Heavy metal-resistant bacterium cn743 and application thereof

Abstract

This invention discloses a heavy metal-resistant bacterium CN743 and its applications, relating to the field of microbial technology. The heavy metal-resistant bacterium CN743, classified as *Thermomonas aquatica* CN743, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20241195, deposited on June 11, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. This strain exhibits high tolerance to various heavy metal ions, enabling it to survive and reproduce in environments with high concentrations of heavy metal pollution, facilitating the rapid formation of microbial communities in aquatic environments. It can efficiently reduce the concentration of heavy metal ions in groundwater within a short time, achieving water remediation and treatment, avoiding the need for long-term field monitoring. It can be applied to the remediation of various heavy metal pollutants in various water bodies, such as manganese and lead, with simple operation, low cost, and significant effects, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a heavy metal resistant bacterium CN743 and its applications. Background Technology

[0002] Human activities have a significant impact on groundwater ecosystems, with heavy metal pollution being a major environmental issue that poses a serious threat to the ecological environment and human health. Heavy metal pollution refers to environmental pollution caused by heavy metals or their compounds. It is mainly caused by anthropogenic factors such as mining, exhaust emissions, wastewater irrigation, and the use of products exceeding heavy metal limits. In-situ groundwater remediation is an environmental protection technology that aims to remediate groundwater directly within the aquifer without extracting the contaminated water. This technology primarily employs physical, chemical, and biological methods to treat contaminated groundwater in situ to achieve remediation goals. In-situ remediation technologies include various methods such as air disturbance, permeable reactive barriers, chemical oxidation, chemical reduction, thermal treatment, biodegradation, and phytoremediation. These methods are applicable to different types of pollutants and various hydrogeological conditions.

[0003] Solving the problem of heavy metal pollution, especially in-situ groundwater remediation, has always been a very urgent task. Traditional heavy metal pollution remediation methods suffer from high costs and poor effectiveness. In-situ bioremediation technology refers to the process of using natural or cultured microorganisms to degrade or transform harmful pollutants in contaminated areas. It falls under the category of environmental remediation and is a branch of bioremediation. Bioremediation originated in the 1970s and has now become an important direction for technological development in the field of environmental engineering. Bioremediation technology is the most valuable and vital biological treatment method of the future. Therefore, finding a microbial strain with high tolerance to heavy metals and bioremediation capabilities is of great significance. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide a heavy metal-resistant bacterium, CN743. Another technical problem this invention aims to solve is to provide an application of the heavy metal-resistant bacterium CN743 for the treatment of heavy metal-polluted water bodies, achieving water body restoration and remediation.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A heavy metal resistant bacterium, CN743, classified as Thermomonas aquatica CN743, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20241195, deposited on June 11, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] Application of heavy metal resistant bacteria CN743 in water remediation.

[0008] The water remediation involves degrading heavy metal pollutants in the water.

[0009] The heavy metal pollutants include one or more of manganese, lead, mercury, and cadmium.

[0010] Application of heavy metal resistant bacteria CN743 in reducing the concentration of heavy metal ions in groundwater.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] 1) The aquatic thermophilic pseudomonas Thermomonas aquatica CN743 disclosed in this invention, which is classified and named Thermomonas aquatica CN743, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20241195, deposit date: June 11, 2024, and deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0013] 2) The aquatic thermomonas CN743 disclosed in this invention has strong tolerance to a variety of heavy metal ions, and can survive and reproduce in environments with high concentrations of heavy metal pollution, which facilitates the rapid formation of microbial communities in aquatic environments.

[0014] 3) The aquatic thermophilic pseudomonas CN743 disclosed in this invention has a strong degradation ability and can efficiently reduce the concentration of heavy metal ions in groundwater in a short time, thereby achieving water body restoration and treatment and avoiding the need for long-term field monitoring.

[0015] 4) The aquatic thermophilic pseudomonas CN743 disclosed in this invention has a wide range of applications and can be used to remediate various heavy metal pollutants in various water bodies, such as manganese and lead. The technology is simple to operate, low in cost, and has significant effects, and has broad application prospects. Attached Figure Description

[0016] Figure 1 A graph showing the number of contigs obtained by comparing the CN743 sequence;

[0017] Figure 2The image shows the growth of Pseudomonas CN743 after adding a manganese aqueous solution (15 mg / L) to the petri dish.

[0018] Figure 3 This image shows the growth of Pseudomonas CN743 after adding a lead solution (0.5 mg / L) to the petri dish. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0020] The groundwater samples used in the following examples were obtained from the Wuli monitoring well in Jiujiang, Jiangxi Province. The manganese content of the groundwater in this area is 0.01-4.66 mg / L and the lead content is 0.01-0.05 mg / L. The bacteria in this environment live in water with high concentrations of heavy metals for a long time.

[0021] Example 1

[0022] 1. Strains Isolation

[0023] Groundwater samples were collected, and 5-10 mL of the sample was added to a 250 mL Erlenmeyer flask containing 100 mL of liquid LB medium. The flask was incubated at 35°C and 150 rpm for 24 h in a shaker. Then, 5 mL of the sample was added to a medium containing 5 mg / L manganese and incubated for 48 h. 20 μL of the final solution was streaked onto a solid medium containing 10 mg / L manganese and incubated upside down in a 35°C incubator. After single colonies grew on the solid plates, different single colonies were picked, isolated, purified, and numbered.

