Microbial decomposing inoculant as well as preparation method and application thereof

Abstract

The invention relates to a microbial decomposition inoculant and preparation and application thereof.The microbial decomposition inoculant comprises a microbial decomposition inoculant A and a microbial decomposition inoculant B. The microbial decomposition inoculant A is composed of bacillus siamensis TA4 and bacillus velezensis L1 according to bacterial liquid; the microbial decomposing inoculant B is composed of bacillus pallidus hb2 bacterial liquid; the microbial inoculum A is added when composting begins, and the microbial inoculum B is added after the temperature of compost materials rises to 45-60 DEG C. The compost microbial decomposition inoculant and the segmented inoculation process thereof provided by the invention can shorten the composting time, promote the decomposition of compost materials and improve the product quality, are suitable for composting treatment of organic solid wastes such as livestock and poultry manure, and have the advantages of small addition amount, high composting efficiency, strong environmental adaptability, wide application range and convenience in use; wide market popularization and application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of preparing organic fertilizer from agricultural biomass, and specifically relates to a microbial decomposition agent and a preparation method and application thereof. Background Art

[0002] my country produces a large amount of agricultural solid waste every year, including about 3.8 billion tons of livestock and poultry manure and about 900 million tons of crop straw. The generation of a large amount of livestock and poultry manure solid waste can be mainly attributed to the continuous growth of global food demand and the subsequent large-scale and intensive development of livestock and poultry farming. Therefore, treating livestock and poultry manure and reducing pollution to the environment are of great practical significance. Composting technology, as an economical, effective and environmentally friendly method, can use microorganisms to convert agricultural solid waste into high-quality and stable organic fertilizer. Traditional natural composting mostly utilizes the metabolic function of indigenous microorganisms in the pile, and often has the problems of long fermentation and decomposition time and low quality of compost products. At present, the method of inoculating microbial agents into the pile at the start of composting is generally used to improve the efficiency of the composting process and the quality of the compost product.

[0003] While scholars both domestically and internationally have conducted extensive research on the development of microbial agents for composting, issues related to microbial inoculation and the study of composting effects remain debatable. First, the impact of acidity, salinity, and high temperature in the composting microbial environment on exogenous agents. Composting processes often exhibit high temperatures, high pH, ​​and high salinity due to the nature of the material. Therefore, screening and evaluating the environmental adaptability of functional microbial strains specifically for livestock and poultry manure compost will enhance the adaptability of developed microbial agents to the composting environment (high temperature, high pH, ​​and high salinity) and reduce their competitiveness with indigenous microorganisms. Second, indigenous microorganisms undergo significant phased functional succession under the selective pressure of composting microbial factors, and different dominant indigenous bacterial communities inevitably play a corresponding role in each stage of composting.

[0004] Chinese patent CN117024210A discloses a method for preparing a mixed microbial inoculant and its use in high-temperature composting. This method employs a single inoculation of a mixed inoculant containing Mucor circinelloides, Bacillus velezensis, and Bacillus tequila at the initial stage of composting. This method fails to consider the impact of changes in the compost microbial environment on the growth of the functional strains in the inoculant and the antagonism between the strains. Consequently, the compost temperature fails to exceed 60°C and the moisture content of the compost product is excessively high (~50%). Therefore, the type of functional microbial inoculant inoculated should be differentiated according to the different composting stages and microbial environment. Inoculating different microbial inoculants in different stages can help minimize antagonism between the different strains in the inoculant and maximize its functionality.

