Method for layered extraction of activated sludge flora signal molecules AHLs
By monitoring the detection time of β-galactosidase in activated sludge samples, the separation time of AHLs in the LB-EPS layer and TB-EPS layer of microbial cells was determined, and the LB-EPS layer, TB-EPS layer and intracellular AHLs were extracted respectively, which solved the problems of cell rupture and AHLs contamination in traditional methods, and achieved efficient stratification extraction of AHLs.
Patent Information
- Application Number
- CN202411530390.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-03-07
AI Technical Summary
When extracting AHLs signal molecules in activated sludge, traditional methods easily destroy the cell structure, leading to leakage of intracellular substances, making it difficult to isolate AHLs in microbial cells, loosely bound extracellular polymers (LB-EPS) and tightly bound extracellular polymers (TB-EPS).
By monitoring the detection time of β-galactosidase in the activated sludge sample, the separation time of AHLs in the LB-EPS layer and TB-EPS layer of microbial cells was determined, and the AHLs in the LB-EPS layer and TB-EPS layer were extracted respectively, and the AHLs in the cell were extracted after the second separation time.
It effectively avoids the rupture of microbial cells during the extraction of extracellular AHLs of microbial cells, solves the problem of AHLs signaling molecule contamination, and realizes the hierarchical separation of extracellular and intracellular AHLs of microbial cells.
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Figure CN119569683A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a method for extracting AHLs, a signal molecule of activated sludge flora, in layers. Background Art
[0002] Activated sludge is one of the most widely used biological treatment systems in wastewater treatment plants. The core of the system is the activity of the microbial community in the sludge. The microbial cells in activated sludge contain a large number of signaling molecules, such as N-acyl homoserine lactones (AHLs), which are important components of bacterial quorum sensing and can regulate intercellular communication and microbial metabolic activities. AHLs signaling molecules exist inside microbial cells, in loosely bound extracellular polymeric substances (LB-EPS) and tightly bound extracellular polymeric substances (TB-EPS) outside microbial cells.
[0003] However, there are many problems with traditional methods for extracting AHLs signal molecules from extracellular polymers, such as high-temperature hydrothermal method and ultrasonic method. During the extraction process, the cell structure is easily destroyed, resulting in leakage of intracellular substances and mixing of intracellular and extracellular AHLs. It is difficult to separate the AHLs inside microbial cells, loosely bound extracellular polymers (LB-EPS) outside microbial cells, and tightly bound extracellular polymers (TB-EPS) outside microbial cells, and thus it is difficult to determine the types and contents of AHLs inside microbial cells, loosely bound extracellular polymers (LB-EPS) outside microbial cells, and tightly bound extracellular polymers (TB-EPS) outside microbial cells. Summary of the invention
[0004] In view of this, the purpose of the present application is to propose a method for layered extraction of activated sludge bacterial signal molecules AHLs to solve the problem of difficulty in separating AHLs inside microbial cells, loosely bound extracellular polymers (LB-EPS) outside microbial cells, and tightly bound extracellular polymers (TB-EPS).
[0005] Based on the above purpose, the present application provides a method for stratified extraction of activated sludge bacterial signal molecules AHLs, comprising the following steps:
[0006] S1: obtaining an activated sludge sample, wherein the activated sludge sample includes a plurality of microbial cells;
[0007] S2: according to the detection time of β-galactosidase in the activated sludge sample, successively determining the first detachment time and the second detachment time of AHLs detaching from the microbial cells in the LB-EPS layer and the TB-EPS layer of the microbial cells;
[0008] S3: extracting AHLs in the LB-EPS layer and the TB-EPS layer at the first detachment time and the second detachment time, respectively, and extracting intracellular AHLs after the second detachment time.
[0009] Optionally, a method for extracting activated sludge bacterial signal molecules AHLs in layers, the S2 step comprises:
[0010] The activated sludge sample is heated once, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the first separation time of AHLs in the microbial cell LB-EPS layer;
[0011] The activated sludge sample after the AHLs in the LB-EPS layer are separated is heated for a second time, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of the maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the second separation time of the TB-EPS layer;
[0012] Among them, the secondary heating temperature is higher than the primary heating temperature.
[0013] Optionally, the detection of the β-galactosidase is based on a color reaction between the supernatant of the activated sludge sample and an ONPG solution.
