CN117187107A - Brevibacillus parabrevis and application thereof - Google Patents
Brevibacillus parabrevis and application thereof Download PDFInfo
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- CN117187107A CN117187107A CN202310911225.1A CN202310911225A CN117187107A CN 117187107 A CN117187107 A CN 117187107A CN 202310911225 A CN202310911225 A CN 202310911225A CN 117187107 A CN117187107 A CN 117187107A
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- 241000534614 Brevibacillus parabrevis Species 0.000 title claims description 16
- 239000002609 medium Substances 0.000 claims abstract description 59
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims abstract description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000001963 growth medium Substances 0.000 claims abstract description 27
- 238000000926 separation method Methods 0.000 claims abstract description 20
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 16
- 241000555281 Brevibacillus Species 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 239000010865 sewage Substances 0.000 claims abstract description 11
- 238000002474 experimental method Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 23
- 238000004321 preservation Methods 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 230000001603 reducing effect Effects 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 230000009467 reduction Effects 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000010802 sludge Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 230000009286 beneficial effect Effects 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 238000002798 spectrophotometry method Methods 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 241001052560 Thallis Species 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 11
- 238000002835 absorbance Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 4
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 229940074439 potassium sodium tartrate Drugs 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 206010059410 Faecaluria Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000001546 nitrifying effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000001476 sodium potassium tartrate Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Abstract
The invention relates to the technical field of sewage environmental treatment, and discloses application of a strain of Brevibacillus parabacter, which comprises the following experimental steps: s1: the preparation of each culture medium and formula required for the experiment comprises the following steps: inorganic salt, enrichment medium I, enrichment medium II and separation medium. According to the Brevibacillus Parabrus and the application thereof, ammonia nitrogen removal bacteria are separated from samples such as water, and after the bacteria are put into sewage, the ammonia nitrogen, nitrogen-containing substances and other organic matters in the sewage can be used as nutrient sources to realize continuous growth and propagation of thalli, and meanwhile, the formation of microbial flora in a water ecological system is promoted, so that nitrogen-containing organic matters in the water are decomposed, the ammonia nitrogen content of the water is reduced, and the effect of purifying water quality is achieved.
Description
Technical Field
The invention relates to the technical field of sewage environmental treatment, in particular to Brevibacillus parabacter and application thereof.
Background
The modern process of China is continuously accelerated, and the living standard of people is increasingly improved. With the problems of sewage containing a large amount of organic matters generated in production and life, a large amount of fecaluria generated in livestock breeding, non-point source pollution caused by chemical fertilizers along with surface water runoff and the like in agricultural planting, water eutrophication caused by intensive and high-density scale cultivation in aquaculture and the like are more and more serious, and the problem of water pollution treatment is more and more prominent.
In the wastewater of livestock and poultry cultivation, aquiculture and the like, the NH4 < + > -N content is obviously higher than that of common polluted water. The culture wastewater with high concentration of ammonia pollutants can cause serious environmental problems if improperly treated, thereby threatening the health of human beings. The method for removing the ammonia nitrogen in the water body mainly comprises a physical method, a chemical method, a biological method and the like, and is significant for improving the sewage treatment efficiency and for human production and life by exploring, researching and developing the high-efficiency and economical ammonia nitrogen removal method. The biological method is used for denitrification, and has the advantages of sustainability, ecology, green, low cost, easy operation, no secondary pollution and the like, so the biological denitrification technology is paid attention to.
The water denitrification and ammonia removal technology mainly realizes the utilization of nitrogen through biochemical reaction performed by microbial catalysis or discharges nitrogen into the air in a nitrogen form. Including nitrifying bacteria, denitrifying bacteria, ammoxidation of ammoxidation bacteria, etc. The ammonia nitrogen-removing microorganisms exist in various habitats, so that the ammonia nitrogen-reducing microorganisms are separated from collected samples under different environmental conditions, and the microbial agent prepared by artificial propagation can be applied to the treatment of denitrification and the like in water.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a Brevibacillus parabrevis and application thereof.
(II) technical scheme
In order to achieve the first object, the present invention adopts the following technical scheme:
the strain of Brevibacillus parabacter (Brevibacillus parabrevis LY) is characterized in that the Brevibacillus parabacter is preserved in China center for type culture collection (CCTCC for short), the preservation number is CCTCC NO: M20231034, the preservation address is in university of Wuhan in Wuhan, hubei province, and the preservation date is 2023, 6 and 15.
