Method for extracting different extracellular polymeric components from activated sludge

Abstract

The invention belongs to the technical field of biological sewage treatment, and particularly relates to a method for extracting different extracellular polymeric components from activated sludge. The method focuses on layered extraction of various components of different extracellular polymeric substances, that is, S-EPS in activated sludge is extracted through a centrifugal method, then LB-EPS is extracted through an ultrasonic method and a high-speed centrifugal method, ionic TB-EPS is extracted through an EDTA method and the high-speed centrifugal method, and finally hydrophobic TB-EPS is extracted through an ethanol method and the high-speed centrifugal method. The methods are sequentially combined to complete the extraction of different components of the extracellular polymeric substance. According to the method for extracting different extracellular polymeric substance components from the activated sludge, provided by the invention, the influence of the extraction method on cell lysis is small as far as possible while high EPS extraction efficiency and high purity are ensured, and accurate quantification and high feasibility of the extracellular polymeric substance extraction method are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage biological treatment, and in particular relates to a method for extracting different extracellular polymer components from activated sludge. Background Art

[0002] In recent years, with the increasing application of biotechnology in the field of sewage treatment technology, it has become the mainstream process technology in sewage treatment plants and has been widely popularized. In the field of biological sewage treatment technology, activated sludge exists in the form of microbial aggregates, providing a stable and suitable ecosystem for microbial communities, which not only helps to separate mud and water and ensure the good quality of effluent, but also maintains the ecological activity of microorganisms, so that different pollutants can achieve good removal effects. Among them, EPS (Extracellular Polymeric Substances) is an important component of activated sludge flocs, widely present in microbial aggregates, and is a high molecular polymer secreted by microorganisms under certain environmental conditions and distributed on the cell surface or outside the cell, including proteins, polysaccharides, humus, deoxyribonucleic acid (DNA), lipids and other macromolecules. At the same time, EPS, as a macromolecular substance with a three-dimensional structure, interacts through electrostatics, hydrophobicity, energy hydrogen bonds, ion bridging connections, etc., to form a mesh-like three-dimensional structure, covering the entire cell surface, thereby promoting cell adhesion, maintaining the stability of microbial aggregates, reducing the damage of toxic substances to cells, and when nutrients are lacking, it can also supplement carbon sources and energy for microorganisms.

[0003] According to the current research and analysis on extracellular polymers (EPS), extracellular polymers are usually divided into soluble extracellular polymers (S-EPS) and bound extracellular polymers (B-EPS). Among them, the binding force between S-EPS and cells is relatively weak, and it is located in the outermost layer of EPS and may even dissolve in the liquid environment; while B-EPS is tightly bound to the cell surface and is located in the innermost layer of EPS. Among them, B-EPS is divided into two layers. The inner layer adheres tightly and stably to the cell surface, which is called tightly bound extracellular polymers (TB-EPS); the outer layer has a loose and dispersed structure, which is a layer of mucus without obvious boundaries, called loosely bound extracellular polymers (LB-EPS). Due to the complex composition and different morphological structures of EPS, the functional role of EPS in the study of sewage biological treatment is still unclear, and the effects of different forms of EPS on microorganisms and sewage treatment have not yet been determined. However, the current extraction of extracellular polymers has problems such as too single method resulting in too low extraction yield, single type of extracted components, and certain effects on cell lysis. Therefore, an accurate and reliable method for EPS extraction is needed, which can extract EPS as completely as possible while reducing the impact of the extraction process on the degree of cell lysis. Summary of the invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a method for extracting different extracellular polymer components from activated sludge. The method can achieve a good extraction yield for various extracellular polymers of sludge, while ensuring that the influence on cell lysis is minimized during the extraction process, thereby ensuring the accuracy and reliability of the extraction, thereby solving the problem that the traditional sludge extracellular polymer extraction method is too single, resulting in too low extraction yield, a single type of extracted components, and a certain influence on cell lysis.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention discloses a method for extracting different extracellular polymer components from activated sludge, the method comprising the following steps:

[0007] S1. Pretreatment: After obtaining activated sludge from the biochemical pool of the urban domestic sewage treatment plant, wash it with buffer 2-4 times and adjust its MLVSS to 5-8g / L;

[0008] S2, treating the pretreated sludge by centrifugation, and then separating to obtain S-EPS supernatant and first sludge sediment;

