Livestock and poultry manure high-temperature rapid composting complex microbial inoculant as well as preparation and application thereof

Abstract

The invention discloses a complex microbial inoculant for high-temperature rapid composting of livestock and poultry manure as well as preparation and application of the complex microbial inoculant. The complex microbial inoculant is prepared by mixing a fermentation product of brevibacillus borsteri and a fermentation product of penicillium chrysogenum. The microbial agent can promote rapid temperature rise of compost, shorten the composting time, remarkably reduce emission of ammonia gas and nitrous oxide in the composting process, improve the content of nitrogen, phosphorus and potassium in the compost and improve the quality of the compost. And the method meets industrial large-scale production requirements and is convenient for large-area popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of solid organic waste disposal and resource utilization, and specifically relates to a composite bacterial agent for high-temperature rapid composting of livestock and poultry manure and a preparation method and application thereof. Background Art

[0002] With the rapid development of animal husbandry in my country, the amount of livestock and poultry waste generated is increasing day by day. According to statistics, the amount of livestock and poultry manure generated in my country is as high as 4 billion tons per year. If the large-scale discharge of livestock and poultry manure is not treated quickly and effectively, it will seriously occupy and pollute farmland and water bodies, produce odor and cause environmental pollution, and also affect the sanitation and epidemic prevention of farms and human health. Therefore, the scientific and rational use of livestock and poultry manure has become a focus of social attention.

[0003] Aerobic composting technology is one of the important technologies for the harmless treatment and resource utilization of organic waste such as livestock and poultry manure and crop straw. However, there are still key technical bottlenecks that need to be solved urgently, such as: long composting cycle, odor and greenhouse gas generation during the composting process, serious nitrogen loss, etc., which make it difficult to ensure the quality of fertilizer (low maturity and high water content) and high production costs.

[0004] Therefore, in order to achieve the harmless treatment and resource utilization of livestock and poultry manure, effectively shorten the composting time, reduce pollutant gas emissions, nitrogen loss and improve compost quality, it is of great significance to develop a high-temperature rapid composting composite bacterial agent for livestock and poultry manure. Summary of the invention

[0005] The main purpose of the present invention is to provide a composite bacterial agent for high-temperature rapid composting of livestock and poultry manure, and a preparation method and application thereof, wherein the bacterial agent can promote rapid temperature rise of the compost, shorten the compost maturity time, and at the same time significantly reduce the emission of ammonia and nitrous oxide during the composting process, increase the content of nitrogen, phosphorus and potassium in the compost, and improve the quality of the compost.

[0006] The technical solution of the present invention is achieved by the following methods:

[0007] The invention discloses a method for preparing a composite bacterial agent for high-temperature rapid composting of livestock and poultry excrement. The compound bacterial agent is prepared by mixing a fermentation product of Brevibacillus Potssteine ​​and a fermentation product of Penicillium chrysogenum.

[0008] Furthermore, the preservation number of the Brevibacillus potsdamiensis is CGMCC No.32115, and the preservation number of the Penicillium chrysogenum is CGMCC No.41529.

[0009] The fermentation product powder of Brevibacillus Potssteine ​​contains 1.0×10 9 ~4.0×10 9cfu / g; the bacterial content of the powder product of Penicillium chrysogenum fermentation product is between 1.0×10 9 ~5.0×10 9 cfu / g;

[0010] Compound microbial agent: 50% to 80% of the fermentation powder of Brevibacillus Potssteine ​​and 20% to 50% of the fermentation powder of Penicillium chrysogenum by weight, and mix thoroughly; the total number of viable bacteria of the microbial agent is not less than 10.0×10 8 cfu / g.

[0011] The preparation method comprises the following steps:

[0012] Preparation of the fermentation product powder of Brevibacillus Potsdamii: The Brevibacillus Potsdamii is activated by slant strains, cultured in shake flasks, cultured in fermenters, and finally solid adsorbed and crushed;

[0013] Preparation of powder of fermented product of Penicillium chrysogenum: The Penicillium chrysogenum is activated by slant strain, cultured in shake flask seeds, and then cultured and crushed by solid fermentation.

