Livestock manure high-temperature rapid composting composite microbial agent and preparation and application thereof

Abstract

The application discloses a kind of livestock and poultry manure high-temperature rapid composting compound microbial inoculum and preparation and application thereof, the microbial inoculum is prepared by the fermentation product of Brevibacillus borsalensis and the fermentation product of Penicillium chrysogenum mixture.The microbial inoculum can promote rapid composting temperature rise, shorten composting time, while significantly reducing ammonia and nitric oxide emissions during composting, increase the content of nitrogen, phosphorus and potassium in composting, and improve composting quality.It is suitable for industrial scale production requirements, and is easy to use in large areas.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of solid organic waste disposal and resource utilization, and particularly relates to a high-temperature rapid composting compound microbial agent for livestock and poultry manure as well as preparation and application thereof. BACKGROUND

[0002] With the rapid development of China's animal husbandry, the generated breeding waste is increasing day by day. According to statistics, the annual livestock and poultry manure production in China is as high as 4 billion tons. If the large-scale discharge of livestock and poultry manure cannot be quickly and effectively treated, it will seriously occupy and pollute farmland and water bodies, produce odor and cause environmental pollution, and also affect the health and epidemic prevention of the breeding farm and human health. Therefore, the scientific and reasonable utilization of livestock and poultry manure has become a focus of social attention.

[0003] The aerobic composting technology is one of the important technologies for harmless treatment and resource utilization of organic waste such as livestock and poultry manure and crop straw, but there are still key technical bottlenecks to be solved, such as long composting period, odor and greenhouse gas generation during composting process, serious nitrogen loss, etc., which leads to difficult guarantee of fertilizer quality (low degree of maturity, high water content) and high production cost.

[0004] Therefore, in order to realize the harmless treatment and resource utilization of livestock and poultry manure, effectively shorten the composting time, reduce the emission of pollution gas and nitrogen loss, and improve the quality of compost, a high-temperature rapid composting compound microbial agent for livestock and poultry manure is developed, which is of great significance. SUMMARY

[0005] The main purpose of the present application is to provide a high-temperature rapid composting compound microbial agent for livestock and poultry manure as well as a preparation method and application thereof. The microbial agent can promote rapid heating of compost, shorten the composting 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 application is realized by the following way:

[0007] The preparation method of the high-temperature rapid composting compound microbial agent for livestock and poultry manure is prepared by mixing the fermentation product of Brevibacillus borsalensis and the fermentation product of Penicillium chrysogenum.

[0008] Further, the Brevibacillus borsalensis has a preservation number of CGMCC No.32115, and the Penicillium chrysogenum has a preservation number of CGMCC No.41529.

[0009] The Brevibacillus borsalensis fermentation product powder product contains bacteria in an amount of 1.0×10 9 ~ 4.0×10 9between 1.0 x 10 9 and 5.0 x 10 9 cfu / g; the Penicillium chrysogenum fermenting powder product contains bacteria in an amount of between 1.0 x 10

[0010] The compound microbial agent is compounded by 50-80% B. pumilus fermenting powder, 20-50% Penicillium chrysogenum fermenting powder, and fully mixed; the total number of viable bacteria of the microbial agent is not less than 10.0 x 10 8 cfu / g.

[0011] The preparation method comprises the following steps:

[0012] The B. pumilus fermenting powder is prepared by the following steps: the B. pumilus is activated by a slant strain, seed culture is carried out in a flask, then fermentation tank culture is carried out, and finally solid adsorption is carried out and then crushing is carried out.

[0013] The Penicillium chrysogenum fermenting powder is prepared by the following steps: the Penicillium chrysogenum is activated by a slant strain, seed culture is carried out in a flask, and then solid fermentation culture is carried out and crushing is carried out.

