Bacillus atrophaeus SCAU-H1 capable of repairing heavy metal pollution and application thereof

Abstract

The present application belongs to the technical field of pollution remediation, and particularly relates to Bacillus atrophaeus SCAU-H1 capable of remediation of heavy metal pollution and application thereof. The present application obtains a strain of Bacillus atrophaeus SCAU-H1, which is preserved in Guangdong Microbial Culture Collection Center on December 22, 2023, with a preservation number of GDMCC NO: 64181. The strain SCAU-H1 can effectively remove As(III), As(V), Cd, Pb and Sb heavy metals in water, and particularly has excellent removal effect on As(V). Moreover, the strain can significantly reduce the effective content of heavy metals Cd and Pb in soil, and has a significant immobilization effect. Therefore, the strain SCAU-H1 can be used for remediation and treatment of heavy metal contaminated water and soil.

Description

Technical Field

[0001] This invention belongs to the field of pollution remediation technology and relates to a Bacillus atrophaeus SCAU-H1 strain capable of remediating heavy metal pollution and its applications. Background Technology

[0002] Soil and water are the environment on which humans depend for survival, and the increasingly serious problem of heavy metal pollution in soil and water has become a global issue.

[0003] Among existing heavy metal remediation technologies, bioremediation has unique advantages compared to physical and chemical remediation technologies. Various microorganisms capable of heavy metal remediation have been reported, including Bacillus subtilis and Pseudomonas sp., and microorganisms are receiving increasing attention in heavy metal remediation.

[0004] Therefore, developing heavy metal remediation technologies and products from the perspective of microorganisms has significant practical value and meaning. Summary of the Invention

[0005] The purpose of this invention is to provide a new microbial bacterium capable of remediating heavy metal pollution, namely Bacillus atrophus SCAU-H1, which has important application value in the remediation of heavy metal polluted environments such as water and soil.

[0006] Another object of the present invention is to provide the application of the Bacillus atrophus SCAU-H1 in the remediation of heavy metal pollution, including bacterial agent products containing the strain and methods for remediating heavy metal pollution based thereon.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] Through research and exploration, this invention isolated a strain of Bacillus atrophaeus, SCAU-H1, from soil collected from arsenic smelting waste areas in Wenshan, Yunnan Province. Research results show that this strain, SCAU-H1, can effectively remove heavy metals from water bodies and significantly reduce the content of available heavy metals in soil, making it suitable for remediating heavy metal-contaminated water and soil. Therefore, this invention claims protection for the following:

[0009] A strain of Bacillus atrophaeus SCAU-H1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2023, with accession number GDMCC NO: 64181.

[0010] A microbial agent containing the aforementioned Bacillus atrophus SCAU-H1 for remediation of heavy metal contamination.

[0011] Application of the above-mentioned Bacillus atrophus SCAU-H1 or the above-mentioned bacterial agent in the removal of heavy metals.

[0012] Application of the above-mentioned Bacillus atrophus SCAU-H1 or the above-mentioned bacterial agent in the remediation or treatment of heavy metal pollution.

[0013] Application of the above-mentioned Bacillus atrophus SCAU-H1 or the above-mentioned bacterial agent in the remediation or treatment of heavy metal contaminated environments.

[0014] The environment is either water or soil.

[0015] Preferably, when the application environment is aquatic, the heavy metal is any one or more of As, Cd, Pb, and Sb; when the application environment is soil, the heavy metal is Cd and / or Pb.

[0016] Preferably, the heavy metal As is As(III) and / or As(V).

[0017] One method for remediating heavy metal-contaminated environments is to treat them using the aforementioned Bacillus atrophus SCAU-H1 or the aforementioned bacterial agent.

[0018] The present invention has the following beneficial effects:

[0019] This invention has yielded a strain of Bacillus atrophaeus, SCAU-H1, which effectively removes heavy metals from water, including As(III), As(V), Cd, Pb, and Sb, with particularly excellent removal efficiency for As(V). Furthermore, strain SCAU-H1 can significantly reduce the available Cd and Pb content in soil. Strain SCAU-H1 can be used for the remediation of heavy metal-contaminated water and soil, demonstrating high application value. Attached Figure Description

[0020] Figure 1 Phylogenetic tree of strain SCAU-H1.

