A method for treating antibiotic-resistant bacteria in wastewater

Abstract

The present invention provides a method for treating antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in wastewater. The present invention adopts nano zero-valent iron (nZVI), chlorite (ClO2 ‑ ) and oxygen ternary reaction system, where ClO2 ‑ Oxidation of nZVI to high-valent iron (Fe IV =O), nZVI reduces O2 to generate superoxide anion (·O2 ‑ ), Fe IV =O can effectively destroy the cell membrane structure of ARB, resulting in extracellular O2 ‑ , iron ions, etc. enter the cell, inducing intracellular oxidative stress and damage, leading to ARB inactivation; at the same time, intracellular ARGs leak out of the cell and are oxidized by ·O2 ‑ The attack is rapid and the degradation is rapid. The two active species work together effectively to quickly kill ARB and ARGs. The treatment method of the present invention has a better disinfecting effect on ARB than active chlorine and the common zero-valent iron-activated peroxide Fenton reaction, and has a wide pH application range.

Description

Technical Field

[0001] The invention relates to the technical field of water treatment, and in particular to a method for treating antibiotic-resistant bacteria in wastewater. Background Art

[0002] In recent years, with the overuse of antibiotics in the fields of medicine, agriculture, industry, aquaculture, etc., a large amount of antibiotics have been discharged into the water environment, exerting selective pressure on microbial populations, inducing the generation and spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), seriously endangering human and animal health and the balance of the ecosystem. Therefore, controlling ARB and ARGs pollution in the water environment is an urgent problem to be solved.

[0003] Although traditional wastewater biochemical treatment and disinfection processes can inactivate ARB, they are greatly affected by various factors such as the type of ARB and ARGs, water quality, and operating conditions. Advanced oxidation processes are currently the most studied deep water treatment method. Studies have shown that they can produce ·OH, ·SO4 - Active species such as oxidative stress and oxidative stress can effectively inactivate ARB. However, this type of free radical-generating advanced oxidation reaction is limited by defects such as short free radical half-life, short migration distance, low steady-state concentration, large dosage and high cost, resulting in low effective utilization of free radicals and ARBs and ARGs, especially limited removal of intracellular resistance genes (iARGs). Therefore, it is crucial to regulate the types of active species in the advanced oxidation system and optimize the reactivity of active species to achieve efficient inactivation of ARB. Summary of the invention

[0004] The present invention aims to solve a problem in the related art at least to a certain extent, and provides a method for treating antibiotic-resistant bacteria in wastewater.

[0005] In a first aspect, the present invention provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0006] Adjust the pH of wastewater containing antibiotic-resistant bacteria to acidic, neutral or slightly alkaline;

[0007] Nano-zero-valent iron and chlorite are added to the wastewater after pH adjustment for sterilization.

[0008] Preferably, the acidic pH is 3 to 7;

[0009] The alkaline pH is 7-9.

[0010] Preferably, the concentration of nano zero-valent iron in the wastewater is 10-20 mg / L.

[0011] Preferably, the concentration of chlorite in the wastewater is 0.01 to 0.05 mmol / L.

[0012] Preferably, the antibiotic-resistant bacteria include Escherichia coli containing tetracycline resistance genes and ampicillin resistance genes.

[0013] Preferably, the chlorite includes at least one of chlorous acid, sodium chlorite and potassium chlorite.

[0014] Preferably, the concentration of nano zero-valent iron in the wastewater is 20 mg / L.

[0015] Preferably, the concentration of chlorite in the wastewater is 0.05 mmol / L.

[0016] In a third aspect, the present invention also provides an application of a preparation for removing antibiotic-resistant bacteria and resistant genes in wastewater, wherein the preparation comprises nano zero-valent iron and chlorite.

