Tailing sand phosphorus-dissolving and potassium-dissolving aeromonas salmonicida CuC5, fermentation product thereof, microbial organic fertilizer and preparation method of microbial organic fertilizer

Abstract

The invention provides tailing sand phosphate and potassium solubilizing aeromonas salmonicida CuC5, a fermentation product thereof, a microbial organic fertilizer and a preparation method of the microbial organic fertilizer, and relates to the field of microbial organic fertilizers. The preservation number of the aeromonas salmonicida CuC5 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO.29265; the invention further discloses a fermentation product corresponding to the aeromonas salmonicida CuC5 and a preparation method. The method also comprises the step of preparing a microbial organic fertilizer from the aeromonas salmonicida CuC5 fermentation product. The microbial organic fertilizer can increase the effective nitrogen, phosphorus and potassium content of copper tailing sand plants, greatly increase the organic matter content of tailing sand and increase the carbon sequestration amount of an ecological system in a tailing area. The method has important significance on comprehensive utilization of the tailing sand and development of functional microbial organic fertilizers.

Description

Technical Field

[0001] The present application relates to the field of microbial organic fertilizers, and in particular to a tailings sand phosphorus- and potassium-dissolving salmonicidal Aeromonas CuC5 and its fermentation products, microbial organic fertilizers and a preparation method thereof. Background Art

[0002] Tailings sand comes from tailings, which are mainly a large amount of waste slag produced by mining enterprises after useful minerals are separated from gangue, surrounding rocks, etc. through ore dressing and other processes during the mining of metal, non-metal, coal and other mineral resources. On the one hand, the accumulation of tailings will occupy a large amount of land resources and affect the sustainable use of land; on the other hand, since tailings contain a large amount of phosphorus and potassium, the large amount of tailings accumulation will also cause a huge burden on the ecological environment. In addition, there is a great demand for ecological restoration in mining areas. In the process of mine restoration, the improved tailings sand will be used as a planting matrix instead of foreign soil, which can not only save soil resources and reduce costs, but also realize the resource utilization of tailings sand. However, tailings sand itself is poor in nutrients, has poor physical and chemical properties, and is not suitable for plant growth and development.

[0003] Microorganisms that dissolve phosphorus and potassium generally refer to those that can decompose the insoluble phosphorus and potassium elements in the soil and convert them into forms that can be absorbed and utilized by crops. These microorganisms include but are not limited to nitrogen-fixing bacteria, phosphorus-dissolving bacteria, potassium-dissolving bacteria, etc., which improve soil fertility and promote crop growth through their own metabolic activities. Therefore, how to make phosphorus-dissolving and potassium-dissolving microorganisms efficiently and environmentally friendly promote the phosphorus and potassium mineralization of tailings sand, promote plant growth in tailings sand, and then make tailings sand close to normal soil has become an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present application is to provide a tailings sand phosphorus- and potassium-dissolving Aeromonas salmonicida CuC5, a fermentation product of Aeromonas salmonicida CuC5 and a preparation method thereof, as well as a microbial organic fertilizer and a preparation method thereof, so as to solve the above-mentioned problems.

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

[0006] In a first aspect, the present application provides a salmonicidal Aeromonas CuC5:

[0007] It was classified and named Aeromonas salmonicida and deposited on December 7, 2023 at the General Microbiology Center of China Culture Collection Administration; Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO.29265.

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

[0009] In a second aspect, the present application provides a method for preparing Aeromonas salmonicida CuC5, comprising:

[0010] The tailings sand is inoculated into the first culture medium for the first culture, and culturable microorganisms are obtained by enrichment and screening.

[0011] The culturable microorganisms are subjected to a second culture to screen out specific phosphate- and potassium-solubilizing strains, which are separated and purified to obtain the salmonicidal Aeromonas CuC5.

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

[0013] Preferably, the second culture comprises the screening of phosphate-solubilizing strains and the screening of potassium-solubilizing strains performed sequentially; or the screening of potassium-solubilizing strains and the screening of phosphate-solubilizing strains performed sequentially.

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

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

[0016] Preferably, the components of the high-density fermentation medium include: 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, and the balance is water.

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

[0018] Preferably, the third culture comprises culturing at 30-35° C. and 140-150 rpm on a shaking platform for 18-24 hours.

[0019] Preferably, the number of activated strains is not less than 8.6*10 10 CFU / mL.

[0020] In a fourth aspect, the present application provides a microbial organic fertilizer, the raw materials of which include a fermentation product of Aeromonas salmonicida CuC5 and an organic material.

[0021] Preferably, the organic material includes any decomposed livestock and poultry manure, agricultural straw, restaurant kitchen waste and / or common organic fertilizer.

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

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

[0024] Preferably, the water content of the organic material is 35-50%.

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

[0026] In a fifth aspect, the present application provides a method for preparing microbial organic fertilizer, which is obtained by solid-state fermentation of the fermentation product of Aeromonas salmonicida CuC5 and the organic material.

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

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

[0029] Preferably, the temperature of the solid-state fermentation is 45-65°C.

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

[0031] Preferably, the solid-state fermentation is carried out for 7 to 10 days.

