Method for extracting amino acid in activated sludge cells

Abstract

The invention provides a method for extracting amino acid in activated sludge cells, and belongs to the technical field of biological sewage treatment. The activated sludge is subjected to freeze-drying treatment to remove moisture, and macromolecular substances are preliminarily separated through cold ethanol precipitation. Then, K2SO4 and pure methanol are used for extraction, K2SO4 contributes to destroying cell membranes, and the pure methanol can be used for dissolving and extracting amino acid. Through mechanical stirring and ultrasonic disruption, cell structures can be effectively destroyed and cell contents can be released, so that the extraction efficiency is maximized. In the whole process, low-temperature treatment is adopted to maintain the stability of amino acid. And finally, separating the extracting solution and the cell debris through centrifugation and filtration of a filter membrane to obtain filtrate, namely an amino acid sample to be analyzed. According to the method, physical and chemical extraction technologies are combined, the intracellular amino acid can be effectively separated and enriched from a complex activated sludge matrix, and a high-quality sample is provided for subsequent gas phase mass spectrometry.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage biological treatment, and in particular relates to a method for extracting amino acids in activated sludge cells. Background Art

[0002] Water eutrophication has become an urgent environmental problem that needs to be solved worldwide. The main reason is the excessive discharge of nutrients, especially nitrogen and phosphorus, into the aquatic environment. Improving the efficiency of nutrient removal is the key to alleviating this environmental challenge. At the same time, considering the need for energy conservation and emission reduction, it is imperative to optimize the treatment process.

[0003] There are many unknown material transformations in biological treatment, among which amino acids are a key research object. Amino acids play an important role in wastewater treatment. They are not only the basic units of protein synthesis, but also play an important role in nitrogen and carbon metabolism. By deeply studying the transformation of amino acids in the process of biological treatment of wastewater and their metabolic pathways, new perspectives and methods can be provided for optimizing wastewater treatment processes.

[0004] Understanding the amino acid transformation process helps reveal the metabolic activities and ecological functions of microbial communities, which is crucial for improving treatment efficiency, reducing energy consumption and reducing by-product generation. In addition, the metabolism of amino acids may be closely related to the removal of certain refractory organic matter and the stability of microbial communities.

[0005] Modern analytical techniques, such as liquid chromatography-mass spectrometry (LC-MS), can achieve high-precision quantitative analysis of amino acids, thus providing reliable data support for process optimization. This will not only help improve nitrogen and phosphorus removal efficiency, but may also lay the foundation for more efficient and economical wastewater treatment processes, and ultimately contribute to the protection and sustainable development of the water environment.

[0006] However, current technologies lack effective methods to extract amino acids from activated sludge cells, which limits the in-depth study and understanding of the metabolic roles and transformation pathways of amino acids in wastewater treatment. Summary of the invention

[0007] The purpose of the present invention is to provide a method for extracting amino acids in activated sludge cells, which solves the problem of being unable to conduct in-depth research and understanding on the metabolic role and conversion pathway of amino acids in the sewage treatment process.

[0008] To achieve the above object, the present invention adopts a method for extracting amino acids in activated sludge cells, comprising the following steps:

[0009] Take the freeze-dried sludge sample and put it into a 15 ml centrifuge tube;

[0010] The precipitate was added with 10 mL of cold ethanol (-20 degrees), stored at -20 degrees for 1 hour, and then centrifuged in a centrifuge to remove the supernatant;

[0011] Add 0.5 mol / L K2SO4 solution and pure methanol, mix well, then transfer the solution to a 2 ml centrifuge tube and stir at 200 rpm for 1.5 hours at room temperature. A mini shaker or vortex mixer can be used to ensure that the sample is fully mixed.

[0012] The mixed suspension was placed in a 4-degree metal bath and the cells were broken using an ultrasonic disruptor. The treated mixed solution was placed in a -20-degree refrigerator for 1 hour. The metal bath was controlled by a microcomputer and aluminum material was used as a heat-conducting medium to control the temperature at 4 degrees.

[0013] The sample was taken out and placed in a centrifuge to remove the supernatant, and then filtered with a membrane with a pore size of 0.2 μm;

[0014] The filtrate was collected in a gas phase vial and stored at -80 degrees until analyzed by gas phase mass spectrometry.