[0021] Figure 2 This is a schematic diagram of the operation procedure for removing SCAU-H1 strain in aqueous solution.

[0022] Figure 3 This is a schematic diagram of the operation for the tolerance test of strain SCAU-H1.

[0023] Figure 4 The growth changes of the activated strain SCAU-H1 under different As / Cd / Pb concentrations.

[0024] Figure 5 The growth of strain SCAU-H1 in LB medium containing different concentrations of As after 72 h is shown.

[0025] Figure 6 The growth of strain SCAU-H1 in LB medium containing different concentrations of Cd after 72 h is shown.

[0026] Figure 7 The growth of strain SCAU-H1 in LB medium containing different concentrations of Pb after 72 h is shown.

[0027] Figure 8 This is a schematic diagram of the soil culture experiment for strain SCAU-H1. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents, materials and microorganisms used in the following examples are commercially available.

[0030] Luria-Bertani medium (LB medium) composition: 10g peptone, 5g yeast extract, 10g sodium chloride, water to 1000mL.

[0031] Example 1: Isolation and identification of strain SCAU-H1

[0032] 1. Sample:

[0033] Soil samples were collected from the arsenic smelting waste slag mining area in Wenshan, Yunnan (E103°55'46"; N23°24'46").

[0034] 2. Methods for strain isolation

[0035] Take 5g of collected soil, sieve it and place it in 95mL of sterile LB medium. Add filtered and sterilized standard arsenic (As) / cadmium (Cd) / lead (Pb) solutions to make the As(III) / As(V) concentration 50mg / L, the Cd concentration 1mg / L and the Pb concentration 100mg / L.

[0036] Then, under sterile conditions and in a constant-temperature shaker, the culture was carried out at 30°C for 24 hours. Afterward, a 1% inoculum was transferred to a higher concentration of As / Cd / Pb medium, and cultured under the same conditions, gradually increasing the As / Cd / Pb concentration. After 5 transfers, the medium was diluted and plated. 1 mL of the supernatant was then used to... -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Perform serial dilutions, with a dilution factor of 10. -5 and 10 -6 The culture medium was spread, and colonies with good growth were selected for streaking and isolation until single colonies with high tolerance to As / Cd / Pb were obtained and recorded as strain SCAU-H1.

[0037] 3. Strain identification

[0038] Strain SCAU-H1 exhibits relatively regular rod-shaped and spherical colonies in petri dishes, forming dense but small colonies with a rough, opaque surface and relatively neat edges. Its morphology is largely consistent with that of the previously discovered Bacillus atrophaeus.

[0039] The SCAU-H1 strain was sent to a biotechnology company for 16S rDNA identification. DNA was extracted using the TSINGKE Plant DNA Extraction Kit (Universal) (catalog number: TSP101). PCR amplification was performed using universal primers for species identification as shown in Table 1. The amplified products were analyzed by agarose gel electrophoresis to determine if the PCR product bands matched the target size, were singular, and showed no banding. After the PCR products passed the test, the target band was cut and purified. The recovered products were then subjected to Sanger sequencing. The sequencing results were spliced ​​using ContigExpress software, and inaccurate ends were removed. Batch alignment of the spliced ​​sequences with a nucleic acid database using BLASTN (latest version v2.13) was performed. The latest version of the nt library was selected as the nucleic acid database. By comparing the nt library with the BLASTN database, the Accession Number of homologous sequences, species identification, and annotation could be obtained.

[0040] Results: A phylogenetic tree was constructed based on the 16S rDNA sequence of strain SCAU-H1 as follows: Figure 1 As shown in Table 2, strain SCAU-H1 and the existing Bacillus atrophaeus strain NX-12 have the same homology (100%), indicating that this species belongs to Bacillus atrophaeus.

