A method for extracting different extracellular polymeric substance components from activated sludge

Abstract

The present application belongs to the field of sewage biological treatment technology, and particularly relates to a method for extracting different extracellular polymer components from activated sludge. The method focuses on layered extraction of various components of different extracellular polymers, that is, S-EPS in the activated sludge is first extracted by centrifugation, then LB-EPS is extracted by ultrasonic method and high-speed centrifugation, ion TB-EPS is extracted by EDTA method and high-speed centrifugation, and finally, hydrophobic TB-EPS is extracted by ethanol method and high-speed centrifugation. The combination of the above methods can complete the extraction of different components of extracellular polymers. The method for extracting different extracellular polymer components from activated sludge provided by the present application can ensure high extraction efficiency and high purity of EPS, and at the same time, the influence of the extraction method on cell lysis is as small as possible, and the quantitative accuracy and feasibility of the extracellular polymer extraction method are ensured.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of sewage biological treatment, and particularly relates to a method for extracting different extracellular polymer components from activated sludge. BACKGROUND

[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 ecological system for microbial communities, which not only helps to separate sludge and water and ensure good water quality, but also maintains the ecological activity of microorganisms, thereby achieving good removal effect of different pollutants. EPS (Extracellular Polymeric Substances) is an important component of activated sludge flocs and widely exists in microbial aggregates. It is a kind of high molecular polymer distributed on the cell surface or outside the cell, which is secreted by microorganisms under certain environmental conditions, including proteins, polysaccharides, humus, deoxyribonucleic acid (DNA), lipids and other macromolecular substances. At the same time, as a kind of three-dimensional macromolecular substance, EPS forms a network of three-dimensional structure through electrostatic, hydrophobic, energy hydrogen bond, ion bridge connection and other interactions, covering the surface of the whole cell, thereby promoting cell adhesion, maintaining the stability of microbial aggregates and reducing the damage of toxic substances to cells. When nutrients are lacking, it can also supplement carbon source and energy for microorganisms.

[0003] At present, the analysis of extracellular polymer substance EPS research, the extracellular polymer substance is generally divided into soluble extracellular polymer substance (S-EPS) and bound extracellular polymer substance (B-EPS). Among them, S-EPS and the binding force between cells are weak, which is in the outermost layer of EPS, and even dissolved in the liquid phase environment; and B-EPS is closely combined with the cell surface, which is in the innermost layer of EPS. Among them, B-EPS is divided into two layers, the inner layer is closely adhered to the cell surface and stable, which is called tightly bound extracellular polymer substance (TB-EPS); the outer layer structure is loose and dispersed, which is a layer of mucous layer without obvious boundary, called loosely bound extracellular polymer substance (LB-EPS). Because EPS has complex composition and different morphological structures, the function of EPS in sewage biological treatment research cannot be determined, and the influence of different forms of EPS on microorganisms and sewage treatment has not been determined. However, the extraction method of extracellular polymer substance is too single at present, which leads to low extraction yield, single type of extracted components, and certain influence on cell lysis. Therefore, an accurate and reliable method for extracting EPS is needed, which can extract EPS as completely as possible while reducing the influence of extraction process on cell lysis. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the present application provides a method for extracting different extracellular polymer substance components from activated sludge, which can achieve good extraction yield of various extracellular polymer substances of sludge, while ensuring that the influence on cell lysis is reduced as much as possible during the extraction process, ensuring the accuracy and reliability of extraction, thereby solving the problems of low extraction yield, single type of extracted components and certain influence on cell lysis caused by the single traditional sludge extracellular polymer substance extraction method.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0006] The present application discloses a method for extracting different extracellular polymer substance components from activated sludge, which comprises the following steps:

[0007] S1, pretreatment: after obtaining the activated sludge from the biochemical tank of the municipal domestic sewage treatment plant, washing it with buffer solution for 2-4 times, and adjusting the MLVSS to 5-8 g / L;

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

[0009] S3, after taking out the S-EPS supernatant of S2, adding an equal volume of buffer into the first sludge sediment and restoring to the original volume, then sequentially treating by ultrasonic method and high-speed centrifugation method, separating to 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 into the second sludge sediment and restoring to the original volume, then adding EDTA solution for treatment, and then performing high-speed centrifugation treatment, separating to obtain ionic TB-EPS supernatant and third sludge sediment;

[0011] S5, after taking out the ionic TB-EPS supernatant of S4, sequentially treating by ethanol method and high-speed centrifugation method, separating to obtain hydrophobic TB-EPS supernatant.

