Extraction method of sphere-like biological aggregate nucleus

Abstract

A method for extracting nucleuses of sphere-like biological aggregates relates to the technical field of biological sewage treatment, and comprises the following steps: collecting and treating a sample, collecting a core separation device and a sample, collecting a mixed solution by using a centrifugal tube through a sampling port in a uniform mixing stage of the sample in a reactor, settling, and washing for three times by using deionized water. According to the aggregate inner core separation operation device, a rectangular organic glass plate on the lower layer serves as a bottom plate to be fixed, and four quadrangular frustum pyramid organic glass blocks with the same size are oppositely spliced to form the upper layer. Capillary glass tubes with different inner diameters are penetrated to extract and separate biological aggregate nucleuses with different particle sizes; after the nucleuses of the biological aggregates are separated, the biological aggregates can be subjected to analysis researches such as high-throughput sequencing. The method can effectively extract the inner core of the sphere-like biological aggregate, and has the advantages of high applicability, convenience and simplicity in operation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage biological treatment, and relates to a method for extracting the inner core of a biological aggregate, in particular to a method for extracting the inner core of a spherical biological aggregate. Background Art

[0002] With the rapid development of my country's economy, the scale of urban construction has continued to expand, and the level of urbanization has become increasingly higher. my country's sewage treatment capacity must also be matched with the amount of sewage discharged. At present, most sewage treatment plants use the activated sludge method to treat sewage, but during their operation, it is found that the sludge output is high, the area occupied is large, and sludge swelling is prone to occur.

[0003] Granular sludge technology is an emerging technology in recent years. It is formed by microbial self-flocculation and is a spherical microbial aggregate formed by orderly self-organization of different microbial populations.

[0004] Aerobic granular sludge has the advantages of large sludge biomass, fast sedimentation rate, and rich microbial species, and has attracted attention in recent years. Compared with traditional activated sludge, it has the advantages of reduced floor space, dense structure, and zoning colonization of microbial flora. It can effectively improve mud-water separation, pollutant removal, excess sludge reduction, and reduce energy consumption. It can simultaneously remove organic matter and remove nitrogen and phosphorus, and is considered to be one of the promising sewage biological treatment technologies. Current studies have shown that the outer layer of aerobic granular sludge is mainly composed of common foreign microorganisms, and the outer layer structure is conducive to the supply of oxygen by participating in aerobic metabolic processes. Studies have shown that the main factors that help promote sludge granulation and maintain the stable operation of granular sludge are slow-growing bacteria: polyphosphate bacteria PAOs, polysaccharide bacteria GAOs, nitrite bacteria AOB and nitrifying bacteria NOB, etc. Stable aerobic granular sludge is cultivated by enriching these types of slow-growing bacteria.

[0005] Anaerobic granular sludge is a self-aggregate composed of methanogens, acetogens, and hydrolytic fermentation bacteria. It has the advantages of good precipitation performance and methanogenic activity, regular spherical structure, and high specific methanogenic rate. Anaerobic granular sludge is also an aggregate of various microorganisms, mainly composed of various acidogenic bacteria and methanogenic bacteria. The acidogenic bacteria are outside the particles, and the methanogenic bacteria are inside the granular sludge. The anaerobic bacteria involved in the decomposition of complex organic matter and the production of methane in granular sludge can be divided into the following three categories: The first category is hydrolytic fermentation bacteria, which initially decompose organic matter to produce organic acids and alcohol. The second category is acetogens, which further decompose and utilize organic acids and alcohol. The third category is methanogens, which convert acetic acid and some other simple compounds into methane.

[0006] The inner core of granular sludge usually refers to the core structure inside the granules, which can be composed of different microorganisms. In the formation process of granular sludge, the concept of inner core is related to a variety of formation mechanisms. For example, the crystal nucleus hypothesis believes that the formation of granular sludge is similar to the crystallization process. The inorganic salt precipitation or inert organic matter produced during the inoculation sludge or reactor operation serves as the crystal nucleus. The granular sludge continues to develop on the basis of the crystal nucleus and eventually forms mature granular sludge. The microbial community structure inside the granules is crucial to the treatment effect of the sludge. However, in order to apply granular sludge technology to actual sewage treatment plants, it is very important to understand the structure and distribution characteristics of the inner and outer microbial flora of the sludge in the early stage of research. For granular sludge of different particle sizes, the distribution of the inner and outer flora is significantly different.

