A Cold-Tolerant Brevibacterium and Its Application of Deodorant in the Treatment of Livestock and Poultry Manure

Abstract

The present invention discloses a cold-tolerant Brevibacterium and its application in the treatment of livestock and poultry manure in the development and utilization of biological strains. The cold-tolerant Brevibacterium TW009 was deposited at the China Center for Type Culture Collection on June 1, 2022, and was classified and named Brevibacterium frigoritolerans TW009, with the deposit number CCTCC NO: M 2022770, and the deposit address is Wuhan, China. A cold-tolerant Brevibacterium disclosed by the present invention can be made into a deodorant and applied to deodorize the air in poultry houses and manure composting.

Description

Technical Field

[0001] The present invention relates to the technical field of utilization and development of biological strains, and particularly to a cold-resistant Brevibacterium and its application in deodorant for treating livestock and poultry manure. Background Art

[0002] With the large-scale and intensive development of the livestock and poultry breeding industry, environmental pollution problems have become increasingly serious, and the management difficulty has increased. During the stacking process of livestock and poultry manure, malodors are generated, leading to environmental deterioration. The odor components generated by livestock and poultry manure are complex and can generally be divided into the following four categories: First, volatile sulfur-containing compounds, such as H 2 S, CH 4 S, thioethers, etc. These substances mainly come from the reduction of sulfates by microorganisms and the metabolism of sulfur-containing amino acids; second, nitrogen-containing compounds, such as NH 3 , trimethylamine, etc. These substances mainly come from the deamination of proteins and amino acids by microorganisms; third, indole and phenol, which mainly come from the decomposition of phenylalanine and tyrosine by microorganisms; fourth, volatile fatty acids, such as acetic acid, propionic acid, butyric acid, valeric acid, etc. These substances mainly come from the deamination of amino acids and the decomposition of carbohydrates.

[0003] Currently, the treatment of livestock and poultry manure odors at home and abroad mainly includes three major types of technologies: physical method, chemical method, and biological method. Physical deodorization technology uses adsorbents and masking agents for temporary adsorption and masking of odors to achieve deodorization effects, but has disadvantages such as high cost and limited adsorption capacity. The masking method uses substances with strong aromatic odors, such as vanillic acid, piperonal, and cedrol, to mask odors, which is applicable to industries such as aquaculture, oil, leather, and fiber processing. It cannot deodorize fundamentally and has a high cost, with limited applications. Chemical deodorization technology achieves the purpose of deodorization by adding chemical substances to react with malodorous substances, including oxidation method, neutralization method, sterilization method, etc. The oxidation method is a process of using strong oxidants to oxidize reducing malodorous substances into harmless and odorless substances. Commonly used oxidants such as ozone, ferrate, etc. The neutralization method is to spread a layer of superphosphate on the bedding in the livestock and poultry house. The superphosphate reacts with the ammonia generated in the manure to form odorless solid ammonium phosphate salts, which can reduce the emission of ammonia in the manure and lower the ammonia concentration. The sterilization method is to spray the bedding with chemical substances such as hydrogen peroxide, potassium permanganate, copper sulfate, acetic acid, benzoic acid, etc. By sterilizing and disinfecting, the activities of harmful bacteria are inhibited, achieving the inhibition and reduction of the generation of harmful gases in the livestock and poultry house. The chemical method has good deodorization effects, but has disadvantages such as high cost, complex reactions, secondary pollution, and increasing the difficulty of resource utilization of manure, making it difficult to promote and apply.

[0004] Biological deodorization technology is a technology developed in the 1950s of the last century, and significant achievements have been made in countries such as Japan, Germany, and the Netherlands. The biological method mainly uses the metabolic function of microorganisms to degrade malodorous components into odorless and harmless end products, such as CO2 , H 2 O, etc., to achieve the purpose of deodorization. Biological deodorization has the advantages of high efficiency, no secondary pollution, simple equipment, low operating cost, convenient management and maintenance, etc., and has become one of the main technical development directions for treating malodors. Commonly used strains currently include photosynthetic bacteria, nitrifying bacteria, denitrifying bacteria, Bacillus, Streptomyces, yeast, lactic acid bacteria, Aspergillus oryzae, etc. SUMMARY OF THE INVENTION

[0005] Object of the Invention: In order to overcome the deficiencies in the prior art, the present invention provides a cold-tolerant Brevibacterium and the application of its deodorant in the treatment of livestock and poultry manure.