[0009] S3, after taking out the S-EPS supernatant of S2, adding an equal volume of buffer to the first sludge sediment to restore the original volume, and then sequentially treating by ultrasonic method and high-speed centrifugation method to separate and obtain LB-EPS supernatant and second sludge sediment;

[0010] S4, after taking out the LB-EPS supernatant of S3, adding an equal volume of buffer to the second sludge sediment to restore the original volume, then adding EDTA solution for treatment, and then performing high-speed centrifugation to separate the ionic TB-EPS supernatant and the third sludge sediment;

[0011] S5. After taking out the ionic TB-EPS supernatant of S4, it was treated by ethanol method and high-speed centrifugation method in sequence to separate and obtain the hydrophobic TB-EPS supernatant.

[0012] As a macromolecular substance with a three-dimensional structure, extracellular polymers are mainly formed by interactions such as electrostatics, hydrophobicity, energy hydrogen bonds, and ion bridging connections to form such a three-dimensional framework. To extract extracellular polymers, it is necessary to break the functional bonds of these connections to separate them from the cell surface, thereby obtaining extracellular polymers. At present, extracellular polymers are usually divided into soluble extracellular polymers (S-EPS) and bound extracellular polymers (B-EPS). To this end, the present invention discloses a method for extracting different extracellular polymer components from activated sludge. First, soluble extracellular polymers (S-EPS) are separated. Since S-EPS is in the outermost layer of EPS, the binding force between cells is relatively weak and even dissolved in the liquid phase environment. Therefore, to separate S-EPS, only a certain degree of low-temperature speed centrifugation is required, here at 4 ° C, 5000g centrifugation for 15 minutes. Then, the bound extracellular polymers (B-EPS) are separated because they are tightly bound to the cell surface and are in the innermost layer of EPS. It is divided into two layers, and the outer structure is loose and dispersed, and it is a mucus layer without obvious boundaries, which is called loosely bound extracellular polymer (LB-EPS). The connection of this LB-EPS is relatively loose, mainly with low-energy bonds such as molecular hydrogen bonds and various forces between molecules to play a connecting role, so it is necessary to adopt physical means to provide corresponding energy to promote bond separation and fracture for this effect. Therefore, the present invention adopts physical method "ultrasound method + high-speed centrifugation method" to extract, and in order to verify the practicality and reliability of this method, several different physical methods are also selected for comparison and comparison to verify the effectiveness of this method. Finally, the inner layer of B-EPS, which is tightly and stably adhered to the cell surface, is called tightly bound extracellular polymer (TB-EPS). This layer is very tightly combined with the cell surface, and it has multiple properties such as hydrophobicity, electrostatics, ions, and general energy physics means cannot be accurately separated, and here it will be separated and extracted for its hydrophobicity and ionicity. Therefore, the present invention selectively divides TB-EPS into ionic TB-EPS and hydrophobic TB-EPS, and extracts these two types of TB-EPS separately, optimizes them from three aspects: different ion extractants, surfactants and different extraction sequences, and then determines the best extraction method and extraction steps.

[0013] Preferably, the pretreatment of S1 is as follows: first, the activated sludge is allowed to stand for 30-40 minutes, then the supernatant is poured off, and a buffer solution is added to restore the original volume, this operation is repeated 2-4 times, and then the MLVSS concentration of the activated sludge is detected, and its MLVSS is adjusted to 5-8 g / L with a buffer solution.

[0014] Preferably, the buffer in S1, S3 and S4 refers to a PBS buffer with a concentration of 0.1 mol / L, a pH of 7.2-7.4 and a NaCl concentration of 1.36 mol / L.

[0015] Preferably, the centrifugation condition of S2 is: centrifugation at 4000-5000 g for 10-15 min at 4°C.

[0016] Preferably, S3 is specifically as follows: the first sludge sediment is restored to its original volume with a buffer solution and then placed in an ultrasonic machine, and then ultrasonicated at 20-40KHZ and a power density of 0.75-1.5w / mL, each ultrasonication lasting 2-4min, for a total of 3 times, with an interval of 2min between each ultrasonication, and intermittent ultrasonication is performed, and then the ultrasonically treated activated sludge is placed at 4°C and centrifuged at 10000-15000g for 15-20min, and finally solid-liquid separation is performed through a 0.45μm filter membrane.