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

[0015] 1) Preparation of B. pumilus fermenting powder

[0016] Firstly, the B. pumilus is activated by a slant strain, seed culture is carried out in a flask;

[0017] Then, fermentation tank culture is carried out: the medium loading amount in the fermentation tank is 50-70% of the total volume, the inoculation amount accounts for 0.1-0.3% of the medium volume, the fermentation temperature is 50-55℃, the air is stirred at 180-200 rpm, the ratio of the air volume to the fermentation liquid volume is 0.9:1-1:1, the tank pressure is 0.03-0.05 MPa; the end point of fermentation is that the spore number is not less than 90% of the total bacteria number, and the viable bacteria number of the fermentation liquid is between 1.0 x 10 10 and 1.5 x 10 10 cfu / mL; the mass percentage of each component of the medium in the fermentation tank is as follows: 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 0.14 MPa, 121℃ sterilization for 30 min;

[0018] Finally, solid adsorption is carried out: the fermentation liquid is adsorbed by wheat bran at a weight ratio of 1:3-1:5, low-temperature dehumidification drying is carried out after adsorption, crushing is carried out, and the bacterial content of the powder product is between 1.0 x 10 9 and 4.0 x 10 9 cfu / g;

[0019] 2) Preparation of Penicillium chrysogenum ferment powder

[0020] Firstly, the Penicillium chrysogenum is activated by a slant strain, and seed culture is carried out in a flask;

[0021] Then, solid fermentation culture is carried out: the inoculation amount is 3%-5% of the weight of the culture medium, inoculation is carried out in a solid fermentation culture medium, mixing is carried out, and the culture is placed in a culture condition of 28-35℃ for 5-7d, and then dried at 45-50℃, and then crushed, and the powder product contains a bacterial content of 1.0×10 9 -5.0×10 9 cfu / g; the solid fermentation culture medium is: corn flour 5-15g, wheat bran 85-95g, inorganic salt nutrient solution ((NH4)2SO40.5g, K2HPO40.1g, MgSO4.7H2O 0.025g, pH value is natural, and the volume is constant to 1L) 75-85ml, the corn flour, the wheat bran and the inorganic salt nutrient solution are fully stirred and mixed, sterilization is carried out at 1.05kg / cm 2 , 121℃ for 30min.

[0022] The culture medium for the slant strain activation in step 1) has the following mass percentage: peptone 1%, NaCl 0.5%, yeast paste 0.5%, agar 1.5%-2.0%, and the rest is water, and the pH value is 7.2-7.4;

[0023] The seed culture medium in step 1) is the slant culture medium in step 1) without agar, and the specific operation is as follows: a ring of activated fresh slant strain is inoculated in a seed liquid culture medium, the loading amount is 100mL / 500mL, the rotation speed is 180-200rpm, and the constant temperature culture is carried out at 50-55℃ for 20-24h, and then the water bath heat treatment is carried out at 80℃ for 10min.

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

[0025] The seed culture medium in step 2) is the slant culture medium in step 2) without agar, and the specific operation is as follows: a ring of activated fresh slant strain is inoculated in a seed liquid culture medium, the loading amount is 200mL / 500mL, the rotation speed is 180-200rpm, and the constant temperature culture is carried out at 28-35℃ for 48-72h, and then the culture is terminated.

[0026] The application further provides a livestock and poultry manure high-temperature rapid composting composite microbial agent prepared by the above method.

[0027] The application further provides application of the composite microbial agent in livestock and poultry manure composting.

[0028] The Brevibacillus borstelensis KY1 and the Penicillium chrysogenum MT2 are preserved in China General Microbiological Culture Collection Center on September 29, 2024, and the preservation numbers are CGMCC No. 32115 and CGMCC No. 41529 respectively.

[0029] The beneficial effects of the present application are:

[0030] The Brevibacillus borstelensis and the Penicillium chrysogenum in the present application are isolated from high-temperature livestock manure compost samples, and there is no antagonism between the two strains. The Brevibacillus borstelensis is high-temperature resistant (the growth temperature range is 35-75℃, and the optimum growth temperature is 55-60℃), and has strong protease production ability, with protease activity as high as 183.8 U / ml; the Penicillium chrysogenum is high-temperature resistant (the growth temperature range is 25-55℃, and the optimum growth temperature is 35-40℃), and has strong cellulase production ability, with cellulase as high as 437.2 U / ml.

