Extremely thermophilic biochar complex microbial inoculant and application thereof in aerobic composting of pig manure

Abstract

The invention discloses an extreme thermophilic charcoal complex microbial inoculant and application thereof in aerobic composting of pig manure, and belongs to the technical field of microorganisms. The invention firstly discloses a strain of thermophilic thermophilic bacteria N1209, and secondly discloses an extreme thermophilic biochar complex microbial inoculant, which comprises geobacillus thermoglucosidase BGSC 95A1, bacillus licheniformis ZR-1, the thermophilic thermophilic thermophilic bacteria N1209 and geobacillus stearothermophilus L5. The extremely thermophilic biochar complex microbial inoculant has the characteristic of high temperature resistance, compared with a single microbial inoculant, produced amylase, protease and lipase are higher in activity and show synergistic advantages, decomposition of pig manure can be remarkably promoted, the extremely thermophilic biochar complex microbial inoculant has advantages in the aspects of nitrogen element retention and CH4 and N2O emission reduction in the pig manure composting process, and the extremely thermophilic biochar complex microbial inoculant can be applied to the field of pig manure composting. And a new method is provided for treatment of livestock breeding wastes.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an extremely thermophilic biochar composite bacterial agent and application thereof in aerobic composting of pig manure. Background Art

[0002] The continuous increase in the scale of pig farming has not only met the market demand for livestock and poultry products, but also produced a large amount of pig manure. According to statistics, about 3.8 billion tons of pig manure are produced each year, but the comprehensive utilization rate is less than 60%. A large amount of livestock and poultry manure contains various pathogenic microorganisms, heavy metals and other harmful substances. If it is discharged without treatment, it will not only cause serious pollution to the environment, but also produce a large amount of greenhouse gases, exacerbating the greenhouse effect. Therefore, accelerating the development of animal husbandry requires accelerating the reduction, harmlessness and resource utilization of livestock and poultry manure.

[0003] Aerobic composting is the most commonly used method for treating livestock and poultry manure. It can not only reduce the accumulation of manure produced by intensive animal husbandry, but also convert it into organic fertilizer for use in agricultural planting. Compared with other treatment methods, composting has low cost, simple operation, and low environmental requirements. However, traditional composting methods have obvious disadvantages. The composting process volatilizes a large amount of odor and greenhouse gases, the compost body is not fully fermented, and the fertilizer obtained by fermentation has low fertilizer efficiency. It has long been unable to keep up with the pace of modern agricultural development.

[0004] Inoculating fast-growing and highly active microorganisms into the compost can not only shorten the composting cycle, but also help retain nutrients in the compost. Studies have shown that compared with inoculating a single bacterial agent, inoculating a composite bacterial agent has more advantages in promoting compost maturity and improving fertilizer efficiency. Inoculating a composite bacterial agent in the compost can accelerate the temperature rise, extend the duration of high temperature, reduce the toxicity of the compost, improve the maturity of the compost and the fertilizer efficiency of the product, and shorten the composting cycle. However, the existing bacterial agents are affected by factors such as the weak high temperature resistance of the microorganisms, slow reproduction speed, and low production capacity, and the application effect in the composting process is not ideal. Summary of the invention

[0005] The purpose of the present invention is to provide an extremely thermophilic biochar composite bacterial agent and its application in aerobic composting of pig manure to solve the problems existing in the above-mentioned prior art. The extremely thermophilic biochar composite bacterial agent of the present invention has high temperature resistance. Compared with a single bacterial agent, the amylase, protease and lipase produced have higher activities, show synergistic advantages, can significantly promote the maturity of pig manure, and has advantages in the retention of nitrogen elements and the reduction of CH4 and N2O emissions during the pig manure composting process, providing a new method for the treatment of livestock and poultry waste.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Thermus thermophilus N1209, which is deposited in China Center for Type Culture Collection on January 13, 2025, and has a deposit number of CCTCC M 2025109.

[0008] The present invention also provides the use of the Thermus thermophilus N1209 in preparing aerobic composting fermentation bacterial agent for pig manure.

[0009] The present invention also provides an extremely thermophilic biochar composite bacterial agent, and a preparation method of the extremely thermophilic biochar composite bacterial agent comprises the following steps:

[0010] (1) mixing a bacterial agent of Geobacillus thermoglucosidase BGSC 95A1, a bacterial agent of Bacillus licheniformis ZR-1, a bacterial agent of Thermus thermophilus N1209 and a bacterial agent of Geobacillus stearothermophilus L5 to obtain an extremely thermophilic composite bacterial agent;

[0011] (2) The extremely thermophilic composite bacterial agent is inoculated into a biochar culture medium, cultured, centrifuged, and resuspended to obtain the extremely thermophilic biochar composite bacterial agent.

[0012] In the present invention, the deposit number of the Parageobacillus thermoglucosidasiu BGSC 95A1 is CCTCC NO: M 2020324, and the deposit number of the Bacillus licheniformis ZR-1 is CCTCC NO: M 2017384.

[0013] In the present invention, the thermoglucosidase Geobacillus BGSC 95A1, Bacillus licheniformis ZR-1, Thermus thermophilus N1209 and Geobacillus stearothermophilus L5 all have high temperature resistance and can stably secrete substances such as amylase, protease and lipase. There is no antagonistic inhibitory effect between the strains, and after mixing, a synergistic effect can be generated to improve the activity of the secretions of the strains.

[0014] In the present invention, after each bacterial species in the extremely thermophilic biochar composite agent is co-cultured with biochar, the bacterial cells can be loaded onto the surface and internal pores of the biochar, providing effective protection for the survival of the bacterial cells and being beneficial to the growth and metabolism of the bacterial cells.

[0015] Preferably, the OD of the inoculum of the thermoglucosidase Geobacillus sp. BGSC 95A1 is 600 The value is 1.6-1.8, the OD of the inoculum of Bacillus licheniformis ZR-1 600 The value is 1.6-1.8, and the OD of the inoculum of Thermus thermophilus N1209 is600 The value is 1.6-1.8, and the OD of the inoculum of the thermophilic Bacillus L5 600 The value is 1.6-1.8.

