P.megatherium and application of P.megatherium in pig farm manure treatment

Abstract

The invention relates to the field of microorganisms, and discloses a P.megaterium and an application thereof in pig farm manure treatment, the P.megaterium is preserved in China Center for Type Culture Collection on December 13, 2022, the preservation address is Wuhan, China, the classification name of the P.megaterium is Pristia megaterium 3144, and the preservation number is CCTCC NO: M20221962. The content of pollutants such as total nitrogen, chemical oxygen demand and the like in pig farm feces is reduced by secreting various degrading enzymes such as protease, lipase and the like through the provided Priestia megaterium 3144 strain, and meanwhile, fecal coliforms and roundworm eggs are killed by the strain through metabolism heat production and antibacterial substance secretion.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to Priesteria gigantea and application thereof in treating manure in pig farms. Background Art

[0002] Traditional microbial strains used for pig farm manure treatment usually have characteristics such as high salinity, high ammonia nitrogen, and large pH fluctuations. Under such environmental conditions, the enzyme activity of most commonly used microbial strains is inhibited, and the cell osmotic pressure is unbalanced, resulting in a significant decrease in strain activity or even death. It is difficult to continuously and efficiently degrade manure, resulting in low manure treatment efficiency and unable to meet the needs of effective manure treatment in actual production. Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the present invention provides a Priesteria gigantea and its application in the treatment of pig farm manure and sewage, which solves the problem that traditional microbial strains used for pig farm manure and sewage treatment are difficult to continuously and efficiently degrade manure and sewage, resulting in low manure and sewage treatment efficiency and unable to meet the demand for effective manure and sewage treatment in actual production.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions: a Priestia megaterium, characterized in that: the Priestia megaterium was deposited in the China Center for Type Culture Collection on December 13, 2022, with the deposit address being: Wuhan, China, and its classification name is Priestia megaterium 3144, and the deposit number is CCTCCNO: M20221962.

[0005] Preferably, the 16S rDNA sequence of Priestia megaterium 3144 is shown as SEQ ID NO: 1.

[0006] Preferably, the colony morphology of Priestia megaterium 3144 is round, white, opaque, 1-2 mm in diameter, with a smooth and moist surface and neat edges.

[0007] Preferably, the physiological and biochemical characteristics of the Priestia megaterium 3144 are: Gram staining positive, aerobic growth, culture temperature of 25-35° C., and pH of 6.5-8.0.

[0008] Preferably, the storage method of Priestia megaterium 3144 comprises: freezing at -80°C using a 20% glycerol aqueous solution as a protective agent.

[0009] A method for culturing Priesteria gigantea, applied to the aforementioned Priesteria gigantea, comprises the following steps:

[0010] Priestia megaterium 3144 was inoculated into solid culture medium for activation;

[0011] The activated strain was inoculated into liquid culture medium and cultured with shaking at 150 rpm until OD 600 =1.5-1.8, then add corn starch solution to make the glucose concentration ≥2g / L, and continue to culture until OD 600 ≥3.0, bacterial cells were obtained;

[0012] The bacterial cells were mixed with sodium alginate-calcium carbonate to prepare immobilized particles, which were then placed in a reactor containing 1% simulated feces solution and cultured for 6 hours to obtain functionally enhanced immobilized strains, thus completing the culture.

[0013] Preferably, the activation is carried out by culturing at 37°C for 4 hours and then reducing the temperature to 30°C for 20 hours. The solid culture medium comprises 10g / L corn starch, 8g / L soybean meal peptone, 5% v / v pig manure extract, 0.5g / L MgSO4·7H2O, 1g / L KH2PO4, and 0.1mmol / L sodium selenate. The composition of the liquid culture medium is the same as that of the solid culture medium but does not contain agar. The concentration of the added corn starch solution is 20% w / v, and the addition rate is 0.5-1mL / min, the preparation of immobilized particles is to mix the bacteria with 4% w / v sodium alginate solution in a volume ratio of 1:3, drip into 2% w / v calcium chloride solution through a syringe, and collect particles with a diameter of 2-3 mm after curing for 2 hours. The manure simulation liquid is: fresh pig manure and deionized water are mixed in a ratio of 1:10 w / v, sterilized at 121°C for 20 minutes, filtered after cooling, and the supernatant is used. Its total nitrogen content is 200-500 mg / L, chemical oxygen demand is 1000-3000 mg / L, and pH is 7-8.5.

