Phosphorus release enhancing type compound fertilizer based on fungal extracellular polymer, preparation method and application

Abstract

The invention provides a phosphorus release improving type compound fertilizer based on a fungal extracellular polymer, a preparation method and application, and belongs to the technical field of microorganisms, the phosphorus release improving type compound fertilizer is prepared by compounding the extracellular polymer generated by specific rhodotorula mucilaginosa WTSX01 and inorganic phosphate according to the mass ratio of 1: 9; the preparation method comprises the following steps: extracting and purifying high-activity EPS from a strain fermentation broth by a low-temperature dialysis method, separating lipid from EPS as a natural adhesive by a cosolvent extraction method, and finally mixing EPS, lipid and inorganic phosphate. The invention also discloses a synergistic application of the fertilizer and a phosphorus-solubilizing fungus aspergillus niger (CCTCC No. M2023240), so that the release efficiency of phosphorus in soil is improved; the fertilizer provided by the invention provides exclusive nutrition for phosphate-solubilizing microorganisms, effectively overcomes indigenous microorganism competition, remarkably promotes microorganism growth and phosphorus release, and realizes reduction and synergism of phosphate fertilizer and yield increase of crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a phosphorus release improving type compound fertilizer based on fungal extracellular polysaccharide, a preparation method and application. BACKGROUND

[0002] Phosphorus is one of the indispensable macroelements for plant growth and development. However, more than 30% of the world's soils are facing the problem of effective phosphorus deficiency. This is not due to the lack of total soil phosphorus content, but because most of the phosphorus (more than 99%) exists in the form of insoluble inorganic phosphate (such as tricalcium phosphate) or organic phosphorus, which cannot be directly absorbed and utilized by plants. The traditional inorganic phosphorus fertilizer applied to the soil is also easily fixed, and its utilization rate in the current season is usually only 10%-25%. This low utilization rate not only causes a huge waste of phosphorus ore resources, an non-renewable resource, but also brings the risk of non-point source pollution such as water eutrophication.

[0003] In order to overcome the problem of phosphorus fixation, the biological strengthening strategy, especially the use of phosphorus-solubilizing microorganisms (such as bacteria and fungi) to activate the fixed state phosphorus in the soil, has become a research hotspot in the field of agriculture. Phosphorus-solubilizing microorganisms can dissolve insoluble phosphate by secreting organic acids, enzymes and other substances, thereby improving the effectiveness of phosphorus. However, in practical application, the efficiency of phosphorus-solubilizing microorganisms is often limited by factors such as unsuitable soil environment, competition with indigenous microorganisms, and insufficient supply of carbon source and nutrients, resulting in difficulties in colonization and unstable phosphorus-solubilizing effect.

[0004] On the other hand, microbial extracellular polymeric substance (EPS) as a high molecular polymer secreted by microorganisms outside the cell, mainly contains polysaccharides, proteins, nucleic acids and lipids, etc., and plays a key role in the aggregation, adhesion and nutrient storage of microorganisms. Especially the EPS from fungi, due to its high biomass and diverse functions, shows great application potential. EPS itself is rich in phosphorus elements, which can act as a phosphorus pool in the environment and play an important role in the adsorption and release of phosphorus. At present, there are various extraction methods for EPS, such as centrifugation, heating, ultrasonic method, etc., but these methods generally have some pain points: for example, the extraction process may cause the destruction of the large molecular structure of EPS and the loss of biological activity; although the commonly used ethanol precipitation method can obtain crude extract, it is often accompanied by pollution of small molecule impurities (such as salt, residual ethanol) and intracellular components (such as DNA), resulting in low purity of EPS, damage to the diversity of organic matter, and thus affecting the uniformity and effect of its subsequent application.

[0005] Therefore, there is an urgent need in this field for a novel fertilizer technology that can synergistically utilize microbial products and inorganic phosphorus sources, effectively improving phosphorus release efficiency and sustainability while providing a favorable growth environment for phosphate-solubilizing microorganisms. Currently, there is a lack of a technical solution that specifically utilizes high-purity EPS derived from specific fungi and precisely combines it with inorganic phosphates to form an organic-inorganic synergistic system, thereby systematically solving the aforementioned problems. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a phosphorus-releasing compound fertilizer based on fungal extracellular polymers, its preparation method, and its application. The compound fertilizer prepared by this invention can provide exclusive nutrition for phosphorus-solubilizing microorganisms and overcome competition from native microorganisms. Furthermore, the preparation method is simple, efficient, and environmentally friendly, and can better maintain the biological activity of fungal extracellular polymers.