[0005] Chinese patent CN1434003A discloses a two-stage inoculation method for composting with a microbial composite agent. It proposes that inoculating two different microbial agents at a 1% ratio during the initial and high-temperature stages (55-60°C) of organic waste composting can improve the reaction process of traditional natural composting and shorten the fermentation time. Chinese patent CN102424614A discloses a three-stage inoculation process and microbial agent for composting lignocellulosic raw materials. It proposes that inoculating three different microbial agents at a 0.5-1% ratio during the initial and cooling stages (35-40°C) of lignocellulosic composting can promote the degradation of composted materials and accelerate the decomposition process. Although these examples improve composting efficiency through multiple staged inoculations of microbial agents, the high inoculation rate (>0.1%) results in high investment and operating costs during the composting process, making it uneconomical and environmentally unsuitable. Therefore, research and development of staged inoculation of microbial agents for livestock and poultry manure composting requires continued exploration and optimization to accelerate composting and significantly improve the efficiency of industrialized organic fertilizer plants. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a microbial composting agent and its preparation method and application. According to the changing characteristics of compost temperature and the interaction between microbial strains, a method of inoculating two different microbial agents in the initial stage and high-temperature stage of composting is adopted to improve the efficiency of composting and the quality of compost.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: A microbial decomposition agent, comprising a microbial decomposition agent A and a microbial decomposition agent B, wherein the microbial decomposition agent A is composed of a bacterial liquid of Bacillus siamese TA4 and Bacillus velezensis L1; and the microbial decomposition agent B is composed of a bacterial liquid of Bacillus pallidum hb2. The deposit number of the siam Bacillus TA4 is CGMCC NO.32827, the deposit number of the velez Bacillus L1 is CGMCC NO.32828, and the deposit number of the pallid Bacillus hb2 is CGMCC NO.32829.

[0008] As a preferred method, the cell concentration of Bacillus siamensis is 3×10 9 ~7×10 9 cfu / mL, the bacterial concentration of Bacillus velezensis was 5×10 8 ~4×10 9 cfu / mL, the bacterial concentration of Bacillus pallidum hb2 is 10 8 ~10 9 cfu / mL.

[0009] Preferably, the microbial decomposition agent A is composed of a mixture of two bacteria, Bacillus siamese TA4 and Bacillus velezensis L1, in a volume ratio of 1:1.

[0010] The present invention also provides a method for preparing the above-mentioned microbial decomposition agent, comprising the following steps: (1) Activation of bacterial strains: Bacillus siamese TA4, Bacillus velezensis L1 and Bacillus pallidum hb2 were inoculated into LB solid medium respectively and activated to activate the bacterial strains; (2) Expansion culture: The activated Bacillus siamensis TA4, Bacillus velezensis L1 and Bacillus pallidum hb2 were inoculated into LB liquid culture medium for primary expansion culture and secondary expansion culture respectively; (3) The expanded cultured Bacillus siamese TA4 and Bacillus velezensis L1 were mixed in proportion to obtain a microbial decomposition agent A, wherein the effective viable bacteria count of the microbial decomposition agent A was not less than 10 8 cfu / mL; the bacillus pallidus hb2 culture solution after expansion is used as the microbial decomposition agent B, and the effective viable count of the microbial decomposition agent B is not less than 10 8 cfu / mL.

[0011] Preferably, step (1) is specifically as follows: the original strains of Bacillus siameensis TA4, Bacillus velezensis L1 and Bacillus pallidus hb2 are inoculated into LB solid culture medium under sterile conditions, Bacillus siameensis TA4 and Bacillus velezensis L1 are cultured at 30-37°C for 24 hours, and Bacillus pallidus hb2 is cultured at 45°C for 48 hours.

[0012] Preferably, step (2) is specifically as follows: (i) Primary expansion culture: The activated strains were inoculated into LB liquid culture medium under sterile conditions. Bacillus siamese TA4 and Bacillus velezensis L1 were cultured at 30-37℃ and 160 rpm for 24-48 h, and Bacillus pallidum hb2 was cultured at 45-65℃ and 160 rpm for 48-72 h. When the OD value of the liquid culture was 600 When the value is greater than 1.0, the culture is stopped and the first-level seed solution is obtained; (ii) Secondary expansion culture: 2-5% (volume ratio) of the primary seed liquid of Bacillus siamese TA4 and Bacillus velezensis L1 were inoculated into LB liquid medium, and cultured at 30-37°C and 160 rpm for 24-48 h to obtain an effective viable cell count of 10 8 ~10 9cfu / mL of bacterial liquid was used to prepare Bacillus siamese TA4 and Bacillus velezensis L1 bacterial liquid; the first-level seed liquid of Bacillus pallidum hb2 was inoculated into LB liquid medium at a ratio of 5% (volume ratio), and cultured at 45-65°C and 160r / min shaking for 36-72h to obtain an effective viable cell count of 10 8 ~10 9 cfu / mL of bacterial solution to prepare the Bacillus pallidum hb2 bacterial solution.

