CN111500492B - Thermophilic bacterium BV-2 and application thereof - Google Patents
Thermophilic bacterium BV-2 and application thereof Download PDFInfo
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- CN111500492B CN111500492B CN202010328942.8A CN202010328942A CN111500492B CN 111500492 B CN111500492 B CN 111500492B CN 202010328942 A CN202010328942 A CN 202010328942A CN 111500492 B CN111500492 B CN 111500492B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to the technical field of microorganisms, and particularly discloses a thermophilic bacterium BV-2 and application thereof, wherein the preservation number of a strain is CGMCC No. 18652. The thermophilic bacteria BV-2 can be applied to the decomposition fermentation of edible fungus matrixes. The thermophilic bacteria BV-2 provided by the invention has the characteristics of strong high-temperature adaptability, wide temperature range suitable for growth, capability of secreting a large amount of high-activity cellulase, and capability of killing and inhibiting other pathogenic bacteria. The edible fungus substrate obtained by the thermophilic fungus BV-2 decomposing fermentation treatment provided by the invention can completely replace the substrate obtained by high-temperature steam sterilization treatment, and has the characteristics of high nutrient content, low contamination rate and obvious increase of the fungus yield of the edible fungus.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to thermophilic bacteria BV-2 and application thereof.
Background
China is a large edible fungus producing country, the annual edible fungus yield is about 4000 ten thousand tons, which accounts for more than 70% of the total edible fungus yield in the world, and the method is one of important prop industries of China's agriculture. The fruiting rate of the culture medium of the mushrooms in the edible mushrooms is about 5: 1, namely about 4000 million tons of edible fungi are produced per year, and the dosage of the matrix is about 2 hundred million tons. At present, no matter the raw material culture medium or the clinker culture medium of the edible fungi is used, the edible fungi culture medium needs to be inoculated after high-temperature sterilization treatment. This process requires a large amount of heat, which not only increases the cost of producing the edible fungi, but also increases the environmental stress.
Microbial agents are added into organic matters such as livestock and poultry manure, so that the temperature of the pile can be raised, the organic materials are decomposed, pathogenic microorganisms in the organic materials are inhibited, and the organic materials are recycled and harmless. However, if the microbial fermentation method is applied to the decomposing fermentation of the edible fungus substrate, the step of heating and high-temperature sterilization cannot be omitted, and the direct edible fungus inoculation of the decomposed and fermented substrate cannot be realized.
Disclosure of Invention
The invention provides a thermophilic bacterium BV-2 and application thereof, aiming at the problems that the microorganisms disclosed in the existing microbial fermentation method can not realize direct edible fungus inoculation on an edible fungus substrate through rotten fermentation, can not avoid a high-temperature sterilization treatment step, and has high energy consumption and high treatment cost.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a thermophilic bacterium BV-2, the preservation number of the strain is CGMCC No.18652, the strain belongs to Bacillus velezensis, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms 10 months and 10 days in 2019, and the preservation address is located in the institute of microbiology of China academy of sciences No. 3 of West Lu No.1 of the sunward region in Beijing.
Compared with the prior art, the thermophilic bacteria BV-2 provided by the invention has the characteristics of strong high-temperature adaptability, wide temperature range suitable for growth (suitable for growth in the range of 25-75 ℃ and still capable of growth at 85 ℃), capability of secreting a large amount of high-activity cellulase, and capability of killing and inhibiting other pathogenic bacteria. The thermophilic bacteria BV-2 is inoculated into the edible fungus matrix within 24 hours, so that the temperature of the edible fungus matrix is raised to be more than 50 ℃, the maximum temperature of the rotten temperature in the edible fungus matrix reaches 85 ℃, and the temperature can be lowered to the normal temperature within 5 days. And the edible fungus substrate treated by the thermophilic fungus BV-2 decomposing fermentation has low contamination rate, can be directly inoculated with the edible fungus, and can completely avoid the high-temperature steam sterilization treatment process of the edible fungus substrate. On the other hand, the edible fungus substrate subjected to the decomposing fermentation treatment by the thermophilic bacteria BV-2 has high nutrient content, can obviously increase the fungus yield of the edible fungus, and has extremely high economic benefit.
The invention also provides the application of the thermophilic bacteria BV-2 in the decomposition fermentation of the edible fungus matrix.