[0012] Extracellular polymer substance as a three-dimensional structure macromolecular substance, its main is through electrostatic, hydrophobic, energy hydrogen bond, ion bridge connection and so on interaction to constitute such three-dimensional framework, and to extract extracellular polymer substance is to break the function of these connected key, make it separate from the cell surface, so as to obtain extracellular polymer substance. And at present, extracellular polymer substance is usually divided into soluble extracellular polymer substance (S-EPS) and bound extracellular polymer substance (B-EPS). Therefore, the present application discloses a method for extracting different extracellular polymer components from activated sludge, first, the soluble extracellular polymer substance (S-EPS) is separated, because S-EPS is in the outermost layer of EPS, and the binding force between cells is relatively weak, and even can be dissolved in liquid phase environment, therefore, the separation of S-EPS only needs to adopt a certain degree of low speed centrifugation, here, 5000g centrifugation is carried out at 4 DEG C for 15 min. Then, the bound extracellular polymer substance (B-EPS) is separated, because it is tightly bound to the cell surface and is in the innermost layer of EPS. It is divided into two layers, the outer layer structure is loose and dispersed, which is a layer of mucous layer without obvious boundary, and is called loosely bound extracellular polymer substance (LB-EPS). The connection of this LB-EPS is relatively loose, and mainly plays a connecting role by means of low energy bond such as molecular hydrogen bond and intermolecular force, therefore, physical means is needed to provide corresponding energy to promote bond separation and breakage. Therefore, the present application adopts the physical method of "ultrasonic method + high speed centrifugation method" to extract, and in order to verify the practicability and reliability of the method, several different physical methods are also selected for comparison to verify the effectiveness of the method. Finally, the inner layer of B-EPS is tightly and stably adhered to the cell surface, and is called tightly bound extracellular polymer substance (TB-EPS). The combination of this layer and the cell surface is very tight, and it has the characteristics of hydrophobicity, electrostaticity, ion and the like, and general energy physical means cannot accurately separate it, here, the hydrophobicity and ion property of the TB-EPS are used for separation and extraction. Therefore, the present application selectively divides the TB-EPS into ionic TB-EPS and hydrophobic TB-EPS, and extracts the two kinds of TB-EPS respectively, and optimizes the three aspects of 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 specifically: first, the activated sludge is placed for 30-40 min, then the supernatant is poured off, and the buffer solution is added to restore the original volume, the operation is repeated 2-4 times, and then the MLVSS concentration of the activated sludge is detected, and the MLVSS of the activated sludge is adjusted to 5-8 g / L by the buffer solution.

[0014] Preferably, the buffer solution in S1, S3 and S4 refers to a PBS buffer solution 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 4000-5000g at 4℃ for 10-15min.

[0016] Preferably, S3 is specifically: after the first sludge sediment is restored to the original volume with the buffer solution, the first sludge sediment is placed in an ultrasonic machine, and then ultrasonic treatment is performed at a power density of 20-40KHZ, 0.75-1.5w / mL, each ultrasonic treatment lasts for 2-4min, the total ultrasonic treatment lasts for 3 times, and each ultrasonic treatment is separated by 2min, so intermittent ultrasonic treatment is performed, and then the activated sludge treated by ultrasonic treatment is centrifuged at 10000-15000g at 4℃ for 15-20min, and finally solid-liquid separation is performed through a filter membrane with a pore size of 0.45μm.

[0017] Preferably, S4 is specifically: the prepared EDTA solution and the second sludge sediment restored to the original volume are mixed uniformly according to a volume ratio of 1:1, and then placed on a constant-rotation oscillator, oscillated at a speed of 200-300rpm / min at 4℃ for 2-3h, centrifuged at 10000-15000g at 4℃ for 15-20min, and finally subjected to solid-liquid separation through a filter membrane with a pore size of 0.45μm, so ion-type TB-EPS and the third sludge sediment are obtained.

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

[0019] Preferably, S5 is specifically: the ethanol solution is added to the third sludge sediment to restore the original volume, and then the mixed activated sludge is placed on a shaking table and oscillated at a speed of 200-250rpm / min at a temperature of 4℃ for 2-3h, and then the treated sludge is centrifuged at 10000-15000g at 4℃ for 15-20min, and finally subjected to solid-liquid separation through a filter membrane with a pore size of 0.45μm, so hydrophobic TB-EPS is obtained.

