CN108129547B - Method for extracting extracellular polymeric substance of zoogloea - Google Patents
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Abstract
The invention provides a method for efficiently extracting activated sludge extracellular polymeric substances, which comprises the steps of cleaning, extracting a mucus layer by a centrifugal method, extracting loosely bound extracellular polymeric substances (LB-EPS) by a high-speed centrifugal method, and extracting tightly bound extracellular polymeric substances (TB-EPS) by a formaldehyde-acetic acid-potassium acetate heating method. The invention has the beneficial effects that: the method can simply, conveniently and quickly extract extracellular polymeric substances of the activated sludge, can avoid the dissolution of cell contents into an extracting solution and the decomposition of protein and polysaccharide caused by the extraction, and can ensure the purity of the extract to the maximum extent.
Description
Technical Field
The invention relates to an extraction method of activated sludge zoogloea extracellular polymeric substances, which can be applied to extraction of activated sludge extracellular polymeric substances and research of properties. In particular to the aspect of sewage treatment, belonging to the technical field of biological engineering.
Background
Extracellular Polymers (EPS) are a general term for Extracellular Polymeric Substances secreted by microbial cells during the metabolic process, and mainly include polysaccharides, nucleic acids, lipids, and the like. The extracellular polymer is an important component of activated sludge, has certain hydrophobicity, can be combined with cations, and has important influence on the surface charge, sedimentation, flocculation and dehydration of the sludge. To study the influence of extracellular polymeric substances on the sludge performance and to quantitatively analyze the influence, a method suitable for extracting the extracellular polymeric substances from the sludge is needed. There are many methods for extracting extracellular polymeric substances, mainly including physical extraction and chemical extraction. In the extraction process, the total amount of extracellular polymeric substances is ensured to be extracted, and biological cells are ensured not to be damaged, so that the extraction of the sludge extracellular polymeric substances is influenced by a plurality of factors.
Extraction of EPS is an important prerequisite for component and functional analysis of EPS, and a standard method is not established yet. The extraction efficiency of the same method for different samples of EPS is different, and the extraction efficiency of the same sample of EPS by different methods is also greatly different. There is no method for extracting the whole sample of EPS, all of which cause cell disruption to some extent. And whether an extraction method is appropriate is evaluated, not only considering the high and low EPS yield, but also considering the damage degree of the extraction method to cells and macromolecular substances in EPS and whether subsequent measurement is interfered and other related researches.
The invention content is as follows:
the invention aims to overcome the defects of the prior art and provide the method for extracting the extracellular polymeric substances of the activated sludge, which does not destroy the microstructure of cells, has less interference of chemical reagents, high extract purity and no decomposition.
The invention provides an extracellular polymer extracting solution which comprises the following components in parts by weight: 5-6 parts of titanium dioxide, 5-6 parts of 3.6% formaldehyde solution, 10-15 parts of acetic acid-potassium acetate solution with pH of 5.5 and 20-25 parts of quartz sand.
The use method of the extracellular polymeric substance extracting solution comprises the following steps of adding the extracellular polymeric substance extracting solution into sludge per liter: 40-52g/L of sludge.
The invention provides a method for efficiently extracting an extracellular polymer of a zoogloea, which comprises the following steps:
(1) cleaning of
Acquiring a sludge sample in situ, measuring the volume of the sludge sample, wetting filter paper, covering the filter paper on a Buchner funnel, placing the sludge on the filter paper, opening a suction filter, and adding a sodium chloride solution into the funnel to clean the sludge until the leaked solution becomes clear;
(2) extracting mucus layer by centrifugation
Adding sodium chloride solution into the treated sludge to restore the original volume, subpackaging the sludge into a centrifugal tube, centrifuging the sludge in a low-speed centrifuge, and extracting supernatant to be a mucus layer.
