CN110964639A - Strain screening method applied to pig raising fermentation bed and application - Google Patents
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- 238000000855 fermentation Methods 0.000 title claims abstract description 37
- 230000004151 fermentation Effects 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000010871 livestock manure Substances 0.000 claims abstract description 26
- 238000012258 culturing Methods 0.000 claims abstract description 25
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- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
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- 238000006731 degradation reaction Methods 0.000 claims abstract description 15
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- 235000015278 beef Nutrition 0.000 claims description 22
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 238000002474 experimental method Methods 0.000 claims description 9
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- 229940041514 candida albicans extract Drugs 0.000 claims description 6
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- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 244000057717 Streptococcus lactis Species 0.000 claims description 4
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 4
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
A strain screening method applied to a pig raising fermentation bed (1) of the invention collects a pig manure urine sample; (2) culturing by a simulated fermentation bed; (3) primary screening; (4) and re-screening to obtain excellent strains. The invention screens out the bacterial strains with better degradation effect on the pig manure from the pig manure sample, and the bacterial strains are respectively expanded and cultured to form mixed bacterial liquid which can be directly applied to the pig raising fermentation bed; the strain screening method provided by the invention can be suitable for ecological environments in different areas, can provide strain sources for local fermentation bed pig raising technology, is not influenced by external strains, and does not destroy local micro-ecological balance.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a strain screening method and application applied to a pig-raising fermentation bed.
Background
With the development of large-scale, intensive and commercialized pig breeding industry, the problem of environmental pollution caused by pig manure is more and more serious, and the problem is an urgent need to be solved by the current rural pig farms. With the advent of ten items of water, detailed requirements are made on pollution prevention and control of rural livestock and poultry breeding and the like by using definite quantitative indexes, which marks the coming of a new environment-friendly era with environmental quality and environmental effect as the core in China.
A fermentation bed pig raising technology is a novel breeding technology for realizing in-situ degradation of pig manure, and the technical principle is that beneficial microbial floras are added into padding such as sawdust and rice husks to serve as a biological fermentation bed, pigs are only raised on the fermentation bed, the excreted manure and the padding are fully mixed under the arch turning and artificial auxiliary rake turning of the pigs, and the manure is decomposed under the action of microorganisms, so that the novel environment-friendly pig raising technology which is free of pollution, near zero emission and virtuous cycle is basically achieved.
The core of the fermentation bed pig raising technology is a microbial agent, wherein the dominant flora plays a key role and has important influence on the fermentation temperature, the degradation of livestock manure and urine, the inhibition of pathogenic bacteria and the like, so that the breeding of the dominant bacteria in the fermentation bed has important significance. The fermentation process of the fermentation bed comprises 3 stages of temperature rise, high temperature and temperature reduction, and related functional microorganisms play a role in decomposing organic matters in different stages. Therefore, the dominant strain of the fermentation bed must be able to withstand a certain high temperature to complete its decomposition of the organic matter. Currently, commercially available fermentation bed strains mainly comprise bacillus, acinetobacter, actinomycete, yeast, lactobacillus and the like, and since commercially available microbial agents are different in quality and correspond to foreign strains in specific regions, once the microbial agents are lost, the local microecological balance is easily damaged.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background art and provide a strain screening method applied to a pig-raising fermentation bed.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a strain screening method applied to a pig raising fermentation bed comprises the following steps:
(1) collecting a pig manure urine sample;
(2) and (3) simulated fermentation bed culture:
s1: mixing the collected 0.1-0.5 part of pig manure urine sample with 0.1-0.5 part of sterilized wheat bran, and uniformly stirring;
s2: mixing 2-4 parts of sterilized sawdust and 2-4 parts of sterile rice hulls, adding 0.2-0.5 part of sterile brown sugar water, uniformly stirring, and simultaneously adding sterile water to enable the water content of a mixed sample to reach 40-55%;
s3: mixing the material obtained in the step S1 with the material obtained in the step S2, uniformly stirring, and culturing at constant temperature for 10-60 days;
(3) primary screening;
(4) and re-screening to obtain excellent strains.