[0007] The particle core separation technology can help researchers better understand the formation mechanism and stability of aerobic granular sludge. Through this technology, researchers can observe and analyze the microstructure inside aerobic granular sludge, identify the redox environment in different areas, and how it affects the composition and function of the microbial community. In addition, particle core separation helps to evaluate the impact of different operating conditions on the stability of aerobic granular sludge, thereby optimizing sewage treatment process parameters, improving treatment efficiency and the system's ability to resist shock loads. For anaerobic granular sludge, particle core separation is equally important because it can reveal the formation process, internal structural characteristics and microbial interactions of anaerobic granular sludge. By analyzing the separated core and peripheral parts, the stability and functional changes of anaerobic granular sludge under different treatment conditions, such as methane production efficiency and organic pollutant degradation efficiency, can be understood. This is of great significance for designing and improving anaerobic digestion processes, improving energy recovery efficiency and reducing environmental pollution.

[0008] In the actual sewage treatment process, due to the small particle size of granular bioaggregates and relatively low particle strength, there is always a lack of an effective means of extracting the particle core. The core structure of granular sludge has an important influence on its overall performance. For example, aerobic granular sludge with a reinforced core and skeleton can maintain better stability during long-term operation. Even after the particles are broken, the core can be reused to help the system recover faster. In summary, in spherical bioaggregates represented by granular sludge, the core is its core structure, which can be composed of a variety of substances and plays a key role in the formation and stability of granular sludge. Granular core separation technology is crucial for a deep understanding of the biological and engineering characteristics of aerobic and anaerobic granular sludge, and helps promote the advancement and sustainable development of sewage treatment technology. Summary of the invention

[0009] In order to overcome the shortcomings of the prior art and solve the problem of difficulty in extracting and separating the core components of the inner core of spherical bioaggregates, the present invention provides a method for extracting the inner core of spherical bioaggregates, extracting samples through a core separation device, and providing a basis for subsequent microbial community analysis and structural analysis. By using capillary glass tubes with different inner diameters and needles to fix granular sludge, the inner core of spherical bioaggregates with a particle size greater than 1000 μm can be effectively extracted. Thereby ensuring that the extracted core sample can accurately reflect the intrinsic characteristics of the aggregate inner core. Extracting the inner core of spherical bioaggregates is used to study their structure and composition, and understanding the characteristics of the inner core of spherical bioaggregates is also of great significance for improving wastewater treatment processes and improving treatment efficiency.

[0010] The technical solution adopted by the present invention to solve its technical problem is:

[0011] A method for extracting the inner core of a spherical bioaggregate comprises the following steps:

[0012] 1) Sample collection and processing

[0013] The uniformly mixed particles are taken out from the uniform mixing stage of the microbial aggregate reactor, and the particles are taken out from the sampling port of the reactor using a 50 ml centrifuge tube, and then the muddy water mixture is separated using a low-speed centrifuge; the supernatant and part of the light floccules after centrifugation are discharged, and the particles are rinsed with deionized water to complete the sample collection;

[0014] 2) Core separation

[0015] The main body of the spherical bioaggregate core separation device used is rectangular and divided into two layers, the bottom of the reactor is made of a glass plate, and bolts are used at four right angles of the bottom plate of the extraction device to fix the bottom plate plane;

[0016] 3) Sample collection.

[0017] Furthermore, in the step 2), the upper layer of the fixed extraction device is composed of four four-sided pyramid organic glass blocks of the same size, which are relatively combined and placed on the bottom plate in four directions: upper left, lower left, upper right, and lower right.

[0018] In the step 2), a bioaggregate core separation device is used to separate the inside and outside of the bioaggregate core, and the method is as follows:

[0019] 2.1) Place the sample particles at the center of four custom-made quadrangular pyramid glass blocks;

[0020] 2.2) Insert two 0.12 mm microneedles in two perpendicular directions to fix the particles;

[0021] 2.3) After the sample is fixed, four capillary glass tubes with an inner diameter of 500-2000 μm are sequentially inserted along the needle according to the spherical bioaggregates, and the four capillary glass tubes are slowly pushed until the inner core of the particles is separated from the outer part.

[0022] In the step 3), after the kernel is extracted by the capillary glass tubes with different inner diameters, the extracted particle kernel is placed in the capillary glass tube, and an oxygen pump with a flow rate of 35 L / min, a power of 20 W, and a pressure of 0.01 MPa is used to adjust the flow rate by connecting the glass rotor flowmeter with a range of 0.1-1 L / min through a 4 mm air tube. The glass rotor flowmeter is adjusted to 0.1 L / min, and the capillary glass tube is connected to blow the kernel into a 50 ml glass beaker containing 30 ml deionized water for collection;

[0023] Furthermore, after separating and extracting the inner core of particles with a particle size greater than 1000 μm from the outer core, it is convenient to analyze the distribution of the microbial community structure and further understand its formation mechanism and mechanism of action.

