Sphingomonas NT-1 and application thereof

Abstract

The invention provides a sphingomonas NT-1 and application thereof, the preservation number of the sphingomonas NT-1 is CCTCC NO: M 20252237, and the sphingomonas NT-1 is high in environmental adaptability and has multiple growth promoting functions of dissolving phosphorus, producing auxin and exopolysaccharide and the like. By applying the strain, the exfoliated substances in the mining area can be converted into an active matrix suitable for plant growth, the content of key nutrients such as nitrogen, phosphorus and potassium in the exfoliated substances and the microbial diversity are effectively improved, and resource reutilization of waste is achieved. In addition, the strain also significantly promotes the growth and nutrient accumulation of plants, and enhances the planting ability of vegetation. The invention provides an efficient and green microbial solution for in-situ utilization of the exfoliated substances in the mining area and rapid recovery of soil vegetation, and has important application value for promoting ecological restoration of mines and regeneration of land resources.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Sphingomonas NT-1 and its applications. Background Technology

[0002] Coal-fired power generation is one of the main sources of electricity supply in my country. While providing energy, open-pit coal mining also poses serious ecological and environmental challenges. To obtain underground coal resources, it is necessary to remove the overburden material (including slag, peat, mudstone, coal gangue, and all non-target minerals) above the ore body; this removed overburden is known as overburden. Large amounts of overburden occupy land resources, severely limiting subsequent mining operations in open-pit coal mines. Therefore, effectively utilizing overburden is crucial to solving the problems of soil scarcity and land resource occupation by overburden in open-pit mines. Furthermore, the excavated and piled surfaces formed during mining are much larger than the original surface area, leading to a lack of topsoil during ecological restoration and severely restricting the subsequent restoration of surface vegetation in the mining area. Relying on the destruction of topsoil in other areas for reclamation causes secondary damage to the ecological environment. Therefore, effectively utilizing overburden is key to solving the problem of solid waste resource utilization and restoring the vegetation and ecological environment of mining areas.

[0003] Overburden from coal mining areas is primarily peat. Peat consists of incompletely decomposed plant remains. While it has a high organic matter content, its mineral nutrient content (nitrogen, phosphorus, potassium, and trace elements) is extremely low, and most of it is locked in difficult-to-decompose organic matter, making it impossible for plants to directly absorb. This results in extremely difficult germination and root development after sowing. Currently, existing technologies largely focus on researching topsoil substitutes using overburden as a matrix to address the problem of topsoil deficiency. Attempts include modifying peat or mixing it with organic fertilizers, wood ash, vermiculite, etc., but the amount of peat added is usually very small, making it difficult to widely promote and apply in-situ restoration of surface vegetation in mining areas. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides a strain of Sphingomonas NT-1 and its application, achieving the dual purpose of effectively utilizing mining strippings and restoring vegetation in mining areas.

[0005] In a first aspect, the present invention provides a strain of Sphingomonas (… Sphingomonas sp.) NT-1, the Sphingosomalidobacterium (sp.) NT-1, the Sphingosomalidobacterium ( Sphingomonas sp.)NT-1 was deposited at the China Center for Type Culture Collection (CCTCC) on October 17, 2025, with accession number CCTCC NO: M 20252237 and accession name: Sphingomonassp. NT-1, deposited at: Wuhan University, Wuhan, China, specifically No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0006] Secondly, the present invention provides a microbial inoculant containing the aforementioned Sphingomonas (… Sphingomonas sp.)NT-1, or its freeze-dried cells, or its bacterial suspension, or the supernatant or precipitate resuspension after centrifugation of the bacterial suspension.

[0007] Preferably, the sphingosine monocytogenes in the microbial agent is... Sphingomonas (sp.) NT-1 viable bacteria content ≥1×10 8 cfu / mL.

[0008] The preparation method of the microbial agent should also be within the scope of protection of this invention. The preparation method includes the following steps: culturing the *Sphingosporium* (…). Sphingomonas sp.)NT-1.

[0009] Preferably, the sphingosine monocytogenes (SMP) is cultured. Sphingomonas The temperature range for sp.)NT-1 is 20℃~40℃.

[0010] Preferably, the sphingosine monocytogenes (SMP) is cultured. Sphingomonas The pH of sp.)NT-1 is 5 to 9.

[0011] Preferably, the concentration of NaCl is used to culture the *Sphingosomalidone* (S). Sphingomonas The salinity of sp.)NT-1 is 0 to 1%.

[0012] Thirdly, the present invention provides the aforementioned Sphingomonas ( Sphingomonas The use of sp.)NT-1 or the microbial agent in phosphorus solubilization, production of indoleacetic acid and / or production of extracellular polysaccharides.

[0013] The Sphingosomalidone ( Sphingomonas The application of sp.)NT-1 or the microbial agent described herein in the preparation of microbial agents having any one or more functions such as phosphorus solubilization, production of indoleacetic acid, or production of extracellular polysaccharides should also be within the scope of protection of this invention.

[0014] Fourthly, the present invention provides the aforementioned Sphingosine Monoclonal bacteria ( Sphingomonas The use of sp.)NT-1 or the microbial agent in promoting plant growth and / or preparing fertilizers.

[0015] Preferably, the plant includes pasture grass.

