Nanometer zero-valent iron-tubificidae synergistic removal technology aiming at antibiotic resistance gene

Abstract

The invention relates to the technical field of environment restoration, in particular to a nanometer zero-valent iron-tubificidae synergistic removal technology aiming at antibiotic resistance genes. According to the method, the nanoscale zero-valent iron-tubificidae complex with a certain combination and proportion is constructed, and the optimal parameters are obtained: the nanoscale zero-valent iron is 0.8 g / L, and the tubificidae is 2000 pieces / L. Then, the conversion rate of the ARG in the solution after different treatment times is measured, and the result shows that the conversion rate of the ARG is greatly reduced after 24 hours, which shows that the nano zero-valent iron-tubificidae complex has better ecological safety when being used for removing the ARG. Compared with the traditional technology, the method has the advantages of high ecological safety and excellent ARG removal effect.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental remediation, and particularly relates to a nano zero-valent iron-aquatic oligochaete synergistic removal technology for antibiotic resistance genes. Background Art

[0002] Since the discovery of penicillin in 1928, antibiotics have played an important role in helping humans resist infectious diseases. However, the long-term use of antibiotics has led to the widespread spread of antibiotic resistance genes (ARGs), which diffuse in the environment through plasmid horizontal transfer, vertical transfer, conjugation and other pathways, and even form super bacteria under the action of natural selection, resulting in an increased risk of antibiotic treatment failure. The problem of antibiotic resistance has become a global challenge, and millions of people die from diseases caused by microbial infections every year. Since 2006, ARGs have been recognized as emerging pollutants and have received extensive attention. At present, ARGs have been detected in soil, water and even air, and high-risk pollution areas include hospital wastewater, sewage treatment plants, farms, surface water and sediments. Due to the nature difference between ARGs as DNA molecules and traditional organic pollutants, existing sewage treatment technologies (such as chlorine disinfection) have poor effects in removing ARGs and low removal rates. Therefore, it is urgent to develop efficient ARG removal technologies.

[0003] Nano zero-valent iron (nZVI) can covalently bind to the phosphate backbone of ARGs through Fe-O-P, but the active Fe sites on its surface are limited, resulting in low actual removal efficiency. Therefore, a high dosage of nano zero-valent iron is required in practical applications, leading to an increase in remediation costs. In order to reduce costs and improve effects, it is particularly important to find green and sustainable remediation means. Existing studies have shown that nanomaterials can stimulate organisms to secrete biomolecules such as enzymes and metabolites as exogenous substances, thereby enhancing the degradation ability of pollutants. Therefore, combining nano zero-valent iron with indigenous organisms in water to construct a material-biological composite system may become a low-cost and high-efficiency synergistic remediation technology.

[0004] Tubificidae is an important freshwater benthic animal and also a dominant species in many polluted water bodies (such as aquaculture wastewater). It has been applied to sludge reduction and pollution removal. Tubificidae has strong tolerance, is not easy to die, can play an ecological role for a long time, and has a low breeding cost. Tubificidae can degrade ARG in the environment by secreting nuclease. However, due to the negatively charged surface and DNA of Tubificidae, the lack of effective contact and interaction limits the efficiency of its degradation of ARG. Our research found that nano zero-valent iron can not only adsorb ARG, enhance the interfacial interaction between Tubificidae and ARG, but also promote Tubificidae to secrete more nuclease. In addition, the interaction between surface-passivated nano zero-valent iron and Tubificidae may stimulate the secretion of macromolecular proteins and small molecule metabolites such as organic acids. These substances can adhere nano zero-valent iron to the surface of Tubificidae and corrode the passivation layer through organic acids, exposing more active Fe sites, thereby enhancing its adsorption and removal performance of ARG. Therefore, constructing a nano zero-valent iron-Tubificidae complex is expected to effectively remove ARG in the water environment with a lower dosage of nano zero-valent iron, providing an economical and efficient new technology for the treatment of antibiotic resistance gene pollution. Summary of the Invention

[0005] The present invention provides a nano zero-valent iron-Tubificidae synergistic remediation technology for removing antibiotic resistance genes. Compared with traditional technologies, the material-biological complex in the present invention is easy to prepare, can simultaneously remove multiple antibiotic resistance genes, and has the advantages of high efficiency of nano remediation technology and environmental friendliness of biological remediation technology, and is expected to be used for the efficient in-situ removal of antibiotic resistance genes in water.

[0006] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a nano zero-valent iron-Tubificidae synergistic remediation technology for removing antibiotic resistance genes. The nano zero-valent iron-Tubificidae complex includes nano zero-valent iron and Tubificidae. The concentration of nano zero-valent iron in the nano zero-valent iron-Tubificidae complex is 0.1-1 g / L, and the number of Tubificidae is 300-3000 per liter.