[0015] Among them, the ratio of freeze-dried sludge sample (mg) to mixed liquor suspended solids (MLSS) (mg / L) is 1:50 to 1:100.

[0016] The first centrifugation was performed at 6000 rpm for 4 to 8 min, and the second centrifugation was performed at 12000 rpm for 15 min at 4 degrees.

[0017] The volume ratio of 0.5 mol / L K2SO4 solution to pure methanol is 4:1. K2SO4 solution is mainly used to destroy cell membranes, and pure methanol is used to dissolve and extract amino acids. This volume ratio has been optimized through multiple experiments and has achieved a balance between extraction efficiency and sample stability.

[0018] Among them, when using an ultrasonic disruptor to disrupt cells, the operating conditions are an ultrasonic power of 100W, a pulse of 5 seconds, an interval of 5 seconds, and a total duration of 2 minutes. The ultrasonic power of 100W is to ensure the disruption efficiency while avoiding excessive damage to the sample. The pulse of 5 seconds, the interval of 5 seconds, and the total duration of 2 minutes are the optimal time determined by experiments, which is sufficient to disrupt cells and will not cause amino acids to be destroyed due to overheating of the sample.

[0019] The beneficial effects of the present invention for extracting amino acids from activated sludge cells are as follows: first, it can better optimize the sewage treatment process and achieve efficient removal of nitrogen and phosphorus by accurately analyzing the dynamics of microbial metabolism. This optimization not only provides a new way to improve the treatment capacity of sewage treatment facilities and reduce operating costs, but also has important significance for controlling eutrophication of water bodies on a global scale. In addition, given the key role of amino acid analysis in revealing the functions and metabolic pathways of microbial communities, this technology will have a positive driving effect in academic research and provide new insights and data support for environmental microbiology and sewage treatment technology research.

[0020] In practical applications, this technology can also promote the development of more environmentally friendly and sustainable water treatment solutions. By reducing the use of chemical reagents and energy consumption and reducing by-products in the treatment process, this invention promotes the application of green technology in environmental management and contributes to the sustainable management of water resources. This is not only in line with the trend of modern ecological protection and energy conservation and emission reduction, but also provides a more efficient and economical means to solve global water environment problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a preparation flow chart for extracting amino acids from activated sludge cells. DETAILED DESCRIPTION

[0022] See also Figure 1 The present invention provides a method for extracting amino acids from activated sludge cells, comprising the following steps:

[0023] Take the freeze-dried sludge sample and put it into a 15 ml centrifuge tube;

[0024] The precipitate was added with 10 mL of cold ethanol (-20 degrees), stored at -20 degrees for 1 hour, and then centrifuged in a centrifuge to remove the supernatant;

[0025] Add 0.5 mol / L K2SO4 solution and pure methanol, mix well, then transfer the solution to a 2 ml centrifuge tube and stir at 200 rpm for 1.5 hours at room temperature. A mini shaker or vortex mixer can be used to ensure that the sample is fully mixed.

[0026] The mixed suspension was placed in a 4-degree metal bath and the cells were broken using an ultrasonic disruptor; the treated mixed solution was placed in a -20-degree refrigerator to settle for 1 hour;

[0027] The sample was taken out and placed in a centrifuge to remove the supernatant, and then filtered with a membrane with a pore size of 0.2 μm;

[0028] The filtrate was collected in a gas phase vial and stored at -80 degrees until analyzed by gas phase mass spectrometry.

[0029] Further, the ratio of freeze-dried sludge sample (mg) to mixed liquor suspended solids (MLSS) (mg / L) was 1:50 to 1:100.

[0030] Furthermore, the conditions for the first centrifugation are 6000 rpm, 4 to 8 min, and the conditions for the second centrifugation are 12000 rpm, 4 degrees, and 15 min.

[0031] Furthermore, the volume ratio of 0.5 mol / L K2SO4 solution and pure methanol is 4:1.

[0032] Furthermore, when using an ultrasonic disruptor to disrupt cells, the operating conditions are as follows: an ultrasonic power of 100 W, a pulse of 5 seconds, an interval of 5 seconds, and a total duration of 2 minutes.

