Priesteria gigantea and its application

Abstract

The present invention belongs to the field of microbial treatment technology, and specifically relates to a Priesteria gigantea strain and its application, which has the ability to efficiently dissolve Ca from coal gangue. 2+ The functional strain is used to treat coastal saline soil. The dissolved free metal ions can accurately target the Na + , by virtue of ion exchange, Na + Desorption and discharge of cultivated soil from saline soil bodies can effectively improve the high salinity and single composition problems of coastal saline soil, and provide advanced and reliable technical means for the ecological restoration and agricultural utilization of coastal saline soil.

Description

Technical Field

[0001] The present invention belongs to the field of microbial treatment technology, and specifically relates to a Priesteria gigantea and its application, and in particular to a strain capable of dissolving Ca2+ from coal gangue at a high price. 2+ Functional bacteria that absorb high-valent metal cations such as cations can achieve efficient treatment of coastal saline soil based on soil colloid adsorption and ion replacement. Background Art

[0002] Coastal saline soils are characterized by heavy surface salt accumulation, a salt composition dominated by a single sodium salt, and a high pH. Therefore, traditional saline-alkali soil improvement methods are not fully applicable. However, coal gangue, rich in calcium and other valuable metal elements, has great potential for regulating soil pH, replacing sodium ions, and alleviating salt damage. Furthermore, as a potential driving force for coastal saline soil remediation, coal gangue has a significant stockpile and annual growth rate. If coal gangue resources can be rationally utilized, they could potentially be developed into a valuable resource for coastal saline soil remediation, thereby synergizing coastal saline soil remediation with the efficient and value-added utilization of coal gangue, achieving simultaneous maximum benefits.

[0003] The main factor limiting the application of coal gangue in coastal saline soil remediation is the difficulty in efficiently utilizing high-valent metal elements such as calcium. The calcium in coal gangue can exist in various forms, such as calcium carbonate and calcium silicate, requiring acidic conditions for the slow release of calcium ions. Furthermore, coastal saline soils are inherently high in salinity, and large amounts of sodium salts can undergo ion exchange reactions with calcium in the coal gangue, potentially exchanging calcium ions already transferred to the surface of soil particles, making it difficult for calcium to remain stable and function in the soil. Soil microorganisms play a key role in the transformation and utilization of calcium, but currently, no microbial strains have been reported that can both adapt to the high-salinity environment of coastal saline soils and effectively enhance calcium dissolution from coal gangue. Summary of the Invention

[0004] The purpose of the present invention is to provide a Priesteria gigantea strain and its application, which has the ability to efficiently dissolve Ca from coal gangue, in order to solve the problem of low dissolution capacity of high-valent metal elements in coal gangue in coastal salt soil treatment applications. 2+ The function of high-valent metal cations such as cations can be realized, and based on the soil colloid adsorption ion replacement, efficient treatment of coastal saline soil can be achieved.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides Priestia megaterium, characterized in that the strain is classified and named Priestia megaterium H16, and was deposited in the China Center for Type Culture Collection on December 12, 2024, with a deposit number of CCTCC NO: M 20242801.

[0007] The present invention uses a functional selection culture medium that retains only the basic carbon source, nitrogen source and buffer system, and replaces trace elements with coal gangue as the enrichment condition. Samples are collected from the rhizosphere soil of the artificially designed culture and acclimation system. Through multi-stage enrichment and separation and purification, a salt-tolerant strain with the ability to efficiently dissolve Ca from coal gangue is screened. 2+ Functional microbial strains of high-valent metals (Priesteria gigantea 16, Priestia megaterium H16 ).

[0008] Priesteria gigantea H16 is an aerobic, Gram-negative bacterium. It forms pale white, smooth, round, raised colonies with intact margins on standard beef extract-peptone agar. Priesteria gigantea H16 exhibits good growth at pH values ​​between 4 and 9. Exogenous addition of 1% (w / v) NaCl promotes its growth. When NaCl tolerance is ≤ 8% (w / v), the OD600 / 9-hour interval can reach 0.18-0.35.