(3) Extraction of loosely bound extracellular Polymer (LB-EPS) by high speed centrifugation
The centrifuge tube sludge was returned to the original volume using sodium chloride solution. The sludge is treated for 1.5-2min by 120W ultrasound. Centrifuging at low temperature and high speed in a high speed centrifuge, and extracting supernatant to obtain loosely bound extracellular polymeric substance (LB-EPS);
(4) heating method of formaldehyde-acetic acid-potassium acetate to extract tightly bound extracellular polymeric substance (TB-EPS)
Adding all the sludge obtained in the step (3) into a beaker, and adding a sodium chloride solution to restore the original volume; adding the extracting solution, placing the sludge into a shaking culture box, shaking-culturing at 25-27 ℃ and 200r/min for 15-20min, and taking out. The extracting solution is as follows: 5-6g/L of titanium dioxide sludge, 5-6mL/L of 3.6% formaldehyde solution, 10-15mL/L of acetic acid-potassium acetate solution with pH of 5.5 sludge and 20-25g/L of quartz sand.
(5) Heating sludge in the beaker in water bath for 20min, cooling, subpackaging in a centrifugal tube, and centrifuging at low temperature and high speed for 20-22min at 1-4 deg.C. The supernatant was extracted as (TB-EPS).
(6) And mixing the extracted mucus layer, LB-EPS and TB-EPS uniformly, and filtering by using a filter membrane to obtain the extracellular polymer extract. The extractive solution should be stored at 1-4 deg.C under refrigeration.
Among them, preferably, in the step (4), the preparation method of the acetic acid-potassium acetate solution with pH of 5.5 is: respectively metering 41g of potassium acetate and 29.5mL of glacial acetic acid to 500mL, uniformly mixing a potassium acetate solution and an acetic acid solution according to the volume ratio of 9:1, and sealing and storing to obtain the potassium acetate solution with the pH = 5.5.
Preferably, the sludge in step (1) is preferably granular anaerobic ammonia oxidation sludge or granular anaerobic sludge, or the sludge in the form of a biofilm.
Preferably, the sodium chloride solution in the step (1) is a sodium chloride solution with a concentration of 0.50-0.80%. The pressure of the suction filter is preferably 0.03 to 0.04 MPa.
Preferably, the centrifugation speed of the centrifuge in the step (2) is 550-650r/min, the centrifugation time is 10-15min, and the centrifugation temperature is preferably 28 ℃.
Preferably, the sodium chloride solution in the steps (3) and (4) is a sodium chloride solution with the concentration of 0.05-0.10%.
Preferably, the temperature of the centrifuge in the step (3) is preferably 3-5 ℃, the centrifugation speed is preferably 5000-.
Preferably, the size of the quartz sand in the step (4) is preferably 50-100 meshes, and the addition amount is 20-25g/L of sludge
Preferably, the water bath heating temperature in step (5) is preferably 60-70 ℃.
Preferably, the rotation speed of the centrifuge in the step (5) is 8000-9000r/min, and the centrifugation time period is preferably 18-20 min.
Preferably, in the step (6), the filtration is performed by pressure filtration using a 0.40 to 0.45 μm aqueous membrane.
The specific principle of the invention is as follows:
the invention obtains the easily-fallen bacterial micelle mucus layer by low-speed centrifugation, and extracts the loose and combined extracellular polymer by cold storage and medium-speed centrifugation; the structure in the cell is protected to the maximum extent by controlling the temperature and the centrifugal speed. The innovative combination of the low-concentration formaldehyde solution and the weakly acidic acetic acid-potassium acetate solution helps to dissociate the tightly bound extracellular polymers on the surface of the zoogloea, and quartz sand and titanium dioxide are used as friction agents to strip the extracellular polymers tightly attached to the surface of the zoogloea through shearing and friction effects. The quartz sand with larger particles and the titanium dioxide powder with smaller particles act together to separate extracellular polymers more completely.
