An extremely thermophilic biochar composite microbial agent and application thereof in pig manure aerobic composting

Abstract

The application discloses an extremely thermophilic biochar composite microbial agent and application thereof in pig manure aerobic composting, and belongs to the technical field of microorganisms. The application discloses a strain of thermophilic thermus N1209, and further discloses an extremely thermophilic biochar composite microbial agent, which comprises thermophilic glucosidase geobacillus BGSC 95A1, bacillus licheniformis ZR-1, thermus N1209 and thermophilic adipose geobacillus L5. The extremely thermophilic biochar composite microbial agent has the characteristics of high temperature resistance, higher amylase, protease and lipase activity compared with single microbial agent, shows synergistic advantages, can significantly promote the composting of pig manure, has advantages in the aspects of nitrogen element reservation and CH4 and N2O emission reduction in the pig manure composting process, and provides a new method for the treatment of poultry breeding waste.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an extreme thermophilic biochar compound agent and its application in aerobic composting of pig manure. Background Technology

[0002] The continuous expansion of pig farming has not only met market demand for livestock products but also generated a large amount of pig manure. Statistics show that approximately 3.8 billion tons of pig manure are produced annually, but the comprehensive utilization rate is less than 60%. This large amount of livestock manure contains various pathogenic microorganisms, heavy metals, and other harmful substances. Untreated and indiscriminate discharge not only causes serious environmental pollution but also produces large amounts of greenhouse gases, exacerbating the greenhouse effect. Therefore, accelerating the development of animal husbandry requires accelerating the reduction, harmless treatment, and resource utilization of livestock manure.

[0003] Aerobic composting is a commonly used method for treating livestock and poultry manure. It not only reduces the accumulation of manure from intensive livestock farming but also converts it into organic fertilizer for agricultural use. Compared to other methods, composting is low-cost, simple to operate, and has low environmental requirements. However, traditional composting methods have significant drawbacks: the process releases large amounts of odorous and greenhouse gases, fermentation is incomplete, and the resulting fertilizer has low efficiency, making it outdated and unable to keep pace with modern agricultural development.

[0004] Inoculating the compost pile with fast-reproducing and highly active microorganisms can not only shorten the composting cycle but also help retain nutrients. Studies have shown that inoculating with compound microbial agents is more advantageous in promoting compost maturation and improving fertilizer efficiency compared to inoculating with a single microbial agent. Inoculating compost with compound microbial agents can accelerate temperature rise, prolong the duration of high temperatures, reduce compost toxicity, improve compost maturity and product fertilizer efficiency, and shorten the maturation cycle. However, the application effect of existing microbial agents in the composting process is not ideal due to factors such as the weak heat resistance of microorganisms, slow reproduction rate, and low productivity. Summary of the Invention

[0005] The purpose of this invention is to provide an extreme thermophilic biochar composite microbial agent and its application in aerobic composting of pig manure, thereby solving the problems existing in the prior art. The extreme thermophilic biochar composite microbial agent of this invention has high-temperature resistance and, compared with single microbial agents, produces amylase, protease, and lipase with higher activity, exhibiting synergistic advantages. It can significantly promote the composting of pig manure and has advantages in nitrogen retention and CH4 and N2O emission reduction during pig manure composting, providing a new method for the treatment of livestock and poultry breeding waste.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a strain of thermophilus N1209, which is deposited at the China Center for Type Culture Collection on January 13, 2025, with accession number CCTCC M 2025109.

[0008] The present invention also provides the application of the above-mentioned thermophilic bacterium N1209 in the preparation of aerobic composting fermentation agent for pig manure.

[0009] This invention also provides an extreme thermophilic biochar composite microbial agent, the preparation method of which includes the following steps:

[0010] (1) Mix the bacterial agent of Bacillus thermoglucosidase BGSC 95A1, the bacterial agent of Bacillus licheniformis ZR-1, the bacterial agent of the above-mentioned thermophilic thermobacter N1209 and the bacterial agent of thermophilic steatobacter L5 to obtain an extreme thermophilic compound bacterial agent.

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

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

[0013] In this invention, the thermoglucosidase-producing Bacillus cereus BGSC 95A1, Bacillus licheniformis ZR-1, thermophilic thermobacter N1209, and thermophilic steatobacter L5 all possess heat-resistant properties and can stably secrete substances such as amylase, protease, and lipase. There are no antagonistic or inhibitory effects among the bacterial species; in fact, mixing them can produce a synergistic effect, enhancing the activity of the secretions.

[0014] In this invention, after the various strains in the extreme thermophilic biochar composite microbial agent are co-cultured with biochar, the microbial cells can be loaded onto the surface and internal pores of the biochar, providing effective protection for the survival of the microbial cells and promoting their growth and metabolism.

[0015] Preferably, the OD of the Bacillus cereus BGSC 95A1 inoculum is... 600 The OD value of the Bacillus licheniformis ZR-1 inoculum is 1.6-1.8. 600 The OD value of the thermophilic bacterium N1209 inoculum is 1.6-1.8.600 The OD value of the *Bacillus stearothermophilus* L5 inoculum is 1.6-1.8. 600 The value is 1.6-1.8.