[0032] Preferably, the content of Aeromonas salmonicida CuC5 in the microbial organic fertilizer after solid-state fermentation is 6.9*10 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 salmonicidal Aeromonas CuC5 with the ability to solubilize phosphate and potassium from tailings sand. It is derived from tailings sand, has adaptability to the tailings sand environment, and has the ability to produce extracellular secretions, fix nitrogen, and produce plant growth hormones, without the risk of human and animal diseases.

[0035] The Aeromonas salmonicida CuC5 fermentation product provided in the present application uses a high-density fermentation medium, has low raw material cost, high strain fermentation yield, and a short fermentation cycle.

[0036] The microbial organic fertilizer carrier provided by the present application is an organic fertilizer obtained by ordinary organic waste fermentation, does not require special organic materials, can obtain products after solid-state fermentation, and the carrier is cheap and easy to obtain. And it is dedicated to tailings sand improvement, dedicated to promoting tailings sand soil, greatly increasing the effective phosphorus and potassium content of tailings sand, and increasing the plant biomass of tailings sand to make it close to normal soil. Not only can it promote the mineralization of tailings sand into soil, it can also greatly increase the organic carbon content of tailings sand, improve the carbon fixation potential of tailings sand, and provide an important method for the comprehensive utilization of tailings sand. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0038] Figure 1 This is a colony morphology diagram of Aeromonas salmonicida CuC5 on an LB plate in Example 1;

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

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

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

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

[0043] Figure 6 This is a schematic diagram comparing the appearance of Elymus salsa. DETAILED DESCRIPTION

[0044] In order to better explain the technical solution provided by this application, before the embodiments, the technical solution is first described as a whole, as follows:

[0045] In the first aspect, the present application provides a salmonicidal Aeromonas CuC5: classified and named Aeromonassalmonicida, deposited on December 7, 2023 at the General Microbiology Center of China Culture Collection Administration; Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO.29265.

[0046]

[0047] In a second aspect, the present application provides a method for preparing Aeromonas salmonicida CuC5, comprising:

[0048] The tailings sand is inoculated into the first culture medium for the first culture, and the culturable microorganisms are obtained by enrichment screening. After the enrichment culture, the microorganisms in the culture system can be screened and expanded, and the culture system contains a variety of strains, which can be used for subsequent screening.

[0049] The culturable microorganism is subjected to a second culture to screen for specific phosphate and potassium solubilizing strains, and the salmonicidal Aeromonas CuC5 is separated and purified. After the first culture, the culture system contains a large number of microbial flora after expanded culture. After the specific potassium and phosphorus solubilizing screening, strains with potassium and phosphorus solubilizing abilities can be specifically screened out from the large number of flora in the culture system after the first culture.

[0050] In a preferred embodiment, the first culture medium is LB medium; the first culture includes primary bacterial screening.

[0051] In a preferred embodiment, the second culture includes the screening of phosphate-solubilizing strains and the screening of potassium-solubilizing strains in sequence; or the screening of potassium-solubilizing strains and the screening of phosphate-solubilizing strains in sequence. In the implementation process, there is no special requirement for the screening order of phosphate-solubilizing and potassium-solubilizing. The phosphate-solubilizing strains are screened first, and then the screened phosphate-solubilizing strains are further screened for potassium-solubilizing, so as to obtain the screening of phosphate-solubilizing and potassium-solubilizing strains; on the contrary, the potassium-solubilizing strains are screened first, and then the screened potassium-solubilizing strains are further screened for phosphate-solubilizing, so as to obtain the phosphate-solubilizing and potassium-solubilizing strains.

[0052] In a preferred embodiment, solid inorganic phosphorus culture medium is used to screen the phosphate-solubilizing strains; solid potassium-solubilizing culture medium is used to screen the potassium-solubilizing strains.

[0053] In a third aspect, the present application provides a method for preparing a fermentation product of Aeromonas salmonicida CuC5, wherein the Aeromonas salmonicida CuC5 is inoculated into a high-density fermentation medium, and an activated strain is obtained after a third culture, wherein the activated strain contains the fermentation product of Aeromonas salmonicida CuC5.

[0054] In an optional embodiment, the components of the high-density fermentation medium include: 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, and the balance is water.

[0055] In an 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.

[0056] In an optional embodiment, the third culture includes culturing at 30-35°C, 140-150rpm shaking table for 18-24h. The culture temperature of the third culture can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or any value between 30-35°C. The speed of the shaking table can be 140rpm, 141rpm, 142rpm, 143rpm, 144rpm, 145rpm, 146rpm, 147rpm, 148rpm, 149rpm, 150rpm, or any value between 140-150rpm. The time of shaking table culture can be 18h, 19h, 20h, 21h, 22h, 23h, 24h, or any value between 18-24h.

[0057] In an optional embodiment, the number of activated strains is not less than 8.6*10 10 CFU / mL.

[0058] In a fourth aspect, the present application provides a microbial organic fertilizer, the raw materials of which include a fermentation product of Aeromonas salmonicida CuC5 and an organic material.

