Thermophilic bacteria strain MY-1, and a biochar-based microbial inoculant and application thereof

Abstract

The application provides a strain of thermophilic bacteria MY-1, a biochar-based microbial inoculant and application of the strain, the strain of thermophilic bacteria MY-1 is preserved in China Typical Culture Collection Center (CCTCC for short) on November 11, 2021, the address is Wuhan University in Wuhan, and the preservation number is CCTCC NO: M 20211405; the strain of thermophilic bacteria MY-1 has extreme thermophilic property and can proliferate rapidly in a solid-state fermentation medium with a water content of 50-70%; in the biochar-based microbial inoculant, the effective viable bacterial count of the strain of thermophilic bacteria MY-1 is 8x10 9 ~1.5x10 10 cfu / g; when the microbial inoculant is applied to ultra-high temperature compost, the water content of compost raw materials is controlled to be between 65-70%, the composting time can be effectively shortened, the compost raw materials can be fully composted, meanwhile, the nitrogen retention in the compost product can be effectively improved, and the quality of the compost product is improved.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology application technology, specifically to a thermophilic bacterium MY-1 and its biochar-based inoculant and its application. Background Technology

[0002] Resource utilization of agricultural waste is an important direction for the green and high-quality development of agriculture and rural areas. Agricultural waste mainly includes crop straw, fruit tree branches, edible fungi residue, and livestock manure, with an annual total output exceeding 3.8 billion tons. Nearly 50% of this waste is not efficiently and comprehensively utilized, causing serious pollution to the agricultural and rural environment. Agricultural and forestry waste is high in organic matter and rich in nutrients, making it a high-quality raw material for producing organic fertilizer.

[0003] Aerobic composting is an important method for the resource utilization of agricultural waste. The composting temperature is generally between 55 and 65°C, with the high-temperature period lasting 5 to 7 days, and the composting time typically between 30 and 60 days. However, aerobic composting requires a large area, and the excessively long composting time leads to low site utilization and high costs. Therefore, providing a highly efficient composting microbial agent to improve composting efficiency is of great significance for increasing composting site utilization and reducing composting costs.

[0004] Extremely thermophilic bacteria have strong application potential in ultra-high temperature composting. By adding extremely thermophilic bacteria during aerobic composting, the compost temperature can be raised to over 80℃, reaching the level of ultra-high temperature composting. This accelerates the transformation of organic matter into humus, effectively shortens composting time, rapidly kills pathogenic microorganisms and insect eggs, and achieves full decomposition. However, if the high temperature is maintained for too long, the nitrogen loss rate in the raw materials will exceed 70%. This severe nitrogen loss affects the nutritional value of the compost product. In addition, the lost nitrogen is emitted into the atmosphere in the form of ammonia and nitrogen oxides, which not only wastes resources but also exacerbates the greenhouse effect and acid rain. If the high temperature duration is reduced, the raw materials will not be completely rendered harmless or fully decomposed, affecting product quality. Therefore, under the current technological level, ultra-high temperature composting presents a contradiction between the full decomposition and harmlessness of compost raw materials and the loss of organic nitrogen.

[0005] Therefore, providing a strain and agent that can effectively decompose compost raw materials and reduce nutrient loss in compost, as well as its application method, is of great significance for ultra-high temperature composting. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides a thermophilic bacterium MY-1, its biochar-based inoculant, and its applications. This thermophilic bacterium MY-1 exhibits extreme thermophilicity and can grow well at 70–90°C. It can rapidly proliferate in a solid-state fermentation medium with a moisture content of 50–70%. Applying the biochar-based inoculant prepared using this thermophilic bacterium MY-1 and the biochar-based solid-state fermentation medium to ultra-high temperature composting, while controlling the moisture content of the composting raw materials between 65–70%, effectively shortens the composting time, ensures thorough decomposition of the composting materials, and simultaneously increases the nitrogen retention in the compost product, thereby improving its quality.

[0007] The technical solution of the present invention is as follows:

[0008] A thermophilic bacterium, MY-1, was deposited on November 11, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, 430072, China, with accession number CCTCC NO: M 20211405. The strain is classified as Geobacillus sp.