[0016] In the present invention, the preparation method of the bacterial agent of the thermoglucosidase Geobacillus subtilis BGSC 95A1, the bacterial agent of the licheniformis ZR-1, the bacterial agent of the thermophilic bacterium N1209 and the bacterial agent of the thermophilic Geobacillus stearothermophilus L5 is as follows:

[0017] The thermoglucosidase Geobacillus BGSC 95A1, the Bacillus licheniformis ZR-1, the Thermus thermophilus N1209 and the Geobacillus stearothermophilus L5 are respectively cultured in LB liquid culture medium at 65°C and 180-200rpm for 24h±2h, centrifuged, and resuspended in sterile water to obtain the thermoglucosidase Geobacillus BGSC 95A1 bacterial agent, the Bacillus licheniformis ZR-1 bacterial agent, the Thermus thermophilus N1209 bacterial agent and the Geobacillus stearothermophilus L5 bacterial agent.

[0018] Preferably, the volume ratio of the bacterial agent of the thermoglucosidase Geobacillus subtilis BGSC 95A1, the bacterial agent of the Bacillus licheniformis ZR-1, the bacterial agent of the Thermus thermophilus N1209 and the bacterial agent of the stearothermophilus Geobacillus subtilis L5 when mixed is 2:3:3:1.

[0019] In the present invention, the mixing ratio of each strain affects the growth rate of bacterial cells, and further affects the reproductive metabolic capacity of bacterial cells. When the bacterial agent of thermoglucosidase Geobacillus BGSC 95A1, the bacterial agent of Bacillus licheniformis ZR-1, the bacterial agent of Thermus thermophilus N1209 and the bacterial agent of Geobacillus stearothermophilus L5 are mixed in a ratio of 2:3:3:1, the growth state of each bacterial cell is optimal.

[0020] In the present invention, the biochar culture medium includes biochar and LB liquid culture medium.

[0021] In the present invention, the mass volume ratio of the biochar to the LB liquid culture medium is (40-60) g:1L, preferably 50 g:1L.

[0022] In the present invention, the biochar is selected from wood charcoal, bamboo charcoal, corn straw biochar or wheat straw biochar, preferably corn straw biochar.

[0023] In the present invention, when the extremely thermophilic composite bacterial agent is inoculated, the volume mass ratio of the extremely thermophilic composite bacterial agent to the biochar is 1 mL: (8-12) g, preferably 1 mL: 10 g.

[0024] Preferably, the culture conditions are 65° C. and 120-160 rpm for 1 week.

[0025] The present invention also provides application of the extremely thermophilic biochar composite bacterial agent in aerobic composting of pig manure.

[0026] The present invention also provides application of the extremely thermophilic biochar composite bacterial agent in aerobic composting for accelerating the decomposition of pig manure.

[0027] The present invention also provides the use of the extremely thermophilic biochar composite bacterial agent in reducing pollutant emissions during aerobic composting of pig manure.

[0028] In the present invention, the pollutants include CH4 and N2O.

[0029] The present invention also provides the use of the extremely thermophilic biochar composite bacterial agent in improving the nitrogen retention amount of the compost during aerobic composting of pig manure.

[0030] The present invention discloses the following technical effects:

[0031] The present invention prepares the extremely thermophilic composite bacterial agent by mixing the thermoglucosidase Geobacillus, Bacillus licheniformis, Thermus thermophilus and Geobacillus stearothermophilus with high temperature resistance in a ratio of 2:3:3:1. Compared with a single bacterial agent, the strains in the extremely thermophilic composite bacterial agent act synergistically, and the amylase, protease and lipase produced by the extremely thermophilic composite bacterial agent have higher activities.

[0032] The extremely thermophilic biochar composite bacterial agent prepared by the present invention can significantly promote the decomposition of pig manure, reduce the toxicity of pig manure, and has advantages in the retention of nitrogen and the reduction of CH4 and N2O emissions during the pig manure composting process. It provides a new method for the treatment of poultry and livestock manure and breeding waste, can effectively promote the reduction, harmlessness and resource utilization of livestock and poultry manure, and promote the development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 This is the Gram staining result of Thermus thermophilus;

[0035] Figure 2 The results of the Thermus thermophilus growth curve test;

[0036] Figure 3The results of the pairwise antagonism test of strains; in the figure, A22 represents Geobacillus stearothermophilus, D9 represents Thermus thermophilus, PT represents Geobacillus thermoglucosidase, and X2 represents Bacillus licheniformis;

[0037] Figure 4 is a scanning electron micrograph of the extremely thermophilic biochar composite bacterial agent; wherein A is a scanning electron micrograph of the sterilized biochar, and B is a scanning electron micrograph of the biochar loaded with the extremely thermophilic composite bacterial agent;

[0038] Figure 5 The following are the results of enzyme activity detection of single strain and extremely thermophilic composite bacteria; A is the result of amylase activity determination, B is the standard curve of protease activity, C is the result of protease activity determination, D is the standard curve of lipase activity, and E is the result of lipase activity determination;

[0039] Figure 6 The effect of extremely thermophilic biochar composite bacteria on the physical and chemical properties of pig manure aerobic composting piles; A is the temperature change of the pile during composting, B is the seed germination index of the pile, C is the C / N change of the pile, and D is the change of total nitrogen in the pile;

[0040] Figure 7 The effect of extremely thermophilic biochar composite agent on the physical and chemical properties of pig manure aerobic composting; A is NH4 + -N content changes, B is NO3 during composting - - Changes in N content, C is NO2 during composting - - Changes in N content;

[0041] Figure 8 Effects of extremely thermophilic biochar composite bacteria on the physical and chemical properties of pig manure aerobic composting; A is the real-time emission of CH4 during composting, B is the real-time emission of N2O during composting, C is the cumulative emission of CH4 during composting, and D is the cumulative emission of N2O during composting. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The present invention relates to the following strains:

[0048] Parageobacillus thermoglucosidasiu BGSC 95A1, deposited in China Center for Type Culture Collection on July 16, 2020, with a deposit number of CCTCC NO: M2020324; Bacillus licheniformis ZR-1, deposited in China Center for Type Culture Collection on June 26, 2017, with a deposit number of CCTCC NO: M2017384;

[0049] Thermus thermophilus N1209 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 2025109; Geobacillus stearothermophillus L5 is recorded in the document "Screening of Extreme Thermophilic Bacteria and Their Application in Ultra-High Temperature Composting of Pig Carcasses" and is currently stored in the E402-80℃ refrigerator in the Fourth Comprehensive Building of Huazhong Agricultural University.

