Phosphorus- and potassium-solubilizing aeromonas salmonicida cuc5 from tailings sand and fermentation product thereof, microbial organic fertilizer, and preparation methods therefor

Abstract

Phosphorus- and potassium-solubilizing Aeromonas salmonicida CuC5 from tailings sand and a fermentation product thereof, a microbial organic fertilizer, and preparation methods therefor, relating to the field of microbial organic fertilizers. The Aeromonas salmonicida CuC5 has a deposit number of CGMCC NO. 29265. A fermentation product corresponding to the Aeromonas salmonicida CuC5 and a preparation method therefor are also provided. The preparation of a microbial organic fertilizer from the fermentation product of the Aeromonas salmonicida CuC5 is further included. The microbial organic fertilizer can increase the plant-available nitrogen, phosphorus, and potassium content of copper tailings sand, greatly increasing the organic matter content of the tailings sand, and increasing the carbon sequestration capacity of the ecosystem within the tailings area. The present invention is of great significance for the comprehensive utilization of tailings sand and the development of functional microbial organic fertilizers.

Description

A phosphorus-solubilizing, potassium-solubilizing, salmon-killing Aeromonas cinerea CuC5 from tailings sand, its fermentation products, microbial organic fertilizer, and its preparation method. Technical Field

[0001] This application relates to the field of microbial organic fertilizer, and in particular to a tailings sand-solubilizing and potassium-solubilizing Aeromonas hydrophila CuC5 and its fermentation products, microbial organic fertilizer and its preparation method. Background Technology

[0002] Tailings originate from tailings, primarily the large amounts of waste generated by mining enterprises during the extraction of metallic, non-metallic, and coal mineral resources. This waste is produced after the useful minerals are separated from gangue and surrounding rocks through processes such as mineral processing. On one hand, tailings accumulation occupies significant amounts of land resources, impacting sustainable land use. On the other hand, because tailings contain high levels of phosphorus and potassium, their large-scale accumulation places a heavy burden on the ecological environment. Furthermore, there is a high demand for ecological restoration in mining areas. In the mine restoration process, improved tailings can be used as a planting substrate to replace topsoil, thus saving soil resources, reducing costs, and achieving resource utilization of tailings. However, tailings themselves are nutrient-poor and have poor physical and chemical properties, making them unsuitable for plant growth and development.

[0003] Phosphorus and potassium solubilizing microorganisms typically refer to those that can decompose insoluble phosphorus and potassium elements in the soil, converting them into forms that crops can absorb and utilize. These microorganisms include, but are not limited to, nitrogen-fixing bacteria, phosphorus-solubilizing bacteria, and potassium-solubilizing bacteria. Through their metabolic activities, they improve soil fertility and promote crop growth. Therefore, how to enable phosphorus and potassium solubilizing microorganisms to efficiently and environmentally promote the mineralization of phosphorus and potassium in tailings sand, promote plant growth in tailings sand, and ultimately make tailings sand resemble normal soil has become an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this application is to provide a tailings sand-solubilizing and potassium-solubilizing Aeromonas salmonidiopathiae CuC5, the fermentation product of Aeromonas salmonidiopathiae CuC5 and its preparation method, as well as a microbial organic fertilizer and its preparation method, to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] In a first aspect, this application provides a salmon-killing Aeromonas hydrophila CuC5:

[0007] The specimen, classified as Aeromonas salmonicida, was deposited on December 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC); No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC NO. 29265.

[0008] The nucleotide sequence of Aeromonas salmonidae CuC5 is shown in SEQ ID NO.1.

[0009] Secondly, this application provides a method for preparing Aeromonas salmonidae CuC5, comprising:

[0010] Tailings sand was inoculated into a first culture medium for first culture, and culturable microorganisms were obtained through enrichment and screening.

[0011] The culturable microorganism was subjected to a second culture to screen for specific phosphorus and potassium solubilizing strains, and the salmon-killing Aeromonas choleraesuis CuC5 was isolated and purified.

[0012] Preferably, the first culture medium is LB medium; the first culture includes initial bacterial screening.

[0013] Preferably, the second culture includes sequential screening of phosphate-solubilizing strains and potassium-solubilizing strains; or sequential screening of potassium-solubilizing strains and phosphate-solubilizing strains.

[0014] Preferably, a solid inorganic phosphorus culture medium is used to screen the phosphorus-solubilizing strains; a solid potassium-solubilizing culture medium is used to screen the potassium-solubilizing strains.

[0015] Thirdly, this application provides a method for preparing the fermentation product of Aeromonas salmonidae CuC5, wherein Aeromonas salmonidae CuC5 is inoculated into a high-density fermentation medium, and after a third culture, an activated strain is obtained, wherein the activated strain contains the fermentation product of Aeromonas salmonidae CuC5.

[0016] Preferably, the high-density fermentation medium comprises: 5.0~10.0 g / L yeast extract, 0.1~0.2 g / L histidine, 15.0~20.0 g / L glycerol, 4.00~5.12 g / L Na2HPO4, 1.0~3.0 g / L KH2PO4, 0.3~0.5 g / L NaCl, 0.3~0.5 g / L MgSO4·7H2O, 0.011~0.035 g / L CaCl2, 0.5~1.0 g / L NH4Cl, with the balance being water.

[0017] Preferably, the pH value of the high-density fermentation medium is 7.5.

[0018] Preferably, the third culture includes culture at 30-35℃ and 140-150 rpm on a shaker for 18-24 hours.

[0019] Preferably, the number of activated bacterial strains is not less than 8.6 × 10⁻⁶. 10 • CFU / mL.

[0020] Fourthly, this application provides a microbial organic fertilizer, the raw materials of which include fermentation products of Aeromonas salmonidae CuC5 and organic materials.

