A composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0035] The functional bacteria I is seawater methylbacterium ( Methylobacterium salsuginis ), whose strain number is CGMCC No.1.6474; the functional bacteria II is Bacillus cellulolyticus ( Bacillus cellulosilyticus ), whose strain number is CGMCC No.1.15312.

[0036] The Methylobacterium marinum strain was purchased from the China General Microorganism Collection Center, with an original deposit date of November 1, 2006. The Bacillus cellulolyticus strain was purchased from the China General Microorganism Collection Center, with an original deposit date of June 12, 2015. Both functional microorganisms are commercially available through the China General Microorganism Collection Center and do not require biological deposit.

[0037] The preparation method of the composite porous carrier is: (1) Palm leaves are dried and crushed, and then mixed with diatomaceous earth in a mass ratio of 10:1, and then polylactic acid (PLA) microspheres (30% by mass of the mixture) are added, and after mixing, they are hot-pressed at 180-200°C; in an N2 atmosphere, the temperature is raised to 600°C at a rate of 2°C / min, PLA decomposes and forms pores while biomass is carbonized, and then the temperature is raised to 700°C and CO2 is activated for 1 hour to increase the specific surface area, and then the temperature is naturally lowered to obtain biochar; (2) The biochar was then immersed in nano-silica sol at a solid-liquid ratio of 1 g:10 mL, and vacuum impregnated at -0.1 MPa for 2 h. After the impregnation was completed, it was filtered and dried at 70-80 ° C for 10 h to obtain activated biochar; (3) The activated biochar obtained in step (2) was immersed in an ethanol solution of 3-aminopropyltriethoxysilane (APTES) with a mass concentration of 5 wt% at a solid-liquid ratio of 1 g:15 mL, refluxed at 70 ° C for 3 h, filtered, washed with ethanol three times, and dried at 55-60 ° C to obtain a composite porous carrier.

[0038] The average particle size of the polylactic acid (PLA) microspheres is 10-20 μm, the solid content of the nano-silica sol is 15-20%, and the average particle size is 100-200 nm.

[0039] The microbial activity promoter is sodium alginate, mannitol and sucrose, which are mixed in a mass ratio of 1:1:10.

[0040] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 1:1 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly at a mass ratio of 1:0.5 to obtain a mixed solution, and the composite porous carrier is added. The mixed solution and the composite porous carrier are mixed at a mass ratio of 3:1, and the mixture is treated under negative pressure for 5 hours so that the carrier can fully absorb the mixed solution. Finally, the product is obtained by granulation and drying.

[0041] The seed culture medium is composed of: 20g glucose, 10g peptone, 5g beef extract, 5g sodium chloride, 1000mL distilled water, and a pH value of 7.0-7.2; the fermentation culture medium is composed of: 30g corn flour, 20g soybean meal powder, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g calcium carbonate, 1000mL distilled water, and a pH value of 7.0-7.2.

[0042] The method for preparing organic fertilizer by fermenting the composite bacteria of the present embodiment comprises the following steps: crushing sheep manure and sieving it, the sieved particle size is ≤2 cm, adding straw powder, mixing uniformly according to the mass ratio of sheep manure: straw=7:1, adjusting the water content of the system to 55-58%, adding 5% of the composite bacteria of the sheep manure mass to carry out compost fermentation, fermenting for 15-20 days, heating / cooling period once every 2 days, high temperature period turning once every 3 days, until there is no odor, the mixture is dark brown and loose, and fermentation is terminated.

[0043] During the fermentation process, the pile is in the form of strips with a length, width and height of 2m×1m×0.6m. Water can be added when the pile is turned to ensure that the moisture content of the pile is 50%-60%.

[0044] Comparative Example 1 In this comparative example, only one functional bacterium I was used, and the rest of the raw materials and process steps were the same as in Example 1. That is: A composite bacteria for sheep manure fermentation comprises functional bacteria I, a composite porous carrier and a microbial activity promoter.

[0045] The functional bacteria I is Methylobacterium marinum, with a strain number of CGMCC No. 1.6474. The Methylobacterium marinum was purchased from the China General Microorganism Collection Center, with the original deposit date being November 1, 2006.

[0046] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium. After activation, take a ring of the activated strain of seawater Methylobacterium and inoculate it into a 500mL conical flask filled with seed culture medium, fill it with 20% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain the seed liquid of functional bacteria I. Inoculate the seed liquid of functional bacteria I into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3 to obtain a mixed liquid, and the composite porous carrier is added. The mixed liquid and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3 hours so that the carrier can fully absorb the mixed liquid. Finally, the product is obtained by granulation and drying.