[0016] In this invention, the preparation methods for the bacterial agents of *Bacillus thermoglucosidase* BGSC 95A1, *Bacillus licheniformis* ZR-1, *Thermophilic thermophilic bacteria* N1209, and *Bacillus stearothermophilus* L5 are as follows:

[0017] The thermoglucosidase-producing Bacillus BGSC 95A1, Bacillus licheniformis ZR-1, Thermophilic thermophilic bacteria N1209, and Thermophilic steatobacterium L5 were cultured in LB liquid medium at 65°C and 180-200 rpm for 24 h ± 2 h with shaking. After centrifugation, the cultures were resuspended in sterile water to obtain Bacillus BGSC 95A1, Bacillus licheniformis ZR-1, Thermophilic thermophilic bacteria N1209, and Thermophilic steatobacterium L5 bacterial inoculants.

[0018] Preferably, the volume ratio of the inoculum of *Bacillus thermoglucosidase* BGSC 95A1, the inoculum of *Bacillus licheniformis* ZR-1, the inoculum of *Thermophilic bacterium* N1209, and the inoculum of *Bacillus stearothermophilus* L5 is 2:3:3:1.

[0019] In this invention, the mixing ratio of each strain affects the growth rate of the bacterial cells, and thus the reproductive and metabolic capacity of the bacterial cells. When the bacterial agents of *Bacillus thermoglucosidase* BGSC 95A1, *Bacillus licheniformis* ZR-1, *Thermophilic bacterium* N1209, and *Bacillus stearothermophilus* L5 are mixed in a ratio of 2:3:3:1, the growth state of each bacterial strain is optimal.

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

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

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

[0023] In this invention, when the thermophilic compound microbial agent is used for inoculation, the volume-to-mass ratio of the thermophilic compound microbial 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 the application of the above-mentioned extreme thermophilic biochar composite agent in aerobic composting of pig manure.

[0026] The present invention also provides the application of the above-mentioned extreme thermophilic biochar composite microbial agent in aerobic composting to accelerate the decomposition of pig manure.

[0027] The present invention also provides the application of the above-mentioned extreme thermophilic biochar composite agent in reducing pollutant emissions during the aerobic composting process of pig manure.

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

[0029] The present invention also provides the application of the above-mentioned extreme thermophilic biochar composite agent in improving the nitrogen retention of pig manure composting process.

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

[0031] This invention prepares an extreme thermophilic compound bacterial agent by combining thermoglucosidase Bacillus, Bacillus licheniformis, thermophilic thermophilic bacteria, and thermophilic steatobacterium in a ratio of 2:3:3:1. Compared with single bacterial agents, the extreme thermophilic compound bacterial agent has a synergistic effect among the strains, resulting in higher activity of amylase, protease, and lipase.

[0032] The extreme thermophilic biochar composite microbial agent prepared by this invention can significantly promote the decomposition of pig manure and reduce its toxicity. It has advantages in nitrogen retention and CH4 and N2O emission reduction during the pig manure composting process, providing a new method for the treatment of poultry and livestock manure and breeding waste. It can effectively promote the reduction, harmlessness and resource utilization of livestock and poultry manure and drive the development of animal husbandry. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 Gram staining results for Thermophilic bacterium;

[0035] Figure 2 The results of the growth curve experiment of thermophilic bacteria;

[0036] Figure 3The results of pairwise antagonism tests of the strains are shown; in the figure, A22 represents Bacillus stearothermophilus, D9 represents Bacillus thermophilus, PT represents Bacillus thermoglucosidase, and X2 represents Bacillus licheniformis.

[0037] Figure 4 The images are scanning electron microscope (SEM) images of the extreme thermophilic biochar composite microbial agent; where A is a scanning electron microscope image of sterilized biochar, and B is a scanning electron microscope image of biochar loaded with the extreme thermophilic composite microbial agent.

[0038] Figure 5 The graph shows the enzyme activity detection results of enzymes produced by single strains and extreme thermophilic compound agents; where A is the amylase activity assay result, B is the protease activity standard curve, C is the protease activity assay result, D is the lipase activity standard curve, and E is the lipase activity assay result.

[0039] Figure 6 The effects of extreme thermophilic biochar compound inoculant on the physicochemical properties of aerobic pig manure compost piles were investigated. Among them, A represents the temperature change of the pile during the composting process, B represents the seed germination index of the pile, C represents the C / N ratio change of the pile, and D represents the total nitrogen change of the pile.