[0009] The strain has the ability to efficiently dissolve Ca from coal gangue 2+ The functional strain is used to treat coastal saline soil. The exchangeable metal ions dissolved can accurately target the Na adsorbed by soil colloids. + , by virtue of ion exchange, Na + Desorption and discharge of saline colloids from the cultivated soil can effectively improve the high salinity and single composition problems of coastal saline soil, and provide advanced and reliable technical means for the ecological restoration and agricultural utilization of coastal saline soil.

[0010] A second aspect of the present invention provides a microbial agent containing the aforementioned Priesteria gigantea.

[0011] Furthermore, it includes bacterial strains, bacterial liquid or fermentation liquid.

[0012] The third aspect of the present invention provides the use of the above-mentioned microbial agent in dissolving high-valent metal ions in coal gangue.

[0013] Furthermore, the high-valent metal ions include calcium ions.

[0014] The functional microbial strain of the present invention is used to treat coal gangue slag in a liquid culture medium environment, and Ca 2+The initial concentration was 26.37±1.11 μg·mL -1 , according to the inoculation of Priesteria gigantea H16 ( Priestia megaterium H1 6) After strain, Ca 2+ The concentration of ions increased rapidly, reaching 60.32±2.64 μg·mL at the 9th hour of inoculation. -1 , reaching a peak of 70.11±3.71 μg·mL at the 24th hour -1 , the dissolution efficiency was increased by 130.58 times compared with the uninoculated strain.

[0015] A fourth aspect of the present invention provides the use of the above-mentioned microbial agent in the treatment of coastal saline soil.

[0016] Furthermore, the treatment of coastal saline soil includes promoting the discharge of salt from the coastal saline soil.

[0017] Furthermore, the treatment of coastal saline soil includes reducing the total salinity of coastal saline soil.

[0018] The functional microorganisms of the present invention were used to enhance the improvement of coastal saline soil by coal gangue. Coal gangue was added to the coastal saline soil at a ratio of 10% (w / w) and the mixture was added at a ratio of 5 mL·kg -1 ratio, spraying Priesteria grandis H16 ( Priestia megaterium H1 6) The bacterial solution was added and natural precipitation was simulated for irrigation. The original total salt content of the soil in the plough layer (0-20 cm depth) was 4.27±0.10 g·kg -1 , after treatment with this technology, it can be reduced to 2.03±0.06 g·kg -1 ; The original sodium ion content was 812.85±25.78 mg·kg -1 , after treatment with this technology, it can be reduced to 277.34±15.07 g·kg -1 The Ca / Na ratio increased from 0.60±0.08 to 0.91±0.07. The principle of the present invention is that the bio-enhanced dissolution of high-valent metals in coal gangue leads to the formation of Ca2+ at the soil colloid interface. 2+ etc. for Na + At the same time, the significant increase in the Ca / Na ratio in the soil can not only promote the improvement of coastal saline soil, but also promote its further utilization.

[0019] A fifth aspect of the present invention provides a method for dissolving high-valent metal ions in coal gangue, using the above-mentioned microbial agent for treatment.

[0020] In a sixth aspect, the present invention provides a method for treating high salinity in coastal saline soil, wherein coal gangue and the above-mentioned microbial agent are added to the coastal saline soil to treat the soil.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention relates to a strain of Priesteria gigantea and its application, which has the ability to efficiently dissolve Ca from coal gangue. 2+ The functional strain is used to treat coastal saline soil. The dissolved free metal ions can accurately target the Na + , by virtue of ion exchange, Na + Desorption and discharge of saline colloids from the cultivated soil can effectively improve the high salinity and single composition problems of coastal saline soil, and provide advanced and reliable technical means for the ecological restoration and agricultural utilization of coastal saline soil.