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

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

[0022] The application discloses a method for extracting different extracellular polymer substance components from activated sludge, and focuses on layered extraction of various components of different extracellular polymer substances, that is, S-EPS in the activated sludge is extracted by a centrifugal method, LB-EPS is extracted by an ultrasonic method and a high-speed centrifugal method, ionic TB-EPS is extracted by an EDTA method and a high-speed centrifugal method, and hydrophobic TB-EPS is extracted by an ethanol method and a high-speed centrifugal method, so that the extraction of different components of extracellular polymer substances is completed by sequentially combining the methods.

[0023] The method for extracting different extracellular polymer substance components from activated sludge provided by the application extracts S-EPS, LB-EPS, hydrophobic TB-EPS and ionic TB-EPS in the activated sludge in sequence by maintaining a stable liquid phase environment for EPS extraction, selecting appropriate ion extractants and surfactants, detecting and analyzing different extraction methods and researching various combinations of the extraction methods. BRIEF DESCRIPTION OF DRAWINGS

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

[0025] Figure 2 Contents of various components of total EPS under different extraction methods (1 is a layered extraction method, 2 is a heat extraction method, 3 is an ultrasonic method, 4 is an EDTA method, 5 is a CER method, 6 is an ethanol method, and 7 is a Tween 20 method);

[0026] Figure 3 Contents of various components of LB-EPS under different extraction methods;

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

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

[0029] Figure 6 Contents of various components of hydrophobic TB-EPS extracted by different extraction sequences. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application and is not intended to limit the present application. Furthermore, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as there is no conflict.

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

[0032] In the following specific examples, the detection methods of various indexes are as follows:

[0033] (1) The determination method of EPS protein content: the BCA kit is used to determine the protein concentration. The determination principle is that in an alkaline environment, the peptide bond structure in the protein molecule is complexed with Cu 2+ and Cu 2+ is reduced to Cu 1+ . The BCA reagent specifically binds with Cu 1+ to form a stable purple-blue complex, which has the maximum light absorption value at 562 nm and is proportional to the protein concentration. The protein concentration can be determined according to the absorption value.

[0034] (2) The determination method of EPS polysaccharide concentration: the anthrone-sulfuric acid colorimetric method is used to determine the polysaccharide concentration. The determination principle is that under high temperature, sugars are dehydrated to furfural or furfural derivatives by concentrated H2SO4, and are condensed with anthrone (C 14 H 10 O) to form a blue compound. The absorbance is determined at 620 nm, and the content of polysaccharide can be calculated according to the standard curve of glucose.

[0035] (3) The determination method of DNA concentration in EPS: the nanodrop nucleic acid quantitative analyzer is used to detect the content and purity of DNA.

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

[0037] ①After the 0.45 μm fiber filter (filter paper) and the egg tart cup (or porcelain crucible) used for measuring the sample are cleaned, they are placed in an oven at 105°C for 40-60 min to remove water, and then they are placed in a desiccator to cool, and the mass of the filter paper and the porcelain crucible is weighed, which is recorded as M0 and M1, respectively.

[0038] ②In the reaction system with uniformly distributed sludge, 5 mL of sludge water mixed sample is accurately taken, which is recorded as V, and vacuum air filtration is performed on it using a 0.45 μm pore size fiber filter.

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

[0040] ④ Continue to heat the egg tart cup in step ③ in a box resistance muffle furnace 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 into a desiccator. When the cup cools to room temperature, weigh its mass and record it as M3. (M2-M3-M0) / V is the MLVSS value (unit: mg / L). Set 2-3 parallel samples for each determination.

[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 is as shown in Figure 1 , comprising the following steps:

[0043] (1) Pretreatment of activated sludge sample: The activated sludge is taken from the residual activated sludge in the biological reaction tank of Zhongshan Public Sewage Treatment Plant in Zhongshan, Guangdong Province. First, the activated sludge is allowed to stand for 30 min, then the supernatant is poured off, and PBS buffer (concentration of 0.1 mol / L, pH of 7.2-7.4, NaCl concentration of 1.36 mol / L PBS buffer) is added to restore the original volume. This operation is repeated 2 times. Then, the MLVSS index of the activated sludge is detected to determine the content of volatile organic matter, and the PBS buffer is used to adjust the MLVSS to 5 g / L.