[0016] More preferably, the forage includes alfalfa and / or crested wheatgrass.

[0017] Preferably, the fertilizer is a solid fertilizer or a liquid fertilizer.

[0018] Preferably, the fertilizer is a fast-acting fertilizer or a slow-release fertilizer.

[0019] Fifthly, the present invention provides a fertilizer comprising the aforementioned Sphingomonas bacillus ( Sphingomonas fertilizers containing sp.)NT-1 or the microbial inoculant.

[0020] Preferably, the fertilizer is a solid fertilizer or a liquid fertilizer.

[0021] Preferably, the fertilizer is a fast-acting fertilizer or a slow-release fertilizer.

[0022] Preferably, the sphingosomalidobacterium (Sphingomonas) in the fertilizer Sphingomonas (sp.) NT-1 viable bacteria content ≥1×10 8 cfu / mL.

[0023] Sixthly, the present invention provides the aforementioned Sphingosine Monoclonal bacteria ( Sphingomonas sp.)NT-1, the application of the microbial agent or the fertilizer in improving mining area soil, preparing soil for mining area reclamation and / or restoring mining area vegetation.

[0024] Preferably, the vegetation includes pasture grasses.

[0025] More preferably, the forage includes alfalfa and / or crested wheatgrass.

[0026] In a seventh aspect, the present invention provides the aforementioned Sphingosine Monoclonal bacteria ( Sphingomonas sp.)NT-1, the application of the microbial agent or the fertilizer in any one of the following (1) to (4): (1) Increase the nutrient content of the stripped material in the mining area; (2) Increase the nutrient content of plants grown using mining strips; (3) Promote the germination and / or growth of plants grown from mining strips; (4) Increase the plant height and / or biomass of plants grown from mining strips.

[0027] Preferably, the stripping material from the mining area includes peat.

[0028] More preferably, the peat content in the stripped material from the mining area is not less than 98%wt.

[0029] More preferably, the peat content in the stripped material from the mining area is 100%wt.

[0030] Preferably, the stripping material from the mining area is stripping material from a coal mining area.

[0031] More preferably, the stripping material from the mining area comes from a mining area in Inner Mongolia.

[0032] More preferably, the stripping material from the mining area comes from the mining area of ​​Xilinhot and / or the mining area of ​​Xiwuqi.

[0033] Preferably, the plant includes pasture grass.

[0034] More preferably, the forage includes alfalfa and / or crested wheatgrass.

[0035] Eighthly, the present invention provides a method for restoring vegetation in a mining area, comprising the following steps: planting plants with the stripped material from the mining area, followed by applying the Sphingomonas sphingosine monocytogenes (SMP). Sphingomonas sp.)NT-1, the microbial agent or the fertilizer.

[0036] Preferably, the stripping material from the mining area includes peat.

[0037] More preferably, the peat content in the stripped material from the mining area is not less than 98%wt.

[0038] More preferably, the peat content in the stripped material from the mining area is 100%wt.

[0039] Preferably, the stripping material from the mining area is stripping material from a coal mining area.

[0040] More preferably, the stripping material from the mining area comes from a mining area in Inner Mongolia.

[0041] More preferably, the stripping material from the mining area comes from the mining area of ​​Xilinhot and / or the mining area of ​​Xiwuqi.

[0042] Preferably, the plant includes pasture grass.

[0043] More preferably, the forage includes alfalfa and / or crested wheatgrass.

[0044] Preferably, the method includes the following steps: sowing the seeds of the plant in the stripping material of the mining area.

[0045] Preferably, the sphingosomalidone (Sphingosomalidone) Sphingomonas (sp.) The effective usage of NT-1 is at least 8.0 × 10 8 cfu / mL.

[0046] The present invention also provides a composition comprising microorganisms and a plant substrate, wherein the microorganisms include the sphingosine monocytogenes (Sphingosine monocytogenes). Sphingomonas sp.)NT-1 or the microbial agent, wherein the plant substrate comprises mining strippings.

[0047] Preferably, the stripping material from the mining area includes peat.

[0048] More preferably, the peat content in the stripped material from the mining area is not less than 98%wt.

[0049] More preferably, the peat content in the stripped material from the mining area is 100%wt.

[0050] Preferably, the stripping material from the mining area is stripping material from a coal mining area.

[0051] More preferably, the stripping material from the mining area comes from a mining area in Inner Mongolia.

[0052] More preferably, the stripping material from the mining area comes from the mining area of ​​Xilinhot and / or the mining area of ​​Xiwuqi.

[0053] Preferably, the plant includes pasture grass.

[0054] More preferably, the forage includes alfalfa and / or crested wheatgrass.

[0055] Preferably, the method includes the following steps: sowing the seeds of the plant in the stripping material of the mining area.

[0056] Unless otherwise stated, strain NT-1 in this article refers to Sphingomonas (… Sphingomonas sp.)NT-1.