[0023] 2. The microorganisms in the present invention produce organic acids through their own metabolic activities and react chemically with calcium carbonate, calcium silicate and other substances in coal gangue to release water-soluble and exchangeable Ca 2+ It forms ion exchange with Na+ in coastal saline soil, allowing Na salt to be discharged from the cultivated soil, thus achieving efficient management of coastal saline soil.

[0024] 3. The present invention realizes the resource utilization of coal gangue, reduces waste emissions, improves resource utilization, and has significant economic and environmental benefits. At the same time, the microbial enhanced dissolution technology can avoid the risk of secondary pollution of soil and groundwater; functional microorganisms have the characteristics of strong colonization ability, fast metabolic rate, and good in situ effect. They can continue to play a role, continuously dissolve calcium in coal gangue and improve coastal saline soil, realizing long-term green management of coastal saline soil.

[0025] 4. The microbial enhanced dissolution technology of the present invention does not require complicated pretreatment and processing of the coal gangue. It only needs to add an appropriate amount of microbial agents and create a suitable microbial growth environment to achieve calcium dissolution and saline soil treatment, thereby reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The appearance and developmental tree of the bacterial colonies tested in Example 1 are shown below. Figure 1 a in the figure is the appearance of the colony. Figure 1 b in the figure is the Gram staining picture. Figure 1 The c in the figure is a developmental tree diagram.

[0027] Figure 2 is the free Ca in the gangue leachate in Example 2 2+ Graph showing the temporal variation of ion concentration.

[0028] Figure 3 Actual photos and schematic diagram of the verification device for microbial enhanced coal gangue improvement of coastal saline soil in Example 3.

[0029] Figure 4 This is a data chart for evaluating the effect of microbial enhancement of coal gangue in improving coastal saline soil. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention. Example

[0031] Establishment of functional selection culture medium:

[0032] The functional selection medium for screening microbial strains capable of dissolving high-valent metals from coal gangue has a specific formula of: glucose 10.0 g·L -1 , peptone 5.0 g·L -1 , coal gangue (200 mesh) 2.0 g·L -1 、NaCl8.0 g·L -1 、KCl 0.2 g·L -1 、Na2HPO4 g·L -1 、KH2PO4 g·L -1 Place the functional selection medium in a high-temperature steam sterilizer, set the temperature to 121°C for 30 minutes, cool to room temperature, and then adjust the pH value to 7.0 with NaOH and HCl solution for later use.

[0033] Establishment of artificial cultivation and domestication system:

[0034] Wheat was planted using a mixture of coastal saline soil (collected from Gudao Town, Dongying City, Shandong Province, at a sampling depth of 0–30 cm, after removing rocks, plant and animal debris, and other debris. Properties are shown in Table 1), coal gangue (collected from a gangue dump at Shengquan Coal Mine in Tai'an City, Shandong Province, at a sampling depth of 0–30 cm. Element distribution is shown in Table 2). The mixture was mixed with commercially available nutrient matrix soil in a 1:1:1 ratio. The pots were removed on the 90th day after planting, and the rhizosphere soil was collected.

[0035] Table 1 Basic properties of the tested coastal saline soil

[0036]

[0037] Table 2 Basic properties of the tested coal gangue

[0038]

[0039] Screening and purification of functional microorganisms:

[0040] Enrichment culture: After collecting the rhizosphere soil, 5.0 g of soil was placed in a 250 mL conical flask containing 100 mL of functional selection medium. The temperature was set to 30 °C and the rotation speed was 120 rpm. -1 , cultured in a shaking incubator for 12 hours;

[0041] b. Plate culture and purification: Diethylpyrocarbonate treated water (DEPC water) was used to culture the bacterial solution at 10 -1 -10 -9 Perform serial dilutions, inoculating 100 μL of the dilution into a culture dish, spreading evenly with an L-shaped applicator, and incubating at 37°C for 3 days. After incubation, pick a single colony with a 1 μL inoculating loop and continue to isolate and purify the bacteria on beef extract peptone agar plates multiple times. After each purification, incubate at 37°C for 3 days until a single colony is visible to the naked eye on the plate.

