A method for removing extracellular DNA contamination in wastewater

Abstract

The present invention discloses a method for removing extracellular DNA contamination from wastewater. The method primarily comprises the following steps: filtering the original wastewater and performing ultrasonic treatment to remove impurities and disrupt bacterial membranes to release DNA; mixing a predetermined amount of carbon nanotubes with the wastewater; adsorbing the extracellular DNA onto the carbon nanotube surfaces; and, after adsorption equilibrium has been reached, adding a predetermined concentration of persulfate to initiate an oxidation reaction, generating reactive oxygen species that oxidatively degrade the extracellular DNA in the wastewater. Compared to existing methods for removing extracellular DNA contamination, the present invention offers the advantages of simplicity, high efficiency, low construction cost, and wide application. It can remove extracellular DNA from wastewater, thereby controlling the risk of resistance gene transmission in the wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage treatment, and particularly relates to a method for removing extracellular DNA contamination in wastewater. Background Art

[0002] In recent years, antibiotic resistance genes (ARGs), a type of novel contaminant, have gradually attracted attention. ARGs are genes that encode antibiotic resistance and can be found inside bacterial cells (iARGs) or outside of them (eARGs). iARGs are located within the chromosomes or plasmid DNA of cells (i.e., drug-resistant ARBs). DNA fragments containing ARGs can be released into the extracellular environment through bacterial secretion or cell rupture. Environmental eARGs can be absorbed, assimilated, or transformed and then reenter the bacterial cell. These bacteria and gene fragments carrying multidrug resistance are difficult to kill with antibiotics, allowing resistant bacteria and resistance genes to persist in the environment.

[0003] Studies have reported that wastewater treatment plants are considered the primary anthropogenic source of extracellular DNA (eDNA) carrying ARGs. This is because wastewater from various sources undergoes a series of water treatment processes after entering a wastewater treatment plant. Conventional wastewater treatment technologies can significantly damage bacterial cells, leading to the lysis and death of numerous bacterial communities. However, these processes cannot completely destroy DNA, resulting in the massive release of intracellular DNA carrying ARGs, making eDNA the primary form of ARGs in wastewater treatment plants. Furthermore, studies have shown that compared to the removal efficiency of cell-associated ARGs, eDNA carrying ARGs is more difficult to remove by water treatment processes (including advanced treatment processes). It is released into the downstream environment with wastewater treatment plant effluent, posing not only ecological risks but also increasing the likelihood of human pathogens acquiring eDNA. Therefore, it is necessary to develop a technology that can effectively remove eDNA as a further treatment process for wastewater treatment plant effluent.

[0004] Persulfate advanced oxidation process (PS-AOPs) is a process based on sulfate free radical (SO4· - ) and hydroxyl radical (·OH) oxidation degradation technology, due to SO4· -It is considered to be a technology with potential for oxidative treatment of pollutants in water due to its longer half-life (30-40μs), higher redox potential (E0=2.5-3.1eV), and wider pH adaptability. This process can activate PS by energy (heat, ultraviolet), metal, carbon-based materials, etc. to produce reactive oxygen species, thereby removing pollutants. In recent years, this green and friendly water treatment method has been extended to the removal of resistant bacteria and resistance genes. It has also been found that different activation methods induce PS oxidation to remove resistant bacteria and resistance genes with different effects. For example, the removal of Escherichia coli and ARG in the photocatalytic system is inefficient and easy to resurrect, ultraviolet irradiation has the problem of high energy consumption, and the metal activation system has limiting factors such as ion leaching and low recycling capacity. Carbon-based materials have better development prospects due to their excellent performance and economic and environmental friendliness. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for removing extracellular DNA contamination in wastewater, to make up for the problem of extracellular DNA contamination in the secondary effluent of existing sewage treatment plants, thereby controlling the risk of transmission of resistance genes in downstream water bodies, and facilitating the regeneration and reuse of wastewater.