[0040] Figure 7 The effect of extreme thermophilic biochar compound inoculant on the physicochemical properties of aerobic pig manure compost; where A represents NH4+ during composting. + The change in -N content, where B represents NO3 during composting. - Changes in -N content, C represents NO2 during composting. - Changes in -N content;

[0041] Figure 8 The study investigated the effects of an extreme thermophilic biochar compound inoculant on the physicochemical properties of aerobic compost piles of pig manure. A represents the real-time CH4 emission during composting, B represents the real-time N2O emission during composting, C represents the cumulative CH4 emission during composting, and D represents the cumulative N2O emission during composting. Detailed Implementation

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

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0048] Parageobacillus thermoglucosidasiu BGSC 95A1 is deposited at the China Center for Type Culture Collection (CCTCC) on July 16, 2020, with accession number CCTCC NO: M2020324; Bacillus licheniformis ZR-1 is deposited at the China Center for Type Culture Collection (CCTCC) on June 26, 2017, with accession number CCTCC NO: M2017384.

[0049] Thermus thermophilus N1209 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025109; Geobacillus stearothermophillus L5 is described in the literature "Screening of extreme thermophiles and their application in ultra-high temperature composting of pig carcasses" and is currently deposited in refrigerator E402-80℃ in the Fourth Comprehensive Building of Huazhong Agricultural University.

[0050] The present invention relates to the following culture medium formulation:

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

[0054] Trace element stock 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: 20g peptone, 5g NaCl, 20g olive oil, 7.00g K2HPO4, 3.00g KH2PO4, 0.10g MgSO4·7H2O, 0.05g FeSO4·7H2O, 1000.0mL distilled water, pH 7.0-7.2;

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

[0057] Culture medium for preserving microorganisms: 5g yeast extract, 10g peptone, 10g NaCl, 20g agar, 1000.0mL distilled water, sterilized at 121℃ for 20min;

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

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

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

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

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

[0063] Hydrogen sulfide production test medium: 3g beef extract, 3g yeast extract, 10g peptone, 0.2g FeSO4, 5g NaCl, Na2S2O3·5H2O, 12g agar, pH 7.0-7.2, 1000.0mL distilled water, sterilized at 121℃ for 20min;

[0064] Amylase production liquid culture 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 consists of: (NH4)2SO4 1 g, MgSO4·7H2O 1 g, KH2PO4 1 g, glucose 5 g, peptone 30 g, rapeseed oil emulsion 12 mL, pH 7.0-7.2, and distilled water 1000.0 mL.

[0066] The present invention relates to a solution formulation for the physiological and biochemical identification of bacteria, as follows:

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

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

[0069] Other materials involved in this invention include:

[0070] Biochar: Corn stalk biochar. No special requirements; purchase through online straw charcoal retailers or other channels.

[0071] Example 1: Screening of Thermus thermophilus N1209

[0072] 1. In a clean bench, weigh 5g of animal carcass residue sample processed by a high-temperature pyrolysis processor from the experimental chicken farm of Huazhong Agricultural University. Place it in 100mL of sterile water containing glass beads and shake continuously for 30min to prepare a bacterial suspension. Pipette 1mL of the bacterial suspension into an Erlenmeyer flask containing 100mL of CYS liquid medium and incubate at 65℃ and 180r / min for 72h. Perform three replicates. Take 1mL of the 65℃ enriched bacterial solution and add it to an Erlenmeyer flask containing 100mL of CYS liquid medium. Incubate at 70℃ and 180r / min for 72h. Take 1mL of the 70℃ enriched bacterial solution and add it to an Erlenmeyer flask containing 100mL of CYS liquid medium. Incubate at 75℃ and 180r / min for 72h. Take 1 mL of the 75℃ enriched bacterial solution and add it to an Erlenmeyer flask containing 100 mL of CYS liquid culture medium. Incubate at 80℃ and 180 r / min for 72 h.

[0073] 2. Dilute the enriched bacterial culture solution tenfold to the standard, taking 10 times the amount of each dilution. -5 10 -6 10 -7 100 μL of the bacterial culture was inoculated into selection medium A and cultured in triplicate for each dilution. The culture was carried out at 75°C in a shaker for 48 h.

[0074] 3. Dilute the bacterial suspension cultured in screening medium A tenfold to the standard, and take 10 times dilutions from each dilution. -5 10 -6 10 -7 100 μL of the bacterial culture was inoculated into selection medium B and cultured in triplicate for each dilution. The culture was carried out at 75°C in a shaker for 48 h.

[0075] 4. Dilute the bacterial suspension enriched in screening medium B to a standard 10-fold dilution, taking 10 μL of each dilution. -5 10 -6 10 -7100 μL of the bacterial culture was inoculated into LB solid medium and cultured in triplicate for each dilution. The culture was incubated at 75°C for 48 h.

[0076] 5. Streak the resulting single colonies multiple times, then inoculate the purified strain into preservation medium and store in glycerol at -20°C. Name it strain N1209.

