Disinfection method for removing antibiotic-resistant bacteria in reclaimed water through multi-parameter collaborative optimization

Abstract

The invention discloses a disinfection method for removing antibiotic-resistant bacteria in reclaimed water through multi-parameter collaborative optimization, which comprises the following steps: 1) collecting secondary effluent of a sewage treatment plant, and taking the secondary effluent as reclaimed water after active sand filtration treatment; (2) adding a sodium hypochlorite solution into the reclaimed water for chlorine disinfection, controlling the chloride ion concentration to be 2-4mg / L, maintaining the disinfection time to be 25-35 minutes under a dark condition, and controlling the water temperature to be 20-30 DEG C in the disinfection process; and 3) after disinfection is finished, transferring the water sample into a dark container, and storing the water sample in an environment at the temperature of less than or equal to 25 DEG C. The method has a good removal effect on antibiotic resistant bacteria, effectively reduces the risk of environmental health and sanitation, can be directly adapted to chlorine disinfection equipment such as an existing UASB (Upflow Anaerobic Sludge Blanket), a contact tank and the like, does not need large-scale equipment transformation, is suitable for a sewage treatment plant with daily treatment capacity of 1000-100000 tons, and has remarkable economic and environmental benefits.

Description

Technical Field

[0001] The present invention relates to a disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water, belonging to the technical field of sewage treatment. Background Art

[0002] Chlorine disinfection is a key process for ensuring the hygienic safety of the effluent from urban sewage treatment plants, but there are many technical bottlenecks in its application. The removal rates of traditional chlorine disinfection processes (such as chlorine concentration of 1 mg / L and contact time ≤ 10 min) for antibiotic-resistant bacteria (ARB) such as clindamycin and ciprofloxacin are only 40% - 60%, resulting in the remaining antibiotic-resistant bacteria after disinfection becoming potential sources of transmission. The prior art has not systematically studied the synergistic effects of chlorine concentration, contact time, and temperature on the removal of antibiotic-resistant bacteria, making it difficult to achieve a balance between disinfection efficiency and resistance control, resulting in large fluctuations in the concentration of antibiotic-resistant bacteria in the effluent and high environmental risks.

[0003] Particularly crucial is that the traditional process does not pay attention to the regrowth problem of antibiotic-resistant bacteria after disinfection. The resistance genes carried by antibiotic-resistant bacteria can be transferred to other bacteria in the environment through horizontal gene transfer by means of plasmids, transposons, etc. Even if these bacteria were originally sensitive to antibiotics, there is a possibility of acquiring drug resistance. This situation will cause the rapid spread of drug resistance in the microbial community, thereby giving rise to "superbugs". This phenomenon will not only weaken the therapeutic effect of antibiotics but also make the global "antibiotic crisis" more severe.

[0004] In the prior art, when the chlorine concentration is below 3 mg / L or the contact time is less than 30 min, the abundance of sulfonamide-resistant bacteria in reclaimed water can increase by 1.65 - 2.91 log units after 3 days of disinfection. When the chlorine concentration is higher than 3 mg / L or the contact time exceeds 30 min, the abundance of ciprofloxacin-resistant bacteria in reclaimed water increases by 4.88 log units after 7 days of disinfection. When the environmental temperature is higher than 25 °C, the abundances of various antibiotic-resistant bacteria in reclaimed water except tetracycline-resistant bacteria increase significantly (the abundance of ciprofloxacin-resistant bacteria increases by 4.32 log units) after 1 day of disinfection. Therefore, there is an urgent need to establish a multi-parameter collaborative control technology that takes into account both "efficient removal" and "long-term control of regeneration". Summary of the Invention

[0005] In view of the problems existing in the above prior art, the present invention provides a disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water.

[0006] In order to achieve the above object, a disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water adopted by the present invention includes the following steps:

[0007] 1) Collect the secondary effluent from the sewage treatment plant and use it as reclaimed water after treatment by activated sand filtration;

[0008] 2) Add sodium hypochlorite solution to the reclaimed water for chlorine disinfection, control the chloride ion concentration to be 2 - 4 mg / L, maintain the disinfection time for 25 - 35 min under light - avoidance conditions, and control the water temperature at 20 - 30 °C during the disinfection process;

[0009] 3) After the disinfection is completed, transfer the water sample to a light - avoidance container and store it in an environment of ≤25 °C.