[0021] Preferably, the organic material includes any well-rotted livestock and poultry manure, agricultural straw, kitchen waste, and / or ordinary organic fertilizer.

[0022] Preferably, the organic matter content in the organic material is not less than 45% by mass.

[0023] Preferably, the total mass percentage of nitrogen, phosphorus, and potassium in the organic material is not less than 5%.

[0024] Preferably, the organic material has a moisture content of 35-50%.

[0025] Preferably, the pH value of the organic material is 6.5 to 7.5.

[0026] Fifthly, this application provides a method for preparing microbial organic fertilizer, which is obtained by solid-state fermentation of the fermentation product of Aeromonas salmonidae CuC5 and the organic material.

[0027] Preferably, the moisture content of the organic material is adjusted to 50-60% before solid-state fermentation.

[0028] Preferably, before solid-state fermentation, the fermentation product of Aeromonas salmonidae CuC5 is mixed at a ratio of 20-40L per ton of organic material.

[0029] Preferably, the solid-state fermentation temperature is 45~65℃.

[0030] Preferably, the moisture content is maintained at 45-50% during the solid-state fermentation process.

[0031] Preferably, the solid-state fermentation takes 7 to 10 days.

[0032] Preferably, the content of Aeromonas salmonidae CuC5 in the microbial organic fertilizer after solid-state fermentation is 6.9 × 10⁻⁶. 8 ~7.2×10 9 •CFU.

[0033] Compared with the prior art, the beneficial effects of this application include:

[0034] This application is the first to isolate Aeromonas salmonidae CuC5, which has the ability to solubilize phosphorus and potassium, from tailings sand. It is derived from tailings sand, is adapted to the tailings sand environment, and has the ability to produce extracellular secretions, fix nitrogen, and produce plant growth hormones, without posing any risk of human or animal diseases.

[0035] The Aeromonas salmonidae CuC5 fermentation product provided in this application uses a high-density fermentation medium, which has low raw material costs, high fermentation yield, and a short fermentation cycle.

[0036] The microbial organic fertilizer provided in this application uses a carrier obtained from the fermentation of ordinary organic waste. It requires no special organic materials, and the product is obtained directly after solid-state fermentation. The carrier is inexpensive and readily available. Furthermore, it is specifically designed for tailings sand remediation, promoting the soil formation of tailings sand, significantly increasing the available phosphorus and potassium content of tailings sand, and enhancing its plant biomass, bringing it closer to normal soil. It not only promotes the mineralization of tailings sand into soil but also significantly increases its organic carbon content, enhancing its carbon sequestration potential and providing an important method for the comprehensive utilization of tailings sand. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0038] Figure 1 shows the colony morphology of Aeromonas salmonidae CuC5 on LB agar plates in Example 1.

[0039] Figure 2 is a scanning electron microscope image of Aeromonas salmonidae CuC5 in Example 1;

[0040] Figure 3 is a schematic diagram of the phylogenetic tree constructed from the 16S rDNA gene fragment of CuC5 in Example 1;

[0041] Figure 4 is a scanning electron microscope image of the Aeromonas salmonidae CuC5 microbial organic fertilizer in Example 8;

[0042] Figure 5 is a schematic diagram of the microbial organic fertilizer product prepared in the example;

[0043] Figure 6 is a schematic diagram showing the appearance comparison of crested wheatgrass. Detailed Implementation

[0044] To better illustrate the technical solution provided in this application, the technical solution will be described in its entirety before the embodiments, as follows:

[0045] In the first aspect, this application provides a salmon-killing Aeromonas CuC5: classified as Aeromonas salmonicida, deposited on December 27, 2023 at the China General Microbiological Culture Collection Center (CGMCC); No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC NO. 29265.

[0046] The nucleotide sequence of Aeromonas salmonicida CuC5 is shown in SEQ ID NO.1, and is as follows:

[0047]

[0048] Secondly, this application provides a method for preparing Aeromonas salmonidae CuC5, comprising:

[0049] Tailings sand is inoculated into a first culture medium for initial culture, and culturable microorganisms are obtained through enrichment and screening. After enrichment culture, microorganisms in the culture system can be screened and expanded, at which point the culture system contains multiple strains that can be used for further screening.

[0050] The culturable microorganisms were subjected to a second culture to screen for specific phosphorus and potassium solubilizing strains, and the *Aeromonas salmonidae* CuC5 was isolated and purified. After the first culture, the culture system contained a large number of expanded microbial communities. After specific potassium and phosphorus solubilizing screening, strains with potassium and phosphorus solubilizing abilities could be specifically screened from the large number of microorganisms in the culture system after the first culture.

[0051] In a preferred embodiment, the first culture medium is LB medium; the first culture includes initial bacterial screening.

[0052] In a preferred embodiment, the second culture includes sequential screening of phosphate-solubilizing strains and potassium-solubilizing strains; or sequential screening of potassium-solubilizing strains and phosphate-solubilizing strains. During implementation, there are no special requirements regarding the screening order for phosphate and potassium solubilizing strains. Phosphate-solubilizing strains are screened first, and then the selected strains are further screened for potassium solubilizing to obtain the phosphate-solubilizing and potassium-solubilizing strains. Conversely, screening potassium-solubilizing strains first, and then further screening the selected strains for phosphate solubilizing, will also yield the phosphate-solubilizing and potassium-solubilizing strains.

[0053] In a preferred embodiment, the phosphorus-solubilizing strains are screened using a solid inorganic phosphorus culture medium; the potassium-solubilizing strains are screened using a solid potassium-solubilizing culture medium.