[0009] Furthermore, the thermophilic bacterium MY-1 was screened from high-temperature compost samples of agricultural and forestry waste; this strain is extremely thermophilic and can grow well at 70-90℃; this thermophilic bacterium MY-1 has the ability to reproduce under low oxygen conditions and can proliferate rapidly in solid fermentation medium with a moisture content of 50-70%.

[0010] A biochar-based bacterial agent, wherein the effective viable count of thermophilic bacterium MY-1 in the agent is 8 × 10⁻⁶. 9 ~1.5×10 10 cfu / gram.

[0011] Furthermore, the above-mentioned biochar-based bacterial agent also includes a biochar-based solid-state fermentation culture medium, which is composed of biochar, rice bran, maifanite, glucose and soybean meal.

[0012] Furthermore, the preparation process of the above-mentioned biochar-based bacterial agent is as follows:

[0013] (1) Activation of strain: Take the slant preserved at 4℃, inoculate the strain onto beef extract peptone solid medium plate, and incubate in a constant temperature incubator at 70℃ for 24 hours, ready for use;

[0014] (2) Preparation of seed culture: Select a single colony on the plate in step (1) and inoculate it into a 500 ml Erlenmeyer flask containing 100 ml of beef extract peptone liquid culture medium. Incubate the inoculated Erlenmeyer flask in a 70℃ constant temperature water bath shaker for 24 hours to obtain the seed culture.

[0015] (3) Preparation of fermentation broth: The seed culture obtained in step (2) is inoculated into a sterilized fermenter at an inoculation rate of 5% for expansion; culture conditions: culture temperature 70℃, stirring speed 150 rpm, aeration ratio 0.5 cubic meters / (cubic meters·min), when the number of cells in the fermentation broth is ≥2×10 8 When the concentration of cfu / mL reaches a certain level, stop fermentation and obtain the fermentation broth.

[0016] (4) Preparation of biochar-based solid fermentation medium: Take dry biochar, rice bran, maifanite, glucose and soybean meal, and mix them evenly according to the dry weight ratio of biochar: maifanite: rice bran: glucose: soybean meal = 50:10:1:5:5 to obtain biochar-based solid fermentation medium.

[0017] (5) Preparation of biochar-based bacterial agent: The fermentation broth from step (3) and the biochar-based solid fermentation substrate from step (4) are mixed in a mass ratio of 2 to 8:100 to obtain a mixture; the water content of the mixture is adjusted to 50 to 70%, and then it is cultured at 70°C for 48 to 72 hours to obtain a solid fermentation product; the solid fermentation product is air-dried naturally or dried at 80 to 90°C until the water content is below 30% to obtain a biochar-based bacterial agent.

[0018] In the biochar-based microbial agent of the present invention, the fermentation broth of thermophilic bacterium MY-1 is mixed with a biochar-based solid fermentation medium and fermented. On the one hand, the biochar-based solid fermentation medium can provide the thermophilic bacterium MY-1 with the nutrients required for its propagation, enabling it to multiply rapidly and improve its colonization ability. On the other hand, the biochar-based solid fermentation medium can provide the thermophilic bacterium MY-1 with a space for reproduction, making the distribution of thermophilic bacterium MY-1 in the obtained biochar-based microbial agent more uniform. Thus, when the biochar-based microbial agent is used in composting, the thermophilic bacterium MY-1 can be more uniformly distributed in the compost raw materials, improving the uniformity of compost product quality and the consistency of quality between batches.

[0019] The above-mentioned biochar-based microbial agent is applied specifically in ultra-high temperature composting. The process involves forming a compost pile, introducing high-humidity air into the pile to maintain the moisture content of the compost materials between 65% and 70%. The thermophilic bacterium MY-1 rapidly multiplies in the compost materials, causing the pile temperature to rise rapidly. On the third day of composting, the pile temperature rises above 80°C, thus quickly and effectively killing pests and diseases in the pile. From the third to the ninth day of composting, the pile temperature remains above 80°C. This prolonged ultra-high temperature allows the compost materials to decompose quickly and fully, shortening the composting time. When the moisture content of the compost materials is between 65% and 70%, the nitrogen content in the compost product can be effectively increased, thus improving the quality of the compost product.

