Biochar-loaded phosphate solubilizing bacterial agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to a biochar-loaded phosphate solubilizing microbial inoculum and a preparation method and application thereof, and the biochar-loaded phosphate solubilizing microbial inoculum is obtained by loading Klebsiella PSCa-E on corn straw biochar; the Klebsiella sp. PSCa-E is preserved in the China Center for Type Culture Collection on July 11, 2025, and the preservation number of the Klebsiella sp. PSCa-E is CCTCC NO: M 20251581. The charcoal-loaded phosphate-solubilizing microbial inoculum provided by the invention realizes function synergy of charcoal and thalli, the phosphate solubilizing amount of the loaded microbial inoculum to hydroxyapatite is increased by about 15%, and the stability of the thalli is enhanced. Pot experiments show that compared with addition of pure bacteria, the content of available phosphorus in soil is increased by 45.5% and the content of phosphorus in the overground part is increased by 215% after the charcoal-loaded microbial agent is added, and the charcoal-loaded microbial agent shows a synergistic interaction characteristic.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a biochar-supported phosphate-solubilizing agent, its preparation method, and its application. Background Technology

[0002] Phosphorus is one of the key nutrients limiting crop yield. However, applied phosphate fertilizers easily combine with calcium ions to form insoluble calcium phosphate compounds such as hydroxyapatite, resulting in a phosphate fertilizer utilization rate generally below 20% in the current season. To maintain crop yields, agricultural production has long relied on high amounts of phosphate fertilizer input, which not only increases costs but also causes ecological and environmental problems: on the one hand, excessive phosphorus accumulation leads to soil compaction and ecological degradation; on the other hand, phosphorus enters water bodies through runoff, causing eutrophication. Therefore, developing green technologies that can efficiently activate soil phosphorus pools and achieve reduced phosphate fertilizer application while increasing efficiency is an important direction for sustainable agricultural development.

[0003] Phosphate-solubilizing microorganisms activate insoluble phosphates by secreting organic acids, which is an environmentally friendly solution. However, free-state phosphate-solubilizing agents often suffer from low survival rates, weak colonization, and unstable function in the field due to water stress, nutrient competition, and antagonism with native microorganisms, resulting in significant differences between laboratory and field effects. Summary of the Invention

[0004] To develop a highly efficient phosphorus-solubilizing compound bacterial agent, this invention provides a biochar-supported phosphorus-solubilizing bacterial agent, its preparation method, and its application. The biochar-supported phosphorus-solubilizing bacterial agent provided by this invention achieves functional synergy between biochar and bacterial cells. After loading, the bacterial agent increases the phosphorus solubility of hydroxyapatite by approximately 15%, exhibiting synergistic effects.

[0005] This invention provides a biochar-supported phosphate-solubilizing agent, which is obtained by supporting Klebsiella pneumoniae PSCa-E on corn straw biochar; The Klebsiella PSCa-E strain was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC NO: M 20251581.

[0006] The biochar-loaded phosphorus-solubilizing bacterial agent provided by this invention achieves functional synergy between biochar and bacterial cells. After loading, the bacterial agent increases the phosphorus solubility of hydroxyapatite by about 15%, exhibiting synergistic and enhanced characteristics.

[0007] Furthermore, the mass ratio of Klebsiella PSCa-E to corn straw biochar in the biochar-supported phosphate-solubilizing agent is 8-10:100.

[0008] The present invention also provides a method for preparing the biochar-supported phosphate-solubilizing agent, comprising the following steps: OD 600The bacterial cells collected after centrifuging a Klebsiella PSCa-E bacterial suspension with a concentration of 0.8–1.2 were mixed with corn straw biochar at a mass ratio of 8–10:100, resuspended in sterile physiological saline, and then incubated at 28°C. o C~30 o C. Under conditions of 180 rpm to 200 rpm, the phosphorus-solubilizing agent loaded onto biochar was obtained after 24 h to 30 h of adsorption by shaking.

[0009] Furthermore, the centrifugation conditions were: 8000–10000 rpm for 4–6 min.

