Feces phosphorus-solubilizing functional bacterium JF-P5 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a manure phosphate-solubilizing functional bacterium JF-P5 and application of the manure phosphate-solubilizing functional bacterium JF-P5. The invention relates to a manure phosphorus-solubilizing functional bacterium JF-P5, the taxonomic name of which is staphylococcus saprophyticus, and the manure phosphorus-solubilizing functional bacterium JF-P5 is preserved in Guangdong Microbial Culture Collection Center on June 4, 2025, and the preservation number is GDMCC No: 66464. According to the staphylococcus saprophyticus strain JF-P5 and the application of the staphylococcus saprophyticus strain JF-P5 to the preparation of the staphylococcus saprophyticus strain JF-P5, a phosphorus-solubilizing strain is screened to obtain a strain JF-P5, and the strain JF-P5 is determined to be the staphylococcus saprophyticus through morphological observation and molecular biological identification. Phosphorus-solubilizing ability tests on the strain JF-P5 find that the strain JF-P5 has strong phosphate-solubilizing ability, can convert insoluble phosphate into an absorbable and usable form, and can provide strain resource support for development of related microbial agents, bio-fertilizers or soil conditioners.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a phosphorus-solubilizing functional bacterium JF-P5 for fecal sewage and its application. Background Art

[0002] Phosphorus is a key limiting factor for crop growth. The traditional intensive agriculture's over-reliance on chemical phosphate fertilizers for yield increase is extremely likely to lead to severe environmental problems. The utilization rate of phosphate fertilizers by crops in the current season is only 15-25%. A large amount of phosphate fertilizers accumulates in the soil, and when surface runoff and soil erosion occur, it is extremely easy to cause eutrophication of surrounding water bodies. At the same time, long-term application of chemical phosphate fertilizers will exacerbate soil acidification, hardening, salinization, deficiency of medium and trace elements, and serious heavy metal risks.

[0003] Microbial phosphorus-solubilizing technology is regarded as an important breakthrough for the green development of agriculture. Phosphorus-solubilizing bacteria secrete substances such as organic acids (such as citric acid, oxalic acid), protons (H + ), and phosphatases to dissolve compounds such as calcium phosphate and iron phosphate, releasing available phosphorus that can be absorbed by plants. Since most of the current mainstream phosphorus-solubilizing strains are isolated from soil (such as Bacillus Bacillus sp., Pseudomonas Pseudomonas sp.), their tolerance to extreme environments with high ammonia nitrogen, high salt content, and high organic matter concentration such as fecal sewage treatment, saline-alkali land, or organic solid waste is poor, and their environmental adaptability is weak; in addition, conventional phosphorus-solubilizing strains are difficult to promote the degradation of phosphorus in organic waste and the nutrient cycling utilization, and their strain degradation functions are relatively single; at the same time, in the process of organic waste phosphorus degradation, organic fertilizer production, and soil phosphorus solubilization, there are problems such as low survival rate of viable phosphorus-solubilizing bacteria and short storage period. The above bottlenecks severely limit the application scope of the strains.

[0004] China's livestock and poultry breeding industry is developed, and the amount of fecal sewage resources is large. Fecal sewage resources are rich in organic phosphorus and insoluble phosphates, but there are high-concentration antibiotics, heavy metals, and ammonia nitrogen inhibitors. Conventional phosphorus-solubilizing bacteria are extremely easy to inactivate in such complex matrix environments and have poor viability preservation performance, resulting in low conversion efficiency of phosphorus resources in fecal sewage. The stress-resistant and highly efficient indigenous phosphorus-solubilizing bacteria screened from fecal sewage environments with multiple stress-resistant genes such as salt tolerance, heavy metal tolerance, and organic poison tolerance and naturally domesticated can not only efficiently transform and release phosphorus, but also synergistically degrade organic waste, achieving the dual goals of "phosphorus recycling utilization" and "waste pollution resource utilization". However, at present, strains with both high phosphorus-solubilizing activity, strong environmental adaptability, and fecal sewage matrix specificity are extremely scarce in the publicly reported literature.

[0005] Affected by the climate characteristics, mountain environment and feed in the subtropical mountainous areas of Guangxi, and given the wide adaptability and strong digestive ability of local chickens in the wild, high-quality chickens account for more than 1 / 4 of the national share. The chicken farm manure has a large quantity of resources, high nutrient content, and is rich in indigenous microorganisms, urgently requiring resource utilization. The inventor studied the microorganisms in the environment of matured manure resources from local chicken farms in Nanning, Yulin, Guigang and other places in Guangxi Zhuang Autonomous Region, and screened out an indigenous strain with efficient phosphate-solubilizing function. It has strong phosphate-solubilizing ability and does not have the problem of weak adaptability of phosphate-solubilizing bacteria in the manure environment, which can provide strain resources for the development of microbial agents, biological fertilizers or soil remediation products in the local area.