[0020] Furthermore, in the above applications, the composting material is a mixture of straw and manure, with a C / N ratio of 30:1 to 10:1. In the early stages of composting, the carbon-nitrogen ratio of the composting material is controlled to provide sufficient nutrition for the rapid proliferation of thermophilic bacteria MY-1, thereby causing the temperature of the compost pile to rise rapidly to above 80°C within 3 days.

[0021] Furthermore, in the above applications, introducing air with a humidity greater than 90% RH into the compost pile, so that the moisture content of the compost raw materials in the pile is between 65% and 70%, can effectively reduce the decomposition rate of organic matter, reduce ammonia release, and increase the nitrogen content in the compost product.

[0022] When biochar-based inoculants are used in ultra-high temperature composting, the moisture content of the composting raw materials is controlled, allowing water molecules to be adsorbed onto the surface of various components in the biochar-based solid fermentation medium. During the ultra-high temperature composting process, the adsorption effect prevents the rapid escape of gases and nitrogen loss, thus increasing the nitrogen content in the compost product. At the same time, this nitrogen can also serve as a nitrogen source to provide nutrients for the reproduction of thermophilic bacteria MY-1, thereby maintaining the temperature of the compost pile above 80°C for up to 7 days. This achieves rapid and harmless decomposition of the composting raw materials and rapid and thorough maturation, shortens the composting time, and improves the quality of the compost product.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The thermophilic bacterium MY-1 provided by the present invention is extremely thermophilic and can grow well between 70 and 90°C; the thermophilic bacterium MY-1 has the ability to reproduce under low oxygen conditions and can proliferate rapidly in a solid fermentation medium with a water content of 50 to 70%.

[0025] 2. In this invention, thermophilic bacterium MY-1 is prepared into a biochar-based inoculum by combining it with a biochar-based solid-state fermentation medium. The effective viable count of thermophilic bacterium MY-1 in this inoculum is 8 × 10⁻⁶. 9 ~1.5×10 10 CFU / g; This biochar-based inoculant is applied to ultra-high temperature composting, where the composting temperature reaches up to 94℃, and the compost body temperature remains above 80℃ for 5 to 7 days; it can effectively and quickly render compost raw materials harmless, accelerate the conversion of organic matter into humus, and thus enable the compost raw materials to fully decompose within a short period of time.

[0026] 3. In this invention, biochar-based inoculants are applied to ultra-high temperature composting. Air with a humidity greater than 90% RH is introduced into the compost pile to maintain the moisture content of the compost raw materials between 65% and 70%. Under the premise of ensuring full decomposition and harmlessness, the nitrogen content in the compost product can be effectively increased. This effectively overcomes the technical barrier between nitrogen loss caused by ultra-high temperature composting and full decomposition and harmlessness, and improves the quality of the compost product. Among them, the total nitrogen content in the compost product is increased by more than 70% compared with conventional compost (control group). Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a culture diagram of the strain in Example 1 under temperature conditions of 30–90°C. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0030] Example 1: Thermophilic Bacterial Strains

[0031] (1) Screening and purification:

[0032] Samples were taken from high-temperature composting of poplar bark waste. The sample was initially screened, then screened again, and purified using beef extract peptone medium (3.0 g beef extract, 10.0 g peptone, 5.0 g sodium chloride, 1.0 L water, pH 7.4–7.6) at 65 °C. The sample was then labeled as thermophilic bacterium MY-1.

[0033] (2) Identification

[0034] The thermophilic bacterium MY-1 screened in step (1) was subjected to molecular biological identification, and the 16S rRNA sequencing results were uploaded to GenBank for homology comparison to determine the species of the strain. The results showed that the sequence had a high similarity of 99.39% with the 16S rRNA gene sequence of Bacillus aeruginosa, confirming that thermophilic bacterium MY-1 belongs to Geobacillus sp. The gene sequence (1046 bp) of thermophilic bacterium MY-1 is shown in SEQ ID NO: 1.

