CN115725453A - Application of bacillus amyloliquefaciens and microbial inoculum thereof in treatment of livestock and poultry manure by hermetia illucens - Google Patents
Application of bacillus amyloliquefaciens and microbial inoculum thereof in treatment of livestock and poultry manure by hermetia illucens Download PDFInfo
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Abstract
The invention discloses application of Bacillus amyloliquefaciens and a bacterial agent thereof in treating livestock and poultry manure by black soldier fly, and relates to the technical field of biological strain utilization and development, wherein Bacillus amyloliquefaciens LYM152 is stored in the common microorganism center of China Committee for culture Collection of microorganisms in 2019, 9 and 17 months, is classified and named as Bacillus amyloliquefaciens LYM152, has the storage number of CGMCC No.18497, and has the storage address of the institute of microbiology of China academy of sciences No. 3 of China, north Chen of south China, no.1 North of the Korean district, beijing city. The bacillus amyloliquefaciens disclosed by the invention has the characteristics of antibiosis, deodorization and high digestive enzyme activity, the microbial inoculum has a good deodorization effect in the treatment of the livestock and poultry manure, and the degradation effect of the livestock and poultry manure is more obvious by combining with the black soldier fly to strengthen the degradation effect.
Description
Technical Field
The invention relates to the technical field of biological strain utilization and development, in particular to application of bacillus amyloliquefaciens and a microbial inoculum thereof in treatment of livestock and poultry manure by hermetia illucens.
Background
The domestic livestock and poultry manure discharge amount is large, and the method is a main source of agricultural non-point source pollution. According to the second national pollution source general survey and measurement, the annual output of the livestock and poultry manure in 2019 in China is 30.5 hundred million tons. The daily excrement yield of the intensive farm is huge, the discharged materials contain a large amount of organic matters, pathogenic microorganisms, parasitic ova, heavy metals, additives, nitrogen, phosphorus, odor and the like, and the livestock and poultry excrement causes great ecological pressure on agricultural production and rural life. The traditional treatment method has the advantages of simple technology and low investment, but has the problems of low resource level, secondary pollution, resource waste and the like, and the defects of high investment and low income exist in mainstream aerobic composting and anaerobic biogas fermentation.
In recent years, techniques for treating livestock and poultry manure by using insects represented by hermetia illucens are receiving wide attention. The black soldier fly (Hermetia illucens L.) is a saprophytic insect, and the larva has the advantages of high feeding capacity, high feed intake, strong stress resistance, no disease propagation, rapid propagation, low feeding cost and the like, and has strong ingestion capability on livestock and poultry manure. After the livestock and poultry manure is converted by larvae, the polypide can be used for high-value animal protein feed, and the polypide can be used for producing high-value-added organic fertilizer. In 2013, the FAO lists the hermetia illucens in one of the most main protein feed instead of insects, and the black hermetia illucens are popularized and applied all over the world. At present, the technology for treating the livestock and poultry manure by using hermetia illucens is gradually accepted, but various problems such as heavy odor, pathogenic bacteria elimination and the like still exist in the application, for example, some functional microorganisms are particularly screened, and the technology plays an important role in the treatment process of the livestock and poultry manure.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) belongs to Bacillus, can generate various digestive enzymes such as amylase, protease and the like, can generate lipopeptide antibiotics including iturins (iturins), fengycin (fengycins) and surfactin, has broad-spectrum antibacterial activity, can inhibit the growth of various plant pathogenic bacteria, has the function of promoting plant growth, and is widely applied to the fields of biological control, organic fertilizer production, soil improvement and the like.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the bacillus amyloliquefaciens provided by the invention has the characteristics of antibiosis, deodorization and high digestive enzyme activity, and the application effect of the microbial inoculum in the black soldier fly treatment of livestock and poultry manure is obvious.
The technical scheme is as follows: in order to achieve the aim, the Bacillus amyloliquefaciens LYM152 is separated from the intestinal tract of hermetia illucens, is stored in the common microorganism center of China Committee for culture Collection of microorganisms in 2019, 9 and 17 days, is classified and named as Bacillus amyloliquefaciens LYM152, and has the storage number of CGMCC NO.18497; the 16S rRNA gene sequence of the strain is shown in SEQ NO. 1.
An agent comprising bacillus amyloliquefaciens LYM152 of claim 1.