[0050] The present invention relates to a culture medium formula as follows:

[0051] LB liquid medium: 5.0 g tryptone, 10.0 g yeast extract, 10.0 g NaCl, 1000.0 mL distilled water, pH 7.0-7.2;

[0052] LB solid medium: 5.0 g tryptone, 10.0 g yeast extract, 10.0 g NaCl, 20.0 g agar, 1000.0 mL distilled water, pH 7.0-7.2;

[0053] Improved CYS medium: yeast extract 4.0 g, hydrolyzed casein 6.0 g, soluble starch 3.0 g, NaCl 8.0 g, MgCl2·6H2O 0.27 g, CaCl2 0.025 g, FeSO4·7H2O 0.01 g, trace elements 100 μL, distilled water 1000.0 mL, pH 7.0-7.2;

[0054] Trace element mother solution: Na2MoO4·2H2O 1.2g, VOSO4·xH2O 0.1g, MnCl2 0.5g, ZnSO4·7H2O 0.06g, CuSO4·5H2O 0.015g, CoCl2·6H2O 0.8g, NiCl·6H2O 0.02g, distilled water 100.0mL;

[0055] Screening medium A: peptone 20 g, NaCl 5 g, olive oil 20 g, K2HPO4 7.00 g, KH2PO4 3.00 g, MgSO4·7H2O 0.10 g, FeSO4·7H2O 0.05 g, distilled water 1000.0 mL, pH 7.0-7.2;

[0056] Screening medium B: peptone 20 g, starch 5 g, (NH4)2SO4 1 g, K2HPO4 1 g, MgSO4·7H2O 0.5 g, distilled water 1000.0 mL, pH 7.0-7.2;

[0057] Culture medium for preservation of bacterial strains: 5 g yeast extract, 10 g peptone, 10 g NaCl, 20 g agar, 1000.0 mL distilled water, sterilized at 121°C for 20 min;

[0058] Beef extract peptone solid medium: 3g beef extract, 10g peptone, 5g sodium chloride, 1000.0mL distilled water, 20g agar, sterilized at 121℃ for 20min;

[0059] VP test medium: 5 g glucose, 5 g peptone, 0.5 g potassium dihydrogen phosphate, 1000.0 mL distilled water, pH 7.0-7.2, sterilized at 121°C for 20 min;

[0060] Nitrate reduction reaction medium: peptone 10g, beef extract 10g, sodium chloride 10g, potassium nitrate 10g, distilled water 1000.0mL, pH 7.2-7.4, sterilized at 121℃ for 20min;

[0061] Indole test medium: peptone 10g, sodium chloride 5g, pH 7.2-7.6, distilled water 1000.0mL, sterilized at 121℃ for 20min;

[0062] Gelatin liquefaction test medium: peptone 0.5 g, gelatin 15 g, distilled water 100.0 mL, pH 7.0-7.2, sterilized at 121 °C for 20 min;

[0063] Hydrogen sulfide production test medium: beef extract 3g, yeast extract 3g, peptone 10g, FeSO40.2g, NaCl5g, Na2S2O3·5H2O, agar 12g, pH 7.0-7.2, distilled water 1000.0mL, sterilized at 121℃ for 20min;

[0064] Amylase enzyme production liquid medium: peptone 10.0g, glucose 1.0g, soluble starch 5.0g, KH2PO4 2.0g, MgSO4·7H2O 0.5g, CaCl2·2H2O 0.2g, pH 7.0-7.2, distilled water 1000.0mL;

[0065] The lipase production medium includes: (NH4)2SO41 g, MgSO4·7H2O 1 g, KH2PO41 g, glucose 5 g, peptone 30 g, rapeseed oil emulsion 12 mL, pH 7.0-7.2, distilled water 1000.0 mL;

[0066] The solution formula of the present invention for bacterial physiological and biochemical identification is as follows:

[0067] Iodine solution: iodine tablets 1g, potassium iodide 2g, distilled water 300mL;

[0068] Indole reagent: p-dimethylaminotolualdehyde 2 g, 95% ethanol 190 mL, distilled water 92.6 mL;

[0069] Other materials involved in the present invention are as follows:

[0070] Biochar: Corn straw biochar. No special requirements, purchased through the straw charcoal online store or other channels.

[0071] Example 1 Screening of Thermus thermophilus N1209

[0072] 1. In the clean bench, weigh 5g of animal carcass residue sample from the high-temperature pyrolysis processor of the experimental chicken farm of Huazhong Agricultural University, put it into 100mL sterile water containing glass beads, and shake it continuously for 30min to make a bacterial suspension. Aspirate 1mL of bacterial suspension, add it to a conical flask containing 100mL CYS liquid culture medium, and culture it in a shaker at 65℃ and 180r / min for 72h, and make 3 repeated samples. Take 1mL of 65℃ enriched bacterial solution, add it to a conical flask containing 100mL CYS liquid culture medium, and culture it in a shaker at 70℃ and 180r / min for 72h. Take 1mL of 70℃ enriched bacterial solution, add it to a conical flask containing 100mL CYS liquid culture medium, and culture it in a shaker at 75℃ and 180r / min for 72h. Take 1 mL of 75°C enriched bacterial solution and add it to a conical flask containing 100 mL of CYS liquid culture medium, and culture it in a shaking incubator at 80°C and 180 r / min for 72 h.

[0073] 2. Make a ten-fold dilution of the enriched culture solution, with each dilution being 10 -5 , 10 -6 , 10 -7 100 μL of the diluted bacterial solution was inoculated into screening medium A for culture. Three parallels were made for each dilution and cultured in a shaker at 75°C for 48 h.

[0074] 3. Make a standard ten-fold dilution of the bacterial solution cultured in screening medium A, with each dilution being 10 -5 , 10 -6 , 10 -7 100 μL of the diluted bacterial solution was inoculated into screening medium B for culture. Three parallels were made for each dilution and cultured in a shaker at 75°C for 48 h.