[0041] Table 1. Universal primers for strain identification

[0042]

[0043] Table 2. Identification results of strain SCAU-H1

[0044]

[0045] In summary, strain SCAU-H1 was identified as Bacillus atrophaeus and deposited on December 22, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 64181), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] Example 2: Heavy metal removal experiment by strain SCAU-H1

[0047] 1. Experimental Methods

[0048] Strain SCAU-H1 was activated and then inoculated into 30 mL LB medium containing As(III) (10 mg / L), As(V) (10 mg / L), Cd (1 mg / L), Pb (200 mg / L), and Sb (5 mg / L), respectively. After incubation at 35°C and 150 rpm for 48 h in a shaker, samples were transferred to sterile centrifuge tubes and centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter, and 4.5 mL of the supernatant was accurately transferred to a digestion tube. 0.4 mL of concentrated nitric acid and 0.1 mL of concentrated hydrochloric acid were added, and the mixture was microwave-digested at 160°C for 10 min. After digestion and cooling to room temperature, the digest was transferred to a 10 mL volumetric flask, diluted to 10 mL with deionized water, and shaken well before analysis. The concentration of As in the digest was determined by hydride generation-atomic fluorescence spectrometry, and the concentrations of Cd and Pb in the digest were determined by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0049] A schematic diagram of the SCAU-H1 aqueous solution removal test is shown below. Figure 2 .

[0050] 2. Experimental Results

[0051] To evaluate the removal efficiency of strain SCAU-H1 for As / Cd / Pb / Sb in aqueous solution, we measured the elemental concentrations in the supernatant of each culture medium. Table 3 shows that at the end of the experiment, the As(V) concentration in the supernatant after inoculation with strain SCAU-H1 decreased from 10 mg / L to 0 mg / L, corresponding to a removal rate of 100%, while the removal rate for As(III) was 65.26%. Furthermore, the removal rates of Cd, Pb, and Sb after inoculation with SCAU-H1 were 59.21%, 58.82%, and 41.79%, respectively.

[0052] Therefore, the results show that strain SCAU-H1 can effectively remove heavy metals from aqueous solutions, including As(III), As(V), Cd, Pb and Sb, with particularly excellent removal effect on As(V).

[0053] Table 3. Removal efficiency of strain SCAU-H1 in aqueous solutions with different concentrations of heavy metals.

[0054]

[0055] Example 3: Heavy metal tolerance test of strain SCAU-H1

[0056] 1. Test Methods

[0057] Based on the results of the aqueous solution removal test, the isolated strain SCAU-H1 was activated and cultured in accordance with the above test. It was inoculated into 100 mL of LB medium and cultured in a shaker at 150 rpm and 30 °C. After 72 h of culture, SCAU-H1 was inoculated at a 1% inoculum size (100 μL of bacterial suspension). SCAU-H1 was inoculated into 10 mL of medium containing different concentrations of As(V) (0, 0.5, 1, 2, 5, 10, 20, 30, 50, 100 mg / L), Cd (0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10 mg / L), and Pb (0, 2, 5, 10, 20, 50, 100, 200, 300 mg / L). After 72 h of culture at 150 rpm and 30 °C, the OD of the bacterial suspension was measured using a multi-functional microplate reader. 600 Value. The procedure diagram for the SCAU-H1 strain tolerance test is shown below. Figure 3 As shown in the figure. The growth changes of activated strain SCAU-H1 under different As / Cd / Pb concentrations are shown in the figure. Figure 4 As shown.

[0058] 2. Test Results

[0059] To evaluate the tolerance of Bacillus atrophaeus SCAU-H1, we measured the OD of the strain under different concentrations of As, Cd, and Pb. 600 value. Figure 5-7 The results show that:

[0060] When the initial As(V) concentration is reduced to below 30 mg / L, the growth of the strain is not significantly inhibited, and SCAU-H1 can tolerate As(V) up to 100 mg / L.

[0061] Regarding Cd, strain SCAU-H1 showed good growth when the initial Cd concentration was below 4 mg / L, and its tolerance to Cd was up to 10 mg / L.

[0062] When the initial Pb concentration is below 50 mg / L, SCAU-H1 exhibits good resistance. When the initial Pb concentration is 100 mg / L, although the growth of SCAU-H1 is significantly inhibited, it can still grow. The tolerance of SCAU-H1 to Pb can reach up to 300 mg / L.

[0063] Therefore, the results show that SCAU-H1 can simultaneously exhibit high tolerance to As(V), Cd, and Pb, and compared with strains resistant to a single or two heavy metals, it can show excellent resistance to multiple heavy metals.