[0054] Thirdly, this application provides a method for preparing the fermentation product of Aeromonas salmonidae CuC5, wherein Aeromonas salmonidae CuC5 is inoculated into a high-density fermentation medium, and after a third culture, an activated strain is obtained, wherein the activated strain contains the fermentation product of Aeromonas salmonidae CuC5.

[0055] In an optional embodiment, the high-density fermentation medium comprises: 5.0-10.0 g / L yeast extract, 0.1-0.2 g / L histidine, 15.0-20.0 g / L glycerol, 4.00-5.12 g / L Na2HPO4, 1.0-3.0 g / L KH2PO4, 0.3-0.5 g / L NaCl, 0.3-0.5 g / L MgSO4·7H2O, 0.011-0.035 g / L CaCl2, 0.5-1.0 g / L NH4Cl, with the balance being water.

[0056] In one optional embodiment, the pH value of the high-density fermentation medium is 6.8 to 7.5. The pH value of the high-density fermentation medium can be 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or any value between 6.8 and 7.5.

[0057] In an optional embodiment, the third culture includes incubation at 30-35°C and 140-150 rpm on a shaker for 18-24 hours. The incubation temperature for the third culture can be 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, or any value between 30-35°C. The shaking speed can be 140 rpm, 141 rpm, 142 rpm, 143 rpm, 144 rpm, 145 rpm, 146 rpm, 147 rpm, 148 rpm, 149 rpm, or 150 rpm, or any value between 140-150 rpm. The incubation time on the shaker can be 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or any value between 18-24 hours.

[0058] In an optional implementation, the number of activated strains is not less than 8.6 × 10⁻⁶. 10 • CFU / mL.

[0059] Fourthly, this application provides a microbial organic fertilizer, the raw materials of which include fermentation products of Aeromonas salmonidae CuC5 and organic materials.

[0060] In one alternative implementation, the organic material includes any well-rotted livestock and poultry manure, agricultural straw, kitchen waste, and / or ordinary organic fertilizer.

[0061] In an optional embodiment, the organic matter in the organic material has a mass percentage content of not less than 45%.

[0062] In an optional implementation, the total mass percentage of nitrogen, phosphorus, and potassium in the organic material is not less than 5%.

[0063] In one optional embodiment, the moisture content of the organic material is 35-50%. The moisture content of the organic material can be 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any value between 35% and 50%.

[0064] In one optional embodiment, the pH value of the organic material is 6.5 to 7.5. The pH value of the organic material can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or any value between 6.5 and 7.5.

[0065] Fifthly, this application provides a method for preparing microbial organic fertilizer, which is obtained by solid-state fermentation of the fermentation product of Aeromonas salmonidae CuC5 and the organic material.

[0066] In an optional implementation, the moisture content of the organic material is adjusted to 50-60% before solid-state fermentation. The moisture content of the organic material before fermentation can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or any value between 50-60%.

[0067] In an optional embodiment, the Aeromonas salmonidae CuC5 fermentation product is mixed with the organic material at a ratio of 20-40 L per ton of organic material before the solid-state fermentation. The ratio between the Aeromonas salmonidae CuC5 fermentation product and the organic material can be 20 L per ton of organic material, 25 L per ton of organic material, 30 L per ton of organic material, 35 L per ton of organic material, 40 L per ton of organic material, or any value between 20-40 L per ton of organic material.

[0068] In one optional embodiment, the solid-state fermentation temperature is 45~65℃. The fermentation temperature of the ancient tower can be 45℃, 50℃, 55℃, 60℃, 65℃, or any value between 45~65℃.

[0069] In one optional implementation, the moisture content is maintained at 45-50% during the solid-state fermentation process. The moisture content during solid-state fermentation can be 45%, 46%, 47%, 48%, 49%, 50%, or any value between 45-50%.

[0070] In one optional implementation, the solid-state fermentation period is 7 to 10 days. The solid-state fermentation period can be 7 days, 8 days, 9 days, 10 days, or any value between 7 and 10 days.

[0071] Preferably, the content of Aeromonas salmonidae CuC5 in the microbial organic fertilizer after solid-state fermentation can be 6.9 × 10⁻⁶. 8 ~7.2×10 9 Any value between CFU.

[0072] It should be noted that the composition of the culture system used in each embodiment of this application is as follows:

[0073] Tailings sand: The tailings sand used in this application was collected from a high-altitude copper mining area in Tibet. It has a pH of 7.38, total nitrogen of 0.33 g / kg, total phosphorus of 0.91 g / kg, total potassium of 5.33 g / kg, available nitrogen of 14.8 mg / kg, available phosphorus of 3.12 mg / kg, available potassium of 18.23 mg / kg, and organic carbon of 3.25 g / kg.

[0074] LB medium: LB liquid medium is prepared by mixing LB Broth medium with distilled water. When preparing solid medium, 2% (by weight / volume percentage) of agar powder is added. All LB media are sterilized in a vertical pressure steam sterilizer at 0.11-0.15 MPa and 121°C for 30 min.

[0075] Inorganic phosphorus medium: 10g glucose, 0.5g diammonium sulfate, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 10g calcium phosphate, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 18g agar. Adjust pH to 7.5 and bring volume to 1L with pure water. All media were sterilized in a vertical pressure steam autoclave at 0.11-0.15 MPa and 121℃ for 30 min.

[0076] Potassium-solubilizing medium: disodium hydrogen phosphate 2g, ferric chloride 0.005g, calcium carbonate 0.1g, magnesium sulfate 0.5g, potassium feldspar 1g, sucrose 5g, agar 18g. Adjust pH to 7.5, and bring volume to 1L with pure water. All media were sterilized in a vertical pressure steam autoclave at 0.11-0.15MPa and 121℃ for 30min.