[0075] 4. Make a standard ten-fold dilution of the bacterial solution enriched in screening medium B, with each dilution being 10 -5 , 10 -6 , 10 -7100 μL of the diluted bacterial solution was inoculated into LB solid medium for culture, and three parallels were made for each dilution, and cultured in a constant temperature incubator at 75°C for 48 h.

[0076] 5. The single colony grown was streaked several times, and the purified strain was inoculated into the preservation medium and stored in glycerol at -20°C. It was named strain N1209.

[0077] Example 2 Determination of physiological and biochemical characteristics of strain N1209

[0078] The physiological and biochemical characteristics of strain N1209 were determined as follows:

[0079] 1. Take a glass slide and wipe it dry with gauze. Mark one side of the glass slide with a marker to determine the location of the smear. Bake the smeared area over the flame of an alcohol lamp to remove grease. Hold the bacterial solution test tube with your left hand and open the lid of the bacterial solution test tube about 5 cm near the flame of the alcohol lamp. Bake the inoculation loop in your right hand over the flame to sterilize it. After cooling, take a loop of bacterial solution from the bacterial solution test tube with the inoculation loop and smear it on the glass slide that has been degreased. The diameter is about 2 mm. Finally, bake the inoculation loop over the flame to sterilize it. Let the smear dry naturally in the air. With the bacterial film facing up, fix it with the flame 2-3 times. Add ammonium oxalate crystal violet solution to the fixed smear and stain it for 1 minute. Rinse the staining solution on the smear slowly with water and dry it with absorbent paper. Add a drop of staining solution, stain for 1 minute, and wash with water. Remove the residual water with absorbent paper, add 95% ethanol to decolorize until the effluent is no longer purple, and wash with water. Add safranin solution to re-stain for 5 minutes, wash with water, and dry. Finally, for microscopic examination, move the stained slide to an upright microscope for observation in different fields of view, and select the appropriate field of view for photography.

[0080] 2. Collect fresh bacterial culture solution after 24 hours of culture, rinse the sample with 1×PBS (pH 7.0) 2-3 times, centrifuge at 5000rpm for 3 minutes, and remove the supernatant. Add 1mL of 2.5% glutaraldehyde solution, shake gently to mix thoroughly, resuspend the bacterial solution, and fix at 4℃ for 4 hours. Centrifuge at 5000rpm for 3 minutes and remove the supernatant, add 500μL PBS to rinse the sample. Then dehydrate with ethanol solution of gradient concentration (including five concentrations of 20%, 40%, 60%, 80%, and 100%), treat each concentration for 10 minutes, and centrifuge at 5000rpm for 3 minutes. Vacuum freeze-drying, first pre-cool at ultra-low temperature for 4 hours, and then vacuum freeze-dry for 36 hours. Observe the bacterial powder by field emission scanning electron microscopy after gold spraying.

[0081] 3. Add 400mL LB liquid culture medium to a 1L conical flask, seal the conical flask with a sealing film, and sterilize at 121℃ for 20min. After it cools to room temperature, inoculate the strain into the conical flask at 1% addition, and place the conical flask in a 65℃ constant temperature water bath shaker for culture at a shaking frequency of 200r / min. During the fermentation process, samples were collected every 2h, and finally at OD 600 Under the same conditions, the absorbance of bacterial solution was measured as OD 600 With culture time as the horizontal axis and the culture time as the vertical axis, a mixed fermentation growth curve was drawn.

[0082] 4. Place the bacterial solution in a water bath at 70℃, 75℃, 80℃, 85℃, and 90℃ for 2 hours, then spread the bacterial solution on LB solid culture medium for culture. If colonies grow, it is positive, otherwise it is negative.

[0083] 5. Take 100 μL of Thermus thermophilus strain and inoculate it into a 50mL sterile centrifuge tube containing 10mL V-P test culture medium. Place it in a 65℃ constant temperature water bath shaker and culture it for 24 hours. Then take an appropriate amount of culture medium and add an equal amount of 40% KOH solution. Then add 0.5-1mg creatine and shake it vigorously. If red appears in the solution within 2-10 minutes, it is a positive VP reaction.

[0084] 6. Take 100 μL of Thermus thermophilus strain and inoculate it into a 50mL sterile centrifuge tube containing 10mL indole test medium. Place it in a 65℃ constant temperature water bath shaker and culture it for 24 hours. Then take an appropriate amount of Thermus thermophilus culture solution and add 1mL of ether. Shake it thoroughly to dissolve indole in the ether. Let it stand for a while. When the ether layer is suspended on the upper layer of the culture solution, slowly add 10 drops of indole reagent along the wall of the tube. The formation of a red ring between the ether and the culture is a positive reaction.

[0085] 7. Inoculate the thermophilic bacteria into the gelatin medium by puncture inoculation method, place it in a constant temperature incubator at 28℃ for 48-64 hours, and observe the liquefaction of the gelatin. Liquefaction is positive, and non-liquefaction is negative.

[0086] 8. Inoculate 100 μL of Thermus thermophilus into 10 mL of nitrate reduction medium, take another test tube with sterile medium as control, and place it in a 65°C constant temperature water bath shaker for 48 hours. Take 3 mL of culture medium, add one drop of Griss A and B reagents, shake well, and observe the color change of the culture medium. Immediately or after 10 minutes, if the culture medium turns red, brown, or orange, it means that nitrate is reduced to nitrite, which is a positive reaction. If the above colors do not appear, add another drop of diphenylamine reagent. If the culture medium is red, it is a positive reaction; if it is blue, it is a negative reaction.

[0087] 9. Pick the colonies to be tested on the solid culture medium, place them on a clean glass slide, add a few drops of 3% hydrogen peroxide solution, and observe the results. If bubbles are generated within half a minute, it is positive, and if no bubbles are generated, it is negative.

[0088] 10. Inoculate the strain to be tested into a medium containing sodium thiosulfate, culture at 65°C for 48 hours, use sterilized tweezers to place the filter paper strip containing lead acetate into a test tube and fix it with a rubber stopper. The lower end of the filter paper should be close to the liquid surface of the culture medium but not soaked, and the filter paper should be suspended in the test tube. Observe whether lead sulfide is produced. If the filter paper turns black, it is positive, and if it does not change color, it is negative.

