Application of Bacillus amyloliquefaciens and its bacterial agent in the treatment of livestock and poultry feces by black soldier flies

Abstract

The present invention discloses the application of Bacillus amyloliquefaciens and its bacterial agent in the treatment of livestock and poultry feces with black soldier flies, and relates to the field of biological strain utilization and development technology. The Bacillus amyloliquefaciens LYM152 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on September 17, 2019, and is classified and named Bacillus amyloliquefaciens LYM152, with a deposit number of CGMCC No.18497, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The Bacillus amyloliquefaciens disclosed in the present invention has the characteristics of antibacterial, deodorizing, and high digestive enzyme activity. Its bacterial agent has a good deodorizing effect in the treatment of livestock and poultry feces, and the effect of strengthening the degradation of livestock and poultry feces in combination with black soldier flies is more significant.

Description

Technical Field

[0001] The invention relates to the technical field of biological strain utilization and development, in particular to application of Bacillus amyloliquefaciens and bacterial agents thereof in treating livestock and poultry excrement with black soldier flies. Background Art

[0002] my country has a large amount of livestock and poultry manure emissions, which is the main source of agricultural non-point source pollution. According to the second national pollution source census, my country's annual output of livestock and poultry manure was 3.05 billion tons in 2019. Intensive farms produce a huge amount of manure every day, and the discharge contains a large amount of organic matter, pathogenic microorganisms and parasite eggs, as well as heavy metals, additives, nitrogen, phosphorus, odor and other pollution. Livestock and poultry manure has caused great ecological pressure on agricultural production and rural life. Traditional treatment methods are simple in technology and require small investments, but the level of resource utilization is low and there are problems such as secondary pollution and waste of resources. The mainstream aerobic composting and anaerobic biogas fermentation have the disadvantages of large investments and low returns.

[0003] In recent years, the technology of treating livestock and poultry manure with insects represented by black soldier flies has received widespread attention. Black soldier flies (Hermetia illucens L.) are saprophytic insects. The larvae have the advantages of omnivorous diet, large appetite, strong resistance to adversity, non-disease transmission, rapid reproduction, and low feeding costs. They have a strong ability to absorb livestock and poultry manure. After the livestock and poultry manure is transformed by the larvae, the insect bodies can be used as high-value animal protein feed, and the insect feces can be used to produce high-value-added organic fertilizer. In 2013, FAO listed black soldier flies as one of the most important protein feed substitute insects and promoted their application worldwide. At present, the technology of treating livestock and poultry manure with black soldier flies has gradually been recognized, but there are still many problems in its application, such as heavy odor and elimination of pathogens. For example, some functional microorganisms have been screened, which have played an important role in the treatment of livestock and poultry manure.

[0004] Bacillus amyloliquefaciens belongs to the genus Bacillus. It can not only produce a variety of digestive enzymes such as amylase and protease, but also produce lipopeptide antibiotics including iturins, fengycins and surfactin. It has a broad-spectrum antibacterial activity, can inhibit the growth of a variety of plant pathogens, and has the function of promoting plant growth. It is widely used in biological control, organic fertilizer production, soil improvement and other fields. Summary of the invention

[0005] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides Bacillus amyloliquefaciens which has the characteristics of antibacterial, deodorizing and high digestive enzyme activity, and its bacterial agent has significant application effect in the treatment of livestock and poultry feces by black soldier flies.

[0006] Technical solution: To achieve the above-mentioned purpose, the present invention provides a Bacillus amyloliquefaciens LYM152, which is isolated from the intestine of black soldier flies and has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 17, 2019, and is classified and named Bacillus amyloliquefaciens LYM152, with a deposit number of CGMCC NO.18497; the 16S rRNA gene sequence of the strain is shown in SEQ ID NO: 1.

[0007] A bacterial agent, comprising the above-mentioned Bacillus amyloliquefaciens LYM152.