[0017] The present invention has the following beneficial effects compared with the prior art:

[0018] 1. The method for treating antibiotic resistant bacteria (ARB) in wastewater of the present invention adopts nano zero-valent iron (nZVI), chlorite (ClO2 - ) and oxygen ternary reaction system (nZVI / ClO2 - / O2), where ClO2 - Oxidation of nZVI to high-valent iron (Fe IV =O), while nZVI reduces O2 to generate superoxide anion (·O2 - ).Fe IV =O can effectively destroy the cell membrane structure of ARB, resulting in extracellular O2 - , iron ions, etc. enter the ARB cells, inducing oxidative stress and damage in the ARB cells, leading to ARB inactivation; at the same time, it promotes the leakage of drug resistance genes (ARGs) in the ARB cells to the extracellular space, and is blocked by ·O2 - Attack rapid degradation; two active species (Fe IV =O and ·O2 - ) effectively cooperates to quickly kill ARB and remove the drug-resistant genes carried by ARB; the treatment method of the present invention has a better disinfecting effect on ARB than active chlorine and common zero-valent iron-activated peroxide Fenton reaction, and has a wide pH (pH 3-9) application range;

[0019] 2. The method for treating antibiotic-resistant bacteria in wastewater of the present invention is to treat the antibiotic-resistant bacteria in wastewater at extremely low doses (10-20 mg / L of nZVI and 0.01-0.05 mmol / L of ClO2 -) and acidic (pH 3-7), neutral, or weakly alkaline (pH 7-9) conditions, the initial concentration of which is about 1×10 7 CFU / mL of dual-AR E.col antibiotic-resistant bacteria, and can effectively remove the resistance genes inside and outside the cells of antibiotic-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 This is a bacterial culture diagram of the antibiotic-containing plate used for bacterial resistance identification in Example 1;

[0022] Figure 2 is the agarose gel electrophoresis image of the pBR322 plasmid PCR product in Example 1;

[0023] Figure 3 The bactericidal effect of different contents of nano zero-valent iron in Example 2;

[0024] Figure 4 The bactericidal effect of chlorite at different concentrations in Example 3;

[0025] Figure 5 For nZVI / ClO2 in Example 4 - / O2 system, nZVI and ClO2 alone - The bactericidal effect of the system;

[0026] Figure 6 For nZVI / ClO2 in Example 5 - Flow cytometric analysis scatter plot of inactivation of drug-resistant bacteria by / O2 system and traditional chlorine disinfection;

[0027] Figure 7 For nZVI / ClO2 in Example 6 - Comparison of the inactivation effects of dual-ARE.coli after 10 minutes of treatment with / O2 system and other advanced oxidation systems;

[0028] Figure 8 For nZVI / ClO2 in Example 7 - / O2 system inactivation dual-ARE.coli quenching experiment results;

[0029] Fig. 9 For nZVI / ClO2 in Example 8- The result of removing the extracellular drug resistance gene tetracycline (tetC) by the / O2 system;

[0030] Fig.10 For nZVI / ClO2 in Example 8 - / O2 system removes extracellular drug resistance gene ampicillin (bla TEM ) result graph;

[0031] Fig.11 For nZVI / ClO2 in Example 9 - The result of removing the intracellular drug resistance gene tetracycline (tetC) by the / O2 system

[0032] Fig.12 For nZVI / ClO2 in Example 9 - / O2 system removes intracellular drug resistance gene ampicillin (bla TEM ) result graph;

[0033] Fig.13 For nZVI / ClO2 in Example 10 - The results of the active species quenching experiment of the / O2 system and the control system;

[0034] Fig.14 For nZVI / ClO2 in Example 11 - Sterilization effect of / O2 system at different pH. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0037] The present invention provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0038] Adjust the pH of wastewater containing antibiotic-resistant bacteria to acidic, neutral or slightly alkaline;

[0039] Nano-zero-valent iron and chlorite are added to the wastewater after pH adjustment for sterilization.

