Klebsiella PSCa-E capable of dissolving calcium and phosphorus, bacterial liquid and application of Klebsiella PSCa-E
By using Klebsiella pneumoniae PSCa-E to efficiently dissolve insoluble calcium and phosphorus in calcareous soils, the problems of low phosphate fertilizer utilization and water eutrophication have been solved, resulting in increased effective phosphorus concentration in the soil and reduced use of chemical phosphate fertilizers. This approach demonstrates good environmental adaptability and economic benefits.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUAZHONG AGRI UNIV
- Filing Date
- 2026-01-30
- Publication Date
- 2026-04-24
AI Technical Summary
In calcareous soils, applied phosphate fertilizers easily combine with calcium ions to form insoluble calcium phosphate salts, resulting in low phosphate fertilizer utilization, increased agricultural costs, and eutrophication of water bodies. Existing phosphate-solubilizing strains have weak colonization ability and unstable phosphate-solubilizing activity in this environment.
A calcium and phosphorus-soluble Klebsiella pneumoniae strain PSCa-E was provided, which can rapidly dissolve hydroxyapatite and dicalcium phosphate dihydrate in liquid culture, has a high phosphorus-solubilizing capacity, and has a strong colonization ability in calcareous soils, which can significantly increase the soil's available phosphorus concentration.
Klebsiella PSCa-E showed a phosphorus solubility of 419 mg·L⁻¹ within 24 h in liquid culture, with a phosphorus solubility rate of 17.5 mg·L⁻¹·h⁻¹. It can significantly increase the available phosphorus content in the soil, reduce the amount of chemical phosphate fertilizer by 20%~30%, improve phosphorus utilization, and reduce agricultural non-point source pollution.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural and environmental microbiology, specifically to a calcium- and phosphorus-soluble Klebsiella pneumoniae strain PSCa-E, its bacterial culture, and its applications. Background Technology
[0002] Phosphorus is an essential macronutrient for plant growth and development, participating in key processes such as energy transfer, photosynthesis, and the transmission of genetic information. However, in most soils, especially in the calcareous soils of my country's major grain-producing areas (such as the Huang-Huai-Hai Plain), applied phosphate fertilizers and inherent soil phosphorus easily combine with calcium ions to form insoluble calcium phosphate salts such as hydroxyapatite. The utilization rate of phosphate fertilizers in the current season is typically less than 20%, meaning that over 80% of the phosphorus is fixed and cannot be directly absorbed and utilized by plants.
[0003] This "phosphorus fixation" phenomenon leads to two problems: first, to maintain crop yields, large amounts of chemical phosphate fertilizers are required, increasing agricultural costs; second, phosphorus accumulated in the soil enters water bodies via runoff, causing eutrophication. Therefore, developing green and efficient technologies that can activate fixed phosphorus and reduce dependence on external phosphate fertilizers has become a key direction for sustainable agricultural development.
[0004] Phosphate-solubilizing microorganisms dissolve insoluble phosphates by secreting organic acids, protons, or chelates to lower the pH or complex metal ions. Current research reports primarily focus on Pseudomonas species (…). Pseudomonas ), Bacillus spp. Bacillus However, these strains often exhibit problems such as weak colonization ability and unstable phosphorus-solubilizing activity in calcareous soils, making it difficult to achieve long-term effective effects. Summary of the Invention
[0005] To develop a strain capable of effectively colonizing and efficiently dissolving calcium and phosphate salts in calcareous soils, this invention provides a calcium and phosphate-dissolving Klebsiella pneumoniae strain PSCa-E, its bacterial culture, and its applications. The Klebsiella pneumoniae PSCa-E provided by this invention can rapidly dissolve hydroxyapatite within 24 hours in liquid culture, achieving a phosphorus dissolution rate of 419 mg·L⁻¹. -1 The phosphorus dissolution rate was 17.5 mg·L⁻¹. -1 ·h -1 This strain can efficiently dissolve hydroxyapatite and calcium hydrogen phosphate dihydrate, with a solubility of 863 mg·L⁻¹ for the latter. -1 This indicates that PSCa-E can activate various forms of fixed calcium and phosphorus.
[0006] This invention provides a calcium- and phosphorus-soluble Klebsiella pneumoniae strain ( Klebsiella sp.Klebsiella pneumoniae PSCa-E was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC NO: M20251581.
[0007] The Klebsiella PSCa-E strain can rapidly dissolve hydroxyapatite within 24 hours in liquid culture, with a phosphorus solubility of up to 419 mg·L⁻¹. -1 The phosphorus dissolution rate was 17.5 mg·L⁻¹. -1 ·h -1 This strain can efficiently dissolve hydroxyapatite and calcium hydrogen phosphate dihydrate, with a solubility of 863 mg·L⁻¹ for the latter. -1 This indicates that PSCa-E can activate various forms of fixed calcium and phosphorus.