[0059] In an optional embodiment, the organic material includes any decomposed livestock and poultry manure, agricultural straw, restaurant kitchen waste and / or common organic fertilizer.

[0060] In an optional embodiment, the mass percentage of organic matter in the organic material is not less than 45%.

[0061] In an optional embodiment, the total mass percentage of nitrogen, phosphorus and potassium in the organic material is not less than 5%.

[0062] In an optional embodiment, the water content of the organic material is 35-50%. The water 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%.

[0063] In an 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.

[0064] In a fifth aspect, the present application provides a method for preparing microbial organic fertilizer, which is obtained by solid-state fermentation of the fermentation product of Aeromonas salmonicida CuC5 and the organic material.

[0065] In an optional embodiment, the water content of the organic material is adjusted to 50-60% before the solid-state fermentation. The water 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% and 60%.

[0066] In an optional embodiment, the fermentation product of Aeromonas salmonicida CuC5 is mixed at a ratio of 20 to 40 L per ton of the organic material before the solid-state fermentation. The ratio between the fermentation product of Aeromonas salmonicida CuC5 and the organic material can be 20 L per ton of the organic material, 25 per ton of the organic material, 30 per ton of the organic material, 35 per ton of the organic material, 40 per ton of the organic material, or any value between 20 and 40 per ton of the organic material.

[0067] In an optional embodiment, the temperature of the solid-state fermentation is 45-65° C. The temperature of the gutta-percha fermentation can be 45° C., 50° C., 55° C., 60° C., 65° C., or any value between 45 and 65° C.

[0068] In an optional embodiment, the water content is maintained at 45-50% during the solid-state fermentation process. The water content maintained during the solid-state fermentation process can be 45%, 46%, 47%, 48%, 49%, 50%, or any value between 45% and 50%.

[0069] In an optional embodiment, the number of days for the solid-state fermentation is 7 to 10 days. The number of days for the solid-state fermentation can be 7 days, 8 days, 9 days, 10 days, or any value between 7 and 10 days.

[0070] Preferably, the content of Aeromonas salmonicida CuC5 in the microbial organic fertilizer after the solid-state fermentation can be 6.9*10 8 ~7.2*10 9 Any value between CFU.

[0071] It should be noted that the components of the culture system used in each embodiment of the present application are as follows:

[0072] Tailings sand: The tailings sand used in this application was collected from a high-altitude copper mining area in Tibet, with a pH of 7.38, total nitrogen 0.33 g / kg, total phosphorus 0.91 g / kg, total potassium 5.33 g / kg, available nitrogen 14.8 mg / kg, available phosphorus 3.12 mg / kg, available potassium 18.23 mg / kg, and organic carbon 3.25 g / kg.

[0073] LB medium: LB liquid medium is prepared by mixing LB Broth medium with distilled water, and 2% (mass volume percentage) agar powder is added when preparing solid medium. All LB medium are sterilized in a vertical pressure steam sterilizer at 0.11-0.15MPa and 121°C for 30 minutes.

[0074] Inorganic phosphorus culture 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 dilute to 1L with pure water. All culture media were sterilized in a vertical pressure steam sterilizer at 0.11-0.15MPa and 121℃ for 30min.

[0075] Potassium-dissolving medium: 2g disodium hydrogen phosphate, 0.005g ferric chloride, 0.1g calcium carbonate, 0.5g magnesium sulfate, 1g potassium feldspar, 5g sucrose, 18g agar. Adjust pH to 7.5 and dilute to 1L with pure water. All culture media are sterilized in a vertical pressure steam sterilizer at 0.11-0.15MPa and 121℃ for 30min.

[0076] Ashuber nitrogen-free medium: mannitol 10g, MgSO4·7H2O 0.2g, CaSO4·2H2O0.1g, KH2PO40.2g, CaCO3 5g, agar 20g, pH 7.2-7.4; make up to 1L.

[0077] CAS culture medium: 2 mL of 1 mmol / L MgSO4, 100 μL of 1 mmol / LCaCl2, 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolyzed casein, 1.8 g of agar, slowly add 5 mL each of phosphate buffer and CAS dye at about 60°C.

[0078] Example 1

[0079] This embodiment provides a salmonicidal Aeromonas CuC5, and the preparation method thereof is as follows:

[0080] 1.1 Bacterial screening

[0081] 10g of the collected tailings sand sample was inoculated into 100mL of sterilized LB medium; cultured in a shaking table at 30℃ and 150rpm; fresh LB medium was replaced every 48h; cultured continuously for 7 days for enrichment and screening to obtain culturable bacteria in the tailings sand. 117 strains of culturable microorganisms in tailings sand were initially screened.

[0082] 1.2 Screening of specific phosphate and potassium solubilizing bacteria

[0083] The selected culturable microorganisms were inoculated into solid inorganic phosphorus medium using an inoculation loop, cultured at 30°C for 5 days, and the growth state of the strain and the size of the transparent circle produced on the medium were observed to determine the strain's ability to solubilize phosphorus. The selected culturable microorganisms were inoculated into solid potassium-solubilizing medium using an inoculation loop, cultured at 30°C for 5 days, and the growth state of the strain and the size of the hydrolysis circle were observed to determine the strain's ability to solubilize potassium.

