CN115287212B - Salt-tolerant growth-promoting bacterium ACP81 and application thereof - Google Patents
Salt-tolerant growth-promoting bacterium ACP81 and application thereof Download PDFInfo
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- CN115287212B CN115287212B CN202210390792.2A CN202210390792A CN115287212B CN 115287212 B CN115287212 B CN 115287212B CN 202210390792 A CN202210390792 A CN 202210390792A CN 115287212 B CN115287212 B CN 115287212B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 11
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- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 11
- 239000002699 waste material Substances 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims abstract description 7
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- 235000015334 Phyllostachys viridis Nutrition 0.000 claims abstract description 7
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- 239000002054 inoculum Substances 0.000 description 2
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- 239000011780 sodium chloride Substances 0.000 description 2
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- 108091000130 1-aminocyclopropane-1-carboxylate deaminase Proteins 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
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- 241000282596 Hylobatidae Species 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
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- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
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- 238000003911 water pollution Methods 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/26—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
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- Environmental & Geological Engineering (AREA)
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Abstract
The invention relates to a salt-tolerant growth-promoting strain ACP81 (Bacillus subtilis), which is separated from bamboo shoot waste, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2022, 1 month and 10 days, and a preservation number of CCTCC NO:24288; the morphological characteristics of the salt-tolerant growth-promoting strain ACP81 are as follows: the colony is round, white, 1.0-3.0 mm in diameter, opaque, flat and rough in surface, irregular in edge, smell-generating, and the 16S rDNA nucleotide sequence of the bacterium is shown in the figure. After the salt-tolerant growth-promoting strain ACP81 is inoculated into a proper fermentation medium for shaking fermentation culture, the salt-tolerant concentration of the strain reaches 20%, and the semi-lethal concentration is 6%. The strain provided by the invention can be applied to the restoration of saline-alkali soil and the degradation of bamboo shoot processing waste liquid due to the salt tolerance and safety.
Description
Technical Field
The invention belongs to the technical field of microorganisms and the field of ecological restoration, and relates to salt-tolerant bacillus subtilis ACP81 and application thereof.
Background
Saline-alkali soil is soil with different degrees of salinization and alkalization, has unstable structure and low fertilizer and water retention, can cause plant dysplasia and even death, and prevents the sustainable development of agriculture and forestry. Moreover, people still face a plurality of problems such as insufficient grain reserves, reduced forest resources and the like. Therefore, the restoration of the saline-alkali soil becomes a backup land resource, and the resource problem to be solved is urgent.
Bioremediation of saline-alkali soil is an efficient and clean way to meet technological development and avoid secondary pollution. In one aspect, bioremediation is the use of the physiological metabolic activity of beneficial microorganisms to increase the salt tolerance of the cells, plants themselves. For example, induction of ACC-deaminase production by plants reduces ethylene synthesis rate, alleviating the inhibitory effect of salt stress on plants; the growth hormone such as IAA indoleacetic acid is produced to stimulate the formation of a great deal of root hairs of plants, and the balance of K +、Na+ in plant tissues is regulated, so that the growth of the plants is promoted; or synthesizing intracellular compatible solute molecules such as L-glutamic acid and the like so as to regulate cell osmotic pressure and membrane fluidity and serve as a carbon source or a nitrogen source, thereby enhancing the resistance to salt stress. On the other hand, part of microorganisms have special functions of nitrogen fixation, phosphate dissolution, enzyme production and the like, and can adjust soil structure and soil physicochemical properties. For example, microorganisms secrete organic acids (oxalic acid, succinic acid, acetic acid) to adjust soil pH, inhibit or promote growth and proliferation of other indigenous microorganisms; the physical and chemical properties of soil such as soil organic matter content, nitrate nitrogen content, effective phosphorus content, quick-acting potassium, ammonium nitrogen and the like are changed, or hydrolytic enzymes (amylase, protease, cellulase and xylanase) are generated to promote the physiological metabolism rate of microorganisms and plants, so that the plant growth is directly or indirectly promoted to adapt to bad environments.