[0007] Technical Solution: The present invention discloses a phosphorus release-enhancing compound fertilizer based on fungal extracellular polymers. The effective components of the compound fertilizer are fungal extracellular polymers and inorganic phosphates. The fungal extracellular polymers are derived from Rhodotorula glutinis (Yeast). Rhodotorula The Rhodotorula mucilaginosa WTSX01 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. M20221765 and deposit date of November 11, 2022.

[0008] Furthermore, the mass ratio of fungal extracellular polymeric polymer to inorganic phosphate is 1:(8~10).

[0009] Furthermore, the inorganic phosphate is Ca3(PO4)2.

[0010] Furthermore, the fungal extracellular polymeric material contains lipid components isolated from Rhodotorula mucilaginosa strain WTSX01 via co-solvent extraction. These lipid components act as a natural binder, contributing to the formation and stability of fertilizer granules while providing nutrients to phosphate-solubilizing fungi.

[0011] A method for preparing the compound fertilizer as described above includes the following steps: Step 1: Extract EPS: Culture Rhodotorula glutinis strain WTSX01 to obtain a bacterial suspension, and extract and purify the solid fungal extracellular polymer by dialysis. Step 2, lipid separation: Lipids were separated from the fungal extracellular polymers obtained in Step 1 using a co-solvent extraction method; Step 3, mixing the fungal extracellular polymer obtained in step 1, the lipid obtained in step 2 and the inorganic phosphate in proportion to prepare the compound fertilizer.

[0012] 6. The preparation method of claim 5, wherein the step 1 is specifically: 1.1, centrifuging the Rhodotorula glutinis suspension at 4℃ and 12000 g for 20 minutes to separate the supernatant; 1.2, centrifuging the supernatant obtained in step 1 at 4℃ and 17000 g for 20 minutes to remove cell debris; 1.3, adding three volumes of anhydrous ethanol to the supernatant obtained in step 2, standing at 4℃ for 48 hours, and collecting the precipitate to obtain the crude extract of fungal extracellular polymer; 1.4, placing the crude extract of fungal extracellular polymer in a dialysis bag with a molecular weight cut-off value of 3500 for dialysis to remove small molecular impurities, salts and residual ethanol; 1.5, freeze-drying the product after dialysis to obtain solid fungal extracellular polymer.

[0013] Further, the step 2 is specifically: the obtained solid fungal extracellular polymer is added to an organic solvent extractant composed of petroleum ether and acetone in a mass ratio of 20:1 while stirring, and then vortexed and centrifuged to collect the upper clear liquid to obtain the crude lipid phase; the extraction is repeated once, and the two obtained crude lipid phases are combined, and then the organic solvent is removed by nitrogen blowing to obtain the separated lipid.

[0014] The phosphorus release promoting type compound fertilizer has the advantages that:

[0015] Further, the compound fertilizer is applied together with the phosphorus-solubilizing fungus.

[0016] Further, the phosphorus-solubilizing fungus is Aspergillus niger WTXS02 Aspergillus niger , which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. M 2023240, and the preservation date is March 3, 2023. Aspergillus niger

[0017] Advantages: Compared with the prior art, the advantages of the present application are: ​(1) Synergistic effect: The present application first compounds EPS from a specific fungus (Rhodotorula WTSX01) with inorganic phosphate at a ratio of 1: (8-10), forming an organic-inorganic synergistic system. EPS, as an organic phosphorus source and carbon source easily utilized by microorganisms, synergizes with insoluble inorganic phosphorus sources, producing a "1+1>2" effect, making phosphorus supply more sustainable and efficient; (2) Overcoming competition: The compound fertilizer provides high-quality and exclusive "food package" (EPS) for exogenous phosphorus-solubilizing fungi (such as Aspergillus niger), enabling them to gain a competitive advantage in the fierce competition of indigenous microorganisms, ensuring successful colonization and vigorous growth. Experiments have shown that the biomass of Aspergillus niger increases by 4 times after applying the fertilizer; (3) Advanced extraction process: The low-temperature dialysis method used can gently and efficiently purify EPS, maximizing the retention of its macromolecular activity and structural integrity, while effectively removing small molecular impurities and ethanol residues, ensuring the high purity and high efficiency of the final product; (4) Environmentally friendly and economic benefits: The present application significantly improves the utilization rate of phosphorus fertilizer, providing a feasible path for achieving phosphorus fertilizer reduction and efficiency, which helps to reduce phosphorus fertilizer non-point source pollution. Field experiments show that the fertilizer can significantly improve the yield of crops such as rice, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a scanning electron micrograph of Aspergillus niger cultured for 5 days after adding the compound fertilizer of the present application in Example 1; Figure 2 is a scanning electron micrograph of Aspergillus niger cultured for 5 days without adding the compound fertilizer of the present application (no phosphorus treatment) in Example 1; Figure 3 is the experimental results of Examples 1-5; Figure 4 is the preparation process of the present application. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings, but the scope of protection of the present application is not limited to the described embodiments.