[0010] Furthermore, the ratio of the mixture of bacterial cells and corn straw biochar to sterile physiological saline was 100 mg to 110 mg: 45 mL.

[0011] The present invention also provides the application of the biochar-supported phosphate-solubilizing agent in phosphate solubilization, wherein the biochar-supported phosphate-solubilizing agent is used to improve the phosphate-solubilizing ability of phosphate-solubilizing bacteria.

[0012] Furthermore, the phosphorus dissolution utilizes phosphorus from hydroxyapatite.

[0013] The present invention also provides the application of the biochar-supported phosphate-solubilizing agent in promoting the growth and development of rapeseed, wherein the biochar-supported phosphate-solubilizing agent is used to increase the aboveground dry weight and total phosphorus accumulation of rapeseed.

[0014] Furthermore, the bacterial carbon mass ratio is 0.08–0.1 for the hydroxyapatite system and 0.0015–0.002 for the soil system.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Enhanced phosphorus-solubility: This invention achieves functional synergy between biochar and microbial cells through optimized adsorption processes, forming a stable "microreactor." After loading, the phosphorus-solubility of hydroxyapatite by the microbial agent increases by approximately 15% (from approximately 400 mg·L⁻¹). -1 Increased to 460 mg·L -1 This demonstrates synergistic effects.

[0016] Enhanced cell stability: Biochar, acting as a protective carrier, significantly improves the stress resistance of the microorganisms. The loaded microbial agents exhibit high survival rates after storage and application, and maintain their functionality for a long time, effectively solving the problem of unstable colonization in the field.

[0017] Outstanding growth-promoting effect: Pot experiments showed that biochar-loaded inoculants could increase the available phosphorus content in calcareous alluvial soil and promote rapeseed growth in the long term. The aboveground dry weight and total phosphorus accumulation were higher than those of the pure inoculant control group. Among them, the available phosphorus in the soil increased by 45.5% and the phosphorus content in the aboveground parts increased by 215%, which verified its application potential in phosphorus-fixing soil.

[0018] 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

[0019] 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.

[0020] Figure 1 A comparison of phosphorus solubility under liquid culture conditions for biochar loaded with phosphate-solubilizing bacteria, free bacteria, and pure biochar.

[0021] Figure 2 This is a comparison chart of cell growth curves with and without biochar.

[0022] Figure 3 The curves show the change of available phosphorus content in soil over time under different treatments.

[0023] Figure 4 A comparison chart of phosphorus uptake in plants under different treatments. Detailed Implementation

[0024] 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.

[0025] Example 1: A biochar-supported phosphate-solubilizing agent, its preparation method and application.

[0026] I. Preparation of Biochar-Loaded Phosphate-Solubilizing Bacterial Agent and Verification of its Phosphate-Solubilizing Ability (1) Preparation of free PSCa-E bacterial suspension: Klebsiella PSCa-E with preservation number CCTCC NO: M 20251581 was inoculated into LB liquid medium and incubated at 30°C. oC. Incubate with shaking at 180 rpm until the logarithmic growth phase (OD). 600 (≈1.0), to obtain bacterial suspension. Take 5 mL of bacterial suspension, centrifuge at 8000 rpm for 4 min to collect bacterial cells, and resuspend in 45 mL of sterile physiological saline to obtain PSCa-E free bacterial suspension.

[0027] (2) Preparation of biochar-supported phosphate-solubilizing bacteria: Klebsiella pneumoniae PSCa-E with preservation number CCTCC NO: M 20251581 was inoculated into LB liquid medium and incubated at 30°C. o C. Incubate with shaking at 180 rpm until the logarithmic growth phase (OD). 600 (≈1.0), to obtain bacterial suspension. Take 5 mL of bacterial suspension, centrifuge at 8000 rpm for 4 min to collect bacterial cells. Take 10 mg of the obtained bacterial cells (wet weight) and mix with 100 mg of high-temperature sterilized corn straw biochar, resuspend in 45 mL of sterile physiological saline. Place the mixture in 30... o C. The bacterial cells were loaded by shaking and adsorption at 180 rpm for 24 h to obtain a liquid bacterial agent (i.e., biochar-loaded phosphate-solubilizing bacterial agent).