[0077] Ashbee's nitrogen-free medium: mannitol 10g, MgSO4·7H2O 0.2g, CaSO4·2H2O 0.1g, KH2PO4 0.2g, CaCO3 5g, agar 20g, pH 7.2-7.4; bring to a final volume of 1L.

[0078] CAS medium: 2 mL of 1 mmol / L MgSO4, 100 μL of 1 mmol / L CaCl2, 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, 1.8 g of agar, and 5 mL each of phosphate buffer and CAS staining solution were slowly added at about 60°C.

[0079] Example 1

[0080] This embodiment provides a salmon-killing Aeromonas hydrophila CuC5, which is prepared by the following method:

[0081] 1.1 Initial bacterial screening

[0082] 10g of collected tailings sand samples were inoculated into 100mL of sterile LB medium; cultured at 30℃ and 150rpm on a shaker; the LB medium was replaced with fresh medium every 48 hours; after continuous culture for 7 days, enrichment and screening were performed to obtain culturable bacteria from the tailings sand. 117 culturable microorganisms from the tailings sand were initially screened out.

[0083] 1.2 Screening for specific phosphorus- and potassium-solubilizing bacteria

[0084] The selected culturable microorganisms were inoculated onto solid inorganic phosphate medium using an inoculation loop and cultured at 30°C for 5 days. The growth status of the strains and the size of the clear zones produced on the medium were observed to determine their phosphate-solubilizing ability. Similarly, the selected culturable microorganisms were inoculated onto solid potassium-solubilizing medium using an inoculation loop and cultured at 30°C for 5 days. The growth status of the strains and the size of the hydrolysis zones were observed to determine their potassium-solubilizing ability.

[0085] 1.3 Secondary screening of phosphorus- and potassium-solubilizing bacteria

[0086] 20g of the collected tailings sand sample was added to 100mL of sterile LB medium and inoculated with the selected phosphorus and potassium solubilizing microorganisms. The sample was then cultured at 30℃ and 150 rpm in a shaker. After seven days of culture, the available phosphorus and available potassium in the culture system were measured for re-screening.

[0087] 1.4 Separation and Purification

[0088] Strains were cultured and subcultured on LB solid plate medium to screen strains, and single colonies capable of isolation and purification were obtained.

[0089] 1.5 Morphological observation

[0090] A strain exhibiting regular, round colonies with neat edges and smooth surfaces, an opaque pale yellow color, relatively adherent and moist cells, easy to pick, and turning pale gray after prolonged incubation on LB agar plates was selected and named CuC5. Figure 1 shows the colony morphology of CuC5 on LB agar plates, and Figure 2 is a scanning electron microscope image of the strain. Under the electron microscope, it appears rod-shaped with what appears to be flagella at the tail, and the average cell length is 1.1 μm. Gram staining confirmed that CuC5 is Gram-negative.

[0091] 1.6 Molecular biological identification

[0092] Primers used for amplification and sequencing of the 16S rDNA gene fragment of CuC5 were 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACGACTT-3). The resulting sequence fragment (1380 bp in length, as shown in Sequence 1) was submitted to the GenBank database (accession number: PQ157593). Blastn sequence alignment with the NCBI database showed that CuC5 exhibited high similarity to several strains of *Aeromonas salmonicida*. The highest homology (99.97%) was found between CuC5 and *Aeromonas salmonicida* strain NCIMB 1102 (NR 118945). Furthermore, a phylogenetic tree of the 16S rDNA gene fragment of CuC5 was constructed, as shown in Figure 3. Based on the phylogenetic tree and alignment results, CuC5 was preliminarily identified as *Aeromonas salmonicida*.

[0093] Example 2

[0094] This embodiment provides a salmon-killing Aeromonas hydrophila CuC5, the preparation process of which is as follows:

[0095] 2.1 Initial bacterial screening

[0096] 10g of collected tailings sand samples were inoculated into 100mL of sterile LB medium; cultured at 25℃ and 140rpm on a shaker; the LB medium was replaced with fresh medium every 24 hours; after continuous culture for 5 days, enrichment and screening were performed to obtain culturable bacteria from the tailings sand. A preliminary screening identified 85 culturable microorganisms from the tailings sand.

[0097] 2.2 Screening for specific phosphorus- and potassium-solubilizing bacteria

[0098] The selected culturable microorganisms were inoculated onto solid inorganic phosphate medium using an inoculation loop and cultured at 25°C for 3 days. The growth status of the strains and the size of the clear zones produced on the medium were observed to determine their phosphate-solubilizing ability. Similarly, the selected culturable microorganisms were inoculated onto solid potassium-solubilizing medium using an inoculation loop and cultured at 25°C for 3 days. The growth status of the strains and the size of the hydrolysis zones were observed to determine their potassium-solubilizing ability.

[0099] 2.3 Secondary screening of phosphorus- and potassium-solubilizing bacteria

[0100] 10g of the collected tailings sand sample was added to 100mL of sterile LB medium and inoculated with the selected phosphorus and potassium solubilizing microorganisms. The sample was then cultured in a shaker at 25℃ and 140rpm for seven days. The available phosphorus and available potassium in the culture system were then measured for re-screening.

[0101] 2.4 Separation and Purification

[0102] Strains were cultured and subcultured on LB solid plate medium to screen strains, and single colonies capable of isolation and purification were obtained.

[0103] 2.5 Morphological observation

[0104] A strain that exhibits regular, round colonies with neat edges and smooth surfaces, an opaque pale yellow color, relatively sticky and moist cells, easy to pick up, and whose colonies tend to turn pale gray after a longer period of incubation on LB agar plates was selected and named CuC5. Gram staining identification showed that CuC5 was Gram-negative.

