An organic phosphorus mineralizing composite bacterial consortium with wide pH adaptability and its application

Abstract

The present application provides an organophosphorus mineralization composite bacterial consortium with wide pH adaptability and its application, wherein the organophosphorus mineralization composite bacterial consortium includes subterranean intergenus Bacillus ( Mesobacillus underground ), Stenotrophomonas ( Stenotrophomonas sp) and Cuproplastes ( Cupriavidus sp). The organophosphate mineralizing bacterial consortium described herein is suitable for use in a wide range of acidic and alkaline environments. The phosphatase secreted by the three bacterial strains is not affected by external phosphorus concentrations; it is a constitutive phosphatase, capable of producing large quantities regardless of phosphorus concentration. The organophosphate mineralizing bacterial consortium described herein significantly promotes the mineralization and release of organophosphate in organic fertilizers, improving the nutrient efficiency of organic fertilizers. Furthermore, it can mineralize organophosphate in both acidic and alkaline soils.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms and the technical field of organic fertilizers. Specifically, the present application provides an organophosphorus mineralizing composite bacterial community with wide pH adaptability and its application. Background Art

[0002] Phosphate fertilizer is essential for ensuring crop yields, and its periodic application is crucial for maintaining crop production. However, the large amount of phosphorus applied to the soil is often fixed in the soil, resulting in significantly low phosphorus fertilizer utilization in that season. Accumulated phosphorus in the soil is a major source of agricultural non-point source pollution and a key issue facing the green development of agriculture.

[0003] Reducing agricultural non-point source pollution at its source is one of the primary measures currently being implemented. Replacing conventional chemical fertilizers with new, green fertilizers can reduce the amount of new phosphorus entering farmland, thereby alleviating agricultural non-point source pollution. However, 30%-65% of total phosphorus in soil exists as organic phosphorus, and in soils high in organic matter, this proportion can reach as high as 90%. Reducing fertilizer application does not adequately deplete the phosphorus already held in the soil. Therefore, utilizing microorganisms to mineralize soil organic phosphorus and improve its utilization efficiency by plants is an effective measure to deplete soil organic phosphorus and mitigate agricultural non-point source pollution.

[0004] Soil pH is the parameter that most significantly influences the survival of functional microorganisms and the activity of phosphatase enzymes secreted by them. The wide variability in soil pH significantly limits understanding the application scope and effectiveness of organophosphorus bacteria. Constructing synthetic bacterial consortia using multiple functional strains allows for functional complementarity by combining the pH tolerance of different bacterial species and the pH tolerance range of their secreted phosphatases. Furthermore, cross-feeding between bacterial strains can further enhance consortium stability and efficacy, demonstrating strong application potential.

[0005] At present, studies have found that Bacillus ( Bacillus spp. ), Pseudomonas ( Pseudomonas spp. ) and Marseilles ( Massetia spp. ) and other phosphate-dissolving bacteria have the ability to dissolve organic phosphorus. However, to date, there are no patents or research reports on the construction of a composite bacterial consortium for organophosphate mineralization with broad pH adaptability using diverse bacterial communities. Summary of the Invention

[0006] On the one hand, the present application provides a composite bacterial consortium for organophosphorus mineralization with wide pH adaptability, wherein the composite bacterial consortium for organophosphorus mineralization includes Bacillus subterraneus ( Mesobacillus subterraneus ), Stenotrophomonas ( Stenotrophomonas sp. ) and Cupricobacteria ( Cupriavidus sp. ).

[0007] Furthermore, the underground intergenus Bacillus (Mesobacillus subterraneus ) is the underground intergeneric Bacillus ( Mesobacillus subterraneus ) A7, which was deposited on December 25, 2024 at the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a postal code of 510070, and the deposit number is GDMCC NO: 65672.

[0008] Furthermore, the oligotrophomonas ( Stenotrophomonas sp. ) is Stenotrophomonas ( Stenotrophomonas sp. ) A80, which was deposited on December 25, 2024 at the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a postal code of 510070, with the deposit number being GDMCC NO: 65673.

[0009] Furthermore, the copper-greedy bacteria ( Cupriavidus sp. ) is copper-greedy bacteria ( Cupriavidus sp. ) D34, which was deposited on December 25, 2024 at the Guangdong Provincial Microbiological Culture Collection Center, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a postal code of 510070, with the deposit number being GDMCC NO: 65674.

[0010] On the other hand, the present application provides the above-mentioned subterranean intergenus Bacillus ( Mesobacillus subterraneus )A7, Stenotrophomonas ( Stenotrophomonas sp) A80 or Cuproplastis ( Cupriavidus sp)D34.