[0031] The application of the compound microbial agent (Brevibacillus borstelensis + Penicillium chrysogenum) in pig manure compost fermentation can rapidly promote the composting temperature, and the temperature is quickly started to rise to 55-60℃ within 24 hours, and the composting is completed within 10-18 days, which is 15-30 days shorter than the traditional composting time, and the composting time is significantly shortened. Compared with the control group, the ammonia gas release amount of the treatment group added with the compound microbial agent is reduced by 67.4%, and the release amount of nitric oxide is reduced by 44.3%. At the same time, compared with the control group, the total nitrogen content of the composting treatment group added with the compound microbial agent is increased by 17.1%, the total phosphorus content is increased by 29.7%, and the total potassium content is increased by 33.3%, which improves the composting quality.

[0032] Therefore, the application of the compound microbial agent provided by the present application in the composting process can not only significantly reduce the emission of ammonia gas and nitric oxide, but also significantly shorten the composting time, improve the composting quality, and is suitable for industrialized large-scale production requirements and easy to popularize and use in a large area. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Temperature change of the microbial agent in livestock and poultry manure composting in Example 3;

[0034] Figure 2 Ammonia gas release change of the microbial agent in livestock and poultry manure composting in Example 3;

[0035] Figure 3 Nitric oxide emission change of the microbial agent in livestock and poultry manure composting in Example 3. DETAILED DESCRIPTION

[0036] The following embodiments are intended to further illustrate the present application, but not to limit the application.

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

[0038] 1. Screening of thermophilic strains

[0039] An appropriate amount of pig manure compost sample was taken and added to a conical flask containing sterilized water, and incubated at a constant temperature for 30 min. After mixing evenly, 5 mL of the suspension was taken and added to LB liquid medium, and incubated at 50°C, 180 rpm for 48-120 h for the first round of acclimation of thermophilic bacteria. The first round of acclimated culture was added to new LB liquid medium at a volume ratio of 5% for the second round of acclimation, and incubated at 55°C, 180 rpm for 48-120 h. The second round of acclimated culture was added to new LB medium at a volume ratio of 5% for the third round of acclimation, and incubated at 60°C, 180 rpm for 48-120 h. The acclimation was continued until 80°C, and after multiple rounds of high-temperature acclimation, a high-concentration acclimated bacterial solution that could grow at high temperature was obtained. 10 mL of the acclimated bacterial solution was taken and appropriately diluted and repeatedly smeared on various isolation plates, and incubated at 50°C for 48-96 h. A total of 12 strains were obtained, of which 5 strains with more colonies were selected as target strains, designated as KY1, KY2, KY3, MT1, and MT2, respectively. The strains were purified and preserved on slants.

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

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

[0047] 3.1 KY1 identification

[0048] Morphological identification: The colony is light yellow, smooth and opaque, and the colony is round. Gram staining is positive. The cell is bacillary, and the spore is oval and non-flagellated. The growth temperature range is 35-75℃, and the optimum growth temperature is 55-60℃.

[0049] Physiological and biochemical tests: Contact enzyme, MR test, citrate utilization, nitrate reduction reaction are positive. Oxidase, VP test, starch hydrolysis, glucose utilization, xylose utilization and mannitol utilization reactions are negative.

[0050] 16S rDNA sequence analysis: The genomic DNA of strain KY1 was used as a template, and the universal primer of bacterial 16S rDNA gene was used for PCR amplification, and a 16S rDNA sequence of about 1.4 kb was obtained. The 16S rDNA sequence is shown as SEQ ID NO. 1. The sequence was subjected to homology comparison with the reported sequences in the NCBI GenBank database by BLAST, and the results showed that the homology of KY1 and Brevibacillus borstelensis was more than 99%. Combined with morphological characteristics and physiological and biochemical tests, KY1 was identified as Brevibacillus borstelensis.