In order to achieve the second object, the present invention adopts the following technical scheme: s1: the preparation of each culture medium and formula required for the experiment comprises the following steps: inorganic salt, enrichment medium I, enrichment medium II and separation medium;
s2: enrichment culture is carried out on the enrichment culture medium I;
s3: further enrichment culture on an enrichment medium II;
s4: separating and culturing on a separating culture medium;
s5: separating and purifying on a separating culture medium to obtain an optimal strain, wherein the strain is numbered LY12;
s6: determining ammonia nitrogen concentration reduction of the strain LY12;
s7: repeated verification of ammonia nitrogen reducing effect of the strain LY12;
s8: identification of LY12 strain.
Preferably, in the step S1, the inorganic salt formulation (g/L) comprises: the NaCl content was 5,K 2 HPO 4 Is 15% MgSO 4 Is 5, the enrichment medium one (g/L) comprises: glucose content of 10, (NH) 4 ) 2 SO 4 The content of (2) and the content of inorganic salt are 100, and the enrichment medium II (g/L) comprises: glucose content 5, (NH) 4 ) 2 SO 4 The content of inorganic salt is 100, and the separation medium (g/L) comprises: glucose content of 0.5, (NH) 4 ) 2 SO 4 The content of (2) was 0.75, the content of inorganic salt was 100, and the content of agar was 20.
Preferably, the step S2 includes: the method comprises the steps of (1) collecting samples from multiple environments such as a pond, a culture water body, a sewage treatment plant, lake water, activated sludge and the like, diluting the solid activated sludge samples by sterile water by 10 times at a weight ratio of 1:10 to prepare liquid samples, inoculating 5mL of each sample into a bottle containing enrichment medium (100 mL/bottle), placing the bottle into a shaking table at 37 ℃ and 180rpm for culturing for 24 hours, and repeating each water sample by 2 bottles as parallel samples.
Preferably, the step S3 includes: the enrichment medium increases (NH) 4 ) 2 SO 4 The method can further enrich the proliferation of microorganisms with the utilization capacity for ammonia nitrogen, thereby being beneficial to further selecting and separating bacteria for reducing ammonia nitrogen. The liquid loading amount of the enrichment medium II is 100 mL/bottle, and corresponds to the enrichment medium I one by one, 5mL of the bacterial liquid after enrichment culture in the enrichment medium I is respectively inoculated into the enrichment medium II, and the enrichment medium II is put into a shaking table for culturing for 24 hours at 37 ℃ and 180 rpm.
Preferably, in the step S4, the separation medium is a solid medium, the plate is poured, cooled, 100 mu L of the bacterial liquid enriched in the enrichment medium II is coated on the plate of the separation medium, and the plate is uniformly coated and then placed in a 37 ℃ incubator for culturing for 24-48 hours.
Preferably, in the step S5, a single colony obtained after coating on the separation medium is picked up, inoculated on the separation medium by a plate streaking method to obtain a single colony, separated and purified, and continuously performed 3 times to obtain pure culture, the macroscopic morphology of the colony is observed, the cell morphology is observed by a microscope, the pure strain is preserved, and the best strain obtained is numbered as LY12.
Preferably, in the step S6, the ability of the strain LY12 to reduce ammonia nitrogen is detected by using a Nahner reagent spectrophotometry.
Preferably, in the step S7, the isolated ammonia nitrogen reduction preservation strain LY12 is activated and cultured for 15 hours on an LB culture medium at 37 ℃ to obtain a single colony, the single colony is selected and inoculated into a 5mL LB test tube, the culture is carried out for 12 hours, bacterial liquid in a 2mL test tube is inoculated into a 100mL ammonia nitrogen culture medium shake flask, after the culture is carried out for 24 hours for nitrogen removal, 1mL bacterial liquid is taken to be in an EP tube, centrifugation is carried out for 10 minutes at 8000rpm, supernatant is sucked into the 5mL test tube, pure water is added for dilution to 5mL, and ammonia nitrogen concentration measurement is carried out.
(III) beneficial effects
Compared with the prior art, the invention provides the Brevibacillus parable and the application thereof, and has the following beneficial effects:
according to the Brevibacillus Parabrus and the application thereof, ammonia nitrogen removal bacteria are separated from samples such as water, and after the bacteria are put into sewage, the ammonia nitrogen, nitrogen-containing substances and other organic matters in the sewage can be used as nutrient sources to realize continuous growth and propagation of thalli, and meanwhile, the formation of microbial flora in a water ecological system is promoted, so that nitrogen-containing organic matters in the water are decomposed, the ammonia nitrogen content of the water is reduced, and the effect of purifying water quality is achieved.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph of ammonia nitrogen content in accordance with the present invention;
FIG. 2 is a sequence alignment chart of the present invention.