[0006] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] The object of the present invention is to provide a manure phosphate-solubilizing functional bacterium JF-P5 and its application, which can decompose insoluble phosphates in the soil, improve the availability of soil phosphorus, and contribute to improving the utilization rate of soil phosphorus.

[0008] To achieve the above object, the present invention provides the following technical solutions: The first object of the present invention is to provide a manure phosphate-solubilizing functional bacterium JF-P5, and the taxonomic name of the manure phosphate-solubilizing functional bacterium JF-P5 is Staphylococcus saprophyticus ( Staphylococcus saprophyticus ), which was deposited at the Guangdong Microbial Culture Collection Center on June 4, 2025, with the deposit number GDMCC No: 66464.

[0009] The second object of the present invention is to provide the application of the manure phosphate-solubilizing functional bacterium JF-P5 in decomposing insoluble phosphates in manure.

[0010] Further, the insoluble phosphates include calcium phosphate and iron phosphate.

[0011] The third object of the present invention is to provide the application of the manure phosphate-solubilizing functional bacterium JF-P5 in improving soil phosphorus utilization rate.

[0012] The fourth object of the present invention is to provide the application of the manure phosphate-solubilizing functional bacterium JF-P5 in the preparation of microbial agents.

[0013] The fifth object of the present invention is to provide the application of the manure phosphate-solubilizing functional bacterium JF-P5 in the preparation of biological fertilizers.

[0014] The sixth object of the present invention is to provide the application of the manure phosphate-solubilizing functional bacterium JF-P5 in soil conditioners.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention screens phosphate-solubilizing strains and obtains strain JF-P5. Through morphological observation and molecular biological identification, it is determined that strain JF-P5 is Staphylococcus saprophyticus ( Staphylococcus saprophyticus ). Through the phosphate-solubilizing ability test of strain JF-P5, it is found that strain JF-P5 has strong phosphate-solubilizing ability and can convert insoluble phosphate into an absorbable form, providing strain resource support for the development of related microbial agents, biological fertilizers or soil conditioners.

[0016] Description of preservation information The fecal sewage phosphate-solubilizing functional bacterium JF-P5, with the taxonomic name of Staphylococcus saprophyticus ( Staphylococcus saprophyticus ) was preserved in the Guangdong Provincial Microbial Culture Collection Center on June 4, 2025. Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangdong City, Institute of Microbiology, Guangdong Academy of Sciences. Its preservation number is GDMCC No: 66464. Description of the drawings

[0017] Figure 1 is the colony morphology diagram of strain JF-P5; Figure 2 is the phylogenetic tree of strain JF-P5; Figure 3 is the standard curve graph of soluble phosphorus content; Figure 4 is the molybdenum-antimony anti-colorimetric comparison graph of strain JF-P5; Figure 5 is the line graph of the growth rate of strain JF-P5 at different temperatures; Figure 6 is the line graph of the phosphate-solubilizing rate of strain JF-P5 in different pH environments; Figure 7 is the line graph of the phosphate-solubilizing rate of strain JF-P5 at different culture times; Figure 8 is the phosphate-solubilizing rate of strain JF-P5 for different phosphorus sources. Detailed implementation manners

[0018] The technical solutions of the present invention patent are described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments.

[0019] 1. Strain screening and identification 1.1 Collection of fecal sewage samples In 2023, fecal sewage resource samples from a chicken farm in Guigang City, Guangxi Zhuang Autonomous Region were collected, placed in a sterile sealed bag, and stored at -20°C under low temperature and sealed for screening phosphate-solubilizing strains.

[0020] 1.2 Culture Medium LB Solid Medium: Tryptone 10.0 g / L, Yeast Extract Powder 5.0 g / L, Sodium Chloride 10.0 g / L, Agar 15 g / L.

[0021] LB Broth Medium: Tryptone 10.0 g / L, Yeast Extract Powder 5.0 g / L, Sodium Chloride 10.0 g / L, pH value 7.0 ± 0.1 (30 °C).

[0022] Phosphate-Solubilizing Solid Medium: Glucose 10.0 g / L, Ammonium Sulfate 0.5 g / L, Yeast Extract Powder 0.5 g / L, Sodium Chloride 0.3 g / L, Potassium Chloride 0.3 g / L, Magnesium Sulfate 0.3 g / L, Ferrous Sulfate 0.03 g / L, Manganese Sulfate 0.03 g / L, Tricalcium Phosphate 5.0 g / L, Agar 15 g / L.