[0051] 3.2 MT2 identification

[0052] Colony characteristics: The mycelium is initially white and dense and grows radially, and the spores are dark green. The colony is round and the surface is powdery. The growth temperature range is 25-55℃, and the optimum growth temperature is 35-40℃.

[0053] Morphological characteristics: Under a microscope, it was observed that the top cyst divided into small branches, and the small branch tip divided into spores in a broom shape, which were shed to form single spherical spores after maturation.

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

[0055] Example 2: Preparation of microbial inoculant

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

[0057] Slope strain activation: Take the refrigerator to store the slope strain, and inoculate it in the fresh test tube slope culture medium under aseptic operation. Incubate it at 50°C overnight to make fresh test tube slope strain. The slope culture medium contains 1% proteose peptone, 0.5% NaCl, 0.5% yeast extract, 1.8% agar, and the rest is water, with pH 7.2.

[0058] Seed culture in a shake flask: Take the activated fresh slope strain, and inoculate it in the seed liquid culture medium in a shake flask under aseptic operation. The loading capacity is 100 mL / 500 mL, and the rotation speed is 200 rpm. Incubate it at 55°C for 24 h, and then heat treat it in a water bath at 80°C for 10 min. The seed culture medium in a shake flask is the slope culture medium without agar.

[0059] Culture in a fermentation tank: The medium loading capacity in the fermentation tank is 60% of the total volume, and the inoculation amount is 0.2% of the medium volume. The fermentation temperature is 55°C, and the air is blown at a rotation speed of 200 rpm for stirring culture. The ratio of the air volume to the fermentation liquid volume is 1:1, and the tank pressure is 0.04 MPa. The end point of fermentation is that the spore number is not less than 90% of the total bacterial number, and the viable bacterial number in the fermentation liquid is between 1.0×10 10 and 1.5×10 10 cfu / mL. The mass percentage of each component in the medium in the fermentation tank is as follows: 1.5% yeast powder, 1.0% proteose peptone, 0.2% potassium dihydrogen phosphate, 0.5% magnesium sulfate, the rest is water, with pH 7.2, pressure 0.14 MPa, and sterilization at 121°C for 30 min.

[0060] Solid adsorption: The fermentation liquid is adsorbed with wheat bran at a weight ratio of 1:4. After adsorption, low-temperature extraction, drying, and crushing, the bacterial content in the powder product is between 1.0×10 9 and 4.0×10 9 cfu / g.

[0061] 2. Preparation of the fermentation product of Penicillium chrysogenum CGMCC No. 41529

[0062] Slope strain activation: Take the refrigerator to store the slope strain, and inoculate it in the fresh test tube slope culture medium under aseptic operation. Incubate it at 30°C for 48 h to make fresh test tube slope strain. The slope strain activation culture medium is PDA culture medium.

[0063] Seed culture in a shake flask: Take the activated fresh slope strain, and inoculate it in the seed liquid culture medium in a shake flask under aseptic operation. The loading capacity is 200 mL / 500 mL, and the rotation speed is 200 rpm. Incubate it at 30°C for 48 h, and then terminate the culture. The seed culture medium in a shake flask is PDA culture medium without agar.

[0064] Solid fermentation culture: inoculation amount is 4% of medium weight, inoculated into solid medium, mixed, placed in 30°C culture condition, cultured for 7d, dried at 45°C, then crushed, the powder product contains bacteria between 1.0x10 9 ~5.0x10 9 cfu / g; solid fermentation medium is: corn flour 10g, wheat bran 90g, inorganic salt nutrient solution ((NH4)2SO40.5g, K2HPO40.1g, MgSO4.7H2O 0.025g, pH value is natural, constant volume to 1L) 80ml, corn flour, wheat bran and inorganic salt nutrient solution are fully stirred and mixed, pressure 1.05kg / cm 2 , sterilized at 121°C for 20min.

