Heavy metal resistant bacterium CN743 and application thereof

Abstract

The invention discloses a heavy metal resistant bacterium CN743 and application thereof, and relates to the technical field of microorganisms. The invention discloses a heavy metal resistant bacterium CN743, which is classified and named as Thermomonas aquacea CN743, and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC M20241195, the preservation date is June 11, 2024, and the preservation address is Luojia Hill on eight road, Wuchang District, Wuhan City, Hubei Province. The strain has high tolerance to various heavy metal ions, can survive and breed in a high-concentration heavy metal polluted environment, and is convenient to quickly form a microbial population in a water body environment; the concentration of heavy metal ions in underground water can be efficiently reduced in a short time, water body restoration and treatment are achieved, and long-term field monitoring is avoided; the method can be applied to repair of various heavy metal pollutants such as manganese and lead in various water bodies, is simple in technical operation, low in cost and remarkable in effect, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a heavy metal resistant bacterium CN743 and application thereof. Background Art

[0002] Human activities have a huge impact on the ecological system of groundwater. Among them, heavy metal pollution is one of the important issues in today's environmental problems, posing 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 human factors such as mining, waste gas emissions, sewage irrigation, and the use of products with excessive heavy metals. In-situ groundwater remediation is an environmental protection technology that aims to remediate groundwater directly in the underground aquifer without extracting contaminated groundwater. This technology mainly uses physical, chemical and biological methods to treat contaminated groundwater in situ to achieve the goal of treatment. In-situ treatment technology includes a variety of methods, such as air disturbance, permeable reaction wall, chemical oxidation, chemical reduction, thermal treatment, biodegradation and phytoremediation. These methods are suitable for different types of pollutants and different hydrogeological conditions.

[0003] How to solve the problem of heavy metal pollution, especially the in-situ remediation of groundwater, has always been a very urgent task. Traditional heavy metal pollution remediation methods have problems such as high cost and poor effect. In-situ bioremediation technology refers to a process in which natural or cultured microorganisms are used to degrade or transform harmful pollutants in polluted areas. It belongs to 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 in the future. Therefore, it is of great significance to find a microbial strain with high heavy metal tolerance and bioremediation ability. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a heavy metal resistant bacterium CN743. The technical problem to be solved by the present invention is to provide an application of a heavy metal resistant bacterium CN743 for the treatment of heavy metal polluted water bodies to achieve the restoration and treatment of water bodies.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A heavy metal-resistant bacterium CN743, classified and named Thermomonas aquatica CN743, has been deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number: CCTCC M 20241195, the deposit date: June 11, 2024, and the deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

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

[0008] The water body remediation is to degrade heavy metal pollutants in the water body.

[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 heavy metal ion concentration in groundwater.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

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

[0013] 2) The aquatic thermophilic Pseudomonas CN743 disclosed in the present invention has strong tolerance to a variety of heavy metal ions, can survive and reproduce in a high-concentration heavy metal pollution environment, and is convenient for quickly forming a microbial community in an aquatic environment.

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

[0015] 4) The aquatic thermophilic Pseudomonas CN743 disclosed in the present invention has a wide range of applications and can be used to remediate a variety of heavy metal pollutants in various water bodies, such as manganese, lead, etc. The technical operation is simple, the cost is low, the effect is significant, and it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph of the number of contigs obtained by aligning the CN743 sequence;

[0017] Figure 2The figure is a graph showing the growth of the strain after adding a manganese aqueous solution (concentration 15 mg / L) to a plate inoculated with Pseudomonas CN743;

[0018] Figure 3 This is a graph showing the growth of the strain after adding lead aqueous solution (concentration 0.5 mg / L) to a plate inoculated with Pseudomonas CN743. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

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

[0021] Example 1

[0022] 1. Strain Isolation

[0023] Collect groundwater samples, add 5-10mL of water sample to a 250mL conical flask containing 100mL of liquid LB culture medium, place the conical flask at 35℃, 150r / min, and culture in a shaker for 24h, then take 5mL of it and add it to a culture medium with a manganese concentration of 5mg / L and culture it for 48h. Take 20μL of the culture solution from the final solution and streak it on a solid culture medium containing 10mg / L manganese, invert it in a 35℃ constant temperature incubator, and after a single colony grows on the solid plate, pick different single colonies for separation, purification and numbering.