After the edible fungus matrix is inoculated with thermophilic bacteria BV-2, the highest temperature can reach 85 ℃, the time reaching the high temperature period is 2 days after fermentation, the high temperature period exceeding 50 ℃ is 4 days, and the high temperature period exceeding 65 ℃ is 2 days. Can obviously promote the temperature rise of the edible fungus substrate, improve the temperature of the material, shorten the decomposition time and lead the pathogenic bacteria to be removed more thoroughly.
The invention also provides a decomposing inoculant prepared by using the thermophilic bacteria BV-2, which comprises a thallus adsorption matrix and thermophilic bacteria BV-2 bacteria powder.
Preferably, the bacteria adsorption matrix is light calcium carbonate.
The light calcium carbonate as thallus adsorbing matrix can make the freezing preservation time of the live thermophilic bacteria BV-2 reach more than one year.
Preferably, the viable count of the thermophilic bacteria BV-2 in the decomposing inoculant is more than or equal to 2 multiplied by 108cfu/g。
The invention also provides a preparation method of the decomposing inoculant, which comprises the following process steps:
a. activating the strains;
b. seed culture;
c. fermenting the second-level seeds;
d. carrying out amplification culture;
e. adding an adsorption matrix and drying; the drying method is spray drying.
Compared with the prior art, the preparation method of the decomposing microbial inoculum provided by the invention has the advantages that the obtained decomposing microbial inoculum can be quickly adapted to different temperature environments and can quickly grow in the edible fungus matrix, and the decomposing speed of the edible fungus matrix is accelerated.
Preferably, in step a, the method for activating the bacterial strain comprises the following steps: and streaking the thermophilic bacteria BV-2 strain which is frozen and preserved on an LB plate, and culturing for 20-30h at 40-50 ℃.
Preferably, in step b, the method for seed culture comprises: and (b) inoculating the activated thermophilic bacteria BV-2 lawn in the step (a) into a triangular flask filled with LB liquid culture medium, and carrying out shake cultivation for 20-30h at 40-50 ℃ and 250 r/min.
Preferably, in step c, the method for secondary seed fermentation is as follows: b, inoculating the seed culture solution obtained in the step b into a seed tank filled with an LB liquid culture medium, and performing fermentation culture at 40-50 ℃ and 150-; the inoculation volume of the seed culture solution accounts for 5-10% of the volume of the LB liquid culture medium.
Preferably, in step d, the method for expanding culture comprises: c, inoculating the thermophilic bacteria BV-2 secondary seed fermentation liquid obtained in the step c into a fermentation tank filled with LB liquid culture medium, and fermenting for 2-3d at 45-50 ℃ and 200 r/min; the inoculation volume of the secondary seed fermentation liquid accounts for 2-5% of the volume of the LB liquid culture medium.
Drawings
FIG. 1 is a colony morphology of a thermophilic BV-2 strain in an example of the present invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Examples
1.1 samples and culture media
1.1.1 samples
Samples of hot springs were collected from the hot spring zone of taiwan wenshan, china, at a temperature of 72 ℃.
1.1.2 culture Medium
ZoBell2216E solid medium, Congo red carboxymethyl cellulose medium, LB solid medium and LB liquid medium.
1.2 screening of thermophilic bacteria
Samples of hot springs were collected from the hot spring zone of taiwan wenshan, china, at a temperature of 72 ℃. Gradually diluting the sample, respectively taking 100 μ L of original hot spring water sample, 10-fold diluted sample and 100-fold diluted sample, spreading on ZoBell2216E solid culture medium, sealing the plate in a sealing bag, and culturing in a 60 deg.C forced air drying oven. And selecting the colony which grows rapidly, and carrying out streaking separation and purification for 5 times on an LB plate to obtain the pure culture strain of the thermophilic bacteria. After the strains are subjected to plate passage for 5 times, 5 single colonies of each strain are selected and cultured at 60 ℃, the genetic stability of the high-temperature growth characteristics of the strains is determined, and the thermophilic strains with stable heredity are obtained.