A method for preparing the microbial inoculum, comprising the following steps:
s1, strain activation: picking strains, streaking and inoculating the strains to a solid slant culture medium, and culturing for 16-24 h at 28-37 ℃ in a constant temperature incubator;
s2, liquid seed preparation: inoculating the activated bacillus amyloliquefaciens LYM152 in the step S1 into a container filled with a liquid seed culture medium, sealing the container with gauze, and carrying out shake culture for 16-24 h at the temperature of 28-37 ℃ and the speed of 160-220 r/min to obtain liquid seeds;
s3, liquid fermentation: inoculating the liquid seeds prepared in the step S2 into a fermentation culture medium according to the inoculation amount of 1-10% of the volume ratio, rotating at the speed of 200-700 r/min, controlling the dissolved oxygen at 20-40%, controlling the pH at 6.0-7.5, controlling the temperature at 28-37 ℃, and performing aeration culture in a 10L fermentation tank for 24-48 h to obtain the liquid microbial inoculum of the bacillus amyloliquefaciens LYM152.
Further, the microbial inoculum can be concentrated to prepare liquid microbial inoculum or freeze-dried to prepare solid bacterial powder, and the viable count of the bacillus amyloliquefaciens LYM152 in the microbial inoculum is not less than 10 8 cfu/mL or 10 8 cfu/g。
Further, the solid slant culture medium is: 1-3 g/L of glucose, 3-7 g/L of yeast extract powder, 5-15 g/L of peptone, 2-7 g/L of NaCl, 15-20 g/L of agar, distilled water for dissolving to a constant volume of 1L, pH of 7.0-7.2, and high-pressure steam sterilization at 121 ℃ for 15-20 min.
Further, the liquid seed culture medium is: 5-15 g/L of glucose, 1-5 g/L of beef extract, 3-7 g/L of yeast extract powder and KH 2 PO 2 0.5～2.0g/L，MgSO 4 ·7H 2 O 0.1～1.0g/L，CaCl 2 ·2H 2 O 0.1～0.5g/L，(NH4) 2 SO 2 1-5 g/L, dissolving distilled water to constant volume to 1L, pH 7.0-7.2, and sterilizing with high pressure steam at 121 ℃ for 15-20 min.
Further, the fermentation medium is: 15-25 g/L of glucose, 1-5 g/L of beef extract, 0.5-2.0 g/L of yeast extract powder and KH 2 PO 4 0.5～2.0g/L，MgSO 4 ·7H 2 O 0.2～0.6g/L，CaCl 2 ·2H 2 0.1-0.5 g/L of O, 1-5 g/L of ammonium citrate, 5-15 g/L of corn steep liquor and FeSO 4 0.5-2.0 mg/L vitaminShengsu B 1 1-3 mg/L, dissolving distilled water to constant volume to 1L, pH 7.0-7.2, and sterilizing with high pressure steam at 121 ℃ for 15-20 min.
The microbial inoculum is applied to the treatment of livestock and poultry manure by using hermetia illucens.
The application of the bacillus amyloliquefaciens and the microbial inoculum thereof in the treatment of livestock and poultry manure by using the hermetia illucens at least has the following technical effects:
(1) The bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497) is separated from the intestinal tract of hermetia illucens, and has the advantages of high propagation speed, high fermentation density, rich digestive enzyme systems, broad-spectrum antibacterial property, strong adverse environment tolerance and the like;
(2) The bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497) can be applied to deodorization of livestock and poultry manure, effectively reduces the release amount of malodorous gas and volatile organic compounds in the livestock and poultry manure, and reduces the generation of odor from the source; stable deodorization effect, less influence of temperature and better deodorization effect under normal temperature condition
(3) The bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497) can be applied to black soldier fly treatment of livestock and poultry manure, can stably colonize for a long time in a black soldier fly treatment livestock and poultry manure system, and plays roles in degrading organic matters, inhibiting pathogenic bacteria, reducing odor generation and release and the like, so that the aims of accelerating livestock and poultry manure reduction, reducing the death rate of black soldier fly, improving the growth speed of black soldier fly, shortening the pupation-ahead time of black soldier fly and the like are fulfilled;
(4) The bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497) has the characteristics of simple and efficient preparation process and stable and reliable product, and has the obvious technical advantage of easy commercialization.
Drawings
FIG. 1 shows the colony morphology of Bacillus amyloliquefaciens LYM 152;
FIG. 2 shows a microscopic pattern of Bacillus amyloliquefaciens LYM 152;
FIG. 3 is an electron micrograph of Bacillus amyloliquefaciens LYM 152;
FIG. 4 is a phylogenetic tree of Bacillus amyloliquefaciens LYM152 based on the 16S rRNA sequence.