Compared with the prior art, the invention has the following advantages:
the physical method for extracting extracellular polymers of all parts disclosed by the invention realizes the respective extraction of extracellular polymers of all parts of the activated sludge by methods of cleaning, centrifuging, dissociating and the like, and is convenient for separately researching all components of the extracellular polymers. The method realizes the high-efficiency separation of the extracellular polymeric substance and the bacterial body, does not hurt the cell, and avoids the interference caused by the overflow of various substances such as polysaccharide, protein, DNA and the like in the cell. Meanwhile, the inactivation of bioactive substances such as protein in the extracting solution caused by a large amount of NaOH and methanol is avoided, and the subsequent research and determination are facilitated.
Description of the drawings:
FIGS. 1 to 4 are graphs comparing the amount of extracellular polymeric substances extracted by various methods and the contents of the components thereof.
FIG. 1 is a comparison of the total amount of three EPS extracted.
FIG. 2 is a comparison of the polysaccharide content in three EPS extracted.
FIG. 3 is a comparison of the protein content in three EPS extracted.
FIG. 4 is a comparison of the DNA content in the three EPS extracted.
Detailed Description
The following examples are further illustrative of the present invention, but the present invention is not limited thereto.
Example 1
The method of the patent is adopted to extract extracellular polymeric substances of certain black anaerobic granular sludge and measure the components of the extracellular polymeric substances. The specific operation steps are as follows:
(1) a sludge sample was taken from the reactor and its volume was determined to be 30 mL. Wetting the filter paper, covering the filter paper on a Buchner funnel, placing the sludge on the filter paper, opening the suction filter, and adjusting the pressure of the suction filter to be 0.03 MPa. The sludge was washed by slowly pouring 0.8% sodium chloride solution into the funnel.
(2) The treated sludge was taken out, placed in a beaker, and the volume was restored to 30mL by adding sodium chloride solution. Loading into 1.5mL centrifuge tubes, placing into a centrifuge, adjusting the centrifuge rotation speed to 600r/min, setting time to 15min, and extracting supernatant to obtain mucus layer.
(3) The sludge in the centrifuge tube was removed and the volume of the sludge was returned to 30mL using sodium chloride solution. 120W of ultrasonic treatment is used for treating the sludge for 2 min. Setting the rotation speed of the centrifuge at 5500r/min, the centrifugation temperature at 4 deg.C, and the centrifugation time at 10min, and extracting supernatant as loosely bound extracellular polymeric substance (LB-EPS).
(4) Adding all the sludge obtained in the step (3) into a beaker, and adding a sodium chloride solution to restore to 30 mL. 0.15g of titanium dioxide, 0.15mL of 3.6% formaldehyde, 0.3mL of sludge of acetic acid-potassium acetate solution having a pH of 5.5 and 0.75g of 75 mesh quartz sand were added. Placing the sludge into a shaking incubator, shaking and culturing for 15min at the temperature of 27 ℃ and at the speed of 150r/min, and taking out.
(5) The beaker is put into a water bath at 60 ℃ for heating for 20min, is cooled and then is subpackaged in a 1.5mL centrifugal tube, and is centrifuged for 20min at 9000r/min under the condition of 4 ℃. The supernatant was extracted as (TB-EPS).
(6) And uniformly mixing the extracted mucus layer, LB-EPS and TB-EPS, and filtering by using an injector and a 0.45-micrometer filter membrane to obtain the EPS extract of the anaerobic granular sludge. The extract is stored at 4 deg.C, and the contents of protein, polysaccharide and DNA are determined. Wherein, the protein is measured by taking bovine serum albumin as a standard sample and measuring the content of the bovine serum albumin by a Coomassie brilliant blue method; the determination of the polysaccharide takes glucose as a standard sample and is performed by an anthrone colorimetric method; the content of DNA is determined by taking a standard DNA solution as a standard sample and adopting a diphenylamine-acetaldehyde spectrophotometry. (Note: purification by phenol extraction to remove proteins and polysaccharides from the sample solution before DNA determination.)