Preferably, the preliminary screening method includes the following specific steps:
(a) weighing 2 parts of sample cultured by a simulated fermentation bed, putting the sample into a conical flask filled with bacteria water and small glass beads, and placing the conical flask in a shaking table to shake for 20-30 min;
(b) transferring 2 samples obtained in the step (a) into 2 different enrichment media, and culturing at constant temperature for 5-7d to obtain a sample A and a sample B;
(c) dividing the sample A into two equal parts A1 and A2, dividing the sample B into two equal parts B1 and B2, respectively culturing A1 and A2 in 2 different enrichment media, respectively culturing B1 and B2 in 2 different enrichment media, and culturing at constant temperature for 5-7d to obtain samples C1, C2, C3 and C4;
(d) sample C1 was aliquoted into 10 portions: c11, C12, C13, C14, C15, C16, C17, C18, C19 and C10,
sample C2 was aliquoted into 10 portions: c21, C22, C23, C24, C25, C26, C27, C28, C29 and C20,
sample C3 was aliquoted into 10 portions: c31, C32, C33, C34, C35, C36, C37, C38, C39 and C30, sample C4 was aliquoted into 10 parts: c41, C42, C43, C44, C45, C46, C47, C48, C49 and C40,
placing C11, C12, C13, C14 and C15 in 5 parts of different separation culture media for constant-temperature culture for 1d, placing C21, C22, C23, C24 and C25 in 5 parts of different separation culture media for constant-temperature culture for 1d, placing C31, C32, C33, C34 and C35 in 5 parts of different separation culture media respectively for constant-temperature culture for 1d, placing C41, C42, C43, C44 and C45 in 5 parts of different separation culture media respectively for constant-temperature culture for 1d, picking up single colonies which are well separated and have representativeness on a plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored;
(e) boiling C16, C17, C18, C19 and C10 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C26, C27, C28, C29 and C20 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C36, C37, C38, C39 and C30 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C46, C47, C48, C49 and C40 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
then picking a single colony which is well separated and representative on the plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored.
Preferably, the 2 different enrichment media are respectively:
improving a beef extract peptone culture medium: 5-8 parts of brown sugar, 3-5 parts of beef extract, 10-20 parts of peptone, 5-10 parts of sodium chloride, 7.0 +/-0.2 of pH, 12 parts of agar and 1000 parts of water;
improving an LB culture medium: 5-7 parts of brown sugar, 5-7 parts of yeast extract, 10-15 parts of peptone, 10-12 parts of sodium chloride, 7.0 +/-0.2 of pH, 12-15 parts of agar and 1000 parts of water.
In the above method for screening bacterial species, preferably, the 5 different isolation media are:
1) modified NA 1: 3-6 parts of beef extract, 5-8 parts of peptone, 7.0 +/-0.2 of pH, 12 parts of agar and 1000 parts of water;
2) modified NA 2: 10-15 parts of beef extract, 10-15 parts of peptone, 5-10 parts of sodium chloride, 7.0 +/-0.2 of pH and agar
12-15 parts of water and 1000 parts of water;
3) improved LB 1: 10-15 parts of beef extract, 5-10 parts of yeast extract, 10-20 parts of sodium chloride, 5-15 parts of glucose and pH7.0
+/-0.2 g of agar and 1000 parts of water;
4) FC: 25-40 parts of glucose, 15-20 parts of beef extract, 2-5 parts of dipotassium hydrogen phosphate, 2.5-5 parts of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 7.0 +/-0.2 of pH, 12-15 parts of agar and 1000 parts of water;
5) and (3) improving MRS: 20-30 parts of brown sugar, 4-6 parts of yeast powder, 5-7 parts of sodium acetate, 2-5 parts of dipotassium hydrogen phosphate, 0.2-0.4 part of magnesium sulfate, 0.05-0.1 part of manganese sulfate, 2-5 parts of triammonium hydrogen citrate, 801-2 parts of tween and 10-15 parts of peptone
The beef jerky comprises, by weight, 5-8 parts of beef extract, pH6.2 +/-0.2, 12-15 parts of agar and 1000 parts of water.
