A method for removing antibiotic resistance genes in water using edible pigment erythrosine

Abstract

The invention discloses a method for removing antibiotic resistance genes in water by using erythrosine, an edible pigment. In the method, erythrosine is added to water contaminated by antibiotic resistance genes in the dark, the pH is adjusted to 3.50-5.00, and the mixture is stirred at 10-40°C for 5-30 minutes; then, the antibiotic resistance genes in water are effectively removed under visible light irradiation. The removal rate of intracellular antibiotic resistance genes in sewage by the method is much faster than other new water disinfection technologies, and the method shows obvious cost-effectiveness advantages compared with other water disinfection technologies.

Description

Technical Field

[0001] The invention relates to the technical field of water treatment, in particular to a method for removing antibiotic resistance genes in water by using erythrosine, an edible pigment. Background Art

[0002] The development of antibiotic resistance has led to a serious public health threat. Antibiotic resistance genes (ARGs) are an emerging pollutant, and the spread of ARGs is the main cause of the development of antibiotic resistance. Water is an important medium for the spread of ARGs, and the treatment of ARGs in water is crucial to controlling the development of resistance. However, traditional water disinfection technologies cannot effectively remove ARGs in water. At present, new water disinfection technologies represented by Fenton, Fenton-like, and photocatalysis have partially solved the above problems. They have strong degradation capabilities for extracellular antibiotic resistance genes (eARGs), but these technologies have poor removal capabilities for intracellular antibiotic resistance genes (iARGs), which leads to the inability of these new water disinfection technologies to effectively remove sewage ARGs at low cost in municipal sewage with iARGs as the main ARGs.

[0003] Erythrosin is a cheap edible pigment that has recently been proposed as a potential new water disinfectant and has shown excellent cost-effectiveness in comparison with other water disinfectants. Unlike most new water disinfection technologies that use free radicals as the main active species, water disinfection based on erythrosin uses non-radical singlet oxygen as the main active species. Singlet oxygen is a highly selective reactive oxygen species that has good tolerance to environments containing high concentrations of organic matter (NOM). The bacterial intracellular environment is a typical high NOM environment, which suggests that erythrosin, which can efficiently produce singlet oxygen, may achieve efficient degradation of iARGs. Therefore, photosensitized water disinfection technology based on erythrosin is expected to achieve effective removal of iARGs in sewage. However, there is currently no research on the application of erythrosin in the removal of ARGs. Developing erythrosin as a new water disinfectant that can remove ARGs will provide an important solution to combat the spread of antibiotic resistance. Summary of the invention

[0004] In view of the problem that current water disinfection technology cannot effectively remove intracellular antibiotic resistance genes in sewage at a low cost, the present invention provides a cost-effective solution, namely, using low-concentration edible pigment erythrosine to achieve efficient degradation of intracellular antibiotic resistance genes in sewage under light, and simultaneously remove extracellular antibiotic resistance genes.

[0005] The objective of the present invention is achieved through the following technical scheme: A method for removing antibiotic resistance genes in water using erythrosine, an edible pigment, comprises the following steps:

[0006] (1) In a dark place, a red erythrosine solution is mixed with an aqueous solution containing an antibiotic resistance gene to obtain a mixed solution; the concentration of the red erythrosine solution is 0.5 to 10.0 mg / L; the concentration of the antibiotic resistance gene is 10 2 ~10 6 copies / μL;

[0007] (2) adjusting the pH of the mixed solution to 3.50-5.00;

[0008] (3) stirring the mixed solution at 10-40° C. for 5-30 min;

[0009] (4) The mixed solution was then exposed to light with a wavelength of ≥420 nm and an intensity of 200 mW / cm 2 under visible light to achieve the removal of antibiotic resistance genes.

[0010] Furthermore, the aqueous solution is deionized water or sewage.

[0011] Furthermore, the antibiotic resistance gene is an extracellular antibiotic resistance gene or an intracellular antibiotic resistance gene.

[0012] Furthermore, the antibiotic resistance gene is a kanamycin resistance gene.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The research results of the present invention show that erythrosine can achieve efficient removal of antibiotic resistance genes at low concentrations. The removal rate of intracellular antibiotic resistance genes in sewage by this method is much faster than other new water disinfection technologies, and it shows obvious cost-effectiveness advantages compared with other water disinfection technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the removal effect of erythrosine on iARGs at different pH values;

[0016] Figure 2 This is a diagram showing the effect of simultaneous removal of iARGs and eARGs by erythrosine in deionized water;

[0017] Figure 3 This is a diagram showing the effect of simultaneous removal of iARGs and eARGs by erythrosine in real sewage;

[0018] Figure 4 The effect of different concentrations of erythrosine on the removal of iARGs is shown;

[0019] Figure 5 This is a graph showing the relationship between erythrosine concentration and iARGs degradation rate constant. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1: Visible light-excited erythrosine degrades iARGs at different pH values

[0022] The iARGs used in this example are kanamycin resistance genes located on plasmids in antibiotic-resistant bacteria. 7 cfu / mL of antibiotic-resistant bacteria were introduced at an initial concentration of 10 6 copies / μL of iARGs. Erythrosine was purchased from Beijing Solebow Technology Co., Ltd. at a concentration of 5.0 mg / L. The light source was a Zhongjiao Jinyuan xenon lamp with an additional filter to provide visible light with a wavelength of ≥420 nm and a light intensity of 200 mW / cm 2 .