Example 2
The method of the patent is adopted to extract extracellular polymers of certain red anaerobic ammonia oxidation granular sludge and determine the components of the extracellular polymers. The specific operation steps are as follows:
(1) a sludge sample was taken from the anammox reactor and its volume was measured to be 30 mL. Wetting the filter paper, covering the filter paper on a Buchner funnel, placing the sludge on the filter paper, opening the suction filter, and adjusting the pressure of the suction filter to be 0.03 MPa. The sludge was washed by slowly pouring 0.8% sodium chloride solution into the funnel.
(2) The treated sludge was taken out, placed in a beaker, and the volume was restored to 30mL by adding sodium chloride solution. Loading into 1.5mL centrifuge tubes, placing into a centrifuge, adjusting the centrifuge rotation speed to 600r/min, setting time to 15min, and extracting supernatant to obtain mucus layer.
(3) The sludge in the centrifuge tube was removed and the volume of the sludge was returned to 30mL using sodium chloride solution. 120W of ultrasonic treatment is used for treating the sludge for 2 min. Setting the rotation speed of the centrifuge at 5500r/min, the centrifugation temperature at 4 deg.C, and the centrifugation time at 10min, and extracting supernatant as loosely bound extracellular polymeric substance (LB-EPS).
(4) Adding all the sludge obtained in the step (3) into a beaker, and adding a sodium chloride solution to restore to 30 mL. 0.16g of titanium dioxide, 0.15mL of 3.6% formaldehyde, 0.3mL of sludge of an acetic acid-potassium acetate solution having a pH of 5.5, and 0.9g of 75 mesh quartz sand were added. Placing the sludge into a shaking incubator, shaking and culturing for 15min at 27 ℃ and 180r/min, and taking out.
(5) The beaker is put into a water bath at 60 ℃ for heating for 20min, is cooled and then is subpackaged in a 1.5mL centrifugal tube, and is centrifuged for 20min at 9000r/min under the condition of 4 ℃. The supernatant was extracted as (TB-EPS).
(6) And uniformly mixing the extracted mucus layer, LB-EPS and TB-EPS, and filtering by using an injector and a 0.45-micrometer water system filter membrane to obtain the EPS extracting solution of the anaerobic granular sludge. The extract is stored at 4 deg.C, and the contents of protein, polysaccharide and DNA are determined. Wherein, the protein is measured by taking bovine serum albumin as a standard sample and measuring the content of the bovine serum albumin by a Coomassie brilliant blue method; the determination of the polysaccharide takes glucose as a standard sample and is performed by an anthrone colorimetric method; the content of DNA is determined by taking a standard DNA solution as a standard sample and adopting a diphenylamine-acetaldehyde spectrophotometry. (Note: purification by phenol extraction to remove proteins and polysaccharides from the sample solution before DNA determination.)
Comparative example 1
The same extracellular polymers of the red anaerobic granular sludge as in example were extracted and the composition thereof was measured using the conventional centrifugal-ultrasonic method and the formaldehyde-NaOH method. The specific operation steps are as follows:
(1) and centrifuging the cleaned sludge sample for 15min at the speed of 600r/min, and filtering the supernatant through a 0.45-micron filter membrane to obtain a mucus layer.
(2) Suspending the residual sludge to the original volume with 0.85% NaCl solution, treating with 120W ultrasound for 2min, shake culturing at 28 deg.C and 180r/min for 10min, centrifuging at 5000r/min and 4 deg.C for 20min, and collecting supernatant as LB-EPS.
(3) Pouring the residual sludge into a beaker, suspending the residual sludge to the original volume by using 0.85% sodium chloride, treating the residual sludge for 2min by using 120W ultrasonic waves, and performing shake culture for 10min at the temperature of 28 ℃ and at the speed of 180 r/min.
(4) And (3) collecting all the sludge treated in the step (3) into a beaker, adding 0.06 mL of 36.5% formaldehyde into the beaker, oscillating for 1h in a shaking table at 28 ℃ and 180r/min, adding 1 mol/L of NaOH solution to adjust the pH value to 11, oscillating for 3h in the shaking table, centrifuging for 20min at 20000 r/min and 4 ℃, and collecting the supernatant to be TB-EPS.