[0105] Example 3

[0106] This embodiment provides a salmon-killing Aeromonas hydrophila CuC5, the preparation process of which is as follows:

[0107] 3.1 Initial bacterial screening

[0108] 10g of collected tailings sand samples were inoculated into 100mL of sterile LB medium; cultured at 28℃ and 145rpm on a shaker; the LB medium was replaced with fresh medium every 36 hours; after continuous culture for 6 days, enrichment and screening were performed to obtain culturable bacteria from the tailings sand. A preliminary screening identified 90 culturable microorganisms from the tailings sand.

[0109] 3.2 Screening for specific phosphorus- and potassium-solubilizing bacteria

[0110] The selected culturable microorganisms were inoculated onto solid inorganic phosphate medium using an inoculation loop and cultured at 28°C for 4 days. The growth status of the strains and the size of the clear zones produced on the medium were observed to determine their phosphate-solubilizing ability. Similarly, the selected culturable microorganisms were inoculated onto solid potassium-solubilizing medium using an inoculation loop and cultured at 28°C for 4 days. The growth status of the strains and the size of the hydrolysis zones were observed to determine their potassium-solubilizing ability.

[0111] 3.3 Secondary screening of phosphorus- and potassium-solubilizing bacteria

[0112] 15g of the collected tailings sand sample was added to 100mL of sterile LB medium and inoculated with the selected phosphorus and potassium solubilizing microorganisms. The sample was then cultured in a shaker at 28℃ and 145rpm for seven days. The available phosphorus and available potassium in the culture system were then measured for rescreening.

[0113] 3.4 Separation and Purification

[0114] Strains were cultured and subcultured on LB solid plate medium to screen strains, and single colonies capable of isolation and purification were obtained.

[0115] 3.5 Morphological observation

[0116] A strain that exhibits regular, round colonies with neat edges and smooth surfaces, an opaque pale yellow color, relatively sticky and moist cells, easy to pick up, and whose colonies tend to turn pale gray after a longer period of incubation on LB agar plates was selected and named CuC5. Gram staining identification showed that CuC5 was Gram-negative.

[0117] Example 4

[0118] This embodiment describes the determination of other potential functions of Aeromonas salmonidae CuC5 based on Example 1, using the following specific methods:

[0119] Detection of nitrogen fixation activity of the strain: Aeromonas salmonicidae CuC5 was inoculated onto Assumption nitrogen-free medium, sealed, inverted, and cultured at 28°C for 5 days. The strain grew and showed nitrogen fixation activity.

[0120] Siderophore production activity detection of the strain: Aeromonas salmonidae CuC5 was inoculated on a CAS test plate and incubated at 28℃ for 48h. The growth status of the strain and whether a yellow halo appeared around the colony were observed. This strain has the ability to produce siderophores.

[0121] IAA production activity assay: The IAA production activity of the strain was detected using a spectrophotometer by reacting Salkowski colorimetric solution with bacterial culture shaken to the logarithmic growth phase. This strain possesses IAA production activity.

[0122] Detection of ACC dehydrogenase production by the strain: Single colonies of Aeromonas salmonicidae CuC5 were picked and enriched in beef extract peptone medium until the logarithmic growth phase. The bacterial cells were collected by centrifugation and treated twice with 0.1 mol / L Tris-HCl (pH 7.6), centrifuged at 10000 rpm for 5 min, and the supernatant was discarded. The bacterial cells were resuspended in 600 μL of 0.1 mol / L Tris-HCl (pH 8.5). 30 μL of toluene was added, and the mixture was stirred for 30 s. 200 μL of the solution was transferred to a 1.5 mL centrifuge tube, and 200 μL of 0.5 mol / L ACC was added, stirred for 5 s, and incubated at 30 °C for 15 min. 0.56 mol / L HCl was added, stirred, and centrifuged at 10000 rpm for 5 min at room temperature. 1 mL of the supernatant was taken, and 800 μL of 0.56 mol / L HCl was added, stirred, and 300 mL of the supernatant was added. Mix 2,4-dinitrophenylhydrazine (2% 2,4-dinitrophenylhydrazine added to 2 mol / L HCl) and incubate at 30℃ for 30 min; add 2 mol / L NaOH; measure the OD450 value. This strain has the ability to produce ACC dehydrogenase.

[0123] Hemolytic reaction test of the strain: Aeromonas salmonidae CuC5 was inoculated onto sheep blood agar plates and incubated upside down at 37°C for 24 hours. No hemolytic zone was observed. The hemolytic reaction of this strain was negative, indicating no risk of pathogenicity in humans or animals.

[0124] The results showed that Aeromonas salmonidae CuC5 possesses the ability to fix nitrogen, produce siderophores, produce IAA, and produce ACC dehydrogenase. It is a phosphorus-solubilizing and methyl-solubilizing bacterium that also promotes plant growth and poses no risk of human or animal disease.

[0125] Example 5

[0126] This embodiment provides a fermentation product prepared from Aeromonas salmonidae CuC5 obtained in Example 1, and the preparation method is as follows:

[0127] The strain Aeromonas salmonidae CuC5 was inoculated into a high-density fermentation medium for fermentation.

[0128] The solutes used in the high-density fermentation medium and their concentrations in the high-density fermentation medium were as follows: 10.0 g / L yeast extract, 0.2 g / L histidine, 20.0 g / L glycerol, 5.12 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L sodium chloride, 0.5 g / L MgSO4·7H2O, 0.035 g / L CaCl2, and 1.0 g / L NH4Cl.

