A method for removing antibiotic resistance genes by using nano zero-valent iron and limnadia cooperatively

Abstract

The present application relates to the technical field of environmental remediation, and in particular to a method for removing antibiotic resistance genes by using nano zero-valent iron and water earthworms in cooperation, wherein a nano zero-valent iron-water earthworm composite with a certain combination and proportion is constructed, and the optimal parameters are obtained, i.e., the nano zero-valent iron is 0.8 g / L, and the water earthworms are 2000 pieces / L. Then, the conversion rate of antibiotic resistance genes in the solution after different treatment times is determined, and the results show that the conversion rate of antibiotic resistance genes decreases significantly after 24 hours, indicating that the nano zero-valent iron-water earthworm composite has good ecological safety in removing antibiotic resistance genes. Compared with the traditional method, the present method has higher ecological safety and excellent antibiotic resistance gene removal effect.

Description

Technical Field

[0001] This invention relates to the field of environmental remediation technology, specifically to a method for the synergistic removal of antibiotic resistance genes by nano-zero-valent iron and tubifex worms. Background Technology

[0002] Since the discovery of penicillin in 1928, antibiotics have played a vital role in helping humans fight infectious diseases. However, long-term antibiotic use has led to the widespread dissemination of antibiotic resistance genes. These genes spread through the environment via horizontal plasmid transfer, vertical transfer, and conjugation, and can even lead to the formation of superbugs under natural selection, exacerbating the risk of antibiotic treatment failure. Antibiotic resistance has become a global challenge, with millions dying each year from diseases caused by microbial infections. Since 2006, antibiotic resistance genes have been identified as an emerging pollutant and have received widespread attention. Currently, antibiotic resistance genes have been detected in soil, water, and even air, with high-risk areas including hospital water, wastewater treatment plants, livestock farms, surface water, and sediments. Due to the differences in properties between antibiotic resistance genes as DNA molecules and traditional organic pollutants, existing wastewater treatment methods (such as chlorination) are ineffective in removing antibiotic resistance genes, resulting in low removal rates. Therefore, there is an urgent need to develop efficient methods for removing antibiotic resistance genes.

[0003] Nano-zero-valent iron can covalently bind to the phosphate backbone of antibiotic resistance genes via Fe-OP, but the limited number of active Fe sites on its surface leads to low actual removal efficiency. Therefore, high dosages of nano-zero-valent iron are required in practical applications, increasing remediation costs. To reduce costs and improve effectiveness, finding green and sustainable remediation methods is crucial. Existing research shows that nanomaterials can act as exogenous substances to stimulate organisms to secrete enzymes and metabolites, thereby enhancing their ability to degrade pollutants. Therefore, combining nano-zero-valent iron with native aquatic organisms to construct a material-biocomposite system may become a low-cost, high-efficiency synergistic remediation method.

[0004] Tubificidae are important freshwater benthic animals and a dominant species in many polluted water bodies (such as aquaculture water), and have been applied to sludge reduction and pollution removal. Tubificidae are highly resilient, not easily killed, and can play a long-term ecological role, with low farming costs. Tubificidae can degrade antibiotic resistance genes in the environment by secreting nucleases, but because their body surface and DNA both carry a negative charge, there is a lack of effective contact and interaction, limiting their efficiency in degrading antibiotic resistance genes. Our research found that nano-zero-valent iron can not only adsorb antibiotic resistance genes and enhance the interfacial interaction between Tubificidae and antibiotic resistance genes, but also promote the secretion of more nucleases by Tubificidae. Furthermore, the interaction between surface-passivated nano-zero-valent iron and Tubificidae may stimulate the secretion of small molecule metabolites such as large proteins and organic acids. These substances can adhere nano-zero-valent iron to the surface of Tubificidae, and the organic acids corrode the passivation layer, exposing more active Fe sites, thereby enhancing its adsorption and removal performance for antibiotic resistance genes. Therefore, constructing a nano-zero-valent iron-aquatic earthworm complex is expected to effectively remove antibiotic resistance genes in the aquatic environment with a relatively low amount of nano-zero-valent iron, providing an economical and efficient new method for the treatment of antibiotic resistance gene pollution. Summary of the Invention

[0005] This invention provides a nano-zero-valent iron-aquatic earthworm synergistic remediation method for removing antibiotic resistance genes. Compared with traditional technologies, the material-biocomposite in this invention is simple to prepare, can simultaneously remove multiple antibiotic resistance genes, and combines the advantages of high efficiency of nano-remediation technology with the environmental friendliness of bioremediation technology, making it promising for the efficient in-situ removal of antibiotic resistance genes in water.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for the synergistic repair of antibiotic resistance genes using nano-zero-valent iron and tubifex worms. The nano-zero-valent iron-tubifex worm complex includes nano-zero-valent iron and tubifex worms. The concentration of nano-zero-valent iron in the nano-zero-valent iron-tubifex worm complex is 0.1-1 g / L, and the concentration of tubifex worms is 300-3000 worms / L (or 0.3-3.0 g / L).