[0008] The present invention also provides a Klebsiella PSCa-E bacterial suspension containing the aforementioned calcium-phosphate-soluble Klebsiella PSCa-E.
[0009] Furthermore, the OD of the Klebsiella PSCa-E bacterial suspension... 600 The value ranges from 0.8 to 1.2.
[0010] The present invention also provides the application of the aforementioned Klebsiella PSCa-E for dissolving calcium and phosphorus, or the aforementioned Klebsiella PSCa-E bacterial solution, in dissolving insoluble inorganic phosphates.
[0011] Furthermore, the sparingly soluble inorganic phosphate is a calcium phosphate compound.
[0012] Furthermore, the calcium phosphate compound includes hydroxyapatite and calcium hydrogen phosphate dihydrate.
[0013] The present invention also provides the application of the aforementioned calcium- and phosphorus-soluble Klebsiella PSCa-E or the aforementioned Klebsiella PSCa-E bacterial solution in promoting the growth and development of rapeseed.
[0014] Furthermore, the Klebsiella PSCa-E is used to activate soil phosphorus fixation.
[0015] Furthermore, promoting the growth and development of rapeseed involves increasing the aboveground dry weight and total phosphorus accumulation of rapeseed.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The Klebsiella PSCa-E strain provided by this invention can rapidly dissolve hydroxyapatite within 24 hours in liquid culture, with a phosphorus solubility of up to 419 mg·L⁻¹. -1 The phosphorus dissolution rate was 17.5 mg·L⁻¹. -1 ·h -1This strain can efficiently dissolve hydroxyapatite and calcium hydrogen phosphate dihydrate, with a solubility of 863 mg·L⁻¹ for the latter. -1 This indicates that PSCa-E can activate various forms of fixed calcium and phosphorus.
[0017] Klebsiella pneumoniae PSCa-E has strong colonization ability and stable function: In a simulated soil system, PSCa-E can continuously increase the concentration of available phosphorus in the soil, and the available phosphorus content increases by more than 100% within 30 days, proving that it has good environmental adaptability and persistence.
[0018] Klebsiella pneumoniae PSCa-E can be used to reduce the application of chemical phosphate fertilizers by 20% to 30%, improve phosphorus utilization, and reduce agricultural non-point source pollution, thus having high ecological and economic application value.
[0019] Information on the Preservation of Biological Materials PSCa-E, referred to as Klebsiella PSCa-E in this application, was deposited on July 11, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251581. The address of the depository is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, 430072, China. It is classified as Klebsiella PSCa-E. Klebsiella sp PSCa-E. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 Morphological and Gram staining identification of strain PSCa-E; In the figure, A shows the colony morphology of strain PSCa-E on LB solid medium; B represents the Gram staining results of strain PSCa-E; C is a micrograph of strain PSCa-E.
[0022] Figure 2 A phylogenetic tree constructed based on the 16S rDNA sequence.
[0023] Figure 3 The phosphorus solubility curve of strain PSCa-E in liquid inorganic phosphorus medium is shown.
[0024] Figure 4This is a comparison of the phosphorus solubility of hydroxyapatite and calcium hydrogen phosphate dihydrate by strain PSCa-E.
[0025] Figure 5 The graph shows the change of available phosphorus in soil over time for strain PSCa-E in a sterilized wet soil system.
[0026] Figure 6 This is a schematic diagram showing the changes in available phosphorus in the soil of strain PSCa-E under different treatment groups in a pot experiment.
[0027] Figure 7 This is a schematic diagram showing the changes in phosphorus content in the aboveground parts of plants in different treatment groups of strain PSCa-E during a pot experiment. Detailed Implementation
[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0029] Example 1: Isolation and identification of strain PSCa-E.
[0030] 1. Isolation and screening of strains Sample collection: 0 cm to 20 cm of surface brown-red soil samples were collected from paddy fields in Qingfeng Village, Jiangxia District, Wuhan City, Hubei Province (latitude and longitude 30°06′55″N, 114°09′39″E).
[0031] Enrichment and initial screening: Weigh 0.5 g of fresh soil sample and inoculate it with 100 mL of insoluble calcium phosphate (hydroxyapatite, 5.0 g·L⁻¹). -1 In NBRIP liquid medium with [a specific phosphorus source] as the sole phosphorus source, the culture was carried out at 30 °C and 180 rpm for 5 days with shaking to enrich calcium- and phosphorus-soluble microorganisms. The enrichment solution was then serially diluted (10... -3 ~10 -9 Take 100 μL of bacterial culture at each dilution and spread it on NBRIP solid medium with hydroxyapatite as the sole phosphorus source. Incubate at 30 °C upside down for 7 days.