[0014] Further, the method specifically includes the following steps:

[0015] 1) Preparation of powder of fermentation product of Brevibacillus Potsdam

[0016] First, Brevibacillus Potssteine ​​was activated by slant culture and seed cultured in shake flasks;

[0017] Then, the fermentation tank is cultured: the volume of the culture medium in the fermentation tank is 50-70% of the total volume, the inoculation volume accounts for 0.1-0.3% of the volume of the culture medium, the fermentation temperature is 50-55°C, the air is ventilated at 180-200rpm, the ratio of the ventilation volume to the fermentation liquid volume is 0.9:1-1:1, and the tank pressure is 0.03-0.05MPa; the fermentation end point is that the spore count is not less than 90% of the total bacterial count, and the number of viable bacteria in the fermentation liquid is 1.0×10 10 ~1.5×10 10 cfu / mL; the mass percentage of each component of the fermentation tank culture medium is: yeast powder 1.5-2.0%, peptone 0.7-1.0%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.5%, and the rest is water, pH 7.2-7.4, pressure 0.14MPa, 121℃ sterilization for 30min;

[0018] Finally, solid adsorption is performed: the fermentation liquid is adsorbed with wheat bran at a weight ratio of 1:3 to 1:5, and then low-temperature dehumidification and drying are performed after adsorption, and the powder product contains bacteria in an amount of 1.0×10 9 ~4.0×10 9 cfu / g;

[0019] 2) Preparation of powder of Penicillium chrysogenum fermentation product

[0020] First, the Penicillium chrysogenum was activated by slant culture and seed cultured in shake flasks;

[0021] Then, solid fermentation culture is carried out: the inoculation amount accounts for 3%-5% of the weight of the culture medium, inoculated into the solid fermentation medium, mixed, placed under 28-35℃ culture conditions, cultured for 5-7 days, dried at 45-50℃, and then crushed. The powder product contains 1.0×10 9 ~5.0×10 9 cfu / g; solid fermentation medium: corn flour 5-15g, wheat bran 85-95g, inorganic salt nutrient solution ((NH4)2SO4 0.5g, K2HPO4 0.1g, MgSO4.7H2O 0.025g, pH value natural, fixed volume to 1L) 75-85ml, corn flour, wheat bran and inorganic salt nutrient solution are fully stirred and mixed, and the pressure is 1.05kg / cm 2 , sterilize at 121℃ for 30min.

[0022] The components of the culture medium for activating the slant strain in step 1) are as follows: 1% peptone, 0.5% NaCl, 0.5% yeast extract, 1.5% to 2.0% agar, and the rest is water, with a pH of 7.2 to 7.4;

[0023] The culture medium of the shake flask seeds described in step 1) is the slant culture medium described in step 1) without adding agar. The specific operation is as follows: take a loop of activated fresh slant strains and inoculate it into the shake flask seed liquid culture medium with a filling volume of 100mL / 500mL, a rotation speed of 180-200rpm, and a constant temperature culture of 50-55°C for 20-24h, followed by heat treatment in a water bath at 80°C for 10 minutes.

[0024] The culture medium for activating the slant strains in step 2) is a PDA solid culture medium;

[0025] The culture medium of the seeds described in step 2) is the slant culture medium described in step 2) without adding agar. The specific operation is: take a loop of activated fresh slant culture, inoculate it into the seed liquid culture medium, the filling volume is 200mL / 500mL, the rotation speed is 180-200rpm, and the constant temperature culture is 28-35℃ for 48-72h, and the culture is terminated.

[0026] The present invention also provides a composite bacterial agent for high-temperature rapid composting of livestock and poultry excrement prepared by the above method.

[0027] The invention also provides application of the composite bacterial agent in livestock and poultry excrement composting.

[0028] The Brevibacillus borstelensis KY1 and Penicillium chrysogenum MT2 described in the present invention were deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 29, 2024, with the deposit numbers CGMCC No.32115 and CGMCC No.41529, respectively.