[0011] On the other hand, the present application provides a composite bacterial agent, which comprises the above-mentioned organophosphorus mineralizing composite bacterial community.

[0012] Those skilled in the art can prepare the three strains provided by the present invention into various conventional microbial preparations according to conventional preparation methods of microbial preparations. The composite microbial preparation can be in the form of freeze-dried bacterial powder, liquid bacterial solution, glycerol tube, sand tube, etc. known in the art, which may include ingredients such as culture medium in addition to bacteria.

[0013] Furthermore, the CFU ratio of Bacillus subterraneus, Stenotrophomonas and Cupriavidus in the composite bacterial agent is 1-2:1-2:1-2.

[0014] Furthermore, the CFU ratio of Bacillus subterraneus, Stenotrophomonas and Cupriavidus in the composite bacterial agent is 1:1:1.

[0015] Furthermore, the preparation method of the composite bacterial agent comprises the following steps:

[0016] (1) Preparation of seed solution: The three bacteria in the composite microbial agent were inoculated into LB liquid medium respectively, cultured at 30°C, 200 r / min, and cultured for 8 h to prepare seed solution;

[0017] (2) Fermentation: The seed liquid was inoculated into a larger volume of LB medium for fermentation. The fermentation conditions were as follows: inoculation volume was 1% of the fermentation volume, liquid volume was 200 mL / 500 mL, initial pH value of the fermentation medium was 7.0-7.2, 30°C, 200 r / min, and fermentation time was 1-2 days to obtain single-bacteria fermentation broth of the three bacteria.

[0018] (3) Mixing: Adjust the fermentation broth of the three bacteria to the same OD with sterile water. 600 The composite bacterial agent is obtained by spray drying the fermentation liquid of the three bacteria into a single bacterial powder and then mixing them in equal volumes.

[0019] Furthermore, the LB liquid culture medium is formulated as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.1-7.5; and is sterilized by high-pressure steam at 121° C. for 20 minutes.

[0020] On the other hand, the present application provides the above-mentioned subterranean intergenus Bacillus ( Mesobacillus subterraneus )A7, Stenotrophomonas ( Stenotrophomonas sp) A80, Cupricobacterium ( Cupriavidus sp) D34, application of organophosphorus mineralizing complex bacteria or complex bacterial agents in promoting the release of organophosphorus in organic fertilizers.

[0021] Furthermore, the organic fertilizer is a neutral to weakly alkaline chicken manure organic fertilizer with a pH of 6.5-8, a neutral to weakly acidic sheep manure organic fertilizer with a pH of 6.0-7.5, or an alkaline mixed organic fertilizer with a pH of 8-10.

[0022] On the other hand, the present application provides the above-mentioned subterranean intergenus Bacillus ( Mesobacillus subterraneus )A7, Stenotrophomonas ( Stenotrophomonas sp) A80, Cupricobacterium ( Cupriavidus sp) D34, application of complex bacterial consortia or complex bacterial agents for mineralizing soil organic phosphorus, improving plant phosphorus absorption and promoting plant growth.

[0023] Furthermore, the soil used in the application is calcareous soil with a pH greater than 7 or red soil with a pH less than 7.

[0024] Furthermore, the mineralized soil organic phosphorus includes increasing the acid phosphatase activity and alkaline phosphatase activity in the plant rhizosphere soil and improving the content of available phosphorus in the soil.

[0025] In the composite bacterial agent of the present invention, the subterranean intergenic bacillus ( Mesobacillus subterraneus ) The phosphate secreted by A7 adapts to alkaline environments, and oligotrophic monocytogenes ( Stenotrophomonas sp) A80 secreted phosphatase that adapts to both acidic and alkaline environments; while Cupriphae sp Cupriavidus The phosphatase secreted by D34 is most effective in acidic environments. Furthermore, the phosphatase secreted by these three bacterial strains is not affected by external phosphorus concentrations; it is a constitutive phosphatase, capable of producing large quantities regardless of phosphorus concentration. This significantly promotes the mineralization and release of organic phosphorus in organic fertilizers, improving their nutrient efficiency. Furthermore, it can mineralize organic phosphorus in both acidic and alkaline soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Phosphatase activities of bacteria under two pH conditions (pH 5.4 and pH 9.4) are shown.

[0027] Figure 2 Phosphatase activities of bacteria under two pH conditions (pH 5.4 and pH 9.4) are shown.