[0035] The thermophilic bacterium MY-1 was deposited on November 11, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, 430072, China, accession number: CCTCC NO: M 20211405; the strain was classified as Geobacillus sp.

[0036] (3) Optimal growth temperature of the strain

[0037] The thermophilic bacterium MY-1 preserved in step (1) was activated at 65℃. After activation, it was inoculated onto beef extract peptone agar medium using the spread plating method, resulting in 21 samples. These were divided into seven groups of three samples each. The seven groups were then statically incubated at constant temperatures of 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, respectively. The growth of the strain was observed after 24 hours of incubation. The results are shown below. Figure 1 . Figure 1 In the table below, A represents the growth status of the strain at 30℃, B represents the growth status at 40℃, C represents the growth status at 50℃, D represents the growth status at 60℃, E represents the growth status at 70℃, F represents the growth status at 80℃, and G represents the growth status at 90℃. The growth results of the strain under different temperature conditions are recorded in Table 1. Where: "-" indicates no growth; "+" indicates slight growth; "++" indicates moderate growth; and "++++" indicates good growth.

[0038] Table 1 Effects of different temperatures on microbial growth

[0039] Incubation temperature (°C) 30 40 50 60 70 80 90 Growth status - - - ++ +++ +++ +++

[0040] As shown in Table 1, the thermophilic bacterium MY-1 can grow at temperatures ranging from 60 to 90°C, and grows well at temperatures ranging from 70 to 90°C, further demonstrating that MY-1 is an extremely thermophilic bacterium.

[0041] Example 2: Preparation of Biochar-Based Inoculant

[0042] The preparation process of biochar-based inoculants is as follows:

[0043] (1) Activation of strain: Take the slant preserved at 4℃ in Example 1, inoculate the strain onto beef extract peptone solid medium plate, and incubate in a constant temperature incubator at 70℃ for 24 hours to activate the strain.

[0044] (2) Preparation of seed culture: The activated single colonies from step (1) were inoculated into two 500 ml Erlenmeyer flasks using an inoculation loop. Each Erlenmeyer flask contained 100 ml of beef extract peptone liquid culture medium. The two Erlenmeyer flasks were then incubated in a 70°C constant temperature water bath shaker for 24 hours to obtain the seed culture. The shaking frequency of the shaker was 150-200 rpm.

[0045] (3) Preparation of fermentation broth: The seed liquid obtained in step (2) was inoculated into a sterilized fermenter at an inoculation rate of 5% for expansion culture; the culture conditions were: culture temperature of 70℃, stirring speed of 150 rpm, aeration ratio of 0.5 cubic meters / (cubic meters·min), culture time of 24 hours, and the number of cells in the fermentation broth was 2×10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth;

[0046] (4) Preparation of biochar-based solid fermentation medium: Take dry biochar, rice bran, maifanite, glucose and soybean meal, and mix them evenly according to the dry weight ratio of biochar: maifanite: rice bran: glucose: soybean meal = 50:10:1:5:5 to obtain biochar-based solid fermentation medium.

[0047] (5) Preparation of biochar-based inoculant: The fermentation broth and biochar-based solid fermentation substrate were mixed at a mass ratio of 2:100 to obtain a mixture; the moisture content of the mixture was adjusted to 50%, and then cultured at 70°C for 48 hours to obtain a solid fermentation product. The solid fermentation product was dried at 80-90°C until the moisture content was below 30% to obtain the biochar-based inoculant.

[0048] The viable bacteria count in this biochar-based inoculant is 8 × 10⁸. 9 cfu / gram.

[0049] Example 3: Preparation of Biochar-Based Inoculant

[0050] The difference from Example 2 is that in step (5), the fermentation broth and the biochar-based solid fermentation substrate are mixed at a mass ratio of 5:100 to obtain a mixture; the water content of the mixture is adjusted to 60%, and then it is cultured at 70°C for 60 hours; the solid fermentation product is dried at 80-90°C to a water content of less than 30% to obtain a biochar-based inoculant.

[0051] The viable bacteria count in this biochar-based inoculant is 1×10⁻⁶. 10 cfu / gram.