[0006] Technical Solution: To achieve the above object, a cold-tolerant Brevibacterium TW009 of the present invention is characterized in that the strain was deposited at the China Center for Type Culture Collection on June 1, 2022, and is classified and named Brevibacterium frigoritolerans TW009, with the deposit number CCTCC NO: M 2022770; the deposit address is Wuhan, China.

[0007] Furthermore, the 16S rDNA of the strain is as shown in SEQ NO.1.

[0008] A deodorant, which comprises the above-mentioned cold-tolerant Brevibacterium TW009.

[0009] Furthermore, the deodorant further comprises one or a combination of two or more of yeast, lactic acid bacteria, Bacillus subtilis, and photosynthetic bacteria.

[0010] Furthermore, the viable bacteria content in the deodorant is not less than 10 9 cfu / mL or 10 9 cfu / g, and the viable bacteria of cold-tolerant Brevibacterium TW009 are not less than 50% of the total viable bacteria content.

[0011] A method for preparing the deodorant, comprising the following steps:

[0012] (S1) Activate cold-tolerant Brevibacterium TW009 on a beef extract peptone medium, and then inoculate it into a liquid beef extract peptone medium, and culture it at 30°C for 16 h;

[0013] (S2) Prepare a liquid fermentation medium: tryptone 15 - 20 g, soy peptone 10 - 15 g, yeast extract 1 - 5 g, NaCl 1 - 8 g, K 2 HPO 4 1 - 5 g, MgSO 4 ·7H 2 O 0.1 - 0.5 g, MnSO 4 ·H2 O 0.005 - 0.0015 g, Na 2 MoO 4 ·2H 2 O 0.005 - 0.015 g, dissolved in distilled water and made up to 1 L in volume, pH 7.0, sterilized at 121 °C for 15 - 20 min;

[0014] (S3)Inoculate the activated Brevibacterium frigoritolerans TW009 at an inoculum size of 1 - 10% into the liquid fermentation medium, and shake - culture at 28 - 37 °C and 160 - 220 rpm for 16 - 24 h. When the bacterial content in the culture broth reaches 10 9 cfu / mL, collect the bacterial liquid, and it can be concentrated into a liquid bacterial agent or freeze - dried into a solid bacterial powder as needed.

[0015] Application of the said deodorant in deodorizing the air in poultry houses and the compost of livestock and poultry manure.

[0016] Application of a strain of Brevibacterium frigoritolerans and its deodorant in the present invention in the treatment of livestock and poultry manure has at least the following technical effects:

[0017] (1)The Brevibacterium frigoritolerans TW009 (CCTCC NO: M 2022770) in the present invention is screened from the farmland soil in Yantai. It belongs to non - pathogenic bacteria, is harmless to humans and animals, does not pollute the environment, and does not harm the ecological balance, meeting the biosafety regulations;

[0018] (2)The Brevibacterium frigoritolerans TW009 (CCTCC NO: M 2022770) in the present invention can be applied to the biological deodorization of livestock and poultry manure, effectively reducing the release of malodorous gases and volatile organic compounds in livestock and poultry manure, reducing the generation of odors from the source; the deodorization effect is stable, less affected by temperature, and still has a good deodorization effect under normal temperature conditions;

[0019] (3)The Brevibacterium frigoritolerans TW009 (CCTCC NO: M 2022770) in the present invention can be used alone or made into a mixed bacterial agent with other bacteria such as yeast, lactic acid bacteria, bacillus, photosynthetic bacteria, etc. When used, it can be treated in - situ or ex - situ. Spray the bacterial liquid or bacterial powder directly onto the manure at an inoculum size of 0.1% - 1.0%. Because it can produce spores and has strong stress resistance, it can grow well in manure, occupy a favorable ecological niche, inhibit the growth and reproduction of malodor - producing microorganisms, and reduce the release amount of manure malodorous gases. Each spraying can continuously play the deodorization role for many days;

[0020] (4)The Brevibacterium frigoritolerans TW009 (CCTCC NO: M 2022770) in the present invention has the characteristics of simple and efficient preparation process and stable and reliable products, and has obvious technical advantages for easy commercialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the microscopic morphology of Brevibacterium frigoritolerans TW009;

[0022] Figure 2 It is the phylogenetic tree of Brevibacterium frigoritolerans TW009 based on the 16S rDNA sequence. DETAILED DESCRIPTION OF THE INVENTION

[0023] The following is combined with Figures 1 to 2 to describe the principles and features of the present invention. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0024] Example 1: Isolation and identification of Brevibacterium frigoritolerans TW009 (CCTCC NO: M 2022770).