Detailed Description
The principles and features of the present invention will be described with reference to fig. 1-4, which are provided for illustration only and are not intended to limit the scope of the invention.
Example 1: separating and screening Bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497).
1. Separation: the strain LYM152 is obtained by separation by a dilution coating method, and the specific separation method comprises the following steps: selecting 20-30 black soldier fly larvae of 7 days old fed by livestock and poultry manure, dissecting and taking intestinal tracts, adding 1mL of sterile normal saline to prepare bacterial suspension, and inoculating 100mL of sterilized livestock and poultry manure: clear rumen fluid =1:1 for 5 days, taking 1mL of gradient dilution, coating on an LB medium plate, placing at 30 ℃ for 3 days, carrying out plate streaking on colonies with different forms on the plate medium, purifying the obtained strains, and respectively numbering and storing. Wherein the LB culture medium comprises the following components: 5g/L yeast extract powder, 10g/L tryptone, 10g/L NaCl, 18g/L agar, pH7.2, and high pressure steam sterilization at 121 deg.C for 20min.
2. Primary screening: respectively inoculating the separated strains into an LB liquid culture medium, placing the LB liquid culture medium in a shaking table with the temperature of 30 ℃ and the speed of 180r/min for shaking culture for 48h, respectively inoculating 10mL of bacterial liquid into a plastic box filled with 200g of livestock and poultry manure according to the inoculation amount of 5%, uniformly mixing the bacterial liquid with an aseptic glass rod, sealing a preservative film, standing and culturing for 1d at the temperature of 30 ℃, replacing the bacterial liquid with sterile water for a control group, judging the deodorization effect of microorganisms by a sensory method, dividing the deodorization effect into 5 odor grades according to non-odor 'M0', micro-odor 'M1', more-odor 'M2', very-odor 'M3' and foul-odor 'M4', and selecting the strains with obvious deodorization effect (the effect reaches non-odor 'M0' and micro-odor 'M1') to carry out a rescreening test.
3. Re-screening: weighing 200g of livestock and poultry manure, placing the livestock and poultry manure into a plastic box, inoculating a culture solution of a primary-screened strain according to an inoculation amount of 5%, fully and uniformly mixing, replacing a bacterial solution with equivalent sterile water as a blank control, placing a 50mL small beaker filled with a 20mL 2% boric acid solution in each plastic box for absorbing ammonia, sealing the plastic boxes by using a preservative film, standing and culturing at 30 ℃, measuring the release amount of the ammonia by using an acid-base titration method on day 3, and repeating the steps for 3 times. And comparing and analyzing with a blank control group, calculating the removal rate of ammonia gas, and screening the strain with the best deodorization effect for purification culture and preservation.
The ammonia gas removal rate calculation formula is as follows: the ammonia gas removal rate = difference between the ammonia gas release amount of the blank control group and the ammonia gas release amount of the test group/ammonia gas release amount of the blank control group multiplied by 100%, and a strain LYM152 which is derived from black soldier fly intestinal tracts and can obviously deaminate and deodorize is obtained through screening.
Example 2: identification of Bacillus amyloliquefaciens LYM152 (CGMCC NO. 18497).
1. Morphological characteristics and physiological and biochemical characteristics
After the strain LYM152 is cultured on LB culture medium for 48h, colonies are white, round, opaque, flat and slightly raised on the surface, sticky when wetted, and do not produce pigments. Gram staining is positive, the thallus is rod-shaped, two ends are blunt, the length is different, no capsule exists, endogenic spores can be formed, the spores are oval, and the thallus is mesogenic or subterminal and has motility. The strain LYM152 can grow in 6.5% sodium chloride and can decompose and utilize various carbon sources such as glucose, mannose, mannitol, trehalose and the like, and beta-mannosidase, N-acetyl-beta-D-glucamine enzyme, kanamycin tolerance and the like are negative through identification of a VITEK2 Compact full-automatic microbiological analysis system. The colony morphology of strain LYM152 is shown in FIG. 1, the microscopic morphology is shown in FIG. 2, and the electron micrograph is shown in FIG. 3.
The physiological and biochemical detection results show that: can decompose glucose, maltose, mannose, mannitol and inositol, and cannot utilize lactose and arabinose. The results of the starch hydrolysis, gelatin liquefaction, nitrate reduction, and 6.5% NaCl growth tests were positive, and the results of the acetomethyl methanol (V-P) test, the phenylalanine deaminase test, the indole test, the Methyl Red (MR) test, and the hydrogen sulfide test were negative, as shown in Table 1.