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

[0045] The specific steps are as follows:

[0046] 1) Take 50 mL of pretreated sludge and treat it with centrifugation at 5000g for 15 min at 4℃ in a low-temperature centrifuge. Then separate the S-EPS supernatant and the first sludge sediment;

[0047] 2) After the S-EPS supernatant was taken out, 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 for ultrasonic treatment at a power of 20 KHZ, 0.75 w / mL, 2 min each time, a total of 3 times, with an interval of 2 min between each ultrasonic treatment. After that, the activated sludge treated by ultrasonic was centrifuged at 10000 g at 4℃ for 20 min, and finally solid-liquid separation was performed through a 0.45 μm filter membrane to obtain the LB-EPS supernatant and the second sludge sediment;

[0048] 3) For the second sludge sediment after centrifugation in step 2), first, it was placed in PBS buffer and restored to the original volume of 50 mL. Then the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) was mixed with the second sludge sediment restored to the original volume at a volume ratio of 1:1, and then the obtained solution was placed on a orbital shaker and shaken at 200 rpm / min at 4℃ for 3 h, then centrifuged at 10000 g at 4℃ for 20 min, and finally solid-liquid separation was performed through a 0.45 μm filter membrane to obtain the ionic TB-EPS and the third sludge sediment;

[0049] 4) Add diluted ethanol solution (absolute ethanol diluted with PBS buffer until the concentration reaches 40%) to the third sludge sediment of step 3) until it is restored to the original volume of 50 mL. Then the mixed activated sludge was placed on a shaker and shaken at 250 rpm / min at 4℃ for 2 h. Then the treated sludge was centrifuged at 15000 g at 4℃ for 20 min, and finally solid-liquid separation was performed through a 0.45 μm filter membrane to obtain the hydrophobic TB-EPS supernatant.

[0050] 2、Single extraction method for extracting activated sludge EPS

[0051] Under the same conditions (i.e. the collection and pretreatment of activated sludge samples are the same as the layered extraction method), the heat extraction method, ultrasonic method, EDTA method, CER method, ethanol method and Tween 20 method were compared with the layered extraction method. At the same time, the commonly used heat extraction method was used as a standard to judge the improvement effect of other methods on the extraction yield of extracellular polymers, in order to illustrate the necessity of the layered extraction method.

[0052] (1) Heat extraction method: After the sludge was pretreated, the supernatant was discarded, and 0.05% NaCl solution was added to resuspend the residual pellets to the original volume of 50 mL. The sludge was heated at 60°C for 30 min, then centrifuged at 10,000 g for 15 min at 4°C. The obtained supernatant was filtered through a 0.45 μm membrane, and the obtained supernatant was EPS.

[0053] (2) Ultrasonic method: After the pretreated sludge was poured, the supernatant was discarded, and the sludge was resuspended to the original volume of 50 mL with PBS buffer. The sludge was treated with ultrasonic waves at a power of 20 KHZ and 0.75 w / mL for 2 min each time, and the total ultrasonic time was 3 times, with an interval of 2 min between each ultrasonic treatment. Then the sludge was centrifuged at 10,000 g for 20 min at 4°C, and the obtained supernatant was filtered through a 0.45 μm membrane to obtain the EPS supernatant.

[0054] (3) EDTA method: 50 mL of pretreated activated sludge was prepared, and then the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) and activated sludge were mixed uniformly at a volume ratio of 1:1. The obtained solution was placed on a constant-temperature oscillator and oscillated at a speed of 200 rpm / min for 3 h at 4°C. Then the sludge was centrifuged at 10,000 g for 20 min at 4°C, and the obtained supernatant was filtered through a 0.45 μm membrane to obtain the EPS supernatant.

[0055] (4) CER method: 50 mL of pretreated sludge sample was taken, and 70 g / g VSS of CER was added. The obtained solution was mixed in a 500 mL conical flask, and then placed on a shaking bed and shaken for 3 h at a speed of 250 rpm / min and a temperature of 4°C. The obtained suspension was centrifuged at 10,000 g and 4°C for 20 min, and then filtered through a 0.45 μm membrane to obtain the EPS supernatant.

[0056] (5) Ethanol method: 50 mL of pretreated activated sludge was taken, and the supernatant was discarded. Diluted ethanol solution (absolute ethanol diluted with PBS buffer to a concentration of 40%) was added to the activated sludge precipitate until the original volume of 50 mL was restored. Then the mixed activated sludge was placed on a shaking bed and shaken for 2 h at a speed of 250 rpm / min and a temperature of 4°C. The treated sludge was centrifuged at 15,000 g for 20 min at 4°C, and then the obtained supernatant was filtered through a 0.45 μm membrane to obtain the EPS supernatant.