[0047] Comparative Example 2 In this comparative example, only one functional bacterium II was used, and the rest of the raw materials and process steps were the same as in Example 1. A composite bacteria for sheep manure fermentation comprises functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0048] The functional bacteria II is Bacillus cellulolyticus, and its strain number is CGMCC No. 1.15312. The Bacillus cellulolyticus was purchased from China General Microorganism Collection Center, and the original deposit date was June 12, 2015.

[0049] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen Bacillus cellulolyticus. After activation, take a loop of the activated strain of Bacillus cellulolyticus and inoculate it into a 500 mL conical flask filled with seed culture medium, fill it with 20% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 h to obtain the seed liquid of functional bacteria II. Inoculate the seed liquid of functional bacteria II into a 1 L conical flask filled with fermentation medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3 to obtain a mixed liquid, and the composite porous carrier is added. The mixed liquid and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3 hours so that the carrier can fully absorb the mixed liquid. Finally, the product is obtained by granulation and drying.

[0050] Comparative Example 3 In this comparative example, except for changing the dosage ratio of functional bacteria I and functional bacteria II (the seed solution of functional bacteria I and functional bacteria II is mixed at a mass ratio of 2:1), the remaining raw materials and process steps are the same as those in Example 1. That is: A composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0051] The functional bacteria I is Methylobacterium marinum, and its strain number is CGMCC No. 1.6474; the functional bacteria II is Bacillus cellulolyticus, and its strain number is CGMCC No. 1.15312.

[0052] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 2:1 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3 to obtain a mixed liquid, and the composite porous carrier is added. The mixed liquid and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3 hours so that the carrier can fully absorb the mixed liquid. Finally, the product is obtained by granulation and drying.

[0053] Comparative Example 4 In this comparative example, except for changing the dosage ratio of functional bacteria I and functional bacteria II (the seed liquid of functional bacteria I and functional bacteria II is mixed at a mass ratio of 1:2), the remaining raw materials and process steps are the same as those in Example 1. That is: A composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

[0054] The functional bacteria I is Methylobacterium marinum, and its strain number is CGMCC No. 1.6474; the functional bacteria II is Bacillus cellulolyticus, and its strain number is CGMCC No. 1.15312.

[0055] The preparation method of composite bacteria for sheep manure fermentation comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 h to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 1:2 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3 to obtain a mixed liquid, and the composite porous carrier is added. The mixed liquid and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3 hours so that the carrier can fully absorb the mixed liquid. Finally, the product is obtained by granulation and drying.

[0056] Comparative Example 5 In this comparative example, except for the conventional anoxic pyrolysis technology to prepare biochar, the rest of the raw materials and process steps are the same as those in Example 1. That is, a composite bacteria for sheep manure fermentation comprises functional bacteria I, functional bacteria II, a biochar carrier, and a microbial activity promoter.

[0057] The preparation method of the biochar carrier is: (1) Palm leaves were dried and crushed, and then mixed with diatomaceous earth in a mass ratio of 10:1. In an N2 atmosphere, the temperature was raised to 600°C at a rate of 2°C / min, and then raised to 700°C and activated with CO2 for 1 h to increase the specific surface area. The biochar carrier was then naturally cooled to obtain the biochar carrier.

[0058] Comparative Example 6 Commercially available microbial composting fermentation agent was purchased from Jining Alida Bioengineering Co., Ltd., model hdwsw ALD, with main components including actinomycetes, Bacillus, yeast, etc.

[0059] Performance Testing Test of cellulose degradation ability of strains: The frozen seawater Methylobacterium and cellulolyticus were activated by Congo red staining. After activation, a ring of activated strains of seawater Methylobacterium and cellulolyticus were taken and inoculated into 500mL conical flasks filled with seed culture medium, with a liquid volume of 20%, and placed in a shaking incubator at 30-37°C and 150-200r / min for 10-12h to obtain seed liquid of functional bacteria I and functional bacteria II.

[0060] Use a pipette to inoculate 200 μL of the test bacterial solution into the center of a sodium carboxymethylcellulose culture medium. Incubate at 30-37°C for 48 hours. Stain the sodium carboxymethylcellulose culture medium with 0.2% Congo red stain and decolorize with 0.5% NaCl solution. Observe the colonies for the presence of a clear hydrolysis zone. If a hydrolysis zone is present, the strain is capable of degrading cellulose. Measure the colony diameter (d) and the clear zone diameter (D) to calculate the D / d ratio.