Biological material preservation information
The culture name and the noted identification characteristics are Brevibacillus parabrevis LY, the culture is preserved in China center for type culture collection, the preservation number is CCTCCNO: M20231034, the preservation address is in university of Wuhan in Wuhan, hubei province, and the preservation date is 2023, 6 and 15.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Example 1
The strain of Brevibacillus parabacter (Brevibacillus parabrevis LY) is characterized in that the Brevibacillus parabacter is preserved in China center for type culture collection (CCTCC for short), the preservation number is CCTCC NO: M20231034, the preservation address is in university of Wuhan in Wuhan, hubei province, and the preservation date is 2023, 6 and 15.
Example two
As shown in figures 1-2, the invention provides application of a strain of Brevibacillus parabacter, which comprises the following experimental steps:
s1: the preparation of each culture medium and formula required for the experiment comprises the following steps: inorganic salt, enrichment medium I, enrichment medium II and separation medium;
s2: enrichment culture is carried out on the enrichment culture medium I;
s3: further enrichment culture on an enrichment medium II;
s4: separating and culturing on a separating culture medium;
s5: separating and purifying on a separating culture medium to obtain an optimal strain, wherein the strain is numbered LY12;
s6: determining ammonia nitrogen concentration reduction of the strain LY12;
s7: repeated verification of ammonia nitrogen reducing effect of the strain LY12;
s8: identification of LY12 strain.
In an embodiment of the present invention, in S1, an inorganic salt formulation (g/L) includes: the NaCl content was 5,K 2 HPO 4 Is 15% MgSO 4 Is 5, the enrichment medium one (g/L) comprises: glucose content of 10, (NH) 4 ) 2 SO 4 The content of (2) and the content of inorganic salt are 100, and the enrichment medium II (g/L) comprises: glucose content 5, (NH) 4 ) 2 SO 4 The content of inorganic salt is 100, and the separation medium (g/L) comprises: glucose content of 0.5, (NH) 4 ) 2 SO 4 The content of (2) was 0.75, the content of inorganic salt was 100, and the content of agar was 20.
In the embodiment of the present invention, S2 includes: the method comprises the steps of (1) collecting samples from multiple environments such as a pond, a culture water body, a sewage treatment plant, lake water, activated sludge and the like, diluting the solid activated sludge samples by sterile water by 10 times at a weight ratio of 1:10 to prepare liquid samples, inoculating 5mL of each sample into a bottle containing enrichment medium (100 mL/bottle), placing the bottle into a shaking table at 37 ℃ and 180rpm for culturing for 24 hours, and repeating each water sample by 2 bottles as parallel samples.
In the embodiment of the present invention, S3 includes: the enrichment medium increases (NH) 4 ) 2 SO 4 The method can further enrich the proliferation of microorganisms with the utilization capacity for ammonia nitrogen, thereby being beneficial to further selecting and separating bacteria for reducing ammonia nitrogen. The liquid loading amount of the enrichment medium II is 100 mL/bottle, and corresponds to the enrichment medium I one by one, 5mL of the bacterial liquid after enrichment culture in the enrichment medium I is respectively inoculated into the enrichment medium II, and the enrichment medium II is put into a shaking table for culturing for 24 hours at 37 ℃ and 180 rpm.
In the embodiment of the invention, in S4, the separation medium is a solid medium, the plate is poured, cooled, 100 mu L of the bacteria liquid enriched in the enrichment medium II is coated on the plate of the separation medium, and the plate is uniformly coated and then placed in a 37 ℃ incubator for culturing for 24-48 hours.
In the embodiment of the invention, in S5, single colony obtained after coating on a separation culture medium is picked, the single colony is obtained by inoculating on the separation culture medium by a plate streaking method, separation and purification are carried out for 3 times continuously, pure culture is obtained, macroscopic morphology of the colony is observed, cell morphology is observed by a microscope, pure strain is preserved, and the best strain obtained is numbered LY12.
In the embodiment of the invention, in S6, a Nahner reagent spectrophotometry is used for detecting the ammonia nitrogen reducing capacity of the strain LY12.