(5) And measuring the contents of protein, polysaccharide and DNA in the extract. Wherein, the protein is measured by taking bovine serum albumin as a standard sample and measuring the content of the bovine serum albumin by a Coomassie brilliant blue method; the determination of the polysaccharide takes glucose as a standard sample and is performed by an anthrone colorimetric method; the content of DNA is determined by taking a standard DNA solution as a standard sample and adopting a diphenylamine-acetaldehyde spectrophotometry. (Note: purification by phenol extraction to remove proteins and polysaccharides from the sample solution before DNA determination.)
In comparison with the above cases, the extracellular polymeric substance extracted by the method of the present invention has a small DNA content of 2.32 mg-gss-1And 2.51 mg. gss-1The extracellular polymer DNA of the anaerobic ammonium oxidation sludge extracted by the traditional method is up to 7.9 mmg. gss-1This demonstrates that the traditional methods are more destructive to the cell mass than the methods described in this patent. The total amount of the anaerobic ammonium oxidation sludge extracellular extract obtained by the method is 33.65 mg-gss-1Although the amount of extracellular polymeric substances extracted is less than that extracted by the traditional formaldehyde-NaOH method (45.31 mg-gss)-1) However, it causes little damage to the cells and contains little intracellular material. Because the anaerobic ammonium oxidation bacteria are cultured by using artificial wastewater, the living environment has very little organic matters, and the polysaccharide and the protein contained in the extracellular polymer are low. And the common anaerobic sludge has high growth speed and can survive in a high organic matter environment, so the contents of protein and polysaccharide are higher, which are respectively 35.23% and 26.01% higher.
Most of the common anaerobic sludge is heterotrophic bacteria, so that the propagation is fast, and the common anaerobic sludge is used for treating high-concentration organic wastewater, so that the synthesis speed of extracellular polymers is high, and the content of organic matters is high; the anaerobic ammonium oxidation bacteria are autotrophic bacteria, are slow in propagation and are used for treating inorganic wastewater, so that the synthesis speed of extracellular polymers is low, and the content of organic matters is low. Although the extraction amount of extracellular polymers of the anammox activated sludge extracted by the traditional method is large, the content of DNA in the extract is high, which indicates that the cell structure is damaged, the contents in the cells flow out, and the extract of the extracellular polymers is polluted, so that the measurement result is inaccurate. Compared with the traditional method, the extracellular polymeric substance extracted by using the novel method provided by the patent has the advantages that although the content and the total amount of each component are slightly lower, the content of DNA is only one third to one fourth of the content of the extracting solution of the traditional method, so that the method provided by the patent can effectively protect the cell structure while extracting the extracellular polymeric substance, ensure the purity of the extracted extracellular polymeric substance and minimize the influence on the experiment.
Claims (7)
1. An extracellular polymer extract, which consists of the following components: according to the weight ratio, 5-6 parts of titanium dioxide, 5-6 parts of 3.6% formaldehyde solution, 10-15 parts of acetic acid-potassium acetate solution with pH of 5.5 and 20-25 parts of 75-mesh quartz sand.
2. The method for using extracellular polymeric substance extracting solution according to claim 1, wherein the amount of extracellular polymeric substance extracting solution added to the sludge is: 40-52g/L of sludge.