[0084] 1.3 Rescreening of Phosphate and Potassium Solubilizing Bacteria

[0085] 20 g of the collected tailings sand sample was added to 100 mL of sterilized LB medium, and the screened phosphate- and potassium-solubilizing microorganisms were inoculated and cultured in a shaking incubator at 30°C and 150 rpm. After seven days of culture, the available phosphorus and available potassium in the culture system were determined for rescreening.

[0086] 1.4 Separation and purification

[0087] Use LB solid plate medium to culture and subculture the strains to obtain single colonies that can be isolated and purified.

[0088] 1.5 Morphological observation

[0089] The strain whose single colony on LB plate medium is regular circle, has neat edges and smooth surface, is opaque light yellow in color, is relatively sticky, moist, easy to pick, and tends to be light gray after a long period of culture was selected and named CuC5; Figure 1 This is the LB plate colony morphology of CuC5. Figure 2 Scanning electron micrograph of the strain. Under an electron microscope, it is rod-shaped, with flagella at the tail, and the average length of the bacteria is 1.1 μm. Gram staining identified CuC5 as Gram-negative.

[0090] 1.6 Molecular Biological Identification

[0091] The primers used for amplification and sequencing of the 16S rDNA gene fragment of CuC5 were 27F: (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R: (5-GGTTACCTTGTTACGACT T-3). The obtained sequence fragment (length 1380bp, as shown in sequence 1) was submitted to the GenBank database with the accession number: PQ157593. After Blastn sequence alignment with the NCBI database, CuC5 had a high degree of similarity with multiple strains of Aeromonas salmonicida. Among them, the homology between CuC5 and Aeromonas salmonicida strain NCIMB 1102 (NR 118945) reached 99.97%. In addition, a phylogenetic tree was constructed for the 16S rDNA gene fragment of CuC5, as shown in Figure 1. Figure 3 As shown, combined with the tree construction and comparison results, CuC5 was preliminarily identified as Aeromonas salmonicida.

[0092] Example 2

[0093] This embodiment provides a salmonicidal Aeromonas CuC5, the preparation process of which is as follows:

[0094] 2.1 Bacterial screening

[0095] 10g of the collected tailings sand sample was inoculated into 100mL of sterilized LB medium; cultured in a shaking table at 25℃ and 140rpm; fresh LB medium was replaced every 24h; cultured continuously for 5 days for enrichment screening to obtain culturable bacteria in the tailings sand. 85 strains of culturable microorganisms in tailings sand were initially screened.

[0096] 2.2 Screening of specific phosphate and potassium solubilizing bacteria

[0097] The selected culturable microorganisms were inoculated into solid inorganic phosphorus medium using an inoculation loop, cultured at 25°C for 3 days, and the growth state of the strain and the size of the transparent circle produced on the medium were observed to determine the strain's ability to solubilize phosphorus. The selected culturable microorganisms were inoculated into solid potassium-solubilizing medium using an inoculation loop, cultured at 25°C for 3 days, and the growth state of the strain and the size of the hydrolysis circle were observed to determine the strain's ability to solubilize potassium.

[0098] 2.3 Rescreening of Phosphate and Potassium Solubilizing Bacteria

[0099] Add 10 g of the collected tailings sand sample into 100 mL of sterilized LB medium, and inoculate the screened phosphate- and potassium-solubilizing microorganisms. Culture in a shaking incubator at 25°C and 140 rpm. After seven days of culture, measure the available phosphorus and available potassium in the culture system for rescreening.

[0100] 2.4 Separation and purification

[0101] Use LB solid plate medium to culture and subculture the strains to obtain single colonies that can be isolated and purified.

[0102] 2.5 Morphological observation

[0103] A strain was selected whose single colony on the LB plate culture medium was a regular circle with neat edges and a smooth surface, an opaque light yellow color, a relatively sticky, moist, and easy-to-pick bacterial body, and whose colony tended to be light gray after a long period of culture. It was named CuC5 and Gram staining was performed to identify CuC5 as Gram-negative.

[0104] Example 3

[0105] This embodiment provides a salmonicidal Aeromonas CuC5, the preparation process of which is as follows:

[0106] 3.1 Bacterial screening

[0107] 10g of the collected tailings sand sample was inoculated into 100mL of sterilized LB medium; cultured in a shaking incubator at 28°C and 145rpm; fresh LB medium was replaced every 36h; cultured continuously for 6 days for enrichment screening to obtain culturable bacteria in the tailings sand. 90 strains of culturable microorganisms in tailings sand were initially screened.

[0108] 3.2 Screening of specific phosphate and potassium solubilizing bacteria

[0109] The selected culturable microorganisms were inoculated into solid inorganic phosphorus medium using an inoculation loop, cultured at 28°C for 4 days, and the growth state of the strain and the size of the transparent circle produced on the medium were observed to determine the strain's ability to solubilize phosphorus. The selected culturable microorganisms were inoculated into solid potassium-solubilizing medium using an inoculation loop, cultured at 28°C for 4 days, and the growth state of the strain and the size of the hydrolysis circle were observed to determine the strain's ability to solubilize potassium.