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

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

[0079] 1. Wipe the glass slide dry with gauze. Mark one side of the slide with a marker pen to determine the location for smearing bacteria. Heat the smeared area over an alcohol lamp flame to remove grease. Hold the bacterial culture tube in your left hand and open the cap about 5cm from the alcohol lamp flame. Sterilize the inoculation loop in your right hand by flaming it over the flame. After cooling, use the inoculation loop to take a loopful of bacterial culture from the bacterial culture tube and smear it onto the degreased glass slide, approximately 2mm in diameter. Finally, sterilize the inoculation loop by flaming it over the flame. Allow the smear to air dry naturally. With the bacterial film facing up, fix it by passing it over the flame 2-3 times. Add ammonium oxalate crystal violet solution to the fixed smear and stain for 1 minute. Slowly rinse the staining solution off the smear with water and blot dry with absorbent paper. Add one drop of staining solution and stain for 1 minute, then rinse with water. Blot away any remaining water with absorbent paper, add 95% ethanol to decolorize until the effluent is no longer purple, then rinse with water. Counterstain with safranin solution for 5 minutes, rinse with water, and air dry. Finally, the stained slide was moved to an upright microscope for observation in different fields of view, and photographs were taken from the appropriate field of view.

[0080] 2. Collect fresh bacterial culture after 24 hours of culture. Wash the sample 2-3 times with 1×PBS (pH 7.0), centrifuge at 5000 rpm for 3 min, and discard the supernatant. Add 1 mL of 2.5% glutaraldehyde solution, gently shake to mix thoroughly, resuspend the bacterial culture, and fix at 4℃ for 4 h. After centrifugation at 5000 rpm for 3 min, discard the supernatant and add 500 μL of PBS to wash the sample. Then dehydrate with a gradient concentration of ethanol solution (including five concentrations: 20%, 40%, 60%, 80%, and 100%), treating for 10 min at each concentration, centrifuging at 5000 rpm for 3 min. Vacuum freeze-dry, first pre-cooling at ultra-low temperature for 4 h, then vacuum freeze-drying for 36 h. Observe the bacterial powder after gold sputtering using a field emission scanning electron microscope.

[0081] 3. Add 400 mL of LB liquid culture medium to a 1 L Erlenmeyer flask, seal the flask with sealing film, and sterilize at 121 °C for 20 min. After cooling to room temperature, inoculate the bacterial strain into the Erlenmeyer flask at a 1% addition rate, and place the flask in a 65 °C constant temperature water bath shaker with a shaking frequency of 200 rpm. During fermentation, take samples every 2 hours, and finally measure the OD value. 600 Under the specified conditions, the absorbance of the bacterial suspension was measured, expressed as OD. 600 Plot the mixed fermentation growth curve with the vertical axis as the ordinate and the cultivation time as the horizontal axis.

[0082] 4. Incubate the bacterial suspension in water baths at 70℃, 75℃, 80℃, 85℃, and 90℃ for 2 hours respectively. Then spread the bacterial suspension on LB solid medium for further incubation. The presence of colonies indicates a positive result, while the absence of colonies indicates a negative result.

[0083] 5. Take 100 μL of thermophilic thermobacter bacterium inoculation into a 50 mL sterile centrifuge tube containing 10 mL of LV-P test medium, and incubate in a 65℃ constant temperature water bath shaker for 24 h. Take an appropriate amount of culture medium, add an equal amount of 40% KOH solution, and then add 0.5-1 mg of creatine. Shake vigorously. If a red color appears in the solution within 2-10 min, it is a positive VP reaction.

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

[0085] 7. Inoculate *Thermophilus thermophilus* into gelatin medium using the puncture inoculation method, and incubate at 28°C for 48-64 hours. Observe the liquefaction of the gelatin. Liquefaction indicates a positive result, while non-liquefaction indicates a negative result.

[0086] 8. Inoculate 100 μL of *Thermophilus thermophilus* into 10 mL of nitrate-reducing medium. Use a sterile medium as a control in another test tube. Incubate both in a 65°C water bath with a shaker for 48 hours. Take 3 mL of the culture medium and add one drop each of Griss A and B reagents. Shake well and observe the color change. Immediately or after 10 minutes, if the culture medium turns red, brown, or orange, it indicates that nitrate has been reduced to nitrite, which is a positive reaction. If no such color appears, add one drop of diphenylamine reagent. A red color indicates a positive reaction; a blue color indicates a negative reaction.

[0087] 9. Pick up the colonies to be tested from the solid culture medium, place them on a clean glass slide, add a few drops of 3% hydrogen peroxide solution, and observe the results. A positive result is indicated by the production of bubbles within half a minute, while a negative result is indicated by the absence of bubbles.

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

[0089] Gram staining results of strain N1209 are shown below. Figure 1 Growth curves are shown Figure 2 The results of the physiological and biochemical characteristic measurements are shown in Tables 1-2. Figure 1 The results show that the bacterium stains red with Gram, confirming it as a Gram-negative bacterium. Tables 1 and 2 show that *Thermophilic thermophilic bacteria* is positive for the VP test, nitrate reduction test, and catalase test; negative for the indole test, gelatin liquefaction test, and hydrogen sulfide test; and it can withstand temperatures up to 90℃. Table 2 shows that *Thermophilic thermophilic bacteria* enters the logarithmic phase at 6 hours of culture and reaches the plateau phase at 22 hours.