In addition, the waste liquid of bamboo shoot processing contains complex substances such as salt, sugar, lignin, cellulose and the like. The water resource waste is serious, the water pollution and the soil pollution are irreversibly destroyed, and the surrounding air and the plant growth are influenced. Therefore, the waste liquid from bamboo shoot processing is treated by using a bioremediation technology, salt-tolerant strains are purposefully screened to degrade the waste liquid so as to meet the forest land emission standard, and the soil self environment can be improved, namely, a complete circulation system is formed in water-soil-plants. Therefore, screening microorganisms with salt tolerance and high enzyme production efficiency becomes a key breakthrough in the technical field.
Disclosure of Invention
The first object of the present invention is to provide a salt-tolerant growth-promoting bacterium Bacillus subtilis ACP81.
The second object of the invention is to provide an application of salt-tolerant growth-promoting bacterium Bacillus subtilis ACP81 which is used for repairing saline-alkali soil and degrading waste liquid.
Wherein the strain is preserved: the salt-tolerant growth-promoting bacillus subtilis Bacillus subtilis ACP is obtained by self-screening by the inventor, is preserved in the China general microbiological culture Collection center, the preservation unit address, the Chinese Wuhan, the preservation date of 2022, 1 month and 10 days, and the preservation number of CCTCC NO:24288.
The salt-tolerant growth-promoting bacterium Bacillus subtilis ACP is of an irregular round shape, rough in colony, opaque, white, 1.0-3.0 mm in diameter, serrated in edge, dry in texture and high in strain order of magnitude of more than 10 7 cfu/g, and the nucleotide sequence of the salt-tolerant growth-promoting strain 16S rDNA is shown in the figure.
The salt-tolerant growth-promoting bacterium Bacillus subtilis ACP provided by the invention can normally grow under the condition of salt stress (the NaCl concentration is 1% -20%), and the NaCl concentration is 6% of the semi-lethal concentration of the bacterium.
The salt-tolerant growth-promoting bacterium Bacillus subtilis ACP81 provided by the invention has the functions of producing amylase, protease and cellulase under high salt stress.
The invention has the advantages that: 1. the culture is convenient, and the growth speed is high; 2. salt-tolerant and high-efficiency enzyme production; 3. the strain can produce amylase, cellulase and protease functions.
Drawings
FIG. 1 is a view of a salt-tolerant growth-promoting bacterium ACP81 after being cultured in a medium.
Detailed Description
Example one isolation and screening of salt-tolerant growth-promoting Strain
1G of bamboo shoot waste is weighed and placed in 100mL of LB liquid culture solution (peptone 10g/L, naCl 10g/L, yeast extract 5g/L and the balance water, pH 7.0 and sterilization at 121 ℃ for 20 min), a constant temperature incubator at 28 ℃ and shaking by a 180r/min shaking table for 48h, and a suspension is obtained. Transferring 100 mu L of bamboo shoot waste suspension into LB liquid culture medium, culturing at 180r/min at 28 ℃ for 24 hours, diluting bacterial liquid 10 -1、10-2,10-3、10-4、10-5、10-6 with sterile raw water to obtain six concentration gradients, respectively transferring 100 mu L of bacterial liquid into LB solid culture medium by selecting three gradients of 10 -4、10-5、10-6, coating a plate by using a sterile coating rod, and finally pouring into a constant-temperature incubator at 28 ℃.
The LB solid culture is based on 48 hours of culture, and then a single colony is obtained. Single bacterial colonies with different forms are picked by using an inoculating needle, and are respectively and continuously streaked and cultured on the surface of an LB solid culture medium, and the steps are repeated for 3 to 5 times, so that the purified bacterial strain with single bacterial colony can be obtained.