[0029] Beneficial effects of the present invention:

[0030] The inventors separated the Brevibacillus Potsdamiensis and Penicillium Chrysogenum from high-temperature livestock and poultry manure compost samples, and there is no antagonism between the two strains. Brevibacillus Potsdamiensis is resistant to high temperatures (the growth temperature range is 35-75°C, and the optimum growth temperature is 55-60°C), has a strong ability to produce protease, and the protease activity is as high as 183.8U / ml; Penicillium Chrysogenum is resistant to high temperatures (the growth temperature range is 25-55°C, and the optimum growth temperature is 35-40°C), has a strong ability to produce cellulase, and the cellulase activity reaches 437.2U / ml.

[0031] The composite microbial agent (Bacillus brevispotensiformis + Penicillium chrysogenum) of the present invention is applied to the composting fermentation of pig manure, which can quickly promote the temperature rise of the compost, quickly start the temperature rise to 55°C to 60°C in 24 hours, and complete the composting maturity in 10 to 18 days, which is 15 to 30 days shorter than the traditional composting time, and significantly shortens the composting time. Compared with the control group, the ammonia release of the treatment group added with the composite microbial agent is reduced by 67.4%, and the release of nitrous oxide is reduced by 44.3%. At the same time, the composting treatment group added with the composite microbial agent has a total nitrogen content increased by 17.1%, a total phosphorus content increased by 29.7%, and a total potassium content increased by 33.3% compared with the control group, thereby improving the quality of the compost.

[0032] It can be seen that the application of the composite microbial agent provided by the present invention in the composting process can not only significantly reduce the emission of ammonia and nitrous oxide, but also significantly shorten the composting maturity time and improve the compost quality, which is suitable for industrial-scale production requirements and is convenient for large-scale promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Temperature changes of microbial agents in livestock and poultry manure composting in Example 3;

[0034] Figure 2 : Changes in ammonia release from the microbial agent in livestock and poultry manure composting in Example 3;

[0035] Figure 3 : Changes in nitrous oxide emissions from microbial agents in livestock and poultry manure composting in Example 3. DETAILED DESCRIPTION

[0036] The following embodiments are intended to further illustrate the present invention, rather than to limit the present invention.

[0037] Example 1: Isolation and identification of thermophilic enzyme-producing strains

[0038] 1. Screening of thermophilic strains

[0039] Take an appropriate amount of pig manure high-temperature compost sample, add it to a conical flask filled with sterilized water, shake it at a constant temperature for 30 minutes, mix it evenly, then draw 5 mL of the suspension, add it to an LB liquid culture medium, and culture it at 50° C. and 180 rpm for 48 to 120 hours to perform a first round of domestication on the thermophilic bacteria; add the first round of domestication culture solution to a new LB liquid culture medium at a volume ratio of 5% for a second round of domestication, and culture it at 55° C. and 180 rpm for 48 to 120 hours; add the second round of domestication culture solution to a new LB culture medium at a volume ratio of 5% for a third round of domestication, and culture it at 60° C. and 180 rpm for 48 to 120 hours, and domesticate it to 80° C. After multiple rounds of high-temperature domestication, a high-concentration domesticated bacterial solution adapted to high-temperature growth is obtained. Take 10 ml of the domesticated bacterial solution, choose an appropriate dilution and repeatedly smear it on various separation plates, and culture it at 50°C for 48 to 96 hours. A total of 12 strains were obtained, among which 5 strains with a large number of bacteria and large colonies were selected as target strains, named KY1, KY2, KY3, MT1, and MT2 respectively. The strains were purified and stored on slant surfaces.

[0040] 2. Rescreening of strains with strong enzyme production ability

[0041] Single colonies of KY1, KY2, KY3, MT1, and MT2 were selected and inoculated into protease medium (casein 1.75%, beef extract 0.5%, starch 0.15%) and cellulase medium (sodium carboxymethyl cellulose 0.75%, peptone 2%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.2%), and samples were taken at 48h and 72h, respectively, to determine the activity of protease and cellulase produced by each strain at different times. The results showed that KY1 had strong protease production ability, with a protease activity of 183.8U / mL at 48h, and MT2 had strong cellulase activity, with a cellulase activity of 437.2U / mL at 72h. KY1 and MT2 were selected as target strains for further identification.