[0090] Table 1. Thermophilic bacteria heat resistance test

[0091]

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

[0093] Table 2 Physiological and biochemical tests of thermophilic bacteria

[0094]

[0095]

[0096] Example 3: Identification of 16S rDNA in strains

[0097] 1. Extract bacterial DNA using the TIAN GEN bacterial DNA extraction kit. The specific steps are as follows:

[0098] (1) Take 5 mL of bacterial culture medium, 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 bacterial cells 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 s, place in a 70 ℃ water bath for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0100] (3) Add 220 μL of anhydrous ethanol and shake thoroughly for 15 s. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. Add the solution and flocculent precipitate to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12000 r / min for 30 s, discard the waste liquid, and place the adsorption column CB3 into 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 into a collection tube. Add 600 μL of wash buffer PW to the adsorption column CB3, centrifuge at 12000 r / min for 30 s, discard the waste liquid, and place the adsorption column CB3 into a collection tube. Repeat once.

[0102] (5) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid. Let the adsorption column sit at room temperature for several minutes to thoroughly dry any remaining wash solution in the adsorption material. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE dropwise to the center of the adsorption membrane, let it sit at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, and collect the solution in the centrifuge tube. Determine the DNA concentration using a Nanodrop 2000, and store the DNA at -20℃ for later use.

[0103] 2. The extracted bacterial DNA was amplified by PCR using primers 27F and 1492R. The PCR reaction system was as follows: template DNA: 2 μL, PCR master mix: 10 μL, 27F: 1 μL, 1492R: 1 μL, double-distilled water: 21 μL; the PCR reaction program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 53℃ annealing for 45 s, 72℃ extension for 45 s, 35 cycles, and a supplementary extension at 72℃ for 10 min.

[0104] The PCR products were separated by 1% agarose gel electrophoresis, and a band of approximately 1500 bp was excised. 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 analyzed for homology using NCBI BLAST. Analysis revealed that strain N1209 was highly homologous to *Thermus thermophilus*, and strain N1209 was identified as *Thermus thermophilus*. This strain was deposited at the China Center for Type Culture Collection on January 13, 2025, with accession number CCTCC M 2025109.

[0105] Example 4: Preparation of an Extremely Thermophilic Biochar Composite Inoculum

[0106] 1. Take 100 μL each of the bacterial suspensions of *Bacillus thermoglucosidase* BGSC 95A1, *Bacillus licheniformis* ZR-1, *Thermophilic bacterium* N1209, and *Bacillus thermophilicus* L5 into sterile LB broth. Incubate at 65℃ and 180-200 rpm with shaking for 24 h ± 2 h. Centrifuge at 4℃ and 3000 rpm for 5 min, discard the supernatant, collect the precipitate, wash three times with sterile PBS, and resuspend in 10 mL of sterile water to obtain the bacterial suspensions of *Bacillus thermoglucosidase*, *Bacillus licheniformis*, *Thermophilic bacterium*, and *Bacillus thermophilicus*. The OD values ​​of each bacterial suspension are... 600 The values ​​are all between 1.60 and 1.80.

[0107] 2. Bacillus thermoglucosidase inoculants, Bacillus licheniformis inoculant, Bacillus thermophilus inoculant, and Bacillus thermophilus steatosis inoculant were streaked in pairs onto LB solid medium and incubated upside down in a 65℃ incubator for 24h ± 2h. The colony growth at the crossover point between the two strains was observed. Figure 3 .from Figure 3 It can be seen that the four strains can grow in contact with each other without the formation of sterile zones, indicating that there is no inhibitory effect among the strains.

[0108] 3. Take *Bacillus thermophilus* inoculum, *Bacillus licheniformis* inoculum, *Thermophilic thermophilus* inoculum, and *Bacillus stearothermophilus* inoculum and mix them in LB liquid medium for culture. The inoculum size is based on a 4-factor, 3-level L9(3) inoculum. 4 Nine different combinations were determined by orthogonal experiments, as shown in Tables 3 and 4 below. The mixed bacterial suspension was successively diluted 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10-7 10 -8 Shake to thoroughly mix the bacterial culture. Use a 1mL sterile pipette tip to aspirate 10 mL of the culture. -6 10 -7 and 10 -8 100 μL of each diluted bacterial suspension was added to LB solid medium, and the bacterial suspension was spread evenly using a sterile glass spreader. The media were then incubated upside down at 65°C for 24 hours. Three replicates were performed for each dilution. The viable count was calculated using the following formula:

[0109] N=∑C / (n1+0.1n2+0.01n3)d

[0110] In the formula:

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

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

[0113] n1——10 -6 Dilution factor and number of plates;

[0114] n2——10 -7 Dilution factor and number of plates;

[0115] n3——10 -8 Dilution factor and number of plates;

[0116] d——10 -6 Dilution factor;

[0117] Table 3. Levels of different strains of inoculant addition in mixed fermentation medium

[0118]

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

[0120]

[0121]

[0122] The fermentation cell counts for each combination were statistically analyzed, and the results are shown in Table 5. Range analysis showed that the ranges for A (Bacillus licheniformis), B (Bacillus thermoglucosidase), C (Bacillus thermophilus), and D (Thermophilic bacteria) were ranked as A>D>B>C. Therefore, the amount of Bacillus licheniformis added is the main factor in mixed fermentation. The ranges for Bacillus thermoglucosidase and Thermophilic bacteria were 36.87 and 38.85, respectively, which are not significantly different, indicating a certain degree of equivalence between the two. Therefore, the optimal treatment is T8, i.e., A3B2C1D3 (Bacillus licheniformis addition of 0.75%, Bacillus thermoglucosidase addition of 0.5%, Bacillus thermophilus addition of 0.25%, and Thermophilic bacteria addition of 0.75%), achieving a viable cell count of 5.65 × 10⁻⁶. 9 cfu / mL, this combination was used as the test bacterial agent formulation scheme.

