The invention relates to a talaromyces pseudoaceae hzt-3apos strain and a preparation method thereof. Preparation method and application thereof

Abstract

The invention relates to talaromyces pseudomonas hzt-3'as well as a preparation method and application of the talaromyces pseudomonas hzt-3 ', and belongs to the technical field of application of microorganisms. The strain is preserved in the China Center for Type Culture Collection, the preservation number of the strain is CCTCC NO: M 20251797, and the strain is classified and named as (Talaromyces pseudomonas hzt-3 '). The method comprises the following steps: collecting moss and vegetation soil samples from basalt, diabase and dolomite; diluting bacterial colonies in the sample, and separating and purifying the bacterial strains by using a Czapek culture medium; anorthite and serpentine are used as inorganic salt sources for strain screening, and strains which grow stably and have a good leaching effect on calcium magnesium silicate minerals are obtained; the strain is applied to efficient leaching of anorthite, serpentine and wollastonite, and compared with a sterile control group, the effect of improving the leaching rate of calcium and magnesium ions is remarkable. The method solves the problem that the reaction rate of mineral sequestration carbon dioxide is slow due to the fact that ions in natural silicate minerals are difficult to leach, and has great significance.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a bacterium called *H. t. 3'*, its preparation method, and its application in the efficient bioleaching of calcium magnesium silicate minerals. Background Technology

[0002] Mineral sequestration, as one of the most stable and long-lasting sequestration methods in CCUS (Carbonized Carbonate Sequestration System) technology, is a process that permanently solidifies CO2 into carbonate minerals through chemical reactions, making it particularly suitable for areas lacking geological sequestration conditions. The raw materials used are widely available, including natural silicate minerals (wollastonite, serpentine, pyroxene, olivine, anorthite, etc.) and alkaline solid wastes (iron slag, fly ash, etc.). Alkaline solid wastes have high reactivity and easily achieve high carbon sequestration effects, but their annual production only contributes about 5% of the total CO2 required for sequestration. Therefore, naturally distributed near-surface silicate minerals have become a more ideal raw material choice for mineral sequestration of CO2. However, these raw materials have poor reactivity and difficulty in leaching calcium and magnesium ions, thus limiting the mineral carbonation reaction rate. Mechanical grinding, calcination, and acid-base-salt leaching are commonly used to activate calcium and magnesium silicate minerals to promote the release of calcium and magnesium ions. However, these traditional physicochemical activation methods have drawbacks such as high pollution, high cost, and high energy consumption. Microbial weathering accelerates the leaching of calcium and magnesium ions from minerals, offering advantages such as low energy consumption, environmental friendliness, and mature technology compared to traditional physical and chemical activation treatments.

[0003] Therefore, developing microbial-enhanced calcium and magnesium ion leaching methods is particularly important for improving the CO2 fixation capacity of silicate minerals. Summary of the Invention

[0004] The purpose of this invention is to provide a *Bacillus simulans* (…). Talaromyces pseudofuniculosus The strain hzt-3' can be used for the bioleaching of calcium magnesium silicate minerals, and can stably and efficiently dissolve calcium and magnesium ions.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a type of *Bacillus simulans* ( Talaromyces pseudofuniculosus The strain hzt-3' has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251797, dated August 7, 2025, at Wuhan University. Morphological characteristics: Colonies are dark green, soft in texture, with clear edges; the reverse side is pale yellow; the mycelium grows densely, spreading in a flat, cotton-like pattern.

[0006] The strain provided by this invention was isolated and purified from diabase vegetation soil using Czapek medium; the specific preparation method includes the following steps: Step 1, Strain source sample collection: Collect vegetation and moss soil samples from the areas surrounding diabase, dolomite, and basalt. Step 2, isolation and purification of strains: The supernatant of the soil sample dissolved in water was diluted and spread on Czapek solid medium, inverted in a constant temperature incubator, and streaked with an inoculation loop; Step 3, screening of leaching strains: The isolated and purified strains are inoculated into a culture medium containing calcium feldspar, serpentine, and wollastonite for leaching and screening. Step 4, Identification of the target strain: Observe the morphological characteristics of the target strain using an optical microscope, and identify the microbial species through molecular biological analysis.