[0010] As an improvement, the water quality indexes of the reclaimed water in step 1) are: total nitrogen < 15 mg / L, total phosphorus < 1 mg / L, COD < 50 mg / L, pH 6 - 9, meeting the first - level A discharge standard in the "Discharge Standard of Pollutants for Municipal Wastewater Treatment Plants" (GB 18918—2002).

[0011] As an improvement, in step 2), control the chloride ion concentration to be 3 mg / L, maintain the disinfection time for 30 min under light - avoidance conditions, and control the water temperature at 25 °C during the disinfection process.

[0012] As an improvement, in step 2), maintain the water temperature stability through a constant - temperature device.

[0013] As an improvement, the sewage treatment plant is a sewage treatment plant with a daily treatment capacity of 1000 - 100000 tons.

[0014] As an improvement, the antibiotic - resistant bacteria include at least one of clindamycin - resistant bacteria, sulfonamide - resistant bacteria, ciprofloxacin - resistant bacteria, and tetracycline - resistant bacteria.

[0015] Compared with the prior art, when the present invention performs chlorine disinfection on the reclaimed water from the sewage treatment plant, by limiting the chloride ion concentration to 3 mg / L, the disinfection time to 30 min, and the temperature to 25 °C, a "concentration - time - temperature" synergistic effect is formed, so that the removal rate of clindamycin - resistant bacteria is increased from 75.14% of traditional chlorine disinfection to 93.89%, and the removal rate of ciprofloxacin - resistant bacteria is increased from 48.46% to 85%. It has a good removal effect on antibiotic - resistant bacteria, effectively reduces the environmental health risk, and can be directly adapted to existing chlorine - disinfection equipment such as UASB and contact tanks without large - scale equipment transformation. It is applicable to sewage treatment plants with a daily treatment capacity of 1000 - 100000 tons, having significant economic and environmental benefits. Description of the Drawings

[0016] Figure 1 It shows the removal of ARB in water under different chloride ion concentrations of the present invention;

[0017] Figures 2A to 2E Effect of different dosages of disinfectants on the regrowth of ARB in the present invention ( Figure 2A 、 2B 、2C, 2D, and 2E are respectively added to a chloride ion concentration of 0, 0.5, 1, 3, 5 mg / L);

[0018] Figure 3 Removal of ARB in water under different disinfection reaction times in the present invention;

[0019] Figure 4 Effect of different disinfection reaction times on the regrowth of ARB in the present invention ((a) to (d) in the figure are reaction times of 5, 15, 30, 60 min respectively);

[0020] Figure 5 Removal of ARB in water under different disinfection reaction temperatures in the present invention;

[0021] Figure 6 Effect of different disinfection reaction temperatures on the regrowth of ARB in the present invention ((a) to (d) in the figure are reaction temperatures of 4, 15, 25, 37 °C respectively). Detailed implementation method

[0022] The following examples are further explanations of the content of the present invention to illustrate the technical content of the present invention. However, the substantial content of the present invention is not limited to what is described in the following examples. Those of ordinary skill in the art can and should know that any simple changes or substitutions based on the substantial spirit of the present invention should fall within the scope of protection required by the present invention.

[0023] Example 1

[0024] A disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water, comprising the following steps:

[0025] 1. Take 20 L of the effluent from the activated sand filter of Zhuzhuanjing Wastewater Treatment Plant in Hefei. The water quality indicators are total nitrogen 5.81 mg / L, total phosphorus 0.15 mg / L, COD 28 mg / L, and pH 6.79. Take 200, 199.9, 199.8, 199.4, 199 mL as experimental water samples;

[0026] 2. Dilute the sodium hypochlorite solution of 100 g / L to 1 g / L;

[0027] 3. Add 0, 0.1, 0.2, 0.6, 1 mL of the 1 g / L sodium hypochlorite solution to the experimental water samples respectively, so that the chloride ion concentration in the water samples is 0, 0.5, 1, 3, 5 mg / L;

[0028] 4. Place the above chlorine - disinfected water sample system in a 25°C constant - temperature incubator and store it in the dark without hydraulic disturbance for 30 min of disinfection.

[0029] 5. After the disinfection is completed, take out the water sample system, transfer the water sample to a light - proof container, store it in an environment of ≤25°C, and take samples on the 1st, 3rd, and 7th days after the disinfection is completed.

[0030] Verify the parameters of the present invention:

[0031] The plate - counting method is used to determine the number of resistant bacteria in the sample. R2A medium is a lean medium. Since low - concentration nutrients can enable a wider range of microorganisms to grow, avoiding the inhibitory effect of fast - growing microorganisms on slow - growing microorganisms during cultivation, using this medium can obtain more sufficient data and be closer to the actual situation.