[0023] Phosphate-Solubilizing Medium: Glucose 10.0 g / L, Ammonium Sulfate 0.5 g / L, Yeast Extract Powder 0.5 g / L, Sodium Chloride 0.3 g / L, Potassium Chloride 0.3 g / L, Magnesium Sulfate 0.3 g / L, Ferrous Sulfate 0.03 g / L, Manganese Sulfate 0.03 g / L, Tricalcium Phosphate 5.0 g / L, pH value 7.0 - 7.5 (30 °C).

[0024] Calcium Phosphate / Aluminum Phosphate / Iron Phosphate Medium: Glucose 10.0 g / L, Ammonium Sulfate 0.5 g / L, Yeast Extract Powder 0.5 g / L, Sodium Chloride 0.3 g / L, Potassium Chloride 0.3 g / L, Magnesium Sulfate 0.3 g / L, Ferrous Sulfate 0.03 g / L, Manganese Sulfate 0.03 g / L, Calcium Phosphate / Aluminum Phosphate / Iron Phosphate 5.0 g / L, pH value 7.0 - 7.5 (30 °C).

[0025] 1.3 Strain Isolation and Purification Weigh 5.0 g of the decomposed chicken manure sample, place it in a 150 mL conical flask containing about 10 sterile glass beads, add 45 mL of sterile water, and shake it evenly at 30 °C and 140 r / min for 30 min. After standing and precipitating for 15 min, use a sterilized pipette to suck 100 μL of the above soil suspension into a 1 mL centrifuge tube, and add 900 μL of sterile water to obtain a 10 -1 -fold dilution. Subsequently, suck 100 μL of the 10 -1 -fold dilution into a test tube containing 900 μL of sterile water, and perform the operations successively to obtain a series of dilutions with concentrations of 10 -6 , 10 -7 , 10 -8 . Suck 100 μL of the dilution and spread it evenly on the phosphate-solubilizing solid medium, then invert it and culture it in an incubator at 30 °C for 3 d. Purify the single colonies by three-zone streaking on the LB solid medium three times and then store them for later use.

[0026] 1.4 Screening of phosphate-solubilizing strains The purified strains were respectively spread on the phosphate-solubilizing solid medium with the bacterial liquid dilutions at the concentrations of 10 -6 and 10 -7 . After the colonies grew out, their growth conditions were observed.

[0027] Nine strains with clear zones around the colonies were screened and judged to have the phosphate-solubilizing function. One of them was named JF-P5.

[0028] 1.5 Morphological observation of strain JF-P5 The strain JF-P5 was inoculated on the LB solid medium and cultured at a constant temperature of 30 °C. The colony morphology at 3 d of culture is shown in Figure 1 .

[0029] As can be seen from Figure 1 , the colony morphology of strain JF-P5 is round, white, opaque, with a convex and moist surface, easy to pick out, and the color does not change in the later stage of growth.

[0030] 1.6 Molecular biological identification of strain JF-P5 The Sanger method was used to sequence strain JF-P5. The universal bacterial primers 27F and 1492R were selected as primers to PCR amplify the 16S rRNA coding gene of the target strain. The sequences of the universal primers are shown in Table 1, and the PCR amplification system is shown in Table 2.

[0031]

[0032]

[0033] The PCR amplification program was: 95 °C, 5 min; 95 °C for 30 s, 56 °C for 30 s, 72 °C for 90 s, with 25 cycles; 72 °C, 10 min.

[0034] The purified PCR product was subjected to 16S RNA sequencing, and the results are shown in Sequence Listing SEQ ID No.1. The 16S rRNA gene sequence was compared with the NCBI database to obtain the species information of the similar sequences and construct an evolutionary tree (see Figure 2 ). With the aid of the homologous alignment method to assist in judging the species information, it was determined that JF-P5 is Staphylococcus saprophyticus ( Staphylococcus saprophyticus ), and it was named Staphylococcus saprophyticus JF-P5.

[0035] 1.7 Phosphate-solubilizing test of strain JF-P5 1.7.1 Preparation of bacterial liquid Inoculate the strain JF-P5 into LB broth medium and culture it in a shaker at 28°C and 140 r / min until the logarithmic growth phase (about 12 h). Centrifuge at 4°C and 3000 r / min for 10 min, wash the precipitate with sterile deionized water, resuspend and centrifuge 3 times, and finally resuspend with 0.9% sodium chloride solution to make its OD 600 value reach 1.0 to obtain the bacterial solution of strain JF-P5.