[0024] The beneficial effects of the present invention are mainly manifested in that the outer layer structure and the inner core structure of the particles can be effectively separated, providing a basis for subsequent high-throughput sequencing analysis of the inner core microbial community structure. The present invention has the advantages of simple operation and high accuracy. The ability to effectively understand the characteristics of the inner core of the bioaggregate is also of great significance for improving wastewater treatment processes and improving treatment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a diagram of a biological aggregate core separation device, wherein 3 represents a tetrahedral organic glass block in the separation device, 4 represents a placed spherical biological aggregate, 5 represents a 0.12 mm needle, and 6 represents a capillary glass tube.

[0026] Figure 2 for Figure 1 A partial enlarged view of the spherical bioaggregate placed in the middle, wherein 5 represents a 0.12 mm needle, 6 represents a capillary glass tube, and 7 represents the extracted core.

[0027] Figure 3 Figure 1 is a diagram of the steps for extracting the inner core of the bioaggregate. In Figure a, 1 represents the bolts for fixing the extraction device, 2 represents the bottom plate for fixing the extraction device, the next step, Figure b, represents placing the particles at the center of the extraction device, and further Figure c represents using a needle to vertically insert the particles, and finally Figure d represents using a capillary glass tube to extract the inner core. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings.

[0029] Reference Figure 1 to Figure 3, a method for extracting the inner core of a spherical bioaggregate, comprising the following steps:

[0030] 1) Sample collection and processing,

[0031] The uniformly mixed particles are taken out from the uniform mixing stage of the microbial aggregate reactor, and the particles are taken out from the sampling port of the reactor using a 50 ml centrifuge tube, and then the muddy water mixture is separated using a low-speed centrifuge; the supernatant and part of the light floccules after centrifugation are discharged, and the particles are rinsed three times with deionized water to complete the sample collection;

[0032] 2) Core separation;

[0033] The main body of the spherical bioaggregate core separation device used is rectangular and divided into two layers, the bottom of the reactor is made of a glass plate with a size of 10 cm in length, 8 cm in width and 1.5 cm in thickness. At the same time, four bolts with a diameter of 5 mm and a length of 12 mm are used at four right angles of the bottom plate to fix the bottom plate plane;

[0034] In the step 2), the upper layer of the fixed extraction device is composed of four square pyramid organic glass blocks of the same size, which are relatively assembled and placed on the bottom plate at the upper left, lower left, upper right and lower right directions, and the size is 20*25mm on the top surface, 40*50mm on the bottom surface, and 20mm in height;

[0035] In the step 2), a bioaggregate core separation device is used to separate the inner and outer parts of the bioaggregate core, and the method is as follows:

[0036] 2.1) Place the sample particles at the center of four custom-made quadrangular pyramid glass blocks;

[0037] 2.2) Insert two 0.12 mm microneedles in two perpendicular directions to fix the particles;

[0038] 2.3) After the sample is fixed, four capillary glass tubes with an inner diameter of 500-2000 μm are inserted along the needle in sequence according to the spherical bioaggregates, and the four capillary glass tubes are slowly pushed until the inner core of the particles is separated from the outer part;

[0039] 3) Sample collection: After the kernel is extracted from the capillary glass tubes with different inner diameters, the extracted particle kernel is placed in the capillary glass tube, and an oxygen pump with a flow rate of 35 L / min, a power of 20 W, and a pressure of 0.01 MPa is used to adjust the flow rate through a 4 mm air tube connected to a glass rotor flow meter with a range of 0.1-1 L / min. The glass rotor flow meter is adjusted to 0.1 L / min, and the capillary glass tube is connected to blow the kernel into a 50 ml glass beaker filled with 30 ml deionized water for collection;

[0040] Furthermore, after separating and extracting the inner core of particles with a particle size greater than 1000 μm from the outer core, it is convenient to analyze the distribution of the microbial community structure and gain a deeper understanding of its formation mechanism and mechanism of action.

[0041] This embodiment takes aerobic granular sludge as an example. The aerobic granular sludge is produced under the conditions of complete granulation and stable removal of pollutants after 60 days of laboratory operation. The chemical oxygen demand of the reactor inlet water is CODcr = 1000±25mg / L, the ammonia nitrogen is 24±1.0mg / L, the total nitrogen is 27±1.5mg / L, artificial simulated wastewater with sodium acetate as the main carbon source, and the SBR reactor is batch-indicated. The aerobic granular sludge is cultivated for a period of 4h. The particle size of the aerobic granular sludge is about 2500-3000μm.