[0006] The technical solution of the present invention is:

[0007] A method for removing extracellular DNA contamination in wastewater, the method comprising the following steps:

[0008] (1) The original wastewater is filtered, pH adjusted, and ultrasonically treated;

[0009] (2) adding carbon nanotubes to the wastewater treated in step (1) and stirring;

[0010] (3) adding persulfate to the wastewater treated in step (2) and stirring to achieve oxidative degradation of extracellular DNA in the wastewater.

[0011] Furthermore, in step (1), the filtration is that the raw wastewater needs to be filtered through a 0.45 μm mixed cellulose filter membrane.

[0012] Furthermore, in step (1), the pH value is adjusted to ≤7.

[0013] Furthermore, in step (1), the parameters of the ultrasonic treatment are: 100 Hz, and the time is not less than 10 min.

[0014] Furthermore, in step (2), the ratio of the added mass concentration of carbon nanotubes to the mass concentration of extracellular DNA in the wastewater is 50:1.

[0015] Furthermore, in step (2), the stirring time is not less than 60 minutes.

[0016] Furthermore, in step (3), the added mass concentration of persulfate is twice the added mass concentration of carbon nanotubes.

[0017] Furthermore, in step (3), the stirring time is not less than 6 hours.

[0018] Furthermore, in step (1), the raw wastewater is secondary effluent from a domestic, industrial, medical or comprehensive sewage treatment plant.

[0019] Raw wastewater is filtered to remove impurities and suspended matter. To improve the removal efficiency of extracellular DNA, filtration is performed using a 0.45μm mixed cellulose membrane. This not only removes impurities and suspended matter, but also removes most pathogenic bacteria (existing advanced treatment processes can already address this issue), thereby mitigating competitive adsorption between bacteria and extracellular DNA on CNTs. Ultrasonic treatment is used to destroy a small number of unfiltered microorganisms, releasing intracellular DNA.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a method for removing extracellular DNA contamination in wastewater, which releases intracellular DNA through ultrasound, adds carbon nanotubes for adsorption, and then adds persulfate for oxidation, thereby generating surface-bound free radicals and singlet oxygen, and directly degrading extracellular DNA contamination in wastewater through electron transfer pathways, thereby compensating for the problem of extracellular DNA contamination in the secondary effluent of existing sewage treatment plants, thereby controlling the risk of resistance gene transmission in downstream water bodies, and facilitating the regeneration and reuse of wastewater.

[0022] (2) The present invention provides a method for removing extracellular DNA contamination in wastewater, which has mild reaction conditions, simple and easy operation, is environmentally friendly, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Adsorption effect of different concentrations of CNT on eDNA;

[0024] Figure 2 The removal effect of eDNA by different concentrations of CNT-activated PS;

[0025] Figure 3 Effects of different initial eDNA concentrations on eDNA removal;

[0026] Figure 4 Effects of different pH values on eDNA removal;

[0027] Figure 5 Effects of different water media on eDNA removal;

[0028] Figure 6 ARG in composite wastewater media Amp and gel electrophoresis of eDNA before and after the reaction;

[0029] Figure 7 ARG in different water media amp removal effect. DETAILED DESCRIPTION

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0031] Example 1: This example provides the effect of carbon nanotubes in adsorbing extracellular DNA contamination. The specific experimental process is as follows:

[0032] (1) Commercial multi-walled carbon nanotubes (CNTs) (Nanjing Pioneer Nanomaterials Technology Co., Ltd., China) were used as adsorption materials without further treatment. Calf thymus DNA (double-stranded, protein-free, CAS No. 91080-16-9) (Sigma-Aldrich, USA) was used as an extracellular DNA (eDNA) model in the experiment. Powdered eDNA was dissolved in enzyme-free sterile water to prepare an eDNA homogenate solution (1.0 mg / mL). Enzyme-free sterile water was used for the preparation of reagents and reaction systems. The treated wastewater was collected from the secondary treatment effluent of domestic, industrial, medical, and comprehensive sewage treatment plants (Zhejiang).

