CN113717902A - Sphingobacterium multivorum and application thereof - Google Patents
Sphingobacterium multivorum and application thereof Download PDFInfo
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- CN113717902A CN113717902A CN202111182074.8A CN202111182074A CN113717902A CN 113717902 A CN113717902 A CN 113717902A CN 202111182074 A CN202111182074 A CN 202111182074A CN 113717902 A CN113717902 A CN 113717902A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/40—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting fertiliser dosage or release rate; for affecting solubility
- C05G3/44—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting fertiliser dosage or release rate; for affecting solubility for affecting solubility
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- 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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- 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
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Abstract
The invention discloses a sphingosine bacillus Sphingobacterium multivorum and application thereof, wherein the bacterial strain is Sphingobacterium multivorum 6 which is preserved in China center for type culture collection with the preservation date of 2021, 5 and 31 days and the preservation number of CCTCC NO: M2021648. The bacterial strain can convert insoluble inorganic phosphate into soluble phosphorus capable of being directly absorbed and utilized by plants, and the phosphorus-dissolving amount of the bacterial strain cultured in four insoluble phosphate liquid culture media of calcium phosphate, iron phosphate, aluminum phosphate and powdered rock phosphate for 7 days is 47.2 mu g mL respectively‑1、6.1μg mL‑1、3.8μgmL‑1、9.5μg mL‑1Provides a certain reference for the later research of the wild strainHas important value for the development of microbial fertilizers such as phosphate solubilizing bacterial manure and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a sphingosine bacillus Sphingobacterium multivorum and application thereof.
Background
Phosphorus is one of the most important nutrients required for plant growth. The inorganic phosphorus in the soil has three forms, namely a water-soluble state, a mineral state and an adsorption state, wherein the inorganic phosphorus mainly exists in the mineral state. Mineral phosphorus is mainly apatite in alkaline soil and ferric aluminum phosphate in acid soil. Adsorbed phosphorus refers to phosphate held by soil solids, such as minerals or organic matter, in the soil, and is generally present in very low levels. The water soluble phosphorus can be dissolved in water, can be directly absorbed and utilized by plants, has very low content in soil, and is mainly generated by dissolution of mineral phosphorus and release of adsorbed phosphorus. The acid-base environment of the soil has direct influence on the form of phosphorus in the soil, so that the effectiveness of the phosphorus in the soil and the phosphorus supply capacity of the soil are closely related to the acid-base environment of the soil. The content of active iron and aluminum in the acid soil is high, and the active iron and aluminum are easily combined with soluble phosphorus and converted into forms of insoluble iron phosphate, aluminum phosphate and the like to be fixed. When the phosphate fertilizer is applied, phosphorus in the fertilizer is often acted with active iron and aluminum in the soil solution to be fixed, and then is hydrolyzed and converted into phosphate such as foscarnite and foscarnite, and further converted into the occluded phosphate, so that the effectiveness and phosphorus supply capacity of phosphorus in the acid soil are usually low.
The circulation of the soil phosphorus mainly takes microorganisms as a center, and the microorganisms which can convert ineffective phosphorus into plants and can absorb and utilize the phosphorus are called soil phosphorus conversion microorganisms and also called phosphorus dissolving microorganisms, wherein bacteria are called phosphorus dissolving bacteria or phosphorus dissolving bacteria, but the solubility of the traditional phosphorus dissolving bacteria to insoluble phosphate is lower. Therefore, there is a need for a Sphingobacterium multivorum and its use.
Disclosure of Invention
The invention provides a sphingosine bacillus Sphingobacterium multivorum with phosphorus dissolving capacity and application thereof.
The strain is Sphingobacterium multivorum p6, is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2021, 5 and 31 months and the preservation number of M2021648, and has the nucleotide sequence shown in SEQ ID NO. 1.
In addition, the invention also provides a method for separating the sphingosine rod strain, which comprises the following steps:
4) weighing 1g of soil sample in sterile environment, placing in a triangular flask (containing sterile glass beads) containing 9mL of sterile water, and oscillating at 28 deg.C for 30min at 160 r.min to select 3 serial dilutions (10)-4,10-5,10-6) And inoculating the modified PKO plate culture medium by using a surface coating method.