[0057] The present invention has the following beneficial effects: The Sphingosomalidobacterium provided by this invention ( Sphingomonas The strain NT-1, with strong environmental adaptability, possesses multiple growth-promoting functions, including phosphorus solubilization, production of plant growth hormones, and extracellular polysaccharides. Applying this strain can transform mining waste—exfoliated material—into a suitable active substrate for plant growth, effectively increasing the content of key nutrients such as nitrogen, phosphorus, and potassium, as well as microbial diversity in the waste, fundamentally improving its infertile state and achieving resource reuse of the waste. Furthermore, this strain significantly promotes plant growth and nutrient accumulation, enhancing vegetation establishment capacity. This invention provides an efficient and green microbial solution for the in-situ utilization of mining waste and rapid restoration of soil vegetation, possessing significant application value for promoting mine ecological restoration and land resource regeneration. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 The sphingosine monocytogenes provided in Example 1 of this invention ( SphingomonasColony growth diagram of sp.)NT-1 bacteria when peat is used as the sole carbon source.

[0060] Figure 2 The sphingosine monocytogenes provided in Example 1 of this invention ( Sphingomonas Phylogenetic tree of the 16S rRNA gene of sp. NT-1.

[0061] Figure 3 This is a photo of plant growth under different irrigation treatments provided in Example 3 of the present invention; A is a photo of potted alfalfa in the control group and the treatment group; B is a photo of whole alfalfa plants in the control group and the treatment group; C is a photo of potted crested wheatgrass in the control group and the treatment group; D is a photo of whole crested wheatgrass plants in the control group and the treatment group; the control group is the control group irrigated with sterile water, and NT-1 is the treatment group irrigated with bacterial suspension of strain NT-1. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0063] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0064] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0065] The inorganic salt liquid culture medium used in the following examples has the following formulation: 1000 mL dH2O, 1.0 g ammonium sulfate, 0.1 g dipotassium hydrogen phosphate trihydrate, 0.1 g potassium dihydrogen phosphate, 0.04 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 0.01 g calcium chloride, pH 7.2–7.4.

[0066] The inorganic salt solid culture medium used in the following examples has the following formula: 1000 mL dH2O, 1.0 g ammonium sulfate, 0.1 g dipotassium hydrogen phosphate trihydrate, 0.1 g potassium dihydrogen phosphate, 0.04 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 0.01 g calcium chloride, 15 g agar, pH 7.2-7.4.

[0067] The inorganic salt culture medium with peat as the sole carbon source used in the following examples has the following formulation: 1000 mL dH2O, 10 g peat, 1.0 g ammonium sulfate, 0.1 g dipotassium hydrogen phosphate trihydrate, 0.1 g potassium dihydrogen phosphate, 0.04 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 0.01 g calcium chloride, pH 7.2–7.4; wherein, the peat is derived from the stripping material from the mining area in the suburbs of Xilinhot, Inner Mongolia Autonomous Region.

[0068] The R2A liquid culture medium used in the following examples is a synthetic culture medium purchased from Beijing Coollab Technology Co., Ltd., with the following formula: 1000mL dH2O, 0.5g glucose, 0.5g starch, 0.5g acid-hydrolyzed casein peptone, 0.5g bacteriological peptone, 0.5g yeast extract, 0.3g sodium pyruvate, 0.3g dipotassium hydrogen phosphate, 0.05g magnesium sulfate heptahydrate, 0.3g dipotassium hydrogen phosphate, pH 7.

[0069] The main component of the stripped material used in the following examples is ≥98%wt peat.

[0070] Example 1 Sphingosine monocytogenes (Sphingosine monocytogenes) Sphingomonas Isolation, purification and identification of sp. NT-1 1. Enrichment culture, isolation and purification of bacterial strains In March 2025, peat was collected from mining strippings in the suburbs of Xilinhot, Inner Mongolia Autonomous Region, and the target strain was obtained through enrichment, screening, separation and purification.

[0071] At the beginning of the enrichment culture, 0.4g of ground peat was placed in a 150mL Erlenmeyer flask containing 40mL of inorganic salt liquid culture medium, and placed in a 30℃ incubator. The mixture was shaken (200rpm) for 30 days to obtain the enriched stock solution.

[0072] Then, the above-mentioned enrichment stock solution was diluted to 10. -5 , and its 10 -5 The diluted culture medium was spread onto 100 ml of inorganic salt solid culture medium containing 2 g of peat (i.e., containing 2% peat) and incubated in a 30°C incubator.

[0073] After the bacteria have grown on the petri dish, pick a single colony and isolate and purify the bacteria with different colony morphologies. The purification method is as follows: pick a single colony and streak it on a new inorganic salt solid medium using the streak plate method. Repeat 5 times to obtain a pure culture.

[0074] The purified strain was retained and designated as strain NT-1.

[0075] 2. Identification of strains (1) Colony morphology identification Strain NT-1 was cultured on an inorganic salt medium with peat as the sole carbon source, such as... Figure 1 As shown, the colonies of this strain are white.

[0076] (2) Gram staining identification Gram staining of strain NT-1 showed that strain NT-1 was Gram-negative.

[0077] (3) Molecular biological identification DNA of strain NT-1 was extracted using the TIANamp Bacteria DNA Kit. The 16S rRNA gene sequence of strain NT-1 was amplified by PCR using primer 27F (sequence: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:1)) and primer 1492R (sequence: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:2)).

[0078] The PCR amplification reaction system (25 μL) consisted of: 12.5 μL of 2×Taq Plus Master Mix (Takara, Japan), 1 μL of DNA template, 1 μL of primer 27F (SEQ ID NO:1), 1 μL of primer 1492R (SEQ ID NO:2), and ddH2O to a final volume of 25 μL.