[0042] Table 1 Determination of protease activity of strains (u / ml)

[0043]

[0044] Table 2 Determination of cellulase activity of strains (u / ml)

[0045]

[0046] 3. Identification of strains

[0047] 3.1 KY1 identification

[0048] Morphological identification: The colonies are light yellow, smooth, opaque, and round; Gram staining is positive; the bacteria are rod-shaped, the spores are oval, and there are no flagella. The growth temperature range is 35-75℃, and the optimal growth temperature is 55-60℃.

[0049] Physiological and biochemical tests: catalase, MR test, nitrate reduction reaction using citrate was positive; oxidase, VP test, starch hydrolysis, acid production using glucose, acid production using xylose, acid production using mannitol were negative.

[0050] 16S rDNA sequence analysis: Using the genomic DNA of strain KY1 as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA genes to obtain a 16S rDNA sequence of about 1.4 kb in length, and its 16S rDNA sequence is shown in SEQ ID NO. 1. The sequence was compared with the reported sequences in the NCBI GenBank database by BLAST. The results showed that the homology between KY1 and Brevibacillus borstelensis was more than 99%. Combining morphological characteristics with physiological and biochemical tests, KY1 was identified as Brevibacillus borstelensis.

[0051] 3.2MT2 Identification

[0052] Colony characteristics: The hyphae are initially white, dense and velvety and grow radially, the spores are dark green, the colonies are round and the surface is powdery. The growth temperature range is 25-55℃, and the optimal growth temperature is 35-40℃.

[0053] Morphological characteristics: Under a microscope, it was observed that the apical capsule was divided into stalks, and the conidia at the top of the stalks were broom-shaped, which fell off after maturity to form single spherical spores.

[0054] ITS sequence analysis: Using the genomic DNA of strain MT2 as a template and ITS1 and ITS4 as primers, PCR amplification was performed to obtain an ITS sequence of about 0.5 kb in length, as shown in SEQ ID NO. 2. The sequence was compared with the reported sequences in the NCBI GenBank database, and the results showed that the similarity between MT2 and Penicillium chrysogenum was as high as 99.8%. Combined with the morphological characteristics, MT2 was identified as Penicillium chrysogenum.

[0055] Example 2: Preparation of microbial agents

[0056] 1. Preparation of fermentation product of Brevibacillus Potssteine ​​CGMCC No.32115

[0057] Activation of slant culture: Take the slant culture stored in the refrigerator, inoculate it into a fresh test tube slant culture medium aseptically, and culture it at 50℃ overnight to make a fresh test tube slant culture medium. Slant culture medium: peptone 1%, NaCl 0.5%, yeast extract 0.5%, agar 1.8%, and the rest is water, pH 7.2.

[0058] Shake flask seed culture: Take a loop of activated fresh slant strains and inoculate it into the shake flask seed liquid culture medium with aseptic operation, the filling volume is 100mL / 500mL, the rotation speed is 200rpm, and after constant temperature culture at 55℃ for 24h, heat treatment is carried out in a water bath at 80℃ for 10 minutes. The culture medium of the shake flask seed is the slant culture medium without adding agar.

[0059] Fermentation tank culture: The volume of culture medium in the fermentation tank is 60% of the total volume, the inoculation volume accounts for 0.2% of the culture medium volume, the fermentation temperature is 55°C, the air is ventilated at 200rpm, the ratio of ventilation volume to fermentation liquid volume is 1:1, and the tank pressure is 0.04MPa; the fermentation endpoint is that the spore count is not less than 90% of the total bacterial count, and the number of viable bacteria in the fermentation liquid is 1.0×10 10 ~1.5×10 10 cfu / mL; the mass percentages of the components of the fermentation tank culture medium are: yeast powder 1.5%, peptone 1.0%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.5%, and the rest is water, pH 7.2, pressure 0.14MPa, and sterilization at 121°C for 30min.