[0008] A method for preparing the bacterial agent comprises the following steps:

[0009] S1. Strain activation: Pick the strain and inoculate it onto the solid slant medium, and culture it in a constant temperature incubator at 28-37℃ for 16-24h;

[0010] S2. Preparation of liquid seeds: inoculate the activated Bacillus amyloliquefaciens LYM152 in step S1 into a container filled with liquid seed culture medium, seal the container with gauze, and culture under shaking conditions of 28-37° C. and 160-220 r / min for 16-24 h to obtain liquid seeds;

[0011] S3, liquid fermentation: the liquid seeds prepared in step S2 are inoculated into the fermentation medium at a volume ratio of 1-10%, the rotation speed is 200-700 r / min, the dissolved oxygen is controlled at 20-40%, the pH is 6.0-7.5, the temperature is 28-37° C., and the 10L fermenter is aerated and cultured for 24-48 hours to obtain a liquid bacterial agent of Bacillus amyloliquefaciens LYM152.

[0012] Furthermore, the bacterial agent can be concentrated to form a liquid bacterial agent or freeze-dried to form a solid bacterial powder, and the amount of live bacteria of Bacillus amyloliquefaciens LYM152 in the bacterial agent is not less than 10 8 cfu / mL or 10 8 cfu / g.

[0013] Furthermore, the solid slant culture medium is: glucose 1-3 g / L, yeast extract 3-7 g / L, peptone 5-15 g / L, NaCl 2-7 g / L, agar 15-20 g / L, dissolved in distilled water to 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0014] Furthermore, the liquid seed culture medium is: glucose 5-15 g / L, beef extract 1-5 g / L, yeast extract 3-7 g / L, KH2PO2 0.5-2.0 g / L, MgSO4·7H2O 0.1-1.0 g / L, CaCl2·2H2O0.1-0.5 g / L, (NH4)2SO2 1-5 g / L, dissolved in distilled water to 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0015] Furthermore, the fermentation medium is: glucose 15-25 g / L, beef extract 1-5 g / L, yeast extract powder 0.5-2.0 g / L, KH2PO4 0.5-2.0 g / L, MgSO4·7H2O 0.2-0.6 g / L, CaCl2·2H2O0.1-0.5 g / L, ammonium citrate 1-5 g / L, corn steep liquor 5-15 g / L, FeSO4 0.5-2.0 mg / L, vitamin B1 1-3 mg / L, dissolved in distilled water to 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0016] The bacterial agent is used in treating livestock and poultry excrement with black soldier flies.

[0017] The application of the Bacillus amyloliquefaciens and its bacterial agent in the treatment of livestock and poultry feces by black soldier flies has at least the following technical effects:

[0018] (1) The Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497) in the present invention is isolated from the intestine of black soldier flies and has the advantages of fast reproduction speed, high fermentation density, rich digestive enzyme system, broad-spectrum antibacterial, and strong tolerance to adverse environments;

[0019] (2) The Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497) of the present invention can be used for deodorization of livestock and poultry manure, effectively reducing the release of malodorous gases and volatile organic compounds in livestock and poultry manure, and reducing the generation of odor from the source; the deodorization effect is stable and is less affected by temperature, and still has a good deodorization effect under normal temperature conditions

[0020] (3) The Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497) of the present invention can be applied to the treatment of livestock and poultry manure by black soldier flies, and can stably colonize in the livestock and poultry manure treatment system by black soldier flies for a long time and play the role of degrading organic matter, inhibiting pathogens, and reducing the generation and release of odor, thereby achieving the purpose of accelerating the reduction of livestock and poultry manure, reducing the mortality rate of black soldier flies, increasing the growth rate of black soldier flies, and shortening the prepupa time of black soldier flies;

[0021] (4) The Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497) in the present invention has the characteristics of simple and efficient preparation process, stable and reliable products, and has the obvious technical advantage of being easy to commercialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of Bacillus amyloliquefaciens LYM152;

[0023] Figure 2 The microscopic morphology of Bacillus amyloliquefaciens LYM152;

[0024] Figure 3 This is an electron microscope photo of Bacillus amyloliquefaciens LYM152;

[0025] Figure 4 Phylogenetic tree of Bacillus amyloliquefaciens LYM152 based on 16S rRNA sequences. DETAILED DESCRIPTION

[0026] The following combination Figures 1 to 4 The principles and features of the present invention are described, and the examples given are only used to explain the present invention, rather than to limit the scope of the present invention.