[0057] (6) Tween 20 method: 50 mL of pretreated activated sludge was taken, the supernatant was poured out, then a surfactant Tween 20 solution (0.25% Tween 20 solution prepared by PBS buffer) was added until the original volume was restored, then it was placed in a shaking bed and shaken for 1 h at a speed of 250 rpm / min and a temperature of 4℃. The obtained suspension was then centrifuged at 15000g and 4℃ 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 and the several single extraction methods were compared, and the results are shown in Table 1. Figure 2 As shown in Table 1, the total content of EPS extracted by the several single extraction methods was low, and the total amount of EPS extracted by the heat extraction method was 6.568 mg / g VSS, and the other single extraction methods (ultrasonic method, EDTA method, CER method, ethanol method, Tween 20 method) were 6.5%, 228%, 144.1%, 352.3%, and 89.2% higher than the heat extraction method, respectively. Obviously, the EDTA method and the ethanol method in the single extraction method have the best extraction effect. However, the content of EPS extracted by these methods is not very high, which shows that the single extraction method cannot extract the accurate content of extracellular polymeric substance. Compared with the heat extraction method, the layered extraction method of the present application can extract EPS with a total amount of 79.971 mg / g VSS, which is increased by 1117.5%. Obviously, compared with other single extraction methods, the layered extraction method of the present application can extract the content of EPS as much as possible, improve the extraction yield of EPS, and also does not have a great impact on cell lysis.

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

[0060] 1. Layered extraction method for extracting LB-EPS

[0061] The layered extraction method of Example 1 was used to extract LB-EPS, steps 1) and 2) (ultrasonic + high-speed centrifugation method):

[0062] 1) 50 mL of pretreated sludge was taken, and the centrifugation method was used for treatment, and the S-EPS supernatant and the first sludge precipitate were separated by centrifuging at 5000g and 4℃ for 15 min in a low-temperature centrifuge;

[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 for ultrasonic treatment at a power of 20 KHZ, 0.75 w / mL, 2 min each time, a total of 3 times, with an interval of 2 min between each ultrasonic treatment. After that, the activated sludge treated by ultrasonic was centrifuged at 10000g at 4℃ for 20 min, and finally solid-liquid separation was carried out through a 0.45μm filter membrane to obtain the LB-EPS supernatant and the second sludge sediment.

[0064] 2, extract LB-EPS by conventional method (water bath heating + high-speed centrifugation method)

[0065] Step 1) of which is the same as step 1) in the layered extraction method of example 1, and step 2) thereof is:

[0066] 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 for ultrasonic treatment at a power of 20 KHZ, 0.75 w / mL, 2 min each time, a total of 3 times, with an interval of 2 min between each ultrasonic treatment. After that, the activated sludge treated by ultrasonic was centrifuged at 10000g at 4℃ for 20 min, and finally solid-liquid separation was carried out through a 0.45μm filter membrane to obtain the LB-EPS supernatant and the second sludge sediment.

[0067] The results of analyzing the two extraction methods are shown in Figure 3 The contents of LB-EPS extracted by the combination method of "ultrasonic + high-speed centrifugation" and "water bath heating + high-speed centrifugation" are 3.019 mg / g VSS and 6.994 mg / g VSS, respectively. From the total amount of LB-EPS extraction, the extraction yield of the combination treatment of ultrasonic + high-speed centrifugation is relatively high, and the ratio of eDNA in LB-EPS is only 6.07%, which also indicates that the combination treatment method of "ultrasonic + high-speed centrifugation" has a small effect on cell damage, and the comprehensive extraction effect is better.