[0042] c. Preservation of bacterial strains: Pick a single colony and inoculate it into 5 mL of Luria-Bertani medium (Solarbio, Product No. L1010) using a 1 μL inoculation loop. Then, incubate at 37°C and 120 rpm for 1 h. -1 After culturing for 12 hours, 800 μL of culture medium was added to a 2 mL sterile cryopreservation tube, and 200 μL of sterile glycerol (80% v / v) was added to mix well. The tube was then stored in a -80°C refrigerator. Meanwhile, the single colonies on the plate were stored in a 4°C refrigerator.

[0043] d. Bacteria identification:

[0044] Transfer an appropriate amount of bacterial cells to a 2ml centrifuge tube and add 567 µL of TE buffer. Resuspend by repeated pipetting. Then, add 30 µL of 10% SDS and 15 µL of proteinase K and mix thoroughly. Incubate at 55°C for 30 minutes. Add an equal volume of chloroform:isoamyl alcohol (24:1 volume ratio) and centrifuge at 12,000 rpm for 4-5 minutes. Transfer the supernatant to a fresh tube and add 0.6-0.8 times the volume of isopropanol. Gently mix until the DNA precipitates and centrifuge at 12,000 rpm for 10 minutes. Wash with 1 mL of 70% ethanol and centrifuge at 12,000 rpm for 10 minutes. Discard the ethanol. Dry in a clean bench and resuspend in 50 µL of TE buffer or deionized water.

[0045] The sequenced strains were identified by SeqMan splicing, vector sequence removal and chimera detection, and compared and classified at the National Center for Biotechnology Information (NCBI). A total of one bacterial strain, Priesteria gigantea H16 ( Priestia megaterium H1 6) belongs to Priestia It is a facultative anaerobic bacterium, Gram-negative, and forms white, round, smooth and moist colonies on ordinary beef extract-peptone agar medium. Figure 1 shown. Figure 1 a in the figure is the appearance of the colony. Figure 1 b in the figure is the Gram staining picture. Figure 1 The c in the figure is a developmental tree diagram.

[0046] Priesteria gigantea H16 ( Priestia megaterium H1 6) It has been deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, the deposit number is CCTCC M 20242801, and the deposit date is December 12, 2024.

[0047] In the laboratory, the long-term storage method of the strain is to add the strain to MB 2216 medium (Difco, product number 279110) containing 30% glycerol and store it at -80°C; the short-term storage method is to inoculate the strain on a slant of beef extract peptone agar medium and store it at 4°C until use.

[0048] The 16S rRNA sequence is shown in the sequence listing SEQ ID NO.1:

[0049] Example

[0050] Example 2 conducted an experiment on the bacterial strain promoting the dissolution of high-valent metal ions from coal gangue, as follows:

[0051] Establishment of culture medium for dissolution of high-valent metal ions from solid waste:

[0052] The method used to verify the dissolution of Ca from coal gangue 2+ Functional verification medium for microbial strains with high-valent metal ion efficacy, the specific formula is: glucose 10.0 g·L -1 , peptone 5.0 g·L -1 , coal gangue powder (100 mesh) 2.0 g·L -1 、NaCl 8.0 g·L -1 、KCl 0.2 g·L -1 、Na2HPO4 g·L -1 、KH2PO4 g·L -1 Place the functional selection medium in a high-temperature steam sterilizer, set the temperature to 121°C for 30 minutes, cool to room temperature, and then adjust the pH value to 7.0 with NaOH and HCl solution for later use.

[0053] Establishment of functional verification experimental system:

[0054] This implementation case is divided into two groups: experimental group and control group.

[0055] The experimental group was treated with Priesteria gigantea H16 ( Priestia megaterium H1 6) glycerol frozen solution (Example 1) was inoculated into the above-mentioned functional verification medium (250 mL Erlenmeyer flask, containing 100 mL medium liquid) at a ratio of 1% (v / v), set up 3 replicates, and incubated at 30 ° C, 120 r·min -1 The mixture was cultured for 7 days to obtain the coal gangue dissolution solution.