[0061] Table 1 Results of strain cellulose degradation test The data in Table 1 show that both strains selected in the present invention have strong cellulose degradation ability, and the cellulolytic Bacillus is relatively good. Figure 4 shown.

[0062] Antibiotic degradation ability test: Obtain seed liquid of Methylobacterium marinum and Bacillus cellulolyticus according to the aforementioned method. Transfer the seed liquid to a degradation medium containing 150 mg / L of the target antibiotic. After culturing for a period of time, remove the bacteria by centrifugation, collect the supernatant, and determine the antibiotic concentration by HPLC. The details are as follows: 1. Degradation test of different types of antibiotics by test strains LB medium (pH 7.0) containing 150 mg / L of different tetracycline antibiotics was prepared as a degradation medium: (1) oxytetracycline; (2) tetracycline; (3) chlortetracycline; and (4) tylosin. The seed liquid of the strain was inoculated into the degradation medium at a 3% inoculum and cultured at 180 rpm, 30-37°C for 8 days. The antibiotic concentration was measured and the degradation rate was calculated as the antibiotic concentration at the end of the culture / the antibiotic concentration before the experiment * 100%. The results are shown in Table 2.

[0063] Table 2 Test results of the ability of strains to degrade antibiotics Composting experiment: Using sheep manure as raw material, antibiotic residue simulated composting was performed by exogenously adding antibiotics such as oxytetracycline and tylosin, and the composting effect of the composite bacteria obtained in the examples and comparative examples was tested. Composting method: The sheep manure was crushed and sieved to a particle size of ≤2 cm. Straw powder was added and mixed evenly at a mass ratio of sheep manure: straw = 7:1. The water content of the system was adjusted to 55-58%, the antibiotic addition amount was 150 mg / kg, and the test bacteria agent was added at 5% by weight of the sheep manure for composting and fermentation. The composting was fermented for 15-20 days, with the compost turned every 2 days during the heating / cooling period and every 3 days during the high temperature period. Fermentation was completed when there was no odor and the mixture was dark brown and loose.

[0064] During the fermentation process, the pile is in the form of strips with a length, width and height of 2m×1m×0.6m. Water can be added when the pile is turned to ensure that the moisture content of the pile is 50%-60%.

[0065] The samples collected after the composting is completed are multi-point mixed samples. The national agricultural standard "NY525-2021 Organic Fertilizer" was used to determine the pH value, electrical conductivity (EC), organic matter, C / N content, fecal coliform count, ascarid mortality rate and other indicators in each mixed sample. Antibiotic detection: refer to "GB / T 3787-2020 Synchronous detection method for the content of tetracyclines, fluoroquinolones, sulfonamides, macrolides and chloramphenicol antibiotics in soil - high performance liquid chromatography". Determination of seed germination index (GI): First, take 10 g of compost sample and distilled water in a ratio of 1:10 and put it into a 250 mL conical flask, and then place it at 150 r·min. -1 The mixture was shaken on a shaker for 30 min. The supernatant was filtered and 5 mL of the filtrate was placed in a culture dish covered with filter paper. Twenty cabbage seeds were placed in the dish and cultured in the dark at 25°C for 48 h. Finally, the germination rate and root length of the seeds were measured, and deionized water was used as a blank control.

[0066] Each indicator was measured three times and the results were averaged.

[0067] Table 3 Composting experiment results Table 4 Composting experiment results According to the data analysis of Tables 3-4, the composite bacterial preparation of the present invention can effectively promote the fermentation process of sheep manure, significantly increase the organic matter content in the compost, reduce electrical conductivity, maintain a suitable pH value, enhance the seed germination index, reduce the C / N ratio, and ensure that the fecal coliform count and ascarid mortality rate meet the standards. In addition, in terms of antibiotic degradation, the present invention performs well and can significantly reduce the residues of tetracycline antibiotics and tylosin in sheep manure. However, when the dominant relationship between the two strains changes, the fermentation and decomposition effect of sheep manure is significantly reduced. This is because the two functional strains of the present invention can achieve efficient decomposition of sheep manure and effective degradation of antibiotics when mixed in equal proportions. After changing the microbial composition ratio, the synergistic balance between the two functional strains is destroyed, resulting in a weakening of the fermentation and degradation effect. Comparative Example 4 using ordinary biochar is inferior to the embodiment of the present invention in terms of microbial protection and adsorption capacity. Although it can still achieve the fermentation purpose, the decomposition and degradation effect is significantly reduced. Whether it is its internal structure or pore structure, it cannot promote the full reproduction and function of microorganisms. As for Comparative Example 6 using a common commercially available bacterial agent, although its composting effect can meet basic needs, its effect on antibiotic degradation is significantly insufficient and cannot meet the needs of practical applications.