[0007] Furthermore, the source samples for the strain were collected from the distribution zone of basic to ultrabasic rocks and carbonate rocks in the Lüliang Mountains region of Shanxi Province, China, administratively mainly located within Jiaocheng County and Fangshan County of Lüliang City. Sampling activities were conducted on typical rock types in this area (including diabase, dolomite, or basalt). Each sample was 50 grams and sealed for preservation.

[0008] Further, the isolation and purification of the strains: the collected samples were dissolved in sterile water and stirred thoroughly until the soil samples and bacteria were completely separated. After standing, the supernatant was taken out and inoculated onto Czapek solid medium using the 10-fold dilution method. The medium was then inverted and cultured in a 30 ℃ constant temperature incubator. Strains with different morphologies and colors were selected for streak purification culture.

[0009] Furthermore, the Czapek solid culture medium consists of: 30 g / L sucrose, 3 g / L sodium nitrate, 20 g agar, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 1000 mL water, adjusted to pH 7.4-7.6, and sterilized at 121°C for 20 min.

[0010] Further, the strains were screened: the purified strains were inoculated into a solid culture medium containing calcium feldspar, serpentine, and wollastonite, and incubated upside down in a 30°C constant temperature incubator for 2–7 days. The strains with superior growth were then re-screened in a liquid culture medium containing calcium feldspar, serpentine, and wollastonite.

[0011] Furthermore, the ITS1 sequence of the strain was amplified by PCR and analyzed by gene sequencing. The obtained sequence was assembled using DNAMAN software and compared and analyzed in the Blast system of the NCBI database to determine the species of the strain.

[0012] This invention provides the above-mentioned *Pseudomonas spp.* ( Talaromyces pseudofuniculosus Application of hzt-3' in bioleaching of calcium- and magnesium-rich silicate minerals (calcium feldspar, serpentine, wollastonite).

[0013] Furthermore, calcium- and magnesium-rich silicate minerals are crushed and screened to a particle size of 0.038 mm to 0.074 mm before being added to the leaching system.

[0014] Furthermore, the leaching system uses a modified Czapek liquid medium, the components of which include: sucrose 5~30 g / L, sodium nitrate 1~3 g / L, potassium chloride 0.1~0.5 g / L, dipotassium hydrogen phosphate 0.2~1 g / L, pH 7.4~7.6, sterilized at 121℃ for 20 min.

[0015] Furthermore, the initial pH of the leaching system was 3–10.5, the solid-liquid ratio of the slurry was 0.5%–5% (mass percentage), and the inoculum size was 1×10⁻⁶. 6 9 × 10⁹ spores / mL 6 spores / mL.

[0016] Furthermore, the leaching time is 2 to 14 days, and the leaching temperature is 25 to 40°C.

[0017] The beneficial effects of this invention are: (1) This invention screens a strain that has a good and stable leaching effect on silicate minerals from vegetation and moss soil. Talaromyces pseudofuniculosus hzt-3', deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251797, deposited on August 7, 2025; (2) The biochemical effects of the strain's metabolites and the biophysical effects of the strain significantly enhance the leaching of calcium and magnesium ions in silicate minerals such as anorthite, serpentine, and wollastonite, thus solving the problem of slow carbon dioxide reaction rate in mineral sequestration caused by the difficulty of ion leaching in natural silicate minerals. Attached Figure Description

[0018] Figure 1 This is a colony morphology diagram of *H. t. 3'*. Figure 2 Phylogenetic tree of *H. t. 3'*. Figure 3 The leaching rate of calcium and magnesium ions in anorthite after bioleaching in Example 2; Figure 4 The leaching rate of magnesium ions in serpentine after the bioleaching process in Example 3; Figure 5 The calcium ion leaching rate in wollastonite after the bioleaching process in Example 4 is shown. Detailed Implementation

[0019] The following examples are intended to further illustrate the present invention, but not to limit it.