[0052] Example 4: Preparation of Biochar-Based Inoculant

[0053] The difference from Example 2 is that in step (5), the fermentation broth and the biochar-based solid fermentation substrate are mixed at a mass ratio of 8:100 to obtain a mixture; the water content of the mixture is adjusted to 70%, and then it is cultured at 70°C for 72 hours; the solid fermentation product is dried at 80-90°C to a water content of less than 30% to obtain a biochar-based inoculant.

[0054] The viable bacteria count in this biochar-based inoculant is 1.5 × 10⁻⁶. 10 cfu / gram.

[0055] Comparative Example 1

[0056] The difference from Example 3 is that the thermophilic bacteria used is thermophilic bacterium (China General Microbiological Culture Collection Center CGMCC 1.15099);

[0057] In step (3), the seed culture obtained in step (2) is inoculated into a sterilized fermenter at an inoculation rate of 5% for expansion culture. The culture conditions are: culture temperature of 70℃, stirring speed of 150 rpm, aeration ratio of 0.5 m³ / (m³·min), culture time of 24 hours, and the number of cells in the fermentation broth is 2×10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth;

[0058] The viable count of the carbon-based bacterial agent obtained after cultivation was 1.2 × 10⁻⁶. 10 cfu / gram.

[0059] Comparative Example 2

[0060] The difference from Example 3 is that in the biochar-based solid-state fermentation medium, biochar was replaced with zeolite, and the resulting biochar-based inoculum contained 5.8 × 10⁻⁶ viable bacteria. 9 cfu / gram.

[0061] Comparative Example 3

[0062] The difference from Example 3 is that in the biochar-based solid-state fermentation medium, maifanite was replaced with zeolite, and the resulting biochar-based inoculum contained 6.4 × 10⁻⁶ viable bacteria. 9 cfu / gram.

[0063] Comparative Example 4

[0064] The difference from Example 3 is that in the biochar-based solid-state fermentation medium, rice bran, glucose, and soybean meal were replaced with wheat bran, starch, and peanut meal, resulting in a biochar-based inoculum with a viable count of 5.0 × 10⁻⁶ cells. 9 cfu / gram.

[0065] Experiment 1: Application of microbial agents in ultra-high temperature composting

[0066] Experimental Design:

[0067] (1) Composting raw materials: Mix wheat straw and chicken manure to adjust the C / N ratio to 10:1, with a moisture content of 68%, and each pile weighs about 2000 kg;

[0068] Stack group:

[0069] After mixing the composting raw materials with the corresponding microbial agent, they are made into windrow piles, as follows:

[0070] Experimental Group A: The weight ratio (g / g) of the microbial agent to the composting raw material in Example 3 was 1:1000;

[0071] Experimental Group B: The weight ratio (g / g) of the microbial agent to the composting raw material in Example 3 was 2:1000;

[0072] Experimental Group C: The weight ratio (g / g) of the microbial agent to the composting raw material in Example 3 was 3:1000;

[0073] Experimental Group D: The weight ratio (g / g) of the microbial agent to the composting raw material in Example 2 was 2:1000;

[0074] Experimental Group E: The weight ratio (g / g) of the microbial agent to the composting raw material in Example 4 was 2:1000;

[0075] Control group: The group without the bacterial agent of Example 3 served as the blank experimental group;

[0076] Control group 1: The difference from experimental group B is that the bacterial agent of control group 1 was added;

[0077] Control group 2: The difference from experimental group B is that the bacterial agent of control group 2 was added;

[0078] Control group 3: The difference from experimental group B is that the bacterial agent of control group 3 was added;

[0079] Control group 4: The difference from experimental group B is that the bacterial agent of control group 4 was added;

[0080] The composting process for experimental groups A, B, C, D, E, F, G, control group, control group 1, control group 2, control group 3, and control group 4 is as follows:

[0081] The compost pile is a windrow type, about 1 meter high. The composting cycle is 15 days. The pile is ventilated twice a day, once in the morning and once in the evening, with air at 96% RH. Each ventilation is 3 cubic meters per minute for 15 minutes, so that the moisture content of the compost material in the pile is maintained between 67% and 68%.