[0110] 3.3 Rescreening of Phosphate and Potassium Solubilizing Bacteria

[0111] 15 g of the collected tailings sand sample was added into 100 mL of sterilized LB medium, and the screened phosphate- and potassium-solubilizing microorganisms were inoculated and cultured in a shaking incubator at 28°C and 145 rpm. After seven days of culture, the available phosphorus and available potassium of the culture system were determined for rescreening.

[0112] 3.4 Separation and purification

[0113] Use LB solid plate medium to culture and subculture the strains to obtain single colonies that can be isolated and purified.

[0114] 3.5 Morphological observation

[0115] A strain was selected whose single colony on the LB plate culture medium was a regular circle with neat edges and a smooth surface, an opaque light yellow color, a relatively sticky, moist, and easy-to-pick bacterial body, and whose colony tended to be light gray after a long period of culture. It was named CuC5 and Gram staining was performed to identify CuC5 as Gram-negative.

[0116] Example 4

[0117] This example is directed to Example 1 to determine other potential functions of Aeromonas salmonicida CuC5, and the specific method is as follows:

[0118] Detection of nitrogen-fixing activity of the strain: The strain Aeromonas salmonicida CuC5 was inoculated onto the Axube nitrogen-free medium, sealed, inverted, and cultured at 28°C for 5 days. The strain grew and had nitrogen-fixing activity.

[0119] Detection of the activity of producing siderophores by the strain: The strain Aeromonas salmonicida CuC5 was inoculated on a CAS detection plate and cultured at 28°C for 48 hours. The growth status of the strain was observed and whether a yellow halo appeared around the colony. The strain had the ability to produce siderophores.

[0120] IAA production activity detection of strains: The IAA production activity of strains was detected by spectrophotometry, and the Salkowski colorimetric solution was reacted with the bacterial solution shaken to the logarithmic phase. The strain has the activity of producing IAA.

[0121] Detection of ACC dehydrogenase produced by the strain: Pick a single colony of the strain Aeromonas salmonicida CuC5 for enrichment culture in beef extract peptone medium and culture it to the logarithmic growth phase; collect the bacteria by centrifugation, treat the bacteria twice with 0.1mol / L Tris-HCl (pH7.6), centrifuge at 10000r / min for 5min, and discard the supernatant; suspend the bacteria in 600μL 0.1mol / L Tris-HCl (PH8.5); add 30μL toluene and mix for 30s; draw 200μL into a 1.5mL centrifuge tube, add 200μL 0.5mol / L ACC, mix for 5s, warm at 30℃ for 15min, add 0.56mol / L HCl, mix, and centrifuge at room temperature at 10000r / min for 5min; take 1mL supernatant and add 800μL 0.56mol / L HCl, mix in a test tube, add 300mL 2,4-dinitrophenylhydrazine (2% 2,4-dinitrophenylhydrazine added to 2 mol / L HCl) was mixed and incubated at 30°C for 30 min; 2 mol / L NaOH was added; and the OD450 value was measured. The strain has the ability to produce ACC dehydrogenase.

[0122] Hemolytic reaction test of the strain: Inoculate the strain Aeromonas salmonicida CuC5 on a sheep blood plate and incubate it at 37℃ for 24 hours. No hemolytic circle is observed. The hemolytic reaction of this strain is negative, and there is no risk of disease to humans and animals.

[0123] The results showed that Aeromonas salmonicida CuC5 has the ability to fix nitrogen, produce siderophores, produce IAA, and produce ACC dehydrogenase. It is a phosphate-solubilizing and acetyl-solubilizing bacterium with plant growth-promoting function and has no risk of causing disease to humans and animals.

[0124] Example 5

[0125] This embodiment provides a fermentation product prepared by Aeromonas salmonicida CuC5 obtained in Example 1, and the preparation method thereof is as follows:

[0126] The strain Aeromonas salmonicida CuC5 was inoculated into high-density fermentation medium for fermentation.

[0127] The high-density medium solutes used for fermentation and their concentrations in the high-density fermentation medium were: 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.

[0128] The specific steps for preparing the fermentation product are as follows:

[0129] (1) Take the isolated, purified and identified Aeromonas salmonicida CuC5 stored in a refrigerator and inoculate it into a conical flask containing 100 ml of high-temperature sterilized LB liquid culture medium;

[0130] (2) placing the inoculated conical flask in a constant temperature shaking incubator and culturing at 150 rpm and 30° C. for 24 hours to obtain an activated salmonicidal Aeromonas CuC5 seed solution;

[0131] (3) The seed liquid of Aeromonas salmonicida CuC5 was inoculated into a fermenter containing a high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.5 with 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, the temperature was 30°C, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation liquid to 10, and appropriate feed was added during the fermentation process. The aeration volume was combined with the agitator to control the dissolved oxygen in the fermentation tank to 6 mg / L. During the fermentation period, samples were taken every 6 hours to detect the OD value and viable bacterial count (CFU / ml) of the fermentation liquid. The viable bacterial count was determined by the gradient dilution plate spread counting method, and the colony forming unit (CFU) was calculated to count the viable bacterial count. After 48 hours, the growth of strain CuC5 entered a stable period, with a maximum CFU of 8.6*10 10 / mL.