1.3 screening of cellulose-degrading thermophilic bacteria
And (3) performing activated culture on the screened thermophilic bacterial strain on an LB culture medium in a 60 ℃ culture box for 24 hours, then respectively inoculating the thermophilic bacterial strain on a Congo red carboxymethyl cellulose culture medium plate, and measuring the colony diameter and the hydrolysis ring diameter of the thermophilic bacterial strain after the thermophilic bacterial strain is cultured for 3 days at a constant temperature of 60 ℃. The measurement shows that the BV-2 strain has the colony diameter of 0.42cm, the hydrolysis ring diameter of 1.04cm and the D/D value of 2.47 when cultured on a Congo red carboxymethyl cellulose plate for 3 days, and has the capacity of secreting cellulase at high temperature.
1.4 identification of the thermophilic BV-2 Strain
The BV-2 strain obtained by screening is cultured for 2 days at 50 ℃ on an LB solid culture medium. The bacterial colony is round, has smooth surface and thick texture, and the diameter of the bacterial colony is 1.45 mm; short rods or spindle-shaped, slightly bent, single, paired or short chain-shaped cells, G +, as shown in FIG. 1; alkalescence, chemoheterotrophic and aerobic; catalase is positive; a homoserine auxotrophic strain; producing L-lysine.
Taking the DNA of the BV-2 strain as a template, taking an upstream primer 27f of a 16S rRNA universal primer: 5'-AGAGTTTGATCCTGGCTC-3' and a downstream primer 1492r: 5'-GGTTACCTTGTTACG ACTT-3', and the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 60s for 30 cycles; extension at 72 ℃ for 8 min. And (3) detecting the purity and the size of the amplified product by electrophoresis, sending the PCR product with the correct amplified length to Shanghai workers for sequencing, carrying out BLAST on NCBI, and according to the BLAST result, ensuring that the similarity of the 16S rRNA gene sequence of the strain and the Bacillus velezensis strain is up to 99%.
By combining the colony morphology and physiological characteristics and the 16S rRNA sequence, the strain BV-2 of the invention can be determined to belong to Bacillus velezensis, is named as B.velezensis BV-2, and is stored in the common microorganism center of China general microbiological culture Collection center in 2019, 10 months and 10 days, and the preservation number is CGMCC number 18652.
1.5 method for preparing decomposing inoculant by using thermophilic bacteria BV-2
The specific method comprises the following steps:
(1) activating strains: the BV-2 strain stored at-80 ℃ is streaked and inoculated on an LB plate, and cultured for 24h at 50 ℃.
(2) Seed culture: scraping off thallus Porphyrae of BV-2 strain on LB plate with inoculating loop, inoculating into triangular flask containing LB liquid culture medium, and shake culturing at 50 deg.C and 200rpm/min for 24 hr.
(3) Secondary seed fermentation: inoculating the seed culture solution into a seed tank containing 10L LB liquid culture medium at an inoculum size of 5%, and fermenting at 50 deg.C and 200rpm/min for 24 h.
(4) And (3) amplification culture: the obtained thermophilic bacteria BV-2 secondary seed fermentation liquor is inoculated in a fermentation tank filled with LB liquid culture medium according to the inoculation amount of 3 percent, and the fermentation is carried out for 2d at 50 ℃ and 200 rpm/min.
(5) Spray drying: adding light calcium carbonate into the fermentation liquor after the enlarged culture, and then carrying out spray drying on the fermentation liquor to obtain the decomposing microbial inoculum containing thermophilic bacteria BV-2.
(6) And (3) product quality detection: the detection of thermophilic bacteria BV-2 decomposition inoculant is carried out by referring to the method of agricultural microbial inoculant standard (GB 20287-8cfu/g, which meets the regulation of agricultural microbial agent standard (GB 20287-2006).
1.6 application of thermophilic bacteria BV-2 decomposing inoculant in decomposing fermentation of oyster mushroom culture medium
Preparing an oyster mushroom culture medium according to the growth requirement of oyster mushrooms, adjusting the water content of materials to 60%, adding the prepared thermophilic bacteria BV-2 decomposed inoculant according to the inoculation amount of 0.5%, and performing stacking fermentation, wherein the width of the bottom of a stack is 1.5 meters, the height of the bottom of the stack is 1.2 meters, and the top of the stack is in a turtle back shape. Taking an oyster mushroom culture medium without inoculated decomposed microbial inoculum as a control. And measuring the stacking temperature every day, raising the temperature of the stack to 50 ℃ after inoculating the decomposed microbial inoculum for 24 hours, raising the maximum temperature to 85 ℃, and then, beginning to reduce the temperature. After thermophilic bacteria BV-2 are inoculated in the compost, the highest temperature can reach 85 ℃, the time reaching the high temperature period is 2d after fermentation, the high temperature period exceeding 50 ℃ is 4d, and the high temperature period exceeding 65 ℃ is 2 d. The highest temperature of fermentation of the control group without inoculating the microbial inoculum is 64 ℃, and the high temperature period exceeding 50 ℃ is 3 days. The BV-2 decomposing microbial inoculum can obviously promote the fermentation and temperature rise of the oyster mushroom culture medium, improve the temperature of materials and accelerate the curing and sterilization of the materials.
