Thiamine-releasing bacillus miguli and application thereof

Abstract

The invention provides a thiamine-releasing bacillus miguli and application of the thiamine-releasing bacillus miguli. According to the invention, a strain of Aneurinibacillus migulanus X3-3 with resistance to a plurality of heavy metals is screened and separated from soil subjected to combined pollution of heavy metals, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the registration number of the preservation center is CGMCC No.33798. The invention also discloses a method for preparing the Aneurinibacillus migulanus X3-3 with resistance to a plurality of heavy metals and application of the Aneurinibacillus migulanus X3-3. The strain can tolerate high-concentration heavy metal pollution, shows good heavy metal removal and solidification capabilities in water and soil, provides an efficient and environment-friendly microbial solution for remediation of a heavy metal combined pollution environment, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a thiamine-decomposing Bacillus mikkelsen and applications thereof. Background Art

[0002] With the rapid development of industrialization and agricultural modernization, heavy metal pollution has become a global environmental problem. Mining, metallurgy, chemical production, and the excessive use of fertilizers and pesticides have led to increasingly severe heavy metal contamination of soil and water bodies. The combined pollution of heavy metals such as cadmium (Cd), arsenic (As), and lead (Pb) is particularly severe. These heavy metals not only damage soil ecosystems but also enter water bodies through surface runoff and groundwater infiltration, further exacerbating aquatic pollution. Heavy metal contamination of water bodies not only threatens the survival of aquatic organisms but also accumulates through drinking water and the food chain, ultimately endangering human health.

[0003] At present, the remediation technologies for heavy metal pollution mainly include physical, chemical and biological methods. Although physical and chemical methods are quick to take effect, they are costly, prone to secondary pollution, and difficult to apply on a large scale. In contrast, bioremediation technology, especially microbial remediation technology, has gradually become a research hotspot due to its advantages such as environmental friendliness, low cost and strong sustainability. Microbial remediation technology mainly removes or solidifies heavy metals from soil or water bodies through the adsorption, precipitation, redox and other effects of microorganisms, thereby reducing their bioavailability and toxicity. However, existing microbial remediation technologies still face many challenges in practical applications: (1) Single resistance limitation: Most microorganisms only have resistance or remediation capabilities to a single heavy metal and are difficult to cope with complex polluted environments; (2) Insufficient tolerance: Many strains have insufficient tolerance when heavy metal concentrations are high, and the remediation efficiency is limited; (3) Adaptability limitations: The adaptability of strains to different media with large differences in environmental conditions, such as soil and water environments, is limited, making it difficult to promote them widely. Therefore, screening and developing microbial strains with multiple heavy metal resistance, wide adaptability and high remediation efficiency has become an important research direction in the current field of heavy metal pollution remediation. Summary of the Invention

[0004] The present invention provides a thiamine-soluting Bacillus mikkelsen and an application thereof. The thiamine-soluting Bacillus mikkelsen is resistant to multiple heavy metals and has the function of repairing heavy metal pollution.

[0005] In a first aspect, the present invention provides Bacillus milleis thiamine-solubilizing bacteria or its progeny, wherein the Bacillus milleis thiamine-solubilizing bacteria is deposited in the General Microorganism Center of China Culture Collection Administration Committee, and the registration number of the Bacillus milleis thiamine-solubilizing bacteria in the General Microorganism Center of China Culture Collection Administration Committee is CGMCC No. 33798.

[0006] The progeny of Bacillus milleis thiamine-lytics as described above refers to daughter cells produced by the growth of the microorganism (eg, culture growth in a culture medium). It is understood that the progeny of Bacillus milleis thiamine-lytics still retains the function of Bacillus milleis thiamine-lytics.

[0007] In a second aspect, the present invention provides a composition comprising the above-mentioned Bacillus mielii or its progeny.

[0008] The composition described above is a bacterial agent. The bacterial agent described above is a live bacterial preparation prepared from the above-mentioned Bacillus milleis thiamine-soluble bacteria. The formulation of the bacterial agent can be various formulations, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0009] The composition as described above may further include other biological components or non-biological components in addition to the thiamine-lytic Bacillus mielii or its progeny.