[0027] Example 1: Isolation and screening of Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497).

[0028] 1. Isolation: The strain LYM152 of the present invention is isolated by a dilution coating method. The specific isolation method is: select 20 to 30 7-day-old black soldier fly larvae fed with livestock and poultry feces, dissect the intestines, add 1 mL of sterile physiological saline to make a bacterial suspension, inoculate 100 mL of sterilized livestock and poultry feces: clarified bovine rumen fluid = 1:1 mixed solution, shake culture for 5 days, take 1 mL of gradient dilution and spread it on the LB culture medium plate, place it at 30°C for 3 days, streak the colonies of different forms on the plate culture medium, purify the obtained strains, and store them by number. The LB culture medium components are: yeast extract powder 5g / L, tryptone 10g / L, NaCl 10g / L, agar 18g / L, pH7.2, and high-pressure steam sterilization at 121°C for 20min.

[0029] 2. Preliminary screening: The isolated strains were inoculated into LB liquid culture medium, placed in a shaking incubator at 30°C and 180r / min for 48h, and 5% inoculation volume, i.e. 10mL, of the bacterial solution was inoculated into a plastic box containing 200g of livestock and poultry manure. The solution was mixed evenly with a sterile glass rod, sealed with plastic wrap, and cultured at 30°C for 1d. The bacterial solution was replaced with sterile water in the control group. The deodorizing effect of the microorganisms was judged by the sensory method. The odor was divided into 5 levels, i.e., no odor "M0", slightly odor "M1", relatively odorous "M2", very odorous "M3", and foul odor "M4". The strains with obvious deodorizing effect (the effect reached no odor "M0" and slightly odorous "M1") were selected for re-screening test.

[0030] 3. Rescreening: Weigh 200g of livestock and poultry manure and place it in a plastic box. Inoculate the culture solution of the primary screening strain at a 5% inoculum, mix thoroughly, replace the bacterial solution with an equal amount of sterile water as a blank control, place a 50mL beaker containing 20mL of 2% boric acid solution in each plastic box to absorb ammonia, seal the plastic box with plastic wrap, and culture it at 30℃. On the third day, determine the release of ammonia by acid-base titration, and repeat 3 times for each group. Compare and analyze with the blank control group, calculate the removal rate of ammonia, and select the strain with the best deodorization effect for purification, culture and preservation.

[0031] The ammonia removal rate was calculated by the formula: ammonia removal rate = the difference between the ammonia release of the blank control group and the ammonia release of the test group / the ammonia release of the blank control group × 100%. Through screening, a strain LYM152 derived from the intestine of black soldier flies was obtained that can significantly deammonify and deodorize.

[0032] Example 2: Identification of Bacillus amyloliquefaciens LYM152 (CGMCC NO.18497).

[0033] 1. Morphological characteristics and physiological and biochemical characteristics

[0034] After strain LYM152 was cultured on LB medium for 48 hours, the colonies were white, round, opaque, with a flat and slightly raised surface, moist and sticky, and no pigment was produced. Gram staining was positive, the bacteria were rod-shaped, with blunt ends, different lengths, no capsule, and could form endospores. The spores were oval, mesogenic or sub-terminal, and motile. Identification by the VITEK2 Compact fully automatic microbial analysis system showed that strain LYM152 was able to grow in 6.5% sodium chloride, and was able to decompose and utilize a variety of carbon sources such as glucose, mannose, mannitol and trehalose. β-mannosidase, N-acetyl-β-D-glucosaminidase, and kanamycin tolerance were all negative. The colony morphology of strain LYM152 is as follows Figure 1 As shown, the microscopic morphology Figure 2 As shown in the electron microscope photos Figure 3 shown.