[0028] (3) Phosphorus dissolution capacity test: To verify the loading effect, the following four treatments were set up: NBRIP liquid medium: C6H 12 O6 10.0 g / L, hydroxyapatite 5.0 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.

[0029] ① Blank control: NBRIP liquid medium (with hydroxyapatite as the sole phosphorus source); ②Biochar control group: NBRIP medium + equal volume of sterilized biochar; ③PSCa-E free bacteria group: NBRIP medium + 1% (v / v) PSCa-E free bacteria suspension (bacterial volume equivalent to the loaded group); ④ Biochar-loaded phosphate-solubilizing bacteria group: Biochar-loaded phosphate-solubilizing bacteria prepared in NBRIP medium + 1% (v / v).

[0030] All processing was performed within 30 seconds. o C. After shaking culture at 180 rpm for 24 h, samples were taken, centrifuged, and filtered through a 0.22 μm filter membrane. The soluble phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method (HJ 632-2011).

[0031] The results are as follows Figure 1 As shown, after 24 hours of cultivation, the phosphorus solubility in the biochar-loaded phosphate-solubilizing bacteria group was 460 mg·L⁻¹. -1 The levels were higher than those in the PSCa-E free bacteria group (400 mg·L⁻¹). -1 The results, along with a biochar control group, indicate that the phosphorus-solubility of strain PSCa-E is enhanced after loading using the method of this invention.

[0032] II. Effects of biochar loading on cell growth stability To investigate the effect of biochar on the long-term survival of bacterial cells, growth stability was monitored. 5 mL of PSCa-E bacterial culture (OD2000) was taken... 600 ≈1.0) Bacterial cells were collected by centrifugation at 8000 rpm for 4 min. The obtained bacterial cells were mixed with 100 mg of hyperthermically sterilized corn straw biochar and resuspended in 45 mL of sterile physiological saline to obtain the bacterial culture and biochar mixture, which was designated as the biochar-added group. The obtained bacterial cells were directly dispersed in 45 mL of sterile physiological saline as a blank control without biochar, designated as the non-biochar-added group. After shaking in saline for 24 h, 200 μL of the bacterial culture and biochar mixture or PSCa-E bacterial culture was aspirated into a disposable sterile 96-well plate. The absorbance (OD) of the culture was monitored in real time at 600 nm using a microbial growth curve analyzer (FLUOstar Omega). 600 ), and record continuously for 24 hours to plot the growth curve.

[0033] The results are as follows Figure 2 As shown, the cell density of the experimental group with added biochar was higher than that of the control group during both the logarithmic and stationary phases, and the density remained constant after the stationary phase. This indicates that biochar can serve as a physical carrier to provide attachment sites and a protective environment for the cells, delaying cell death and maintaining high metabolic activity.

[0034] III. Verification of the growth-promoting effect of biochar-supported microbial agents in potted plant systems (1) Experimental design: The test soil was collected from the Yuanyang Base of Henan Agricultural University (longitude: 113°57′10.65″, latitude: 35°6′24.75″). The soil type was alluvial soil, pH 8.3, and available phosphorus 12 mg·kg -1 After being air-dried and sieved through a 2 mm sieve, it was used. The experiment included four treatments:

[0035] Blank control (CK): No bacterial agent or biochar added; Biochar Group (C): 1% (w / w) corn stalk biochar added; Phosphate-solubilizing bacteria group (PSCa-E): 5 mL of PSCa-E bacterial culture (OD) 600≈1.0) was mixed with 45 mL of sterile physiological saline, shaken for 24 h, and then added to 500 g of soil. The mixture was stirred and mixed thoroughly, and the ratio of bacteria to soil was controlled at 1% (v / w). Biochar-supported phosphate-solubilizing bacteria group (C-PSCa-E): 5 mL of PSCa-E bacterial culture (OD) was added to the culture medium. 600 ≈1.0) was mixed with 5 g of biochar and 45 mL of sterile physiological saline, shaken for 24 h, and then added to 500 g of soil. The mixture was stirred and mixed thoroughly, and the ratio of bacteria to soil was controlled at 1% (v / w).