[0079] The PCR amplification reaction conditions were: ① 94℃ for 5 min; ② 94℃ denaturation for 30 s; ③ 55℃ annealing for 30 s; ④ extension at 72℃ for 80 s; steps ② to ④ were repeated 28 times; 72℃ for 10 min; 4℃ for 10 min.

[0080] The PCR amplification products were sent to General Biotech for sequencing, and the sequence of the 16S rRNA gene of strain NT-1 was determined as follows: TCGCTGCCTCTCTTGCGAGTTAGCGCAACGCCTTCGGGTGAACCCAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCTGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGCCTTCATGCTCTCGAGTTGCAGAGAACAATCCGAACTGAGACGGCTTTTGGAGATTAGCGCACTCTCGCGAGTTTGCTGCCCACTGTCACCGCCATTGTAGCACGTGTGTAGCCCAGCGCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGCTTATCACCGGCGGTTCCTTTAGAGTCCCCAACTAAATGATGGTAACTAAAGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCAGTCCCCGAAGGGAAGAGATCCATCTCTGGAAATCGTCCGGACATGTCAAACGCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCAGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGATAACTTAATGCGTTAGCTGCGCCACCCAAAGACCAAGTCCCCGGACAGCTAGTTATCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAATACCAGTCCAGTGAGCCGCCTTCGCCACTGGTGTTCTTCCGAATATCTACGAATTTCACCTCTACACTCGGAATTCCACTCACCTCTCCTGGATTCAAGCGATGCAGTCTTAAAGGCAGTTCTGGAGTTGAGCTCCAGGCTTTCACCTCTAACTTACAAAGCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAACGCTAGCTCCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGAGCTTATTCTCCCGGTACTGTCA(SEQ ID NO:3)。

[0081] The obtained 16S rRNA gene sequence (SEQ ID NO:3) was compared with NCBI's BLASTN, and closely related species were screened to construct a phylogenetic tree of the 16S rRNA gene sequence, as shown below. Figure 2 As shown, strain NT-1 and Sphingomonas spp. Sphingomonas aquatilis The 16S rRNA gene sequence similarity of NBRC 16722 is 99.8%, therefore strain NT-1 belongs to the genus Sphingomonas (…). Sphingomonas sp.).

[0082] 3. Preservation of bacterial strains Strain NT-1 was deposited at the China Center for Type Culture Collection (CCTCC) on October 17, 2025, with accession number CCTCC NO: M 20252237 and accession name: Sphingomonas sp. NT-1, deposited at: Wuhan University, Wuhan, China, specifically No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0083] Example 2 Sphingosine monocytogenes (Sphingosine monocytogenes) Sphingomonas Physiological and biochemical experiments of sp.)NT-1 1. Phosphorus solubilization capacity test To evaluate the ability of strain NT-1 to degrade insoluble phosphorus and release soluble phosphorus, inorganic phosphorus culture medium and organic phosphorus culture medium were used for testing by molybdenum antimony spectrophotometry.

[0084] (1) Reagent preparation The formula for PKO inorganic phosphorus liquid medium is as follows: 1000 mL dH2O, 10.0 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 0.3 g MgSO4·7H2O, 5 g Ca3(PO4)2, pH 7 ± 0.2.

[0085] The formula for the Mongkina organophosphorus liquid culture medium is as follows: 1000 mL dH2O, 10.0 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 0.3 g MgSO4·7H2O, 0.2 g egg yolk lecithin, 5 g CaCO3, 0.4 g yeast extract, pH 7±0.2.

[0086] Weigh 1.5g of ascorbic acid and dissolve it in 100mL of molybdenum-antimony stock solution to obtain the molybdenum-antimony reagent, which should be prepared fresh before use.

[0087] (2) Test method Add 50 mL of PKO inorganic phosphorus liquid culture medium or 50 mL of Monkina organic phosphorus liquid culture medium to a 150 mL Erlenmeyer flask, and sterilize at 121 °C for 20 min.

[0088] Strain NT-1 was cultured to the logarithmic growth phase using R2A medium, and the seed culture was collected. The seed culture was then inoculated at 5% v / v into PKO inorganic phosphorus liquid medium and Monkina organic phosphorus liquid medium as experimental groups, with three replicates for each medium. The corresponding medium without NT-1 inoculation served as the control (CK). All cultures were incubated for 10 days in a constant temperature shaker at 30℃ and 150 rpm.

[0089] On days 2, 4, 7, and 10 of culture, 10 mL of culture medium was placed in 50 mL centrifuge tubes and centrifuged at 10000 rpm for 15 min at 4°C using a refrigerated centrifuge. Then, 5 mL of the supernatant, 45 mL of NaHCO3 solution (0.5 mol / L), and 1.9 g of phosphorus-free activated carbon were added to a 150 mL Erlenmeyer flask. The flask was sealed and shaken for 30 min (180 rpm). The mixture was then filtered through phosphorus-free filter paper, and the filtrate was collected. 1 mL of the filtrate, 5 mL of NaHCO3 (0.5 mol / L), 30 mL of dH2O, and 5 mL of colorimetric reagent (molybdenum antimony reagent) were added to a 50 mL volumetric flask and mixed well. After 30 min of color development, the absorbance of the culture medium was measured using the molybdenum antimony reagent spectrophotometric method, and the result was calculated according to the standard curve formula (y = 0.0524x - 0.0011, where x is the phosphorus concentration, y is the absorbance, R0 = ...). 2 The concentration of phosphorus was calculated using the formula (=0.9999), and the concentration difference between the experimental group and the control group was then used as the water-soluble phosphorus content in the bacterial solution.