[0033] (2) The collected original wastewater was filtered through a 0.45 μm mixed cellulose filter membrane and placed in an ultrasonic cleaning instrument for ultrasonic treatment at 25°C and 100 Hz for 10 minutes. In order to more intuitively reflect the experimental results, eDNA was artificially added to the wastewater to make the total eDNA content reach 10 μg / mL, and then stored at -4°C for use. A certain amount of CNT was weighed and placed in sterile water, ultrasonically dispersed for 5 minutes, and a certain concentration of adsorbent mother solution was prepared for use. The adsorption experiment was carried out in a sterile tube. 1 mL of the ultrasonic wastewater was taken, and different concentrations of CNT solution were added to form different concentration ratios with eDNA. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were used to adjust the pH (pH = 7.0 ± 0.1) and the adsorption experiment was carried out. The water sample without CNT was used as a blank control. All treatment groups were shaken and mixed at 25°C and 170 rpm. 100 μL of the mixture solution was taken at different time intervals, centrifuged at 12000 rpm for 5 minutes, and 3 μL of the supernatant was taken. The OD value (A260) was measured to determine the eDNA concentration. The concentration of eDNA was determined using a multifunctional microplate reader (Thermo Fisher Scientific, USA).

[0034] (3) The experimental results are as follows Figure 1As shown, it shows that the adsorption effect is obvious only when the concentration ratio of CNT to eDNA is at least 50, among which the best adsorption effect under economic conditions is achieved when CNT:eDNA=50:1.

[0035] Example 2: This example provides the effect of carbon nanotubes catalyzing persulfate oxidation to remove extracellular DNA contamination in water. The specific experimental process is as follows:

[0036] (1) The CNTs, eDNA, and raw wastewater used in this experiment were the same as those described in Example 1. All chemicals were of laboratory analytical grade. Enzyme-free sterile water was used in the preparation of reagents and reaction systems.

[0037] (2) Oxidative degradation experiments were conducted in filtered, ultrasonically treated wastewater adjusted to pH 7.0 ± 0.1 with NaOH and HCl. The total volume of the reaction system was 1 mL. eDNA was added to achieve an initial eDNA concentration of 10 μg / mL in the wastewater. CNT and sodium persulfate (PS) solutions at varying concentrations were added. All treatment groups were shaken and mixed at 25°C and 170 rpm. At different sampling times, 100 μL of the mixed reaction solution was collected and centrifuged at 12,000 rpm for 5 min. 3 μL of the supernatant was then collected and the OD value (A260) of the supernatant was measured using a multifunctional microplate reader to determine the eDNA concentration.

[0038] (3) The experimental results are as follows Figure 2 As shown in the figure, the best removal effect can be achieved when the eDNA contamination concentration is 10 μg / mL and the CNT to PS addition ratio is 0.5:1. Therefore, the PS addition concentration should be twice that of CNT.

[0039] Example 3: This example provides the effects of different factors on the degradation of extracellular DNA contamination in water by persulfate oxidation catalyzed by carbon nanotubes. The specific experimental process is as follows:

[0040] (1) The materials and reagents used in this experiment are the same as those described in Example 2.

[0041] (2) This experiment investigated the effects of different initial eDNA concentrations, different pH values, and different water media on the degradation of extracellular DNA pollution. In the experiment with different initial eDNA concentrations, the CNT dosage was 0.5 mg / mL, the PS dosage was 1 mg / mL, and the initial eDNA concentration in the wastewater was controlled by adding eDNA. In the experiment with different pH effects, the initial eDNA concentration was 10 μg / mL, the CNT dosage was 0.5 mg / mL, and the PS dosage was 1 mg / mL. HCl and NaOH were used to adjust the initial pH to 3, 5, 7, 9, and 11, respectively. In the experiment with different water media, degradation experiments were conducted using secondary effluent from domestic, industrial, medical, and comprehensive sewage treatment plants, and the other experimental conditions remained unchanged.