[0082] Control group 5: The difference from experimental group B is that dry air was introduced into the compost pile during the composting process.

[0083] (2) Experimental group F: The difference from experimental group B is that the C / N ratio is 20:1, and air with a humidity of 100%RH is introduced into the compost pile to maintain the moisture content of the compost raw materials between 69% and 70%.

[0084] Experimental Group G: The difference from Experimental Group B is that the C / N ratio is 30:1, and air with a humidity of 92%RH is introduced into the compost pile to maintain the moisture content of the compost raw materials between 65% and 66%.

[0085] Test 1: Temperature changes in the compost pile during the composting process

[0086] The temperature of the pile was measured before ventilation in the morning. The temperature was measured at the perimeter and the middle of the pile, with a depth of 40 cm. The average value was taken. The results are shown in Tables 2-5.

[0087] Table 2. Temperature changes (°C) of compost piles in experimental groups A, B, C, and the control group during composting treatment.

[0088]

[0089] As shown in Table 2, using the microbial agent provided in Example 3, the temperature of the compost pile reached above 80°C on the third day, and this high temperature could be maintained until the ninth day. This indicates that the thermophilic bacterium MY-1 can rapidly proliferate and has a vigorous metabolic capacity under high temperature conditions, thereby achieving rapid and pollution-free composting and shortening the composting time. In addition, Table 2 also shows that changes in the amount of microbial agent added have a certain impact on the temperature of the compost pile. When the weight ratio of microbial agent to composting raw materials is 2:1000 (Experimental Group B), the highest temperature of the compost pile reaches 94°C. When the weight ratio of microbial agent to composting raw materials is 3:1000 (Experimental Group C), the highest temperature of the compost pile is 92°C. When the weight ratio of microbial agent to composting raw materials is 1:1000 (Experimental Group A), the highest temperature of the compost pile is 90°C. This shows that the amount of microbial agent added is not necessarily better the more there is.

[0090] Table 3. Temperature changes (°C) of compost piles in experimental groups B, F, G, and control group 5 during composting treatment.

[0091]

[0092]

[0093] Based on the temperature changes of experimental groups B, F, and G in Table 3, it can be seen that changing the moisture content of the composting raw materials will produce certain differences in temperature changes, but the temperature changes will still be within the range of 3 days after composting. When the temperature of the compost pile was above 80℃, and the high temperature above 80℃ could be maintained until the 9th day, it indicates that the thermophilic bacterium MY-1 has strong adaptability to carbon-nitrogen ratio and moisture content. Moreover, when the moisture content of the compost raw materials is between 65% and 70%, the thermophilic bacterium MY-1 has vigorous metabolic capacity, which can achieve rapid pollution-free composting and shorten the composting time. In control group 5, dry air was introduced into the compost pile, and the temperature on the 3rd day of composting was 75℃, with the highest composting temperature being 83℃, which was significantly lower than that of experimental groups B, F, G, A, and C (refer to Table 2). This indicates that the thermophilic bacterium MY-1 provided by this invention is more suitable for rapid propagation under the condition of compost raw material moisture content between 65% and 70%, indicating that the thermophilic bacterium MY-1 has the ability to reproduce under low oxygen conditions.

[0094] Table 4. Temperature changes (°C) of compost piles in experimental groups B, D, E, and control group 1 during composting treatment.

[0095]

[0096] Based on the temperature changes of experimental groups B, D, and E in Table 4, it can be seen that using the microbial agent provided by this invention, when the moisture content of the composting raw materials is between 65% and 70%, the temperature of the compost pile can be >80℃ on the 3rd day, and the high temperature above 80℃ can be maintained until the 9th day. Based on the temperature changes of the control group 1, it can be seen that the number of viable bacteria in the microbial agent used in control group 1 is about 1.5 times that of the microbial agent used in experimental group D. However, the highest temperature of the control group 1 is 73℃, while the highest temperature of the control group D is 90℃. This indicates that the thermophilic bacteria in the microbial agent of control group 1 cannot adapt to rapid reproduction in an environment with a moisture content of 65% to 70%.