[0014] Optionally, the color development of the activated sludge sample supernatant and ONPG solution includes: adding the activated sludge sample to a modified PBS buffer to dilute and fully shake, centrifuging, collecting the supernatant to react with the ONPG solution, and measuring the absorbance of the solution after color development at an ultraviolet light wavelength of 405nm.
[0015] Optionally, the modified PBS buffer comprises PBS buffer, β-mercaptoethanol and magnesium sulfate.
[0016] Optionally, the temperature of the first heating is 60° C., and the first separation time is 10 minutes.
[0017] Optionally, the secondary heating temperature is 70° C., and the second separation time is 30 minutes.
[0018] Optionally, the extracting of intracellular AHLs includes: ultrasonically disrupting the microbial cells in the activated sludge sample, and determining the cell disruption by monitoring the color development of β-galactosidase in the activated sludge sample; after the microbial cells are completely disrupted, extracting intracellular AHLs from the activated sludge sample.
[0019] Optionally, the extracted LB-EPS layer, TB-EPS layer and intracellular AHLs are purified separately, different types of AHLs are separated, and the concentrations of different types of AHLs are analyzed.
[0020] Optionally, obtaining the activated sludge sample includes: centrifuging the activated sludge to obtain an activated sludge precipitate, washing the activated sludge precipitate, and placing it in a PBS buffer to obtain the activated sludge sample.
[0021] From the above, it can be seen that since the β-galactosidase in the microbial cells cannot pass through the intact cell structure, the integrity of the microbial cells in the activated sludge sample can be judged by judging the detection and activity of β-galactosidase in the activated sludge sample. The moment when β-galactosidase is detected in the activated sludge sample is the moment when the microbial cells in the activated sludge sample begin to rupture. At this moment, the extracellular AHLs and intracellular AHLs of the microbial cells begin to mix, which is not conducive to separation. Therefore, before β-galactosidase is detected in the activated sludge sample, the AHLs in the extracellular LB-EPS layer and the TB-EPS layer of the microbial cells are successively separated. Ls are detached from the microbial cells, and the detached AHLs are extracted from the activated sludge samples respectively, and then the microbial cells are broken, and finally the intracellular AHLs are extracted; in summary, the present application uses extracellular β-galactosidase activity as an indicator of cell structural integrity to avoid microbial cell rupture during the extraction of extracellular AHLs of microbial cells, which affects the extraction of AHLs in the extracellular EPS layer, thereby facilitating the hierarchical separation of loosely bound extracellular polymers (LB-EPS) outside of microbial cells, tightly bound extracellular polymers (TB-EPS) and AHLs inside microbial cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0023] Figure 1 This is the technical process for the hierarchical extraction of AHLs from activated sludge microbial cells in the examples of this application;
[0024] Figure 2 These are the concentrations of the LB-EPS layer, TB-EPS layer and intracellular AHLs in the examples of this application. DETAILED DESCRIPTION
[0025] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.
[0026] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should be the usual meanings understood by people with ordinary skills in the field to which the present application belongs. The "first", "second" and similar words used in the embodiments of the present application do not represent any order, quantity or importance, but are only used to distinguish different components. "Including" or "comprising" and similar words mean that the elements or objects appearing in front of the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.
[0027] Based on the background, activated sludge is one of the most widely used biological treatment systems in wastewater treatment plants, and its core lies in the activity of microbial communities in sludge. Microbial cells in activated sludge contain a large number of signaling molecules, such as N-acyl homoserine lactones (AHLs), which are important components of bacterial quorum sensing and can regulate intercellular communication and microbial metabolic activities. AHLs signaling molecules exist inside microbial cells, in loosely bound extracellular polymers (LB-EPS) and tightly bound extracellular polymers (TB-EPS) outside microbial cells.
[0028] However, there are many problems with traditional methods for extracting AHLs signal molecules from extracellular polymers, such as high-temperature hydrothermal method and ultrasonic method. During the extraction process, the cell structure is easily destroyed, resulting in leakage of intracellular substances and mixing of intracellular and extracellular AHLs. It is difficult to separate the AHLs in loosely bound extracellular polymers (LB-EPS) and tightly bound extracellular polymers (TB-EPS) inside and outside microbial cells, respectively, and thus it is difficult to determine the types and contents of AHLs in loosely bound extracellular polymers (LB-EPS) and tightly bound extracellular polymers (TB-EPS) inside and outside microbial cells, respectively.