[0020] Example 1: A bacterial strain for bioleaching calcium magnesium silicate minerals, the strain being deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251797, and deposit date August 7, 2025. The screening method for the strain includes the following steps: 1. Sample collection Soil samples were collected using a five-point sampling method. The source samples for the bacterial strains were collected from the distribution zone of basic to ultrabasic rocks and carbonate rocks in the Lüliang Mountains region of Shanxi Province, China, mainly located within Jiaocheng County and Fangshan County of Lüliang City. Sampling activities were conducted targeting typical rock types in this area (including diabase, dolomite, and basalt). Each sample was taken in 50 g portions, placed in a sample bag, and stored in a laboratory refrigerator at 4°C.

[0021] The first sampling point (diabase and dolomite) was located in Shuiyuguan Town, Jiaocheng County, Lüliang City, Shanxi Province (N 37°39′, E111°56′), and the second sampling point (basalt) was located in Jialiangshan, Fangshan County, Lüliang City (N 37°53′, E 111°14′). 50g samples were taken, placed in sample bags, and stored in a 4°C refrigerator in the laboratory.

[0022] 2. Isolation and purification of strains Take 5 g of the collected vegetation and moss soil sample and pour it into a beaker containing 50 mL of sterile distilled water. Stir for 15 min to mix thoroughly. After standing for 2 h, take the supernatant and prepare 3 dilutions (10-10). -2 10 -4 10 -6 Suspensions of different concentrations were spread into Czapek solid medium (30 g / L sucrose, 3 g / L sodium nitrate, 20 g agar, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 1000 mL water, pH adjusted to 7.4–7.6) and incubated at 30°C for 4 days. Individual colonies with differences in shape, color, and surface properties were streaked five times until pure colonies were obtained. Finally, the pure colonies were inoculated into liquid medium for further culture and stored at 4°C.

[0023] 3. Screening of strains The purified strain was inoculated into a solid culture medium containing anorthite and serpentine, and incubated upside down in a 30 ℃ constant temperature incubator for 2–7 days. Selected strains with excellent and stable growth were inoculated into Czapek liquid medium containing 2.0 g anorthite and serpentine (30 g / L sucrose, 3 g / L sodium nitrate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, and 1000 mL deionized water). The ore was leached for 7 days in a constant temperature shaker at 30 ℃ and 150 r / min, and the ion concentration was measured. The total calcium and magnesium concentration in the anorthite fermentation system was 9.72 mmol / L, and the total calcium and magnesium concentration in the serpentine fermentation system was 20.62 mmol / L.

[0024] 4. Identification of strains The morphological characteristics of strain hzt-3' observed under a microscope were as follows: the colonies were dark green, soft in texture, with clear edges, and pale yellow on the reverse side. The mycelia grew densely, spreading in a flat, cotton-like pattern (see...). Figure 1 The ITS1 sequence of the strain was amplified by PCR and analyzed by gene sequencing. The obtained sequence was assembled using DNAMAN software and compared with the Blast system in the NCBI database to identify strain hzt-3' as... Talaromyces pseudofuniculosus (See Figure 2 ).

[0025] Example 2: Application of the strain hzt-3' of the present invention in the leaching of calcium and magnesium elements from anorthite. The method described in this embodiment is mainly carried out according to the following steps: 1. The anorthite was crushed and sieved to a particle size of 0.038 mm to 0.074 mm. The main chemical composition of the anorthite was determined by X-ray fluorescence spectrometry (XRF) to be SiO2 46.81%, Al2O3 33.62%, CaO 10.78%, MgO 5.91%, and Na2O 0.78%.

[0026] 2. Add 0.5 g of calcium feldspar to 97 mL of modified Czapek liquid medium (30 g / L sucrose, 3 g / L sodium nitrate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, pH 7.4~7.6), adjust the pH to 7.5, and sterilize in a high-temperature autoclave at 121℃ for 20 min.