TABLE 1 physiological and biochemical identification results of the Strain LYM152
Note: "+" indicates a positive reaction, and "-" indicates a negative reaction
2. Phylogenetic analysis
The 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing has a length of 1491bp, BLAST comparison is carried out on GenBank, relevant sequence information of homologous strains is obtained by screening, phylogenetic analysis is carried out, a phylogenetic tree based on the 16S rRNA sequence is shown in figure 4, the base similarity of the strain and Bacillus amyloliquefaciens (EF 423607) reaches 99 percent, and the strain LYM152 is identified as the Bacillus amyloliquefaciens by combining morphological and physiological and biochemical indexes.
3. Genetic stability
The strain LYM152 is continuously subcultured on a solid slant culture medium for 50 times by adopting a streak method, and the morphology, the growth speed and the ammonia gas removal capacity of the strain are measured, so that the strain has no obvious difference from primary strains, and has good genetic stability.
4. The strain is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms (CCM) in 2019, 9 and 17 months, is classified and named as Bacillus amyloliquefaciens LYM152, has the preservation number of CGMCC No.18497, and has the preservation address of China academy of sciences institute of microbiology No. 3 of North Chen Xilu No.1 of the open area of Beijing City.
The 16S rRNA sequence of the Bacillus amyloliquefaciens LYM152 is as follows:
GAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGG
example 3: a method for preparing bacterial preparation containing Bacillus amyloliquefaciens LYM152 is provided.
Specifically, the preparation method of the microbial inoculum comprises the following steps:
s1, strain activation: selecting strains, streaking and inoculating the strains to a solid slant culture medium, culturing for 18h in a constant-temperature incubator at 30 ℃, and transferring liquid seeds after a large number of somatic cells grow out of the slant;
the solid slant culture medium is 2g/L of glucose, 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl and 18g/L of agar, the volume is up to 1L when the distilled water is dissolved, the pH value is 7.0, and the solid slant culture medium is sterilized by high-pressure steam at 121 ℃ for 20min.
S2, preparing liquid seeds: selecting activated Bacillus amyloliquefaciens LYM152, inoculating into a container filled with liquid seed culture medium, sealing with eight layers of gauze, shake-culturing at 30 deg.C and 180r/min for 18h to obtain large amount of thallus, and collecting liquid seed;
the liquid seed culture medium comprises 10g/L glucose, 3g/L beef extract, 5g/L yeast extract powder and KH 2 PO 2 1g/L，MgSO 4 ·7H 2 O 0.5g/L，CaCl 2 ·2H 2 O 0.3g/L，(NH4) 2 SO 2 3g/L, dissolving with distilled water to constant volume of 1L, pH7.0, high pressure at 121 deg.CSteam sterilizing for 20min.
S3, liquid fermentation: inoculating the liquid seeds prepared in the step 2 into a fermentation culture medium according to the inoculation amount of 5 percent of the volume ratio, rotating speed is 200-700 r/min, dissolved oxygen is controlled to be 30 percent, pH is 7.0, the temperature is 30 ℃, aeration culture is carried out for 36 hours, and the thallus concentration reaches 1 multiplied by 10 8 collecting the fermentation liquid above cfu/mL to obtain liquid microbial inoculum of Bacillus amyloliquefaciens LYM152, subpackaging, sealing, and refrigerating at 4 deg.C for use.
The fermentation medium comprises 20g/L of glucose, 3g/L of beef extract, 1g/L of yeast extract powder and KH 2 PO 4 1g/L，MgSO 4 ·7H 2 O 0.4g/L，CaCl 2 ·2H 2 0.3g/L of O, 3g/L of ammonium citrate, 10g/L of corn steep liquor and FeSO 4 1mg/L, vitamin B 1 2mg/L, dissolving with distilled water to a constant volume of 1L, pH7.0, and sterilizing with high pressure steam at 121 deg.C for 20min.
When in use, the microbial inoculum can be concentrated into liquid microbial inoculum or freeze-dried into solid bacterial powder according to requirements, and the viable count of the bacillus amyloliquefaciens LYM152 in the microbial inoculum is not less than 1 multiplied by 10 8 cfu/mL or 10 8 cfu/g。
Example 4: the application of the microbial inoculum containing the bacillus amyloliquefaciens LYM152 in deodorizing the livestock and poultry manure.