[0089] The Gram staining results of strain N1209 are shown in Figure 1 , growth curve see Figure 2 The results of physiological and biochemical characteristics are shown in Table 1-Table 2. Figure 1 It can be seen from Table 1 that the bacteria is Gram-stained red, and it can be determined that the bacteria is Gram-negative. From Tables 1 and 2, it can be seen that the results of the VP test, nitrate reduction test, and catalase test of Thermus thermophilus are positive; the results of the indole test, gelatin liquefaction test, and hydrogen sulfide test are negative; and the bacteria can withstand a high temperature of 90°C. From Table 2, it can be seen that Thermus thermophilus enters the logarithmic phase at the 6th hour of culture and enters the plateau phase at the 22nd hour of culture.

[0090] Table 1 High temperature resistance test of Thermus thermophilus

[0091]

[0092] Note: "+" in the table indicates positive or available, "-" indicates negative or available

[0093] Table 2 Physiological and biochemical tests of Thermus thermophilus

[0094]

[0095]

[0096] Example 3 Identification of strain 16S rDNA

[0097] 1. Use TIAN GEN bacterial DNA extraction kit to extract strain DNA. The specific steps are as follows:

[0098] (1) Take 5 mL of bacterial culture medium and centrifuge at 1000 r / min for 1 min. Aspirate the supernatant as much as possible, add 200 μL of buffer GA to the bacterial pellet, and shake until the bacteria are completely suspended;

[0099] (2) Add 20 μL of Proteinase K solution to the tube, mix well, add 220 μL of buffer GB, shake for 15 seconds, and place in a 70°C water bath for 10 minutes. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap;

[0100] (3) Add 220 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitates may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap. Add the solution and flocculent precipitates to an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12000 r / min for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0101] (4) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 r / min for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube. Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 r / min for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube. Repeat once.

[0102] (5) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000r / min for 2min, and discard the waste liquid. Place the adsorption column at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material. Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200μL of elution buffer TE to the middle part of the adsorption membrane, place at room temperature for 2-5min, centrifuge at 12000r / min for 2min, and collect the solution in a centrifuge tube. Measure the DNA concentration using Nanodrop 2000 and store the DNA at -20℃ for later use.

[0103] 2. Use primers 27F and 1492R to perform PCR amplification on the extracted strain DNA. The PCR reaction system is: template DNA: 2μL, PCR master mix: 10μL, 27F: 1μL, 1492R: 1μL, double distilled water: 21μL; PCR reaction program is: 94℃ pre-denaturation for 5min, 94℃ denaturation for 45s, 53℃ annealing for 45s, 72℃ extension for 45s, reaction for 35 cycles, 72℃ supplementary extension for 10min.

[0104] The PCR product was separated by 1% agarose electrophoresis, and the band of about 1500bp was cut off, and the DNA was recovered using the TIAN GEN DNA fragment rapid purification and recovery kit. Sequencing was performed at Beijing Qingke Biotechnology Co., Ltd. The 16S rDNA sequence of the strain was put into NCBI's BLAST for homology analysis. Analysis found that strain N1209 was highly homologous to Thermus thermophilus, and strain N1209 was identified as Thermusthermophilus. The strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC M 2025109.

[0105] Example 4 Preparation of extremely thermophilic biochar composite bacterial agent

[0106] 1. Take 100 μL of each bacterial solution of Geobacillus thermoglucosidase BGSC 95A1, Bacillus licheniformis ZR-1, Thermus thermophilus N1209, and Geobacillus stearothermophilus L5 in sterile LB liquid culture medium, shake and culture at 65°C and 180-200 rpm for 24 h±2 h, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant and collect the precipitate, wash it 3 times with sterile PBS, and resuspend it with 10 mL of sterile water to obtain Geobacillus thermoglucosidase, Bacillus licheniformis, Thermus thermophilus, and Geobacillus stearothermophilus agents. The OD of each agent is 600 The values ​​are all between 1.60-1.80.

[0107] 2. Inoculate the thermoglucosidase Geobacillus, licheniformis, Thermus thermophilus, and Geobacillus stearothermophilus in pairs on LB solid medium, invert and culture in a 65℃ constant temperature incubator for 24h±2h, and observe the colony growth at the intersection of the two strains. Figure 3 .from Figure 3 It can be seen that the four strains can grow in contact with each other, and no sterile zone is formed, indicating that there is no inhibitory effect between the strains.

[0108] 3. Thermoglucosidase Geobacillus, licheniformis, Thermus thermophilus, and Geobacillus stearothermophilus were mixed and cultured in LB liquid medium. The inoculation amount was L9 (3 at 4 factors and 3 levels) 4 ) Orthogonal test was used to determine 9 different combinations, as shown in Tables 3 and 4 below. The mixed bacterial suspension was diluted 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10-7 , 10 -8 Use a 1mL sterile pipette tip to draw 10 -6 , 10 -7 and 10 -8 100 μL of each diluted bacterial suspension was added to LB solid culture medium, and the bacterial solution was evenly spread with a sterile glass coating rod. The suspension was placed in a 65°C constant temperature incubator and inverted for 24 hours. Three replicates were set for each dilution concentration. The number of viable bacteria was calculated according to the following formula:

[0109] N=∑C / (n1+0.1n2+0.01n3)d

[0110] Where:

[0111] N——number of viable bacteria in the sample;

[0112] ∑C——Total number of colonies on the plate;

[0113] n1——10 -6 Number of plates with dilution multiple;

[0114] n2——10 -7 Number of plates with dilution multiple;

[0115] n3——10 -8 Number of plates with dilution multiple;

[0116] d——10 -6 Dilution factor:

[0117] Table 3 Levels of different bacterial strains added in mixed fermentation medium

[0118]

[0119] Table 4L9(3 4 )Orthogonal test table

[0120]

[0121]

[0122] The number of fermentation bacteria in each combination was counted, and the results are shown in Table 5. Through range analysis, the range of A (Bacillus licheniformis), B (GeoBacillus thermoglucosidase), C (GeoBacillus stearothermophilus), and D (Thermus thermophilus) is ranked as A>D>B>C, so the amount of Bacillus licheniformis added in mixed fermentation is the main factor. The ranges of GeoBacillus thermoglucosidase and Thermus thermophilus are 36.87 and 38.85, respectively, which are not much different, indicating that the two have a certain equivalence. Therefore, the optimal treatment is T8, that is, A3B2C1D3 (the addition of Bacillus licheniformis is 0.75%, the addition of GeoBacillus thermoglucosidase is 0.5%, the addition of GeoBacillus stearothermophilus is 0.25%, and the addition of Thermus thermophilus is 0.75%), and the number of viable bacteria can reach 5.65×10 9 cfu / mL, and this combination was used as the experimental bacterial agent combination scheme.

