A method for removing bacterial biofilm using enhanced advanced oxidation technology

Abstract

This invention provides a method for removing bacterial biofilm using enhanced advanced oxidation technology. The method uses persulfate or permonsulfate as the advanced oxidation system, Fe(II) as the activator, and epigallocatechin gallate as the enhancer. The advanced oxidation system, activator, and enhancer react in a container containing bacterial biofilm to remove it. This method achieves good biofilm removal while reducing the amount of chemical reagents added to the system, eliminating the need for additional post-treatment methods. Therefore, this technology can be applied not only to wastewater treatment systems but also to water supply networks and secondary water supply equipment.

Description

Technical Field

[0001] This invention belongs to the field of biofilm removal technology and relates to a method for removing bacterial biofilms using enhanced advanced oxidation technology. Background Technology

[0002] Biofilms are the most common form of bacteria in the natural environment. Because bacteria within a biofilm are surrounded by their own secreted extracellular polymers, it becomes much more difficult for substances from the external environment to come into contact with them. This makes it difficult for bacteria within a biofilm to be completely inactivated by sterilizing agents, thus giving biofilms a very strong survival ability. Due to the ubiquitous presence of biofilms in the environment, their formation in food processing equipment, medical devices, and water supply networks has led to various food safety incidents, patient infections, and disease transmission events, posing a significant threat to human health. Shigella flexneri, a pathogen that can be transmitted through water and food, has caused widespread bacterial dysentery infections and has a strong ability to form biofilms. Furthermore, Shigella flexneri exhibits multidrug resistance, with only a few antibiotics capable of inactivating it. If Shigella flexneri forms a biofilm in a water supply system, it will cause enormous harm. Common methods for controlling and removing biofilms include physical removal (mechanical wiping and scraping) and chemical removal (using antibiotics and bactericides, etc.), but these methods all have drawbacks such as low efficiency and incomplete removal of biofilms. Therefore, there is a need to find an effective method for controlling and removing Shigella flexneri biofilm.

[0003] Advanced oxidation technologies (AOs) have attracted attention as an emerging pollutant control technology. By activating the free radicals generated by oxidants in the system through various methods, AOs can efficiently and thoroughly remove organic matter and also have good inactivation capabilities against free bacteria in water. Clearly, AOs show great promise in the field of bacterial inactivation in water; however, research on the removal of mature biofilms using AOs is limited. Among the many methods for generating free radicals, common activation methods such as light, sound, electricity, and magnetism suffer from high energy consumption, complex operation, and the need for additional equipment. Methods using transition metal ions (including ferrous and ferric ions) as activators to generate free radicals in the system have received widespread attention. Although ferrous ions are inexpensive and readily available, large amounts can easily generate large amounts of iron sludge when added, increasing secondary treatment costs. Furthermore, the poor pH adaptability of ferrous ions limits the application conditions of ferrous-activated AO systems. Since biofilm removal requires more efficient methods, improving the activation efficiency of ferrous ions and expanding the application conditions of ferrous-activated AO systems are key issues that need to be addressed in applying ferrous-activated AO technology to the field of biofilm removal.

[0004] Chinese patent document CN 116495867 A, published on July 28, 2023, utilizes pyrite to enhance the activation of an advanced oxidation system with iron ions and applies it to remove pollutants from secondary effluent. Chinese patent document CN 116573746 A, published on August 11, 2023, improves the efficiency of the advanced oxidation system by adding fluoride ions or utilizing fluoride ions present in wastewater. However, these methods still have problems. Both methods require additional means to remove the added substances, complicating the water treatment process and increasing costs. Furthermore, neither method is applicable to water supply networks, as they cannot remove biofilms formed in these networks. Therefore, a method that better applies advanced oxidation technology to remove biofilms in water supply networks is still needed. Summary of the Invention

[0005] The purpose of this invention is to solve the problems in the prior art and provide a method for removing bacterial biofilm using enhanced advanced oxidation technology. This method achieves good biofilm removal while reducing the amount of chemical reagents added to the system and eliminating the need for additional post-treatment methods. This technology can be applied not only to wastewater treatment systems but also to water supply network systems.

[0006] This invention is achieved through the following technical solution:

[0007] A method for removing bacterial biofilm using enhanced advanced oxidation technology involves using persulfate or permonsulfate as the advanced oxidation system, Fe(II) as the activator, and epigallocatechin gallate as the enhancer. The advanced oxidation system, activator, and enhancer are reacted in a container containing bacterial biofilm to remove the bacterial biofilm.