[0014] Application of Priesteria gigantea in pig farm manure treatment.

[0015] The invention provides Priesteria gigantea and application thereof in treating manure in pig farms.

[0016] It has the following beneficial effects:

[0017] 1. The Priestia megaterium 3144 strain provided by the present invention reduces the content of pollutants such as total nitrogen and chemical oxygen demand in pig farm manure by secreting multiple degradative enzymes such as protease and lipase. At the same time, the strain produces heat through metabolism and secretes antibacterial substances to kill fecal coliforms and ascaris eggs, so that the content of pathogenic microorganisms and parasite eggs in manure meets the "Pollutant Emission Standards for Livestock and Poultry Farming", effectively reducing the pollution risk of manure to the environment, ensuring the safety of surrounding water bodies and soil, and meeting the demand for effective treatment of manure in actual production.

[0018] 2. The present invention uses sodium alginate-calcium carbonate immobilization technology to embed the bacterial strain in gel particles with a diameter of 2-3 mm, forming a functional unit with high mechanical strength and strong anti-pollution ability, reducing strain loss and lowering the strain cost in industrial applications. At the same time, low-cost raw materials such as corn starch, soybean meal peptone and pig manure extract are used in the cultivation process. The pig manure extract not only provides natural growth factors, but also enhances the adaptability of the strain to the manure environment, forming a "fertilizer-growing" cycle model. The treated manure can be converted into organic fertilizer raw materials rich in bacterial protein and humus, achieving the dual benefits of "pollution control + resource recovery".

[0019] 3. The present invention adopts a pure biological treatment process without the need to add chemical agents, thus avoiding the risks of heavy metal residues or excessive agents caused by traditional chemical methods. The strain itself is non-pathogenic, and the immobilized particles can be physically recovered to reduce environmental release. In addition, the strain can gradually decay naturally in the natural environment, without the risk of biological invasion. It meets the requirements of green agriculture and sustainable development, and provides a safe and reliable technical path for the pollution control of livestock and poultry breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention provides a flow chart of a method for culturing Priesteria gigantea. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] An embodiment of the present invention provides Priestia megaterium, characterized in that: Priestia megaterium was deposited with the China Center for Type Culture Collection on December 13, 2022, in Wuhan, China, with a classification name of Priestia megaterium 3144 and a deposit number of CCTCC NO: M20221962. Postal code: 430072.

[0023] The 16S rDNA sequence of Priestia megaterium 3144 is shown in SEQ ID NO: 1.

[0024] The colonies of Priestia megaterium 3144 are round, white, opaque, 1-2 mm in diameter, with a smooth and moist surface and neat edges.

[0025] The physiological and biochemical characteristics of Priestia megaterium 3144 are: Gram staining positive, aerobic growth, culture temperature of 25-35℃, pH 6.5-8.0.

[0026] The storage method of Priestia megaterium 3144 includes freezing at -80°C using 20% ​​glycerol aqueous solution as a protective agent.

[0027] Specifically, 500 mL of sludge sample was collected from the pig farm manure treatment pool (pH 7.5, total nitrogen content 800 mg / L), placed in a sterile sampling bottle, stored at 4°C and brought back to the laboratory.

[0028] 10 mL of sludge sample was inoculated into 100 mL of liquid culture medium (ingredients: peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, pH 7.3) and cultured at 30°C and 150 rpm for 24 h;

[0029] Take 1 mL of bacterial solution and transfer it to liquid culture medium containing 3% NaCl. Repeat the subculture 5 times to screen salt-tolerant strains. After the last subculture, the survival rate of the bacterial solution under 3% NaCl conditions is ≥85% (plate count method), indicating successful acclimation.

[0030] The acclimated bacterial solution was diluted to 10 -5 times, 100 μL was spread on solid culture medium (containing 3% NaCl), cultured at 30°C for 48 hours, and a single colony was picked for streak purification to obtain a pure strain (numbered Niuwu 3144).