[0090] (3) Test results The test results showed that when cultured with PKO inorganic phosphorus liquid medium, the water-soluble phosphorus content of strain NT-1 in the bacterial culture on days 2, 4, 7 and 10 was 0.05 μg / mL, 0.06 μg / mL, 1.78 μg / mL and 2.42 μg / mL, respectively; when cultured with Monkina organic phosphorus liquid medium, the water-soluble phosphorus content of strain NT-1 in the bacterial culture on days 2, 4, 7 and 10 was 0.03 μg / mL, 0.03 μg / mL, 0.04 μg / mL and 0.04 μg / mL, respectively.

[0091] This indicates that strain NT-1 has the ability to degrade both inorganic and organic phosphorus, especially in the efficient degradation of insoluble inorganic phosphorus into soluble inorganic phosphorus.

[0092] 2. Indoleacetic acid (IAA) production capacity test (1) Reagent preparation The King liquid culture medium formula is as follows: tryptophan 100 mg / L, tryptone 20 g, glycine 15 mL, K2HPO4 1.15 g, MgSO4·7H2O 1.5 g, dH2O 1000 mL, pH 7.

[0093] Prepare a 10.8M H2SO4 solution, then weigh 4.5g of FeCl3 and dissolve it in the 10.8M H2SO4 solution. After cooling, dilute to 1L with ultrapure water to obtain the S2 colorimetric solution.

[0094] (2) Test method Add 50 mL of King liquid culture medium to a 150 mL Erlenmeyer flask and sterilize at 121 °C for 20 min.

[0095] The strain NT-1 was cultured to the logarithmic growth phase using R2A medium. Seed culture was collected and inoculated into King liquid medium at an inoculation rate of 5% v / v. Three parallel replicates were set up. After inoculation, the culture was placed on a shaker at 30°C and 150 r / min for 5 days.

[0096] On days 2, 3, 4, and 5 of culture, 10 mL of culture medium was taken, centrifuged at 10000 r / min and 4℃ for 15 min, and 5 mL of the supernatant was collected and mixed with 5 mL of S2 colorimetric solution. The mixture was then incubated in the dark for 30 min, and the absorbance (wavelength 530 nm) of each culture medium was measured. The results were then analyzed using the standard curve formula (y = 0.0142x + 0.0327, where x is the IAA concentration, y is the absorbance, and R0 is the absorbance). 2 The concentration of IAA was calculated using the formula (=0.9984).

[0097] (3) Test results The test results showed that the indoleacetic acid (IAA) production of strain NT-1 was 19.74 mg / L, 20.94 mg / L, 24.08 mg / L, and 32.40 mg / L on days 2, 3, 4, and 5 of cultivation, respectively. This indicates that strain NT-1 has excellent IAA production capacity.

[0098] 3. Extracellular polysaccharide production experiment (1) Reagent preparation The fermentation medium formula is as follows: 25.0g sucrose, 10.0g NaCl, 0.5g NaNO3, 0.5g MgSO4, 0.5g K2HPO4, 3g yeast extract, 3g tryptone, 1000mL dH2O, pH 7.

[0099] (2) Experimental methods Prepare 50 mL of fermentation medium and sterilize it at high temperature in a 250 mL Erlenmeyer flask, then cool it. Inoculate strain NT-1 into the flask and incubate it on a shaker (30℃, 150 r / min) until it reaches the logarithmic growth phase (about 36 h), then collect the seed culture.

[0100] The seed culture was transferred into 100 mL of fermentation medium at an inoculation rate of 5% v / v, and three parallel replicates were set up. The culture was carried out on a shaker at 30℃ and 150 r / min for 4 days.

[0101] On days 2, 3 and 4 of culture, the fermentation broth was collected, centrifuged at 3600 r / min for 5 min, and 5 mL of the supernatant was collected. 20 mL of anhydrous ethanol was added to precipitate the polysaccharide. After standing at 4℃ for 1 h, the mixture was centrifuged at 3000 r / min for 10 min, the supernatant was discarded, and the precipitate was dissolved in 2 mL of distilled water to prepare the crude polysaccharide extract.

[0102] Prepare a 1 g / L glucose solution. Pipette 16 μL, 24 μL, 32 μL, 40 μL, 48 μL, 56 μL, 64 μL, and 72 μL of the 1 g / L glucose solution into 1.5 mL centrifuge tubes. Add distilled water to each tube to a final volume of 80 μL. Then add 40 μL of freshly prepared phenol solution (6% phenol concentration) and 200 μL of concentrated sulfuric acid. Mix well, cool, and incubate at room temperature for 20 min. Measure the absorbance (wavelength 490 nm). The fitted standard curve formula is y = 0.0381x + 0.2256, where x is the glucose concentration, y is the absorbance, and R0 is the absorbance. 2 =0.9924.