[0008] In the embodiments of this invention, it was found that tubifex worms and nano-zero-valent iron interact, and the composite system achieves a higher degradation efficiency of antibiotic resistance genes compared to the single system. The reason for this phenomenon may be that nano-zero-valent iron may promote the secretion of nucleases in tubifex worms. Furthermore, the interaction between the surface-passivated nano-zero-valent iron and tubifex worms may stimulate the production of large-molecule proteins and small-molecule metabolites such as organic acids, adhering the nano-zero-valent iron to the body surface. The organic acids then corrode the passivation layer on the surface of the nano-zero-valent iron, thereby activating it.

[0009] The particle size of the nano-zero valent iron is 100 nm.

[0010] Preferably, the concentration of nano-zero valent iron in the nano-earthworm composite is 0.8 g / L, and the concentration of earthworms is 2000 worms / L.

[0011] Preferably, the antibiotic resistance gene in the water is a chloramphenicol antibiotic resistance gene.

[0012] The present invention also provides a method for removing antibiotic resistance genes in water, wherein the nano-zero-valent iron-water earthworm complex is added to water containing antibiotic resistance genes.

[0013] This invention discloses a method for constructing a nano-zero-valent iron-aquatic earthworm complex to remove antibiotic resistance genes in water, comprising the following steps:

[0014] (1) Construction of nano-zero-valent iron-water earthworm complex

[0015] A water-medium simulation experiment was set up, and different doses of nano-zero valent iron were mixed with tubifex worms of different biomass. The adsorption of nano-zero valent iron by the tubifex worm epidermis was used to prepare nano-zero valent iron-tubifex worm complex. The synergistic removal effect on antibiotic resistance genes under different ratios was measured, and the best ratio was selected to construct the nano-zero valent iron-tubifex worm complex.

[0016] According to the above plan,

[0017] In step (1), the water medium simulation experiment is carried out in a 6-well plate with a volume of 10 mL per well;

[0018] In step (1), the antibiotic resistance gene is a chloramphenicol resistance gene, and the concentration is 1 ng / μL;

[0019] In step (1), the amount of nano-zero valent iron used in the simulation study is 0, 0.1, 0.3, 1 g / L, the amount of tubifex worms used is 0, 300, 1000, 3000 worms / L (or 0, 0.3, 1.0, 3.0 g / L), and the exposure period is 3 days;

[0020] (2) Transformation experiment

[0021] Transformation experiments were conducted using competent E. coli cells that originally did not contain antibiotic resistance genes to determine the activity of antibiotic resistance genes carried by plasmids after treatment with nano-zero-valent iron-earthworm complexes, and to confirm the ecological safety of degradation products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention constructs a nano-zero-valent iron-aquatic earthworm composite with a specific ratio. The nano-zero-valent iron is activated through nano-biological interface interaction, and the nano-zero-valent iron stimulates the aquatic earthworms to secrete nucleases, significantly improving the material's removal effect on antibiotic resistance genes in water. The optimal repair combination was finally determined: 0.8 g / L of nano-zero-valent iron and 2000 aquatic earthworms / L, which can remove antibiotic resistance genes in water while ensuring ecological safety. Attached Figure Description

[0024] Figure 1 The removal rate of antibiotic resistance genes by different concentrations of nano-zero valent iron and tubifex worms under single exposure in Example 1 of this invention;

[0025] Figure 2 The removal rate of antibiotic resistance genes by different concentrations of nano-zero valent iron and tubifex worms under combined exposure in Example 1 of this invention;

[0026] Figure 3 Response surface methodology analysis of the removal rate of antibiotic resistance genes by nano-zero-valent iron-earthworm in Example 1 of this invention;

[0027] Figure 4 This shows the plasmid transformation of the nano-zero-valent iron-earthworm composite group after removing antibiotic resistance genes in water in Example 1 of the present invention. Detailed Implementation

[0028] The present invention will now be described with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0029] The specific steps of the method of this invention are as follows:

[0030] (1) Construction of nano-zero-valent iron-water earthworm complex

[0031] This embodiment uses the chloramphenicol resistance gene as a representative antibiotic resistance gene. Based on a factorial experimental design, an aqueous medium simulation experiment was conducted in a 6-well plate. The specific steps were as follows: In the single treatment group, a 1 ng / μL aqueous solution of the antibiotic resistance gene was prepared, and 10 mL of the solution was added to one well. Then, 0.1, 0.3, and 1 g / L of 100 nm zero-valent iron nanoparticles and 300, 1000, and 3000 tubifex worms / L were added, respectively. In the combined treatment group, solutions containing 1 ng / μL of the antibiotic resistance gene and 0.1, 0.3, and 1 g / L of 100 nm zero-valent iron nanoparticles were prepared, and 10 mL of each solution was added to one well. Three replicates were set up, with each group containing 300, 1000, and 3000 tubifex worms / L (or 0.3, 1.0, and 3.0 g / L), respectively. The adsorption of the zero-valent iron nanoparticles by the tubifex worm epidermis was utilized to form nano-armored tubifex worms. 50 μL of supernatant was collected at 12 h, 24 h, 36 h, 48 h, and 72 h of exposure.

[0032] Amplicons were designed to determine the concentration of antibiotic resistance genes in solution samples using qPCR. Primers were designed using the NCBI Primer-BLAST primer design tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The concentration of antibiotic resistance genes was measured using qPCR (QuantStudio 3 Real-Time PCR System, Thermo Fisher Scientific, CA, USA). All qPCR reactions were performed in triplicate, with a cycle threshold (C0). T The standard deviation was <0.5, and the mean copy number was used for subsequent calculations. Standard qPCR curves for antibiotic resistance genes were created by serially diluting the plasmid 10-fold in background solution. 8 -10 2 copies / μL), and found to have model fit (R0). 2 The amplification efficiency was 114.1% (value > 0.99).

[0033] The results showed that, under single degradation conditions, tubifex worms and nano-zero-valent iron had low removal rates of antibiotic resistance genes. Figure 1 At the same concentration, the nano-zero-valent iron-earthworm complex significantly improved the removal rate of antibiotic resistance genes. Figure 2 Then, the optimal parameters were obtained using response surface methodology: nano-zero valent iron was 0.8 g / L, and tubifex worms were 2000 worms / L. Figure 3 ).

[0034] (2) Antibiotic resistance gene transformation experiment

[0035] To confirm the ecosafety of the degradation products, transformation experiments were performed using competent *E. coli* DH5α cells that originally did not contain the antibiotic resistance gene to determine the activity of the antibiotic resistance gene carried by the plasmid after treatment with the nano-zero-valent iron-butterfly complex. Briefly, 10 μL of the antibiotic resistance gene sample treated with the nano-zero-valent iron-butterfly complex was added to 100 μL of thawed competent *E. coli* DH5α cells, gently mixed, and then placed on ice for 30 minutes. The mixture was then subjected to heat shock at 42 °C for 90 seconds, followed by another 2 minutes on ice. 890 μL of LB medium was then added to the mixture, and the mixture was incubated at 37 °C for 1 hour (180 rpm). Subsequently, serial dilutions were performed from the original suspension, and 100 μL of each dilution was inoculated onto selective LB agar plates containing chloramphenicol (25 mg / L). After incubation at 37 °C for 36 hours, colonies on the agar plates were counted. The transformation efficiency was calculated by dividing the number of colonies grown on the selective plate by the mass of plasmid DNA added. The results showed that the transformation efficiency of the antibiotic resistance gene decreased significantly after 24 h. Figure 4 This indicates that the nano-zero-valent iron-earthworm complex has good ecological safety in removing antibiotic resistance genes.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. All such modifications and substitutions should be covered within the scope of the claims of the present invention and are within the scope of protection of the present invention.

Claims

1. A method for the synergistic removal of antibiotic resistance genes by nano-zero-valent iron and tubifex worms, characterized in that, Includes the following steps: Nano-zero valent iron and tubifex worms are added to the water body containing antibiotic resistance genes to form a nano-zero valent iron-tubifex worm composite system in the water body. The water body is then treated by contact treatment to reduce the concentration of antibiotic resistance genes in the water body. The concentration of the nano-zero-valent iron is 0.1-1.0 g / L based on the volume of the water to be treated, and the density of the tubifex worms is 300-3000 worms / L or 0.3-3.0 g / L.

2. The method according to claim 1, characterized in that, The concentration of the nano-zero valent iron is 0.8 g / L, and the density of the tubifex worms is 2000 worms / L.

3. The method according to claim 1 or 2, characterized in that, The particle size of the nano-zero valent iron is 100 nm.

4. The method according to claim 1, characterized in that, The antibiotic resistance gene is a chloramphenicol antibiotic resistance gene.

5. The method according to claim 1, characterized in that, The contact treatment time is 24-72 hours.

6. The method according to claim 1, characterized in that, The water worms mentioned are freshwater benthic water worms.

Images