[0031] The genomic DNA of the strain was extracted by the CTAB method. 16S rDNA was amplified using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The reaction conditions were as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 1 min, for a total of 30 cycles, and a final extension at 72°C for 10 min.

[0032] The PCR product was sequenced and a 1500 bp sequence (SEQ ID NO: 1) was obtained. BLAST comparison in the NCBI database showed a 99.8% sequence identity with Priestia megaterium strain B1 (CP064275.1). Combined with the physiological and biochemical characteristics (Gram-positive, aerobic), the strain was identified as Priestia megaterium.

[0033] The purified strain was inoculated into liquid culture medium and cultured until the logarithmic growth phase (OD 600 =1.0), mixed with bacterial solution and 20% glycerol aqueous solution in a volume ratio of 1:1, divided into cryovials, and stored at -80°C.

[0034] Please see the attached Figure 1 A method for culturing Priesteria gigantea, applied to the aforementioned Priesteria gigantea, comprises the following steps:

[0035] Priestia megaterium 3144 was inoculated into solid culture medium for activation;

[0036] The activated strain was inoculated into liquid culture medium and cultured with shaking at 150 rpm until OD 600 =1.5-1.8, then add corn starch solution to make the glucose concentration ≥2g / L, and continue to culture until OD 600 ≥3.0, bacterial cells were obtained;

[0037] The bacterial cells were mixed with sodium alginate-calcium carbonate to prepare immobilized particles, which were then placed in a reactor containing 1% simulated feces solution and cultured for 6 hours to obtain functionally enhanced immobilized strains, thus completing the culture.

[0038] The activation process is carried out by culturing at 37°C for 4 hours and then reducing the temperature to 30°C for 20 hours. The solid culture medium includes 10g / L corn starch, 8g / L soybean meal peptone, 5% v / v pig manure extract, 0.5g / L MgSO4·7H2O, 1g / L KH2PO4, and 0.1mmol / L sodium selenate. The liquid culture medium composition is the same as the solid culture medium but does not contain agar. The concentration of the corn starch solution added is 20% w / v, and the feeding rate is 0.5-1mL / min. To prepare immobilized particles, the bacteria were mixed with a 4% w / v sodium alginate solution in a volume ratio of 1:3, and the mixture was dripped into a 2% w / v calcium chloride solution via a syringe. After curing for 2 hours, particles with a diameter of 2-3 mm were collected. The manure simulation liquid was: fresh pig manure was mixed with deionized water in a ratio of 1:10 w / v, sterilized at 121°C for 20 minutes, filtered after cooling, and the supernatant was used. Its total nitrogen content was 200-500 mg / L, chemical oxygen demand was 1000-3000 mg / L, and pH was 7-8.5.

[0039] Application of Priesteria gigantea in pig farm manure treatment.

[0040] Specifically, the solid culture medium is configured as follows: 10 g / L corn starch, 8 g / L soybean meal peptone, 5% (v / v) pig manure extract, 0.5 g / L MgSO4·7H2O, 1.0 g / L KH2PO4, 0.1 mmol / L sodium selenate, 15 g / L agar, pH 7.2, and sterilized at 121°C for 20 minutes.

[0041] Then, the preserved bacterial liquid was taken out from -80℃, 100 μL was inoculated on a solid culture medium plate, and cultured at 37℃ for 4 hours, then reduced to 30℃ for 20 hours to obtain a single colony (colony morphology: white, round, 1-2 mm in diameter, with neat edges).

[0042] Liquid culture medium configuration: The composition is the same as solid culture medium, without agar, and sterilized at 121℃ for 20 minutes.

[0043] Then, a single colony was picked and inoculated into a 500 mL shake flask (containing 300 mL of liquid culture medium) and cultured with shaking at 150 rpm and 30°C for 12 hours until the OD 600 =1.6; transferred to 5L fermenter (inoculation volume 10%), maintained dissolved oxygen 30%, pH 7.0, when OD 600 =1.0, add 1mmol / L PTG to induce salt-tolerance gene expression; monitor glucose concentration in real time, add 20% corn starch solution at a rate of 0.8mL / min when it is lower than 2g / L, and culture until OD 600 =3.2, obtained bacteria (viable bacteria count 2.8×10 9 CFU / mL).