3. A method for efficiently extracting extracellular polymeric substances of zoogloea adopts the following steps:
(1) cleaning of
Acquiring a sludge sample in situ, measuring the volume of the sludge sample, wetting filter paper, covering the filter paper on a Buchner funnel, placing the sludge on the filter paper, opening a suction filter, and adding a sodium chloride solution into the funnel to clean the sludge until the leaked solution becomes clear;
(2) extracting mucus layer by centrifugation
Adding sodium chloride solution into the treated sludge to restore the original volume, subpackaging the sludge into a centrifugal tube, centrifuging the sludge in a low-speed centrifuge, and extracting supernatant to be a mucus layer; the centrifugal speed of the centrifugal machine is 550-650r/min, the centrifugal time is 10-15min, and the centrifugal temperature is 28 ℃;
(3) extraction of loosely bound extracellular Polymer (LB-EPS) by high speed centrifugation
Restoring the centrifugal tube sludge to the original volume by using a sodium chloride solution; treating the sludge for 1.5-2min by using 120W ultrasound; centrifuging at low temperature and high speed in a high speed centrifuge, and extracting supernatant to obtain loosely bound extracellular polymeric substance (LB-EPS); the temperature of the centrifuge is 3-5 ℃, the centrifugation speed is 5000-;
(4) heating method of formaldehyde-acetic acid-potassium acetate to extract tightly bound extracellular polymeric substance (TB-EPS)
Adding all the sludge obtained in the step (3) into a beaker, and adding a sodium chloride solution to restore the original volume; adding the extracting solution, placing the sludge into a shaking culture box, shaking-culturing for 15-20min at 25-27 ℃ and 200r/min, and taking out; the extracting solution is as follows: 5-6g/L of titanium dioxide sludge, 5-6mL/L of 3.6% formaldehyde solution, 10-15mL/L of acetic acid-potassium acetate solution with pH of 5.5 sludge and 20-25g/L of 75-mesh quartz sand;
(5) heating sludge in the beaker in water bath for 20min, cooling, subpackaging in a centrifugal tube, and centrifuging at low temperature and high speed for 20-22min at 1-4 deg.C; extracting the supernatant to obtain (TB-EPS); the rotating speed of the centrifuge is 8000-;
(6) mixing the extracted mucus layer, LB-EPS and TB-EPS, and filtering with filter membrane to obtain extracellular polymer extractive solution; the extractive solution should be stored at 1-4 deg.C under refrigeration.
4. The method for efficiently extracting the zoogloea extracellular polymeric substance as claimed in claim 3, wherein in the step (4), the preparation method of the acetic acid-potassium acetate solution with pH of 5.5 comprises the following steps: respectively metering 41g of potassium acetate and 29.5mL of glacial acetic acid to 500mL, uniformly mixing a potassium acetate solution and an acetic acid solution according to the volume ratio of 9:1, and sealing and storing to obtain the potassium acetate solution with the pH = 5.5.
5. The method for efficiently extracting the zoogloea extracellular polymeric substance according to claim 3, wherein the concentration of the sodium chloride solution in the step (1) is 0.50% -0.80%; the pressure of the suction filter is 0.03-0.04 MPa; the concentration of the sodium chloride solution in the steps (3) and (4) is 0.05-0.10%.
6. The method for efficiently extracting the extracellular polymeric substance of zoogloea as claimed in claim 3, wherein the water bath heating temperature in the step (5) is 60-70 ℃.
7. The method for extracting extracellular polymeric substance of zoogloea with high efficiency as claimed in claim 3, wherein the filtration in the step (6) is performed by pressure filtration using 0.40-0.45 μm water system filter membrane.
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| CN109211738B (en) * | 2018-07-18 | 2021-04-13 | 广西大学 | Method for detecting and judging calcification and activity of anaerobic granular sludge in high-calcium wastewater |
| CN109111499A (en) * | 2018-08-23 | 2019-01-01 | 暨南大学 | A method of extracting ground bacillus Extracellular polymers |
| CN110451762B (en) * | 2019-07-05 | 2020-09-18 | 重庆大学 | Extraction method of activated sludge extracellular polymers |
| CN112898374B (en) * | 2021-02-05 | 2023-02-03 | 中南大学 | Method for extracting extracellular polymer of bacterial-algae symbiotic system |
| CN114797777B (en) * | 2022-04-28 | 2023-06-06 | 南京大学 | Preparation method of sludge-based biochar-loaded nano iron based on extracellular polymer regulation |
| CN114890636A (en) * | 2022-05-19 | 2022-08-12 | 浙江工业大学 | Activated sludge extracellular polymer extraction method based on divalent cation complexation |
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