[0132] Example 6

[0133] This embodiment provides a fermentation product prepared by Aeromonas salmonicida CuC5 obtained in Example 2, and the preparation method thereof is as follows:

[0134] The strain Aeromonas salmonicida CuC5 was inoculated into high-density fermentation medium for fermentation.

[0135] The high-density culture medium solutes used for fermentation and their concentrations in the high-density fermentation medium were: 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.

[0136] The specific steps for preparing the fermentation product are as follows:

[0137] (1) Take the isolated, purified and identified Aeromonas salmonicida CuC5 stored in a refrigerator and inoculate it into a conical flask containing 100 ml of high-temperature sterilized LB liquid culture medium;

[0138] (2) placing the inoculated conical flask in a constant temperature shaking incubator and culturing at 140 rpm and 35° C. for 18 hours to obtain an activated salmonicidal Aeromonas CuC5 seed solution;

[0139] (3) The seed liquid of Aeromonas salmonicida CuC5 was inoculated into a fermenter containing a high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.4 with 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, the temperature was 35°C, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation liquid to 10, and appropriate feeding was performed during the fermentation process. The aeration volume was combined with the agitator to control the dissolved oxygen in the fermentation tank to 5 mg / L. During the fermentation period, samples were taken every 5 hours to detect the OD value and viable bacterial count (CFU / ml) of the fermentation liquid. The viable bacterial count was determined by the gradient dilution plate spread counting method, and the colony forming unit (CFU) was calculated to count the viable bacterial count. After 52 hours, the growth of strain CuC5 entered a stable period, with a maximum CFU of 7.6*10 10 / mL.

[0140] Example 7

[0141] This embodiment provides a fermentation product prepared by Aeromonas salmonicida CuC5 obtained in Example 3, and the preparation method thereof is as follows:

[0142] The strain Aeromonas salmonicida CuC5 was inoculated into high-density fermentation medium for fermentation.

[0143] The high-density medium solutes used for fermentation and their concentrations in the high-density fermentation medium were: 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.

[0144] The specific steps for preparing the fermentation product are as follows:

[0145] (1) Take the isolated, purified and identified Aeromonas salmonicida CuC5 stored in a refrigerator and inoculate it into a conical flask containing 100 ml of high-temperature sterilized LB liquid culture medium;

[0146] (2) placing the inoculated conical flask in a constant temperature shaking incubator and culturing at 140 rpm and 35° C. for 18 hours to obtain an activated salmonicidal Aeromonas CuC5 seed solution;

[0147] (3) The seed liquid of Aeromonas salmonicida CuC5 was inoculated into a fermenter containing a high-density fermentation medium at a volume percentage of 5%. The fermentation conditions were controlled as follows: pH was controlled at 7.2 with 0.5 mol / L sodium hydroxide and 0.5 mol / L hydrochloric acid solution, the temperature was 32°C, sodium citrate was added as an additional carbon source to adjust the C / N ratio of the fermentation liquid to 10, and appropriate feeding was performed during the fermentation process. The aeration volume was combined with the agitator to control the dissolved oxygen in the fermentation tank to 4.5 mg / L. During the fermentation period, samples were taken every 4 hours to detect the OD value and viable bacterial count (CFU / ml) of the fermentation liquid. The viable bacterial count was determined by the gradient dilution plate spread counting method, and the colony forming unit (CFU) was calculated to count the viable bacterial count. After 36 hours, the growth of strain CuC5 entered a stable period, with a maximum CFU of 7.9*10 10 / mL.

[0148] Example 8

[0149] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonicida CuC5 obtained in Example 5, and the preparation method is as follows:

[0150] The organic materials used to prepare microbial organic fertilizer are composted cow dung and highland barley rape straw from a certain area, with an organic matter content of 46.2%, a total nitrogen content of 1.2%, a total phosphorus content of 2.7%, a total potassium content of 1.3%, a water content of 38.1%, and a pH of 7.2. The water content of the organic fertilizer is adjusted to 50% before secondary fermentation. Mix thoroughly and keep it loose and breathable.

[0151] The fermentation product of Aeromonas salmonicida CuC5 prepared in Example 5 was sprayed into the organic material at an inoculation rate of 40 L per ton of organic material, mixed evenly, and formed a mixed material. The secondary solid-state fermentation cycle was 10 days, and the compost was turned once on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. After testing, the number of viable bacteria in the microbial organic fertilizer was 7.2*10 9 CFU, scanning electron microscope image of Aeromonas salmonicida CuC5 microbial organic fertilizer Figure 4 As shown, the final product is Figure 5 shown.