[0044] Immobilization reagent configuration: 4% (w / v) sodium alginate solution, 2% (w / v) calcium chloride solution.

[0045] The bacteria were mixed with sodium alginate solution in a volume ratio of 1:3, and calcium chloride solution was added dropwise through a 1mm needle syringe. The mixture was solidified for 2 hours to form particles with a diameter of 2-3mm. The particles were washed with sterile water 3 times, drained and placed in a reactor containing 1% fecal simulated liquid. The mixture was shaken and cultured at 30°C and 100 rpm for 6 hours to obtain a functionally enhanced immobilized strain.

[0046] Fresh manure was collected from the same pig farm, with an initial total nitrogen of 1200 mg / L, COD of 5000 mg / L, pH of 8.0, and a moisture content of 85%.

[0047] Experimental group: 1×10 8 The immobilized strain particles were added with an inoculum of CFU / g manure and left to stand at 25°C for 7 days, with stirring once a day.

[0048] Control group: no strain was added, and other conditions were the same.

[0049] Table 1:

[0050]

[0051] As shown in Table 1, all indicators of the experimental group were better than those of the control group, indicating that the immobilized strain had a significant degradation and harmless effect on pig farm manure.

[0052] By screening out the Priestia megaterium 3144 strain that is salt-tolerant, resistant to high ammonia nitrogen, and adaptable to pH fluctuations, and optimizing its culture and immobilization treatment, it is able to maintain high activity and metabolic capacity in complex and harsh environments such as high salinity (salt content can reach 1.5-3%), high ammonia nitrogen (ammonia nitrogen concentration can reach 200-500 mg / L), and large pH fluctuations (pH range is about 7.5-8.5) in pig farms. This strain can secrete a variety of degradative enzymes to synergistically and efficiently degrade organic matter, nitrogen, phosphorus and other pollutants in manure. In this way, efficient treatment of pig farm manure is achieved, solving the problem that traditional microbial strains used for pig farm manure treatment are difficult to continuously and efficiently degrade manure, resulting in low manure treatment efficiency and inability to meet the demand for effective manure treatment in actual production.

[0053] Example 1

[0054] The strain Priestia megaterium 3144 was isolated from the sludge of a manure treatment pool in a pig farm. After salt tolerance acclimation (survival rate ≥85% under 3% NaCl conditions) and solid plate streaking purification, a pure culture was obtained.

[0055] The genomic DNA of the strain was extracted using the CTAB method. The specific steps are as follows:

[0056] Take 2 mL of logarithmic growth phase bacterial solution (OD 600 =1.0), centrifuge at 12000 rpm for 5 min, and discard the supernatant;

[0057] The precipitate was added with 500 μL C CTAB extraction buffer (containing 1% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl), mixed and incubated in a 65°C water bath for 30 min, inverting several times during the process;

[0058] An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, mixed gently by inversion, centrifuged at 12000 rpm for 10 min, and the upper aqueous phase was collected;

[0059] Add 0.8 times the volume of isopropanol, let it stand at -20°C for 30 min, centrifuge at 12000 rpm for 10 min, and discard the supernatant;

[0060] The precipitate was washed twice with 75% ethanol, dried, dissolved in 50 μL sterile water, and stored at -20°C until use.

[0061] Universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) amplified a fragment of approximately 1500 bp;

[0062] Reaction system (50 μL): 2× TaqPCR Master Mix 25 μL, 1 μL each of upstream and downstream primers, 2 μL of genomic DNA template, and sterile water to 50 μL;

[0063] Pre-denaturation at 95°C for 5 min;

[0064] Denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles;

[0065] Final extension was performed at 72°C for 10 min.

[0066] The PCR product was detected by 1% agarose gel electrophoresis (clear bands, no debris bands), and sent to a biological company for Sanger sequencing to obtain forward and reverse sequences. After splicing, the complete 16S rDNA sequence (SEQ ID NO: 1) was obtained, which was 1469 bp in length.