Transferring the purified strain into LB liquid culture medium containing 1% NaCl at an inoculum size of 2%, culturing at a rotating speed of 180r/min at 28 ℃ for 24 hours, transferring the bacterial liquid into LB liquid culture medium containing 3% NaCl again at an inoculum size of 2%, repeating the steps until the NaCl content in the LB culture liquid is 20%, respectively streaking the obtained bacterial culture liquid, and picking out single bacterial colonies with good growth, thus obtaining the salt-tolerant growth-promoting strain.
The strain with salt tolerance is respectively absorbed into 5 mu L of bacterial liquid and respectively dripped into a skimmed milk powder culture medium, a starch culture medium and a cellulose culture medium, is cultured for 2d in a constant temperature incubator at 28 ℃, is subjected to dyeing treatment, is repeated for 3 times, and is provided with 3 obvious transparent water rings, and is named as ACP81 as shown in the figure.
0.7-0.8 ML of bacterial culture solution and 0.2-0.3 mL of glycerin are added into a sterile strain preservation tube, and the mixture is fully and uniformly mixed and preserved in a refrigerator at the temperature of minus 80 ℃. As shown in fig. 1.
Example two, identification of salt-tolerant growth-promoting Strain
(1) Morphological features
The liquid culture medium is uniformly turbid and accompanied by micro-precipitation, and the precipitation is dispersed by gentle shaking. On LB solid culture medium, the colony diameter is 1.0-3.0 mm, the bacteria are irregular round, the colony surface is rough, opaque, white, sawtooth-shaped at the edge and dry in texture.
(2) 16S rRNA sequence analysis
Further 16S rRNA sequence analysis and alignment were performed on the isolated strain ACP 81. Cloning and sequence determination were performed according to a conventional method, and the results were compared with 16S rRNA sequences registered in GenBank (accession numbers: NC-000964.3 and NZ-CP 034943.1, etc.), and the gene homology was 99% or more. Strain ACP81 was identified as B.subtilis by morphological characterization and 16S rRNA sequence analysis according to Bergey' sMannualof Determinative Bacteriology (Holt, J.G., gibbons, N.E., 1994) and the handbook of common bacterial System identification (Dongxiu bead and Cai Miaoying et al, 2001).
(3) Research on salt-tolerant growth characteristics of strains
Activating the strain stored on the inclined surface of the test tube, inoculating the strain on an LB liquid culture medium for activation, culturing for 24 hours, taking 100 mu L of seed liquid as seed liquid, transferring the seed liquid into the LB liquid culture medium containing different salt concentrations, oscillating for 5min, and uniformly mixing. The absorbance (OD 600 nm) was measured by incubating at 28℃for 20 hours without inoculating liquid LB as a control. The strain has a maximum salt tolerance concentration of 20% NaCl (containing the NaCl content in LB culture solution). Growth curves of ACP81 strains at different salt concentrations are shown in table 1:
TABLE 1 growth curves of ACP81 strains at different salt concentrations
Example III, functional identification of protease produced by Strain
The strain preserved at the temperature of minus 80 ℃ is inoculated on LB liquid medium for activation, the strain is used as seed liquid after being cultured for 24 hours, 100 mu L of seed liquid is absorbed and transferred to LB liquid medium for activation for 24 hours, the process is repeated for 3 times, 5 mu L of seed liquid is absorbed and inoculated on a skim milk powder culture medium flat plate (15.0 g/L of skim milk powder, 2.5g/L of beef extract, 5.0g/L of peptone, 2.5g/L of sodium chloride, 10.0g/L of agar, pH value of 7.0-7.4 and sterilization at the temperature of 115 ℃ for 20 minutes), and each culture dish is inoculated with three points for three times. And (3) placing the strain in a constant temperature box at 28 ℃ in an inverted manner, culturing for 3-5d, and observing that a clear water ring appears around a colony, namely the strain has the function of producing protease.