[0123] Table 5 Results of orthogonal test of strains

[0124]

[0125]

[0126] 4. Take 75 μL of Bacillus licheniformis, 50 μL of Geobacillus thermoglucosidase, 25 μL of Geobacillus stearothermophilus, and 75 μL of Thermus thermophilus and add them to sterile LB liquid culture medium, and culture at 65°C and 180-200 rpm for 24 h±2 h. Centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant and retain the precipitate, wash with sterile PBS buffer 2-3 times, and resuspend with 10 mL of sterile water to obtain an extremely thermophilic composite bacterial agent.

[0127] Weigh 50g corn straw biochar and add it to 1L LB liquid culture medium, adjust the pH to 7.2-7.4, and sterilize at 121℃ for 30min. After cooling, take 5mL of extreme thermophilic composite bacteria and add it to the sterile LB liquid culture medium, and culture it in a water bath shaker at 65℃ and 120-160rpm for one week. Centrifuge at 4℃ and 5000rpm for 10min, discard the supernatant, wash with sterile PBS buffer 2-3 times, collect the precipitate, and obtain the extreme thermophilic biochar composite bacteria. Observe by scanning electron microscopy. Figure 4 , A is a scanning electron microscope image of corn straw biochar, and B is a scanning electron microscope image of biochar loaded with extreme thermophilic composite bacteria (i.e., extreme thermophilic biochar composite bacteria). Figure 4 It can be seen that the bacteria are well loaded on the biochar, indicating that the extremely thermophilic biochar composite agent was successfully prepared.

[0128] Example 5 Determination of enzyme activity of single bacterial agent and composite bacterial agent

[0129] 1. Preparation of crude enzyme solution:

[0130] (1) 100 μL of thermoglucosidase Geobacillus, licheniformis, Thermus thermophilus, Geobacillus stearothermophilus and composite bacteria were inoculated into 10 mL of sterile LB liquid medium, and the shaking culture conditions were 65°C and the rotation speed was 180-200 rpm for about 24 hours until the bacterial OD 600 The value is 1.6-1.8. The culture solution of each bacterial agent was centrifuged at 4°C and 3000 rpm for 5 minutes, and resuspended with 10 mL of sterile water to obtain the thermoglucosidase Geobacillus seed solution, the licheniformis seed solution, the thermophilic Thermus seed solution, the stearothermophilic Geobacillus seed solution and the composite bacterial agent seed solution.

[0131] 100 μL of the seed liquid of thermoglucosidase Geobacillus, Bacillus licheniformis, Thermus thermophilus, Geobacillus stearothermophilus and the composite bacterial agent were inoculated into 10 mL of the amylase enzyme production liquid medium and the lipase enzyme production medium, respectively, and cultured at 65°C for 24 hours to obtain the fermentation liquid. Each fermentation liquid was centrifuged at 4°C and 10000 rpm for 10 minutes, and the supernatant was taken to obtain the crude enzyme liquid.

[0132] 2. The specific steps of amylase activity determination are as follows: add 5mL of 1% starch substrate solution to a clean 50mL centrifuge tube, preheat in a water bath at 65℃ for 10min, and then add 1mL of thermoglucosidase Geobacillus crude enzyme solution, Bacillus licheniformis crude enzyme solution, Thermus thermophilus crude enzyme solution, Geobacillus stearothermophilus crude enzyme solution and composite bacterial agent crude enzyme solution respectively. React in a water bath at 65℃ for 5min, add 5mL of 0.1mol / L sulfuric acid solution to terminate the reaction, take 0.5mL of reaction solution and mix with 5mL of dilute iodine solution, and measure the absorbance at 620nm. Add 1mL of deionized water instead of crude enzyme solution and perform the above operation as a control, replace the reaction solution with 0.5mL of deionized water and mix with iodine solution as a reference solution, and repeat three times in each group. Calculate amylase activity according to the following formula.

[0133] Enzyme activity (U) = (R0-R) × 50 × D / R0

[0134] Where R0 and R represent the absorbance of the control and reaction solution respectively, and D is the dilution factor of the enzyme. D is adjusted so that (R0-R) / R0 is between 0.2-0.7.

[0135] The results are as follows Figure 5As shown in A, the amylase activity of the composite bacterial agent group (T8) was the highest at 16.567±0.236U / mL, and the amylase activities of Bacillus licheniformis (X2), Thermus thermophilus (D9), Bacillus stearothermophilus (A22) and Geobacillus thermoglucosidase (PT) were 15.352±0.527U / mL, 13.708±2.78U / mL, 14.037±1.063U / mL and 11.835±0.131U / mL, respectively, indicating that the composite bacterial agent has a stronger ability to decompose starch than a single bacterial agent.

[0136] 3. The specific steps of protease activity determination are as follows: weigh 0.1000g±0.0002g of L-tyrosine dried to constant weight at 105℃, dissolve it with 1mol / L hydrochloric acid solution and dilute to 100mL, which is 1mg / mL tyrosine solution; take 10mL of 1mg / mL tyrosine solution, dilute to 100mL with 0.1mol / L hydrochloric acid solution, which is 100μg / mL L-tyrosine standard solution. Take 0, 1, 2, 3, 4, 5mL of tyrosine standard solution and add distilled water to 10mL. Take 1mL of each of the above solutions, add 5mL of 42.4g / L sodium carbonate solution and 1mL of Folin reagent solution, shake well, keep warm at 40℃ for 20min, cool, measure the absorbance at 680nm, and draw a standard curve. Using the regression equation, calculate the amount of tyrosine when the absorbance is 1, which is the absorption constant K. The K value should be in the range of 95 to 100.