[0040] The method for treating antibiotic-resistant bacteria in wastewater of the present invention adopts nano zero-valent iron (nZVI), chlorite (ClO2 - ) and oxygen ternary reaction system (nZVI / ClO2 - / O2), where ClO2 - nZVI is oxidized to high-valent iron (Fe IV =O)(Fe IV =O has a higher redox potential and can selectively attack pollutants and amide bonds, and is more effective than ·OH and ·SO4 - The half-life of Fe is longer. The surface of bacteria usually has more electron-rich components, such as polysaccharides, proteins, phospholipids, etc. IV =O is more favorable than free radical active species in inactivating ARB), while nZVI reduces O2 to generate superoxide anion (·O2 - ).Fe IV =O can effectively destroy the cell membrane structure of ARB, resulting in increased cell permeability and ARB extracellular O2 -, iron ions, etc. enter the ARB cells, inducing oxidative stress and damage in the ARB cells. The combined effects of exogenous damage and endogenous oxidative stress lead to the inactivation of ARB. The cell membrane structure is destroyed, promoting the leakage of drug resistance genes (ARGs) in the ARB cells to the outside of the ARB cells and adsorption on the surface of nZVI. nZVI activates dissolved oxygen molecules in water to generate superoxide anions (·O2 - ), which has a high reactivity with ARGs adsorbed on the surface of nZVI, resulting in ARGs being absorbed by ·O2 - Attack rapid degradation; two active species (Fe IV =O and ·O2 - ) work together effectively to quickly kill ARB and remove the drug-resistant genes carried by ARB.

[0041] Specifically, nZVI generates active species ·O2 by single-electron reduction of dissolved oxygen molecules. - , O2 - Attacking the phosphodiester bonds of ARGs adsorbed on the surface of nZVI leads to the effective degradation of ARGs and the subsequent removal of ARGs.

[0042] In some embodiments, nano zero-valent iron is prepared by sodium borohydride or potassium borohydride reduction method. Specifically, the preparation method of nano zero-valent iron comprises the following steps:

[0043] Dissolve 1.5 g of FeCl3·6H2O in 500 mL of deionized water to obtain a FeCl3·6H2O solution;

[0044] Add 3 g of NaBH4 solid to 200 mL of deionized water to obtain a NaBH4 solution;

[0045] NaBH4 solution was added to FeCl3·6H2O solution and reacted for 10 minutes to obtain a suspension;

[0046] The suspension was allowed to stand for 2 h, the black precipitate at the bottom was collected, and washed three times with 5 mL of deionized water and anhydrous ethanol respectively. The obtained black solid was placed in a freeze dryer and dried for 12 h to obtain a dry black solid, which is nano zero-valent iron (nZVI).

[0047] In some embodiments, the acidic pH is 3 to 7, i.e., 3 ≤ acidic pH < 7;

[0048] The weak alkaline pH is 7-9, that is, 7<weak alkaline pH≤9.

[0049] In some embodiments, the concentration of nano zero-valent iron in wastewater is 10-20 mg / L.

[0050] In some embodiments, the concentration of chlorite in the wastewater is 0.01-0.05 mmol / L.

[0051] In some embodiments, antibiotic-resistant bacteria include but are not limited to Escherichia coli containing tetracycline resistance genes and ampicillin resistance genes.

[0052] Escherichia coli containing tetracycline resistance gene and ampicillin resistance gene is a dual-resistant bacterium (dual-ARE.coli) carrying pBR322 plasmid.

[0053] The experimental results show that the present invention adopts nZVI / ClO2 - / O2 system at very low doses (10-20 mg / L nZVI and 0.01-0.05 mmol / L ClO2 - ) and acidic (pH 3-7), neutral or weakly alkaline (pH 7-9) conditions, the initial concentration of which is about 1×10 7 CFU / mL of dual-ARE.col, and can effectively remove intracellular and extracellular drug resistance genes; Fe IV =O can effectively destroy the cell membrane structure of ARB, leading to increased cell permeability, which in turn leads to extracellular superoxide anions (·O2 - ), iron ions, etc. enter the bacterial cells, inducing oxidative stress and damage in the cells. The combined effects of exogenous damage and endogenous oxidative stress lead to the inactivation of ARB, while promoting the leakage of intracellular drug-resistant genes to the extracellular space and their rapid removal. The treatment method of the present invention has a better disinfecting effect on ARB than active chlorine and the common zero-valent iron-activated peroxide Fenton reaction, and has a wide pH (pH 3-9) application range.

[0054] In some embodiments, the chlorite includes at least one of chlorous acid, sodium chlorite, and potassium chlorite.

[0055] In some embodiments, the concentration of nano zero-valent iron in the wastewater is 20 mg / L.

[0056] In some embodiments, the concentration of chlorite in the wastewater is 0.05 mmol / L.