[0025] (S1) Sampling: Take 10 g of farmland soil, dissolve it in 90 mL of sterile water, shake it at 200 rpm for 30 min, and let it stand for 10 min. Take the supernatant as the bacterial suspension for standby.

[0026] (S2) Enrichment: Take 1 mL of the bacterial suspension and inoculate it into a 100 mL Erlenmeyer flask of liquid enrichment medium. Incubate it at 180 rpm and 30 °C for 5 days. Take 1 mL of the culture solution for gradient dilution, and take 10 -3 、10 -4 、10 -5 、10 -6 Four gradients, treat it in a boiling water bath for 3 min, coat it on a solid enrichment plate medium, and culture it at 30 °C for 72 hours. Pick single colonies from the plate for microscopic examination, record their microscopic morphology, and transfer the single colonies with different colony morphologies and microscopic morphologies to a slant LB medium, culture it at 30 °C for 48 hours, and store it at 4 °C for standby. The components and their contents in 1 L of enrichment medium: glucose 10 g, (NH 4 ) 2 SO 4 2 g, NaCl 1 g, MgSO 4· 7H 2 O 1 g, K 2 HPO 4· 2H 2 O 1 g, FeSO 4· 7H 2 O 0.4 g, complex trace elements 1 mL, pH 7.2, sterilize it with high-temperature steam at 121 °C for 20 min. Compound trace element formula (1 L): EDTA 10 g, ZnSO 4 1.2 g, CaCl 2 1.5 g, MgCl 2 ·4H 2O1g, (NH 4 ) 6 Mo 2 O 24 ·4H 2 O 1g, CuSO 4 ·5H 2 O 1g, CoCl 2 ·6H 2 O 1g. 15 g of agar is added to every 1 L of the solid enrichment medium, and no agar is added to the liquid enrichment medium.

[0027] (S3)Primary screening: The isolated strains are respectively inoculated into 100 mL of the liquid enrichment medium, placed on a shaker at 30 °C and 180 r / min for 48 h. Then, 10 mL of the culture solution is taken and inoculated into a plastic lunch box containing 200 g of fresh chicken manure and mixed evenly. The control group adds an equal amount of sterile water to replace the bacterial solution. The plastic lunch box is sealed with plastic wrap and statically cultured at 30 °C for 7 d. The deodorization effect of the microorganisms is judged by the sensory method and divided into 5 levels according to no odor "M0", slightly odorous "M1", relatively odorous "M2", very odorous "M3", and extremely odorous "M4". The strains with deodorization effects reaching no odor and slightly odorous are subjected to re-screening.

[0028] (S4)Re-screening: Weigh 200 g of fresh chicken manure and place it in a plastic box. Inoculate the cultured primary screening strains according to an inoculation amount of 5% (10 mL), and mix well with a glass rod. At the same time, add an equal amount of sterile water as a blank control, and set 3 parallels for each strain. Place a 50 mL small beaker containing 20 mL of boric acid absorption solution in the plastic box to absorb ammonia. Seal the plastic box and statically culture at 30 °C. The release amount of ammonia is measured by acid-base titration, and each group is repeated 3 times. Perform a significant difference analysis with the strains in the blank control group, and calculate the ammonia removal rate. The specific calculation formula is: Removal rate = (Difference in release amounts between the blank control group and the experimental group) / Release amount of the blank control group × 100%. Screen the strain TW009 with the best deodorization effect for purification culture and preservation in a glycerol tube at -80 °C.