The invention can separate different strains by improving different enrichment culture media and separation culture media.
In the above method for screening strains, preferably, the temperature for constant temperature culture is 30-32 ℃.
Preferably, the bacterial strain screening method comprises the following specific operation steps:
①, respectively carrying out degradation experiments of pig manure and urine on the single strains selected in the primary screening steps (d) and (e), measuring the weight change of the samples before and after treatment, and screening out strains with better deodorization effect on the pig manure;
② the degradation effect was tested by culturing a mixture of strains of the strain obtained in step ①.
The strain screening method preferably re-screens to finally obtain a plurality of single strains with better degradation efficiency, namely 6 strains of alcaligenes faecalis, acinetobacter, lactococcus lactis, bacillus megaterium, bacillus subtilis and bacillus licheniformis.
As a general inventive concept, the invention also provides an application of the strain obtained by the method in a pig-raising fermentation bed, and specifically, a plurality of single strains obtained by the method are respectively subjected to amplification culture to form a mixed bacterial liquid which is directly applied to the pig-raising fermentation bed.
Compared with the prior art, the invention has the advantages that:
(1) the invention screens out the bacterial strains with better degradation effect on the pig manure from the pig manure sample, and the bacterial strains are respectively expanded and cultured to form mixed bacterial liquid which can be directly applied to the pig raising fermentation bed; the strain screening method provided by the invention can be suitable for ecological environments in different areas, can provide strain sources for local fermentation bed pig raising technology, is not influenced by external strains, and does not destroy local micro-ecological balance.
(2) The composite strain obtained by screening has strong adaptability and high propagation speed, and can be directly applied to a pig raising fermentation bed after the strain is subjected to enlarged culture, so that pig manure is decomposed, and pollution-free and near zero emission is basically realized.
Drawings
FIG. 1 shows the colony morphology of 6 single species screened in the example of the present invention.
FIG. 2 shows the identification results (based on the phylogenetic tree analysis of 16S rDNA sequences) of 6 individuals of strains selected in this example of the present invention.
Detailed Description
In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
the enrichment medium adopted in the embodiment respectively comprises the following components:
improving a beef extract peptone culture medium: 5g of brown sugar, 3g of beef extract, 10gg of peptone, 5g of sodium chloride, pH7.0 +/-0.2, 12 parts of agar and 1L of water.
Improving an LB culture medium: 5g of brown sugar, 5g of yeast extract, 10g of peptone, 10g of sodium chloride, pH7.0 +/-0.2, 12g of agar and 1L of water.
The formula of the separation culture medium adopted in the embodiment is respectively as follows:
1) modified NA 1: 4g of beef extract, 6g of peptone, pH7.0 +/-0.2, 12g of agar and 1L of water.
2) Modified NA 2: 10g of beef extract, 12g of peptone, 8g of sodium chloride, pH7.0 +/-0.2, 12g of agar and 1L of water.
3) Improved LB 1: 10-g of beef extract, 5g of yeast extract, 15g of sodium chloride, 10g of glucose, 7.0 +/-0.2 of pH, 12g of agar and 1L of water.
4) FC: 25g of glucose, 15g of beef extract, 2-g of dipotassium hydrogen phosphate, 2.5g of monopotassium phosphate, 1g of magnesium sulfate and pH7.0
+ -0.2, agar 12g, water 1L.
5) And (3) improving MRS: 20g of brown sugar, 4g of yeast powder, 5g of sodium acetate, 2g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2g of triammonium hydrogen citrate, 801 mL of tween, 10g of peptone, 5g of beef extract, pH6.2 +/-0.2 and agar
12g of water and 1L of water.