[0065] 3. Microbial inoculant compound

[0066] According to weight percentage, Bacillus pumilus fermentation powder 60%, Penicillium chrysogenum fermentation powder 40%, fully mixed. 8 The total number of viable bacteria of the microbial inoculant is not less than 10.0x10

[0067] Example 3: Application of microbial inoculant in livestock and poultry manure composting

[0068] 1. Pig manure composting

[0069] Pig manure, sawdust, rice husk and water were mixed uniformly to make C / N ratio between 20-25, water content about 60%, pH value natural, 20d aerobic composting fermentation test was carried out in a 100L composting reactor, the properties of the composting raw materials are shown in Table 3. Forced oxygen supply was carried out by vortex air pump, and the air flow was set to 0.5L / min -1 kg -1 by gas rotor flowmeter, aeration for 10min, stop for 30min. Composting for 7d, open the stirring once a day, composting for 8d-15d, open the stirring once every 3d, speed 20rpm / min, each time 30min, no stirring in later period. Gas samples were collected every 1d, solid compost samples were collected on 0, 5, 10, 15 and 20d. When sampling, first start the stirrer for 30min, then take about 500g samples from the upper, middle and lower sampling ports respectively, mix uniformly. Divide the samples into two parts, one stored in a 4°C refrigerator, the other dried and ground through a 1mm sieve for standby.

[0070] 2. Test grouping

[0071] The CK group served as the control group, with no microbial inoculants added to the compost. In experiment T1, 0.3% of the *Bacillus brevis* ferment prepared in Example 2 was added to the compost. In experiment T2, 0.3% of the *Penicillium chrysogenum* ferment prepared in Example 2 was added to the compost. In experiment T3, 0.3% of the compound microbial inoculant prepared in Example 2 (composed of *Bacillus brevis* and *Penicillium chrysogenum*) was added to the compost. The physicochemical properties of the compost raw materials are shown in Table 3.

[0072] Table 3 Physicochemical properties of compost raw materials

[0073]

[0074] 3. Experimental Results

[0075] 1) Temperature change

[0076] like Figure 1 As shown, all treatments in the composting process went through four stages: heating period, high-temperature period, cooling period, and maturation period. The experimental groups T1, T2, and T3, inoculated with microbial agents, reached temperatures of 60.8℃, 55.3℃, and 61.5℃ on day 1, respectively, while the control (CK) treatment heated up more slowly, reaching the high-temperature period (55.6℃) on day 4. The duration of high temperature for CK, T1, T2, and T3 was 6 days, 9 days, 8 days, and 10 days, respectively, with peak temperatures of 59.4℃ (day 7), 74.5℃ (day 5), 67.2℃ (day 6), and 77.3℃ (day 6), respectively. Therefore, compared to the control, the experimental groups with added microbial agents promoted the earlier entry into the high-temperature period and prolonged its duration, which is beneficial for the decomposition of organic matter in the compost and accelerates the composting process.

[0077] 2) Ammonia release

[0078] NH3 release amounts in each treatment group are as follows Figure 2 As shown in the figure, the peak NH3 release in experimental groups T1, T2, and T3 during composting occurred on day 5, with maximum NH3 release values ​​of 32.1 ppm, 26.2 ppm, and 21.7 ppm, respectively. In contrast, the peak NH3 release in the control group occurred on day 7, at 66.5 ppm. At day 13 of composting, the ammonia release in T1, T2, and T3 was zero, while the ammonia release in the control group (CK) was 3.2 ppm at day 15. Throughout the composting process, compared to the control group, the experimental groups with added microbial agents effectively reduced ammonia volatilization. The maximum NH3 release in experimental groups T1, T2, and T3 was reduced by 51.7%, 60.6%, and 67.4% compared to the CK group, respectively.