[0017] Preferably, S4 is specifically as follows: the prepared EDTA solution and the second sludge sediment restored to its original volume are mixed evenly in a volume ratio of 1:1, and then placed on an orbital oscillator, oscillated at 200-300 rpm / min at 4°C for 2-3 hours, and then placed at 4°C and centrifuged at 10000-15000g for 15-20 minutes, and finally solid-liquid separation is performed through a 0.45 μm filter membrane to obtain ionic TB-EPS and the third sludge sediment.

[0018] Preferably, the EDTA solution described in S4 is an EDTA solution dissolved in Tris-HCl buffer with a concentration of 0.5-2%.

[0019] Preferably, S5 is specifically as follows: adding ethanol solution to the third sludge sediment to restore it to its original volume, then placing the mixed activated sludge on a shaker for 2-3 hours at a speed of 200-250 rpm / min and a temperature of 4°C, then centrifuging the treated sludge at 10000-15000g for 15-20 minutes at 4°C, and finally performing solid-liquid separation through a 0.45 μm filter membrane to obtain a hydrophobic TB-EPS.

[0020] Preferably, the ethanol solution in S5 is an ethanol solution diluted with phosphate buffered saline (PBS buffer) to a concentration of 20-40%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention discloses a method for extracting different extracellular polymer components from activated sludge. The method focuses on performing layered extraction on various components of different extracellular polymers, that is, firstly extracting S-EPS in the activated sludge by centrifugation, then extracting LB-EPS by ultrasound and high-speed centrifugation, then extracting ionic TB-EPS by EDTA and high-speed centrifugation, and finally extracting hydrophobic TB-EPS by ethanol and high-speed centrifugation, and the above methods are sequentially combined to complete the extraction of different components of extracellular polymers.

[0023] The method for extracting different extracellular polymer components from activated sludge provided by the present invention is to maintain a stable liquid phase environment for EPS extraction, select suitable ion extractants and surfactants, detect and analyze different extraction methods, and study various combinations of extraction methods, and finally extract four different types of EPS, S-EPS, LB-EPS, hydrophobic TB-EPS, and ionic TB-EPS in the activated sludge in sequence. While ensuring high extraction efficiency and high purity of EPS, the method also minimizes the effect of the extraction method on cell lysis, ensuring that the extracellular polymer extraction method is quantitatively accurate and feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A process flow chart for extracting different extracellular polymer components from activated sludge;

[0025] Figure 2 is the content of each component of the overall EPS under different extraction methods (① is the layered extraction method, ② is the thermal extraction method, ③ is the ultrasonic method, ④ is the EDTA method, ⑤ is the CER method, ⑥ is the ethanol method, and ⑦ is the Tween 20 method);

[0026] Figure 3 is the content of each component of LB-EPS under different extraction methods;

[0027] Figure 4 The contents of various components of ionic TB-EPS extracted by different ion extractants;

[0028] Figure 5 The contents of various components of hydrophobic TB-EPS extracted by different surfactants;

[0029] Figure 6 The contents of various components of hydrophobic TB-EPS extracted in different extraction orders. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0032] In the following specific embodiments, the detection method of each indicator is:

[0033] (1) Determination of EPS protein content: The protein concentration was determined using a BCA kit. The determination principle is that the peptide bond structure in the protein molecule reacts with Cu in an alkaline environment. 2+ Complexing and Cu 2+ Reduction to Cu 1+ BCA reagent binds specifically to Cu 1+ The complex combines to form a stable purple-blue complex with a maximum light absorption value at 562nm, which is proportional to the protein concentration. The protein concentration can be determined based on the absorption value.

[0034] (2) Determination of EPS polysaccharide concentration: The anthrone-sulfuric acid colorimetric method is used to determine the polysaccharide concentration. The determination principle is that at high temperature, sugars will be dehydrated by concentrated H2SO4 to generate furfural or furfural derivatives, and then react with anthrone (C 14 H 10 The absorbance was measured at 620 nm and the polysaccharide content was calculated using the glucose standard curve.

[0035] (3) Method for determining DNA concentration in EPS: The content and purity of DNA were detected using nanodrop nucleic acid quantitative analyzer.

[0036] (4) The specific method of MLSS / MLVSS of activated sludge concentration is as follows:

[0037] ① After cleaning, place the 0.45μm fiber filter membrane (filter paper) and egg tart cup (or porcelain crucible) used for the test in a drying oven and heat at 105℃ for 40-60min to remove moisture. Then place them in a desiccator and wait for them to cool. Weigh the mass of the filter paper and porcelain crucible, and record them as M0 and M1 respectively;

[0038] ② In the reaction system where the sludge is evenly distributed, accurately take 5 mL of the sludge-water mixture sample, record it as V, and vacuum filter it with a fiber filter membrane with a pore size of 0.45 μm.