[0028] Figure 3 The results show the mineralization effect of composite microbial agent on organic phosphorus in organic fertilizers with different pH values ​​(chicken manure pH 6.5, sheep manure pH 7.5, mixed organic fertilizer pH 9.4).

[0029] Figure 4 The results show that the composite bacterial agent promotes the growth of corn in soils with different pH values.

[0030] Figure 5 The results show that the composite bacterial agent promotes the growth of corn in soils with different pH values. DETAILED DESCRIPTION

[0031] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0032] Example 1 Phosphatase secretion characteristics of three bacterial strains in the composite bacterial agent

[0033] Several strains secreting phosphatase were screened in the rhizosphere soil of corn. Some strains with better effects were selected, identified and deposited in the Guangdong Provincial Microbial Culture Collection Center. The address of the Guangdong Provincial Microbial Culture Collection Center is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, zip code 510070. The specific strains include Bacillus subtilis ( Mesobacillus subterraneus ) A7, deposit number is GDMCC NO.65672; Stenotrophomonas ( Stenotrophomonas sp) A80, deposit number 65673; Cupriphora sp. D34 (Cupriavidus sp), accession number 65674.

[0034] Strains of Intergeneric Bacillus subtilis A7, Stenotrophomonas aeruginosa A80, and Cuprilobacter glutinosus D34 were streaked onto solid LB medium and cultured in a 30°C incubator. After single colonies grew, they were picked and inoculated into 5 mL of liquid LB medium. The culture was shaken at 30°C and 180 rpm for 18 hours. Then, 1 mL of the bacterial suspension was aspirated and inoculated into 100 mL of liquid LB medium. The culture was shaken at 30°C and 180 rpm for 18 hours. Phosphatase assays were performed in triplicate for each strain. For comparison, the phosphatase activity of five other phosphate-solubilizing bacteria was compared.

[0035] Pipette 0.5 mL of bacterial fermentation broth into a 2 mL centrifuge tube, centrifuge at 8000 rpm at 4°C for 3-5 min, discard the supernatant, and resuspend the tube twice with 0.48 mL of acidic Tris buffer (pH 5.4) for later use.

[0036] Treatment group: Add 20 µL of 100 mM pNPP (final concentration 4 mM) and incubate in a 30°C water bath for 1 hour. Then, add 0.5 mL of 0.5 M NaOH for 10 minutes to terminate the enzyme reaction. Centrifuge at 8000 rpm for 3 minutes, and measure the absorbance of the supernatant at 405 nm.

[0037] Control group: 0.5 mL NaOH (0.5 M) was added to terminate the enzyme activity reaction in advance, and then 20 μL 100 mM pNPP (final concentration 4 mM) was added. The cells were incubated at 30°C for 1 h, centrifuged at 8000 rpm for 3 min, and the absorbance of the supernatant was measured at 405 nm.

[0038] In addition, a separate resuspended bacterial suspension was used to measure OD600 for standardization.

[0039] Calculate the acid phosphatase activity = (the unit of the phosphatase activity of the treatment group is the amount of p-NPP catalyzed per gram of soil per minute - the unit of the phosphatase activity of the control group is the amount of p-NPP catalyzed per gram of soil per minute) / OD 600 value.

[0040] In addition, the alkaline phosphatase activity assay procedure was the same as above, except that the acidic Tris buffer (pH 5.4) was replaced with alkaline Tris (pH 9.4).

[0041] Test results: Figure 1 As shown, the acid phosphatase activity of Bacillus subterraneus A7 was 22.16 pKatalOD600 -1, alkaline phosphatase activity was 128.57 pKatal OD600 -1 ; The acid phosphatase activity of Stenotrophomonas sp. A80 was 50.51 pKatal OD600 -1 , alkaline phosphatase activity was 374.76 pKatal OD600 -1 The acid phosphatase activity of Cuprifolia D34 was 102.47 pKatal OD600 -1 , alkaline phosphatase activity was only 7.97 pKatal OD600 -1 Compared with the phosphatase activities of other bacteria, the activities of these three bacterial strains are the strongest.

[0042] Example 2 Test of pH growth tolerance of three bacterial strains in the composite bacterial agent

[0043] Strains of Intergeneric Bacillus subtilis A7, Stenotrophomonas A80, and Cuprilobacter sp. D34 were streaked onto solid LB medium and cultured in a 30°C incubator. After single colonies grew, they were selected and inoculated into 5 mL of liquid LB medium. The culture was shaken at 30°C and 180 rpm for 18 hours to prepare the seed solution.