[0056] The control group was a function verification culture medium without inoculation of functional microorganisms.

[0057] Determination of dissolution efficiency (high-valent metal ion concentration):

[0058] The enhanced effect of functional strains on the dissolution of high-valent metal ions from coal gangue was verified by the free Ca in the culture medium (coal gangue leachate). 2+ Ion concentration performance. At the 9th hour, 1, 3, 5, and 7th day of culture, 10 mL of liquid was collected and the culture medium was heated at 4°C and 8000 r·min. -1After centrifugation for 10 min, the supernatant was collected and filtered through a 0.45 μm aqueous polyethersulfone filter membrane and a C18 column to remove suspended particulate matter and organic matter, respectively. Finally, the supernatant was diluted 5 times with 2.5% HNO3 and the Ca content was determined according to the national environmental protection standard method "Determination of 32 elements in water by inductively coupled plasma optical emission spectrometry" (HJ 776-2015). 2+ concentration.

[0059] Evaluation of dissolution efficiency:

[0060] Priesteria gigantea H16 ( PriestiamegateriumH1 6) Ca in coal gangue 2+ showed good leaching efficiency (e.g. Figure 2 In the functional verification medium, Ca 2+ The initial concentrations of -1 The control group never showed significant differences. After inoculation of functional strains, Ca 2+ The concentration increased rapidly and reached 60.32±2.64 μg·mL at 9 hours after inoculation. -1 , reaching a peak of 70.11±3.71 μg·mL at the 24th hour -1 , the dissolution efficiency increased by 130.58 times.

[0061] Example 3

[0062] Example 3 conducted an experiment on improving coastal saline soil by microbial enhancement of coal gangue, as follows:

[0063] Soil column verification test materials and equipment:

[0064] The coastal saline soil samples for testing were collected from Gudao Town, Dongying City, Shandong Province. Detailed soil properties are listed in Table 3. Soil profiles with typical characteristics were selected, and soil samples were collected from a depth of 0–30 cm. After thorough mixing and air-drying, the soil samples were passed through a 4 mm sieve and packed into soil columns.

[0065] Priestia megaterium H16 was first cultured in Luria-Bertani medium (Solarbio, Product No. L1010) for 10 hours at a constant temperature. -1 The cells were centrifuged at 4 °C for 10 min to obtain cell pellets. The cell pellets were washed three times with phosphate buffered saline (PBS). The pellets were redissolved with 0.01 M phosphate buffered saline (PBS) and the concentration was adjusted to 10 8 CFU / mL.

[0066] Transparent acrylic columns with an inner diameter of 20 cm and a height of 40 cm were used as the experimental apparatus. During the construction process, a 1 cm diameter hole was precisely reserved at the bottom of each soil column to facilitate the smooth drainage of leachate. This ensured that excess water and soluble substances in the soil system could be promptly discharged during the experiment, maintaining the stability of experimental conditions. A 5 cm thick layer of quartz sand was laid at the bottom of the soil column to serve as a filter layer. With its fine particle structure and porosity, it effectively prevented soil particles at the bottom of the column from agglomerating and clogging the column outlet due to water impact or gravity, ensuring smooth leachate discharge. A layer of nylon mesh was carefully laid on top of the quartz sand to further refine the filtration effect, preventing fine soil particles from penetrating the quartz sand layer and entering the drainage holes. It also provided support and dispersion for the soil layer above, ensuring a more even distribution of soil within the column.

[0067] According to the actual bulk density value (1.4 g・cm⁻³) obtained from the actual measurement of the soil at the sampling site, the coastal saline soil required for a height of 30 cm was accurately weighed. According to the design plan, the pre-prepared coal gangue slag and the bacterial solution were mixed or sprayed in a specific way to make them evenly distributed in the soil. After sufficient mixing and stirring to ensure that all components were evenly distributed, the soil filling operation in the soil column began. In order to ensure the uniformity and compactness of the soil filling, a batch filling strategy was adopted. Each soil column was filled a total of 6 times, with each filling height of 5 cm. After the filling operation was completed, a 5 cm water storage layer was reserved above the soil column (see for details). Figure 3 ), this aquifer can, on the one hand, provide a buffer space for water replenishment in the subsequent experimental process, and on the other hand, help simulate the water retention conditions in the upper soil layer under natural conditions, ensuring the compatibility of the experimental conditions with the actual soil ecological environment.