5) Each plate was inoculated with 0.1ml of soil solution. The inoculated plates were incubated at 28 ℃ for about 4 days in an incubator. After the bacterial colony grows out, the dominant bacterial colony is further screened according to the growth condition and the size of the phosphorus-solubilizing bacteria.
6) And (3) selecting the strain with the transparent phosphorus-dissolving ring, continuously carrying out streak culture for 3 times, and only taking the strain which can still grow on the PKO plate after the continuous streak culture for 3 times as the effective phosphorus-dissolving bacterium.
Further, the improved PKO culture medium comprises 3g of calcium phosphate, 10g of cane sugar, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate, 0.5g of yeast extract, 15g of agar, 1000mL of water and pH of 7.0-7.2.
The invention also provides a dissolving agent of the insoluble phosphate, which comprises the fermentation liquor of Sphingobacterium multivorum P6 or Sphingobacterium multivorum P6.
Further, a method for preparing the Sphingobacterium Sphingobacterium Multivorum fermentation broth, comprising the steps of:
1) preparing a seed solution: inoculating Sphingobacterium multivorum into Gao's I liquid culture medium, culturing at 28 deg.C and 240r/min for 5-7d to obtain seed solution;
2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1 vol%, the bottling amount is 200mL/250mL, the initial pH value of a fermentation medium is 7.0-7.2, the fermentation temperature is 28 ℃, 180r/min, and the fermentation time is 10 days, so that the sphingosine bacillus multivorum fermentation liquid is obtained.
Further, preparation of a sterilized fermentation broth of Sphingobacterium multivorum P6: centrifuging Sphingobacterium multivorum fermentation liquor for 5min at 5000r/min, and filtering and sterilizing by using a 0.4-micron sterilization microporous filter membrane to obtain the Sphingobacterium multivorum sterile fermentation liquor.
An application of Sphingobacterium multivorum in dissolving insoluble phosphate is provided.
The invention has the beneficial effects that:
the bacterial strain has the capacity of dissolving insoluble phosphate, and the phosphorus-dissolving amount of the bacterial strain cultured in four insoluble phosphate liquid culture media of calcium phosphate, iron phosphate, aluminum phosphate and ground phosphate rock for 7 days is 47.2 mu g mL-1、6.1μg mL-1、3.8μg mL-1、9.5μg mL-1The strain and the soil phosphorus dissolving capacity are combined for the first time, a certain reference is provided for the later research of wild strains, and the strain has important value on the development of microbial fertilizers such as phosphate solubilizing bacterial fertilizers and the like.
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FIG. 1 is a schematic diagram showing the dynamic change of the available phosphorus content of a liquid medium according to the present invention;
FIG. 2 is a schematic diagram showing the dynamic variation of the pH of the liquid culture medium according to the present invention;
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
As shown in figure 1 of the drawings, in which,
EXAMPLE 1 selection of phosphate solubilizing bacteria
Selecting heat-ground Stylosanthes guianensis cv.Reyan No.2 in vegetative stage, oven drying and sieving (2mm) as green manure sample, and seaBrick red soil of a test base of a tropical crop variety resource research institute of tropical agricultural science institute of southern province, delirium city, is used as a soil sample, an indoor soil column culture method is adopted, one end of a wax cylinder is sealed by two layers of filter paper, the soil to be tested is uniformly added into the wax cylinder, and the soil column is enabled to uniformly absorb water to reach saturated water capacity by utilizing capillary action. And sealing the whole wax cylinder by using a preservative film, and vertically placing the wax cylinder in an incubator to be protected from light and balanced for 48 hours. After the soil column is balanced, uniformly mixing a phosphate fertilizer or a green manure, putting the mixture into a nylon bag, putting the nylon bag on the top of the soil column, sealing the nylon bag by using a preservative film, and culturing the mixture in a biological incubator at 30 ℃ (simulating similar field environment and selecting the normal temperature). After 15 days and 30 days, taking out a top green manure sample for analyzing indexes such as decay rate and the like, and then sequentially cutting 25 soil pieces with the thickness of 2mm from the top end of the wax column by using a soil cutting device. Weighing 1g of the soil sample in a sterile environment, placing in a triangular flask (containing sterile glass beads) containing 9mL of sterile water, and oscillating at 28 deg.C for 30min at 160 r.min) to select 3 serial dilutions (10 ℃-4,10-5,10-6) Surface-coating was used for plating. The inoculation culture Medium is a modified PKO culture Medium (Pikovskaya's Medium), and comprises 3g of calcium phosphate, 10g of sucrose, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate, 0.5g of yeast paste, 15g of agar, 1000mL of water and pH of 7.0-7.2. Each plate was inoculated with 0.1ml of soil solution. The inoculated plates were incubated at 28 ℃ for about 4 days in an incubator. After the bacterial colony grows out, the dominant bacterial colony is further screened according to the growth condition and the size of the phosphorus-solubilizing bacteria. And (3) selecting the strain with the transparent phosphorus-dissolving ring, continuously carrying out streak culture for 3 times, and only taking the strain which can still grow on the PKO plate after the continuous streak culture for 3 times as the effective phosphorus-dissolving bacterium.
