Pseudomonas aeruginosa T1 resistant to multiple heavy metals and application thereof

Abstract

The present application relates to a kind of heavy metal-resistant pseudomonas aeruginosa T1 and its application, can effectively solve the problem of lacking functional strain capable of growing in multiple heavy metals at present, its solution technical scheme is, the pseudomonas aeruginosa T1 described in the present application, its classification designation is pseudomonas aeruginosa ( Pseudomonas aeruginosa ), has been preserved in China Microbial Culture Collection Center, preservation number is CGMCC No.28029, preservation date is July 27, 2023, and the address is No.3, Xili, Beichen, Beijing, Chaoyang District, the 16S rDNA gene sequence of the strain is as shown in SEQ ID No.1, the present application pseudomonas aeruginosa T1 is resistant to multiple types of heavy metals, and has strong resistance, and is suitable for application in multiple heavy metal-polluted water or soil treatment, and has good popularization and application value.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Pseudomonas aeruginosa T1 resistant to multiple heavy metals and its applications. Background Technology

[0002] With the rapid development of industrial technology, such as in mining, metallurgy, electroplating, and leather tanning, the discharge of waste and wastewater containing various heavy metals is increasing, causing widespread pollution of soil and water bodies and seriously endangering the ecological environment and human health. Heavy metal pollution in soil and water refers to the result of concentrations of metal ions such as copper, zinc, cadmium, chromium, and lead exceeding certain ranges. Heavy metals can cause direct or indirect harm to human health through various forms of deposition, including contamination of groundwater, agricultural products, and aquatic products.

[0003] Heavy metal pollution typically contains multiple metal elements and exists in complex forms, making it difficult to remediate. Traditional methods for heavy metal pollution remediation mainly include physical, chemical, and bioremediation methods. Physical remediation methods include soil replacement, leaching, electrokinetic remediation, and thermal treatment. Chemical remediation methods involve adding chemical substances, such as calcium carbonate, phosphates, and metal oxides, to oxidize, reduce, precipitate, or inhibit heavy metal elements, thereby reducing their bioavailability. Although physical and chemical remediation technologies are relatively mature, they suffer from complex processes, high energy consumption, high costs, and serious secondary pollution. Under increasingly stringent environmental protection requirements, they do not align with my country's sustainable development strategy and are not a long-term solution for heavy metal pollution remediation. Bioremediation technology, due to its advantages of safe and simple operation, low treatment costs, and no secondary pollution, has great development potential in heavy metal pollution remediation. However, the remediation of heavy metal-contaminated areas by microorganisms generally requires a long period of time, and most studies have found that the types and concentrations of heavy metals that microorganisms can tolerate are often very limited, which is difficult to meet the current urgent needs for the remediation of heavy metal pollution in water and soil. Therefore, this has greatly limited the promotion and application of microbial technology, which is related to the lack of functional strains that can tolerate the growth of multiple heavy metals. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide a strain of Pseudomonas aeruginosa T1 that is resistant to multiple heavy metals and its application, which can effectively solve the problem of the lack of functional strains that can grow in a variety of heavy metals.

[0005] The technical solution provided by this invention is that the *Pseudomonas aeruginosa* T1 described in this invention is classified and named *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosaThe strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28029, deposit date July 27, 2023, and address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The 16S rDNA gene sequence of this strain is shown in SEQ ID No. 1.

[0006] The application of Pseudomonas aeruginosa T1 in the remediation of heavy metal pollution, wherein the heavy metal is at least one of copper, zinc, chromium, cadmium, and lead.

[0007] The highest concentrations of heavy metals that Pseudomonas aeruginosa T1 can tolerate are 200 mg / L for copper, 1000 mg / L for zinc, 1000 mg / L for chromium, 300 mg / L for cadmium, and 800 mg / L for lead.

[0008] The application of the aforementioned Pseudomonas aeruginosa T1 bacterial solution in the remediation of various heavy metal contaminations.

[0009] The method for preparing Pseudomonas aeruginosa T1 bacterial suspension is as follows: Pseudomonas aeruginosa T1 is inoculated into 100 mL of sterilized NB liquid culture medium at an inoculation rate of 1%, and cultured at 30°C with shaking for 24 h to obtain T1 bacterial suspension.

[0010] The *Pseudomonas aeruginosa* T1 strain of this invention exhibits strong tolerance to a wide variety of heavy metals, making it suitable for application in the remediation of water or soil contaminated with multiple heavy metals. It demonstrates the ability to tolerate the growth of various heavy metals, such as Cu. 2+ Zn 2+ Cd 2+ Cr 6+ Pb 2+ They all exhibit strong tolerance and can be used to remediate soil and water bodies contaminated with heavy metals. They have advantages such as simple operation, green efficiency, safety and convenience, and low cost, and have good promotion and application value. Attached Figure Description

[0011] Figure 1 This is the phylogenetic tree of Pseudomonas aeruginosa of the present invention.