[0042] The method for extracting the core of the spherical bioaggregate of the present embodiment comprises the following steps:

[0043] 1) Sample collection and pretreatment: During the uniform mixing stage of the aerobic granular sludge reactor, the granular sludge mixture was taken out through the sampling port using a 50 ml centrifuge tube. After standing for 5 minutes, the upper liquid and some light bulk sludge were poured out; 3-5 particles were sucked with a large-caliber pipette and rinsed three times with deionized water.

[0044] 2) Core separation: The main body of the core separation device for spherical bioaggregates is rectangular and divided into two layers. First, the bottom plate 2 of the extraction device is placed on a horizontal table, and then the four corners are fixed with bolts 1. 3-5 particles are taken out from the outlet of the aeration stage of the aerobic granular sludge reactor, and the particles are rinsed three times with deionized water. The particle size is measured using a wet-method fully automatic laser particle size analyzer; a large-caliber plastic straw (more than 5 mm) is used to place the particles at the center end position 4 of the particle extraction device, and two 0.12 mm microneedles 5 are vertically inserted to fix the particles. Finally, a capillary glass tube 6 is slowly inserted from the outside of the extraction device to the inside of the particle along the microneedle, and the inner part of the capillary glass tube is the core of the particle 7;

[0045] 3) Sample collection: Connect one end of the inner core in the capillary glass tube to the air pipe, use an oxygen pump to connect the glass rotor flowmeter, adjust its parameters to 0.1L / min, and then use a plastic adapter to connect the air pipe, and blow the particle inner core of the capillary glass tube into a 50ml glass beaker filled with deionized water.

[0046] The device for implementing the method comprises a bolt 1 for fixing the extraction device, a bottom plate 2 for fixing the extraction device, a tetrahedral organic glass block 3 in the separation device, a spherical biological aggregate 4, a 0.12 mm needle 5, a capillary glass tube 6, and an extracted kernel 7.

[0047] The contents described in the embodiments of this specification are merely enumerations of implementation forms of the inventive concept and are for illustrative purposes only. The protection scope of the present invention should not be considered to be limited to the specific forms described in this embodiment, and the protection scope of the present invention also extends to equivalent technical means that can be thought of by ordinary technicians in this field based on the inventive concept.

Claims

1. A method for extracting the inner core of a spherical bioaggregate, characterized in that: The method comprises the following steps: 1) Sample collection and processing The uniformly mixed particles are taken out from the uniform mixing stage of the microbial aggregate reactor, and the particles are taken out from the sampling port of the reactor using a 50 ml centrifuge tube, and then the muddy water mixture is separated using a low-speed centrifuge; the supernatant and part of the light floccules after centrifugation are discharged, and the particles are rinsed with deionized water to complete the sample collection; 2) Core separation The main body of the spherical bioaggregate core separation device used is rectangular and divided into two layers, the bottom of the reactor is made of a glass plate, and bolts are used at four right angles of the bottom plate of the extraction device to fix the bottom plate plane; 3) Sample collection.

2. The method for extracting the core of a spherical bioaggregate according to claim 1, characterized in that: In the step 2), the upper layer of the fixed extraction device is composed of four four-sided pyramid organic glass blocks of the same size, which are relatively assembled and placed at the upper left, lower left, upper right and lower right positions on the bottom plate.

3. A method for extracting the inner core of a spherical bioaggregate according to claim 1 or 2, characterized in that: In the step 2), a bioaggregate core separation device is used to separate the inside and outside of the bioaggregate core, and the method is as follows: 2.1) Place the sample particles at the center of four custom-made quadrangular pyramid glass blocks; 2.2) Insert two 0.12 mm microneedles in two perpendicular directions to fix the particles; 2.3) After the sample is fixed, four capillary glass tubes with an inner diameter of 500-2000 μm are sequentially inserted along the needle according to the spherical bioaggregates, and the four capillary glass tubes are slowly pushed until the inner core of the particles is separated from the outer part.

4. The method for extracting the inner core of a spherical bioaggregate according to claim 3, characterized in that: In the step 3), after the core is extracted by capillary glass tubes with different inner diameters, the extracted particle core is placed in the capillary glass tube, and an oxygen pump with a flow rate of 35 L / min, a power of 20 W, and a pressure of 0.01 MPa is used to adjust the flow rate through a 4 mm air tube connected to a glass rotor flowmeter with a range of 0.1-1 L / min. The glass rotor flowmeter is adjusted to 0.1 L / min, and the capillary glass tube is connected to blow the core into a 50 ml glass beaker containing 30 ml of deionized water for collection.

5. The method for extracting the inner core of a spherical bioaggregate according to claim 4, characterized in that: After separating and extracting the inner core of particles with a particle size greater than 1000 μm from the outer core, it is convenient to analyze the distribution of the microbial community structure and gain a deeper understanding of its formation mechanism and mechanism of action.

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