Specifically, reagents required for the experiment include:
standard ammonia solution: 0.05g (NH) 4 ) 2 SO 4 Adding pure water to fix the volume to 100mL to prepare a standard ammonia solution with the concentration of 1 mg/mL;
0.5g/mL sodium potassium tartrate: 25g of potassium sodium tartrate is dissolved in 50mL of water, and the mixture is heated and boiled to remove ammonia;
a Nahner reagent;
ammonia nitrogen culture medium with initial concentration of 10 mg/L:
| composition of the components | Glu | (NH 4 ) 2 SO 4 | NaCl | K 2 HPO 4 ·2H 2 O | MgSO 4 ·7H 2 O |
| Quantity (g/L) | 1.25 | 0.005 | 1.0 | 0.5 | 0.25 |
The experimental steps of this embodiment further include:
k1, drawing a standard curve;
k101: preparing a standard solution: adding 0.00, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL of standard ammonia solution into 8 5mL test tubes respectively, and adding pure water to 5mL;
k102: color development: adding 0.1mL of potassium sodium tartrate solution into the test tube, and shaking uniformly; adding 0.15mL of Nahner reagent, and shaking uniformly; standing for 10 minutes;
k103: colorimetric measurement: at a wavelength of 420mm, absorbance was read;
k104: drawing a standard curve: drawing a standard curve of ammonia nitrogen content to absorbance;
k2: measuring the ammonia nitrogen content of the strain LY12 after the ammonia nitrogen liquid culture medium is treated;
k201: sampling: performing activation culture on the separated ammonia nitrogen reduction preservation strain LY12 on an LB culture medium at 37 ℃ for 15 hours to obtain single colonies, selecting the single colonies, inoculating the single colonies into a 5mL LB test tube, culturing for 12 hours, taking bacterial liquid in a 1mL test tube, inoculating the bacterial liquid into a 100mL ammonia nitrogen culture medium shake flask, culturing for 24 hours to remove nitrogen, taking the 1mL bacterial liquid into an EP tube, centrifuging at 8000rpm for 10 minutes, sucking the supernatant into the 5mL test tube, adding pure water to dilute the supernatant to 5mL, and determining the ammonia nitrogen concentration;
k202: color development: adding 0.1mL of potassium sodium tartrate solution into the test tube, and uniformly mixing; adding 0.15mL of Nahner reagent, and uniformly mixing; standing for 10 minutes;
k203: colorimetric measurement: reading absorbance at a wavelength of 420 nm;
k204: and recording the absorbance of each sample liquid, and obtaining parameters through an ammonia nitrogen content calculation formula.
Determination of ammonia nitrogen content:
firstly, preparing a standard curve, and calculating the ammonia nitrogen content according to the absorbance corresponding to the standard curve in the solution after the strain treatment.
Drawing a standard curve:
relationship between ammonia nitrogen content and absorbance (420 nm):
Y＝16.245X+0.1679
y: absorbance of the solution at wavelength 420nm
X: ammonia nitrogen concentration of solution
The determination results of the ammonia nitrogen after LY12 treatment of the ammonia nitrogen solution are shown in the table:
results: the ammonia nitrogen reducing capacity (degradation rate) of LY12 to ammonia nitrogen is 48.88%.
In the embodiment of the invention, in S7, the separated ammonia nitrogen reduction preservation strain LY12 is activated and cultured for 15 hours on an LB culture medium at 37 ℃ to obtain a single colony, the single colony is selected and inoculated into a 5mL LB test tube, the culture is carried out for 12 hours, bacterial liquid in 2mL test tubes is inoculated into a 100mL ammonia nitrogen culture medium shake flask, after nitrogen removal is carried out for 24 hours, 1mL bacterial liquid is taken to be placed in an EP tube, centrifugation is carried out for 10 minutes at 8000rpm, supernatant liquid is absorbed into the 5mL test tube, pure water is added for dilution to 5mL, ammonia nitrogen concentration measurement is carried out, the method is the same as above, and the results show that the ammonia nitrogen concentration in untreated stock solution is 450mg/L, the ammonia nitrogen concentration in treated sample 1 is 55mg/L, the ammonia nitrogen removal rate is 87.7%, the ammonia nitrogen concentration in treated sample 2 is 70mg/L, and the ammonia nitrogen removal rate is 84.4%.
Further, 16S rRNA gene sequence identification is carried out on LY12 strain;
LY1216S rDNA sequence:
GGCGGCGTGCCTAATACATGCAAGTCGAGCGAGGGTTTTCGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACCGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATTGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGTGCCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGAGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGAGGACGCCAATCTCTGAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAGCCGCCGAAGGTGGGGTAGATGACTGGGGGTGAATCTAAGGGGG。
a Blast search alignment was performed using this sequence, and as a result, brevibacillus parabacter (Brevibacillus parabrevis).