[0020] Example 1 Preparation of compound fertilizer (1) Extraction of EPS: The activated Rhodotorula glutinis WTSX01 strain was subjected to liquid fermentation to obtain a bacterial suspension. The bacterial suspension was centrifuged at 12000 g for 20 minutes at 4°C to collect the supernatant. The supernatant was further centrifuged at 17000 g for 20 minutes at 4°C to completely remove the cell debris. Three times the volume of ice ethanol was added to the supernatant, which was then placed at 4°C for 48 hours to collect the precipitate to obtain the EPS crude extract. The crude extract was loaded into a pretreated 3500 MWCO dialysis bag, which was dialyzed against deionized water for 72 hours to remove small molecular impurities, salts and residual ethanol. Finally, the dialysate was freeze-dried to obtain white flocculent solid EPS.

[0021] (2) Lipid separation: After the EPS was dissolved, petroleum ether: acetone mixed solvent was added, vortexed for 10 seconds, and centrifuged at 4000 g for 15 minutes to collect the upper organic phase. An equal volume of extractant was added to the lower aqueous phase, and the operation was repeated once. The two upper organic phases were combined and dried under nitrogen to obtain the lipid extract.

[0022] (3) Compounding: The EPS obtained in step 1, the lipid obtained in step 2 and the tricalcium phosphate (Ca3(PO4)2) powder were mixed uniformly at a mass ratio of 1 (EPS): 0.05 (lipid): 9 (Ca3(PO4)2), thereby obtaining the novel compound fertilizer of the present application.

[0023] Example 2 The difference between this example and Example 1 is that in the compounding step, the EPS obtained in step 1, the lipid obtained in step 2 and the tricalcium phosphate (Ca3(PO4)2) powder are mixed uniformly at a mass ratio of 1 (EPS): 0.05 (lipid): 8 (Ca3(PO4)2), thereby obtaining the novel compound fertilizer of Example 2.

[0024] Example 3 The difference between this example and Example 1 is that in the compounding step, the EPS obtained in step 1, the lipid obtained in step 2 and the tricalcium phosphate (Ca3(PO4)2) powder are mixed uniformly at a mass ratio of 1 (EPS): 0.05 (lipid): 8.5 (Ca3(PO4)2), thereby obtaining the novel compound fertilizer of Example 3.

[0025] Example 4 The difference between this example and Example 1 is that in the compounding step, the EPS obtained in step 1, the lipid obtained in step 2 and the tricalcium phosphate (Ca3(PO4)2) powder are mixed uniformly at a mass ratio of 1 (EPS): 0.05 (lipid): 9.5 (Ca3(PO4)2), thereby obtaining the novel compound fertilizer of Example 4.

[0026] Example 5 The difference between the present embodiment and embodiment 1 is that in the compounding step, the EPS obtained in step 1, the lipid obtained in step 2 and the tricalcium phosphate (Ca3(PO4)2) powder are mixed uniformly at a mass ratio of 1 (EPS): 0.05 (lipid): 10 (Ca3(PO4)2), thereby obtaining the novel compound fertilizer of embodiment 5.

[0027] The compound fertilizers prepared in embodiments 1-5 are applied to field experiments, and the test crops are Huiliangyou Yeheso seedlings, and the results are shown in Table 1. Figure 3

[0028] It can be seen that the use of the ratio provided in the present application is significantly high in improving the yield of rice.

[0029] Test Example 1: Effect of compound fertilizer on growth and morphology of Aspergillus niger The compound fertilizer prepared in embodiment 1 is mixed with Aspergillus niger spore suspension, 10 ml of Aspergillus niger spore suspension is mixed with 1 kg of compound fertilizer, the effective viable bacteria number in the Aspergillus niger spore suspension is ≥1.0*10 7 cfu·ml -1 ; and then inoculated into a culture medium lacking effective phosphorus (treatment group). A blank control without adding any phosphorus source and an inorganic phosphorus control with only an equal amount of tricalcium phosphate are used. The Aspergillus niger WTXS02 used in the present test example is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. M 2023240. Aspergillus niger

[0030] After 5 days of culture, it is found that in the blank control group (corresponding to Figure 2 ), most of the Aspergillus niger spores do not germinate or only form very short germ tubes, and the mycelium development is severely poor. In the inorganic phosphorus control group, the mycelium growth is improved, but it is still not vigorous. In the treatment group (corresponding to Figure 1 ), the Aspergillus niger mycelium is dense, thick and well-developed, and under scanning electron microscope, the mycelium surface is rough due to the action of EPS, which is beneficial to the contact with soil and fertilizer. The biomass determination shows that the fungal biomass of the treatment group is more than 4 times that of the blank control group, proving that the fertilizer of the present application can greatly promote the growth of phosphorus-solubilizing fungi.