[0029] The following is combined with Figure 1-2 The embodiments of the present application will be described in detail.
[0030] A method for extracting activated sludge bacterial signal molecules AHLs by stratification, comprising the following steps:
[0031] S1: obtaining an activated sludge sample, wherein the activated sludge sample includes a plurality of microbial cells;
[0032] S2: according to the detection time of β-galactosidase in the activated sludge sample, successively determining the first detachment time and the second detachment time of AHLs detaching from the microbial cells in the LB-EPS layer and the TB-EPS layer of the microbial cells;
[0033] S3: extracting AHLs in the LB-EPS layer and the TB-EPS layer at the first detachment time and the second detachment time, respectively, and extracting intracellular AHLs after the second detachment time.
[0034] In this embodiment, since the β-galactosidase in the microbial cells cannot pass through the intact cell structure, the integrity of the microbial cells in the activated sludge sample can be judged by judging the detection and activity of β-galactosidase in the activated sludge sample. The moment when β-galactosidase is detected in the activated sludge sample is the moment when the microbial cells in the activated sludge sample begin to rupture, and the extracellular AHLs and intracellular AHLs of the microbial cells begin to mix, which is not conducive to separation. Therefore, before β-galactosidase is detected in the activated sludge sample, the AHLs in the extracellular LB-EPS layer and the TB-EPS layer of the microbial cells are successively separated. Ls are detached from the microbial cells, and the detached AHLs are extracted from the activated sludge samples respectively, and then the microbial cells are broken, and finally the intracellular AHLs are extracted; in summary, the present application uses extracellular β-galactosidase activity as an indicator of cell structural integrity to avoid microbial cell rupture during the extraction of extracellular AHLs of microbial cells, which affects the extraction of AHLs in the extracellular EPS layer, thereby facilitating the sequential layered separation of loosely bound extracellular polymers (LB-EPS) outside of microbial cells, tightly bound extracellular polymers (TB-EPS) and AHLs inside microbial cells.
[0035] In some embodiments, obtaining the activated sludge sample includes: centrifuging the activated sludge to obtain an activated sludge precipitate, washing the activated sludge precipitate, and placing it in a PBS buffer to obtain the activated sludge sample.
[0036] Specifically, 50 mL of activated sludge (sampled from the sludge in the secondary sedimentation tank of a municipal sewage treatment plant) was placed in a centrifuge and centrifuged at 4000 rpm / min for 5 min. The supernatant was removed to obtain an activated sludge precipitate. The activated sludge precipitate was repeatedly washed three times with 50 mL of PBS buffer (containing a phosphate buffer at pH 7.4). The washed activated sludge precipitate was resuspended in 50 mL of PBS buffer (containing a phosphate buffer at pH 7.4), and an activated sludge sample was obtained after the sludge was completely dispersed.
[0037] In this embodiment, the activated sludge sample is obtained by centrifuging and washing the activated sludge to ensure that the activated sludge sample is clean and free of impurities. The PBS buffer solution can provide a suitable pH environment for the microbial cells, avoid cell rupture, and provide a stable growth environment for the cells.
[0038] In some embodiments, the β-galactosidase is detected based on a color reaction between the activated sludge sample supernatant and ONPG solution.
[0039] In addition, the color development of the activated sludge sample supernatant and the ONPG solution includes: adding the activated sludge sample to a modified PBS buffer for dilution and sufficient shaking, centrifuging, collecting the supernatant for reaction with the ONPG solution, and measuring the absorbance of the solution after color development at a wavelength of 405 nm ultraviolet light.