[0013] Preferably, in step (3), the two bacterial strains of Bacillus siamese TA4 and Bacillus velezensis L1 are mixed in a volume ratio of 1:1.

[0014] In addition, the present invention also provides the use of the above-mentioned microbial decomposition agent in composting livestock and poultry manure.

[0015] Preferably, the application comprises the following steps: (a) Pretreatment of composting materials: Adjust the carbon-nitrogen ratio of livestock and poultry manure to 20-25, the moisture content to 55%-60%, and the pH value to 6-7.5; (b) Two-stage inoculation of microbial decomposition agents: at the beginning of composting, microbial decomposition agent A is inoculated at a concentration of less than 0.1% by mass of the compost material. When the compost temperature rises to 45℃~60℃, microbial decomposition agent B is inoculated at a concentration of less than 0.1% by mass of the compost material.

[0016] The present invention's microbial agent A is added at the beginning of composting, and microbial agent B is added after the temperature of the compost material rises to 45°C to 60°C. It is applied to the aerobic composting process of livestock and poultry manure, reducing the antagonism between functional microbial strains and between them and indigenous microorganisms, enhancing the adaptability of microbial agents to the composting environment and promoting their dominant growth, thereby accelerating the temperature of the pile to exceed 60°C, effectively promoting the degradation of the compost material, shortening the decomposition cycle, and improving the maturity and yield of the compost product. Compared with the one-time mixing and adding of two microbial agents, the present invention has the advantages of strong adaptability and stable performance, is easy to use, simple in process, and low in cost, and has broad application prospects in the rapid composting and resource utilization of organic waste.

[0017] Preferably, the mass percentage of microbial decomposing agent A is 0.066%, and the mass percentage of microbial decomposing agent B is 0.034%; composting is carried out aerobically, the pile height is 0.6m to 0.8m, the composting cycle is 30d to 47d, and the pile is turned 1 to 2 times a week.

[0018] The composting microbial decomposing agent and the segmented inoculation process thereof provided by the present invention can shorten the composting time, promote the decomposition of compost materials, and improve the quality of products. The composting microbial decomposing agent is suitable for the composting treatment of organic solid wastes such as livestock and poultry manure. The addition amount is small and the composting efficiency is high. At the same time, the composting microbial decomposing agent has strong environmental adaptability, a wide range of applications, is easy to use, and has broad market promotion and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The temperature effect diagrams of Bacillus siamensis TA4 (abbreviated as TA4), Bacillus velez L1 (abbreviated as L1) and Aerobacillus pallidum hb2 (abbreviated as hb2) strains are shown; Figure 2 This is the effect diagram of pH and salinity for the hb2 strain; Figure 3 The effect of pH and salinity on TA4 and L1 strains; Figure 4 The results of enzyme activity assays of TA4, L1 and hb2 strains for hydrolyzing cellulose, xylan and starch are shown; Figure 5 This is a diagram showing temperature changes during the composting process in Example 2; Figure 6 This is a graph showing changes in the germination index of materials during the composting process in Example 2; Figure 7 This is a diagram showing temperature changes during the composting process in Example 3; Figure 8 This is a graph showing changes in the material germination index during the composting process of Example 3. DETAILED DESCRIPTION

[0020] The technical solutions of the present invention are further specifically described below through examples. These examples are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. All other examples obtained by persons of ordinary skill in the art based on the examples in this application without creative work are intended to fall within the scope of protection of this application.