[0057] In some embodiments, the pH of wastewater containing antibiotic-resistant bacteria is adjusted to acidic or weakly alkaline. Specifically, 0.1 mol / L NaOH solution and HCl solution are used to adjust the pH value of the wastewater.

[0058] In some embodiments, nano zero-valent iron and chlorite are added to the wastewater after pH adjustment, and sterilized at 100-600 rpm for 1-20 min.

[0059] Traditional sterilization technology is economical, but the sterilization effect is affected by dosage, contact time, and characteristics of bacteria themselves. On the one hand, traditional disinfection technology may not be able to completely inactivate ARB, and often induces ARB to be in a dormant state (VBNC state), which can be restored to a culturable wild-type state under favorable conditions, and is an important way for bacteria to acquire antibiotic resistance. On the other hand, traditional disinfection technology cannot effectively remove ARGs. Zero-valent iron can inactivate microorganisms through multiple pathways such as adsorption, activation of oxidants to generate active species, and induction of intracellular oxidative stress in bacteria, but it is still limited by low reactivity, high dosage and cost of agents, and intrinsic defects of free radical active species, such as short half-life, small migration distance, and low steady-state concentration. The present invention constructs a new type of zero-valent iron advanced oxidation reaction system, regulates the types and yields of active species, so as to enhance their reactivity with typical ARBs and ARGs, achieve their efficient removal, and reduce the dosage of agents in the treatment method to save costs.

[0060] Based on the same concept, the present invention also provides an application of a preparation in removing antibiotic-resistant bacteria and resistant genes in wastewater, wherein the preparation comprises nano zero-valent iron and chlorite.

[0061] The following further illustrates the method for treating antibiotic-resistant bacteria in wastewater of the present invention with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] In the following examples, the preparation method of nano zero-valent iron (nZVI) is prepared according to the above method.

[0063] In the following examples, the bacterial solution preparation method is to add the drug-resistant bacteria into water; the initial concentration of the bacterial solution is about 1×10 7 CFU / mL.

[0064] In the following examples, N represents the concentration of drug-resistant bacteria in the sample (unit: CFU / mL; CFU: colony forming unit), and log(N) is used to represent the bactericidal effect on drug-resistant bacteria.

[0065] In the following examples, N represents: the concentration of resistant bacteria in the sample (unit: CFU / mL; CFU: colony forming unit); N0: the concentration of untreated resistant bacteria (i.e., initial resistant bacteria) as the control group, and log (N / N0) represents the inactivation effect of resistant bacteria.

[0066] Example 1

[0067] The present application example provides the construction of dual-ARE.coli carrying the pBR322 plasmid, which specifically includes the following steps:

[0068] 1.1 Preparation of LB solid selective medium

[0069] (1) According to the formula of LB medium (brand: BIOBOMEI), weigh the corresponding drugs into a 500 mL conical flask, add 200 mL of ultrapure water, seal the flask, and sterilize it with high pressure steam at 121°C for 20 min;

[0070] (2) This step is carried out in a clean bench: when the temperature of the culture medium drops to about 50-60°C, add 0.4 mL of tetracycline (5 mg / mL) and 0.2 mL of ampicillin (100 mg / mL) solution to the culture medium, mix thoroughly and then slowly pour into a sterile culture dish and wait for natural cooling and solidification.

[0071] 1.2 ARE.coli transformation

[0072] (1) Take out the plasmid (specifically pBR322 plasmid DNA, Thermo Fisher, Catalog No. SD0041, which has two antibiotic resistance genes - ampicillin resistance gene and tetracycline resistance gene) from -20°C and put it into the clean bench. Then take out 100 μL of competent cells E. coli DH5α (Competent Cells E. coli DH5α Competent Cells, Takara, Code No. 9057) from the ultra-low temperature refrigerator (-80°C), transfer it to the clean bench, and place it on ice to melt (about 2 to 3 minutes);

[0073] (2) After mixing, add 1 μL of pBR322 plasmid, place on ice for 30 min, heat shock in a 42°C electric constant temperature water bath for 45 s, and quickly return to ice for 1-2 min;

[0074] (3) Add 900 μL of SOC medium (McLean brand) to a final volume of 1 mL in a clean bench and culture at 37°C in a shaker (220 rpm) for 1 h;

