Strain for increasing available phosphorus content of coal-based solid waste and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a strain for increasing the available phosphorus content of coal-based solid waste and application of the strain. The strain is Acinetobacter sp., and is preserved in the China General Microbiological Culture Collection Center on September 26, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.36083. The invention further discloses a preparation method of the acinetobacter sp. The strain provided by the invention has excellent phosphate solubilizing capacity, and provides a new way for ecological restoration of the abandoned mining area.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that increases the effective phosphorus content of coal-based solid waste and its application. Background Technology

[0002] In recent years, renewable energy has gradually gained global attention and is widely regarded as a potential alternative to fossil fuels. However, fossil fuels such as coal still dominate the world's energy landscape. Coal mining, washing, and processing generate a large amount of coal-based solid waste, with coal gangue being the most abundant. Coal gangue is a dark gray rock with a lower carbon content and calorific value than coal, accounting for approximately 15% to 20% of coal production. With the increasing use of fossil fuels such as coal, the stockpile of coal gangue is also growing rapidly.

[0003] Because coal gangue contains elements such as carbon and phosphorus, its development and utilization have made some progress. However, most of the nutrients in coal gangue exist in a form that is difficult to decompose, resulting in low nutrient release efficiency. The nutrients released into the soil are difficult for plants to absorb. Therefore, the utilization of coal gangue often involves applying microorganisms to provide attachment sites for microorganisms, thereby improving the soil microbial community structure and utilizing the metabolism of microorganisms to promote the nutrient conversion of coal gangue.

[0004] Numerous studies have explored the use of microorganisms to release available nutrients from coal gangue, aiming to address gangue accumulation and improve soil fertility. However, few studies have considered the ecological cycle following nutrient release. Therefore, utilizing phosphorus-solubilizing microorganisms to promote nutrient release from coal gangue, enhance soil fertility, and boost plant growth would significantly contribute to the ecological restoration of abandoned mining areas. Summary of the Invention

[0005] The purpose of this invention is to provide a strain that increases the effective phosphorus content of coal-based solid waste, using microorganisms to release effective nutrients from coal gangue to solve the problem of coal gangue accumulation and improve soil fertility.

[0006] The technical solution adopted in this invention is: a strain that increases the effective phosphorus content of coal-based solid waste, which is Acinetobacter bacillus. Acinetobacter sp. It was deposited on September 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 36083.

[0007] Preferably, the strain is used in the dissolution of insoluble phosphates in soil or in the soil-based utilization of coal-based solid waste.

[0008] The present invention also provides a method for dissolving insoluble phosphates in soil or for the soil-based utilization of coal-based solid waste, wherein the method comprises adding the culture medium of the strain to the soil or coal-based solid waste.

[0009] The present invention also provides a product for the soil-based utilization of insoluble phosphates or coal-based solid waste in soil, characterized in that it contains a culture of the strain.

[0010] The strain provided by this invention has excellent phosphorus solubility, offering a new approach for the ecological restoration of abandoned mining areas. Attached Figure Description

[0011] Figure 1 The degradation of strain SXLF09 on inorganic phosphorus plates; Figure 2 The growth of strain SXLF09 on LB plates; Figure 3 The effect of adding strain SXLF09 on the available phosphorus content in inorganic phosphorus medium; Figure 4 The effect of adding strain SXLF09 on the available phosphorus content in soil; Figure 5 The effect of adding strain SXLF09 on alfalfa biomass. Detailed Implementation

[0012] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following embodiments or drawings are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0013] I. Screening of Inorganic Phosphorus-Relieving Microorganisms 10 g of soil from a coal gangue pile was added to 500 ml of an inorganic salt culture medium containing a small amount of nutrients and incubated at 37℃ and 120 rpm for 48 hours. The coal gangue powder had a particle size of 300 mesh. The inorganic salt culture medium contained the following components: 2.5 g / L coal gangue powder, 1 g / L glucose, 1 g / L peptone, 1 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 2.5 g / L sodium chloride, 2 g / L calcium chloride, and pH 7.0. The culture medium was contained in 1000 ml Erlenmeyer flasks. Before adding the inoculum, the flasks containing the culture medium were covered with sterile paper and sterilized at 120℃ for 20 minutes. After cooling to room temperature, the inoculum was added, and the culture was carried out under the conditions described above.