[0068] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A composite bacteria for sheep manure fermentation, characterized in that: It comprises functional bacteria I, functional bacteria II, a composite porous carrier and a microbial activity promoter.

2. The composite bacteria for sheep manure fermentation according to claim 1, characterized in that: The functional bacteria I is seawater methylbacterium ( Methylobacterium salsuginis ), whose strain number is CGMCC No.1.6474; the functional bacteria II is Bacillus cellulolyticus ( Bacillus cellulosilyticus ), whose strain number is CGMCC No.1.15312.

3. The composite bacteria for sheep manure fermentation according to claim 1, characterized in that: The preparation method of the composite porous carrier is: (1) Palm leaves are dried and crushed, and then mixed with diatomaceous earth in a mass ratio of 10:1, and then polylactic acid (PLA) microspheres (30% by mass of the mixture) are added, and after mixing, they are hot-pressed at 180-200°C; in an N2 atmosphere, the temperature is raised to 600°C at a rate of 2°C / min, PLA decomposes and forms pores while biomass is carbonized, and then the temperature is raised to 700°C and CO2 is activated for 1 hour to increase the specific surface area, and then the temperature is naturally lowered to obtain biochar; (2) The biochar was then immersed in nano-silica sol at a solid-liquid ratio of 1 g:10 mL, and vacuum impregnated at -0.1 MPa for 1-2 h. After impregnation, it was filtered and dried at 70-80 ° C for 6-10 h to obtain activated biochar; (3) The activated biochar obtained in step (2) was immersed in an ethanol solution of 3-aminopropyltriethoxysilane (APTES) with a mass concentration of 5 wt% at a solid-liquid ratio of 1 g:15 mL, refluxed at 70 ° C for 3 h, filtered, washed with ethanol three times, and dried at 55-60 ° C to obtain a composite porous carrier.

4. The composite bacteria for sheep manure fermentation according to claim 3, characterized in that: The average particle size of the polylactic acid (PLA) microspheres is 10-20 μm, the solid content of the nano-silica sol is 15-20%, and the average particle size is 100-200 nm.

5. The composite bacteria for sheep manure fermentation according to claim 1, characterized in that: The microbial activity promoter is sodium alginate, mannitol and sucrose, which are mixed in a mass ratio of 1:1:

10.

6. The method for preparing composite bacteria for sheep manure fermentation according to claim 1, characterized in that: The method comprises the following preparation steps: (1) Activate the frozen seawater Methylobacterium and Bacillus cellulolyticus. After activation, take a ring of the activated strains of seawater Methylobacterium and Bacillus cellulolyticus, inoculate them into 500mL conical flasks filled with seed culture medium, fill them with 20% liquid, and culture them in a shaking incubator at 30-37°C and 150-200 r / min for 10-12 hours to obtain seed liquids of functional bacteria I and functional bacteria II. Mix the seed liquids of functional bacteria I and functional bacteria II in a mass ratio of 1:1 to obtain a mixed seed liquid. Inoculate the mixed seed liquid into a 1L conical flask filled with fermentation culture medium at a 10% inoculation rate, fill it with 50% liquid, and culture it in a shaking incubator at 30-37°C and 150-200 r / min. Ferment and culture until the effective viable bacteria count is 3×10 9 -5×10 9 cfu / mL, and the pH value was adjusted to 6.5-7.5 to obtain a functional microbial culture solution; (2) preparing a composite porous carrier; (3) The functional microbial liquid and the microbial activity promoter are mixed evenly in a mass ratio of 1:0.3-0.5 to obtain a mixed solution, and the composite porous carrier is added. The mixed solution and the composite porous carrier are mixed in a mass ratio of 3:1, and the mixture is treated under negative pressure for 3-5 hours so that the carrier can fully absorb the mixed solution. Finally, the product is obtained by granulation and drying.

7. A method for preparing organic fertilizer by fermenting the composite bacteria according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: crushing sheep manure and screening the crushed sheep manure to obtain a particle size of ≤2 cm, adding straw powder, and evenly mixing the crushed sheep manure and straw at a mass ratio of 7:1, adjusting the water content of the system to 55-58%, adding composite bacteria in an amount of 5% by mass of the sheep manure for composting and fermentation, fermenting the compost for 15-20 days, turning the compost every 2 days during a heating / cooling period, and turning the compost every 3 days during a high temperature period, and completing the fermentation process until there is no odor and the mixture is dark brown and loose.

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