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

[0069] 1, extract ionic TB-EPS by layered extraction method

[0070] Extract LB-EPS by steps 1), 2) and 3) in the layered extraction method of example 1:

[0071] 1) Take 50 mL of pretreated sludge, and treat it by centrifugation at 5000g at 4℃ for 15 min in a low-temperature centrifuge, and then separate to obtain S-EPS supernatant and first sludge sediment;

[0072] 2) After the S-EPS supernatant was taken out, 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 for ultrasonic treatment at a power of 20 KHZ, 0.75 w / mL, 2 min each time, a total of 3 times, with an interval of 2 min between each ultrasonic treatment. After that, the activated sludge treated by ultrasonic was centrifuged at 10000g at 4℃ for 20 min, and finally solid-liquid separation was performed by using a 0.45 μm filter membrane to obtain the LB-EPS supernatant and the second sludge sediment;

[0073] 3) For the second sludge sediment after centrifugation in step 2), first, it was placed in PBS buffer and restored to the original volume of 50 mL. Then the prepared EDTA solution (2% EDTA solution dissolved in Tris-HCl buffer) was mixed with the second sludge sediment restored to the original volume at a volume ratio of 1:1, and then the obtained solution was placed on a constant-temperature oscillator and oscillated at 200 rpm / min at 4℃ for 3h, and then centrifuged at 10000g at 4℃ for 20 min, and finally solid-liquid separation was performed by using a 0.45 μm filter membrane to obtain the ionic TB-EPS and the third sludge sediment.

[0074] 2, the ionic TB-EPS was extracted by using a conventional method

[0075] Step 1) and step 2) of the layered extraction method in example 1 are the same, and 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 of CER was added to the second sludge sediment in a 500 mL conical flask, and then the obtained solution was placed on a shaking table and shaken at 250 rpm / min at 4℃ for 3h, and then the obtained suspension was centrifuged at 10000g at 4℃ for 20 min, and finally solid-liquid separation was performed by using a 0.45 μm filter membrane to obtain the ionic TB-EPS.

[0077] Data analysis and comparison of the two different ion extractants EDTA and CER were carried out respectively, and the results were as follows Figure 4As shown, the contents of the ionic TB-EPS extracted by the two ion extractants of EDTA and CER are 38.529 mg / g VSS and 20.227 mg / g VSS, respectively. In terms of the total amount of ionic TB-EPS extracted, the extraction yield of the ion extractant of EDTA is the highest, and it has a significant advantage for the extraction of polysaccharides. At the same time, the proportions of eDNA in the ionic TB-EPS extracted by the two extractants are 15.67% and 24.78%, respectively. At present, the eDNA / EPS index is generally used to measure the degree of cell damage, and the yield of TB-EPS extracted by EDTA is not only higher, but also the eDNA / EPS ratio is still within the normal proportion range (2% to 15%), which also indirectly shows that the ion extractant of EDTA has a relatively small effect on the degree of cell damage after being configured at a corresponding concentration, and the overall extraction effect is better.

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

[0079] 1. Layered extraction method for extracting hydrophobic TB-EPS

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

[0081] 2. Influence of different surfactants and extraction sequences on extraction yield of hydrophobic TB-EPS

[0082] Under the same conditions as in Example 4, the types of surfactants and the extraction sequences were changed respectively for comparison and analysis to study the influence of different surfactants and extraction sequences on the extraction yield of hydrophobic TB-EPS.

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

[0084] A non-surfactant Tween 20 solution (0.25% Tween 20 solution prepared with PBS buffer) was added to the third sludge sediment in step 3), and then the resulting solution was 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 15000g 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 (hydrophobic TB-EPS first): steps 1) to step 2) are the same as steps 1) to step 2) of the layered extraction method of Example 1, and step 3) is:

[0086] To the second sludge sediment of step 2), dilute ethanol solution (absolute ethanol diluted with PBS buffer until the concentration reaches 40%) was added until the original volume of 50 mL was restored. Then the mixed activated sludge was placed in a shaking bed for 2 h at a speed of 250 rpm / min and a temperature of 4°C. Then the treated sludge was centrifuged at 10000 g and 4°C for 20 min, and finally the solid-liquid separation was performed by using a 0.45 μm filter membrane to obtain the 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:

[0088] To the second sludge sediment of step 2), non-surfactant Tween 20 solution (Tween 20 solution with a concentration of 0.25% prepared with PBS buffer) was added, and then the resulting solution was placed in a shaking bed for 1 h at a speed of 250 rpm / min and a temperature of 4°C. Then the resulting suspension was centrifuged at 10000 g and 4°C for 20 min, and finally the solid-liquid separation was performed by using a 0.45 μm filter membrane to obtain the hydrophobic TB-EPS supernatant.