[0060] Solid adsorption: The fermentation liquid is adsorbed with wheat bran in a weight ratio of 1:4. After adsorption, it is dehumidified and dried at low temperature and crushed. The bacterial content of the powder product is 1.0×10 9 ~4.0×10 9 cfu / g;

[0061] 2. Preparation of Fermentation Product of Penicillium chrysogenum CGMCC No.41529

[0062] Activation of slant strains: Take the slant strains stored in the refrigerator, inoculate them into fresh test tube slant culture medium under aseptic operation, and culture them at 30℃ for 48h to make fresh test tube slant strains. The slant strain activation medium is PDA medium;

[0063] Shake flask seed culture: Take a loop of activated fresh slant strains and inoculate it into a shake flask seed liquid culture medium with a filling volume of 200mL / 500mL, a rotation speed of 200rpm, and a constant temperature culture of 30℃ for 48h, then terminate the culture. The shake flask seed culture medium is PDA culture medium without agar.

[0064] Solid fermentation culture: The inoculation amount accounts for 4% of the weight of the culture medium, inoculated into the solid culture medium, mixed, placed at 30℃ culture conditions, cultured for 7 days, dried at 45℃, and then crushed. The powder product contains 1.0×10 9 ~5.0×10 9 cfu / g; solid fermentation medium: corn flour 10g, wheat bran 90g, inorganic salt nutrient solution ((NH4)2SO4 0.5g, K2HPO40.1g, MgSO4.7H2O 0.025g, pH value natural, fixed volume to 1L) 80ml, corn flour, wheat bran and inorganic salt nutrient solution are fully stirred and mixed, the pressure is 1.05kg / cm 2 , sterilize at 121℃ for 20min.

[0065] 3. Compounding of microbial agents

[0066] According to the weight percentage, 60% of the fermentation powder of Brevibacillus Potssteine ​​and 40% of the fermentation powder of Penicillium chrysogenum are mixed thoroughly. The total number of viable bacteria of the microbial agent is not less than 10.0×10 8 cfu / g.

[0067] Example 3: Application of microbial agents in livestock and poultry feces composting

[0068] 1. Pig manure composting

[0069] Pig manure, sawdust, and rice husk were mixed with water to make the C / N ratio between 20 and 25, the moisture content was about 60%, and the pH value was natural. The aerobic composting fermentation test was carried out in a 100L composting reactor for 20 days. The properties of the compost raw materials are shown in Table 3. Forced oxygen was supplied by a vortex air pump, and the ventilation volume was set to 0.5L min by a gas rotor flowmeter. -1 kg -1 , ventilate for 10 minutes, and stop for 30 minutes. Stir once a day for the first 7 days of composting, and stir every 3 days from 8 to 15 days of composting, with a speed of 20rpm / min, each time for 30 minutes, and no stirring in the later period. Gas samples are collected once every 1 day, and solid compost samples are collected at 0, 5, 10, 15 and 20 days. When sampling, start the agitator and mix for 30 minutes, then take about 500g of sample from the upper, middle and lower sampling ports respectively, and mix them evenly. The sample is divided into 2 parts, one is stored in a 4℃ refrigerator, and the other is air-dried and ground through a 1mm sieve for later use.

[0070] 2. Experimental Grouping

[0071] The CK group was a control group, and no microbial agent was added to the compost; the T1 group was added with 0.3% of the fermented product of Brevibacillus Potsdam prepared in Example 2; the T2 group was added with 0.3% of the fermented product of Penicillium chrysogenum prepared in Example 2; the T3 group was added with 0.3% of the composite microbial agent (composed of Brevibacillus Potsdam and Penicillium chrysogenum) prepared in Example 2. The physical and chemical properties of the compost raw materials are shown in Table 3.

[0072] Table 3 Physical and chemical properties of compost raw materials

[0073]

[0074] 3. Experimental results

[0075] 1) Temperature changes

[0076] like Figure 1 As shown in the figure, all treatments experienced four stages during the composting process: temperature rise period, high temperature period, temperature drop period, and maturity period. The temperatures of the experimental groups T1, T2, and T3 inoculated with microbial agents reached 60.8℃, 55.3℃, and 61.5℃ on the first day, respectively, while the temperature of the CK treatment rose slowly and entered the high temperature period (55.6℃) on the fourth day. The high temperature was maintained for 6d, 9d, 8d, and 10d for CK, T1, T2, and T3, respectively, and the peak temperatures of the piles were 59.4℃ (7th day), 74.5℃ (5th day), 67.2℃ (6th day), and 77.3℃ (6th day), respectively. It can be seen that compared with the control, the experimental group with the addition of microbial agents can promote the pile to enter the high temperature period in advance and prolong the duration of high temperature, which is beneficial to the decomposition of organic matter in the pile and accelerates the composting process.