[0044] The pH buffer 2-morpholinoethanesulfonic acid (MES) was added to the liquid LB at a final concentration of 100 mM, and the pH of the culture medium was adjusted to 4.5, 7.0, and 9.5. The seed solution was then added to LB culture medium at different pH values ​​at a 1% inoculum, with three replicates for each treatment. The culture was shaken at 200 rpm and 30°C for 48 hours. The final OD was measured at the end of the experiment. 600 value.

[0045] All three bacterial strains can grow in a pH environment of 4.5-9.5. The OD 600 There was no significant difference in the values ​​between the cultures under different pH conditions.

[0046] Example 3 Effect of composite bacterial agent on the mineralization of organic phosphorus in organic fertilizers with different pH values

[0047] The strains of Bacillus subtilis A7, Stenotrophomonas A80, and Cupribothrium glutinosum D34 were streaked on solid LB medium and cultured in a constant temperature incubator at 30°C. After the strains grew single colonies, they were picked and inoculated into 5 mL of liquid LB medium, cultured in a shaker at 30°C and 180 rpm for 18 h, and then 3 mL of bacterial liquid was aspirated and inoculated into 300 mL of liquid LB medium, and cultured in a constant temperature shaker at 180 rpm and 30°C for 24 h. The fermentation broth after culture was centrifuged in a 50 mL centrifuge tube at 4000 rpm and 4°C, and the supernatant was discarded. The three bacterial strains were re-selected with sterile water to adjust the OD600 =1 and then mixed in equal volumes to form a composite bacterial agent, and the inoculation amount was set to 250mL / kg organic fertilizer.

[0048] This experiment included six treatments: JF (chicken manure), JF+B (chicken manure + a compound inoculant), YF (sheep manure), YF+B (sheep manure + a compound inoculant), 5F (mixed organic fertilizer), and 5F+B (mixed organic fertilizer + a compound inoculant). An equal volume of sterile water was added to the control treatment. Each treatment was replicated three times. After inoculation with bacteria, the inoculum was incubated at 30°C for 30 days and then the changes in organic phosphorus were measured. The pH at a fertilizer-water ratio of 1:5 was 6.5 for chicken manure, 7.5 for sheep manure, and 9.4 for mixed organic fertilizer.

[0049] Specific results: The average content of available phosphorus in chicken manure, sheep manure and mixed organic fertilizer treated with compound bacterial agents were 546.34μg / kg, 410.84μg / kg and 2020.60μg / kg, respectively, which were significantly higher than those in the control group without compound bacterial agents. The available phosphorus increased by 35.3%, 33.4% and 18.8% respectively compared with the control group.

[0050] Example 4: Compound microbial agent for the mineralization of organic phosphorus in high organic phosphorus soil

[0051] A composite bacterial agent composed of Bacillus subtilis A7, Stenotrophomonas A80, and Cupriphaga glutinosus D34 was prepared as in Example 3. High organic phosphorus accumulation is a key characteristic of agricultural non-point source pollution, particularly in the Erhai Lake basin, which is a typical area affected by non-point source pollution. Therefore, the test soil was collected from Gusheng Village, Erhai, Dali, Yunnan. The soil was air-dried and sieved. The basic physical and chemical characteristics were: organic matter 52.6 g kg -1 , alkaline nitrogen 153 mg kg -1 , available phosphorus 164.0 mg kg -1 , fast-acting potassium 169 mg / kg -1 , pH value is 6.02.

[0052] The composite bacterial agent was inoculated into black soil at a concentration of 250 mL / kg and cultivated in the greenhouse of China Agricultural University for 30 days at a soil moisture content of 18%.

[0053] Specific results: The compound microbial agent can increase the content of available phosphorus in black soil. After being treated with phosphate-solubilizing bacteria, the content of available phosphorus in the soil increased significantly by 9.2% ( P <0.05).

[0054] Table 1 Effect of composite microbial agent on mineralization of organic phosphorus in black soil

[0055]

[0056] Example 5: Effect of the composite microbial agent on organic phosphorus mineralization in soils with two pH values ​​and phosphorus absorption in corn

[0057] The experimental plant was corn (Zhengdan 958). Two soils with different pH values ​​were used. Acidic red soil was collected from the Qiyang Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences. The soil was air-dried and passed through a 2 mm sieve. Its basic physical and chemical properties were: pH (water: soil = 5:1) 6.4, organic matter 5.5 g kg⁻¹, Olsen-Pi 10.11 mg kg⁻¹, Olsen-Po 0.57 mg kg⁻¹, and exchangeable potassium 37.61 mg kg⁻¹. Alkaline fluvo-aquic soil was collected from the Shangzhuang Experimental Station of China Agricultural University and had a pH (water: soil = 5:1) of 7.94, organic matter 11.12 g kg⁻¹, Olsen-Pi 24.97 mg kg⁻¹, Olsen-Po 6.78 mg kg⁻¹, and exchangeable potassium 54.37 mg kg⁻¹. Each pot was filled with 1 kg of soil and 2% organic fertilizer was applied to provide basic nutrients.