[0123] Table 5 Results of orthogonal experiments on strains

[0124]

[0125]

[0126] 4. Take 75 μL of Bacillus licheniformis inoculum, 50 μL of Bacillus thermoglucosidase inoculum, 25 μL of Bacillus stearothermophilus inoculum, and 75 μL of Bacillus thermophilus inoculum and add them to sterile LB liquid medium. Incubate at 65℃ and 180-200 rpm for 24 h ± 2 h. Centrifuge at 4℃ and 3000 rpm for 5 min, discard the supernatant and retain the precipitate. Wash 2-3 times with sterile PBS buffer and resuspend in 10 mL of sterile water to obtain the extreme thermophilic compound inoculum.

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

[0128] Example 5 Enzyme Activity Determination of Single and Compound Microbial Agents

[0129] 1. Preparation of crude enzyme solution:

[0130] (1) Inoculate 100 μL of each of the following bacterial agents into 10 mL of sterile LB liquid medium: Bacillus thermophilus, Bacillus licheniformis, Bacillus thermophilus, Bacillus stearothermophilus, and a compound agent. Incubate at 65 °C with shaking at 180-200 rpm for approximately 24 h until bacterial OD occurs. 600 The value was 1.6-1.8. The culture solutions of each bacterial agent were centrifuged at 3000 rpm for 5 min at 4℃, and resuspended in 10 mL of sterile water to obtain seed solutions of *Bacillus thermoglucosidase*, *Bacillus licheniformis*, *Thermophilic thermophilic bacteria*, *Bacillus steatophilus*, and a compound bacterial agent.

[0131] 100 μL of seed culture of *Bacillus thermoglucosidase*, *Bacillus licheniformis*, *Thermophilic Bacillus*, *Bacillus steatophilus*, and a compound inoculum were inoculated into 10 mL of amylase-producing liquid medium and lipase-producing liquid medium, respectively, and cultured at 65 °C for 24 h to obtain fermentation broth. Each fermentation broth was centrifuged at 4 °C and 10,000 rpm for 10 min, and the supernatant was collected to obtain crude enzyme solution.

[0132] 2. The specific steps for amylase activity determination are as follows: Add 5 mL of 1% starch substrate solution to a clean 50 mL centrifuge tube, preheat in a water bath at 65℃ for 10 min, then add 1 mL of crude enzyme solution from *Bacillus cereus* (thermosinophilic glucosidase), *Bacillus licheniformis*, *Thermophilic thermophilic bacteria*, *Bacillus steatophilic*, and a compound bacterial agent. React in a water bath at 65℃ for 5 min, then add 5 mL of 0.1 mol / L sulfuric acid solution to terminate the reaction. Mix 0.5 mL of the reaction solution with 5 mL of dilute iodine solution and measure the absorbance at 620 nm. A solution with 1 mL of deionized water added instead of the crude enzyme solution and subjected to the same procedure is used as a control. A solution with 0.5 mL of deionized water added instead of the reaction solution and mixed with iodine solution is used as a reference solution. Each group is repeated three times. Calculate the amylase activity according to the following formula.

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

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

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

[0136] 3. The specific steps for protease activity determination are as follows: Weigh 0.1000g ± 0.0002g of L-tyrosine dried to constant weight at 105℃, dissolve it in 1mol / L hydrochloric acid solution, and bring the volume to 100mL to obtain a 1mg / mL tyrosine solution; take 10mL of the 1mg / mL tyrosine solution and bring the volume to 100mL with 0.1mol / L hydrochloric acid solution to obtain a 100μg / mL L-tyrosine standard solution. Take 0, 1, 2, 3, 4, and 5mL of the tyrosine standard solution and make up to 10mL with distilled water. 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, incubate at 40℃ for 20min for color development, cool, and measure the absorbance at 680nm to plot a standard curve. Using the regression equation, the amount of tyrosine when the absorbance is 1 is calculated, which is the absorbance constant K. The value of K should be in the range of 95 to 100.