[0103] Add 80 μL of crude polysaccharide extract to each tube, measure the absorbance value according to the above-described phenol-sulfuric acid method, and calculate the extracellular polysaccharide content based on the standard curve formula.

[0104] (2) Experimental results The results showed that the extracellular polysaccharide content in the NT-1 bacterial culture was 186 mg / L, 211 mg / L, and 224 mg / L on days 2, 3, and 4, respectively. This indicates that strain NT-1 has excellent extracellular polysaccharide production capacity.

[0105] 4. Growth temperature test (1) Test method Strain NT-1 was cultured to the logarithmic growth phase on R2A medium, and the seed culture was collected. The seed culture was then inoculated into R2A liquid medium (pH 7, NaCl concentration 0%) at an inoculum rate of 5% v / v. The culture was then incubated at 4℃, 10℃, 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃ for 72 h, respectively. After the incubation period, the OD of the bacterial culture was measured. 600 .

[0106] (2) Test results The results showed that the OD of strain NT-1 cultured at 4℃, 10℃, 20℃, 25℃, 30℃, 35℃, 40℃ and 45℃ were significantly different. 600 The values ​​were 0.20, 0.35, 0.75, 0.90, 1.20, 0.85, 0.75 and 0.40, respectively, indicating that strain NT-1 can grow at temperatures ranging from 20℃ to 40℃, with the optimal growth temperature being 30℃.

[0107] 5. Growth pH test (1) Test method Strain NT-1 was cultured to the logarithmic growth phase on R2A medium, and the seed culture was collected. The seed culture was then inoculated at a rate of 5% v / v into R2A liquid medium (0% NaCl) at different pH values ​​(4, 5, 6, 7, 8, 9, 10, 11, and 12). The culture was then incubated at 30°C for 72 h. After incubation, the OD value of the bacterial culture was measured. 600 .

[0108] (2) Test results The results showed that the OD of strain NT-1 cultured under pH conditions of 4, 5, 6, 7, 8, 9, 10, 11, and 12 was significantly lower. 600 The values ​​were 0.10, 0.80, 0.78, 1.20, 0.88, 0.75, 0.10, 0.10 and 0.10, respectively, indicating that strain NT-1 can grow well under pH conditions of 5 to 9, with the optimal growth pH being 7.

[0109] 6. Salinity test for growth (1) Test method Strain NT-1 was cultured to the logarithmic growth phase on R2A medium, and the seed culture was collected. The seed culture was then inoculated at a rate of 5% v / v into R2A liquid medium (pH 7) with different salinities (NaCl concentrations of 0%, 1%, 2%, 3%, 4%, and 5%). The culture was then incubated at 30°C for 72 h. After incubation, the OD value of the bacterial culture was measured. 600 .

[0110] (2) Test results The results showed that the OD of strain NT-1 cultured under NaCl concentrations of 0%, 1%, 2%, 3%, 4%, and 5% were significantly different. 600 The values ​​were 1.20, 0.90, 0.40, 0.30, 0.10, and 0.10, respectively, indicating that strain NT-1 can grow under conditions with a salinity (NaCl concentration) of 0–1%.

[0111] The above results indicate that strain NT-1 is a potential growth-promoting strain with the ability to solubilize phosphorus, produce indoleacetic acid and extracellular polysaccharides, and grow using peat as the sole carbon source. It can grow under conditions of 20℃ to 40℃, pH 5 to 9, and salinity (NaCl concentration) of 0 to 1%.

[0112] Example 3 Sphingosine monocytogenes (Sphingosine monocytogenes) Sphingomonas The growth-promoting effect of sp.)NT-1 on plants To clarify the growth-promoting effect of strain NT-1 on plants, this example uses alfalfa and crested wheatgrass as test plants, and peat collected from mining strips in the Xilinhot suburbs and Xiwuqi mining area as test substrates for pot experiments.

[0113] 1. Experimental Methods (1) Preparation of planting substrate Crush the peat moss to an average particle size of about 0.5 mm, and pour 2 kg of peat moss evenly into the flower pot for later use.

[0114] (2) Preparation of bacterial suspension The strain NT-1 was cultured in R2A medium to the logarithmic growth phase. The seed culture was collected and inoculated into sterilized R2A liquid medium at an inoculation rate of 5% v / v. The medium was then placed on a shaker and cultured at 30°C and 150 r / min until the logarithmic growth phase (approximately 36 h).

[0115] Collect the culture medium, centrifuge at 5000 r / min for 10 min, discard the supernatant, add sterile water to the remaining bacterial cells to prepare a bacterial suspension, and prepare 400 mL of bacterial suspension for each 1 L of culture medium for later use.

[0116] (3) Sowing 1) Alfalfa Select alfalfa seeds of the same size and plumpness, and evenly scatter 20 alfalfa seeds into each flowerpot at a depth of 1.5cm.

[0117] 2) Leymus chinensis Select Winged Leymus seeds of the same size and plumpness, and evenly scatter 20 Winged Leymus seeds into each flowerpot at a depth of 1.5cm.

[0118] (4) Grouping and irrigation treatment Two plant species were each set up as a treatment group (NT-1) and a control group, with three parallel replicates in each group.