[0032] NBRIP liquid medium with hydroxyapatite as the sole phosphorus source: C6H 12 O610.0 g·L -1 Hydroxyapatite 5.0 g·L -1 MgCl2·6H2O 5.0 g·L -1MgSO4·7H2O 0.25 g·L -1 KCl 0.2 g·L -1 0.1 g·L⁻¹ of (NH₄)₂SO₄ -1 pH 7.0; The NBRIP solid medium formula with hydroxyapatite as the sole phosphorus source is based on NBRIP liquid medium with the addition of 18 g·L⁻¹ agar. -1 .
[0033] Purification and rescreening: Single colonies forming a clear lysozyme zone on the plate were picked, repeatedly streaked for purification, and then inoculated into NBRIP liquid medium. The culture was incubated at 30 °C and 180 rpm with shaking for 4 days. -1 Centrifuge for 10 min, collect the supernatant, filter through a 0.22 μm microporous membrane, and determine the soluble phosphorus content using the molybdenum antimony colorimetric method. The strain with the strongest ability to dissolve hydroxyapatite was obtained through screening and named PSCa-E.
[0034] 2. Identification of strains Morphological characteristics: After culturing on LB solid medium for 24 h, strain PSCa-E forms round, smooth, moist, translucent colonies, approximately 1–2 mm in diameter. Under a microscope, the bacteria are short rod-shaped and Gram-negative. Figure 1 ).
[0035] Molecular biological identification: Genomic DNA was extracted from the strain, and the 16S rDNA fragment was amplified using universal primers 27F and 1492R. After sequencing the PCR products, BLAST alignment was performed in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA11 software.
[0036] The results are as follows Figure 2 As shown, this strain shares 94% homology with Klebsiella quasivariicola and has been identified as belonging to the genus Klebsiella. Klebsiella sp ).
[0037] Strain preservation: Strain PSCa-E was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC NO: M 20251581.
[0038] Example 2: Phosphate solubility dynamics of strain PSCa-E in liquid culture.
[0039] The activated strain PSCa-E was inoculated into LB liquid medium and cultured until the logarithmic growth phase. The bacterial concentration was then adjusted to OD. 600≈1.0. The inoculum was transferred at a rate of 1% (v / v) into NBRIP medium with hydroxyapatite as the sole phosphorus source and cultured at 30°C with shaking at 180 rpm. Samples were taken at 4 h, 8 h, 15 h, 24 h, 36 h, 48 h, and 72 h to determine the soluble phosphorus content in the supernatant.
[0040] The results are as follows Figure 3 As shown, strain PSCa-E exhibits rapid and efficient phosphorus solubility. The phosphorus solubility rate increases significantly in the early stages of cultivation (4 h–8 h), reaching a peak phosphorus solubility of 419 mg·L⁻¹ at 24 h. -1 The maximum phosphorus dissolution rate reached 17.5 mg·L⁻¹. -1 ·h -1 The results showed that this strain has an excellent ability to dissolve insoluble calcium phosphate.
[0041] Example 3: Solubility characteristics of strain PSCa-E for different calcium and phosphorus compounds.
[0042] At 5.0 g·L -1 Hydroxyapatite [Ca5(PO4)3OH] or 5.0 g·L -1 NBRIP liquid medium was prepared using dicalcium phosphate dihydrate [CaHPO4·2H2O] as the sole phosphorus source. Inoculation conditions were the same as in Example 2: culture at 30°C with shaking at 180 rpm for 5 days, with soluble phosphorus content measured daily.
[0043] NBRIP liquid medium with calcium phosphate dihydrate as the sole phosphorus source: C6H 12 O610.0 g·L -1 5.0 g·L of dicalcium phosphate dihydrate -1 MgCl2·6H2O 5.0 g·L -1 MgSO4·7H2O 0.25 g·L -1 KCl 0.2 g·L -1 0.1 g·L⁻¹ of (NH₄)₂SO₄ -1 pH 7.0; NBRIP solid medium formulation is based on NBRIP liquid medium with the addition of 18 g / L agar. -1 .
[0044] The results are as follows Figure 4 As shown, PSCa-E exhibits significant solubility for both calcium and phosphorus compounds. The strongest solubility is observed in CaHPO4·2H2O, with a phosphorus solubility of 863 mg·L⁻¹. -1 The phosphorus solubility of hydroxyapatite is 422 mg·L⁻¹. -1 This indicates that the strain can efficiently activate various forms of insoluble calcium phosphate.
[0045] Example 4: Phosphorus solubilization effect of strain PSCa-E in calcareous soil.