1.7 detection of fruiting rate and contamination rate of Pleurotus Ostreatus culture medium fermented by thermophilic bacteria BV-2 decomposing inoculant
Three oyster mushroom culture substrates which are treated by thermophilic bacteria BV-2 mature microbial inoculum, treated without microbial inoculum (contrast) and sterilized by high-temperature steam are bagged and inoculated with oyster mushrooms, the inoculation method and the inoculation amount are the same, the fruiting rate and the contamination rate of the substrate materials after different treatments are measured, and the results are shown in Table 1. The fungus-staining rate is the percentage of the number of fungus-staining oyster mushroom culture medium bags to the total number of test bags, and the fruiting rate is the percentage of the weight of oyster mushrooms growing on the oyster mushroom culture medium to the dry material of the oyster mushroom culture medium.
TABLE 1 contamination and fruiting rates of substrates with different treatments
Wherein the contamination rate of the substrate treated by thermophilic bacteria BV-2 decomposing inoculant is 2.5 percent, which is lower than that of the substrate treated by high-temperature sterilization (4.2 percent) and the contrast (10.4 percent). The method proves that the thermophilic bacteria BV-2 decomposing inoculant can effectively kill and inhibit the mixed bacteria in the oyster mushroom culture medium and reduce the contamination rate of oyster mushroom.
The fruiting rate of the substrate treated by the thermophilic bacteria BV-2 decomposing inoculant is 118.2 percent and is higher than that of high-temperature sterilization (108.6 percent) and blank control (94.8 percent). The thermophilic bacteria BV-2 can effectively improve the fruiting condition of the oyster mushroom culture medium.
Comparative example
The inoculant prepared by the thermophilic bacteria Bacillus smithii Ths1 in the patent CN109609412A with the invention name of "a thermophilic bacteria Bacillus smithii Ths1 and application thereof" is used for replacing the thermophilic bacteria BV-2 decomposing inoculant in the embodiment of the invention to carry out fermentation and decomposition of the oyster mushroom culture medium, and the using amount inoculation method for inoculating the thermophilic bacteria Bacillus smithii Ths1 inoculant into the oyster mushroom culture medium is the same as that in the embodiment of the invention.
The fruiting rate and the contamination rate of the pleurotus ostreatus culture medium fermented and decomposed by the inoculant of the thermophilic Bacillus smithii Ths1 are detected, and the detection results are shown in table 2.
TABLE 2 contamination and fruiting rates of Pleurotus ostreatus culture medium
The contamination rate of the substrate treated by the thermophilic bacteria Bacillus smithii Ths1 inoculant is 6.7 percent, which is higher than the contamination rate of the substrate treated by the thermophilic bacteria BV-2 decomposing inoculant in the embodiment of the invention. The thermophilic bacteria BV-2 decomposing inoculant can effectively kill and inhibit the mixed bacteria in the oyster mushroom culture medium, and reduce the contamination rate of oyster mushroom.