[0036] 1.7.2 Soluble phosphorus standard curve 1.7.2.1 Related reagents Potassium antimonyl tartrate solution: Weigh 0.3 g of potassium antimonyl tartrate and dissolve it in water, then dilute it to 100 mL.

[0037] Molybdenum-antimony stock solution: Weigh 10.0 g of ammonium molybdate and dissolve it in 300 mL of water at 60°C, and then cool it. Separately, slowly inject 181 mL of concentrated H2SO4 into 800 mL of water, stir well, and cool; then inject the dilute H2SO4 into the ammonium molybdate solution, stir well, cool, and then add 100 mL of 3 g / L potassium antimonyl tartrate solution, and finally dilute it to 2 L with water and store it in a brown bottle for later use.

[0038] Molybdenum-antimony-ascorbic acid color reagent: Weigh 0.5 g of ascorbic acid (C6H8O6, levorotatory, specific rotation +21 - 22°) and dissolve it in 100 mL of molybdenum-antimony stock solution.

[0039] Standard phosphorus stock solution: Dry KH2PO4 (guaranteed reagent) at 105°C for 2 h, then weigh 0.4390 g, dissolve it in water, add 5 mL of concentrated H2SO4, and then make up the volume to 1000 mL with water, and store it in the refrigerator for long-term use.

[0040] Standard phosphorus solution: Pipette 5 mL of the standard phosphorus stock solution into a 100 mL volumetric flask and make up the volume.

[0041] 1.7.2.2 Plot the soluble phosphorus standard curve Pipette 0, 1.00, 2.00, 3.00, 4.00, 5.00 mL of the standard phosphorus solution (5 mg / L) into 25 mL colorimetric tubes respectively, add 5.00 mL of molybdenum-antimony-ascorbic acid color reagent, shake well, make up the volume with distilled water to obtain a series of phosphorus standard solutions with concentrations of 0.00, 0.20, 0.40, 0.60, 0.80, 1.00 mg / L. After standing at room temperature of 20 - 25°C for 30 min, using the 0 mg / L phosphorus standard solution as the reference solution, measure the absorbance value at a wavelength of 880 nm on a spectrophotometer. Finally, plot the standard curve of soluble phosphorus with the absorbance value as the ordinate and the concentration of the standard phosphorus solution as the abscissa. The results are shown in Figure 3 .

[0042] From Figure 3It can be known that the standard curve equation of soluble phosphorus is: y = 0.7073x + 0.0199, and the determination coefficient R of the regression equation 2 = 0.9928.

[0043] 1.7.3 Quantitative test of strain JF-P5 Prepare 50 mL of phosphorus-solubilizing medium. After sterilization, add 1 mL of strain JF-P5 bacterial solution. The experiment is set with 3 replicates, and the one without adding bacterial solution is used as the blank control. Transfer it to culture at 30 °C and 140 r / min. After 72 h, take 1 mL of the culture solution and centrifuge it at 12,000 r / min for 5 min. Take part of the supernatant for dilution, and use the molybdenum antimony anti-colorimetric method to measure the available phosphorus content. The result shows that the available phosphorus content in the culture solution is 553.65 mg / L, that is, the phosphorus-solubilizing amount of strain JF-P5 is 553.65 mg / L. The results are shown in Figure 4 .

[0044]

[0045] 1.7.3.1 Effect of different temperatures on the growth rate of strain JF-P5 Prepare 50 mL of phosphorus-solubilizing medium, set 5 temperature gradients of 30 °C, 35 °C, 40 °C, 45 °C and 50 °C. After sterilization, add 1 mL of strain JF-P5 bacterial solution and culture it at 140 r / min for 120 h. Sampling is carried out every 24 h, and the absorbance value at a wavelength of 600 nm is measured with a spectrophotometer. The results Figure 5 .

[0046] It can be Figure 5 known that the growth rate of strain JF-P5 is the fastest at 30 - 40 °C, and the bacterial solution concentration is higher; it can enter the logarithmic growth phase faster at 45 - 50 °C, which is suitable for short-term breeding. It shows that the strain JF-P5 of the present invention can grow at high temperatures.

[0047] 1.7.3.2 Effect of different pH environments on the phosphorus-solubilizing rate of strain JF-P5 Prepare 50 mL of phosphorus-solubilizing medium, and use 0.1 mol / L NaOH solution and 0.1 mol / L HCl solution to adjust the pH value of the phosphorus-solubilizing medium to 4.0 ± 0.1, 5.0 ± 0.1, 6.0 ± 0.1, 7.0 ± 0.1, 8.0 ± 0.1, 9.0 ± 0.1 respectively. After sterilizing the above series of phosphorus-solubilizing media with different pH values, add 1 mL of strain JF-P5 bacterial solution. Each treatment is set with 3 replicates, and a blank control is set. Transfer it to culture at 30 °C and 140 r / min. After 72 h, take 1 mL of the culture solution and centrifuge it at 12,000 r / min for 5 min. Take part of the supernatant for dilution, use the molybdenum antimony anti-colorimetric method to measure the available phosphorus content, and calculate the phosphorus-solubilizing rate. The results are shown in Figure 6 .