[0097] Table 5. Temperature changes (°C) of compost piles in experimental group B, control group 2, control group 3, and control group 4 during composting treatment.

[0098]

[0099]

[0100] As shown in Table 5, the highest composting temperature in control groups 2, 3, and 4 reached 87℃. This indicates that changing the composition of the biochar-based solid-state fermentation medium affects the propagation ability of thermophilic bacteria MY-1. For example, in control group 4, the compost temperature was only 63℃ on the third day of composting. It is evident that the biochar-based solid-state fermentation medium has a significant impact on the propagation of thermophilic bacteria MY-1.

[0101] As can be seen from Tables 2-5, the highest composting temperature in experimental group B reached 94℃, indicating that the composting materials had a C / N ratio of 10:1, a moisture content maintained between 67% and 68%, and a viable bacteria count of 1×10⁻⁶ in the microbial agent. 10 Under conditions of cfu / g, the metabolic activity of thermophilic bacteria MY-1 is most vigorous.

[0102] Test 2: Determination of total nitrogen content in compost products

[0103] After composting, samples were taken from the perimeter and center of the compost pile, mixed thoroughly, and the total nitrogen content was measured. The total nitrogen was determined using the potassium persulfate oxidation method. The results are shown in Tables 6 and 7.

[0104] Table 6. Total nitrogen content in compost products from the experimental and control groups.

[0105]

[0106] As can be seen from Table 6, when the microbial agents provided in Examples 2, 3, and 4 of this invention are applied to ultra-high temperature composting, and the carbon-to-nitrogen ratio of the composting raw materials is between 10:1 and 30:1, and the moisture content of the composting raw materials is between 65% and 70%, the total nitrogen content in the compost product is between 13.8 and 14.6 g / kg. Compared with the initial value, the nitrogen loss rate is between 9.9% and 14.8%, while the loss rate of the control group is 50%. It can be seen that using the biochar-based microbial agents provided by this invention for ultra-high temperature composting can effectively improve the nitrogen retention in the compost product and improve the quality of the compost product.

[0107] Table 7. Total nitrogen content in compost products from experimental group B, experimental group F, and control group 5.

[0108]

[0109] As can be seen from Table 7, the total nitrogen loss rate in the compost product of control group 1 was 33.3% compared to the initial value. In the case of the microbial agent provided by the present invention, the loss rate of experimental group F was 14.8% compared to the initial value. Compared with control group 1, the nitrogen loss rate of experimental group F was reduced by 55.6%. It can be seen that the microbial agent prepared by thermophilic bacterium MY-1 of the present invention can effectively reduce nitrogen loss in compost products and improve the quality of compost products when applied to ultra-high temperature composting.

[0110] Compared with the initial value, the total nitrogen loss rate in the compost products of control groups 2-4 was between 21.6% and 26.5%. This indicates that the thermophilic bacterium MY-1 and the biochar-based solid fermentation medium in the microbial agent provided by the present invention have a synergistic effect. When the composition of the biochar-based solid fermentation medium is changed, the propagation ability and nitrogen holding capacity of thermophilic bacterium MY-1 are affected, thereby increasing the nitrogen loss rate in the compost products.

[0111] Compared with the initial value, the total nitrogen content in the compost product of control group 5 was 37.7% lower. Although the highest temperature of the compost pile in control group 5 was only 83℃ (Table 3), a large amount of nitrogen in the compost pile still leaked out. It can be seen that the moisture content of the compost raw materials in the compost pile also has a certain impact on the retention of nitrogen.

[0112] It can be seen that when the microbial agent provided by this invention is applied to ultra-high temperature composting, the C / N ratio of the composting raw materials is between 10:1 and 30:1, the moisture content is maintained between 65% and 70%, and the number of viable bacteria in the microbial agent is 8 × 10⁻⁶. 9 ~1.5×10 10 Under the condition of CFU / g, it can effectively reduce nitrogen loss rate, increase nitrogen retention in compost products, and ensure the quality of compost products.

[0113] Test 3: Quality testing of compost products

[0114] The compost products were tested according to the organic fertilizer standard NY / T 525-2021, and the results are shown in Table 8.