[0075] (4) Pipette 500 μL of bacterial solution and add it to 50 mL of LB selection medium (100 μL of tetracycline (5 mg / mL) and 50 μL of ampicillin (100 μg / mL), and culture in a shaker (220 rpm) at 37 °C for 12 h;

[0076] (5) labeling the cultured E. coli as ARE.coli, and preserving the strain in a volume ratio of ARE.coli:50% glycerol = 1:1;

[0077] (6) Spread the overnight cultured bacterial solution (ARE.coli) onto a plate containing both tetracycline and ampicillin resistance and incubate for 12 hours to observe whether colonies grow. If colonies grow, it means that both resistances have been generated (i.e., dual-resistant bacteria (dual-ARE.coli) carrying the pBR322 plasmid have been obtained). The results are as follows: Figure 1 .

[0078] Figure 1 The left side in the middle shows the dual-AR E. coli carrying the pBR322 plasmid. Figure 1 The right side of the middle shows no colony growth. According to the method in Example 1, dual-ARE.coli carrying the pBR322 plasmid can be screened.

[0079] Plasmid identification

[0080] The plasmid was extracted from the overnight culture solution (ARE.coli) using a plasmid extraction kit and used as a template to amplify the resistance genes tetC (tetracycline resistance gene) and bla TEM (Ampicillin resistance gene). Primer sequences and PCR reaction system are shown in Tables 1 and 2. The PCR amplification program was set as follows: (1) 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s; (2) return to step 2 and cycle 25 times; (3) 72°C extension for 10 min; (4) 4°C cooling.

[0081] Prepare agarose gel with a 2% agarose percentage, set the electrophoresis instrument voltage to 110mV and the current to 150mA, and perform agarose gel electrophoresis on the PCR product. The results are as follows: Figure 2 .

[0082] Table 1-PCR reaction system

[0083]

[0084] Table 2 - tetC and bla TEM PCR primers

[0085]

[0086] from Figure 2 It can be seen that the screened bacterial solution contains the resistance genes tetC and bla TEM .from Figure 2 It can be seen that the screened bacterial solution contains the resistance genes tetC and bla TEM .

[0087] The dual-ARE.coli bacteria used in the following Examples 2 to 9 were all constructed using the method in Example 1.

[0088] Example 2 (Nano-Zero-Valent Iron Dosage Optimization)

[0089] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0090] Take 100mL, 100mL, and 50mL of the prepared dual-ARE.coli bacterial solution (the bacterial solution is prepared by adding the drug-resistant bacteria to water, and the initial concentration of the bacterial solution is about 1×10 7 CFU / mL) in a conical flask, add 0.0010g, 0.0015g, and 0.0010g nZVI (i.e., the concentrations of nZVI are 10, 15, and 20mg / L, respectively), and then add ClO2 - To the initial concentration of 0.05mmol / L (in the form of NaClO2 to make ClO2 - The concentration was 0.05mmol / L), the pH was adjusted to 6.8, and the mixture was placed in a rotary shaker. After reacting at 200rpm for 10min, 1mL of the mixture was taken and 100μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows Figure 3 shown. Figure 3 Where N represents the concentration of drug-resistant bacteria in the sample (unit: CFU / mL; CFU: colony forming unit), and log(N) is used to represent the bactericidal effect of drug-resistant bacteria. The results are as follows: Figure 3 shown.

[0091] from Figure 3 It can be seen that when ClO2 - When the initial concentration is 0.05mmol / L, adding 10mg / L nZVI can inactivate 5.3logARB within 10 minutes, and when adding 20mg / L nZVI, 6.2logARB is inactivated within 10 minutes. The present invention selects 20mg / L nZVI as the condition for subsequent experimental exploration.

[0092] Example 3 (Optimization of Dosage of Chlorite)

[0093] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0094] Measure 50mL of the prepared dual-ARE.coli bacterial solution into a conical flask, add 0.0010g (20mg / L) nZVI, and then add ClO2 -(Added in the form of NaClO2) to initial concentrations of 0.01mmol / L, 0.025mmol / L, and 0.05mmol / L, respectively. The pH was adjusted to 6.8 and placed in a rotary shaker. After reacting at 200rpm for 10min, 1mL of the mixture was taken and 100μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows Figure 4 shown.