[0021] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0022] The three strains used in this invention were obtained from the composting habitat of livestock and poultry manure (pig manure, chicken manure, and sheep manure) and screened under different culture conditions such as temperature, pH value, and salt concentration. They are classified and named as follows: Bacillus siamensis ( Bacillus siamensis) TA4 (Bacillus siamese TA4), deposited with the accession number CGMCC NO.32827, was deposited on November 28, 2024, at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus velez ( Bacillus velezensis ) L1 (Bacillus velezensis L1), deposited with CGMCC NO.32828, on November 28, 2024, at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus pallidum ( Aeribacillus pallidus )hb2 (Bacillus pallidum hb2), the deposit number is CGMCC NO.32829, and it was deposited in the General Microbiology Center of China Culture Collection Administration on November 28, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] Example 1 Bacillus siamensis ( Bacillus siamensis )TA4, Bacillus velezinoffii ( Bacillus velezensis ) L1 and Aerobacillus pallidum ( Aeribacillus pallidus ) hb2, all from livestock and poultry manure (pig manure, chicken manure and sheep manure) compost habitats and screened under different culture conditions such as temperature, pH value and salt concentration. At the same time, the cellulolytic metabolic function of these strains was analyzed and verified. The results of the temperature effect are as follows Figure 1 As shown, from Figure 1 It can be seen that at 30℃, the OD600 of Bacillus siamese TA4 is the largest and the growth state is the best. As the temperature rises, its OD600 gradually decreases and its OD value returns to zero at 60℃. However, the OD value of Bacillus pallidus hb2 gradually increases with the increase of temperature and reaches the best growth state at 60℃. The OD600 value of Bacillus velezensis L1 reaches the highest at 45℃. When the temperature is higher than 60℃, the strain cannot grow. The experimental results of the optimal pH and salinity of Bacillus pallidus hb2 are shown in Figure 2 .Depend on Figure 2 It can be seen that the growth of the hb2 strain increases with the increase of pH value. When the pH value is 7.0, the OD600 value of the bacteria is the largest and the growth condition is the best. The strain has a certain tolerance to salinity. When the mass fraction of NaCl is greater than 1%, the OD600 of the strain increases with the increase of the NaCl mass fraction. When the mass fraction of NaCl is 4%, its OD value increases to 0.79, and the growth condition of the strain is not affected. The optimal pH and salinity experimental results of Bacillus siamese TA4 and Bacillus velezini L1 are shown in Figure 2. Figure 3 As shown, from Figure 3 It can be seen that the growth of the two strains showed different results in response to pH value. The OD600 value of the L1 strain gradually decreased with the increase of pH value, while the activity of the TA4 strain increased with the increase of pH value. The OD600 of the strain tended to be smooth when the pH value was between 7.0 and 9.0, so this was the optimal pH range for the growth of the strain. From the salinity test results, it was found that TA4 had good salt tolerance. When the mass fraction of NaCl was less than 2%, the growth of the strain was almost unaffected; when the mass fraction of NaCl was greater than 2%, the OD600 of the strain gradually decreased with the increase of the NaCl mass fraction. The L1 strain had poor tolerance to salinity, and the greater the mass fraction of NaCl, the stronger the growth inhibition of the strain. The results of the cellulase, xylanase and amylase metabolic capacity tests of the TA4, L1 and hb2 strains are shown in Figure 4 .Depend on Figure 4 The hb2 strain had the highest cellulase activity, reaching 34.42 U / ml. The TA4 strain was superior in starch hydrolysis, reaching an activity of 2.22 U / ml. The three strains were similar in their ability to hydrolyze xylan. The optimal temperature, pH, and salinity conditions for these strains indicate that they can grow well during composting and maintain good enzyme activity, providing a basis for subsequent composting experiments.

[0024] The preparation method of the above-mentioned microbial decomposition agent comprises the following steps: (1) Activation of bacterial strains: Bacillus siamese TA4 and Bacillus velezensis L1 were streaked onto LB solid medium and cultured at 30°C for 24 h; Bacillus pallidum hb2 was streaked onto LB solid medium and cultured at 45°C for 48 h.

[0025] (2) Expansion culture: The activated Bacillus siamensis TA4, Bacillus velezensis L1 and Bacillus pallidum hb2 were inoculated into LB liquid culture medium for primary expansion culture and secondary expansion culture respectively; (i) Primary expansion culture: The activated strains were inoculated into LB liquid medium under sterile conditions. Bacillus siamese TA4 and Bacillus velezensis L1 were cultured at 30℃ and 160r / min in a shaking incubator for 48h, and Bacillus pallidum hb2 was cultured at 60℃ and 160r / min in a shaking incubator for 72h. When the OD 600 When the value is greater than 1.0, the culture is stopped and the first-level seed solution is obtained; (ii) Secondary expansion culture: 2% to 5% of the first-stage seed liquid of Bacillus siamese TA4 and Bacillus velez L1 were inoculated into LB liquid medium, and cultured at 30°C and 160 rpm for 48 h to obtain Bacillus siamese TA4 and Bacillus velez L1 bacterial liquids; 5% of the first-stage seed liquid of Bacillus pallidus hb2 was inoculated into LB liquid medium, and cultured at 60°C and 160 rpm for 72 h to obtain Bacillus pallidus hb2 bacterial liquid.