[0008] Preferably, methods for removing bacterial biofilms using enhanced advanced oxidation technologies include:

[0009] S1, Fe(II) and epigallocatechin gallate are added to a container containing background solution and covered with bacterial biofilm to obtain Fe(II) / EGCG mixed solution;

[0010] S2, by adding persulfate or permonose to the Fe(II) / EGCG mixed solution, an enhanced advanced oxidation system is obtained;

[0011] S3 enhances the reaction of the advanced oxidation system to remove bacterial biofilm.

[0012] Furthermore, prior to S1, the process includes adding antibiotics to a container containing a background solution and covered with bacterial biofilm to treat the bacterial biofilm.

[0013] Furthermore, the concentration of antibiotics in the background solution is 25–50 mg / L.

[0014] Furthermore, in S1, the pH of the background solution is adjusted to 2–7.

[0015] Furthermore, in S1, the Fe(Ⅱ) is FeSO4.

[0016] Furthermore, in S1, the dosage of Fe(II) is 10–100 μmol / L, and the dosage of epigallocatechin gallate is 5–40 μmol / L.

[0017] Furthermore, in S2, the dosage of persulfate or permonose is 0.5–2.5 mmol / L.

[0018] Furthermore, in S3, the enhanced advanced oxidation system is reacted at room temperature for 20–60 min.

[0019] Preferably, the bacterial biofilm is a Shigella flexneri biofilm.

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

[0021] This invention utilizes the antioxidant and complexing properties of epigallocatechin gallate (EGCG) to enhance the Fe(II)-activated advanced oxidation system. On one hand, EGCG creates a reducing environment in the system, preventing Fe(II) from being easily oxidized and losing its activating effect. On the other hand, EGCG can act as a multi-base ligand to complex with Fe(II) in the system, forming a Fe(II) / Fe(III) redox cycle. This reduces ions that have been converted to Fe(III) back to Fe(II), allowing an equal amount of Fe(II) to activate more oxidants, thus reducing the amount of chemical reagents required. Simultaneously, due to the complexing effect of EGCG, iron ions in the system do not precipitate too quickly due to increased pH. Compared to traditional Fe(II)-activated advanced oxidation systems, this enhanced advanced oxidation system can function well in neutral pH water. The EGCG used in this invention is a major functional component of tea and an environmentally friendly natural organic compound. As a fortifier, only a small amount is needed to enhance the Fe(II) activation of the advanced oxidation system, without causing additional pollution to the water body due to excessive dosage. This invention only requires the addition of chemical reagents to remove biofilms, eliminating the need for additional reagent treatment or recycling, which facilitates the widespread application of this technology at the end of water supply systems.

[0022] Furthermore, this invention first treats the bacterial biofilm with antibiotics, and then uses the enhanced advanced oxidation technology of this invention to remove the bacterial biofilm. The combination of the two can improve the removal rate of bacterial biofilm. At the same time, the enhanced advanced oxidation technology can further remove antibiotics, avoiding the pollution of the environment by antibiotics. Therefore, the combination of antibiotics and enhanced advanced oxidation technology has a synergistic effect. Attached Figure Description

[0023] Figure 1 The effect of different EGCG dosages on the removal of Shigella flexneri biofilm;

[0024] Figure 2 The effect of different Fe(II) dosages on the removal of Shigella flexneri biofilm;

[0025] Figure 3 The effect of different PDS dosages on the removal of Shigella flexneri biofilm;

[0026] Figure 4 The effect of different pH values ​​on the removal of Shigella flexneri biofilm;

[0027] Figure 5 The relative content of Shigella flexneri biofilm in different systems changed over time;

[0028] Figure 6 The remaining amount of Shigella flexneri biofilm and the remaining amount of antibiotics when antibiotics and enhanced advanced oxidation technology are used in combination. Detailed Implementation

[0029] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0030] This invention uses EGCG as an enhancer for Fe(II) activated advanced oxidation systems to remove bacterial biofilms.

[0031] The present invention describes a method for removing bacterial biofilms using enhanced advanced oxidation technology, comprising the following steps:

[0032] Step 1: Adjust the pH of the background solution in the container with bacterial biofilm to 2-7;

[0033] Step 2: Fe(II) and EGCG are added sequentially to the background solution to obtain a Fe(II) / EGCG mixed solution. The dosage of Fe(II) is 10-100 μmol / L and the dosage of EGCG is 5-40 μmol / L.

[0034] Step 3: Add persulfate (PDS) to the Fe(II) / EGCG mixed solution at a concentration of 0.5–2.5 mmol / L;

[0035] Step 4: Allow the obtained enhanced advanced oxidation system to react at room temperature (25°C) for 20–60 minutes to achieve a good removal effect on biofilm.