[0007] In the examples of the present invention, it was found that there is an interaction between Tubificidae and nano zero-valent iron, and the composite system can obtain a higher ARG degradation efficiency compared with the single system. The possible reasons for analyzing the above phenomenon are as follows: Nano zero-valent iron may have the effect of promoting the secretion of nuclease by Tubificidae. In addition, the interaction between surface-passivated nano zero-valent iron and Tubificidae may also stimulate the production of macromolecular proteins and small molecule metabolites such as organic acids, adhere nano zero-valent iron to the body surface, and corrode the surface passivation layer of nano zero-valent iron through the action of organic acids to achieve the activation of nano zero-valent iron.

[0008] The particle size of the nano zero-valent iron is 100 nm.

[0009] Preferably, the concentration of nano zero-valent iron in the nano zero-valent iron-tubifex complex is 0.8 g / L, and the number of tubifex is 2000 per liter.

[0010] Preferably, the antibiotic resistance gene in the wastewater is a chloramphenicol antibiotic resistance gene.

[0011] The present invention also provides a method for removing antibiotic resistance genes from wastewater, which comprises adding the nano zero-valent iron-tubifex complex into the wastewater containing antibiotic resistance genes.

[0012] The method for constructing a nano zero-valent iron-tubifex complex for removing antibiotic resistance genes from wastewater according to the present invention comprises the following steps: (1) Constructing a nano zero-valent iron-tubifex complex Set up a water medium simulation experiment, mix different doses of nano zero-valent iron with different biomasses of tubifex, and use the adsorption of nano zero-valent iron on the epidermis of tubifex to prepare nano-armored tubifex. Measure the synergistic removal effect on antibiotic resistance genes under different ratios, and preferably construct a nano zero-valent iron-tubifex complex with the best performance ratio.

[0013] According to the above scheme, Further, in step (1), the water medium simulation experiment is carried out in a 6-well plate, and the volume of each well is 10 mL; Further, in step (1), the antibiotic resistance gene is a chloramphenicol resistance gene, and the configured concentration is 1 ng / μL; Further, in step (1), the dosage of nano zero-valent iron in the simulation study is 0, 0.1, 0.3, 1 g / L, the dosage of tubifex is 0, 300, 1000, 3000 per liter, and the exposure period is 3 days; (2) Transformation experiment Use the original Escherichia coli competent cells without ARG to carry out the transformation experiment to determine the activity of ARG carried by the plasmid after treatment with the nano zero-valent iron-tubifex complex, and further confirm the ecological safety of the degradation products.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs a nano zero-valent iron-tubifex complex with a certain ratio, which can activate nano zero-valent iron through nano-biological interface interaction, and use nano zero-valent iron to stimulate tubifex to secrete nuclease, significantly improving the removal effect of the material on antibiotic resistance genes in wastewater. Finally, the optimized repair combination is determined: nano zero-valent iron is 0.8 g / L, and tubifex is 2000 per liter, which can achieve the removal of antibiotic resistance genes in wastewater while ensuring ecological safety. Description of the drawings

[0015] Figure 1 Removal rates of ARGs under single exposure of nano-zero-valent iron and tubificids at different concentrations in Example 1 of the present invention; Figure 2 Removal rates of ARGs under combined exposure of nano-zero-valent iron and tubificids at different concentrations in Example 1 of the present invention; Figure 3 Response surface analysis of the nano-zero-valent iron-tubificid and ARG removal rate in Example 1 of the present invention; Figure 4 Plasmid transformation after the removal of ARGs in water by the nano-zero-valent iron-tubificid composite group in Example 1 of the present invention. Detailed implementation mode

[0016] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0017] Example: The method steps of the present invention are specifically as follows: (1) Construct a nano-zero-valent iron-tubificid complex In this case, the chloramphenicol resistance gene was used as a representative ARG, and a water medium simulation experiment was carried out in a 6-well plate according to the factorial experimental design. The specific steps were as follows: In the single treatment group, an ARG aqueous solution with a concentration of 1 ng / μL was prepared. 10 mL of the ARG aqueous solution was added to one well, and then 0.1, 0.3, 1 g / L of 100 nm nano-zero-valent iron and 300, 1000, 3000 tubificids / L were added respectively. In the combined treatment group, a solution containing 1 ng / μL ARG and 0.1, 0.3, 1 g / L of 100 nm nano-zero-valent iron was prepared, and 10 mL was added to one well respectively. Three replicates were set, and 300, 1000, 3000 tubificids / L were placed in each group. The nano-armored tubificids were formed by the adsorption of nano-zero-valent iron on the epidermis of the tubificids. At 12 h, 24 h, 36 h, 48 h, and 72 h of exposure, 50 μL of the supernatant was taken respectively.