The method for screening the strains applied to the pig-raising fermentation bed comprises the following steps:
(1) collecting a pig manure urine sample;
(2) and (3) simulated fermentation bed culture:
(a) firstly, a 5L barrel with a cover is adopted, 75% alcohol is used for disinfection, 300g of sterile sawdust and 300g of sterile rice hulls are added, and the mixture is fully stirred, uniformly mixed and slightly compressed;
(b) adding 50mL of sterile brown sugar water, stirring uniformly, and simultaneously adding a proper amount of sterile water to finally enable the water content of the mixture to reach 40%;
(c) taking 10g of pig manure, uniformly mixing with 10g of sterile wheat bran, uniformly mixing with the mixture in the step (b), placing in a ventilated place, and naturally fermenting for 15 days.
(3) Primary screening:
a: weighing 1 part of sample cultured by a simulated fermentation bed, putting the sample into a conical flask filled with a proper amount of sterile water and small glass beads, and placing the conical flask in a shaking table to shake for 30min to disperse microbial cells;
b: transferring a proper amount of a sample into an enrichment medium, and culturing at constant temperature of 30 ℃ for 6 d;
c: transferring a proper amount of the sample b into an enrichment medium, and culturing at the constant temperature of 30 ℃ for 6 d;
d: transferring 1 part of b sample to perform a gradient dilution inversion experiment of 10 times, culturing at constant temperature of 30 ℃ for 1d, then selecting a single colony which is well separated and has a representative on a flat plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored;
e: transferring 1 part of sample c, boiling for 10min with 100 ℃ boiling water to kill bacteria which do not form spores, performing 10-time gradient dilution and pouring into a flat plate experiment, culturing at the constant temperature of 30 ℃ for about 1d, selecting a single colony which is well separated and representative on the flat plate, and purifying and separating by using a flat plate scribing method until a pure culture body is obtained and stored;
(5) re-screening:
(a) weighing 2 parts of a sample cultured by a simulated fermentation bed, putting the sample into a conical flask filled with bacteria water and small glass beads, and placing the conical flask in a shaking table to shake for 30 min;
(b) transferring 2 samples obtained in the step (a) into 2 different enrichment media, and culturing at constant temperature for 6d to obtain a sample A and a sample B;
(c) dividing the sample A into two equal parts A1 and A2, dividing the sample B into two equal parts B1 and B2, respectively placing A1 and A2 in 2 different enrichment media, respectively placing B1 and B2 in 2 different enrichment media, and culturing at constant temperature for 6d to obtain samples C1, C2, C3 and C4;
(d) sample C1 was aliquoted into 10 portions: c11, C12, C13, C14, C15, C16, C17, C18, C19 and C10,
sample C2 was aliquoted into 10 portions: c21, C22, C23, C24, C25, C26, C27, C28, C29 and C20,
sample C3 was aliquoted into 10 portions: c31, C32, C33, C34, C35, C36, C37, C38, C39 and C30, sample C4 was aliquoted into 10 parts: c41, C42, C43, C44, C45, C46, C47, C48, C49 and C40,
placing C11, C12, C13, C14 and C15 in 5 different separation culture media for constant-temperature culture for 1d, placing C21, C22, C23, C24 and C25 in 5 different separation culture media for constant-temperature culture for 1d, placing C31, C32, C33, C34 and C35 in 5 different separation culture media for constant-temperature culture for 1d, placing C41, C42, C43, C44 and C45 in 5 different separation culture media for constant-temperature culture for 1d, picking up single colonies which are well separated and have representativeness on a plate, and carrying out purification and separation by using a plate-scribing method until a pure culture body is obtained and stored;
(e) boiling C16, C17, C18, C19 and C10 with 100 ℃ boiling water for 10min, performing gradient dilution by 10 times, pouring into a flat plate experiment, and respectively placing in 5 different separation culture media for constant temperature culture for 3 d;
boiling C26, C27, C28, C29 and C20 with 100 ℃ boiling water for 10min, performing gradient dilution by 10 times, pouring into a flat plate experiment, and respectively placing in 5 different separation culture media for constant temperature culture for 3 d;
boiling C36, C37, C38, C39 and C30 with 100 ℃ boiling water for 10min, performing gradient dilution by 10 times, pouring into a flat plate experiment, and respectively placing in 5 different separation culture media for constant temperature culture for 3 d;
boiling C46, C47, C48, C49 and C40 with 100 ℃ boiling water for 10min, performing gradient dilution by 10 times, pouring into a flat plate experiment, and respectively placing in 5 different separation culture media for constant temperature culture for 3 d;
then picking a single colony which is well separated and representative on the plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored.