[0014] The cultured bacterial suspension was spread onto an inorganic phosphorus solid medium for separation and purification. First, 100 μL of the bacterial suspension (sterilized at 120°C for 20 minutes) was pipetted into 900 μL of sterile deionized water and vortexed thoroughly. The sterile deionized water was then placed in a sterile 2 mL centrifuge tube, thus diluting the bacterial suspension 10-fold. Then, 100 μL of the 10-fold diluted bacterial suspension was added to 900 μL of sterile deionized water and vortexed thoroughly, thus diluting the bacterial suspension 100-fold. This process was repeated until the bacterial suspension was diluted 10-fold. -9 Diluted 10 times. The solution was aspirated from the tip of a 200µL pipette, which had been sterilized at 120°C for 20 minutes. -1 10 -3 10 -5 10 -7 10 -9 100 μL of bacterial suspension was spread onto inorganic phosphorus solid medium and incubated at 37°C for 3 days. The inorganic phosphorus solid medium consisted of: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.5 g / L yeast extract, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, 0.03 g / L manganese sulfate, 5 g / L tricalcium phosphate, 20 g / L agar powder, and a pH of 7.0. Disposable sterile petri dishes with a diameter of 10 cm were used for the solid medium. After incubation, single colonies were picked using a 1 μL sterile inoculation loop and repeatedly isolated and purified on the solid inorganic phosphorus medium using the streak plating method. Each purification was incubated at 37°C for 3 days until single colonies of the same species were visible to the naked eye on the plate.

[0015] II. Identification of the inorganic phosphorus solubility of the strain

[0016] To assess the inorganic phosphorus solubility, a single colony from (1) was picked using a 1 μL sterile inoculation loop and streaked onto a solid inorganic phosphorus medium. After inoculation, the medium was incubated at 37°C for 3 days. The solid inorganic phosphorus medium used was composed of: 10 g / L glucose, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L calcium triphosphate, 20 g / L agar powder, and a pH of 7.0. The culture dish used for the inorganic phosphorus medium was a disposable sterile culture dish with a diameter of 10 cm.

[0017] Observe the colony for signs of degradation after 3 days. Figure 1The presence of a distinct hydrolysis zone in the inorganic phosphorus medium indicates that the bacterium possesses phosphorus-solubilizing ability. Strains exhibiting the ability to dissolve insoluble inorganic phosphorus were picked using a 1 μL sterile inoculation loop and briefly preserved on LB agar. The selected LB agar composition was: 10 g / L beef extract peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0018] Among the bacterial strains capable of dissolving insoluble phosphates, strain SXLF09 exhibits the best solubility. After verification in LB medium for 18-24 hours, the selected strains typically form circular colonies approximately 1-3 mm in diameter. The colony edges are intact and smooth. The colonies are opaque, pale yellow, with a smooth, moist, and glossy surface. The colonies usually exhibit a low-convex to convex shape, such as... Figure 2 As shown in the figure. 16S rRNA gene sequencing was performed on the pure culture in LB medium, and the sequence is shown in SEQ ID NO. 1 of the sequence listing. The PCR product was sequenced, and the sequencing results were compared with the NCBI database. The results showed that the strain belonged to the genus Acinetobacter. Furthermore, comparison with existing known Acinetobacter gene sequences (see Table 1) revealed differences in the base sequences. Therefore, strain SXLF09 was identified as a novel strain.

[0019] Table 1. Alignment results of 16S rRNA with strain SXLF09 in Genebank .

[0020] The strain SXLF09 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 26, 2025, with accession number CGMCC NO. 36083. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] III. Determination of Phosphate Solubilization Capacity of Strains Using a Flow Analyzer

[0022] To investigate the phosphate-solubilizing ability of strain SXLF09, a seed culture of the strain was first prepared. 7 ml of liquid LB medium was added to a 15 ml glass tube and sterilized at 120 °C for 20 minutes. Colonies from the LB solid medium in (3) were picked up using a 1 μl sterile inoculation loop and inoculated into a 15 ml test tube containing 7 ml of liquid LB medium. After inoculation, the tube was incubated at 37 °C and 150 rpm for 12 hours. 1.5 ml of the cultured bacterial solution was transferred to a 2 ml centrifuge tube sterilized at 120 °C for 20 minutes using a disposable sterile dropper. The tube was centrifuged at 6000 rpm for 3 minutes, and the supernatant was discarded. This process was repeated 3 times until cells the size of soybeans appeared. 2 ml of PBS buffer was added, and the tube was centrifuged at 6000 rpm for 3 minutes. The supernatant was discarded, and this process was repeated 2-3 times. The last time, the buffer was retained, thus obtaining the seed culture of the strain.

[0023] Next, the seed culture needs to be inoculated into liquid inorganic phosphorus medium for fermentation. First, add 30 ml of liquid inorganic phosphorus medium to a 50 ml centrifuge tube and sterilize at 120°C for 20 minutes. Using a sterilized 1000 μL pipette tip, aspirate 1000 μL of the bacterial culture from the seed culture and inoculate into the fermentation medium. Incubate at 37°C and 150 rpm for 3 days. The composition of the inorganic phosphorus liquid medium is: glucose 10 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, tricalcium phosphate 5 g / L, pH 7.0.