[0077] 2) Ammonia release

[0078] The NH3 release of each treatment group is as follows Figure 2 As shown. The peak of NH3 release during composting in the experimental groups T1, T2, and T3 occurred on the 5th day, with the maximum NH3 release values ​​being 32.1ppm, 26.2ppm, and 21.7ppm, respectively. The peak of NH3 release in the control treatment was 66.5ppm on the 7th day. At 13 days of composting, the ammonia release of T1, T2, and T3 was 0, while the ammonia release of the control group CK was 3.2ppm at 15 days of composting. During the entire composting process, compared with the control group, the experimental group with the addition of microbial agents effectively reduced the volatilization of ammonia. The maximum NH3 release of the experimental groups T1, T2, and T3 was reduced by 51.7%, 60.6%, and 67.4% respectively compared with the CK group.

[0079] 3) Nitrous oxide emissions

[0080] N2O emissions during composting Figure 3As shown in the figure, the N2O peaks of each treatment appeared in the cooling or maturity stage of the compost. As the composting process progressed, the N2O release increased first and then decreased. The maximum N2O emissions of the CK, T1, T2, and T3 treatments were 47.1, 29.3, 33.8, and 26.2 mg / d, respectively. Among them, the T3 test group inoculated with composite microbial agents had the best inhibitory effect on N2O emissions. Compared with the control group, the maximum N2O release was reduced by 44.3%. During the entire test, the N2O release rate of the control group that was not inoculated with microbial agents was higher than that of the test group, indicating that the inoculation of microbial agents can effectively reduce the emission of N2O during the composting process.

[0081] 4) Seed germination index

[0082] As can be seen from Table 4, the seed germination index (GI) of different treatment groups showed a gradual increase trend throughout the composting process. At 10 days of composting, the GI values ​​of the experimental groups T1, T2, and T3 reached 78.5%, 73.4%, and 98.7%, respectively. At 15 days of composting, the GI values ​​of the experimental groups T1, T2, and T3 were 99.7%, 92.5%, and 123.8%, respectively. The GI values ​​of the experimental groups far exceeded the compost maturity standard (GI>80%), while the control group was only 70.3%. This shows that adding microbial agents to the compost can accelerate the maturity of the pile, especially the T3 experimental group with the addition of composite microbial agents had the fastest compost maturity, reaching the compost maturity standard at 10 days of composting, significantly shortening the compost maturity time.

[0083] Table 4 Effect of adding microbial agents on seed germination index

[0084]

[0085] 5) Analysis of nutrient elements at 20 days of composting

[0086] The content of nutrient elements is shown in Table 5. It can be seen from Table 5 that after 20 days of composting, the total nitrogen content of CK, T1, T2, and T3 was 1.81%, 2.03%, 1.97%, and 2.12%, respectively. Compared with the control group, the total nitrogen content of the T3 test group increased by 17.1%; the total phosphorus content of CK, T1, T2, and T3 was 1.92%, 2.31%, 2.23%, and 2.49%, respectively. Compared with the control group, the total phosphorus content of the T3 test group increased by 29.7%; the total potassium content of CK, T1, T2, and T3 was 1.47%, 1.65%, 1.53%, and 1.96%, respectively. Compared with the control group, the total potassium content of the T3 test group increased by 33.3%. The inoculation of microbial agents in the test group can effectively increase the content of nitrogen, phosphorus, and potassium in compost.

[0087] At the end of composting, the carbon-nitrogen ratio of all test groups was less than 20, which met the compost maturity standard, while the carbon-nitrogen ratio of the control group was 21.54, which did not meet the compost maturity requirement. At 20 days of composting, the organic matter content of CK, T1, T2, and T3 was 67.24%, 64.32%, 65.71%, and 61.85%, respectively, indicating that inoculation of microbial agents can promote the degradation of organic matter in compost.