[0040] Specifically, a modified PBS buffer, an o-nitrophenyl β-D-galactopyranoside (ONPG) solution and a Na2CO3 solution were prepared; the modified PBS buffer was prepared by mixing 50 mL of PBS buffer, 0.1955 g of β-mercaptoethanol and 0.006 g of MgSO4; the o-nitrophenyl β-D-galactopyranoside (ONPG) solution was prepared by mixing 0.1 g of ONPG and 25 mL of deionized water; the Na2CO3 solution was prepared by mixing 10.6 g of Na2CO3 and 100 mL of deionized water;
[0041] Take 50 μL of activated sludge sample in a 1.5 mL centrifuge tube, dilute it with 200 μL of modified PBS buffer, and shake it thoroughly for 2 minutes. Centrifuge it at 10,000 rpm / min for 5 minutes to obtain the supernatant. Pipette 50 μL of the supernatant and add it to a 96-well cell culture plate, then add 110 μL of modified PBS buffer, and incubate it at 37°C for 5 minutes. Then, add 50 μL of ONPG solution as the β-galactosidase reactant, incubate it at 37°C for 6 hours until the solution turns bright yellow, then add 90 μL of Na2CO3 solution to terminate the reaction, and finally use a microplate reader to measure the absorbance (OD405nm) at a wavelength of 405 nm.
[0042] In this embodiment, the ONPG solution reacts sensitively with the β-galactosidase in the supernatant of the activated sludge sample, and is suitable for on-site monitoring and real-time monitoring. The detection results of the ONPG solution and β-galactosidase colorimetric test are very intuitive, and the activity of the β-galactosidase can also be judged by observing the color change of the reaction solution. When the reaction solution turns yellow, it indicates that the β-galactosidase exists and is active; when the reaction solution remains colorless, it indicates that the β-galactosidase does not exist or has low activity; in addition, in this embodiment, a modified PBC buffer is also used to dilute the sludge active sample, mainly because the mercaptoethanol in the modified PBC buffer is a reducing agent to prevent other substances from oxidative damage to the microbial cells, and MgSO4 mainly plays a lubricating role, allowing the microbial cells to be dispersed as much as possible to avoid cell agglomeration affecting the experimental results.
[0043] In some embodiments, the S2 step includes:
[0044] The activated sludge sample is heated once, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the first separation time of AHLs in the microbial cell LB-EPS layer;
[0045] The activated sludge sample after the AHLs in the LB-EPS layer are separated is heated for a second time, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of the maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the second separation time of the TB-EPS layer;
[0046] Among them, the secondary heating temperature is higher than the primary heating temperature.
[0047] In addition, the extraction of intracellular AHLs includes: ultrasonically disrupting the microbial cells in the activated sludge sample, and determining the cell disruption by monitoring the color development of β-galactosidase in the activated sludge sample; after the microbial cells are completely disrupted, intracellular AHLs are extracted from the activated sludge sample.
[0048] Specifically, the LB-EPS layer, TB-EPS and intracellular AHLs were extracted respectively:
[0049] (1) Extraction of AHLs in the LB-EPS layer: The activated sludge sample was heated once at 60°C, and the AHLs signal molecules in the LB-EPS layer were hydrothermally extracted at 60°C. During the extraction process, samples were collected at 0 min, 5 min, 10 min, 30 min, 60 min, and 120 min, and 50 μL was sampled each time to detect the activity of extracellular β-galactosidase to monitor the integrity of the cell structure;
[0050] like Figure 1 As shown in the figure, the experimental results show that the first detachment time is 10 minutes. When the hydrothermal treatment is carried out at 60℃ for 10 minutes, the activity of β-galactosidase remains basically unchanged, indicating that the cell membrane is not damaged and the cell structure is intact. At this time, the AHLs signal molecules in the LB-EPS have been fully released, and further increasing the hydrothermal time will cause cell rupture. Therefore, at a temperature of 60℃, 10 minutes is the optimal extraction time for AHLs in the LB-EPS layer. After hydrothermal treatment at 60℃ for 10 minutes, the treated activated sludge sample was placed in a centrifuge and centrifuged at 10,000 rpm / min for 5 minutes to separate the primary sludge precipitate and the primary supernatant.
[0051] (2) Extraction of AHLs in the LB-EPS layer: The above-mentioned primary sludge sediment was resuspended in 50 mL PBS buffer (containing phosphate buffer at pH 7.4) to ensure that the sludge groups were completely dispersed, and the activated sludge sample was obtained again. The activated sludge sample was then placed in a water bath and heated again to extract the AHLs signal molecules in the TB-EPS layer. The temperature of the second heating was 70°C. During the extraction process, samples were collected at 0 min, 5 min, 10 min, 30 min, 60 min and 120 min of heating, and 50 μL of the sample was used to detect the activity of extracellular β-galactosidase to monitor the integrity of the cell structure.