[0039] ③Put the fiber filter membrane with the sludge sample after filtering out the water into the egg tart cup, and heat it in a drying oven at 105℃ for 2h. Then take out the egg tart cup and put it in a dryer. After the temperature drops to room temperature, weigh its mass and record it as M2. (M2-M1) / V is the MLSS value (note the unit: mg / L).

[0040] ④ Place the egg tart cup in step ③ in a box-type resistance muffle furnace and heat it at 605℃ for 2h. When the temperature in the muffle furnace drops below 100℃, take out the egg tart cup with gloves and put it in a dryer. After the cup cools to room temperature, weigh the mass and record it as M3. (M2-M3-M0) / V is the MLVSS value (unit: mg / L). Set up 2-3 parallel samples for each measurement.

[0041] Example 1: Effect of different extraction methods on EPS yield of activated sludge

[0042] 1. A method for extracting different extracellular polymer components from activated sludge (layered extraction method), the process flow of which is as follows: Figure 1 As shown, the following steps are included:

[0043] (1) Activated sludge sample pretreatment: This activated sludge was taken from the residual activated sludge in the biological reactor of Zhongshan Public Wastewater Treatment Plant in Zhongshan City, Guangdong Province. First, the activated sludge was allowed to stand for 30 minutes, then the supernatant was poured off and PBS buffer (PBS buffer with a concentration of 0.1 mol / L, pH 7.2-7.4, and NaCl concentration of 1.36 mol / L) was added to restore it to its original volume. This operation was repeated twice. Then, the MLVSS index of the activated sludge was detected to determine its volatile organic matter content, and its MLVSS was adjusted to 5 g / L with PBS buffer.

[0044] (2) Layered extraction and graded extraction analysis of EPS from activated sludge:

[0045] The specific steps are as follows:

[0046] 1) Take 50 mL of the pretreated sludge and centrifuge it at 5000 g for 15 min at 4 °C in a low-temperature centrifuge to separate the S-EPS supernatant and the first sludge sediment;

[0047] 2) After taking out the S-EPS supernatant, suspend the first sludge sediment after centrifugation in step 1) in PBS buffer and restore it to the original volume of 50mL, then place the buffer of the first sludge sediment in an ultrasonic machine, and perform ultrasonic treatment at 20KHZ and 0.75w / mL power, 2 minutes each time, 3 times in total, 2 minutes between each ultrasound, and so on. Then, place the ultrasonically treated activated sludge at 4°C and centrifuge it at 10000g for 20 minutes, and finally perform solid-liquid separation through a 0.45μm filter membrane to obtain LB-EPS supernatant and the second sludge sediment;

[0048] 3) For the second sludge sediment after centrifugation in step 2), first place it in PBS buffer and restore it to its original volume of 50mL. Then, mix the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) and the second sludge sediment restored to its original volume in a volume ratio of 1:1, and then place the resulting solution on an orbital oscillator, oscillate at 200rpm / min for 3h at 4°C, and then centrifuge at 10000g for 20min at 4°C, and finally perform solid-liquid separation through a 0.45μm filter membrane to obtain ionic TB-EPS and the third sludge sediment;

[0049] 4) Add the diluted ethanol solution (dilute anhydrous ethanol with PBS buffer until the concentration reaches 40%) to the third sludge sediment in step 3) until the original volume is restored to 50 mL. Then, place the mixed activated sludge on a shaker for 2 hours at a speed of 250 rpm / min and a temperature of 4°C. Then, centrifuge the treated sludge at 15000g for 20 minutes at 4°C, and finally perform solid-liquid separation through a 0.45 μm filter membrane to separate the hydrophobic TB-EPS supernatant.

[0050] 2. Extracting EPS from activated sludge using a single extraction method

[0051] Under the same conditions (i.e., the collection and pretreatment of activated sludge samples are the same as the stratified extraction method), the thermal extraction method, ultrasonic method, EDTA method, CER method, ethanol method, and Tween 20 method are used to extract extracellular polymers and are compared with the stratified extraction method. At the same time, the commonly used thermal extraction method is used as the standard to determine the effect of other methods on improving the extraction yield of extracellular polymers, in order to illustrate the necessity of the stratified extraction method.