[0036] In this invention, the enhanced advanced oxidation system mainly targets mature bacterial biofilms for removal, with Shigella flexneri biofilms being the preferred bacterial biofilm.

[0037] This invention can also combine antibiotics with enhanced advanced oxidation technology. That is, before step 1, the bacterial biofilm is treated with antibiotics for a period of time.

[0038] In this invention, the persulfate can be replaced with permonose.

[0039] Example 1

[0040] This embodiment investigated the effect of EGCG dosage on the removal of Shigella flexneri biofilm by an EGCG-enhanced Fe(II) activated PDS system. The specific steps are as follows:

[0041] (1) A container with Shigella flexneri biofilm was used as the workplace for enhancing the Fe(II) activated advanced oxidation system. A 5 mmol / L Na2SO4 solution prepared with ultrapure water was added to the reaction vessel as the background solution.

[0042] (2) The pH of the background solution was adjusted to 3 using H2SO4 solution and NaOH solution;

[0043] (3) FeSO4 and EGCG were added to the background solution in sequence. The amount of FeSO4 added was 60 μmol / L, and the amount of EGCG added was 5, 10, 15, 20 and 40 μmol / L, respectively, to obtain Fe(II) / EGCG mixed solution.

[0044] (4) Add Na2S2O8 to the Fe(II) / EGCG mixed solution at a dosage of 2.0 mmol / L;

[0045] (5) The obtained enhanced advanced oxidation system was reacted at room temperature (25℃) for 60 min. The remaining amount of Shigella flexneri biofilm was measured and the removal rate was calculated. The results are as follows: Figure 1 As shown.

[0046] The experimental results show that when the EGCG dosage in the system is 10 μmol / L, the removal rate of Shigella flexneri biofilm is the highest, reaching 67.79%, after 60 min of reaction. Therefore, 10 μmol / L was chosen as the dosage of EGCG.

[0047] Example 2

[0048] This embodiment investigated the effect of Fe(II) dosage on the removal of Shigella flexneri biofilm by an EGCG-enhanced Fe(II)-activated PDS system. The specific steps are as follows:

[0049] (1) A container with Shigella flexneri biofilm was used as the workplace for enhancing the Fe(II) activated advanced oxidation system. A 5 mmol / L Na2SO4 solution prepared with ultrapure water was added to the reaction vessel as the background solution.

[0050] (2) The pH of the background solution was adjusted to 3 using H2SO4 solution and NaOH solution;

[0051] (3) FeSO4 and EGCG were added to the background solution in sequence. The dosage of FeSO4 was 10, 20, 40, 60 and 100 μmol / L, and the dosage of EGCG was 10 μmol / L, to obtain Fe(II) / EGCG mixed solution.

[0052] (4) Add Na2S2O8 to the Fe(II) / EGCG mixed solution at a dosage of 2.0 mmol / L;

[0053] (5) The obtained enhanced advanced oxidation system was reacted at room temperature (25°C) for 60 min, and the remaining amount of Shigella flexneri biofilm was measured. The results are as follows: Figure 2 As shown.

[0054] The experimental results show that when the Fe(II) dosage in the system is 60 μmol / L, the removal rate of Shigella flexneri biofilm is the highest, reaching 64.70%, after 60 min of reaction. Therefore, 60 μmol / L was chosen as the dosage of Fe(II).

[0055] Example 3

[0056] This embodiment investigated the effect of PDS dosage on the removal of Shigella flexneri biofilm by an EGCG-enhanced Fe(II)-activated PDS system. The specific steps are as follows:

[0057] (1) A container with Shigella flexneri biofilm was used as the workplace for enhancing the Fe(II) activated advanced oxidation system. A 5 mmol / L Na2SO4 solution prepared with ultrapure water was added to the reaction vessel as the background solution.

[0058] (2) The pH of the background solution was adjusted to 3 using H2SO4 solution and NaOH solution;

[0059] (3) FeSO4 and EGCG were added to the background solution in sequence. The amount of FeSO4 added was 60 μmol / L and the amount of EGCG added was 10 μmol / L, to obtain a Fe(II) / EGCG mixed solution.

[0060] (4) Add Na2S2O8 to the Fe(II) / EGCG mixed solution at concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 mmol / L, respectively.

[0061] (5) The obtained enhanced advanced oxidation system was reacted at room temperature (25°C) for 60 min, and the remaining amount of Shigella flexneri biofilm was measured. The results are as follows: Figure 3 As shown.