[0035] The results of physiological and biochemical tests showed that it could decompose glucose, maltose, mannose, mannitol and inositol, but could not utilize lactose and arabinose. The results of starch hydrolysis, gelatin liquefaction, nitrate reduction and 6.5% NaCl growth tests were positive, while the acetylmethylcarbinol (VP) test, phenylalanine deaminase test, indole test, methyl red (MR) test and hydrogen sulfide test were all negative, as shown in Table 1.

[0036] Table 1 Physiological and biochemical identification results of strain LYM152

[0037]

[0038]

[0039] Note: "+" indicates a positive reaction, "-" indicates a negative reaction

[0040] 2. Phylogenetic analysis

[0041] The 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing was 1491 bp in length. BLAST comparison was performed on GenBank to screen the relevant sequence information of homologous strains and perform phylogenetic analysis. The phylogenetic tree based on the 16S rRNA sequence is shown in the figure. Figure 4 As shown, it was found that the strain had a base similarity of 99% with Bacillus amyloliquefaciens (EF423607). Combined with morphological and physiological and biochemical indicators, the strain LYM152 was identified as Bacillus amyloliquefaciens.

[0042] 3. Genetic stability

[0043] The strain LYM152 was subcultured 50 times on solid slant medium by streak method. The bacterial morphology, growth rate and ability to remove ammonia were measured. There was no significant difference with the original strain and the genetic stability was good.

[0044] 4. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection on September 17, 2019, and was classified as Bacillus amyloliquefaciens LYM152, with the deposit number CGMCC No.18497. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] The 16S rRNA sequence of the Bacillus amyloliquefaciens LYM152 is:

[0046]

[0047] Example 3: A method for preparing a bacterial agent containing Bacillus amyloliquefaciens LYM152.

[0048] Specifically, the preparation method of the bacterial agent is:

[0049] S1. Strain activation: Pick the strain and streak it onto the solid slant medium, and culture it in a constant temperature incubator at 30°C for 18 hours. When a large number of bacterial cells grow on the slant, they can be used to transfer liquid seeds.

[0050] The solid slant culture medium is composed of 2 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and 18 g / L agar, which are dissolved in distilled water to a volume of 1 L, pH 7.0, and sterilized by high-pressure steam at 121° C. for 20 min.

[0051] S2. Preparation of liquid seeds: Pick activated Bacillus amyloliquefaciens LYM152 and inoculate it into a container filled with liquid seed culture medium, seal it with eight layers of gauze, and culture it in a shaking incubator at 30°C and 180 rpm for 18 hours to grow a large number of bacteria to obtain liquid seeds;

[0052] The liquid seed culture medium comprises 10 g / L glucose, 3 g / L beef extract, 5 g / L yeast extract powder, 1 g / L KH2PO2, 0.5 g / L MgSO4·7H2O, 0.3 g / L CaCl2·2H2O, and 3 g / L (NH4)2SO2, which are dissolved in distilled water to a constant volume of 1 L, pH value is 7.0, and high-pressure steam sterilization is performed at 121° C. for 20 min.

[0053] S3, liquid fermentation: inoculate the fermentation medium with the liquid seeds prepared in step 2 at a volume ratio of 5%, rotate at 200-700 r / min, control the dissolved oxygen at 30%, pH 7.0, temperature 30°C, and culture for 36 h with aeration until the bacterial concentration reaches 1×10 8 cfu / mL or above, collect the fermentation broth to obtain the liquid inoculum of Bacillus amyloliquefaciens LYM152, seal it in portions, and refrigerate it at 4°C for later use.

[0054] The fermentation medium comprises 20 g / L glucose, 3 g / L beef extract, 1 g / L yeast extract powder, 1 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.3 g / L CaCl2·2H2O, 3 g / L ammonium citrate, 10 g / L corn steep liquor, 1 mg / L FeSO4, and 2 mg / L vitamin B1, which is dissolved in distilled water to a constant volume of 1 L, pH value is 7.0, and high-pressure steam sterilization is performed at 121° C. for 20 min.