[0064] Example 4: Soil culture experiment to study the effect of strain SCAU-H1 on soil available As / Cd / Pb.

[0065] 1. Test Methods

[0066] Based on the results of the aqueous solution removal experiment, a soil experiment was conducted to study the effect of strain SCAU-H1 on the available As / Cd / Pb in soil. Soil samples were collected from the surface layer (0-20 cm) of an arsenic smelting waste mine in Wenshan, Yunnan Province. After air-drying, the soil was passed through a 2 mm sieve and ground. Soil samples with different heavy metal concentrations (Total As: 91.384 mg / kg) were prepared for the experiment. Two treatments were administered: untreated soil (CK) and soil treated with 1 mL of SCAU-H1 bacterial solution. All treatments were replicated in triplicate, with 10 g of soil used for each treatment. The soil was watered daily to maintain 60% of field capacity. After 20 days, soil samples were taken and passed through a 2 mm sieve. Available Cd and Pb were extracted from the soil using 0.005 M DTPA, and available As was extracted using 0.05 M (NH4)2SO4. The concentration of As in the solution was determined using hydride generation-atomic fluorescence spectrometry, and the concentrations of Cd and Pb in the solution were determined using ICP-OES. The operation diagram of soil culture experiment for strain SCAU-H1 is shown below. Figure 8 As shown.

[0067] 2. The test results are shown in Table 4:

[0068] Table 4. Effects of SCAU-H1 inoculation on available As, Cd, and Pb in soil 1

[0069]

[0070] Note: Different lowercase letters indicate significant differences (p<0.05) among available As / available Cd / available Pb under different treatments at the same soil concentration.

[0071] The immobilization capacity of *Bacillus atrophaeus* SCAU-H1 was assessed by measuring the available As, Cd, and Pb content in contaminated soil. Table 4 shows the effects of strain SCAU-H1 on the immobilization of As, Cd, and Pb. The results show that SCAU-H1 had no immobilization effect on As. For Cd and Pb, the inoculated strain had a significant effect. Strain SCAU-H1 significantly reduced the available Cd concentration in the soil from 0.132 mg / kg to 0.110 mg / kg, with a Cd immobilization rate of 17%. Inoculation bacteria can affect the composition of Cd in soil by influencing the binding of Cd with functional groups (such as thiol, carboxyl, and amide groups) and the binding of bacterial extracellular polymers to soil organic acids (Shou et al., 2018). SCAU-H1 reduced the available Pb concentration in the soil from 1.015 mg / kg to 0.890 mg / kg, with a Pb immobilization rate of 12%. The results showed that SCAU-H1 could effectively reduce the concentration of available Cd and Pb in the soil, demonstrating a significant immobilization effect.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus atrophaeus SCAU-H1, characterized in that, The bacteria were deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 22, 2023, with accession number GDMCC NO: 64181.

2. A microbial agent capable of remediating heavy metal pollution, characterized in that, Contains the Bacillus atrophus SCAU-H1 as described in claim 1.

3. The application of Bacillus atrophus SCAU-H1 of claim 1 or the bacterial agent of claim 2 in the removal of heavy metals.

4. The application of Bacillus atrophus SCAU-H1 as described in claim 1 or the bacterial agent as described in claim 2 in the remediation or treatment of heavy metal pollution.

5. The application of Bacillus atrophus SCAU-H1 as described in claim 1 or the bacterial agent as described in claim 2 in the remediation or treatment of heavy metal contaminated environments.

6. The application according to claim 5, characterized in that, The environment is either water or soil.

7. The application according to any one of claims 3-6, characterized in that, When the application environment is aquatic, the heavy metal is any one or more of As, Cd, Pb, and Sb; when the application environment is soil, the heavy metal is Cd and / or Pb.

8. The application according to claim 7, characterized in that, The heavy metal As is As(III) and / or As(V).

9. A method for remediating heavy metal-contaminated environments, characterized in that, Treatment is performed using the Bacillus atrophus SCAU-H1 of claim 1 or the bacterial agent of claim 2.

10. The method according to claim 9, characterized in that, The environment is either water or soil.

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