[0024] The isolated strains were subjected to resistance experiments against various heavy metals. The method involved adding various heavy metal-contaminated water samples (manganese concentration 15 mg / L, lead concentration 0.5 mg / L, etc.) to petri dishes inoculated with the strains, and culturing them at 35℃ for 24 h. Each concentration was repeated three times. Strains with high tolerance to heavy metals were selected as pre-selected strains. The purified pre-selected strains were inoculated into aqueous solutions of various heavy metals (such as manganese and lead), and cultured at 35℃ and 150 rpm for 48 h. Changes in heavy metal concentrations were then measured. The strain CN743, with the highest manganese removal rate, was selected.

[0025] 2. Strain identification and preservation

[0026] 1) Morphological identification: When this strain was cultured on LB plates at 35°C for 24 hours, round, light brown colonies with smooth surfaces and regular edges were observed.

[0027] 2) Molecular biological identification: Using universal primers for 16S rDNA gene amplification, PCR amplification was performed with CN743 bacterial culture as a template. The 16S rDNA nucleotide sequence of strain CN743 was obtained as shown in SEQ ID NO.1, totaling 1495 bp.

[0028] The 16S rDNA nucleotide sequence of strain CN743 was analyzed for sequence homology using the BLAST search system of the National Center for Biotechnology Information (NCBI). The sequence showed 100% similarity to the 16S rDNA gene sequence of *Thermomonas aquatica*. Based on the morphological and molecular biological characteristics of strain CN743, it was identified as belonging to *Thermomonas aquatica* and named *Thermomonas aquatica* CN743. The strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20241195, deposit date June 11, 2024, and address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0029] Example 2

[0030] The culture conditions in the initial culture system were optimized (all of the following are single-factor experimental methods) to screen the optimal growth conditions for the strain, as follows:

[0031] 1) Screening using different pH values

[0032] Using a metagenomic database established at various sampling sites in Jiangxi Province, the number of contigs was obtained by comparing the CN743 sequence. Figure 1 ), and statistical analysis of habitat pH values ​​was performed on samples with high abundance.

[0033] The strains were inoculated at a 2% inoculum into liquid LB medium with different pH values ​​(6.5, 7, 7.5, 8, 8.5) and cultured at 35°C for 24 h for preliminary screening. The obtained target strains exhibited good fermentation performance and tolerance. The absorbance at OD600 nm was measured using a spectrophotometer, and the medium was zeroed.

[0034] 2) Screening using different temperatures

[0035] The strains were inoculated at a 2% inoculum in liquid LB medium and cultured at different temperatures (30℃, 33℃, 35℃, 38℃, and 40℃) for 24 h to perform preliminary screening, ensuring that the obtained target strains exhibited good fermentation performance and tolerance. The absorbance at OD600nm was measured using a spectrophotometer, and the medium was zeroed.

[0036] Table 1 Different tolerance tests

[0037] Maximum tolerance 35℃ 7-7.5

[0038] Example 3

[0039] 1. Determination of the tolerance of *Pseudomonas aeruginosa* CN743 to different heavy metal ions

[0040] Because the bacterial strains in this environment have long lived in water bodies with high concentrations of heavy metals, based on 6 years of historical monitoring data, the maximum measured concentrations were: manganese 4.66 mg / L (Class V) and lead 0.05 mg / L (Class IV). Tolerance testing was conducted only for these high concentration limits. The testing method involved adding various heavy metal-contaminated water samples to Petri dishes inoculated with Pseudomonas CN743. Strains were selected based on the limits for Class V water bodies in the "Groundwater Quality Standard" (GBT 14848-2017) at 5-10 times the specified limits. The samples were incubated at 35℃ for 24 hours, with each concentration repeated three times. Strains exhibiting high tolerance to heavy metals were selected as pre-selected strains.

[0041] The results are as follows Figure 2 As shown, when manganese aqueous solution (concentration 15 mg / L) was added to a Petri dish inoculated with Pseudomonas CN743, the strain grew normally and multiplied in large quantities.

[0042] The results are as follows Figure 3 As shown, when lead aqueous solution (concentration 0.5 mg / L) was added to the Petri dish inoculated with Pseudomonas CN743, the strain grew normally.

[0043] 2. Determination of the degradation capacity of heavy metal pollutants by *Pseudomonas aeruginosa* CN743

[0044] Strains CN743 were cultured to the logarithmic growth phase and then inoculated at a 2% inoculum into various heavy metal aqueous solutions as shown in Table 1. Adsorption was carried out at 35℃ with shaking at 150 rpm for 48 hours, with each group repeated three times. The changes in heavy metal content before and after treatment were measured, and the removal efficiency of strain CN743 for high-concentration metal solutions was recorded.

[0045] The results are shown in Table 2. The manganese removal rate reached 88.2% and the lead removal rate reached 78.7%.

[0046] Table 2. Removal efficiency of different heavy metals in aqueous solutions by the strains after 48 hours.

[0047] manganese 10mg / L 88.16 copper 10mg / L 33.31 lead 0.05 mg / L 78.67 mercury 0.02 mg / L 21.88 Chromium (hexavalent) 1mg / L 10.05

[0048] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A heavy metal-resistant bacterium, CN743, is classified and named... Thermomonas aquatica It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20241195, deposit date: June 11, 2024, and deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application of the heavy metal resistant bacteria CN743 as described in claim 1 in water remediation; wherein the water remediation is the degradation of heavy metal pollutants in the water; wherein the heavy metal pollutants are one or more of manganese, lead, mercury, and cadmium.

3. The application of the heavy metal resistant bacteria CN743 according to claim 1 in reducing the concentration of heavy metal ions in groundwater; wherein the heavy metal is one or more of manganese, lead, mercury, and cadmium.

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