[0052] like Figure 1 As shown, the experimental results show that the second detachment time is 30 minutes. At 30 minutes, the extracellular β-galactosidase activity did not change significantly, indicating that the cell structure remained intact, no intracellular material leakage occurred, and the AHLs concentration was the highest. The extracellular β-galactosidase activity increased significantly with the continued increase in hydrothermal time. Therefore, 30 minutes is the optimal heating time for the extraction of signal molecules AHLs in the TB-EPS layer. After the heating, the activated sludge sample was placed in a centrifuge and centrifuged at 10,000 rpm / min for 5 minutes to separate the secondary sludge precipitate and the secondary supernatant.
[0053] (3) Extraction of intracellular AHLs: The secondary sludge precipitate was resuspended in 50 mL of PBS buffer (containing phosphate buffer at pH 7.4) and fully dispersed to obtain the activated sludge sample. The activated sludge sample was placed in an ultrasonic crusher for treatment at an ultrasonic frequency of 40 kHz. Samples were collected at 0 min, 5 min, 10 min, 30 min, 60 min and 120 min of ultrasound. 50 μL of the sample was taken to detect the activity of extracellular β-galactosidase to monitor the damage of the cell structure.
[0054] like Figure 1As shown in the figure, the experimental results show that the extracellular β-galactosidase activity reaches a peak value when the ultrasonic treatment is 60 minutes, indicating that the cell structure has been completely broken and the intracellular signal molecules are fully released. Therefore, 60 minutes is selected as the optimal ultrasonic treatment time for the extraction of intracellular signal molecules. After ultrasonic treatment, the activated sludge sample is placed in a centrifuge again and centrifuged at 10,000 rpm / min for 5 minutes to separate three activated sludges and three supernatants.
[0055] In this embodiment, the extracellular β-galactosidase activity is used as an indicator of the integrity of the cell structure, combined with hydrothermal treatment and ultrasonic disruption technology, to gradually extract loosely bound extracellular polymers (LB-EPS), tightly bound extracellular polymers (TB-EPS) and intracellular signaling molecules AHLs in the sludge, thereby ensuring the hierarchical separation and precise extraction of the signaling molecules AHLs. In the process of extracting loosely bound extracellular polymers (LB-EPS), the first heating temperature of 60°C and the first detachment time of 10min were determined to be the optimal extraction conditions. In the process of extracting tightly bound extracellular polymers (TB-EPS), the second heating temperature of 70°C and the second detachment time of 30min were determined to be the optimal extraction conditions. In the process of extracting intracellular cells, the ultrasonic frequency of 40kHz and the ultrasonic time of 60min were determined to be the optimal extraction conditions.
[0056] In some embodiments, the extracted LB-EPS layer, TB-EPS layer and intracellular AHLs are purified separately, different types of AHLs are separated, and the concentrations of different types of AHLs are analyzed.
[0057] Specifically, the purification includes: using liquid phase extraction to mix 50 mL of the first supernatant, 50 mL of the second supernatant, and 50 mL of the third supernatant with 50 mL of ethyl acetate, respectively, concentrating the organic phases by rotary evaporator, and drying the ethyl acetate by nitrogen blowing to obtain the dried LB-EPS layer, TB-EPS layer, and intracellular AHLs, respectively. Finally, the dried LB-EPS layer, TB-EPS layer, and intracellular AHLs were dissolved in 1 mL of acetonitrile, respectively, and stored at -20°C for later use;
[0058] The LB-EPS layer, TB-EPS layer and intracellular AHLs signal molecule samples dissolved in acetonitrile were respectively sent to liquid chromatography-mass spectrometry (LC-MS) for quantitative analysis. The analysis experiment used a Shim-pack GIST C18 column, and the mobile phase was a mixture of 5% (v / v) buffer, 55% (v / v) acetonitrile, and 40% (v / v) isopropanol. The analysis was performed using a gradient elution program. Each sample was calibrated with a gradient concentration of AHLs standards, and the concentrations of different AHLs signal molecules, including C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL and 3OC12-HSL, were determined by MRM mode (multiple reaction monitoring). The concentrations of each signal molecule were referenced to Figure 2 shown.