Example 2 identification of Phosphosolubilizing strains
Selecting a purified and cultured strain, sequencing by 16S rDNA, comparing the sequence obtained by sequencing in NCBI, and selecting the sequence with the maximum similarity as a species identification result. The similarity of the sequence and the 16SrDNA gene sequence of the Sphingobacterium multivorum is up to 99 percent through comparison.
EXAMPLE 3 ability of phosphorus-solubilizing bacteria to solubilize poorly soluble phosphates
A PKO liquid culture medium taking calcium phosphate, iron phosphate, aluminum phosphate and powdered rock phosphate as phosphorus sources is inoculated with a bacterial solution (OD600 is 0.5) prepared from an equivalent strain, the bacterial solution is placed in a constant-temperature shaking table (160r/min,28 ℃) for culture for 7d, the culture solution is centrifuged (10000r/min) for 15min at 4 ℃, the effective phosphorus content of the supernatant is measured by a molybdenum-antimony colorimetric method, meanwhile, the test is set for 3 times by taking a suspension without inoculation of bacteria as a control. The measured available phosphorus content is as follows:
effective phosphorus content of Sphingobacterium Multivorum p6 in four phosphorus source cultures (μ g mL)-1)
The phosphorus dissolution amount is calculated by the formula:
calculating the formula: p ═ K-Kck)×V/V1
Wherein: p is the amount of dissolved phosphorus (mug/mL); k is the content of effective phosphorus measured by the bacterial culture solution to be measured; kckEffective phosphorus content (μ g/mL) was determined for non-inoculated bacterial suspensions; v is the volume (mL) of the solution for constant volume during color development; v1Volume of supernatant (mL) was aspirated for color development. The phosphorus-dissolving amount of Sphingobacterium Multivorum P6 was calculated to be 47.2. mu.g mL of calcium phosphate-16.1. mu.g mL of iron phosphate-13.8. mu.g mL of aluminum phosphate-19.5 mu g mL of powdered rock phosphate-1。
EXAMPLE 4 dynamic variation of the available phosphorus content of the liquid Medium
Using the method of example 3, the available phosphorus content in different culture solutions was measured at intervals of 24h as shown in FIG. 1, and the resulting 7d available phosphorus content was dynamically changed.
Example 5 dynamic variation of amino acid basicity in liquid culture
The pH value in different culture solutions was measured at intervals of 24h as shown in FIG. 2, and the resulting pH value was dynamically changed for 7 d.
EXAMPLE 6 bacterial liquid organic acid content
After culturing in a liquid culture medium taking calcium phosphate as a phosphorus source for 7 days, the culture solution passes through a sterile filter membrane with the diameter of 0.45 mu m, and the contents of oxalic acid, tartaric acid, malic acid, citric acid and succinic acid in the solution are measured by a high performance liquid chromatograph, which is shown in the following table:
content of organic acid (mg L) in culture medium with calcium phosphate as phosphorus source-1)
Chromatographic conditions are as follows: an analytical column RP-ODS-C18 with a column temperature of 30 ℃ and a mobile phase of 0.02mol/LKH2PO4 buffer solution-methanol solution (99: 1), pH adjusted to 2.60 with 1mol/L phosphoric acid, an ultraviolet detection wavelength of 210nm, a sample size of 20. mu.L, and a flow rate of 0.5 mL/min.