[0095] from Figure 4 It can be seen that when the initial concentration of nZVI is 20 mg / L, ClO2 - The greater the dose, the better the inactivation effect of dual-AR E.coli. my country's drinking water hygiene standards stipulate that the limit of free chlorine in factory water and terminal water is 2mg / L. Therefore, under the premise of cost saving and environmental friendliness, the present invention selects 0.05mmol / L (1.78mg / L) ClO2 - as a condition for subsequent experimental investigation.

[0096] Example 4 (nZVI / ClO2 - / O2 system inactivates ARB)

[0097] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0098] Take 50 mL of the prepared dual-AR E. coli bacterial solution, add 0.0010 g of nZVI, and then add ClO2 - The initial concentration was 0.05mmol / L, the pH was adjusted to 6.8, and the mixture was placed in a rotary shaker and reacted at 200rpm for 0min, 2.5min, 5min, 10min, and 15min. 1mL of the mixed solution was transferred with a pipette and 100μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows: Figure 5 shown. Figure 5 Medium ZVI / ClO2 - Indicates adding nZVI and ClO2 at the same time - , nZ VI means adding nZVI alone, ClO2 - Indicates the addition of nZVI alone.

[0099] from Figure 5 It can be seen that the individual nZVI and ClO2 - The inactivation effect of the system on dual-AR E.coli was almost negligible, while nZVI / ClO2 - The bactericidal effect of the system was significantly improved, reaching 6.3 log after 10 minutes of treatment.

[0100] Example 5 (nZVI / ClO2 - Comparison of the performance of dual-AR E.coli disinfection by O2 system and traditional chlorine disinfection)

[0101] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0102] 50 mL of the prepared dual-AR E. coli bacterial solution was measured, sodium hypochlorite solution was added to an initial concentration of 0.05 mmol / L, pH was adjusted to 6.8, and placed in a rotary shaker. After reacting at 200 rpm for 10 minutes, 1 mL of the mixed solution was removed and 5% sodium thiosulfate solution was added to terminate the reaction. The bactericidal effect was determined by the plate count method.

[0103] At the same time, take 50 mL of the prepared dual-AR E. coli bacterial solution, add 0.0010 g of nZVI, and then add ClO2 - The initial concentration was 0.05 mmol / L, the pH was adjusted to 6.8, and the mixture was placed in a rotary oscillator. After reacting at 200 rpm for 10 min, 1 mL of the mixed solution was transferred with a pipette and 100 μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The flow cytometer was combined with the Live / Dead fluorescent kit cell staining to count the intact cells according to the membrane integrity, thereby evaluating the efficacy of traditional chlorine disinfection and nZVI / ClO2. - The status of drug-resistant bacteria after treatment with the / O2 system, the results are as follows Figure 6 shown.

[0104] Figure 6 where represent traditional chlorine disinfection (ClO-) and nZVI / ClO2 - The system shows three states of drug-resistant bacteria at 0min, 10min, and 15min: Dead, Injury, and Live;

[0105] from Figure 6 It can be seen that traditional chlorine disinfection will induce bacteria to enter the VBNC state, nZVI / ClO2 - After 15 minutes of treatment with the system, the mortality rate of drug-resistant bacteria reached 100%.

[0106] Example 6 (nZVI / ClO2 - Comparison of the performance of the / O2 system and other advanced oxidation systems in inactivating dual-AR E.coli

[0107] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0108] Take 50 mL of the prepared dual-AR E.coli bacterial solution, add 0.0010 g of nZVI, and then add H2O2, PMS (peroxymonosulfate), PDS (peroxydisulfate), ClO2 - The initial concentration was 0.05 mmol / L, the pH was adjusted to 6.8, and the mixture was placed in a rotary shaker. After reacting for 10 min at 200 rpm, 1 mL of the mixed solution was transferred with a pipette and 100 μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows: Figure 7 shown.

[0109] from Figure 7 It can be seen that after 10 minutes of treatment, nZVI / ClO2 - The system inactivates resistant bacteria by up to 6.2 log, while the inactivation effects of other advanced oxidation systems are not significant.