[0058] Selected corn seeds were soaked in a 2.5% sodium hypochlorite solution for 10 minutes, then surface-disinfected with 75% alcohol for 1 minute. After rinsing 6-7 times with sterile water, the seeds were placed on a petri dish and incubated for 24 hours. After germination, the seeds were sown in pots, with five seeds per pot, and covered with soil. The experimental group was irrigated with 100 ml of the inoculum, while the control group was irrigated with an equal volume of deionized water. Each group was replicated three times. After 30 days of growth, the plant height, stem diameter, and aboveground phosphorus content of the corn were measured to determine whether the composite inoculum promoted growth and phosphorus absorption in the corn.

[0059] Results showed that for corn grown in fluvo-aquic soil, the stem diameter and plant height of the treatments inoculated with phosphate-solubilizing bacteria and applied with organic fertilizer were significantly higher than those of the treatment applied with organic fertilizer alone. The stem diameter of the JF+B, YF+B, and 5F+B treatments increased by 11.4%, 34.5%, and 6.5% compared with the JF, YF, and 5F treatments, respectively. There was no significant difference in stem diameter between the JF treatment and the control, while plant height and stem diameter of the other treatments were significantly higher than the control. For corn grown in red soil, there was no significant difference in stem diameter among the JF, JF+B, YF, and YF+B treatments, and the stem diameter was significantly lower than that of the CK.

[0060] In all treatments, the aboveground phosphorus content of plants was significantly increased after adding the compound bacterial agent. Specifically, the increase in red soil was 17.09-47.71%; while in moist soil, the inoculation of the compound bacterial agent increased plant phosphorus absorption by 8.78-42.61%.

[0061] Table 2 Effects of compound microbial agents on phosphorus content in aboveground parts of plants

[0062]

[0063] Note: The indicators marked with * show significant differences between the control group and the group with the addition of compound microbial agent when the same soil and the same organic fertilizer are applied.

Claims

1. An organophosphorus mineralizing composite bacterial consortium with wide pH adaptability, characterized in that: The organophosphorus mineralization complex bacterial group includes subterranean intergeneric Bacillus ( Mesobacillus subterraneus ), Stenotrophomonas ( Stenotrophomonas sp) and Cuproplastes ( Cupriavidus sp); the underground intergeneric Bacillus is underground intergeneric Bacillus A7, which was deposited in Guangdong Provincial Microorganism Culture Collection Center on December 25, 2024, with a deposit number of GDMCCNO.65672; the Stenotrophomonas is Stenotrophomonas A80, which was deposited in Guangdong Provincial Microorganism Culture Collection Center on December 25, 2024, with a deposit number of GDMCC NO.65673; the copper-greedy bacteria is copper-greedy bacteria D34, which was deposited in Guangdong Provincial Microorganism Culture Collection Center on December 25, 2024, with a deposit number of GDMCC NO.65674.

2. A composite bacterial agent, characterized in that: The composite bacterial agent comprises the organic phosphorus mineralizing composite bacterial community according to claim 1.

3. The composite bacterial agent according to claim 2, wherein the CFU ratio of Intergeneric Bacillus subterraneus, Stenotrophomonas and Cupriavidus in the composite bacterial agent is 1-2:1-2:1-2.

4. Use of the organophosphorus mineralizing composite bacterial community according to claim 1 in promoting the release of organophosphorus in organic fertilizer.

5. application according to claim 4, the organic fertilizer is a neutral to weakly alkaline chicken manure organic fertilizer with a pH of 6.5-8, a neutral to weakly acidic sheep manure organic fertilizer with a pH of 6.0-7.5, or an alkaline mixed organic fertilizer with a pH of 8-10.

6. Use of the organophosphorus mineralizing composite bacterial consortium according to claim 1 in mineralizing soil organic phosphorus, improving plant phosphorus absorption and promoting plant growth.

7. The use according to claim 6, wherein the soil in the use is calcareous soil with a pH>7 or red soil with a pH<7.

8. The use according to claim 6, wherein the mineralization of soil organic phosphorus comprises increasing the acid phosphatase activity and alkaline phosphatase activity in the rhizosphere soil of plants and increasing the content of available phosphorus in the soil.

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