[0032] ① 15 ml / plate. Calculate the amount of R2A agar required according to the number of plates. Boil the R2A agar twice. Sterilize the boiled medium, culture dishes, required pipette tips, and several 5 - ml centrifuge tubes (the specific number depends on the dilution factor) in an autoclave at 121°C for 20 min. After the sterilization is completed, to prevent the medium from cooling and solidifying, place it in a 60°C oven to maintain the temperature.

[0033] ② Dilute the sample; first, inject 1.8 ml of normal saline into all 5 - ml centrifuge tubes used for dilution, close the lids to avoid bacterial contamination, and then prepare for dilution; take out 0.2 mL of the sample from the shaken conical flask and put it into a 5 - ml centrifuge tube (diluted 10 times), and then dilute it step - by - step to a reasonable multiple (diluted 10 times each time). After each dilution, shake it well and wait for about 2 minutes before proceeding to the next - gradient dilution (the bacteria will evenly distribute themselves).

[0034] ③ Label the plates; during the dilution process, place the culture dishes on the table and mark them on the lids with a marker pen.

[0035] ④ Add the sample; after the dilution and plate - labeling are both completed, wash your hands with alcohol spray. Use a pipette to take 0.1 mL of the sample and place it in the culture dish, and then arrange the culture dishes with added samples according to the category. Take the medium out of the oven. Do not add antibiotics to one of them, which is used to measure the HPC (total heterotrophic bacteria count), and add tetracycline, clindamycin, sulfamethoxazole, and ciprofloxacin to the other 4 media respectively to make the antibiotic concentration in the medium reach the minimum inhibitory concentration. Pour the medium into the culture dish to a thickness of about 15 mm, shake it slowly (clockwise and counter - clockwise plus up - and - down shaking), and the speed should not be too fast to avoid the medium sticking to the top cover. For plates that have been standing for a long time, shake them for a longer time. After shaking, place them aside and wait for them to cool and solidify. After solidification, place them upside - down in a 37°C incubator and culture for 36 h.

[0036] In the experiment, the concentrations of the four antibiotics were based on the minimum inhibitory concentration (MIC) established by the Clinical and Laboratory Standards Institute of the United States as the experimental concentration. The MIC of sulfamethoxazole was 50.4 mg / L, the MIC of tetracycline was 16.0 mg / L, the MIC of ciprofloxacin was 4.0 mg / L, and the MIC of clindamycin was 1.0 mg / L.

[0037] As Figure 1 shown, the addition of the disinfectant (sodium hypochlorite solution) significantly reduced the abundance of HPC (total heterotrophic bacteria) and various ARBs (antibiotic-resistant bacteria) in the water, and with the increase in the concentration of the disinfectant, the abundance of various bacteria showed a continuous downward trend. When the disinfectant was added until the chloride ion concentration reached 3 mg / L, the abundance of HPC was 1.13×10 4 CFU / mL, the removal amount was 0.80 log unit, and the removal rate was 84.33%; the abundance of CLI-class resistant bacteria (clindamycin-class resistant bacteria) was 4.67×10 3 CFU / mL, the removal amount was 1.21 log unit, and the removal rate was 93.89%; the abundance of SMX-class resistant bacteria (sulfonamide-resistant bacteria) was 5.17×10 2 CFU / mL, the removal amount was 0.49 log unit, and the removal rate was 67.34%; the removal amount of TET-class resistant bacteria (tetracycline-class resistant bacteria) was 0.67 log unit, and the removal rate was 78.4%; the abundance of CIP-class resistant bacteria (ciprofloxacin-class resistant bacteria) was 2.85×10 2 CFU / mL, the removal amount was 0.82 log unit, and the removal rate was 85%. When the chloride ion concentration increased to 5 mg / L, although the abundance of various bacteria continued to decline, the trend was not significant. Therefore, it can be considered that when the dosage of the disinfectant reached a chloride ion concentration of 3 mg / L, a high level of removal effect of HPC and various ARBs in the water was achieved.