[0110] Example 7 (nZVI / ClO2 - / O2 system to inactivate active species of dual-AR E.coli)

[0111] Dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), furfuryl alcohol (FFA), and benzoic acid (BA) were used as quenchers to quench the high-valent iron (Fe IV =O) and hydroxyl radicals (·OH), superoxide radicals (·O2 - ) and hydroxyl radicals (·OH) and chlorine radicals (·Cl), singlet oxygen ( 1 O2), hydroxyl radical (·OH) and chlorine radical (·Cl).

[0112] 50 mL of the prepared dual-AR E. coli bacterial solution was measured and added with the above different quenchers and without quencher ( Figure 8 After adding 0.0010g of nZVI and ClO2 to an initial concentration of 0.05mmol / L, the samples without - The solution was adjusted to pH 6.8 and placed in a rotary shaker. After reacting at 200 rpm for 15 min, 1 mL of sample was taken and 100 μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows: Figure 8 shown.

[0113] from Figure 8 It can be seen that the addition of SOD inhibited the inactivation of drug-resistant bacteria by about 1.8 log·O2 - The addition of FFA and BA had no effect on the inactivation of drug-resistant bacteria, indicating that nZVI / ClO2 -Does not exist in the system 1 O2 and ·OH; the inactivation effect of resistant bacteria was inhibited by about 5.8 log after adding DMSO, indicating that Fe IV =O is the main active species in the process of inactivation of drug-resistant bacteria.

[0114] Example 8 (nZVI / ClO2 - / O2, nZVI system to remove extracellular drug resistance genes)

[0115] The dual-AR E. coli cultured overnight was used to extract the drug resistance gene using the Tiangen Bio high-purity plasmid extraction kit and the initial concentration was 1×10 10 Measure 50 mL of the gene solution with 100 copies / mL, add 0.0010 g nZVI and 250 μL 10 mmol / L ClO2 - The solution was adjusted to pH 6.8 and placed on a rotary oscillator at 200 rpm. 1 mL of sample was taken at 0 min, 2.5 min, 5 min, 10 min, and 15 min, and then 100 μL of DMSO solution was added to terminate the reaction. Only 0.0010 g ZVI or 250 μL of 10 mmol / L ClO2 was added. - The solution system was used as the control system. Real-time fluorescence quantitative PCR (qPCR) was used to determine the gene abundance. The results are shown in Figures 11-12 shown.

[0116] from Fig. 9 It can be seen that after 15 minutes of treatment, nZVI / ClO2 - The removal effect of the system on tetC reached 3.86log, the nZVI system alone could reach 2.88log, and the ClO2 alone could reach 2.86log. - The removal effect of tetC in the system is negligible; Fig.10 As shown, ClO2 - Extracellular bla TEM The concentration of ZVI / ClO2 fluctuates within the error range and can be ignored. - After 15 minutes of treatment, extracellular bla TEM The concentration decreased by 4.2 log, and the extracellular bla TEM The concentration decreased by 2.67 log.

[0117] Example 9 (nZVI / ClO2 - / O2 system to remove intracellular drug resistance genes)

[0118] Take 50mL dual-AR E.coli culture, add 0.0010g nZVI, and then add ClO2 - The system solution was adjusted to an initial concentration of 0.05mmol / L, the pH was adjusted to 6.8, and placed in a rotary oscillator. The reaction was carried out at 200rpm. 2mL samples were taken at 0min, 15min, 30min, 45min, and 60min, and 200μL DMSO solution was added to terminate the reaction. The bacterial solution was filtered with a 0.22μm filter membrane, and then the filter membrane was rinsed with 2mL of deionized water. The resistant bacteria were resuspended in the aqueous solution, and the intracellular drug-resistant genes were extracted using the Tiangen Bio high-purity plasmid extraction kit. The gene abundance was determined using the qPCR system and procedure in the extracellular drug-resistant gene removal experiment. The results are shown in Figures 11-12 As shown. Fig.11 It can be seen that nZVI or ClO2 alone - After 60 minutes of treatment, the intracellular tetC concentration did not change significantly, while nZVI / ClO2 - After 60 minutes of treatment, intracellular tetC can be degraded by 2.49 log. Fig.12 As shown, intracellular bla TEM The removal effect of nZVI or ClO2 alone - The system cannot effectively degrade intracellular bla T EM , and nZVI / ClO2 - After 60 minutes of treatment, the system was able to degrade 2.90 log intracellular bla TEM .