[0067] Database search: SEQ ID NO: 1 was submitted to the NCBI BLAST platform (https: / / blast.ncbi.nlm.nih.gov) and the "16S ribosomal RNA sequences (Bacteria and Archaea)" database was selected for comparison. The results are shown in Table 2:

[0068] Table 2:

[0069]

[0070]

[0071] As shown in Table 2, the sequence identity of the strain with Bacillus and Priestia strains reached 99.8%, query cover 100%, and E value 0, indicating that it is highly homologous to the strains of the two genera at the 16S rRNA gene level.

[0072] The genus Priestia was once part of the genus Bacillus, but later became an independent new genus due to genomic differences, but retained a highly conserved 16S sequence. Combined with the strain's physiological and biochemical characteristics (Gram-positive, aerobic, and salt-tolerant) and genomic comparison results with type strains (such as Priestia megaterium B1), the strain was identified as Priestia (Chinese name unknown) and presumably Priestia megaterium (Chinese name unknown).

[0073] sequence:

[0074] SEQ ID NO: 1

[0075]

[0076] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A Priesterol bacterium characterized by: The Priestia megaterium was deposited in the China Center for Type Culture Collection on December 13, 2022, with the collection address being: Wuhan, China. Its classification name is Priestiamegaterium 3144, and the collection number is CCTCC NO: M20221962.

2. The Priesteria gigantea according to claim 1, characterized in that: The 16S rDNA sequence of Priestiamegaterium 3144 is shown in SEQ ID NO:

1.

3. The Priesteria gigantea according to claim 1, characterized in that: The colony morphology of Priestiamegaterium 3144 is round, white, opaque, 1-2 mm in diameter, with a smooth and moist surface and neat edges.

4. The Priesteria gigantea according to claim 1, characterized in that: The physiological and biochemical characteristics of the Priestiamegaterium 3144 are: Gram staining positive, aerobic growth, culture temperature of 25-35° C., and pH of 6.5-8.

0.

5. The Priesteria gigantea according to claim 1, characterized in that: The storage method of Priestiamegaterium 3144 includes: freezing at -80°C using a 20% glycerol aqueous solution as a protective agent.

6. A method for culturing Priesteria gigantea, characterized in that: The method for applying the Priesteria gigantea according to any one of claims 1 to 5, comprising the following steps: Priestia megaterium 3144 was inoculated into solid culture medium for activation; The activated strain was inoculated into liquid culture medium and cultured with shaking at 150 rpm until OD 600 =1.5-1.8, then add corn starch solution to make the glucose concentration ≥2g / L, and continue to culture until OD 600 ≥3.0, bacterial cells were obtained; The bacterial cells were mixed with sodium alginate-calcium carbonate to prepare immobilized particles, which were then placed in a reactor containing 1% simulated feces solution and cultured for 6 hours to obtain functionally enhanced immobilized strains, thus completing the culture.

7. The method for culturing Priesteria gigantea according to claim 6, wherein: The activation is performed by culturing at 37°C for 4 hours and then decreasing the temperature to 30°C for 20 hours. The solid culture medium comprises 10 g / L corn starch, 8 g / L soybean meal peptone, 5% v / v pig manure extract, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, and 0.1 mmol / L sodium selenate. The liquid culture medium has the same composition as the solid culture medium but does not contain agar. The concentration of the corn starch solution added is 20% w / v, and the addition rate is 0.5-1 mL / min. n, the preparation of immobilized particles is to mix the bacteria with a 4% w / v sodium alginate solution in a volume ratio of 1:3, drip the mixture into a 2% w / v calcium chloride solution via a syringe, and collect particles with a diameter of 2-3 mm after curing for 2 hours. The manure simulation liquid is: fresh pig manure and deionized water are mixed in a ratio of 1:10 w / v, sterilized at 121°C for 20 minutes, filtered after cooling, and the supernatant is used. Its total nitrogen content is 200-500 mg / L, chemical oxygen demand is 1000-3000 mg / L, and pH is 7-8.

5.

8. Use of Priesteria gigantea according to claim 1 in treating manure in pig farms.

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