[0052] (1) Thermal extraction method: After the sludge is pretreated, the supernatant is discarded and 0.05% NaCl solution is added to resuspend the residual pellets to the original volume of 50 mL. The pellets are heated at 60 °C for 30 min and then centrifuged at 10,000 g for 15 min. Finally, the supernatant is filtered through a 0.45 μm membrane. The supernatant obtained after solid-liquid separation is EPS.

[0053] (2) Ultrasonic method: Pour the supernatant of the pretreated sludge, restore the original volume to 50 mL with PBS buffer, and then place it in an ultrasonic machine for ultrasonic treatment at 20KHZ and 0.75w / mL power. Ultrasonic treatment lasts for 2 minutes each time, and a total of 3 times, with an interval of 2 minutes between each ultrasound. Then, the ultrasonically treated activated sludge is placed at 4°C and centrifuged at 10,000g for 20 minutes. Finally, solid-liquid separation is performed through a 0.45μm filter membrane to obtain EPS supernatant.

[0054] (3) EDTA method: Prepare 50 mL of pretreated activated sludge, then mix the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) and activated sludge in a volume ratio of 1:1, and then place the resulting solution on an orbital oscillator and oscillate at 200 rpm / min for 3 h at 4°C. Then centrifuge at 10,000 g for 20 min at 4°C, and finally filter through a 0.45 μm filter membrane for solid-liquid separation to obtain EPS supernatant.

[0055] (4) CER method: Take 50 mL of pretreated sludge sample, add 70 g / g VSS CER, mix the resulting solution in a 500 mL conical flask, and then place it on a shaker for 3 h at a speed of 250 rpm / min and a temperature of 4°C. Then, centrifuge the resulting suspension at 10,000 g and 4°C for 20 min, and finally filter it using a 0.45 μm filter membrane. The collected supernatant is EPS.

[0056] (5) Ethanol method: First, take out 50 mL of pretreated activated sludge, pour off the supernatant, and add the diluted ethanol solution (dilute anhydrous ethanol with PBS buffer until the concentration reaches 40%) to the activated sludge sediment until the original volume is restored to 50 mL. Then, place the mixed activated sludge on a shaker for 2 hours at a speed of 250 rpm / min and a temperature of 4°C. Then, place the treated sludge at 4°C and centrifuge it at 15,000g for 20 minutes. Finally, separate the solid and liquid through a 0.45 μm filter membrane to obtain the EPS supernatant.

[0057] (6) Tween 20 method: Take 50 mL of pretreated activated sludge, discard the supernatant, and then add the surfactant Tween 20 solution (0.25% Tween 20 solution prepared with PBS buffer) until the original volume is restored, and then place it on a shaker for 1 h at a speed of 250 rpm / min and a temperature of 4°C. The resulting suspension is then centrifuged at 15000g and 4°C for 20 min, and finally filtered using a 0.45 μm filter membrane to separate the hydrophobic TB-EPS supernatant.

[0058] The layered extraction method of Example 1 was compared with these single extraction methods. The results are as follows: Figure 2 As shown, the overall content of EPS extracted by several single extraction methods is low. Taking the thermal extraction method as the standard, the total amount of EPS extracted is 6.568 mg / gVSS, while other single extraction methods (ultrasound method, EDTA method, CER method, ethanol method, Tween 20 method) are 6.5%, 228%, 144.1%, 352.3%, and 89.2% higher than the thermal extraction method, respectively. Obviously, the EDTA method and ethanol method have the best extraction effect among the single extraction methods. However, the EPS content extracted by these methods is not very high, which shows that the single extraction method cannot extract the accurate content of extracellular polymers. Compared with the thermal extraction method, the stratified extraction method of the present invention is used to extract EPS, which can make the total amount of EPS reach 79.971 mg / gVSS, an increase of 1117.5%. Obviously, compared with other single extraction methods, the stratified extraction method of the present invention can extract the content of EPS as much as possible, improve the extraction yield of EPS, and will not cause a significant impact on cell lysis.