[0055] When used, the bacterial agent can be concentrated into a liquid bacterial agent or freeze-dried into a solid bacterial powder as needed, and the amount of live bacteria of Bacillus amyloliquefaciens LYM152 in the bacterial agent is not less than 1×10 8 cfu / mL or 10 8 cfu / g.

[0056] Example 4: Application of a bacterial agent containing Bacillus amyloliquefaciens LYM152 in deodorizing livestock and poultry manure.

[0057] 10 mL of the Bacillus amyloliquefaciens LYM152 bacterial agent (the bacterial agent in Example 2) was inoculated into a 2L volume lunch box containing 1000 g of chicken manure, pig manure, cow manure and kitchen waste, respectively, and the mixture was evenly mixed and sealed with plastic wrap. 10 mL of sterile water was added to the control group instead of the bacterial solution. Three replicates were set for each group. The ammonia concentration was measured after being placed at room temperature for 5 days. The results are shown in Table 2. As can be seen from Table 1, after the livestock and poultry manure was inoculated with the strain LYM152 for 5 days, the ammonia concentration produced by chicken manure, pig manure, cow manure and kitchen waste was significantly reduced, showing a good deodorizing effect.

[0058] Table 2 Deodorization effect of Bacillus amyloliquefaciens LYM152 on livestock and poultry manure

[0059] Measurement index (ammonia concentration: ppm) Chicken manure Pig manure Cow dung Food waste Comparison 134 129 116 93 Treatment Group 31 40 23 39 Ammonia removal rate (%) 77 69 80 58

[0060] Example 5: Application of a bacterial agent containing Bacillus amyloliquefaciens LYM152 in deodorization of livestock and poultry manure composting

[0061] Take 2000 kg of chicken manure, add sawdust and rice husk powder to adjust the moisture content to 60% and mix evenly, divide it into 2 parts, add Bacillus amyloliquefaciens LYM152 bacterial agent (the bacterial agent in Example 2) at an inoculum amount of 0.5% to the experimental group, add an equal amount of sterile water instead of the bacterial solution to the control group, compost it for 7 days, and use sodium hypochlorite-salicylic acid spectrophotometry (HJ 534-2009) to determine the ammonia concentration, methylene blue spectrophotometry to determine the hydrogen sulfide concentration, three-point comparison odor bag method (GB / T14675-1993) to determine the odor concentration, and adsorption tube sampling-thermal desorption / gas chromatography-mass spectrometry (HJ644-2013) to determine volatile organic compounds (VOCs). The results are shown in Table 3. After inoculation with Bacillus amyloliquefaciens LYM152, the odor concentration and VOCs produced by chicken manure composting were significantly lower than those in the control group. It not only had a significant effect on the removal of ammonia and hydrogen sulfide, but also significantly reduced trichloromethane, tetrachloroethylene and benzene series in volatile organic compounds, showing a good deodorization effect overall.

[0062] Table 3 Deodorization effect of Bacillus amyloliquefaciens LYM152 on livestock and poultry manure composting

[0063]

[0064]

[0065] Note: ND means not detected.

[0066] Example 6: Application of a bacterial agent containing Bacillus amyloliquefaciens LYM152 in combination with black soldier flies to enhance the degradation of livestock and poultry manure

[0067] The livestock and poultry feces were divided into 2 portions, each with 1 kg, and were recorded as Group A and Group B. Group A was inoculated with 10 mL of Bacillus amyloliquefaciens LYM152 bacterial agent (the bacterial agent in Example 2) and mixed, and Group B was added with 10 mL of sterile water, and then 3000 3-year-old black soldier fly larvae were inoculated in the two groups, and both were treated for 3 days under natural light, ventilation, and 30°C. The material digestion capacity, water content and pH value were detected, the weight and length of the black soldier fly larvae were measured, and the ammonia release was detected by boric acid absorption titration. The results are shown in Table 4. The material digestion capacity of the experimental group added with Bacillus amyloliquefaciens LYM152 bacterial agent was increased by 17% compared with the control group, the material water content was reduced by 14%, the pH value was higher than the blank control group but the difference was not significant, and the ammonia release was reduced by 85%; the survival rate of the black soldier fly larvae was increased by 15%, the weight increased by 24%, and the length of the larvae increased by 17%.