[0129] The specific steps for preparing the fermentation product are as follows:

[0130] (1) Take the isolated, purified and identified Aeromonas salmonidae CuC5 that has been frozen in the refrigerator and inoculate it into an Erlenmeyer flask containing 100 ml of high-temperature sterilized LB liquid medium;

[0131] (2) The inoculated conical flask was placed in a constant temperature shaking incubator and cultured at 150 rpm and 30 ℃ for 24 hours to obtain activated Aeromonas salmonidae CuC5 seed liquid;

[0132] (3) The seed liquid of Aeromonas salmonicidae CuC5 was inoculated into a fermenter containing high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.5 using 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, temperature was 30℃, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation broth to 10, and appropriate feeding was added during the fermentation process. The dissolved oxygen in the fermenter was controlled at 6 mg / L using an aerator. During the fermentation period, samples were taken every 6 hours to detect the OD value and viable cell count (CFU / ml) of the fermentation broth. The viable cell count was determined by the gradient dilution plate count method, and the viable cell count was counted by calculating the colony forming unit (CFU). After 48 hours, the growth of strain CuC5 entered the stationary phase, with a maximum CFU of 8.6 × 10⁻⁶. 10 / mL.

[0133] Example 6

[0134] This embodiment provides a fermentation product prepared from Aeromonas salmonidae CuC5 obtained in Example 2, and the preparation method is as follows:

[0135] The strain Aeromonas salmonidae CuC5 was inoculated into a high-density fermentation medium for fermentation.

[0136] The solutes used in the high-density fermentation medium and their concentrations in the high-density fermentation medium were as follows: 5.0 g / L yeast extract, 0.1 g / L histidine, 15.0 g / L glycerol, 4.0 g / L Na2HPO4, 1.0 g / L KH2PO4, 0.3 g / L sodium chloride, 0.3 g / L MgSO4·7H2O, 0.011 g / L CaCl2, and 0.5 g / L NH4Cl.

[0137] The specific steps for preparing the fermentation product are as follows:

[0138] (1) Take the isolated, purified and identified Aeromonas salmonidae CuC5 that has been frozen in the refrigerator and inoculate it into an Erlenmeyer flask containing 100 ml of high-temperature sterilized LB liquid medium;

[0139] (2) The inoculated conical flask was placed in a constant temperature shaking incubator and cultured at 140 rpm and 35 ℃ for 18 hours to obtain activated Aeromonas salmonidae CuC5 seed liquid;

[0140] (3) The seed liquid of Aeromonas salmonicidae CuC5 was inoculated into a fermenter containing high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.4 using 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, temperature was 35℃, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation broth to 10, and appropriate feeding was added during the fermentation process. The dissolved oxygen in the fermenter was controlled at 5 mg / L using an aerator. During the fermentation period, samples were taken every 5 hours to detect the OD value and viable cell count (CFU / ml) of the fermentation broth. The viable cell count was determined by the gradient dilution plate count method, and the viable cell count was counted by calculating the colony forming unit (CFU). After 52 hours, the growth of strain CuC5 entered the stationary phase, with a maximum CFU of 7.6 × 10⁻⁶. 10 / mL.

[0141] Example 7

[0142] This embodiment provides a fermentation product prepared from Aeromonas salmonidae CuC5 obtained in Example 3, and the preparation method is as follows:

[0143] The strain Aeromonas salmonidae CuC5 was inoculated into a high-density fermentation medium for fermentation.

[0144] The solutes used in the high-density fermentation medium and their concentrations in the high-density fermentation medium were as follows: 10.0 g / L yeast extract, 0.15 g / L histidine, 17.0 g / L glycerol, 4.56 g / L Na2HPO4, 1.5 g / L KH2PO4, 0.4 g / L sodium chloride, 0.4 g / L MgSO4·7H2O, 0.025 g / L CaCl2, and 0.7 g / L NH4Cl.

[0145] The specific steps for preparing the fermentation product are as follows:

[0146] (1) Take the isolated, purified and identified Aeromonas salmonidae CuC5 that has been frozen in the refrigerator and inoculate it into an Erlenmeyer flask containing 100 ml of high-temperature sterilized LB liquid medium;

[0147] (2) The inoculated conical flask was placed in a constant temperature shaking incubator and cultured at 140 rpm and 35 ℃ for 18 hours to obtain activated Aeromonas salmonidae CuC5 seed liquid;

[0148] (3) The seed liquid of Aeromonas salmonicidae CuC5 was inoculated into a fermenter containing high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.2 using 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, temperature was 32℃, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation broth to 10, and appropriate feeding was added during the fermentation process. The dissolved oxygen in the fermenter was controlled at 4.5 mg / L using an aerator. During fermentation, samples were taken every 4 hours to test the OD value and viable cell count (CFU / ml) of the fermentation broth. The viable cell count was determined by the gradient dilution plate count method, and the viable cell count was counted by calculating the colony forming unit (CFU). After 36 hours, the growth of strain CuC5 entered the stationary phase, with a maximum CFU of 7.9 × 10⁻⁶. 10 / mL.

[0149] Example 8

[0150] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonidae CuC5 obtained in Example 5. The preparation method is as follows:

[0151] The organic materials used to prepare microbial organic fertilizer were well-rotted cow manure and barley and rapeseed straw compost from a certain region. The compost contained 46.2% organic matter, 1.2% total nitrogen, 2.7% total phosphorus, 1.3% total potassium, 38.1% moisture, and a pH of 7.2. Before secondary fermentation, the moisture content of the organic fertilizer was adjusted to 50%. It was thoroughly mixed to maintain a loose and aerated texture.

[0152] The fermentation product of Aeromonas salmonidae CuC5 strain prepared in Example 5 was sprayed onto the organic material at an inoculation rate of 40 L per ton of organic material, and mixed evenly to form a mixture. The secondary solid-state fermentation cycle was 10 days, with turning over on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. Testing showed that the viable bacteria count in the microbial organic fertilizer was 7.2 × 10⁻⁶. 9 The scanning electron microscope image of CFU (Aeromonas salmonidae CuC5) microbial organic fertilizer is shown in Figure 4, and the final product is shown in Figure 5.