[0068] Table 3 Basic properties of the tested coastal saline soil

[0069]

[0070] This implementation case is divided into experimental group 1, experimental group 2 and control group, a total of 3 groups.

[0071] Experimental group 1 was to add coal gangue to coastal saline soil at a ratio of 10% (w / w) and fill the soil column;

[0072] Experimental group 2 was to add coal gangue to coastal saline soil at a ratio of 10% (w / w) and at the same time add 5 mL·kg -1 The ratio of spraying Priesteria gigantea H16 ( Priestia megaterium H1 6) Add bacterial solution and fill the soil column;

[0073] The control group was simply filled with the same mass of coastal saline soil as that of the experimental groups without any other treatment.

[0074] Testing and analysis of the efficiency of microbial enhanced coal gangue improvement:

[0075] To simulate the salt leaching caused by natural precipitation, an intermittent leaching method was used. Leaching was started every 12 hours, and 300 mL of leaching water was added each time. The experiment lasted for 21 days. After the experiment, the soil in the soil column was removed, dried naturally in the shade, and thoroughly mixed. The exchangeable base ion content (K) of the soil was determined according to the "Determination of Cation Exchange Capacity and Exchangeable Base of Neutral Soils" (NY / T 295-1995). + 、Na + , Ca 2+ Mg 2+ ); Refer to "Soil Testing Part 18: Determination of Soil Sulfate Ion Content" (NY / T1121.18-2006), determine the soil sulfate ion content (SO4 2- ); Refer to "Soil Testing Part 17: Determination of Soil Chloride Content" (NY / T 1121.17-2006), determine the chloride ion content in the soil (Cl - ) content; refer to "Analysis of Water-Soluble Salt in Forest Soil" (LY / T 1251-1999), determine the content of carbonate (CO3 2- ) and bicarbonate (HCO3 - ) ion content.

[0076] Evaluation of the efficiency of microbial enhanced coal gangue improvement:

[0077] Only the addition of coal gangue blasting and simulated precipitation leaching to the soil column of coastal saline soil reduced the total salt content and improved the composition of salt ions to a certain extent, while the inoculation of functional microorganisms significantly enhanced this process, such as Figure 4 As shown. Ca in coal gangue 2+ In situ bioaugmented dissolution leads to the formation of Ca at the soil colloidal interface 2+ To Na + At the same time, the significant increase in the Ca / Na ratio in the soil can not only promote the improvement of coastal saline soil, but also promote its further utilization.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Priesterol bacterium characterized by: The strain was classified and named Priestia megaterium H16, and was deposited in the China Center for Type Culture Collection on December 12, 2024, with the deposit number CCTCC NO: M 20242801.

2. A microbial agent containing Priesteria gigantea as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that Including strains, bacterial liquid or fermentation liquid.

4. The use of the microbial agent as claimed in claim 2 in dissolving high-valent metal ions in coal gangue, characterized in that: The high-valent metal ions are calcium ions.

5. The use of the microbial agent in treating coastal saline soil according to claim 2, characterized in that: The purpose of controlling coastal saline soil is to promote the discharge of salt from coastal saline soil or reduce the total salinity of coastal saline soil.

6. A method for dissolving high-valent metal ions in coal gangue, characterized in that: The microbial agent according to claim 2 is used to treat coal gangue, wherein the high-valent metal ions are calcium ions.

7. A method for treating high salinity in coastal saline soil, characterized in that: Coal gangue and the microbial agent as claimed in claim 2 are added to coastal saline soil to treat the soil.

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