[0010] In a third aspect, the present invention provides a use of the above-mentioned Bacillus mielii thiamine-solubilizing or progeny thereof, wherein the use is selected from any one of A1) to A4);

[0011] A1) Application in remediation of heavy metal pollution in water environment;

[0012] A2) Application in the preparation of products for remediation of heavy metal pollution in water environments;

[0013] A3) Application in remediation of heavy metal contaminated soil;

[0014] A4) Application in the preparation of products for repairing heavy metal contamination in soil.

[0015] In a fourth aspect, the present invention provides a use of any of the above-described compositions, wherein the use is selected from any one of A1) to A4);

[0016] A1) Application in remediation of heavy metal pollution in water environment;

[0017] A2) Application in the preparation of products for remediation of heavy metal pollution in water environments;

[0018] A3) Application in remediation of heavy metal contaminated soil;

[0019] A4) Application in the preparation of products for repairing heavy metal contamination in soil.

[0020] In a fifth aspect, the present invention provides a method for repairing heavy metal contamination in a sample, comprising: contacting the sample to be treated with the above-mentioned Bacillus mikkelsen or its progeny or any of the above-mentioned compositions, reducing the content of heavy metals in the sample to be treated, and achieving repair of heavy metal contamination in the sample.

[0021] In the method as described above, the sample to be processed is selected from at least one of water samples and soil.

[0022] In the above method, the heavy metal includes at least one of cadmium, arsenic, lead and antimony.

[0023] According to the above method, when the sample to be processed is a water sample, the amount of the thiamine-decomposing Bacillus mikkelsen or its progeny added is not less than 5×10 10 [cfu]; when the sample to be treated is soil, the amount of the thiamine-decomposing Bacillus mielii or its progeny added is not less than 8×10 9 [cfu].

[0024] The present invention screened and isolated a strain of Aneurinibacillus migulanus X3-3, resistant to multiple heavy metals, from soil contaminated with multiple heavy metal compounds. The strain has been deposited with the China General Microbiological Culture Collection (CGMCC) under the registration number CGMCC No. 33798. This strain can tolerate high concentrations of heavy metal pollution and exhibits significant heavy metal removal and solidification capabilities in water and soil. This provides a highly efficient and environmentally friendly microbial solution for the remediation of soil and water contaminated with multiple heavy metal compounds, with broad application prospects.

[0025] Preservation Instructions

[0026] Bacterial species: Aneurinibacillus migulanus X3-3

[0027] Depository: General Microbiology Center of China Culture Collection Administration

[0028] Abbreviation of depository institution: CGMCC

[0029] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0030] Deposit date: March 12, 2025

[0031] CGMCC registration number: CGMCC No.33798 BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the growth curve of Bacillus mikkelsenii X3-3 in LB medium containing different concentrations of heavy metal As;

[0033] Figure 2 This is the growth curve of Bacillus mikkelsenii X3-3 in LB medium containing different concentrations of heavy metal Cd;

[0034] Figure 3 This is the growth curve of Bacillus mikkelsenii X3-3 in LB medium containing different concentrations of heavy metal Pb. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0036] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0037] Example 1. Acquisition, isolation and identification of strains

[0038] 1. Acquisition and isolation of strains

[0039] On August 7, 2019, surface soil (0-20 cm depth) was collected using a sterile spatula from a mining area severely contaminated by heavy metal compounds in Sima Village, Nanzhou Town, Lukou District, Zhuzhou City, Hunan Province, China. The collected surface soil was placed in sterile plastic bags, sealed, and labeled. The collected soil samples were stored at 4°C to minimize loss of microbial activity. The soil samples were sieved through a 2 mm sieve to remove stones and plant debris.

[0040] Weigh 10g of soil sample, add sterile water, filter, and evenly smear 100μL of the clarified soil solution containing microorganisms onto a LB plate containing heavy metals using a streak technique. Invert the plate and incubate in a 30°C incubator for 24-48 hours to enrich for heavy metal-resistant strains. The LB plate contains 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, and 1.5% agar at a pH of 7.0-7.2. Cd, As, and Pb are added at concentrations of 50mg / kg, 200mg / kg, and 200mg / kg, based on the weight of the LB medium.