[0036] Each treatment was replicated three times. All treatments were treated with the same nitrogen fertilizer (urea 0.2 gN·kg⁻¹). -1 ) and potassium fertilizer (potassium sulfate 0.24g K2O·kg) -1 No phosphate fertilizer was applied to assess the phosphorus-releasing and growth-promoting effects.

[0037] (2) Plant cultivation: Rapeseed seeds were selected, and after disinfection and germination, seedlings with uniform growth were transplanted into pots (500g soil per pot) with a diameter of 11cm, a bottom diameter of 8.8cm, and a height of 12cm, with 3 seedlings per pot. The seedlings were then grown in a controlled greenhouse (25°C). o Cultured at C (12h light / 12h dark) for 60 days, maintaining soil moisture content at approximately 40%.

[0038] (3) Index determination: Samples were taken 30 days and 60 days after transplanting, and the available phosphorus content in the soil was determined by 0.5 M NaHCO3 extraction-molybdenum antimony colorimetric method.

[0039] The results are as follows Figure 3 As shown, the C-PSCa-E treatment group had the highest available phosphorus content in the soil throughout the entire incubation period, and maintained a significant advantage until 60 days.

[0040] After 60 days of cultivation, the aboveground parts of the plants were harvested, and the dry weight was measured. The total phosphorus content was determined using the molybdenum blue method. The results are as follows: Figure 4 As shown, the aboveground dry weight and total phosphorus accumulation of the C-PSCa-E treatment group were higher than those of other treatments, indicating that the inoculant can effectively improve crop phosphorus absorption and promote growth in calcareous soils.

[0041] 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.

[0042] 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 biochar-supported phosphate-solubilizing agent, characterized in that, The biochar-loaded phosphate-solubilizing agent was obtained by loading Klebsiella PSCa-E onto corn straw biochar. The Klebsiella PSCa-E strain was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC NO: M 20251581.

2. The biochar-supported phosphate-solubilizing agent according to claim 1, characterized in that, The mass ratio of Klebsiella pneumoniae PSCa-E to corn straw biochar in the biochar-loaded phosphate-solubilizing agent is 8-10:

100.

3. The method for preparing the biochar-supported phosphate-solubilizing agent according to claim 1 or 2, characterized in that, Includes the following steps: OD 600 The bacterial cells collected after centrifuging a Klebsiella PSCa-E bacterial suspension with a concentration of 0.8–1.2 were mixed with corn straw biochar at a mass ratio of 8–10:100, resuspended in sterile physiological saline, and then incubated at 28°C. o C~30 o C. Under conditions of 180 rpm to 200 rpm, the phosphorus-solubilizing agent loaded onto biochar was obtained after 24 h to 30 h of adsorption by shaking.

4. The method for preparing biochar-supported phosphate-solubilizing bacteria according to claim 3, characterized in that, Centrifugation conditions: 8000-10000 rpm for 4-6 min.

5. The method for preparing biochar-supported phosphate-solubilizing bacteria according to claim 3, characterized in that, The ratio of the mixture of bacterial cells and corn straw biochar to sterile physiological saline was 100 mg to 110 mg: 45 mL.

6. The application of the biochar-supported phosphate-solubilizing agent according to claim 1 or 2 in phosphate solubilization, characterized in that, The biochar-loaded phosphate-solubilizing agent is used to enhance the phosphate-solubilizing ability of phosphate-solubilizing bacteria.

7. The application according to claim 6, characterized in that, The phosphorus dissolution is achieved by utilizing phosphorus in hydroxyapatite.

8. The application of the biochar-supported phosphate-solubilizing agent according to claim 1 or 2 in promoting the growth and development of rapeseed, characterized in that, The biochar-loaded phosphate-solubilizing agent is used to increase the aboveground dry weight and total phosphorus accumulation of rapeseed.

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