[0038] As Figures 2A to 2E shown, in the case of a lower concentration of the disinfectant, the regrowth ability of SMX-class resistant bacteria in the water was the strongest. The abundance on the 3rd day after treatment with 1 mg / L of the disinfectant was 4.53×10 5 CFU / mL, an increase of 2.91 log units, and the abundance on the 7th day was 5.77×10 5 CFU / mL, with only an increase of 0.11 log unit compared to the 3rd day. In contrast, after the action of a high-concentration disinfectant, the regrowth ability of CIP-class resistant bacteria was greatly enhanced and reached the maximum abundance on the 7th day. Among them, the abundance after the action of 3 mg / L of the disinfectant reached 4.4×10 5CFU / mL, showing an increase of 3.19 log units; while after the action of 5 mg / L disinfectant, the abundance reached 2.03×10 6 CFU / mL, showing an increase of 4.88 log units. It can be seen from this that after the action of disinfectants with different concentrations, the regrowth ability of ARB in water was enhanced to varying degrees, and low-concentration disinfectants played a better screening role in the regrowth of SMX-resistant bacteria, while high-concentration disinfectants had a stronger screening effect on CIP-resistant bacteria.

[0039] As Figure 3 shown, when the disinfection reaction time was 15 min, the removal effects of HPC and ARB in water were poor. At this time, the numbers of HPC, CLI-class, and SMX-class resistant bacteria were significantly higher than those in the other three groups. At this time, the abundance of HPC in the effluent was 2.52×10 5 CFU / mL, the abundance of CLI-class resistant bacteria was 2.32×10 5 CFU / mL, and the abundance of SMX-class resistant bacteria was 1.2×10 3 CFU / mL, and the removal rates of HPC and various ARB were all below 20%. In contrast, when the disinfection reaction time was 30 min, the removal rate of SMX-class resistant bacteria in the effluent reached 81.11%, the removal rate of CIP-class resistant bacteria reached 86.77%, and at the same time, the removal rate of TE-class resistant bacteria increased to 88.0%. The removal rates of HPC and CLI-class resistant bacteria remained at 82.72% and 82.88%.

[0040] As Figure 4 shown, all types of ARB were effectively removed under short disinfection reaction time (5 min), and showed a strong regrowth trend within the following 7 days. Among them, the abundance of CLI-class resistant bacteria increased to 8.67×10 5 CFU / mL, showing an increase of 1.69 log units; the abundance of SMX-class resistant bacteria increased to 9.33×10 4 CFU / mL, showing an increase of 2.10 log units; the abundance of TET-class resistant bacteria increased to 4.0×10 3 CFU / mL, showing an increase of 1.56 log units; the abundance of CIP-class resistant bacteria increased to 6.37×10 4CFU / mL, an increase of 2.72 log units. At long disinfection reaction times (30 min and 60 min), various ARBs maintained a similar regrowth trend. However, as the disinfection reaction time increased, there was also a positive correlation trend in the regrowth ability and magnitude change of various ARBs. On the 7th day, the absolute abundance of CLI-type resistant bacteria increased by 1.74 log units; that of SMX-type resistant bacteria increased by 2.51 log units; that of TET-type resistant bacteria increased by 2.02 log units; and that of CIP-type resistant bacteria increased by 3.60 log units, and the abundance increased by as much as 3.68 log units on the 3rd day. Thus, it can be seen that as the disinfection reaction time increases, various ARBs in water will show a strong regrowth trend, and their regrowth ability is positively correlated with the disinfection reaction time.

[0041] It can be seen from Figure 5 that there is no significant difference in the magnitude of the numbers of HPC and ARBs in water after disinfection at different temperatures. The removal effect of HPC shows a downward trend as the environmental temperature increases, with the removal rate decreasing from 94.52% (4℃) to 86.95% (37℃). There is a similar trend for SMX-type resistant bacteria, and as the environmental temperature increases, its removal rate decreases from 77.42% (4℃) to 40.32% (37℃). However, CLI-type, TET-type, and CIP-type resistant bacteria maintain stable removal effects at different environmental temperatures, which are 91.82% - 93.08% (CLI-type resistant bacteria), 79.09% - 83.64% (TET-type resistant bacteria), and 78.16% - 84.38% (CIP-type resistant bacteria) respectively, indicating that there is no significant correlation between the removal effects of these types of ARBs and the disinfection environmental temperature (p > 0.05).