[0119] The treatment group was irrigated daily with 400 mL of bacterial suspension to ensure that each pot had an inoculum quantity of 8.0 × 10⁻⁶. 8 The concentration of cfu / mL was above 100 mg / mL, and the solution did not overflow the flowerpot but completely seeped into the soil. The control group was watered with the same amount of sterile water.

[0120] (5) Observation and index measurement Observe the seedling emergence in the flowerpots daily, record the number of germinations each day after emergence, and calculate the germination rate. The experiment lasted for 40 days after emergence.

[0121] After one month of cultivation, three plants were randomly selected from each group to measure their plant height and biomass (i.e., the dry weight of the whole plant).

[0122] To investigate the growth-promoting mechanism of strain NT-1 and clarify the changes in carbon and nitrogen in peat, plants and peat from each group were collected for physicochemical analysis after the pot experiment. The peat and plants were dried (60℃, 5h) and ground, then sent to the testing platform of the College of Ecology and Environment, Inner Mongolia University to determine the total carbon (TC), total nitrogen (TN), and ammonia nitrogen (NH4) in the peat. + ), nitrate nitrogen (NO3) - The contents of available phosphorus and available potassium were measured, and the contents of total carbon (TC) and total nitrogen (TN) in the aboveground and underground parts of the plant were determined.

[0123] 2. Experimental Results (1) Plant growth like Figure 3 As shown in A to D, compared with the control group, the irrigation strain NT-1 significantly promoted the growth of alfalfa and crested wheatgrass. The plants grew more vigorously overall, with thicker stems, more leaves and darker green color, and a more developed root system. Not only were the taproots thicker, but the number of fibrous roots and root hairs were also dense, forming a huge absorption network, showing a stronger ability to absorb nutrients and water.

[0124] Table 1. Plant height and biomass of plants in different mining areas (mean ± standard deviation)

[0125] Note: , , The values ​​indicate that the differences between the treatment group and the control group were statistically significant at the levels of P<0.05, P<0.01, and P<0.001, respectively.

[0126] As shown in Table 1, irrigation with strain NT-1 significantly increased the plant height and biomass of alfalfa and crested wheatgrass grown on peat substrate in the two mining areas.

[0127] For alfalfa, the plant height of the treatment group was 2.9 to 3.7 times that of the control group, and the biomass was 9.7 to 13 times that of the control group. Among them, the plant height and biomass of alfalfa grown using peat in the mining area of ​​Xilinhot suburbs increased by 2.7 times and 12 times, respectively, while the plant height and biomass of alfalfa grown using peat in the mining area of ​​Xiwuqi increased by 1.9 times and 8.7 times, respectively.

[0128] For Leymus chinensis, the plant height and biomass of the treatment groups were 2.6–2.7 times and 2.5–3.9 times that of the control group, respectively. Among them, the plant height and biomass of alfalfa planted on peat in the Xilinhot suburban mining area increased by 1.6 times and 1.5 times, respectively, while the plant height and biomass of alfalfa planted on peat in the Xiwuqi mining area increased by 1.7 times and 2.9 times, respectively.

[0129] Overall, when pure peat derived from mining strippings was used as a substrate, application of strain NT-1 significantly promoted plant growth.

[0130] (2) Nutrient analysis of peat Table 2. Effects of strain NT-1 on peat nutrients in the mining area (mean ± standard deviation)

[0131] Note: , , The values ​​indicate that the differences between the treatment group and the control group were significant at the P<0.05, P<0.01, and P<0.001 levels, respectively.

[0132] As shown in Table 2, the NT-1 strain irrigation treatment significantly increased the content of total carbon, total nitrogen, available potassium and available phosphorus in peat in the two mining areas, and significantly reduced the content of ammonia nitrogen and nitrate nitrogen.

[0133] For alfalfa, compared with the control group, the total carbon in the peat of the treatment group in Xilinhot mining area increased by 31.53%, total nitrogen increased by 45.83%, available phosphorus increased by 22.67%, available potassium increased by 21.24%, ammonia nitrogen decreased by 25.29%, and nitrate nitrogen changed from positive to negative, decreasing by 24 times. Compared with the control group, the total carbon in the peat of the treatment group in Xiwuqi mining area increased by 23.53%, total nitrogen increased by 81.25%, available phosphorus increased by 27.47%, available potassium increased by 10.97%, ammonia nitrogen decreased by 21.52%, and nitrate nitrogen also changed to negative, decreasing by 10 times.

[0134] For *Leymus chinensis*, compared with the control group, the treatment group in Xilinhot mining area showed an increase of 11.68% in total carbon, 12.00% in total nitrogen, 71.05% in available phosphorus, 48.13% in available potassium, a decrease of 26.86% in ammonia nitrogen, and a decrease of 433.33% in nitrate nitrogen. Compared with the control group, the treatment group in Xiwuqi mining area showed an increase of 18.52% in total carbon, 90.91% in total nitrogen, 101.82% in available phosphorus, 41.54% in available potassium, a decrease of 35.97% in ammonia nitrogen, and a decrease of 360.00% in nitrate nitrogen.

[0135] The above changes in peat nutrient content indicate that the application of strain NT-1 significantly increased the nutrient content of the peat, especially the content of available phosphorus, available potassium, and total nitrogen. Nitrogen, phosphorus, and potassium are key nutrients required for plant growth, and the increase in these nutrients ensured plant growth.