[0119] Example 10 (Superoxide Anion Quenching Experiment)

[0120] The dual-AR E. coli cultured overnight was used to extract the drug resistance gene using the Tiangen Bio high-purity plasmid extraction kit and the initial concentration was 1×10 10 50 mL of the gene solution containing 100 copies / mL of the solution was measured, and 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL), tert-butyl alcohol (TBA), and DMSO were added as quenchers, and no quencher was added (i.e. Fig.13 No scavengers) as a comparison, 0.0010g nZVI and 250μL 10mmol / L ClO2 - The solution was adjusted to pH 6.8 and placed on a rotary shaker. After reacting at 200 rpm for 10 min, 1 mL of sample was taken and 100 μL of DMSO solution was added to terminate the reaction. Real-time fluorescence quantitative PCR (qPCR) was used to determine blaTEM Gene abundance, results such as Fig.13 shown.

[0121] Depend on Fig.13 It can be seen that nZVI / ClO2 - In the system, the addition of TBA has an effect on the expression of extracellular drug resistance genes (eARGs) bla TEM The removal of ·O2 had no effect, but was slightly inhibited by the addition of DMSO and significantly inhibited by the addition of TEMPOL, indicating that the main active oxygen species removed from eARGs is ·O2 - , instead of Fe IV =O.

[0122] Example 11 (nZVI / ClO2 at different pH - / O2 system sterilization effect)

[0123] This embodiment provides a method for treating antibiotic-resistant bacteria in wastewater, comprising the following steps:

[0124] Measure 50mL of the prepared dual-ARE.coli bacterial solution into a conical flask, add 0.0010g (20mg / L) nZVI, and then add ClO2 - (Added in the form of NaClO2) to an initial concentration of 0.05mmol / L, the pH was adjusted to 3, 5, 7, and 9 respectively, and placed in a rotary oscillator. After reacting at 200rpm for 10min, 1mL of the mixed solution was transferred with a pipette and 100μL of DMSO was added to terminate the reaction. The bactericidal effect was determined by the plate count method. The results are as follows Fig.14 As shown, Fig.14 ND in the middle means nZVI / ClO2 - All bacteria in the system were killed (no colonies on the plate).

[0125] from Fig.14 It can be seen that in the pH range of 3 to 9, the nZVI / ClO2 - / O2 systems all have good bactericidal effects, and when the pH is between 3 and 5, there are no colonies on the plate, and the bactericidal effect is optimal.

[0126] 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 protection scope of the present invention.

Claims

1. A method for treating antibiotic-resistant bacteria in wastewater, characterized in that: The following steps are involved: Adjust the pH of wastewater containing antibiotic-resistant bacteria to acidic, neutral or slightly alkaline; Adding nano zero-valent iron and chlorite to the wastewater after pH adjustment for sterilization; The acidic pH value is 3 to 7; The weakly alkaline pH is 7 to 9; The concentration of nano zero-valent iron in the wastewater is 10-20 mg / L; The concentration of chlorite in the wastewater is 0.01 to 0.05 mmol / L; The antibiotic-resistant bacteria include Escherichia coli containing tetracycline resistance genes and ampicillin resistance genes; The chlorite includes at least one of chlorous acid, sodium chlorite and potassium chlorite.

2. The method for treating antibiotic-resistant bacteria in wastewater according to claim 1, characterized in that: The concentration of nano zero-valent iron in the wastewater is 20 mg / L; The concentration of chlorite in the wastewater is 0.05 mmol / L.

3. Use of a preparation for removing antibiotic-resistant bacteria and drug-resistant genes in wastewater, the preparation comprising nano zero-valent iron and chlorite; The antibiotic-resistant bacteria include Escherichia coli containing tetracycline resistance genes and ampicillin resistance genes; The wastewater pH is adjusted to be acidic, neutral or weakly alkaline; The acidic pH value is 3 to 7; The weakly alkaline pH is 7-9.

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