[0088] The above data show that adding homemade microbial agents to compost is not only beneficial to increasing the compost temperature, accelerating compost maturity, and reducing the emission of ammonia and nitrous oxide, but also beneficial to improving the quality of compost. In particular, the T3 test group with the addition of composite microbial agents showed the best effect, with the fastest compost maturity. After 10 days of composting, the compost maturity standard was reached (commercially available compost microbial agents generally take about 30 days to reach the compost maturity standard), and the nutrient content was the highest.

[0089] Table 5 Analysis of nutrient elements at 20 days of composting

[0090]

[0091] SEQ ID NO.1: KY1 16SrDNA sequence:

[0092] CAGGCGGTGCCTATTCATTGCAGTTCGAGCGAGTCCCTTTCCGGGGGCTTCGCGGCGG

[0093] AAGGGGTTGAAGTAACACGTAGGCAACCTTGCCCGTAAGCTTCGGGATAACATTGGG

[0094] GAAACTCATGCTAATACCGGATAGGGTCTTCTCTCGCATGAGAGGAGACGGAAAGGT

[0095] GGCGCAAGCTACCACTTACGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAA

[0096] CGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGG

[0097] ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGG

[0098] ACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGT

[0099] TCTGTTGTCAGAGACGAACAAGTACCGTTCGAACAGGGCGGTACCTTGACGGTACCT

[0100] GACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA

[0101] AGCGTTGTCCGGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGT

[0102] GTTAAAGCCCGGGCTCAACCCCGGTTCGCATCGGAAACTGTGTAGCTTTGAGTGCAG

[0103] AAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAC

[0104] ACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGG

[0105] GGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGT

[0106] GTTGGGGGTTTCCAATCCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGG

[0107] GAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT

[0108] GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTTGACATCCC

[0109] GCTGACCGTCCTAGAGATAGGGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATG

[0110] GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCCGCAACGAGCGCAACCT

[0111] TATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACG

[0112] GAGGAAGGCGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG

[0113] TGCTACAATGGCTGGTACAACGGGAAGCTAGCTCGCGAGAGTATGCCAATCTCTTAA

[0114] AACCAGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCT

[0115] AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC

[0116] CCGTCACACCACGGGAGTTTGCAACACCCGGAAGTCGGTGAGGGAAACCGCAAGGA

[0117] GCCAGCCGCCGAAGTGCG

[0118] SEQ ID NO.2: MT2 ITS sequence

[0119] AAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAG

[0120] TGAGGGCCCTCTGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGG

[0121] GCCCGCCTTAACTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGA

[0122] CACCCTCGAACTCTGTCTGAAGATTGTAGTCTGAGTGAAAATATAAATTATTTAAAAC

[0123] TTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATA

[0124] CGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCC

[0125] CTGGTATTCCGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGT

[0126] GTGTTGGGCCCCGTCCTCCGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACC

[0127] GCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGC

[0128] TTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTAGGGATACCCGC

[0129] TGAACTTAAGCATA。

Claims

1. A method for preparing a composite bacterial agent for high-temperature rapid composting of livestock and poultry manure, characterized in that: The fermentation product is prepared by mixing the fermentation product of Brevibacillus Potssteine ​​and the fermentation product of Penicillium chrysogenum.

2. The preparation method according to claim 1, characterized in that: The preservation number of the Brevibacillus Potsdam is CGMCC No.32115, and the preservation number of the Penicillium chrysogenum is CGMCC No.41529.

3. The preparation method according to claim 1 or 2, characterized in that: The fermentation product of Brevibacillus Potssteine ​​contains 1.0×10 9 ~4.0×10 9 cfu / g; The bacterial content of the powder product of Penicillium chrysogenum fermentation product is 1.0×10 9 ~5.0×10 9 cfu / g; Compound microbial agent: 50% to 80% of the fermentation powder of Brevibacillus Potssteine ​​and 20% to 50% of the fermentation powder of Penicillium chrysogenum by weight, and mix thoroughly; the total number of viable bacteria of the microbial agent is not less than 10.0×10 8 cfu / g.