[0153] Example 9

[0154] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonidae CuC5 obtained in Example 6. The preparation method is as follows:

[0155] The organic materials used to prepare microbial organic fertilizer were well-rotted cow manure and barley and rapeseed straw compost from a certain region. The compost contained 45.0% organic matter, 1.1% total nitrogen, 2.2% total phosphorus, 1.7% total potassium, 45.2% moisture, and a pH of 7.5. Before secondary fermentation, the moisture content of the organic fertilizer was adjusted to 5%. It was thoroughly mixed to maintain a loose and aerated texture.

[0156] The fermentation product of Aeromonas salmonidae CuC5 strain prepared in Example 6 was sprayed onto the organic material at an inoculation rate of 30 L per ton of organic material, and mixed evenly to form a mixture. The secondary solid-state fermentation cycle was 7 days, with turning over on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. Testing showed that the viable bacteria count in the microbial organic fertilizer was 6.9 × 10⁻⁶. 9 •CFU.

[0157] Example 10

[0158] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonidae CuC5 obtained in Example 7. The preparation method is as follows:

[0159] The organic materials used to prepare microbial organic fertilizer were well-rotted cow manure and barley and rapeseed straw compost from a certain region. The compost contained 45.5% organic matter, 1.3% total nitrogen, 2.2% total phosphorus, 1.5% total potassium, 42.3% moisture, and a pH of 6.9. Before secondary fermentation, the moisture content of the organic fertilizer was adjusted to 60%. It was thoroughly mixed to maintain a loose and aerated texture.

[0160] The fermentation product of Aeromonas salmonidae CuC5 strain prepared in Example 7 was sprayed onto the organic material at an inoculation rate of 35 L per ton of organic material, and mixed evenly to form a mixture. The secondary solid-state fermentation cycle was 9 days, with turning over on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. Testing showed that the viable bacteria count in the microbial organic fertilizer was 7.0 × 10⁻⁶. 9 •CFU.

[0161] Example 11

[0162] This example involves a pot experiment conducted from late June to early August 2024 to evaluate the effect of microbial organic fertilizer on tailings sand improvement.

[0163] The potting soil used in the pot experiment was pure tailings sand, with a dosage of 3 kg; the fertilizer was the microbial organic fertilizer prepared in Example 8, with an application rate of 60 g of the microbial organic fertilizer from Example 8; after mixing the potting soil and microbial organic fertilizer, the mixture was placed in flowerpots, and seeds of *Elymus sibiricum* were sown. Water was applied at 200 mL every two days, and three parallel experiments were set up. After the plants matured, plant and soil samples were collected, and plant biomass and soil physicochemical properties were measured.

[0164] Comparative Example 1

[0165] This comparative study involved a pot experiment conducted from late June to early August 2024 to evaluate the effect of microbial organic fertilizer on the improvement of tailings sand.

[0166] The potting soil used in the experiment was pure tailings sand, with a dosage of 3 kg. The fertilizer was organic fertilizer made from well-rotted cow manure and barley and rapeseed straw compost from a certain region, with an organic matter content of 46.2%, total nitrogen content of 1.2%, total phosphorus content of 2.7%, total potassium content of 1.3%, water content of 38.1%, and pH of 7.2, with an application rate of 60 g. After the potting soil and organic fertilizer were mixed evenly, the mixture was placed in flowerpots, and seeds of *Leymus chinensis* were sown. Water was applied at 200 mL every two days, and three parallel experiments were set up. After the plants matured, plant and soil samples were collected, and plant biomass and soil physicochemical indicators were measured.

[0167] Comparative Example 2

[0168] This comparative study involved a pot experiment conducted from late June to early August 2024 to evaluate the effect of microbial organic fertilizer on the improvement of tailings sand.

[0169] The potting soil used in the pot experiment was pure tailings sand, with a dosage of 3 kg, and no fertilizer was applied. The soil was filled into flowerpots, and seeds of *Elymus sibiricum* were sown. Water was applied with 200 mL every two days, and three parallel experiments were set up. After the plants matured, plant and soil samples were collected, and plant biomass and soil physicochemical properties were measured.

[0170] Comparative Example 3

[0171] This comparative study involved a pot experiment conducted from late June to early August 2024 to evaluate the effect of microbial organic fertilizer on the improvement of tailings sand.

[0172] The potting soil used in the pot experiment was pure tailings sand, with a dosage of 3 kg. The fertilizer was commercial organic fertilizer purchased from Genlido Biotechnology Co., Ltd., specifically fermented livestock and poultry manure organic fertilizer (product number: 10047929946683), with an application rate of 60 g. After mixing the potting soil and microbial organic fertilizer, the mixture was placed in flowerpots, and seeds of *Leymus chinensis* were sown. Water was applied with 200 mL of water every two days, and three parallel experiments were set up. After the plants matured, plant and soil samples were collected to determine plant biomass and soil physicochemical properties.

[0173] Comparative Example 4

[0174] This comparative study involved a pot experiment conducted from late June to early August 2024 to evaluate the effect of microbial organic fertilizer on the improvement of tailings sand.

[0175] The potting soil used in the experiment was normal soil, 3 kg in volume, without fertilizer. The soil was placed in flowerpots, and seeds of *Elymus sibiricum* were sown. Water was applied at 200 mL every two days, and three parallel experiments were set up. After the plants matured, plant and soil samples were collected to determine plant biomass and soil physicochemical properties.