[0026] (3) Preparation of microbial decomposition agent by two-stage inoculation: The cultured Bacillus siamese TA4 and Bacillus velezensis L1 were mixed evenly in a volume ratio of 1:1 to obtain microbial decomposition agent A for inoculation in the initial stage of composting. The effective viable bacteria count of the microbial decomposition agent A was 10 8 cfu / mL; the pale aerobic bacillus hb2 bacterial solution after expansion culture is used alone as a microbial decomposition agent B for inoculation in the high temperature stage of composting, and the effective viable count of the microbial decomposition agent B is 10 8 cfu / mL.

[0027] Example 2 Two-stage inoculation of pig manure and mushroom residue straw compost 1. Materials Pig manure and mushroom residue straw were obtained from organic fertilizer factories in the vicinity of Tongxiang City, Zhejiang Province; the preparation of the microbial decomposition agent was according to Example 1.

[0028] 2. Methods Pig manure and mushroom residue straw were mixed at a ratio of 4:5 by weight, with an initial carbon-nitrogen ratio of 20. The initial moisture content was adjusted to 60%. Microbial composting agent A prepared in Example 1 was inoculated into the mixture of pig manure and mushroom residue straw at a rate of 0.066% at the initial stage of composting and mixed thoroughly. On the fourth day of composting, microbial composting agent B prepared in Example 1 was inoculated into the pile at a rate of 0.034% and mixed thoroughly. Composting was then continued at room temperature for 47 days (designated as treatment group M). A control group (CK) (uninoculated) and a single-inoculation treatment group (S) (inoculated simultaneously with 0.066% microbial composting agent A and 0.034% microbial composting agent B at the beginning of composting) were also established. During the composting period, the pile temperature was measured daily, and the compost was turned on days 0, 4, 6, 13, 18, 23, 28, 30, 32, 35, 39, 42, and 47 to increase the oxygen supply within the pile. Compost samples were collected at multiple locations on days 0, 6, 13, 23, 35, 42, and 47, mixed, and stored at -20°C. The germination index and related physical and chemical parameters of samples from each treatment were measured according to the standard for organic fertilizer (NY525-2021).

[0029] 3. Results (1) Temperature changes during composting Temperature changes during composting Figure 5 As shown, from Figure 5 As can be seen, neither the control group CK nor the treatment group S entered the high-temperature phase (<50°C) during the first three days of composting. Meanwhile, the pile temperature in the treatment group M, which received two staged inoculations of microbial decomposition agents, reached 50.3°C (>50°C) by the third day of composting. Furthermore, significant differences were observed in the pile temperatures on the fourth day among the treatments. The temperatures in the CK and S treatments were only 52.4°C and 53.2°C, respectively, while the pile temperature in the M treatment reached 58.6°C. This indicates that the staged inoculation of microbial decomposition agents significantly increased the pile temperature during the initial high-temperature phase (P < 0.05). Temperature is an important indicator of microbial metabolic intensity, indicating that the two staged inoculations of microbial decomposition agents enhanced microbial activity during the initial composting phase and promoted the increase in pile temperature. After the high-temperature phase, the pile temperatures in the CK and S treatments continued to decrease. The pile temperatures in the M treatments dropped below 50°C by the 43rd and 44th days of composting, respectively, while the pile temperature in the CK and S treatments dropped below 50°C only by the 41st day of composting. These results indicate that the two-stage inoculation of microbial composting agents in treatment M can promote the degradation of substances, accelerate the composting process, and shorten the composting time by 2 to 3 days compared with the CK and S treatments.