Claims (9)
1. The strain of Brevibacillus parabacter (Brevibacillus parabrevis LY) is characterized in that the Brevibacillus parabacter is preserved in China center for type culture collection (CCTCC for short), the preservation number is CCTCC NO: M20231034, the preservation address is in university of Wuhan in Wuhan, hubei province, and the preservation date is 2023, 6 and 15.
2. The application of the Brevibacillus parabrevis is characterized by comprising the following experimental steps:
s1: the preparation of each culture medium and formula required for the experiment comprises the following steps: inorganic salt, enrichment medium I, enrichment medium II and separation medium;
s2: enrichment culture is carried out on the enrichment culture medium I;
s3: further enrichment culture on an enrichment medium II;
s4: separating and culturing on a separating culture medium;
s5: separating and purifying on a separating culture medium to obtain an optimal strain, wherein the strain is numbered LY12;
s6: determining ammonia nitrogen concentration reduction of the strain LY12;
s7: repeated verification of ammonia nitrogen reducing effect of the strain LY12;
s8: identification of LY12 strain.
3. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: in the S1, the inorganic salt formula (g/L) comprises: the NaCl content was 5,K 2 HPO 4 Is 15% MgSO 4 Is 5, the enrichment medium one (g/L) comprises: glucose content of 10, (NH) 4 ) 2 SO 4 The content of (2) and the content of inorganic salt are 100, and the enrichment medium II (g/L) comprises: glucose content 5, (NH) 4 ) 2 SO 4 The content of inorganic salt is 100, and the separation medium (g/L) comprises: glucose content of 0.5, (NH) 4 ) 2 SO 4 The content of (2) was 0.75, the content of inorganic salt was 100, and the content of agar was 20.
4. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: the step S2 comprises the following steps: the method comprises the steps of (1) collecting samples from multiple environments such as a pond, a culture water body, a sewage treatment plant, lake water, activated sludge and the like, diluting the solid activated sludge samples by sterile water by 10 times at a weight ratio of 1:10 to prepare liquid samples, inoculating 5mL of each sample into a bottle containing enrichment medium (100 mL/bottle), placing the bottle into a shaking table at 37 ℃ and 180rpm for culturing for 24 hours, and repeating each water sample by 2 bottles as parallel samples.
5. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: the step S3 comprises the following steps: the enrichment medium increases (NH) 4 ) 2 SO 4 The method can further enrich the proliferation of microorganisms with the utilization capacity for ammonia nitrogen, thereby being beneficial to further selecting and separating bacteria for reducing ammonia nitrogen. The liquid loading amount of the enrichment medium II is 100 mL/bottle, and corresponds to the enrichment medium I one by one, 5mL of the bacterial liquid after enrichment culture in the enrichment medium I is respectively inoculated into the enrichment medium II, and the enrichment medium II is put into a shaking table for culturing for 24 hours at 37 ℃ and 180 rpm.
6. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: in the step S4, the separation medium is a solid medium, the plate is poured, cooled, 100 mu L of bacteria liquid enriched in the enrichment medium II is coated on the plate of the separation medium, and the plate is uniformly coated and then placed in a 37 ℃ incubator for culturing for 24-48 hours.
7. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: in the step S5, single colonies obtained after coating on a separation culture medium are picked, inoculated on the separation culture medium by a plate streaking method to obtain single colonies, separated and purified, and continuously cultured for 3 times to obtain pure culture, the macroscopic morphology of the colonies is observed, the cell morphology is observed by a microscope, pure strains are preserved, and the best strain is numbered as LY12.
8. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: in the step S6, a Navier reagent spectrophotometry method is used for detecting the ammonia nitrogen reducing capacity of the strain LY12.
9. The use of a strain of Brevibacillus parabrevis as claimed in claim 2, wherein: in the S7, the separated ammonia nitrogen reduction preservation strain LY12 is activated and cultured for 15 hours on an LB culture medium at 37 ℃ to obtain a single colony, the single colony is selected and inoculated into a 5mL LB test tube, the culture is carried out for 12 hours, bacterial liquid in a 2mL test tube is inoculated into a 100mL ammonia nitrogen culture medium shake flask, after the culture is carried out for 24 hours for nitrogen removal, 1mL bacterial liquid is taken out in an EP tube, centrifugation is carried out for 10 minutes at 8000rpm, supernatant is sucked into the 5mL test tube, pure water is added for dilution to 5mL, and ammonia nitrogen concentration measurement is carried out.
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