Example IV, functional identification of amylase produced by Strain
The strain preserved at the temperature of minus 80 ℃ is inoculated on an LB liquid culture medium for activation, the strain is used as seed liquid after being cultured for 24 hours, 100 mu L of seed liquid is absorbed and transferred to the LB liquid culture medium for activation for 24 hours, the process is repeated for 3 times, 5 mu L of seed liquid is absorbed and is inoculated on a soluble starch culture medium flat plate (10.0 g/L of soluble starch, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of magnesium sulfate, 1.0g/L of sodium chloride, 2.0g/L of ammonium sulfate, 0.001g/L of ferrous sulfate, 0.001g/L of manganese chloride, 0.001g/L of zinc sulfate, 10.0g/L of agar, the pH value is 7.0-7.4, and the sterilization is performed at the temperature of 121 ℃ for 15 minutes), and each culture dish is inoculated with two points and repeated for three times. The mixture is placed in a constant temperature cabinet at 28 ℃ for 2d of culture, and the constant temperature culture is continued by using 1% iodine solution for dyeing under the aseptic condition. The edge of the colony producing amylase has obvious decoloring halo, i.e. the strain has the function of producing amylase.
Example five, functional identification of cellulase produced by Strain
The strain preserved at the temperature of minus 80 ℃ is inoculated on LB liquid medium for activation, the strain is used as seed liquid after being cultured for 24 hours, 100 mu L of seed liquid is absorbed and transferred to fresh LB liquid medium for activation for 24 hours, the process is repeated for 3 times, 5 mu L of seed liquid is absorbed and is inoculated on a cellulose screening culture medium (CMC-Na) plate (2.5 g/L of monopotassium phosphate, 2.5g/L of disodium hydrogen phosphate, 20.0g/L of sodium carboxymethyl cellulose, 2.0g/L of peptone, 0.5g/L of yeast extract powder, 14.0g/L, pH of agar, 7.0-7.4 of value of 7.4 and 15 minutes of sterilization at 121 ℃) and each culture dish is inoculated with two points for three times. The mixture was placed in an incubator at 28℃and cultured for 2d. Under the aseptic condition, 1% Congo red solution is used for dyeing for 30min, 1mg/mL NaCl solution is added for rinsing for 20min, bacterial colonies generating cellulase series are dyed and rinsed, and the edges of the bacterial colonies have obvious decoloration halos, namely the bacterial strains have the function of generating cellulase.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions can be made without departing from the spirit of the invention, and these are considered to be within the scope of the invention.
Sequence listing
<120> A salt-tolerant growth-promoting bacterium ACP81 and application thereof
<141> 2022-03-01
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1445
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
tgggaggtgc tataatgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg 60
cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120
ggggctaata ccggatggtt gtttgaaccg catggttcaa acataaaagg tggcttcggc 180
taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag 240
gcaacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300
agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360
aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420
gtaccgttcg aatagggcgg taccttgacg gtacctaacc agaaagccac ggctaactac 480
gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540
gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600
attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660
aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720
gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780
aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840
gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900
cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960
acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg 1020
catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080
cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg 1140
gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200
tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt 1260
tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320
ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380
gagagtttgt aacacccgaa gtcggtgagg taacctttta ggagccagcc gccgaagggt 1440
ggaac 1445
Claims (2)
1. The salt-tolerant growth-promoting bacterium ACP81 is characterized in that the salt-tolerant growth-promoting bacterium ACP81 is bacillus subtilis, the Latin chemical name is Bacillus subtilis, the preservation name is Bacillus subtilis ACP81, and the salt-tolerant growth-promoting bacterium ACP81 is preserved in China general microbiological culture collection center (CCTCC) with the preservation number of CCTCC NO:24288.
2. The use of salt tolerant growth-promoting bacterium ACP81 of claim 1, wherein the use is for repairing saline-alkali soil and degrading bamboo shoot processing waste.
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