[0012] Figure 2 The growth effect of Pseudomonas aeruginosa of the present invention on culture plates with a combined heavy metal concentration of 40 mg / L and 60 mg / L (in the figure, (a) heavy metal concentration is 40 mg / L, (b) heavy metal concentration is 60 mg / L).

[0013] Figure 3 This is a graph showing the adsorption rate of various heavy metals by *Pseudomonas aeruginosa* of this invention. Detailed Implementation

[0014] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0015] The *Pseudomonas aeruginosa* T1 strain described in this invention was isolated from heavy metal-contaminated soil. This strain grew on NA solid medium plates at 30°C, producing light green colonies with moist and irregular surfaces. Using selective mediums with specific concentration gradients of heavy metals (copper, zinc, chromium, cadmium, and lead), the maximum concentration of each heavy metal ion that this bacterium could tolerate was determined. The selected pure strain underwent 16SRNA sequencing to obtain the 16SRNA gene sequence. Comparison with the NCBI nucleic acid database and phylogenetic tree construction using BLAST search and MEGA software revealed a 96.56% similarity to *Pseudomonas aeruginosa* (DSM50071). Based on growth morphology and culture conditions, this strain was confirmed as *Pseudomonas aeruginosa* and named *Pseudomonas aeruginosa* T1. Adding solutions containing copper, zinc, chromium, cadmium, and lead to the culture medium of *Pseudomonas aeruginosa* T1 created a complex heavy metal environment. Experimental culture showed that it exhibited good adsorption effects under conditions where multiple heavy metals were present.

[0016] This invention identified Pseudomonas aeruginosa T1 and assessed its tolerance to heavy metal Cu. 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ The maximum concentration was studied, and the results showed that Pseudomonas aeruginosa T1 is resistant to heavy metal Cu. 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ The maximum concentrations were 200 mg / L, 1000 mg / L, 1000 mg / L, 300 mg / L, and 800 mg / L, respectively. The relevant experimental data are as follows:

[0017] Five g of soil from a region in northern Henan province contaminated with multiple heavy metals was placed in an Erlenmeyer flask containing 60 mL of sterile distilled water and incubated at 30°C in a shaker for 30 minutes. Two mL of the supernatant was inoculated into 100 mL of sterile NB liquid medium and then incubated at 30°C with shaking at 150 rpm for 24 hours to obtain an enriched microbial strain capable of surviving in heavy metal-contaminated soil.

[0018] After serially diluting the culture medium containing the enriched microbial strains, 100 μL of each medium was spread onto samples containing Cu. 2+ Zn 2+ Cr 6+ Cd 2+ Pb2+ On NA solid culture medium plates, Cu 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ The concentration in the culture medium was 100 mg / L, and the cultures were incubated at a constant temperature of 30°C. Then, vigorous single colonies were selected and repeatedly strewn under the same culture conditions to obtain pure strains resistant to heavy metals such as copper, zinc, cadmium, chromium, and lead.

[0019] The formula for NB liquid culture medium is: 10 g / L peptone, 3 g / L beef meal, 5 g / L NaCl, 1 L deionized water, pH 7.0 ± 0.5;

[0020] The formula for NA solid culture medium is as follows: 10 g / L peptone, 3 g / L beef meal, 5 g / L NaCl, 15 g / L agar, 1 L deionized water, pH 7.0±0.5.

[0021] Cu in the experiment 2+ Derived from Cu(NO3)2·3H2O, Zn 2+ Derived from Zn(NO3)2·6H2O, Cr 6+ Derived from K2Cr2O7, Cd 2+ It originates from Cd(NO3)2·4H2O, and Pb originates from Pb(CH3COO)2·3H2O.

[0022] Selected purebred strains tolerant to heavy metals were inoculated onto NA solid agar plates containing different concentration gradients of copper, zinc, chromium, cadmium, and lead, and incubated at a constant temperature of 30℃. The heavy metal Cu... 2+ The concentrations in NA solid medium were 50, 100, 200, 300, and 400 mg / L; Zn 2+ The concentrations in NA solid medium were 400, 600, 800, 1000, and 1200 mg / L; Cr 6+ The concentrations in NA solid medium were 400, 600, 800, 1000, and 1200 mg / L; Cd 2+ The concentrations in NA solid medium were 50, 100, 200, 300, and 400 mg / L; Pb 2+ After comparing heavy metal tolerance levels of 400, 600, 800, 1000, and 1200 mg / L in NA solid medium, the strain with the best heavy metal tolerance was identified. This strain could tolerate a maximum Cu concentration of 200 mg / L on NA solid medium. 2+ 1000 mg / L Zn 2+ 1000 mg / L Cr 6+300 mg / L of Cd 2+ 800 mg / L of Pb 2 + It grows.