[0027] 3. After cooling to room temperature, inoculate with 3 mL of spore suspension (1×10⁻⁶). 6 The ore was soaked in a constant temperature shaker at 30℃ and 150 r / min for 7 days (spores / mL), and the concentration of calcium and magnesium ions in the fermentation broth was measured. A sterile control group was set up under the same conditions.

[0028] See results Figure 3 Ca in anorthite leaching suspension 2+ The concentration was 5.39 mmol / L, and the leaching rate was 56.14%, which was 12.53 times higher than that of the sterile control group; Mg 2+ The concentration was 4.41 mmol / L, corresponding to a leaching rate of 59.64%, which was 13.53 times higher than that of the sterile control group.

[0029] Example 3: Application of the strain hzt-3' of the present invention in the leaching of magnesium from serpentine. The method described in this embodiment is mainly carried out according to the following steps: 1. The serpentine was crushed and sieved to a particle size of 0.038 mm to 0.074 mm. The main chemical composition of the serpentine was determined by X-ray fluorescence spectrometry (XRF) to be SiO2 53.43%, MgO 37.90%, Fe2O3 5.01%, Al2O3 1.56%, CaO 1.04%, NiO 0.51%, and Cr2O3 0.15%.

[0030] 2. Add 0.5 g of serpentine to 99 mL of modified Czapek liquid medium (30 g / L sucrose, 3 g / L sodium nitrate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, pH 7.4~7.6), adjust the pH to 6.0, and sterilize in a high-temperature autoclave at 121℃ for 20 min.

[0031] 3. After cooling to room temperature, inoculate with 1 mL of spore suspension (1×10⁻⁶). 6 The ore was soaked in a constant temperature shaker at 25℃ and 150 r / min for 7 days (spores / mL), and the concentration of calcium and magnesium ions in the fermentation broth was measured. A sterile control group was set up under the same conditions.

[0032] See results Figure 4 Mg in serpentine leaching suspension 2+ The concentration was 13.03 mmol / L, and the leaching rate was 27.49%, which was 8.98 times higher than that of the sterile control group.

[0033] Example 4: Application of the strain hzt-3' of the present invention in the leaching of calcium from wollastonite The method described in this embodiment is mainly carried out according to the following steps: 1. The wollastonite was crushed and sieved to a particle size of 0.038 mm to 0.074 mm. The main chemical composition of the wollastonite was determined by X-ray fluorescence spectrometry (XRF) to be CaO 56.69%, SiO2 40.23%, Al2O3 0.87%, MgO 0.78%, Fe2O3 0.64%, and Na2O 0.31%.

[0034] 2. Add 0.5 g of wollastonite to 98 mL of modified Czapek liquid medium (30 g / L sucrose, 3 g / L sodium nitrate, 0.5 g / L potassium chloride, 1 g / L dipotassium hydrogen phosphate, pH 7.4~7.6) to adjust the pH to 7.5, and sterilize in a high-temperature autoclave at 121℃ for 20 min.

[0035] 3. After cooling to room temperature, inoculate with 2 mL of spore suspension (1×10⁻⁶). 6 The ore was soaked in a constant temperature shaker at 35℃ and 150 r / min for 7 days (spores / mL), and the concentration of calcium and magnesium ions in the fermentation broth was measured. A sterile control group was set up under the same conditions.

[0036] See results Figure 5 Ca in wollastonite leaching suspension 2+ The concentration was 43.79 mmol / L, corresponding to a leaching rate of 86.51%, which was 320.41 times higher than that of the sterile control group.

Claims

1. A Catenasphaeria hzt-3' characterized in that, The strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251797, and the preservation date is August 7, 2025; and the preservation place is Wuhan University.

2. The C. zeylanoides hzt-3' of claim 1, wherein, The morphological characteristics of the strain: the colony color is dark green, the colony texture is soft, the edge is clear, the back color is light yellow, and the mycelium grows densely and spreads in a flat cotton-like manner.