[0024] The isolated strains were subjected to resistance tests for various heavy metals. The determination method was to add various heavy metal-contaminated water samples (manganese pollution concentration 15 mg / L, lead concentration 0.5 mg / L, etc.) to the plate inoculated with the strains, and cultured in a 35°C culture medium for 24 hours, with each concentration repeated 3 times. The strains with higher tolerance to heavy metals were selected as pre-selected strains, and the isolated and purified pre-selected strains were inoculated in various heavy metal aqueous solutions (such as manganese, lead, etc.), cultured at 35°C and 150r / min for 48 hours, and the changes in heavy metal concentrations were determined. The strain CN743 with the highest manganese removal rate was screened.

[0025] 2. Strain identification and preservation

[0026] 1) Morphological identification: The strain was cultured on LB plates at 35°C for 24 h and circular colonies were observed. They were light brown in color, with smooth surfaces and neat edges.

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

[0028] The 16S rDNA nucleotide sequence of strain CN743 was analyzed by the BLAST search program system of the National Center for Biotechnology Information (NCBI) of the United States for sequence homology analysis, and the similarity with the 16SrDNA gene sequence of aquatic thermogenic Pseudomonas was 100%. According to the morphological characteristics and molecular biological identification characteristics of the CN743 strain, the CN743 strain was identified as aquatic thermogenic Pseudomonas and named as Thermomonas aquatica CN743. Its classification name is Thermomonas aquatica CN743, which has been deposited in the China Center for Type Culture Collection (CCTCC), with a deposit number of CCTCC M20241195, a deposit date of June 11, 2024, and a deposit address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0029] Example 2

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

[0031] 1) Screening using different pH values

[0032] Using the metagenomic database established at each sampling point in Jiangxi Province, the CN743 sequence was compared to obtain the contig number ( Figure 1 ), and statistically analyze the measured values ​​of habitat pH at the sampling points with higher abundance.

[0033] The strains were inoculated in liquid LB medium with different pH values ​​(6.5, 7, 7.5, 8, 8.5) at 2% inoculum, cultured at 35°C for 24 hours, and the strains were preliminarily screened to obtain target strains with good fermentation performance and tolerance. The absorbance at OD600nm was measured with a spectrophotometer, and the medium was zeroed.

[0034] 2) Use different temperatures for screening

[0035] The strains were inoculated into liquid LB medium at 2% inoculation, and cultured at different temperatures (30°C, 33°C, 35°C, 38°C, 40°C) for 24 hours to perform preliminary screening of the strains, so that the fermentation performance and tolerance of the target strains were good. The absorbance at OD600nm was measured with a spectrophotometer, and the culture medium was zeroed.

[0036] Table 1 Different tolerance tests

[0037] category temperature pH Highest tolerance 35℃ 7-7.5

[0038] Example 3

[0039] 1. Determination of tolerance of aquatic thermophilic Pseudomonas CN743 to different heavy metal ions

[0040] Because the bacteria in this environment have lived in high-concentration heavy metal water for a long time, according to the statistics of 6 years of historical monitoring data, the maximum measured concentrations are: manganese content 4.66mg / L (Class V), lead content 0.05mg / L (Class IV), and tolerance is only measured for high concentration limits. The determination method is to add various heavy metal contaminated water samples to the plate inoculated with Pseudomonas CN743, and screen according to the "Groundwater Quality Standard" (GBT 14848-2017 evaluation standard 5-10 times the limit of five types of water bodies for screening, culture in 35℃ medium for 24h, and repeat 3 times for each concentration. The strains with higher tolerance to heavy metals are selected as pre-selected strains.

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

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

[0043] 2. Degradation ability of Pseudomonas aquaticus CN743 on heavy metal pollutants

[0044] Strain CN743 was cultured to the logarithmic phase and inoculated into various heavy metal aqueous solutions as shown in Table 1 at an inoculum of 2%. The culture was carried out at 35°C, 150 r / min, and oscillation adsorption was carried out for 48 hours. Each group was repeated 3 times. The changes in the content of heavy metals 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 strains for different heavy metal aqueous solutions for 48 h

[0047] Heavy metal aqueous solution Initial concentration Heavy metal removal efficiency (%) manganese 10mg / L 88.16 copper 10mg / L 33.31 lead 0.05mg / L 78.67 mercury 0.02mg / L 21.88 Chromium (hexavalent) 1mg / L 10.05

[0048] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

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

2. Use of the heavy metal resistant bacteria CN743 described in claim 1 in water body remediation.

3. The use according to claim 2, characterized in that: The water body remediation is to degrade heavy metal pollutants in the water body.

4. The use according to claim 3, characterized in that: The heavy metal pollutants include one or more of manganese, lead, mercury and cadmium.

5. Use of the heavy metal resistant bacteria CN743 described in claim 1 in reducing the concentration of heavy metal ions in groundwater.

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