Respectively inoculating 10mL of Bacillus amyloliquefaciens LYM152 microbial inoculum (microbial inoculum in example 2) into a 2L volume lunch box filled with 1000g of chicken manure, pig manure, cow manure and kitchen waste, uniformly mixing, sealing by using a preservative film, adding 10mL of sterile water into a control group to replace the bacterial liquid, setting 3 times for each group, standing at room temperature for 5 days, and measuring the ammonia concentration, wherein the results are shown in Table 2. As can be seen from Table 1, after 5 days of treatment by inoculating the strain LYM152 in the livestock and poultry manure, the concentration of ammonia gas generated by the chicken manure, the pig manure, the cow manure and the kitchen waste is obviously reduced, and a good deodorization effect is shown.
TABLE 2 deodorizing effect of Bacillus amyloliquefaciens LYM152 on livestock and poultry feces
| Measurement index (Ammonia concentration: ppm) | Chicken manure | Pig manure | Cow dung | Kitchen waste |
| Control | 134 | 129 | 116 | 93 |
| Treatment group | 31 | 40 | 23 | 39 |
| Removal rate of Ammonia (%) | 77 | 69 | 80 | 58 |
Example 5: application of microbial inoculum containing bacillus amyloliquefaciens LYM152 in livestock and poultry manure compost deodorization
2000kg of chicken manure is taken, sawdust and rice hull powder are added to adjust the water content to 60%, the mixture is uniformly mixed and evenly divided into 2 parts, an experimental group is added with a bacillus amyloliquefaciens LYM152 microbial inoculum (the microbial inoculum in example 2) according to the inoculation amount of 0.5%, a control group is added with an equal amount of sterile water to replace the microbial inoculum, composting treatment is carried out for 7 days, the ammonia gas concentration is respectively measured by adopting a sodium hypochlorite-salicylic acid spectrophotometry (HJ 534-2009), the hydrogen sulfide concentration is measured by adopting a methylene blue spectrophotometry, the odor gas concentration is measured by adopting a three-point comparison type odor bag method (GB/T14675-1993), volatile Organic Compounds (VOCs) are measured by adopting an adsorption tube sampling-thermal desorption/gas chromatography-mass spectrometry (HJ 644-2013), and the results are shown in the following table 3. After the bacillus amyloliquefaciens LYM152 microbial inoculum is inoculated for treatment, the odor concentration and VOCs generated by the chicken manure compost are obviously lower than those of a control group, the removal effect on ammonia gas and hydrogen sulfide is obvious, trichloromethane, tetrachloroethylene and benzene series in volatile organic matters are obviously reduced, and the whole deodorizing effect is good.
TABLE 3 deodorizing effect of Bacillus amyloliquefaciens LYM152 on compost of livestock and poultry manure
Note: ND means not detected.
Example 6: application of fungicide containing bacillus amyloliquefaciens LYM152 in combination with hermetia illucens to strengthening degradation of livestock and poultry manure
The livestock manure is divided into 2 parts on average, each 1kg, and the parts are respectively marked as A group and B group. Group A was inoculated with 10mL of Bacillus amyloliquefaciens LYM152 (the bacterial agent in example 2) and mixed well, group B was added with 10mL of sterile water, and then two groups were inoculated with 3000 black soldier fly larvae of 3 ages, both groups were treated under natural illumination, ventilation and 30 ℃ for 3 days, the amount of absorption, water content and pH value of the material were measured, the weight and length of the black soldier fly larvae were measured, and the ammonia release amount was measured by boric acid absorption titration, the results are shown in Table 4. The consumption of the experimental group material added with the bacillus amyloliquefaciens LYM152 microbial inoculum is improved by 17 percent compared with the control group, the water content of the material is reduced by 14 percent, the pH value is higher than that of the blank control group but the difference is not obvious, and the ammonia release amount is reduced by 85 percent; the survival rate of the hermetia illucens larvae is improved by 15 percent, the weight is increased by 24 percent, and the length of the hermetia illucens is increased by 17 percent.