[0024] Take 20 mL of the above fermentation broth and place it into a 50 mL centrifuge tube that has been sterilized at 120°C for 20 minutes. Centrifuge at 6000 rpm for 12 minutes. After centrifugation, collect the supernatant and filter it through phosphorus-free filter paper. The sample preparation is now complete. Finally, calculate the available phosphorus content based on the absorbance value in the flow analyzer. A standard curve needs to be plotted before the calculation.

[0025] The principle of flow analyzer determination of available phosphorus: The sample forms an antimony-phosphate-molybdate complex in acidic ammonium molybdate and potassium antimony tartrate solution. Ascorbic acid and vitamin C are used as the colorimetric reagent, and the result is measured at 880 nm. The standard curve is plotted as follows: 0 ml, 300 ml, 600 ml, 900 ml, 1200 ml, 1500 ml, and 1800 ml of a 100 ppm phosphorus standard solution are added to 50 ml volumetric flasks, and deionized water is added to bring the volume to 50 ml. The standard curve is determined using a flow analyzer with different concentrations of phosphorus solutions, and a regression equation is plotted. The 100 ppm phosphorus standard solution is prepared as follows: 0.4394 g of dipotassium hydrogen phosphate is dried at 150°C for 2 hours, then dissolved in 800 ml of deionized water. The solution is transferred to a 1 L volumetric flask and diluted to 1 L with water. This yields a 100 ppm phosphorus standard solution, which should not be stored for extended periods.

[0026] Solution preparation: Sulfuric acid solution, ammonium molybdate tetrahydrate solution: 40 ml concentrated sulfuric acid diluted in 800 ml distilled water, add 4.8 g ammonium molybdate tetrahydrate, and make up to 1000 ml; Potassium antimony tartrate stock solution: 300 mg potassium antimony tartrate dissolved in 80 ml distilled water, and make up to 100 ml; Ascorbic acid solution: 4.5 g L(+) ascorbic acid vitamin C dissolved in 200 ml distilled water, and make up to 250 ml.

[0027] The phosphate-solubilizing ability of strain SXLF09 was statistically analyzed using an Excel spreadsheet and plotted using Origin 2021. The results are as follows: Figure 3 As shown, this indicates that it can dissolve insoluble phosphates in the culture medium and release available phosphorus.

[0028] IV. Determination of the solubility of strain SXLF09 in insoluble phosphates from coal gangue using a flow analyzer After verifying that strain SXLF09 has good phosphorus-solubilizing ability, its ability to release available phosphorus from coal gangue was investigated. First, sterile coal gangue soil was prepared. Coal gangue that had passed through a 10-mesh sieve was mixed with soil at a 1:1 ratio and sterilized at 120℃ for 20 minutes. A soil experiment was conducted for 7 days, and samples were taken on day 0, day 3, and day 7. The experiment was designed with two treatments: one with and one without inoculation, and 12 replicates for each treatment. Following the method for preparing the seed solution in (3), the strain seed solution was prepared and inoculated into the inoculated treatment. The control treatment was given the same volume of PBS solution to keep the variables consistent. After sampling, the available phosphorus in the soil was extracted using the KCl extraction method, and the available phosphorus content in the soil was determined by a flow analyzer. The phosphorus-solubilizing ability of strain SXLF09 was statistically analyzed using an Excel spreadsheet and plotted using Origin 2021. The results are as follows. Figure 4 As shown, the addition of strain SXLF09 can effectively increase the available phosphorus content in the soil.

[0029] V. Application of strain SXLF09 in the soil utilization of coal-based solid waste First, sterile coal-based solid waste was prepared. The coal-based solid waste, which had passed through a 10-mesh sieve, was mixed with soil at a 1:1 ratio. The composition of the coal-based solid waste was soil: coal gangue: fly ash: desulfurized gypsum = 46:1.85:3.15. The mixture was sterilized at 120℃ for 20 minutes. Alfalfa was planted on the soil substrate for a two-month soil experiment. After the experiment, the plants were harvested and their biomass was measured. The phosphorus solubilization capacity of strain SXLF09 was statistically analyzed using Excel and plotted using Origin 2021. The results are shown below. Figure 5 As shown, strain SXLF09 can promote the growth of alfalfa and increase its biomass.

Claims

1. A bacterial strain that enhances the available phosphorus content in coal-based solid waste, characterized in that, This strain is Acinetobacter. Acinetobacter sp. It was deposited on September 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 36083.

2. The application of the strain described in claim 1 in the soil-based utilization of insoluble phosphates or coal-based solid waste.

3. A method for dissolving insoluble phosphates in soil or utilizing coal-based solid waste in soil, characterized in that, Add the culture medium of the strain described in claim 1 to soil or coal-based solid waste.

4. A product for the soil-based utilization of insoluble phosphates or coal-based solid waste, characterized in that... A culture containing the strain of claim 1.

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