[0030] (2) Changes in the germination index (GI) of materials during composting The cucumber seed germination index was used to determine the maturity of the compost products under each treatment. The germination index of the material changed during the composting process. Figure 6 As shown by Figure 6 As can be seen, the M treatment achieved the highest GI value (152.77%), followed by the S treatment (135.67%) and the CK treatment (108.04%). Furthermore, the M treatment maintained GI values ​​greater than 70% throughout the composting period (ranging from 72.71% to 152.77%), meeting the requirements of NY / T 525-2021. Therefore, staged inoculation of microbial composting agents can consistently improve compost maturity.

[0031] (3) Yield of materials under each treatment After 47 days of full fermentation, the compost weights for the CK, S, and M treatments were 372.62 kg, 400.14 kg, and 453.98 kg, respectively, corresponding to organic fertilizer yields of 37.26%, 40.01%, and 45.40%, respectively (compost yield = compost weight divided by raw material weight × 100%, with raw material weight being 1000 kg). The M treatment had the highest organic fertilizer yield, indicating that the two-stage inoculation process effectively reduced material loss and increased compost yield.

[0032] (4) Basic physical and chemical properties of each treatment before and after composting The basic physical and chemical properties of each treatment before and after composting are shown in Table 1: Table 1

[0033] Note: CK means natural composting without any microbial agents; S means microbial decomposition agents A and B were inoculated at the initial stage of composting; M means microbial decomposition agents A and B were inoculated at the initial stage of composting and when the temperature rose to 45℃, respectively.

[0034] As shown in Table 1, the pH values ​​of all treatments before and after composting ranged from 6.57 to 7.52, generally meeting the requirements of NY / T525-2021. The essence of composting is the mineralization and degradation of organic matter under the action of microbial communities. The organic matter content of each treatment decreased significantly after composting, with average organic matter degradation rates of 21.92%, 23.58%, and 37.31% for CK, S, and M, respectively. Therefore, the two-stage inoculation of microbial decomposition agents can promote the degradation of organic materials during composting and accelerate the composting process. Furthermore, the total nitrogen, total phosphorus, total potassium, and total nutrient contents of the compost product from the M treatment were higher than those from the CK and S treatments, respectively. From day 0 to day 47, the M treatment showed the highest increase in nutrient content, demonstrating the enhanced fertility of organic fertilizers through staged inoculation. These results clearly demonstrate that the new two-stage inoculation of microbial decomposition agents process can effectively promote the degradation of organic matter in compost and improve the fertility of compost products.

[0035] The comparison results of the limited heavy metal contents in the finished compost products of each treatment are shown in Table 2: Table 2

[0036] As can be seen from the results in Table 2, the final products after composting of each treatment have a consistent distribution of limited heavy metal content, all showing a trend of "total chromium content > total lead content > total arsenic content > total cadmium content > total mercury content", and all tested indicators meet the standards of NY / T 525-2021.

[0037] Example 3 Two-stage inoculation of chicken manure and rice husk straw compost 1. Materials Chicken manure and rice husk straw were obtained from organic fertilizer factories in the vicinity of Tongxiang City, Zhejiang Province; the preparation of the microbial decomposition agent was according to Example 1.

[0038] 2. Methods Chicken manure and rice husk straw were mixed at a ratio of 7.5:1 by weight. The initial carbon-nitrogen ratio and pH of the mixed material were 25 and 6.18, respectively. The initial moisture content was adjusted to 58%. Microbial composting agent A prepared in Example 1 was inoculated into the mixture of chicken manure and rice husk straw at a rate of 0.066% at the beginning of composting and mixed thoroughly. On the fourth day of composting, microbial composting agent B prepared in Example 1 was inoculated into the pile at a rate of 0.034% and mixed thoroughly. The compost was then composted at room temperature for 30 days (designated treatment group T3). A control group (uninoculated) and a single-inoculation treatment group (T2) were also established (inoculated simultaneously with 0.066% microbial composting agent A and 0.034% microbial composting agent B at the beginning of composting). The pile temperature was measured daily, and the pile was turned on days 0, 4, 7, 13, 17, 23, and 30 to increase oxygen supply. On the 0th, 7th, 27th and 30th day of composting, compost samples were collected from multiple points on the pile and mixed, then stored at -20°C. The relevant indicators of the samples were measured according to the standards for organic fertilizers (NY525-2021).