[0042] (3) The experimental results are as follows Figure 3-5 As shown, in the experiments with different initial eDNA concentrations ( Figure 3 ), when the dosage of CNT and PS is constant, the degradation rate gradually decreases with the increase of the initial concentration of eDNA. When it reaches 100 μg / mL, the degradation rate is less than 40%. In the experiment of different pH effects ( Figure 4 ), the maximum degradation effect was achieved when pH = 3, and the eDNA degradation rate gradually decreased with the increase of pH value. Figure 5 ), the removal rate of eDNA in the secondary effluent of various types of sewage treatment plants by CNT-activated PS can reach more than 80%. The method of the present invention can be applied to the eDNA treatment process of the secondary effluent of different types of sewage treatment plants.

[0043] Example 4: This example provides a comparison of the effects of carbon nanotubes catalyzing persulfate oxidation to remove eDNA and DNA carrying resistance genes in water. The specific experimental process is as follows:

[0044] (1) The CNTs and eDNA used in this experiment were the same as those described in Example 1. Resistant E. coli carrying an ampicillin (Amp)-resistant plasmid was used as a model for ARG-carrying resistance gene DNA. All chemicals were of laboratory analytical grade. Enzyme-free sterile water was used for the preparation of reagents and reaction systems.

[0045] (2) Contains ARG AmpThe detailed extraction process for the plasmid DNA stock solution is as follows: 100 μL of a resistant E. coli suspension (F1 generation) stored at -4°C was incubated with 500 mL of LB broth containing 100 μg / mL Amp at 37°C, 180 rpm for 16 hours. The resulting bulk resistant E. coli solution was then centrifuged at 4000 rpm for 10 minutes, and plasmid DNA was extracted and purified according to the specific procedures of a plasmid extraction kit (Tiangen, Beijing). The plasmid DNA concentration was measured at A260 using a multifunctional microplate reader. The prepared sample was stored at -20°C until use.

[0046] (3) The oxidative degradation experimental system and process adopted in this experiment are the same as those described in Example 2.

[0047] (4) In this experiment, ARG was evaluated before and after the reaction by agarose gel electrophoresis. Amp Quantitative PCR (qPCR) was used to detect ARG before and after the reaction. Amp The qPCR mixture (10 μL) consisted of 0.3 μL of front-end and back-end primers, 5 μL of 2x M5 HiPer SYBR Premix Es Taq, 1 μL of DNA template, and 3.4 μL of sterile deionized water. After the mixture was prepared, it was mixed thoroughly, centrifuged, and added to a 384-well Roche fluorescent quantitative PCR plate. The plate was then placed in a fluorescent quantitative PCR instrument for reaction and measurement. The qPCR reaction was performed using a two-step method.

[0048] (5) The results of this experiment are as follows Figure 6-7 As shown. Taking composite wastewater as an example, gel electrophoresis experiment was carried out by gel electrophoresis ( Figure 6 ) It can be seen that ARG before degradation Amp and eDNA have obvious bands, indicating that the DNA structure is intact. After the reaction is completed, the ARG Amp and eDNA bands disappeared, indicating that ARG Amp The ARGs were further determined by qPCR before and after the reaction with different water media. Amp The concentration changes, such as Figure 7 As shown, ARG was found before and after the reaction in all water media. Amp The above results indicate that the CNT-catalyzed PS oxidation treatment system can remove one or more combinations of extracellular free double-stranded or cyclic deoxyribonucleotides (including genomes with antibiotic resistance genes or plasmids carrying antibiotic resistance genes).

Claims

1. A method for removing extracellular DNA contamination in wastewater, characterized in that: The method comprises the following steps: (1) The raw wastewater is filtered through a 0.45 μm mixed cellulose membrane, the pH value is adjusted to ≤7, and then ultrasonically treated; the raw wastewater is the secondary effluent from a domestic, industrial, medical or comprehensive sewage treatment plant, and the ultrasonic treatment parameters are: 100 Hz, for not less than 10 minutes; (2) adding carbon nanotubes to the wastewater treated in step (1), wherein the ratio of the added mass concentration of carbon nanotubes to the mass concentration of extracellular DNA in the wastewater is 50:1, and stirring for not less than 60 minutes; (3) adding persulfate to the wastewater treated in step (2) at a concentration twice that of the carbon nanotubes, stirring for at least 6 hours to achieve oxidative degradation of the extracellular DNA in the wastewater.

Images