[0062] The experimental results show that when the PDS dosage in the system is 1.5 mmol / L, the removal rate of Shigella flexneri biofilm is the highest, reaching 67.46%, after 60 min of reaction. Therefore, 1.5 mmol / L was chosen as the dosage of PDS.

[0063] Example 4

[0064] This embodiment investigated the effect of pH on the removal of Shigella flexneri biofilm from an EGCG-enhanced Fe(II)-activated PDS system. The specific steps are as follows:

[0065] (1) A container with Shigella flexneri biofilm was used as the workplace for enhancing the Fe(II) activated advanced oxidation system. A 5 mmol / L Na2SO4 solution prepared with ultrapure water was added to the reaction vessel as the background solution.

[0066] (2) The pH values ​​of the background solution were adjusted to 2, 3, 4, 5 and 7 using H2SO4 solution and NaOH solution, respectively;

[0067] (3) FeSO4 and EGCG were added to the background solution in sequence. The amount of FeSO4 added was 60 μmol / L and the amount of EGCG added was 10 μmol / L, to obtain a Fe(II) / EGCG mixed solution.

[0068] (4) Add Na2S2O8 to the Fe(II) / EGCG mixed solution at a dosage of 1.5 mmol / L;

[0069] (5) The obtained enhanced advanced oxidation system was reacted at room temperature (25°C) for 60 min, and the remaining amount of Shigella flexneri biofilm was measured. The results are as follows: Figure 4 As shown.

[0070] The experimental results show that as the pH of the system gradually increases, the removal rate of Shigella flexneri biofilm decreases slightly after 60 minutes of reaction. The removal rate reaches its highest level of 67.76% at pH 3, but at pH 7, the biofilm removal rate is only about 13% lower than at pH 3. Therefore, the optimal reaction pH for this enhanced system is 3, but good removal of biofilm can also be achieved under neutral conditions.

[0071] Example 5

[0072] This example compares the changes in the relative content of Shigella flexneri biofilm over time in different systems. The specific steps are as follows:

[0073] (1) A container with Shigella flexneri biofilm was used as the workplace for enhancing the Fe(II) activated advanced oxidation system. A 5 mmol / L Na2SO4 solution prepared with ultrapure water was added to the reaction vessel as the background solution.

[0074] (2) The pH of the background solution was adjusted to 7 using H2SO4 solution and NaOH solution;

[0075] (3) FeSO4 and EGCG were added sequentially to the background solution, resulting in two systems: EGCG / Fe(II) / PDS and Fe(II) / PDS, depending on the dosage. In the EGCG / Fe(II) / PDS system, EGCG was added at a dosage of 10 μmol / L and FeSO4 at a dosage of 60 μmol / L, resulting in a Fe(II) / EGCG mixed solution. In the Fe(II) / PDS system, only FeSO4 was added at a dosage of 0.8 mmol / L.

[0076] (4) Add Na2S2O8 to the two systems obtained in step (3), wherein the amount of Na2S2O8 added in the EGCG / Fe(Ⅱ) / PDS system is 1.5 mmol / L and the amount of Na2S2O8 added in the Fe(Ⅱ) / PDS system is 2.0 mmol / L;

[0077] (5) The two advanced oxidation systems obtained in step (4) were reacted at room temperature (25℃) for 60 min. The remaining amount of Shigella flexneri biofilm was measured at 0, 5, 10, 15, 20, 30, 45, and 60 min, respectively. The results are as follows: Figure 5 As shown.

[0078] The experimental results show that when the pH of the system is 7, both systems reach equilibrium after 30 minutes of reaction, and the biofilm removal rate tends to stabilize. However, after 60 minutes of reaction, the biofilm removal rate of the EGCG / Fe(II) / PDS system reaches 54.53%, which is 21.25% higher than the 33.28% of the Fe(II) / PDS system. At the same time, the dosage of Fe(II) in the EGCG / Fe(II) / PDS system is reduced by 92.5% and the dosage of PDS is reduced by 25% compared to the Fe(II) / PDS system.

[0079] In summary, compared to the traditional Fe(II) / PDS system without EGCG, the enhanced Fe(II) activated persulfate system with EGCG has a higher biofilm removal rate and requires less Fe(II) and PDS dosage, making it more suitable for widespread use in real-world environments.