The statistics are carried out according to the fruiting situation, wherein the fruiting rate of the substrate treated by the inoculant of the thermophilic bacteria Bacillus smithii Ths1 is 103.3 percent and is higher than that of a blank control (94.8 percent). But the contamination rate of the treated substrate is higher and lower than the fruiting rate of the substrate treated by the thermophilic bacteria BV-2 decomposing inoculant in the application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Cangzhou Wang Producer technology research institute, Inc
<120> thermophilic bacterium BV-2 and application thereof
<130> 2020
<160> 3
<170> PatentIn version 3.5
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<213> Artificial sequence (27 f)
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agagtttgat cctggctc 18
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<213> Artificial sequence (1492 r)
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ggttaccttg ttacgactt 19
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<213> 16S rDNA
<400> 3
ttgctttttg aaagttagcg gcggacgggt gagtaacacg tgggcaacct gcctgcaaga 60
cggggataac tccgggaaac cggggctaat accggataat atcttccttc gcatgaagga 120
aggttgaaag gcggcgcaag ctgccgcttg cagatgggcc cgcggcgcat tagctagttg 180
gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac 240
actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 300
tggacgaaag tctgacggag caacgccgcg tgagcgaaga aggtcttcgg atcgtaaagc 360
tctgttgtca gggaagaaca agtaccgttc gaacagggcg gtaccttgac ggtacctgac 420
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 480
tccggaatta ttgggcgtaa agcgcgcgca ggcggtctct taagtctgat gtgaaagccc 540
acggctcaac cgtggagggt cattggaaac tgggagactt gagtgcagaa gaggagagcg 600
gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 660
gctctctggt ctgtaactga cgctgaggcg cgaaagcgtg gggagcgaac aggattagat 720
accctggtag tccacgccgt aaacgatgag tgctaagtgt tagagggctt ccacccttta 780
gtgctgcagc taacgcatta agcactccgc ctggggagta cggccgcaag gctgaaactc 840
aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 900
gaagaacctt accaggtctt gacatccttc gctacctcta gagatagagg gttccccttc 960
gggggacgga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1020
taagtcccgc aacgagcgca acccttgacc ttagttgcca gcattcagtt gggcactcta 1080
aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1140
tatgacctgg gctacacacg tgctacaatg gatggtacaa agggtcgcga aaccgcgagg 1200
tggagccaat cccaaaaaac cattctcagt tcggattgca ggctgcaact cgcctgcatg 1260
aagccggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1320
gtacacaccg cccgtcacac cacgagagtt t 1351
Claims (10)
1. A thermophilic bacterium BV-2, characterized in that: the preservation number of the strain is CGMCC No. 18652.
2. The use of the thermophilic bacterium BV-2 of claim 1 in the composting fermentation of edible fungus substrates.
3. The decomposing inoculant prepared by using the thermophilic bacteria BV-2 of claim 1, which is characterized in that: comprises an adsorption matrix and thermophilic bacteria BV-2 bacteria powder.
4. The decomposing inoculant according to claim 3, wherein: the adsorption substrate is light calcium carbonate.
5. The decomposing inoculant according to claim 3, wherein: the number of the thermophilic bacteria BV-2 in the decomposing inoculant is more than or equal to 2 multiplied by 108cfu/g。
6. The method for producing a decomposing inoculant according to any one of claims 3 to 5, wherein: the method comprises the following process steps:
a. activating the strains;
b. seed culture;
c. fermenting the second-level seeds;
d. carrying out amplification culture;
e. the adsorption matrix is added and dried.
7. The method for preparing a decomposing inoculant according to claim 6, wherein: in the step a, the method for activating the strains comprises the following steps: and streaking the thermophilic bacteria BV-2 strain which is frozen and preserved on an LB plate, and culturing for 20-30h at 40-50 ℃.
8. The method for preparing a decomposing inoculant according to claim 6, wherein: in the step b, the seed culture method comprises the following steps: and (b) inoculating the thermophilic bacteria BV-2 lawn obtained by activation in the step a into a triangular flask filled with LB liquid culture medium, and carrying out shake cultivation for 20-30h at 40-50 ℃ and 250 r/min.
9. The method for preparing a decomposing inoculant according to claim 6, wherein: in the step c, the secondary seed fermentation method comprises the following steps: b, inoculating the seed culture solution obtained in the step b into a seed tank filled with an LB liquid culture medium, and performing fermentation culture at 40-50 ℃ and 150-; the inoculation volume of the seed culture solution accounts for 5-10% of the volume of the LB liquid culture medium.
10. The method for preparing a decomposing inoculant according to claim 6, wherein: in the step d, the method for expanding culture comprises the following steps: c, inoculating the thermophilic bacteria BV-2 secondary seed fermentation liquid obtained in the step c into a fermentation tank filled with LB liquid culture medium, and fermenting for 2-3d at 45-50 ℃ and 200 r/min; the inoculation volume of the secondary seed fermentation liquid accounts for 2-5% of the volume of the LB liquid culture medium.
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