[0033] Implementation Cases

[0034] The sludge at the end of the anaerobic stage in the continuous flow anaerobic / aerobic / anoxic (AOA) reactor for treating domestic sewage was collected and freeze-dried, wherein the MLSS was 4000 mg / L;

[0035] Take out 40 mg of freeze-dried sludge sample and put it into a 15 ml centrifuge tube;

[0036] The precipitate was added with 10 mL of cold ethanol (-20 degrees), stored at -20 degrees for 1 hour, and then centrifuged in a centrifuge (6000 rpm, 8 min) to remove the supernatant;

[0037] Add 0.8 ml of 0.5 mol / L K2SO4 solution and 0.2 ml of pure methanol, mix well, then transfer the solution to a 2 ml centrifuge tube and stir at 200 rpm for 1.5 hours at room temperature. A mini shaker or vortex mixer can be used to ensure that the sample is fully mixed.

[0038] The mixed suspension was placed in a 4-degree metal bath and the cells were broken using an ultrasonic disruptor (ultrasonic power 100 W, pulse 5 seconds, interval 5 seconds, total duration 2 minutes); the treated mixed solution was placed in a -20-degree refrigerator for 1 hour;

[0039] The sample was taken out and placed in a centrifuge for centrifugation (12000 rpm, 4 degrees, 15 min) to remove the supernatant, and then filtered with a membrane with a pore size of 0.2 μm;

[0040] The filtrate was collected in a gas phase vial and quantitatively analyzed by a liquid mass spectrometer. 18 amino acids were detected, including serine (SER), valine (VAL), threonine (THR), leucine (LEU), ornithine (ORN), asparagine (ASN), lysine (LYS), glutamic acid (GLU), phenylalanine (PHE), arginine (ARG), isoleucine (ILE), glutamine (GLN), proline (PRO), histidine (HIS), methionine (MET), tryptophan (TRY), cysteine ​​(CYS), and aspartic acid (ASP). The contents of the 18 amino acids are shown in Table 1.

[0041] Table 1 Amino acid content after quantitative analysis by liquid chromatography mass spectrometry

[0042]

[0043] The above are specific embodiments of the present invention, which are convenient for technicians in this technical field to better understand and apply the present invention. The implementation of the present invention is not limited to this, so simple improvements made to the present invention by technicians in this technical field are within the scope of the present invention.

Claims

1. A method for extracting amino acids from activated sludge cells, characterized in that: The following steps are involved: Take the freeze-dried sludge sample and put it into a 15ml centrifuge tube; the ratio of the freeze-dried sludge sample to the mixed liquid suspended solids MLSS is 1:50-1:100mg / L; The precipitate was added to 10 mL of cold ethanol at -20 degrees, stored at -20 degrees for 1 hour, and then centrifuged in a centrifuge to remove the supernatant; Add 0.5 mol / L K2SO4 solution and methanol, mix well, then transfer the solution to a 2 ml centrifuge tube, stir at 200 rpm for 1.5 hours at room temperature to mix thoroughly; the volume ratio of 0.5 mol / L K2SO4 solution to pure methanol is 4:1; The mixed suspension was placed in a 4-degree metal bath and the cells were broken using an ultrasonic disruptor; the treated mixed solution was placed in a -20-degree refrigerator to settle for 1 hour; The sample was taken out and placed in a centrifuge to remove the supernatant, and then filtered with a membrane with a pore size of 0.2 μm; The filtrate was collected in a gas phase vial and stored at -80 degrees until analyzed by gas phase mass spectrometry.

2. A method for extracting amino acids from activated sludge cells as claimed in claim 1, characterized in that: The conditions for the first centrifugation were 6000 rpm, 4-8 min; the conditions for the second centrifugation were 12000 rpm, 4 degrees, 15 min.

3. A method for extracting amino acids from activated sludge cells as claimed in claim 1, characterized in that: When using an ultrasonic disruptor to disrupt cells, the operating conditions are as follows: an ultrasonic power of 100 W, a pulse of 5 seconds, an interval of 5 seconds, and a total duration of 2 minutes.

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