4. The preparation method according to claim 1, 2 or 3, characterized in that: The following steps are involved: Preparation of the fermentation product powder of Brevibacillus Potsdamii: The Brevibacillus Potsdamii is activated by slant strains, cultured in shake flasks, cultured in fermenters, and finally solid adsorbed and crushed; Preparation of powder of fermented product of Penicillium chrysogenum: The Penicillium chrysogenum is activated by slant strain, cultured in shake flask seeds, and then cultured and crushed by solid fermentation.

5. The preparation method according to claim 4, characterized in that: The following steps are involved: 1) Preparation of powder of fermentation product of Brevibacillus Potsdam First, Brevibacillus Potssteine ​​was activated by slant culture and seed cultured in shake flasks; Then, the fermentation tank is cultured: the volume of the culture medium in the fermentation tank is 50-70% of the total volume, the inoculation volume accounts for 0.1-0.3% of the volume of the culture medium, the fermentation temperature is 50-55°C, the air is ventilated at 180-200rpm, the ratio of the ventilation volume to the fermentation liquid volume is 0.9:1-1:1, and the tank pressure is 0.03-0.05MPa; the fermentation end point is that the spore count is not less than 90% of the total bacterial count, and the number of viable bacteria in the fermentation liquid is 1.0×10 10 ~1.5×10 10 cfu / mL; the mass percentage of each component of the fermentation tank culture medium is: yeast powder 1.5-2.0%, peptone 0.7-1.0%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.5%, and the rest is water, pH 7.2-7.4, pressure 0.14MPa, 121℃ sterilization for 30min; Finally, solid adsorption is performed: the fermentation liquid is adsorbed with wheat bran at a weight ratio of 1:3 to 1:5, and then low-temperature dehumidification and drying are performed after adsorption, and the powder product contains bacteria in an amount of 1.0×10 9 ~4.0×10 9 cfu / g; 2) Preparation of powder of Penicillium chrysogenum fermentation product First, the Penicillium chrysogenum was activated by slant culture and seed cultured in shake flasks; Then, solid fermentation culture is carried out: the inoculation amount accounts for 3%-5% of the weight of the culture medium, inoculated into the solid fermentation medium, mixed, placed under 28-35℃ culture conditions, cultured for 5-7 days, dried at 45-50℃, and then crushed. The powder product contains 1.0×10 9 ~5.0×10 9 cfu / g; solid fermentation medium: 5-15g corn flour, 85-95g wheat bran, 75-85ml inorganic salt nutrient solution, stir corn flour, wheat bran and inorganic salt nutrient solution thoroughly, pressure 1.05kg / cm 2 , sterilize at 121℃ for 30min.

6. The preparation method according to claim 5, characterized in that: The components of the culture medium for activating the slant strain in step 1) are as follows: 1% peptone, 0.5% NaCl, 0.5% yeast extract, 1.5% to 2.0% agar, and the rest is water, with a pH of 7.2 to 7.4; The culture medium of the shake flask seeds described in step 1) is the slant culture medium described in step 1) without adding agar. The specific operation is as follows: take a loop of activated fresh slant strains, inoculate it into the shake flask seed liquid culture medium, the filling volume is 100mL / 500mL, the rotation speed is 180-200rpm, and after constant temperature cultivation at 50-55°C for 20-24h, heat treatment is performed in a water bath at 80°C for 10 minutes.

7. The preparation method according to claim 5, characterized in that: The culture medium for activating the slant strains in step 2) is a PDA solid culture medium; The culture medium of the seeds described in step 2) is the slant culture medium described in step 2) without adding agar. The specific operation is: take a loop of activated fresh slant culture, inoculate it into the seed liquid culture medium, the filling volume is 200mL / 500mL, the rotation speed is 180-200rpm, and the constant temperature culture is 28-35℃ for 48-72h, and the culture is terminated.

8. A composite bacterial agent for high-temperature rapid composting of livestock and poultry manure prepared by the method according to any one of claims 1 to 7.

9. Use of the composite bacterial agent according to claim 8 in livestock and poultry manure composting.

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