The research on bacillus Sphingobacterium multivorum has focused on the decomposition of cellulose and the decomposition of petroleum hydrocarbons, and so on, and no reports on the phosphorus dissolution have been reported at present.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (8)
1. A Sphingobacterium multivorum characterized in that: the strain is Sphingobacterium multivorum P6, is preserved in China center for type culture Collection with the preservation date of 2021 year, 5 months and 31 days and the preservation number of CCTCC NO: M2021648, and the nucleotide sequence of the strain is shown in SEQ ID NO. 1.
2. The bacterium sphingosine according to claim 1, wherein the strain has the following properties:
a) sphingobacterium multivorum
b) Is gram negative.
3. The sphingosine bacterium according to claim 1, wherein the method for isolation of a strain of Sphingobacterium comprises the steps of:
1) weighing 1g of soil sample in sterile environment, placing in a triangular flask (containing sterile glass beads) containing 9mL of sterile water, and oscillating at 28 deg.C for 30min at 160 r.min to select 3 serial dilutions (10)-4,10-5,10-6) And inoculating the modified PKO plate culture medium by using a surface coating method.
2) Each plate was inoculated with 0.1ml of soil solution. The inoculated plates were incubated at 28 ℃ for about 4 days in an incubator. After the bacterial colony grows out, the dominant bacterial colony is further screened according to the growth condition and the size of the phosphorus-solubilizing bacteria.
3) And (3) selecting the strain with the transparent phosphorus-dissolving ring, continuously carrying out streak culture for 3 times, and only taking the strain which can still grow on the PKO plate after the continuous streak culture for 3 times as the effective phosphorus-dissolving bacterium.
4. The sphingosine bacillus according to claim 1, wherein the modified PKO medium comprises 3g of calcium phosphate, 10g of sucrose, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate, 0.5g of yeast extract, 15g of agar, 1000mL of water, pH 7.0-7.2.
5. A sparingly soluble phosphate solubilizing agent characterized by: the lytic reagent comprises a strain of Sphingobacterium multivorum P6 fermentation broth or Sphingobacterium multivorum P6 sterile fermentation broth.
6. A sparingly soluble phosphate solubilizing agent according to claim 5, characterized in that: the preparation method of the sphingosine bacillus Sphingobacterium multivorum fermentation liquor comprises the following steps:
1) preparing a seed solution: inoculating Sphingobacterium multivorum into Gao's I liquid culture medium, culturing at 28 deg.C and 240r/min for 5-7d to obtain seed solution;
2) fermentation: inoculating the seed liquid into a fermentation culture medium, wherein the fermentation conditions are as follows: the inoculation amount is 1 vol%, the bottling amount is 200mL/250mL, the initial pH value of a fermentation medium is 7.0-7.2, the fermentation temperature is 28 ℃, 180r/min, and the fermentation time is 10 days, so that the sphingosine bacillus multivorum fermentation liquid is obtained.
7. A sparingly soluble phosphate solubilizing agent according to claim 6, characterized in that: preparation of the Sphingobacterium multivorum thallus-free fermentation liquor: centrifuging Sphingobacterium multivorum fermentation liquor for 5min at 5000r/min, and filtering and sterilizing by using a 0.4-micron sterilization microporous filter membrane to obtain the Sphingobacterium multivorum sterile fermentation liquor.
8. An application of Sphingobacterium multivorum in dissolving insoluble phosphate is provided.
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Cited By (2)
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| CN115820490A (en) * | 2022-11-29 | 2023-03-21 | 江西省科学院生物资源研究所 | Sphingobacterium multivorum, microbial inoculum and application of microbial inoculum in prevention and treatment of kiwifruit soft rot |
| CN116515716A (en) * | 2023-06-20 | 2023-08-01 | 云南大学 | Sphingobacterium faecium and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820490A (en) * | 2022-11-29 | 2023-03-21 | 江西省科学院生物资源研究所 | Sphingobacterium multivorum, microbial inoculum and application of microbial inoculum in prevention and treatment of kiwifruit soft rot |
| CN115820490B (en) * | 2022-11-29 | 2023-06-27 | 江西省科学院生物资源研究所 | Sphingobacterium polycephalum, microbial inoculum and application thereof in preventing and treating kiwi soft rot |
| CN116515716A (en) * | 2023-06-20 | 2023-08-01 | 云南大学 | Sphingobacterium faecium and application thereof |
| CN116515716B (en) * | 2023-06-20 | 2024-03-08 | 云南大学 | Sphingobacterium faecium and application thereof |
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