[0137] Take 1 mL each of the following crude enzyme solutions: *Bacillus thuringiensis*, *Bacillus licheniformis*, *Thermophilic Bacillus*, *Bacillus stearothermophilus*, and the compound bacterial agent. Preheat each solution in a 40°C water bath for 2 minutes. Then add 1 mL of a similarly preheated 10.0 g / L casein solution to each solution. Incubate precisely for 10 minutes. Immediately add 2 mL of 65.4 g / L trichloroacetic acid to each solution to terminate the reaction. Continue incubation in a water bath for 20 minutes, then filter. Take 1 mL of each filtrate, add 5 mL of 42.4 g / L sodium carbonate and 1 mL of Folin-Ciocalteu reagent solution, shake well, and incubate at 40°C for 20 minutes. After cooling, measure the absorbance. The blank experiment is performed using the same method, except that trichloroacetic acid solution is added before adding casein to inactivate the enzyme. Calculate the protease activity using a standard curve and the following formula.

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

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

[0140] standard curve of protease activity as follows Figure 5 As shown in B, the protease activities of each bacterial agent are as follows: Figure 5 As shown in Figure C, the composite microbial agent group (T8) exhibited the highest protease activity at 4.133±1.049 U / mL. The protease activities of *Bacillus licheniformis* (X2), *Thermophilic bacillus* (D9), *Bacillus stearothermophilus* (A22), and *Bacillus thermoglucosidase* (PT) were 0.144±0.130 U / mL, 1.689±1.550 U / mL, 0.901±0.367 U / mL, and 0.914±0.045 U / mL, respectively. The protease activity of the composite microbial agent group was significantly higher than that of the single microbial agent group, indicating that the composite microbial agent has a superior protein-degrading ability compared to the single microbial 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, and dilute 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 dilute to 2 mL, add 1 mL of 95% ethanol solution to each test tube, mix well, and measure the absorbance at 410 nm. Plot a standard curve of p-nitrophenol solution concentration.

[0142] Mix 1 mL of 10 mmol / L p-nitrophenol palmitate and 9 mL of 50 mmol / L Tris-HCl thoroughly, then add 1% Triton-X and 0.1% gum arabic powder to prepare a mixture. Add 1 mL each of the following crude enzyme solutions: *Bacillus cereus* (thermosinophilic), *Bacillus licheniformis*, *Thermophilic thermophilic*, *Bacillus steatophilic*, and a compound bacterial agent, to 9 mL of the mixture. Finally, add 95% ethanol solution to terminate the reaction and measure the absorbance at 410 nm. Using the inactivated enzyme solution after a 95°C water bath as a control, 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] In the formula, 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 reaction liquid volume, mL; V2 is the enzyme solution volume, mL; and t is the reaction time.

[0145] p-Nitrophenol standard curve as follows Figure 5 As shown in D, the lipase activities of each bacterial agent are as follows: Figure 5As shown in Figure E, the compound bacterial agent group (T8) exhibited the highest protease activity at 33.267±6.069 U / mL. The protease activities of *Bacillus licheniformis* (X2), *Thermophilic Bacillus* (D9), *Bacillus stearothermophilus* (A22), and *Bacillus thermoglucosidase* (PT) were 20.555±3.376 U / mL, 9.076±4.802 U / mL, 15.496±7.364 U / mL, and 18.220±3.169 U / mL, respectively. The higher lipase activity in the compound bacterial agent group compared to the single bacterial agent group indicates that the compound bacterial agent has a superior ability to decompose fats compared to the single bacterial agent.

[0146] Example 6: Application of Extremely Thermophilic Biochar Composite Inoculant in Aerobic Composting Experiment of Pig Manure

[0147] The aerobic composting experiment of pig manure was divided into a control group (CK) and a group with added thermophilic biochar compound microbial agent (HTC), with three parallel replicates in each group. First, wheat straw was chopped into 3-5 cm lengths using a chaff cutter. Then, pig manure and wheat straw were mixed evenly at a mass ratio of 4:1, with 4.8 t of pig manure and 1.2 t of wheat straw. The completely mixed pile was divided into 6 piles, each 1 t in size. The thermophilic biochar compound microbial agent (actual inoculum amount of biochar was 0.1% (w / w), and the actual inoculum amount of compound microbial agent was 0.01% (v / w)) was inoculated into the HTC group, while the CK group was inoculated with an equal volume of clean water. The initial moisture content of the piles was adjusted to approximately 60% ± 5%, and the initial C / N ratio was adjusted to 20-25. The piles were turned over every 3 days using a forklift. Every morning from 8:00 to 10:00, the temperature at five different locations on the compost pile was measured using a thermometer. Samples were collected from different locations on days 0, 1, 3, 6, 9, 12, 15, 18, 24, and 30 of the composting process to test the physicochemical properties of the compost pile.

[0148] Temperature changes in the reactor body are as follows Figure 6 As shown in Figure A, the temperature rose rapidly after composting began. The HTC group reached its highest temperature of 81.2℃ on day 6 of composting, while the CK group reached its highest temperature of 59.13℃ on day 3. The duration of the high-temperature period (>50℃) for the CK and HTC groups was 7 days and 23 days, respectively, meeting the temperature requirements for ultra-high temperature composting. The experiment demonstrates that inoculation with extreme thermophilic biochar composite inoculant has a good effect on increasing the composting temperature.