[0080] Example 6

[0081] This example investigated the change in the relative content of Shigella flexneri biofilm in a system where antibiotics were used in combination with EGCG-enhanced Fe(II)-activated PDS, as a function of antibiotic concentration. The specific steps are as follows:

[0082] (1) The container with Shigella flexneri biofilm was used as the workplace for the antibiotic / advanced oxidation combined system. 1 / 2 LB liquid medium was added to the reaction container as background solution and antibiotics were added to treat the biofilm for 12 h. The antibiotic selected in this experiment was ciprofloxacin (CIP), and the dosages were 25, 50, 100, 150 and 200 mg / L.

[0083] (2) After the antibiotic treatment was completed, the culture medium in the container was removed and a 5 mmol / L Na2SO4 solution prepared with ultrapure water was added as a background solution.

[0084] (3) Adjust the pH of the background solution to 3 using H2SO4 solution and NaOH solution;

[0085] (4) Add CIP to the background solution at concentrations of 25, 50, 100, 150, and 200 mg / L.

[0086] (5) FeSO4 and EGCG were added to the background solution in sequence. The amount of EGCG added was 10 μmol / L and the amount of FeSO4 added was 60 μmol / L, to obtain a CIP / Fe(II) / EGCG mixed solution.

[0087] (6) Add Na2S2O8 to the mixed solution obtained in step (5) at a dosage of 2.0 mmol / L;

[0088] (7) The advanced oxidation system obtained in step (6) was reacted at room temperature (25°C) for 60 min. After the reaction, the remaining amount of Shigella flexneri biofilm and CIP was measured. The results are as follows: Figure 6 As shown.

[0089] In step (1) of this embodiment, antibiotics are added to treat the Shigella flexneri biofilm. The background solution in this step is the culture medium, which is removed after treatment. The purpose is to obtain an antibiotic-treated Shigella flexneri biofilm that does not contain other substances. Then, the background solution and related reactants required for the advanced oxidation reaction are added. At this time, the solution does not contain antibiotics, so antibiotics are added again to simulate the original antibiotic concentration, allowing the advanced oxidation system to treat both the antibiotics in the solution and the antibiotic-pretreated biofilm simultaneously.

[0090] The experimental results show that adding a certain concentration of CIP can improve the removal rate of Shigella flexneri biofilm. Simultaneously, the added CIP can be degraded and removed by the EGCG-enhanced Fe(II)-activated PDS system. When the CIP dosage is only 25 mg / L, the biofilm removal rate reaches 77.68%, higher than the highest biofilm removal rate (67.76%) of the EGCG / Fe(II) / PDS system, at which point the CIP removal rate is 93.74%. The biofilm removal rate reaches its highest point (80.81%) when the CIP dosage is 50 mg / L, at which point the CIP removal rate is 86.07%. However, when the CIP dosage is greater than or equal to 100 mg / L, the biofilm removal rate decreases (not exceeding 65%), and the CIP removal rate also decreases slightly. This indicates that when the CIP dosage is too high, the free radicals generated by the advanced oxidation system will be consumed by the CIP, only ensuring removal to a certain extent and not further acting on the biofilm. Considering the biofilm removal rate and the content of residual antibiotics in the system after the reaction, when antibiotics are used in combination with the EGCG-enhanced Fe(II) activated PDS system, the dosage of antibiotics should not exceed 50 mg / L, i.e., preferably 25-50 mg / L.

Claims

1. A method for removing bacterial biofilm using enhanced advanced oxidation technology, characterized in that, The application relates to a method for removing bacterial biofilm, which comprises the following steps: S1, adding antibiotics into a container containing a background solution and attached with bacterial biofilm, wherein the concentration of the antibiotics in the background solution is 25-50 mg / L, so as to treat the bacterial biofilm; then Fe (II) and epigallocatechin gallate are added, so as to obtain a Fe (II) / EGCG mixed solution; S2, adding peroxysulphate or persulphate into the Fe (II) / EGCG mixed solution, so as to obtain a reinforced advanced oxidation system; S3, making the reinforced advanced oxidation system react, so as to remove the bacterial biofilm.

2. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, In S1, the pH value of the background solution is adjusted to 2-7.

3. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, In S1, the Fe (II) is FeSO4.

4. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, In S1, the adding amount of Fe (II) is 10-100 micromol / L, and the adding amount of epigallocatechin gallate is 5-40 micromol / L.

5. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, In S2, the adding amount of peroxysulphate or persulphate is 0.5-2.5 mmol / L.

6. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, In S3, the reinforced advanced oxidation system is made to react at normal temperature for 20-60 min.

7. The method of removing bacterial biofilm using enhanced advanced oxidation technology of claim 1, wherein, The bacterial biofilm is Shigella flexneri biofilm.

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