[0089] For the effect of different surfactants on the extraction yield of hydrophobic TB-EPS, the results are shown in Figure 5 Tween 20 solution (Tween 20 solution with a concentration of 0.25% prepared with PBS buffer) and ethanol solution (absolute ethanol diluted with PBS buffer until the concentration reaches 40%) were used to extract hydrophobic TB-EPS, and the contents of hydrophobic TB-EPS were 17.799 mg / g VSS and 32.876 mg / g VSS, respectively, and the ratios of eDNA / EPS were 18.87% and 12.62%, respectively. Therefore, ethanol solution is selected to extract hydrophobic TB-EPS, which can extract more EPS under the condition of ensuring cell integrity.

[0090] For the effect of different extraction sequences on the extraction yield of hydrophobic TB-EPS, the results are shown in Figure 6 Tween 20 solution and ethanol solution were used to extract hydrophobic TB-EPS first, and the contents of hydrophobic TB-EPS were 15.105 mg / g VSS and 27.987 mg / g VSS, respectively. The extraction sequence of extracting ionic TB-EPS first and then extracting hydrophobic TB-EPS was used, and the contents of hydrophobic TB-EPS were 17.799 mg / g VSS and 32.876 mg / g VSS, respectively. Figure 5), and the ratio of eDNA / EPS was also similar. This indicated that the order of first extracting ion TB-EPS by EDTA method and then extracting hydrophobic TB-EPS by ethanol solution was more effective than other extraction orders, which was beneficial to improve the extraction yield of TB-EPS while ensuring the integrity of cells.

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

Claims

1. A method for extracting different exopolymer components from activated sludge, characterized in that, Includes the following steps: S1. Pretreatment: After obtaining activated sludge from the biochemical tank of the municipal sewage treatment plant, wash it 2-4 times with buffer solution and adjust its MLVSS to 5-8 g / L. S2. The pretreated sludge is processed by centrifugation, and then the S-EPS supernatant and the first sludge settling are separated. S3. After removing the S-EPS supernatant from S2, add an equal volume of buffer solution to the first sludge sediment and restore it to its original volume. Then, process it sequentially using ultrasonic and high-speed centrifugation methods to separate the LB-EPS supernatant and the second sludge sediment. S4. After removing the LB-EPS supernatant from S3, add an equal volume of buffer solution to the second sludge sediment to restore it to its original volume. Mix the 0.5-2% EDTA solution dissolved in Tris-HCl buffer and the restored second sludge sediment at a 1:1 volume ratio. Then place it on a fixed-track shaker and shake at 200-300 rpm / min at 4 ℃ for 2-3 h. Then centrifuge at 10000-15000g at 4 ℃ for 15-20 min. Finally, perform solid-liquid separation through a 0.45μm filter membrane to obtain ionic TB-EPS and the third sludge sediment. S5. After removing the supernatant of the ionic TB-EPS from S4, add an ethanol solution diluted to a concentration of 20-40% with phosphate buffer solution to the third sludge sediment to restore the original volume. Then, place the mixed activated sludge in a shaker and shake for 2-3 hours at a speed of 200-250 rpm / min and a temperature of 4℃. Then, centrifuge the treated sludge at 10000-15000 g for 15-20 min at 4℃. Finally, perform solid-liquid separation through a 0.45μm filter membrane to obtain hydrophobic TB-EPS.

2. The method for extracting different exopolymer components from activated sludge according to claim 1, characterized in that, The pretreatment of S1 is as follows: First, let the activated sludge stand for 30-40 minutes, then pour off the supernatant and add buffer to restore the original volume. Repeat this operation 2-4 times. Then, detect the MLVSS concentration of the activated sludge and adjust its MLVSS to 5-8 g / L with buffer.

3. The method for extracting different exopolymer components from activated sludge according to claim 1, characterized in that, The buffer solutions in S1, S3, and S4 refer to PBS buffer solutions 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. The method for extracting different exopolymer components from activated sludge according to claim 1, characterized in that, The centrifugation conditions for S2 are: centrifuge at 4000-5000g for 10-15 minutes at 4℃.

5. The method for extracting different exopolymer components from activated sludge according to claim 1, characterized in that, S3 specifically involves: restoring the first sludge sediment to its original volume with buffer solution and then placing it in an ultrasonic machine. Ultrasonication is then performed at a power density of 20-40 kHz and 0.75-1.5 w / mL for 2-4 minutes each time, for a total of 3 times, with a 2-minute interval between each ultrasound session. This intermittent ultrasound treatment is then followed by centrifugation at 10000-15000 g for 15-20 minutes at 4°C. Finally, solid-liquid separation is performed through a 0.45 μm filter membrane.

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