[0029] The colonies of strain TW009 on beef extract peptone medium were milky white, nearly round, smooth and moist, with regular edges, and easy to pick; observed under a microscope, they were rod-shaped, Gram-positive, and oval spores were observed inside the bacteria after 72 hours of culture. Physiological and chemical tests were performed on strain TW009, and the test results are shown in Table 1. The results showed that strain TW009 was positive for leucine arylaminease, phenylalanine arylaminease, alanine arylaminease, tyrosine arylaminease, alanine-phenylalanine-proline arylaminease, ELLMAN, D-mannitol, red tetrazolium, and esculin hydrolysis tests, and did not grow in 6.5% NaCl medium. The 16S rDNA of strain TW009 was amplified and sequenced by PCR to obtain a 1394 bp 16S rDNA sequence. The BLAST program was used to compare it with the gene sequences registered in GenBank. The relevant sequence information of strains with high similarity and named were selected for sequence alignment using ClustalX 1.83. The Neighbor-Joining method in MEGA 7.0 was used to construct a phylogenetic tree, and Bootstrap (repeated sampling 1000 times) was used to analyze and evaluate the stability of the tree. It was found that TW009 had a strong affinity with known strains. Brevibacterium frigoritolerans The highest similarity with NR_115064 was 100%. Combined with the comparison results of morphology, physiological and biochemical characteristics and 16S rDNA gene sequence, strain TW009 was identified as Brevibacterium frostii ( Brevibacterium frigoritolerans ).

[0030] Table 1 Physiological and biochemical characteristics of strain TW009 (Note: “+” indicates a positive reaction, “-” indicates a negative reaction)

[0031]

[0032] Strain TW009 was deposited in the China Center for Type Culture Collection on June 1, 2022 and was classified and named Brevibacterium frigoritolerans TW009, the deposit number is CCTCC NO: M 2022770: the deposit address is Wuhan, China. The 16S rDNA sequence of Brevibacterium frostii TW009 is:

[0033]

[0034] Example 2: A deodorant containing Brevibacterium frost-tolerant TW009 and a method for preparing the deodorant.

[0035] Specifically, the preparation method of the deodorant is:

[0036] (S1) Brevibacterium frostii TW009 was activated on beef extract peptone medium, then inoculated into liquid beef extract peptone medium and cultured at 30°C for 16 h;

[0037] (S2) Preparation of liquid fermentation medium: 17 g tryptone, 13 g soy peptone, 3 g yeast extract, 5 g NaCl, K 2 HPO 4 2.5 g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·H 2 O 0.01g, Na 2 MoO 4 ·2H 2 O 0.01g, distilled water 1L, pH 7.0, sterilized at 121℃ for 15min;

[0038] (S3) The activated cold-resistant Brevibacterium TW009 was inoculated into the liquid fermentation medium at a rate of 5%, and cultured under shaking conditions of 30°C and 180 rpm for 18-24 h. When the bacterial count in the culture medium reached 10 9 cfu / mL, the bacterial liquid is collected and can be concentrated into a liquid bacterial agent or freeze-dried into a solid bacterial powder as needed, or it can be combined with one or two or more of yeast, lactic acid bacteria, Bacillus subtilis and photosynthetic bacteria to form a composite bacterial agent.

[0039] The deodorant should contain at least Brevibacterium frostii TW009. If yeast, lactic acid bacteria, Bacillus subtilis, photosynthetic bacteria, etc. are added, it should also contain the above-mentioned bacteria. At the same time, the amount of live bacteria in the deodorant should not be less than 10 9 cfu / mL or 10 9 cfu / g, wherein the viable bacteria of Brevibacterium frostii TW009 is not less than 50% of the total viable bacteria. Specifically, three bacterial agents are disclosed in this embodiment, and the components of the liquid fermentation medium of each bacterial agent and the type of bacterial agent are shown in Table 2.

[0040] Example 3: Use of a deodorant containing Brevibacterium frost-tolerant TW009 in deodorizing poultry house air.

[0041] Select two poultry houses with the same breeding scale and the same management conditions. Uniformly spray the deodorant diluted 100 times (bacterial agent 2 in Example 2) into one poultry house as the experimental group. Spray the same amount of sterile water into the other poultry house in the same way as the control group. After spraying for 12 hours, select 5 sampling points at different positions. Use the sodium hypochlorite-salicylic acid spectrophotometric method (HJ 534-2009) to measure the ammonia concentration, the methylene blue spectrophotometric method to measure the hydrogen sulfide concentration, and the three-point comparison odor bag method (GB / T 14675-1993) to measure the odor concentration. The results are averaged and summarized in Table 3. It can be seen that after spraying the deodorant, the odor concentration in the poultry house is significantly reduced, and the ammonia, hydrogen sulfide, and odor concentrations are all lower than the detection limit. The deodorizing effect is significantly better than that of the control group, indicating that Brevibacterium frigoritolerans TW009 has an obvious deodorizing effect on the air in the poultry house.