[0137] Take 1 mL of each of the crude enzyme solution of thermoglucosidase Geobacillus, crude enzyme solution of Bacillus licheniformis, crude enzyme solution of Thermus thermophilus, crude enzyme solution of Geobacillus stearothermophilus and crude enzyme solution of composite bacterial agent, place in a 40℃ water bath for preheating for 2 minutes, then add 1 mL of 10.0 g / L casein solution that has been preheated in the same way, keep warm for 10 minutes, and immediately add 2 mL of 65.4 g / L trichloroacetic acid to terminate the reaction, continue to keep warm in a water bath for 20 minutes, and then filter. Take 1 mL of each filtrate, add 5 mL of 42.4 g / L sodium carbonate, 1 mL of Folin reagent solution, shake well, keep warm at 40℃ for 20 minutes, and measure the absorbance after cooling. The blank test method is the same as above, except that the enzyme is inactivated by adding trichloroacetic acid solution before adding casein. The protease activity is calculated using the standard curve and the following formula.

[0138] Enzyme activity (U) = A1 × 4 × n / 10

[0139] Wherein, A1 is the enzyme activity of the final dilution of the sample obtained from the standard curve, U / mL; n is the dilution multiple of the sample; 4 is the total volume of the reaction reagent, mL; 10 is the reaction time 10 min.

[0140] Protease activity standard curve Figure 5 As shown in B, the protease activity of each bacterial agent is as follows Figure 5 As shown in C, it can be seen from the figure that the protease activity of the composite bacterial agent group (T8) is the highest at 4.133±1.049U / mL, and the protease activities of Bacillus licheniformis (X2), Thermus thermophilus (D9), Bacillus stearothermophilus (A22) and Geobacillus thermoglucosidase (PT) are 0.144±0.130U / mL, 1.689±1.550U / mL, 0.901±0.367U / mL, and 0.914±0.045U / mL, respectively. The protease activity of the composite bacterial agent group is significantly higher than that of the single bacterial agent group, indicating that the composite bacterial agent has better ability to decompose protein than the single bacterial agent.

[0141] 4. The specific steps for lipase activity determination are as follows: weigh 0.08346 g of p-nitrophenol, dissolve it in 95% ethanol, make up to 100 mL with distilled water, take 0, 2.5, 5, 7.5, 10, 15, 20, 30, 40, and 50 μL of p-nitrophenol standard solution into each test tube, add 50 mmol / L Tris-HCl to make up to 2 mL, add 1 mL of 95% ethanol solution to each test tube, mix well, measure the absorbance at 410 nm, and draw a standard curve of p-nitrophenol solution concentration.

[0142] Take 1mL 10mmol / L p-nitrophenol palmitate and 9mL 50mmol / L Tris-HCl and mix them evenly, then add 1% Triton-X and 0.1% gum arabic powder to prepare a mixed solution. Take 1mL of crude enzyme solution of Geobacillus thermoglucosidase, crude enzyme solution of Bacillus licheniformis, crude enzyme solution of Thermus thermophilus, crude enzyme solution of Geobacillus stearothermophilus and crude enzyme solution of composite bacterial agent and add them to 9mL of the mixed solution, finally add 95% ethanol solution to terminate the reaction, and measure the absorbance at 410nm. Take the inactivated enzyme solution after 95℃ water bath as the control, and calculate the lipase activity according to the standard curve and the following formula.

[0143] Enzyme activity (U) = ([A1-A0] × K + C0) × V1 × n / (V2 × t)

[0144] Wherein, A1 is the absorbance of the sample; A0 is the blank absorbance of the corresponding sample; K is the slope of the p-nitrophenol standard curve; C0 is the intercept of the p-nitrophenol standard curve; n is the dilution factor; V1 is the volume of the reaction solution, mL; V2 is the volume of the enzyme solution, mL; t is the reaction time.

[0145] The standard curve of p-nitrophenol is as follows Figure 5 As shown in D, the lipase activity of each bacterial agent is as follows Figure 5As shown in E, it can be seen that the protease activity of the composite bacterial agent group (T8) is the highest at 33.267±6.069U / mL, and the protease activities of Bacillus licheniformis (X2), Thermus thermophilus (D9), Bacillus stearothermophilus (A22) and Geobacillus thermoglucosidase (PT) are 20.555±3.376U / mL, 9.076±4.802U / mL, 15.496±7.364U / mL and 18.220±3.169U / mL, respectively. The lipase activity of the composite bacterial agent group is higher than that of the single bacterial agent group, indicating that the composite bacterial agent has a better ability to decompose fat than the single bacterial agent.

[0146] Example 6 Application of extremely thermophilic biochar composite bacterial agent in aerobic composting test of pig manure

[0147] The aerobic composting test of pig manure was divided into a control group (CK) and a group with extreme thermophilic biochar composite bacteria (HTC), with three parallel replicates in each group. First, wheat straw was processed into 3-5 cm length with a hay cutter, and then pig manure and wheat straw were evenly mixed in a mass ratio of 4:1, with 4.8t of pig manure and 1.2t of wheat straw. The completely mixed pile was divided into 6 piles, each with a size of 1t. The extreme thermophilic biochar composite bacteria (the actual inoculation amount of biochar was 0.1% (w / w), and the actual inoculation amount of the composite bacteria was 0.01% (v / w)) was inoculated into the HTC group, and the CK group was inoculated with an equal volume of clean water. The initial moisture content of the pile was adjusted to about 60% ± 5%, the initial C / N ratio was adjusted to 20-25, and the pile was turned by a forklift every 3 days. From 8:00 to 10:00 every morning, the temperature of five different positions of the pile was measured using a thermometer. Samples were collected from different positions of the pile on the 0th, 1st, 3rd, 6th, 9th, 12th, 15th, 18th, 24th, and 30th days of composting to test the physical and chemical properties of the pile.