[0023] Set up 8 experimental groups: in the dark, add erythrosine to deionized water to prepare an erythrosine solution with a concentration of 5.0 mg / L, and then add an initial concentration of 10 6 The mixed solution was prepared by mixing 1000 copies / μL of iARGs, and then the pH of the mixed solution was adjusted to 3.50, 3.75, 4.00, 4.10, 4.25, 4.50, 4.75 and 5.00 in the dark; the mixed solution was stirred in a 30°C constant temperature water bath and reacted in the dark for 15 min; the mixed solution was then exposed to visible light (wavelength ≥ 420 nm, light intensity 200 mW / cm 2 ), 1 mL of sample was taken at 0, 2.5, 5, 10, 15, 20, 30, and 40 min, and 1 mL of sample was centrifuged at 11500g for 1 min in a high-speed centrifuge to obtain supernatant and precipitate; the genome in the precipitate was extracted and the concentration of iARGs was quantified by real-time fluorescent polymerase chain reaction.

[0024] This example includes 8 treatment groups, and the experimental parameters are shown in Table 1.

[0025] Table 1: Experimental parameters of the 8 experimental groups

[0026]

[0027] The experimental results are as follows Figure 1 As shown in the figure, the degradation ability of erythrosine on iARGs is significantly pH-dependent. Considering the cost of adding acidifiers, the recommended pH value for practical application is 4.00-4.10.

[0028] Example 2: Removal of ARGs in deionized water and sewage by erythrosine excited by visible light

[0029] Collection and use of sewage: The effluent from the secondary sedimentation tank of Hangzhou Chengxi Sewage Treatment Plant (referred to as sewage) was collected and filtered through a 0.22 μm polyethersulfone water filter membrane to remove the original microorganisms in the sewage, and stored at 4°C.

[0030] The experiments of simultaneous degradation of iARGs and eARGs by erythrosine were carried out in deionized water and sewage, respectively. 6 copies / μL, eARGs: 10 4 The number of iARGs added to real sewage was 10 4 copies / μL, eARGs: 10 2 copies / μL.

[0031] In this example, two treatment groups and one control group were set up, and the experimental parameters are shown in Table 2. The specific experimental steps and other parameters are the same as those in Example 1. After the experiment, the genome in the precipitate was extracted and the concentration of iARGs was quantified by real-time fluorescence polymerase chain reaction; at the same time, the concentration of eARGs in the supernatant was measured by real-time fluorescence polymerase chain reaction.

[0032] Table 2: Experimental parameters of the treatment and control groups

[0033]

[0034]

[0035] like Figure 2 As shown. In the figure, VL refers to visible light. In deionized water, the iARGs in the control group under visible light did not degrade, and the eARGs were slightly degraded. In contrast, the method provided by the present invention can achieve six orders of magnitude of iARGs degradation (removal efficiency of 99.9999%) and four orders of magnitude of eARGs degradation (removal efficiency of 99.99%) within 40 minutes. Therefore, erythrosine in deionized water at low concentrations can achieve simultaneous and effective removal of iARGs and eARGs, that is, erythrosine in deionized water at low concentrations can achieve efficient removal of ARGs.

[0036] like Figure 3As shown in the data, in real sewage, photosensitized erythrosine can still complete the degradation of four orders of magnitude of iARGs within 40 minutes, and the eARGs in the supernatant are also reduced to below the detection limit (10 copies / μL) within 40 minutes, indicating that photosensitized erythrosine can achieve the simultaneous and effective removal of iARGs and eARGs in real sewage, that is, photosensitized erythrosine can achieve the efficient removal of ARGs in real sewage.

[0037] Example 3: Ability of erythrosine stimulated by visible light to remove iARGs at different concentrations

[0038] The iARGs removal experiment was carried out in deionized water with erythrosine concentrations of 0.5, 1.0, 2.5, 4.0, 5.0, 7.5, and 10 mg / L, respectively. The pH was 4.00, and the specific steps and other parameters of the experiment were the same as those in Example 1.

[0039] The experimental results are as follows Figure 4-5 As shown in the figure, for iARGs degradation, when the concentration of erythrosine is lower than 5.0 mg / L, there is a linear relationship between the concentration of erythrosine and the iARGs degradation rate constant k(iARGs). When the concentration of erythrosine is greater than 5.0 mg / L, k(iARGs) no longer increases with the increase of erythrosine concentration. In summary, for iARGs degradation, the optimal concentration of erythrosine solution is 5.0 mg / L.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for removing antibiotic resistance genes in water using erythrosine, a food coloring, characterized in that: The following steps are involved: (1) In a dark place, a red erythrosine solution is mixed with an aqueous solution containing an antibiotic resistance gene to obtain a mixed solution; the concentration of the red erythrosine solution is 0.5 to 10.0 mg / L; the concentration of the antibiotic resistance gene is 10 2 ~10 6 copies / μL; (2) adjusting the pH of the mixed solution to 3.50-5.00; (3) stirring the mixed solution at 10-40° C. for 5-30 min; (4) The mixed solution was then exposed to light with a wavelength of ≥420 nm and an intensity of 200 mW / cm 2 under visible light to achieve the removal of antibiotic resistance genes.

2. the method for removing antibiotic resistance genes in water from a kind of food pigment erythrosine according to claim 1, is characterized in that, The aqueous solution is deionized water or sewage.

3. a method for removing antibiotic resistance genes in water from a kind of food pigment erythrosine according to claim 1, is characterized in that, The antibiotic resistance gene is an extracellular antibiotic resistance gene or an intracellular antibiotic resistance gene.

4. a kind of food coloring erythrosine removes the method for antibiotic resistance gene in water according to claim 1, it is characterized in that, The antibiotic resistance gene is a kanamycin resistance gene.

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