(6) According to the purpose of realizing the rapid decomposition of the pig manure by the pig raising fermentation bed, carrying out a pig manure degradation experiment on the single bacterial strains screened in the steps (d) and (e), measuring the weight change of the sample before and after treatment, and screening 6 bacterial strains with better deodorization effect on the pig manure, wherein the data with better degradation data effect are shown in the table 1;
(7) and (3) performing mixed culture on the 6 strains obtained by screening in the step (6), and testing the degradation effect of the strains on pig manure, wherein the degradation data are shown in table 2.
TABLE 1 preliminary screening data
TABLE 2 rescreening data
Experimental results show that the 6 single strains have a good degradation effect on pig manure, the colony morphology of the 6 single strains is shown in figure 1, molecular identification is carried out on the 6 single strains, the identification results (based on 16S rDNA sequence phylogenetic tree analysis) are shown in figure 2, and the 6 strains are respectively Alcaligenes faecalis, Acinetobacter, lactococcus lactis, Bacillus megaterium, Bacillus subtilis and Bacillus licheniformis.
The alcaligenes faecalis, acinetobacter, lactococcus lactis, bacillus megaterium, bacillus subtilis and bacillus licheniformis obtained in the embodiment are subjected to amplification culture respectively to form mixed bacteria liquid, and the mixed bacteria liquid is directly applied to a local pig raising fermentation bed.
Claims (8)
1. A strain screening method applied to a pig raising fermentation bed is characterized by comprising the following steps:
(1) collecting a pig manure urine sample;
(2) and (3) simulated fermentation bed culture:
s1: mixing the collected 0.1-0.5 part of pig manure urine sample with 0.1-0.5 part of sterilized wheat bran, and uniformly stirring;
s2: mixing 2-4 parts of sterilized sawdust and 2-4 parts of sterile rice hulls, adding 0.2-0.5 part of sterile brown sugar water, uniformly stirring, and simultaneously adding sterile water to enable the water content of a mixed sample to reach 40-55%;
s3: mixing the material obtained in the step S1 with the material obtained in the step S2, uniformly stirring, and culturing at constant temperature for 10-60 days;
(3) primary screening;
(4) and re-screening to obtain excellent strains.
2. The strain screening method as claimed in claim 1, wherein the preliminary screening comprises the following steps:
(a) weighing 2 parts of sample cultured by a simulated fermentation bed, putting the sample into a conical flask filled with bacteria water and small glass beads, and placing the conical flask in a shaking table to shake for 20-30 min;
(b) transferring 2 samples obtained in the step (a) into 2 different enrichment media, and culturing at constant temperature for 5-7d to obtain a sample A and a sample B;
(c) dividing the sample A into two equal parts A1 and A2, dividing the sample B into two equal parts B1 and B2, respectively placing A1 and A2 in 2 different enrichment media, respectively placing B1 and B2 in 2 different enrichment media, and culturing at constant temperature for 5-7d to obtain samples C1, C2, C3 and C4;
(d) sample C1 was aliquoted into 10 portions: c11, C12, C13, C14, C15, C16, C17, C18, C19 and C10,
sample C2 was aliquoted into 10 portions: c21, C22, C23, C24, C25, C26, C27, C28, C29 and C20,
sample C3 was aliquoted into 10 portions: c31, C32, C33, C34, C35, C36, C37, C38, C39 and C30,
sample C4 was aliquoted into 10 portions: c41, C42, C43, C44, C45, C46, C47, C48, C49 and C40,
placing C11, C12, C13, C14 and C15 in 5 different kinds of separation culture media for constant-temperature culture for 1d, placing C21, C22, C23, C24 and C25 in 5 different kinds of separation culture media for constant-temperature culture for 1d, placing C31, C32, C33, C34 and C35 in 5 different kinds of separation culture media respectively for constant-temperature culture for 1d, placing C41, C42, C43, C44 and C45 in 5 different kinds of separation culture media respectively for constant-temperature culture for 1d, picking up single colonies which are well separated and have representativeness on a plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored;
(e) boiling C16, C17, C18, C19 and C10 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C26, C27, C28, C29 and C20 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C36, C37, C38, C39 and C30 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
boiling C46, C47, C48, C49 and C40 with 100 deg.C boiling water for 8-10min, performing gradient dilution by 10 times, pouring into flat plate, and culturing in 5 different separation culture media at constant temperature for 2-3 days;
then picking a single colony which is well separated and representative on the plate, and purifying and separating by using a plate marking method until a pure culture body is obtained and stored.