[0031] Test Example 2: Field application test The implementation site is a rice field in Jiangning District, Nanjing City.

[0032] The test crops are Changyoujing No. 10, Huiliangyou Yeheso seedlings and Jiuyou Yeheso seedlings.

[0033] The test design is to set a treatment group applying the compound fertilizer of the present application and a control group applying conventional fertilization, randomly arrange in a block, and repeat 3 times. The application amount of the fertilizer of the present application is 50 kg / mu. ​​

[0034] The results are shown in Table 1. After applying the compound fertilizer of the present application, the yield of the three rice varieties is significantly higher than that of the control group, with an average yield increase of about 8%, which proves the effectiveness and application value of the fertilizer in actual agricultural production.

[0035] Table 1 As described above, although the present application has been indicated and expressed with reference to specific preferred embodiments, it should not be construed as limiting the present application itself. Various changes can be made in form and details without departing from the spirit and scope of the present application defined by the appended claims.

Claims

1. A phosphorus release enhancing compound fertilizer based on fungal extracellular polymers, characterized by: The effective components of the compound fertilizer are fungal extracellular polymers and inorganic phosphates, wherein the fungal extracellular polymers are derived from Rhodotorula glutinis (Rhodotorula glutinis). Rhodotorul The Rhodotorula mucilaginosa WTSX01 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo. M20221765.

2. The phosphorus release enhancing compound fertilizer based on fungal extracellular polymers according to claim 1, characterized in that: The mass ratio of the fungal extracellular polymer and the inorganic phosphate is 1: (8-10).

3. The phosphorus release enhancing compound fertilizer based on fungal exopolysaccharides according to claim 2, characterized in that: The inorganic phosphate is Ca3 (PO4) 2.

4. The phosphorus release enhancing compound fertilizer based on fungal exopolysaccharides according to claim 2, characterized in that: The fungal extracellular polymer comprises a lipid component isolated from Rhodotorula mucilaginosa WTSX01 strain by a co-solvent extraction method, which serves as a natural adhesive and provides nutrients for phosphorus-solubilizing fungi at the same time.

5. A method of preparing a composite fertilizer according to any one of claims 1 to 4, characterized in that The method comprises the following steps: Step 1, extracting EPS: culturing Rhodotorula mucilaginosa WTSX01 strain to obtain a bacterial suspension, and extracting and purifying the bacterial suspension by a dialysis method to obtain solid fungal extracellular polymer; Step 2, separating lipids: separating lipids from the fungal extracellular polymer obtained in step 1 by a co-solvent extraction method; Step 3, mixing the fungal extracellular polymer obtained in step 1, the lipids obtained in step 2 and inorganic phosphate according to a proportion to prepare the compound fertilizer.

6. A method of manufacture as claimed in claim 5, characterised in that: The step 1 specifically comprises: 1.1, centrifuging the Rhodotorula mucilaginosa bacterial suspension at 4℃ and 12000 g for 20 minutes to separate supernatant; 1.2, centrifuging the supernatant obtained in step 1 at 4℃ and 17000 g for 20 minutes again to remove cell debris; 1.3, adding three volumes of anhydrous ethanol to the supernatant obtained in step 2, and standing at 4℃ for 48 hours to collect precipitate to obtain crude fungal extracellular polymer extract; 1.4, dialyzing the crude fungal extracellular polymer extract in a dialysis bag with a molecular weight cut-off value of 3500 to remove small molecular impurities; 1.5, freeze-drying the product after dialysis to obtain solid fungal extracellular polymer.

7. The method of claim 5, wherein: The step 2 specifically comprises: preparing a 1g / L fungal extracellular polymer solution by adding and stirring the obtained solid fungal extracellular polymer, adding an organic solvent extractant composed of petroleum ether and acetone to the solution, the mass ratio of the petroleum ether and the acetone being 20:1, vortexing and mixing uniformly, centrifuging, collecting supernatant to obtain crude lipid phase, repeating the extraction once, combining the two obtained crude lipid phases, removing the organic solvent by nitrogen blowing to obtain separated lipids.

8. The phosphorus release promoting compound fertilizer according to any one of claims 1-4 is applied to promote soil phosphorus release efficiency and / or promote growth of phosphorus-solubilizing fungi.

9. Use according to claim 8, characterized in that: The compound fertilizer is applied together with the phosphorus-solubilizing fungi.

10. Use according to claim 9, characterized in that: The phosphorus-solubilizing fungus is Aspergillus niger WTXS02 ( Aspergillus niger ), which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. M 2023240. Aspergillus niger ), which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. M 2023240.

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