TABLE 4 influence of Bacillus amyloliquefaciens LYM152 on the treatment of livestock and poultry manure by hermetia illucens
| Detecting items | (A) Without adding bacteria | (B) Adding strain LYM152 |
| Consumption of material (g) | 424.8±35.4 | 497.5±7.1 |
| Moisture content of the Material (%) | 57.0±1.6 | 49.2±2.0 |
| pH of the material | 7.4±0.6 | 7.9±0.1 |
| Heisui river horsefly survival rate (%) | 75.8±13.6 | 90.5±7.3 |
| Weight of black soldier fly (g/100) | 10.73±1.50 | 13.34±2.39 |
| Black soldier fly length (cm) | 1.40±0.15 | 1.64±0.05 |
| Ammonia gas release amount (× 10) -5 mol) | 7.70±0.92 | 1.12±0.23 |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. The Bacillus amyloliquefaciens LYM152 is characterized in that the strain is separated from the intestinal tract of hermetia illucens, is preserved in the common microorganism center of China general microbiological culture Collection center in 2019, 9 and 17 days, is classified and named as Bacillus amyloliquefaciens LYM152, and has the preservation number of CGMCC No.18497; the 16S rRNA gene sequence of the strain is shown in SEQ NO. 1.
2. An agent comprising the bacillus amyloliquefaciens LYM152 of claim 1.
3. A method for preparing the microbial preparation according to claim 2, comprising the steps of:
s1, strain activation: picking strains, streaking and inoculating the strains to a solid slant culture medium, and culturing for 16-24 h at 28-37 ℃ in a constant temperature incubator;
s2, preparing liquid seeds: inoculating the activated bacillus amyloliquefaciens LYM152 in the step S1 into a container filled with a liquid seed culture medium, sealing the container with gauze, and carrying out shake culture for 16-24 h at the temperature of 28-37 ℃ and the speed of 160-220 r/min to obtain liquid seeds;
s3, liquid fermentation: inoculating the liquid seeds prepared in the step S2 into a fermentation culture medium according to the inoculation amount of 1-10% of the volume ratio, rotating at the speed of 200-700 r/min, controlling the dissolved oxygen at 20-40%, controlling the pH at 6.0-7.5, controlling the temperature at 28-37 ℃, and performing aeration culture in a 10L fermentation tank for 24-48 h to obtain the liquid microbial inoculum of the bacillus amyloliquefaciens LYM152.
4. The method of claim 3, wherein the solid slant medium is: 1-3 g/L of glucose, 3-7 g/L of yeast extract powder, 5-15 g/L of peptone, 2-7 g/L of NaCl, 15-20 g/L of agar, distilled water for dissolving to a constant volume of 1L, pH of 7.0-7.2, and high-pressure steam sterilization at 121 ℃ for 15-20 min.
5. The method of claim 3, wherein the liquid seed medium is: 5-15 g/L of glucose, 1-5 g/L of beef extract, 3-7 g/L of yeast extract powder and KH 2 PO 2 0.5～2.0g/L，MgSO 4 ·7H 2 O 0.1～1.0g/L，CaCl 2 ·2H 2 O 0.1～0.5g/L，(NH4) 2 SO 2 1-5 g/L, dissolving distilled water to constant volume to 1L, pH 7.0-7.2, and sterilizing with high pressure steam at 121 ℃ for 15-20 min.
6. The method of claim 3, wherein the fermentation medium is: 15-25 g/L of glucose, 1-5 g/L of beef extract, 0.5-2.0 g/L of yeast extract powder and KH 2 PO 4 0.5～2.0g/L，MgSO 4 ·7H 2 O 0.2～0.6g/L，CaCl 2 ·2H 2 0.1-0.5 g/L of O, 1-5 g/L of ammonium citrate, 5-15 g/L of corn steep liquor and FeSO 4 0.5-2.0 mg/L of vitamin B 1 1-3 mg/L, dissolving distilled water to constant volume to 1L, pH 7.0-7.2, and sterilizing with high pressure steam at 121 ℃ for 15-20 min.
7. The microbial inoculum of claim 2, wherein the microbial inoculum can be concentrated to prepare a liquid microbial inoculum or freeze-dried to prepare solid bacterial powder, and the viable count of the bacillus amyloliquefaciens LYM152 in the microbial inoculum is not less than 10 8 cfu/mL or 10 8 cfu/g。
8. The use of the microbial inoculum according to claim 2 for the treatment of livestock and poultry manure by hermetia illucens.
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| CN202211181648.4A CN115725453A (en) | 2022-09-27 | 2022-09-27 | Application of bacillus amyloliquefaciens and microbial inoculum thereof in treatment of livestock and poultry manure by hermetia illucens |
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| CN202211181648.4A CN115725453A (en) | 2022-09-27 | 2022-09-27 | Application of bacillus amyloliquefaciens and microbial inoculum thereof in treatment of livestock and poultry manure by hermetia illucens |
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