3. A method of screening bacterial species according to claim 2, wherein the 2 different enrichment media are:
improving a beef extract peptone culture medium: 5-8 parts of brown sugar, 3-5 parts of beef extract, 10-20 parts of peptone, 5-10 parts of sodium chloride, 7.0 +/-0.2 of pH, 12 parts of agar and 1000 parts of water;
improving an LB culture medium: 5-7 parts of brown sugar, 5-7 parts of yeast extract, 10-15 parts of peptone, 10-12 parts of sodium chloride, 7.0 +/-0.2 of pH, 12-15 parts of agar and 1000 parts of water.
4. A method of strain screening as claimed in claim 2 wherein the 5 different isolation media are:
1) modified NA 1: 3-6 parts of beef extract, 5-8 parts of peptone, 7.0 +/-0.2 of pH, 12 parts of agar and 1000 parts of water;
2) modified NA 2: 10-15 parts of beef extract, 10-15 parts of peptone, 5-10 parts of sodium chloride, 7.0 +/-0.2 of pH, 12-15 parts of agar and 1000 parts of water;
3) improved LB 1: 10-15 parts of beef extract, 5-10 parts of yeast extract, 10-20 parts of sodium chloride, 5-15 parts of glucose, 7.0 +/-0.2 of pH, 12g of agar and 1000 parts of water;
4) FC: 25-40 parts of glucose, 15-20 parts of beef extract, 2-5 parts of dipotassium hydrogen phosphate, 2.5-5 parts of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 7.0 +/-0.2 of pH, 12-15 parts of agar and 1000 parts of water;
5) and (3) improving MRS: 20-30 parts of brown sugar, 4-6 parts of yeast powder, 5-7 parts of sodium acetate, 2-5 parts of dipotassium hydrogen phosphate, 0.2-0.4 part of magnesium sulfate, 0.05-0.1 part of manganese sulfate, 2-5 parts of triammonium hydrogen citrate, 801-2 parts of tween, 10-15 parts of peptone, 5-8 parts of beef extract, pH6.2 +/-0.2, 12-15 parts of agar and 1000 parts of water.
5. A method for screening a bacterial species according to any one of claims 1 to 4, wherein said incubation is carried out at a temperature of 30 to 32 ℃.
6. The strain screening method as claimed in claim 2, wherein the re-screening comprises the following steps:
①, respectively carrying out degradation experiments of pig manure and urine on the single strains selected in the primary screening steps (d) and (e), measuring the weight change of the samples before and after treatment, and screening out strains with better deodorization effect on the pig manure;
② the degradation effect was tested by culturing a mixture of strains of the strain obtained in step ①.
7. The strain screening method as claimed in claim 6, wherein the single strain with better degradation effect is obtained by re-screening, and comprises Alcaligenes faecalis, Acinetobacter, lactococcus lactis, Bacillus megaterium, Bacillus subtilis and Bacillus licheniformis.
8. The application of the strain obtained in claim 7 in a pig-raising fermentation bed is characterized in that a plurality of single strains obtained in claim 7 are subjected to amplification culture respectively to form a mixed bacterial liquid, and the mixed bacterial liquid is directly applied to the pig-raising fermentation bed.
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