[0059] Example 2: Effect of different extraction methods on the extraction of LB-EPS

[0060] 1. Extraction of LB-EPS by layered extraction method

[0061] LB-EPS was extracted by using steps 1) and 2) (ultrasound + high-speed centrifugation) in the layered extraction method of Example 1:

[0062] 1) Take 50 mL of the pretreated sludge and centrifuge it at 5000 g for 15 min at 4 °C in a low-temperature centrifuge to separate the S-EPS supernatant and the first sludge sediment;

[0063] 2) After taking out the S-EPS supernatant, the first sludge sediment after centrifugation in step 1) was suspended in PBS buffer and restored to the original volume of 50 mL, and then the buffer of the first sludge sediment was placed in an ultrasonic machine, and ultrasonic treatment was performed at 20KHZ and 0.75w / mL power, each ultrasonic treatment for 2 minutes, a total of 3 ultrasonic treatments, and each ultrasonic treatment was separated by 2 minutes, and intermittent ultrasonic treatment was performed. Then, the ultrasonically treated activated sludge was placed at 4°C and centrifuged at 10000g for 20 minutes, and finally solid-liquid separation was performed through a 0.45μm filter membrane to obtain LB-EPS supernatant and second sludge sediment.

[0064] 2. Use conventional method to extract LB-EPS (water bath heating + high-speed centrifugation)

[0065] The step 1) is the same as the step 1) in the layered extraction method of Example 1, and the step 2) is:

[0066] After taking out the S-EPS supernatant, the first sludge sediment after centrifugation in 1) was suspended in a buffer solution and restored to the original volume of 50 mL, and then heated in a water bath at 60°C for 30 min, and then centrifuged at 10000g for 20 min at 4°C, and finally solid-liquid separation was performed through a 0.45 μm filter membrane to obtain the LB-EPS supernatant.

[0067] The two extraction methods were analyzed and the results were as follows Figure 3 As shown, the contents of LB-EPS extracted by the combined methods of "ultrasound + high-speed centrifugation" and "water bath heating + high-speed centrifugation" were 3.019 mg / gVSS and 6.994 mg / gVSS, respectively. From the total amount of LB-EPS extracted, the extraction yield of the combined treatment of ultrasound + high-speed centrifugation was relatively high. At the same time, the ratio of eDNA to LB-EPS was only 6.07%, which also indirectly shows that the combined treatment method of "ultrasound + high-speed centrifugation" has little effect on the degree of cell damage and has a better comprehensive extraction effect.

[0068] Example 3: Effects of different extraction methods on the extraction of ionic TB-EPS

[0069] 1. Layered extraction method to extract ionic TB-EPS

[0070] LB-EPS was extracted using steps 1), 2) and 3) of the layered extraction method of Example 1:

[0071] 1) Take 50 mL of the pretreated sludge and centrifuge it at 5000 g for 15 min at 4 °C in a low-temperature centrifuge to separate the S-EPS supernatant and the first sludge sediment;

[0072] 2) After taking out the S-EPS supernatant, suspend the first sludge sediment after centrifugation in step 1) in PBS buffer and restore it to the original volume of 50mL, then place the buffer of the first sludge sediment in an ultrasonic machine, and perform ultrasonic treatment at 20KHZ and 0.75w / mL power, 2 minutes each time, 3 times in total, 2 minutes between each ultrasound, and so on. Then, place the ultrasonically treated activated sludge at 4°C and centrifuge it at 10000g for 20 minutes, and finally perform solid-liquid separation through a 0.45μm filter membrane to obtain LB-EPS supernatant and the second sludge sediment;

[0073] 3) For the second sludge sediment after centrifugation in step 2), first place it in PBS buffer and restore it to its original volume of 50mL. Then, the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) and the second sludge sediment restored to its original volume are mixed evenly in a volume ratio of 1:1, and then the resulting solution is placed on an orbital oscillator, oscillated at 200rpm / min for 3h at 4°C, and then centrifuged at 10000g for 20min at 4°C, and finally solid-liquid separation is performed through a 0.45μm filter membrane to obtain ionic TB-EPS and the third sludge sediment.

[0074] 2. Extraction of ionic TB-EPS using conventional methods

[0075] The step 1) is the same as the steps 1) and 2) in the hierarchical extraction method of Example 1, and the step 3) is:

[0076] The second sludge sediment in step 2) was mixed with PBS buffer and restored to the original volume of 50 mL. Then, 70 g / g VSS CER was added to the second sludge sediment and mixed in a 500 mL conical flask. The resulting solution was then placed on a shaker for 3 h at a speed of 250 rpm / min and a temperature of 4 ° C. The resulting suspension was then centrifuged at 10000 g and 4 ° C for 20 min, and finally filtered through a 0.45 μm filter membrane for solid-liquid separation to obtain ionic TB-EPS.