[0136] Furthermore, strain NT-1 possesses plant-promoting abilities such as phosphorus solubilization, indoleacetic acid production, and polysaccharide production, which also supports the increase in peat nutrients. Rapid plant growth leading to nutrient uptake from the peat may have resulted in a decrease in nitrate and ammonia nitrogen levels in the peat.

[0137] Meanwhile, the increase in total carbon and total nitrogen in peat may be due to rapid plant growth, which releases carbon and nitrogen through secretions, thus increasing the total carbon and total nitrogen content in peat.

[0138] (3) Nutrient analysis of plants Table 3. Effects of strain NT-1 on plant nutrients (mean ± standard deviation)

[0139] Note: , , The values ​​indicate that the differences between the treatment group and the control group were significant at the P<0.05, P<0.01, and P<0.001 levels, respectively.

[0140] As shown in Table 3, irrigation treatment with strain NT-1 significantly increased the total aboveground carbon, total aboveground nitrogen, and total underground nitrogen of peat-grown plants in the two mining areas, while significantly reducing the total underground carbon of the plants.

[0141] For alfalfa, compared with the control group in the Xilinhot suburban mining area, the treatment group showed a 38.64% increase in total aboveground carbon, a 143.64% increase in total aboveground nitrogen, a 61.21% increase in total underground nitrogen, and a 3.47% decrease in total underground carbon. Compared with the control group in the Xiwuqi mining area, the treatment group in the peat-grown alfalfa showed a 51.46% increase in total aboveground carbon, a 121.80% increase in total aboveground nitrogen, a 66.67% increase in total underground nitrogen, and a 3.86% decrease in total underground carbon.

[0142] For *Leymus chinensis*, compared with the control group in the Xilinhot suburban mining area, the treatment group showed a 33.51% increase in total aboveground carbon, a 192.52% increase in total aboveground nitrogen, a 63.89% increase in total underground nitrogen, and a 6.36% decrease in total underground carbon. Compared with the control group in the Xiwuqi mining area, the treatment group showed a 38.63% increase in total aboveground carbon, a 240.43% increase in total aboveground nitrogen, an 80.53% increase in total underground nitrogen, and an 8.86% decrease in total underground carbon.

[0143] The above changes in plant nutrients indicate that the application of strain NT-1 significantly increased the nutrient content of peat-grown plants and increased the accumulation of total nitrogen in both aboveground and belowground parts.

[0144] Based on the plant growth and peat nutrient status, it can be explained that the decrease in ammonia nitrogen and nitrate nitrogen in peat is due to the rapid growth of plants, during which they absorb and utilize large amounts of ammonia nitrogen and nitrate nitrogen from peat and fix them within the plant body.

[0145] In addition, the application of strain NT-1 increased the total carbon in the aboveground parts of the plant, which may be related to enhanced photosynthesis, while the decrease in total carbon in the underground parts may be related to the release of some carbon into the peat as root exudates. This carbon released in the form of root exudates also increased the total carbon content in the peat.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Sphingosine monocytogenes (SSM-1) Sphingomonas sp.)NT-1, characterized in that, The Sphingosomalidone ( Sphingomonas The accession number for sp.)NT-1 is CCTCC NO: M 20252237.

2. A microbial inoculant, characterized in that, Contains the Sphingosine Monoclonal strain of claim 1 ( Sphingomonas sp.)NT-1, or its freeze-dried cells, or its bacterial suspension, or the supernatant or precipitate resuspension after centrifugation of the bacterial suspension.

3. The Sphingosine Monoclonal strain as described in claim 1 ( Sphingomonas The use of the microbial agent of sp.)NT-1 or claim 2 in phosphorus solubilization, production of indoleacetic acid and / or production of extracellular polysaccharides.

4. The Sphingosine Monoclonal strain as described in claim 1 ( Sphingomonas The use of the microbial agent described in sp.)NT-1 or claim 2 in promoting plant growth and / or preparing fertilizer.

5. A fertilizer, characterized in that, Contains the Sphingosine Monoclonal strain of claim 1 ( Sphingomonas sp.)NT-1 or the microbial agent of claim 2.

6. The Sphingosine Monoclonal strain according to claim 1 ( Sphingomonas The application of the microbial agent of claim 2 or the fertilizer of claim 5 in improving soil in mining areas, preparing soil for reclamation in mining areas and / or restoring vegetation in mining areas.

7. The Sphingosine Monoclonal strain according to claim 1 ( Sphingomonas sp.)NT-1, the microbial agent of claim 2 or the fertilizer of claim 5, in any one of the following (1) to (4): (1) Increase the nutrient content of the stripped material in the mining area; (2) Increase the nutrient content of plants grown using mining strips; (3) Promote the germination and / or growth of plants grown from mining strips; (4) Increase the plant height and / or biomass of plants grown from mining strips.

8. The application according to claim 7, characterized in that, The strippings from the mining area include peat.

9. The application according to claim 7 or 8, characterized in that, The plants include pasture grasses.

10. A method for restoring vegetation in a mining area, characterized in that, Includes the following steps: Plants were grown using stripped material from the mining area, followed by application of the Sphingomonas strain described in claim 1. Sphingomonas sp.)NT-1, the microbial agent of claim 2, or the fertilizer of claim 5.

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