[0059] pass Figure 1 and Figure 2 The analysis results show that the layered extraction method adopted in the present invention can effectively and accurately separate the signal molecules of each layer of activated sludge microbial cells, avoid the rupture of microbial cells during the extraction of extracellular AHLs of microbial cells, affect the extraction of AHLs in the extracellular EPS layer, and solve the problem of signal molecule contamination caused by leakage of intracellular substances, thereby facilitating the layered separation of loosely bound extracellular polymers (LB-EPS) outside microbial cells, tightly bound extracellular polymers (TB-EPS) and AHLs inside microbial cells in sequence.
[0060] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.
[0061] In addition, to simplify the description and discussion, and in order not to make the embodiments of the present application difficult to understand, the known power supply / ground connection with the integrated circuit (IC) chip and other components may or may not be shown in the provided drawings. In addition, the device can be shown in the form of a block diagram to avoid making the embodiments of the present application difficult to understand, and this also takes into account the fact that the details of the implementation of these block diagram devices are highly dependent on the platform to be implemented in the embodiments of the present application (that is, these details should be fully within the scope of understanding of those skilled in the art). In the case of elaborating specific details (e.g., circuits) to describe exemplary embodiments of the present application, it is obvious to those skilled in the art that the embodiments of the present application can be implemented without these specific details or when these specific details are changed. Therefore, these descriptions should be considered to be illustrative rather than restrictive.
[0062] Although the present application has been described in conjunction with specific embodiments of the present application, many replacements, modifications and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. For example, other memory architectures (e.g., dynamic RAM (DRAM)) may use the embodiments discussed.
[0063] The embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present application should be included in the protection scope of the present application.
Claims
1. A method for extracting activated sludge bacterial signal molecules AHLs by stratification, characterized in that: The following steps are involved: S1: obtaining an activated sludge sample, wherein the activated sludge sample includes a plurality of microbial cells; S2: according to the detection time of β-galactosidase in the activated sludge sample, successively determining the first detachment time and the second detachment time of AHLs detaching from the microbial cells in the LB-EPS layer and the TB-EPS layer of the microbial cells; S3: extracting AHLs in the LB-EPS layer and the TB-EPS layer at the first detachment time and the second detachment time, respectively, and extracting intracellular AHLs after the second detachment time.
2. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 1, characterized in that: The S2 step includes: The activated sludge sample is heated once, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the first separation time of AHLs in the microbial cell LB-EPS layer; The activated sludge sample after the AHLs in the LB-EPS layer are separated is heated for a second time, and the detection time of the extracellular β-galactosidase and the AHLs concentration therein are monitored, and the time of the maximum AHLs concentration before the initial detection time of the extracellular β-galactosidase is determined as the second separation time of the TB-EPS layer; Among them, the secondary heating temperature is higher than the primary heating temperature.
3. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 2, characterized in that: The detection basis of the β-galactosidase is that the supernatant of the activated sludge sample and the ONPG solution show a color reaction.
4. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 3, characterized in that: The color development of the activated sludge sample supernatant and the ONPG solution includes: adding the activated sludge sample to a modified PBS buffer for dilution and sufficient shaking, centrifuging, collecting the supernatant for reaction with the ONPG solution, and measuring the absorbance of the solution after color development at a wavelength of 405 nm ultraviolet light.
5. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 4, characterized in that: The modified PBS buffer comprises PBS buffer, β-mercaptoethanol and magnesium sulfate.
6. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 2, characterized in that: The primary heating temperature is 60° C., and the first separation time is 10 minutes.
7. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 2, characterized in that: The secondary heating temperature is 70° C., and the second separation time is 30 minutes.
8. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 1, characterized in that: The extraction of intracellular AHLs includes: ultrasonically disrupting the microbial cells in the activated sludge sample, and judging the cell disruption by monitoring the color development of β-galactosidase in the activated sludge sample; after the microbial cells are completely disrupted, intracellular AHLs are extracted from the activated sludge sample.
9. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 1, characterized in that: The extracted AHLs from the LB-EPS layer, TB-EPS layer and cells were purified, and different types of AHLs were separated and their concentrations were analyzed.
10. The method for extracting activated sludge bacterial signal molecules AHLs by stratification according to claim 1, characterized in that: The method of obtaining the activated sludge sample comprises: centrifuging the activated sludge to obtain an activated sludge precipitate, washing the activated sludge precipitate, and placing the activated sludge precipitate in a PBS buffer to obtain the activated sludge sample.