[0079] 3) Nitrous oxide emissions

[0080] N2O emissions during composting, such as Figure 3The N2O peak of each treatment appeared in the cooling or composting stage of the compost. With the progress of the composting process, the N2O emission showed a trend of first increasing and then decreasing. The maximum N2O emission of CK, T1, T2, and T3 was 47.1, 29.3, 33.8, and 26.2 mg / d, respectively. The T3 test group with inoculation of compound microbial inoculant had the best N2O emission inhibition effect, and the maximum N2O emission was reduced by 44.3% compared with the control group. During the entire test process, the N2O emission rate of the control group without microbial inoculant was higher than that of the test group, indicating that inoculation of microbial inoculant could effectively reduce N2O emission during composting.

[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 increasing trend during the entire composting process. At 10 days of composting, the GI values of test groups T1, T2, and T3 were 78.5%, 73.4%, and 98.7%, respectively. At 15 days of composting, the GI values of test groups T1, T2, and T3 were 99.7%, 92.5%, and 123.8%, respectively, and the GI values of the test groups were much higher than the composting maturity standard (GI > 80%), while the control group was only 70.3%. This indicates that the addition of microbial inoculant in composting can accelerate the composting maturity, especially the addition of compound microbial inoculant T3 test group, which reaches the composting maturity standard at 10 days of composting, significantly shortening the composting time.

[0083] Table 4 Effect of microbial inoculant addition on seed germination index

[0084]

[0085] 5) Nutrient element analysis at 20 days of composting

[0086] The nutrient element content is shown in Table 5. As can be seen from Table 5, at 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, which was 17.1% higher than that of the control group. The total phosphorus content of CK, T1, T2, and T3 was 1.92%, 2.31%, 2.23%, and 2.49%, respectively, which was 29.7% higher than that of the control group. The total potassium content of CK, T1, T2, and T3 was 1.47%, 1.65%, 1.53%, and 1.96%, respectively, which was 33.3% higher than that of the control group. Inoculation of microbial inoculant can effectively increase the content of nitrogen, phosphorus, and potassium in composting.

[0087] At the end of composting, the C / N ratio of all test groups was less than 20, meeting the composting maturity standard, while the C / N ratio of the control group was 21.54, which did not meet the 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 inoculating microbial inoculants can promote the degradation of organic matter in compost.

[0088] The above data shows that adding self-made microbial inoculants to compost not only helps to increase the composting temperature, accelerate composting maturity, reduce ammonia and nitrous oxide emissions, but also helps to improve compost quality, especially the T3 test group with the addition of compound microbial inoculants has the best effect, the fastest composting speed, and the highest nutrient content.

[0089] Table 5 Nutrient element analysis at 20 days of composting

[0090]

[0091] SEQ ID NO. 1: 16S rDNA sequence of KY1:

[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] GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT

[0111] TATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACG

[0112] GAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG

[0113] TGCTACAATGGCTGGTACAACGGGAAGCTAGCTCGCGAGAGTATGCCAATCTCTTAA

[0114] AACCAGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCT

[0115] AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC

[0116] CCGTCACACCACGGGAGTTTGCAACACCCGGAAGTCGGTGAGGGAAACCGCAAGGA

[0117] GCCAGCCGCCGAAGTGCG

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

[0119] AAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAG

[0120] TGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGG

[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 livestock and poultry manure high-temperature rapid composting composite microbial agent, characterized in that, is prepared by mixing the fermentation product of Brevibacillus pozeanensis and the fermentation product of Penicillium chrysogenum; the Brevibacillus pozeanensis has the preservation number of CGMCC No.32115, and the Penicillium chrysogenum has the preservation number of CGMCC No.41529; the bacterial content of the Brevibacillus pozeanensis fermentation product powder is between 1.0×10 9 ~4.0×10 9 cfu / g; The Penicillium chrysogenum fermentation product powder contains bacteria in an amount of 1.0×10 9 ~5.0×10 9 cfu / g; the compound microbial agent is compounded by 50%~80% Bacillus pumilus fermentation product powder, 20%~50% Penicillium chrysogenum fermentation product powder, and fully mixed; the total number of live bacteria of the microbial agent is not less than 10.0×10 8 cfu / g.