[0176] The appearance of *Leymus chinensis* in Example 11 and Comparative Examples 1-4 is shown in Figure 6. The microbial organic fertilizer disclosed in this application can effectively increase the biomass of tailings plants, approaching that of normal soil. The biomass indicators of *Leymus chinensis* are shown in Table 1. The results show that the microbial organic fertilizer disclosed in this application can effectively increase the biomass of tailings plants, with a significantly stronger effect than commercial microbial organic fertilizer, approaching that of normal soil.

[0177] Table 1 Plant biomass indicators for different treatments

[0178]

[0179] The results of soil physicochemical property testing are shown in Table 2. The results indicate that the microbial organic fertilizer disclosed in this application can significantly increase the available phosphorus and potassium content of tailings sand, effectively promoting tailings sand mineralization and accelerating the soilification process. Furthermore, the microbial organic fertilizer disclosed in this application can increase the organic carbon content of tailings sand, which is of great significance for improving the carbon sequestration potential of tailings.

[0180] Table 2 Soil physicochemical properties for different treatments

[0181]

[0182] The above results indicate that, after the Aeromonas salmonidae strain CuC5 bio-organic fertilizer is introduced into tailings sand, compared with commercial microbial organic fertilizer and single organic fertilizer carriers, it effectively promotes the soilification of tailings sand, significantly increases the available phosphorus and potassium content of tailings sand, and promotes the yield of vegetation in tailings sand. The microbial organic fertilizer disclosed in this application can be used for the comprehensive utilization and reclamation of tailings, and is of great significance in solving the problem of tailings stock and improving the efficiency of tailings comprehensive utilization.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0184] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

A CuC5 for killing Aeromonas salmonicida, characterized by, The specimen, classified as Aeromonas salmonicida, was deposited on December 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC); No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC NO. 29265. The Aeromonas salmonicida CuC5 according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1. A preparation method of a CuC5 for killing Aeromonas salmonicida, characterized by comprising the following steps: include: Tailings sand was inoculated into a first culture medium for first culture, and culturable microorganisms were obtained by enrichment and screening. The culturable microorganism was subjected to a second culture to screen for specific phosphorus and potassium solubilizing strains, and the salmon-killing Aeromonas choleraesuis CuC5 was isolated and purified. The method for preparing Aeromonas salmonicidae CuC5 according to claim 3 is characterized in that, The first culture medium is LB medium; the first culture includes initial bacterial screening; The second culture includes the sequential screening of phosphate-solubilizing strains and potassium-solubilizing strains; or, The screening of potassium-solubilizing strains and then the screening of phosphorus-solubilizing strains were carried out in sequence. The method for preparing Aeromonas salmonicidae CuC5 according to any one of claims 2 to 4 is characterized in that, The phosphate-solubilizing strains were screened using a solid inorganic phosphate culture medium. The potassium-solubilizing strains were screened using solid potassium-solubilizing medium. A method for preparing fermentation products of Aeromonas salmonidae CuC5, characterized in that, Aeromonas salmonicidae CuC5 of claim 1 was inoculated into a high-density fermentation medium and subjected to a third culture to obtain an activated strain, wherein the activated strain contains the fermentation product of Aeromonas salmonicidae CuC5. The method for preparing the fermentation product of Aeromonas salmonidae CuC5 according to claim 6 is characterized in that, One or more of the following conditions a~d are satisfied: a. The high-density fermentation medium comprises: 5.0~10.0 g / L yeast extract, 0.1~0.2 g / L histidine, 15.0~20.0 g / L glycerol, 4.00~5.12 g / L Na2HPO4, 1.0~3.0 g / L KH2PO4, 0.3~0.5 g / L NaCl, 0.3~0.5 g / L MgSO4·7H2O, 0.011~0.035 g / L CaCl2, 0.5~1.0 g / L NH4Cl, with the balance being water; b. The pH value of the high-density fermentation medium is 6.8~7.5; c. The third culture includes culture at 30-35℃ and 140-150rpm on a shaker for 18-24 hours; d. the number of the activated strain is not less than 8.6 x 10 10 CFU / mL. A fermentation product of Aeromonas salmonidae CuC5, characterized in that, The product was prepared according to the method for preparing Aeromonas salmonidae CuC5 fermentation product according to any one of claims 6 to 7. A microbial organic fertilizer, characterized in that, Its raw materials include the fermentation product of Aeromonas salmonidae CuC5 as described in claim 8 and organic materials. The microbial organic fertilizer according to claim 9 is characterized in that, The organic material satisfies one or more of the following conditions e~i: e. The organic materials include any well-rotted livestock and poultry manure, agricultural straw, kitchen waste and / or ordinary organic fertilizer; f. The organic matter content in the organic material is not less than 45% by mass; g. The total mass percentage of nitrogen, phosphorus, and potassium in the organic material is not less than 5%; h. The moisture content of the organic material is 35-50%; i. The pH value of the organic material is 6.5~7.

5. A method for preparing microbial organic fertilizer, characterized in that: The product of Aeromonas salmonidae CuC5 fermentation is obtained by solid-state fermentation of the organic material, wherein the solid-state fermentation satisfies one or more of the following conditions: j. Before solid-state fermentation, the moisture content of the organic material is adjusted to 50-60%; k. Before the solid-state fermentation, the fermentation product of Aeromonas salmonidae CuC5 is mixed at a ratio of 20-40L per ton of the organic material; l. The temperature for solid-state fermentation is 45~65℃; m. During the solid-state fermentation process, the moisture content is maintained at 45-50%; n. The solid-state fermentation period is 7-10 days; o. The content of the P. piscicida CuC5 in the microbial organic fertilizer after the solid-state fermentation is 6.9 x 10 8 7.2 x 10 9 CFU.

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