[0046] To verify its function in actual soil, a microcosm experiment was conducted. Alluvial soil (pH 8.3) from the Yuanyang base of Henan Agricultural University was selected, air-dried, and sterilized with gamma rays. A soil suspension was prepared using NBRIP liquid medium without hydroxyapatite (phosphorus-free NBRIP liquid medium), obtained by mixing soil and phosphorus-free NBRIP medium at a ratio of 1 g: 2 mL. OD... 600 Activated bacterial solution with a concentration of approximately 1.0 was inoculated into soil suspension at 1% (v / v), with an uninoculated system serving as a control. The system was incubated at 30 °C and 180 rpm, and samples were taken at 6 h, 12 h, 24 h, 48 h, 72 h, and 30 d to determine soluble phosphorus content.
[0047] NBRIP liquid medium without hydroxyapatite: C6H 12 O610.0 g·L -1 MgCl2·6H2O 5.0 g·L -1 MgSO4·7H2O 0.25 g·L -1 KCl 0.2 g·L -1 0.1 g·L⁻¹ of (NH₄)₂SO₄ -1 pH 7.0; the NBRIP solid medium formula is based on the NBRIP liquid medium with the addition of 18 g / L agar.
[0048] The results are as follows Figure 5 As shown, the amount of soluble phosphorus in the PSCa-E inoculation group was significantly higher than that in the control group. After 24 h of culture, the soluble phosphorus content in the treatment group reached 0.76 mg·L⁻¹. -1 The amount of phosphorus dissolved at 30 days was more than twice that of the control, indicating that this strain can significantly activate phosphorus fixation in the soil and has potential for agricultural and environmental remediation applications.
[0049] Example 5: Growth-promoting effect of strain PSCa-E on rapeseed.
[0050] To evaluate its growth-promoting effect, Henan alluvial soil (pH 8.3, available phosphorus 12 mg·kg⁻¹) was selected, air-dried and sieved, and two treatments were set up: uninoculated (CK); PSCa-E treatment, with 1% (v / w) inoculated bacterial solution (OD). 600 ≈1.0, 1 x 10 9 CFU / mL). Each treatment was repeated in triplicate, with uniform nitrogen application (urea 0.2 gN·kg). -1 ) and potassium (potassium sulfate 0.24 g K2O·kg -1No phosphate fertilizer was applied. Disinfected and pre-germinated rapeseed seedlings were sown and cultured in a temperature-controlled greenhouse for 60 days. Soil available phosphorus content was measured at 30 and 60 days.
[0051] The results are as follows Figure 6 As shown, the available phosphorus content in the soil treated with PSCa-E was consistently higher than that in the control (CK), reaching 7.70 mg·kg⁻¹ at 60 days. -1 With 6.48 mg·kg -1 The result is as follows Figure 7 As shown, after 60 days of cultivation, the plants were harvested. The aboveground dry weight and total phosphorus accumulation in the PSCa-E treatment group were significantly increased, with the total phosphorus accumulation increasing by 171%. This result indicates that strain PSCa-E can significantly promote crop phosphorus uptake and growth in phosphorus-deficient calcareous soils.
[0052] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.
[0053] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A strain of Klebsiella pneumoniae that dissolves calcium and phosphorus ( Klebsiella sp. PSCa-E, characterized in that, The Klebsiella PSCa-E strain was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC NO: M20251581.
2. A Klebsiella pneumoniae PSCa-E bacterial suspension, characterized in that, Klebsiella pneumoniae PSCa-E containing the calcium-soluble phosphorus as described in claim 1.
3. The Klebsiella PSCa-E bacterial suspension according to claim 2, characterized in that, The OD of the Klebsiella PSCa-E bacterial suspension 600 The value ranges from 0.8 to 1.
2.
4. The use of Klebsiella PSCa-E as described in claim 1 or the Klebsiella PSCa-E bacterial solution as described in any one of claims 2 to 3 in the dissolution of insoluble inorganic phosphates.
5. The application according to claim 4, characterized in that, The sparingly soluble inorganic phosphate is a calcium phosphate compound.
6. The application according to claim 5, characterized in that, The calcium phosphate compounds include hydroxyapatite and calcium hydrogen phosphate dihydrate.
7. The application of the calcium- and phosphorus-soluble Klebsiella PSCa-E of claim 1 or the Klebsiella PSCa-E bacterial solution of any one of claims 2 to 3 in promoting the growth and development of rapeseed.
8. The application according to claim 7, characterized in that, The Klebsiella PSCa-E strain is used to activate soil phosphorus fixation.
9. The application according to claim 7, characterized in that, The method of promoting rapeseed growth and development is to increase the aboveground dry weight and total phosphorus accumulation of rapeseed.