2. The production method according to claim 1, characterized by, It comprises the following steps: Preparation of B. pottsani ferment powder: B. pottsani is activated by slant strain, seed culture in a flask, then fermented in a fermentor, and finally crushed after solid adsorption; Preparation of P. chrysogenum ferment powder: P. chrysogenum is activated by slant strain, seed culture in a flask, then solid fermentation culture and crushing.

3. The preparation method according to claim 2, characterized in that, It comprises the following steps: 1) Preparation of B. pottsani ferment powder First, B. pottsani is activated by slant strain, seed culture in a flask; Then the fermentation tank culture is carried out: the medium loading amount in the fermentation tank is 50-70% of the total volume, the inoculation amount accounts for 0.1-0.3% of the medium volume, the fermentation temperature is 50-55℃, the culture is stirred by air at 180-200 rpm, the ratio of the air volume to the fermentation liquid volume is 0.9:1-1:1, and the tank pressure is 0.03-0.05 MPa; the fermentation end point is that the spore number is not less than 90% of the total bacteria number, and the viable bacteria number in the fermentation liquid is between 1.0×10 10 and 1.5×10 10 cfu / mL; the mass percentage of each component in the medium of the fermentation tank is as follows: 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 0.14 MPa, sterilization at 121℃ for 30 min; Finally, solid adsorption: the fermentation broth is adsorbed by wheat bran at a weight ratio of 1:3-1:5, and after adsorption, low-temperature dehumidification drying, crushing, the powder contains 1.0×10 9 ~4.0×10 9 cfu / g of bacteria; 2) Preparation of P. chrysogenum ferment powder First, P. chrysogenum is activated by slant strain, seed culture in a flask; Then solid fermentation culture is carried out: inoculation amount is 3%-5% of medium weight, inoculation is carried out in solid fermentation medium, mixing is uniform, and culture is carried out under 28-35℃ culture condition for 5-7d, 45-50℃ drying is carried out, then crushing is carried out, and the powder product contains 1.0×10 9 -5.0×10 9 cfu / g; the solid fermentation medium is: corn flour 5-15g, wheat bran 85-95g, inorganic salt nutrient solution 75-85ml, corn flour, wheat bran and inorganic salt nutrient solution are fully stirred and mixed, pressure is 1.05kg / cm 2 , 121℃ sterilization is carried out for 30min.

4. The preparation method according to claim 3, characterized in that, In step 1), the culture medium for slant strain activation comprises the following components: 1% of proteose peptone, 0.5% of NaCl, 0.5% of yeast extract, 1.5-2.0% of agar, and the rest is water, with pH being 7.2-7.4; In step 1), the seed culture medium is prepared by not adding agar to the slant culture medium in step 1), and the specific operation is as follows: one ring of activated fresh slant strain is inoculated into a seed liquid culture medium in a flask, with a loading amount of 100 mL / 500 mL, a rotation speed of 180-200 rpm, and constant temperature culture at 50-55℃ for 20-24 h, and then hot treatment at 80℃ for 10 min.

5. The preparation method according to claim 3, characterized in that, In step 2), the culture medium for slant strain activation is PDA solid culture medium; In step 2), the seed culture medium is prepared by not adding agar to the slant culture medium in step 2), and the specific operation is as follows: one ring of activated fresh slant strain is inoculated into a seed liquid culture medium in a flask, with a loading amount of 200 mL / 500 mL, a rotation speed of 180-200 rpm, and constant temperature culture at 28-35℃ for 48-72 h.

6. The compound microbial agent for high-temperature rapid composting of livestock and poultry manure prepared by the method of any one of claims 1-5.

7. The application of the compound microbial agent of claim 6 in livestock and poultry manure composting.

Images