[0068] Table 4 Effect of Bacillus amyloliquefaciens LYM152 on the treatment of livestock and poultry manure by black soldier flies

[0069]

[0070]

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens LYM152, characterized in that The strain was isolated from the intestine of black soldier fly and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 17, 2019. It was classified and named Bacillusamyloliquefaciens LYM152 and the deposit number was CGMCC No.18497. The 16S rRNA gene sequence of the strain is shown in SEQ ID NO:

1.

2. A bacterial agent, characterized in that The bacterial agent comprises the Bacillus amyloliquefaciens LYM152 described in claim 1.

3. A method for preparing the bacterial agent according to claim 2, characterized in that: The following steps are involved: S1. Strain activation: Pick Bacillus amyloliquefaciens LYM152 and streak it onto a solid slant medium, and culture it in a constant temperature incubator at 28-37°C for 16-24h; S2. Preparation of liquid seeds: inoculate the activated Bacillus amyloliquefaciens LYM152 in step S1 into a container filled with liquid seed culture medium, seal the container with gauze, and culture under shaking conditions of 28-37° C. and 160-220 r / min for 16-24 h to obtain liquid seeds; S3, liquid fermentation: the liquid seeds prepared in step S2 are inoculated into the fermentation medium at a volume ratio of 1-10%, the rotation speed is 200-700 r / min, the dissolved oxygen is controlled at 20-40%, the pH is 6.0-7.5, the temperature is 28-37° C., and the 10L fermenter is aerated and cultured for 24-48 hours to obtain a liquid bacterial agent of Bacillus amyloliquefaciens LYM152.

4. The method for preparing a bacterial agent according to claim 3, characterized in that: The solid slant culture medium comprises: 1-3 g / L glucose, 3-7 g / L yeast extract, 5-15 g / L peptone, 2-7 g / L NaCl, 15-20 g / L agar, dissolved in distilled water to a volume of 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121° C. for 15-20 min.

5. The method for preparing a bacterial agent according to claim 3, characterized in that: The liquid seed culture medium comprises: 5-15 g / L glucose, 1-5 g / L beef extract, 3-7 g / L yeast extract, 0.5-2.0 g / L KH2PO2, 0.1-1.0 g / L MgSO4·7H2O, 0.1-0.5 g / L CaCl2·2H2O, 1-5 g / L (NH4)2SO2, dissolved in distilled water to a constant volume of 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121°C for 15-20 min.

6. The method for preparing a microbial agent according to claim 3, characterized in that: The fermentation medium comprises: 15-25 g / L glucose, 1-5 g / L beef extract, 0.5-2.0 g / L yeast extract, 0.5-2.0 g / L KH2PO4, 0.2-0.6 g / L MgSO4·7H2O, 0.1-0.5 g / L CaCl2·2H2O, 1-5 g / L ammonium citrate, 5-15 g / L corn steep liquor, 0.5-2.0 mg / L FeSO4, 1-3 mg / L vitamin B1, dissolved in distilled water to a volume of 1 L, pH 7.0-7.2, and sterilized by high-pressure steam at 121° C. for 15-20 min.

7. The bacterial agent according to claim 2, characterized in that The bacterial agent can be concentrated to form a liquid bacterial agent or freeze-dried to form a solid bacterial powder, and the amount of live bacteria of Bacillus amyloliquefaciens LYM152 in the bacterial agent is not less than 10 8 cfu / mL or 10 8 cfu / g.

8. Use of the bacterial agent according to claim 2 in treating livestock and poultry excrement with black soldier flies.

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