[0039] 3. Results (1) Temperature changes during composting Temperature changes during composting Figure 7 As shown, all treatments entered the high-temperature phase within the first five days of composting, followed by a gradual increase in temperature. Treatment T3 reached 60°C on the seventh day, 4 and 2 days earlier than treatments T1 and T2, respectively. The high-temperature phases in treatments T1, T2, and T3 lasted 22, 18, and 18 days, respectively, meeting the hygienic and harmless requirements for eliminating potential pathogens and parasite eggs in the compost. Furthermore, treatments T2 and T3 entered the cooling phase one day earlier than T1. In particular, treatment T3 experienced a more rapid overall temperature decrease within the 24-30 days of the cooling phase, promoting stable composting maturity. Therefore, the novel process of double-stage inoculation of microbial composting agents can enhance microbial activity in the early stages of the composting process, accelerate the rise in compost temperature, and accelerate composting maturity.

[0040] (2) Changes in the germination index (GI) of materials during composting The changes of germination index GI during composting process are as follows: Figure 8 As shown, from Figure 8 As can be seen from the data, the germination index of each treatment showed a trend of first decreasing and then gradually increasing to a stable state. Compared with treatments T1 and T2, treatment T3 preferentially reached a higher germination index (81.94%-98.50%) between days 27 and 30 of the composting maturity stage, indicating that the two-stage inoculation of microbial decomposition agents promoted the safety of the compost product.

[0041] (3) Comparison of pH, moisture content, conversion rate of materials into organic fertilizer, and nutrient content of the compost after each treatment The comparison results of pH, moisture content and material yield after composting under different treatments are shown in Table 3: Table 3

[0042] Note: T1 is natural composting without adding any microbial agents; T2 is a one-time inoculation of microbial decomposition agents A and B at the initial stage of composting; T3 is a one-time inoculation of microbial decomposition agents A and B at the initial stage of composting and when the temperature rises to 45°C.

[0043] As shown in Table 3, the pH of the compost products from all treatments met the NY / T 525-2021 standard. The final moisture content of the T1 compost was high, but the organic fertilizer yield was low. The final moisture content of the T2 and T3 composts was below 30%, and the organic fertilizer yield was higher than that of the T1 compost, especially the T3 treatment. Furthermore, the T3 treatment had the lowest carbon-nitrogen ratio (12.13) and the highest total nitrogen, phosphorus, and potassium content (10.59%). Therefore, the new process of double-stage inoculation of microbial decomposition agents is beneficial for reducing carbon and nitrogen losses during composting, increasing the yield of organic fertilizer conversion, and improving the quality of the compost product.

[0044] The present invention adds agent A at the beginning of composting and agent B after the temperature of the compost material reaches 45°C to 60°C. When applied to the aerobic composting process of livestock and poultry manure, it reduces the antagonism between functional microbial strains and between them and indigenous microorganisms, enhances the adaptability of the microbial agents to the composting environment and promotes their dominant growth, thereby accelerating the temperature of the pile to exceed 60°C, effectively promoting the degradation of the compost material, shortening the decomposition cycle, and improving the maturity and yield of the compost product. Therefore, compared with the one-time mixed addition of two microbial agents, the new staged inoculation process has the advantages of strong adaptability and stable performance, is easy to use, simple in process, and low in cost, and has broad application prospects in the rapid composting and resource utilization of organic waste.

[0045] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A microbial decomposition agent, characterized in that: The microbial decomposition agent includes a microbial decomposition agent A and a microbial decomposition agent B, wherein the microbial decomposition agent A is composed of a bacterial liquid of Bacillus siamese TA4 and Bacillus velez L1; the microbial decomposition agent B is composed of a bacterial liquid of Bacillus pallidum hb2; The deposit number of the siam Bacillus TA4 is CGMCC NO.32827, the deposit number of the velez Bacillus L1 is CGMCC NO.32828, and the deposit number of the pallid Bacillus hb2 is CGMCC NO.32829.

2. The microbial decomposition agent according to claim 1, characterized in that The concentration of Bacillus siamese bacteria was 3×10 9 ~7×10 9 cfu / mL, the bacterial concentration of Bacillus velezensis was 5×10 8 ~4×10 9 cfu / mL, the bacterial concentration of Bacillus pallidum hb2 is 10 8 ~10 9 cfu / mL.