[0148] The temperature change of the pile is as follows Figure 6 As shown in A, after the composting started, the temperature rose rapidly, with the HTC group reaching the highest temperature of 81.2°C on the 6th day of composting, and the CK group reaching the highest temperature of 59.13°C on the 3rd day of composting. The high temperature period (>50°C) of the CK group and the HTC group lasted for 7 days and 23 days, respectively, meeting the temperature requirements of ultra-high temperature composting. The experiment proved that inoculation of extreme thermophilic biochar composite bacteria has a good effect in increasing the composting temperature.

[0149] The seed germination index (GI) of the pile is as follows: Figure 6As shown in B, the GI values ​​of each treatment generally showed a trend of first increasing and then stabilizing. During most of the composting period, the GI value of the HTC group was higher than that of the CK group. At the end of composting, the GI of the CK group was 97.86%±0.45, and the GI of the HTC group was 120.85%±0.563, indicating that inoculation of extreme thermophilic biochar composite bacteria has a good effect in promoting the maturity of the pile and reducing the toxicity of the pile.

[0150] The C / N changes of the pile are as follows: Figure 6 As shown in C, the changes in total nitrogen in the pile are as follows Figure 6 As shown in D, the C / N change showed a downward trend. This was due to the rapid degradation of organic matter, and the carbon decomposition rate was faster than the nitrogen loss in the same period, which led to a decrease in C / N. After the composting was completed, the TN content of the HTC group was higher than that of the CK group, indicating that the inoculation of composite bacteria can improve nitrogen retention.

[0151] NH4 in the composting process + -N content changes as Figure 7 As shown in A, during the composting process, NO3 - -N content changes as Figure 7 As shown in B, during the composting process, NO 2- -N content changes as Figure 7 As shown in C. During the composting process, the HTC pile NH4 + -N content was significantly higher than that of the CK group, indicating that the microbial activity in the HTC pile was stronger and a large amount of nitrogen-containing organic matter was converted into NH4 + -N. NO in the pile 2- -N content first increased rapidly, then decreased and stabilized. 2- -N content is significantly lower than that of CK. This may be due to NO2 - -N is unstable and is more likely to transform at high temperatures. - -N content showed a trend of continuous increase, and NO3 - -N was significantly higher than that of the CK group (P<0.05). It is generally believed that nitrification in aerobic composting is nitrogen oxidation (NH4 + -N→NO2 - / NO3 - ) is the main process. NO3 in compost - -N content is considered an important indicator of compost maturity. 3- -N formation is also thought to contribute to nitrogen retention in compost. This suggests that the extreme thermophilic bacterial inoculant has a high efficacy in promoting compost maturation and nitrogen sequestration.

[0152] The real-time emission of CH4 during composting is as follows: Figure 8As shown in A, the real-time emission of N2O during composting is as follows Figure 8 As shown in B, the cumulative CH4 emissions during the composting process are as follows Figure 8 As shown in C, the cumulative N2O emissions during the composting process are as follows Figure 8 As shown in D. It can be seen from the figure that during most of the composting time, the real-time CH4 and N2O emissions of the HTC group were lower than those of the CK group. After the composting was completed, compared with the CK group, the cumulative CH4 emissions of the HTC group decreased by 42.88%, and the cumulative N2O emissions decreased by 46.22%, indicating that the addition of the extreme thermophilic biochar composite agent has a significant effect in reducing CH4 and N2O emissions.

[0153] In summary, adding extremely thermophilic biochar composite bacteria to the pig manure composting process can not only increase the decomposition rate of organic matter in the pile, promote the maturity of the compost, and improve the nitrogen fixation effect, but also reduce the emission of CH4 and N2O.

[0154] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Thermus thermophilus N1209, deposited in China Center for Type Culture Collection (CCTCC) on January 13, 2025, with a deposit number of CCTCC M 2025109.

2. Use of the thermophilic bacterium N1209 according to claim 1 in preparing aerobic composting fermentation agent for pig manure.

3. An extremely thermophilic biochar composite bacterial agent, characterized in that: The preparation method of the extremely thermophilic biochar composite bacterial agent comprises the following steps: (1) mixing a bacterial agent of Geobacillus thermoglucosidase BGSC 95A1, a bacterial agent of Bacillus licheniformis ZR-1, a bacterial agent of Thermus thermophilus N1209 according to claim 1, and a bacterial agent of Geobacillus stearothermophilus L5 to obtain an extremely thermophilic composite bacterial agent; (2) The extremely thermophilic composite bacterial agent is inoculated into a biochar culture medium, cultured, centrifuged, and resuspended to obtain the extremely thermophilic biochar composite bacterial agent.

4. The extremely thermophilic biochar composite bacterial agent according to claim 3, characterized in that: The OD of the inoculum of the thermoglucosidase Geobacillus sp. BGSC 95A1 was 600 The value is 1.6-1.8, the OD of the inoculum of Bacillus licheniformis ZR-1 600 The value is 1.6-1.8, and the OD of the inoculum of Thermus thermophilus N1209 is 600 The value is 1.6-1.8, and the OD of the inoculum of the thermophilic Bacillus L5 600 The value is 1.6-1.

8.

5. The extremely thermophilic biochar composite bacterial agent according to claim 3, characterized in that: The volume ratio of the bacterial agent of the thermoglucosidase Geobacillus subtilis BGSC 95A1, the bacterial agent of the licheniformis ZR-1, the bacterial agent of the thermophilic bacterium N1209 and the bacterial agent of the stearothermophilus Geobacillus subtilis L5 when mixed is 2:3:3:

1.

6. The extremely thermophilic biochar composite bacterial agent according to claim 3, characterized in that: The culture conditions are: 65° C. and 120-160 rpm for 1 week.

7. Use of the extremely thermophilic biochar composite bacterial agent according to any one of claims 3 to 6 in aerobic composting of pig manure.

8. Use of the extremely thermophilic biochar composite bacterial agent according to any one of claims 3 to 6 in aerobic composting to accelerate the decomposition of pig manure.

9. Use of the extremely thermophilic biochar composite bacterial agent according to any one of claims 3 to 6 in reducing pollutant emissions during aerobic composting of pig manure.

10. Use of the extremely thermophilic biochar composite bacterial agent according to any one of claims 3 to 6 in increasing the nitrogen retention in the aerobic composting process of pig manure.

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