[0041] After strain enrichment is completed, single colonies with different morphologies are selected and streaked onto new LB solid culture medium plates containing heavy metals for purification and culture. Repeat streaking 2-3 times until pure colonies are obtained. At the same time, pick the purified single colonies and inoculate them into LB liquid culture medium containing heavy metals. Culture at 30°C and 150rpm with shaking for 24-48 hours, and observe the growth of the bacterial liquid. Inoculate the purified strain onto LB slant culture medium, culture for 24 hours, and store it at 4°C for a short term. At the same time, mix the bacterial liquid with 20%-30% glycerol, divide it into cryopreservation tubes, and store it in a -80°C ultra-low temperature refrigerator for long-term storage.

[0042] 2. Identification of strains

[0043] The purified colonies were subjected to morphological observation and molecular biological identification. DNA was extracted using a rapid bacterial genome extraction kit, and the 16S rDNA sequence was amplified by PCR to confirm its taxonomic status. The 16S rDNA sequence of this strain is shown in SEQ ID NO:1. By BLAST comparison with the NCBI database and construction of a phylogenetic tree, combined with morphological analysis, the strain was identified as Aneurinibacillus migulanus and named Aneurinibacillus migulanus X3-3, hereinafter referred to as strain X3-3.

[0044] SEQ ID NO: 1 is specifically as follows:

[0045]

[0046] The strain X3-3 was deposited in the China General Microorganism Culture Collection (CGMCC) on March 12, 2025, and the registration number of the China General Microorganism Culture Collection Center is CGMCC No. 33798.

[0047] Example 2: Heavy Metal Tolerance Detection of Strain X3-3

[0048] LB liquid culture medium containing heavy metals Cd, As, and Pb at different concentration gradients was prepared. Specifically, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water were first mixed to obtain an LB liquid culture medium with a pH of 7.0-7.2; heavy metal Cd was added to the LB liquid culture medium to obtain an LB liquid culture medium containing heavy metal Cd with a Cd concentration gradient of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 20, and 30 mg / L. B liquid culture medium; adding heavy metal As(V) to the LB liquid culture medium to obtain an LB liquid culture medium containing heavy metal As with an As(V) concentration gradient of 0, 0.5, 1, 2, 5, 10, 20, 30, 50, 100, 200, 300, and 400 mg / L; adding heavy metal Pb to the LB liquid culture medium to obtain an LB liquid culture medium containing heavy metal Pb with a Pb concentration gradient of 0, 2, 5, 10, 20, 50, 100, 200, 300, and 400 mg / L.

[0049] The strain X3-3 was inoculated into LB liquid medium containing different concentrations of As, Cd, and Pb, and cultured at 150 rpm and 300 °C for 72 h. The OD600 value of the bacterial solution was measured using a multifunctional microplate reader, and the growth curve of the strain under different concentrations of heavy metals was drawn to evaluate its heavy metal tolerance. The results are as follows: Figure 1-3 As shown. Figure 1-3 It can be seen that strain X3-3 has multiple resistance to heavy metals such as As, Cd, and Pb, and can grow in an environment with a certain concentration of heavy metals.

[0050] Example 3: Determination of heavy metal removal ability of strain X3-3

[0051] 1. Water experiment

[0052] Prepare a 0.05M KNO3 solution (buffer) with a total cadmium concentration of 1.0ppm, a trivalent arsenic As(III) concentration of 10ppm, a pentavalent arsenic As(V) concentration of 10ppm, a total lead concentration of 200ppm, and a total antimony concentration of 5ppm. Inoculate the strain in the above solution with an inoculum size of 5×10 10[cfu] / L, with six replicates. After incubation at 37°C and 180 rpm for 72 hours, the supernatant was centrifuged at 12,000g for 5 minutes, and the heavy metal concentrations in the supernatant were measured. The cell adsorption capacity was calculated by subtracting the initial heavy metal concentration in the solution from the concentration of the heavy metal after inoculation. The removal rate was calculated as the cell adsorption capacity / initial heavy metal concentration in the solution * 100%. The results are shown in Table 1.