[0152] Example 9

[0153] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonicida CuC5 obtained in Example 6, and the preparation method is as follows:

[0154] The organic materials used to prepare microbial organic fertilizer are composted cow dung and highland barley rape straw from a certain area, with an organic matter content of 45.0%, a total nitrogen content of 1.1%, a total phosphorus content of 2.2%, a total potassium content of 1.7%, a water content of 45.2%, and a pH of 7.5. The water content of the organic fertilizer is adjusted to 5% before the secondary fermentation. Mix thoroughly and keep it loose and breathable.

[0155] The fermentation product of Aeromonas salmonicida CuC5 prepared in Example 6 was sprayed into the organic material at an inoculation rate of 30 L per ton of organic material, and mixed evenly to form a mixed material. The secondary solid-state fermentation cycle was 7 days, and the compost was turned once on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. After testing, the number of viable bacteria in the microbial organic fertilizer was 6.9*10 9 CFU.

[0156] Example 10

[0157] This embodiment provides a microbial organic fertilizer prepared from the fermentation product of Aeromonas salmonicida CuC5 obtained in Example 7, and the preparation method is as follows:

[0158] The organic materials used to prepare microbial organic fertilizer are composted cow dung and highland barley rape straw from a certain area, with an organic matter content of 45.5%, a total nitrogen content of 1.3%, a total phosphorus content of 2.2%, a total potassium content of 1.5%, a water content of 42.3%, and a pH of 6.9. The water content of the organic fertilizer is adjusted to 60% before the secondary fermentation. Mix thoroughly and keep it loose and breathable.

[0159] The fermentation product of Aeromonas salmonicida CuC5 prepared in Example 7 was sprayed into the organic material at an inoculation rate of 35 L per ton of organic material, mixed evenly, and formed a mixed material. The secondary solid-state fermentation cycle was 9 days, and the compost was turned once on the 5th day. After the secondary solid-state fermentation, the fermentation product was cooled and aged to obtain the final microbial organic fertilizer. After testing, the number of viable bacteria in the microbial organic fertilizer was 7.0*10 9 CFU.

[0160] Embodiment 11

[0161] This embodiment is a potted plant experiment conducted from late June 2024 to early August 2024 to evaluate the improvement effect of microbial organic fertilizer on tailings sand.

[0162] The potting soil used in the potted experiment was pure tailings, with an amount of 3 kg; the fertilizer was the microbial organic fertilizer prepared in Example 8, with an application amount of 60 g of the microbial organic fertilizer in Example 8; the potting soil and the microbial organic fertilizer were mixed and placed in a flower pot, and Elymus salsa seeds were sown, and 200 mL of water was poured every two days, and three parallel experiments were set up. Plant and soil samples were collected after the plants matured, and plant biomass and soil physical and chemical indicators were measured.

[0163] Comparative Example 1

[0164] This comparative example is a potted plant experiment conducted from late June 2024 to early August 2024, which is used to evaluate the improvement effect of microbial organic fertilizer on tailings sand.

[0165] The potting soil used in the potting experiment was pure tailings, with a dosage of 3kg; the fertilizer was organic fertilizer made from composted cow dung and highland barley rape straw in a certain area, with an organic matter content of 46.2%, a total nitrogen content of 1.2%, a total phosphorus content of 2.7%, a total potassium content of 1.3%, a water content of 38.1%, and a pH of 7.2. The fertilizer application amount was 60g; the potting soil and organic fertilizer were mixed and placed in a flowerpot, and Elymus salsa seeds were sown. 200mL of water was poured every two days, and three parallel experiments were set up. Plant and soil samples were collected after the plants matured to measure plant biomass and soil physical and chemical indicators.

[0166] Comparative Example 2

[0167] This comparative example is a potted plant experiment conducted from late June 2024 to early August 2024, which is used to evaluate the improvement effect of microbial organic fertilizer on tailings sand.

[0168] The potting soil used in the potting experiment was pure tailings, with a dosage of 3 kg and no fertilizer. The potting soil was placed in a flowerpot, and Elymus seeds were sown. 200 mL of water was poured every two days, and three parallel experiments were set up. Plant and soil samples were collected after the plants matured to measure plant biomass and soil physical and chemical indicators.

[0169] Comparative Example 3

[0170] This comparative example is a potted plant experiment conducted from late June 2024 to early August 2024, which is used to evaluate the improvement effect of microbial organic fertilizer on tailings sand.

[0171] The culture soil used in the potted experiment was pure tailings, with a dosage of 3 kg; the fertilizer was commercial organic fertilizer purchased from Genliduo Biotechnology Co., Ltd., which was organic fertilizer fermented from livestock and poultry manure (product number: 10047929946683), with a fertilizer dosage of 60 g; the culture soil and microbial organic fertilizer were mixed and placed in a flowerpot, and Elymus seeds were sown. 200 mL of water was poured every two days, and three parallel experiments were set up. Plant and soil samples were collected after the plants matured, and plant biomass and soil physical and chemical indicators were measured.

[0172] Comparative Example 4

[0173] This comparative example is a potted plant experiment conducted from late June 2024 to early August 2024, which is used to evaluate the improvement effect of microbial organic fertilizer on tailings sand.