[0115] Table 8 Quality Inspection of Compost Products

[0116]

[0117] As shown in Table 8, the experimental group using the biochar-based inoculant provided by this invention for ultra-high temperature composting had higher GI index and organic matter content than the control and comparison groups. This demonstrates that the biochar-based inoculant and composting process provided by this invention can significantly increase the organic matter content, GI index, and organic matter content of compost products, enabling them to reach organic fertilizer standards more quickly.

[0118] SEQ ID NO: 1

[0119]

[0120] Although the present invention has been described in detail with reference to preferred embodiments, it is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the claims.

Claims

1. A thermophilic bacterium MY-1, characterized in that, This strain was deposited on November 11, 2021, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, 430072, China, with accession number CCTCC NO: M20211405. The strain is classified as follows: Geobacillus sp.; This strain is extremely thermophilic and can grow well at 70-90℃. This thermophilic bacterium MY-1 has the ability to reproduce under low oxygen conditions and can proliferate rapidly in solid fermentation medium with a water content of 50-70%.

2. The thermophilic bacterium MY-1 as described in claim 1, characterized in that, The thermophilic bacterium MY-1 was screened from high-temperature composting samples of agricultural and forestry waste.

3. A biochar-based bacterial agent, characterized in that, The microbial agent comprises the thermophilic bacterium MY-1 as described in claim 1 and a biochar-based solid-state fermentation medium; wherein the effective viable count of the thermophilic bacterium MY-1 in the microbial agent is 8 × 10⁻⁶. 9 ~1.5×10 10 cfu / gram.

4. The biochar-based bacterial agent as described in claim 3, characterized in that, The biochar-based solid-state fermentation medium is composed of biochar, rice bran, maifanite, glucose, and soybean meal.

5. The biochar-based bacterial agent as described in claim 4, characterized in that, The preparation process is as follows: (1) Activation of strain: Take the slant preserved at 4℃, inoculate the strain onto beef extract peptone solid medium plate, and incubate in a constant temperature incubator at 70℃ for 24 hours, ready for use; (2) Preparation of seed culture: Select a single colony on the plate in step (1) and inoculate it into a 500 ml Erlenmeyer flask containing 100 ml of beef extract peptone liquid culture medium. Incubate the inoculated Erlenmeyer flask in a 70℃ constant temperature water bath shaker for 24 hours to obtain the seed culture. (3) Preparation of fermentation broth: The seed liquid obtained in step (2) is inoculated into a sterilized fermenter at an inoculation rate of 5% for expansion culture; Culture conditions: culture temperature 70℃, stirring speed 150 rpm, aeration ratio 0.5 cubic meters / (cubic meters·min), when the number of cells in the fermentation broth is ≥2×10 8 When the concentration of cfu / mL reaches a certain level, stop fermentation and obtain the fermentation broth. (4) Preparation of biochar-based solid fermentation medium: Take dry biochar, rice bran, maifanite, glucose and soybean meal, and mix them evenly according to the dry weight ratio of biochar:maifanite:rice bran:glucose:soybean meal = 50:10:1:5:5 to obtain biochar-based solid fermentation medium. (5) Preparation of biochar-based bacterial agent: The fermentation broth of step (3) and the biochar-based solid fermentation substrate of step (4) are mixed in a mass ratio of 2~8:100 to obtain a mixture; the water content of the mixture is adjusted to 50~70%, and then it is cultured at 70℃ for 48~72 hours to obtain a solid fermentation product; the solid fermentation product is air-dried naturally or dried at 80~90℃ until the water content is below 30% to obtain a biochar-based bacterial agent.

6. The application of the biochar-based bacterial agent according to claim 4, characterized in that, The biochar-based microbial agent was applied in ultra-high temperature composting. The process involved forming a compost pile and introducing air with a humidity greater than 90% RH into the pile to maintain the moisture content of the compost raw materials between 65% and 70%.

7. The application as described in claim 6, characterized in that, The composting material is a mixture of straw and manure, and the C / N ratio of the composting material is 30:1 to 10:1.

Images