[0023] The selected strain was identified using molecular biology methods, and its 16SRNA sequence is shown in the sequence listing. Its sequence was compared with the NCBI nucleic acid database. A BLAST search revealed that *Pseudomonas aeruginosa* DSM50071 had the highest sequence similarity to this bacterium, reaching 96.56%. A phylogenetic tree was constructed using MEGA software, as shown below. Figure 1 As shown, the strain was identified as Pseudomonas aeruginosa and named Pseudomonas aeruginosa T1. It was deposited on July 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28029 and address at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing.

[0024] Pseudomonas aeruginosa is widely distributed in nature and is one of the common bacteria found in soil. It can also survive in water sources, air, human skin, and the human body. Under normal circumstances, it poses little threat to the ecological environment and human health.

[0025] The anti-multiple heavy metal growth activities of the screened *Pseudomonas aeruginosa* T1 and the purchased *Pseudomonas aeruginosa* were compared experimentally. The purchased *Pseudomonas aeruginosa* was obtained from the China General Microbiological Culture Collection Center (CGMCC), CGMCC number 1.10452. Using a sterilized inoculation loop, *Pseudomonas aeruginosa* T1 and the purchased *Pseudomonas aeruginosa* (denoted as S and M respectively in the experiment) were streaked onto a surface containing the complex heavy metal Cu. 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ The culture was performed at 30°C on solid NA medium plates with concentrations of 40 mg / L and 60 mg / L, and the results are as follows. Figure 2 As shown in the figure, it is clear that the screened Pseudomonas aeruginosa T1 has higher anti-heavy metal activity compared to the purchased conventional Pseudomonas aeruginosa M.

[0026] The cultured *Pseudomonas aeruginosa* T1 bacteria were inoculated at a rate of 1% into 100 mL of sterilized NB liquid medium and cultured in a shaker at 30°C and 150 rpm for 24 h to obtain a new generation of highly active T1 bacterial culture. Cu (Cu) was then added to the T1 bacterial culture. 2+ Zn 2+ Cr6+ Cd 2+ Pb 2+ The solution was prepared so that the concentration of each component in the T1 bacterial culture was 50 mg / L. After thorough mixing, the mixture was incubated for 2 days in a shaker at 30℃ and 150 rpm. The T1 bacteria were then removed by centrifugation and filtration. The Cu content in the solution was analyzed. 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ The remaining concentration was used to calculate the adsorption rate of each heavy metal ion by Pseudomonas aeruginosa T1, and the results are as follows: Figure 3 As shown. In composite Cu 2+ Zn 2+ Cr 6+ Cd 2+ Pb 2+ Under the presence of five heavy metal ions, Pseudomonas aeruginosa showed an adsorption rate of over 20% for each heavy metal element, indicating a good overall adsorption effect.

[0027] In summary, the *Pseudomonas aeruginosa* T1 provided by this invention is tolerant to a wide variety and high concentration of heavy metals, and also exhibits good adsorption effects on complex pollution caused by multiple heavy metals. Furthermore, it is simple to operate, safe, efficient, and low-cost, showing great application potential in the remediation of multiple heavy metal pollution in soil and water. If there is an opportunity for large-scale promotion of *Pseudomonas aeruginosa* T1 and its application technology in the future, the culture medium of *Pseudomonas aeruginosa* T1 can be prepared into bacterial powder through freeze-drying or spray drying, and mixed with a nutrient carrier to ensure the long-term activity of the bacterial T1. This will be more beneficial for industrial production and practical applications, and has broad market prospects.

Claims

1. A multi-heavy-metal resistant *Pseudomonas aeruginosa* strain T1, characterized in that, Its classification name is Pseudomonas aeruginosa ( Pseudomonas aeruginosa The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28029. The 16S rDNA gene sequence of this strain is shown in SEQ ID No.

1.

2. The application of the multi-heavy-metal-resistant Pseudomonas aeruginosa T1 as described in claim 1 in the remediation of heavy metal pollution, characterized in that, The heavy metals mentioned are at least one of copper, zinc, chromium, cadmium, and lead.

3. The application of the multi-heavy-metal-resistant Pseudomonas aeruginosa T1 according to claim 2 in the remediation of heavy metal pollution, characterized in that, The maximum tolerable concentrations of heavy metals are 200 mg / L for copper, 1000 mg / L for zinc, 1000 mg / L for chromium, 300 mg / L for cadmium, and 800 mg / L for lead.

4. The application of the Pseudomonas aeruginosa T1 bacterial solution as described in claim 1 in the remediation of various heavy metal contaminations.

5. The application of the *Pseudomonas aeruginosa* T1 bacterial suspension according to claim 4 in the remediation of various heavy metal contaminations, characterized in that, The method for preparing Pseudomonas aeruginosa T1 bacterial suspension is as follows: Pseudomonas aeruginosa T1 is inoculated into 100 mL of sterilized NB liquid culture medium at an inoculation rate of 1%, and cultured at 30°C with shaking for 24 h to obtain T1 bacterial suspension.

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