[0174] The potting experiment used normal soil, 3 kg of soil, and no fertilizer. The soil was placed in the pot, and Elymus seeds were sown. Water was applied every two days with 200 mL, and three parallel experiments were set up. Plant and soil samples were collected after the plants matured to measure plant biomass and soil physical and chemical indicators.

[0175] The appearance of the Elymus dahliae in the above-mentioned Example 11 and Comparative Examples 1-4 is as follows: Figure 6 As shown, the microbial organic fertilizer disclosed in this application can effectively increase the biomass of plants in tailings, which is close to normal soil. The biomass index of Elymus dahliae is shown in Table 1. The results show that the microbial organic fertilizer disclosed in this application can effectively increase the biomass of plants in tailings, and the effect is significantly stronger than that of commercial microbial organic fertilizer, which is close to normal soil.

[0176] Table 1 Plant biomass indicators under different treatments

[0177]

[0178] The results of soil physical and chemical property test are shown in Table 2. The results show that the microbial organic fertilizer disclosed in this application can significantly increase the quick-acting phosphorus and potassium content of tailings sand, effectively promote the mineralization of tailings sand, and accelerate the soilization process of tailings sand. In addition, the microbial organic fertilizer disclosed in this application can increase the organic carbon of tailings sand, which is of great significance to improving the carbon fixation potential of tailings.

[0179] Table 2 Physical and chemical indicators of soil under different treatments

[0180]

[0181]

[0182] The above results show that after the strain Aeromonas salmonicida CuC5 biological organic fertilizer is introduced into the tailings sand, compared with commercial microbial organic fertilizers and single organic fertilizer carriers, it effectively promotes the soilification of tailings sand, greatly increases the effective phosphorus and potassium content of tailings sand, and promotes the plant yield of tailings sand. The microbial organic fertilizer disclosed in this application can be used for the comprehensive utilization and reclamation of tailings, which is of great significance in solving the tailings inventory and improving the comprehensive utilization efficiency of tailings.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0184] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. A salmonicidal Aeromonas CuC5, characterized in that It was classified and named Aeromonas salmonicida and was deposited in the General Microbiology Center of China Culture Collection Administration on December 7, 2023; Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO.29265.

2. Aeromonas salmonicida CuC5 according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

3. A method for preparing Aeromonas salmonicida CuC5, characterized in that: include: Inoculating the tailings sand into the first culture medium for first culture, and enriching and screening to obtain culturable microorganisms; The culturable microorganisms are subjected to a second culture to screen out specific phosphate- and potassium-solubilizing strains, which are separated and purified to obtain the salmonicidal Aeromonas CuC5.

4. The method for preparing Aeromonas salmonicida CuC5 according to claim 3, characterized in that: The first culture medium is LB culture medium; the first culture includes primary bacterial screening; The second culture includes the screening of phosphate-solubilizing strains and the screening of potassium-solubilizing strains in sequence; or, The screening of potassium-solubilizing strains and the screening of phosphate-solubilizing strains were carried out sequentially.

5. A method for preparing a fermentation product of Aeromonas salmonicida CuC5, characterized in that: The Aeromonas salmonicida CuC5 of claim 1 is inoculated into a high-density fermentation medium, and an activated strain is obtained after a third culture, wherein the activated strain contains a fermentation product of Aeromonas salmonicida CuC5.

6. The method for preparing the fermentation product of Aeromonas salmonicida CuC5 according to claim 5, characterized in that: Satisfy one or more of the following conditions a to d: a. The components of the high-density fermentation medium include: 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, and the balance is water; b. The pH value of the high-density fermentation medium is 6.8 to 7.5; c. The third culture comprises culturing at 30 to 35 ° C and 140 to 150 rpm on a shaker for 18 to 24 h; d. The number of activated strains is not less than 8.6*10 10 CFU / mL.

7. A fermentation product of Aeromonas salmonicida CuC5, characterized in that: The product is prepared according to the method for preparing the fermentation product of Aeromonas salmonicida CuC5 according to any one of claims 5 to 6.

8. A microbial organic fertilizer, characterized in that: The raw materials include the fermentation product of Aeromonas salmonicida CuC5 as claimed in claim 7 and organic materials.

9. The microbial organic fertilizer according to claim 8, characterized in that The organic material meets one or more of the following conditions e to i: e. The organic materials include any decomposed livestock and poultry manure, agricultural straw, kitchen waste and / or ordinary organic fertilizer; f. The mass percentage of organic matter in the organic material is not less than 45%; 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.

10. A method for preparing microbial organic fertilizer, characterized in that The solid-state fermentation is performed by subjecting the fermentation product of Aeromonas salmonicida CuC5 to the organic material, wherein the solid-state fermentation satisfies one or more of the following conditions j to o: j. Before the 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 salmonicida CuC5 is mixed at a ratio of 20 to 40 L per ton of the organic material; 1. The solid-state fermentation temperature is 45 to 65°C; m. The water content is maintained at 45 to 50% during the solid-state fermentation process; n. The solid-state fermentation is carried out for 7 to 10 days; o. The content of the salmon-killing Aeromonas CuC5 in the microbial organic fertilizer after the solid-state fermentation is 6.9*10 8 ~7.2*10 9 CFU.

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