[0077] Data analysis and comparison of two different ion extractants, EDTA and CER, were performed. The results are as follows: Figure 4As shown in the figure, the contents of ionic TB-EPS extracted by EDTA and CER were 38.529 mg / gVSS and 20.227 mg / gVSS respectively. From the total amount of ionic TB-EPS extracted, the extraction yield of EDTA as an ionic extractant was the highest, especially for the extraction of polysaccharides. At the same time, the proportion of eDNA extracted by these two extractants to ionic TB-EPS was 15.67% and 24.78% respectively. At present, the degree of cell damage is generally measured by the index of eDNA / EPS. The yield of TB-EPS extracted by EDTA is not only higher, but also its eDNA / EPS ratio is still within the normal ratio range (2% to 15%). This also indirectly shows that the influence of EDTA as an ionic extractant on the degree of cell damage is relatively small after the corresponding concentration is configured, and the comprehensive extraction effect is better.

[0078] Example 4: Effects of different extraction methods on the extraction of hydrophobic TB-EPS

[0079] 1. Extraction of hydrophobic TB-EPS by layered extraction method

[0080] The hydrophobic LB-EPS was extracted using the layered extraction method of Example 1 (step 1) to step 4)), which is referred to as method 1.

[0081] 2. Effects of different surfactants and extraction sequence on the extraction yield of hydrophobic TB-EPS

[0082] Under the same conditions as in Example 4, the types of surfactants and the extraction sequence were changed to analyze and compare, and the effects of different surfactants and extraction sequences on the extraction yield of hydrophobic TB-EPS were studied.

[0083] (1) Method 2 (ionic TB-EPS first, then hydrophobic TB-EPS): Steps 1) to 3) are the same as steps 1) to 3) of the layered extraction method of Example 1, and step 4) is as follows:

[0084] To the third sludge sediment in step 3), a non-surfactant Tween 20 solution (a 0.25% Tween 20 solution prepared with PBS buffer) was added, and the resulting solution was then placed on a shaker for 1 h at a speed of 250 rpm / min and a temperature of 4°C. The resulting suspension was then centrifuged at 15,000 g and 4°C for 20 min, and finally filtered using a 0.45 μm filter membrane to separate the hydrophobic TB-EPS supernatant.

[0085] (2) Method 3 (extracting hydrophobic TB-EPS first): Steps 1) to 2) are the same as steps 1) to 2) of the layered extraction method of Example 1, and step 3) is as follows:

[0086] Add the diluted ethanol solution (dilute anhydrous ethanol with PBS buffer until the concentration reaches 40%) to the second sludge sediment in step 2) until the original volume is restored to 50 mL. Then place the mixed activated sludge on a shaker for 2 hours at a speed of 250 rpm / min and a temperature of 4°C. Then centrifuge the treated sludge at 10000g for 20 minutes at 4°C, and finally separate the solid and liquid through a 0.45 μm filter membrane to obtain a hydrophobic TB-EPS supernatant.

[0087] (3) Method 4 (extracting hydrophobic TB-EPS first): Steps 1) to 2) are the same as steps 1) to 2) of the layered extraction method of Example 1, and step 3) is as follows:

[0088] To the second sludge sediment in step 2), a non-surfactant Tween 20 solution (a 0.25% Tween 20 solution prepared with PBS buffer) was added, and the resulting solution was then placed on a shaker for 1 h at a speed of 250 rpm / min and a temperature of 4°C. The resulting suspension was then centrifuged at 10,000 g and 4°C for 20 min, and finally filtered using a 0.45 μm filter membrane to separate the hydrophobic TB-EPS supernatant.

[0089] The effects of different surfactants on the extraction yield of hydrophobic TB-EPS are shown in the following table. Figure 5 As shown in the figure, the contents of hydrophobic TB-EPS extracted by Tween 20 solution (0.25% Tween 20 solution prepared with PBS buffer) and ethanol solution (anhydrous ethanol diluted with PBS buffer until the concentration reaches 40%) are 17.799 mg / gVSS and 32.876 mg / gVSS, respectively, and the ratios of eDNA / EPS are 18.87% and 12.62%, respectively. Therefore, using ethanol solution to extract hydrophobic TB-EPS can extract more EPS while ensuring the integrity of cells.