[0042] It can be seen from Figure 6 that the regrowth of various ARBs is greatly affected by the increase in environmental temperature. At 37℃, various ARBs all have strong regrowth abilities. On the 1st day of the regrowth process, except for the increase in the abundance of TET-type resistant bacteria by only 0.67 log units, the abundances of the other ARBs all increased by at least 2.03 log units in magnitude, and the abundance of CIP-type resistant bacteria increased by 4.32 log units, with the absolute number reaching 6.90×10 5 CFU / mL. As the regrowth process progresses, on the 3rd day, the abundances of various ARBs in water reach the maximum. At this time, the abundance of CLI-type resistant bacteria is 1.10×10 7 CFU / mL, an increase of 3.48 log units compared to the end of disinfection; the abundance of SMX-type resistant bacteria is 6.50×10 5CFU / mL, with a 3.02 log unit increase; the abundance of TET-resistant bacteria was 1.77×10 3 CFU / mL, with a 1.77 log unit increase; the abundance of CIP-resistant bacteria was 2.83×10 6 CFU / mL, with a 4.93 log unit increase.

[0043] Example 2

[0044] A disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water, comprising the following steps:

[0045] 1. Take 20 L of the effluent from the activated sand filter of Zhuzhuanjing Wastewater Treatment Plant in Hefei. The water quality indexes are total nitrogen 5.81 mg / L, total phosphorus 0.15 mg / L, COD 28 mg / L, and pH 6.79. Take 199.4 mL as the experimental water sample;

[0046] 2. Dilute the 100 g / L sodium hypochlorite solution to 1 g / L;

[0047] 3. Add 0.6 mL of the 1 g / L sodium hypochlorite solution to 199.4 mL of the water sample;

[0048] 4. Place the above chlorine disinfection system in a 25°C constant temperature incubator for dark storage, and disinfect without hydraulic disturbance for 5, 15, 30, and 60 min;

[0049] 5. After disinfection, take out the water sample system, transfer the water sample to a light-proof container, store it in an environment of ≤25°C, and take samples on the 1st, 3rd, and 7th days after disinfection.

[0050] Example 3

[0051] A disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water, comprising the following steps:

[0052] 1. Take 20 L of the effluent from the activated sand filter of Zhuzhuanjing Wastewater Treatment Plant in Hefei. The water quality indexes are total nitrogen 5.81 mg / L, total phosphorus 0.15 mg / L, COD 28 mg / L, and pH 6.79. Take 199.4 mL as the experimental water sample;

[0053] 2. Dilute the 100 g / L sodium hypochlorite solution to 1 g / L;

[0054] 3. Add 0.6 mL of the 1 g / L sodium hypochlorite solution to 199.4 mL of the water sample;

[0055] 4. Place the above chlorine disinfection system in a constant temperature incubator at 4, 15, 25, and 37°C for dark storage, and disinfect without hydraulic disturbance for 30 min;

[0056] 5. After the disinfection is completed, take out the water sample system, transfer the water sample to a light-proof container, store it in an environment of ≤25°C, and take samples on the 1st, 3rd, and 7th days after the disinfection is completed.

[0057] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A disinfection method for removing antibiotic-resistant bacteria in reclaimed water through multi-parameter collaborative optimization, characterized in that, It includes the following steps: 1) Collect the secondary effluent of the sewage treatment plant and use it as reclaimed water after treatment by activated sand filtration; 2) Add sodium hypochlorite solution to the reclaimed water for chlorine disinfection, control the chloride ion concentration to be 2 - 4 mg / L, maintain the disinfection time for 25 - 35 min under light - avoiding conditions, and control the water temperature at 20 - 30 °C during the disinfection process; 3) After the disinfection is completed, transfer the water sample to a light - avoiding container and store it in an environment of ≤25 °C.

2. The disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water according to claim 1, characterized in that The water quality indexes of the reclaimed water in step 1) are: total nitrogen < 15 mg / L, total phosphorus < 1 mg / L, COD < 50 mg / L, pH 6 - 9.

3. A disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water according to claim 1, characterized in that, In step 2), control the chloride ion concentration to be 3 mg / L, maintain the disinfection time for 30 min under light - avoiding conditions, and control the water temperature at 25 °C during the disinfection process.

4. A disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water according to claim 1, characterized in that In step 2), maintain the water temperature stability through a constant - temperature device.

5. A disinfection method for multi-parameter collaborative optimization to remove antibiotic-resistant bacteria in reclaimed water according to claim 1, characterized in that, The sewage treatment plant is a sewage treatment plant with a daily treatment capacity of 1000 - 100000 tons.

6. The disinfection method for multi-parameter collaborative optimization of removing antibiotic-resistant bacteria in reclaimed water according to claim 1, wherein The antibiotic - resistant bacteria include at least one of clindamycin - resistant bacteria, sulfonamide - resistant bacteria, ciprofloxacin - resistant bacteria, and tetracycline - resistant bacteria.

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