[0042] Table 2 Composition of the deodorant liquid medium and types of bacterial strains (+ indicates containing this bacterial agent, - indicates not containing this bacterial agent)

[0043]

[0044] Table 3 Deodorizing effect of Brevibacterium frigoritolerans TW009 on the air in livestock and poultry houses (Note: ND means not detected)

[0045]

[0046] Example 4: Application of the deodorant containing Brevibacterium frigoritolerans TW009 in deodorizing livestock and poultry manure compost.

[0047] Respectively take 2000 kg of chicken manure, pig manure, and cow manure, add sawdust and rice husk powder to adjust the water content to 60% and mix evenly. Divide them into 2 equal parts on average. The experimental group is added with the deodorant (bacterial agent 2 in Example 2), and the control group is added with the same amount of sterile water instead of the bacterial solution. Compost for 7 days. Use the sodium hypochlorite-salicylic acid spectrophotometric method (HJ 534-2009) to measure the ammonia concentration, the methylene blue spectrophotometric method to measure the hydrogen sulfide concentration, the three-point comparison odor bag method (GB / T 14675-1993) to measure the odor concentration, and the adsorption tube sampling-thermal desorption / gas chromatography-mass spectrometry method (HJ644-2013) to measure volatile organic compounds (VOCs, including chloroform, tetrachloroethylene, etc.). The results are shown in Table 4 below. After treatment with Brevibacterium frigoritolerans TW009, the odor concentration and VOCs generated by chicken manure, pig manure, and cow manure are significantly lower than those of the control group. Especially, the removal effects on hydrogen sulfide and chloroform are significant, showing an overall good deodorizing effect.

[0048] Table 4 Deodorizing effect of Brevibacterium frigoritolerans TW009 on livestock and poultry manure compost (Note: ND means not detected)

[0049]

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Brevibacterium frigoritolerans TW009, characterized in that, This strain was deposited at the China Center for Type Culture Collection on June 1, 2022, and its taxonomic name is Brevibacterium frigoritolerans TW009, with the deposit number CCTCC NO: M 2022770, and the deposit address is Wuhan, China.

2. The Brevibacterium frigoritolerans TW009 according to claim 1, characterized in that, the 16S rDNA of the strain is as shown in SEQ ID NO.

1.

3. A deodorant, characterized in that: the deodorant comprises the Brevibacterium frigoritolerans TW009 according to claim 1.

4. The deodorant according to claim 3, characterized in that, The viable count of live bacteria in the deodorant is not less than 10 9 cfu / mL or 10 9 cfu / g, and the viable count of Brevibacterium frigoritolerans TW009 is not less than 50% of the total viable count of live bacteria.

5. A method for preparing the deodorant according to claim 3, characterized in that, comprises the following steps: (S1) Activate Brevibacterium frigoritolerans TW009 on a beef extract peptone medium, then inoculate it into a liquid beef extract peptone medium and culture at 30 °C for 16 h; (S2) Prepare a liquid fermentation medium: 15 - 20 g of tryptone, 10 - 15 g of soy peptone, 1 - 5 g of yeast extract, 1 - 8 g of NaCl, K 2 HPO 4 1 - 5 g, MgSO 4 ·7H 2 O 0.1 - 0.5 g, MnSO 4 ·H 2 O 0.005 - 0.0015 g, Na 2 MoO 4 ·2H 2 O 0.005 - 0.015 g, dissolve in distilled water and make up the volume to 1 L, pH 7.0, sterilize at 121 °C for 15 - 20 min; (S3) Inoculate the activated Brevibacterium frigoritolerans TW009 at an inoculation amount of 1-10% into a liquid fermentation medium, and culture it by shaking at 28-37 °C and 160-220 rpm for 16-24 h. When the bacterial content in the culture solution reaches 10 9 cfu / mL, collect the bacterial liquid, and it can be concentrated into a liquid bacterial agent or freeze-dried into a solid bacterial powder as needed.

6. Use of the deodorant according to claim 3 for deodorizing the air in a poultry house.

7. Use of the deodorant according to claim 3 for deodorizing livestock and poultry manure compost.

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