3. The microbial decomposition agent according to claim 1, characterized in that The microbial decomposition agent A is composed of a mixture of two bacteria, Bacillus siamese TA4 and Bacillus velezensis L1, in a volume ratio of 1:

1.

4. A method for preparing the microbial decomposition agent according to claim 1, characterized in that: The steps include: (1) Activation of bacterial strains: Bacillus siamese TA4, Bacillus velezensis L1 and Bacillus pallidum hb2 were inoculated into LB solid medium respectively and activated to activate the bacterial strains; (2) Expansion culture: The activated Bacillus siamensis TA4, Bacillus velezensis L1 and Bacillus pallidum hb2 were inoculated into LB liquid culture medium for primary expansion culture and secondary expansion culture respectively; (3) The expanded cultured Bacillus siamese TA4 and Bacillus velezensis L1 were mixed in proportion to obtain a microbial decomposition agent A, wherein the effective viable bacteria count of the microbial decomposition agent A was not less than 10 8 cfu / mL; the bacillus pallidus hb2 culture solution after expansion is used as the microbial decomposition agent B, and the effective viable count of the microbial decomposition agent B is not less than 10 8 cfu / mL.

5. The method for preparing the microbial decomposition agent according to claim 4, characterized in that: Step (1) is specifically as follows: the original strains of Bacillus siamese TA4, Bacillus velez L1 and Bacillus pallidus hb2 are inoculated into LB solid culture medium under sterile conditions, Bacillus siamese TA4 and Bacillus velez L1 are cultured at 30-37°C for 24 hours, and Bacillus pallidus hb2 is cultured at 45°C for 48 hours.

6. The method for preparing the microbial decomposition agent according to claim 4, characterized in that: Step (2) is as follows: (i) Primary expansion culture: The activated strains were inoculated into LB liquid culture medium under sterile conditions. Bacillus siamese TA4 and Bacillus velezensis L1 were cultured at 30-37℃ and 160 rpm for 24-48 h, and Bacillus pallidum hb2 was cultured at 45-65℃ and 160 rpm for 48-72 h. When the OD value of the liquid culture was 600 When the value is greater than 1.0, the culture is stopped and the first-level seed solution is obtained; (ii) Secondary expansion culture: The first-level seed liquid of Bacillus siamese TA4 and Bacillus velezensis L1 was inoculated into LB liquid medium at a volume ratio of 2-5%, and cultured at 30-37°C and 160 rpm for 24-48 h to obtain an effective viable cell count of 10 8 ~10 9 cfu / mL of bacterial liquid was used to prepare Bacillus siamese TA4 and Bacillus velezensis L1 bacterial liquid; the first-level seed liquid of Bacillus pallidum hb2 was inoculated into LB liquid medium at a volume ratio of 5%, and cultured at 45-65℃ and 160r / min shaking for 36-72h to obtain an effective viable cell count of 10 8 ~10 9 cfu / mL of bacterial solution to prepare the Bacillus pallidum hb2 bacterial solution.

7. The method for preparing the microbial decomposition agent according to claim 4, characterized in that: In step (3), the two bacterial strains of Bacillus siamese TA4 and Bacillus velezensis L1 are mixed in a volume ratio of 1:

1.

8. Use of the microbial decomposition agent according to claim 1 in composting livestock and poultry manure.

9. The use according to claim 8, characterized in that The steps include: (a) Pretreatment of composting materials: Adjust the carbon-nitrogen ratio of livestock and poultry manure to 20-25, the moisture content to 55%-60%, and the pH value to 6-7.5; (b) Two-stage inoculation of microbial decomposition agents: at the beginning of composting, microbial decomposition agent A is inoculated at a concentration of less than 0.1% by mass of the compost material. When the compost temperature rises to 45℃~60℃, microbial decomposition agent B is inoculated at a concentration of less than 0.1% by mass of the compost material.

10. The use according to claim 9, characterized in that The mass percentage of microbial composting agent A is 0.066%, and the mass percentage of microbial composting agent B is 0.034%. Composting is carried out aerobically, the pile height is 0.6m to 0.8m, the composting cycle is 30d to 47d, and the pile is turned 1 to 2 times a week.

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