3. A process for the preparation of Cytospora hypoloides hzt-3' according to claim 1 or 2, characterized in that, The Talaromyces stipitatus hzt-3' is obtained by isolation and purification from soil by Czapek medium, including the following steps: Step one, sample collection: vegetation and moss soil samples around diabase, dolomite and basalt are taken respectively; Step two, isolation and purification of the strain: the supernatant of the soil sample dissolved in water is diluted and coated on the Czapek solid medium, and then inverted in a constant temperature incubator for culture, and streak culture is performed with an inoculation loop; Step three, screening of leaching strains: the strains after isolation and purification are inoculated in the medium containing calcium feldspar, serpentine and wollastonite for leaching screening; Step four, identification of the target strain: the morphological characteristics of the target strain are observed by optical microscope, and the species of the microorganism are identified by molecular biology analysis.

4. The method of claim 3, wherein the C. zeylanoides hzt-3' is prepared by the steps of: The source sample of the strain is collected from the basic-ultrabasic rock and carbonate rock distribution belt in Lüliang Mountain area of Shanxi Province, China, mainly located in Jiaocheng County and Fangshan County of Lüliang City; the sampling activity is carried out for the typical rock types in the region, including diabase, dolomite or basalt; each sample is 50 grams and is sealed for preservation.

5. The method of claim 3, wherein the C. zeylanoides hzt-3' is prepared by the steps of: The isolation and purification of the strain: the collected sample is dissolved in sterile water, stirred thoroughly until the soil sample and the bacterial body are completely separated, and then the supernatant is taken after standing and inoculated on the Czapek solid medium by 10-fold dilution method, inverted in a 30℃ constant temperature incubator for culture, and the strains with different morphologies and colors are picked and streaked for purification culture.

6. The method of claim 5, wherein the C. zeylanoides hzt-3' is prepared by the steps of: The components of the Czapek solid medium include: sucrose 30 g / L, sodium nitrate 3 g / L, 20 g agar, magnesium sulfate 0.5 g / L, potassium chloride 0.5 g / L, potassium phosphate 1 g / L, ferrous sulfate 0.01 g / L, 1000 mL water, pH 7.4-7.6, 121℃ sterilization for 20 min.

7. The method of claim 3, wherein the C. zeylanoides hzt-3' is prepared by the steps of: Strain screening: the purified strain is inoculated in the solid medium containing calcium feldspar, serpentine and wollastonite, inverted in a 30℃ constant temperature incubator for culture for 2-7 days; the well-grown strains are selected for re-screening in the liquid medium containing calcium feldspar, serpentine and wollastonite; the ITS1 sequence of the strain is subjected to PCR amplification and gene sequencing analysis, the obtained sequence is spliced by using DNAMAN software, and the comparison analysis is performed in the Blast system in the NCBI database to determine the species of the strain.

8. The Talaromyces stipitatus hzt-3' of claim 1 or 2 in the application of bioleaching of silicate minerals rich in calcium and magnesium.

9. Use according to claim 8, characterized in that, The silicate mineral is one of anorthite, serpentine and wollastonite; the silicate mineral rich in calcium and magnesium is crushed and sieved to a particle size of 0.038 mm to 0.074 mm and then added to the ore leaching system; the initial pH value of the ore leaching system is 3 to 10.5, the solid-liquid ratio concentration of the ore pulp is 0.5% to 5%, the inoculation amount is 1×10 6 spores / mL to 9×10 6 spores / mL; the ore leaching time is 2 to 14 days, and the ore leaching temperature is 25 to 40 DEG C.

10. Use according to claim 9, characterized in that, The leaching system adopts improved Czapek liquid culture medium, and the composition of the culture medium includes: sucrose 5-30 g / L, sodium nitrate 1-3 g / L, potassium chloride 0.1-0.5 g / L, dipotassium hydrogen phosphate 0.2-1 g / L, pH 7.4-7.6, and sterilization at 121 DEG C for 20 min.

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