[0053] Table 1 Heavy metal removal efficiency of strain X3-3 in solution

[0054]

[0055] 2. Soil experiment

[0056] Contaminated soil from the arsenic smelting waste slag mining area in Wenshan, Yunnan was collected and its heavy metal content was determined. The soil contained 0.1 mg / kg of available cadmium, 0.5 mg / kg of available arsenic, and 1.0 mg / kg of available lead. The strain X3-3 was inoculated into the heavy metal contaminated soil at an inoculum size of 8 × 10 9 [cfu] / kg. All treatments were repeated 6 times, and 10g of soil was used for each treatment. The soils were watered once a day to maintain 60% of the maximum field water holding capacity. After 20 days, soil samples were sieved through a 2mm sieve, and 0.005M DTPA was used to extract the available Cd and Pb in the soil, and 0.05M (NH4)2SO4 was used to extract the available As in the soil. The concentration of As in the solution was determined by hydride generation-atomic fluorescence spectrometry, and the concentration of Cd and Pb in the solution was determined by ICP-OES. The heavy metal solidification rate was calculated according to (the original content of available heavy metals in the soil - the content of available heavy metals in the soil after strain treatment) / the original content of available heavy metals in the soil * 100%, and the results are shown in Table 2.

[0057] Table 2 Heavy metal fixation efficiency of strain X3-3 in soil

[0058]

[0059] Note: Different lowercase letters in the table indicate significant differences in available As / available Cd / available Pb among different treatments at the same soil concentration (p<0.05).

[0060] According to the above experiments, the strain X3-3 provided by the present invention has the function of heavy metal remediation, and provides an excellent strain resource for subsequent heavy metal remediation in soil and water bodies.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Bacillus mielii thiamine-solubilizing or its progeny, characterized in that The thiamine-solubilizing Bacillus mikkelsen is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and the registration number of the thiamine-solubilizing Bacillus mikkelsen in the General Microbiology Center of China Culture Collection Administration of Microorganisms is CGMCC No.33798.

2. A composition comprising the thiaminolytic Bacillus mielii or its progeny according to claim 1.

3. The composition according to claim 2, characterized in that The composition is a bacterial agent.

4. The use of the thiaminolytic Bacillus mielii or its progeny according to claim 1, characterized in that: The application is selected from any one of A1) to A4); A1) Application in remediation of heavy metal pollution in water environment; A2) Application in the preparation of products for remediation of heavy metal pollution in water environments; A3) Application in remediation of heavy metal contaminated soil; A4) Application in the preparation of products for repairing heavy metal contamination in soil.

5. The use of the composition according to any one of claims 2 to 3, characterized in that: The application is selected from any one of A1) to A4); A1) Application in remediation of heavy metal pollution in water environment; A2) Application in the preparation of products for remediation of heavy metal pollution in water environments; A3) Application in remediation of heavy metal contaminated soil; A4) Application in the preparation of products for repairing heavy metal contamination in soil.

6. A method for repairing heavy metal contamination in a sample, characterized in that: include: The sample to be treated is contacted with the thiamine-solubilizing Bacillus mielii or its progeny according to claim 1 or the composition according to any one of claims 2-3 to reduce the content of heavy metals in the sample to be treated and achieve the repair of heavy metal contamination in the sample.

7. The method according to claim 6, characterized in that The sample to be processed is selected from at least one of water samples and soil samples. The method according to claim 6 , wherein the heavy metal comprises at least one of cadmium, arsenic, lead and antimony.

9. The method for culturing the thiaminolytic Bacillus mielii according to claim 1, characterized in that: The method comprises culturing the thiamine-lytic Bacillus mielii or its progeny in a culture medium for culturing microorganisms.

10. A method for preparing the composition according to claim 2 or 3, characterized in that: The composition is prepared by using the thiaminolytic Bacillus mielii or its progeny according to claim 1.

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