[0048]

[0049] It can be seen from Figure 6 that when the pH is 5.0 - 9.0, the phosphate-solubilizing rate of strain JF-P5 in the solution is relatively obvious, being 17.71 - 38.52%; and when the pH value is 6.0 - 9.0, the phosphate-solubilizing rate of strain JF-P5 is greater than 30%, and when the pH value is 9.0, the phosphate-solubilizing rate of strain JF-P5 is the highest.

[0050] 1.7.3.3 Effect of Different Culture Times on the Phosphate-Solubilizing Rate of Strain JF-P5 Prepare 50 mL of phosphate-solubilizing medium, add 1 mL of strain JF-P5 bacterial solution after sterilization, set 3 replicates, and use the one without bacterial solution as the blank control. Transfer to culture at 30 °C and 140 r / min. Take 1 mL of the culture solution at 24 h, 48 h, 72 h, 96 h, and 120 h, centrifuge at 12000 r / min for 5 min, then take a part of the supernatant for dilution, and use the molybdenum antimony anti-colorimetric method to measure the available phosphorus content and calculate the phosphate-solubilizing rate. The results are shown in Figure 7 .

[0051] It can be seen from Figure 7 that within 24 - 120 h, the phosphate-solubilizing rate of strain JF-P5 in the solution is 20.05 - 34.57%; among them, when the culture time reaches 48 h, the phosphate-solubilizing rate of strain JF-P5 is greater than 30%, and it increases slowly with the increase of time. The phosphate-solubilizing rate of strain JF-P5 reaches 34.57% at 120 h.

[0052] 1.7.3.4 Phosphate-Solubilizing Rate of Strain JF-P5 on Different Phosphorus Sources Prepare 50 mL of medium with calcium phosphate, aluminum phosphate, and iron phosphate as phosphorus sources respectively, add 1 mL of strain JF-P5 bacterial solution after sterilization, set 3 replicates for the test, and set the corresponding blank control. Transfer to culture at 30 °C and 140 r / min. After 72 h, take 1 mL of the culture solution, centrifuge at 12000 r / min for 5 min, take a part of the supernatant for dilution, use the molybdenum antimony anti-colorimetric method to measure the available phosphorus content, and calculate the phosphate-solubilizing rate. The results are shown in Figure 8 .

[0053] It can be seen from Figure 8 that when the phosphorus source is iron phosphate and calcium phosphate, the strain has phosphate-solubilizing efficacy, and the phosphate-solubilizing rates are 0.04% and 29.97% respectively; among them, strain JF-P5 has the greatest phosphate-solubilizing efficacy on calcium phosphate.

[0054] In summary, the strain JF-P5 of the present invention has strong phosphate-solubilizing ability in a thermophilic and acid-tolerant environment, and can provide strain resource support for the development of related microbial inoculants, biological fertilizers or soil conditioners.

[0055] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A phosphorus-solubilizing functional bacterium JF-P5 for fecal sewage, characterized in that, The fecal sewage phosphate-solubilizing functional bacterium JF-P5 is taxonomically named Staphylococcus saprophyticus ( Staphylococcus saprophyticus ), and was deposited in the Guangdong Provincial Culture Collection of Microorganisms on June 4, 2025, with the deposit number GDMCC No: 66464.

2. Use of the fecal sewage phosphate-solubilizing functional bacterium JF-P5 as claimed in claim 1 in decomposing insoluble phosphates in fecal sewage.

3. The application according to claim 2, characterized in that The insoluble phosphates include calcium phosphate and iron phosphate.

4. Use of the fecal sewage phosphate-solubilizing functional bacterium JF-P5 as claimed in claim 1 in improving soil phosphorus utilization rate.

5. Use of the fecal sewage phosphate-solubilizing functional bacterium JF-P5 as claimed in claim 1 in preparing microbial inoculants.

6. Use of the fecal sewage phosphate-solubilizing functional bacterium JF-P5 as claimed in claim 1 in preparing biological fertilizers.

7. Use of the fecal sewage phosphate-solubilizing functional bacterium JF-P5 as claimed in claim 1 in preparing soil conditioners.

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