[0018] Design amplicons and use qPCR to determine the ARG concentration in solution samples. Primers were designed using the NCBI Primer-BLAST primer design tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The concentration of ARG was measured using qPCR (QuantStudio 3 Real-Time PCR System, ThermoFisher Scientific, CA, USA). All qPCR reactions were performed in triplicate, and the standard deviation of the cycle threshold (C T ) was <0.5, and the average copy number was used for subsequent calculations. A standard qPCR curve of ARG was created by serially diluting a plasmid 10-fold in the background solution (10 8 -10 2 copies / μL), and it was found to have a model fit (R 2 ) > 0.99 and an amplification efficiency of 114.1%.

[0019] The results showed that in the case of single degradation, the degradation rates of ARG by Tubifex tubifex and nano-zero-valent iron were relatively low ( Figure 1 ). While at the same concentration, the degradation rate of ARG by the nano-zero-valent iron-Tubifex tubifex complex was significantly increased ( Figure 2 ). Then, the optimal parameters were obtained using the response surface method: nano-zero-valent iron was 0.8 g / L and Tubifex tubifex was 2000 individuals / L ( Figure 3 ).

[0020] (2) ARG transformation experiment To further confirm the ecological safety of the degradation products, a transformation experiment was conducted using Escherichia coli DH5α competent cells that originally did not contain ARG to determine the activity of ARG carried by the plasmid after treatment with the nano-zero-valent iron-Tubifex tubifex complex. Briefly, 10 μL of the ARG sample treated with the nano-zero-valent iron-Tubifex tubifex complex was added to 100 μL of thawed competent Escherichia coli DH5α, gently mixed, and then the mixture was placed on ice for 30 minutes. Then the mixture was kept at 42 °C for 90 seconds to apply heat shock, and then placed on ice for 2 minutes. Then 890 μL of LB medium was added to the mixture, and then cultured at 37 °C for 1 hour (180 rpm). Subsequently, serial dilutions were made from the original suspension, and 100 μL of each dilution was inoculated onto selective LB agar plates containing chloramphenicol (25 mg / L). After culturing at 37 °C for 36 hours, the colonies on the agar plates were counted. The transformation efficiency was calculated as the number of colonies growing on the selective plates divided by the initial number of plasmids. The results showed that the ARG conversion rate decreased significantly after 24 h (Figure 4 ), indicating that the nano-zero-valent iron-aquatic earthworm complex has good ecological safety in removing ARGs.

[0021] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Those skilled in the art can modify or equivalently replace the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention and fall within the scope of protection of the present invention.

Claims

1. A nano-zero-valent iron-aquatic oligochaete synergistic removal technology for antibiotic resistance genes, characterized in that it includes the following steps: (1) Construct a nano-zero-valent iron-aquatic oligochaete complex for removing ARGs Using the plasmid containing ARGs as the target pollutant, set up a water medium simulation degradation experiment, mix different doses of nano-zero-valent iron with different biomasses of aquatic oligochaetes, and use the adsorption of nano-zero-valent iron by the epidermis of aquatic oligochaetes to prepare the material-aquatic oligochaete complex. Measure the synergistic removal effect on antibiotic resistance genes under different ratios, and preferably construct a nano-zero-valent iron-aquatic oligochaete complex with the best performance. According to the above scheme, Further, in step (1), the antibiotic resistance gene is the chloramphenicol resistance gene, and referring to the environmentally relevant concentration, the concentration is configured to be 1 ng / μL; Further, in step (1), the water medium simulation experiment is carried out in a 6-well plate, and the volume of each well is 10 mL; Further, in step (1), the dosage of nano-zero-valent iron in the simulation study is 0, 0.1, 0.3, 1 g / L, the dosage of aquatic oligochaetes is 0, 300, 1000, 3000 pieces / L, and the degradation period is 3 days; (2) A transformation experiment for verifying the inhibitory effect on ARG activity Use Escherichia coli competent cells without ARGs to carry out a transformation experiment to determine the activity of ARGs carried by the plasmid after treatment with the nano-zero-valent iron-aquatic oligochaete complex, and further confirm the ecological safety of the degradation products.

2. The nano zero-valent iron-aquatic oligochaete synergistic removal technology for antibiotic resistance genes in water according to claim 1, wherein In the nano-zero-valent iron-aquatic oligochaete complex described in step (1), the concentration of nano-zero-valent iron is: nano-zero-valent iron is 0.8 g / L, and aquatic oligochaetes are 2000 pieces / L.

3. A nano zero-valent iron-aquatic oligochaete synergistic removal technology for antibiotic resistance genes in water, characterized in that, In step (1), the particle size of the nano-zero-valent iron is 100 nm.

4. A nano-zero-valent iron-aquatic oligochaete synergistic removal technology for antibiotic resistance genes in water, characterized in that, In step (1), the antibiotic resistance gene in the wastewater is the chloramphenicol antibiotic resistance gene.

Images