[0149] Seed germination index (GI) of the heap, such as Figure 6As shown in Figure 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 value of the CK group was 97.86% ± 0.45, and the GI value of the HTC group was 120.85% ± 0.563. This indicates that inoculation with the extreme thermophilic biochar compound agent has a good effect on promoting composting and reducing compost toxicity.

[0150] The C / N ratio of the stack is as follows Figure 6 As shown in C, the variation of total nitrogen in the reactor body is as follows: Figure 6 As shown in Figure D, the C / N ratio shows a decreasing trend. This is because the rapid degradation of organic matter leads to faster carbon decomposition than nitrogen loss during the same period, resulting in a decrease in the C / N ratio. After composting, the TN content in the HTC group was higher than that in the CK group, indicating that inoculation with the compound microbial agent can improve nitrogen retention.

[0151] NH4 during composting + The change in -N content is as follows Figure 7 As shown in A, NO3 during composting - The change in -N content is as follows Figure 7 As shown in B, NO during composting 2- The change in -N content is as follows Figure 7 As shown in C. During the composting process, HTC's NH4+ content in the compost pile... + The -N content was significantly higher than that of the CK group, indicating that the microbial activity within the HTC reactor was stronger, and a large amount of nitrogen-containing organic matter was converted into NH4. + -N. NO in the heap 2- -N content initially rose rapidly, then decreased and stabilized; NO in the HTC group 2- -N content was significantly lower than CK. This may be due to NO2 - The instability of -N makes it more prone to transformation at high temperatures. Throughout the composting process, NO3... - -N content showed a continuous increasing trend, with NO3 in the HTC group - -N was significantly higher in the control group than in the control group (P<0.05). It is generally believed that nitrification in aerobic composting is primarily nitrogen oxidation (NH4+). + -N→NO2 - / NO3 - The main process of NO3 in composting. - -N content is considered an important indicator of compost maturity. 3- The formation of -N is also believed to contribute to nitrogen retention in compost. This indicates that the extreme thermophilic compound microbial agent has high efficacy in promoting compost maturation and nitrogen fixation.

[0152] Real-time CH4 emissions during composting, such as Figure 8As shown in A, the real-time N2O emissions during composting are 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 Figure D, the real-time CH4 and N2O emissions of the HTC group were lower than those of the CK group for most of the composting process. At the end of composting, compared to 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 compound inoculant had a significant effect on reducing CH4 and N2O emissions.

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

[0154] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of thermophilus N1209, deposited at the China Center for Type Culture Collection (CCTCC) on January 13, 2025, with accession number CCTCC M 2025109.

2. The application of the thermophilic bacterium N1209 as described in claim 1 in the preparation of aerobic composting fermentation agent for pig manure.

3. An extreme thermophilic biochar compound microbial agent, characterized in that, The preparation method of the extreme thermophilic biochar composite microbial agent includes the following steps: (1) The bacterial agent of Parageobacillus thermoglucosidasius BGSC 95A1, the bacterial agent of Bacillus licheniformis ZR-1, the bacterial agent of thermophilic thermobacter N1209 as described in claim 1 and the bacterial agent of thermophilic lipid Bacillus L5 are mixed to obtain an extreme thermophilic compound bacterial agent; (2) The extreme thermophilic compound bacterial agent was inoculated into a biochar culture medium, cultured, centrifuged, and resuspended to obtain the extreme thermophilic biochar compound bacterial agent; The preservation number of the thermoglucosidase Bacillus cereus BGSC 95A1 is CCTCC NO: M2020324; the preservation number of the Bacillus licheniformis ZR-1 is CCTCC NO: M 2017384. The OD of the Bacillus cereus BGSC 95A1 inoculum was... 600 The OD value of the Bacillus licheniformis ZR-1 inoculum is 1.6-1.

8. 600 The OD value of the thermophilic bacterium N1209 inoculum is 1.6-1.

8. 600 The OD value of the *Bacillus stearothermophilus* L5 inoculum is 1.6-1.

8. 600 The value is 1.6-1.8; The volume ratio of the inoculum of *Bacillus thermoglucosidase* BGSC 95A1, the inoculum of *Bacillus licheniformis* ZR-1, the inoculum of *Thermophilic bacterium* N1209, and the inoculum of *Bacillus stearothermophilus* L5 is 2:3:3:

1.

4. The extreme thermophilic biochar composite microbial agent according to claim 3, characterized in that, The culture conditions were 65℃ and 120-160 rpm for 1 week.

5. The application of the extreme thermophilic biochar composite microbial agent as described in claim 3 or 4 in aerobic composting of pig manure.

6. The application of the extreme thermophilic biochar composite microbial agent as described in claim 3 or 4 in aerobic composting to accelerate the decomposition of pig manure.

7. The application of the extreme thermophilic biochar composite microbial agent as described in claim 3 or 4 in reducing pollutant emissions during the aerobic composting process of pig manure.

8. The application of the extreme thermophilic biochar composite microbial agent as described in claim 3 or 4 in improving the nitrogen retention of pig manure compost during the aerobic composting process.

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