[0090] The results of the effects of different extraction sequences on the extraction yield of hydrophobic TB-EPS are shown in Figure 2. Figure 6 As shown in the figure, the hydrophobic TB-EPS was first extracted with Tween 20 solution and ethanol solution, and the contents of the extracted hydrophobic TB-EPS were 15.105 mg / gVSS and 27.987 mg / gVSS respectively. The contents of the hydrophobic TB-EPS extracted in the order of extracting ionic TB-EPS first and then hydrophobic TB-EPS were 17.799 mg / gVSS and 32.876 mg / gVSS respectively. Figure 5), and the ratio of eDNA / EPS was not much different. This shows that the extraction of ionic TB-EPS by EDTA method first and then the extraction of hydrophobic TB-EPS by ethanol solution is more effective than other extraction sequences. It is more conducive to improving the extraction yield of TB-EPS while ensuring the integrity of cells.

[0091] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for extracting different extracellular polymer components from activated sludge, characterized in that: The following steps are involved: S1. Pretreatment: After obtaining activated sludge from the biochemical pool of the urban domestic sewage treatment plant, wash it with buffer 2-4 times and adjust its MLVSS to 5-8g / L; S2, treating the pretreated sludge by centrifugation, and then separating to obtain S-EPS supernatant and first sludge sediment; S3, after taking out the S-EPS supernatant of S2, adding an equal volume of buffer to the first sludge sediment to restore the original volume, and then sequentially treating by ultrasonic method and high-speed centrifugation method to separate and obtain LB-EPS supernatant and second sludge sediment; S4, after taking out the LB-EPS supernatant of S3, adding an equal volume of buffer to the second sludge sediment to restore the original volume, then adding EDTA solution for treatment, and then performing high-speed centrifugation to separate the ionic TB-EPS supernatant and the third sludge sediment; S5. After taking out the ionic TB-EPS supernatant of S4, it was treated by ethanol method and high-speed centrifugation method in sequence to separate and obtain the hydrophobic TB-EPS supernatant.

2. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: The pretreatment of S1 is as follows: first, the activated sludge is allowed to stand for 30-40 minutes, then the supernatant is poured off and buffer is added to restore the original volume. This operation is repeated 2-4 times, and then the MLVSS concentration of the activated sludge is detected and adjusted to 5-8 g / L with buffer.

3. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: The buffer in S1, S3, and S4 refers to a PBS buffer with a concentration of 0.1 mol / L, a pH of 7.2-7.4, and a NaCl concentration of 1.36 mol / L.

4. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: The centrifugation conditions for S2 are: centrifugation at 4000-5000g for 10-15 min at 4°C.

5. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: S3 is specifically as follows: the first sludge sediment is restored to its original volume with a buffer solution and then placed in an ultrasonic machine, and then ultrasonicated at a power density of 20-40KHZ and 0.75-1.5w / mL, with each ultrasonication lasting 2-4min, for a total of 3 times, with a 2min interval between each ultrasonication. Intermittent ultrasonication is then performed, and the ultrasonically treated activated sludge is then placed at 4°C and centrifuged at 10000-15000g for 15-20min, and finally solid-liquid separation is performed through a 0.45μm filter membrane.

6. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: S4 is specifically as follows: the prepared EDTA solution and the second sludge sediment restored to its original volume are mixed evenly in a volume ratio of 1:1, and then placed on an orbital oscillator, oscillated at 200-300 rpm / min at 4°C for 2-3 hours, and then placed at 4°C and centrifuged at 10000-15000g for 15-20 minutes, and finally solid-liquid separation is performed through a 0.45μm filter membrane to obtain ionic TB-EPS and the third sludge sediment.

7. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: The EDTA solution described in S4 is an EDTA solution dissolved in Tris-HCl buffer with a concentration of 0.5-2%.

8. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: S5 is specifically as follows: adding ethanol solution to the third sludge sediment to restore it to its original volume, then placing the mixed activated sludge on a shaker for 2-3 hours at a speed of 200-250rpm / min and a temperature of 4°C, then centrifuging the treated sludge at 10000-15000g for 15-20min at 4°C, and finally performing solid-liquid separation through a 0.45μm filter membrane to obtain hydrophobic TB-EPS.

9. A method for extracting different extracellular polymer components from activated sludge according to claim 1, characterized in that: The ethanol solution described in S5 is an ethanol solution diluted with phosphate buffered saline to a concentration of 20-40%.

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